SUMMARY/ABSTRACT Heart failure (HF) is a leading cause of death worldwide. Although the pathophysiology of HF is complex and remains incompletely understood, defects in mitochondrial function have been implicated in the progression and outcomes of HF, and emerged as an important target for HF therapy. One well-known contributing factor to adverse cardiac remodeling in the failing hearts is excess mitochondrial-derived reactive oxygen species (mtROS). Accordingly, a variety of antioxidant-based therapies have been developed for HF treatment over the last decades. However, despite promising outcomes in preclinical studies, translation of these therapies to the clinic has not succeeded to date, suggesting that alternative or complementary mitochondrial therapeutic targets are needed. In addition to excess mtROS, profound loss of mitochondrial membrane potential (m) is another key hallmark of HF. In the cardiomyocyte (CM), m disruption affects not only energy production, but also a variety of signaling pathways crucial for cell function and survival, such as redox balance, calcium homeostasis and mitochondrial quality control. We hypothesize that synergistically targeting mitochondria, i.e., concurrently preserving mitochondrial m and scavenging excess mtROS, is a viable therapeutic strategy for HF treatment. However, assessing the therapeutic potential of m preservation is challenging, due to a lack of tools for dynamic and specific control of CM m in live animals. To overcome this technical barrier, we developed an innovative mitochondrial-targeted luminoptogenetic (named mLumOpto) technology by integrating luciferase- luciferin-emitted endogenous bioluminescence with the mitochondrial optogenetics we recently published. Our preliminary data indicate that mLumOpto can induce dynamic CM m control in the absence of external light illumination. The primary goal of this project is to employ this advanced mLumOpto technology to manipulate CM m in vivo to evaluate the efficacy of CM m preservation alone, or in synergism with a mtROS scavenger (i.e., mitochondrial-specific antioxidant), in HF treatment in preclinical mouse models. The translational potential of the proposed synergistic mitochondrial-targeted HF therapy will also be assessed in human-like large animals (i.e., pigs). Three Specific Aims are proposed to accomplish our objectives. Aim 1 will determine the role of sustained CM m depolarization in pathological cardiac remodeling and HF development in mice, and dissect the underlying molecular mechanisms. Aim 2 will evaluate the efficacy of synergistic m preservation and mtROS scavenging in improving adverse cardiac remodeling and contractile dysfunction in two well-established mouse HF models (i.e., pressure overload and myocardial ischemia-reperfusion). Aim 3 will assess the translational potential of mLumOpto-mediated HF therapy in pigs. Successful completion of this project will lead to not only an innovative technology c...