Histone demethylase KDM5 regulates cardiomyocyte maturation by promoting fatty acid oxidation, oxidative phosphorylation and myofibrillar organization

Rationale: The molecular basis of cardiomyocyte (CM) maturation is not fully known. Human pluripotent stem cell-derived CMs (iPSC-CMs) are a valuable tool to reconstruct the CM maturation process and thus offer the opportunity to identify and characterize factors that regulate maturation. The transition from immature fetal to adult CM entails coordinated regulation of the mature gene program which is characterized by the induction of myofilament and OXPHOS gene expression. Recent studies in Drosophila, C. elegans and C2C12 myoblast cell lines have implicated the histone H3K4me3 demethylase KDM5 and its homologs, as a potential regulator of developmental gene program and OXPHOS gene expression. We speculated that KDM5 may potentiate maturation of iPSC-CMs through a similar conserved yet cell type specific epigenetic program.Objectives: The purpose of this study is to determine the role of KDM5 in iPSC-CM maturation.Methods and Results: Immunoblot analysis revealed that KDM5A, B, and C expression was progressively downregulated in postnatal cardiomyocytes and absent in adult hearts and CMs. Additionally, KDM5 proteins were found to be persistently expressed in iPSC-CMs up to 60 days after the onset of myogenic differentiation, consistent with the immaturity of these cells. Inhibition of KDM5 by KDM5-C70 -a pan-KDM5 inhibitor- resulted in differential regulation of 2,372 genes (q<0.01), including upregulation of Fatty acid oxidation (FAO), OXPHOS and myogenic gene program in iPSC-CMs. Likewise, genome-wide profiling of H3K4me3 binding sites by the CUT&RUN assay revealed enriched H3K4me3 peaks at the promoter regions of FAO, OXPHOS and sarcomere genes. Consistent with the chromatin and gene expression data, expression of multiple sarcomere proteins was induced, myofibrillar organization was enhanced and calcium handling was improved in KDM5-C70 treated iPSC-CMs. Furthermore, inhibition of KDM5 increased H3K4me3 deposits at the promoter region of the ESRRA gene, which is known to regulate OXPHOS and cardiomyocyte maturation, and resulted in its increased RNA and protein levels. Finally, KDM5 inhibition increased baseline, peak, and spare oxygen consumption rates in iPSC-CM.Conclusions: KDM5 regulates the maturation of iPSC-CMs by epigenetically regulating the expression of ESRRA, OXPHOS, FAO, and sarcomere genes and enhancing myofibril organization and mitochondrial function.