Abstract: During development, cell division (proliferation or hyperplasia) is tightly coupled to the accumulation of cell mass (hypertrophy) to ensure that myocyte size is constant; however, in adult cardiac myocytes (ACMs), similar growth signals primarily induce hypertrophic growth without proliferation even though many of the same signaling pathways are activated. At a molecular level, while hyperplastic growth is associated with the expression of a panel of cell cycle genes regulated by the E2F family of transcription factors, these genes are not upregulated in hypertrophic myocytes. Despite numerous descriptive studies characterizing the limited ability of ACMs to exit G1 or divide in response to various stimuli, almost no data exists to explain why the majority of ACMs do not enter S phase when stimulated. We have identified a novel mechanism for silencing G2M/cytokinesis genes in ACMs; namely, histone methylation of Rb-E2F regulated cell cycle genes. We show that the two major histone modifications associated with stable gene silencing are upregulated in ACMs and targeted to E2F-dependent cell cycle genes. We propose to test if the importance of these epigenetic marks and if they are targeted to E2F-dependent cell cycle genes by Rb family members in vivo. Genetically reactivating cell cycle genes in transgenic mice is associated with the reexpression of specific histone demethylases, something normally seen only in proliferating fetal cardiac myocytes not hypertrophy. Interestingly, the fact that these epigenetic changes might be reversible suggests that this might be a therapeutic avenue to "remodel" or "reprogram" ACMs to restore their proliferative potential. We will explore the importance of histone methylation in limiting ACM proliferation by determining if reversing H3K9 and H3K27 histone methylation converts a hypertrophic reposnse to hyperplasia in adult cardiac myocytes (Aim 1), determining the factors that target histone methylations in ACMs and their role in silencing cell cycle genes and preventing proliferation (Aim2) and determining how histone methylation remodeling occurs in ACMs and its physiologic significance (Aim 3).

Relevance to Public Health: Myocardial regeneration to restore cardiac muscle mass after injury has been proposed as a means to prevent the development of congestive heart failure for decades. Developing strategies that promote dedifferentiation and proliferation of the endogenous cardiac myocytes holds great promise as a therapeutic strategy. The studies in this application will address critical deficiencies in our current knowledge of cardiac growth and will identify specific molecular pathways amenable to directed therapies.