Administrative Appointments

  • Founder and President, SPARK Global (2015 - Present)
  • Fellow, Stanford University Institute for Chemical Biology (2013 - Present)
  • Steering Committee Member, Cardiovascular Institute (2013 - Present)
  • Associate Director, Cardiovascular Institute (2007 - 2011)
  • SPARK Founder and co-Director, Stanford University, School of Medicine (2006 - Present)
  • Senior Associate Dean for Research, Stanford University School of Medicine (2006 - 2013)
  • Member, Cancer Institute at Stanford (2005 - Present)
  • Professor, by courtesy, Department of Neurosurgery (2004 - 2009)
  • Chair, Department of Molecular Pharmacology AKA Department of Chemical and Systems Biology (2002 - 2006)
  • Professor, Department of Chemical and Systems Biology (2001 - Present)
  • Chief, Division of Chemical Biology (2001 - 2002)

Honors & Awards

  • Reed-Hodgson Professor in Human Biology, Stanford University (1996-2001)
  • The George D Smith Professor of Translational Medicine, School of medicine (2005)
  • Janice Pfeffer Distinguished Lecture Award, International Society for Heart Research (ISHR) (2012)

Professional Education

  • Ph.D., Weizmann Institute, Israel, Chemical Immunology (1983)
  • B.S., Tel Aviv University, Israel, Life Sciences (1977)

Community and International Work

  • SPARK Global


    Translational Research

    Partnering Organization(s)

    SPARK prgrams in other academic institutions



    Ongoing Project


    Opportunities for Student Involvement


Research & Scholarship

Current Research and Scholarly Interests

We are a multi-disciplinary research lab that includes chemists, biochemists, biologists and physician scientists. We develop pharmacological agents and apply them to understand molecular and cellular events under basal and disease conditions using in vitro, in culture and in vivo models.

There are several research areas: two use peptide inhibitors and two to small molecules.
1. We study how protein-protein interactions govern cell signaling (Science, 1995). Using rational approaches, we identify short peptide inhibitors of intracellular protein-protein interactions to interfere with signal transduction under basal and pathological conditions (Nature Biotechnology, 2008). This rational approach led to the discovery of the only highly selective protein kinase C (PKC) inhibitors and activators. These peptide regulators of PKC identified the role of this family of enzymes in a number of cellular responses. Importantly, these peptide regulators are useful as therapeutics in a variety of animal models of human diseases, including myocardial infarction and heart failure (Nature Review Drug Discovery, 2013). A phase IIa study in humans demonstrated that one of the peptide inhibitors is efficacious in reducing cardiac damage in myocardial infarction patients. The study was carried out by KAI Pharmaceuticals that was co-founded with Dr. Leon Chen (a graduate student from the lab) in 2002. The company was acquired by Amgen in 2012 and one of KAI's drug was approved in Europe (2016). Current lab efforts focus on rationally generating substrate-specific inhibitors of the multi-substrate kinase, delta PKC (Qvit, J Am Chem Soc 2016; Qvit, Angewante 2016).

2. Recent effort focuses on rational design of inhibitors and activators of large GTPases that regulate mitochondrial dynamics (fusion and fission; Kornfeld, Circ Res 2015). One peptide inhibitor of pathological mitochondrial fission (Qi, JCS 2013; Guo, JCI 2013) is now being developed in Mitoconix (founded in 2016), as a treatment for Huntington's disease and other neurodegenerative diseases (Distanik, J Exp Med, 2016). Another peptide may provide a treatment for Charcot-Marie-Tooth II (Franco, Nature, 2016).

3. We unexpectedly identified aldehyde dehydrogenase 2 (ALDH2), the rate determining enzyme in ethanol metabolism, as a key regulator of cell survival under oxidative stress. We designed a novel assay to screen for activators of ALDH2, called Aldas (for ALDH activators) Science, 2008). Importantly, Aldas correct a structural mutation in ALDH2 found in ~0.5 billion East Asians and therefore represents a new class of drugs that serve as molecular chaperons (Nature Structure and Molecular Biology, 2010). Aldas also prevent nitroglycerin-induced tolerance and improves outcome after myocardial infarction(Science Translational Medicine, 2011). Very few selective activators of enzymes have been described. This research led to founding ALDEA Pharma with Dr. Che-Hong Chen, a senior scientist in the lab (2011); licensed to Foresee (2016). We also founded STAR, an international research organization for ALDH2 enzymopathy (Gross, Ann Rev Tox, 2015). Because defense from oxidative stress is determining cell survival, we examines the benefit of activating different ALDHs in a variety of diseases, including in Fanconi Anemia and radiation disease. Using a small molecule, we also 'hijacked' ALDH3A1 to metabolize the substrate of the mutated ALDH2 (Chen, PNAS, 2016).

4. Current efforts focus also on identifying small molecules that correct genetic defects in another critical enzyme for cell protection, glucose-6-phosphate dehydrogenase (G6PD). Mutations in G6PD lead to the second most common enzymopathy (~350 million people). Using high-throughput screening, in silico design and synthetic organic chemistry and X-ray crystallography, small molecule activators that increase the catalytic activity of the most common G6PD mutations are under development.


Stanford Advisees


All Publications

  • Aldehyde dehydrogenase 2*2 knock-in mice show increased reactive oxygen species production in response to cisplatin treatment. Journal of biomedical science Kim, J., Chen, C., Yang, J., Mochly-Rosen, D. 2017; 24 (1): 33-?


    The aldehyde dehydrogenase (ALDH) enzyme family metabolizes and detoxifies both exogenous and endogenous aldehydes. Since chemotherapeutic agents, such as cisplatin, generate cytotoxic aldehydes and oxidative stress, and chemoresistant cancer cells express high levels of ALDH enzymes, we hypothesized that different ALDH expression within cells may show different chemosensitivity. ALDH2 has the lowest Km for acetaldehyde among ALDH isozymes and detoxifies acetaldehydes in addition to other reactive aldehydes, such as 4-hydroxy-nonenal, malondialdehyde and acrolein produced from lipid peroxidation by reactive oxygen species (ROS). Thus, cells with an ALDH2 variant may sensitize them to these ROS-inducing chemotherapy drugs.Here, we used wild type C57BL/6 mice and ALDH2*2 knock-in mutant mice and compared the basal level of ROS in different tissues. Then, we treated the mice with cisplatin, isolated cells from organs and fractionated them into lysates containing mitochondrial and cytosolic fractions, treated with cisplatin again in vitro, and compared the level of ROS generated.We show that overall ROS production increases with cisplatin treatment in cells with ALDH2 mutation. The treatment of cisplatin in the wild type mice did not change the level of ROS compared to PBS treated controls. In contrast, ALDH2*2 knock-in mutant mice showed a significantly increased level of ROS compared to wild type mice in tongue, lung, kidney and brain tissues without any treatment. ALDH2*2 mutant mice showed 20% of the ALDH2 activity in the kidney compared to wild type mice. Treatment of ALDH2*2 mutant mice with cisplatin showed increased ROS levels in the mitochondrial fraction of kidney. In the cytosolic fraction, treatment of mutant mice with cisplatin increased ROS levels in lung and brain compared to PBS treated controls. Furthermore, ALDH2*2 mutant mice treated with cisplatin showed increased cytotoxicity in the kidney cells compared to PBS treated mutant controls.These data indicate that deficiency in ALDH2 activity may contribute to increased cisplatin sensitivity and cytotoxicity by producing more ROS by the treatment. Based on these data, the amount of cisplatin used in patients may need to be adjusted based on their ALDH2 variant profile.

    View details for DOI 10.1186/s12929-017-0338-8

    View details for PubMedID 28532411

  • Targeting aldehyde dehydrogenase activity in head and neck squamous cell carcinoma with a novel small molecule inhibitor. Oncotarget Kim, J., Ho Shin, J., Chen, C., Cruz, L., Farnebo, L., Yang, J., Borges, P., Kang, G., Mochly-Rosen, D., Sunwoo, J. B. 2017


    Chemoresistant cancer cells express high levels of aldehyde dehydrogenases (ALDHs), particularly in head and neck squamous cell carcinoma (HNSCC). The ALDH family of enzymes detoxify both exogenous and endogenous aldehydes. Since many chemotherapeutic agents, such as cisplatin, result in the generation of cytotoxic aldehydes and oxidative stress, we hypothesized that cells expressing high levels of ALDH may be more chemoresistant due to their increased detoxifying capacity and that inhibitors of ALDHs may sensitize them to these drugs. Here, we show that overall ALDH activity is increased with cisplatin treatment of HNSCC and that ALDH3A1 protein expression is particularly enriched in cells treated with cisplatin. Activation of ALDH3A1 by a small molecule activator (Alda-89) increased survival of HNSCC cells treated with cisplatin. Conversely, treatment with a novel small molecule ALDH inhibitor (Aldi-6) resulted in a marked decrease in cell viability, and the combination of Aldi-6 and cisplatin resulted in a more pronounced reduction of cell viability and a greater reduction in tumor burden in vivo than what was observed with cisplatin alone. These data indicate that ALDH3A1 contributes to cisplatin resistance in HNSCC and that the targeting of ALDH, specifically, ALDH3A1, appears to be a promising strategy in this disease.

    View details for DOI 10.18632/oncotarget.17017

    View details for PubMedID 28455980

  • Coupling between Protein Stability and Catalytic Activity Determines Pathogenicity of G6PD Variants CELL REPORTS Cunningham, A. D., Colavin, A., Huang, K. C., Mochly-Rosen, D. 2017; 18 (11): 2592-2599


    G6PD deficiency, an enzymopathy affecting 7% of the world population, is caused by over 160 identified amino acid variants in glucose-6-phosphate dehydrogenase (G6PD). The clinical presentation of G6PD deficiency is diverse, likely due to the broad distribution of variants across the protein and the potential for multidimensional biochemical effects. In this study, we use bioinformatic and biochemical analyses to interpret the relationship between G6PD variants and their clinical phenotype. Using structural information and statistical analyses of known G6PD variants, we predict the molecular phenotype of five uncharacterized variants from a reference population database. Through multidimensional analysis of biochemical data, we demonstrate that the clinical phenotypes of G6PD variants are largely determined by a trade-off between protein stability and catalytic activity. This work expands the current understanding of the biochemical underpinnings of G6PD variant pathogenicity and suggests a promising avenue for correcting G6PD deficiency by targeting essential structural features of G6PD.

    View details for DOI 10.1016/j.celrep.2017.02.048

    View details for Web of Science ID 000397330000006

    View details for PubMedID 28297664

  • Peptidomimetic therapeutics: scientific approaches and opportunities. Drug discovery today Qvit, N., Rubin, S. J., Urban, T. J., Mochly-Rosen, D., Gross, E. R. 2017; 22 (2): 454-462


    Natural endogenously occurring peptides exhibit desirable medicinal properties, but are often limited in application by rapid proteolysis and inadequate membrane permeability. However, editing naturally occurring peptide sequences to develop peptidomimetic analogs created a promising class of therapeutics that can augment or inhibit molecular interactions. Here, we discuss a variety of chemical modifications, including l to d isomerization, cyclization, and unnatural amino acid substitution, as well as design strategies, such as attachment to cell-penetrating peptides, which are used to develop peptidomimetics. We also provide examples of approved peptidomimetics and discuss several compounds in clinical trials.

    View details for DOI 10.1016/j.drudis.2016.11.003

    View details for PubMedID 27856346

  • Aldehyde dehydrogenase 2 activation and coevolution of its epsilon PKC-mediated phosphorylation sites JOURNAL OF BIOMEDICAL SCIENCE Nene, A., Chen, C., Disatnik, M., Cruz, L., Mochly-Rosen, D. 2017; 24


    Mitochondrial aldehyde dehydrogenase 2 (ALDH2) is a key enzyme for the metabolism of many toxic aldehydes such as acetaldehyde, derived from alcohol drinking, and 4HNE, an oxidative stress-derived lipid peroxidation aldehyde. Post-translational enhancement of ALDH2 activity can be achieved by serine/threonine phosphorylation by epsilon protein kinase C (εPKC). Elevated ALDH2 is beneficial in reducing injury following myocardial infarction, stroke and other oxidative stress and aldehyde toxicity-related diseases. We have previously identified three εPKC phosphorylation sites, threonine 185 (T185), serine 279 (S279) and threonine 412 (T412), on ALDH2. Here we further characterized the role and contribution of each phosphorylation site to the enhancement of enzymatic activity by εPKC.Each individual phosphorylation site was mutated to a negatively charged amino acid, glutamate, to mimic a phosphorylation, or to a non-phosphorylatable amino acid, alanine. ALDH2 enzyme activities and protection against 4HNE inactivation were measured in the presence or absence of εPKC phosphorylation in vitro. Coevolution of ALDH2 and its εPKC phosphorylation sites was delineated by multiple sequence alignments among a diverse range of species and within the ALDH multigene family.We identified S279 as a critical εPKC phosphorylation site in the activation of ALDH2. The critical catalytic site, cysteine 302 (C302) of ALDH2 is susceptible to adduct formation by reactive aldehyde, 4HNE, which readily renders the enzyme inactive. We show that phosphomimetic mutations of T185E, S279E and T412E confer protection of ALDH2 against 4HNE-induced inactivation, indicating that phosphorylation on these three sites by εPKC likely also protects the enzyme against reactive aldehydes. Finally, we demonstrate that the three ALDH2 phosphorylation sites co-evolved with εPKC over a wide range of species. Alignment of 18 human ALDH isozymes, indicates that T185 and S279 are unique ALDH2, εPKC specific phosphorylation sites, while T412 is found in other ALDH isozymes. We further identified three highly conserved serine/threonine residues (T384, T433 and S471) in all 18 ALDH isozymes that may play an important phosphorylation-mediated regulatory role in this important family of detoxifying enzymes.εPKC phosphorylation and its coevolution with ALDH2 play an important role in the regulation and protection of ALDH2 enzyme activity.

    View details for DOI 10.1186/s12929-016-0312-x

    View details for Web of Science ID 000391701200003

    View details for PubMedID 28056995

  • Peptides and peptidomimetics as regulators of protein-protein interactions. Current opinion in structural biology Cunningham, A. D., Qvit, N., Mochly-Rosen, D. 2017; 44: 59-66


    Protein-protein interactions are essential for almost all intracellular and extracellular biological processes. Regulation of protein-protein interactions is one strategy to regulate cell fate in a highly selective manner. Specifically, peptides are ideal candidates for inhibition of protein-protein interactions because they can mimic a protein surface to effectively compete for binding. Additionally, peptides are synthetically accessible and can be stabilized by chemical modifications. In this review, we survey screening and rational design methods for identifying peptides to inhibit protein-protein interactions, as well as methods for stabilizing peptides to effectively mimic protein surfaces. In addition, we discuss recent applications of peptides to regulate protein-protein interactions for both basic research and therapeutic purposes.

    View details for DOI 10.1016/

    View details for PubMedID 28063303

  • Disruption of mitochondrial quality control in peripheral artery disease: New therapeutic opportunities PHARMACOLOGICAL RESEARCH Ueta, C. B., Gomes, K. S., Ribeiro, M. A., Mochly-Rosen, D., Ferreira, J. C. 2017; 115: 96-106


    Peripheral artery disease (PAD) is a multifactorial disease initially triggered by reduced blood supply to the lower extremities due to atherosclerotic obstructions. It is considered a major public health problem worldwide, affecting over 200 million people. Management of PAD includes smoking cessation, exercise, statin therapy, antiplatelet therapy, antihypertensive therapy and surgical intervention. Although these pharmacological and non-pharmacological interventions usually increases blood flow to the ischemic limb, morbidity and mortality associated with PAD continue to increase. This scenario raises new fundamental questions regarding the contribution of intrinsic metabolic changes in the distal affected skeletal muscle to the progression of PAD. Recent evidence suggests that disruption of skeletal muscle mitochondrial quality control triggered by intermittent ischemia-reperfusion injury is associated with increased morbidity in individuals with PAD. The mitochondrial quality control machinery relies on surveillance systems that help maintaining mitochondrial homeostasis upon stress. In this review, we describe some of the most critical mechanisms responsible for the impaired skeletal muscle mitochondrial quality control in PAD. We also discuss recent findings on the central role of mitochondrial bioenergetics and quality control mechanisms including mitochondrial fusion-fission balance, turnover, oxidative stress and aldehyde metabolism in the pathophysiology of PAD, and highlight their potential as therapeutic targets.

    View details for DOI 10.1016/j.phrs.2016.11.016

    View details for Web of Science ID 000392790400009

    View details for PubMedID 27876411

  • The Role of Mitochondrial Aldehyde Dehydrogenase 2 (ALDH2) in Neuropathology and Neurodegeneration. Acta neurologica Taiwanica Chen, C., Joshi, A. U., Mochly-Rosen, D. 2016; 25(4): 111-123


    Aldehydes-induced toxicity has been implicated in many neurodegenerative diseases. Exposure to reactive aldehydes from (1) alcohol and food metabolism; (2) environmental pollutants, including car, factory exhausts, smog, pesticides, herbicides; (3) metabolism of neurotransmitters, amino acids and (4) lipid peroxidation of biological membrane from excessive ROS, all contribute to 'aldehydic load' that has been linked to the pathology of neurodegenerative diseases. In particular, the α, β-unsaturated aldehydes derived from lipid peroxidation, 4-hydroxynonenal (4-HNE), DOPAL (MAO product of dopamine), malondialdehyde, acrolein and acetaldehyde, all readily form chemical adductions with proteins, DNA and lipids, thus causing neurotoxicity. Mitochondrial aldehyde dehydrogenase 2 (ALDH 2) is a major aldehyde metabolizing enzyme that protects against deleterious aldehyde buildup in brain, a tissue that has a particularly high mitochondrial content. In this review, we highlight the deleterious effects of increased aldehydic load in the neuropathology of ischemic stroke, Alzheimer's disease and Parkinson's disease. We also discuss evidence for the association between ALDH2 deficiency, a common East Asianspecific mutation, and these neuropathologies. A novel class of small molecule aldehyde dehydrogenase activators (Aldas), represented by Alda-1, reduces neuronal cell death in models of ischemic stroke, Alzheimer's disease and Parkinson's disease. Together, these data suggest that reducing aldeydic load by enhancing the activity of aldehyde dehydrogenases, such as ALDH2, represents as a therapeutic strategy for neurodegenerative diseases.

    View details for PubMedID 28382610

  • Genetic variations of aldehyde dehydrogenase 2 and alcohol dehydrogenase 1B are associated with the etiology of atrial fibrillation in Japanese JOURNAL OF BIOMEDICAL SCIENCE Nakano, Y., Ochi, H., Onohara, Y., Sairaku, A., Tokuyama, T., Matsumura, H., Tomomori, S., Amioka, M., Hironomobe, N., Motoda, C., Oda, N., Chayama, K., Chen, C., Gross, E. R., Mochly-Rosen, D., Kihara, Y. 2016; 23


    Alcohol consumption and oxidative stress are well-known risk factors for developing atrial fibrillation (AF). Single nucleotide polymorphisms (SNPs) of alcohol dehydrogenase (ADH1B) and aldehyde dehydrogenase 2 (ALDH2) genes encoding enzymes of alcohol and reactive aldehyde metabolism, respectively, are prevalent among East Asians. Here, we examined whether these SNPs were associated with AF in Japanese patients.Five hundred seventy-seven Japanese patients with AF undergoing catheter ablation and 1935 controls at Hiroshima University Hospital were studied. Alcohol consumption habits, medical history, electrocardiogram (EKG), electrophysiology and cardiac echocardiography were reviewed. Patients were also genotyped for ALDH2 (rs671) and ADH1B (rs1229984). A significant linear correlation was found between ALDH2 genotype and mean alcohol intake (P = 1.7 × 10(-6)). Further, ALDH2 (rs671) was associated with AF (P = 7.6 × 10(-4), odds ratio [OR] = 0.6). Frequency of the ALDH2 SNP allele A which limits acetaldehyde metabolism was lower in patients with AF (18.8%) than in controls (23.5%). In contrast, we found that the frequencies of the ADH1B SNP genotypes were similar in patients with AF and in controls. Subset analysis among the 182 patients with lone AF and 914 controls (control II) (<60 years of age and without hypertension), both ALDH2 and ADH1B SNPs were significantly associated with AF (P = 0.013, OR = 0.7; P = 0.0007, OR = 1.4, respectively). The frequency of the dysfunctional allele A of ALDH2 was significantly lower and the dysfunctional allele G of ADH1B was significantly higher in patients with lone AF than in control II (ALDH2 A allele frequency = 0.176 vs 0.235, OR = 1.3, P = 0.013, ADH1B SNP G allele frequency = 0.286 vs 0.220, OR = 1.4, P = 0.0007).When considering all patients enrolled, the dysfunctional ALDH2 allele was negatively associated with AF. When examining a subset of patients with lone AF, the dysfunctional ALDH2 allele was negatively associated with AF and the slower metabolizing ADH1B allele was positively associated with AF. Hence, prolonged metabolic conversion of alcohol to acetaldehyde may be associated with the occurrence of AF in the Japanese and other East Asian populations.

    View details for DOI 10.1186/s12929-016-0304-x

    View details for Web of Science ID 000390284900001

    View details for PubMedID 27927211

    View details for PubMedCentralID PMC5142328

  • Engineered Substrate-Specific Delta PKC Antagonists to Enhance Cardiac Therapeutics ANGEWANDTE CHEMIE-INTERNATIONAL EDITION Qvit, N., Kornfeld, O. S., Mochly-Rosen, D. 2016; 55 (50): 15672-15679


    Most protein kinases phosphorylate multiple substrates, each of which induces different and sometimes opposing functions. Determining the role of phosphorylation of each substrate following a specific stimulus is challenging but is essential to elucidate the role of that substrate in the signaling event. Here we describe a rational approach to identify inhibitors of delta protein kinase C (δPKC), each inhibiting the phosphorylation of only one of δPKC's substrates. δPKC regulates many signaling events and we hypothesized that a docking inhibitor of a given substrate to δPKC should selectively abrogate the phosphorylation of only that substrate, without affecting the phosphorylation of the other δPKC substrates. Here we report the development of selective inhibitors of three δPKC substrates (in vitro Kd ≈3 nm); two greatly reduced ischemia-induced cardiac injury with an IC50 of ≈200 nm and the third had no effect, indicating that its respective substrate phosphorylation by δPKC has no role in the response to cardiac ischemia and reperfusion. The three inhibitors are highly specific; even at 1 μm, the phosphorylation of other δPKC protein substrates was unaffected. The rationale we describe is likely applicable for the development of other substrate-specific inhibitors as well.

    View details for DOI 10.1002/anie.201605429

    View details for Web of Science ID 000389246400039

    View details for PubMedID 27860071

    View details for PubMedCentralID PMC5308906

  • Potential biomarkers to follow the progression and treatment response of Huntington's disease. journal of experimental medicine Disatnik, M., Joshi, A. U., Saw, N. L., Shamloo, M., Leavitt, B. R., Qi, X., Mochly-Rosen, D. 2016


    Huntington's disease (HD) is a rare genetic disease caused by expanded polyglutamine repeats in the huntingtin protein resulting in selective neuronal loss. Although genetic testing readily identifies those who will be affected, current pharmacological treatments do not prevent or slow down disease progression. A major challenge is the slow clinical progression and the inability to biopsy the affected tissue, the brain, making it difficult to design short and effective proof of concept clinical trials to assess treatment benefit. In this study, we focus on identifying peripheral biomarkers that correlate with the progression of the disease and treatment benefit. We recently developed an inhibitor of pathological mitochondrial fragmentation, P110, to inhibit neurotoxicity in HD. Changes in levels of mitochondrial DNA (mtDNA) and inflammation markers in plasma, a product of DNA oxidation in urine, mutant huntingtin aggregates, and 4-hydroxynonenal adducts in muscle and skin tissues were all noted in HD R6/2 mice relative to wild-type mice. Importantly, P110 treatment effectively reduced the levels of these biomarkers. Finally, abnormal levels of mtDNA were also found in plasma of HD patients relative to control subjects. Therefore, we identified several potential peripheral biomarkers as candidates to assess HD progression and the benefit of intervention for future clinical trials.

    View details for PubMedID 27821553

  • Correcting mitochondrial fusion by manipulating mitofusin conformations. Nature Franco, A., Kitsis, R. N., Fleischer, J. A., Gavathiotis, E., Kornfeld, O. S., Gong, G., Biris, N., Benz, A., Qvit, N., Donnelly, S. K., Chen, Y., Mennerick, S., Hodgson, L., Mochly-Rosen, D., Dorn, G. W. 2016


    Mitochondria are dynamic organelles that exchange contents and undergo remodelling during cyclic fusion and fission. Genetic mutations in MFN2 (the gene encoding mitofusin 2) interrupt mitochondrial fusion and cause the untreatable neurodegenerative condition Charcot-Marie-Tooth disease type 2A (CMT2A). It has not yet been possible to directly modulate mitochondrial fusion, in part because the structural basis of mitofusin function is not completely understood. Here we show that mitofusins adopt either a fusion-constrained or a fusion-permissive molecular conformation, directed by specific intramolecular binding interactions, and demonstrate that mitofusin-dependent mitochondrial fusion can be regulated in mouse cells by targeting these conformational transitions. On the basis of this model, we engineered a cell-permeant minipeptide to destabilize the fusion-constrained conformation of mitofusin and promote the fusion-permissive conformation, reversing mitochondrial abnormalities in cultured fibroblasts and neurons that harbour CMT2A-associated genetic defects. The relationship between the conformational plasticity of mitofusin 2 and mitochondrial dynamism reveals a central mechanism that regulates mitochondrial fusion, the manipulation of which can correct mitochondrial pathology triggered by defective or imbalanced mitochondrial dynamics.

    View details for DOI 10.1038/nature20156

    View details for PubMedID 27775718

    View details for PubMedCentralID PMC5315023

  • In Vivo Post-Cardiac Arrest Myocardial Dysfunction Is Supported by Ca2+/Calmodulin-Dependent Protein Kinase II-Mediated Calcium Long-Term Potentiation and Mitigated by Alda-1, an Agonist of Aldehyde Dehydrogenase Type 2. Circulation Woods, C. E., Shang, C., Taghavi, F., Downey, P., Zalewski, A., Rubio, G. R., Liu, J., Homburger, J. R., Grunwald, Z., Qi, W., Bollensdorff, C., Thanaporn, P., Ali, A., Riemer, R. K., Kohl, P., Mochly-Rosen, D., Gerstenfeld, E., Large, S., Ali, Z. A., Ashley, E. A. 2016; 134 (13): 961-977


    Survival after sudden cardiac arrest is limited by postarrest myocardial dysfunction, but understanding of this phenomenon is constrained by a lack of data from a physiological model of disease. In this study, we established an in vivo model of cardiac arrest and resuscitation, characterized the biology of the associated myocardial dysfunction, and tested novel therapeutic strategies.We developed rodent models of in vivo postarrest myocardial dysfunction using extracorporeal membrane oxygenation resuscitation followed by invasive hemodynamics measurement. In postarrest isolated cardiomyocytes, we assessed mechanical load and Ca(2) (+)-induced Ca(2+) release (CICR) simultaneously using the microcarbon fiber technique and observed reduced function and myofilament calcium sensitivity. We used a novel fiberoptic catheter imaging system and a genetically encoded calcium sensor, GCaMP6f, to image CICR in vivo.We found potentiation of CICR in isolated cells from this extracorporeal membrane oxygenation model and in cells isolated from an ischemia/reperfusion Langendorff model perfused with oxygenated blood from an arrested animal but not when reperfused in saline. We established that CICR potentiation begins in vivo. The augmented CICR observed after arrest was mediated by the activation of Ca(2+)/calmodulin-dependent protein kinase II (CaMKII). Increased phosphorylation of CaMKII, phospholamban, and ryanodine receptor 2 was detected in the postarrest period. Exogenous adrenergic activation in vivo recapitulated Ca(2+) potentiation but was associated with lesser CaMKII activation. Because oxidative stress and aldehydic adduct formation were high after arrest, we tested a small-molecule activator of aldehyde dehydrogenase type 2, Alda-1, which reduced oxidative stress, restored calcium and CaMKII homeostasis, and improved cardiac function and postarrest outcome in vivo.Cardiac arrest and reperfusion lead to CaMKII activation and calcium long-term potentiation, which support cardiomyocyte contractility in the face of impaired postarrest myofilament calcium sensitivity. Alda-1 mitigates these effects, normalizes calcium cycling, and improves outcome.

    View details for DOI 10.1161/CIRCULATIONAHA.116.021618

    View details for PubMedID 27582424

    View details for PubMedCentralID PMC5040468

  • Transient Receptor Potential Vanilloid 1 Regulates Mitochondrial Membrane Potential and Myocardial Reperfusion Injury. Journal of the American Heart Association Hurt, C. M., Lu, Y., M Stary, C., Piplani, H., Small, B. A., Urban, T. J., Qvit, N., Gross, G. J., Mochly-Rosen, D., Gross, E. R. 2016; 5 (9)


    The transient receptor potential vanilloid 1 (TRPV1) mediates cellular responses to pain, heat, or noxious stimuli by calcium influx; however, the cellular localization and function of TRPV1 in the cardiomyocyte is largely unknown. We studied whether myocardial injury is regulated by TRPV1 and whether we could mitigate reperfusion injury by limiting the calcineurin interaction with TRPV1.In primary cardiomyocytes, confocal and electron microscopy demonstrates that TRPV1 is localized to the mitochondria. Capsaicin, the specific TRPV1 agonist, dose-dependently reduced mitochondrial membrane potential and was blocked by the TRPV1 antagonist capsazepine or the calcineurin inhibitor cyclosporine. Using in silico analysis, we discovered an interaction site for TRPV1 with calcineurin. We synthesized a peptide, V1-cal, to inhibit the interaction between TRPV1 and calcineurin. In an in vivo rat myocardial infarction model, V1-cal given just prior to reperfusion substantially mitigated myocardial infarct size compared with vehicle, capsaicin, or cyclosporine (24±3% versus 61±2%, 45±1%, and 49±2%, respectively; n=6 per group; P<0.01 versus all groups). Infarct size reduction by V1-cal was also not seen in TRPV1 knockout rats.TRPV1 is localized at the mitochondria in cardiomyocytes and regulates mitochondrial membrane potential through an interaction with calcineurin. We developed a novel therapeutic, V1-cal, that substantially reduces reperfusion injury by inhibiting the interaction of calcineurin with TRPV1. These data suggest that TRPV1 is an end-effector of cardioprotection and that modulating the TRPV1 protein interaction with calcineurin limits reperfusion injury.

    View details for PubMedID 27671317

    View details for PubMedCentralID PMC5079036

  • The entangled ER-mitochondrial axis as a potential therapeutic strategy in neurodegeneration: A tangled duo unchained. Cell calcium Joshi, A. U., Kornfeld, O. S., Mochly-Rosen, D. 2016; 60 (3): 218-234


    Endoplasmic reticulum (ER) and mitochondrial function have both been shown to be critical events in neurodegenerative diseases. The ER mediates protein folding, maturation, sorting as well acts as calcium storage. The unfolded protein response (UPR) is a stress response of the ER that is activated by the accumulation of misfolded proteins within the ER lumen. Although the molecular mechanisms underlying ER stress-induced apoptosis are not completely understood, increasing evidence suggests that ER and mitochondria cooperate to signal cell death. Similarly, calcium-mediated mitochondrial function and dynamics not only contribute to ATP generation and calcium buffering but are also a linchpin in mediating cell fate. Mitochondria and ER form structural and functional networks (mitochondria-associated ER membranes [MAMs]) essential to maintaining cellular homeostasis and determining cell fate under various pathophysiological conditions. Regulated Ca(2+) transfer from the ER to the mitochondria is important in maintaining control of pro-survival/pro-death pathways. In this review, we summarize the latest therapeutic strategies that target these essential organelles in the context of neurodegenerative diseases.

    View details for DOI 10.1016/j.ceca.2016.04.010

    View details for PubMedID 27212603

    View details for PubMedCentralID PMC5012927

  • Aldehyde dehydrogenase 2 in aplastic anemia, Fanconi anemia and hematopoietic stem cells MOLECULAR GENETICS AND METABOLISM Van Wassenhove, L. D., Mochly-Rosen, D., Weinberg, K. I. 2016; 119 (1-2): 28-36


    Maintenance of the hematopoietic stem cell (HSC) compartment depends on the ability to metabolize exogenously and endogenously generated toxins, and to repair cellular damage caused by such toxins. Reactive aldehydes have been demonstrated to cause specific genotoxic injury, namely DNA interstrand cross-links. Aldehyde dehydrogenase 2 (ALDH2) is a member of a 19 isoenzyme ALDH family with different substrate specificities, subcellular localization, and patterns of expression. ALDH2 is localized in mitochondria and is essential for the metabolism of acetaldehyde, thereby placing it directly downstream of ethanol metabolism. Deficiency in ALDH2 expression and function are caused by a single nucleotide substitution and resulting amino acid change, called ALDH2*2. This genetic polymorphism affects 35-45% of East Asians (about ~560 million people), and causes the well-known Asian flushing syndrome, which results in disulfiram-like reactions after ethanol consumption. Recently, the ALDH2*2 genotype has been found to be associated with marrow failure, with both an increased risk of sporadic aplastic anemia and more rapid progression of Fanconi anemia. This review discusses the unexpected interrelationship between aldehydes, ALDH2 and hematopoietic stem cell biology, and in particular its relationship to Fanconi anemia.

    View details for DOI 10.1016/j.ymgme.2016.07.004

    View details for Web of Science ID 000384631000005

    View details for PubMedID 27650066

  • PKC-ALDH2 Pathway Plays a Novel Role in Adipocyte Differentiation PLOS ONE Yu, Y., Liao, P., Guo, C., Chen, C., Mochly-Rosen, D., Chuang, L. 2016; 11 (8)


    The ALDH2 gene encodes the mitochondrial aldehyde dehydrogenase 2 (ALDH2), a critical enzyme involved in ethanol clearance through acetaldehyde metabolism. ALDH2 also catalyzes the metabolism of other bioreactive aldehydes, including propionaldehyde, butyraldehyde, and 4-hydroxykenals (4-HNE). Increased levels of 4-HNE in adipose tissue positively correlate with obesity and insulin resistance. However, it remains unclear whether ALDH2 is involved in regulation of adipocyte differentiation. Here, we found that ALDH2 protein levels were lower in white adipose tissue of high-fat diet-fed mice and ob/ob mice relative to lean mice. Knockdown of ALDH2 expression in 3T3-L1 preadipocytes caused an increase in intracellular 4-HNE, thereby attenuated adipocyte differentiation. By contrast, an ALDH2 activator, Alda-1, significantly accelerated adipogenesis, which was accompanied by an increase in adipogenic gene expression. Consistently, adipogenesis was reduced when protein kinase C ε (PKCε), an ALDH2 phosphorylating activator, was silenced in 3T3-L1 preadipocytes, whereas treatment with a PKCε agonist in 3T3-L1 preadipocytes enhanced adipogenesis. Whole-genome microarray profiling of Alda-1-treated cells demonstrated several upregulated transcripts encoding proteins involved in metabolism and the majority of these transcripts are for proteins involved in PPAR signaling pathways. Furthermore, PKCε-ALDH2 interaction alleviates 4-HNE induced aberrant PPARγ regulation on adipogenesis. Taken together, these results demonstrate that ALDH2 activation enhances adipogenesis and signaling pathways involving PPARγ. Thus, activation of PKCε-ALDH2 regulatory axis may be a therapeutic target for treating obesity and type 2 diabetes.

    View details for DOI 10.1371/journal.pone.0161993

    View details for Web of Science ID 000382877200052

    View details for PubMedID 27575855

  • Glyceraldehyde-3-Phosphate Dehydrogenase (GAPDH) Protein-Protein Interaction Inhibitor Reveals a Non-catalytic Role for GAPDH Oligomerization in Cell Death JOURNAL OF BIOLOGICAL CHEMISTRY Qvit, N., Joshi, A. U., Cunningham, A. D., Ferreira, J. C., Mochly-Rosen, D. 2016; 291 (26): 13608-13621


    Glyceraldehyde-3-phosphate dehydrogenase (GAPDH), an important glycolytic enzyme, has a non-catalytic (thus a non-canonical) role in inducing mitochondrial elimination under oxidative stress. We recently demonstrated that phosphorylation of GAPDH by delta protein kinase C (PKC) inhibits this GAPDH-dependent mitochondrial elimination. deltaPKC phosphorylation of GAPDH correlates with increased cell injury following oxidative stress, suggesting that inhibiting GAPDH phosphorylation should decrease cell injury. Using rational design, we identified pseudoGAPDH peptide, an inhibitor of deltaPKC-mediated GAPDH phosphorylation that does not inhibit the phosphorylation of other deltaPKC substrates. Unexpectedly, pseudoGAPDH decreased mitochondrial elimination and increased cardiac damage in an animal model of heart attack. Either treatment with pseudoGAPDH or direct phosphorylation of GAPDH by deltaPKC decreased GAPDH tetramerization, which corresponded to reduced GAPDH glycolytic activity in vitro and ex vivo. Taken together, our study identified the potential mechanism by which oxidative stress inhibits the protective GAPDH-mediated elimination of damaged mitochondria. Our study also identified a pharmacological tool, pseudoGAPDH peptide, with interesting properties. pseudoGAPDH peptide is an inhibitor of the interaction between deltaPKC and GAPDH, and of the resulting phosphorylation of GAPDH by deltaPKC. pseudoGAPDH peptide is also an inhibitor of GAPDH oligomerization and thus an inhibitor of GAPDH glycolytic activity. Finally, we found that pseudoGAPDH peptide is an inhibitor of the elimination of damaged mitochondria. We discuss how this unique property of increasing cell damage following oxidative stress suggests a potential use for pseudoGAPDH peptide-based therapy.

    View details for DOI 10.1074/jbc.M115.711630

    View details for Web of Science ID 000379771100018

    View details for PubMedID 27129213

  • Selective Phosphorylation Inhibitor of Delta Protein Kinase C-Pyruvate Dehydrogenase Kinase Protein-Protein Interactions: Application for Myocardial Injury in Vivo JOURNAL OF THE AMERICAN CHEMICAL SOCIETY Qvit, N., Disatnik, M., Sho, E., Mochly-Rosen, D. 2016; 138 (24): 7626-7635


    Protein kinases regulate numerous cellular processes, including cell growth, metabolism, and cell death. Because the primary sequence and the three-dimensional structure of many kinases are highly similar, the development of selective inhibitors for only one kinase is challenging. Furthermore, many protein kinases are pleiotropic, mediating diverse and sometimes even opposing functions by phosphorylating multiple protein substrates. Here, we set out to develop an inhibitor of a selective protein kinase phosphorylation of only one of its substrates. Focusing on the pleiotropic delta protein kinase C (δPKC), we used a rational approach to identify a distal docking site on δPKC for its substrate, pyruvate dehydrogenase kinase (PDK). We reasoned that an inhibitor of PDK's docking should selectively inhibit the phosphorylation of only PDK without affecting phosphorylation of the other δPKC substrates. Our approach identified a selective inhibitor of PDK docking to δPKC with an in vitro Kd of ∼50 nM and reducing cardiac injury IC50 of ∼5 nM. This inhibitor, which did not affect the phosphorylation of other δPKC substrates even at 1 μM, demonstrated that PDK phosphorylation alone is critical for δPKC-mediated injury by heart attack. The approach we describe is likely applicable for the identification of other substrate-specific kinase inhibitors.

    View details for DOI 10.1021/jacs.6b02724

    View details for Web of Science ID 000378584600035

    View details for PubMedID 27218445

  • Glucose-6-Phosphate Dehydrogenase Deficiency and the Need for a Novel Treatment to Prevent Kernicterus CLINICS IN PERINATOLOGY Cunningham, A. D., Hwang, S., Mochly-Rosen, D. 2016; 43 (2): 341-?


    Hyperbilirubinemia occurs frequently in newborns, and in severe cases can progress to kernicterus and permanent developmental disorders. Glucose-6-phosphate dehydrogenase (G6PD) deficiency, one of the most common human enzymopathies, is a major risk factor for hyperbilirubinemia and greatly increases the risk of kernicterus even in the developed world. Therefore, a novel treatment for kernicterus is needed, especially for G6PD-deficient newborns. Oxidative stress is a hallmark of bilirubin toxicity in the brain. We propose that the activation of G6PD via a small molecule chaperone is a potential strategy to increase endogenous defense against bilirubin-induced oxidative stress and prevent kernicterus.

    View details for DOI 10.1016/j.clp.2016.01.010

    View details for Web of Science ID 000378367300012

    View details for PubMedID 27235212

  • Scaffold proteins LACK and TRACK as potential drug targets in kinetoplastid parasites: Development of inhibitors. International journal for parasitology. Drugs and drug resistance Qvit, N., Schechtman, D., Pena, D. A., Berti, D. A., Soares, C. O., Miao, Q., Liang, L. A., Baron, L. A., Teh-Poot, C., Martínez-Vega, P., Ramirez-Sierra, M. J., Churchill, E., Cunningham, A. D., Malkovskiy, A. V., Federspiel, N. A., Gozzo, F. C., Torrecilhas, A. C., Manso Alves, M. J., Jardim, A., Momar, N., Dumonteil, E., Mochly-Rosen, D. 2016; 6 (1): 74-84


    Parasitic diseases cause ∼500,000 deaths annually and remain a major challenge for therapeutic development. Using a rational design based approach, we developed peptide inhibitors with anti-parasitic activity that were derived from the sequences of parasite scaffold proteins LACK ( L eishmania's receptor for activated C-kinase) and TRACK ( T rypanosoma receptor for activated C-kinase). We hypothesized that sequences in LACK and TRACK that are conserved in the parasites, but not in the mammalian ortholog, RACK (Receptor for activated C-kinase), may be interaction sites for signaling proteins that are critical for the parasites' viability. One of these peptides exhibited leishmanicidal and trypanocidal activity in culture. Moreover, in infected mice, this peptide was also effective in reducing parasitemia and increasing survival without toxic effects. The identified peptide is a promising new anti-parasitic drug lead, as its unique features may limit toxicity and drug-resistance, thus overcoming central limitations of most anti-parasitic drugs.

    View details for DOI 10.1016/j.ijpddr.2016.02.003

    View details for PubMedID 27054066

  • Scaffold proteins LACK and TRACK as potential drug targets in kinetoplastid parasites: Development of inhibitors INTERNATIONAL JOURNAL FOR PARASITOLOGY-DRUGS AND DRUG RESISTANCE Qvit, N., Schechtman, D., Pena, D. A., Berti, D. A., Soares, C. O., Miao, Q., Liang, L. (., Baron, L. A., Teh-Poot, C., Martinez-Vega, P., Ramirez-Sierra, M. J., Churchill, E., Cunningham, A. D., Malkovskiy, A. V., Federspiel, N. A., Gozzo, F. C., Torrecilhas, A. C., Manso Alves, M. J., Jardim, A., Momar, N., Dumonteil, E., Mochly-Rosen, D. 2016; 6 (1): 74-84
  • VCP recruitment to mitochondria causes mitophagy impairment and neurodegeneration in models of Huntington's disease. Nature communications Guo, X., Sun, X., Hu, D., Wang, Y., Fujioka, H., Vyas, R., Chakrapani, S., Joshi, A. U., Luo, Y., Mochly-Rosen, D., Qi, X. 2016; 7: 12646-?


    Mutant Huntingtin (mtHtt) causes neurodegeneration in Huntington's disease (HD) by evoking defects in the mitochondria, but the underlying mechanisms remains elusive. Our proteomic analysis identifies valosin-containing protein (VCP) as an mtHtt-binding protein on the mitochondria. Here we show that VCP is selectively translocated to the mitochondria, where it is bound to mtHtt in various HD models. Mitochondria-accumulated VCP elicits excessive mitophagy, causing neuronal cell death. Blocking mtHtt/VCP mitochondrial interaction with a peptide, HV-3, abolishes VCP translocation to the mitochondria, corrects excessive mitophagy and reduces cell death in HD mouse- and patient-derived cells and HD transgenic mouse brains. Treatment with HV-3 reduces behavioural and neuropathological phenotypes of HD in both fragment- and full-length mtHtt transgenic mice. Our findings demonstrate a causal role of mtHtt-induced VCP mitochondrial accumulation in HD pathogenesis and suggest that the peptide HV-3 might be a useful tool for developing new therapeutics to treat HD.

    View details for DOI 10.1038/ncomms12646

    View details for PubMedID 27561680

  • Impaired GAPDH-induced mitophagy contributes to the pathology of Huntington's disease EMBO MOLECULAR MEDICINE Hwang, S., Disatnik, M., Mochly-Rosen, D. 2015; 7 (10): 1307-1326

    View details for Web of Science ID 000362414000008

    View details for PubMedID 26268247

  • Mitochondrial Reactive Oxygen Species at the Heart of the Matter New Therapeutic Approaches for Cardiovascular Diseases CIRCULATION RESEARCH Kornfeld, O. S., Hwang, S., Disatnik, M., Chen, C., Qvit, N., Mochly-Rosen, D. 2015; 116 (11): 1783-1799
  • Mitochondrial reactive oxygen species at the heart of the matter: new therapeutic approaches for cardiovascular diseases. Circulation research Kornfeld, O. S., Hwang, S., Disatnik, M., Chen, C., Qvit, N., Mochly-Rosen, D. 2015; 116 (11): 1783-1799


    Reactive oxygen species (ROS) have been implicated in a variety of age-related diseases, including multiple cardiovascular disorders. However, translation of ROS scavengers (antioxidants) into the clinic has not been successful. These antioxidants grossly reduce total levels of cellular ROS including ROS that participate in physiological signaling. In this review, we challenge the traditional antioxidant therapeutic approach that targets ROS directly with novel approaches that improve mitochondrial functions to more effectively treat cardiovascular diseases.

    View details for DOI 10.1161/CIRCRESAHA.116.305432

    View details for PubMedID 25999419

    View details for PubMedCentralID PMC4443500

  • Pharmacological recruitment of aldehyde dehydrogenase 3A1 (ALDH3A1) to assist ALDH2 in acetaldehyde and ethanol metabolism in vivo PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Chen, C., Cruz, L. A., Mochly-Rosen, D. 2015; 112 (10): 3074-3079


    Correcting a genetic mutation that leads to a loss of function has been a challenge. One such mutation is in aldehyde dehydrogenase 2 (ALDH2), denoted ALDH2*2. This mutation is present in ∼ 0.6 billion East Asians and results in accumulation of toxic acetaldehyde after consumption of ethanol. To temporarily increase metabolism of acetaldehyde in vivo, we describe an approach in which a pharmacologic agent recruited another ALDH to metabolize acetaldehyde. We focused on ALDH3A1, which is enriched in the upper aerodigestive track, and identified Alda-89 as a small molecule that enables ALDH3A1 to metabolize acetaldehyde. When given together with the ALDH2-specific activator, Alda-1, Alda-89 reduced acetaldehyde-induced behavioral impairment by causing a rapid reduction in blood ethanol and acetaldehyde levels after acute ethanol intoxication in both wild-type and ALDH2-deficient, ALDH2*1/*2, heterozygotic knock-in mice. The use of a pharmacologic agent to recruit an enzyme to metabolize a substrate that it usually does not metabolize may represent a novel means to temporarily increase elimination of toxic agents in vivo.

