Dnmt3a-mediated inhibition of Wnt in cardiac progenitor cells improves differentiation and remote remodeling after infarction
Aurelia De Pauw, … , Denise Hilfiker-Kleiner, Jean-Luc Balligand
Aurelia De Pauw, … , Denise Hilfiker-Kleiner, Jean-Luc Balligand
Published June 15, 2017
Citation Information: JCI Insight. 2017;2(12):e91810. https://doi.org/10.1172/jci.insight.91810.
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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

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Figure 3

miR-29a increases Wif1 through repression of Dnmt3a and its de novo methylation of Wif1 gene.

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miR-29a increases Wif1 through repression of Dnmt3a and its de novo meth...
(A) Cultured cardiac progenitor cells (CPCs) were treated with 5-azacytidine/TGF-β (DIFF) or control medium (CTL) for 3 to 11 days. Expression of miR-29a was measured by qRT-PCR and normalized to 5S. *P < 0.05 vs. CTL at day 5; Mann-Whitney test. miR-29a in DIFF at day 5 is also different from DIFF at days 3, 8, and 11 by Kruskal-Wallis with Bonferroni correction; n = 3 experiments. (B) Untreated CPCs were transfected with either a miR-29a mimic construct (Mm-29a) or LNA-miR-29a (LNA-29a) or respective scramble constructs (Scr). Dnmt3a proteins (by Western Blot) and (C) Wif1 transcripts (by qRT-PCR), normalized by GAPDH protein or mRNA abundance, are reported as fold changes from CTL (set at 1). *P < 0.05 vs. mimic/LNA-scr; n = 4–8 experiments; unpaired t test. (D) Untreated CPCs were cotransfected with si-Dnmt3a and LNA-29a (or Scr controls). Relative expression of Wif1 mRNA (qRT-PCR, normalized to GAPDH) is expressed as fold change from CTL (transfected with both Scr constructs, set at 1). *P < 0.05 vs. LNA-Scr+si-Scr, 1-way ANOVA, Bonferroni’s test; n = 3 experiments. (E) Untreated CPCs were transfected with LNA-29a (or LNA-Scr) for 72 hours, and CpG methylation of the Wif1 gene region extending from –88 to +102 bp was assessed by bisulfite sequencing. Representative image of (un)methylated CpG in corresponding Wif1 region. Each vertical line of boxes below corresponds to multiple clones of the corresponding sequence of the Wif1 gene (white box: unmethylated; black box: methylated). Percentage of methylated CpG in the Wif1 DNA fragments. *P < 0.05 vs. LNA-Scr; n = 4 experiments; unpaired t test.