As critical regulators of cellular metabolism, mitochondria activate various pathways in response to stressors (e.g., aging) and dysfunction (e.g., unfolded proteins). However, little is known about the in vivo pathways mitochondria use to communicate impaired energy production. Mitochondrial energy dysfunction is a hallmark of a range of degenerative diseases affecting tissues with high energy demands, thus understanding how mitochondria respond to energy dysfunction and direct the cellular response to energetic crisis in vivo is critical for the design of targeted strategies to ameliorate these diseases. Here, we will leverage a unique model of in vivo mitochondrial energy impairment that we engineered by inducible deletion of the cardiac mitochondrial phosphate carrier (SLC25A3) in adult mouse cardiomyocytes. This model offers a novel system to model mitochondrial energy impairment in a terminally differentiated tissue with high energy demands. Intriguingly, despite the cardiac disease exhibited by these mice, SLC25A3 deficiency does not engage canonical mitochondrial energy dysfunction pathways like AMPK and ROS signaling, nor is cell death or fibrosis exhibited by deficient hearts. Instead, loss of SLC25A3 in adult hearts causes a striking increase in mitochondria-specific protein acylations, particularly acetylation and malonylation. Acylations are dynamic post-translational modifications derived from metabolic intermediates and subject to removal by sirtuin deacylases. Importantly, acylations harbor the potential to link metabolism to protein functional regulation, while altered acylation is associated with disease pathogenesis. Our preliminary data suggest that, in particular, two aspects of the acylome—the acetylome and the malonylome—are remodeled in response to mitochondrial energy dysfunction. While the acylome is well known to regulate mitochondrial metabolism, our work suggests that the converse is also possible: that mitochondrial energy dysfunction directs acylome remodeling. We hypothesize that acylome modifications represent a mitochondria-intrinsic mechanism to coordinate the cellular response to energy stress. Using the SLC25A3 deletion mice together with cell biology, biochemistry, proteomics, and innovative in vivo gene therapy approaches, we will 1) identify the mechanisms underlying SLC25A3 deletion- mediated acylome remodeling, 2) define how acylations regulate the mitochondrial permeability transition pore cell death pathway, and 3) determine the physiological impact of aberrant acylations on the mitochondrial energy-impaired heart. The proposed studies will provide novel insight on the link between mitochondrial bioenergetics and acylome remodeling and position acylations as an arm of the mitochondrial stress response that is activated upon mitochondrial energy dysfunction. Ultimately, identification of pathways regulating mitochondrial dysfunction will facilitate the development of new therapies targeting mitochondrial...