Survival and maturation of human embryonic stem cell-derived cardiomyocytes in rat hearts

Human embryonic stem cell (hESC)-derived cardiomyocytes are a promising cell source for cardiac repair. Whether these cells can be transported long distance, survive, and mature in hearts subjected to ischemia/reperfusion with minimal infarction is unknown. Taking advantage of a constitutively GFP-expressing hESC line we investigated whether hESC-derived cardiomyocytes could be shipped and subsequently form grafts when transplanted into the left ventricular wall of athymic nude rats subjected to ischemia/reperfusion with minimal infarction. Co-localization of GFP-epifluorescence and cardiomyocyte-specific marker staining was utilized to analyze hESC-derived cardiomyocyte fate in a rat ischemia/reperfused myocardium. Differentiated, constitutively green fluorescent protein (GFP)-expressing hESCs (hES3-GFP; Envy) containing about 13% cardiomyocytes were differentiated in Singapore, and shipped in culture medium at 4 degrees C to Los Angeles (shipping time approximately 3 days). The cells were dissociated and a cell suspension (2 x 10(6) cells for each rat, n=10) or medium (n=10) was injected directly into the myocardium within the ischemic risk area 5 min after left coronary artery occlusion in athymic nude rats. After 15 min of ischemia, the coronary artery was reperfused. The hearts were harvested at various time points later and processed for histology, immunohistochemical staining, and fluorescence microscopy. In order to assess whether the hESC-derived cardiomyocytes might evade immune surveillance, 2 x 10(6) cells were injected into immune competent Sprague-Dawley rat hearts (n=2), and the hearts were harvested at 4 weeks after cell injection and examined as in the previous procedures. Even following 3 days of shipping, the hESC-derived cardiomyocytes within embryoid bodies (EBs) showed active and rhythmic contraction after incubation in the presence of 5% CO(2) at 37 degrees C. In the nude rats, following cell implantation, H&E, immunohistochemical staining and GFP epifluorescence demonstrated grafts in 9 out of 10 hearts. Cells that demonstrated GFP epifluorescence also stained positive (co-localized) for the muscle marker alpha-actinin and exhibited cross striations (sarcomeres). Furthermore, cells that stained positive for the antibody to GFP (immunohistochemistry) also stained positive for the muscle marker sarcomeric actin and demonstrated cross striations. At 4 weeks engrafted hESCs expressed connexin 43, suggesting the presence of nascent gap junctions between donor and host cells. No evidence of rejection was observed in nude rats as determined by inspection for lymphocytic infiltrate and/or giant cells. In contrast, hESC-derived cardiomyocytes injected into immune competent Sprague-Dawley rats resulted in an overt lymphocytic infiltrate. hESCs-derived cardiomyocytes can survive several days of shipping. Grafted cells survived up to 4 weeks after transplantation in hearts of nude rats subjected to ischemia/reperfusion with minimal infarction. They continued to express cardiac muscle markers and exhibit sarcomeric structure and they were well interspersed with the endogenous myocardium. However, hESC-derived cells did not escape immune surveillance in the xenograft setting in that they elicited a rejection phenomenon in immune competent rats.     

Direct and acute cardiotoxic effects of ultrafine air pollutants in spontaneously hypertensive rats and Wistar--Kyoto rats

It is hypothesized that preexisting cardiovascular disease could affect the susceptibility to direct and acute cardiotoxic effects of ultrafine air pollutants. Ultrafine particles (UFP) isolated from 12.5 mg of diesel particulate matter (National Institute of Standards and Technology) were infused into isolated Langendorffperfused hearts obtained from spontaneously hypertensive rats (SHR) and normotensive control Wistar- Kyoto rats (WKY). Perfusion for 30 minutes with UFP reduced cardiac function in both groups-but to a greater extent in WKY. In SHR, developed pressure was reduced by 24.1 +/- 4.4% of baseline and maximal dP/dt was reduced by 19.8 +/- 4.9%; in WKY, developed pressure was reduced by 43.5 +/- 7.3% and maximal dP/dt by 41.8 +/- 8.2% (P < .05 for maximal dP/dt in SHR vs WKY). Coronary flow was decreased by 30.3% versus 53.7% in SHR versus WKY ( P < .05). The results of this study suggest that although UFP depress myocardial contractile response and coronary flow in both SHR and WKY the underlying hypertension does not necessarily worsen the response.     