    View details for DOI 10.1073/pnas.1414657112

    View details for Web of Science ID 000350646500050

    View details for PubMedID 25713355

  • New therapeutics to modulate mitochondrial dynamics and mitophagy in cardiac diseases. Journal of molecular medicine (Berlin, Germany) Disatnik, M., Hwang, S., Ferreira, J. C., Mochly-Rosen, D. 2015; 93 (3): 279-287


    The processes that control the number and shape of the mitochondria (mitochondrial dynamics) and the removal of damaged mitochondria (mitophagy) have been the subject of intense research. Recent work indicates that these processes may contribute to the pathology associated with cardiac diseases. This review describes some of the key proteins that regulate these processes and their potential as therapeutic targets for cardiac diseases.

    View details for DOI 10.1007/s00109-015-1256-4

    View details for PubMedID 25652199

  • Aldehydic load and aldehyde dehydrogenase 2 profile during the progression of post-myocardial infarction cardiomyopathy: Benefits of Alda-1 INTERNATIONAL JOURNAL OF CARDIOLOGY Gomes, K. M., Bechara, L. R., Lima, V. M., Ribeiro, M. A., Campos, J. C., Dourado, P. M., Kowaltowski, A. J., Mochly-Rosen, D., Ferreira, J. C. 2015; 179: 129-138


    We previously demonstrated that reducing cardiac aldehydic load by aldehyde dehydrogenase 2 (ALDH2), a mitochondrial enzyme responsible for metabolizing the major lipid peroxidation product, protects against acute ischemia/reperfusion injury and chronic heart failure. However, time-dependent changes in ALDH2 profile, aldehydic load and mitochondrial bioenergetics during progression of post-myocardial infarction (post-MI) cardiomyopathy are unknown and should be established to determine the optimal time window for drug treatment.Here we characterized cardiac ALDH2 activity and expression, lipid peroxidation, 4-hydroxy-2-nonenal (4-HNE) adduct formation, glutathione pool and mitochondrial energy metabolism and H₂O₂ release during the 4 weeks after permanent left anterior descending (LAD) coronary artery occlusion in rats.We observed a sustained disruption of cardiac mitochondrial function during the progression of post-MI cardiomyopathy, characterized by >50% reduced mitochondrial respiratory control ratios and up to 2 fold increase in H₂O₂ release. Mitochondrial dysfunction was accompanied by accumulation of cardiac and circulating lipid peroxides and 4-HNE protein adducts and down-regulation of electron transport chain complexes I and V. Moreover, increased aldehydic load was associated with a 90% reduction in cardiac ALDH2 activity and increased glutathione pool. Further supporting an ALDH2 mechanism, sustained Alda-1 treatment (starting 24h after permanent LAD occlusion surgery) prevented aldehydic overload, mitochondrial dysfunction and improved ventricular function in post-MI cardiomyopathy rats.Taken together, our findings demonstrate a disrupted mitochondrial metabolism along with an insufficient cardiac ALDH2-mediated aldehyde clearance during the progression of ventricular dysfunction, suggesting a potential therapeutic value of ALDH2 activators during the progression of post-myocardial infarction cardiomyopathy.

    View details for DOI 10.1016/j.ijcard.2014.10.140

    View details for Web of Science ID 000346089400044

    View details for PubMedID 25464432

  • A personalized medicine approach for asian americans with the aldehyde dehydrogenase 2*2 variant. Annual review of pharmacology and toxicology Gross, E. R., Zambelli, V. O., Small, B. A., Ferreira, J. C., Chen, C., Mochly-Rosen, D. 2015; 55: 107-127


    Asian Americans are one of the fastest-growing populations in the United States. A relatively large subset of this population carries a unique loss-of-function point mutation in aldehyde dehydrogenase 2 (ALDH2), ALDH2*2. Found in approximately 560 million people of East Asian descent, ALDH2*2 reduces enzymatic activity by approximately 60% to 80% in heterozygotes. Furthermore, this variant is associated with a higher risk for several diseases affecting many organ systems, including a particularly high incidence relative to the general population of esophageal cancer, myocardial infarction, and osteoporosis. In this review, we discuss the pathophysiology associated with the ALDH2*2 variant, describe why this variant needs to be considered when selecting drug treatments, and suggest a personalized medicine approach for Asian American carriers of this variant. We also discuss future clinical and translational perspectives regarding ALDH2*2 research.

    View details for DOI 10.1146/annurev-pharmtox-010814-124915

    View details for PubMedID 25292432

  • Neuroprotective effects of aldehyde dehydrogenase 2 activation in rotenone-induced cellular and animal models of parkinsonism EXPERIMENTAL NEUROLOGY Chiu, C., Yeh, T., Lai, S., Wu-Chou, Y., Chen, C., Mochly-Rosen, D., Huang, Y., Chen, Y., Chen, C., Chang, Y., Wang, H., Lu, C. 2015; 263: 244-253


    Many studies have shown that mitochondrial aldehyde dehydrogenase 2 (ALDH2) functions as a cellular protector against oxidative stress by detoxification of cytotoxic aldehydes. Within dopaminergic neurons, dopamine is metabolized by monoamine oxidase to yield 3,4-dihydroxyphenylacetaldehyde (DOPAL) then converts to a less toxic acid product by ALDH. The highly toxic and reactive DOPAL has been hypothesized to contribute to the selective neurodegeneration in Parkinson's disease (PD). In this study, we investigated the neuroprotective mechanism and therapeutic effect of ALDH2 in rotenone models for parkinsonism. Overexpression of wild-type ALDH2 gene, but not the enzymatically deficient mutant ALDH2*2 (E504K), reduced rotenone-induced cell death. Application of a potent activator of ALDH2, Alda-1, was effective in protecting against rotenone-induced apoptotic cell death in both SH-SY5Y cells and primary cultured substantia nigra (SN) dopaminergic neurons. In addition, intraperitoneal administration of Alda-1 significantly reduced rotenone- or MPTP-induced death of SN tyrosine hydroxylase (TH)-positive dopaminergic neurons. The attenuation of rotenone-induced apoptosis by Alda-1 resulted from decreasing ROS accumulation, reversal of mitochondrial membrane potential depolarization, and inhibition of activation of proteins related to mitochondrial apoptotic pathway. The present study demonstrates that ALDH2 plays a crucial role in maintaining normal mitochondrial function to protect against neurotoxicity and that Alda-1 is effective in ameliorating mitochondrial dysfunction and inhibiting mitochondria-mediated apoptotic pathway. These results indicate that ALDH2 activation could be a neuroprotective therapy for PD.

    View details for DOI 10.1016/j.expneurol.2014.09.016

    View details for Web of Science ID 000346626100025

    View details for PubMedID 25263579

    View details for PubMedCentralID PMC4415848

  • The many hats of protein kinase C delta: one enzyme with many functions BIOCHEMICAL SOCIETY TRANSACTIONS Qvit, N., Mochly-Rosen, D. 2014; 42: 1529-1533
  • Characterization of the molecular mechanisms underlying increased ischemic damage in the aldehyde dehydrogenase 2 genetic polymorphism using a human induced pluripotent stem cell model system SCIENCE TRANSLATIONAL MEDICINE Ebert, A. D., Kodo, K., Liang, P., Wu, H., Huber, B. C., Riegler, J., Churko, J., Lee, J., de Almeida, P., Lan, F., Diecke, S., Burridge, P. W., Gold, J. D., Mochly-Rosen, D., Wu, J. C. 2014; 6 (255)
  • Characterization of the molecular mechanisms underlying increased ischemic damage in the aldehyde dehydrogenase 2 genetic polymorphism using a human induced pluripotent stem cell model system. Science translational medicine Ebert, A. D., Kodo, K., Liang, P., Wu, H., Huber, B. C., Riegler, J., Churko, J., Lee, J., de Almeida, P., Lan, F., Diecke, S., Burridge, P. W., Gold, J. D., Mochly-Rosen, D., Wu, J. C. 2014; 6 (255): 255ra130-?


    Nearly 8% of the human population carries an inactivating point mutation in the gene that encodes the cardioprotective enzyme aldehyde dehydrogenase 2 (ALDH2). This genetic polymorphism (ALDH2*2) is linked to more severe outcomes from ischemic heart damage and an increased risk of coronary artery disease (CAD), but the underlying molecular bases are unknown. We investigated the ALDH2*2 mechanisms in a human model system of induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) generated from individuals carrying the most common heterozygous form of the ALDH2*2 genotype. We showed that the ALDH2*2 mutation gave rise to elevated amounts of reactive oxygen species and toxic aldehydes, thereby inducing cell cycle arrest and activation of apoptotic signaling pathways, especially during ischemic injury. We established that ALDH2 controls cell survival decisions by modulating oxidative stress levels and that this regulatory circuitry was dysfunctional in the loss-of-function ALDH2*2 genotype, causing up-regulation of apoptosis in cardiomyocytes after ischemic insult. These results reveal a new function for the metabolic enzyme ALDH2 in modulation of cell survival decisions. Insight into the molecular mechanisms that mediate ALDH2*2-related increased ischemic damage is important for the development of specific diagnostic methods and improved risk management of CAD and may lead to patient-specific cardiac therapies.

    View details for DOI 10.1126/scitranslmed.3009027

    View details for PubMedID 25253673

  • Aldehyde dehydrogenase 2 activation in heart failure restores mitochondrial function and improves ventricular function and remodelling CARDIOVASCULAR RESEARCH Gomes, K. M., Campos, J. C., Bechara, L. R., Queliconi, B., Lima, V. M., Disatnik, M., Magno, P., Chen, C., Brum, P. C., Kowaltowski, A. J., Mochly-Rosen, D., Ferreira, J. C. 2014; 103 (4): 498-508


    We previously demonstrated that pharmacological activation of mitochondrial aldehyde dehydrogenase 2 (ALDH2) protects the heart against acute ischaemia/reperfusion injury. Here, we determined the benefits of chronic activation of ALDH2 on the progression of heart failure (HF) using a post-myocardial infarction model.We showed that a 6-week treatment of myocardial infarction-induced HF rats with a selective ALDH2 activator (Alda-1), starting 4 weeks after myocardial infarction at a time when ventricular remodelling and cardiac dysfunction were present, improved cardiomyocyte shortening, cardiac function, left ventricular compliance and diastolic function under basal conditions, and after isoproterenol stimulation. Importantly, sustained Alda-1 treatment showed no toxicity and promoted a cardiac anti-remodelling effect by suppressing myocardial hypertrophy and fibrosis. Moreover, accumulation of 4-hydroxynonenal (4-HNE)-protein adducts and protein carbonyls seen in HF was not observed in Alda-1-treated rats, suggesting that increasing the activity of ALDH2 contributes to the reduction of aldehydic load in failing hearts. ALDH2 activation was associated with improved mitochondrial function, including elevated mitochondrial respiratory control ratios and reduced H2O2 release. Importantly, selective ALDH2 activation decreased mitochondrial Ca(2+)-induced permeability transition and cytochrome c release in failing hearts. Further supporting a mitochondrial mechanism for ALDH2, Alda-1 treatment preserved mitochondrial function upon in vitro aldehydic load.Selective activation of mitochondrial ALDH2 is sufficient to improve the HF outcome by reducing the toxic effects of aldehydic overload on mitochondrial bioenergetics and reactive oxygen species generation, suggesting that ALDH2 activators, such as Alda-1, have a potential therapeutic value for treating HF patients.

    View details for DOI 10.1093/cvr/cvu125

    View details for Web of Science ID 000342917500009

    View details for PubMedID 24817685

  • Aldehyde dehydrogenase-2 regulates nociception in rodent models of acute inflammatory pain. Science translational medicine Zambelli, V. O., Gross, E. R., Chen, C., Gutierrez, V. P., Cury, Y., Mochly-Rosen, D. 2014; 6 (251): 251ra118-?


    Exogenous aldehydes can cause pain in animal models, suggesting that aldehyde dehydrogenase-2 (ALDH2), which metabolizes many aldehydes, may regulate nociception. To test this hypothesis, we generated a knock-in mouse with an inactivating point mutation in ALDH2 (ALDH2*2), which is also present in human ALDH2 of ~540 million East Asians. The ALDH2*1/*2 heterozygotic mice exhibited a larger response to painful stimuli than their wild-type littermates, and this heightened nociception was inhibited by an ALDH2-selective activator (Alda-1). No effect on inflammation per se was observed. Using a rat model, we then showed that nociception tightly correlated with ALDH activity (R(2) = 0.90) and that reduced nociception was associated with less early growth response protein 1 (EGR1) in the spinal cord and less reactive aldehyde accumulation at the insult site (including acetaldehyde and 4-hydroxynonenal). Further, acetaldehyde- and formalin-induced nociceptive behavior was greater in the ALDH2*1/*2 mice than in the wild-type mice. Finally, Alda-1 treatment was even beneficial when given after the inflammatory agent was administered. Our data in rodent models suggest that the mitochondrial enzyme ALDH2 regulates nociception and could serve as a molecular target for pain control, with ALDH2 activators, such as Alda-1, as potential non-narcotic, cardiac-safe analgesics. Furthermore, our results suggest a possible genetic basis for East Asians' apparent lower pain tolerance.

    View details for DOI 10.1126/scitranslmed.3009539

    View details for PubMedID 25163478

  • Aldehyde dehydrogenase-2 regulates nociception in rodent models of acute inflammatory pain. Science translational medicine Zambelli, V. O., Gross, E. R., Chen, C., Gutierrez, V. P., Cury, Y., Mochly-Rosen, D. 2014; 6 (251): 251ra118-?

    View details for DOI 10.1126/scitranslmed.3009539

    View details for PubMedID 25163478

  • Peripheral Sensitization Increases Opioid Receptor Expression and Activation by Crotalphine in Rats PLOS ONE Zambelli, V. O., de Oliveira Fernandes, A. C., Gutierrez, V. P., Batista Ferreira, J. C., Parada, C. A., Mochly-Rosen, D., Cury, Y. 2014; 9 (3)


    Inflammation enhances the peripheral analgesic efficacy of opioid drugs, but the mechanisms involved in this phenomenon have not been fully elucidated. Crotalphine (CRP), a peptide that was first isolated from South American rattlesnake C.d. terrificus venom, induces a potent and long-lasting anti-nociceptive effect that is mediated by the activation of peripheral opioid receptors. Because the high efficacy of CRP is only observed in the presence of inflammation, we aimed to elucidate the mechanisms involved in the CRP anti-nociceptive effect induced by inflammation. Using real-time RT-PCR, western blot analysis and ELISA assays, we demonstrate that the intraplantar injection of prostaglandin E2 (PGE2) increases the mRNA and protein levels of the µ- and κ-opioid receptors in the dorsal root ganglia (DRG) and paw tissue of rats within 3 h of the injection. Using conformation state-sensitive antibodies that recognize activated opioid receptors, we show that PGE2, alone does not increase the activation of these opioid receptors but that in the presence of PGE2, the activation of specific opioid receptors by CRP and selective µ- and κ-opioid receptor agonists (positive controls) increases. Furthermore, PGE2 down-regulated the expression and activation of the δ-opioid receptor. CRP increased the level of activated mitogen-activated protein kinases in cultured DRG neurons, and this increase was dependent on the activation of protein kinase Cζ. This CRP effect was much more prominent when the cells were pretreated with PGE2. These results indicate that the expression and activation of peripheral opioid receptors by opioid-like drugs can be up- or down-regulated in the presence of an acute injury and that acute tissue injury enhances the efficacy of peripheral opioids.

    View details for DOI 10.1371/journal.pone.0090576

    View details for Web of Science ID 000332475500070

    View details for PubMedID 24594607

  • Development of Selective Inhibitors for Aldehyde Dehydrogenases Based on Substituted Indole-2,3-diones JOURNAL OF MEDICINAL CHEMISTRY Kimble-Hill, A. C., Parajuli, B., Chen, C., Mochly-Rosen, D., Hurley, T. D. 2014; 57 (3): 714-722


    Aldehyde dehydrogenases (ALDH) participate in multiple metabolic pathways and have been indicated to play a role in several cancerous disease states. Our laboratory is interested in developing novel and selective ALDH inhibitors. We looked to further work recently published by developing a class of isoenzyme-selective inhibitors using similar indole-2,3-diones that exhibit differential inhibition of ALDH1A1, ALDH2, and ALDH3A1. Kinetic and X-ray crystallography data suggest that these inhibitors are competitive against aldehyde binding, forming direct interactions with active-site cysteine residues. The selectivity is precise in that these compounds appear to interact directly with the catalytic nucleophile, Cys243, in ALDH3A1 but not in ALDH2. In ALDH2, the 3-keto group is surrounded by the adjacent Cys301/303. Surprisingly, the orientation of the interaction changes depending on the nature of the substitutions on the basic indole ring structure and correlates well with the observed structure-activity relationships for each ALDH isoenzyme.

    View details for DOI 10.1021/jm401377v

    View details for Web of Science ID 000331493000014

    View details for PubMedID 24444054

  • The challenge in translating basic research discoveries to treatment of Huntington disease. Rare diseases (Austin, Tex.) Mochly-Rosen, D., Disatnik, M., Qi, X. 2014; 2


    Huntington disease is a rare neurodegenerative disease resulting from insertion and/or expansion of a polyglutamine repeats close to the N-terminal of the huntingtin protein. Although unequivocal genetic tests have been available for about 20 years, current pharmacological treatments do not prevent or slow down disease progression. Recent basic research identified potential novel drug targets for the treatment of Huntington disease. However, there are clear challenges in translating these discoveries into treatment strategies for these patients. The following is a brief discussion of these challenges using our recent experience as an example.

    View details for DOI 10.4161/rdis.28637

    View details for PubMedID 25054095



    A family of detoxifying enzymes called aldehyde dehydrogenases (ALDHs) has been a subject of recent interest, as its role in detoxifying aldehydes that accumulate through metabolism and to which we are exposed from the environment has been elucidated. Although the human genome has 19 ALDH genes, one ALDH emerges as a particularly important enzyme in a variety of human pathologies. This ALDH, ALDH2, is located in the mitochondrial matrix with much known about its role in ethanol metabolism. Less known is a new body of research to be discussed in this review, suggesting that ALDH2 dysfunction may contribute to a variety of human diseases including cardiovascular diseases, diabetes, neurodegenerative diseases, stroke, and cancer. Recent studies suggest that ALDH2 dysfunction is also associated with Fanconi anemia, pain, osteoporosis, and the process of aging. Furthermore, an ALDH2 inactivating mutation (termed ALDH2*2) is the most common single point mutation in humans, and epidemiological studies suggest a correlation between this inactivating mutation and increased propensity for common human pathologies. These data together with studies in animal models and the use of new pharmacological tools that activate ALDH2 depict a new picture related to ALDH2 as a critical health-promoting enzyme.

    View details for DOI 10.1152/physrev.00017.2013

    View details for Web of Science ID 000329194000001

    View details for PubMedID 24382882

  • Inhibition of mitochondrial fragmentation diminishes Huntington's disease-associated neurodegeneration JOURNAL OF CLINICAL INVESTIGATION Guo, X., Disatnik, M., Monbureau, M., Shamloo, M., Mochly-Rosen, D., Qi, X. 2013; 123 (12): 5371-5388


    Huntington's disease (HD) is the result of expression of a mutated Huntingtin protein (mtHtt), and is associated with a variety of cellular dysfunctions including excessive mitochondrial fission. Here, we tested whether inhibition of excessive mitochondrial fission prevents mtHtt-induced pathology. We developed a selective inhibitor (P110-TAT) of the mitochondrial fission protein dynamin-related protein 1 (DRP1). We found that P110-TAT inhibited mtHtt-induced excessive mitochondrial fragmentation, improved mitochondrial function, and increased cell viability in HD cell culture models. P110-TAT treatment of fibroblasts from patients with HD and patients with HD with iPS cell-derived neurons reduced mitochondrial fragmentation and corrected mitochondrial dysfunction. P110-TAT treatment also reduced the extent of neurite shortening and cell death in iPS cell-derived neurons in patients with HD. Moreover, treatment of HD transgenic mice with P110-TAT reduced mitochondrial dysfunction, motor deficits, neuropathology, and mortality. We found that p53, a stress gene involved in HD pathogenesis, binds to DRP1 and mediates DRP1-induced mitochondrial and neuronal damage. Furthermore, P110-TAT treatment suppressed mtHtt-induced association of p53 with mitochondria in multiple HD models. These data indicate that inhibition of DRP1-dependent excessive mitochondrial fission with a P110-TAT-like inhibitor may prevent or slow the progression of HD.

    View details for DOI 10.1172/JCI70911

    View details for Web of Science ID 000327826100040

    View details for PubMedID 24231356

    View details for PubMedCentralID PMC3859413

  • Acute Inhibition of Excessive Mitochondrial Fission After Myocardial Infarction Prevents Long-term Cardiac Dysfunction JOURNAL OF THE AMERICAN HEART ASSOCIATION Disatnik, M., Ferreira, J. C., Campos, J. C., Gomes, K. S., Dourado, P. M., Qi, X., Mochly-Rosen, D. 2013; 2 (5)


    Ischemia and reperfusion (IR) injury remains a major cause of morbidity and mortality and multiple molecular and cellular pathways have been implicated in this injury. We determined whether acute inhibition of excessive mitochondrial fission at the onset of reperfusion improves mitochondrial dysfunction and cardiac contractility postmyocardial infarction in rats.We used a selective inhibitor of the fission machinery, P110, which we have recently designed. P110 treatment inhibited the interaction of fission proteins Fis1/Drp1, decreased mitochondrial fission, and improved bioenergetics in three different rat models of IR, including primary cardiomyocytes, ex vivo heart model, and an in vivo myocardial infarction model. Drp1 transiently bound to the mitochondria following IR injury and P110 treatment blocked this Drp1 mitochondrial association. Compared with control treatment, P110 (1 μmol/L) decreased infarct size by 28 ± 2% and increased adenosine triphosphate levels by 70+1% after IR relative to control IR in the ex vivo model. Intraperitoneal injection of P110 (0.5 mg/kg) at the onset of reperfusion in an in vivo model resulted in improved mitochondrial oxygen consumption by 68% when measured 3 weeks after ischemic injury, improved cardiac fractional shortening by 35%, reduced mitochondrial H2O2 uncoupling state by 70%, and improved overall mitochondrial functions.Together, we show that excessive mitochondrial fission at reperfusion contributes to long-term cardiac dysfunction in rats and that acute inhibition of excessive mitochondrial fission at the onset of reperfusion is sufficient to result in long-term benefits as evidenced by inhibiting cardiac dysfunction 3 weeks after acute myocardial infarction.

    View details for DOI 10.1161/JAHA.113.000461

    View details for Web of Science ID 000326343200009

    View details for PubMedID 24103571

  • PKC epsilon activation promotes FGF-2 exocytosis and induces endothelial cell proliferation and sprouting JOURNAL OF MOLECULAR AND CELLULAR CARDIOLOGY Monti, M., Donnini, S., Morbidelli, L., Giachetti, A., Mochly-Rosen, D., Mignatti, P., Ziche, M. 2013; 63: 107-117


    Protein kinase C epsilon (PKCε) activation controls fibroblast growth factor-2 (FGF-2) angiogenic signaling. Here, we examined the effect of activating PKCε on FGF-2 dependent vascular growth and endothelial activation. ψεRACK, a selective PKCε agonist induces pro-angiogenic responses in endothelial cells, including formation of capillary like structures and cell growth. These effects are mediated by FGF-2 export to the cell membrane, as documented by biotinylation and immunofluorescence, and FGF-2/FGFR1 signaling activation, as attested by ERK1/2-STAT-3 phosphorylation and de novo FGF-2 synthesis. Similarly, vascular endothelial growth factor (VEGF) activates PKCε in endothelial cells, and promotes FGF-2 export and FGF-2/FGFR1 signaling activation. ψεRACK fails to elicit responses in FGF-2(-/-) endothelial cells, and in cells pretreated with methylamine (MeNH2), an exocytosis inhibitor, indicating that both intracellular FGF-2 and its export toward the membrane are required for the ψεRACK activity. In vivo ψεRACK does not induce angiogenesis in the rabbit cornea. However, ψεRACK promotes VEGF angiogenic responses, an effect sustained by endothelial FGF-2 release and synthesis, since anti-FGF-2 antibody strongly attenuates VEGF responses. The results demonstrate that PKCε stimulation promotes angiogenesis and modulates VEGF activity, by inducing FGF-2 release and autocrine signaling.

    View details for DOI 10.1016/j.yjmcc.2013.07.006

    View details for Web of Science ID 000325387300012

    View details for PubMedID 23880610

  • Nociceptive-induced myocardial remote conditioning is mediated by neuronal gamma protein kinase C. Basic research in cardiology Gross, E. R., Hsu, A. K., Urban, T. J., Mochly-Rosen, D., Gross, G. J. 2013; 108 (5): 381-?


    Deciphering the remote conditioning molecular mechanism may provide targets to develop therapeutics that can broaden the clinical application. To further investigate this, we tested whether two protein kinase C (PKC) isozymes, the ubiquitously expressed epsilon PKC (εPKC) and the neuronal-specific gamma PKC (γPKC), mediate nociceptive-induced remote myocardial conditioning. Male Sprague-Dawley rats were used for both in vivo and ex vivo myocardial ischemia-reperfusion protocols. For the in vivo studies, using a surgical abdominal incision for comparison, applying only to the abdomen either bradykinin or the εPKC activator (ψεRACK) reduced myocardial infarct size (45 ± 1, 44 ± 2 %, respectively, vs. incision: 43 ± 2 %, and control: 63 ± 2 %, P < 0.001). Western blot showed only εPKC, and not γPKC, is highly expressed in the myocardium. However, applying a selective γPKC inhibitor (γV5-3) to the abdominal skin blocked remote protection by any of these strategies. Using an ex vivo isolated heart model without an intact nervous system, only selective εPKC activation, unlike a selective classical PKC isozyme activator (activating α, β, βII, and γ), reduced myocardial injury. Importantly, the classical PKC isozyme activator given to the abdomen in vivo (with an intact nervous system including γPKC) during myocardial ischemia reduced infarct size as effectively as an abdominal incision or ψεRACK (45 ± 1 vs. 45 ± 2 and 47 ± 1 %, respectively). The classical PKC activator-induced protection was also blocked by spinal cord surgical transection. These findings identified potential remote conditioning mimetics, with these strategies effective even during myocardial ischemia. A novel mechanism of nociceptive-induced remote conditioning, involving γPKC, was also identified.

    View details for DOI 10.1007/s00395-013-0381-x

    View details for PubMedID 23982492

  • A Novel Aldehyde Dehydrogenase-3 Activator (Alda-89) Protects Submandibular Gland Function from Irradiation without Accelerating Tumor Growth. Clinical cancer research Xiao, N., Cao, H., Chen, C., Kong, C. S., Ali, R., Chan, C., Sirjani, D., Graves, E., Koong, A., Giaccia, A., Mochly-Rosen, D., Le, Q. 2013; 19 (16): 4455-4464


    To determine the effect of Alda-89 (an ALDH3 activitor) on (i) the function of irradiated (radiotherapy) submandibular gland (SMG) in mice, (ii) its toxicity profile, and (iii) its effect on the growth of head and neck cancer (HNC) in vitro and in vivo.Adult mice were infused with Alda-89 or vehicle before, during, and after radiotherapy. Saliva secretion was monitored weekly. Hematology, metabolic profile, and postmortem evaluation for toxicity were examined at the time of sacrifice. Alda-89 or vehicle was applied to HNC cell lines in vitro, and severe combined immunodeficient (SCID) mice transplanted with HNC in vivo with or without radiation; HNC growth was monitored. The ALDH3A1 and ALDH3A2 protein expression was evaluated in 89 patients with HNC and correlated to freedom from relapse (FFR) and overall survival (OS).Alda-89 infusion significantly resulted in more whole saliva production and a higher percentage of preserved acini after radiotherapy compared with vehicle control. There was no difference in the complete blood count, metabolic profile, and major organ morphology between the Alda-89 and vehicle groups. Compared with vehicle control, Alda-89 treatment neither accelerated HNC cell proliferation in vitro, nor did it affect tumor growth in vivo with or without radiotherapy. Higher expression of ALDH3A1 or ALDH3A2 was not significantly associated with worse FFR or OS in either human papillomavirus (HPV)-positive or HPV-negative group.Alda-89 preserves salivary function after radiotherapy without affecting HNC growth or causing measurable toxicity in mice. It is a promising candidate to mitigate radiotherapy-related xerostomia.

    View details for DOI 10.1158/1078-0432.CCR-13-0127

    View details for PubMedID 23812668

    View details for PubMedCentralID PMC3745542

  • A Novel Aldehyde Dehydrogenase-3 Activator (Alda-89) Protects Submandibular Gland Function from Irradiation without Accelerating Tumor Growth CLINICAL CANCER RESEARCH Xiao, N., Cao, H., Chen, C., Kong, C. S., Ali, R., Chan, C., Sirjani, D., Graves, E., Koong, A., Giaccia, A., Mochly-Rosen, D., Quynh-Thu Le, Q. T. 2013; 19 (16): 4455-4464
  • Glyceraldehyde-3-phosphate Dehydrogenase (GAPDH) Phosphorylation by Protein Kinase C delta (PKC delta) Inhibits Mitochondria Elimination by Lysosomal-like Structures following Ischemia and Reoxygenation-induced Injury JOURNAL OF BIOLOGICAL CHEMISTRY Yogalingam, G., Hwang, S., Ferreira, J. C., Mochly-Rosen, D. 2013; 288 (26): 18947-18960


    After cardiac ischemia and reperfusion or reoxygenation (I/R), damaged mitochondria propagate tissue injury by promoting cell death. One possible mechanism to protect from I/R-induced injury is the elimination of damaged mitochondria by mitophagy. Here we identify new molecular events that lead to mitophagy using a cell culture model and whole hearts subjected to I/R. We found that I/R induces glyceraldehyde-3-phosphate dehydrogenase (GAPDH) association with mitochondria and promotes direct uptake of damaged mitochondria into multi-organellar lysosomal-like (LL) structures for elimination independently of the macroautophagy pathway. We also found that protein kinase C delta (δPKC) inhibits GAPDH-driven mitophagy by phosphorylating mitochondrial-associated GAPDH at threonine 246 following I/R. Phosphorylated GAPDH promotes the accumulation of mitochondria at the periphery of LL structures, which coincides with increased mitochondrial permeability. Either inhibition of δPKC or expression of a phosphorylation-defective GAPDH mutant during I/R promotes a reduction in mitochondrial mass and apoptosis, thus indicating rescued mitophagy. Taken together, we identified a GAPDH/δPKC signaling switch, which is activated during oxidative stress to regulate the balance between cell survival by mitophagy and cell death due to accumulation of damaged mitochondria.

    View details for DOI 10.1074/jbc.M113.466870

    View details for Web of Science ID 000321335800032

    View details for PubMedID 23653351

  • Selective activation of protein kinase C? in mitochondria is neuroprotective in vitro and reduces focal ischemic brain injury in mice. Journal of neuroscience research Sun, X., Budas, G. R., Xu, L., Barreto, G. E., Mochly-Rosen, D., Giffard, R. G. 2013; 91 (6): 799-807


    Activation of protein kinase C∊ (PKC∊) confers protection against neuronal ischemia/reperfusion. Activation of PKC∊ leads to its translocation to multiple intracellular sites, so a mitochondria-selective PKC∊ activator was used to test the importance of mitochondrial activation to the neuroprotective effect of PKC∊. PKC∊ can regulate key cytoprotective mitochondrial functions, including electron transport chain activity, reactive oxygen species (ROS) generation, mitochondrial permeability transition, and detoxification of reactive aldehydes. We tested the ability of mitochondria-selective activation of PKC∊ to protect primary brain cell cultures or mice subjected to ischemic stroke. Pretreatment with either general PKC∊ activator peptide, TAT-Ψ∊RACK, or mitochondrial-selective PKC∊ activator, TAT-Ψ∊HSP90, reduced cell death induced by simulated ischemia/reperfusion in neurons, astrocytes, and mixed neuronal cultures. The protective effects of both TAT-Ψ∊RACK and TAT-Ψ∊HSP90 were blocked by the PKC∊ antagonist ∊V1-2 , indicating that protection requires PKC∊ interaction with its anchoring protein, TAT-∊RACK. Further supporting a mitochondrial mechanism for PKC∊, neuroprotection by TAT-Ψ∊HSP90 was associated with a marked delay in mitochondrial membrane depolarization and significantly attenuated ROS generation during ischemia. Importantly, TAT-Ψ∊HSP90 reduced infarct size and reduced neurological deficit in C57/BL6 mice subjected to middle cerebral artery occlusion and 24 hr of reperfusion. Thus selective activation of mitochondrial PKC∊ preserves mitochondrial function in vitro and improves outcome in vivo, suggesting potential therapeutic value clinically when brain ischemia is anticipated, including neurosurgery and cardiac surgery.

    View details for DOI 10.1002/jnr.23186

    View details for PubMedID 23426889

  • In vivo measurement of aldehyde dehydrogenase-2 activity in rat liver ethanol model using dynamic MRSI of hyperpolarized [1-(13) C]pyruvate. NMR in biomedicine Josan, S., Xu, T., Yen, Y., Hurd, R., Ferreira, J., Chen, C., Mochly-Rosen, D., Pfefferbaum, A., Mayer, D., Spielman, D. 2013; 26 (6): 607-612


    To date, measurements of the activity of aldehyde dehydrogenase-2 (ALDH2), a critical mitochondrial enzyme for the elimination of certain cytotoxic aldehydes in the body and a promising target for drug development, have been largely limited to in vitro methods. Recent advancements in MRS of hyperpolarized (13) C-labeled substrates have provided a method to detect and image in vivo metabolic pathways with signal-to-noise ratio gains greater than 10 000-fold over conventional MRS techniques. However aldehydes, because of their toxicity and short T1 relaxation times, are generally poor targets for such (13) C-labeled studies. In this work, we show that dynamic MRSI of hyperpolarized [1-(13) C]pyruvate and its conversion to [1-(13) C]lactate can provide an indirect in vivo measurement of ALDH2 activity via the concentration of NADH (nicotinamide adenine dinucleotide, reduced form), a co-factor common to both the reduction of pyruvate to lactate and the oxidation of acetaldehyde to acetate. Results from a rat liver ethanol model (n = 9) show that changes in (13) C-lactate labeling following the bolus injection of hyperpolarized pyruvate are highly correlated with changes in ALDH2 activity (R(2)  = 0.76). Copyright © 2012 John Wiley & Sons, Ltd.

    View details for DOI 10.1002/nbm.2897

    View details for PubMedID 23225495

    View details for PubMedCentralID PMC3634870

  • Mitochondrial aldehyde dehydrogenase-2 activation prevents beta-amyloid-induced endothelial cell dysfunction and restores angiogenesis JOURNAL OF CELL SCIENCE Solito, R., Corti, F., Chen, C., Mochly-Rosen, D., Giachetti, A., Ziche, M., Donnini, S. 2013; 126 (9): 1952-1961


    Amyloid β peptides (Aβ1-40 and Aβ1-42) cause cerebral degeneration by impairing the activity of angiogenic factors and inducing apoptosis and senescence in the endothelium. Amyloid peptides are known to induce oxidative stress. Impairment of mitochondrial aldehyde dehydrogenase 2 (ALDH2) following oxidative stress, results in accumulation of toxic aldehydes, particularly 4-hydroxynoneal (4-HNE). We sought to determine the role of mitochondrial ALDH2 in Aβ-related impairment of angiogenesis. We hypothesized that by increasing the detoxification activity of ALDH2 we would reduce Aβ-driven endothelial injuries and restore angiogenesis. We used a selective ALDH2 activator, Alda-1, assessing its ability to repair mitochondrial dysfunction in the endothelium. Treatment of human endothelial cells with Aβ1-40 (5-50 µM) induced loss of mitochondrial membrane potential, increased cytochrome c release and ROS accumulation. These events were associated with 4-HNE accumulation and decrease in ALDH2 activity (40%), and resulted in disassembly of endothelial junctions, as evidenced by β-catenin phosphorylation, disorganization of adherens and tight junctions, and by disruption of pseudocapillary formation. Alda-1 (10-40 µM) abolished Aβ-induced 4-HNE accumulation, apoptosis and vascular leakiness, fully restoring the pro-angiogenic endothelial phenotype and responses to FGF-2. Our data document that mitochondrial ALDH2 in the endothelium is a target for the vascular effect of Aβ, including loss of barrier function and angiogenesis. ALDH2 activation, by restoring mitochondrial functions in the endothelium, prevents Aβ-induced dysfunction and anti-angiogenic effects. Thus, agents activating ALDH2 may reduce endothelial injuries including those occurring in cerebral amyloid angiopathy, preserving the angiogenic potential of the endothelium.

    View details for DOI 10.1242/jcs.117184

    View details for Web of Science ID 000319561700007

    View details for PubMedID 23447675

  • A novel Drp1 inhibitor diminishes aberrant mitochondrial fission and neurotoxicity JOURNAL OF CELL SCIENCE Qi, X., Qvit, N., Su, Y., Mochly-Rosen, D. 2013; 126 (3): 789-802


    Excessive mitochondrial fission is associated with the pathology of a number of neurodegenerative diseases. Therefore, inhibitors of aberrant mitochondrial fission could provide important research tools in addition to potential leads for drug development. Using a rational approach, we designed a novel and selective peptide inhibitor, P110, of excessive mitochondrial fission. P110 inhibits Drp1 enzyme activity and blocks Drp1/Fis1 interaction in vitro and in cultured neurons, whereas it has no effect on the interaction between Drp1 and other mitochondrial adaptors, as demonstrated by co-immunoprecipitation. Furthermore, using a model of Parkinson's disease (PD) in culture, we demonstrated that P110 is neuroprotective by inhibiting mitochondrial fragmentation and reactive oxygen species (ROS) production and subsequently improving mitochondrial membrane potential and mitochondrial integrity. P110 increased neuronal cell viability by reducing apoptosis and autophagic cell death, and reduced neurite loss of primary dopaminergic neurons in this PD cell culture model. We also found that P110 treatment appears to have minimal effects on mitochondrial fission and cell viability under basal conditions. Finally, P110 required the presence of Drp1 to inhibit mitochondrial fission under oxidative stress conditions. Taken together, our findings suggest that P110, as a selective peptide inhibitor of Drp1, might be useful for the treatment of diseases in which excessive mitochondrial fission and mitochondrial dysfunction occur.

    View details for DOI 10.1242/jcs.114439

    View details for Web of Science ID 000317281700009

    View details for PubMedID 23239023

    View details for PubMedCentralID PMC3619809

  • Exercise Training Restores Cardiac Protein Quality Control in Heart Failure PLOS ONE Campos, J. C., Queliconi, B. B., Dourado, P. M., Cunha, T. F., Zambelli, V. O., Bechara, L. R., Kowaltowski, A. J., Brum, P. C., Mochly-Rosen, D., Ferreira, J. C. 2012; 7 (12)


    Exercise training is a well-known coadjuvant in heart failure treatment; however, the molecular mechanisms underlying its beneficial effects remain elusive. Despite the primary cause, heart failure is often preceded by two distinct phenomena: mitochondria dysfunction and cytosolic protein quality control disruption. The objective of the study was to determine the contribution of exercise training in regulating cardiac mitochondria metabolism and cytosolic protein quality control in a post-myocardial infarction-induced heart failure (MI-HF) animal model. Our data demonstrated that isolated cardiac mitochondria from MI-HF rats displayed decreased oxygen consumption, reduced maximum calcium uptake and elevated H₂O₂ release. These changes were accompanied by exacerbated cardiac oxidative stress and proteasomal insufficiency. Declined proteasomal activity contributes to cardiac protein quality control disruption in our MI-HF model. Using cultured neonatal cardiomyocytes, we showed that either antimycin A or H₂O₂ resulted in inactivation of proteasomal peptidase activity, accumulation of oxidized proteins and cell death, recapitulating our in vivo model. Of interest, eight weeks of exercise training improved cardiac function, peak oxygen uptake and exercise tolerance in MI-HF rats. Moreover, exercise training restored mitochondrial oxygen consumption, increased Ca²⁺-induced permeability transition and reduced H₂O₂ release in MI-HF rats. These changes were followed by reduced oxidative stress and better cardiac protein quality control. Taken together, our findings uncover the potential contribution of mitochondrial dysfunction and cytosolic protein quality control disruption to heart failure and highlight the positive effects of exercise training in re-establishing cardiac mitochondrial physiology and protein quality control, reinforcing the importance of this intervention as a non-pharmacological tool for heart failure therapy.

    View details for DOI 10.1371/journal.pone.0052764

    View details for Web of Science ID 000312829100078

    View details for PubMedID 23300764

  • Protein kinase C, an elusive therapeutic target? NATURE REVIEWS DRUG DISCOVERY Mochly-Rosen, D., Das, K., Grimes, K. V. 2012; 11 (12): 937-957


    Protein kinase C (PKC) has been a tantalizing target for drug discovery ever since it was first identified as the receptor for the tumour promoter phorbol ester in 1982. Although initial therapeutic efforts focused on cancer, additional indications--including diabetic complications, heart failure, myocardial infarction, pain and bipolar disorder--were targeted as researchers developed a better understanding of the roles of eight conventional and novel PKC isozymes in health and disease. Unfortunately, both academic and pharmaceutical efforts have yet to result in the approval of a single new drug that specifically targets PKC. Why does PKC remain an elusive drug target? This Review provides a short account of some of the efforts, challenges and opportunities in developing PKC modulators to address unmet clinical needs.

    View details for DOI 10.1038/nrd3871

    View details for Web of Science ID 000311895800021

    View details for PubMedID 23197040

  • Common ALDH2 genetic variants predict development of hypertension in the SAPPHIRe prospective cohort: Gene-environmental interaction with alcohol consumption BMC CARDIOVASCULAR DISORDERS Chang, Y., Chiu, Y., Lee, I., Ho, L., Hung, Y., Hsiung, C. A., Quertermous, T., Donlon, T., Lee, W., Lee, P., Chen, C., Mochly-Rosen, D., Chuang, L. 2012; 12


    Genetic variants near/within the ALDH2 gene encoding the mitochondrial aldehyde dehydrogenase 2 have been associated with blood pressure and hypertension in several case-control association studies in East Asian populations.Three common tag single nucleotide polymorphisms (tagSNP) in the ALDH2 gene were genotyped in 1,134 subjects of Chinese origin from the Stanford Asia-Pacific Program for Hypertension and Insulin Resistance (SAPPHIRe) family cohort. We examined whether the ALDH2 SNP genotypes predicted the development of hypertension in the prospective SAPPHIRe cohort.Over an average follow-up period of 5.7  years, carriers homozygous for the rs2238152 T allele in the ALDH2 gene were more likely to progress to hypertension than were non-carriers (hazard ratio [HR], 2.88, 95% confidence interval [CI], 1.06-7.84, P = 0.03), corresponding to a population attributable risk of ~7.1%. The risk associated with the rs2238152 T allele were strongest in heavy/moderate alcohol drinkers and was reduced in non-drinkers, indicating an interaction between ALDH2 genetic variants and alcohol intake on the risk of hypertension (P for interaction = 0.04). The risk allele was associated with significantly lower ALDH2 gene expression levels in human adipose tissue.ALDH2 genetic variants were associated with progression to hypertension in a prospective Chinese cohort. The association was modified by alcohol consumption.

    View details for DOI 10.1186/1471-2261-12-58

    View details for Web of Science ID 000308988000001

    View details for PubMedID 22839215

    View details for PubMedCentralID PMC3476438

  • Mitigation of Radiation-Induced Dermatitis by Activation of Aldehyde Dehydrogenase 2 Using Topical Alda-1 in Mice RADIATION RESEARCH Ning, S., Budas, G. R., Churchill, E. N., Chen, C., Knox, S. J., Mochly-Rosen, D. 2012; 178 (1): 69-74


    Radiation-induced dermatitis is a debilitating clinical problem in cancer patients undergoing cancer radiation therapy. It is also a possible outcome of exposure to high levels of radiation due to accident or hostile activity. We report that activation of aldehyde dehydrogenase 2 (ALDH2) enzymatic activity using the allosteric agonist, Alda-1, significantly reduced 4-hydroxynonenal adducts accumulation, delayed the onset of radiation dermatitis and substantially reduced symptoms in a clinically-relevant model of radiation-induced dermatitis. Importantly, Alda-1 did not radioprotect tumors in mice. Rather, it increased the sensitivity of the tumors to radiation therapy. This is the first report of reactive aldehydes playing a role in the intrinsic radiosensitivity of normal and tumor tissues. Our findings suggest that ALDH2 represents a novel target for the treatment of radiation dermatitis without reducing the benefit of radiotherapy.

    View details for DOI 10.1667/RR2861.1

    View details for Web of Science ID 000306940600008

    View details for PubMedID 22404739

    View details for PubMedCentralID PMC3417825

  • Identification of epsilon PKC Targets During Cardiac Ischemic Injury CIRCULATION JOURNAL Budas, G., Costa, H. M., Batista Ferreira, J. C., da Silva Ferreira, A. T., Perales, J., Krieger, J. E., Mochly-Rosen, D., Schechtman, D. 2012; 76 (6): 1476-1485
  • Proteins kinase C epsilon is required for non-small cell lung carcinoma growth and regulates the expression of apoptotic genes ONCOGENE Caino, M. C., Lopez-Haber, C., Kim, J., Mochly-Rosen, D., Kazanietz, M. G. 2012; 31 (20): 2593-2600


    Protein kinase C (PKC)ɛ, a member of the novel PKC family, has key roles in mitogenesis and survival in normal and cancer cells. PKCɛ is frequently overexpressed in epithelial cancers, particularly in lung cancer. Using a short-hairpin RNA approach, here we established that PKCɛ is required for non-small cell lung carcinoma (NSCLC) growth in vitro as well as tumor growth when inoculated into athymic mice. Moreover, sustained delivery of a PKCɛ-selective inhibitor peptide, ɛV1-2, reduced xenograft growth in mice. Both RNA interference depletion and pharmacological inhibition of PKCɛ caused a marked elevation in the number of apoptotic cells in NSCLC tumors. PKCɛ-depleted NSCLC cells show elevated expression of pro-apoptotic proteins of the Bcl-2 family, caspase recruitment domain-containing proteins and tumor necrosis factor ligands/receptor superfamily members. Moreover, a Gene Set Enrichment Analysis revealed that a vast majority of the genes changed in PKCɛ-depleted cells were also deregulated in human NSCLC. Our results strongly suggest that PKCɛ is required for NSCLC cell survival and maintenance of NSCLC tumor growth. Therefore, PKCɛ may represent an attractive therapeutic target for NSCLC.

