PROJECT SUMMARY/ABSTRACT Induced pluripotent stem cell (iPSC)-derived cardiomyocytes (iCMs) have generated great excitement for their promise to regenerate the injured myocardium. In pre-clinical studies, we have demonstrated that iCMs have significant functional benefit; however, substantial challenges remain, including ventricular arrhythmia, teratoma formation, and poor engraftment in the host myocardium. Furthermore, reliable regeneration of the injured myocardium has yet to be seen. While no effective strategy for permanent restoration has emerged, paracrine factors appear to underlie the beneficial effects of iCM therapy. Recently, we discovered the mitochondria-rich extracellular vesicles (M-EVs), which are secreted from the iCMs. These M-EVs effectively repair the injured cardiomyocytes and myocardium through restoration of intracellular bioenergetics. The paracrine effect is achieved by mitochondrial transfer and biogenesis to augment ATP production. This proposal will re-shape the future of heart failure (HF) therapeutics. There is clear clinical indication and need to improve the high mortality and morbidity of HF patients. The shortcoming of current standard of care may be due to the unmet need in understanding the bioenergetic imbalance in HF. The disruption of the balance between energy supply and demand underlies the pathogenesis of HF. Cardiac tissues from patients with hypertrophic, dilated, or ischemic cardiomyopathy all exhibit structural abnormalities of mitochondria and diminished ATP production despite increased metabolic energy demands in the failing heart. Although peroxisome proliferator-activated receptor γ coactivator-1α (PGC-1α) serves as a master regulator of mitochondrial biogenesis and function, PGC-1α levels are decreased in the myocardium of the HF patients. Insufficient energy generation results in the loss of cardiomyocyte contractility, myocardial dysfunction, and, ultimately, decompensated HF. Proteomic analysis of M-EVs demonstrated a novel cluster of 6 enriched M-EV proteins (PC), which interact with PGC-1α. PC was found to up-regulate energy metabolism, including oxidative phosphorylation, fatty acid metabolism, and glycolysis. Therefore, we hope to develop an innovative therapy that targets the intracellular bioenergetics directly through the following 3 Specific Aims: Specific Aim 1 – Confirm the role of enriched M-EV protein cluster (PC) in mitochondrial biogenesis. Specific Aim 2: Determine the mechanism of the protective effects of M-EVs in an in vitro iCM model of hypoxic injury. Specific Aim 3: Assess the functional benefits of mitochondrial augmentation and/or biogenesis in an in vivo mouse model of chronic myocardial injury. Upon conclusion of this study, the bioenergetic mechanism of mitochondrial augmentation and biogenesis will be confirmed in M-EVs for significant and sustained restoration of the injured myocardium.