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 2

Dnmt3a downregulation upregulates Wif1 expression in cardiac progenitor cells and promotes their differentiation to the myocyte lineage.

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Dnmt3a downregulation upregulates Wif1 expression in cardiac progenitor ...
(A) Cultured cardiac progenitor cells (CPCs) were treated with 5-azacytidine and TGF-β (DIFF medium) for 5 days, and Dnmt3a and Dnmt3b abundance was measured by immunoblotting (corrected for equal protein loading by GAPDH immunodetection). *P < 0.05 vs. CTL; n = 4 experiments; unpaired t test. (B and C) Untreated CPCs were transfected with siRNA targeting Dnmt3a for 72 hours, and (B) the effect on Dnmt3a protein level was measured by Western Blot and (C) the effect on Wif1 transcripts was measured by qRT-PCR. Results were normalized by GAPDH protein or mRNA abundance, respectively. *P < 0.05 vs. Si-Scr; n = 5–7 experiments; unpaired t test. (D) Analysis of the methylation pattern of the Wif1 gene in CPCs transfected with Si-Dnmt3a for 72 hours. CpG methylation in the Wif1 gene region extending from –88 to +102 bp was assessed by bisulfite sequencing. Representative images of (un)methylated CpG in corresponding Wif1 region. Vertical bars correspond to the location of CpG sites. Vertical lines of boxes below correspond to multiple clones of the corresponding sequences of Wif1 (white box: unmethylated; black box: methylated). Percentage of methylated CpGs in the Wif1 DNA fragments, calculated as a ratio of the number of methylated CpG to the total number of CpG sites from CPCs treated with Si-Dnmt3a (or Si-Scr). *P < 0.05 vs. Si-Scr; n = 3 experiments; unpaired t test. (E) CM-Dil–stained CPCs were first transfected with si-Dnmt3a (or si-Scramble) and then cocultured with neonatal cardiomyocytes (NNCM) for 14 days. Differentiation was assessed from the percentage of cardiac troponin T–positive (cTnT-positive) CPC. Dnmt3a inhibition increases the efficiency of CPC differentiation in coculture with NNCMs. *P < 0.05 vs. Si-Scr; n > 3 experiments; unpaired t test.