Pten Regulates Cardiomyocyte Differentiation by Modulating Non‐CG Methylation via Dnmt3

The regulation of cardiomyocyte differentiation is a fundamental aspect of cardiac development and regenerative medicine. PTEN plays important roles during embryonic development. However, its role in cardiomyocyte differentiation remains unknown. In this study, a low‐cost protocol for cardiomyocyte differentiation from mouse embryonic stem cells (ESCs) is presented and it is shown that Pten deletion potently suppresses cardiomyocyte differentiation. Transcriptome analysis shows that the expression of a series of cardiomyocyte marker genes is downregulated in Pten−/− cardiomyocytes. Pten ablation induces Dnmt3b expression via the AKT/FoxO3a pathway and regulates the expression of a series of imprinted genes, including Igf2. Double knockout of Dnmt3l and Dnmt3b rescues the deficiency of cardiomyocyte differentiation of Pten−/− ESCs. The DNA methylomes from wild‐type and Pten−/− embryoid bodies and cardiomyocytes are analyzed by whole‐genome bisulfite sequencing. Pten deletion significantly promotes the non‐CG (CHG and CHH) methylation levels of genomic DNA during cardiomyocyte differentiation, and the non‐CG methylation levels of cardiomyocyte genes and Igf2 are increased in Pten−/− cardiomyocytes. Igf2 or Igf1r deletion also suppresses cardiomyocyte differentiation through the MAPK/ERK signaling pathway, and IGF2 supplementation partially rescues the cardiomyocyte differentiation. Finally, Pten conditional knockout mice are generated and the role of PTEN in cardiomyocyte differentiation is verified in vivo. Pten deletion suppresses cardiomyocyte differentiation by reducing the expression of cardiomyocyte genes. A series of imprinted genes are regulated by Pten, and Pten ablation induces the expression of Dnmt3 by activating AKT. The non‐CG DNA methylation levels of cardiomyocyte genes are higher in Pten−/− cardiomyocytes, and Pten regulates cardiomyocyte differentiation by modulating non‐CG DNA methylation via Dnmt3.