Promotion of Differentiating Bone Marrow Mesenchymal Stromal Cells (BMSCs) into Cardiomyocytes via HCN2 and HCN4 Cotransfection

Aim. Investigation of the influences HCN2 and HCN4 has on bone marrow mesenchymal stromal cells (BMSCs) on cardiomyocyte differentiation. Methods. Miniature adult pigs were used for bone marrow extraction and isolation of BMSCs. The identification of these BMSCs was done by using flow cytometry for the detection of expressed surface antigens CD45, CD11B, CD44, and CD90. Using HCN2 and HCN4 genes cotransfected into BMSCs as group HCN2+HCN4 while myocardial induction solution was used to induced BMSC differentiation in the BMSC induction group. Myocardial marker proteins α-actin and cTnT were detected by immunofluorescence staining, while α-actin, cTnT, and Desmin myocardial marker proteins expressed were detected by Western blot. The whole-cell patch-clamp technique was used to identify and detect cellular HCN2 channels, HCN4 channel current activation curve, and the inhibitory effect of CsCl on heterologous expression currents. Results. Flow cytometry results showed that CD45 and CD11B were expressed negatively while CD90 and CD44 were positive. Post HCN2 and HCN4 gene transfection, immunofluorescence staining, and Western blot showed significantly increased HCN2, HCN4, α-actin, and cTnT expressed in group HCN2+HCN4 were, which could be compared to the expression levels in the BMSC-induced group. The HCN2+HCN4 group was able to document cell membrane channel ion currents that were similar to If properties. Conclusion. HCN2 and HCN4 overexpression can considerably enhance the MSC ability to differentiate into cardiomyocytes in vitro and restore the ionic current.

Abstract 22: Identification of the Nature of Cardiac Resident c-kit + Cells

Identifying a bona fide population of cardiac stem cells (CSCs) is a critical step for developing cell-based therapies for heart failure patients. For more than a decade, c-kit, a receptor tyrosine kinase expressed in certain types of hematopoietic stem cells, has been recognized as a marker of resident CSCs in mammals. It was shown that c-kit + cells are multipotent, with differentiation potential to become cardiomyocytes, endothelial, and smooth muscle cells in vitro and after cardiac injury. Here, we provide new insights into the nature of cardiac resident c-kit + cells. By targeting the c-kit locus with several reporter genes in mice, we unexpectedly found that c-kit + cells rarely co-localizes with cardiac progenitor marker Nkx2.5 or myocardial marker cTnT. Instead, c-kit labels an endocardial population from embryonic stage to adulthood. After acute cardiac injury, the c-kit + cells still retain their endothelial identity and do not become cardiomyocytes. Our study supports the notion that cardiac c-kit + cells are in fact endothelial cells and not CSCs. This finding suggests an urgent need to re-evaluate the mechanisms by which c-kit + cells contribute to heart repair or regeneration given their endothelial identity.

Transmural Strains in the Ovine Left Ventricular Lateral Wall During Diastolic Filling

Rapid early diastolic left ventricular (LV) filling requires a highly compliant chamber immediately after systole, allowing inflow at low driving pressures. The transmural LV deformations associated with such filling are not completely understood. We sought to characterize regional transmural LV strains during diastole, with focus on early filling, in ovine hearts at 1 week and 8 weeks after myocardial marker implantation. In seven normal sheep hearts, 13 radiopaque markers were inserted to silhouette the LV chamber and a transmural beadset was implanted into the lateral equatorial LV wall to measure transmural strains. Four-dimensional marker dynamics were obtained 1 week and 8 weeks thereafter with biplane videofluoroscopy in closed-chest, anesthetized animals. LV transmural strains in both cardiac and fiber-sheet coordinates were studied from filling onset to the end of early filling (EOEF, 100 ms after filling onset) and at end diastole. At the 8 week study, subepicardial circumferential strain (ECC) had reached its final value already at EOEF, while longitudinal and radial strains were nearly zero at this time. Subepicardial ECC and fiber relengthening (Eff) at EOEF were reduced to 1 compared with 8 weeks after surgery (ECC:0.02±0.01 to 0.08±0.02 and Eff:0.00±0.01 to 0.03±0.01, respectively, both P<0.05). Subepicardial ECC during early LV filling was associated primarily with fiber-normal and sheet-normal shears at the 1 week study, but to all three fiber-sheet shears and fiber relengthening at the 8 week study. These changes in LV subepicardial mechanics provide a possible mechanistic basis for regional myocardial lusitropic function, and may add to our understanding of LV myocardial diastolic dysfunction.

Abstract 2362: Myonecrosis is Predicted by Lack of Platelet Inhibition Measured in Response to Multiple Agonists in Patients Undergoing Elective Stenting:The Risk of A Global High Platelet Reactivity Phenotype

Background: Small studies have demonstrated the relation of clopidogrel nonresponsiveness to adverse clinical events. We sought to determine: the relation between inhibition of ADP-induced platelet aggregation and inhibition of aggregation induced by other agonists and the relation of clopidogrel responsiveness to periprocedural myonecrosis in patients undergoing stenting treated with a 600mg clopidogrel loading dose and bivalirudin. Methods: Platelet inhibition by low and high dose ADP, collagen and TRAP by conventional aggregometry (LTA) was determined at 2 hr and 24 hr post-stenting in 50 patients treated with aspirin, bivalirudin and 600 mg clopidogrel. Patients were stratified into low clopidogrel responders (LR)[≤10% relative platelet inhibition (RPI)], moderate responders (>10 – <30% RPI) and high responders (>30% RPI)]. Results: Inhibition measured by 5uM ADP correlated with inhibition by 20uM ADP, collagen and TRAP (r =0.98, 0.91, and 0,83, respectively, p < 0.001 for all). Myocardial marker release occurred in the LR and MR groups only. (Table ) Conclusions: 600 mg clopidogrel markedly inhibits platelet reactivity to multiple agonists in selected patients who are in turn protected from periprocedural MI during elective stenting. These data suggest that low and high risk patients can be identified by platelet function testing that may enhance decision making for adjunctive antithrombotic therapy in elective stenting.