All Publications

  • Critical Role of Coaptive Strain in Aortic Valve Leaflet Homeostasis: Use of a Novel Flow Culture Bioreactor to Explore Heart Valve Mechanobiology. Journal of the American Heart Association Maeda, K., Ma, X., Hanley, F. L., Riemer, R. K. 2016; 5 (8)


    Aortic valve (AV) disease presents critical situations requiring surgery in over 2% of the US population and is increasingly the reason for cardiac surgery. Throughout the AV cycle, mechanical forces of multiple types and varying intensities are exerted on valve leaflets. The mechanisms whereby forces regulate leaflet homeostasis are incompletely understood. We used a novel flow bioreactor culture to investigate alteration of AV opening or closure on leaflet genes.Culture of rat AV was conducted in a flow bioreactor for 7 days at 37°C under conditions approximating the normal stroke volume. Three force condition groups were compared: Cycling (n=8); always open (Open; n=3); or always closed (Closed; n=5). From each culture, AV leaflets were pooled by force condition and RNA expression evaluated using microarrays. Hierarchical clustering of 16 transcriptome data sets from the 3 groups revealed only 2 patterns of gene expression: Cycling and Closed groups clustered together, whereas Open AV were different (P<0.05). Sustained AV opening induced marked changes in expression (202 transcripts >2-fold; P<0.05), whereas Closed AV exhibited similar expression pattern as Cycling (no transcripts >2-fold; P<0.05). Comparison with human sclerotic and calcific AV transcriptomes demonstrated high concordance of >40 Open group genes with progression toward disease.Failure of AV to close initiates an extensive response characterized by expression changes common to progression to calcific aortic valve disease. AV coaptation, whether phasic or chronic, preserved phenotypic gene expression. These results demonstrate, for the first time, that coaptation of valve leaflets is a fundamentally important biomechanical cue driving homeostasis.

    View details for DOI 10.1161/JAHA.116.003506

    View details for PubMedID 27464792

  • Tetralogy of Fallot: aorto-pulmonary collaterals and pulmonary arteries have distinctly different transcriptomes PEDIATRIC RESEARCH Ma, X., Barboza, L. A., Siyahian, A., Reinhartz, O., Maeda, K., Reddy, V. M., Hanley, F. L., Riemer, R. K. 2014; 76 (4): 341-346
  • Dynamics of human myocardial progenitor cell Populations in the neonatal period ANNALS OF THORACIC SURGERY Amir, G., Ma, X., Reddy, V. M., Hanley, F. L., Reinhartz, O., Ramamoorthy, C., Riemer, R. K. 2008; 86 (4): 1311-1320


    Pluripotent cardiac progenitor cells resident in myocardium offer a potentially promising role in promoting recovery from injury. In pediatric congenital heart disease (CHD) patients, manipulation of resident progenitor cells may provide important new approaches to improving outcomes. Our study goals were to identify and quantitate populations of progenitor cells in human neonatal myocardium during the early postnatal period and determine the proliferative capacity of differentiated cardiac myocytes.Immunologic markers of cell lineage (stage-specific embryonic antigen 4 [SSEA-4], islet cell antigen 1 [Isl1], c-kit, Nkx2.5, sarcoplasmic reticulum calcium-regulated ATPase type 2 [SERCA2]) and proliferation (Ki67) were localized in right ventricular biopsies from 32 CHD patients aged 2 to 93 days.Neonatal myocardium contains progenitor cells and transitional cells expressing progenitor and differentiated myocyte marker proteins. Some cells expressed the pluripotent cell marker c-kit and also coexpressed the myocyte marker SERCA2. Multipotent progenitor cells, identified by the expression of Isl1, were found. Ki67 was expressed in some myocytes and in nonmyocyte cells. A few cells expressing SSEA-4 and Isl1 were observed during the early postnatal period. Cells expressing c-kit, the premyocyte marker Nkx2.5, and Ki67 were found throughout the first postnatal month. A progressive decline in cell density during the first postnatal month was observed for c-kit+ cells (p = 0.0013) and Nkx2.5+ cells (p = 0.0001). The percentage of cells expressing Ki67 declined during the first 3 postnatal months (p = 0.0030).Cells in an incomplete state of cardiomyocyte differentiation continue to reside in the infant heart. However, the relative density of progenitor cells declines during the first postnatal month.

    View details for DOI 10.1016/j.athoracsur.2008.06.058

    View details for Web of Science ID 000259848000036

    View details for PubMedID 18805183

  • 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

  • Is Continuous Flow Superior to Pulsatile Flow in Single Ventricle Mechanical Support? Results from a Large Animal Pilot Study ASAIO JOURNAL Fujii, Y., Ferro, G., Kagawa, H., Centola, L., Zhu, L., Ferrier, W. T., Talken, L., Riemer, R. K., Maeda, K., Nasirov, T., Hodges, B., Amirriazi, S., Lee, E., Sheff, D., May, J., May, R., Reinhartz, O. 2015; 61 (4): 443-447


    Durable mechanical support in situations of physiologic single ventricle has been met with little success so far, particularly in small children. We created an animal model to investigate whether pulsatile or continuous flow would be superior. Three 1 month old sheep (10-16 kg) were instrumented. Via sternotomy and with cardiopulmonary bypass, a large ventricular septal defect and atrial septal defect were created. The left ventricle was cannulated using a Berlin Heart inflow cannula. This was connected sequentially to a continuous flow device (Thoratec HeartMate X, Pleasanton, CA) and to a pulsatile device (Berlin Heart Excor, The Woodlands, TX). Outflow was via a Y-graft to both aorta and pulmonary artery, striving for equal flow to both. Atrial filling pressures were controlled with volume infusions over a wide range. Under comparable loading conditions, significantly higher maximum flow was obtained by HeartMate X than by Excor (4.95 ± 1.27 L/min [range, 3.84-6.34 L/min] for HeartMate X vs. 1.80 ± 0.85 L/min [range, 1.01-2.7 L/min] for Excor; p < 0.05). Judging from this limited animal study, in single ventricle scenarios, continuous flow devices may achieve higher pump flows than pulsatile devices when provided with similar filling pressures. Their clinical use should be investigated. More extensive experimental studies are needed.

    View details for DOI 10.1097/MAT.0000000000000220

    View details for Web of Science ID 000358285100013

  • Hemodynamic Monitoring of Large Animal Chronic Studies After Median Sternotomy: Experiences With Different Telemetric Physiological Devices ASAIO JOURNAL Fujii, Y., Pitsillides, K., Ferro, G., Kagawa, H., Centola, L., Kinouchi, K., Zhu, L., Ferrier, W. T., Talken, L., Nasirov, T., Riemer, R. K., Reinhartz, O. 2015; 61 (3): 332-338


    Telemetric physiological monitoring systems (TPMS) have enabled accurate continuous measurement of animal blood pressures and flows. However, few studies describe approaches for use of TPMS in the great vessels or inside the heart. We describe our initial experiences using two types of TPMSs. Twelve lambs (20-37 kg) underwent sternotomy. Two lambs were not instrumented and were killed at 14 days to confirm normal sternal wound healing (sham group, n = 2). Ten lambs underwent placement of either standard indwelling pressure-monitoring catheter and perivascular-flow-probe (CFP group, n = 3) or TPMS implantation (TPMS group, n = 7). The TPMS used were EG1-V3S2T-M2 (EG1, n = 5; Transonic Endogear Inc.) and Physio Tel Digital L21 (PTD, n = 2; Data Sciences Inc.). Two deaths because of respiratory problems occurred in TPMS group, attributed to lung compression by the implanted device. In TPMS group, more consistent trends of blood pressures and flows were recorded, and management of animals was easier and less labor-intensive. Comparing the two TPMSs, the initiation and renewal costs for each case was $28 K vs. $20 K and $1,700 vs. $0, (PTD versus EG1, respectively). In conclusion, TPMS implantation was feasible via median sternotomy in lambs. Telemetric physiological monitoring systems significantly improve reliability of hemodynamic monitoring in chronic survival animal study. EG1 was less costly than PTD.

    View details for DOI 10.1097/MAT.0000000000000202

    View details for Web of Science ID 000353811800018

    View details for PubMedID 25635937

  • Embryology and Anatomy of Intrapulmonary Shunts ECHOCARDIOGRAPHY-A JOURNAL OF CARDIOVASCULAR ULTRASOUND AND ALLIED TECHNIQUES McMullan, D. M., Riemer, R. K. 2015; 32: S190-S194


    Pulmonary vascular shunting poses a major clinical risk. In this brief overview, we discuss the morphological aspects of shunting vessels in the lung, their development, and the regulation of their patency.

    View details for DOI 10.1111/echo.12888

    View details for Web of Science ID 000349966000002

    View details for PubMedID 25693623

  • Fetal cardiac intervention: Improved results of fetal cardiac bypass in immature fetuses using the TinyPump device. journal of thoracic and cardiovascular surgery Sebastian, V. A., Ferro, G., Kagawa, H., Nasirov, T., Maeda, K., Ferrier, W. T., Takatani, S., Riemer, R. K., Hanley, F. L., Reddy, V. M. 2013; 145 (6): 1460-1464


    Fetal cardiac surgery is a potential innovative treatment for certain congenital heart defects that have significant mortality and morbidity in utero or after birth, but it has been limited by placental dysfunction after fetal cardiac bypass. We have used the TinyPump device for fetal cardiac bypass in sheep fetuses at 90 to 110 days gestation.Ten mixed-breed pregnant ewes were used over a period of 6 months, and 10 fetuses were placed on bypass for 30 minutes. Five fetuses with a mean gestational age of 104 ± 4.5 days and mean weight of 1.4 ± 0.4 kg were placed on bypass using the TinyPump device, and 5 fetuses with a mean gestational age of 119 ± 4.5 days and mean weight of 3.4 ± 0.4 kg were placed on bypass using the roller head pump. The fetuses were monitored for up to 3 hours after bypass or until earlier demise.Progressive respiratory and metabolic acidosis developed in all fetuses. The TinyPump group had a lower gestational age and weight compared with the roller head pump group. However, the rate of postbypass deterioration in the TinyPump group, as measured with blood gases, was noted to be significantly slower compared with the roller head pump group.We demonstrate the feasibility of the TinyPump device for fetal cardiac bypass in a fetal sheep model. The TinyPump group showed improved results compared with the roller head group despite more immature fetuses. The TinyPump device seems to be a promising device for future studies of fetal cardiac bypass in immature fetal sheep and in primates.

    View details for DOI 10.1016/j.jtcvs.2012.08.014

    View details for PubMedID 22944083

  • Reconstruction of pulmonary artery with porcine small intestinal submucosa in a lamb surgical model: Viability and growth potential JOURNAL OF THORACIC AND CARDIOVASCULAR SURGERY Boni, L., Chalajour, F., Sasaki, T., Snyder, R. L., Boyd, W. D., Riemer, R. K., Reddy, V. M. 2012; 144 (4): 963-?


    This study investigated the time-dependent remodeling and growth potential of porcine small intestine submucosa as a biomaterial for the reconstruction of pulmonary arteries in a lamb model.Left pulmonary arteries were partially replaced with small intestine submucosal biomaterial in 6 lambs. Two animals each were humanely killed at 1, 3, and 6 months. Computed tomographic angiography, macroscopic examination of the implanted patch, and microscopic analysis of tissue explants were performed.All animals survived without complications. Patency and arborization of the pulmonary arteries were detected 6 months after implantation. There was no macroscopic narrowing or aneurysm formation in the patch area. The luminal appearance of the patch was similar to the intimal layer of the adjacent native pulmonary artery. Scanning electron microscopy showed that the luminal surface of the patch was covered by confluent cells. Immunohistochemical examination confirmed endothelialization of the luminal side of the patch in all of the explanted patches. The presence of smooth muscle cells in the medial layer was confirmed at all time points; however, expression of elastin, growth of the muscular layer, and complete degradation of patch material were detectable only after 6 months. The presence of c-Kit-positive cells suggests migration of multipotent cells into the patch, which may play a role in remodeling the small intestine submucosal biomaterial.Our data confirmed that remodeling and growth potential of the small intestine submucosal biomaterial are time dependent. Additional experiments are required to investigate the stability of the patch material over a longer period.

