Abstract Body: Background: The adult mammalian heart exhibits limited regenerative capacity due to restricted cardiomyocyte (CM) proliferation following injuries like ischemia. Foxk1 and Foxk2, members of the Foxk family, are pivotal in cell proliferation and metabolic regulation. However, the roles of Foxk1 and Foxk2 in CM proliferation and heart regeneration remain elusive.
Hypothesis: We propose that Foxk1 and Foxk2 are master regulators of CM proliferation and heart regeneration.
Methods: Three genetic mouse lines, Myh6-CreER, Foxk1^fl/fl, and Foxk2^fl/fl were used to generate CM-specific Foxk1 or Foxk2 deficient mice. Myocardial infarction was performed in neonatal and adult mice. Cleavage Under Targets and Tagmentation sequencing (CUT&Tag-seq), RNA sequencing (RNA-seq), qRT-PCR, Western blotting, immunostaining, and Seahorse metabolic assays were conducted to investigate the intrinsic mechanisms by which Foxk1 and Foxk2 regulate CM proliferation and heart regeneration. Masson’s staining and echocardiography were conducted to evaluate cardiac fibrosis and function, respectively.
Results: Our results showed that the expression of Foxk1 and Foxk2 gradually decreased in CMs during postnatal heart development. CM-specific knockout of Foxk1 or Foxk2 inhibited neonatal heart regeneration after MI. AAV9-mediated CM-specific overexpression of Foxk1 or Foxk2 prolonged the postnatal proliferative window of CMs in mice. AAV9-mediated overexpression of Foxk1 or Foxk2 in adult mice improved heart regeneration by promoting endogenous CM proliferation after MI. Through joint analysis of RNA-Seq and CUT&Tag-seq, we found that Foxk1 and Foxk2 regulated Ccnb1 and Cdk1 transcription respectively. Knockdown of Ccnb1 hindered CM proliferation induced by Foxk1 overexpression, and the same effect was observed when Cdk1 was knocked down in Foxk2-overexpressing CMs. Additionally, Foxk1 and Foxk2 induced CM metabolic reprogramming, suppressing mitochondrial oxidative phosphorylation (OXPHOS) via Hif1α upregulation and reducing oxidative DNA damage, thereby modulating the cell cycle.
Conclusions: Foxk1 and Foxk2 promote CM proliferation and heart regeneration. Mechanistically, Foxk1 and Foxk2 promote CM proliferation via dual effects that directly promote cell cycle gene transcription and induce metabolic reprogramming. Our findings suggest these transcription factors as potential novel targets in cardiac regeneration post-injury.