PROJECT SUMMARY Heart failure, measured at the subcellular level, is the result of impaired cardiomyocyte excitation-contraction (E-C) coupling. One key structural component of E–C coupling is the myocyte transverse (T)-tubule system. T- tubules play an essential role in coordinating membrane excitation with muscle contraction by facilitating the synchronized release of Ca2+ from the sarcoplasmic reticulum. Our group, as well as those of others, have provided strong evidence that failing myocytes from patients and animal models are characterized by a degenerated and disorganized T-tubule system resulting in impaired intracellular Ca2+ dynamics and myofilament contraction. The long-term goal of our research is to discern and take advantage of the fundamental mechanisms underlying T-tubule remodeling processes in heart disease toward restoring T-tubule integrity and slowing, if not reversing, heart failure progression. We have identified Mitsugumin 53 (MG53/TRIM72) as a potential T-tubule repair enzyme. We find MG53, known to be involved in injury-induced membrane vesicle trafficking and repair in striated muscle, localizes to T-tubules and is upregulated in human failing hearts and animal models of chronic heart failure. Our preliminary data indicate MG53 possesses apparent divergent functions in the heart, MG53 deletion of MG53 exacerbates T-tubule degeneration in stressed hearts while exogenous MG53 overexpression promotes progressive and severe T-tubule disruption. Interestingly, we have mapped overexpression effects to its little-studied E3-ligase domain. It is our hypothesis, therefore, that the membrane repair versus E3-ubiquitin ligase activities of MG53 determine cardiomyocyte T-tubule integrity and E-C coupling function during health and in disease. We have recently generated two novel knockin animals for separately examining endogenous MG53 E3-ligase and membrane repair functions during cardiac stress responses. We will test our hypothesis by first determining the physiological (Aim 1) and molecular (Aim 2) actions of the MG53 E3-ubiquitin ligase, as well as its membrane repair (Aim 3), domains in T-tubule structure and Ca2+ handling at baseline and in response to cardiac stress. We will attribute apparently discordant ubiquitin-dependent proteolysis and membrane healing functions of MG53 to overall T-tubule integrity, E-C coupling and cardiac remodeling processes. Understanding the mechanisms by which MG53 facilitates T-tubule remodeling versus repair will allow us design highly targeted and efficacious therapeutics for heart failure treatment.