    View details for DOI 10.1038/onc.2011.428

    View details for Web of Science ID 000304191800009

    View details for PubMedID 21996750

  • Protein quality control disruption by PKCbetaII in heart failure Experimental Biology Meeting 2012 Ferreira, J. C., Mochly-Rosen, D., Brum, P. C. FEDERATION AMER SOC EXP BIOL. 2012
  • Protein Quality Control Disruption by PKC beta II in Heart Failure; Rescue by the Selective PKC beta II Inhibitor, beta IIV5-3 PLOS ONE Ferreira, J. C., Boer, B. N., Grinberg, M., Brum, P. C., Mochly-Rosen, D. 2012; 7 (3)
  • Identification of ePKC targets during cardiac ischemic injury. Circulation journal Budas, G., Costa, H. M., Ferreira, J. C., Teixeira da Silva Ferreira, A., Perales, J., Krieger, J. E., Mochly-Rosen, D., Schechtman, D. 2012; 76 (6): 1476-1485


    Epsilon-protein kinase C (εPKC) protects the heart from ischemic injury. However, the mechanism(s) of εPKC cardioprotection is still unclear. Identification of the εPKC targets may aid in elucidating the εPKC-mediated cardioprotective mechanisms. Previous studies, using εPKC transgenic mice and difference in gel electrophoresis, identified proteins involved in glucose metabolism, the expression of which was modified by εPKC. Those studies were accompanied by metabolomic analysis, suggesting that increased glucose oxidation may be responsible for the cardioprotective effect of εPKC. Whether these εPKC-mediated alterations were because of differences in protein expression or phosphorylation was not determined.In the present study, we used an εPKC -specific activator peptide, ψεRACK, combined with phosphoproteomics, to find εPKC targets, and identified that the proteins whose phosphorylation was altered by selective activation of εPKC were mostly mitochondrial proteins. Analysis of the mitochondrial phosphoproteome led to the identification of 55 spots, corresponding to 37 individual proteins, exclusively phosphorylated, in the presence of ψεRACK. The majority of the proteins identified were involved in glucose and lipid metabolism, components of the respiratory chain as well as mitochondrial heat shock proteins.The protective effect of εPKC during ischemia involves phosphorylation of several mitochondrial proteins involved in glucose and lipid metabolism and oxidative phosphorylation. Regulation of these metabolic pathways by εPKC phosphorylation may lead to εPKC-mediated cardioprotection induced by ψεRACK.

    View details for PubMedID 22453000

  • Nitroglycerin Use in Myocardial Infarction Patients - Risks and Benefits CIRCULATION JOURNAL Ferreira, J. C., Mochly-Rosen, D. 2012; 76 (1): 15-21
  • Protein quality control disruption by PKCßII in heart failure; rescue by the selective PKCßII inhibitor, ßIIV5-3. PloS one Ferreira, J. C., Boer, B. N., Grinberg, M., Brum, P. C., Mochly-Rosen, D. 2012; 7 (3)


    Myocardial remodeling and heart failure (HF) are common sequelae of many forms of cardiovascular disease and a leading cause of mortality worldwide. Accumulation of damaged cardiac proteins in heart failure has been described. However, how protein quality control (PQC) is regulated and its contribution to HF development are not known. Here, we describe a novel role for activated protein kinase C isoform βII (PKCβII) in disrupting PQC. We show that active PKCβII directly phosphorylated the proteasome and inhibited proteasomal activity in vitro and in cultured neonatal cardiomyocytes. Importantly, inhibition of PKCβII, using a selective PKCβII peptide inhibitor (βIIV5-3), improved proteasomal activity and conferred protection in cultured neonatal cardiomyocytes. We also show that sustained inhibition of PKCβII increased proteasomal activity, decreased accumulation of damaged and misfolded proteins and increased animal survival in two rat models of HF. Interestingly, βIIV5-3-mediated protection was blunted by sustained proteasomal inhibition in HF. Finally, increased cardiac PKCβII activity and accumulation of misfolded proteins associated with decreased proteasomal function were found also in remodeled and failing human hearts, indicating a potential clinical relevance of our findings. Together, our data highlights PKCβII as a novel inhibitor of proteasomal function. PQC disruption by increased PKCβII activity in vivo appears to contribute to the pathophysiology of heart failure, suggesting that PKCβII inhibition may benefit patients with heart failure. (218 words).

    View details for DOI 10.1371/journal.pone.0033175

    View details for PubMedID 22479367

  • Nitroglycerin use in myocardial infarction patients. Circulation journal Ferreira, J. C., Mochly-Rosen, D. 2012; 76 (1): 15-21


    Acute myocardial infarction (MI) and its sequelae are leading causes of morbidity and mortality worldwide. Nitroglycerin (glyceryl trinitrate [GTN]) remains a first-line treatment for angina pectoris and acute MI. Nitroglycerin achieves its benefit by giving rise to nitric oxide (NO), which causes vasodilation and increases blood flow to the myocardium. However, continuous delivery of GTN results in tolerance, limiting the use of this drug. Nitroglycerin tolerance is caused, at least in part, by inactivation of aldehyde dehydrogenase 2 (ALDH2), an enzyme that converts GTN to the vasodilator, NO. We recently found that in a MI model in animals, in addition to GTN's effect on the vasculature, sustained treatment negatively affected cardiomyocyte viability following ischemia, thus resulting in increased infarct size. Coadministration of Alda-1, an activator of ALDH2, with GTN improves metabolism of reactive aldehyde adducts and prevents the GTN-induced increase in cardiac dysfunction following MI. In this review, we describe the molecular mechanisms associated with the benefits and risks of GTN administration in MI.

    View details for PubMedID 22040938

  • Regulation of cardiac excitability by protein kinase C isozymes. Frontiers in bioscience (Scholar edition) Ferreira, J. C., Mochly-Rosen, D., Boutjdir, M. 2012; 4: 532-546


    Cardiac excitability and electrical activity are determined by the sum of individual ion channels, gap junctions and exchanger activities. Electrophysiological remodeling during heart disease involves changes in membrane properties of cardiomyocytes and is related to higher prevalence of arrhythmia-associated morbidity and mortality. Pharmacological and genetic manipulation of cardiac cells as well as animal models of cardiovascular diseases are used to identity changes in electrophysiological properties and the molecular mechanisms associated with the disease. Protein kinase C (PKC) and several other kinases play a pivotal role in cardiac electrophysiological remodeling. Therefore, identifying specific therapies that regulate these kinases is the main focus of current research. PKC, a family of serine/threonine kinases, has been implicated as potential signaling nodes associated with biochemical and biophysical stress in cardiovascular diseases. In this review, we describe the role of PKC isozymes that are involved in cardiac excitability and discuss both genetic and pharmacological tools that were used, their attributes and limitations. Selective and effective pharmacological interventions to normalize cardiac electrical activities and correct cardiac arrhythmias will be of great clinical benefit.

    View details for PubMedID 22202075

  • Discovery of a Novel Class of Covalent Inhibitor for Aldehyde Dehydrogenases JOURNAL OF BIOLOGICAL CHEMISTRY Khanna, M., Chen, C., Kimble-Hill, A., Parajuli, B., Perez-Miller, S., Baskaran, S., Kim, J., Dria, K., Vasiliou, V., Mochly-Rosen, D., Hurley, T. D. 2011; 286 (50): 43486-43494


    Human aldehyde dehydrogenases (ALDHs) comprise a family of 17 homologous enzymes that metabolize different biogenic and exogenic aldehydes. To date, there are relatively few general ALDH inhibitors that can be used to probe the contribution of this class of enzymes to particular metabolic pathways. Here, we report the discovery of a general class of ALDH inhibitors with a common mechanism of action. The combined data from kinetic studies, mass spectrometric measurements, and crystallographic analyses demonstrate that these inhibitors undergo an enzyme-mediated β-elimination reaction generating a vinyl ketone intermediate that covalently modifies the active site cysteine residue present in these enzymes. The studies described here can provide the basis for rational approach to design ALDH isoenzyme-specific inhibitors as research tools and perhaps as drugs, to address diseases such as cancer where increased ALDH activity is associated with a cellular phenotype.

    View details for DOI 10.1074/jbc.M111.293597

    View details for Web of Science ID 000298351300068

    View details for PubMedID 22021038

  • A Novel Aldehyde Dehydrogenase-3 Activator Leads to Adult Salivary Stem Cell Enrichment In Vivo CLINICAL CANCER RESEARCH Banh, A., Xiao, N., Cao, H., Chen, C., Kuo, P., Krakow, T., Bavan, B., Khong, B., Yao, M., Ha, C., Kaplan, M. J., Sirjani, D., Jensen, K., Kong, C. S., Mochly-Rosen, D., Koong, A. C., Quynh-Thu Le, Q. T. 2011; 17 (23): 7265-7272


    To assess aldehyde dehydrogenase (ALDH) expression in adult human and murine submandibular gland (SMG) stem cells and to determine the effect of ALDH3 activation in SMG stem cell enrichment.Adult human and murine SMG stem cells were selected by cell surface markers (CD34 for human and c-Kit for mouse) and characterized for various other stem cell surface markers by flow cytometry and ALDH isozymes expression by quantitative reverse transcriptase PCR. Sphere formation and bromodeoxyuridine (BrdUrd) incorporation assays were used on selected cells to confirm their renewal capacity and three-dimensional (3D) collagen matrix culture was applied to observe differentiation. To determine whether ALDH3 activation would increase stem cell yield, adult mice were infused with a novel ALDH3 activator (Alda-89) or with vehicle followed by quantification of c-Kit(+)/CD90(+) SMG stem cells and BrdUrd(+) salispheres.More than 99% of CD34(+) huSMG stem cells stained positive for c-Kit, CD90 and 70% colocalized with CD44, Nestin. Similarly, 73.8% c-Kit(+) mSMG stem cells colocalized with Sca-1, whereas 80.7% with CD90. Functionally, these cells formed BrdUrd(+) salispheres, which differentiated into acinar- and ductal-like structures when cultured in 3D collagen. Both adult human and murine SMG stem cells showed higher expression of ALDH3 than in their non-stem cells and 84% of these cells have measurable ALDH1 activity. Alda-89 infusion in adult mice significantly increased c-Kit(+)/CD90(+) SMG population and BrdUrd(+) sphere formation compared with control.This is the first study to characterize expression of different ALDH isozymes in SMG stem cells. In vivo activation of ALDH3 can increase SMG stem cell yield, thus providing a novel means for SMG stem cell enrichment for future stem cell therapy.

    View details for DOI 10.1158/1078-0432.CCR-11-0179

    View details for Web of Science ID 000298133600009

    View details for PubMedID 21998334

    View details for PubMedCentralID PMC3544360

  • Pharmacological inhibition of beta IIPKC is cardioprotective in late-stage hypertrophy JOURNAL OF MOLECULAR AND CELLULAR CARDIOLOGY Ferreira, J. C., Koyanagi, T., Palaniyandi, S. s., Fajardo, G., Churchill, E. N., Budas, G., Disatnik, M., Bernstein, D., Brum, P. C., Mochly-Rosen, D. 2011; 51 (6): 980-987


    We previously found that in the hearts of hypertensive Dahl salt-sensitive rats, βIIPKC levels increase during the transition from compensated cardiac hypertrophy to cardiac dysfunction. Here we showed that a six-week treatment of these hypertensive rats with a βIIPKC-specific inhibitor, βIIV5-3, prolonged their survival by at least 6weeks, suppressed myocardial fibrosis and inflammation, and delayed the transition from compensated hypertrophy to cardiac dysfunction. In addition, changes in the levels of the Ca(2+)-handling proteins, SERCA2 and the Na(+)/Ca(2+) exchanger, as well as troponin I phosphorylation, seen in the control-treated hypertensive rats were not observed in the βΙΙPKC-treated rats, suggesting that βΙΙPKC contributes to the regulation of calcium levels in the myocardium. In contrast, treatment with the selective inhibitor of βIPKC, an alternative spliced form of βIIPKC, had no beneficial effects in these rats. We also found that βIIV5-3, but not βIV5-3, improved calcium handling in isolated rat cardiomyocytes and enhanced contractility in isolated rat hearts. In conclusion, our data using an in vivo model of cardiac dysfunction (late-phase hypertrophy), suggest that βIIPKC contributes to the pathology associated with heart failure and thus an inhibitor of βIIPKC may be a potential treatment for this disease.

    View details for DOI 10.1016/j.yjmcc.2011.08.025

    View details for Web of Science ID 000296943800014

    View details for PubMedID 21920368

    View details for PubMedCentralID PMC3418885

  • ALDH2 activator inhibits increased myocardial infarction injury by nitroglycerin tolerance. Science translational medicine Sun, L., Ferreira, J. C., Mochly-Rosen, D. 2011; 3 (107): 107ra111-?


    Nitroglycerin, which treats impaired cardiac function through vasodilation as it is converted to nitric oxide, is used worldwide for patients with various ischemic and congestive cardiac diseases, including angina pectoris. Nevertheless, after continuous treatment, the benefits of nitroglycerin are limited by the development of tolerance to the drug. Nitroglycerin tolerance is a result of inactivation of aldehyde dehydrogenase 2 (ALDH2), an enzyme essential for cardioprotection in animals subjected to myocardial infarction. Here, we tested the hypothesis that the tolerance that develops as a result of sustained nitroglycerin treatment increases cardiac injury by subsequent myocardial infarction. In a rat model of myocardial infarction, 16 hours of prior, sustained nitroglycerin treatment resulted in infarcts that were twice as large as those in untreated control animals and in diminished cardiac function at 3 days and 2 weeks after the myocardial infarction. We also sought to identify a potential treatment to protect against this increased cardiac damage. Nitroglycerin inhibited ALDH2 activity in vitro, an effect that was blocked by Alda-1, an activator of ALDH2. Co-administration of Alda-1 with the nitroglycerin prevented the nitroglycerin-induced increase in cardiac dysfunction after myocardial infarction in rats, at least in part by enhancing metabolism of reactive aldehyde adducts that impair normal protein functions. If our animal studies showing that nitroglycerin tolerance increases cardiac injury upon ischemic insult are corroborated in humans, activators of ALDH2 such as Alda-1 may help to protect patients with myocardial infarction from this nitroglycerin-induced increase in cardiac injury while maintaining the cardiac benefits of the increased nitric oxide concentrations produced by nitroglycerin.

    View details for DOI 10.1126/scitranslmed.3002067

    View details for PubMedID 22049071

  • ALDH2 Activator Inhibits Increased Myocardial Infarction Injury by Nitroglycerin Tolerance SCIENCE TRANSLATIONAL MEDICINE Sun, L., Cesar, J., Ferreira, B., Mochly-Rosen, D. 2011; 3 (107)
  • beta 2-adrenergic receptors mediate cardioprotection through crosstalk with mitochondrial cell death pathways JOURNAL OF MOLECULAR AND CELLULAR CARDIOLOGY Fajardo, G., Zhao, M., Berry, G., Wong, L., Mochly-Rosen, D., Bernstein, D. 2011; 51 (5): 781-789


    β-adrenergic receptors (β-ARs) modulate cardiotoxicity/cardioprotection through crosstalk with multiple signaling pathways. We have previously shown that β2-ARs are cardioprotective during exposure to oxidative stress induced by doxorubicin (DOX). DOX cardiotoxicity is mediated in part through a Ca(2+)-dependent opening of the mitochondrial permeability transition (MPT), however the signals linking a cell surface receptor like the β2-AR to regulators of mitochondrial function are not clear. The objective of this study was to assess mechanisms of crosstalk between β2-ARs and mitochondrial cell death pathways. DOX administered to WT mice resulted in no acute mortality, however 85% of β2-/- mice died within 30 min. Several pro- and anti-survival pathways were altered. The pro-survival kinase, εPKC, was decreased by 64% in β2-/- after DOX vs WT (p<0.01); the εPKC activator ψεRACK partially rescued these mice (47% reduction in mortality). Activity of the pro-survival kinase Akt decreased by 76% in β2-/- after DOX vs WT (p<0.01). The α1-antagonist prazosin restored Akt activity to normal and also partially reversed the mortality (45%). Deletion of the β2-AR increased rate of Ca(2+) release by 75% and peak [Ca(2+)](i) by 20% respectively in isolated cardiomyocytes; the Ca(2+) channel blocker verapamil also partially rescued the β2-/- (26%). Mitochondrial architecture was disrupted and complex I and II activities decreased by 40.9% and 34.6% respectively after DOX only in β2-/-. The MPT blocker cyclosporine reduced DOX mortality by 41% and prazosin plus cyclosporine acted synergistically to decrease mortality by 85%. β2-ARs activate pro-survival kinases and attenuate mitochondrial dysfunction during oxidative stress; absence of β2-ARs enhances cardiotoxicity via negative regulation of survival kinases and enhancement of intracellular Ca(2+), thus predisposing the mitochondria to opening of the MPT.

    View details for DOI 10.1016/j.yjmcc.2011.06.019

    View details for Web of Science ID 000295604500018

    View details for PubMedID 21756913

    View details for PubMedCentralID PMC3184305

  • Myocardial salvage in acute myocardial infarction - Challenges in clinical translation JOURNAL OF MOLECULAR AND CELLULAR CARDIOLOGY Mochly-Rosen, D., Grimes, K. V. 2011; 51 (4): 451-453

    View details for DOI 10.1016/j.yjmcc.2011.08.002

    View details for Web of Science ID 000295302900006

    View details for PubMedID 21851825

  • beta IIPKC and epsilon PKC isozymes as potential pharmacological targets in cardiac hypertrophy and heart failure JOURNAL OF MOLECULAR AND CELLULAR CARDIOLOGY Batista Ferreira, J. C., Brum, P. C., Mochly-Rosen, D. 2011; 51 (4): 479-484
  • ßIIPKC and ePKC isozymes as potential pharmacological targets in cardiac hypertrophy and heart failure. Journal of molecular and cellular cardiology Ferreira, J. C., Brum, P. C., Mochly-Rosen, D. 2011; 51 (4): 479-484


    Cardiac hypertrophy is a complex adaptive response to mechanical and neurohumoral stimuli and under continual stressor, it contributes to maladaptive responses, heart failure and death. Protein kinase C (PKC) and several other kinases play a role in the maladaptative cardiac responses, including cardiomyocyte hypertrophy, myocardial fibrosis and inflammation. Identifying specific therapies that regulate these kinases is a major focus of current research. PKC, a family of serine/threonine kinases, has emerged as potential mediators of hypertrophic stimuli associated with neurohumoral hyperactivity in heart failure. In this review, we describe the role of PKC isozymes that is involved in cardiac hypertrophy and heart failure. This article is part of a special issue entitled "Key Signaling Molecules in Hypertrophy and Heart Failure".

    View details for DOI 10.1016/j.yjmcc.2010.10.020

    View details for PubMedID 21035454

  • PKCßII inhibition attenuates myocardial infarction induced heart failure and is associated with a reduction of fibrosis and pro-inflammatory responses. Journal of cellular and molecular medicine Palaniyandi, S. S., Ferreira, J. C., Brum, P. C., Mochly-Rosen, D. 2011; 15 (8): 1769-1777


    Protein kinase C βII (PKCβII) levels increase in the myocardium of patients with end-stage heart failure (HF). Also targeted overexpression of PKCβII in the myocardium of mice leads to dilated cardiomyopathy associated with inflammation, fibrosis and myocardial dysfunction. These reports suggest a deleterious role of PKCβII in HF development. Using a post-myocardial infarction (MI) model of HF in rats, we determined the benefit of chronic inhibition of PKCβII on the progression of HF over a period of 6 weeks after the onset of symptoms and the cellular basis for these effects. Four weeks after MI, rats with HF signs that were treated for 6 weeks with the PKCβII selective inhibitor (βIIV5-3 conjugated to TAT(47-57) carrier peptide) (3 mg/kg/day) showed improved fractional shortening (from 21% to 35%) compared to control (TAT(47-57) carrier peptide alone). Formalin-fixed mid-ventricle tissue sections stained with picrosirius red, haematoxylin and eosin and toluidine blue dyes exhibited a 150% decrease in collagen deposition, a two-fold decrease in inflammation and a 30% reduction in mast cell degranulation, respectively, in rat hearts treated with the selective PKCβII inhibitor. Further, a 90% decrease in active TGFβ1 and a significant reduction in SMAD2/3 phosphorylation indicated that the selective inhibition of PKCβII attenuates cardiac remodelling mediated by the TGF-SMAD signalling pathway. Therefore, sustained selective inhibition of PKCβII in a post-MI HF rat model improves cardiac function and is associated with inhibition of pathological myocardial remodelling.

    View details for DOI 10.1111/j.1582-4934.2010.01174.x

    View details for PubMedID 20874717

  • PKC beta II inhibition attenuates myocardial infarction induced heart failure and is associated with a reduction of fibrosis and pro-inflammatory responses JOURNAL OF CELLULAR AND MOLECULAR MEDICINE Palaniyandi, S. S., Batista Ferreira, J. C., Brum, P. C., Mochly-Rosen, D. 2011; 15 (8): 1769-1777
  • PKC delta Activation Mediates Angiogenesis via NADPH Oxidase Activity in PC-3 Prostate Cancer Cells PROSTATE Kim, J., Koyanagi, T., Mochly-Rosen, D. 2011; 71 (9): 946-954


    PKCδ is generally known as a pro-apoptotic and anti-proliferative enzyme in human prostate cancer cells.Here, we investigated the role of PKCδ on the growth of PC-3 human prostate cancer cells in vivo and in vitro.We found that sustained treatment with a specific PKCδ activator (ψδ receptor for active C kinase, ψδRACK) increased growth of PC-3 xenografts. There was increased levels of HIF-1α, vascular endothelial growth factor and CD31-positive cells in PC-3 xenografts, representative of increased tumor angiogenesis. Mechanistically, PKCδ activation increased the levels of reactive oxygen species (ROS) by binding to and phosphorylating NADPH oxidase, which induced its activity. Also, PKCδ-induced activation of NADPH oxidase increased the level of HIF-1α.Our results using tumors from the PC-3 xenograft model suggest that PKCδ activation increases angiogenic activity in androgen-independent PC-3 prostate cancer cells by increasing NADPH oxidase activity and HIF-1α levels and thus may partly be responsible for increased angiogenesis in advanced prostate cancer.

    View details for DOI 10.1002/pros.21310

    View details for Web of Science ID 000290264400004

    View details for PubMedID 21541971

    View details for PubMedCentralID PMC3544470

  • Therapeutic Potential for Protein Kinase C Inhibitor in Vascular Restenosis JOURNAL OF CARDIOVASCULAR PHARMACOLOGY AND THERAPEUTICS Ding, R. Q., Tsao, J., Chai, H., Mochly-Rosen, D., Zhou, W. 2011; 16 (2): 160-167


    Vascular restenosis, an overreaction of biological response to injury, is initialized by thrombosis and inflammation. This response is characterized by increased smooth muscle cell migration and proliferation. Available pharmacological treatments include anticoagulants, antiplatelet agents, immunosuppressants, and antiproliferation agents. Protein kinase C (PKC), a large family of serine/threonine kinases, has been shown to participate in various pathological stages of restenosis. Consequently, PKC inhibitors are expected to exert a wide range of pharmacological activities therapeutically beneficial for restenosis. In this review, the roles of PKC isozymes in platelets, leukocytes, endothelial cells, and smooth muscle cells are discussed, with emphasis given to smooth muscle cells. We will describe cellular and animal studies assessing prevention of restenosis with PKC inhibitors, particularly targeting -α, -β, -δ, and -ζ isozymes. The delivery strategy, efficacy, and safety of such PKC regulators will also be discussed.

    View details for DOI 10.1177/1074248410382106

    View details for Web of Science ID 000290562100005

    View details for PubMedID 21183728

    View details for PubMedCentralID PMC3527091

  • Activation of aldehyde dehydrogenase type 2 (ALDH2) by Alda-1 increases maximum running distance in a hindlimb ischemia model of peripheral arterial disease Thanaporn, P., Ferreira, J. C., Lee, J. C., Okagbaa, J., Mochly-Rosen, D., Cooke, J. P. SAGE PUBLICATIONS LTD. 2011: 219–19
  • Matrix metalloproteinases modulated by protein kinase C epsilon mediate resistin-induced migration of human coronary artery smooth muscle cells JOURNAL OF VASCULAR SURGERY Ding, Q., Chai, H., Mahmood, N., Tsao, J., Mochly-Rosen, D., Zhou, W. 2011; 53 (4): 1044-1051


    Emerging evidence showed that resistin induces vascular smooth muscle cell (VSMC) migration, a critical step in initiating vascular restenosis. Adhesion molecule expression and cytoskeletal rearrangement have been observed in this progress. Given that matrix metalloproteinases (MMPs) also regulate cell migration, we hypothesized that MMPs may mediate resistin-induced VSMC migration.Human VSMCs were treated with recombinant human resistin at physiologic (10 ng/mL) and pathologic (40 ng/mL) concentrations for 24 hours. Cell migration was determined by the Boyden chamber assay. MMP and tissue inhibitor metalloproteinase (TIMP) mRNA and protein levels were measured with real-time PCR and ELISA. MMP enzymatic activity was measured by zymography. In another experiment, neutralizing antibodies against MMP-2 and MMP-9 were coincubated with resistin in cultured VSMCs. The regulation of MMP by protein kinase C (PKC) was determined by εV1-2, a selective PKCε inhibitor.Resistin-induced smooth muscle cell (SMC) migration was confirmed by the Boyden chamber assay. Forty nanograms/milliliter resistin increased SMC migration by 3.7 fold. Additionally, resistin stimulated MMP-2 and -MMP9 mRNA and protein expressions. In contrast, the TIMP-1 and TIMP-2 mRNA levels were inhibited by resistin. Neutralizing antibodies against MMP-2 and MMP-9 effectively reversed VSMC migration. Furthermore, resistin activated PKCε, but selective PKCε inhibitor suppressed resistin-induced MMP expression, activity, and cell migration.Our study confirmed that resistin increased vascular smooth muscle cell migration in vitro. In terms of mechanism, resistin-stimulated cell migration was associated with increased MMP expression, which was dependent on PKCε activation.

    View details for DOI 10.1016/j.jvs.2010.10.117

    View details for Web of Science ID 000289012600023

    View details for PubMedID 21277149

    View details for PubMedCentralID PMC3538810

  • Aberrant mitochondrial fission in neurons induced by protein kinase C delta under oxidative stress conditions in vivo MOLECULAR BIOLOGY OF THE CELL Qi, X., Disatnik, M., Shen, N., Sobel, R. A., Mochly-Rosen, D. 2011; 22 (2): 256-265


    Neuronal cell death in a number of neurological disorders is associated with aberrant mitochondrial dynamics and mitochondrial degeneration. However, the triggers for this mitochondrial dysregulation are not known. Here we show excessive mitochondrial fission and mitochondrial structural disarray in brains of hypertensive rats with hypertension-induced brain injury (encephalopathy). We found that activation of protein kinase Cδ (PKCδ) induced aberrant mitochondrial fragmentation and impaired mitochondrial function in cultured SH-SY5Y neuronal cells and in this rat model of hypertension-induced encephalopathy. Immunoprecipitation studies indicate that PKCδ binds Drp1, a major mitochondrial fission protein, and phosphorylates Drp1 at Ser 579, thus increasing mitochondrial fragmentation. Further, we found that Drp1 Ser 579 phosphorylation by PKCδ is associated with Drp1 translocation to the mitochondria under oxidative stress. Importantly, inhibition of PKCδ, using a selective PKCδ peptide inhibitor (δV1-1), reduced mitochondrial fission and fragmentation and conferred neuronal protection in vivo and in culture. Our study suggests that PKCδ activation dysregulates the mitochondrial fission machinery and induces aberrant mitochondrial fission, thus contributing to neurological pathology.

    View details for DOI 10.1091/mbc.E10-06-0551

    View details for Web of Science ID 000286185600008

    View details for PubMedID 21119009

  • Sustained inhibition of PKC alpha reduces intravasation and lung seeding during mammary tumor metastasis in an in vivo mouse model ONCOGENE Kim, J., Thorne, S. H., Sun, L., Huang, B., Mochly-Rosen, D. 2011; 30 (3): 323-333


    Metastasis is the major reason for breast cancer-related deaths. Although there is a host of indirect evidence for a role of protein kinase C (PKC) α in primary breast cancer growth, its role in the molecular pathways leading to metastasis has not been studied comprehensively. By treating mice with αV5-3, a novel peptide inhibitor selective for PKCα, we were able to determine how PKCα regulates metastasis of mammary cancer cells using a syngeneic and orthotopic model. The primary tumor growth was not affected by αV5-3 treatment. However, the mortality rate was reduced and metastasis in the lung decreased by more than 90% in the αV5-3-treated mice relative to the control-treated mice. αV5-3 treatment reduced intravasation by reducing matrix metalloproteinase-9 activities. αV5-3 treatment also reduced lung seeding of tumor cells and decreased cell migration, effects that were accompanied by a reduction in nuclear factor kappa B activity and cell surface levels of the CXCL12 receptor, CXCR4. αV5-3 treatment caused no apparent toxicity in non-tumor-bearing naïve mice. Rather, inhibiting PKCα protected against liver damage and increased the number of immune cells in tumor-bearing mice. Importantly, αV5-3 showed superior efficacy relative to anti-CXCR4 antibody in reducing metastasis in vivo. Together, these data show that pharmacological inhibition of PKCα effectively reduces mammary cancer metastasis by targeting intravasation and lung seeding steps in the metastatic process and suggest that PKCα-specific inhibitors, such as αV5-3, can be used to study the mechanistic roles of PKCα specifically and may provide a safe and effective treatment for the prevention of lung metastasis of breast cancer patients.

    View details for DOI 10.1038/onc.2010.415

    View details for Web of Science ID 000286418800007

    View details for PubMedID 20856202

  • Mitochondrial import of PKC epsilon is mediated by HSP90: a role in cardioprotection from ischaemia and reperfusion injury CARDIOVASCULAR RESEARCH Budas, G. R., Churchill, E. N., Disatnik, M., Sun, L., Mochly-Rosen, D. 2010; 88 (1): 83-92


    Protein kinase C epsilon (PKCepsilon) is critical for cardiac protection from ischaemia and reperfusion (IR) injury. PKCepsilon substrates that mediate cytoprotection reside in the mitochondria. However, the mechanism enabling mitochondrial translocation and import of PKCepsilon to enable phosphorylation of these substrates is not known. Heat shock protein 90 (HSP90) is a cytoprotective protein chaperone that participates in mitochondrial import of a number of proteins. Here, we investigated the role of HSP90 in mitochondrial import of PKCepsilon.Using an ex vivo perfused rat heart model of IR, we found that PKCepsilon translocates from the cytosol to the mitochondrial fraction following IR. Immunogold electron microscopy and mitochondrial fractionation demonstrated that following IR, mitochondrial PKCepsilon is localized within the mitochondria, on the inner mitochondrial membrane. Pharmacological inhibition of HSP90 prevented IR-induced interaction between PKCepsilon and the translocase of the outer membrane (Tom20), reduced mitochondrial import of PKCepsilon, and increased necrotic cell death by approximately 70%. Using a rational approach, we designed a 7-amino acid peptide activator of PKCepsilon, derived from an HSP90 homologous sequence located in the C2 domain of PKCepsilon (termed psiepsilonHSP90). Treatment with this peptide (conjugated to the cell permeating TAT protein-derived peptide, TAT(47-57)) increased PKCepsilon-HSP90 protein-protein interaction, enhanced mitochondrial translocation of PKCepsilon, increased phosphorylation and activity of an intra-mitochondrial PKCepsilon substrate, aldehyde dehydrogenase 2, and reduced cardiac injury in ex vivo and in vivo models of myocardial infarction.Our results suggest that HSP90-mediated mitochondrial import of PKCepsilon plays an important role in the protection of the myocardium from IR injury.

    View details for DOI 10.1093/cvr/cvq154

    View details for Web of Science ID 000281714600012

    View details for PubMedID 20558438

  • Mitochondrial aldehyde dehydrogenase and cardiac diseases CARDIOVASCULAR RESEARCH Chen, C., Sun, L., Mochly-Rosen, D. 2010; 88 (1): 51-57


    Numerous conditions promote oxidative stress, leading to the build-up of reactive aldehydes that cause cell damage and contribute to cardiac diseases. Aldehyde dehydrogenases (ALDHs) are important enzymes that eliminate toxic aldehydes by catalysing their oxidation to non-reactive acids. The review will discuss evidence indicating a role for a specific ALDH enzyme, the mitochondrial ALDH2, in combating oxidative stress by reducing the cellular 'aldehydic load'. Epidemiological studies in humans carrying an inactive ALDH2, genetic models in mice with altered ALDH2 levels, and small molecule activators of ALDH2 all highlight the role of ALDH2 in cardioprotection and suggest a promising new direction in cardiovascular research and the development of new treatments for cardiovascular diseases.

    View details for DOI 10.1093/cvr/cvq192

    View details for Web of Science ID 000281714600008

    View details for PubMedID 20558439

    View details for PubMedCentralID PMC2936126

  • Sustained Inhibition of epsilon Protein Kinase C Inhibits Vascular Restenosis After Balloon Injury and Stenting 82nd National Conference and Exhibitions and Scientific Sessions of the American-Heart-Association Deuse, T., Koyanagi, T., Erben, R. G., Hua, X., Velden, J., Ikeno, F., Reichenspurner, H., Robbins, R. C., Mochly-Rosen, D., Schrepfer, S. LIPPINCOTT WILLIAMS & WILKINS. 2010: S170–S178


    ε protein kinase C (εPKC) is involved in vascular smooth muscle cell (VSMC) activation, but little is known about its function in vascular pathology. We aimed at assessing the role of εPKC in the development of restenosis.Rat models of aortic balloon injury with or without subsequent stenting were used. Rats were treated with the selective ψεPKC activator ε receptor for activated protein kinase C (ψεRACK), the selective εPKC inhibitor εV1-2, or saline. Both down-stream cascades of the platelet-derived growth factor receptor via extracellular signal-regulated kinase and Akt, respectively, were evaluated in vivo and in VSMC cultures. Intimal hyperplasia with luminal obliteration developed in saline-treated balloon-injured rat aortas (20.3±8.0%), and ψεRACK significantly promoted neointima development (32.4±4.9%, P=0.033), whereas εV1-2 significantly inhibited luminal narrowing (9.2±4.3%, P=0.039). εPKC inhibition led to significantly reduced VSMC extracellular signal-regulated kinase phosphorylation in vivo, whereas Akt phosphorylation was not markedly affected. Neointimal proliferation in vivo and platelet-derived growth factor-induced VSMC proliferation/migration in vitro were significantly inhibited by εV1-2. The inhibition of the platelet-derived growth factor pathway was mediated by inhibiting down-stream extracellular signal-regulated kinase and Akt phosphorylation. In vitro, εV1-2 showed inhibitory properties on endothelial cell proliferation, but that did not prevent reendothelialization in vivo. εV1-2 showed proapoptotic effects on VSMC in vitro. After stent implantation, luminal restenosis (quantified by optical coherence tomography imaging) was significantly reduced with εV1-2 (8.0±2.0%) compared with saline (20.2±9.8%, P=0.028).εPKC seems to be centrally involved in the development of neointimal hyperplasia. We suggest that εPKC inhibition may be mediated via inhibition of extracellular signal-regulated kinase and Akt activation. εPKC modulation may become a new therapeutic target against vascular restenosis.

    View details for DOI 10.1161/CIRCULATIONAHA.109.927640

    View details for Web of Science ID 000282294800025

    View details for PubMedID 20837910

  • Aldehyde Dehydrogenase Activation Prevents Reperfusion Arrhythmias by Inhibiting Local Renin Release From Cardiac Mast Cells CIRCULATION Koda, K., Salazar-Rodriguez, M., Corti, F., Chan, N. Y., Estephan, R., Silver, R. B., Mochly-Rosen, D., Levi, R. 2010; 122 (8): 771-U51


    Renin released by ischemia/reperfusion from cardiac mast cells activates a local renin-angiotensin system (RAS). This exacerbates norepinephrine release and reperfusion arrhythmias (ventricular tachycardia and fibrillation), making RAS a new therapeutic target in myocardial ischemia.We investigated whether ischemic preconditioning (IPC) prevents cardiac RAS activation in guinea pig hearts ex vivo. When ischemia/reperfusion (20 minutes of ischemia/30 minutes of reperfusion) was preceded by IPC (two 5-minute ischemia/reperfusion cycles), renin and norepinephrine release and ventricular tachycardia and fibrillation duration were markedly decreased, a cardioprotective anti-RAS effect. Activation and blockade of adenosine A(2b)/A(3) receptors and activation and inhibition of protein kinase Cepsilon (PKCepsilon) mimicked and prevented, respectively, the anti-RAS effects of IPC. Moreover, activation of A(2b)/A(3) receptors or activation of PKCepsilon prevented degranulation and renin release elicited by peroxide in cultured mast cells (HMC-1). Activation and inhibition of mitochondrial aldehyde dehydrogenase type-2 (ALDH2) also mimicked and prevented, respectively, the cardioprotective anti-RAS effects of IPC. Furthermore, ALDH2 activation inhibited degranulation and renin release by reactive aldehydes in HMC-1. Notably, PKCepsilon and ALDH2 were both activated by A(2b)/A(3) receptor stimulation in HMC-1, and PKCepsilon inhibition prevented ALDH2 activation.The results uncover a signaling cascade initiated by A(2b)/A(3) receptors, which triggers PKCepsilon-mediated ALDH2 activation in cardiac mast cells, contributing to IPC-induced cardioprotection by preventing mast cell renin release and the dysfunctional consequences of local RAS activation. Thus, unlike classic IPC in which cardiac myocytes are the main target, cardiac mast cells are the critical site at which the cardioprotective anti-RAS effects of IPC develop.

    View details for DOI 10.1161/CIRCULATIONAHA.110.952481

    View details for Web of Science ID 000281193100004

    View details for PubMedID 20697027

  • Regulation of mitochondrial processes: a target for heart failure. Drug discovery today. Disease mechanisms Palaniyandi, S. S., Qi, X., Yogalingam, G., Ferreira, J. C., Mochly-Rosen, D. 2010; 7 (2): e95-e102


    Cardiac mitochondria, the main source of energy as well as free radicals, are vital organelles for normal functioning of the heart. Mitochondrial number, structure, turnover and function are regulated by processes such as mitochondrial protein quality control, mitochondrial fusion and fission and mitophagy. Recent studies suggest that abnormal changes in these mitochondrial regulatory processes may contribute to the pathology of heart failure (HF). Here we discuss these processes and their potential as therapeutic targets.

    View details for PubMedID 21278905

  • Highly Specific Modulators of Protein Kinase C Localization: Applications to Heart Failure. Drug discovery today. Disease mechanisms Qvit, N., Mochly-Rosen, D. 2010; 7 (2): e87-e93


    Heart failure (HF) in which the blood supply does not match the body's needs, affects 10% of the population over 65 years old. The protein kinase C (PKC) family of kinases has a key role in normal and disease states. Here we discuss the role of PKC in HF and focus on the use of specific PKC regulators to identify the mechanism leading to this Pathology and potential leads for therapeutics.

    View details for PubMedID 21151743

  • Non-volume-loaded heart provides a more relevant heterotopic transplantation model TRANSPLANT IMMUNOLOGY Tang-Quan, K. R., Bartos, J., Deuse, T., Churchill, E., Schaefer, H., Reichenspurner, H., Mochly-Rosen, D., Robbins, R. C., Schrepfer, S. 2010; 23 (1-2): 65-70


    We aimed to compare two techniques of heterotopic heart transplantation in rats. Non-volume-loaded (NL) and volume-loaded (VL) models were tested for their physiologic and immunologic properties to assess their suitability for transplant studies.Syngeneic heterotopic heart transplants were performed according to the techniques previously described by Ono (NL) and Yokoyama (VL). Grafts were followed over 90 days with sequential echocardiography. Ex-vivo Langendorff perfusion was used to gain functional data. Allogeneic heart transplants were done to determine whether chronic allograft vasculopathy (CAV) develops at a different pace in both transplant models.The ischemic time during surgery was significantly longer using the VL model (p<0.001). The LV diameter of NL hearts decreased over time while that of the VL model significantly increased (p=0.004 on POD 90). Mean LV developed pressure and (dP/dt)max were significantly higher with the NL model (61.1+/-8.5 mmHg and 4261.7+/-419.6 mmHg/s) than with VL hearts (19.9+/-16.5 mmHg; p=0.011 and 924.8+/-605.6 mmHg/s; p<0.001). The mean weight of NL hearts (0.45+/-0.03 g) was significantly less than that of VL hearts (1.21+/-0.16 g, p<0.001). Histology of syngeneic NL grafts showed healthy, but partly atrophic myocardium, whereas the LV myocardium of VL hearts showed dilation and scarring typical for chronic ischemic injury. Heart allografts similarly developed CAV with luminal narrowing of 37.2+/-16.6% (NL) and 34.4+/-21.4% (VL), respectively by POD 90 (p=0.807).Since the coronary arteries in the VL model get perfused with partly deoxygenated blood, the myocardium suffers from chronic ischemic injury. We recommend using the NL model in preclinical transplant studies.

    View details for DOI 10.1016/j.trim.2010.04.005

    View details for Web of Science ID 000280052500012

    View details for PubMedID 20403439

  • delta PKC inhibition or epsilon PKC activation repairs endothelial vascular dysfunction by regulating eNOS post-translational modification JOURNAL OF MOLECULAR AND CELLULAR CARDIOLOGY Monti, M., Donnini, S., Giachetti, A., Mochly-Rosen, D., Ziche, M. 2010; 48 (4): 746-756


    The balance between endothelial nitric oxide synthase (eNOS)-derived nitric oxide (NO) and reactive oxygen species (ROS) production determines endothelial-mediated vascular homeostasis. Activation of protein kinase C (PKC) has been linked to imbalance of the eNOS/ROS system, which leads to endothelial dysfunction. We previously found that selective inhibition of delta PKC (deltaPKC) or selective activation of epsilon PKC (varepsilonPKC) reduces oxidative damage in the heart following myocardial infarction. In this study we determined the effect of these PKC isozymes in the survival of coronary endothelial cells (CVEC). We demonstrate here that serum deprivation of CVEC increased eNOS-mediated ROS levels, activated caspase-3, reduced Akt phosphorylation and cell number. Treatment with either the deltaPKC inhibitor, deltaV1-1, or the varepsilonPKC activator, psivarepsilonRACK, inhibited these effects, restoring cell survival through inhibition of eNOS activity. The decrease in eNOS activity coincided with specific de-phosphorylation of eNOS at Ser1179, and eNOS phosphorylation at Thr497 and Ser116. Furthermore, deltaV1-1 or psivarepsilonRACK induced physical association of eNOS with caveolin-1, an additional marker of eNOS inhibition, and restored Akt activation by inhibiting its nitration. Together our data demonstrate that (1) in endothelial dysfunction, ROS and reactive nitrogen species (RNS) formation result from uncontrolled eNOS activity mediated by activation of deltaPKC or inhibition of varepsilonPKC; (2) inhibition of deltaPKC or activation of varepsilonPKC corrects the perturbed phosphorylation state of eNOS, thus increasing cell survival. Since endothelial health ensures better tissue perfusion and oxygenation, treatment with a deltaPKC inhibitor and/or an varepsilonPKC activator in diseases of endothelial dysfunction should be considered.

    View details for DOI 10.1016/j.yjmcc.2009.11.002

    View details for Web of Science ID 000275835700022

    View details for PubMedID 19913548

  • Activation of aldehyde dehydrogenase 2 (ALDH2) confers cardioprotection in protein kinase C epsilon (PKC epsilon) knockout mice JOURNAL OF MOLECULAR AND CELLULAR CARDIOLOGY Budas, G. R., Disatnik, M., Chen, C., Mochly-Rosen, D. 2010; 48 (4): 757-764


    Acute administration of ethanol can reduce cardiac ischemia/reperfusion injury. Previous studies demonstrated that the acute cytoprotective effect of ethanol on the myocardium is mediated by protein kinase C epsilon (PKCvarepsilon). We recently identified aldehyde dehydrogenase 2 (ALDH2) as a PKCvarepsilon substrate, whose activation is necessary and sufficient to confer cardioprotection in vivo. ALDH2 metabolizes cytotoxic reactive aldehydes, such as 4-hydroxy-2-nonenal (4-HNE), which accumulate during cardiac ischemia/reperfusion. Here, we used a combination of PKCvarepsilon knockout mice and a direct activator of ALDH2, Alda-44, to further investigate the interplay between PKCvarepsilon and ALDH2 in cardioprotection. We report that ethanol preconditioning requires PKCvarepsilon, whereas direct activation of ALDH2 reduces infarct size in both wild type and PKCvarepsilon knockout hearts. Our data suggest that ALDH2 is downstream of PKCvarepsilon in ethanol preconditioning and that direct activation of ALDH2 can circumvent the requirement of PKCvarepsilon to induce cytoprotection. We also report that in addition to ALDH2 activation, Alda-44 prevents 4-HNE induced inactivation of ALDH2 by reducing the formation of 4-HNE-ALDH2 protein adducts. Thus, Alda-44 promotes metabolism of cytotoxic reactive aldehydes that accumulate in ischemic myocardium. Taken together, our findings suggest that direct activation of ALDH2 may represent a method of harnessing the cardioprotective effect of ethanol without the side effects associated with alcohol consumption.

    View details for DOI 10.1016/j.yjmcc.2009.10.030

    View details for Web of Science ID 000275835700023

    View details for PubMedID 19913552

  • Alda-1 is an agonist and chemical chaperone for the common human aldehyde dehydrogenase 2 variant NATURE STRUCTURAL & MOLECULAR BIOLOGY Perez-Miller, S., Younus, H., Vanam, R., Chen, C., Mochly-Rosen, D., Hurley, T. D. 2010; 17 (2): 159-U4


    In approximately one billion people, a point mutation inactivates a key detoxifying enzyme, aldehyde dehydrogenase (ALDH2). This mitochondrial enzyme metabolizes toxic biogenic and environmental aldehydes, including the endogenously produced 4-hydroxynonenal (4HNE) and the environmental pollutant acrolein, and also bioactivates nitroglycerin. ALDH2 is best known, however, for its role in ethanol metabolism. The accumulation of acetaldehyde following the consumption of even a single alcoholic beverage leads to the Asian alcohol-induced flushing syndrome in ALDH2*2 homozygotes. The ALDH2*2 allele is semidominant, and heterozygotic individuals show a similar but less severe phenotype. We recently identified a small molecule, Alda-1, that activates wild-type ALDH2 and restores near-wild-type activity to ALDH2*2. The structures of Alda-1 bound to ALDH2 and ALDH2*2 reveal how Alda-1 activates the wild-type enzyme and how it restores the activity of ALDH2*2 by acting as a structural chaperone.

