Cardiac myocyte (CM) proliferation is required for the heart regeneration seen in lower vertebrates and neonatal mammalian injury models. However, mammalian CMs stop proliferating soon after birth and subsequent heart growth comes from hypertrophy, limiting the adult heart’s regenerative potential after injury. The molecular events blocking CM proliferation in the adult heart remain poorly understood. We hypothesized repressive epigenetic mechanisms are responsible for the stable silencing of cell cycle genes in adult CMs (ACMs). Studies from our lab and others have suggested trimethylation of Lysine 9 of Histone H3 (H3K9me3) and H3K27me3, histone modifications associated with heterochromatin, are associated with permanent cell cycle exit. To test if depleting these repressive methylations in ACMs could relieve the silencing of cell cycle genes, we developed an adenoviral-gene-transduction model for combined H3K9me3- and H3K27me3-depletion in vitro. We tested this hypothesis in vivo using a transgenic mouse model where H3K9me3 is specifically removed by histone demethylase KDM4D in CMs. Loss of H3K9me3 in CMs disrupts ACM cell cycle gene silencing preferentially and results in increased CM cycling. Normalized heart mass was increased by postnatal day 14 (P14) and continued to increase until 9-weeks of age. ACM number, but not size, was significantly increased in BiTg hearts, suggesting CM hyperplasia accounts for the increased heart mass. Challenging H3K9me3-depleted hearts with a hypertrophic growth signal stimulated ACM mitotic activity. Thus, we demonstrated that H3K9me3 is required for cell cycle gene silencing in ACMs and depletion of H3K9me3 allows hyperplastic growth in vivo. To gain mechanistic understanding of the observed proliferation-competence we examined global chromatin structure and loci-specific DNA accessibility in H3K9me3-depleted and control ACMs. Combined with DNA methylation bisulfite sequencing (DNAme-Seq) and chromatin-immunoprecipitation (ChIP) studies, these data suggest a model where cell cycle genes have a unique chromatin signature, where the gene bodies are heterochromatinized and the gene promoters are regulated by canonical cell cycle transcription factor pathways that are modulated by H3K9me3
