Abstract During the development of heart failure cardiac fuel metabolism switches from predominantly fatty acid oxidation (FAO) to increased reliance on glucose, especially glycolysis. This metabolic remodeling is generally recognized and considered ultimately maladaptive for sustaining myocardial energetics and function. The mechanisms responsible for the switch are poorly understood but appear to be coupled with impaired mitochondrial function. Downregulations of multiple transcriptional mechanisms, such as PPARa or PGC-1a, for FAO have been identified in heart failure. Reduced FAO could release the inhibition of glucose use through Randle cycle and thus promote myocardial glucose utilization. However, this hypothesis does not explain why reduced FAO leads to predominantly glycolysis uncoupled with glucose oxidation, a phenomenon similar to Warburg effect. In addition to decreased FAO, multiple aspects of mitochondrial function, in particular, oxidative phosphorylation, oxidative stress, and redox balance, are also altered in hearts with pathological hypertrophy. These observations raise an intriguing possibility that increased glycolysis is driven by mitochondrial dysfunction although the molecular mediator(s) in the switch are elusive. Recently, we found that the expression of mitochondrial ATPase inhibitor factor 1 (ATPIF1) was increased in rodent hearts or cardiomyocytes (CMs) with pathological hypertrophy. Upregulation of ATPIF1 in non-cardiomyocytes has been shown to increase glycolysis, to trigger mitochondrial hyperpolarization and increase the production of mitochondrial reactive oxygen species (mtROS). In our preliminary study, ATPIF1 overexpression also shifted energy metabolism from mitochondrial oxidation to glycolysis in CMs. Therefore, we asked whether and how ATPIF1 connects mitochondrial function and glycolysis in the heart undergoing pathological hypertrophy. The ATPIF1 is well conserved from yeast to human, and it is known to inhibit the reversed operation of FoF1-ATPase in Complex V (normally functions as ATP synthase) to hydrolyze ATP and thus maintain the proton gradient during reduced membrane potential, such as ischemia. The consequence of ATPIF1 upregulation in the non-ischemic heart is unknown. In the proposed study, we will determine the interaction of ATPIF1 with Complex V under normal and stress conditions and test the hypothesis that increased ATPIF1 inhibits ATP synthase and triggers the metabolic switch to glycolysis via stimulation of HIF1a signaling during pathological hypertrophy. We have generated preliminary data and research tools for the following three specific aims: 1) To test the hypothesis that up-regulation of ATPIF1 increases myocardial glycolysis through enhancing the HIF1α signaling. 2) To determine the molecular interaction of ATPIF...