    View details for DOI 10.1038/nsmb.1737

    View details for Web of Science ID 000274228400006

    View details for PubMedID 20062057

  • Ischaemic preconditioning improves proteasomal activity and increases the degradation of delta PKC during reperfusion CARDIOVASCULAR RESEARCH Churchill, E. N., Ferreira, J. C., Brum, P. C., Szweda, L. I., Mochly-Rosen, D. 2010; 85 (2): 385-394


    The response of the myocardium to an ischaemic insult is regulated by two highly homologous protein kinase C (PKC) isozymes, delta and epsilonPKC. Here, we determined the spatial and temporal relationships between these two isozymes in the context of ischaemia/reperfusion (I/R) and ischaemic preconditioning (IPC) to better understand their roles in cardioprotection.Using an ex vivo rat model of myocardial infarction, we found that short bouts of ischaemia and reperfusion prior to the prolonged ischaemic event (IPC) diminished deltaPKC translocation by 3.8-fold and increased epsilonPKC accumulation at mitochondria by 16-fold during reperfusion. In addition, total cellular levels of deltaPKC decreased by 60 +/- 2.7% in response to IPC, whereas the levels of epsilonPKC did not significantly change. Prolonged ischaemia induced a 48 +/- 11% decline in the ATP-dependent proteasomal activity and increased the accumulation of misfolded proteins during reperfusion by 192 +/- 32%; both of these events were completely prevented by IPC. Pharmacological inhibition of the proteasome or selective inhibition of epsilonPKC during IPC restored deltaPKC levels at the mitochondria while decreasing epsilonPKC levels, resulting in a loss of IPC-induced protection from I/R. Importantly, increased myocardial injury was the result, in part, of restoring a deltaPKC-mediated I/R pro-apoptotic phenotype by decreasing pro-survival signalling and increasing cytochrome c release into the cytosol.Taken together, our findings indicate that IPC prevents I/R injury at reperfusion by protecting ATP-dependent 26S proteasomal function. This decreases the accumulation of the pro-apoptotic kinase, deltaPKC, at cardiac mitochondria, resulting in the accumulation of the pro-survival kinase, epsilonPKC.

    View details for DOI 10.1093/cvr/cvp334

    View details for Web of Science ID 000273118900016

    View details for PubMedID 19820255

  • Focus on: the cardiovascular system: what did we learn from the French (paradox)? Alcohol research & health Mochly-Rosen, D., Zakhari, S. 2010; 33 (1): 76-86


    Although heavy alcohol consumption has deleterious effects on heart health, moderate drinking is thought to have cardioprotective effects, reducing the risk of coronary artery disease and improving prognosis after a myocardial infarction. It still is unclear, however, if this effect can be achieved with all types of alcoholic beverages and results from the alcohol itself, from other compounds found in alcoholic beverages, or both. For example, the polyphenolic compound resveratrol, which is found particularly in red wine, can reduce the risk of atherosclerosis; however, it is not clear if the resveratrol levels present in wine are sufficient to achieve this result. Alcohol itself contributes to cardioprotection through several mechanisms. For example, it can improve the cholesterol profile, increasing the levels of "good" cholesterol and reducing the levels of "bad" cholesterol. Alcohol also may contribute to blood clot dissolution and may induce a phenomenon called pre-conditioning, whereby exposure to moderate alcohol levels (like short bouts of blood supply disruption [i.e., ischemia]), and result in reduced damage to the heart tissue after subsequent prolonged ischemia. Finally, the enzyme aldehyde dehydrogenase (ALDH) 2, which is involved in alcohol metabolism, also may contribute to alcohol-related cardioprotection by metabolizing other harmful aldehydes that could damage the heart muscle.

    View details for PubMedID 23579938

  • Sustained PKCbetaII inhibition restores both proteasome activity and protein quality control in two different heart failure models Ferreira, J. C., Brum, P. C., Mochly-Rosen, D. OXFORD UNIV PRESS. 2009: 574–575
  • Aldehyde Dehydrogenase 2 in Cardiac Protection: A New Therapeutic Target? TRENDS IN CARDIOVASCULAR MEDICINE Budas, G. R., Disatnik, M., Mochly-Rosen, D. 2009; 19 (5): 158-164


    Mitochondrial aldehyde dehydrogenase 2 (ALDH2) is emerging as a key enzyme involved in cytoprotection in the heart. ALDH2 mediates both the detoxification of reactive aldehydes such as acetaldehyde and 4-hydroxy-2-nonenal and the bioactivation of nitroglycerin to nitric oxide. In addition, chronic nitrate treatment results in ALDH2 inhibition and contributes to nitrate tolerance. Our laboratory recently identified ALDH2 to be a key mediator of endogenous cytoprotection. We reported that ALDH2 is phosphorylated and activated by the survival kinase protein kinase C epsilon and found a strong inverse correlation between ALDH2 activity and infarct size. We also identified a small molecule ALDH2 activator which reduces myocardial infarct size induced by ischemia/reperfusion in vivo. In this review, we discuss evidence that ALDH2 is a key mediator of endogenous survival signaling in the heart, suggest possible cardioprotective mechanisms mediated by ALDH2 and discuss potential clinical implications of these findings.

    View details for Web of Science ID 000279988500003

    View details for PubMedID 20005475

  • Protein kinase C in heart failure: a therapeutic target? CARDIOVASCULAR RESEARCH Palaniyandi, S. S., Sun, L., Batista Ferreira, J. C., Mochly-Rosen, D. 2009; 82 (2): 229-239


    Heart failure (HF) afflicts about 5 million people and causes 300,000 deaths a year in the United States alone. An integral part of the pathogenesis of HF is cardiac remodelling, and the signalling events that regulate it are a subject of intense research. Cardiac remodelling is the sum of responses of the heart to causes of HF, such as ischaemia, myocardial infarction, volume and pressure overload, infection, inflammation, and mechanical injury. These responses, including cardiomyocyte hypertrophy, myocardial fibrosis, and inflammation, involve numerous cellular and structural changes and ultimately result in a progressive decline in cardiac performance. Pharmacological and genetic manipulation of cultured heart cells and animal models of HF and the analysis of cardiac samples from patients with HF are all used to identify the molecular and cellular mechanisms leading to the disease. Protein kinase C (PKC) isozymes, a family of serine-threonine protein kinase enzymes, were found to regulate a number of cardiac responses, including those associated with HF. In this review, we describe the PKC isozymes that play critical roles in specific aspects of cardiac remodelling and dysfunction in HF.

    View details for DOI 10.1093/cvr/cvp001

    View details for Web of Science ID 000265095400008

    View details for PubMedID 19168855

    View details for PubMedCentralID PMC2675930

  • Preserved coronary endothelial function by inhibition of delta protein kinase C in a porcine acute myocardial infarction model INTERNATIONAL JOURNAL OF CARDIOLOGY Kaneda, H., Ikeno, F., Inagaki, K., Mochly-Rosen, D. 2009; 133 (2): 256-259


    Previous studies demonstrate impairment of endothelial-dependent vasodilation after ischemia/reperfusion (I/R). Though we have demonstrated that inhibition of delta protein kinase C (delta PKC) at reperfusion reduces myocyte damage and improves cardiac function in a porcine acute myocardial infarction (AMI) model, impact of the selective delta PKC inhibitor on epicardial coronary endothelial function remains unknown.Either delta PKC inhibitor (delta V1-1, n=5) or saline (n=5) was infused into the left anterior descending artery at the last 1 min of the 30-min ischemia by balloon occlusion. In vivo responses to bradykinin (endothelium-dependent vasodilator) or nitroglycerin (endothelium-independent vasodilator) were analyzed at 24 h after I/R using intravascular ultrasound. Vascular responses were calculated as the ratio of vessel area at each time point (30, 60, 90 and 120 s after the infusion), divided by values at baseline (before the infusion).In control pigs, endothelial-dependent vasodilation following bradykinin infusion in infarct-related epicardial coronary artery was impaired, whereas in delta PKC inhibitor-treated pigs the endothelial-dependent vasodilation was preserved. Nitroglycerin infusion caused similar vasodilatory responses in the both groups.This is the first demonstration that a delta PKC inhibitor preserves vasodilator capacity in epicardial coronary arteries in an in vivo porcine AMI model. Because endothelial dysfunction correlates with worse outcome in patients with AMI, this preserved endothelial function in epicardial coronary arteries might result in a better clinical outcome.

    View details for DOI 10.1016/j.ijcard.2007.11.021

    View details for Web of Science ID 000264284500023

    View details for PubMedID 18242734

  • Time-dependent and ethanol-induced cardiac protection from ischemia mediated by mitochondrial translocation of epsilon PKC and activation of aldehyde dehydrogenase 2 JOURNAL OF MOLECULAR AND CELLULAR CARDIOLOGY Churchill, E. N., Disatnik, M., Mochly-Rosen, D. 2009; 46 (2): 278-284


    The cardioprotective effects of moderate alcohol consumption have been well documented in animal models and in humans. Protection afforded against ischemia and reperfusion injury (I/R) proceeds through an ischemic preconditioning-like mechanism involving the activation of epsilon protein kinase C (varepsilonPKC) and is dependent on the time and duration of ethanol treatment. However, the substrates of varepsilonPKC and the molecular mechanisms by which the enzyme protects the heart from oxidative damage induced by I/R are not fully described. Using an open-chest model of acute myocardial infarction in vivo, we find that intraperitoneal injection of ethanol (0.5 g/kg) 60 min prior to (but not 15 min prior to) a 30-minute transient ligation of the left anterior descending coronary artery reduced I/R-mediated injury by 57% (measured as a decrease of creatine phosphokinase release into the blood). Only under cardioprotective conditions, ethanol treatment resulted in the translocation of varepsilonPKC to cardiac mitochondria, where the enzyme bound aldehyde dehydrogenase-2 (ALDH2). ALDH2 is an intra-mitochondrial enzyme involved in the detoxification of toxic aldehydes such as 4-hydroxy-2-nonenal (4-HNE) and 4-HNE mediates oxidative damage, at least in part, by covalently modifying and inactivating proteins (by forming 4-HNE adducts). In hearts subjected to I/R after ethanol treatment, the levels of 4-HNE protein adducts were lower and JNK1/2 and ERK1/2 activities were diminished relative to the hearts from rats subjected to I/R in the absence of ethanol. Together, this work provides an insight into the mitochondrial-dependent basis of ethanol-induced and varepsilonPKC-mediated protection from cardiac ischemia, in vivo.

    View details for DOI 10.1016/j.yjmcc.2008.09.713

    View details for Web of Science ID 000262707500020

    View details for PubMedID 18983847

  • Activating delta PKC antagonizes the protective effect of ERK1/2 inhibition against stroke in rats BRAIN RESEARCH Castaneda, D., Zhao, H., Mochly-Rosen, D., Steinberg, G. K. 2009; 1251: 256-261


    Two pathways that have been shown to mediate cerebral ischemic damage are the MEK/ERK cascade and the pro-apoptotic deltaPKC pathway. We investigated the relationship between these pathways in a rat model of focal ischemia by observing and modifying the activation state of each pathway. The ERK1/2 inhibitor, U0126, injected at ischemia onset, attenuated the increase in phosphorylated ERK1/2 (P-ERK1/2) after reperfusion. The deltaPKC inhibitor, deltaV1-1, delivered at reperfusion, did not significantly change P-ERK1/2 levels. In contrast, the deltaPKC activator, psi deltaRACK, injected at reperfusion, reduced ERK1/2 phosphorylation measured 4 h after reperfusion. Additionally, U0126 pretreatment at ischemia onset reduced infarct size compared with vehicle, but U0126 injected at the onset of reperfusion had no protection. Finally, combination of U0126 injection at ischemia onset plus deltaV1-1 injection at reperfusion further reduced infarct size, while combination of U0126 delivered at ischemia onset with psi deltaRACK injected at reperfusion increased infarct size compared with U0126 alone. In conclusion, we find that inhibiting both the MEK/ERK and the deltaPKC pathways offers greater protection than either alone, indicating they likely act independently.

    View details for DOI 10.1016/j.brainres.2008.11.051

    View details for Web of Science ID 000263304400025

    View details for PubMedID 19063870

  • Rationally designed peptide regulators of protein kinase C TRENDS IN ENDOCRINOLOGY AND METABOLISM Churchill, E. N., Qvit, N., Mochly-Rosen, D. 2009; 20 (1): 25-33


    Protein-protein interactions sequester enzymes close to their substrates. Protein kinase C (PKC) is one example of a ubiquitous signaling molecule with effects that are dependent upon localization. Short peptides derived from interaction sites between each PKC isozyme and its receptor for activated C kinase act as highly specific inhibitors and have become available as selective drugs in basic research and animal models of human diseases, such as myocardial infarction and hyperglycemia. Whereas the earlier inhibitory peptides are highly specific, we believe that peptides targeting additional interactions between PKC and selective substrates will generate even more selective tools that regulate different functions of individual isozymes. Here, we discuss the methodologies and applications for identifying selective regulators of PKC.

    View details for DOI 10.1016/j.tem.2008.10.002

    View details for Web of Science ID 000262898600004

    View details for PubMedID 19056296

  • Ethanol for cardiac ischemia: the role of protein kinase c. Therapeutic advances in cardiovascular disease Churchill, E. N., Disatnik, M., Budas, G. R., Mochly-Rosen, D. 2008; 2 (6): 469-483


    The physiological effects of ethanol are dependent upon the amount and duration of consumption. Chronic excessive consumption can lead to diseases such as liver cirrhosis, and cardiac arrhythmias, while chronic moderate consumption can have therapeutic effects on the cardiovascular system. Recently, it has also been observed that acute administration of ethanol to animals prior to an ischemic event provides significant protection to the heart. This review focuses on the different modalities of chronic vs. acute ethanol consumption and discusses recent evidence for a protective effect of acute ethanol exposure and the possible use of ethanol as a therapeutic agent.

    View details for DOI 10.1177/1753944708094735

    View details for PubMedID 19124442

  • Mast cells and epsilon PKC: A role in cardiac remodeling in hypertension-induced heart failure JOURNAL OF MOLECULAR AND CELLULAR CARDIOLOGY Palaniyandi, S. S., Inagaki, K., Mochly-Rosen, D. 2008; 45 (6): 779-786


    Heart failure (HF) is a chronic syndrome in which pathological cardiac remodeling is an integral part of the disease and mast cell (MC) degranulation-derived mediators have been suggested to play a role in its progression. Protein kinase C (PKC) signaling is a key event in the signal transduction pathway of MC degranulation. We recently found that inhibition of epsilonPKC slows down the progression of hypertension-induced HF in salt-sensitive Dahl rats fed a high-salt diet. We therefore determined whether epsilonPKC inhibition affects MC degranulation in this model. Six week-old male Dahl rats were fed with a high-salt diet to induce systemic hypertension, which resulted in concentric left ventricular hypertrophy at the age of 11 weeks, followed by myocardial dilatation and HF at the age of 17 weeks. We administered epsilonV1-2, an epsilonPKC-selective inhibitor peptide (3 mg/kg/day), deltaV1-1, a deltaPKC-selective inhibitor peptide (3 mg/kg/day), TAT (negative control; at equimolar concentration; 1.6 mg/kg/day) or olmesartan (angiotensin receptor blocker [ARB] as a positive control; 3 mg/kg/day) between 11 weeks and 17 weeks. Treatment with epsilonV1-2 attenuated cardiac MC degranulation without affecting MC density, myocardial fibrosis, microvessel patency, vascular thickening and cardiac inflammation in comparison to TAT- or deltaV1-1-treatment. Treatment with ARB also attenuated MC degranulation and cardiac remodeling, but to a lesser extent when compared to epsilonV1-2. Finally, epsilonV1-2 treatment inhibited MC degranulation in isolated peritoneal MCs. Together, our data suggest that epsilonPKC inhibition attenuates pathological remodeling in hypertension-induced HF, at least in part, by preventing cardiac MC degranulation.

    View details for DOI 10.1016/j.yjmcc.2008.08.009

    View details for Web of Science ID 000261529000011

    View details for PubMedID 18804478

  • Sustained Pharmacological beta IIPKC Inhibition Is Cardioprotective In Late-stage Hypertrophy And End-stage Heart Failure In Two Rat Models 81st Annual Scientific Session of the American-Heart-Association Ferreira, J. C., Koyanagi, T., Inagaki, K., Fajardo, G., Churchill, E. N., Budas, G., Disatnik, M., Tsutsui, J. M., Kihara, Y., Bernstein, D., Brum, P. C., Mochly-Rosen, D. LIPPINCOTT WILLIAMS & WILKINS. 2008: S535–S535
  • Ischemic Preconditioning Regulates Myocardial Viability Through The Proteasomal Regulation Of Two Opposing PKC Isozymes 81st Annual Scientific Session of the American-Heart-Association Churchill, E., Ferreira, J., Brum, P., Szweda, L., Mochly-Rosen, D. LIPPINCOTT WILLIAMS & WILKINS. 2008: S294–S294
  • beta 2-Adrenergic Receptor Signaling Positively Modulates Pro-Survival Kinases and Ameliorates Mitochondrial Dysfunction during Doxorubicin Cardiotoxicity 81st Annual Scientific Session of the American-Heart-Association Fajardo, G., Zhao, M., Barry, G., Mochly-Rosen, D., Bernstein, D. LIPPINCOTT WILLIAMS & WILKINS. 2008: S485–S486
  • Activation of aldehyde dehydrogenase-2 reduces ischemic damage to the heart SCIENCE Chen, C., Budas, G. R., Churchill, E. N., Disatnik, M., Hurley, T. D., Mochly-Rosen, D. 2008; 321 (5895): 1493-1495


    There is substantial interest in the development of drugs that limit the extent of ischemia-induced cardiac damage caused by myocardial infarction or by certain surgical procedures. Here, using an unbiased proteomic search, we identified mitochondrial aldehyde dehydrogenase 2 (ALDH2) as an enzyme whose activation correlates with reduced ischemic heart damage in rodent models. A high-throughput screen yielded a small-molecule activator of ALDH2 (Alda-1) that, when administered to rats before an ischemic event, reduced infarct size by 60%, most likely through its inhibitory effect on the formation of cytotoxic aldehydes. In vitro, Alda-1 was a particularly effective activator of ALDH2*2, an inactive mutant form of the enzyme that is found in 40% of East Asian populations. Thus, pharmacologic enhancement of ALDH2 activity may be useful for patients with wild-type or mutant ALDH2 who are subjected to cardiac ischemia, such as during coronary bypass surgery.

    View details for DOI 10.1126/science.1158554

    View details for Web of Science ID 000259121800042

    View details for PubMedID 18787169

  • Centrosomal PKC beta II and pericentrin are critical for human prostate cancer growth and angiogenesis CANCER RESEARCH Kim, J., Choi, Y., Vallentin, A., Hunrichs, B. S., Hellerstein, M. K., Peehl, D. M., Mochly-Rosen, D. 2008; 68 (16): 6831-6839


    Angiogenesis is critical in the progression of prostate cancer. However, the interplay between the proliferation kinetics of tumor endothelial cells (angiogenesis) and tumor cells has not been investigated. Also, protein kinase C (PKC) regulates various aspects of tumor cell growth, but its role in prostate cancer has not been investigated in detail. Here, we found that the proliferation rates of endothelial and tumor cells oscillate asynchronously during the growth of human prostate cancer xenografts. Furthermore, our analyses suggest that PKCbetaII was activated during increased angiogenesis and that PKCbetaII plays a key role in the proliferation of endothelial cells and tumor cells in human prostate cancer; treatment with a PKCbetaII-selective inhibitor, betaIIV5-3, reduced angiogenesis and tumor cell proliferation. We also find a unique effect of PKCbetaII inhibition on normalizing pericentrin (a protein regulating cytokinesis), especially in endothelial cells as well as in tumor cells. PKCbetaII inhibition reduced the level and mislocalization of pericentrin and normalized microtubule organization in the tumor endothelial cells. Although pericentrin has been known to be up-regulated in epithelial cells of prostate cancers, its level in tumor endothelium has not been studied in detail. We found that pericentrin is up-regulated in human tumor endothelium compared with endothelium adjacent to normal glands in tissues from prostate cancer patients. Our results suggest that a PKCbetaII inhibitor such as betaIIV5-3 may be used to reduce prostate cancer growth by targeting both angiogenesis and tumor cell growth.

    View details for DOI 10.1158/0008-5472.CAN-07-6195

    View details for Web of Science ID 000258548200042

    View details for PubMedID 18701509

  • epsilon PKC confers acute tolerance to cerebral ischemic reperfusion injury NEUROSCIENCE LETTERS Bright, R., Sun, G., Yenari, M. A., Steinberg, G. K., Mochly-Rosen, D. 2008; 441 (1): 120-124


    In response to mild ischemic stress, the brain elicits endogenous survival mechanisms to protect cells against a subsequent lethal ischemic stress, referred to as ischemic tolerance. The molecular signals that mediate this protection are thought to involve the expression and activation of multiple kinases, including protein kinase C (PKC). Here we demonstrate that epsilonPKC mediates cerebral ischemic tolerance in vivo. Systemic delivery of psiepsilonRACK, an epsilonPKC-selective peptide activator, confers neuroprotection against a subsequent cerebral ischemic event when delivered immediately prior to stroke. In addition, activation of epsilonPKC by psiepsilonRACK treatment decreases vascular tone in vivo, as demonstrated by a reduction in microvascular cerebral blood flow. Here we demonstrate the role of acute and transient epsilonPKC in early cerebral tolerance in vivo and suggest that extra-parenchymal mechanisms, such as vasoconstriction, may contribute to the conferred protection.

    View details for DOI 10.1016/j.neulet.2008.05.080

    View details for Web of Science ID 000258174400025

    View details for PubMedID 18586397

  • Alteration of gene expression during progression of hypertension-induced cardiac dysfunction in rats AMERICAN JOURNAL OF PHYSIOLOGY-HEART AND CIRCULATORY PHYSIOLOGY Koyanagi, T., Wong, L. Y., Inagaki, K., Petrauskene, O. V., Mochly-Rosen, D. 2008; 295 (1): H220-H226


    Hypertension induced by high-salt diet in Dahl salt-sensitive rats leads to compensatory cardiac hypertrophy by approximately 11 wk, cardiac dysfunction at approximately 17 wk, and death from cardiac dysfunction at approximately 21 wk. It is unclear what molecular hallmarks distinguish the compensatory hypertrophy from the decompensated cardiac dysfunction phase. Here we compared the gene expression in rat cardiac tissue from the compensatory hypertrophic phase (11 wk, n = 6) with the cardiac dysfunction phase (17 wk, n = 6) and with age-matched normotensive controls. Messenger RNA levels of 93 genes, selected based on predicted association with cardiac dysfunction, were measured by quantitative real-time PCR. In the hypertrophic phase, the expression of three genes, atrial natriuretic peptide (ANP; P = 0.0089), brain natriuretic peptide (P = 0.0012), and endothelin-1 precursor (P = 0.028), significantly increased, whereas there was decreased expression of 24 other genes including SOD2 (P = 0.0148), sarco(endo)plasmic reticulum Ca(2+)-ATPase 2a (P = 0.0002), and ryanodine receptor 2 (P = 0.0319). In the subsequent heart cardiac dysfunction phase, the expression of an additional 20 genes including inducible nitric oxide synthase (NOS; P = 0.0135), angiotensin I-converting enzyme (P = 0.0082), and IL-1beta (P < 0.0001) increased, whereas the expression of seven genes decreased compared with those of age-matched controls. Furthermore, the expression of 22 genes, including prepro-endothelin-1, ANP, angiotensin I-converting enzyme, beta(1)-adrenergic receptor, SOD2, and endothelial NOS, significantly changed in the cardiac dysfunction phase compared with the compensatory hypertrophic phase. Finally, principal component analysis successfully segregated animals with decompensatory cardiac dysfunction from controls, as well as from animals at the compensated hypertrophy phase, suggesting that we have identified molecular markers for each stage of the disease.

    View details for DOI 10.1152/ajpheart.00289.2008

    View details for Web of Science ID 000257593800027

    View details for PubMedID 18487446

  • Pharmacological inhibition of epsilon-protein kinase C attenuates cardiac fibrosis and dysfunction in hypertension-induced heart failure HYPERTENSION Inagaki, K., Koyanagi, T., Berry, N. C., Sun, L., Mochly-Rosen, D. 2008; 51 (6): 1565-1569


    Studies on genetically manipulated mice suggest a role for epsilon-protein kinase C (epsilonPKC) in cardiac hypertrophy and in heart failure. The potential clinical relevance of these findings was tested here using a pharmacological inhibitor of epsilonPKC activity during the progression to heart failure in hypertensive Dahl rats. Dahl rats, fed an 8% high-salt diet from the age of 6 weeks, exhibited compensatory cardiac hypertrophy by 11 weeks, followed by heart failure at approximately 17 weeks and death by the age of approximately 20 weeks (123+/-3 days). Sustained treatment between weeks 11 and 17 with the selective epsilonPKC inhibitor epsilonV1-2 or with an angiotensin II receptor blocker olmesartan prolonged animal survival by approximately 5 weeks (epsilonV1-2: 154+/-7 days; olmesartan: 149+/-5 days). These treatments resulted in improved fractional shortening (epsilonV1-2: 58+/-2%; olmesartan: 53+/-2%; saline: 41+/-6%) and decreased cardiac parenchymal fibrosis when measured at 17 weeks without lowering blood pressure at any time during the treatment. Combined treatment with epsilonV1-2, together with olmesartan, prolonged animal survival by 5 weeks (37 days) relative to olmesartan alone (from 160+/-5 to 197+/-14 days, respectively) and by approximately 11 weeks (74 days) on average relative to saline-treated animals, suggesting that the pathway inhibited by epsilonPKC inhibition is not identical to the olmesartan-induced effect. These data suggest that an epsilonPKC-selective inhibitor such as epsilonV1-2 may have a potential in augmenting current therapeutic strategies for the treatment of heart failure in humans.

    View details for DOI 10.1161/HYPERTENSIONAHA.107.109637

    View details for Web of Science ID 000256053500029

    View details for PubMedID 18413490

    View details for PubMedCentralID PMC3646632

  • Dopamine and ethanol cause translocation of epsilon PKC associated with epsilon RACK: Cross-talk between cAMP-dependent protein kinase A and protein kinase C signaling pathways MOLECULAR PHARMACOLOGY Yao, L., Fan, P., Jiang, Z., Gordon, A., Mochly-Rosen, D., Diamond, I. 2008; 73 (4): 1105-1112


    We found previously that neural responses to ethanol and the dopamine D2 receptor (D2) agonist 2,10,11-trihydroxy-N-propylnorapomorphine hydrobromide (NPA) involve both epsilon protein kinase C (epsilonPKC) and cAMP-dependent protein kinase A (PKA). However, little is known about the mechanism underlying ethanol- and D2-mediated activation of epsilonPKC and the relationship to PKA activation. In the present study, we used a new epsilonPKC antibody, 14E6, that selectively recognized active epsilonPKC when not bound to its anchoring protein epsilonRACK (receptor for activated C-kinase), and PKC isozyme-selective inhibitors and activators to measure PKC translocation and catalytic activity. We show here that ethanol and NPA activated epsilonPKC and induced translocation of both epsilonPKC and its anchoring protein, epsilonRACK to a new cytosolic site. The selective epsilonPKC agonist, pseudo-epsilonRACK, activated epsilonPKC but did not cause translocation of the epsilonPKC/epsilonRACK complex to the cytosol. These data suggest a step-wise activation and translocation of epsilonPKC after NPA or ethanol treatment, where epsilonPKC first translocates and binds to its RACK and subsequently the epsilonPKC/epsilonRACK complex translocates to a new subcellular site. Direct activation of PKA by adenosine-3',5'-cyclic monophosphorothioate, Sp-isomer (Sp-cAMPS), prostaglandin E1, or the adenosine A2A receptor is sufficient to cause epsilonPKC translocation to the cytosolic compartment in a process that is dependent on PLC activation and requires PKA activity. These data demonstrate a novel cross-talk mechanism between epsilonPKC and PKA signaling systems. PKA and PKC signaling have been implicated in alcohol rewarding properties in the mesolimbic dopamine system. Cross-talk between PKA and PKC may underlie some of the behaviors associated with alcoholism.

    View details for DOI 10.1124/mol.107.042580

    View details for Web of Science ID 000254064900008

    View details for PubMedID 18202306

  • cPKC isozyme specific substrates in murine embryonic stem cells Costa Junior, H. M., Andrade, A., Labate, C., Krieger, J. E., Mochly-Rosen, D., Schechtman, D. FEDERATION AMER SOC EXP BIOL. 2008
  • The PKC delta-Abl complex communicates ER stress to the mitochondria - an essential step in subsequent apoptosis JOURNAL OF CELL SCIENCE Qi, X., Mochly-Rosen, D. 2008; 121 (6): 804-813


    Conditions that compromise protein folding in the endoplasmic reticulum trigger the unfolded protein response (UPR), which either restores proper protein folding or results in cellular demise through apoptosis. In this study, we found that, in response to ER stress in vivo and in vitro, PKCdelta translocates to the ER where it binds to the tyrosine kinase Abl. Tyrosine phosphorylation and kinase activity of PKCdelta are required for PKCdelta binding to Abl in the ER. Moreover, we found that inhibition of PKCdelta by the PKCdelta-specific peptide inhibitor deltaV1-1 or by silencing of PKCdelta reduces ER-stress-induced JNK activation and inhibits ER-stress-mediated apoptosis. Furthermore, the inhibitor of PKCdelta kinase activity rottlerin blocks the translocation of the PKCdelta-Abl complex from the ER to the mitochondria and confers protection against apoptosis. Thus, PKCdelta communicates ER stress to the mitochondria by binding to ER-localized Abl. The PKCdelta-Abl complex then translocates to the mitochondria, communicating ER stress to this organelle, thereby, triggering apoptosis.

    View details for DOI 10.1242/jcs.024653

    View details for Web of Science ID 000254660200009

    View details for PubMedID 18285444

  • Islet cell survival during isolation improved through protein kinase C epsilon activation Joint Meeting of the International-Xenotransplantation-Association/International-Pancreas-and-Islet-Transplant-Association/Cell-Transplant-Society Kvezereli, M., Vallentin, A., Mochly-Rosen, D., Busque, S., Fontaine, M. J. ELSEVIER SCIENCE INC. 2008: 375–78


    Strategies inhibiting cell death signaling pathways may enhance the availability of islet transplantation for patients with type 1 diabetes mellitus. The epsilon isoform of protein kinase C (PKC epsilon) has been shown to have an anti-apoptotic effect in many cell types. The present study investigated whether activation of PKC epsilon may improve the yield of functional islet cells for transplantation. Islet cells were isolated from wild-type BALB/c mice preconditioned with either a PKC epsilon activator (psi epsilon RACK) or a TAT carrier control peptide and further treated with the same agents during isolation and in vitro for either 0, 1, 16, or 40 hours. Islet cells were assessed at each time point for viability, apoptosis, and function. psi epsilon RACK-treated islets showed significantly decreased islet cell death up to 40 hours after isolation compared with TAT-treated control islets. Beta-cell function in response to high glucose challenge remained unchanged.

    View details for DOI 10.1016/j.transproceed.2008.01.014

    View details for Web of Science ID 000254695600014

    View details for PubMedID 18374073

  • Intracoronary KAI-9803 as an adjunct to primary percutaneous coronary intervention for acute ST-segment elevation myocardial infarction CIRCULATION Roe, M. T., Hartmann, F., LINS, J., Batchelor, W., Ruzyllo, W., Kochman, J., Armstrong, B., Buszman, P., Buszman, P., Leisch, F., Baran, K., Roubin, G., Zenni, M., Bilodeau, L., Caputo, R., Chu, A., Lombardi, W., Adamus, J., Hermiller, J., Darius, H., Krzeminska-Pakula, M., Saucedo, J., Simek, S., Zmudka, K., Farah, T., Hauptmann, K., Lee, D., Lemos, P., Rohrbeck, S., Moshage, W., Strasser, R., Albirini, A., Anderson, E., Bode, C., Caramori, P., Eaton, G., Hoffman, S., Huber, K., Khoury, S., Miller, J., Peterson, J., Porizka, V., Rivera, E., Roe, M., Schuster, P., Stasek, J., Zago, A. 2008; 117 (7): 886-896


    KAI-9803, a delta-protein kinase C inhibitor, has been shown to ameliorate injury associated with ischemia and reperfusion in animal models of acute myocardial infarction (MI).Direct Inhibition of delta-Protein Kinase C Enzyme to Limit Total Infarct Size in Acute Myocardial Infarction (DELTA MI) was a "first-in-human," dose-escalation study that evaluated the safety, tolerability, and activity of KAI-9803 for patients with acute anterior ST-segment elevation MI undergoing primary percutaneous coronary intervention. Patients who presented within 6 hours of symptom onset and had an occluded left anterior descending infarct artery on angiography were randomized in a 2:1 fashion to receive 1 of 4 doses of KAI-9803 (cohort 1, 0.05 mg; cohort 2, 0.5 mg; cohort 3, 1.25 mg; cohort 4, 5.0 mg) versus blinded concurrent placebo delivered in 2 divided doses via intracoronary injection before and after reestablishment of antegrade epicardial flow with percutaneous coronary intervention. Safety and biomarker end points were assessed. Overall, 154 patients were randomized and treated with study drug (37 in cohort 1, 38 in cohort 2, 38 in cohort 3, 41 in cohort 4). The incidence of serious adverse events was similar between patients treated with KAI-9803 versus placebo. Other safety end points, including changes in QT intervals and standard laboratory values after study drug administration, were similar between treatment groups. Although the study was not powered to demonstrate efficacy with the biomarker end points assessed, signs of drug activity with KAI-9803 were suggested by trends for consistent, nonsignificant reductions in creatine kinase-MB area under the curve and ST-recovery area under the curve values across all dosing cohorts with KAI-9803 compared with concurrent placebo, and similar trends were demonstrated for improvements in (99m)technetium sestamibi infarct size values with active study drug in cohorts 1, 2, and 3.KAI-9803 had an acceptable safety and tolerability profile when delivered via intracoronary injection during primary percutaneous coronary intervention for ST-segment elevation MI. Signs of potential drug activity were demonstrated with biomarker end points in this small exploratory study, indicating that further testing of KAI-9803 as an adjunctive therapy for ST-segment elevation MI is warranted.

    View details for DOI 10.1161/CIRCULATIONAHA.107.759167

    View details for Web of Science ID 000253428100005

    View details for PubMedID 18250271

  • Sustained pharmacological inhibition of delta PKC protects against hypertensive encephalopathy through prevention of blood-brain barrier breakdown in rats JOURNAL OF CLINICAL INVESTIGATION Qi, X., Inagaki, K., Sobel, R. A., Mochly-Rosen, D. 2008; 118 (1): 173-182


    Hypertensive encephalopathy is a potentially fatal condition associated with cerebral edema and the breakdown of the blood-brain barrier (BBB). The molecular pathways leading to this condition, however, are unknown. We determined the role of deltaPKC, which is thought to regulate microvascular permeability, in the development of hypertensive encephalopathy using deltaV1-1 - a selective peptide inhibitor of deltaPKC. As a model of hypertensive encephalopathy, Dahl salt-sensitive rats were fed an 8% high-salt diet from 6 weeks of age and then were infused s.c. with saline, control TAT peptide, or deltaV1-1 using osmotic minipumps. The mortality rate and the behavioral symptoms of hypertensive encephalopathy decreased significantly in the deltaV1-1-treated group relative to the control-treated group, and BBB permeability was reduced by more than 60%. Treatment with deltaV1-1 was also associated with decreased deltaPKC accumulation in capillary endothelial cells and in the endfeet of capillary astrocytes, which suggests decreased microvasculature disruption. Treatment with deltaV1-1 prevented hypertension-induced tight junction disruption associated with BBB breakdown, which suggests that deltaPKC may specifically act to dysregulate tight junction components. Together, these results suggest that deltaPKC plays a role in the development of hypertension-induced encephalopathy and may be a therapeutic target for the prevention of BBB disruption.

    View details for DOI 10.1172/JCI32636

    View details for Web of Science ID 000252122900019

    View details for PubMedID 18097471

  • PKC Isozymes in chronic cardiac disease: Possible therapeutic targets? ANNUAL REVIEW OF PHARMACOLOGY AND TOXICOLOGY Churchill, E., Budas, G., Vallcntin, A., Koyanag, T., Mochly-Rosen, D. 2008; 48: 569-599


    Cardiovascular disease is the leading cause of death in the United States. Therefore, identifying therapeutic targets is a major focus of current research. Protein kinase C (PKC), a family of serine/threonine kinases, has been identified as playing a role in many of the pathologies of heart disease. However, the lack of specific PKC regulators and the ubiquitous expression and normal physiological functions of the 11 PKC isozymes has made drug development a challenge. Here we discuss the validity of therapeutically targeting PKC, an intracellular signaling enzyme. We describe PKC structure, function, and distribution in the healthy and diseased heart, as well as the development of rationally designed isozyme-selective regulators of PKC functions. The review focuses on the roles of specific PKC isozymes in atherosclerosis, fibrosis, and cardiac hypertrophy, and examines principles of pharmacology as they pertain to regulators of signaling cascades associated with these diseases.

    View details for DOI 10.1146/annurev.pharmtox.48.121806.154902

    View details for Web of Science ID 000253396900020

    View details for PubMedID 17919087

  • Protection against mammary tumor metastasis by inhibition of PKC alpha Kim, J., Thorne, S. H., Sun, L., Mochly-Rosen, D. AMER ASSOC CANCER RESEARCH. 2007: 3434S–3434S
  • Competitive inhibitors and allosteric activators of protein kinase C isoenzymes: a personal account and progress report on transferring academic discoveries to the clinic Focus Topic at Life Sciences 2007 Conference Budas, G. R., Koyanagi, T., Churchill, E. N., Mochly-Rosen, D. PORTLAND PRESS LTD. 2007: 1021–1026


    PKC (protein kinase C) isoenzymes are related protein kinases, involved in many signalling events in normal state and in disease. Basic research into identifying the molecular basis of PKC selectivity led to simple strategies to identify selective competitive inhibitor peptides and allosteric agonist peptides of individual PKC isoenzymes. The strategies and rationale used to identify these peptide regulators of protein-protein interaction may be applicable to other signalling events. Importantly, the PKC-regulating peptides proved to be useful pharmacological tools and may serve as drugs or drug leads for a variety of human diseases.

    View details for DOI 10.1042/BST0351021

    View details for Web of Science ID 000251279800040

    View details for PubMedID 17956268

  • The roles of PKC delta and epsilon isoenzymes in the regulation of myocardial ischaemia/reperfusion injury Focus Topic at Life Sciences 2007 Conference Churchill, E. N., Mochly-Rosen, D. PORTLAND PRESS LTD. 2007: 1040–1042


    Reperfusion of ischaemic cardiac tissue is associated with increased apoptosis and oncosis, resulting in diminished heart function. Short bouts of ischaemia before the prolonged ischaemic event (ischaemic preconditioning) protect the heart from injury mediated by reperfusion. The PKC (protein kinase C) family of serine/threonine kinases are involved in many different signalling processes. Two calcium-insensitive isoforms of the novel PKC subfamily, PKCdelta and epsilon, play opposing roles in ischaemia/reperfusion injury. Activation of PKCdelta during reperfusion induces cell death through the regulation of mitochondrial function and induction of apoptosis and oncosis. In contrast, activation of PKCepsilon before ischaemia protects mitochondrial function and diminishes apoptosis and oncosis. How can two highly homologous PKC isoenzymes play such opposing roles through the regulation of mitochondrial function? This review will highlight what is known about PKCdelta and epsilon function during ischaemia/reperfusion injury and will suggest a novel regulatory pathway which determines the fate of the cell following ischaemic stress.

    View details for DOI 10.1042/BST0351040

    View details for Web of Science ID 000251279800045

    View details for PubMedID 17956273

  • Mitochondrial protein kinase C epsilon (PKC epsilon): emerging role in cardiac protection from ischaemic damage Focus Topic at Life Sciences 2007 Conference Budas, G. R., Mochly-Rosen, D. PORTLAND PRESS LTD. 2007: 1052–1054


    Mitochondria mediate diverse cellular functions including energy generation and ROS (reactive oxygen species) production and contribute to signal transduction. Mitochondria are also key regulators of cell viability and play a central role in necrotic and apoptotic cell death pathways induced by cardiac ischaemia/reperfusion injury. PKC (protein kinase C) epsilon plays a critical role in cardioprotective signalling pathways that protect the heart from ischaemia/reperfusion. Emerging evidence suggests that the cardioprotective target of PKCepsilon resides at the mitochondria. Proposed mitochondrial targets of PKCepsilon include mitoK(ATP) (mitochondrial ATP-sensitive K(+) channel), components of the MPTP (mitochondrial permeability transition pore) and components of the electron transport chain. This review highlights mitochondrial targets of PKCepsilon and their possible role in cardioprotective signalling in the setting of ischaemia/reperfusion injury.

    View details for DOI 10.1042/13ST03S1052

    View details for Web of Science ID 000251279800049

    View details for PubMedID 17956277

  • delta PKC participates in the endoplasmic reticulum stress-induced response in cultured cardiac myocytes and ischemic heart JOURNAL OF MOLECULAR AND CELLULAR CARDIOLOGY Qi, X., Vallentin, A., Churchill, E., Mochly-Rosen, D. 2007; 43 (4): 420-428


    The cellular response to excessive endoplasmic reticulum (ER) stress includes the activation of signaling pathways, which lead to apoptotic cell death. Here we show that treatment of cultured cardiac myocytes with tunicamycin, an agent that induces ER stress, causes the rapid translocation of deltaPKC to the ER. We further demonstrate that inhibition of deltaPKC using the deltaPKC-specific antagonist peptide, deltaV1-1, reduces tunicamycin-induced apoptotic cell death, and inhibits expression of specific ER stress response markers such as CHOP, GRP78 and phosphorylation of JNK. The physiological importance of deltaPKC in this event is further supported by our findings that the ER stress response is also induced in hearts subjected to ischemia and reperfusion injury and that this response also involves deltaPKC translocation to the ER. We found that the levels of the ER chaperone, GRP78, the spliced XBP-1 and the phosphorylation of JNK are all increased following ischemia and reperfusion and that deltaPKC inhibition by deltaV1-1 blocks these events. Therefore, ischemia-reperfusion injury induces ER stress in the myocardium in a mechanism that requires deltaPKC activity. Taken together, our data show for the first time that deltaPKC activation plays a critical role in the ER stress-mediated response and the resultant cell death.

    View details for DOI 10.1016/j.yjmc.2007.07.061

    View details for Web of Science ID 000250439800006

    View details for PubMedID 17825316

  • Pharmacological inhibition of epsilon PKC suppresses chronic inflammation in murine cardiac transplantation model JOURNAL OF MOLECULAR AND CELLULAR CARDIOLOGY Koyanagi, T., Noguchi, K., Ootani, A., Inagaki, K., Robbins, R. C., Mochly-Rosen, D. 2007; 43 (4): 517-522


    Epsilon protein kinase C (epsilonPKC) plays pivotal roles in myocardial infarction and in heart failure. Although cardiac transplantation is a well-established therapy for severe heart failure, allograft rejection and host inflammatory responses limit graft function and reduce life expectancy. Here we determined whether sustained epsilonPKC inhibition beginning 3 days after transplantation suppress allograft rejection and improve cardiac transplantation using a murine heterotopic transplantation model. Hearts of FVB mice (H-2(q)) were transplanted into C57BL/6 mice (H-2(b)). Delivery of the epsilonPKC inhibitor, TAT(47-57)-epsilonV1-2 (epsilonV1-2, n=9, 20 mg/kg/day), or the carrier control peptide, TAT(47-57) (TAT, n=8), by osmotic pump began 3 days after transplantation and continued for the remaining 4 weeks. epsilonV1-2 treatment significantly improved the beating score throughout the treatment. Infiltration of macrophages and T cells into the cardiac grafts was significantly reduced and parenchymal fibrosis was decreased in animals treated with epsilonV1-2 as compared with control treatment. Finally, the rise in pro-fibrotic cytokine, TGF-beta and monocyte recruiting chemokine MCP-1 levels was almost abolished by epsilonV1-2 treatment, whereas the rise in PDGF-BB level was unaffected. These data suggest that epsilonPKC activity contributes to the chronic immune response in cardiac allograft and that an epsilonPKC-selective inhibitor, such as epsilonV1-2, could augment current therapeutic strategies to suppress inflammation and prolong graft survival in humans.

    View details for DOI 10.1016/j.yjmcc.2007.06.003

    View details for Web of Science ID 000250439800016

    View details for PubMedID 17655859

  • Suppression of delta PKC activation after focal cerebral ischemia contributes to the protective effect of hypothermia JOURNAL OF CEREBRAL BLOOD FLOW AND METABOLISM Shimohata, T., Zhao, H., Sung, J. H., Sun, G., Mochly-Rosen, D., Steinberg, G. K. 2007; 27 (8): 1463-1475


    Mild hypothermia is a robust neuroprotective treatment for stroke. Understanding the mechanisms underlying hypothermia's benefits will lead to more effective treatments to prevent stroke damage. Delta protein kinase C (deltaPKC) is a kinase that has been strongly implicated in executing ischemic damage. We investigated the effects of hypothermia on deltaPKC activation, as determined by its subcellular translocation, proteolytic cleavage, and phosphorylation in a focal cerebral ischemia model. The amount of constitutively activated C-terminal catalytic fragment of deltaPKC (CF-deltaPKC) increased after stroke. Both hypothermia (30 degrees C) and the caspase-3-specific inhibitor, Z-DQMD-FMK, blocked the accumulation of activated deltaPKC in the penumbra. Other hallmarks of deltaPKC activation, its translocation to the mitochondria, and nucleus were observed in the penumbra as early as 10 mins after reperfusion. These events were blocked by hypothermia. Hypothermia also blocked CF-deltaPKC increases in the mitochondria and nuclei. Conversely, a specific deltaPKC activator, psideltaRACK, decreased the neuroprotective effect of hypothermia. Finally, deltaPKC activity may lead to mitochondrial injury and cytochrome c release, as the timing of cytochrome c release corresponded to the time course of deltaPKC translocation. Both cytochrome c release and deltaPKC translocation were blocked by hypothermia. In conclusion, hypothermia protects against ischemic damage in part by suppressing deltaPKC activation after stroke.

