PROJECT SUMMARY/ABSTRACT Currently, more than 5 million Americans suffer from heart failure with approximately 500,000 new diagnoses each year. In response to cardiac injury, a fibrotic response arises in an effort to maintain the structural and functional integrity of the heart. Central to this healing response is the differentiation of fibroblasts to highly specialized synthetic and contractile myofibroblasts. Unfortunately, the chronic nature of many cardiac diseases is accompanied by the persistence of myofibroblasts and progressive fibrosis contributing to cardiac decompensation and eventual failure. Therefore, it is important to identify the molecular mechanisms necessary for myofibroblast formation and maintenance. Recent studies suggest that changes in cellular metabolism are required for cellular remodeling and are associated with the induction of gene programs to support differentiation in numerous cell types. Interestingly, changes in intermediary metabolism can alter the abundance of various metabolites, some of which are essential to the function of epigenetic-modifying enzymes, such as DNA and histone methyltransferases and demethylases. Indeed, epigenetic modifications are known to both silence and promote gene transcription and differentiation programs, which may instigate the induction of the fibrotic gene program. Therefore, we hypothesize that changes in intermediary metabolism are necessary for the epigenetic reprogramming required for myofibroblast differentiation. Our preliminary data suggest that increased glycolytic activity, as occurs during differentiation, is sufficient to promote myofibroblast differentiation, even in the absence of pro-fibrotic stimuli. In addition, these changes in glycolysis are associated with alterations in the abundance of key metabolites, some of which are required cofactors for specific epigenetic modifiers. In particular, α- ketoglutarate levels are significantly increased upon TGF stimulation (canonical fibrotic agonist) and in correlation we find a loss of H3K27me2 within the regulatory regions of known fibrotic genes. Preliminary data suggests that H3K27 demethylation is dependent on the activity of a specific class of αKG-dependent, histone KDMs (Jumonji C-domain containing histone demethylases). Here we will employ both metabolomic and epigenomic approaches in gain- and loss-of-function model systems to examine the centrality of this process in myofibroblast differentiation. This project will be the first to define how changes in intermediary metabolism are directly linked to the transcriptional regulation mediating fibroblast differentiation.