Joan Dow

Allogeneic Mesenchymal Stem Cell Transplantation in Postinfarcted Rat Myocardium Short- and Long-Term Effects

Background—Mesenchymal stem cells (MSCs) have the potential to replace infarct scar, but the long-term effects are unknown. We studied short- and long-term effects of MSC transplantation on left ventricular (LV) function in a rat myocardial infarction model. Methods and Results—Saline (n=46) or MSCs labeled with 1,1′-dioctadecyl-3,3,3′3′-testramethylindocarbocyanine perchlorate (DiI; n=49, 2×106 cells each) were injected into the scar of a 1-week-old myocardial infarction in Fischer rats. The presence and differentiation of engrafted cells and their effect on LV ejection fraction was assessed. At 4 weeks, LV stroke volume was significantly greater in the MSC-treated group (145±9 &mgr;L) than in the saline group (122±3 &mgr;L, P=0.032), and LV ejection fraction was significantly greater in MSC-treated animals (43.8±1.0%) than in the saline group (38.8±1.1%, P=0.0027). However, at 6 months, these benefits of MSC treatment were lost. DiI-positive cells were observed in the MSC group at 2 weeks and at 3 and 6 months. Expression of the muscle-specific markers α-actinin, myosin heavy chain, phospholamban, and tropomyosin was not observed at 2 weeks in DiI-positive cells. At 3 and 6 months, the DiI-positive cells were observed to express the above muscle-specific markers, but they did not fully evolve into an adult cardiac phenotype. Some of the DiI-positive cells expressed von Willebrand factor. Conclusions—Allogeneic MSCs survive in infarcted myocardium as long as 6 months and express markers that suggest muscle and endothelium phenotypes. MSCs improved global LV function at 4 weeks; however, this benefit was transient, which suggests a possible early paracrine effect.

Rebuilding a Damaged Heart Long-Term Survival of Transplanted Neonatal Rat Cardiomyocytes After Myocardial Infarction and Effect on Cardiac Function

Background—The long-term effects of cardiac cell transplantation on cardiac function are unknown. Therefore, we tested the survival and functional impact of rat neonatal cardiac myocytes up to 6 months after transplantation into infarcted hearts. Methods and Results—Cardiomyocytes from male neonatal Fischer 344 rats (1 to 2 days, 3 to 5×106) or medium was injected into the infarcts of adult syngeneic female animals 1 week after left coronary artery ligation. Six months later, implanted cardiomyocytes were still present by quantitative TaqMan polymerase chain reaction and histology. In all treated hearts, discrete lumps of cells were present within the infarct scar, which was not observed in media-injected hearts typified by a transmural infarct scar. Infarct thickness was greater in treated animals versus control animals (909±97 versus 619±43 &mgr;m, P <0.02), whereas infarct size and left ventricular volumes were similar. By biplane angiography, left ventricular ejection fractions at 6 months were greater (0.36±0.03 versus 0.25±0.02, P <0.01) and significantly less infarct zone dyskinesis was seen (0.30±0.08 versus 0.55±0.07, P =0.035, lateral projection) in treated animals versus control animals. Conclusions—Grafted neonatal cardiomyocytes were present in infarcts 6 months after transplantation; they thickened the wall of the left ventricle and were associated with enhanced ejection fraction and reduced paradoxical systolic bulging of the infarct. Therefore, neonatal cardiac cell transplants exhibit long-term survival in a myocardial infarct model and contribute to long-term improved cardiac function. These results suggest that a damaged heart can be rebuilt.

No-reflow phenomenon persists long-term after ischemia/reperfusion in the rat and predicts infarct expansion.

