Project Summary/Abstract The goal of this project is to elucidate mechanisms responsible for gap junction remodeling in arrhythmogenic cardiomyopathy (ACM), a leading cause of sudden death in the young and especially in athletes. Gap junction remodeling occurs early in ACM, well before development of fibro-fatty scar tissue and contractile dysfunction. Arrhythmias occur frequently during this so-called “concealed phase” and appear to arise through interactions between altered cell-cell electrical coupling and reduced INa and IK1. Here, we propose studies to define, at a level of detail never before achieved, how gap junction remodeling occurs in this highly arrhythmogenic human heart disease and how it promotes sudden death. Moreover, we have a small molecule that reverses gap junction remodeling in ACM. Thus, we will not only define the mechanism of gap junction remodeling in ACM, but will also define the mode of action of a mechanism-based therapy that may eventually be used in patients. In the proposed research, we will use state-of-the-art methods to rigorously quantify changes in Cx43 expression at intercalated discs and measure cell-cell conductance in ventricular myocytes expressing ACM- causing desmosomal mutations (such measurements. We will characterize changes in impulse propagation at a subcellular level of resolution in precisely patterned tissue preparations and in intact adult mouse hearts using multiple models that faithfully recapitulate the ACM disease phenotype seen in patients. We will use powerful computational tools to define interactions between reduced cell-cell electrical coupling and reduced INa and IK1current densities, and precisely determine their individual contributions to conduction abnormalities and arrhythmogenesis. We will perform detailed studies in collaboration with Robin Shaw, a leading authority in connexin trafficking, to define molecular mechanisms responsible for gap junction remodeling in ACM. We will define the role of EB1-based forward Cx43 trafficking in ACM, and investigate the role of changes in Cx43 phosphorylation and internalization. We will also elucidate molecular mechanisms by which abnormal translocation of the GSK3β/APC/Axin complex to the intercalated disc interferes with Cx43 trafficking in ACM. The ultimate objective is to develop new ways to prevent sudden death in ACM by correcting the trafficking defect that appears to be at the root of lethal rhythm disorders in this disease spectrum.