Abstract
Mesenchymal stem cells (MSCs) have begun to manifest themselves as a safe and beneficial therapy for restoring cardiac function in failing hearts. However, the mechanism underlying this function is unclear. The present study was initiated to investigate the role of Connexin 43 (Cx43) in hMSC integration, migration and differentiation in co-culture with neonatal rat cardiomyocyte (NRVMs) in vitro. Cx43 is a gap junction connexin which is required for proper heart development and heart electrophysiology. We generated lentiviral constructs for Cx43 knockdown and Cx43 overexpression and used them to alter the level of Cx43 in hMSCs. The effectiveness of these vectors was confirmed by assessing Cx43 levels in hMSCs by Western Blot analysis and by Real Time-PCR. These hMSCs were co-cultured with NRVMs for up to 1 month on poly-lysine and collagen I coated glass cover slips. Co-cultures containing MSCs overexpressing Cx43 exhibited coordinated beating and three-dimensional tube formation in 10 days, whereas 14 days were required for control cells. The hMSCs were integrated into these beating three dimensional tubes. No tubes were observed in co-cultures with Cx43 knockdown hMSCs, rather we observed the formation of small, unconnected beating spheres after 30 days in co-culture. Human MSC-Cx43 knockdown cells were integrated into these spheres and immunofluorescence staining demonstrated that Cx43 expression in hMSC-Cx43 knockdown remained reduced when compared to the control group. Real Time-PCR analysis using human specific primers showed significant upregulation of KDR, smooth muscle actin, Pecam1, CD34, CDH2, and CaCNA1C. Several genes, not initially seen in hMSCs, including Gata4, CDH5, SCN5A, SLC8A1, and KCNQ1, appeared in co-cultures with hMSCs with normal and elevated expression levels of Cx43. However, in co-culture of cardiomyocytes with Cx43 knockdown MSCs we observed downregulation of KDR, CHD2, SCN5A, SLC8A1, KCNQ1 compare to control. These results suggest a strong correlation between the presence of Cx43 and the ability of hMSCs to differentiate into cardiac and endothelial lineages.