Background—No-reflow after reperfusion therapy for myocardial infarction is a strong predictor of clinical outcome. But its fate on a long-term basis and potential significance for infarct healing are not yet known. Methods and Results—Twenty-nine female Fisher rats were subjected to 60 minutes of coronary occlusion followed by reperfusion. At 4 weeks, 15 survivors were euthanized after measurement of regional myocardial blood flow (radioactive microspheres) and in vivo staining of perfused tissue (0.5 mL 50% Uniperse blue IV). Infarct size (34.3±3.4%), scar thickness (1.19±0.10 mm), and infarct expansion index (0.51±0.04) were assessed from histological sections (2 additional exclusions because of failed occlusion). Regional myocardial blood flow in the reperfused infarct was reduced significantly compared with noninfarcted tissue (1.98±0.47 versus 4.55±0.86 mL · min−1 · g−1, P <0.003, apical slice, and 1.77±0.44 versus 5.34±0.38 mL · min−1 · g−1, P <0.0001, second slice), accompanied by a striking reduction of perfused capillaries within the infarct (n=23±4 versus 163±8 in the noninfarcted tissue, P <0.0001, microscopically assessed as capillaries containing blue particles per high-power field). Macroscopically, no-reflow areas were visible in 9 of 13 hearts. The number of perfused capillaries within the infarct correlated significantly with infarct expansion index (r =−0.76, P <0.003), infarct thickness (r =0.60, P <0.03), and the ratio of infarct to septum thickness (r =0.74, P <0.004). Conclusions—The no-reflow phenomenon persists for 1 month after reperfusion and predicts worse scar thinning and infarct expansion. Thus, one might shift the “open-artery” hypothesis downstream to an “open-microvessel” hypothesis, relating infarct healing, infarct expansion, and outcome to the completeness of microvascular reperfusion above and beyond epicardial artery patency.

Ranolazine, an antianginal agent, markedly reduces ventricular arrhythmias induced by ischemia and ischemia-reperfusion.

We tested the effect of the antianginal agent ranolazine on ventricular arrhythmias in an ischemic model using two protocols. In protocol 1, anesthetized rats received either vehicle or ranolazine (10 mg/kg, iv bolus) and were subjected to 5 min of left coronary artery (LCA) occlusion and 5 min of reperfusion with electrocardiogram and blood pressure monitoring. In protocol 2, rats received either vehicle or three doses of ranolazine (iv bolus followed by infusion) and 20 min of LCA occlusion. With protocol 1, ventricular tachycardia (VT) occurred in 9/12 (75%) vehicle-treated rats and 1/11 (9%) ranolazine-treated rats during reperfusion (P = 0.003). Sustained VT occurred in 5/12 (42%) vehicle-treated but 0/11 in ranolazine-treated rats (P = 0.037). The median number of episodes of VT during reperfusion in vehicle and ranolazine groups was 5.5 and 0, respectively (P = 0.0006); median duration of VT was 22.2 and 0 s in vehicle and ranolazine rats, respectively (P = 0.0006). With protocol 2, mortality in the vehicle group was 42 vs. 17% (P = 0.371), 10% (P = 0.162) and 0% (P = 0.0373) with ranolazine at plasma concentrations of 2, 4, and 8 microM, respectively. Ranolazine significantly reduced the incidence of ventricular fibrillation [67% in controls vs. 42% (P = 0.414), 30% (P = 0.198) and 8% (P = 0.0094) in ranolazine at 2, 4, and 8 microM, respectively]. Median number (2.5 vs. 0; P = 0.0431) of sustained VT episodes, incidence of sustained VT (83 vs. 33%, P = 0.0361), and the duration of VT per animal (159 vs. 19 s; P = 0.0410) were also significantly reduced by ranolazine at 8 microM. Ranolazine markedly reduced ischemia-reperfusion induced ventricular arrhythmias. Ranolazine demonstrated promising anti-arrhythmic properties that warrant further investigation.

Long-term outcome of fetal cell transplantation on postinfarction ventricular remodeling and function