Correlates of reperfusion ventricular fibrillation in dogs

To elucidate determinants of reperfusion ventricular fibrillation (VF), regional myocardial blood flow, ATP, creatine phosphate (CP), heart rate and blood pressure were compared in 2 groups of anesthetized dogs: those that fibrillated spontaneously upon release of a 15-minute coronary artery occlusion (VF group, n = 8) and those that did not fibrillate when reperfused (No VF group, n = 27). Arterial pressure and heart rate before and during coronary artery occlusion were similar in both groups. Ischemie endo- and epicardial ATP values, measured at the end of the occlusion period, were reduced approximately 20% of nonischemic values in both groups. In contrast, CP (nmohmg protein^?1) within the ischemie zone was significantly lower in the VF group in both the epicardium (14.3 ± 1.6 in the VF group vs 22.8 ± 2.5 in the No VF group, p < 0.01) and the endocardium (9.0 ± 2.0 in the VF group vs 18.7 ± 1.8 in the No VF group, p < 0.01). Furthermore, epi- and endocardial regional myocardial blood flow in the center of the ischemic zone during occlusion was significantly lower in VF dogs than in No VF dogs. Epicardial flow was 0.06 ± 0.03 ml·min^?1·g^?1in VFdogsvs 0.44 ± 0.06 in No VF dogs (p < 0.001) and endocardial flow was 0.03 ± 0.02 ml·min^?1·g^?1 in VF dogs vs 0.23 ± 0.04 ·ml-min^?1·g^?1 in No VF dogs (p < 0.001). These data suggest that low levels of regional myocardial blood flow and CP during coronary artery occlusion are associated with an increased risk of VF on reperfusion. Thus, the severity of ischemia in the center of the ischemie zone may be a determinant of reperfusion VF.     

Direct and acute cardiotoxicity of ultrafine particles in young adult and old rat hearts

Abstract Background Air pollution is associated with significant increases in cardiac morbidity and mortality in the general population. The elderly cohort within the general population is considered at high risk for cardiac diseases. However the degree to which air pollutants affect cardiac responses in old hearts vs. their young adult counterparts has not been systematically addressed. Objectives We sought to investigate the response of young adult vs. old rat hearts to the direct exposure of ultrafine particles (UFP); i.e. when the UFP are directly instilled into the cardiac vasculature, and their effects are not dependent upon UFP inhalation. Methods The study was performed in isolated Langendorff-perfused rat hearts obtained from young adult (4 months old) and aged (26 months old) Fisher 344/Brown Norway rats. Two treatment groups (control and UFP-treated) were studied, and two ages (young adult and old) were studied within each group. Control hearts were perfused with buffer only, UFP-treated hearts were perfused with buffer containing ultrafine particles isolated from industrial diesel reference particulate matter. Systolic and end-diastolic pressures, positive and negative dP/dt, and coronary flow were measured. Results Young adult and old hearts demonstrated equal functional deterioration in response to direct infusion of UFP. Developed pressure in young adult UFP-treated hearts fell from 101±4 to 68±8 mmHg (a decrease by 33%, p<0.05). In the old UFP-treated hearts developed pressure fell by 35% (from 101±7 to 67±9mmHg, p<0.05). Positive dP/dt was equally affected in the young adult and old UFP-treated hearts and was decreased by 28% in both groups. Conclusion Ultrafine particles when instilled directly into the cardiac vasculature were equally cardiotoxic in young adult and old rat hearts.     

Postconditioning does not reduce myocardial infarct size in an in vivo regional ischemia rodent model

In our laboratory, postconditioning reliably reduces lethal ventricular arrhythmias in an in vivo rat model but its effect on necrosis in our model is unknown. In the present analysis, we tested a variety of postconditioning regimens in anesthetized rats subjected to 45 minutes of coronary occlusion and 120 minutes of reperfusion or 30 minutes of coronary occlusion and 120 minutes of reperfusion. In all studies, area at risk was determined by the blue dye technique and area of necrosis was assessed with triphenyl tetrazolium chloride staining and computerized planimetry of ventricular slices. Postconditioning regimens included 4 cycles of 10 seconds of reperfusion/10 seconds of reocclusion, 4 cycles of 20 seconds of reperfusion/20 seconds of reocclusion, 8 cycles of 30 seconds of reperfusion/30 seconds of reocclusion, and 20 cycles of 10 seconds of reperfusion/10 seconds of reocclusion. Postconditioning did not reduce myocardial infarct size with any of these regimens.