    View details for DOI 10.1016/j.jtcvs.2012.07.024

    View details for Web of Science ID 000309111600034

    View details for PubMedID 22917684

  • Reconstruction of Pulmonary Artery in a Newborn Using a Porcine Small Intestinal Submucosal Patch ANNALS OF THORACIC SURGERY Chalajour, F., Barboza, L. A., Boni, L., Snyder, R., Hanley, F. L., Reddy, V. M., Riemer, R. K. 2012; 93 (4): 1311-1315


    In this case report, we evaluated cellular structure and the growth potential of a porcine small intestinal submucosal patch used for pulmonary artery augmentation in a 20-day-old newborn with pulmonary atresia. The patch was resected 2 months postoperatively due to apparent abnormal wall thickening and evaluated by histologic and immunohistologic staining.

    View details for DOI 10.1016/j.athoracsur.2011.08.055

    View details for Web of Science ID 000302120200064

    View details for PubMedID 22450088

  • Maintenance Dose of Warfarin in Sheep and Effect of Diet: A Preliminary Report JOURNAL OF INVESTIGATIVE SURGERY Sasaki, T., Tsuda, S., Trujillo, M., Riemer, R. K., Reinhartz, O. 2012; 25 (1): 29-32


    Sheep models are widely used to evaluate the feasibility of various cardiac assist devices. Anticoagulation therapy postoperatively, however, is seldomly reported on. Continuous heparin infusion is often used, but is cumbersome due to long-term line management with the risk of infection and dislodgement. We contemplated using warfarin instead and started a pilot dose-finding study. Three sheep were given oral warfarin between 0.1 and 0.3 mg/kg/day. Prothrombin time was monitored and INR was calculated daily. If the INR did not reach a target of 2.5-3.5, warfarin dose was doubled. We found that sheep required a dose of warfarin between 1.6 and 2.4 mg/kg/day to raise the INR to the target zone. In a subsequent study to evaluate the effect of diet on INR in sheep, three sheep were fed alfalfa hay or alfalfa pellets in a crossover design. All the animals were given warfarin at the dose of 1.6 mg/kg. The diet was switched when the INR reached the target zone of 2.5-3.5. Hay-fed animals reached the target INR on days 6 and 7. On the other hand, pellet-fed animals did not reach the target value by day 7 with the initial dose and required 2.4 mg/kg of warfarin to achieve the goal. Hay raised the INR faster and higher than pellets with the same warfarin dose. Hay may be advantageous when using oral warfarin therapy in sheep.

    View details for DOI 10.3109/08941939.2011.598219

    View details for Web of Science ID 000299424500006

    View details for PubMedID 22272634

  • Challenges in Longer-Term Mechanical Support of Fontan Circulation in Sheep ASAIO JOURNAL Boni, L., Sasaki, T., Ferrier, W. T., Yeung, J. T., Reichenbach, S. H., Riemer, R. K., Reinhartz, O. 2012; 58 (1): 60-64


    Single ventricle congenital heart defects are usually palliated with the end result of a Fontan circulation. Despite improving results, this circulation is still associated with long-term failure. We previously developed an animal model of mechanical cavopulmonary circulation support that was successful in the acute and mid-term period. In the current study, we evaluated longer support durations in five Western-breed sheep. Through a right thoracotomy we instituted mechanical support from the inferior vena cava to the pulmonary artery, using a Heartmate II axial flow pump (Thoratec Corp., Pleasanton, CA). Postoperatively, the animals were anticoagulated with heparin iv. Hemodynamics, pump flow, anticoagulation, and hepatic and renal function were monitored daily. All animals survived the operation. Signs of moderate liver and kidney injury in general reversed quickly. Two animals had a fatal pump thrombosis. When anticoagulation was effective, hemodynamics and pump flow were maintained to normal values. Effective anticoagulation was difficult to achieve because of the high variability in response to heparin. Survival up to 18 days was accomplished. This study is the longest reported survival of animals with a mechanically assisted cavopulmonary circulation. The performance of the Thoratec Heartmate II has been good, but the issue of effective anticoagulation has not yet been solved.

    View details for DOI 10.1097/MAT.0b013e31823c0aa4

    View details for Web of Science ID 000298643200011

    View details for PubMedID 22210652

  • Cerebral Oxygen Metabolism During Total Body Flow and Antegrade Cerebral Perfusion at Deep and Moderate Hypothermia ARTIFICIAL ORGANS Sasaki, T., Boni, L., Riemer, R. K., Yeung, J. T., Ramamoorthy, C., Beckman, R., Gisner, C., Shuttleworth, P., Hanley, F. L., Reddy, V. M. 2010; 34 (11): 980-986


    The aim of this study is to evaluate the effect of temperature on cerebral oxygen metabolism at total body flow bypass and antegrade cerebral perfusion (ACP). Neonatal piglets were put on cardiopulmonary bypass (CPB) with the initial flow rate of 200mL/kg/min. After cooling to 18°C (n=6) or 25°C (n=7), flow was reduced to 100mL/kg/min (half-flow, HF) for 15min and ACP was initiated at 40mL/kg/min for 45min. Following rewarming, animals were weaned from bypass and survived for 4h. At baseline, HF, ACP, and 4 h post-CPB, cerebral blood flow (CBF) was measured using fluorescent microspheres. Cerebral oxygen extraction (CEO(2) ) and cerebral metabolic rate of oxygen (CMRO(2) ) were monitored. Regional cranial oxygen saturation (rSO(2) ) was continuously recorded throughout the procedure using near-infrared spectroscopy. At 18°C, CBF trended lower at HF and ACP and matched baseline after CPB. CEO(2) trended lower at HF and ACP, and trended higher after CPB compared with baseline. CMRO(2) at ACP matched that at HF. Cranial rSO(2) was significantly greater at HF and ACP (P<0.001, P<0.001) and matched baseline after CPB. At 25°C, CBF trended lower at HF, rebounded and trended higher at ACP, and matched baseline after CPB. CEO(2) was equal at HF and ACP and trended higher after CPB compared with baseline. CMRO(2) at ACP was greater than that at HF (P=0.001). Cranial rSO(2) was significantly greater at HF (P=0.01), equal at ACP, and lower after CPB (P=0.03). Lactate was significantly higher at all time points (P=0.036, P<0.001, and P<0.001). ACP provided sufficient oxygen to the brain at a total body flow rate of 100mL/kg/min at deep hypothermia. Although ACP provided minimum oxygenation to the brain which met the oxygen requirement, oxygen metabolism was altered during ACP at moderate hypothermia. ACP strategy at moderate hypothermia needs further investigation.

    View details for DOI 10.1111/j.1525-1594.2010.01131.x

    View details for Web of Science ID 000284588300019

    View details for PubMedID 21092040

  • Optimal flow rate for antegrade cerebral perfusion JOURNAL OF THORACIC AND CARDIOVASCULAR SURGERY Sasaki, T., Tsuda, S., Riemer, R. K., Ramamoorthy, C., Reddy, V. M., Hanley, F. L. 2010; 139 (3): 530-535


    Antegrade cerebral perfusion is widely used in neonatal heart surgery, yet commonly used flow rates have never been standardized. The objective of this study was to determine the antegrade cerebral perfusion flow rate that most closely matches standard cardiopulmonary bypass conditions.Nine neonatal piglets underwent deep hypothermic cardiopulmonary bypass at a total body flow of 100 mL/kg/min (baseline). Antegrade cerebral perfusion was conducted via innominate artery cannulation at perfusion rates of 10, 30, and 50 mL/kg/min in random order. Cerebral blood flow was measured using fluorescent microspheres. Regional oxygen saturation and cerebral oxygen extraction were monitored.Cerebral blood flow was as follows: baseline, 60 +/- 17 mL/100 g/min; antegrade cerebral perfusion at 50 mL/kg/min, 56 +/- 17 mL/100 g/min; antegrade cerebral perfusion at 30 mL/kg/min, 36 +/- 9 mL/100 g/min; and antegrade cerebral perfusion at 10 mL/kg/min, 13 +/- 6 mL/100 g/min. At an antegrade cerebral perfusion rate of 50 mL/kg/min, cerebral blood flow matched baseline (P = .87), as did regional oxygen saturation (P = .13). Antegrade cerebral perfusion at 30 mL/kg/min provided approximately 60% of baseline cerebral blood flow (P < .002); however, regional oxygen saturation was equal to baseline (P = .93). Antegrade cerebral perfusion at 10 mL/kg/min provided 20% of baseline cerebral blood flow (P < .001) and a lower regional oxygen saturation than baseline (P = .011). Cerebral oxygen extraction at antegrade cerebral perfusion rates of 30 and 50 mL/kg/min was equal to baseline (P = .53, .48) but greater than baseline (P < .0001) at an antegrade cerebral perfusion rate of 10 mL/kg/min. The distributions of cerebral blood flow and regional oxygen saturation were equal in each brain hemisphere at all antegrade cerebral perfusion rates.Cerebral blood flow increased with antegrade cerebral perfusion rate. At an antegrade cerebral perfusion rate of 50 mL/kg/min, cerebral blood flow was equal to baseline, but regional oxygen saturation and cerebral oxygen extraction trends suggested more oxygenation than baseline. An antegrade cerebral perfusion rate of 30 mL/kg/min provided only 60% of baseline cerebral blood flow, but cerebral oxygen extraction and regional oxygen saturation were equal to baseline. An antegrade cerebral perfusion rate that closely matches standard cardiopulmonary bypass conditions is between 30 and 50 mL/kg/min.

    View details for DOI 10.1016/j.jtcvs.2009.12.005

    View details for Web of Science ID 000274735400002

    View details for PubMedID 20176202

  • Deep Brain Hyperthermia While Rewarming from Hypothermic Circulatory Arrest 41st Annual Meeting of the Society-of-Thoracic-Surgeons Amir, G., Ramamoorthy, C., Riemer, R. K., Hanley, F. L., Reddy, V. M. WILEY-BLACKWELL PUBLISHING, INC. 2009: 606–10


    Neurologic injury is a feared and serious long-term complication of cardiopulmonary bypass (CPB) and deep hypothermic circulatory arrest (DHCA). Postoperative hyperthermia was found to enhance postischemic neurologic injury. The use of core temperature as the reference point through CPB assumes parallel changes in brain temperature. We tested the hypothesis that regional and deep brain temperature (DBT) differ during cooling, DHCA, and rewarming.Neonatal piglets (n = 9) were subject to CPB and cooled to rectal temperature (RT) of 18 degrees C, 30 minutes of DHCA were initiated, and subsequently the piglets were rewarmed to RT of 36.5 degrees C and weaned from CPB. Temperature probes were inserted into the DBT targeting the caudate and thalamic nuclei, their position confirmed by pathology. Superficial brain temperature was measured by a temperature probe inserted extradurally. RT, nasopharyngeal (NPT), and tympanic (TT) temperatures were recorded.During cooling the deep brain cooled faster and to lower temperatures compared to RT and TT; NPT reflected DBT accurately. During rewarming DBT was significantly higher than RT and TT. By the end of rewarming the difference between the deep brain and the RT reached statistical significance (30 minutes: 35.1 +/- 0.7 vs. 32.3 +/- 0.7 p < 0.05, respectively, 40 minutes: 37.5 +/- 0.3 vs. 34.7 +/- 0.8 p < 0.05, respectively).Deep brain hyperthermia routinely occurs during the last stages of rewarming following DHCA. DBT is accurately reflected by NPT and is directly correlated with inflow temperature. Therefore, during rewarming inflow temperatures should not exceed 36 degrees C and NPT should be closely monitored.