OBJECTIVES The purpose of this study was to determine the long-term outcome of fetal cell transplantation into myocardial infarction on left ventricular (LV) function and remodeling. BACKGROUND While neonatal cell transplantation improved function for acute myocardial infarction, long-term data on the effects of cell-transplant therapy using a more primitive cell on ventricular remodeling and function are needed.Methods. - Therefore, we injected 4 x 10(6) Fischer 344 fetal cardiac cells or medium into 1-week old infarcts in adult female Fischer rats to assess long-term outcome. RESULTS Ten months after transplantation histologic analysis showed that cell implants were readily visible within the infarct scar. Infarct wall thickness was greater in cell-treated at 0.69 +/- 0.05 mm (n = 11) vs. medium-treated hearts at 0.33 +/- 0.01 mm (n = 19; P = 0.0001). Postmortem LV volume was 0.41 +/- 0.04 ml in cell-treated vs. 0.51 +/- 0.03 ml in medium-treated hearts (P < 0.04). Ejection fraction assessed by LV angiography was 0.40 +/- 0.02 in cell-treated (n = 16) vs. 0.33 +/- 0.02 in medium-treated hearts (n = 24; P < 0.03) with trends towards smaller in vivo end-diastolic and end-systolic volumes in cell-treated vs. medium-treated hearts. Polymerase chain reaction analysis of the Sry gene of the Y chromosome was positive in four of five cell-treated and zero of five medium-treated hearts confirming viability of male cells in female donors. CONCLUSION Over the course of 10 months, fetal cardiac cell transplantation into infarcted hearts increased infarct wall thickness, reduced LV dilatation, and improved LV ejection fraction. Thus, fetal cell-transplant therapy mitigated the longer-term adverse effects of LV remodeling following a myocardial infarction.

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.

Postconditioning Markedly Attenuates Ventricular Arrhythmias After Ischemia-Reperfusion

Background: Brief periods of reocclusion (postconditioning) during early reperfusion reduce myocardial infarct size. Whether postconditioning has an effect on lethal ventricular arrhythmias independent of infarction in an in-vivo regional ischemia model is unknown. The purpose of this study was to determine if postconditioning limited reperfusion arrhythmias in a necrosis-free model. Methods: Anesthetized rats were subjected to 5 minutes of proximal coronary artery occlusion; they were randomized to a control group (n = 15) that underwent reperfusion alone or a postconditioning group (n = 15) that received four cycles of 20 seconds reperfusion, 20 seconds reocclusion before final reperfusion. Results: During the final reperfusion phase, ventricular arrhythmias occurred in 14 of 15 control rats and 8 of 15 postconditioning rats (P = .017). Ventricular tachycardia occurred in 10 of 15 control rats vs 4 of 15 postconditioning rats (P = .028). Control rats demonstrated 1.3 runs of ventricular tachycardia per minute vs 0.4 runs in postconditioning rats (P = .026). The average duration of ventricular tachycardia runs was 8.8 ± 3.2 seconds in the control group vs 5.0 ± 3.9 seconds in postconditioning rats (P = NS). Conclusion: This in-vivo study showed that postconditioning markedly attenuates ventricular arrhythmia after regional ischemia in a noninfarct model.

Transplantation of neonatal cardiomyocytes after permanent coronary artery occlusion increases regional blood flow of infarcted myocardium.

BACKGROUND Cellular cardiomyoplasty is a promising approach for rebuilding scar tissue after acute myocardial infarction. However, the angiogenic potential of transplanted immature cardiomyocytes and their effect on regional myocardial blood flow (RMBF) after coronary artery occlusion remain to be evaluated. METHODS AND RESULTS Intramyocardial injection of cultured neonatal cardiomyocytes (4 x 10(6) cells/50-70 microliter) into the scar 1 week after permanent coronary occlusion in rats resulted in improved RMBF in the infarct 4 weeks after transplantation (radioactive microspheres, 0.97 +/- 0.18 ml/min/g) in comparison to medium-injected hearts (0.61 +/- 0.11 ml/min/g, P < 0.047). The macroscopic perfusion defect after in vivo staining with the blue dye 50% Uniperse blue was significantly smaller in the cell transplantation group (1.5 +/- 0.3% of the heart) compared to the medium group (3.0 +/- 0.6%, P < 0.017). Clusters of engrafted cells within the scar demonstrated a high capillary density (1217 +/- 114 perfused (blue) capillaries/mm(2)); however, in the scar tissue itself capillary density in the cell group (156 +/- 62/mm(2)) did not significantly differ from the medium group (125 +/- 10/mm(2)), suggesting that neo-angiogenesis was confined to regions of successful engraftment (non-infarcted tissue: 1924 +/- 114 perfused capillaries/mm(2)). The transplantation group was characterized by smaller diastolic and systolic left ventricular volumes, as assessed by intravenous ventriculography, along with thickened infarcts (0.93 +/- 0.07 vs. 0.75 +/- 0.04 mm, P < 0.020) and lower infarct expansion indices (0.64 +/- 0.07 vs. 0.83 +/- 0.06, P < 0.023), as determined by post-mortem morphometry of histologic slides. CONCLUSIONS Transplantation of neonatal cardiomyocytes induced neo-angiogenesis in zones of successful cell engraftment within the scar, which effectively enhanced tissue perfusion.