    View details for DOI 10.1038/sj.jcbfm.9600450

    View details for Web of Science ID 000248266700005

    View details for PubMedID 17293847

  • Happy birthday protein kinase C: past, present and future of a superfamily. Pharmacological research Battaini, F., Mochly-Rosen, D. 2007; 55 (6): 461-466

    View details for PubMedID 17582783

  • Identification of mitochondria aldehyde dehydrogenase-selective activators as potent protective agents for injury from cardiac ischemia 30th Annual Meeting of the Research-Society-on-Alcoholism Chen, C., Budas, G., Corti, F., Solow-Cordero, D., Ouyang, X., Mochly-Rosen, D. WILEY-BLACKWELL. 2007: 179A–179A
  • Cardioprotective mechanisms of PKC isozyme-selective activators and inhibitors in the treatment of ischemia-reperfusion injury PHARMACOLOGICAL RESEARCH Budas, G. R., Churchill, E. N., Mochly-Rosen, D. 2007; 55 (6): 523-536


    Current treatment for acute myocardial infarction (AMI) is aimed at limiting the duration of ischemia by either mechanical (balloon catheters) or enzymatic (thrombolytics) means to disrupt the occlusion. While these treatments are effective in limiting the duration of ischemia, no therapeutic treatment is currently available to prevent ischemic injury and to reduce reperfusion injury, which occurs after these interventions. The development of rationally designed PKC isozyme-selective regulator peptides has permitted investigation into the role of specific PKC isozymes in ischemia-reperfusion (IR) injury. Based on these studies, it is now evident that epsilon and deltaPKC have distinct temporal and opposing roles in regulating myocardial damage induced by IR. Activation of epsilonPKC before ischemia protects the heart by mimicking preconditioning, whereas inhibition of deltaPKC during reperfusion protects the heart from reperfusion-induced damage. These cardioprotective effects have been observed in isolated cardiomyocytes, isolated perfused hearts and in vivo in all species tested including mouse, rat and pig and may provide the basis for future therapeutic agents. Having established the efficacy of PKC isozyme-specific regulators in reducing IR injury, the next challenge is to outline the molecular mechanisms regulated by delta and epsilonPKC isozymes that result in enhanced tolerance to IR. In this review, we discuss progress that has been made in establishing cytoprotective mechanisms, which arise as a consequence of epsilonPKC activation or deltaPKC inhibition, and how they may lead to protection in the setting of myocardial ischemia reperfusion.

    View details for DOI 10.1016/j.phrs.2007.04.005

    View details for Web of Science ID 000248160400007

    View details for PubMedID 17576073

  • Insight into intra- and inter-molecular interactions of PKC: Design of specific modulators of kinase function PHARMACOLOGICAL RESEARCH Kheifets, V., Mochly-Rosen, D. 2007; 55 (6): 467-476


    Protein kinase C (PKC) is a family of kinases that are critical in many cellular events. These enzymes are activated by lipid-derived second messengers, are dependent on binding to negatively charged phospholipids and some members also require calcium to attain full activation. The interaction with lipids and calcium activators is mediated by binding to the regulatory domains C1 and C2. In addition, many protein-protein interactions between PKC and other proteins have been described. These include interactions with adaptor proteins, substrates and cytoskeletal elements. Regulation of the interactions between PKC, small molecules and other proteins is essential for signal transduction to occur. Finally, a number of auto-inhibitory intra-molecular protein-protein interactions have also been identified in PKC. This chapter focuses on mapping the sites for many of these inter- and intra-molecular interactions and how this information may be used to generate selective inhibitors and activators of PKC signaling.

    View details for DOI 10.1016/j.phrs.2007.04.014

    View details for Web of Science ID 000248160400002

    View details for PubMedID 17580120

  • delta PKC mediates microcerebrovascular dysfunction in acute ischemia and in chronic hypertensive stress in vivo BRAIN RESEARCH Bright, R., Steinberg, G. K., Mochly-Rosen, D. 2007; 1144: 146-155


    Maintaining cerebrovascular function is a priority for reducing damage following acute ischemic events such as stroke, and under chronic stress in diseases such as hypertension. Ischemic episodes lead to endothelial cell damage, deleterious inflammatory responses, and altered neuronal and astrocyte regulation of vascular function. These, in turn, can lead to impaired cerebral blood flow and compromised blood-brain barrier function, promoting microvascular collapse, edema, hemorrhagic transformation, and worsened neurological recovery. Multiple studies demonstrate that protein kinase C (PKC), a widely expressed serine/threonine kinase, is involved in mediating arterial tone and microvascular function. However, there is no clear understanding about the role of individual PKC isozymes. We show that intraperitoneal injection of deltaV1-1-TAT(47-57) (0.2 mg/kg in 1 mL), an isozyme-specific peptide inhibitor of deltaPKC, improved microvascular pathology, increased the number of patent microvessels by 92% compared to control-treated animals, and increased cerebral blood flow by 26% following acute focal ischemia induced by middle cerebral artery occlusion in normotensive rats. In addition, acute delivery of deltaV1-1-TAT(47-57) in hypertensive Dahl rats increased cerebral blood flow by 12%, and sustained delivery deltaV1-1-TAT(47-57) (5 uL/h, 1 mM), reduced infarct size by 25% following an acute stroke induced by MCA occlusion for 90 min. Together, these findings demonstrate that deltaPKC is an important therapeutic target for protection of microvascular structure and function under both acute and chronic conditions of cerebrovascular stress.

    View details for DOI 10.1016/j.brainres.2007.01.113

    View details for Web of Science ID 000246034100017

    View details for PubMedID 17350602

  • Rational design of a selective antagonist of epsilon protein kinase C derived from the selective allosteric agonist, pseudo-RACK peptide JOURNAL OF MOLECULAR AND CELLULAR CARDIOLOGY Liron, T., Chen, L. E., Khaner, H., Vallentin, A., Mochly-Rosen, D. 2007; 42 (4): 835-841


    We have previously shown that domains involved in binding of protein kinase C (PKC) isozymes to their respective anchoring proteins (RACKs) and short peptides derived from these domains are PKC isozyme-selective antagonists. We also identified PKC isozyme-selective agonists, named psiRACK peptides, derived from a sequence within each PKC with high homology to its respective RACK. We noted that all the psiRACK sequences within each PKC isozyme have at least one non-homologous amino acid difference from their corresponding RACK that constitutes a charge change. Based on this information, we have devised here a new approach to design an isozyme-selective PKC antagonist, derived from the psiRACK sequence. We focused on epsilonPKC psiRACK peptide, where the pseudo-epsilonRACK sequence (psiepsilonRACK; HDAPIGYD; corresponding to epsilonPKC85-92) is different in charge from the homologous RACK-derived sequence (NNVALGYD; corresponding to epsilonRACK285-292) in the second amino acid. Here we show that changing the charge of the psiepsilonRACK peptide through a substitution of only one amino acid (aspartate to asparagine) resulted in a peptide with an opposite activity on the same cell function and a substitution for aspartate with an alanine resulted in an inactive peptide. These data support our hypothesis regarding the mechanism by which pseudo-RACK peptide activates PKC in heart cells and suggest that this approach is applicable to other signaling proteins with inducible protein-protein interactions.

    View details for DOI 10.1016/j.yjmcc.2007.01.007

    View details for Web of Science ID 000246287600016

    View details for PubMedID 17337000

  • Impaired perfusion after myocardial infarction is due to reperfusion-induced delta PKC-mediated myocardial damage CARDIOVASCULAR RESEARCH Ikeno, F., Inagaki, K., Rezaee, M., Mochly-Rosen, D. 2007; 73 (4): 699-709


    To improve myocardial flow during reperfusion after acute myocardial infarction and to elucidate the molecular and cellular basis that impedes it. According to the AHA/ACC recommendation, an ideal reperfusion treatment in patients with acute myocardial infarction (AMI) should not only focus on restoring flow in the occluded artery, but should aim to reduce microvascular damage to improve blood flow in the infarcted myocardium.Transgenic mouse hearts expressing the deltaPKC (protein kinase C) inhibitor, deltaV1-1, in their myocytes only were treated with or without the deltaPKC inhibitor after ischemia in an ex vivo AMI model. deltaV1-1 or vehicle was also delivered at reperfusion in an in vivo porcine model of AMI. Microvascular dysfunction was assessed by physiological and histological measurements.deltaPKC inhibition in the endothelial cells improved myocardial perfusion in the transgenic mice. In the porcine in vivo AMI model, coronary flow reserve (CFR), which is impaired for 6 days following infarction, was improved immediately following a one-minute treatment at the end of the ischemic period with the deltaPKC-selective inhibitor, deltaV1-1 ( approximately 250 ng/kg), and was completely corrected by 24 h. Myocardial contrast echocardiography, electron microscopy studies, and TUNEL staining demonstrated deltaPKC-mediated microvascular damage. epsilonPKC-induced preconditioning, which also reduces infarct size by >60%, did not improve microvascular function.These data suggest that deltaPKC activation in the microvasculature impairs blood flow in the infarcted tissue after restoring flow in the occluded artery and that AMI patients with no-reflow may therefore benefit from treatment with a deltaPKC inhibitor given in conjunction with removal of the coronary occlusion.

    View details for DOI 10.1016/j.cardiores.2006.12.011

    View details for Web of Science ID 000245301800012

    View details for PubMedID 17234167

  • Peptides derived from the C2 domain of protein kinase C epsilon (epsilon PKC) modulate epsilon PKC activity and identify potential protein-protein interaction surfaces JOURNAL OF BIOLOGICAL CHEMISTRY Brandman, R., Disatnik, M., Churchill, E., Mochly-Rosen, D. 2007; 282 (6): 4113-4123


    Peptides derived from protein kinase C (PKC) modulate its activity by interfering with critical protein-protein interactions within PKC and between PKC and PKC-binding proteins (Souroujon, M. C., and Mochly-Rosen, D. (1998) Nat. Biotechnol. 16, 919-924). We previously demonstrated that the C2 domain of PKC plays a critical role in these interactions. By focusing on epsilonPKC and using a rational approach, we then identified one C2-derived peptide that acts as an isozyme-selective activator and another that acts as a selective inhibitor of epsilonPKC. These peptides were used to identify the role of epsilonPKC in protection from cardiac and brain ischemic damage, in prevention of complications from diabetes, in reducing pain, and in protecting transplanted hearts. The efficacy of these two peptides led us to search for additional C2-derived peptides with PKC-modulating activities. Here we report on the activity of a series of 5-9-residue peptides that are derived from regions that span the length of the C2 domain of epsilonPKC. These peptides were tested for their effect on PKC activity in cells in vivo and in an ex vivo model of acute ischemic heart disease. Most of the peptides acted as activators of PKC, and a few peptides acted as inhibitors. PKC-dependent myristoylated alanine-rich C kinase substrate phosphorylation in epsilonPKC knock-out cells revealed that only a subset of the peptides were selective for epsilonPKC over other PKC isozymes. These epsilonPKC-selective peptides were also protective of the myocardium from ischemic injury, an epsilonPKC-dependent function (Liu, G. S., Cohen, M. V., Mochly-Rosen, D., and Downey, J. M. (1999) J. Mol. Cell. Cardiol. 31, 1937-1948), and caused selective translocation of epsilonPKC over other isozymes when injected systemically into mice. Examination of the structure of the C2 domain from epsilonPKC revealed that peptides with similar activities clustered into discrete regions within the domain. We propose that these regions represent surfaces of protein-protein interactions within epsilonPKC and/or between epsilonPKC and other partner proteins; some of these interactions are unique to epsilonPKC, and others are common to other PKC isozymes.

    View details for DOI 10.1074/jbc.M608521200

    View details for Web of Science ID 000244481900075

    View details for PubMedID 17142835

  • Protection against hypertension-induced stroke and encephalopathy in hypertensive rats by sustained pharmacological inhibition of delta protein kinase C 32nd International Stroke Conference Qi, X., Inagaki, K., Mochly-Rosen, D. LIPPINCOTT WILLIAMS & WILKINS. 2007: 584–84
  • RBCK1, a protein kinase C beta I (PKC beta I)-interacting protein, regulates PKC beta-dependent function JOURNAL OF BIOLOGICAL CHEMISTRY Vallentin, A., Mochly-Rosen, D. 2007; 282 (3): 1650-1657


    RBCK1 (RBCC protein interacting with PKC 1) has originally been identified as a protein kinase CbetaI (PKCbetaI)-binding partner by a two-hybrid screen and as one of the gene transcripts that increases during adult cardiac hypertrophy. To address whether RBCK1 and PKCbetaI functions are interconnected, we used cultured neonatal myocytes where we previously found that the activity of PKCbetaI is required for an increase in cell size, also called hypertrophy. In this study, we showed that acute treatment of cardiac myocytes with phenylephrine, a prohypertrophic stimulant, transiently increased the association of RBCK1 with PKCbetaI within 1 min. A prolonged phenylephrine treatment also resulted in an increase of the interaction of the two proteins. Endogenous RBCK1 protein levels increased upon phenylephrine-induced hypertrophy. Further, adenovirus-based RBCK1 overexpression in the absence of phenylephrine increased cardiac cell size. This RBCK1-mediated hypertrophy required PKCbeta activity, since the increase in cell size was inhibited when the RBCK1-expressing cells were treated with PKCbeta-selective antagonists, supporting our previous observation that both PKCbetaI and PKCbetaII are required for hypertrophy. Unexpectedly, RBCK1-induced increased cell size was inhibited by phenylephrine. This effect correlated with a decrease in the level of both PKCbeta isoforms. Most importantly, RNA interference for RBCK1 significantly inhibited the increase in cell size of cardiac myocytes following phenylephrine treatment. Our results suggest that RBCK1 binds PKCbetaI and is a key regulator of PKCbetaI function in cells and that, together with PKCbetaII, the three proteins are essential for developmental hypertrophy of cardiac myocytes.

    View details for DOI 10.1074/jbc.M601710200

    View details for Web of Science ID 000243451300016

    View details for PubMedID 17121852

  • Peptide modulators of Src activity in G(1) regulate entry into S phase and proliferation of NIH 3T3 cells BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS Mamidipudi, V., Miller, L. D., Mochly-Rosen, D., Cartwright, C. A. 2007; 352 (2): 423-430


    Cascades of kinases and phosphatases are regulated by selective protein-protein interactions that are essential for signal transduction. Peptide modulators of these interactions have been used to dissect the function of individual components of the signaling cascade, without relying on either the over- or underexpression of proteins. Previously, we identified RACK1 as an endogenous substrate, binding partner and inhibitor of Src tyrosine kinases. Here, we utilized cell-permeable peptides that selectively disrupt or enhance the interaction of RACK1 and Src to further examine the function of RACK1. Our results provide direct physiologic evidence that RACK1 regulates growth of NIH3T3 cells by suppressing the activity of Src and other cell cycle regulators in G1, and delaying entry into S phase. They also demonstrate the potential for using peptide modulators of Src activity as a tool for regulating cell growth, and for designing new strategies for cancer therapy that target specific protein-protein interactions.

    View details for DOI 10.1016/j.bbrc.2006.11.034

    View details for Web of Science ID 000243196300023

    View details for PubMedID 17118337

  • Induced fit and wit: Celebrating the life of Daniel E. Koshland, Jr. (1920-2007) - Obituary IUBMB LIFE Newton, A. C., Mochly-Rosen, D. 2007; 59 (12): 741-743

    View details for DOI 10.1080/15216540701697438

    View details for Web of Science ID 000251714500001

    View details for PubMedID 18085473

  • Use of a novel method to find substrates of protein kinase C delta identifies M2 pyruvate kinase INTERNATIONAL JOURNAL OF BIOCHEMISTRY & CELL BIOLOGY Siwko, S., Mochly-Rosen, D. 2007; 39 (5): 978-987


    Protein kinase C (PKC) family members have been implicated in numerous cellular processes. However, identifying the substrates of each PKC isozyme remains a challenge. Here, we describe a method using two-dimensional (2D) isoelectric focusing gel electrophoresis to identify substrates of delta PKC (deltaPKC) in MCF-7 breast carcinoma cells. We show that M2 pyruvate kinase is a substrate of deltaPKC, and further characterize the interaction between M2 pyruvate kinase and deltaPKC in MCF-7 cells by immunoprecipitation. deltaPKC activation in vitro or in cells did not appear to alter the enzyme activity or polymerization of M2 pyruvate kinase.

    View details for DOI 10.1016/j.biocel.2007.01.018

    View details for Web of Science ID 000246535900014

    View details for PubMedID 17337233

  • Protein kinase C delta (delta PKC)-annexin V interaction - A required step in delta PKC translocation and function JOURNAL OF BIOLOGICAL CHEMISTRY Kheifets, V., Bright, R., Inagaki, K., Schechtman, D., Mochly-Rosen, D. 2006; 281 (32): 23218-23226


    Protein kinase C (PKC) plays a critical role in diseases such as cancer, stroke, and cardiac ischemia, and participates in a variety of signal transduction pathways such as apoptosis, cell proliferation, and tumor suppression. Though much is known about PKC downstream signaling events, the mechanisms of regulation of PKC activation and subsequent translocation have not been elucidated. Protein-protein interactions regulate and determine the specificity of many cellular signaling events. Such a specific protein-protein interaction is described here between deltaPKC and annexin V. We demonstrate, at physiologically relevant conditions, that a transient interaction between annexin V and deltaPKC occurs in cells after deltaPKC stimulation, but before deltaPKC translocates to the particulate fraction. Evidence of deltaPKC-annexin V binding is provided also by FRET and by in vitro binding studies. Dissociation of the deltaPKC-annexin V complex requires ATP and microtubule integrity. Furthermore, depletion of endogenous annexin V, but not annexin IV, with siRNA inhibits deltaPKC translocation following PKC stimulation. A rationally designed eight amino acid peptide, corresponding to the interaction site for deltaPKC on annexin V, inhibits deltaPKC translocation and deltaPKC-mediated function as evidenced by its protective effect in a model of myocardial infarction. Our data indicate that translocation of deltaPKC is not simply a diffusion-driven process, but is instead a multi-step event regulated by protein-protein interactions. We show that following cell activation, deltaPKC-annexin V binding is a transient and an essential step in the function of deltaPKC, thus identifying a new role for annexin V in PKC signaling and a new step in PKC activation.

    View details for DOI 10.1074/jbc.M602075200

    View details for Web of Science ID 000239542600084

    View details for PubMedID 16785226

  • Epsilon protein kinase C as a potential therapeutic target for the ischemic heart CARDIOVASCULAR RESEARCH Inagaki, K., Churchill, E., Mochly-Rosen, D. 2006; 70 (2): 222-230


    Ischemic heart disease is the leading cause of morbidity and mortality in the western world. Ischemic damage can occur by acute myocardial infarction, stable angina, cardiac stunning, and myocardial hibernation. In addition, 'scheduled' ischemic events, occurring during cardiac surgery, heart transplantation, and elective angioplasty, can also result in cardiac damage. Ischemic or pharmacological preconditioning can decrease the extent of damage to the myocardium. Although the mechanism of preconditioning-mediated cardioprotection is not fully understood, epsilonPKC has been implicated as a critical mediator of this process in animal studies. The use of isozyme-specific pharmacological tools has permitted a better elucidation of the upstream stimuli and the downstream transducers of epsilonPKC in the pathways leading to cardioprotection. While little is known about the role of epsilonPKC in these pathways in humans, animal studies suggest a potential therapeutic role of epsilonPKC. This review will focus on the role of epsilonPKC in cardiac protection and on the signal transduction cascades that have been implicated in this protection.

    View details for DOI 10.1016/j.cardiores.2006.02.015

    View details for Web of Science ID 000237622300008

    View details for PubMedID 16635641

  • The role of protein kinase C in cerebral ischemic and reperfusion injury STROKE Bright, R., Mochly-Rosen, D. 2005; 36 (12): 2781-2790


    Stroke is a leading cause of disability and death in the United States, yet limited therapeutic options exist. The need for novel neuroprotective agents has spurred efforts to understand the intracellular signaling pathways that mediate cellular response to stroke. Protein kinase C (PKC) plays a central role in mediating ischemic and reperfusion damage in multiple tissues, including the brain. However, because of conflicting reports, it remains unclear whether PKC is involved in cell survival signaling, or mediates detrimental processes.This review will examine the role of PKC activity in stroke. In particular, we will focus on more recent insights into the PKC isozyme-specific responses in neuronal preconditioning and in ischemia and reperfusion-induced stress.Examination of PKC isozyme activities during stroke demonstrates the clinical promise of PKC isozyme-specific modulators for the treatment of cerebral ischemia.

    View details for DOI 10.1161/01.STR.0000189996.71237.f7

    View details for Web of Science ID 000233452400057

    View details for PubMedID 16254221

  • PKC-epsilon-dependent survival signals in diabetic hearts 76th Annual Scientific Session of the American-Heart-Association Malhotra, A., Begley, R., Kang, B. P., Rana, I., Liu, J., Yang, G. P., Mochly-Rosen, D., Meggs, L. G. AMER PHYSIOLOGICAL SOC. 2005: H1343–H1350


    Diabetes mellitus is complicated by the development of a primary cardiomyopathy, which contributes to the excess morbidity and mortality of this disorder. The protein kinase C (PKC) family of isozymes plays a key role in the cardiac phenotype expressed during postnatal development and in response to pathological stimuli. Hyperglycemia is an activating signal for cardiac PKC isozymes that modulate a myriad of cell events including cell death and survival. The epsilon-isozyme of the PKC family transmits a powerful survival signal in cardiac muscle cells. Accordingly, to test the hypothesis that endogenous activation of cardiac PKC-epsilon will protect against hyperglycemic cell injury and left ventricular dysfunction, diabetes mellitus was induced using streptozotocin in genetically engineered mice with cardiac-specific expression of the PKC-epsilon translocation activator [psiepsilon-receptors for activated C kinase (psiepsilon-RACK)]. The results demonstrate a striking PKC-epsilon cardioprotective phenotype in diabetic psiepsilon-RACK (epsilon-agonist) mice that is characterized by inhibition of the hyperglycemia apoptosis signal, attenuation of hyperglycemia-mediated oxidative stress, and preservation of parameters of left ventricular pump function. Hearts of diabetic epsilon-agonist mice exhibited selective trafficking of PKC-epsilon to membrane and mitochondrial compartments, phosphorylation/inactivation of the mitochondrial Bad protein, and inhibition of cytochrome c release. We conclude that activation of endogenous PKC-epsilon in hearts of diabetic epsilon-agonist mice promotes the survival phenotype, attenuates markers of oxidative stress, and inhibits the negative inotropic properties of chronic hyperglycemia.

    View details for DOI 10.1152/ajpheart.01200.2004

    View details for Web of Science ID 000231875300004

    View details for PubMedID 15894568

  • RSA 2004: combined basic research satellite symposium - session three: alcohol and mitochondrial metabolism: at the crossroads of life and death. Alcoholism, clinical and experimental research Szabo, G., Hoek, J. B., Darley-Usmar, V., Hajnoczky, G., Knudsen, T., Mochly-Rosen, D., Zakhari, S. 2005; 29 (9): 1749-1752


    This article summarizes the proceedings of the RSA 2004 Combined Basic Research Satellite Meeting convened at the Westin Bayshore Resort and Marina, Vancouver, CA. One of the sessions "Alcohol and mitochondrial metabolism: At the crossroads of life and death" featured five speakers and was chaired by Drs. Jan Hoek and Sam Zakhari. The presentations were 1) Introduction: Alcohol and cellular energy metabolism by Jan Hoek, 2) Ethanol-dependent dysfunction of mitochondrial energy metabolism: the role of NO by Victor Darley-Usmar, 3) Ethanol and apoptosis in the heart by Gyorgy Hajnoczky, 4) Alcohol and mitochondrial biogenesis in development by Thomas Knudsen, and 5) Alcohol, mitochondrial function and cardiac preconditioning by Daria Mochly-Rosen.

    View details for PubMedID 16205376

  • delta PKC-mediated activation of epsilon PKC in ethanol-induced cardiac protection from ischemia JOURNAL OF MOLECULAR AND CELLULAR CARDIOLOGY Inagaki, K., Mochly-Rosen, D. 2005; 39 (2): 203-211


    Previous studies have demonstrated that acute ethanol exposure induces activation of delta protein kinase C (deltaPKC) and epsilonPKC, and mimics ischemic preconditioning via epsilonPKC activation. However, the role of deltaPKC isozyme in ischemia and reperfusion is still controversial. Here, we investigated the role of deltaPKC in ethanol-induced cardioprotection using a selective deltaPKC activator (psideltaRACK), or inhibitor (deltaV1-1), and a selective epsilonPKC inhibitor (epsilonV1-2) in isolated mouse hearts. Mice were injected intraperitoneally or by gavage with ethanol, regulators of delta and epsilonPKC or an adenosine A1 receptor blocker (DPCPX). Isolated perfused mouse hearts were subjected to a 30-min global ischemia and a 120-min reperfusion, ex vivo. Injection of 0.5 g/kg ethanol 1 h, but not 10 min, before ischemia reduced infarct size and CPK release. Pretreatment with epsilonV1-2 abolished this ethanol-induced cardioprotection. Pretreatment with deltaV1-1 induced cardioprotection when injected with ethanol (0.5 g/kg) 10 min before ischemia, but deltaV1-1 partly inhibited ethanol-induced cardioprotection when injected with ethanol 1-h before the onset of ischemia. psideltaRACK injection 1 h, but not 10 min, before ischemia induced cardioprotection and translocation of epsilonPKC from the cytosol to the particulate fraction. Pretreatment with DPCPX or epsilonV1-2 inhibited psideltaRACK-induced cardioprotection and translocation of epsilonPKC. Therefore, activation of deltaPKC-induced by ethanol or by the deltaPKC activator is cardioprotective, provided that sufficient time passes to allow deltaPKC-induced activation of epsilonPKC, an A1 adenosine receptor-dependent process.

    View details for DOI 10.1016/j.yjmcc.2005.04.014

    View details for Web of Science ID 000231155800001

    View details for PubMedID 15990110

  • Ethanol withdrawal-associated allodynia and hyperalgesia: Age-dependent regulation by protein kinase C epsilon and gamma isozymes JOURNAL OF PAIN Shumilla, J. A., Liron, T., Mochly-Rosen, D., Kendig, J. J., Sweitzer, S. M. 2005; 6 (8): 535-549


    Ethanol (EtOH) withdrawal increases sensitivity to painful stimuli in adult rats. In this study, withdrawal from a single, acute administration of EtOH dose-dependently produced mechanical allodynia and thermal hyperalgesia in postnatal day 7 (P7) rats. In contrast, P21 rats exhibited earlier and more prolonged mechanical allodynia but not thermal hyperalgesia. For both P7 and P21 rats, blood and spinal cord EtOH levels peaked at 30 minutes after administration, with P7 rats achieving overall higher spinal cord concentrations. Protein kinase C (PKC) has been implicated in mediating pain responses. Inhibitory PKC- and gamma-specific peptides attenuated mechanical allodynia and thermal hyperalgesia in P7 rats, whereas only the PKCgamma inhibitor prevented mechanical allodynia in P21 rats. Immunoreactive PKC in dorsal root ganglion and PKCgamma in lumbar spinal cord increased at 6 hours after EtOH administration in P7 rats. In P21 rats, the density of PKC immunoreactivity remained unchanged, whereas the density of PKCgamma immunoreactivity increased and translocation occurred. These studies demonstrate developmental differences in neonatal nociceptive responses after withdrawal from acute EtOH and implicate a role for specific PKC isozymes in EtOH withdrawal-associated allodynia and hyperalgesia.This study examines age-specific nociceptive responses after ethanol exposure by using 2 different ages of rats. The results suggest that ethanol age-dependently alters sensitivity to mechanical and thermal stimuli via specific protein kinase C isozymes. These results begin to ascertain the mechanisms that produce abnormal pain after alcohol exposure.

    View details for DOI 10.1016/j.jpain.2005.03.005

    View details for Web of Science ID 000231546000007

    View details for PubMedID 16084468

  • Reperfusion-induced translocation of delta PKC to cardiac mitochondria prevents pyruvate dehydrogenase reactivation CIRCULATION RESEARCH Churchill, E. N., Murriel, C. L., Chen, C. H., Mochly-Rosen, D., Szweda, L. I. 2005; 97 (1): 78-85


    Cardiac ischemia and reperfusion are associated with loss in the activity of the mitochondrial enzyme pyruvate dehydrogenase (PDH). Pharmacological stimulation of PDH activity improves recovery in contractile function during reperfusion. Signaling mechanisms that control inhibition and reactivation of PDH during reperfusion were therefore investigated. Using an isolated rat heart model, we observed ischemia-induced PDH inhibition with only partial recovery evident on reperfusion. Translocation of the redox-sensitive delta-isoform of protein kinase C (PKC) to the mitochondria occurred during reperfusion. Inhibition of this process resulted in full recovery of PDH activity. Infusion of the deltaPKC activator H2O2 during normoxic perfusion, to mimic one aspect of cardiac reperfusion, resulted in loss in PDH activity that was largely attributable to translocation of deltaPKC to the mitochondria. Evidence indicates that reperfusion-induced translocation of deltaPKC is associated with phosphorylation of the alphaE1 subunit of PDH. A potential mechanism is provided by in vitro data demonstrating that deltaPKC specifically interacts with and phosphorylates pyruvate dehydrogenase kinase (PDK)2. Importantly, this results in activation of PDK2, an enzyme capable of phosphorylating and inhibiting PDH. Thus, translocation of deltaPKC to the mitochondria during reperfusion likely results in activation of PDK2 and phosphorylation-dependent inhibition of PDH.

    View details for DOI 10.1161/01.RES.0000173896.32522.6e

    View details for Web of Science ID 000230334900011

    View details for PubMedID 15961716

  • Protein kinase C delta cleavage initiates an aberrant signal transduction pathway after cardiac arrest and oxygen glucose deprivation JOURNAL OF CEREBRAL BLOOD FLOW AND METABOLISM Raval, A. P., Dave, K. R., Prado, R., Katz, L. M., Busto, R., Sick, T. J., Ginsberg, M. D., Mochly-Rosen, D., Perez-Pinzon, M. A. 2005; 25 (6): 730-741


    Protein kinase C (PKC) isozymes have been known to mediate a variety of complex and diverse cellular functions. deltaPKC has been implicated in mediating apoptosis. Using two models of cerebral ischemia, cardiac arrest in rats and oxygen glucose deprivation (OGD) in organotypic hippocampal slices, we tested whether an ischemic insult promoted deltaPKC cleavage during the reperfusion and whether the upstream pathway involved release of cytochrome c and caspase 3 cleavage. We showed that cardiac arrest/OGD significantly enhanced deltaPKC translocation and increased its cleavage at 3 h of reperfusion. Since deltaPKC is one of the substrates for caspase 3, we next determined caspase 3 activation after cardiac arrest and OGD. The maximum decrease in levels of procaspase 3 was observed at 3 h of reperfusion after cardiac arrest and OGD. We also determined cytochrome c release, since it is upstream of caspase 3 activation. Cytochrome c in cytosol increased at 1 h of reperfusion after cardiac arrest/OGD. Inhibition of either deltaPKC/caspase 3 during OGD and early reperfusion resulted in neuroprotection in CA1 region of hippocampus. Our results support the deleterious role of deltaPKC in reperfusion injury. We propose that early cytochrome c release and caspase 3 activation promote deltaPKC translocation/cleavage.

    View details for DOI 10.1038/sj.jcbfm.9600071

    View details for Web of Science ID 000229245900007

    View details for PubMedID 15716854

  • Inhibition of heart transplant injury and graft coronary artery disease after prolonged organ ischemia by selective protein kinase C regulators JOURNAL OF THORACIC AND CARDIOVASCULAR SURGERY Tanaka, M., Gunawan, F., Terry, R. D., Inagaki, K., Caffarelli, A. D., Hoyt, G., Tsao, P. S., Mochly-Rosen, D., Robbins, R. C. 2005; 129 (5): 1160-1167


    Transplanted hearts subjected to prolonged ischemia develop ischemia-reperfusion injury and graft coronary artery disease. To determine the effect of delta-protein kinase C and -protein kinase C on ischemia-reperfusion injury and the resulting graft coronary artery disease induced by prolonged ischemia, we used a delta-protein kinase C-selective inhibitor peptide and an -protein kinase C-selective activator peptide after 30 or 120 minutes of ischemia.Hearts of piebald viral glaxo (PVG) rats were heterotopically transplanted into allogeneic August Copenhagen Irish (ACI) rats. After cardioplegic arrest of the donor heart, -protein kinase C activator was injected antegrade into the coronary arteries. Hearts were procured and bathed in -protein kinase C activator, and before reperfusion, delta-protein kinase C inhibitor was injected into the recipient inferior vena cava. Controls were treated with saline. To analyze ischemia-reperfusion injury, grafts were procured at 4 hours after transplantation and analyzed for superoxide generation; myeloperoxidase activity; tumor necrosis factor alpha, interleukin 1beta, and monocyte/macrophage chemoattractant protein 1 production; and cardiomyocyte apoptosis by terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling and caspase 2, 3, 8, and 9 activity. To analyze graft coronary artery disease, another set of animals underwent equal ischemic times and treatment strategies and then after 90 days were analyzed for graft coronary artery disease indexes.All measures of ischemia-reperfusion injury and graft coronary artery disease after 120 minutes of ischemia in the saline-treated group were significantly increased relative to those observed after 30 minutes of ischemia. It is important to note that all ischemia-reperfusion injury parameters and graft coronary artery disease indexes decreased significantly in the protein kinase C regulator-treated group in comparison to saline-treated controls; additionally, these values were equivalent to those in saline-treated controls with 30 minutes of ischemia.Combined treatment with -protein kinase C activator and delta-protein kinase C inhibitor reduces ischemia-reperfusion injury and decreases the resulting graft coronary artery disease induced by prolonged ischemia.

    View details for DOI 10.1016/j.jtcvs.2004.09.015

    View details for Web of Science ID 000228947200029

    View details for PubMedID 15867794

  • Protein kinase C gamma mediates ethanol withdrawal hyper-responsiveness of NMDA receptor currents in spinal cord motor neurons BRITISH JOURNAL OF PHARMACOLOGY Li, H. F., Mochly-Rosen, D., Kendig, J. J. 2005; 144 (3): 301-307


    The present studies were designed to test the hypothesis that neuronal-specific protein kinase Cgamma (PKCgamma) plays a critical role in acute ethanol withdrawal hyper-responsiveness in spinal cord. Patch-clamp studies were carried out in motor neurons in neonatal rat spinal cord slices. Postsynaptic currents were evoked by brief pulses of 2 mM N-methyl-D-aspartic acid (NMDA) in the presence of bicuculline methiodide 10 microM; strychnine 5 microM and tetrodotoxin 0.5 microM. Both ethanol depression and withdrawal hyper-responsiveness of NMDA-evoked currents are dependent on increases in intracellular Ca(2+). Blocking intracellular increase in Ca(2+) by 30 mM 1,2-bis(2-aminophenoxy)-ethane-N,N,N',N'-tetraacetic acid (BAPTA) not only decreased the ethanol-induced depression of NMDA-evoked currents (33+/-5% in control vs 20+/-3% in BAPTA, P<0.05) but also eliminated acute ethanol withdrawal hyper-responsiveness. Immunohistochemistry studies revealed that neonatal spinal cord motor neurons contain an abundance of nuclear PKCgamma. Exposure to ethanol (100 mM) induced PKCgamma translocation from the nucleus to cytoplasm in motor neurons. Pretreatment with the gamma-isozyme-specific peptide PKC inhibitor, gammaV5-3, blocked ethanol-induced translocation and also blocked withdrawal hyper-responsiveness. The results show that PKCgamma mediates ethanol withdrawal hyper-responsiveness in spinal motor neurons; the results may be relevant to some symptoms of ethanol withdrawal in vivo.

    View details for DOI 10.1038/sj.bjp.0706033

    View details for Web of Science ID 000226848700001

    View details for PubMedID 15655532

  • Cardioprotection by epsilon-protein kinase C activation from ischemia - Continuous delivery and antiarrhythmic effect of an epsilon-protein kinase C-activating peptide CIRCULATION Inagaki, K., Begley, R., Ikeno, F., Mochly-Rosen, D. 2005; 111 (1): 44-50


    We previously showed that a selective activator peptide of epsilon-protein kinase C (PKC), psi(epsilon)RACK, conferred cardioprotection against ischemia-reperfusion when delivered ex vivo before the ischemic event. Here, we tested whether in vivo continuous systemic delivery of psi(epsilon)RACK confers sustained cardioprotection against ischemia-reperfusion in isolated mouse hearts and whether psi(epsilon)RACK treatment reduces infarct size or lethal arrhythmias in porcine hearts in vivo.After psi(epsilon)RACK was systemically administered in mice either acutely or continuously, hearts were subjected to ischemia-reperfusion in an isolated perfused model. Whereas psi(epsilon)RACK-induced cardioprotection lasted 1 hour after a single intraperitoneal injection, continuous treatment with psi(epsilon)RACK induced a sustained preconditioned state during the 10 days of delivery. There was no desensitization to the therapeutic effect, no downregulation of epsilonPKC, and no adverse effects after sustained psi(epsilon)RACK delivery. Porcine hearts were subjected to ischemia-reperfusion in vivo, and psi(epsilon)RACK was administered by intracoronary injection during the first 10 minutes of ischemia. psi(epsilon)RACK treatment reduced infarct size (34+/-2% versus 14+/-1%, control versus psi(epsilon)RACK) and resulted in fewer cases of ventricular fibrillation during ischemia-reperfusion (87.5% versus 50%, control versus psi(epsilon)RACK).The epsilonPKC activator psi(epsilon)RACK induced cardioprotection both in vivo and ex vivo, reduced the incidence of lethal arrhythmia during ischemia-reperfusion, and did not cause desensitization or downregulation of epsilonPKC after sustained delivery. Thus, psi(epsilon)RACK may be useful for patients with ischemic heart disease. In addition, the psi(epsilon)RACK peptide should be a useful pharmacological agent for animal studies in which systemic and sustained modulation of epsilonPKC in vivo is needed.

    View details for DOI 10.1161/01.CIR.0000151614.22282.F1

    View details for Web of Science ID 000226065300008

    View details for PubMedID 15611364

  • Protein kinase C delta activation induces apoptosis in response to cardiac ischemia and reperfusion damage - A mechanism involving bad and the mitochondria JOURNAL OF BIOLOGICAL CHEMISTRY Murriel, C. L., Churchill, E., Inagaki, K., Szweda, L. I., Mochly-Rosen, D. 2004; 279 (46): 47985-47991


    Heart attacks caused by occlusion of coronary arteries are often treated by mechanical or enzymatic removal of the occlusion and reperfusion of the ischemic heart. It is now recognized that reperfusion per se contributes to myocardial damage, and there is a great interest in identifying the molecular basis of this damage. We recently showed that inhibiting protein kinase Cdelta (PKCdelta) protects the heart from ischemia and reperfusion-induced damage. Here, we demonstrate that PKCdelta activity and mitochondrial translocation at the onset of reperfusion mediates apoptosis by facilitating the accumulation and dephosphorylation of the pro-apoptotic BAD (Bcl-2-associated death promoter), dephosphorylation of Akt, cytochrome c release, PARP (poly(ADP-ribose) polymerase) cleavage, and DNA laddering. Our data suggest that PKCdelta activation has a critical proapoptotic role in cardiac responses following ischemia and reperfusion.

    View details for DOI 10.1074/jbc.M405071200

    View details for Web of Science ID 000224957000069

    View details for PubMedID 15339931

  • Suppression of graft coronary artery disease by a brief treatment with a selective epsilon PKC activator and a delta PKC inhibitor in murine cardiac allografts CIRCULATION Tanaka, M., Terry, R. D., Mokhtari, G. K., Inagaki, K., Koyanagi, T., Kofidis, T., Mochly-Rosen, D., Robbins, R. C. 2004; 110 (11): II194-II199


    Inhibiting delta protein kinase C (deltaPKC) during reperfusion and activating epsilon PKC (epsilonPKC) before ischemia each limits cardiac ischemic injury. Here, we examined whether limiting ischemia-reperfusion injury inhibits graft coronary artery disease (GCAD) and improves murine cardiac allografting.Hearts of FVB mice (H-2q) were transplanted into C57BL/6 mice (H-2b). epsilonPKC activator (psiepsilonRACK) was injected intraperitoneally (20 nmol) into donor mice 20 minutes before procurement. Hearts were then perfused with psiepsilonRACK (1.5 nmol) through the inferior vena cava (IVC) and subsequently submerged in psiepsilonRACK (0.5 micromol/L) for 20 minutes at 4 degrees C. Before reperfusion, the peritoneal cavity of recipients was irrigated with deltaPKC inhibitor (deltaV1-1, 300 nmol); control animals were treated with normal saline. The total ischemic time to the organ was 50 minutes. Two hours after transplantation, production of inflammatory cytokines and adhesion molecules, cardiomyocyte apoptosis, and caspase-3 and caspase-9 (but not caspase-8) activities were significantly reduced in the PKC regulator-treated group. Fas ligand levels (but not Fas) were also significantly reduced in this group. Importantly, GCAD indices, production of inflammatory cytokines, and adhesion molecules were significantly decreased and cardiac allograft function was significantly better as measured up to 30 days after transplantation.An epsilonPKC activator and a deltaPKC inhibitor together reduced GCAD. Clinically, these PKC isozyme regulators may be useful for organ preservation and prevention of ischemia-reperfusion injury and graft coronary artery disease in cardiac transplantation.

    View details for DOI 10.1161/01.CIR.0000138389.22905.62

    View details for Web of Science ID 000224023600034

    View details for PubMedID 15364862

  • Protein kinase C delta mediates cerebral reperfusion injury in vivo JOURNAL OF NEUROSCIENCE Bright, R., Raval, A. P., Dembner, J. M., Perez-Pinzon, M. A., Steinberg, G. K., Yenari, M. A., Mochly-Rosen, D. 2004; 24 (31): 6880-6888


    Protein kinase C (PKC) has been implicated in mediating ischemic and reperfusion damage in multiple organs. However, conflicting reports exist on the role of individual PKC isozymes in cerebral ischemic injury. Using a peptide inhibitor selective for deltaPKC, deltaV1-1, we found that deltaPKC inhibition reduced cellular injury in a rat hippocampal slice model of cerebral ischemia [oxygen-glucose deprivation (OGD)] when present both during OGD and for the first 3 hr of reperfusion. We next demonstrated peptide delivery to the brain parenchyma after in vivo delivery by detecting biotin-conjugateddeltaV1-1 and by measuring inhibition of intracellular deltaPKC translocation, an indicator of deltaPKC activity. Delivery of deltaV1-1 decreased infarct size in an in vivo rat stroke model of transient middle cerebral artery occlusion. Importantly, deltaV1-1 had no effect when delivered immediately before ischemia. However, delivery at the onset, at 1 hr, or at 6 hr of reperfusion reduced injury by 68, 47, and 58%, respectively. Previous work has implicated deltaPKC in mediating apoptotic processes. We therefore determined whether deltaPKC inhibition altered apoptotic cell death or cell survival pathways in our models. We found that deltaV1-1 reduced numbers of terminal deoxynucleotidyl transferase-mediated biotinylated UTP nick end labeling-positive cells, indicating decreased apoptosis, increased levels of phospho-Akt, a kinase involved in cell survival pathways, and inhibited BAD (Bcl-2-associated death protein) protein translocation from the cell cytosol to the membrane, indicating inhibition of proapoptotic signaling. These data support a deleterious role for deltaPKC during reperfusion and suggest that deltaV1-1 delivery, even hours after commencement of reperfusion, may provide a therapeutic advantage after cerebral ischemia.

    View details for DOI 10.1523/JNEUROSCI.4474-03.2004

    View details for Web of Science ID 000223102500004

    View details for PubMedID 15295022

  • RACK1 regulates Src-mediated Sam68 and p190RhoGAP signaling ONCOGENE Miller, L. D., Lee, K. C., Mochly-Rosen, D., Cartwright, C. A. 2004; 23 (33): 5682-5686


    RACK1 is the founding member of a family of receptors for activated C kinase collectively called RACKs. Upon activation of PKC, RACK1 co-localizes with the Src tyrosine kinase at the plasma membrane and functions as a substrate, binding partner and inhibitor of Src (as measured in vitro), and a growth inhibitor in NIH 3T3 cells. To further analyze the function of RACK1 in Src and PKC signaling, we utilized cell-permeable peptides that modulate the interaction of RACK1 and betaIIPKC, thereby affecting betaIIPKC translocation and function. We found that the association of betaIIPKC and RACK1 is necessary for Src phosphorylation of RACK1. Src activity is required for tyrosine phosphorylation of RACK1, and for RACK1 binding to Src, but not to betaIIPKC. Endogenous Src kinase activity, as measured by phosphorylation of Sam68 (a mitotic-specific Src substrate involved in cell cycle regulation and RNA splicing) or p190RhoGAP (a Src substrate and GTPase-activating protein involved in actin reorganization), increases with disruption of the Src-RACK1 complex, and decreases with enhanced complex formation. RACK1 inhibits Src-mediated p190RhoGAP signaling and actin cytoskeleton rearrangement. Thus, RACK1 functions as an endogenous inhibitor of the Src kinase in diverse signaling pathways that regulate distinct cellular functions. Our results demonstrate the potential for using peptide modulators of Src activity as a tool for uncovering the function of Src in cells.

    View details for DOI 10.1038/sj.onc.1207735

    View details for Web of Science ID 000222629500013

    View details for PubMedID 15184885

  • Cell-specific role for epsilon- and beta I-protein kinase C isozymes in protecting cortical neurons and astrocytes from ischemia-like injury NEUROPHARMACOLOGY Wang, J., Bright, R., Mochly-Rosen, D., Giffard, R. G. 2004; 47 (1): 136-145


    Activation of epsilon protein kinase C (epsilonPKC) has been shown to protect cardiac myocytes against ischemia and reperfusion injury. However, the role of PKC in ischemic brain injury is less well defined. Western blot analysis of murine neurons and astrocytes in primary culture demonstrated epsilon- and betaIPKC expression in both cell types. Activation of epsilonPKC increased in neuronal cultures in response to the ischemia-like insult of oxygen-glucose deprivation (OGD). Isozyme-specific peptide activators or inhibitors of PKC were applied at various times before, during and after the OGD period. Neuron-astrocyte mixed cultures pretreated with a selective epsilonPKC activator peptide showed a significant reduction in neuronal injury after OGD and reperfusion, compared to cultures pretreated with control peptide. The epsilonPKC activator peptide counteracted the increased damage induced by pretreatment with the epsilonPKC-selective inhibitor peptide in relatively pure neuronal cultures subjected to OGD. Neither epsilonPKC activator nor inhibitor peptides affected injury of neurons when applied after OGD onset. In contrast, the betaIPKC-selective inhibitor peptide increased injury in astrocyte cultures exposed to OGD at all application times tested. Our data demonstrate protection of neurons by selective activation of epsilonPKC but enhanced astrocyte cell death with selective inhibition of betaIPKC. Thus PKC isozymes exhibit cell type-specific effects on ischemia-like injury.

