Enhancing the cardiomyogenic potential of human mesenchymal stem cells by modulation of miR expression.

Introduction: it has been shown that mesenchymal stem cells (MSC) can differentiate into cardiomyocytes (CMC) both in vitro and in vivo. However, the efficiency of cardiac differentiation is very limited. Recently, it has been suggested that miR1, 133 and 499 are involved in cardiac development and stem cell differentiation. The role of these miR in MSC differentiation is currently unknown. Accordingly, we tested if the overexpression of miR1, 133 and 499 may favour the differentiation of MSC into CMC. Methods: miR1, 133, and 499 were transiently overexpressed, one by one or in different combinations, in human MSC. Cardiac differentiation was evaluated by measuring the expression of the cardiac specific markers GATA4, Nkx2.5, Tbx5, MEF2c, MLC2v, Cx43, cTnT and α-MHC. Western Blot and immunocytochemistry (ICC) for cTnT and Cx43 were also performed. Results: miR499 alone increased the expression of the early cardiac genes GATA4, Tbx5, MLC2v. In particular, miR499 exerted more pronounced effects compared with miR1 and miR133. Concomitant overexpression of miR499 and miR133 further increased the expression of Tbx5 and MEF2c. Western Blot analysis showed that miR499 increased the expression of late cardiac genes when overexpressed alone or in combination with miR133. Finally, ICC staining confirmed that miR499 in association with miR133 increase Cx43 and cTnT protein expression. Finally, the expression of cardiac specific protein α-MHC was more pronounced in MSC overexpressing miR499 alone or in combination with miR133. Conclusions: concomitant overexpression of miR499 and miR133 successfully commit AMSC into CMC. Our data suggests that miR modulation can represent a promising approach to improve the efficiency of cardioregnerative therapy with multipotent stem cells.