    View details for DOI 10.1111/j.1540-8191.2009.00883.x

    View details for Web of Science ID 000269540900034

    View details for PubMedID 19740304

  • Recovery During Mid-Term Mechanical Support of Fontan Circulation in Sheep ASAIO JOURNAL Tsuda, S., Sasaki, T., Maeda, K., Riemer, R. K., Reichenbach, S. H., Reinhartz, O. 2009; 55 (4): 406-411


    Total cavopulmonary connection (CPC) has a significant incidence of late failure due to increased systemic venous pressure and low cardiac output. Mechanical support could prevent failure by correcting hemodynamics. We established a model of inferior CPC using an axial flow pump (Thoratec HeartMate II, Thoratec Corp. Pleasanton, CA) in a group of ten 47-57 kg sheep and assessed hemodynamics and metabolism as a potential chronic treatment option for failed Fontan circulation. After pilot studies (n = 7), three animals underwent pump-supported inferior CPC to assess hemodynamic and metabolic responses. Pump inflow was connected to the inferior vena cava (IVC) and outflow to the main pulmonary artery. The IVC was ligated at the right atrium. Hemodynamic and biochemical parameters were recorded over four days. The first seven animals died from pump-related causes (graft kinking, three; pump thrombosis, one) or other causes (GI bleeding, one; suspected stroke, two). The subsequent three animals were electively euthanized on postoperative day four due to IRB requirements. Over the four day postoperative period, pump flow was 3.43 +/- 0.62 L/min and IVC pressure 4.05 +/- 3.21 mm Hg (mean +/- SD). Lactate levels remained normal. Low pressure and high-volume IVC flow was sustained by mechanical support. We will next attempt chronic pump implantation.

    View details for DOI 10.1097/MAT.0b013e3181a0a570

    View details for Web of Science ID 000267559100016

    View details for PubMedID 19471161

  • Morphological studies of pulmonary arteriovenous shunting in a lamb model of superior cavopulmonary anastomosis PEDIATRIC CARDIOLOGY McMullan, D. M., Reddy, V. M., Gottliebson, W. M., Silverman, N. H., Perry, S. B., Chan, F., Hanley, F. L., Riemer, R. K. 2008; 29 (4): 706-712


    We sought to identify and characterize the abnormal vascular structures responsible for pulmonary arteriovenous shunting following the Glenn cavopulmonary shunt. Superior cavopulmonary shunt is commonly performed as part of the staged pathway to total cavopulmonary shunt to treat univentricular forms of congenital heart disease, however, clinically significant pulmonary arteriovenous malformations develop in some patients after the procedure. The causes of pulmonary arteriovenous malformations and other pulmonary vascular changes that occur after cavopulmonary shunt are not known. Using a juvenile lamb model of superior cavopulmonary anastomosis that reliably produces pulmonary arteriovenous malformations, we performed echocardiography and morphological analyses to determine the anatomic site of shunting and to identify the vascular structures involved. Pulmonary arteriovenous shunting was identified by contrast echocardiography in all surviving animals (n = 40) following superior cavopulmonary anastomosis. Pulmonary vascular corrosion casts revealed abnormal tortuous vessels joining pulmonary arteries and veins in cavopulmonary shunt animals but not control animals. In conclusion, unusual channels that bridged pulmonary arteries and veins were identified. These may represent the vascular structures responsible for arteriovenous shunting following the classic Glenn cavopulmonary shunt. Detailed analysis of these structures may elucidate factors responsible for their development.

    View details for DOI 10.1007/s00246-007-9152-5

    View details for Web of Science ID 000257393800003

    View details for PubMedID 18043856

    View details for PubMedCentralID PMC2970565

  • Identification and characterization of cells with high angiogenic potential and transitional phenotype in calcific aortic valve EXPERIMENTAL CELL RESEARCH Chalajour, F., Treede, H., Gehling, U. M., Ebrahimnejad, A., Boehm, D. H., Riemer, R. K., Ergun, S., Reichenspurner, H. 2007; 313 (11): 2326-2335


    Recent data suggest that angiogenesis plays an important role in the pathogenesis of valvular disease. However, the cellular mechanisms underlying this process remain unknown. This study aimed at identifying and characterizing the cellular components responsible for pathological neovascularization in calcific aortic valves (CAV). Immunohistochemical analysis of uncultured CAV tissues revealed that smooth muscle alpha-actin (alpha-SMA)-positive cells, which coexpressed Tie-2 and vascular endothelial growth factor receptor-2 (VEGFR-2), can be identified prior to the initiation of capillary-like tube formation. In a second step, leaflets of CAV and non-calcific aortic valves (NCAV) were cultured and the cells involved in capillary-like tube formation were isolated. The majority of these cells displayed the same phenotype as non-cultured cells identified in CAV tissues, i.e., expression of alpha-SMA, Tie-2, and VEGFR-2. In comparison to cells isolated from cultures of NCAV leaflets, these cells showed enhanced angiogenic activity as demonstrated by migration and tube assays. The coexpression of VEGFR-2 and Tie-2 together with alpha-SMA suggests both endothelial and mesenchymal properties of the angiogenically activated cells involved in valvular neovascularization. Hence, our findings might provide new insights into the process of pathological angiogenesis in cardiac valves.

    View details for DOI 10.1016/j.yexcr.2007.02.033

    View details for Web of Science ID 000247606800005

    View details for PubMedID 17475245

  • Visual light spectroscopy reflects flow-related changes in brain oxygenation during regional low-flow perfusion and deep hypothermic circulatory arrest 31st Annual Meeting of the Western-Thoracic-Surgical-Association Amir, G., Ramamoorthy, C., Riemer, R. K., Davis, C. R., Hanley, F. L., Reddy, V. M. MOSBY-ELSEVIER. 2006: 1307–13


    Regional low-flow perfusion has been used to minimize ischemic brain injury during complex heart surgery in children. However, optimal regional low-flow perfusion remains undetermined. Visible light spectroscopy is a reliable method for continuous determination of capillary oxygen saturation (SgvO2). We used visible light spectroscopy to follow deep and superficial brain SgvO2 during cardiopulmonary bypass, regional low-flow perfusion, and deep hypothermic circulatory arrest.Visible light spectroscopy probes were inserted into the superficial and deep brain of neonatal (3.9-4.5 kg) piglets, targeting the caudate and thalamic nuclei. The piglets were subjected to cardiopulmonary bypass and cooled to a rectal temperature of 18 degrees C using pH stat. Regional low-flow perfusion was initiated through the innominate artery at 18 degrees C, and pump flows were adjusted to 40, 30, 20, and 10 mL/kg/min for 10-minute intervals followed by 30 minutes of deep hypothermic circulatory arrest. Regional low-flow perfusion was reestablished, and flows were increased in a stepwise manner from 10 to 40 mL/kg/min. SgvO2 was continuously monitored. Carotid flow was measured using a flow probe, and cerebral blood flow (milliliters per kilogram body weight per minute) was calculated.There were no significant differences between the deep and superficial brain tissue oxygenation during regional low flow brain perfusion before deep hypothermic circulatory arrest. However, after deep hypothermic circulatory arrest, the superficial brain SgvO2 was lower than the deep brain SgvO2 (24 +/- 12 vs 55.3 +/- 8, P = .05, at flows of 30 mL/kg/min, and 34.2 +/- 17 vs 62.5 + 8, P = .06, at a flow rate of 40 mL/kg/min). During regional low-flow perfusion, SgvO2 was maintained at flows of 30 to 40 mL/kg/min (cerebral blood flows of 15 to 21 mL/kg/min and 19 to 24 mL/kg/min, respectively), but was significantly lower at pump flows of 20 mL/kg/min (cerebral blood flow of 10 to 14 mL/kg/min) and 10 mL/kg/min (cerebral blood flow of 5 to 9 mL/kg/min) compared with the values obtained just before regional low-flow perfusion (pre-deep hypothermic circulatory arrest, 37 +/- 6 vs 65.5 +/- 4.4, P < .05, and 21.6 +/- 3.7 vs 65.5 +/- 4.4, P < .01, respectively; and post-deep hypothermic circulatory arrest, 32 +/- 4.5 vs 65.5 +/- 4.4, P < .05, and 16.6 +/- 4.7 vs 65.5 +/- 4.4, P < .01, respectively).Regional low-flow perfusion at pump flows of 30 to 40 mL/kg/min with resulting cerebral blood flows of 14 to 24 mL/kg/min was adequate in maintaining both deep and superficial brain oxygenation. However, lower pump flows of 20 and 10 mL/kg/min, associated with cerebral blood flow of 9 to 14 mL/kg/min, resulted in significantly reduced SgvO2 values.

    View details for DOI 10.1016/j.jtcvs.2006.04.056

    View details for Web of Science ID 000242626200012

    View details for PubMedID 17140947

  • Antegrade cerebral perfusion reduces apoptotic neuronal injury in a neonatal piglet model of cardiopulmonary bypass JOURNAL OF THORACIC AND CARDIOVASCULAR SURGERY Chock, V. Y., Amir, G., DAVIS, C. R., Ramamoorthy, C., Riemer, R. K., Ray, D., Giffard, R. G., Reddy, V. M. 2006; 131 (3): 659-665


    Neonates with congenital heart disease might require surgical repair with deep hypothermic circulatory arrest, a technique associated with adverse neurodevelopmental outcomes. Antegrade cerebral perfusion is thought to minimize ischemic brain injury, although there are no supporting experimental data. We sought to evaluate and compare the extent of neurologic injury in a neonatal piglet model of deep hypothermic circulatory arrest and antegrade cerebral perfusion.Neonatal piglets undergoing cardiopulmonary bypass were randomized to deep hypothermic circulatory arrest or antegrade cerebral perfusion for 45 minutes. Animals were killed after 6 hours of recovery, and brain tissue was stained for evidence of cellular injury and for the apoptotic markers activated caspase 3 and cytochrome c translocation from mitochondria to cytosol.Piglets from the antegrade cerebral perfusion group exhibited less apoptotic or necrotic injury (4 +/- 3 vs 29 +/- 12 cells per field, P = .03). The piglets undergoing antegrade cerebral perfusion also had less evidence of apoptosis, with fewer cells staining for activated caspase 3 (57 +/- 8 vs 93 +/- 9 cells per field, P = .001) or showing cytochrome c translocation (6 +/- 2 vs 15 +/- 4 cells per field, P = .02).The use of antegrade cerebral perfusion in place of deep hypothermic circulatory arrest reduces evidence of apoptosis and histologic injury in neonatal piglets. Neonates with congenital heart disease might benefit from antegrade cerebral perfusion during complex cardiac surgery to improve their overall neurologic outcome.

    View details for DOI 10.1016/j.jtcvs.2005.09.005

    View details for Web of Science ID 000235940600024

    View details for PubMedID 16515920

  • Neonatal brain protection and deep hypothermic circulatory arrest: pathophysiology of ischemic neuronal injury and protective strategies ANNALS OF THORACIC SURGERY Amir, G., Ramamoorthy, C., Riemer, K., Reddy, V. M., Hanley, F. L. 2005; 80 (5): 1955-1964


    Deep hypothermic circulatory arrest (DHCA) has been used for the past 50 years in the surgical repair of complex congenital cardiac malformations and operations involving the aortic arch; it enables the surgeon to achieve precise anatomical reconstructions by creating a bloodless operative field. Nevertheless, DHCA has been associated with immediate and late neurodevelopmental morbidities. This review provides an overview of the pathophysiology of neonatal hypoxic brain injury after DHCA, focusing on cellular mechanisms of necrosis, apoptosis, and glutamate excitotoxicity. Techniques and strategies in neonatal brain protection include hypothermia, acid base blood gas management during cooling, and pharmacologic interventions such as the use of volatile anesthetics. Surgical techniques consist of intermittent cerebral perfusion during periods of circulatory arrest and continuous regional brain perfusion.

