Abstract Body (Do not enter title and authors here): Background: Cardiovascular disease remains a leading cause of mortality, resulting in irreversible cardiomyocyte loss due to their limited proliferative capacity. While embryonic and neonatal hearts retain the ability to regenerate through cardiomyocyte proliferation, this capacity is lost in adulthood. De novo DNA methyltransferases (DNMTs), may play a crucial role in modulating cardiomyocyte plasticity. DNMT3B, a de novo DNMT, is highly expressed in early cardiac progenitors but downregulated as cardiomyocytes mature. This study explores DNMT3B’s potential in reprogramming adult cardiomyocytes to re-enter the cell cycle and promote cardiac regeneration post-MI.
Methods and Results: Single-cell RNA sequencing of the developing heart field at embryonic day (E) 7 revealed that DNMT3B is highly expressed in epiblast cells but progressively declines as cells commit to mature cardiac lineages. This decline is pronounced from E10.5 onward, with expression drastically reduced in the adult heart. To investigate DNMT3B’s role in cardiomyocyte plasticity, human iPSC-derived cardiomyocytes (iPSC-CMs) were transduced with DNMT3B overexpression (DNMT3B^OE) constructs. DNMT3B^OE reactivated cardiac progenitor markers (NKX2.5, GATA4) and significantly upregulated key cell cycle regulators (CDK7, CCND2, CDK4), facilitating cell cycle re-entry. DNMT3B ChIP-seq was done in DNMT3B^OE iPSC-CMs to confirm its direct binding to genes associated with cell cycle regulation asserting its role in epitranscriptomal reprogramming. Additionally, DNA methylation sequencing showed CpG methylation at gene regionas associated with cell-cycle inhibitors (eg. RB1) as well as several transcription factors associated with cardiac differentaition. To evaluate the therapeutic potential of DNMT3B^OE in vivo, an AAV9-mediated cardiac-specific DNMT3B^OE system was employed in a murine MI model. Significant improvement was observed in cardiac function. Significantly higher active cardiomyocyte proliferation and cytokinesis were observed in DNMT3B^OE-treated hearts, as further confirmed by Aurora B kinase and PHH3 staining.
Conclusion: DNMT3B is capable of reprogramming adult cardiomyocytes to promote proliferation. DNMT3B^OE restores embryonic gene expression, facilitates cell cycle re-entry, and modulates the epigenetic landscape to suppress inflammation and enhance myocardial regeneration. These findings position DNMT3B as a promising target for regenerative strategies in ischemic heart disease.