Early upregulation of miR-29a mediates differentiation of cardiac stem cells into cardiomyocytes through inhibition of endogenous Wnt/beta-catenin

Abstract

Paracrine signalling within the cardiac stem cell (CSC) niche controls CSC proliferation and differentiation, but the identity of putative mediators is poorly characterized. We isolated and clonally expanded Sca-1+/c-kit+ CSC from murine adult hearts. Upon culture in a differentiation medium (DIFF) containing 5-Azacytidine and TGF-β1, these cells differentiate into cardiomyocytes, as determined by up-regulation of mRNA encoding early (Nkx2.5; 230±3.5%; n=3, p 4-fold; p<0.01; n=3 preps). This was associated with downregulation of the de novo DNA methyltransferase, Dnmt3a, a bona fide target of miR29a. Notably, several Wnt/beta-catenin repressor genes, such as Wif-1, Sfrp1-2-4-5 (secreted frizzled-related protein), and Dkk1 (Dickkopf) exhibit CpG-islands in their promoter region, suggesting an epigenetic control repressing their expression through DNA methylation. Accordingly, early treatment of CSC with a miR-29a mimic (vs Scrambled ctl) decreased Dnmt3a and increased Wif-1 expression; conversely, treatment with Locked Nucleic Acid anti-miR29a (LNA-miR-29a) in DIFF upregulated Dnmt3a, downregulated Wif-1 and significantly decreased CSC differentiation (assessed by cTnt expression; all P<0.05 vs. LNA-scrambled control). We conclude that extinction of the endogenous canonical Wnt/β-catenin pathway is sufficient to allow Sca-1+ CSC differentiation and involves the upregulation of Wnt negative regulators, such as Wif-1, through miR-29a. Our data suggest a new epigenetic regulation of CSC differentiation through Dnmt3a, possibly amenable to therapeutic modulation for cardiac repair.