    View details for DOI 10.1016/j.athoracsur.2004.12.040

    View details for Web of Science ID 000232970500075

    View details for PubMedID 16242503

  • Mechanical support of total cavopulmonary connection with an axial flow pump 50th Annual Conference of the American-Society-for-Artificial-Internal-Organs Riemer, R. K., Amir, G., Reichenbach, S. H., Reinhartz, O. MOSBY-ELSEVIER. 2005: 351–54


    Even under optimal circumstances, total cavopulmonary connection is associated with a continuous late risk of death. Hemodynamics are distinctly abnormal, with increased systemic venous pressures and frequent low cardiac output. Our study uses a sheep model of total cavopulmonary connection to test the response to axial flow pump (Thoratec HeartMate II; Thoratec Corporation (Pleasanton, Calif)) support of total cavopulmonary connection, which might be suitable to treat patients with failing Fontan circulation.Eight sheep (42-48 kg) were studied. After pilot studies in 3 animals, 5 underwent both pump-supported and nonsupported total cavopulmonary connection in alternating sequence for up to 2 hours. This was achieved with a 12-mm polytetrafluoroethylene graft from the (distally ligated) superior vena cava to the main pulmonary artery and a cannula placed in the inferior vena cava with an attached 16-mm Dacron graft to the main pulmonary artery. Pressures (arterial, inferior vena cava, left atrium, and pulmonary artery) and flows (ascending aorta and inferior vena cava) were recorded over 1 hour both with unsupported total cavopulmonary connection and after placing an axial flow pump (Thoratec HeartMate II) between the inferior vena caval inflow cannula and the main pulmonary artery.Under nonsupported total cavopulmonary connection circulation, inferior vena caval and aortic blood flow decreased by nearly 50%. Inferior vena caval pressure nearly doubled, whereas arterial pressure decreased by one third. Pulmonary artery pressure became nonpulsatile; however, mean pulmonary artery pressure and left atrial pressure did not change significantly. With pump-supported Fontan circulation, cardiac output, inferior vena caval flow, and arterial pressure returned to baseline. Inferior vena caval pressure decreased to below baseline levels. Mean pulmonary artery pressure and left atrial pressure again remained unchanged.Axial flow pump support from the inferior vena cava to the pulmonary artery can prevent the substantial decrease of aortic flow and pressure associated with total cavopulmonary connection and can reverse its poor hemodynamics. This is a simple model that can be used to further evaluate the potential of mechanical support as a treatment option in failing Fontan circulation.

    View details for DOI 10.1016/j.jtcvs.2004.12.037

    View details for Web of Science ID 000231069700019

    View details for PubMedID 16077398

  • Preliminary results of fetal cardiac bypass in nonhuman primates 84th Annual Meeting of the American-Association-for-Thoracic-Surgery Ikai, A., Riemer, R. K., Ramamoorthy, C., Malhatra, S., Cassorla, L., Amir, G., Hanley, F. L., Reddy, V. M. MOSBY-ELSEVIER. 2005: 175–81


    Fetal cardiac surgery has potential benefits for treatment of some congenital heart defects. However, placental dysfunction as a result of fetal bypass, fetal stress, and fetal exposure to external milieu needs to be overcome to optimize the outcomes of fetal cardiac bypass. In this study we evaluated the technical feasibility of cardiac bypass in the nonhuman primate fetus and the efficacy of different anesthetic approaches.Twelve baboon fetuses, average gestation 146 +/- 8 days and weight 696 +/- 184 g, were used. Three fetuses were excluded from the study because of nuchal cord presentations. The animals were separated into two anesthesia groups: isoflurane (n = 6) and fentanyl and midazolam (n = 3). A miniature roller pump circuit without oxygenator was used for fetal bypass for 30 minutes. No blood transfusion was performed. Fetal blood gas samples were collected before bypass, during bypass, and at 15 and 60 minutes after bypass.All fetuses in the isoflurane group were successfully placed on the cardiac bypass circuit. However, 2 animals in the fentanyl and midazolam group were not placed on the bypass circuit because of sustained elevation in maternal uterine tone. All maternal baboons survived. Of the 6 fetuses in the isoflurane group, 5 survived for 60 minutes; however, placental function continued to deteriorate after bypass (Pa o 2 33 +/- 3 mm Hg before bypass, 23 +/- 6 mm Hg 15 minutes after, and 18 +/- 9 mm Hg 60 minutes after).The technical feasibility of cardiac bypass in nonhuman primate fetuses weighing less than 1000 g was confirmed. Isoflurane anesthesia appears to be superior to fentanyl and midazolam anesthesia for fetal cardiac surgery because of adequate uterine relaxation.

    View details for DOI 10.1016/j.jtcvs.2004.09.003

    View details for Web of Science ID 000226216600024

    View details for PubMedID 15632840

  • Pulmonary arteriovenous shunting in the normal fetal lung JOURNAL OF THE AMERICAN COLLEGE OF CARDIOLOGY McMullan, D. M., Hanley, F. L., Cohen, G. A., Portman, M. A., Riemer, R. K. 2004; 44 (7): 1497-1500


    We hypothesized that pulmonary arteriovenous shunting (PAVS) is normally present in fetal lungs and that cavopulmonary anastomosis-induced PAVS may represent a return to an earlier morphologic stage of development.The surgical superior cavopulmonary anastomosis is performed as part of the staged Fontan pathway to treat univentricular forms of congenital heart disease; PAVS is a known sequela after superior cavopulmonary anastomosis and may have important clinical consequences. Although the etiology and true morphology of the structures responsible for PAVS are unknown, a leading theory is that PAVS is caused by absence of normal hepatic venous drainage to the pulmonary circulation.To determine whether normal fetal lungs demonstrate PAVS, we performed contrast echocardiograms on 13 fetal lambs, 8 neonatal lambs, 4 juvenile lambs, and 4 adult sheep using a blended mixture of saline and blood injected directly into the proximal pulmonary artery.Pulmonary arteriovenous shunting was detected by direct epicardial echocardiography in all fetal lambs (n = 13) and neonatal animals studied at one and three days of life (n = 4) and in two of four animals studied at six to nine days of life. Pulmonary arteriovenous shunting was not present in animals studied at four weeks of life (n = 2) and in adult sheep (n = 5).These studies demonstrate that PAVS is normally present in late gestation fetal and early neonatal lambs but then disappears during the later neonatal period. Furthermore, these findings suggest that PAVS associated with cavopulmonary anastomosis or other processes affecting the developing pulmonary circulation may represent a return to an earlier morphologic stage of development.

    View details for DOI 10.1016/j.jacc.2004.06.064

    View details for Web of Science ID 000224225600025

    View details for PubMedID 15464334

  • Pulmonary expression of the hepatocyte growth factor receptor c-Met shifts from medial to intimal layer after cavopulmonary anastomosis 83rd Annual Meeting of the American-Association-for-Thoracic-Surgery Ikai, A., Riemer, R. K., Ma, X. Y., Reinhartz, O., Hanley, F. L., Reddy, V. M. MOSBY-ELSEVIER. 2004: 1442–49


    Pulmonary arteriovenous malformations occur in up to 60% of patients after cavopulmonary anastomosis. We compared the effects of cavopulmonary anastomosis and pulmonary artery banding on lung gene expression in an ovine model to study the abnormal pulmonary vascular remodeling after the exclusion of inferior vena caval blood independent of reduced pulmonary blood flow. We previously demonstrated by contrast echocardiography that pulmonary arteriovenous malformations develop by 8 weeks after cavopulmonary anastomosis but not after pulmonary artery banding. Hepatocyte growth factor, a pleiotropic factor with morphogenic, mitogenic, and angiogenic activities, signals via its specific receptor c-Met to induce the antiapoptotic factor Bcl-2. In this study, we examined pulmonary artery expression of these factors and their potential role in pulmonary artery remodeling after cavopulmonary anastomosis and pulmonary artery banding.Eighteen lambs aged 35 to 45 days were placed into 3 groups: cavopulmonary anastomosis, pulmonary artery banding, and control (n = 6/group). In the cavopulmonary anastomosis group, the superior vena cava was anastomosed to the right pulmonary artery in an end-to-end fashion. In the pulmonary artery banding group, the left pulmonary artery was banded to reduce blood flow to 20% of control. The control group had a simple right pulmonary artery clamp for 30 minutes. Lung was harvested for Western blot, reverse transcriptase-polymerase chain reaction, and immunostaining at 2 weeks (n = 3/group) and 5 weeks (n = 3/group) after surgery.The expression of c-Met mRNA after cavopulmonary anastomosis was increased by twofold compared with the control or pulmonary artery banding group. The total lung expression of c-Met by Western blot was also up regulated at 2 weeks (P <.05). However, total lung expression of hepatocyte growth factor and Bcl-2 by Western and reverse transcriptase-polymerase chain reaction was not different from the control and pulmonary artery banding groups at both 2 and 5 weeks after surgery. Immunohistochemical analysis revealed that c-Met expression was localized to the intimal layer of the pulmonary artery in the cavopulmonary anastomosis, while its expression in the control and pulmonary artery banding lungs was localized to the medial layer. Localization of Bcl-2 on the intimal layer in lambs with cavopulmonary anastomosis followed the same pattern as c-Met.After cavopulmonary anastomosis, pulmonary artery expression of the hepatocyte growth factor receptor c-Met and one of its downstream effectors, Bcl-2, had increased in the intimal layer and decreased in the medial layer. Because the hepatocyte growth factor signaling promotes increased endothelial cell survival, it may have a role in pulmonary artery remodeling following cavopulmonary anastomosis. In addition, the change of c-Met expression in the medial layer after cavopulmonary anastomosis suggests a possible mechanism for the smooth muscle cell alteration related to abnormal angiogenesis.

    View details for DOI 10.1016/j.jtcvs.2003.09.009

    View details for Web of Science ID 000221134600031

    View details for PubMedID 15116006

  • A method for selectively limiting lumen diameter in corrosion casting MICROVASCULAR RESEARCH McMullan, D. M., Hanley, F. L., Riemer, R. K. 2004; 67 (3): 215-217


    Corrosion casting is a technique frequently used to evaluate the form and spatial relationship of three dimensional biological structures, such as vascular networks, in vitro [Scann. Microsc. 5 (1991) 1097; Schraufnagel, D.E. The lung microstructure. In: Motta, P.M., Murakami, T. Fujita, H. eds. Scanning Electron Microscopy of Vascular Casts: Methods and Applications. Boston: Kluwer Academic Publishers; 1992:123-137]. However, because corrosion casts tend to reproduce the complexity of surrounding vascular structures as well as the structures of interest, the use of this technique in highly complex vascular systems may obscure important changes such as A-V shunts because they may get lost among the myriad of capillaries. We developed a novel modification for creating vascular corrosion casts by utilizing polystyrene microspheres to selectively embolize normal capillary networks and thereby reduce the overall complexity of the cast. his technique may aid investigators in the evaluation of a variety of vascular beds and is useful in demonstrating non-capillary arteriovenous communications.

