Abstract: This project explores the intersection of two fundamental areas: cellular (mal)adaptation to primary mitochondrial dysfunction and the biology of the mitochondrial Ca2+ uniporter (MCUC). In humans, primary mitochondrial disease arises from mutations in nuclear- or mitochondrial-encoded DNA, or from pharmacological agents or toxins. Skeletal muscle is among the most severely affected organs. Mouse models of mitochondrial myopathy (MM) show that energy deficit, per se, is not the major factor for pathology, but, rather, that mitochondrial dysfunction initiate a progression of adaptive and maladaptive changes in, e.g., metabolism and proteostasis, and also activates endoplasmic reticulum (ER) stress and the Integrated Stress Response (ISR); the result is muscle atrophy, weakness, and diminished exercise capacity. Major cytoplasmic signaling pathways beyond the ISR have been investigated but found to not fully explain MM. Whether processes within mitochondria can impact MM progression has only been narrowly considered. Published data and our preliminary data document an increased abundance of the MCUC and increased mitochondrial Ca2+ uptake in MM and myopathies of other origins. The possibility that mitochondrial Ca2+ uptake contributes to pathology has only been considered in the context of MCUC's ability to cause a sustained opening of the mitochondrial permeability transition pore (mPTP), which can trigger cell death. Yet, our preliminary data suggest the hypothesis that the MCUC serves a beneficial role in MM, by expanding the oxidative phosphorylation capacity of dysfunctional muscle mitochondrial, and blunting the ISR. This hypothesis will be tested in two Specific Aims, by depletion MCUC in two models of MM (mice with depletion of PiC in skeletal muscle; mice with whole-body loss of Frataxin), for in vivo and ex vivo studies. We will also use advanced imaging techniques and genetic sensors to evaluate metabolism, bioenergetics and redox, and calcium, in a compartmentalized manner, including at the ER-mitochondria interface, in cells acutely depleted of MCUC. We will also use sophisticated methods to evaluate protein translation, since this is a key feature of the ISR that can influence muscle mass. Aim 1 will test the hypothesis that mitochondrial Ca2+ uptake improves energetics during the early phase of mitochondrial dysfunction. Aim 2 will determine how MCUC contributes to mitochondrial dysfunction-induced ER stress and the ISR and consequences on cell viability and muscle mass. Aim 3 will test the hypothesis that regulation of MCUC by MICU3 renders mitochondria vulnerable to sustained mPTP opening such that the MCUC becomes a liability for skeletal muscle at later phases of mitochondrial dysfunction. These studies are expected to reveal a novel role for MCUC in the (mal)adaptive response of skeletal muscle to mitochondrial dysfunction and in regulating muscle mass in myopathy, and, broadly, to provide new insight into the regulati...