Alterations in myostatin expression are associated with changes in cardiac left ventricular mass but not ejection fraction in the mouse.

Myostatin (Mst) is a negative regulator of skeletal muscle in humans and animals. It is moderately expressed in the heart of sheep and cattle, increasing considerably after infarction. Genetic blockade of Mst expression increases cardiomyocyte growth. We determined whether Mst overexpression in the heart of transgenic mice reduces left ventricular size and function, and inhibits in vitro cardiomyocyte proliferation. Young transgenic mice overexpressing Mst in the heart (Mst transgenic mice (TG) under a muscle creatine kinase (MCK) promoter active in cardiac and skeletal muscle, and Mst knockout (Mst (-/-)) mice were used. Xiscan angiography revealed that the left ventricular ejection fraction did not differ between the Mst TG and the Mst (-/-) mice, when compared with their respective wild-type strains, despite the decrease in whole heart and left ventricular size in Mst TG mice, and their increase in Mst (-/-) animals. The expected changes in cardiac Mst were measured by RT-PCR and western blot. Mst and its receptor (ActRIIb) were detected by RT-PCR in rat H9c2 cardiomyocytes. Transfection of H9c2 with plasmids expressing Mst under muscle-specific creatine kinase promoter, or cytomegalovirus promoter, enhanced p21 and reduced cdk2 expression, when assessed by western blot. A decrease in cell number occurred by incubation with recombinant Mst (formazan assay), without affecting apoptosis or cardiomyocyte size. Anti-Mst antibody increased cardiomyocyte replication, whereas transfection with the Mst-expressing plasmids inhibited it. In conclusion, Mst does not affect cardiac systolic function in mice overexpressing or lacking the active protein, but it reduces cardiac mass and cardiomyocyte proliferation.

In vivo and in vitro models to test the hypothesis of particle-induced effects on cardiac function and arrhythmias.

Exposure to ultrafine particles (UFPs) by inhalation increases the number and severity of cardiac events. The specific mechanism(s), of action are unknown. This study was designed to examine whether UFPs could exert a direct effect on the cardiovascular system without dependence upon lung-mediated responses. The direct effects of UFPs were determined in normal rats (infused intravenously with UFPs), and in the isolated Langendorff perfused rat heart. UFPs from either ambient air (UFAAs) or diesel engine exhaust (UFDGs) were studied. Infusion of UFDGs prepared in our laboratory caused ventricular premature beats (VPBs) in 2 of 3 rats in vivo. Ejection fraction in creased slightly (∼4.5%) in rats receiving UFPAA and was unchanged in the UFDG and saline groups in vivo. In the isolated rat heart, perfused according to Langendorff, UFDGs caused a marked ncrease in left-ventricular end-diastolic pressure (LVEDP; from 12.0±4.6 mmHg to 24.8±11.2 mmHg, p<0.05) after 30 min of exposure. UFPs isolated from industrial diesel particulate matter (UFIDs), obtained from the National Institute of Standards and Technology, caused a significant decrease in left-ventricular systolic pressure (LVSP; from 85.7±4.0 mmHg to 37.9±20.3 mmHg, p<0.05) and ±dp/dt (from 2365±158 mmHg/s to 1188±858 mmHg/s, p<0.05) at 30 min after the start of infusion. This effect was absent when the soluble fraction (containing no particles) isolated from the UFIDs was studied. These findings indicate that UFPs can have direct effects on the cardiovascular system that are independent of effects of particles on the lungs.