    View details for Web of Science ID 000221443700002

    View details for PubMedID 15121445

  • Induced fibrillation is equally effective as crystalloid cardioplegia in the protection of fetal myocardial function 82nd Annual Meeting of the American-Association-for-Thoracic-Surgery Malhotra, S. P., Thelitz, S., Riemer, R. K., Reddy, V. M., Suleman, S., Hanley, F. L. MOSBY-ELSEVIER. 2003: 1276–82


    Fetal cardiac intervention represents a potential advance in the treatment of congenital cardiac lesions that increase in complexity during development. Prenatal repair of a primary defect might prevent pathologic blood-flow patterns that can result in hypoplasia of a cardiac chamber or great vessel. However, strategies to optimize fetal myocardial protection have not been studied. A biventricular working fetal heart preparation was used to evaluate the cardioprotective properties of induced fibrillation and crystalloid cardioplegia.Hearts from 16 fetal lambs at 115 to 125 days' gestation were harvested and perfused with Krebs-Henseleit solution. The descending aorta was ligated distal to the ductal insertion and the branch pulmonary arteries were ligated to simulate the parallel circulation of the fetus. Hearts were arrested with normothermic fibrillation (n = 8) or hypothermic crystalloid cardioplegia (n = 8) before reperfusion with Krebs-Henseleit solution. Baseline and postarrest myocardial function measurements were obtained from analysis of pressure-dimension relationships.Fibrillatory and cardioplegic arrest were equally effective at preserving postarrest systolic function (left ventricle, 70% +/- 5% vs 68% +/- 15%, P =.52; right ventricle, 68% +/- 4.5% vs 65% +/- 4.5%, P =.26) and preventing increased diastolic stiffness (left ventricle, 32% +/- 5.3% vs 38% +/- 11%, P =.24; right ventricle, 25% +/- 3.3% vs 27% +/- 2.1%, P =.46). Myocardial water content was unchanged in hearts arrested with fibrillation and cardioplegia (84% +/- 1.5% vs 83.7% +/- 0.9%, P =.71).Normothermic fibrillation and hypothermic crystalloid cardioplegia provide equal protection of the fetal myocardium. In the setting of diminished fetal myocardial reserve and because of the limited ability to manipulate the surrounding temperature in the fetus, normothermic fibrillation may be preferable for in utero repairs of selected congenital heart defects.

    View details for DOI 10.1016/S0022-5223(02)73245-5

    View details for Web of Science ID 000183864700012

    View details for PubMedID 12830044

  • Fetal myocardial protection is markedly improved by reduced cardioplegic calcium content ANNALS OF THORACIC SURGERY Malhotra, S. P., Thelitz, S., Riemer, R. K., Reddy, V. M., Suleman, S., Hanley, F. L. 2003; 75 (6): 1937-1941


    Fetal cardiac surgery holds a clear therapeutic benefit in the treatment of lesions that increase in complexity due to pathologic blood flow patterns during development. Fetal and neonatal myocardial physiology differ substantially, particularly in the regulation of myocardial calcium concentration. To examine issues of calcium homeostasis and fetal myocardial protection, a novel isolated biventricular working fetal heart preparation was developed.Hearts from 20 fetal lambs, 115 to 125 days gestation, were harvested and perfused with standard Krebs-Henseleit (K-H) solution. The descending aorta was ligated distal to the ductal insertion and the branch pulmonary arteries were ligated to mimic fetal cardiovascular physiology. Hearts were arrested for 30 minutes with normocalcemic (n = 8), hypocalcemic (n = 6), or hypercalcemic (n = 6) cold crystalloid cardioplegia before reperfusion with K-H solution.Compared with normocalcemic cardioplegia, hypocalcemic cardioplegia improved preservation of left ventricular (LV) systolic function (88% +/- 2.2% vs 64% +/- 15% recovery of end-systolic elastance, p = 0.02), diastolic function (12% +/- 21% vs 38% +/- 11% increase in end-diastolic stiffness, p = 0.04), and myocardial contractility (97% +/- 9.6% vs 75.2% +/- 13% recovery of preload recruitable stroke work [PRSW], p = 0.04). In contrast, the fetal myocardium was sensitive to hypercalcemic arrest with poor preservation of LV systolic function (37.5% +/- 8.4% recovery of elastance), diastolic function (86% +/- 21% increased stiffness), and overall contractility (32% +/- 13% recovery of PRSW). Myocardial water content was reduced in hearts arrested with hypocalcemic cardioplegia (79% +/- 1.8% vs 83.7% +/- 0.9%, p = 0.0006).This study demonstrates the sensitivity of the fetal myocardium to cardioplegic calcium concentration. Hypocalcemic cardioplegia provides superior preservation of systolic, diastolic, and contractile function of the fetal myocardium.

    View details for Web of Science ID 000183311400054

    View details for PubMedID 12822639

  • Robotic-assisted endoscopic thoracic aortic anastomosis in juvenile lambs 5th Annual Meeting of the International-Society-for-Minimally-Invasive-Cardiac-Surgery Malhotra, S. P., Le, D., Thelitz, S., Hanley, F. L., Riemer, R. K., Suleman, S., Reddy, V. M. FORUM MULTIMEDIA PUBLISHING, LLC. 2003: 38–42
  • The role of oxidative stress in the development of pulmonary arteriovenous malformations after cavopulmonary anastomosis JOURNAL OF THORACIC AND CARDIOVASCULAR SURGERY Malhotra, S. P., Reddy, V. M., Thelitz, S., He, Y. P., McMullan, D. M., Hanley, F. L., Riemer, R. K. 2002; 124 (3): 479-485


    Cavopulmonary anastomosis is used for palliation of cyanotic heart disease. Clinically significant pulmonary arteriovenous malformations occur in up to 25% of patients after surgical intervention. Cavopulmonary anastomosis creates several modifications to pulmonary physiology that may contribute to the development of pulmonary arteriovenous malformations, including reduced pulmonary blood flow and the exclusion of inferior vena caval effluent.By comparing the expression of angiogenic and stress-related proteins after cavopulmonary anastomosis and pulmonary artery banding, we sought to determine which genes were upregulated independent of reduced pulmonary blood flow.Lambs aged 35 to 45 days were placed into 1 of 3 groups: cavopulmonary anastomosis (n = 6), pulmonary artery banding (n = 6), and sham control (n = 6) animals. In our model pulmonary arteriovenous malformations are detectable by means of bubble-contrast echocardiography 8 weeks after cavopulmonary anastomosis. Lung tissue was harvested for Western blotting at 2 and 5 weeks after surgery.Cavopulmonary anastomosis and pulmonary artery banding both increased angiogenic gene expression, but only cavopulmonary anastomosis induced the expression of endothelial stress-related genes. Vascular endothelial growth factor was upregulated 2.5-fold after both cavopulmonary anastomosis (P =.002) and pulmonary artery banding (P =.007). Only cavopulmonary anastomosis upregulated 2 stress-related genes, HO1 and GLUT1, 2.7-fold (P =.002) and 3.8-fold (P =.03), respectively. Hypoxia-inducible factor was upregulated 4-fold (P =.003) after cavopulmonary anastomosis. Pulmonary artery banding failed to induce the increased expression of any of these proteins.Reduced pulmonary blood flow induces a pulmonary angiogenic response but not an endothelial stress response. These results suggest that oxidative stress is more relevant to the formation of pulmonary arteriovenous malformations than angiogenic signaling alone because pulmonary artery banding does not result in pulmonary arteriovenous malformations. Oxidative stress of the pulmonary endothelium resulting from cavopulmonary anastomosis may predispose the affected vasculature to arteriovenous shunting.

    View details for DOI 10.1067/mtc.2002.120346

    View details for Web of Science ID 000177840600009

    View details for PubMedID 12202863

  • Cavopulmonary anastomosis induces pulmonary expression of the angiotensin II receptor family 73rd Annual Scientific Sessions of the American-Heart-Association Malhotra, S. P., Reddy, V. M., Thelitz, S., He, Y. P., Hanley, F. L., Suleman, S., Riemer, R. K. MOSBY-ELSEVIER. 2002: 655–60


    Cavopulmonary anastomosis is used for palliation of cyanotic cardiac lesions. Postoperative development of pulmonary arteriovenous malformations can be significant in 10% to 25% of patients. To study the basis for formation of arteriovenous malformations, we developed an ovine model that reliably induces their development 8 weeks after cavopulmonary anastomosis. Previously, we found that cavopulmonary anastomosis inhibits the expression of pulmonary angiotensin-converting enzyme and suppresses angiotensin II production.This study examines the role of the angiotensin II receptors, type 1 and type 2, in this setting of pulmonary vascular remodeling.Lambs, aged 40 to 50 days, underwent cavopulmonary anastomosis. In age-matched control animals, a sham operation was performed. Messenger RNA and protein expression in lung specimens was measured at successive time points after cavopulmonary anastomosis or sham operations (n = 3 at each time point).Angiotensin type 1 mRNA was maximally upregulated 2-fold at 5 weeks after cavopulmonary anastomosis (P =.006). Expression of angiotensin type 1 protein was increased at least 2-fold at 2, 5, and 15 weeks after cavopulmonary anastomosis (P =.005). Cavopulmonary anastomosis also increased angiotensin type 2 mRNA and protein expression at least 2-fold at 2 and 5 weeks (P =.02) after surgical intervention. At 15 weeks, expression of angiotensin type 2 mRNA and protein was unchanged from that seen in control animals. Immunolocalization in pulmonary tissue sections 2 weeks after cavopulmonary anastomosis revealed markedly enhanced staining of angiotensin II receptor type 1 in vascular smooth muscle and angiotensin II receptor type 2 in the endothelium of pulmonary arteries.Rapid elevation in the expression of the type 1 and 2 angiotensin II receptors in the affected pulmonary vasculature after cavopulmonary anastomosis suggests their involvement in the pathologic vascular remodeling that occurs after cavopulmonary anastomosis.

    View details for DOI 10.1067/mtc.2002.119699

    View details for Web of Science ID 000175400100008

    View details for PubMedID 11986592

  • Robotic-assisted endoscopic thoracic aortic anastomosis in juvenile lambs. heart surgery forum Malhotra, S. P., Le, D., Thelitz, S., Hanley, F. L., Riemer, R. K., Suleman, S., Reddy, V. M. 2002; 6 (1): 38-42


    Advances in robotic technology have enabled a wider range of applications for minimally invasive techniques in cardiac surgery, including mitral valve repair and coronary artery bypass grafting. With increased technical sophistication, robotic-assisted techniques can be developed for the endoscopic repair of certain congenital cardiac lesions.The purpose of this study was to assess the feasibility of closed chest thoracic aortic anastomosis in a juvenile ovine model.Lambs, aged 45 to 55 days, underwent surgery that was performed using the da Vinci robotic surgical system. Using 3 ports, the surgeon dissected the descending thoracic aorta and mobilized it free from attachments, using single-lung ventilation and CO2 insufflation. Snares were introduced through 2 stab wounds for aortic occlusion proximally and distally. In 4 lambs, the aorta was completely transected and reanastomosed using interrupted nitinol sutures. One lamb underwent longitudinal aortotomy, and patch aortoplasty was performed with the placement of a Gore-Tex patch. Snares were released and the animals were recovered once hemodynamically stable. Animals were sacrificed at 6 to 12 hours after surgery and the descending aorta was harvested. Burst-pressure testing was performed on the anastomoses.All 5 lambs survived the procedure with stabilization of hemodynamic parameters following surgery. The mean aortic clamp time was 47 +/- 17 minutes, and the anastomosis was completed in 26 +/- 5 minutes. The mean burst pressure was 163 +/- 9 mm Hg.Endoscopic thoracic aortic anastomosis can be performed safely and with adequate exposure in a juvenile large-animal model using computer-assisted surgical techniques. With further refinements, these approaches could be applied to the repair of congenital anomalies of the aorta, including interrupted aortic arch and aortic coarctation.

