PROJECT SUMMARY Diseases that arise from mutations in components of mitochondrial oxidative phosphorylation can be devastating, as mitochondria are crucial for energy synthesis. These diseases occur predominantly in infants and children, with a prevalence of 1 in 5000. Though virtually any organ can be affected, the heart is frequently involved, because cardiac function has such high energy requirements. These mitochondrial cardiomyopathies have a particularly grim prognosis, with mortality rates increased nearly three-fold compared to children without cardiac involvement, and no specific therapies available. In linking cardiac function to mitochondrial metabolism, calcium signaling may be central to the pathological process. Calcium influx into the mitochondria can potently stimulate ATP synthesis. In the initial period of this application, we identified a regulatory mechanism by which dysfunction within Complex I of the electron transport chain causes a compensatory increase in activity of the mitochondrial calcium uniporter channel, preserving ATP synthesis. During normal physiology, Complex I promotes uniporter degradation via an interaction with the uniporter, a mechanism we term Complex I-induced protein turnover (CLIPT). During Complex I dysfunction, interaction with the uniporter is inhibited, preventing degradation and leading to a build-up in functional channels. This mechanism is widespread and was seen in fruit flies, mice, and humans. Moreover, while inhibiting the uniporter led to early demise in Complex I-deficient animals, enhancing uniporter stability rescued survival and function. In this project period, we propose the following three aims to further study this pathway and determine whether it can be exploited for potential therapeutic benefit. We focus on mitochondrial one-carbon (1C) metabolism, a producer of mitochondrial antioxidant species (NAPDH), because this pathway is substantially upregulated in mitochondrial cardiomyopathies. In the first aim, we will examine if adenine dinucleotides (NAD+/NADP+) regulate the uniporter, and if calcium regulates critical 1C metabolism enzymes. In the second aim, we will establish whether uniporter activity in mitochondrial cardiomyopathies extends beyond ATP synthesis to NADPH biology, affecting redox balance and one-carbon metabolism. In the third aim, we will test whether manipulation of the portion of the uniporter that interacts with Complex I, the uniporter N-terminal domain (NTD), can be exploited in mouse models as a potential therapy for mitochondrial cardiomyopathies.