CTCF ChIP-seq in AC16 cell: A Conserved Chromatin Structural Program Underlies Injury-Induced Cardiomyocyte Proliferation

Because adult mammalian cardiomyocytes cannot reactivate their proliferative machinery, the heart does not recover effectively after injury. In contrast, during the early neonatal period, cardiomyocytes retain a transient regenerative capacity. Myocardial injury performed before postnatal day 3 triggers robust cardiomyocyte proliferation and complete recovery within weeks, and apical resection during the first postnatal days leads to full regeneration without scar formation. In pigs, apical resection at day 1 followed by myocardial infarction at day 28 induces strong cardiomyocyte proliferation and complete functional recovery by day 56. To investigate the molecular programs that underlie this transient regenerative competence, we reanalyzed mouse and pig snRNA-seq datasets using a cell cycle–specific autoencoder (CSA) and a chromatin structural factor–specific autoencoder (CSF). Both frameworks consistently identified a distinct cardiomyocyte population (CM1) enriched for mitotic markers after injury. Gene Ontology analysis revealed coordinated activation of cell-cycle pathways together with chromatin organization and remodeling processes, suggesting that cell-cycle re-entry requires extensive epigenetic restructuring. Cross-species comparison identified a hallmark of chromatin structural regulators associated to cardiomytocyte proliferation. Among these factors, CTCF emerged as a central regulator. Its expression declined after birth but was robustly reactivated in injured mouse and pig hearts. Functional assays demonstrated that CTCF overexpression promotes cardiomyocyte-like cell proliferation, and induce targeted redistribution of CTCF binding at key cell-cycle regulatory loci. Together, these findings identify conserved modulation of chromatin structural factors as a key mechanism underlying cardiomyocyte cell-cycle reactivation and position CTCF as a prominent regulator within this architectural program during cardiac regeneration.