    View details for PubMedID 12611730

  • Failing Fontan circulation sheep model and utilization of axial flow pump - Preliminary experiments 8th World Congress on Heart Failure - Mechanisms and Management Reinhartz, O., Reichenbach, S. H., Ikai, A., Riemer, R. K., Karl, T. R. MEDIMOND S R L. 2002: 531–535
  • Superior cavopulmonary anastomosis suppresses the activity and expression of pulmonary angiotensin-converting enzyme 80th Annual Meeting of the American-Association-for-Thoracic-Surgery Malhotra, S. P., Riemer, R. K., Thelitz, S., He, Y. P., Hanley, F. L., Reddy, V. M. MOSBY-ELSEVIER. 2001: 464–69


    Superior cavopulmonary anastomosis is widely used for palliation of various forms of univentricular heart defects. However, clinically significant pulmonary arteriovenous malformations develop in 15% to 25% of patients after surgery.To assess altered regulation of pulmonary vascular tone caused by superior cavopulmonary anastomosis in an ovine model.Lambs, aged 35 to 45 days, underwent an end-to-end anastomosis of the superior vena cava to the right pulmonary artery. In age-matched controls, a sham operation was performed. Arteriovenous malformations were detectable by contrast echocardiography by 8 weeks after surgery. Animals (n = 24) were studied at various time points after the operations. Expression of angiotensin-converting enzyme messenger RNA, protein levels, and enzyme activity were measured in lung homogenates. Levels of angiotensin II were measured by enzyme-linked immunosorbent assay.Expression of angiotensin-converting enzyme messenger RNA and protein was significantly reduced at 1 to 5 weeks after superior cavopulmonary anastomosis. Angiotensin-converting enzyme activity in the right lung of animals subjected to superior cavopulmonary anastomosis was reduced 86% +/- 1% (standard deviation) compared with control values at 1 week (P =.003) and 77% +/- 8.5% at 2 weeks (P <.001) after surgery. This correlated with a 59% +/- 3.5% (P =.007) reduction in angiotensin II levels up to 5 weeks after cavopulmonary anastomosis. By 15 weeks after the operations, angiotensin II levels were equivalent to control levels (P =.19).Superior cavopulmonary anastomosis causes an early reversible reduction in activity and expression of angiotensin-converting enzyme, resulting in decreased circulating levels of the vasoconstrictor angiotensin II. These results suggest that the ability of the pulmonary endothelium to regulate vascular tone is inhibited after superior cavopulmonary anastomosis. Dilation of the affected vasculature induced by cavopulmonary anastomosis may contribute to the disordered vascular remodeling observed in this setting.

    View details for DOI 10.1067/mtc.2001.115698

    View details for Web of Science ID 000171027100010

    View details for PubMedID 11547295

  • Regulation of uterine smooth muscle function during gestation PEDIATRIC RESEARCH Riemer, R. K., Heymann, M. A. 1998; 44 (5): 615-627


    The uterus is unique among smooth muscular organs in that, during pregnancy, it undergoes profound, largely reversible, changes orchestrated by the ovarian hormones. These changes facilitate uterine adaptation to the stretch induced by the growing fetus such that a state of myometrial contractile quiescence can be maintained. This quiescent state usually is maintained until fetal development is sufficient for extrauterine life, at which point unknown mechanisms precipitate conversion to a highly contractile state. Throughout pregnancy, signaling mechanisms for myometrial contractility are altered--first to promote quiescence and then again to promote contractions. The mechanisms responsible for these changes are only partially understood. This review attempts to summarize salient features of many of the changes in uterine contractile signaling and the current state of ongoing investigations of their mechanisms. We have also highlighted some newer information and concepts from nonuterine tissues, which we believe may provide insight into the control of uterine smooth muscle function. Some detail has been omitted, and can be found in the many excellent reviews cited. We hope that this discussion may stimulate the interests of other investigators. The diverse areas of inquiry offer hope that this decade will lead to a fuller understanding of myometrial function and the development of vastly improved approaches for the control of preterm labor.

    View details for Web of Science ID 000076607700001

    View details for PubMedID 9803440

  • Regulation of ductus arteriosus patency by nitric oxide in fetal lambs: The role of gestation, oxygen tension, and vasa vasorum PEDIATRIC RESEARCH Clyman, R. I., Waleh, N., Black, S. M., Riemer, R. K., Mauray, F., Chen, Y. Q. 1998; 43 (5): 633-644


    We hypothesized that nitric oxide (NO) production by the fetal ductus arteriosus is limited because of low fetal PO2, but that at neonatal PO2, NO might be an important regulator of ductus arteriosus tone. We exposed isolated rings of fetal lamb ductus arteriosus to elevated PO2. L-NG-nitro-L-arginine methyl ester (L-NAME), an inhibitor of nitric oxide synthase (NOS), and methylene blue and 6-anilino-5,8-quinolinedione (LY83583), inhibitors of guanylate cyclase, produced constriction of the ductus arteriosus. When ductus arteriosus rings were exposed to low PO2, L-NAME had no effect, and methylene blue and LY83583 had only a small effect on ductus arteriosus tone. Sodium nitroprusside and calcium ionophore A23187 relaxed ductus arteriosus rings more than aortic rings, and relaxed ductus arteriosus rings from immature fetuses more than those from late gestation fetuses. In contrast, ductus arteriosus rings from both early and late gestation were equally sensitive to 8-bromo-cGMP. By both reverse transcriptase-polymerase chain reaction and immunohistochemistry, endothelial cell NOS and inducible calcium-independent NOS, but not nerve cell NOS, were detected in the ductus arteriosus. Inducible NOS was expressed only by endothelial cells lining the ductus arteriosus lumen; in contrast, endothelial cell NOS was expressed by both luminal and vasa vasorum endothelial cells. The role of inducible NOS in the ductus arteriosus is uncertain because the potency of a specific inducible NOS inhibitor in constricting the ductus arteriosus was negligible compared with that of an endothelial cell NOS inhibitor. We speculate that NO may be an important regulator of ductus arteriosus tone at high but not low PO2. The endothelial cell NOS isoform found in vasa vasorum may be an important source of NO because removal of ductus arteriosus luminal endothelium only partially blocks the effects of L-NAME, methylene blue, and LY83583.

    View details for Web of Science ID 000073301600012

    View details for PubMedID 9585010

  • Growth factor induction of nitric oxide synthase in rat pheochromocytoma cells MOLECULAR BRAIN RESEARCH Sheehy, A. M., Phung, Y. T., Riemer, K. R., Black, S. M. 1997; 52 (1): 71-77


    Previous work has suggested that nerve growth factor treatment of PC12 cells induces neuronal nitric oxide synthase, and possibly also endothelial nitric oxide synthase (NOS) and inducible NOS. To further analyze this process we exposed rat pheochromocytoma (PC12) cells to increasing concentrations of basic fibroblast growth factor (bFGF), epidermal growth factor (EGF), nerve growth factor (NGF), and vascular endothelial cell growth factor (VEGF). Changes in NOS expression were then analyzed by Western blotting, using antisera generated against the three isoforms of NOS. Our results demonstrate that neuronal NOS was induced by growth factors that promote both differentiation (bFGF, NGF) and proliferation (EGF). nNOS levels were unaffected by VEGF treatment. In contrast, the levels of endothelial and inducible NOS were undetectable in these same cells, suggesting that different clonal lines of PC12 cells have different isoform complements.

    View details for Web of Science ID A1997YL45800008

    View details for PubMedID 9450679

  • Increased expression of nitric oxide synthase in the myometrium of the pregnant rat uterus AMERICAN JOURNAL OF PHYSIOLOGY-ENDOCRINOLOGY AND METABOLISM Riemer, R. K., Buscher, C., Bansal, R. K., Black, S. M., He, Y. P., NATUZZI, E. S. 1997; 272 (6): E1008-E1015


    Nitric oxide (NO) relaxes uterine smooth muscle and is produced by the pregnant uterus. Our previous studies revealed an increase in rat uterine NO synthase (NOS) activity in pregnancy and a decline at term. In the present study, we have examined the distribution of NOS isoform expression to determine whether their regulation is consistent with a role in the inhibition of uterine contractions before term. At day 17-18 of pregnancy, NOS immunohistochemistry revealed expression of two isoforms: endothelial constitutive form of NOS (ecNOS) in vascular endothelium and inducible form of NOS (iNOS) in myometrial and vascular smooth muscle and in decidual epithelium. Immunoblotting revealed that expression of iNOS declined nearly fivefold, whereas ecNOS declined twofold in laboring rats at term. We conclude that iNOS is expressed in myometrium of pregnant rat uterus but not the virgin rat and that iNOS expression declines at term when labor is present. The pattern of changes in myometrial iNOS expression with advancing gestation suggests that NO could act in an autocrine and/or paracrine manner to inhibit uterine contractions before term.

    View details for Web of Science ID A1997XF78600010

    View details for PubMedID 9227445

  • Decline in myometrial nitric oxide synthase expression is associated with labor and delivery JOURNAL OF CLINICAL INVESTIGATION Bansal, R. K., Goldsmith, P. C., He, Y. P., Zaloudek, C. J., Ecker, J. L., Riemer, R. K. 1997; 99 (10): 2502-2508


    The mechanisms that maintain relative uterine quiescence during pregnancy remain largely unknown. A possible role for nitric oxide has recently emerged, however, the expression of nitric oxide synthase within human myometrium at midgestation, a time when the uterus is normally quiescent, has not been investigated. The purpose of this study was to identify cell types in human myometrium that contain inducible nitric oxide synthase (iNOS), and to examine changes in its expression during pregnancy and labor. We found that iNOS is expressed in smooth muscle cells of pregnant myometrium. Expression of iNOS was highest in myometrium of preterm not-in-labor patients. At term, iNOS expression fell by 75%, and was barely detectable in preterm in-labor or term in-labor specimens. There was no staining in the myocytes of nonpregnant myometrium. Western blotting also revealed a similar pattern of changes in iNOS expression. In summary, iNOS expression in the myocytes of human myometrium is increased greatly during pregnancy, and declines towards term or with labor. Significantly, preterm inlabor patients also had a large decline in iNOS expression. These data suggest that changes in myometrial iNOS expression may participate in the regulation of uterine activity during human pregnancy.

    View details for Web of Science ID A1997XB22600030

    View details for PubMedID 9153294



    Prazosin (an alpha-1-adrenergic blocker) and cromakalim (potassium channel opener), given alone, induced significant fatigue of the urethral sphincter at a concentration of 10(-4) M; both drugs combined achieved a significant sphincteric fatigue at a concentration of 10(-5) M each. To 10(-4) M hexamethonium (ganglionic smooth muscle blocker) and 10(-4) M decamethonium (nicotinic blocker of striated muscle) the striated urethral sphincter responded like striated muscle with no detectable function of its smooth muscle component. Therefore, the striated component seems to play a dominant role in sphincteric function. With calcium depletion or in the presence of a calcium channel blocker (10(-4) M nifedipine) the urethral sphincter showed a relative enhancement of response to electrical field stimulation when compared with smooth and skeletal muscle, whose responses were both significantly reduced. This phenomenon could not be explained with calcium-dependent, inhibitory, nitric oxide-releasing nerves, as the NO-synthase blocker N-nitro-L-arginine (10(-5) M to 5 x 10(-5) M) failed to induce the enhancement of sphincter contraction during electrostimulation found with calcium depletion. Still, NO-releasing nerves might play a role in sphincteric relaxation because sodium nitroprusside (10(-5) M) induced a significant relaxation of the urethral sphincter precontracted with 80 mM potassium. The potential to weaken sphincteric closure with drugs, exemplified by the results obtained in response to prazosin and cromakalim, would represent a therapeutic advance in the patient with neurogenic bladder dysfunction.

    View details for Web of Science ID A1995QP29100006

    View details for PubMedID 7540086

  • GLYCERYL TRINITRATE STOPS ACTIVE LABOUR IN SHEEP 3rd International Meeting on the Biology of Nitric Oxide Heymann, M. A., BOOTSTAYLOR, B., Roman, C., NATUZZI, E. S., Parer, J. T., Harrison, M. R., Riemer, R. K. PORTLAND PRESS LTD. 1994: 201–203
  • UTERINE SMOOTH MUSCLE CELLS EXHIBIT AGONIST-LINKED NITRIC OXIDE SYNTHASE ACTIVITY 3rd International Meeting on the Biology of Nitric Oxide Riemer, R. K., NATUZZI, E. S., Baker, S. M., Heymann, M. A., Musci, T. J. PORTLAND PRESS LTD. 1994: 185–189


    The mechanisms that mediate changes in uterine activity from a quiescent state during pregnancy to active labor at parturition are unknown. Nitric oxide (NO), a potent mediator of smooth muscle relaxation, and its presence in the uterus is the subject of this report. Nitric oxide synthase (NOS) activity was demonstrated in nerves, blood vessels and decidua of gravid rat uterus by the NADPH-diaphorase staining method. Uterine tissue fixed during labor demonstrated markedly less NOS. Quantitation of NOS activity in subcellular fractions of pregnant and laboring uterus revealed its presence in both the cytosolic and the membranous compartments of uterine homogenates. In both cellular subfractions the enzyme activity decreased significantly from pregnancy to term. We conclude NOS is present in multiple structures within the uterus. Its presence in two cellular compartments suggests more than one form of NOS may be present in the uterus. Reduction in NOS activity at parturition suggests NO may contribute to the maintenance of uterine contractile quiescence during gestation.