    View details for DOI 10.1016/j.neuropharm.2004.03.009

    View details for Web of Science ID 000222041600013

    View details for PubMedID 15165841

  • Exaggerated nociceptive responses on morphine withdrawal: roles of protein kinase C epsilon, and gamma PAIN Sweitzer, S. M., Wong, S. M., Tjolsen, A., Allen, C. P., Mochly-Rosen, D., Kendig, J. J. 2004; 110 (1-2): 281-289


    On withdrawal from opioids many patients experience a heightened sensitivity to stimuli and an exaggerated pain response. The phenomenon has been little studied in infants. We present evidence that in postnatal day 7 rats an exaggerated nociceptive ventral root response of spinal cords in vitro and withdrawal-associated thermal hyperalgesia in vivo are dependent on protein kinase C (PKC), and we document the roles of PKC and gamma isozymes. In vitro, the slow ventral root potential (sVRP) is a nociceptive-related response in spinal cord that is depressed by morphine and recovers to levels significantly above control on administration of naloxone. A broad-spectrum PKC antagonist, GF109213X, blocked withdrawal hyperresponsiveness of the sVRP whereas an antagonist specific to Ca(++)-dependent isozymes, Go69076, did not. Consistent with this finding, a specific peptide inhibitor of calcium-independent PKC, but not an inhibitor of calcium-dependent PKC gamma, blocked withdrawal hyperresponsiveness of the sVRP. Similarly, in vivo in 7-day-old rat pups, inhibition of PKC, but not PKC gamma, prevented thermal hyperalgesia precipitated by naloxone at 30 min post-morphine. In contrast, thermal hyperalgesia during spontaneous withdrawal was inhibited by both PKC and gamma inhibitors. The consistency between the in vivo and in vitro findings with respect to naloxone-precipitated withdrawal provides further evidence that the sVRP reflects nociceptive neurotransmission. In addition the difference between naloxone-precipitated and spontaneous withdrawal in vivo suggests that in postnatal day 7 rats, morphine exposure produces an early phase of primary afferent sensitization dependent upon PKC translocation, followed by a later phase involving spinal sensitization mediated by PKC gamma.

    View details for DOI 10.1016/j.pain.2004.04.004

    View details for Web of Science ID 000223232800034

    View details for PubMedID 15275778

  • Biodistribution of intracellularly acting peptides conjugated reversibly to Tat BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS Begley, R., Liron, T., Baryza, J., Mochly-Rosen, D. 2004; 318 (4): 949-954


    Intracellularly acting peptide modulators of signaling enzymes provide a powerful means to regulate signaling events. Delivery of peptides into cells is facilitated by conjugation to carrier peptides, such as Tat. When peptides are irreversibly conjugated to Tat, Tat-mediated subcellular localization may predominate, resulting in mislocalization of the peptide cargo. We have used intracellularly acting peptides, conjugated to Tat by a disulfide bond, to modulate protein kinase C (PKC) signaling; these PKC-modulating peptides are released from Tat upon intracellular delivery. Previously, the distribution of these peptides within tissue and throughout the body had not been demonstrated. We show here intravascular delivery of a PKC-peptide, reversibly conjugated to Tat, resulted in distribution throughout cardiac tissue. In addition, a single injection resulted in selective modulation of PKC activity in many organs. Therefore, intracellularly acting peptide modulators of signaling enzymes, reversibly conjugated to Tat, have extensive biodistribution and can be used to modulate signaling pathways in vivo.

    View details for DOI 10.1016/j.bbrc.2004.04.121

    View details for Web of Science ID 000221776400023

    View details for PubMedID 15147964

  • Epsilon protein kinase C mediated ischemic tolerance requires activation of the extracellular regulated kinase pathway in the organotypic hippocampal slice JOURNAL OF CEREBRAL BLOOD FLOW AND METABOLISM Lange-Asschenfeldt, C., Raval, A. P., Dave, K. R., Mochly-Rosen, D., Sick, T. J., Perez-Pinzon, M. A. 2004; 24 (6): 636-645


    Ischemic preconditioning (IPC) promotes brain tolerance against subsequent ischemic insults. Using the organotypic hippocampal slice culture, we conducted the present study to investigate (1) the role of adenosine A1 receptor (A1AR) activation in IPC induction, (2) whether epsilon protein kinase C (epsilonPKC) activation after IPC is mediated by the phosphoinositol pathway, and (3) whether epsilonPKC protection is mediated by the extracellular signal-regulated kinase (ERK) pathway. Our results demonstrate that activation of A1AR emulated IPC, whereas blockade of the A1AR during IPC diminished neuroprotection. The neuroprotection promoted by the A1AR was also reduced by the epsilonPKC antagonist. To determine whether epsilonPKC activation in IPC and A1AR preconditioning is mediated by activation of the phosphoinositol pathway, we incubated slices undergoing IPC or adenosine treatment with a phosphoinositol phospholipase C inhibitor. In both cases, preconditioning neuroprotection was significantly attenuated. To further characterize the subsequent signal transduction pathway that ensues after epsilonPKC activation, mitogen-activated protein kinase kinase was blocked during IPC and pharmacologic preconditioning (PPC) (with epsilonPKC, NMDA, or A1AR agonists). This treatment significantly attenuated IPC- and PPC-induced neuroprotection. In conclusion, we demonstrate that epsilonPKC activation after IPC/PPC is essential for neuroprotection against oxygen/glucose deprivation in organotypic slice cultures and that the ERK pathway is downstream to epsilonPKC.

    View details for Web of Science ID 000221824000006

    View details for PubMedID 15181371

  • Mechanism of interaction between Annexin V and delta PKC Annual Meeting of the American-Society-for-Biochemistry-and-Molecular-Biology/8th Congress of the International-Union-for-Biochemistry-and-Molecular-Biology Kheifets, V., Bright, R., Wong, M., Kihara, Y., Mochly-Rosen, D. FEDERATION AMER SOC EXP BIOL. 2004: C213–C213
  • Protein kinase C epsilon and gamma: Involvement in formalin-induced nociception in neonatal rats JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS Sweitzer, S. M., Wong, S. M., Peters, M. C., Mochly-Rosen, D., Yeomans, D. C., Kendig, J. J. 2004; 309 (2): 616-625


    The central nervous system undergoes dynamic changes as it matures. However, until recently, very little was known about the impact of these changes on pain and analgesia. This study tested the hypothesis that the epsilon and gamma isozymes of protein kinase C (PKC) contribute to formalin-induced nociception in an age-dependent manner. Expression of epsilon and gamma PKC and the contributions of these isozymes in formalin-induced nociception was examined in postnatal day 7, 15, and 21 rats. epsilonPKC expression in dorsal root ganglion neurons and gammaPKC expression in lamina II of the spinal cord increased from the first to the third postnatal week. Coupling immunohistochemical and Western analysis, translocation of epsilonPKC followed intraplantar formalin in all ages. In contrast, formalin-induced gammaPKC translocation was observed only in postnatal day 21 rats. Behaviorally, intrathecal administration of the epsilonPKC-specific inhibitor (epsilonV1-2) attenuated phase 1 and phase 2 formalin behaviors at all ages. In contrast, intrathecal administration of the gammaPKC-specific inhibitor (gammaV5-3) attenuated only phase 2 responses in postnatal day 15 and 21 rats. Functionally, inhibition of epsilonPKC decreased capsaicin-stimulated release of glutamate and calcitonin gene-related peptide in spinal cords isolated from postnatal day 7 rats. These results suggest that epsilonPKC age independently mediates inflammatory pain produced by intraplantar formalin. In contrast, gammaPKC contributes to formalin-induced nociception in an age-dependent manner. Identifying the molecular mechanisms responsible for age-specific patterns of nociception is necessary for the rational development of novel therapeutic strategies for treating pediatric pain.

    View details for DOI 10.1124/jpet.103.060350

    View details for Web of Science ID 000220972900024

    View details for PubMedID 14762097

  • Protection against reperfusion injury-induced microvascular damage by inhibition of is an element of ' protein kinase C 5th Annual Conference on Arteriosclerosis, Thrombosis, and Vascular Biology Ikeno, F., Inagaki, K., Kaneda, H., Price, E. T., Chen, L., Yeung, A. C., Mochly-Rosen, D., Rezaee, M. LIPPINCOTT WILLIAMS & WILKINS. 2004: E38–E38
  • State-specific monoclonal antibodies identify an intermediate state in epsilon protein kinase C activation JOURNAL OF BIOLOGICAL CHEMISTRY Souroujon, M. C., Yao, L. N., Chen, H. B., Endemann, G., Khaner, H., Geeraert, V., Schechtman, D., Gordon, A. S., Diamond, I., Mochly-Rosen, D. 2004; 279 (17): 17617-17624


    Evaluation of the activation state of protein kinase C (PKC) isozymes relies on analysis of subcellular translocation. A monoclonal antibody, 14E6, specific for the activated conformation of epsilonPKC, was raised using the first variable (V1) domain of epsilonPKC as the immunogen. 14E6 binding is specific for epsilonPKC and is greatly increased in the presence of PKC activators. Immunofluorescence staining by 14E6 of neonatal rat primary cardiac myocytes and the NG108-15 neuroblastoma glioma cell line, NG108-15/D2, increases rapidly following cell activation and is localized to new subcellular sites. However, staining of translocated epsilonPKC with 14E6 is transient, and the epitope disappears 30 min after activation of NG-108/15 cells by a D2 receptor agonist. In contrast, subcellular localization associated with activation, as determined by commercially available polyclonal antibodies, persists for at least 30 min. In vitro, epsilonRACK, the receptor for activated epsilonPKC, inhibits 14E6 binding to epsilonPKC, suggesting that the 14E6 epitope is lost or hidden when active epsilonPKC binds to its RACK. Therefore, the 14E6 antibody appears to identify a transient state of activated but non-anchored epsilonPKC. Moreover, binding of 14E6 to epsilonPKC only after activation suggests that lipid-dependent conformational changes associated with epsilonPKC activation precede binding of the activated isozyme to its specific RACK, epsilonRACK. Further, monoclonal antibody 14E6 should be a powerful tool to study the pathways that control rapid translocation of epsilonPKC from cytosolic to membrane localization on activation.

    View details for DOI 10.1074/jbc.M400962200

    View details for Web of Science ID 000220870400090

    View details for PubMedID 14761958

  • A critical intramolecular interaction for protein kinase C epsilon translocation JOURNAL OF BIOLOGICAL CHEMISTRY Schechtman, D., Craske, M. L., Kheifets, V., Meyer, T., Schechtman, J., Mochly-Rosen, D. 2004; 279 (16): 15831-15840


    Disruption of intramolecular interactions, translocation from one intracellular compartment to another, and binding to isozyme-specific anchoring proteins termed RACKs, accompany protein kinase C (PKC) activation. We hypothesized that in inactive epsilonPKC, the RACK-binding site is engaged in an intramolecular interaction with a sequence resembling its RACK, termed psiepsilonRACK. An amino acid difference between the psiepsilonRACK sequence in epsilonPKC and its homologous sequence in epsilonRACK constitutes a change from a polar non-charged amino acid (asparagine) in epsilonRACK to a polar charged amino acid (aspartate) in epsilonPKC. Here we show that mutating the aspartate to asparagine in epsilonPKC increased intramolecular interaction as indicated by increased resistance to proteolysis, and slower hormone- or PMA-induced translocation in cells. Substituting aspartate for a non-polar amino acid (alanine) resulted in binding to epsilonRACK without activators, in vitro, and increased translocation rate upon activation in cells. Mathematical modeling suggests that translocation is at least a two-step process. Together our data suggest that intramolecular interaction between the psiepsilonRACK site and RACK-binding site within epsilonPKC is critical and rate limiting in the process of PKC translocation.

    View details for DOI 10.1074/jbc.M310696200

    View details for Web of Science ID 000220747900019

    View details for PubMedID 14739299

  • Preservation of base-line hemodynamic function and loss of inducible cardioprotection in adult mice lacking protein kinase C-epsilon JOURNAL OF BIOLOGICAL CHEMISTRY Gray, M. O., Zhou, H. Z., Schafhalter-Zoppoth, I., Zhu, P. L., Mochly-Rosen, D., Messing, R. O. 2004; 279 (5): 3596-3604


    Signaling pathways involving protein kinase C isozymes are modulators of cardiovascular development and response to injury. Protein kinase C epsilon activation in cardiac myocytes reduces necrosis caused by coronary artery disease. However, it is unclear whether protein kinase C epsilon function is required for normal cardiac development or inducible protection against oxidative stress. Protein kinase C delta activation is also observed during cardiac preconditioning. However, its role as a promoter or inhibitor of injury is controversial. We examined hearts from protein kinase C epsilon knock-out mice under physiological conditions and during acute ischemia reperfusion. Null-mutant and wild-type mice displayed equivalent base-line morphology and hemodynamic function. Targeted disruption of the protein kinase C epsilon gene blocked cardioprotection caused by ischemic preconditioning and alpha(1)-adrenergic receptor stimulation. Protein kinase C delta activation increased in protein kinase C epsilon knock-out myocytes without altering resistance to injury. These observations support protein kinase C epsilon activation as an essential component of cardioprotective signaling. Our results favor protein kinase C delta activation as a mediator of normal growth. This study advances the understanding of cellular mechanisms responsible for preservation of myocardial integrity as potential targets for prevention and treatment of ischemic heart disease.

    View details for DOI 10.1074/jbc.M311459200

    View details for Web of Science ID 000188379600057

    View details for PubMedID 14600145

  • Opposing roles of delta and epsilon PKC in cardiac ischemia and reperfusion: targeting the apoptotic machinery ARCHIVES OF BIOCHEMISTRY AND BIOPHYSICS Murriel, C. L., Mochly-Rosen, D. 2003; 420 (2): 246-254


    Heart attacks, or acute myocardial infarctions (AMI), affect more than one million people in the US every year. The damage that occurs to the heart by AMI is often permanent and as a result, the morbidity and mortality rates of patients that experience AMIs continue to be high. Consequently, AMI patients are at significantly increased risks for future myocardial infarctions, decreased heart function, heart failure, and death [Heart and Stroke statistical update. In American Heart Association (2002) 4]. In this review, we discuss the events that lead to cardiac damage by AMI. Specifically, we discuss the current understanding of the role of ischemic damage vs. reperfusion damage, which is induced by the return of blood, oxygen, and nutrients to the organ. We also discuss the role of apoptosis and necrosis in cardiac damage, the means to protect the heart from damage by ischemia and reperfusion, and the role of protein kinase C in these processes.

    View details for DOI 10.1016/

    View details for Web of Science ID 000187459000006

    View details for PubMedID 14654063

  • Inhibition of delta-protein kinase C protects against reperfusion injury of the ischemic heart in vivo CIRCULATION Inagaki, K., Chen, L., Ikeno, F., Lee, F. H., Imahashi, K., Bouley, D. M., Rezaee, M., Yock, P. G., Murphy, E., Mochly-Rosen, D. 2003; 108 (19): 2304-2307


    Current treatment for acute myocardial infarction (AMI) focuses on reestablishing blood flow (reperfusion). Paradoxically, reperfusion itself may cause additional injury to the heart. We previously found that delta-protein kinase C (deltaPKC) inhibition during simulated ischemia/reperfusion in isolated rat hearts is cardioprotective. We focus here on the role for deltaPKC during reperfusion only, using an in vivo porcine model of AMI.An intracoronary application of a selective deltaPKC inhibitor to the heart at the time of reperfusion reduced infarct size, improved cardiac function, inhibited troponin T release, and reduced apoptosis. Using 31P NMR in isolated perfused mouse hearts, we found a faster recovery of ATP levels in hearts treated with the deltaPKC inhibitor during reperfusion only.Reperfusion injury after cardiac ischemia is mediated, at least in part, by deltaPKC activation. This study suggests that including a deltaPKC inhibitor at reperfusion may improve the outcome for patients with AMI.

    View details for DOI 10.1161/01.CIR.0000101682.24138.36

    View details for Web of Science ID 000186475200003

    View details for PubMedID 14597593

  • Protein kinase C-epsilon dependent survival signals in the diabetic heart 76th Annual Scientific Session of the American-Heart-Association Malhotra, A., Begley, R., Kang, B. P., Liu, J., Thaisz, J., Yang, G. P., Frisoli, T., Mochly-Rosen, D., Meggs, L. G. LIPPINCOTT WILLIAMS & WILKINS. 2003: 205–
  • Additive protection of the ischemic heart ex vivo by combined treatment with delta-protein kinase C inhibitor and epsilon-protein kinase C activator CIRCULATION Inagaki, K., Hahn, H. S., Dorn, G. W., Mochly-Rosen, D. 2003; 108 (7): 869-875


    Protein kinase C (PKC) plays a major role in cardioprotection from ischemia/reperfusion injury. Using an HIV-1 Tat protein-derived peptide to mediate rapid and efficient transmembrane delivery of peptide regulators of PKC translocation and function, we examined the cardioprotective effect of selective delta-PKC inhibitor (deltaV1-1) and epsilon-PKC activator (psi(epsilon)RACK) peptides for ischemia/reperfusion damage in isolated perfused rat hearts. Furthermore, we examined the protective effects of these PKC isozymes in isolated perfused hearts subjected to ischemia/reperfusion damage using transgenic mice expressing these peptides specifically in their cardiomyocytes.In isolated perfused rat hearts, administration of deltaV1-1 but not psi(epsilon)RACK during reperfusion improved cardiac function and decreased creatine phosphokinase release. In contrast, pretreatment with psi(epsilon)RACK but not deltaV1-1, followed by a 10-minute washout before ischemia/reperfusion, also improved cardiac function and decreased creatine phosphokinase release. Furthermore, administration of psi(epsilon)RACK before ischemia followed by deltaV1-1 during reperfusion only conferred greater cardioprotective effects than that obtained by each peptide treatment alone. Both the delta-PKC inhibitor and epsilon-PKC activator conferred cardioprotection against ischemia/reperfusion injury in transgenic mice expressing these peptides in the heart, and coexpression of both peptides conferred greater cardioprotective effects than that obtained by the expression of each peptide inhibitor prevents reperfusion injury, and epsilon-PKC activator mimics ischemic preconditioning. Furthermore, treatment with both peptides confers additive cardioprotective effects. Therefore, these peptides mediate cardioprotection by regulating ischemia/reperfusion damage at distinct time points.

    View details for DOI 10.1161/01.CIR.0000081943.93653.73

    View details for Web of Science ID 000184818000017

    View details for PubMedID 12860903

  • Opposing effects of delta- and zeta-protein kinase C isozymes on cardiac fibroblast proliferation: use of isozyme-selective inhibitors JOURNAL OF MOLECULAR AND CELLULAR CARDIOLOGY Braun, M. U., Mochly-Rosen, D. 2003; 35 (8): 895-903


    Neonatal primary cardiac fibroblasts in defined medium continue to proliferate. Here, we show that phorbol ester inhibited and transforming growth factor-beta1 (TGFbeta1) stimulated this fibroblast proliferation. Cardiac fibroblasts contain six protein kinase C (PKC) isozymes: alpha-, delta-, epsilon-, betaI-, betaII-, and zeta-PKC. To evaluate the effect of different PKC isozymes on the proliferation of these cells, we used isozyme-selective PKC inhibitors. Inhibition of endogenous delta-PKC with deltaV1-1, an isozyme-selective translocation inhibitor, resulted in increased basal thymidine incorporation by 58 +/- 12% of control cells, but did not affect TGFbeta1-induced cell growth. Inhibition of endogenous zeta-PKC in neonatal rat cardiac fibroblasts with zeta-pseudosubstrate, a selective inhibitor for the atypical PKC isozymes, revealed an opposite effect; this inhibitor reduced basal growth to 45 +/- 11% and TGFbeta1-induced growth to 61 +/- 10%. Other isozyme-specific inhibitors used in this study did not alter basal or TGFbeta1-stimulated fibroblast growth. Taken together, our data provide evidence that delta-PKC inhibits and zeta-PKC stimulates proliferation of neonatal rat cardiac fibroblasts.

    View details for DOI 10.1016/S0022-2828(03)00142-1

    View details for Web of Science ID 000184527400004

    View details for PubMedID 12878476

  • PKC isozyme selective regulation of cloned human cardiac delayed slow rectifier K current BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS Xiao, G. Q., Mochly-Rosen, D., Boutjdir, M. 2003; 306 (4): 1019-1025


    Delayed rectifying K(+) channel, I(Ks), plays a vital role in normal and arrhythmogenic heart. I(Ks) is modulated by PKC but the identity of which PKC isozymes is involved in this modulation is not known. To dissect the role of individual PKC isozymes in the regulation of I(Ks), human cardiac I(Ks) channel (minK+KvLQT1) was expressed in Xenopus oocytes. Peptide PKC isozyme-specific activator and inhibitors, in addition to the general PKC activator, PMA, were used. Whole-cell I(Ks) was recorded using two-electrode voltage clamp technique. PMA and epsilon PKC specific activator peptide, but not the inactive analog, 4alphaPDD, significantly increased I(Ks). Peptide specific inhibitors for beta(II)PKC, and a general PKC inhibitor, calphostin C antagonized PMA-induced activation of I(Ks). However, control peptide, pentalysine, and specific inhibitor peptide for alphaPKC, beta(I)PKC, deltaPKC, or etaPKC did not alter PMA effect on I(Ks). The present study demonstrates that beta(II)PKC, epsilon PKC but not beta(I)PKC, alphaPKC, deltaPKC, and etaPKC, are involved in PMA-induced activation of the cloned human I(Ks) expressed in Xenopus oocyte. Furthermore, this is the first report to dissect the fine functional role of beta(II)PKC and beta(I)PKC in the regulation of I(Ks). Identification of the particular isozyme(s) that mediates the regulation of I(Ks) channels is of importance for the understanding of the mechanism of ion channel regulation and the development of new therapeutic agents.

    View details for DOI 10.1016/S0006-291X(03)01095-7

    View details for Web of Science ID 000183992400034

    View details for PubMedID 12821145

  • Prevention of NMDA-induced death of cortical neurons by inhibition of protein kinase C zeta JOURNAL OF NEUROCHEMISTRY Koponen, S., Kurkinen, K., Akerman, K. E., Mochly-Rosen, D., Chan, P. H., Koistinaho, J. 2003; 86 (2): 442-450


    Excitotoxicity through stimulation of N-methyl-d-aspartate (NMDA) receptors contributes to neuronal death in brain injuries, including stroke. Several lines of evidence suggest a role for protein kinase C (PKC) isoforms in NMDA excitotoxicity. We have used specific peptide inhibitors of classical PKCs (alpha, beta, and gamma), novel PKCs delta and epsilon, and an atypical PKCzeta in order to delineate which subspecies are involved in NMDA-induced cell death. Neuronal cell cultures were prepared from 15-day-old mouse embryos and plated onto the astrocytic monolayer. After 2 weeks in vitro the neurons were exposed to 100 micro m NMDA for 5 min, and 24 h later the cell viability was examined by measuring the lactate dehydrogenase release and bis-benzimide staining. While inhibitors directed to classical (alpha, beta, and gamma) or novel PKCs (delta or epsilon) had no effect, the PKCzeta inhibitor completely prevented the NMDA-induced necrotic neuronal death. Confocal microscopy confirmed that NMDA induced PKCzeta translocation, which was blocked by the PKCzeta inhibitor. The NMDA-induced changes in intracellular free Ca2+ were not affected by the peptides. In situ hybridization experiments demonstrated that PKCzeta mRNA is induced in the cortex after focal brain ischemia. Altogether, the results indicate that PKCzeta activation is a downstream signal in NMDA-induced death of cortical neurons.

    View details for DOI 10.1046/j.1471-4159.2003.01846.x

    View details for Web of Science ID 000183900300018

    View details for PubMedID 12871585

  • Inhibition of delta protein kinase C protects vascular injury and microcirculation from reperfusion injury in myocardial infarction 52nd Annual Scientific Session of the American-College-of-Cardiology Ikeno, F., Inagaki, K., Chen, L., Price, E. T., Fenn, R. C., Yeung, A. C., Mochly-Rosen, D., Rezaee, M. ELSEVIER SCIENCE INC. 2003: 373A–373A
  • Cytotoxicity of pEGFP vector is due to residues encoded by multiple cloning site ANALYTICAL BIOCHEMISTRY Endemann, G., Schechtman, D., Mochly-Rosen, D. 2003; 313 (2): 345-347
  • epsilon PKC is required for the induction of tolerance by ischemic and NMDA-mediated preconditioning in the organotypic hippocampal slice JOURNAL OF NEUROSCIENCE Raval, A. P., Dave, K. R., Mochly-Rosen, D., Sick, T. J., Perez-Pinzon, M. A. 2003; 23 (2): 384-391


    Glutamate receptors and calcium have been implicated as triggering factors in the induction of tolerance by ischemic preconditioning (IPC) in the brain. However, little is known about the signal transduction pathway that ensues after the IPC induction pathway. The main goals of the present study were to determine whether NMDA induces preconditioning via a calcium pathway and promotes translocation of the protein kinase C epsilon (epsilonPKC) isozyme and whether this PKC isozyme is key in the IPC signal transduction pathway. We corroborate here that IPC and a sublethal dose of NMDA were neuroprotective, whereas blockade of NMDA receptors during IPC diminished IPC-induced neuroprotection. Calcium chelation blocked the protection afforded by both NMDA and ischemic preconditioning significantly, suggesting a significant role of calcium. Pharmacological preconditioning with the nonselective PKC isozyme activator phorbol myristate acetate could not emulate IPC, but blockade of PKC activation with chelerythrine during IPC blocked its neuroprotection. These results suggested that there might be a dual involvement of PKC isozymes during IPC. This was corroborated when neuroprotection was blocked when we inhibited epsilonPKC during IPC and NMDA preconditioning, and IPC neuroprotection was emulated with the activator of epsilonPKC. The possible correlation between NMDA, Ca2+, and epsilonPKC was found when we emulated IPC with the diacylglycerol analog oleoylacetyl glycerol, suggesting an indirect pathway by which Ca2+ could activate the calcium-insensitive epsilonPKC isozyme. These results demonstrated that the epsilonPKC isozyme played a key role in both IPC- and NMDA-induced tolerance.

    View details for Web of Science ID 000180523000004

    View details for PubMedID 12533598

  • Glutathione S-transferase pull-down assay. Methods in molecular biology (Clifton, N.J.) Schechtman, D., Mochly-Rosen, D., Ron, D. 2003; 233: 345-350

    View details for PubMedID 12840520

  • Methods for detecting binding proteins: an introduction. Methods in molecular biology (Clifton, N.J.) Endemann, G., Mochly-Rosen, D. 2003; 233: 307-325

    View details for PubMedID 12840518

  • Overlay method for detecting protein-protein interactions. Methods in molecular biology (Clifton, N.J.) Schechtman, D., Murriel, C., Bright, R., Mochly-Rosen, D. 2003; 233: 351-357

    View details for PubMedID 12840521

  • The beta gamma subunit of Heterotrimeric G proteins interacts with RACK1 and two other WD repeat proteins JOURNAL OF BIOLOGICAL CHEMISTRY Dell, E. J., Connor, J., Chen, S. H., Stebbins, E. G., Skiba, N. P., Mochly-Rosen, D., Hamm, H. E. 2002; 277 (51): 49888-49895


    A yeast two-hybrid approach was used to discern possible new effectors for the betagamma subunit of heterotrimeric G proteins. Three of the clones isolated are structurally similar to Gbeta, each exhibiting the WD40 repeat motif. Two of these proteins, the receptor for activated C kinase 1 (RACK1) and the dynein intermediate chain, co-immunoprecipitate with Gbetagamma using an anti-Gbeta antibody. The third protein, AAH20044, has no known function; however, sequence analysis indicates that it is a WD40 repeat protein. Further investigation with RACK1 shows that it not only interacts with Gbeta(1)gamma(1) but also unexpectedly with the transducin heterotrimer Galpha(t)beta(1)gamma(1). Galpha(t) alone does not interact, but it must contribute to the interaction because the apparent EC(50) value of RACK1 for Galpha(t)beta(1)gamma(1) is 3-fold greater than that for Gbeta(1)gamma(1) (0.1 versus 0.3 microm). RACK1 is a scaffold that interacts with several proteins, among which are activated betaIIPKC and dynamin-1 (1). betaIIPKC and dynamin-1 compete with Gbeta(1)gamma(1) and Galpha(t)beta(1)gamma(1) for interaction with RACK1. These findings have several implications: 1) that WD40 repeat proteins may interact with each other; 2) that Gbetagamma interacts differently with RACK1 than with its other known effectors; and/or 3) that the G protein-RACK1 complex may constitute a signaling scaffold important for intracellular responses.

    View details for DOI 10.1074/jbc.M202755200

    View details for Web of Science ID 000180028900103

    View details for PubMedID 12359736

  • Tissue angiotensin II during progression or ventricular hypertrophy to heart failure in hypertensive rats; Differential effects on PKC epsilon and PKC beta JOURNAL OF MOLECULAR AND CELLULAR CARDIOLOGY Inagaki, K., Iwanaga, Y., Sarai, N., Onozawa, Y., Takenaka, H., Mochly-Rosen, D., Kihara, Y. 2002; 34 (10): 1377-1385


    The protein kinase C (PKC) family has been implicated as second messengers in mechanosensitive modulation of cardiac hypertrophy. However, little information is available on the role of expression and activation of specific cardiac PKC isozymes during development of left ventricular hypertrophy (LVH) and failure (LVF). Dahl salt-sensitive rats fed an 8% salt diet developed systemic hypertension and concentric LVH at 11 weeks of age that is followed by left ventricle (LV) dilatation and global hypokinesis at 17 weeks. Among several PKC isozymes expressed in the LV myocardium, only PKC epsilon showed a 94% increase at the LVH stage. At the LVF stage, however, PKC epsilon returned to the control level, whereas PKC beta I and beta II increased by 158% and 155%, respectively. Hearts were studied at each stage using the Langendorff set-up, and a LV balloon was inflated to achieve an equivalent diastolic wall stress. Following mechanical stretch, PKC epsilon was significantly activated in LVH myocardium in which tissue angiotensin II levels were increased by 59%. Pre-treatment with valsartan, an AT(1)-receptor blocker, abolished the stretch-mediated PKC epsilon activation. Mechanical stretch no longer induced PKC epsilon activation in LVF. Chronic administration of valsartan blunted the progression of LVF and inhibited the increase in PKC beta. Mechanosensitive PKC epsilon activation is augmented and therefore may contribute to the development of compensatory hypertrophy. This effect was dependent on activation of tissue angiotensin II. However, this compensatory mechanism becomes inactive in LVF, where PKC beta may participate in the progression to cardiac dysfunction and LV remodeling.

    View details for DOI 10.1006/jmcc.2002.2089

    View details for Web of Science ID 000179126200019

    View details for PubMedID 12392998

  • Cardioprotection mediated by sphingosine-1-phosphate and ganglioside GM-1 in wild-type and PKC epsilon knockout mouse hearts AMERICAN JOURNAL OF PHYSIOLOGY-HEART AND CIRCULATORY PHYSIOLOGY Jin, Z. Q., Zhou, H. Z., Zhu, P. L., Honbo, N., Mochly-Rosen, D., Messing, R. O., Goetzl, E. J., Karliner, J. S., Gray, M. O. 2002; 282 (6): H1970-H1977


    Sphingosine-1-phosphate (S1P) protects neonatal rat cardiac myocytes from hypoxic damage through unknown signaling pathways. We tested the hypothesis that S1P-induced cardioprotection requires activation by the epsilon-isoform of protein kinase C (PKC epsilon) by subjecting hearts isolated from PKC epsilon knockout mice and wild-type mice to 20 min of global ischemia and 30 min of reperfusion. Pretreatment with a 2-min infusion of 10 nM S1P improved recovery of left ventricular developed pressure (LVDP) in both wild-type and PKC epsilon knockout hearts and reduced the rise in LV end-diastolic pressure (LVEDP) and creatine kinase (CK) release. Pretreatment for 2 min with 10 nM of the ganglioside GM-1 also improved recovery of LVDP and suppressed CK release in wild-type hearts but not in PKC epsilon knockout hearts. Importantly, GM-1 but not S1P, increased the proportion of PKC epsilon localized to particulate fractions. Our results suggest that GM-1, which enhances endogenous S1P production, reduces cardiac injury through PKC epsilon-dependent intracellular pathways. In contrast, extracellular S1P induces equivalent cardioprotection through PKC epsilon-independent signaling pathways.

    View details for DOI 10.1152/ajpheart.01029.2001

    View details for Web of Science ID 000175531800004

    View details for PubMedID 12003800

  • Sequential activation of individual PKC isozymes in integrin-mediated muscle cell spreading: a role for MARCKS in an integrin signaling pathway JOURNAL OF CELL SCIENCE Disatnik, M. H., Boutet, S. C., Lee, C. H., Mochly-Rosen, D., Rando, T. A. 2002; 115 (10): 2151-2163


    To understand how muscle cell spreading and survival are mediated by integrins, we studied the signaling events initiated by the attachment of muscle cells to fibronectin (FN). We have previously demonstrated that muscle cell spreading on FN is mediated by alpha5beta1 integrin, is associated with rapid phosphorylation of focal adhesion kinase and is dependent on activation of protein kinase C (PKC). Here we investigated the role of individual PKC isozymes in these cellular processes. We show that alpha, delta and epsilonPKC are expressed in muscle cells and are activated upon integrin engagement with different kinetics - epsilonPKC was activated early, whereas alpha and deltaPKC were activated later. Using isozyme-specific inhibitors, we found that the activation of epsilonPKC was necessary for cell attachment to FN. However, using isozyme-specific activators, we found that activation of each of three isozymes was sufficient to promote the spreading of alpha5-integrin-deficient cells on FN. To investigate further the mechanism by which integrin signaling and PKC activation mediate cell spreading, we studied the effects of these processes on MARCKS, a substrate of PKC and a protein known to regulate actin dynamics. We found that MARCKS was localized to focal adhesion sites soon after cell adhesion and that MARCKS translocated from the membrane to the cytosol during the process of cell spreading. This translocation correlated with different phases of PKC activation and with reorganization of the actin cytoskeleton. Using MARCKS-antisense cDNA, we show that alpha5-expressing cells in which MARCKS expression is inhibited fail to spread on FN, providing evidence for the crucial role of MARCKS in muscle cell spreading. Together, the data suggest a model in which early activation of epsilonPKC is necessary for cell attachment; the later activation of alpha or deltaPKC may be necessary for the progression from attachment to spreading. The mechanism of PKC-mediated cell spreading may be via the phosphorylation of signaling proteins, such as MARCKS, that are involved in the reorganization of the actin cytoskeleton.

    View details for Web of Science ID 000176109300015

    View details for PubMedID 11973356

  • Isozyme-specific abnormalities of PKC in thyroid cancer: Evidence for post-transcriptional changes in PKC epsilon JOURNAL OF CLINICAL ENDOCRINOLOGY & METABOLISM Knauf, J. A., Ward, L. S., Nikiforov, Y. E., Nikiforova, M., Puxeddu, E., Medvedovic, M., Liron, T., Mochly-Rosen, D., Fagin, J. A. 2002; 87 (5): 2150-2159


    PKC isozymes are the major binding proteins for tumor-promoting phorbol esters, and PKC activity is abnormal in a number of different human cancers. Less is known about putative structural and functional changes of specific PKC isozymes in human neoplasms. A single-point mutation of PKCalpha at position 881 of the coding sequence has been observed in human pituitary adenomas and up to 50% of thyroid follicular neoplasms, and a rearrangement of PKCepsilon was reported in a thyroid follicular carcinoma cell line, suggesting that these signaling proteins may play a role in thyroid tumorigenesis. To explore this possibility, we examined thyroid neoplasms for mutations and changes in expression levels of PKCepsilon or alpha. None of the 57 follicular adenomas, 26 papillary carcinomas (PCs), 7 follicular carcinomas, or the anaplastic carcinoma harbored the PKCalpha 881A>G mutation. Moreover, none of 15 PCs, 10 follicular adenomas, or 6 follicular carcinomas showed evidence of mutations of PKCepsilon. However, 8 of 11 PCs had major isozyme-specific reductions of the PKCepsilon protein, which occurred through either translational or posttranslational mechanisms. These data indicate that post-transcriptional changes in PKCepsilon are highly prevalent in thyroid tumors and may play a significant role in their development.

    View details for Web of Science ID 000175648100039

    View details for PubMedID 11994357

  • Molecular dynamics characterization of the C2 domain of protein kinase C beta JOURNAL OF BIOLOGICAL CHEMISTRY Banci, L., Cavallaro, G., Kheifets, V., Mochly-Rosen, D. 2002; 277 (15): 12988-12997


    Protein kinase C (PKC) isozymes comprise a family of related enzymes that play a central role in many intracellular eukaryotic signaling events. Isozyme specificity is mediated by association of each PKC isozyme with specific anchoring proteins, termed RACKs. The C2 domain of betaPKC contains at least part of the RACK-binding sites. Because the C2 domain contains also a RACK-like sequence (termed pseudo-RACK), it was proposed that this pseudo-RACK site mediates intramolecular interaction with one of the RACK-binding sites in the C2 domain itself, stabilizing the inactive conformation of betaPKC. BetaPKC depends on calcium for its activation, and the C2 domain contains the calcium-binding sites. The x-ray structure of the C2 domain of betaPKC shows that three Ca(2+) ions can be coordinated by two opposing loops at one end of the domain. Starting from this x-ray structure, we have performed molecular dynamics (MD) calculations on the C2 domain of betaPKC bound to three Ca(2+) ions, to two Ca(2+) ions, and in the Ca(2+)-free state, in order to analyze the effect of calcium on the RACK-binding sites and the pseudo-RACK sites, as well as on the loops that constitute the binding site for the Ca(2+) ions. The results show that calcium stabilizes the beta-sandwich structure of the C2 domain and thus affects two of the three RACK-binding sites within the C2 domain. Also, the interactions between the third RACK-binding site and the pseudo-RACK site are not notably modified by the removal of Ca(2+) ions. On that basis, we predict that the pseudo-RACK site within the C2 domain masks a RACK-binding site in another domain of betaPKC, possibly the V5 domain. Finally, the MD modeling shows that two Ca(2+) ions are able to interact with two molecules of O-phospho-l-serine. These data suggest that Ca(2+) ions may be directly involved in PKC binding to phosphatidylserine, an acidic lipid located exclusively on the cytoplasmic face of membranes, that is required for PKC activation.

    View details for DOI 10.1074/jbc.M106875200

    View details for Web of Science ID 000175036300065

    View details for PubMedID 11782454

  • Dopamine-induced exocytosis of Na,K-ATPase is dependent on activation of protein kinase C-epsilon and -delta MOLECULAR BIOLOGY OF THE CELL Ridge, K. M., Dada, L., Lecuona, E., Bertorello, A. M., Katz, A. I., Mochly-Rosen, D., Sznajder, J. I. 2002; 13 (4): 1381-1389


    The purpose of this study was to define mechanisms by which dopamine (DA) regulates the Na,K-ATPase in alveolar epithelial type 2 (AT2) cells. The Na,K-ATPase activity increased by twofold in cells incubated with either 1 microM DA or a dopaminergic D(1) agonist, fenoldopam, but not with the dopaminergic D(2) agonist quinpirole. The increase in activity paralleled an increase in Na,K-ATPase alpha1 and beta1 protein abundance in the basolateral membrane (BLM) of AT2 cells. This increase in protein abundance was mediated by the exocytosis of Na,K-pumps from late endosomal compartments into the BLM. Down-regulation of diacylglycerol-sensitive types of protein kinase C (PKC) by pretreatment with phorbol 12-myristate 13-acetate or inhibition with bisindolylmaleimide prevented the DA-mediated increase in Na,K-ATPase activity and exocytosis of Na,K-pumps to the BLM. Preincubation of AT2 cells with either 2-[1-(3-dimethylaminopropyl)-5-methoxyindol-3-yl]-3-(1H-indol-3-yl)maleimide (Gö6983), a selective inhibitor of PKC-delta, or isozyme-specific inhibitor peptides for PKC-delta or PKC-epsilon inhibited the DA-mediated increase in Na,K-ATPase. PKC-delta and PKC-epsilon, but not PKC-alpha or -beta, translocated from the cytosol to the membrane fraction after exposure to DA. PKC-delta- and PKC-epsilon-specific peptide agonists increased Na,K-ATPase protein abundance in the BLM. Accordingly, dopamine increased Na,K-ATPase activity in alveolar epithelial cells through the exocytosis of Na,K-pumps from late endosomes into the basolateral membrane in a mechanism-dependent activation of the novel protein kinase C isozymes PKC-delta and PKC-epsilon.

    View details for DOI 10.1091/mbc.01-07-0323

    View details for Web of Science ID 000175318400023

    View details for PubMedID 11950946

  • A novel inhibitor of protein kinase-C as a therapeutic for ischemic reperfusion injury in acute coronary syndromes Lee, F., Inagaki, K., Chen, L., Bailey, L., Ikeno, F., Lyons, J., Bouley, D., Yock, P., Carter, A., Yeung, A., Mochly-Rosen, D. ELSEVIER SCIENCE INC. 2002: 445A–445A
  • Isozyme-specific inhibitors and activators of protein kinase C G PROTEIN PATHWAYS: PT C, EFFECTOR MECHANISMS Schechtman, D., Mochly-Rosen, D. 2002; 345: 470-489


    We describe here the methods we have used to generate selective peptide inhibitors and activators of PKC-mediated signaling. These approaches should be applicable to any signaling event that is dependent on protein-protein interaction. Furthermore, targeting downstream enzymes in signal transduction has been notoriously difficult as there are often families of related enzymes in each cell. The approaches we have used overcame this difficulty and may prove useful not only in basic research, but also in drug discovery.

    View details for Web of Science ID 000171866300037

    View details for PubMedID 11665630

  • Intracellular transport mechanisms of signal transducers ANNUAL REVIEW OF PHYSIOLOGY Dorn, G. W., Mochly-Rosen, D. 2002; 64: 407-429


    Recent discoveries have revolutionized our conceptions of enzyme-substrate specificity in signal transduction pathways. Protein kinases A and C are localized to discreet subcellular regions, and this localization changes in an isozyme-specific manner upon activation, a process referred to as translocation. The mechanisms for translocation involve interactions of soluble kinases with membrane-bound anchor proteins that recognize individual kinase isoenzymes and their state of activation. Recently, modulation of kinase-anchor protein interactions has been used to specifically regulate, positively or negatively, the activity of C kinase isozymes. Also described in this review is a role for the Rab family of small G proteins in regulating subcellular protein trafficking. The pathophysiological significance of disrupted subcellular protein transport in cell signaling and the potential therapeutic utility of targeted regulation of these events are in the process of being characterized.

    View details for Web of Science ID 000174713000015

    View details for PubMedID 11826274

  • Molecular transporters for peptides: delivery of a cardioprotective epsilon PKC agonist peptide into cells and intact ischemic heart using a transport system, R-7 CHEMISTRY & BIOLOGY Chen, L., Wright, L. R., Chen, C. H., Oliver, S. F., Wender, P. A., Mochly-Rosen, D. 2001; 8 (12): 1123-1129


    Recently, we reported a novel oligoguanidine transporter system, polyarginine (R(7)), which, when conjugated to spectroscopic probes (e.g., fluorescein) and drugs (e.g., cyclosporin A), results in highly water-soluble conjugates that rapidly enter cells and tissues. We report herein the preparation of the first R(7) peptide conjugates and a study of their cellular and organ uptake and functional activity. The octapeptide (psi)(epsilon)RACK was selected for this study as it is known to exhibit selective epsilon protein kinase C isozyme agonist activity and to reduce ischemia-induced damage in cardiomyocytes. However, (psi)(epsilon)RACK is not cell-permeable.Here we show that an R(7)-(psi)(epsilon)RACK conjugate readily enters cardiomyocytes, significantly outperforming (psi)(epsilon)RACK conjugates of the transporters derived from HIV Tat and from Antennapedia. Moreover, R(7)-(psi)(epsilon)RACK conjugate reduced ischemic damage when delivered into intact hearts either prior to or after the ischemic insult.Our data suggest that R(7) converts a peptide lead into a potential therapeutic agent for the ischemic heart.

    View details for Web of Science ID 000173750400001

    View details for PubMedID 11755391

  • Evidence for functional role of epsilon PKC isozyme in the regulation of cardiac Na+ channels AMERICAN JOURNAL OF PHYSIOLOGY-CELL PHYSIOLOGY Xiao, G. Q., Qu, Y. X., Sun, Z. Q., Mochly-Rosen, D., Boutjdir, M. 2001; 281 (5): C1477-C1486


    Investigation of the role of individual protein kinase C (PKC) isozymes in the regulation of Na(+) channels has been largely limited by the lack of isozyme-selective modulators. Here we used a novel peptide-specific activator (epsilonV1-7) of epsilonPKC and other peptide isozyme-specific inhibitors in addition to the general PKC activator phorbol 12-myristate 13-acetate (PMA) to dissect the role of individual PKCs in the regulation of the human cardiac Na(+) channel hH1, heterologously expressed in Xenopus oocytes. Peptides were injected individually or in combination into the oocyte. Whole cell Na(+) current (I(Na)) was recorded using two-electrode voltage clamp. epsilonV1-7 (100 nM) and PMA (100 nM) inhibited I(Na) by 31 +/- 5% and 44 +/- 8% (at -20 mV), respectively. These effects were not seen with the scrambled peptide for epsilonV1-7 (100 nM) or the PMA analog 4alpha-phorbol 12,13-didecanoate (100 nM). However, epsilonV1-7- and PMA-induced I(Na) inhibition was abolished by epsilonV1-2, a peptide-specific antagonist of epsilonPKC. Furthermore, PMA-induced I(Na) inhibition was not altered by 100 nM peptide-specific inhibitors for alpha-, beta-, delta-, or etaPKC. PMA and epsilonV1-7 induced translocation of epsilonPKC from soluble to particulate fraction in Xenopus oocytes. This translocation was antagonized by epsilonV1-2. In native rat ventricular myocytes, PMA and epsilonV1-7 also inhibited I(Na); this inhibition was antagonized by epsilonV1-2. In conclusion, the results provide evidence for selective regulation of cardiac Na(+) channels by epsilonPKC isozyme.

    View details for Web of Science ID 000171694400009

    View details for PubMedID 11600410

  • Adaptor proteins in protein kinase C-mediated signal transduction ONCOGENE Schechtman, D., Mochly-Rosen, D. 2001; 20 (44): 6339-6347


    Spatial and temporal organization of signal transduction is essential in determining the speed and precision by which signaling events occur. Adaptor proteins are key to organizing signaling enzymes near their select substrates and away from others in order to optimize precision and speed of response. Here, we describe the role of adaptor proteins in determining the specific function of individual protein kinase C (PKC) isozymes. These isozyme-selective proteins were called collectively RACKs (receptors for activated C-kinase). The role of RACKs in PKC-mediated signaling was determined using isozyme-specific inhibitors and activators of the binding of each isozyme to its respective RACK. In addition to anchoring activated PKC isozymes, RACKs anchor other signaling enzymes. RACK1, the anchoring protein for activated betaIIPKC, binds for example, Src tyrosine kinase, integrin, and phosphodiesterase. RACK2, the epsilonPKC-specific RACK, is a coated-vesicle protein and thus is involved in vesicular release and cell-cell communication. Therefore, RACKs are not only adaptors for PKC, but also serve as adaptor proteins for several other signaling enzymes. Because at least some of the proteins that bind to RACKs, including PKC itself, regulate cell growth, modulating their interactions with RACKs may help elucidate signaling pathways leading to carcinogenesis and could result in the identification of novel therapeutic targets.

