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

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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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