SUMMARY/ABSTRACT Heart failure (HF) remains a major health problem and significant mortality in the United States. While defects in mitochondrial function are strongly implicated in the pathophysiology of HF, no mitochondrial-targeted therapy has been successful in the clinic to date, indicating that we still do not possess sufficient understanding of the mechanisms connecting mitochondrial dysfunction and HF development. Normal mitochondrial functions rely on maintaining the inner membrane potential (m). In the cardiomyocyte (CM), m perturbations not only directly affect ATP production, but also influence a variety of signaling pathways that modulate redox balance, Ca2+ homeostasis, and mitochondrial quality control. However, to what extent and how the loss of CM m alone impairs cardiac energetics, contractility, and electrical activity (i.e., hallmarks of HF) remains poorly defined. This is due, in part, to a lack of methods for effectively and selectively manipulating CM mitochondria in the in vivo setting. Existing pharmacological approaches to depolarize m lack both cell (CM versus non-CM) and organ (cardiac versus non-cardiac) specificity. To overcome this technical barrier, we developed an innovative mitochondrial-targeted luminoptogenetic (termed mLumOpto) technology and demonstrated that it can induce dynamic, selective CM m depolarization both in vitro and in vivo, with luciferase-luciferin pair-emitted intracellular bioluminescent illumination. The primary goal of this proposal is to utilize the mLumOpto technology to directly induce CM-specific m depolarization in intact hearts, and delineate the acute (i.e. <24 hours) and chronic (i.e. 2 weeks) impacts on cardiac functions. Our hypothesis is that CM-specific m depolarization alone is sufficient to induce adverse cardiac remodeling and HF development, which will be tested with the following Specific Aims. Specific Aim 1 will fully characterize mLumOpto-mediated m depolarization in isolated adult CMs (ACMs) in vitro, and establish luciferin dose dependency. How the mLumOpto-mediated m depolarization acutely and chronically impacts ACM function and health, respectively, will be dissected. Specific Aim 2 will determine the acute effect of CM-specific m depolarization in intact hearts. First, mLumOpto-mediated in vivo CM-specific m depolarization will be determined. Then, the acute effects of CM m depolarization on cardiac metabolism, pump function, and electrophysiology in intact hearts will be examined both ex vivo and in vivo. Specific Aim 3 will delineate the chronic effects of moderate CM m depolarization on cardiac remodeling (at gravimetric, histologic, biochemical, electrophysiological, and molecular levels) that lead to contractile dysfunction and HF development. The reversibility of CM m depolarization-induced HF will also be examined. Successful completion of this research will lead to not only an innovative technology for dynamic control of CM mitoc...