Abstract 10462: Induction of Cardiomyogenic Differentiation in Human Mesenchymal Stem Cells is Mediated by Gap Junctional Coupling with Cultured Rat Cardiomyocytes
Purpose - Connexin43 (Cx43) expression is important for functional integration of therapeutic cells with host myocardium. However, the role of this gap junction protein in cardiomyogenic differentiation of such cells is still unclear. In this study, the role of Cx43 expression in cardiomyogenic differentiation of human mesenchymal stem cells (hMSCs) was investigated.
Methods - Knockdown of Cx43 (Cx43KD) was established by 2 short hairpins directed against Cx43 in fetal hMSCs. Dye transfer and whole-cell current-clamp recordings were used to determine functional coupling of neonatal rat cardiomyocytes (nrCMCs) with hMSCs. To study cardiomyogenic differentiation, control hMSCs transduced with a short hairpin directed against eGFP and Cx43KD-hMSCs were co-incubated with nrCMCs for 10 days. Differentiation was assessed by immunostaining and whole-cell current-clamp recordings. Cx45 overexpression was introduced in Cx43KD-hMSCs to establish whether the effects of Cx43KD could be restored.
Results - A Cx43 knockdown of 80% was achieved in Cx43KD-hMSCs compared to control hMSCs. Dye transfer and patch clamp analysis showed diminished functional coupling between Cx43KD-hMSCs and nrCMCs compared to control hMSCs and nrCMCs. Ten days after co-incubation, not a single Cx43KD-hMSC expressed the cardiac protein α-actinin, while control hMSCs did (2.18±0.39%, n=5000). Furthermore, functional cardiomyogenic differentiation, based on action potential recordings after gap junctional uncoupling, was shown to occur in control hMSCs but not in Cx43KD-hMSCs. After overexpression of Cx45 in Cx43KD-hMSCs, functional gap junctions were formed with nrCMCs. The ability to undergo functional cardiomyogenic differentiation was restored in these cells. This was confirmed by expression of α-actinin (1.58±0.38%, n=5000) and the presence of intrinsic action potentials in these cells.
Conclusion - Induction of cardiomyogenic differentiation in fetal hMSCs is mediated by gap junctional coupling with nrCMCs. Reduced expression of Cx43 in hMSCs inhibits cardiomyogenic differentiation, which can be rescued by concurrent overexpression of Cx45. These insights may improve our understanding of how the local environment influences stem cell differentiation.
- © 2011 by American Heart Association, Inc.