    View details for Web of Science ID 000171640600010

    View details for PubMedID 11607837

  • Opposing cardioprotective actions and parallel hypertrophic effects of delta PKC and epsilon PKC PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Chen, L., Hahn, H., Wu, G. Y., Chen, C. H., Liron, T., Schechtman, D., Cavallaro, G., Banci, L., Guo, Y. R., Bolli, R., Dorn, G. W., Mochly-Rosen, D. 2001; 98 (20): 11114-11119


    Conflicting roles for protein kinase C (PKC) isozymes in cardiac disease have been reported. Here, deltaPKC-selective activator and inhibitor peptides were designed rationally, based on molecular modeling and structural homology analyses. Together with previously identified activator and inhibitor peptides of epsilonPKC, deltaPKC peptides were used to identify cardiac functions of these isozymes. In isolated cardiomyocytes, perfused hearts, and transgenic mice, deltaPKC and epsilonPKC had opposing actions on protection from ischemia-induced damage. Specifically, activation of epsilonPKC caused cardioprotection whereas activation of deltaPKC increased damage induced by ischemia in vitro and in vivo. In contrast, deltaPKC and epsilonPKC caused identical nonpathological cardiac hypertrophy; activation of either isozyme caused nonpathological hypertrophy of the heart. These results demonstrate that two related PKC isozymes have both parallel and opposing effects in the heart, indicating the danger in the use of therapeutics with nonselective isozyme inhibitors and activators. Moreover, reduction in cardiac damage caused by ischemia by perfusion of selective regulator peptides of PKC through the coronary arteries constitutes a major step toward developing a therapeutic agent for acute cardiac ischemia.

    View details for Web of Science ID 000171237100027

    View details for PubMedID 11553773

  • Binding specificity for RACK1 resides in the V5 region of beta II protein kinase C JOURNAL OF BIOLOGICAL CHEMISTRY Stebbins, E. G., Mochly-Rosen, D. 2001; 276 (32): 29644-29650


    Identification of selective anchoring proteins responsible for specialized localization of specific signaling proteins has led to the identification of new inhibitors of signal transduction, inhibitors of anchoring protein-ligand interactions. RACK1, the first receptor for activated C kinase identified in our lab, is a selective anchoring protein for betaII protein kinase C (betaIIPKC). We previously found that at least part of the RACK1-binding site resides in the C2 domain of betaIIPKC (Ron, D., Luo, J., and Mochly-Rosen, D. (1995) J. Biol. Chem. 270, 24180-24187). Here we show that the V5 domain also contains part of the RACK1-binding site in betaIIPKC. In neonatal rat cardiac myocytes, the betaIIV5-3 peptide (amino acids 645-650 in betaIIPKC) selectively inhibited phorbol 12-myristate 13-acetate (PMA)-induced translocation of betaIIPKC and not betaIPKC. In addition, the betaIIV5-3 peptide inhibited cardiac myocyte hypertrophy in PMA-treated cells. Interestingly, betaIV5-3 (646-651 in betaIPKC), a selective translocation inhibitor of betaIPKC, also inhibited PMA-induced cardiac myocyte hypertrophy, demonstrating that both betaI- and betaIIPKC are essential for this cardiac function. Therefore, the betaIIV5 domain contains part of the RACK1-binding site in betaIIPKC; a peptide corresponding to this site is a selective inhibitor of betaIIPKC and, hence, enables the identification of betaIIPKC-selective functions.

    View details for Web of Science ID 000170558000008

    View details for PubMedID 11387319

  • Localization, anchoring, and functions of protein kinase C isozymes in the heart JOURNAL OF MOLECULAR AND CELLULAR CARDIOLOGY MACKAY, K., Mochly-Rosen, D. 2001; 33 (7): 1301-1307


    Although protein kinase C (PKC) was identified more than 20 years ago, and is involved in a wide variety of essential cellular processes, assigning specific roles to each PKC isozyme has proved difficult. Results over the last few years have suggested that much of the specificity of activated PKC isozymes is attributed to their subcellular localization bringing them into close proximity to a subset of substrates. Our laboratory has taken advantage of the importance of PKC localization and studied the way in which PKC isozymes are anchored. We have identified PKC anchoring proteins (RACKs or Receptors for Activated C Kinase) and used information about interaction sites between PKC isozymes and their respective RACKs to design peptides which modulate translocation of specific PKC isozymes to the functional site. These isozyme-specific peptides can be delivered into isolated or cultured cells or expressed in transgenic mice to determine the role of specific PKC isozymes in particular functions. Here we will describe the isozymes-specific peptide activators and inhibitors that we have developed and the specific functions of each isozyme in cardiac ventricular tissue.

    View details for DOI 10.1006/jmcc.2001.1400

    View details for Web of Science ID 000171322800002

    View details for PubMedID 11437536

  • Arachidonic acid protects neonatal rat cardiac myocytes from ischaemic injury through epsilon protein kinase C CARDIOVASCULAR RESEARCH MACKAY, K., Mochly-Rosen, D. 2001; 50 (1): 65-74


    Arachidonic acid is a second messenger which activates protein kinase C (PKC) and is released from the heart during ischaemic preconditioning. The purpose of this study was to examine the effect of arachidonic acid on activation of PKC in cardiac myocytes and the cellular consequences.Neonatal rat cardiac myocytes were isolated and maintained in culture. Arachidonic acid-induced activation of PKC was examined by cell fractionation and western blot analysis. Contraction frequency was measured by visual inspection under a microscope. Ischaemia was simulated by subjecting cells to an atmosphere of lower than 0.5% oxygen in the absence of glucose and cell damage determined by release of cytosolic lactate dehydrogenase or direct cell viability assay.Arachidonic acid resulted in translocation of delta and epsilonPKC but not alpha, betaII, eta or zetaPKC isozymes, indicating activation of only delta and epsilonPKC. Arachidonic acid induced a dose-dependent decrease in spontaneous contraction rate of cardiac myocytes which was blocked by a selective peptide translocation inhibitor of epsilonPKC. Pretreatment with arachidonic acid partially protected cardiac myocytes against ischaemia. Down-regulation of PKC with 24 h 4beta-phorbol,12-myristate,13-acetate treatment, inhibition of PKC by chelerythrine and selective inhibition of epsilonPKC translocation all decreased the protective effect of arachidonic acid. Pretreatment with eicosapentaenoic acid or oleic acid also protected cardiac myocytes against ischaemia.These results demonstrate that arachidonic acid selectively activates delta and epsilonPKC in neonatal rat cardiac myocytes, leading to protection from ischaemia. We suggest this is a potential mechanism of PKC activation during PC. In addition, our results suggest that different classes of free fatty acid directly exert cardioprotection from ischaemic injury in cardiac myocytes.

    View details for Web of Science ID 000168031500009

    View details for PubMedID 11282079

  • Opposing effects of delta and epsilon PKC in ethanol-induced cardioprotection JOURNAL OF MOLECULAR AND CELLULAR CARDIOLOGY Chen, C. H., Mochly-Rosen, D. 2001; 33 (3): 581-585


    Low amounts of ethanol reduce cardiac damage induced by ischemia. The protection from ischemic damage by acute exposure to low amounts of ethanol in isolated myocytes and intact heart have been attributed to activation of protein kinase C (PKC). We previously found that two PKC isozymes, delta and xi, are activated by ethanol in several cell models. Here, we perfused isozyme-selective agonist and antagonist peptides that we have generated into intact heart to determine the role of these two isozymes in ethanol-induced protection from transient ischemia. Whereas xi PKC activation was required for ethanol-induced protection, delta PKC activation led to further damage. These data explain the conflicting reports on the role of acute exposure to ethanol in protection from cardiac ischemia. The clinical implications of these findings are also discussed.

    View details for DOI 10.1006/jmcc.2000.1330

    View details for Web of Science ID 000167365200018

    View details for PubMedID 11181025

  • Spontaneous occurrence of an inhibitor of protein kinase C localization in a thyroid cancer cell line: Role in thyroid tumorigenesis 41st International Symposium on Regulation of Enzyme Activity and Synthesis in Normal and Neoplastic Tissues Mochly-Rosen, D., Fagin, J. A., Knauf, J. A., Nikiforov, Y., Liron, T., Schechtman, D. PERGAMON-ELSEVIER SCIENCE LTD. 2001: 87–97

    View details for Web of Science ID 000169792600005

    View details for PubMedID 11384739

  • Evidence for functional role of epsilon PKC isozyme in the regulation of cardiac Ca2+ channels AMERICAN JOURNAL OF PHYSIOLOGY-HEART AND CIRCULATORY PHYSIOLOGY Hu, K. L., Mochly-Rosen, D., Boutjdir, M. 2000; 279 (6): H2658-H2664


    Limited information is available regarding the effects of protein kinase C (PKC) isozyme(s) in the regulation of L-type Ca(2+) channels due to lack of isozyme-selective modulators. To dissect the role of individual PKC isozymes in the regulation of cardiac Ca(2+) channels, we used the recently developed novel peptide activator of the epsilonPKC, epsilonV1-7, to assess the role of epsilonPKC in the modulation of L-type Ca(2+) current (I(Ca,L)). Whole cell I(Ca,L) was recorded using patch-clamp technique from rat ventricular myocytes. Intracellular application of epsilonV1-7 (0.1 microM) resulted in a significant inhibition of I(Ca,L) by 27.9 +/- 2.2% (P < 0.01, n = 8) in a voltage-independent manner. The inhibitory effect of epsilonV1-7 on I(Ca,L) was completely prevented by the peptide inhibitor of epsilonPKC, epsilonV1-2 [5.2 +/- 1.7%, not significant (NS), n = 5] but not by the peptide inhibitors of cPKC, alphaC2-4 (31.3 +/- 2.9%, P < 0.01, n = 6) or betaC2-2 plus betaC2-4 (26.1 +/- 2.9%, P < 0.01, n = 5). In addition, the use of a general inhibitor (GF-109203X, 10 microM) of the catalytic activity of PKC also prevented the inhibitory effect of epsilonV1-7 on I(Ca,L) (7.5 +/- 2.1%, NS, n = 6). In conclusion, we show that selective activation of epsilonPKC inhibits the L-type Ca channel in the heart.

    View details for Web of Science ID 000165409300013

    View details for PubMedID 11087218

  • Protein kinase C isozymes and the regulation of diverse cell responses AMERICAN JOURNAL OF PHYSIOLOGY-LUNG CELLULAR AND MOLECULAR PHYSIOLOGY Dempsey, E. C., Newton, A. C., Mochly-Rosen, D., Fields, A. P., Reyland, M. E., Insel, P. A., Messing, R. O. 2000; 279 (3): L429-L438


    Individual protein kinase C (PKC) isozymes have been implicated in many cellular responses important in lung health and disease, including permeability, contraction, migration, hypertrophy, proliferation, apoptosis, and secretion. New ideas on mechanisms that regulate PKC activity, including the identification of a novel PKC kinase, 3-phosphoinositide-dependent kinase-1 (PDK-1), that regulates phosphorylation of PKC, have been advanced. The importance of targeted translocation of PKC and isozyme-specific binding proteins (like receptors for activated C-kinase and caveolins) is well established. Phosphorylation state and localization are now thought to be key determinants of isozyme activity and specificity. New concepts on the role of individual PKC isozymes in proliferation and apoptosis are emerging. Opposing roles for selected isozymes in the same cell system have been defined. Coupling to the Wnt signaling pathway has been described. Phenotypes for PKC knockout mice have recently been reported. More specific approaches for studying PKC isozymes and their role in cell responses have been developed. Strengths and weaknesses of different experimental strategies are reviewed. Future directions for investigation are identified.

    View details for Web of Science ID 000088906900004

    View details for PubMedID 10956616

  • Involvement of a p38 mitogen-activated protein kinase phosphatase in protecting neonatal rat cardiac myocytes from ischemia JOURNAL OF MOLECULAR AND CELLULAR CARDIOLOGY MACKAY, K., Mochly-Rosen, D. 2000; 32 (8): 1585-1588


    Our recent results showed that extended p38 mitogen-activated protein kinase (p38) activation during ischemia leads to cell death, at least partly through apoptosis, in neonatal rat cardiomyocytes. However, other studies have shown that p38 activation during a short preconditioning treatment protects cardiomyocytes from ischemic cell death. This suggests that the duration of p38 activation determines its cellular function and therefore inactivation of p38 by phosphatases may play an important role. In neonatal rat cardiomyocytes, we used the tyrosine phosphatase inhibitor, vanadate, to prevent p38 inactivation, thus extending the strength and length of p38 activation during ischemia. This resulted in higher susceptibility to cell death from ischemia in a dose-dependent manner and over time; the additional damage induced by vanadate was inhibited by SB203580, a selective inhibitor of p38. We conclude that a tyrosine phosphatase is inactivated during ischemia, resulting in prolonged p38 activation which causes cell death.

    View details for Web of Science ID 000088561600020

    View details for PubMedID 10900183

  • Chronic hypersensitivity for inflammatory nociceptor sensitization mediated by the epsilon isozyme of protein kinase C JOURNAL OF NEUROSCIENCE Aley, K. O., Messing, R. O., Mochly-Rosen, D., Levine, J. D. 2000; 20 (12): 4680-4685


    We have identified a mechanism, mediated by the epsilon isozyme of protein kinase C (PKCepsilon) in peripheral neurons, which may have a role in chronic inflammatory pain. Acute inflammation, produced by carrageenan injection in the rat hindpaw, produced mechanical hyperalgesia that resolved by 72 hr. However, for up to 3 weeks after carrageenan, injection of the inflammatory mediators prostaglandin E(2) or 5-hydroxytryptamine or of an adenosine A(2) agonist into the same site induced a markedly prolonged hyperalgesia (>24 hr compared with 5 hr or less in control rats not pretreated with carrageenan). A nonselective inhibitor of several PKC isozymes and a selective PKCepsilon inhibitor antagonized this prolonged hyperalgesic response equally. Acute carrageenan hyperalgesia could be inhibited by PKA or PKG antagonists. However, these antagonists did not inhibit development of the hypersensitivity to inflammatory mediators. Our findings indicate that different second messenger pathways underlie acute and prolonged inflammatory pain.

    View details for Web of Science ID 000087448500035

    View details for PubMedID 10844037

  • Cardiotrophic effects of protein kinase C epsilon - Analysis by in vivo modulation of PKC epsilon translocation CIRCULATION RESEARCH Mochly-Rosen, D., Wu, G. Y., Hahn, H., Osinska, H., Liron, T., Lorenz, J. N., Yatani, A., Robbins, J., Dorn, G. W. 2000; 86 (11): 1173-1179


    Protein kinase C (PKC) is a key mediator of many diverse physiological and pathological responses. Although little is known about the specific in vivo roles of the various cardiac PKC isozymes, activation-induced translocation of PKC is believed to be the primary determinant of isozyme-specific functions. Recently, we have identified a catalytically inactive peptide translocation inhibitor (epsilonV1) and translocation activator (psiepsilonRACK [receptors for activated C kinase]) specifically targeting PKCepsilon. Using cardiomyocyte-specific transgenic expression of these peptides, we combined loss- and gain-of-function approaches to elucidate the in vivo consequences of myocardial PKCepsilon signaling. As expected for a PKCepsilon RACK binding peptide, confocal microscopy showed that epsilonV1 decorated cross-striated elements and intercalated disks of cardiac myocytes. Inhibition of cardiomyocyte PKCepsilon by epsilonV1 at lower expression levels upregulated alpha-skeletal actin gene expression, increased cardiomyocyte cell size, and modestly impaired left ventricular fractional shortening. At high expression levels, epsilonV1 caused a lethal dilated cardiomyopathy. In contrast, enhancement of PKCepsilon translocation with psiepsilonRACK resulted in selectively increased beta myosin heavy chain gene expression and normally functioning concentric ventricular remodeling with decreased cardiomyocyte size. These results identify for the first time a role for PKCepsilon signaling in normal postnatal maturational myocardial development and suggest the potential for PKCepsilon activators to stimulate "physiological" cardiomyocyte growth.

    View details for Web of Science ID 000087571800013

    View details for PubMedID 10850970

  • Pharmacological regulation of network kinetics by protein kinase C localization SEMINARS IN IMMUNOLOGY Mochly-Rosen, D., Kauvar, L. M. 2000; 12 (1): 55-61


    Protein kinase C (PKC) is a conserved family of 11 serine/threonine kinases. Most cell types express multiple members of the family. Because the catalytic sites are homologous, and able to accommodate a broad range of substrates in vitro, specificity in function is dependent on subcellular localization of each isozyme in each cell type. Physiological stimulation can result in major changes in localization of individual PKC isozymes, mediated through binding to specific anchoring proteins. We describe data demonstrating that disruption of such translocations of PKC isozymes by pharmacological agents, peptides, or antibodies, causes profound effects on T cell functions. The pharmacological opportunity provided by distinct kinetic properties of complex assembly is also discussed.

    View details for Web of Science ID 000085469100006

    View details for PubMedID 10723798

  • Evidence for functional regulation of cardiac Ca channels by epsilon PKC using specific peptides activator and inhibitor of epsilon PKC Hu, K., El-Sherif, N., Mochly-Rosen, D., Boutjdir, M. LIPPINCOTT WILLIAMS & WILKINS. 1999: 190–91
  • Sustained in vivo cardiac protection by a rationally designed peptide that causes epsilon protein kinase C translocation PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Dorn, G. W., Souroujon, M. C., Liron, T., Chen, C. H., Gray, M. O., Zhou, H. Z., Csukai, M., Wu, G. Y., Lorenz, J. N., Mochly-Rosen, D. 1999; 96 (22): 12798-12803


    Brief periods of cardiac ischemia trigger protection from subsequent prolonged ischemia (preconditioning). epsilon Protein kinase C (epsilonPKC) has been suggested to mediate preconditioning. Here, we describe an epsilonPKC-selective agonist octapeptide, psiepsilon receptor for activated C-kinase (psiepsilonRACK), derived from an epsilonPKC sequence homologous to its anchoring protein, epsilonRACK. Introduction of psiepsilonRACK into isolated cardiomyocytes, or its postnatal expression as a transgene in mouse hearts, increased epsilonPKC translocation and caused cardio-protection from ischemia without any deleterious effects. Our data demonstrate that epsilonPKC activation is required for protection from ischemic insult and suggest that small molecules that mimic this epsilonPKC agonist octapeptide provide a powerful therapeutic approach to protect hearts at risk for ischemia.

    View details for Web of Science ID 000083373000107

    View details for PubMedID 10536002

  • Cardioprotection from ischemia by a brief exposure to physiological levels of ethanol: Role of epsilon protein kinase C PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Chen, C. H., Gray, M. O., Mochly-Rosen, D. 1999; 96 (22): 12784-12789


    Recent epidemiological studies indicate beneficial effects of moderate ethanol consumption in ischemic heart disease. Most studies, however, focus on the effect of long-term consumption of ethanol. In this study, we determined whether brief exposure to ethanol immediately before ischemia also produces cardioprotection. In addition, because protein kinase C (PKC) has been shown to mediate protection of the heart from ischemia, we determined the role of specific PKC isozymes in ethanol-induced protection. We demonstrated that (i) brief exposure of isolated adult rat cardiac myocytes to 10-50 mM ethanol protected against damage induced by prolonged ischemia; (ii) an isozyme-selective epsilonPKC inhibitor developed in our laboratory inhibited the cardioprotective effect of acute ethanol exposure; (iii) protection of isolated intact adult rat heart also occurred after incubation with 10 mM ethanol 20 min before global ischemia; and (iv) ethanol-induced cardioprotection depended on PKC activation because it was blocked by chelerythrine and GF109203X, two PKC inhibitors. Consumption of 1-2 alcoholic beverages in humans leads to blood alcohol levels of approximately 10 mM. Therefore, our work demonstrates that exposure to physiologically attainable ethanol levels minutes before ischemia provides cardioprotection that is mediated by direct activation of epsilonPKC in the cardiac myocytes. The potential clinical implications of our findings are discussed.

    View details for Web of Science ID 000083373000105

    View details for PubMedID 10536000

  • RACK1, a protein kinase C anchoring protein, coordinates the binding of activated protein kinase C and select pleckstrin homology domains in vitro BIOCHEMISTRY Rodriguez, M. M., Ron, D., Touhara, K., Chen, C. H., Mochly-Rosen, D. 1999; 38 (42): 13787-13794


    The pleckstrin homology (PH) domain, identified in numerous signaling proteins including the beta-adrenergic receptor kinase (betaARK), was found to bind to various phospholipids as well as the beta subunit of heterotrimeric G proteins (Gbeta) [Touhara, K., et al. (1994) J. Biol. Chem. 269, 10217-10220]. Several PH domain-containing proteins are also substrates of protein kinase C (PKC). Because RACK1, an anchoring protein for activated PKC, is homologous to Gbeta (both contain seven repeats of the WD-40 motif), we determined (i) whether a direct interaction between various PH domains and RACK1 occurs and (ii) the effect of PKC on this interaction. We found that recombinant PH domains of several proteins exhibited differential binding to RACK1. Activated PKC and the PH domain of beta-spectrin or dynamin-1 concomitantly bound to RACK1. Although PH domains bind acidic phospholipids, the interaction between various PH domains and RACK1 was not dependent on the phospholipid activators of PKC, phosphatidylserine and 1, 2-diacylglycerol. Binding of these PH domains to RACK1 was also not affected by either inositol 1,4,5-triphosphate (IP(3)) or phosphatidylinositol 4,5-bisphosphate (PIP(2)). Our in vitro data suggest that RACK1 binds selective PH domains, and that PKC regulates this interaction. We propose that, in vivo, RACK1 may colocalize the kinase with its PH domain-containing substrates.

    View details for Web of Science ID 000083288400003

    View details for PubMedID 10529223

  • Protein kinase c-e is responsible for the protection of preconditioning in rabbit cardiomyocytes JOURNAL OF MOLECULAR AND CELLULAR CARDIOLOGY Liu, G. S., Cohen, M. V., Mochly-Rosen, D., Downey, J. M. 1999; 31 (10): 1937-1948


    The role of protein kinase C (PKC) in the protection of ischemic preconditioning (PC) is still controversial, partly because of the multiple isozymes of PKC and the inability to directly measure PKC activity in vivo. In this study we have used novel peptide inhibitors which correspond to part of the amino acid sequence from the isozyme-specific RACK-binding site on the PKC molecule. The peptides prevent binding of a specific activated PKC isozyme to its RACK, thus halting isozyme translocation and function. The inhibitor peptides are cross-linked to the membrane-translocating antennapedia homeodomain peptide that allows their entry into cells. The effect of inhibitors of PKC-beta, -delta, -epsilon and -eta were evaluated. Rabbit adult ventricular myocytes were obtained by enzymatic dissociation. Ischemia was simulated by centrifuging the myocytes into an oxygen-free pellet for 180 min. PC was induced by 10 min of pelleting followed by resuspension in oxygenated medium for 15 min. During simulated ischemia cells undergo a predictable increase in osmotic fragility as judged by determination of the number of stained cells following their incubation in hypotonic (85 mOsm) trypan blue. The percentage of cells experiencing membrane rupture, and thus cell staining, was considered to be an index of ischemic injury. PC significantly delayed the progression of osmotic fragility during simulated ischemia (P<0.01). The protection of PC was abolished by the peptide inhibitor of PKC-epsilon but not by the peptide inhibitors selective for PKC-beta, PKC-delta, or PKC-eta; each was applied at 100 n N. Protection could also be induced by the PKC activator oleoylacetyl glycerol, and that protection was aborted by the inhibitor selective for PKC-epsilon, but not by the inhibitor for PKC-delta. None of the above peptide treatments affected the osmotic fragility in non-PC cells during simulated ischemia. Our studies further support PKC as a critical part of the signal transduction pathway in PC and indicate that PKC-epsilon alone is responsible for the early phase of PC's protection in rabbit cardiomyocytes.

    View details for Web of Science ID 000082885000014

    View details for PubMedID 10525430

  • Involvement of protein kinase C epsilon (PKC epsilon) in thyroid cell death - A truncated chimeric PKC epsilon cloned from a thyroid cancer cell line protects thyroid cells from apoptosis JOURNAL OF BIOLOGICAL CHEMISTRY Knauf, J. A., Elisei, R., Mochly-Rosen, D., Liron, T., Chen, X. N., Gonsky, R., Korenberg, J. R., Fagin, J. A. 1999; 274 (33): 23414-23425


    The protein kinase C (PKC) family has been implicated in the regulation of apoptosis. However, the contribution of individual PKC isozymes to this process is not well understood. We reported amplification of the chromosome 2p21 locus in 28% of thyroid neoplasms, and in the WRO thyroid carcinoma cell line. By positional cloning we identified a rearrangement and amplification of the PKCepsilon gene, that maps to 2p21, in WRO cells. This resulted in the overexpression of a chimeric/truncated PKCepsilon (Tr-PKCepsilon) mRNA, coding for N-terminal amino acids 1-116 of the isozyme fused to an unrelated sequence. Expression of the Tr-PKCepsilon protein in PCCL3 cells inhibited activation-induced translocation of endogenous PKCepsilon, but its kinase activity was unaffected, consistent with a dominant negative effect of the mutant protein on activation-induced translocation of wild-type PKCepsilon and/or displacement of the isozyme to an aberrant subcellular location. Cell lines expressing Tr-PKCepsilon grew to a higher saturation density than controls. Moreover, cells expressing Tr-PKCepsilon were resistant to apoptosis, which was associated with higher Bcl-2 levels, a marked impairment in p53 stabilization, and dampened expression of Bax. These findings point to a role for PKCepsilon in apoptosis-signaling pathways in thyroid cells, and indicate that a naturally occurring PKCepsilon mutant that functions as a dominant negative can block cell death triggered by a variety of stimuli.

    View details for Web of Science ID 000082012800069

    View details for PubMedID 10438519

  • Characterization of the binding and phosphorylation of cardiac calsequestrin by epsilon protein kinase C FEBS LETTERS Rodriguez, M. M., Chen, C. H., Smith, B. L., Mochly-Rosen, D. 1999; 454 (3): 240-246


    In this study, we report the cloning of the rat cardiac isoform of calsequestrin on the basis of its interaction with an epsilonprotein kinase C-unique sequence (epsilonV1) derived form the epsilonprotein kinase C regulatory domain. Calsequestrin binds activated epsilonprotein kinase C holoenzyme better than the inactive enzyme and nearly three times better than other protein kinase C isozymes. The interaction between epsilonprotein kinase C and calsequestrin is mediated by sequences in both the regulatory and kinase domains of the epsilonprotein kinase C. Finally, we show that calsequestrin is an epsilonprotein kinase C substrate in vitro and protein kinase C phosphorylation of calsequestrin leads to a decreased binding of epsilonprotein kinase C to calsequestrin.

    View details for Web of Science ID 000081477900015

    View details for PubMedID 10431815

  • Pharmacologic modulation of protein kinase C isozymes: The role of races and subcellular localisation PHARMACOLOGICAL RESEARCH Csukai, M., Mochly-Rosen, D. 1999; 39 (4): 253-259


    Protein kinase C (PKC) isozymes are highly homologous kinases and several different isozymes can be present in a cell. Each isozyme is likely to mediate unique functions, but pharmacological tools to explore their isozyme-specific roles have not been available until recently. In this review, we describe the development and application of isozyme-selective inhibitors of PKC. The identification of these inhibitors stems from the observation that PKC isozymes are each localised to unique subcellular locations following activation. Inhibitors of this isozyme-unique localisation have been shown to act as selective inhibitors of the functions of individual isozymes. The identification of isozyme-specific inhibitors should allow the exploration of individual PKC isozyme function in a wide range of cell systems.

    View details for Web of Science ID 000079827000001

    View details for PubMedID 10208754

  • An inhibitor of p38 mitogen-activated protein kinase protects neonatal cardiac myocytes from ischemia JOURNAL OF BIOLOGICAL CHEMISTRY MACKAY, K., Mochly-Rosen, D. 1999; 274 (10): 6272-6279


    Cellular ischemia results in activation of a number of kinases, including p38 mitogen-activated protein kinase (MAPK); however, it is not yet clear whether p38 MAPK activation plays a role in cellular damage or is part of a protective response against ischemia. We have developed a model to study ischemia in cultured neonatal rat cardiac myocytes. In this model, two distinct phases of p38 MAPK activation were observed during ischemia. The first phase began within 10 min and lasted less than 1 h, and the second began after 2 h and lasted throughout the ischemic period. Similar to previous studies using in vivo models, the nonspecific activator of p38 MAPK and c-Jun NH2-terminal kinase, anisomycin, protected cardiac myocytes from ischemic injury, decreasing the release of cytosolic lactate dehydrogenase by approximately 25%. We demonstrated, however, that a selective inhibitor of p38 MAPK, SB 203580, also protected cardiac myocytes against extended ischemia in a dose-dependent manner. The protective effect was seen even when the inhibitor was present during only the second, sustained phase of p38 MAPK activation. We found that ischemia induced apoptosis in neonatal rat cardiac myocytes and that SB 203580 reduced activation of caspase-3, a key event in apoptosis. These results suggest that p38 MAPK induces apoptosis during ischemia in cardiac myocytes and that selective inhibition of p38 MAPK could be developed as a potential therapy for ischemic heart disease.

    View details for Web of Science ID 000078902800037

    View details for PubMedID 10037715

  • Evidence of zeta protein kinase C involvement in polymorphonuclear neutrophil integrin-dependent adhesion and chemotaxis JOURNAL OF BIOLOGICAL CHEMISTRY Laudanna, C., Mochly-Rosen, D., Liron, T., Constantin, G., BUTCHER, E. C. 1998; 273 (46): 30306-30315


    Classical chemoattractants and chemokines trigger integrin-dependent adhesion of blood leukocytes to vascular endothelium and also direct subsequent extravasation and migration into tissues. In studies of human polymorphonuclear neutrophil responses to formyl peptides and to interleukin 8, we show evidence of involvement of the atypical zeta protein kinase C in the signaling pathway leading to chemoattractant-triggered actin assembly, integrin-dependent adhesion, and chemotaxis. Selective inhibitors of classical and novel protein kinase C isozymes do not prevent chemoattractant-induced neutrophil adhesion and chemotaxis. In contrast, chelerythrine chloride and synthetic myristoylated peptides with sequences based on the endogenous zeta protein kinase C pseudosubstrate region block agonist-induced adhesion to fibrinogen, chemotaxis and F-actin accumulation. Biochemical analysis shows that chemoattractants trigger rapid translocation of zeta protein kinase C to the plasma membrane accompanied by rapid but transient increase of the kinase activity. Moreover, pretreatment with C3 transferase, a specific inhibitor of Rho small GTPases, blocks zeta but not alpha protein kinase C plasma membrane translocation. Synthetic peptides from zeta protein kinase C also inhibit phorbol ester-induced integrin-dependent adhesion but not NADPH-oxidase activation, and C3 transferase pretreatment blocks phorbol ester-triggered translocation of zeta but not alpha protein kinase C. These data suggest the involvement of zeta protein kinase C in chemoattractant-induced leukocyte integrin-dependent adhesion and chemotaxis. Moreover, they highlight a potential link between atypical protein kinase C isozymes and Rho signaling pathways leading to integrin-activation.

    View details for Web of Science ID 000077008100039

    View details for PubMedID 9804792

  • Peptide modulators of protein-protein interactions in intracellular signaling NATURE BIOTECHNOLOGY Souroujon, M. C., Mochly-Rosen, D. 1998; 16 (10): 919-924


    Signal transduction cascades involve multiple enzymes and are orchestrated by selective protein-protein interactions that are essential for the progression of intracellular signaling events. Modulators of these protein-protein interactions have been used to dissect the role of individual components of each signaling cascade. We describe several methods that have been developed for the identification of peptides that inhibit the interaction between signaling proteins and hence selectively modulate their functions. Such peptide modulators provide important tools for basic research and have great potential as leads for the development of new classes of therapeutic drugs.

    View details for Web of Science ID 000076323300025

    View details for PubMedID 9788346

  • Activation of epsilon protein kinase C correlates with a cardioprotective effect of regular ethanol consumption PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Miyamae, M., Rodriguez, M. M., Camacho, S. A., Diamond, I., Mochly-Rosen, D., Figueredo, V. M. 1998; 95 (14): 8262-8267


    In addition to decreasing the incidence of myocardial infarction, recent epidemiological data suggest that regular alcohol consumption improves survival after myocardial infarction. We recently found that chronic ethanol exposure induces long-term protection against cardiac ischemia-reperfusion injury, which improves myocardial recovery after infarction. Furthermore, this cardioprotection by ethanol is mediated through myocyte adenosine A1 receptors. We now determine the role of protein kinase C (PKC) in ethanol's protective effect against ischemia-reperfusion injury. Using perfused hearts of ethanol-fed guinea pigs, we find that improved contractile recovery and creatine kinase release after ischemia-reperfusion are abolished by PKC inhibition with chelerythrine. Western blot analysis and immunofluorescence localization demonstrate that regular ethanol consumption causes sustained translocation (activation) of epsilonPKC, but not delta or alphaPKC. This same isozyme is directly implicated in ischemic preconditioning's protection against ischemia-reperfusion injury. Our findings suggest (i) that regular ethanol consumption induces long-term cardioprotection through sustained translocation of epsilonPKC and (ii) that PKC activity is necessary at the time of ischemia to mediate ethanol's protective effect against ischemia-reperfusion injury. Studying this selective effect of ethanol on epsilonPKC activation may lead to new therapies to protect against ischemia-reperfusion injury in the heart and other organ systems.

    View details for Web of Science ID 000074717300076

    View details for PubMedID 9653175

  • Modulating protein kinase C signal transduction. Advances in pharmacology (San Diego, Calif.) Mochly-Rosen, D., Kauvar, L. M. 1998; 44: 91-145

    View details for PubMedID 9547885

  • Anchoring proteins for protein kinase C: a means for isozyme selectivity FASEB JOURNAL Mochly-Rosen, D., Gordon, A. S. 1998; 12 (1): 35-42


    Protein kinase C (PKC) isozymes comprise a family of related enzymes. There are only limited differences between these isozymes in substrate specificity or sensitivity to activators. However, there are multiple isozymes within a cell mediating isozyme-specific functions. Differential subcellular localization has been proposed to explain this specificity. When members of the PKC family are activated by lipid-derived second messengers, they translocate from one cell compartment to another. Isozyme specificity appears to be mediated in part by association of each PKC isozyme with specific anchoring proteins. This review will cover the proteins involved in the anchoring of PKC isozymes at specific subcellular sites, the domains in the PKC isozymes that mediate protein-protein interaction with isozyme-specific anchoring proteins, and identification of peptides that interfere with or promote these protein-protein interactions, thus altering the localization and function of individual isozymes.

    View details for Web of Science ID 000071523800004

    View details for PubMedID 9438408

  • Molecular genetic approaches. II. Expression-interaction cloning. Methods in molecular biology (Clifton, N.J.) Csukai, M., Mochly-Rosen, D. 1998; 88: 133-139

    View details for PubMedID 9664302

  • A selective epsilon-protein kinase C antagonist inhibits protection of cardiac myocytes from hypoxia-induced cell death JOURNAL OF BIOLOGICAL CHEMISTRY Gray, M. O., Karliner, J. S., Mochly-Rosen, D. 1997; 272 (49): 30945-30951


    Protein kinase C activation is thought to protect cardiac tissue from subsequent ischemic injury by a process termed preconditioning. The protein kinase C isozyme that mediates preconditioning has not yet been identified. Using a cell culture model of hypoxic preconditioning, we found that cardiac myocyte viability after 9 h of hypoxia was increased by more than 50% over control. Preconditioning activated protein kinase C isozymes as evidenced by translocation from one cell compartment to another as follows: there was a 2.1-fold increase in epsilon-protein kinase C activation, a 2. 8-fold increase in delta-protein kinase C activation, and no increase in betaI-protein kinase C activation. 4beta-Phorbol 12-myristate 13-acetate mimicked hypoxic preconditioning, increasing myocyte survival after prolonged hypoxia by 34% compared with control. We previously identified an epsilon-protein kinase C-selective antagonist, epsilonV1-2 peptide, that inhibits epsilon-protein kinase C translocation and function in cardiac myocytes (Johnson, J. A., Gray, M. O., Chen, C.-H., and Mochly-Rosen, D. (1996) J. Biol. Chem. 271, 24962-24966). epsilonV1-2 peptide abolished hypoxic preconditioning and phorbol ester-mediated cardiac protection. Therefore, preconditioning can be induced in this culture model, and activation of epsilon-protein kinase C is critical for cardiac myocyte protection.

    View details for Web of Science ID 000071640800054

    View details for PubMedID 9388241

  • The coatomer protein beta'-COP, a selective binding protein (RACK) for protein kinase C epsilon JOURNAL OF BIOLOGICAL CHEMISTRY Csukai, M., Chen, C. H., DEMATTEIS, M. A., MOCHLYROSEN, D. 1997; 272 (46): 29200-29206


    Distinct subcellular localization of activated protein kinase C (PKC) isozymes is mediated by their binding to isozyme-specific RACKs (receptors for activated C-kinase). Our laboratory has previously isolated one such protein, RACK1, and demonstrated that this protein displays specificity for PKCbeta. We have recently shown that at least part of the PKCepsilon RACK-binding site on PKCepsilon lies within the unique V1 region of this isozyme (Johnson, J. A., Gray, M. O., Chen, C.-H., and Mochly-Rosen, D. (1996) J. Biol. Chem. 271, 24962-24966). Here, we have used the PKCepsilon V1 region to clone a PKCepsilon-selective RACK, which was identified as the COPI coatomer protein, beta'-COP. Similar to RACK1, beta'-COP contains seven repeats of the WD40 motif and fulfills the criteria previously established for RACKs. Activated PKCepsilon colocalizes with beta'-COP in cardiac myocytes and binds to Golgi membranes in a beta'-COP-dependent manner. A role for PKC in control of secretion has been previously suggested, but this is the first report of direct protein/protein interaction of PKCepsilon with a protein involved in vesicular trafficking.

    View details for Web of Science ID A1997YF68400057

    View details for PubMedID 9360998

  • An inhibitory fragment derived from protein kinase C epsilon prevents enhancement of nerve growth factor responses by ethanol and phorbol esters JOURNAL OF BIOLOGICAL CHEMISTRY Hundle, B., McMahon, T., Dadgar, J., Chen, C. H., MOCHLYROSEN, D., Messing, R. O. 1997; 272 (23): 15028-15035


    We have studied nerve growth factor (NGF)-induced differentiation of PC12 cells to identify PKC isozymes important for neuronal differentiation. Previous work showed that tumor-promoting phorbol esters and ethanol enhance NGF-induced mitogen-activated protein (MAP) kinase activation and neurite outgrowth by a PKC-dependent mechanism. Ethanol also increases expression of PKCdelta and PKCepsilon, suggesting that one these isozymes regulates responses to NGF. To examine this possibility, we established PC12 cell lines that express a fragment encoding the first variable domain of PKCepsilon (amino acids 2-144), which acts as an isozyme-specific inhibitor of PKCepsilon in cardiac myocytes. Phorbol ester-stimulated translocation of PKCepsilon was markedly reduced in these PC12 cell lines. In addition, phorbol ester and ethanol did not enhance NGF-induced MAP kinase activation or neurite outgrowth in these cells. In contrast, phorbol ester and ethanol increased neurite outgrowth and MAP kinase phosphorylation in cells expressing a fragment derived from the first variable domain of PKCdelta. These results demonstrate that PKCepsilon mediates enhancement of NGF-induced signaling and neurite outgrowth by phorbol esters and ethanol in PC12 cells.

    View details for Web of Science ID A1997XC32700076

    View details for PubMedID 9169479

  • C2 region-derived peptides of p-protein kinase C regulate cardiac Ca2+ channels CIRCULATION RESEARCH Zhang, Z. H., Johnson, J. A., Chen, L., ELSHERIF, N., MOCHLYROSEN, D., Boutjdir, M. 1997; 80 (5): 720-729


    We have previously shown that alpha1-adrenergic activation inhibited beta-adrenergic-stimulated L-type Ca2+ current (I(Ca)). To determine the role of protein kinase C (PKC) in this regulation, the inositol trisphosphate pathway was bypassed by direct activation of PKC with 4beta-phorbol 12-myristate 13-acetate (PMA). To minimize Ca2+-induced Ca2+ inactivation, Ba2+ current (I(Ba)) was recorded through Ca2+ channels in adult rat ventricular myocytes. We found that PMA (0.1 micromol/L) consistently inhibited basal I(Ba) by 40.5+/-7.4% and isoproterenol (ISO, 0.1 micromol/L)-stimulated I(Ba) by 48.9+/-7.8%. These inhibitory effects were not observed with the inactive phorbol ester analogue alpha-phorbol 12,13-didecanoate (0.1 micromol/L). To identify the PKC isozymes that mediate these PMA effects, we intracellularly applied peptide inhibitors of a subclass of PKC isozymes, the C2-containing cPKCs. These peptides (betaC2-2 and betaC2-4) specifically inhibit the translocation and function of C2-containing isozymes (alpha-PKC, betaI-PKC, and betaII-PKC), but not the C2-less isozymes (delta-PKC and epsilon-PKC). We first used the pseudosubstrate peptide (0.1 micromol/L in the pipette), which inhibits the catalytic activity of all the PKC isozymes, and found that PMA-induced inhibition of ISO-stimulated I(Ba) was reduced to 16.8+/-7.4% but was not affected by the scrambled pseudosubstrate peptide. The effects of PMA on basal and ISO-stimulated I(Ba) were then determined in the presence of C2-derived peptides or control peptides. When the pipette contained 0.1 micromol/L of betaC2-2 or betaC2-4, PMA-induced inhibition of basal I(Ba) was 26.1+/-4.5% and 23.6+/-2.2%, respectively. Similarly, ISO-stimulated I(Ba) was inhibited by 29.9+/-6.6% and 29.3+/-7.8% in the presence of betaC2-2 and betaC2-4, respectively. In contrast, there was no significant change in the effect of PMA in the presence of control peptides, scrambled betaC2-4, or pentalysine. Finally, PMA-induced inhibition of basal and ISO-stimulated I(Ba) was almost completely abolished in cells dialyzed with both betaC2-2 and betaC2-4. Together, these data suggest a role for C2-containing isozymes in mediating PMA-induced inhibition of L-type Ca2+ channel activity.

    View details for Web of Science ID A1997WX25600013

    View details for PubMedID 9130453

  • Translocation inhibitors define specificity of protein kinase C isoenzymes in pancreatic beta-cells JOURNAL OF BIOLOGICAL CHEMISTRY YEDOVITZKY, M., MOCHLYROSEN, D., Johnson, J. A., Gray, M. O., Ron, D., ABRAMOVITCH, E., Cerasi, E., Nesher, R. 1997; 272 (3): 1417-1420


    The protein kinase C (PKC) family consists of 11 isoenzymes. Following activation, each isoenzyme translocates and binds to a specific receptor for activated C kinase (RACK) (Mochly-Rosen, D. (1995) Science 268, 247-251) that provides an anchoring site in close proximity to the isoenzyme's specific substrate. Pancreatic islet cells contain at least six PKC isoenzymes (Knutson, K. L., and Hoenig, M. (1994) Endocrinology 135, 881-886). Although PKC activation enhances insulin release, the specific function of each isoenzyme is unknown. Here we show that following stimulation with glucose, alphaPKC and epsilonPKC translocate to the cell's periphery, while deltaPKC and zetaPKC translocate to perinuclear sites. betaC2-4, a peptide derived from the RACK1-binding site in the C2 domain of betaPKC, inhibits translocation of alphaPKC and reduces insulin response to glucose. Likewise, epsilonV1-2, an epsilonPKC-derived peptide containing the site for its specific RACK, inhibits translocation of epsilonPKC and reduces insulin response to glucose. Inhibition of islet-glucose metabolism with mannoheptulose blocks translocation of both alphaPKC and epsilonPKC and diminishes insulin response to glucose while calcium-free buffer inhibits translocation of alphaPKC but not epsilonPKC and lowers insulin response by 50%. These findings illustrate the unique ability of specific translocation inhibitors to elucidate the isoenzyme-specific functions of PKC in complex signal transduction pathways.

    View details for Web of Science ID A1997WD05800002

    View details for PubMedID 8999804

  • An improved permeabilization protocol for the introduction of peptides into cardiac myocytes - Application to protein kinase C research CIRCULATION RESEARCH Johnson, J. A., Gray, M. O., Karliner, J. S., Chen, C. H., MOCHLYROSEN, D. 1996; 79 (6): 1086-1099


    We have developed an improved, less disruptive procedure for the transient permeabilization of neonatal cardiac myocytes using saponin. The method allows delivery of peptides to a high percentage of cells in culture without effects on long-term cell viability. Permeation was confirmed microscopically by cellular uptake of a fluorescently labeled peptide and biochemically by uptake of 125I-labeled calmodulin and a 20-kD protein kinase C epsilon fragment into the cells. The intracellular molar concentration of the introduced peptide was approximately 10% of that applied outside. We found no significant effects of permeabilization on spontaneous, phorbol ester-modulated, or norepinephrine-modulated contraction rates. Similarly, the expression of c-fos mRNA (measured 30 minutes after permeabilization) and the incorporation of [-14C]phenylalanine following agonist stimulation (measured 3 days after permeabilization) were not altered by saponin permeabilization. Finally, permeabilization of cells in the presence of a protein kinase C pseudosubstrate peptide, but not a control peptide, inhibited phorbol ester-induced [14C]phenylalanine incorporation into proteins by 80%. Our results demonstrate a methodology for the introduction of peptides into neonatal cardiac myocytes that allows study of their actions without substantial compromises in cell integrity.

    View details for Web of Science ID A1996VV61200004

    View details for PubMedID 8943947

  • A protein kinase C translocation inhibitor as an isozyme-selective antagonist of cardiac function JOURNAL OF BIOLOGICAL CHEMISTRY Johnson, J. A., Gray, M. O., Chen, C. H., MOCHLYROSEN, D. 1996; 271 (40): 24962-24966


    Protein kinase C (PKC) isozymes translocate to unique subcellular sites following activation. We previously suggested that translocation of activated isozymes is required for their function and that in addition to binding to lipids, translocation involves binding of the activated isozymes to specific anchoring proteins (receptors for activated protein kinase C. Using cultured cardiomyocytes we identified inhibitors, the V1 fragment of epsilonPKC (epsilonV1), and an 8-amino acid peptide derived from it that selectively inhibited the translocation of epsilonPKC. Inhibition of epsilonPKC translocation but not inhibition of delta or betaPKC translocation specifically blocked phorbol ester- or norepinephrine-mediated regulation of contraction. These isozyme-selective translocation inhibitors provide novel tools to determine the function of individual PKC isozymes in intact cells.

