Abstract 4138199: Examining the regulating mechanism of the HIF-FOXM1 axis in promoting adult pig cardiomyocyte proliferation

Background: Contractile tissue that is lost to myocardial infarction (MI) in adult mammalian hearts cannot be regenerated, because cardiomyocytes (CMs) undergo cell-cycle arrest within a few days after birth; however, when apical resection (AR) surgery was performed in neonatal pigs on postnatal day (P) 1 (AR P1 ), followed by MI induction on P28 (MI P28 ), the animals completely recovered, and the regenerative process was driven primarily by CM proliferation. Furthermore, analysis of single-nucleus RNA sequencing (snRNAseq) data collected from the hearts of animals studied in our pig AR P1 MI P28 model and in a mouse MI model indicated that FOXM1 expression was upregulated in clusters of CMs that were co-enriched for markers of proliferation, cytokinesis, mitosis, and DNA synthesis. FOXM1 also reactivated cell-cycle progression in CMs differentiated from human-induced pluripotent stem cells (hiPSC-CMs) after the cells had lost their intrinsic cell-cycle activity, and FOXM1 promotes proliferation in cancer cells, where its expression is regulated by HIF signaling. However, the mechanisms that regulate FOXM1-induced cell-cycle reactivation in CMs are not well studied. Hypothesis: The HIF signaling pathway regulates FOXM1 expression in the cycling CMs of AR P1 MI P28 pig hearts. Approach: The expression of FOXM1, four HIF proteins (HIF1A, ARNT, EPAS1, and ARNT2), and markers for reactive oxygen species (ROS) and DNA damage were compared in CMs from regenerating and nonregenerating hearts (i.e., pigs that underwent AR P1 MI P28 or MI P28 alone, respectively) via snRNAseq analysis and immunohistochemistry. Results: Both FOXM1 and EPAS1 were upregulated in AR P1 MI P28 CMs, and these increases were accompanied by significant declines in ROS production and DNA damage, both of which are known downstream effects of HIF pathway activation. Furthermore, the results from Eukaryotic Promoter Database indicated that EPAS1 directly binds the FOXM1 promoter in CMs. Conclusion: Reactivation of the CM cell cycle in AR P1 MI P28 hearts was at least partially induced via the HIF-mediated upregulation of FOXM1 expression, which also protected CMs from ROS and DNA damage.