P512Inhibition of endogenous Wnt/beta-catenin following miR29a downregulation mediates differentiation of Cardiac Stem Cells into cardiomyocytes

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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, characterized and clonally expanded Sca-1 + CSC from murine adult hearts. These cells can differentiate into cardiomyocytes upon treatment with 5-Azacytidine (AZA, 5 µM) and transforming growth factor β1 (TGF-β1, 1ng/ml), as determined by a clear up-regulation of mRNA encoding early (Nkx2.5; 230 ± 3.5%; n=3, p < 0.05), intermediate (cTnT; 40-fold over control), and late (α-MHC; 100-fold over control) cardiomyocyte markers, characteristic of cardiomyocyte differentiation. We previously showed that cardiac differentiation was enhanced in Sca-1 + CSC isolated from mice with inducible deletion of β-catenin. In cloned, undifferentiated Sca1 + CSC, we detected a constitutive activity of canonical Wnt/β-catenin, that could be further stimulated with LiCl. Consistent with our previous results, differentiation of Sca1 + CSC in monoculture with AZA and TGF-β1 induced a parallel down-regulation of the Wnt target genes, Wnt4 (at 2 weeks: 26 ± 11.9 %; n=3; P < 0.05), and Axin2 (at 3 weeks: 51.9 ± 17.9%; n=3; P < 0.05). Accordingly, treatment of Sca-1 + CSC in monoculture with pharmacological drugs inhibiting Wnt signaling (Inhibitor of Wnt Response, IWR, 10µM) activated their cardiac differentiation (assessed by cTnI immunostaining), demonstrating a causal link between extinction of the Wnt pathway and differentiation. Notably, treatment of CSC with DETA-NO (1µM) produced a downregulation of Wnt target genes (Axin2: 78 ± 3.7%; Wnt4: 71 ± 6.4% ; n=3; p < 0.05), suggesting an inhibitory effect of NO on Wnt signaling (as confirmed in a β-catenin activity reporter assay with DETA-NO: 37 ± 4.9%; n=3; p < 0.05). In search of endogenous molecular regulator(s) coordinating theses changes, we found that pre-miR29a (and miR29a) was downregulated in CSC during differentiation (67.3 ± 3.2%; n=3; p < 0.05); accordingly, DETA-NO downregulated the expression of pre-miR 29a (and miR29a) (88 ± 8.5%; n=3; p < 0.05); this was associated with upregulation of HBP1 (155 ± 15.9%; n=3; p < 0.05), a bona fide target of miR29a, that is also a negative regulator of Wnt expression. Finally, DETA-NO treatment increased the proportion of cTnI + cells (from 13.3 ± 9.3% to 37.3 ± 18.2%; n=5; p < 0.05). We conclude that extinction of the endogenous canonical Wnt/β-catenin pathway is sufficient to allow Sca-1 + CSC differentiation and involves the coordinate regulation by miR29a of Wnt negative regulators, such as HBP1, possibly amenable to therapeutic modulation for cardiac repair.

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