    View details for Web of Science ID A1993LM60300001

    View details for PubMedID 7687424



    We describe several characteristics of a novel smooth muscle cell line, SHM (Syrian hamster myometrium) derived from a primary uterine leiomyosarcoma which was induced by chronic estrogen plus androgen treatment of a female Syrian (golden) hamster. To determine the usefulness of the SHM cell line as a model for understanding myometrial function and its regulation, we have examined the morphologic and immunocytochemical properties of these cells, and the ability of uterotonic agonists to activate transmembrane signaling via phosphoinositide hydrolysis. The SHM cells exhibited a spindle-shape, smooth musclelike morphology when subconfluent, and a more compact, stellate shape at confluence. Like primary myocytes, SHM cells expressed the intermediate filament desmin and the contractile protein alpha smooth muscle actin, but not the epithelial antigen cytokeratin. Norepinephrine and bradykinin, which stimulate contraction and inositol polyphosphate production in the uterus, also stimulated inositol polyphosphate production in SHM cells. The maximal phosphoinositide signaling responses were lower in SHM cells compared with primary hamster uterine myocytes. We conclude that the SHM cell line exhibits primary uterine myocyte characteristics, and may therefore be a useful system for examining the mechanisms through which myometrial functions are regulated.

    View details for Web of Science ID A1993LM38300010

    View details for PubMedID 8392508

  • Myometrial characteristics of the syrian hamster uterine smooth muscle cell line, SHM. In vitro cellular & developmental biology. Animal Riemer, R. K., Sadovsky, Y., ROBERTS, J. M. 1993; 29 (6): 478-480


    We describe several characteristics of a novel smooth muscle cell line, SHM (Syrian hamster myometrium) derived from a primary uterine leiomyosarcoma which was induced by chronic estrogen plus androgen treatment of a female Syrian (golden) hamster. To determine the usefulness of the SHM cell line as a model for understanding myometrial function and its regulation, we have examined the morphologic and immunocytochemical properties of these cells, and the ability of uterotonic agonists to activate transmembrane signaling via phosphoinositide hydrolysis. The SHM cells exhibited a spindle-shape, smooth musclelike morphology when subconfluent, and a more compact, stellate shape at confluence. Like primary myocytes, SHM cells expressed the intermediate filament desmin and the contractile protein alpha smooth muscle actin, but not the epithelial antigen cytokeratin. Norepinephrine and bradykinin, which stimulate contraction and inositol polyphosphate production in the uterus, also stimulated inositol polyphosphate production in SHM cells. The maximal phosphoinositide signaling responses were lower in SHM cells compared with primary hamster uterine myocytes. We conclude that the SHM cell line exhibits primary uterine myocyte characteristics, and may therefore be a useful system for examining the mechanisms through which myometrial functions are regulated.

    View details for DOI 10.1007/BF02639382

    View details for PubMedID 27519749



    Although the in vivo effect of estrogen on myometrial differentiation is well documented, estrogen effects on primary myocytes in vitro have been difficult to demonstrate. To construct a stable uterine myocyte system, capable of direct estrogen responsiveness, we used a transformed hamster myometrial cell line. Since these cells expressed a low level of estrogen receptors (ERs), we have stably transfected them with a vector for the human ER. After transfection, ER concentration increased from less than 300 sites per cell to 17,000 +/- 2,000 sites per cell (mean +/- SEM). To test the functional integrity of the transfected receptors, a chloramphenicol acetyltransferase gene linked to an estrogen response element upstream of thymidine kinase promoter was transiently transfected, and the amount of chloramphenicol acetyltransferase activity, an indicator of estrogen responsiveness, was found to increase 20-fold in response to 17 beta-estradiol (1 nM for 48 h). Furthermore, we tested the ability of estrogen to activate endogenous genes by measuring progesterone receptor (PR) induction. PR concentration in the transfected cells was 3,700 +/- 800 and increased 9-fold to 33,000 +/- 6,000 with 17 beta-estradiol (2 nM). This receptor density increase was confirmed by immunoblotting. PR induction was maximal at 16 h, was concentration dependent, and was not elicited by tamoxifen or ICI 164,384. We conclude that transformed hamster myocytes transfected with an ER gene are capable of estrogen-dependent PR expression in vitro and may serve as a useful system to study estrogen effect on myocytes.

    View details for Web of Science ID A1993KV62900025

    View details for PubMedID 8462459



    Prostaglandin E1 (PGE1) has been shown to relax the muscles of the corpora cavernosa and inhibit spontaneous activity, and clinical trials have proved its safety and effectiveness when given intracavernously to induce erection. Through use of a specific PGE1 receptor binding assay, we undertook this study to quantify its receptor density and measure binding affinity. The cavernous tissue of normal and impotent men as well as that of monkeys and dogs was studied in an attempt to understand their different responses to the intracavernous injection of PGE1. Our results showed a lower receptor density in impotent men than in normal men and monkeys and a complete absence of receptors in dogs. These findings correlated well with the clinical response to intracavernous injection of PGE1.

    View details for Web of Science ID A1993KV10000005

    View details for PubMedID 8465481



    The purpose of this study was to determine whether the concentration of estrogen receptors in cultured myocytes is preserved after dispersion.Primary myocytes were prepared from rabbit myometrium by collagenase dispersion after removing the endometrium and were isolated with Percoll density gradients. The cells were assayed for estrogen receptor concentration at intervals after dispersion by means of a whole-cell binding assay. Unpaired t test was used for comparisons.The concentration of estrogen receptors on the first day after dispersion was 12,058 +/- 1096 sites per cell (mean +/- SEM) and decreased to 4389 +/- 1223 site per cell within 9 to 14 days after dispersion (63% decline, p < 0.001). A similar decrease was observed when 2 nmol/L estradiol was present in the medium.The concentration of estrogen receptors in isolated rabbit uterine myocytes decreases after dispersion. This may partly explain the difficulty of demonstrating in vitro estrogen effects on myocytes, which are well established in vivo.

    View details for Web of Science ID A1992KD42900027

    View details for PubMedID 1471678



    We examined the linkage of cholinergic receptors to the phosphoinositide signaling pathway to elucidate one facet of the autonomic response mechanism in fetal and adult sheep. Cholinergic stimulation with carbachol increases the production of 3H-inositol mono-, bis-, and trisphosphates in a time- and concentration-dependent manner in both fetal and adult myocardium. However, the maximal stimulation of inositol polyphosphates above basal activity was much greater in fetal (120 +/- 11%) than in adult (20 +/- 7%) myocardium (mean +/- SEM). Saturation binding analysis of myocardial muscarinic receptors using 3H-N-methylscopolamine revealed significantly higher receptor concentration in fetal (240 +/- 25 fmol/mg protein) than in adult (78 +/- 15 fmol/mg protein) myocardium (mean +/- SEM). Binding competition studies revealed a pattern of selectivity-atropine less than 4-diphenylacetoxy-N-methylpiperidine methiodide less than pirenzepine less than or equal to (4-hydroxy-2-butynyl)-1-trimethylammonium m-chlorocarbanilate chloride less than or equal to 11-2[[2-[(diethylamino)-methyl]-1-piperidinyl]acetyl]-5, 11-dihydro-6H-pyrido[2,3-b][1,4]benzodiazepine-6-one 116-compatible with the presence of muscarinic receptor (MR)2, MR3, and/or MR5 subtypes. Receptor subtype determination by Northern blot analysis revealed mRNA specific for the MR2 subtype in both fetal and adult myocardium, although expression was greater in fetal heart. We conclude that decreases in MR2 subtype protein and mRNA levels parallel the age-related decrease in carbachol-stimulated PLC activity. Our studies demonstrate differences between fetal and adult myocardium in the concentration of muscarinic cholinergic receptors and their linkage to a putative calcium mobilizing signaling pathway and suggest that this pathway may play a different role in the fetus than in the adult.(ABSTRACT TRUNCATED AT 250 WORDS)

    View details for Web of Science ID A1992HV26600013

    View details for PubMedID 1321973



    Estrogen increases the alpha 1-adrenergic contractile sensitivity of the rabbit uterus. Since estrogen treatment increases prostaglandin (PG) production by the perfused rabbit uterus, and PGs contribute to the alpha 1-adrenergic contractile response, we postulated that estrogen's effects on PG production or response may play a role in the increased alpha 1-adrenergic sensitivity induced by estrogen. We studied the effects of the eicosanoid synthesis inhibitor meclofenamate (60 microM) on the response to epinephrine (10(-9)-10(-5) M) of uterine strips from ovariectomized, mature, and estrogen-treated rabbits in terms of both contractile response and PGE2 and PGF2 alpha production. We also measured the contractile response to PGE2 and PGF2 alpha (both 10(-10)-10(-5) M) and KCl (70 mM) of uterine strips from these groups. We found that in the ovariectomized rabbits, meclofenamate decreased PG production, but did not alter the alpha 1-adrenergic sensitivity. In the mature rabbit uterus, meclofenamate decreased both PGE2 and PGF2 alpha production and reduced the alpha 1-adrenergic sensitivity. In the estrogen-treated rabbit uterus, meclofenamate decreased PGF2 alpha, but not PGE2, production and did not alter the alpha 1-adrenergic sensitivity. Finally, meclofenamate reduced the contractile response to KCl in all three groups, and exposure to PGE2 increased the contractile response to KCl in both the mature and estrogen-treated rabbits. We conclude that PGs play a role in the increase in the alpha 1-adrenergic sensitivity of the uterus in mature rabbits, and that this may be the result of an estrogen-mediated alteration in the postreceptor effects of PGs.

    View details for Web of Science ID A1991GD33300043

    View details for PubMedID 1651850



    Cocaine use in pregnancy is associated with a premature labor rate as high as 50%, but little is known about its effect on uterine contractility. To determine whether cocaine directly augments pregnant uterus contractility, uterine strips from 27-day pregnant New Zealand White rabbits (term, 31 days) were exposed to cocaine alone (30 mumol/L) or cocaine plus epinephrine (10(-9) to 10(-5) mol/L) or oxytocin (10(-10) to 10(-6) mol/L). Cocaine alone produced no contractions, but increased the epinephrine sensitivity by 51% and the maximal response by 33%. When beta-adrenoceptors were blocked with DL-propranolol (2 mumol/L), the contractile response to epinephrine was increased, and cocaine's effect was blocked. In the presence of the stereoisomer D-propranolol (2 mumol/L) with no beta-adrenergic antagonist activity, the contractile response to epinephrine was unchanged, but the effect of cocaine was still blocked. We conclude that cocaine directly augments the alpha-adrenergic contractile response of the pregnant rabbit uterus by a mechanism that is blocked by the non-beta-adrenergic effects of propranolol.

