Dnmt3a-mediated inhibition of Wnt in cardiac progenitor cells improves differentiation and remote remodeling after infarction
Aurelia De Pauw, Emilie Andre, Belaid Sekkali, Caroline Bouzin, Hrag Esfahani, Nicolas Barbier, Axelle Loriot, Charles De Smet, Laetitia Vanhoutte, Stéphane Moniotte, Bernhard Gerber, Vittoria di Mauro, Daniele Catalucci, Olivier Feron, Denise Hilfiker-Kleiner, Jean-Luc Balligand
Aurelia De Pauw, Emilie Andre, Belaid Sekkali, Caroline Bouzin, Hrag Esfahani, Nicolas Barbier, Axelle Loriot, Charles De Smet, Laetitia Vanhoutte, Stéphane Moniotte, Bernhard Gerber, Vittoria di Mauro, Daniele Catalucci, Olivier Feron, Denise Hilfiker-Kleiner, Jean-Luc Balligand
View: Text | PDF
Research Article Cardiology Stem cells

Dnmt3a-mediated inhibition of Wnt in cardiac progenitor cells improves differentiation and remote remodeling after infarction

Abstract

Adult cardiac progenitor cells (CPCs) display a low capacity to differentiate into cardiomyocytes in injured hearts, strongly limiting the regenerative capacity of the mammalian myocardium. To identify new mechanisms regulating CPC differentiation, we used primary and clonally expanded Sca-1+ CPCs from murine adult hearts in homotypic culture or coculture with cardiomyocytes. Expression kinetics analysis during homotypic culture differentiation showed downregulation of Wnt target genes concomitant with increased expression of the Wnt antagonist, Wnt inhibitory factor 1 (Wif1), which is necessary to stimulate CPC differentiation. We show that the expression of the Wif1 gene is repressed by DNA methylation and regulated by the de novo DNA methyltransferase Dnmt3a. In addition, miR-29a is upregulated early during CPC differentiation and downregulates Dnmt3a expression, thereby decreasing Wif1 gene methylation and increasing the efficiency of differentiation of Sca-1+ CPCs in vitro. Extending these findings in vivo, transient silencing of Dnmt3a in CPCs subsequently injected in the border zone of infarcted mouse hearts improved CPC differentiation in situ and remote cardiac remodeling. In conclusion, miR-29a and Dnmt3a epigenetically regulate CPC differentiation through Wnt inhibition. Remote effects on cardiac remodeling support paracrine signaling beyond the local injection site, with potential therapeutic interest for cardiac repair.

Authors

Aurelia De Pauw, Emilie Andre, Belaid Sekkali, Caroline Bouzin, Hrag Esfahani, Nicolas Barbier, Axelle Loriot, Charles De Smet, Laetitia Vanhoutte, Stéphane Moniotte, Bernhard Gerber, Vittoria di Mauro, Daniele Catalucci, Olivier Feron, Denise Hilfiker-Kleiner, Jean-Luc Balligand

×

Figure 1

Cardiac progenitor cell differentiation is concomitant with a downregulation of Wnt signaling and upregulation of Wnt inhibitors and is potentiated by inhibition of Wnt/β-catenin.

Options: View larger image (or click on image) Download as PowerPoint
Cardiac progenitor cell differentiation is concomitant with a downregula...
Cultured cardiac progenitor cells (CPCs) were incubated or not (control cells [Ctl]) with 5-azacytidine (AZA) and TGF-β (DIFF) for 5, 8, 11, or 14 days. Gene expression (relative to respective time Ctl) was analyzed using RT-qPCR and normalized to GAPDH. (A) Relative expression of Wnt/β-catenin pathway target genes (Axin2, Wnt4) and Wnt activators (Lef1 and Snai2). Bounds of boxes represent minimum and maximum values, inner line represent means. *P < 0.05 vs. CTL; n = 3 different preparations; Kruskal-Wallis with Bonferroni correction. Expression of β-catenin protein levels in DIFF, relative to Ctl at each time point. *P < 0.05 vs. Ctl; n ≥ 3 different preparations; Mann-Whitney test. (B) CPCs were cultured in Ctl or DIFF medium for 8 days and their supernatant was incubated with HEK cells expressing the TOPflash reporter construct, indicative of Wnt morphogen production by and Wnt activity in donor CPCs. TOPflash signal was normalized for transfection efficiency (cotransfected TKRenilla). *P < 0.05 vs. Ctl; n = 4 different cultures; Mann-Whitney. (C) Relative (to respective time Ctl) expression of Wnt repressors Wif1 and Hbp1. *P < 0.05 vs. Ctl; n = 3 different preparations; Kruskal-Wallis with Bonferroni correction. (D) Representative images of CPCs treated or not with AZA and either TGF-β or the pharmacologic inhibitor of Wnt signaling response (IWR1, 10 μM) for 26 days. Immunocytochemistry was performed using an antibody against cardiac troponin T. Relative gene expression of Nkx2.5 in CPCs treated with IWR1 in absence of AZA for 11 days. *P < 0.05, n ≥ 4 experiments; Mann-Whitney test. Scale bar: 20 μm. (E) Wif1 expression modulates CPC differentiation in coculture. Expression of Wif1 in CPCs transfected with 50 nM siRNA targeting Wif1 (or siRNA scramble) for 48 hours. CPCs transfected with siRNA-Wif1 (si-Wif1) or scramble control (si-Scr) were cocultured with cardiomyocytes and their differentiation monitored by expression of α-sr-actinin. Results are reported as relative to differentiation in si-Scr–transfected CPCs (set at 100%). CPC differentiation is significantly decreased upon Wif1 inhibition. *P < 0.05; n = 4 different preparations; Mann-Whitney test.