    View details for Web of Science ID A1996VM67400099

    View details for PubMedID 8798776

  • Differential activation of protein kinase C isozymes by phorbol ester and collagen in human skin microvascular endothelial cells JOURNAL OF INVESTIGATIVE DERMATOLOGY Zhou, L. Y., Disatnik, M. H., Herron, G. S., Rosen, D. M., Karasek, M. A. 1996; 107 (2): 248-252


    Human dermal microvascular endothelial cells participate in activities including inflammation, wound healing, and angiogenesis (neovascularization). Two stages of angiogenesis can be mimicked in vitro by two models of cultured foreskin human dermal microvascular endothelial cells: the differentiation of epithelioid endothelial cells to spindle-shaped mesenchymal-like cells induced by phorbol ester treatment; and the formation of vascular channels induced by exposing the luminal surface of endothelial cell monolayers to type I collagen gels. The mechanisms underlying these two processes, however, are largely unknown. Protein kinase C isozymes, which are activated by phorbol esters, are important mediators in the angiogenic process. In the current work, we identified the protein kinase C isozymes present in human dermal microvascular endothelial cells and determined which of the isozymes are activated in response to phorbol ester or to collagen treatments. Using western blot analysis, we found that microvascular endothelial cells contain at least six protein kinase C isozymes (alpha, beta, delta, epsilon, zeta, eta). Immunocytochemical studies demonstrated that the isozymes are located in distinct cellular compartments and that following treatment with phorbol 12-myristate 13-acetate or with a collagen gel overlay, most isozymes (protein kinase C alpha, beta1, betaII, delta, epsilon, eta) translocated to different parts of the cell. Moreover, for two of these isozymes (betaII and eta), the localization differs after phorbol 12-myristate 13-acetate treatment as compared with collagen treatment. These results demonstrate that agents that mimic two stages in the angiogenic process in vitro initiate diverse changes in the subcellular localization of specific protein kinase C isozymes and suggest a role for different isozymes in this process.

    View details for Web of Science ID A1996UY64200019

    View details for PubMedID 8757771

  • The HIV Nef protein associates with protein kinase C theta JOURNAL OF BIOLOGICAL CHEMISTRY Smith, B. L., KRUSHELNYCKY, B. W., MOCHLYROSEN, D., Berg, P. 1996; 271 (28): 16753-16757


    Expression of the human immunodeficiency virus (HIV) Nef protein has been linked to both decreased cell surface expression of CD4 and an impairment of signal transduction. The recently reported association of Nef with an unidentified serine kinase provides a clue as to how Nef might exert its effects. Considering the key role of protein kinase C (PKC) in T cell activation, we investigated the possibility that Nef interacts with PKC. Our results, using two approaches for detecting interactions between Nef and PKC isozymes in Jurkat cells, show that Nef interacts preferentially with thetaPKC. The interaction of Nef and thetaPKC is independent of calcium, enhanced by phospholipid activators of PKC and not affected by a PKC pseudosubstrate peptide. Phorbol 12-myristate 13-acetate and phytohemagglutinin stimulation of Jurkat cells expressing Nef fails to produce the usual translocation of thetaPKC from the cytosol to the particulate fraction; translocation of betaPKC and epsilonPKC was unaffected. Indeed, there appears to be a net loss of thetaPKC in Nef-expressing cells following stimulation. The loss of thetaPKC, which may be a result of inhibition of its binding to RACKs due to Nef binding, could contribute to the various impairments of T cell function associated with HIV infection and Nef expression.

    View details for Web of Science ID A1996UX12600053

    View details for PubMedID 8663223

  • Signal transduction by the polymeric immunoglobulin receptor suggests a role in regulation of receptor transcytosis JOURNAL OF CELL BIOLOGY Cardone, M. H., Smith, B. L., Mennitt, P. A., MOCHLYROSEN, D., Silver, R. B., MOSTOV, K. E. 1996; 133 (5): 997-1005


    Many membrane traffic events that were previously thought to be constitutive recently have been found to be regulated by a variety of intracellular signaling pathways. The polymeric immunoglobulin receptor (pIgR) transcytoses dimeric IgA (dIgA) from the basolateral to the apical surface of polarized epithelial cells. Transcytosis is stimulated by binding of dIgA to the pIgR, indicating that the pIgR can transduce a signal to the cytoplasmic machinery responsible for membrane traffic. We report that dIgA binding to the pIgR causes activation of protein kinase C (PKC) and release of inositol 1,4,5-trisphosphate (IP3). The IP3 causes an elevation of intracellular Ca. Artificially activating PKC with phorbol myristate acetate or poisoning the calcium pump with thapsigargin stimulates transcytosis of pIgR, while the intracellular Ca chelator BAPTA-AM inhibits transcytosis. Our data suggest that ligand-induced signaling by the pIgR may regulate membrane traffic via well-known second messenger pathways involving PKC, IP3, and Ca. This may be a model of a general means by which membrane traffic is regulated by receptor-ligand interaction and signaling pathways.

    View details for Web of Science ID A1996UQ15100006

    View details for PubMedID 8655590



    Protein kinase C (PKC) isozymes regulate a number of cardiac functions including contractility, gene expression, and hypertrophy. There are at least six PKC isozymes in neonatal rat ventricular myocytes. We have shown previously that stimulation of cardiac myocytes in culture with norepinephrine (NE) or phorbol 12-myristate 13-acetate (PMA) results in translocation of each isozyme to distinct subcellular sites. In the present work, we demonstrated that PKC isozymes vary in their sensitivity to stimulation by acidic fibroblast growth factor (aFGF) and transforming growth factor-beta 1 (TGF-beta 1). Moreover, immunocytochemical studies indicated differences in the subcellular localization of activated isozymes following stimulation with each growth factor. These data suggest that the site of translocation and the resulting function of individual PKC isozymes are distinct for different PKC activators. Identification of the PKC isozymes that respond to aFGF and TGF-beta 1 and their subcellular localization may provide a molecular basis for the divergent cardiac functions mediated by these two growth factors.

    View details for Web of Science ID A1995TD00200006

    View details for PubMedID 8596198



    RACK1 is a protein kinase C (PKC)-binding protein that fulfills the criteria previously established for a receptor for activated C-kinase (RACK). If binding of PKC to RACK anchors the activated enzyme near its protein substrates, then inhibition of this binding should inhibit translocation and function of the enzyme in vivo. Here, we have identified such inhibitors that mimic the RACK1-binding site on beta PKC. We first found that a C2-containing fragment, but not a C1-containing fragment of beta PKC, bound to RACK1 and inhibited subsequent beta PKC binding. The RACK1-binding site was further mapped; peptides beta C2-1 (beta PKC(209-216), beta C2-2 (beta PKC(186-198)), and beta C2-4 (beta PKC(218-226), but not a number of control peptides, bound to RACK1 and inhibited the C2 fragment binding to RACK1. Peptides beta C2-1, beta C2-2, and beta C2-4 specifically inhibited phorbol ester-induced translocation of the C2-containing isozymes in cardiac myocytes and insulin-induced beta PKC translocation and function in Xenopus oocytes. Therefore, peptides corresponding to amino acids 186-198, and 209-226 within the C2 region of the beta PKC are specific inhibitors for functions mediated by beta PKC.

    View details for Web of Science ID A1995TA21700050

    View details for PubMedID 7592622



    Ataxia-telangiectasia (AT) is an autosomal recessive human genetic disease characterized by immunological, neurological, and developmental defects and an increased risk of cancer. Cells from individuals with AT show sensitivity to ionizing radiation, elevated recombination, cell cycle abnormalities, and aberrant cytoskeletal organization. The molecular basis of the defect is unknown. A candidate AT gene (ATDC) was isolated on the basis of its ability to complement the ionizing radiation sensitivity of AT group D fibroblasts. Whether ATDC is mutated in any AT patients is not known. We have found that the ATDC protein physically interacts with the intermediate-filament protein vimentin, which is a protein kinase C substrate and colocalizing protein, and with an inhibitor of protein kinase C, hPKCI-1. Indirect immunofluorescence analysis of cultured cells transfected with a plasmid encoding an epitope-tagged ATDC protein localizes the protein to vimentin filaments. We suggest that the ATDC and hPKCI-1 proteins may be components of a signal transduction pathway that is induced by ionizing radiation and mediated by protein kinase C.

    View details for Web of Science ID A1995RP74800046

    View details for PubMedID 7644499



    We have determined the effects of chronic exposure to the protein kinase C (PKC) activating drug 4-beta phorbol 12-myristate-13-acetate (PMA) on PKC isozymes and the rate of spontaneous contraction in neonatal rat cardiac myocytes in culture. Western blot analyses revealed that a two day exposure to 0.1-1 nM PMA increased the total amount of delta PKC, whereas, 100 nM PMA concentrations caused a complete down-regulation of the alpha PKC and an 80 kDa zeta PKC-like protein. In addition, 100 nM PMA treatment for 2 days did not result in complete down-regulation of the beta, delta, and epsilon PKC isozymes in Western blot and immunocytochemical studies. We also found a PKC-mediated enhancement of the rate of contraction in these cells following prolonged exposure to PMA (1-100nM). Our studies suggest that this enhancement of contraction rate may be mediated by the beta, delta, or epsilon PKC isozymes. A better understanding of the role(s) of PKC isozymes in the modulation of cardiac functions may reveal selective targets for therapies of cardiac disorders.

    View details for Web of Science ID A1995RN15200002

    View details for PubMedID 7658910


    View details for Web of Science ID A1995RU08500036

    View details for PubMedID 8566424



    A fundamental question in signal transduction is how stimulation of a specific protein kinase leads to phosphorylation of particular protein substrates throughout the cell. Recent studies indicate that specific anchoring proteins located at various sites in the cell compartmentalize the kinases to their sites of action. Inhibitors of the interactions between kinases and their anchoring proteins inhibit the functions mediated by the kinases. These data indicate that the location of these anchoring proteins provides some of the specificity of the responses mediated by each kinase and suggest that inhibitors of the interaction between the kinases and their anchoring proteins may be useful as therapeutic agents.

    View details for Web of Science ID A1995QT18500024

    View details for PubMedID 7716516



    Protein kinase C (PKC) enzymes regulate numerous cardiac functions. In the present study, we determined the effects of the PKC-activating drug 4-beta phorbol 12-myristate 13-acetate (4-beta PMA) on the rate of contraction and correlated these changes with the distribution and levels of alpha-, beta-, delta-, epsilon-, and zeta-PKC isozymes by using neonatal rat cardiac myocytes in culture. Treatment with 0.3 to 100 nmol/L 4-beta PMA caused negative chronotropic effects on contraction. This effect was maximal at a concentration of 3 nmol/L 4-beta PMA and correlated with redistribution of the alpha- and epsilon-PKC isozymes from the cytosolic to the particulate cell fraction. After a 1-hour treatment with 100 nmol/L PMA, the alpha- and beta-PKC isozymes and an 80-kD zeta-like PKC isozyme were greatly diminished (downregulated), yet the negative chronotropic effect was sustained. Therefore, our results are most consistent with a role for the epsilon-PKC isozyme in suppressing the contraction rate of neonatal rat cardiac myocytes. Understanding the role(s) of individual PKC isozymes in the modulation of cardiac functions may ultimately yield more selective targets for therapies of cardiac disorders.

    View details for Web of Science ID A1995QQ51800019

    View details for PubMedID 7895339



    We have previously identified receptors for activated C kinase (RACKs) as components of protein kinase C (PKC) signaling. RACK1, a recently cloned 36-kDa RACK, has short sequences of homology to PKC. A possible explanation for the homologous sequences between the ligand (PKC) and its intracellular receptor (RACK1) may be that, similar to the pseudosubstrate autoregulatory sequence on PKC, there is also a pseudo-RACK1 binding site on the enzyme. If this is the case, peptides with these sequences (derived from either RACK1 or PKC) are expected to affect PKC binding to RACK1 in vitro and PKC-mediated functions in vivo. Here, we show that the PKC-derived peptide (pseudo-RACK1 peptide), but not its RACK1 homologue, modulated PKC function both in vitro and in vivo. Our data suggest that the pseudo-RACK1 peptide binds and activates PKC in the absence of PKC activators and thereby acts as an agonist of PKC function in vivo. Therefore, the pseudo-RACK1 sequence in PKC appears to be another autoregulatory site; when PKC is in an inactive conformation, the pseudo-RACK1 site interacts with the RACK-binding site. Activation of PKC exposes the RACK-binding site, enabling the association of the enzyme with its anchoring RACK. Similar pseudoanchoring sites may regulate the function of other protein kinases.

    View details for Web of Science ID A1995QC87400033

    View details for PubMedID 7831317



    Physical association between proteins involved in signal transduction is required for their functions. Therefore, identification of the interacting sites in the signaling molecules can lead to the development of means to modulate these interactions. We applied this approach to study signal transduction by protein kinase C (PKC). We have previously identified potential PKC binding sites in two PKC binding proteins (annexin I and RACK1). Peptides derived from these sequences inhibit PKC binding to RACK1 in vitro. Here, we tested the ability of two of these peptides, I (KGDYEKILVALCGGN) and rVI (DIINALCF), to affect PKC-mediated function in vivo. The peptides were microinjected into Xenopus oocytes, and insulin-induced beta PKC translocation and oocyte maturation were examined. The peptides had opposite activities on oocyte; peptide I inhibited whereas peptide rVI stimulated insulin-induced Xenopus oocyte maturation. As expected, beta PKC translocation from the cytosol to the particulate fraction of the Xenopus oocytes was inhibited after microinjection of peptide I and induced after microinjection of peptide rVI. Moreover, peptide rVI caused translocation of beta PKC and oocyte maturation without hormone stimulation. In the absence of PKC activators, peptide rVI but not peptide I, activated PKC in vitro as demonstrated in three assays: increased sensitivity to Arg-C endopeptidase, PKC autophosphorylation, and histone phosphorylation. Therefore, although peptides I and rVI have sequence homology, one mimicked hormone-induced PKC-mediated function whereas the other inhibited this hormone-induced function. The molecular mechanisms underlying these opposing effects of the peptides are discussed.

    View details for Web of Science ID A1994PK97300002

    View details for PubMedID 8063768



    We have studied the effect of activation of the c-erbB-2 receptor tyrosine kinase on protein kinase C (PKC) in cultured SKBR-3 human breast cancer cells. Treatment with the agonistic anti-receptor monoclonal antibody TAb 250 induces receptor autophosphorylation and stimulates phospholipase C-gamma 1 (L. K. Shawver et al. Cancer Res., 54: 1367-1373, 1994). TAb 250 induced a rapid and marked translocation of PKC histone phosphorylation activity to the particulate fraction of SKBR-3 cells. By immunoblot, however, this translocation was limited to specific PKC isozymes. beta PKC and zeta PKC translocated to the particulate fraction, whereas epsilon PKC underwent "partial reversed translocation" to the cell soluble fraction after receptor stimulation. Furthermore, beta PKC was rapidly degraded following TAb 250 treatment. By immunocytochemistry, beta IPKC translocated from the perinuclear area to the cytosol and into the nucleus, whereas zeta PKC translocated to the perinuclear region and into the nucleus. Consistent with the Western blot results, epsilon PKC translocated from the nucleus to the perinuclear area and the cytosol. These changes in the localization of PKC isozymes were not observed after addition of normal IgG1 or a nonagonistic anti-c-erbB-2 monoclonal antibody to SKBR-3 cells. alpha, beta II, or delta PKC present in these cells did not translocate following receptor stimulation. These data indicate that c-erbB-2 signal transduction may involve the activation of specific PKC isozymes. The biological role of these enzymes in the phenotype and cellular responses of c-erbB-2-overexpressing carcinoma cells remains to be studied.

    View details for Web of Science ID A1994PB14900009

    View details for PubMedID 7986752



    We have examined the neonatal developmental expression of protein kinase C subspecies (PKCs) in rat brain, pituitary glands and cells by enzymatic activity assays, immunohistochemistry and Western blot analysis with type-specific antibodies. A very large increase (455%) was noticed in brain PKC activity during the first week of life with the particulate fraction (22% of total enzyme activity on day 1) increasing dramatically (900%) during the first week to 50% of enzyme activity. In contrast, the pituitary gland showed high activity on day 1 that decreased progressively to reach the lowest levels at 1 year of age. Paradoxically, the number of pituitary cells immunolabeled for PKC increases as a function of age. Western blot analysis showed only small changes in PKC alpha, PKC beta and PKC epsilon when brains from 6-day-old and 3-month-old female rats were compared, whereas PKC tau and PKC delta increased markedly during this period. On the other hand, brain PKC zeta decreased between 6 days and 3 months of age. Western blot analysis showed no major changes in pituitary PKC alpha, PKC beta and PKC zeta when 6-day-old and 3-month-old female rats were compared, while PKC tau was not detected. The major band of pituitary PKC delta (76 kDa) decreased markedly between 6 days and 3 months of age whereas the minor band (68 kDa) did not change.(ABSTRACT TRUNCATED AT 250 WORDS)

    View details for Web of Science ID A1994NU19800018

    View details for PubMedID 7958391



    We observed that phorbol myristate acetate (PMA) stimulates transcytosis of the polymeric immunoglobulin receptor (pIgR) in MDCK cells. Apical release of pre-endocytosed ligand (dimeric IgA) bound to the pIgR can be stimulated twofold within 7 min of addition of PMA while recycling of the ligand from the basal surface is not affected. In addition, apical surface delivery of pIgR and cleavage of its ectodomain to secretory component (SC) is also stimulated by PMA. The recycling of apically internalized ligand back to the apical surface is similarly stimulated. These results suggest that the stimulation of apical delivery is from an apical recycling compartment. The effect of PMA suggests that protein kinase C (PKC) is involved in the regulation of pIgR trafficking in MDCK cells. To test this we down regulated PKC activity by pre-treating cells with PMA for 16 h and observed that transcytosis could no longer be stimulated by PMA. Western blots show that the PKC isozymes alpha and to a lesser extent epsilon, are depleted from MDCK cells which have been pre-treated with PMA for 16 h and that treatment of MDCK cells with PMA for 5 min causes a dramatic translocation of the PKC alpha isozyme and a partial translocation of the epsilon isozyme from the cytosol to the membrane fraction of cell homogenates. This translocation suggests that the alpha and/or epsilon isozymes may be involved in PMA mediated stimulation of transcytosis. A mutant pIgR in which serines 664 and 726, the major sites of phosphorylation, are replaced by alanine is stimulated to transcytose by PMA, suggesting that phosphorylation of pIgR at these sites is not required for the effect of PMA. These results suggest that PMA-mediated stimulation of pIgR transcytosis may involve the activation of PKC alpha and/or epsilon, and that this stimulation occurs independently of the major phosphorylation sites on the pIgR. Finally, PMA stimulates transcytosis of basolaterally internalized transferrin, suggesting that PMA acts to generally stimulate delivery of endocytosed proteins to the apical surface.

    View details for Web of Science ID A1994MY84600008

    View details for PubMedID 8120094



    Protein kinase C (PKC) translocates from the soluble to the cell particulate fraction on activation. Intracellular receptors that bind activated PKC in the particulate fraction have been implicated by a number of studies. Previous work identified 30- to 36-kDa proteins in the particulate fraction of heart and brain that bound activated PKC in a specific and saturable manner. These proteins were termed receptors for activated C-kinase, or RACKs. In the following study, we describe the cloning of a cDNA encoding a 36-kDa protein (RACK1) that fulfills the criteria for RACKs. (i) RACK1 bound PKC in the presence of PKC activators, but not in their absence. (ii) PKC binding to the recombinant RACK1 was not inhibited by a pseudosubstrate peptide or by a substrate peptide derived from the pseudosubstrate sequence, indicating that the binding did not reflect simply PKC association with its substrate. (iii) Binding of PKC to RACK1 was saturable and specific; two other protein kinases did not bind to RACK1. (iv) RACK1 contains two short sequences homologous to a PKC binding sequence previously identified in annexin I and in the brain PKC inhibitor KCIP. Peptides derived from these sequences inhibited PKC binding to RACK1. Finally, RACK1 is a homolog of the beta subunit of G proteins, which were recently implicated in membrane anchorage of the beta-adrenergic receptor kinase [Pitcher, J., Inglese, L., Higgins, J. B., Arriza, J. A., Casey, P. J., Kim, C., Benovic, J. L., Kwatra, M. M., Caron, M. G. & Lefkowitz, R. J. (1992) Science 257, 1264-1267]. Our in vitro data suggest a role for RACK1 in PKC-mediated signaling.

    View details for Web of Science ID A1994MV27800003

    View details for PubMedID 8302854

    View details for PubMedCentralID PMC521407



    Activation of protein kinase C (PKC) isozymes is associated with their translocation from the cell-soluble fraction to the cell-particulate fraction, presumably near their protein substrates. Therefore, identifying the subcellular localization of each activated PKC isozyme may help to elucidate its role in cardiac functions. In the present work, we have determined the subcellular localization of six PKC isozymes (alpha, beta I, beta II, delta, epsilon, and zeta) in nonstimulated cardiac myocytes and in myocytes stimulated by norepinephrine (2 microM) or phorbol 12-myristate 13-acetate (100 nM). Activated PKC isozymes were localized in various subcellular compartments such as inside the nucleus and on myofibrils. The presence of serum in the growth medium also caused a redistribution of PKC isozymes in the cells distinct from that obtained with cells cultured in defined medium. We suggest that isozyme-specific localization may determine phosphorylation of different protein substrates present at these respective translocation sites and the resulting PKC-mediated cellular responses.

    View details for Web of Science ID A1994MV44000020

    View details for PubMedID 8299726



    Phospholipase C-gamma 1 (PLC-gamma 1; EC hydrolyzes phosphatidylinositol 4,5-bisphosphate to generate diacylglycerol and inositol 1,4,5-trisphosphate and is activated in response to growth factor stimulation and tyrosine phosphorylation. Concomitantly, the enzyme translocates from the cytosol to the particulate cell fraction. A similar process of activation-induced translocation from the cytosol to the cell particulate fraction has also been described for protein kinase C (PKC). We have previously shown that activated PKC binds to specific receptor proteins, receptors for activated C kinase, or RACKs, of approximately 30 kDa. Here, we show that PLC-gamma 1 bound to these RACKs and inhibited subsequent PKC binding to RACKs. However, unlike PKC, the binding of PLC-gamma 1 to RACKs did not require phospholipids and calcium. After epidermal growth factor treatment of intact A-431 cells, the binding of PLC-gamma 1 to RACKs increased as compared with PLC-gamma 1 from control cells. This increase in PLC-gamma 1 binding to RACKs was due to the phosphorylation of PLC-gamma 1. Additional data indicated that PLC-gamma 1 binds to RACKs in solution; epidermal growth factor receptor-dependent PLC-gamma 1 phosphorylation and activation decreased in the presence of RACKs. It is possible that, in vivo, PLC-gamma 1 associates with RACKs or with other PLC-gamma 1-specific anchoring proteins in the particulate cell fraction. Since a PKC C2 homologous region is present in PLC-gamma 1, the C2 region may mediate the activation-induced translocation of the enzyme to the cell particulate fraction and the anchoring protein-PLC-gamma 1 complex may be the active translocated form of PLC-gamma 1.

    View details for Web of Science ID A1994MR98900028

    View details for PubMedID 8290562



    We tested the hypothesis that the translocation and function of protein kinase C (PKC) requires the binding of PKC to its intracellular receptors (RACKs), using insulin-induced maturation of Xenopus oocytes. We show that after exposure of oocytes to insulin, PKC translocated from the cytosol to the particulate fraction. PKC is also required for insulin-induced oocyte maturation: microinjection of a PKC inhibitory peptide delayed maturation. To determine whether translocation of PKC was a result of the binding of PKC to the RACKs in the particulate fraction, we microinjected purified rat brain RACKs into oocytes before insulin exposure. Microinjection of RACKs, but not inactive phosphorylated RACKS, inhibited PKC translocation and delayed oocyte maturation. These results suggest an in vivo role for RACKs in a function mediated by PKC.

    View details for Web of Science ID A1992JY44600039

    View details for PubMedID 1332715



    Adenosine modulation of nicotinic ACh receptor (nAChR) function was studied in primary cultures of rat skeletal muscle. Activation of the nAChR by carbachol increased extracellular adenosine concentration in a dose-dependent manner. Furthermore, carbachol activation of the nicotinic receptor resulted in a twofold increase in cAMP levels in the muscle cells. The carbachol-dependent increase in cAMP levels was inhibited by adenosine receptor antagonists as well as by nicotinic receptor antagonists. These results suggest that the increased cAMP levels were due to adenosine receptor activation by the extracellular adenosine accumulated on nAChR activation. Others have shown that desensitization of the nAChR by agonist is mediated, in part, by phosphorylation. Since we found that nicotinic cholinergic agonists also cause adenosine accumulation with concomitant cAMP increases, we determined whether the accumulated adenosine has a role in desensitization. We found that the adenosine receptor antagonist, BW1434U, significantly inhibited carbachol-induced nAChR desensitization, indicating that extracellular adenosine is involved in nAChR desensitization. Our data suggest that nAChR function is regulated via a feedback mechanism mediated by adenosine released from muscle on activation of the nAChR.

    View details for Web of Science ID A1992JY18800034

    View details for PubMedID 1331363



    Receptors for activated protein kinase C (RACKs) have been isolated from the particulate cell fraction of heart and brain. We previously demonstrated that binding of protein kinase C (PKC) to RACKs requires PKC activators and is via a site on PKC that is distinct from the substrate binding site. Here, we examine the possibility that the C2 region in the regulatory domain of PKC is involved in binding of PKC to RACKs. The synaptic vesicle-specific p65 protein contains two regions homologous to the C2 region of PKC. We found that three p65 fragments, containing either one or two of these PKC C2 homologous regions, bound to highly purified RACKs. Binding of the p65 fragments and PKC to RACKs was mutually exclusive; preincubation of RACKs with the p65 fragments inhibited PKC binding, and preincubation of RACKs with PKC inhibited binding of the p65 fragments. Preincubation of the p65 fragments with a peptide resembling the PKC binding site on RACKs also inhibited p65 binding to RACKs, suggesting that PKC and p65 bind to the same or nearby regions on RACKs. Since the only homologous region between PKC and the p65 fragments is the C2 region, these results suggest that the C2 region on PKC contains at least part of the RACK binding site.

    View details for Web of Science ID A1992JM57200003

    View details for PubMedID 1326322



    Protein kinase C (PKC) isozymes comprise a family of cytosolic enzymes that translocate to different intracellular sites on activation. We have recently characterized at least two intracellular receptor proteins for PKC (termed RACKs for receptors for activated C-kinase) in the Triton-insoluble material of the particulate fraction from neonatal rat heart. Here, we identify a sequence that appears to resemble the PKC binding site on these RACKs. A peptide (peptide I) with the sequence KGDYEKILVALCGGN bound PKC, and binding was markedly increased in the presence of PKC activators. Furthermore, peptide I inhibited PKC binding to RACKs in a dose-dependent manner. These data suggest that these RACKs have a common PKC binding sequence. Since peptide I inhibited PKC binding to RACKs in vitro, it may be a useful tool to inhibit PKC translocation and subsequent function in vivo.

    View details for Web of Science ID A1991GB09700010

    View details for PubMedID 1831196



    Acute exposure to ethanol in culture inhibits adenosine uptake into cells, thereby increasing the concentration of extracellular adenosine. Extracellular adenosine then reacts with adenosine A2 receptors to stimulate intracellular cAMP production. During prolonged exposure to ethanol, the increase in cAMP is followed by the development of heterologous desensitization of receptors coupled to adenylyl cyclase via Gs, the stimulatory GTP-binding protein. Ethanol-induced heterologous desensitization appears to be due to a reduction in mRNA and protein for G alpha s, a subunit of Gs. This is an example of cellular dependence on ethanol. The important implication of these findings is that a selective inhibitory effect of ethanol on adenosine uptake can lead to desensitization of diverse receptors coupled to cAMP production. Such changes could contribute to the pleiotropic effects of ethanol in the brain and other organs. Prolonged exposure to ethanol also alters the nucleoside transport system. While ethanol inhibits adenosine uptake into naive cells, ethanol no longer inhibits adenosine uptake into cells that have adapted to ethanol. This resistance to ethanol inhibition appears to be a form of cellular tolerance to ethanol. Thus, there appears to be a synergism between ethanol-induced heterologous desensitization of receptor-stimulated cAMP production (cellular dependence) and resistance to ethanol inhibition of adenosine uptake (cellular tolerance), because both lead to reduced intracellular levels of cAMP. Our studies on cAMP signal transduction in cell culture are directly relevant to the pathophysiology of human alcoholism. Heterologous desensitization of cAMP production is demonstrable in lymphocytes taken from actively drinking alcoholics; this measurement appears to be a biologic marker of active alcohol consumption. In addition, regulation of adenosine receptor-dependent cAMP production may be altered in patients at risk to develop alcoholism because of genetic factors. Thus, lymphocytes from alcoholics cultured many generations in the absence of ethanol show increased adenosine receptor-dependent cAMP production and increased sensitivity to ethanol-induced heterologous desensitization. These persistent phenotypic abnormalities in cell culture could be used as genetic markers for alcoholism. Studies are under way to test this possibility.

    View details for Web of Science ID A1991FZ45500054

    View details for PubMedID 2058901



    Protein kinase C (PKC) translocates from the cytosol to the particulate fraction on activation. This activation-induced translocation of PKC is thought to reflect PKC binding to the membrane lipids. However, immunological and biochemical data suggest that PKC may bind to proteins in the cytoskeletal elements in the particulate fraction and in the nuclei. Here we describe evidence for the presence of intracellular receptor proteins that bind activated PKC. Several proteins from the detergent-insoluble material of the particulate fraction bound PKC in the presence of phosphatidylserine and calcium; binding was further increased with the addition of diacylglycerol. Binding of PKC to two of these proteins was concentration-dependent, saturable, and specific, suggesting that these binding proteins are receptors for activated C-kinase, termed here "RACKs." PKC binds to RACKs via a site on PKC distinct from the substrate binding site. We suggest that binding to RACKs may play a role in activation-induced translocation of PKC.

    View details for Web of Science ID A1991FK18400100

    View details for PubMedID 1850844



    We have used the Torpedo electric organ to study GTP-binding protein localization since functionally distinct membrane fractions can be isolated from this tissue. Postsynaptic membranes from the innervated face and membranes from the non-innervated face of the electrocyte, as well as presynaptic membranes from the innervating nerve, can be isolated. alpha s was restricted to the innervated face of the postsynaptic cell; alpha i, alpha o, and ras were found only in the presynaptic membrane fraction of the innervating nerve. 21 and 25 kDa GTP-binding proteins were present in all the membrane fractions. These results suggest that specific GTP-binding proteins are differentially restricted to membrane areas specialized in signal transduction.

    View details for Web of Science ID A1990EL63000059

    View details for PubMedID 2124113



    Protein kinase C (PKC)1 isozymes comprise a family of related cytosolic kinases that translocate to the cell particulate fraction on stimulation. The activated enzyme is thought to be on the plasma membrane. However, phosphorylation of protein substrates occurs throughout the cell and is inconsistent with plasma membrane localization. Using an isozyme-specific monoclonal antibody we found that, on activation, this PKC isozyme translocates to myofibrils in cardiac myocytes and to microfilaments in fibroblasts. Translocation of this activated PKC isozyme to cytoskeletal elements may explain some of the effects of PKC on cell contractility and morphology. In addition, differences in the translocation site of individual isozymes--and, therefore, phosphorylation of different substrates localized at these sites--may explain the diverse biological effects of PKC.

    View details for Web of Science ID A1990DZ34000008

    View details for PubMedID 2078573



    Chronic exposure of cultured cell lines to ethanol results in a heterologous desensitization of receptors coupled to adenylate cyclase via GS, the stimulatory guanine nucleotide regulatory protein. This heterologous desensitization is accompanied by a decrease in alpha S, the GTP-binding subunit of GS. Ethanol-induced accumulation of extracellular adenosine is required for the development of heterologous desensitization. To determine the mechanism underlying the ethanol-dependent increase in extracellular adenosine, we investigated the effects of ethanol on the nucleoside transporter responsible for the bidirectional transport of adenosine into and out of the cell. We found that ethanol specifically and non-competitively inhibited nucleoside uptake. Inhibition of adenosine uptake was primarily due to decreased influx via the nucleoside transporter. Thus, ethanol-induced increases in extracellular adenosine appear to be due to inhibition of adenosine influx. After chronic exposure to ethanol, cells became tolerant to the acute effects of ethanol, i.e. ethanol no longer inhibited uptake and, consequently, no longer increased extracellular adenosine concentration. Taken together with our previous studies, these results suggest that acute ethanol inhibition of adenosine influx leads to an increase in extracellular adenosine which in turn activates adenosine A2 receptors to increase cyclic AMP levels, leading to desensitization of receptor-dependent cyclic AMP signal transduction after chronic exposure to ethanol. We next determined whether the same effects of ethanol also occur in alcoholics. We isolated lymphocytes from alcoholics and non-alcoholics and found that alcoholics had a 75% decrease in basal and adenosine receptor-stimulated cyclic AMP production compared with non-alcoholics. To determine whether these differences were due to exposure to ethanol in vivo or to a possible genetic difference between alcoholics and non-alcoholics, we grew lymphocytes in culture in the absence of ethanol. Adenosine receptor-stimulated cyclic AMP levels were higher in alcoholics than non-alcoholics. Moreover, cultured cells from alcoholics were more sensitive to the effects of chronic alcohol on cyclic AMP signal transduction than cells from non-alcoholics. Our results suggest that the cyclic AMP signal transduction system may reflect a genetic alteration in alcoholics and that studies in cultured lymphocytes may allow us to identify individuals at risk of developing alcoholism.

    View details for Web of Science ID A1990GR37100012

    View details for PubMedID 2175189



    One of the biochemical results of ethanol exposure is a change in the amount of the intracellular second messenger cyclic AMP (cAMP) produced in response to receptor stimulation. In general, acute ethanol exposure increases the amount of cAMP produced on stimulation of receptors coupled to the enzyme adenylyl cyclase via the GTP-binding protein Gs, whereas chronic ethanol exposure has the opposite effect (results for receptors coupled via Gi have been more variable). We previously reported that adaptation to continuous ethanol exposure reduces receptor-stimulated cAMP production by 25-35% in a neuroblastoma cell line (NG108-15), and an even greater reduction of 75% was observed in lymphocytes taken from actively-drinking alcoholics. This reduction in receptor-stimulated cAMP levels was recently confirmed in platelets from alcoholics. None of these studies, however, determined whether more than one receptor coupled to adenylyl cyclase activity was affected in the same cell. Here we report that chronic ethanol exposure causes desensitization of heterologous receptors coupled to Gs as cAMP production mediated by prostaglandin E1 as well as by adenosine is reduced by approximately 30% in NG108-15 cells. We show that, after chronic ethanol exposure, the activity of the alpha subunit of Gs is decreased by 29%, the amount of alpha s protein is decreased by 38.5%, and alpha s messenger RNA is decreased by 30%. Thus, cellular adaptation to ethanol involves a reduction in alpha s mRNA and, as a consequence, reduced cAMP production by heterologous receptors coupled to Gs. Such changes in cAMP production may account for the tolerance and physical dependence on ethanol in alcoholism.

    View details for Web of Science ID A1988P029200051

    View details for PubMedID 2838757



    In this communication we describe a microfiltration assay to identify monoclonal antibodies that interfere with the activity of enzymes. This method is quick and sensitive to small changes in the activity of the enzyme and does not require highly purified enzyme or large quantities of antibodies. It has been applied to identify anti-protein kinase C antibodies which would have been impossible to identify by classical assays such as enzyme-linked immunosorbent assay.

    View details for Web of Science ID A1988M799600004

    View details for PubMedID 3291639



    Monoclonal antibodies raised against highly purified protein kinase C were used to localize protein kinase C in the rat brain. Using various monoclonal antibodies, at least three distinct antibody-staining patterns were found. One monoclonal antibody exclusively labeled astroglial elements, including astrocytes, tanycytes, and cerebellar radial glia. Another monoclonal antibody exclusively labeled neural cells, including cortical and hippocampal pyramidal dendrites and Purkinje cells of the cerebellum. A third monoclonal antibody (which inhibited protein kinase C activity) intensely stained more limited brain regions, particularly thalamic neurons, and also stained astroglial structures in brain, spinal cord, and cerebellum. The possibility that the three staining patterns reflect the differential regional and cellular localization of related, but distinct, enzymes of protein kinase C is discussed.

    View details for Web of Science ID A1987J070000065

    View details for PubMedID 3299372



    To gauge similarities between S100b protein and calmodulin, interactions were observed between S100b and melittin and between S100b and tau, the microtubule-associated proteins. The interaction of melittin with S100b protein in the presence and absence of calcium was studied by fluorescence polarization, UV difference spectroscopy, and sulfhydryl derivatization. Whether calcium was present or not in the solution, melittin and S100b form a complex of molar ratios up to 2:1. Further binding of melittin occurred, but it resulted in precipitation of S100b, as is true of the corresponding case of melittin binding to calmodulin. In the absence of calcium, the interaction of melittin and S100b shielded the tryptophan (Trp) of the former protein and exposed cysteine-84 beta (Cys-84 beta) of the latter protein, leaving the tyrosine-16 beta (Tyr-16 beta) of S100b unaffected. Calcium addition to the complex partially restored the exposure of Trp of melittin and caused changes in the environment of Tyr-16 beta (unlike the environmental changes induced for Tyr-16 beta by calcium in the absence of melittin). The conformational changes induced in S100b by interaction with melittin increased its affinity for calcium and offset the inhibition of calcium binding otherwise observed in the presence of potassium ions. This corroborated the previous finding that S100b affinity for calcium greatly depends on the protein conformation. The phenomena described above are similar to the interactions of melittin with calmodulin and thus suggest that S100b and calmodulin have a common structural domain not only that binds melittin but also that may interact with common target proteins.(ABSTRACT TRUNCATED AT 250 WORDS)

    View details for Web of Science ID A1987H415000033

    View details for PubMedID 3111527



    Protein kinase C (Ca2+/phospholipid-dependent protein kinase) purified from rat brain or endogenous to cell-free extracts from HeLa cells stimulates, by a factor of 2-3, HeLa DNA polymerase alpha but not beta or gamma. Monoclonal antibody to the kinase prevents the stimulation, and monoclonal antibody to human DNA polymerase alpha neutralizes the enhanced activity. Reduced DNA polymerase alpha activity is obtained from noncycling HeLa cells and this activity has lower fidelity when copying synthetic primer-templates than that obtained from log phase cultures. After exposure to the kinase, the fidelities and activities of the polymerase from both sources increase by 2- to 3-fold. This improved accuracy is not accompanied by the appearance of triphosphatase or DNase activities. Exposure to the protein kinase reduces the Km for activated DNA and for poly(dA-dT) but not for dNTPs. Moreover, the Vmax for activated DNA but not for poly(dA-dT) is increased approximately 2- to 3-fold. These alterations suggest a role for protein phosphorylation in modulating DNA polymerase alpha.

    View details for Web of Science ID A1987G477800002

    View details for PubMedID 3818650



    The phospholipid- and calcium-dependent protein kinase C has been shown to autophosphorylate on both the catalytic and the regulatory domains. The autophosphorylation displays zero-order kinetics, indicating that it is an intramolecular event. Autophosphorylation increases the activity of protein kinase C by decreasing the Km for the substrate H1 histone. The catalytic fragment obtained by limited proteolysis can no longer autophosphorylate and has a reduced affinity for H1 histone, exhibiting a Km 5-fold higher than that of the intact enzyme. Monoclonal antibodies produced against the enzyme can distinguish between the catalytic fragment and the intact enzyme by inhibiting their activities in a specific manner. Evidence suggesting that dimerization of protein kinase C occurs upon activation is presented.

    View details for Web of Science ID A1987G509800057

    View details for PubMedID 3818597



    The beta subunit of purified insulin receptor is phosphorylated on a serine residue by purified preparations of protein kinase C (ATP: protein phosphotransferase, EC This phosphorylation is inhibited by antibodies to protein kinase C and stimulated by phospholipids, diacylglycerol, and Ca2+. The phosphorylation of the receptor by protein kinase C does not affect its insulin-binding activity but does inhibit by 65% the receptor's intrinsic tyrosine-specific protein kinase activity (ATP: protein-tyrosine O-phosphotransferase, EC These results indicate that activators of protein kinase C, such as phorbol esters, desensitize cells to insulin by direct protein kinase C action on the insulin receptor.

    View details for Web of Science ID A1986D754900016

    View details for PubMedID 3526339

    View details for PubMedCentralID PMC386387



    Monoclonal antibodies (mcAbs) elicited against the nicotinic acetylcholine receptor (AChR) from Torpedo, were used to follow antigenic changes in AChR during muscle development. Newborn rat muscle and denervated mouse muscle were used as sources of extrajunctional AChR; adult innervated rat and mouse muscle were used as sources of junctional AChR. Most of the mcAbs tested reacted preferably, but not exclusively with extrajunctional AChR (EJR), as compared to junctional AChR (JR). None was found to react with only one of the two forms of AChR. We conclude that the anti-AChR monoclonal antibodies used in this study detect antigenic determinants which are shared by EJR and JR, but which probably undergo structural changes during muscle development.

    View details for Web of Science ID A1985AJG3100009

    View details for PubMedID 2581995

  • AN ANTI-ACETYLCHOLINE RECEPTOR MONOCLONAL-ANTIBODY CROSS-REACTS WITH PHOSVITIN FEBS LETTERS Pizzighella, S., Gordon, A. S., Souroujon, M. C., MOCHLYROSEN, D., Sharp, A., Fuchs, S. 1983; 159 (1-2): 246-250


    Rabbit and mouse anti-Torpedo acetylcholine receptor antibodies cross-reacted partially with the highly phosphorylated protein, phosvitin. We have selected an anti-Torpedo acetylcholine receptor monoclonal antibody which binds specifically to phosvitin; this binding is inhibited by acetylcholine receptor. These findings suggest that a phosphorylated amino acid residue may be a part of the determinant on the acetylcholine receptor recognized by this monoclonal antibody.

    View details for Web of Science ID A1983RD45900052

    View details for PubMedID 6192017



    Several monoclonal antibodies (mcAbs) elicited against the nicotinic acetylcholine receptor (AChR) from Torpedo react also with skeletal muscle AChR. Such mcAbs were used to define antigenic determinants on muscle AChR and to elucidate their effect on muscle AChR functions. Primary chick muscle cultures were used as a model for skeletal muscle. Of the four mcAbs studies only mcAb 5.5, which is directed against the cholinergic site in Torpedo AChR, blocks the binding of alpha-bungarotoxin (alpha-Bgt) to AChR in chick muscle cultures and inhibits carbamylcholine-induced sodium transport in these cells. The interaction of mcAb 5.5 with the cholinergic site on muscle AChR demonstrates the conservation of this site. Two mcAbs, 5.5 and 5.34, each of a different antigenic specificity but both directed against conformation-dependent antigenic determinants, accelerate the degradation of AChR in muscle cultures. From the reactivity of the various mcAbs with Triton-solubilized and membranous AChR it appears that there are some antigenic differences between the detergent solubilized and membranous forms of the receptor.

    View details for Web of Science ID A1983QR36400009

    View details for PubMedID 6866010



    Monoclonal antibodies directed against the cholinergic binding site of the acetylcholine receptor were found to alter the ion channel properties in cultured chick "myoballs." Time and dose dependent reduction in acetylcholine sensitivity was observed. Noise analysis experiments indicated a decrease in the mean single channel conductance and an increase in the mean single channel open time.

    View details for Web of Science ID A1983RP34500003

    View details for PubMedID 6315946


    View details for Web of Science ID A1981NE12700008

    View details for PubMedID 6176165

  • Molecular aspects of experimental autoimmune myasthenia gravis. Progress in clinical and biological research Fuchs, S., Bartfeld, D., Mochly-Rosen, D., Schmidt-Hopfeld, I., Tarrab-Hazdai, R. 1981; 63: 405-417


    Structure and function studies on acetylcholine receptor (AChR) were attained by studying various derivatives of the receptor molecule, by analysis of monoclonal antibodies, and by investigating the possible structural relationship between AChR and the thymus. Pharmacologically inactive denatured AChR preparation does not induce myasthenia in rabbits, although it elicits antibodies that cross-react with the intact receptor. Denatured AChR has both preventive and therapeutic effects on experimental autoimmune myasthenia gravis. Mild tryptic digestion of AChR does not abolish the pharmacological specificity and myasthenic activity of the receptor. Trypsinated AChR, which on SDS gel electrophoresis shows one major band with a molecular weight of 27,000, represents an active receptor derivative with a relatively low structural complexity. Monoclonal antibodies against defined determinants of AChR were elicited and characterized. One monoclonal antibody line is directed against the cholinergic binding site. Experiments demonstrating that thymic lymphocytes bear a surface antigen that cross-reacts with AChR are described.

    View details for PubMedID 7031685



    We have isolated 32 hybridoma cell lines producing monoclonal antibodies against the acetylcholine receptor from Torpedo californica. One of these lines, designated 5.5.G.12, secretes antibodies which are directed against the cholinergic binding site of the acetylcholine receptor. This specific antibody blocked the binding of alpha-bungarotoxin to the acetylcholine receptor. The binding of monoclonal antibody 5.5.G.12 to acetylcholine receptor was inhibited by alpha-neurotoxins and by other cholinergic ligands in accordance with their affinities to the nicotinic acetylcholine receptor. None of the other monoclonal antibodies obtained inhibited the binding of alpha-bungarotoxin to acetylcholine receptor, nor was their binding to the acetylcholine receptor inhibited by cholinergic ligands. The monoclonal antibody elicited against the binding site of Torpedo acetylcholine receptor bound also to acetylcholine receptors of various species and organs, demonstrating the wide structural homology between the cholinergic sites of various acetylcholine receptors.

    View details for Web of Science ID A1981MJ45500041

    View details for PubMedID 7295707


    View details for Web of Science ID A1980KF75100006

    View details for PubMedID 6157569



    Experimental autoimmune myasthenia gravis (EAMG) is an appropriate model for studying the molecular origin, immunological mechanism and regulation of myasthenia gravis. Several approaches are being utilised for the regulation of the immune response to AChR and for immunosuppression of EAMG: Corticosteriods and azathioprine can suppress EAMG concomitantly with suppression of immune responses to AChR. High dose cyclophosphamide treatment in mice facilitates the onset of EAMG and results in a selective suppression of the humoral response to AChR whereas the cellular response is enhanced. Specific immunosuppression of EAMG is achieved by using a nonmyasthenic, denatured AChR preparation which cross reacts with the intact receptor. Various degradations and modifications of AChR are being performed in order to identify the smallest molecular entity responsible for the myasthenic activity of AChR. Studies on specific monoclonal antibodies, anti-idiotypes, and on the effect of measles virus on EAMG are being described and their possible significance in regulating myasthenia are being discussed.

    View details for Web of Science ID A1980KC58900010

    View details for PubMedID 7400824