    View details for Web of Science ID A1991ET50000039

    View details for PubMedID 1670909



    Previously, we demonstrated that either putative leukotriene receptor antagonists or a synthesis inhibitor markedly decreased pulmonary vascular resistance in the near-term fetal lamb and concluded that leukotrienes may play a role in maintaining the high pulmonary vascular resistance in the fetus. To further investigate the role of leukotrienes, we measured concentrations of leukotriene (LT) C4, LTD4, and LTE4 in 17 tracheal fluid samples from 8 of 9 near-term (129-139 days, term = 145 days), chronically-catheterized, fetal lambs during normoxia to evaluate their possible role in regulating resting tone and in seven of the nine before and during hypoxia to evaluate their possible role in hypoxic vasoconstriction. The tracheal fluid samples collected by gravity over 1-3 min, on ice, were immediately treated with cold ethanol, centrifuged, and the supernatant covered with N2 and stored in a -70 degrees C freezer for a maximum of 3 weeks. Purification and separation of leukotrienes was done by reverse-phase high performance liquid chromatography using a gradient elution method, and fractions corresponding to LTC4, LTD4, and LTE4 standards were quantified immediately by radioimmunoassay. During normoxia (descending aortic PaO2 2.9 +/- 0.3 kPa [21.5 +/- 2.5 mmHg]; mean +/- SD), all 3 leukotrienes were detected in 16 of the 17 samples: LTC4 29 +/- 28 pg/ml (range 0-119 pg/ml); LTD4 66 +/- 51 pg/ml (range 9-177 pg/ml); and LTE4 43 +/- 50 pg/ml (range 0-204 pg/ml).(ABSTRACT TRUNCATED AT 250 WORDS)

    View details for Web of Science ID A1990EX88600006

    View details for PubMedID 1965438



    The uterine response to adrenergic stimulation is determined by the hormonal milieu. This response is particularly well characterized in the rabbit. In this species, as in humans, the response of the uterus to sympathetic stimulation is alpha-adrenergically mediated contraction with elevated circulating estrogen. However, with progesterone predominance, similar stimulation inhibits uterine contractions, a response mediated by beta-adrenergic receptors acting through their second message, cyclic adenosine monophosphate. We studied the mechanisms by which sex steroids regulate myometrial adrenergic responses. In this study, we questioned whether part of the effect of sex steroids could be explained by an alteration of the coupling of the alpha 2-adrenergic receptor to the inhibition of adenylate cyclase. We found that in the progesterone-treated rabbit, although alpha 2-receptors are present, they are not linked to inhibition of cyclic adenosine monophosphate synthesis. The net synthesis of cyclic adenosine monophosphage in response to endogenous catecholamines is determined by their activation of beta-adrenergic receptors to increase and alpha 2-receptors to decrease cyclic adenosine monophosphate formation. Thus the uncoupling of alpha 2-receptors contributes to increased intracellular cyclic adenosine monophosphate in myometrium of progesterone-treated animals consistent with the reported predominance of beta-adrenergic contractile responses in this setting.

    View details for Web of Science ID A1989U260700009

    View details for PubMedID 2540655



    The contractile response of the uterus is modified by sex steroids. In rabbit uterus, oestrogen promotes alpha-adrenergically-mediated contraction, whilst progesterone treatment results in beta-adrenergic relaxation. Examination of the mechanisms responsible for these changing adrenergic responses with sex steroids reveals multiple sites of regulation. Oestrogen increases alpha 1-receptor concentration and the linkage of the receptor to phospholipase C. In addition to this direct effect to promote contraction, oestrogen also uncouples the beta-receptor from adenylate cyclase. Progesterone, conversely, promotes relaxation through beta-receptors by uncoupling alpha 2-receptors from inhibition of adenylate cyclase. Thus sex steroids can regulate specific agonist responses at and beyond the receptor.

    View details for Web of Science ID A1989AR24700001

    View details for PubMedID 2553793



    The uterine contractile response to adrenergic agonists or sympathetic stimulation is influenced dramatically by the hormonal milieu. Rabbit uterine contraction is mediated by alpha 1-adrenoceptors, whereas relaxation in response to the same stimulus is mediated by beta 2-adrenoceptors. Whether uterine contractility is increased or decreased by adrenergic stimulation is determined by the gonadal steroids estrogen and progesterone: uterine contraction prevails in the estrogen-dominant or the ovariectomized animal, but in the progesterone-dominant rabbit, uterine relaxation is observed. In previous studies, we have demonstrated that changes in the concentration or agonist affinity of these adrenoceptors cannot account for the changes in contractile response. In the present studies, we tested whether sex steroids might alter beta-adrenergic response by acting on events distal to receptor occupancy, and whether this could explain the conversion of contractile response. We found that myometrial cAMP generation is potently stimulated by beta-agonists in progesterone-treated and also in ovariectomized animals, but this stimulation is absent after estrogen treatment. Similar, but smaller, changes were observed for cAMP generation in response to prostaglandin E2 and forskolin. Stimulation of adenylate cyclase in uterine particulates by agents which act on the guanyl nucleotide-sensitive stimulatory transducer, Gs, is unchanged after estrogen treatment. However, specific labeling of Gs catalyzed by cholera toxin is reduced in membrane particulates from estrogen-treated animals. Recombination of extracts of uterine membranes from the differently treated animals also suggested qualitative differences in Gs. We conclude that at least one component of the adenylate cyclase cascade beyond the beta-adrenoceptor, i.e., Gs, is a target for ovarian steroids; estrogen reduces Gs labeling and beta-adrenoceptor-mediated cAMP production. However, uterine Gs labeling and cAMP production are similar in ovariectomized and in progesterone-treated rabbits. Since these uteri exhibit different contractile responses, the observed changes are not sufficient to explain sex steroid-mediated conversion of myometrial contractile response.

    View details for Web of Science ID A1988N033800005

    View details for PubMedID 2833685



    alpha 1-Adrenergic and muscarinic cholinergic stimuli activate uterine contraction. Estrogen increases adrenergic but not cholinergic sensitivity of rabbit myometrium independent of its effects on adrenoceptor concentration. Since both alpha 1-adrenergic and muscarinic receptors are coupled to phosphatidylinositol hydrolysis, we tested the hypothesis that estrogen increases adrenergic- but not cholinergic-mediated inositol triphosphate production. We found that maximal production of inositol phosphates stimulated by norepinephrine was increased approximately 3-fold following estrogen treatment. Cholinergic-stimulated production was not increased by estrogen treatment. These results demonstrate that the effect of estrogen to enhance uterine adrenergic sensitivity is associated with an increased post-receptor response. The nature of the selectivity of estrogen for adrenergic versus cholinergic response remains obscure, but the results suggest the presence of parallel pathways for receptor activation of a common post-receptor response.

    View details for Web of Science ID A1987L110200014

    View details for PubMedID 2824981



    Both myometrial oxytocin and alpha 2-adrenergic receptors are induced by estrogen. To compare the regulation of these two receptor populations by progesterone, we measured myometrial receptor concentration in ovariectomized steroid-treated and in pregnant rabbits. To control for the effects of estrogen withdrawal, we used concomitant rather than sequential presentation of estrogen and progesterone in ovariectomized rabbits. Estradiol increased both myometrial oxytocin and alpha 2-adrenergic receptor concentrations in ovariectomized rabbits after 8 days of treatment. Simultaneous progesterone administration during the last 4 days of estradiol treatment reversed the induction of oxytocin, but not alpha 2-adrenergic, receptors. Similarly, administration of the antiprogestin RU 38486 to pregnant rabbits on day 27 of gestation resulted in premature delivery and evoked an increase in myometrial oxytocin receptor concentration mimicking that observed at term (day 31). However, RU 38486 did not significantly affect alpha 2-adrenergic receptor concentration. Our data provide further support for involvement of oxytocin receptors in parturition, but do not indicate a comparable function for myometrial alpha 2-adrenergic receptors.

    View details for Web of Science ID A1987G193000049

    View details for PubMedID 3026788



    The ability of estrogen to increase the alpha-1 adrenergic sensitivity of rabbit uterine smooth muscle was compared with its effects on the concentration of alpha adrenoceptor subtypes in myometrium. Compared to ovariectomized controls, estrogen treatment was found to increase the adrenergic contractile sensitivity of rabbit uterus determined in vitro. Estrogen treatment increased uterine alpha-2 adrenoceptor concentration nearly 5-fold. Mature rabbits (endogenous estrogen) had the same uterine sensitivity and alpha-2 adrenoceptor concentration as estrogen-treated rabbits. Alpha-1 receptor concentration was increased only in the estrogen-treated group, and was accompanied by increased maximal contractile response. Thus, the increase in adrenergic sensitivity after estrogen was correlated closely with an increased concentration of alpha-2 adrenoceptors. However, enhanced adrenergic sensitivity persisted after alpha-1 and alpha-2 receptor concentration returned to pre-estrogen levels in rabbits pretreated with estrogen before ovariectomy. Studies utilizing subtype-selective competitive antagonists verified that contractile response is mediated primarily by alpha-1 adrenoceptors, with no apparent influence of alpha-2 adrenoceptors. Finally, we found that adrenergic sensitivity is altered by temperature and divalent cation concentration, but these effects do not prevent the expression of the regulatory action of estradiol. We conclude that estrogen increases the alpha-1 adrenergic sensitivity of rabbit uterus without changes in alpha-1 receptors, and thus may act on postreceptor response events.(ABSTRACT TRUNCATED AT 250 WORDS)

    View details for Web of Science ID A1987F649900007

    View details for PubMedID 3027308


    View details for Web of Science ID A1986F781900005

    View details for PubMedID 3105067



    We examined the relation between increased uterine oxytocin receptor concentration and increased in vivo sensitivity of the rabbit uterus to oxytocin at the end of gestation. We determined oxytocin receptor concentrations in myometrium and decidua on different days near term of gestation and postpartum. We also examined the in vitro contractile response to oxytocin on days 30 and 5 days postpartum, when the uterus is unresponsive in vivo, and on day 31 (term), when the uterus is exquisitely sensitive to this hormone in vivo. In addition, we tested the role of endogenous eicosanoids and decidual oxytocin receptors in the myometrial contractile response to oxytocin by examining the contractile response in the presence of the cyclooxygenase/lipoxygenase inhibitor sodium meclofenamate or in muscle strips from which the decidua had been removed by scraping. The concentration of specific binding sites for [3H]oxytocin in myometrial and also decidual membrane preparations was determined. We demonstrate that contractile sensitivity to oxytocin increases at least 4-fold between days 30 and 31 (term) of gestation, and this is accompanied by a nearly 10-fold increase in the concentration of oxytocin-binding sites in both decidua and myometrium. The lesser sensitivity to oxytocin on day 30 was, however, only apparent in the presence of meclofenamate, which suggests that endogenous eicosanoids contribute to the preterm response to oxytocin measured in vitro. The maximal response to oxytocin (integrated area) increased 2-fold between day 30 and term. Thus, an increase in both sensitivity and maximal response to oxytocin could be demonstrated at term in vitro. Five days after parturition, maximal response and uterine sensitivity measured in the presence of meclofenamate had returned to those of the preterm uterus, and the concentration of oxytocin-binding sites had declined. In contrast, sensitivity and maximal response to the cholinergic agonist carbamylcholine declined between day 30 and term. These results support a highly regulated physiological role for oxytocin in parturition which depends primarily on changes in receptor concentration.

    View details for Web of Science ID A1986D342700037

    View details for PubMedID 3015554


    View details for Web of Science ID A1985AEB2000028

    View details for PubMedID 2991309



    In isolated, isometrically contracting left guinea pig atria, sanguinarine, a benzophenanthridine alkaloid from the papaveracea Sanguinaria canadensis, produced a concentration-dependent positive inotropic effect. Between 2.3 x 10(-6) M and 6.5 x 10(-5) M, sanguinarine increased contractility by 108% which was comparable to the maximal inotropic effect of ouabain. Within the same concentration range, sanguinarine caused inhibition of Na+,K+-ATPase isolated from guinea pig myocardium. 100% inhibition of Na+,K+,ATPase activity occurred at 1 x 10(-4) M sanguinarine. The I50 for enzyme inhibition and the ED50 for the inotropic action of sanguinarine were the same (6-6.5 x 10(-6) M) indicating that both effects may be causally related.

    View details for Web of Science ID A1979JB86100016

    View details for PubMedID 230984