Diabetes mellitus (DM) induces a cardiac muscle disorder known as diabetic cardiomyopathy (DMCM) that progresses to heart failure. A hallmark of DMCM is disrupted metabolism, resulting from increased dependency on fatty acid oxidation (FAO) for ATP production. Increased FAO results in mitochondrial stress and ultimately mitochondrial dysfunction, however the underlying molecular mechanisms are unclear. Ketogenesis is a fatty acid oxidation spillover pathway that contributes to mitochondrial damage via the rate limiting enzyme 3-Hydroxy-3-Methylglutaryl-CoA Synthase 2 (HMGCS2). Mitochondrial damage is further compounded by impaired mitophagy, the degradation of damaged mitochondria, in the DM heart. Furthermore, miR-133a, a highly abundant and cardioprotective miRNA, is downregulated in the DM heart. However, it is unclear how miR-133a regulates metabolic remodeling and mitochondrial damage in diabetic cardiomyopathy. My preliminary data shows that overexpression of miR-133a prevents mitochondrial lipid accumulation, upregulates fatty acid transport proteins, and downregulates HMGCS2 expression in the Type 1 DM (T1DM) heart. Moreover, miR-133a upregulated the mitophagy associated protein Parkin, plausibly by targeting Ectoderm Neural Cortex 1 (ENC1) - an inhibitor of mitophagy. Thus, we hypothesize that decreased fatty acid metabolism induces spillover activation of ketogenesis in the T1DM heart contributes to mitochondrial dysfunction, which is exacerbated by impaired mitophagy via upregulated ENC1 and ameliorated by increased expression of miR-133a in diabetic cardiomyopathy. I will examine whether ketogenesis is increased in the T1DM heart, and if miR-133a mitigates ketogenesis and restores mitophagy by formulating two specific Aims. Aim 1: To test the hypothesis that activation of ketogenesis leads to mitochondrial dysfunction in DMCM, which is mitigated in part by increased expression of miR-133a in the T1DM heart. Aim 2: To test the hypothesis that increased ENC1 impairs mitophagy in diabetic cardiomyopathy, which is ameliorated in part by upregulation of miR-133a in the T1DM heart. These studies will unravel a novel regulatory mechanism of ketogenesis and mitochondrial damage in DMCM and provide a miR-133a-based therapeutic approach to ameliorate cardiac metabolic derangement and mitochondrial dysfunction in DMCM.