Project Summary Heart disease remains the leading cause of mortality in the United States and in the developed world. Arrhythmogenic cardiomyopathy (ACM) in particular is a leading cause of sudden cardiac death in both young people and athletes, remains difficult to diagnose, and has no currently effective treatments. ACM is termed a disease of the desmosome, a cell-cell junctional protein complex critical to cardiomyocyte adhesion, as 40-50% of underlying genetic mutations known to be pathologic for ACM affect a core desmosomal gene component. Importantly, the loss or reduction of any singular desmosomal protein component at the intercalated disc is associated with a “domino effect,” where adjacent desmosomal protein expression is lost, compromising cellular attachment and gap junction electrical conductivity in the heart. Our hypothesis is that the dysregulation of desmosomal protein content homeostasis is directly correlated to ACM disease progression and can be leveraged to identify viable therapeutic targets which could be translated into patient care. Here I will employ both molecular and theoretical approaches to screen for differential transcriptomics and proteomics related to reduction and loss of the core desmosomal component plakophilin-2 (PKP2). In addition to these unbiased approaches, I will leverage human induced pluripotent stem cell (iPSC) and mouse models of PKP2-mutant ACM to characterize candidate regulatory pathways of disease progression. These models each contain distinct splice acceptor site mutations, and have displayed sufficiency to recapitulate disease phenotypes, providing first-of- their-kind platforms to address how RNA alternative splicing and post-transcriptional dysregulation may drive ACM. We further show preliminary studies demonstrating that gene therapy reintroduction of the gap junction protein connexin-43 (Cx43) rescues both early- and late- stage disease phenotypes in spite of the continued absence of the desmosomal core component desmoplakin. Here I will assess whether Cx43 itself may serve as a master regulator of desmosome protein content at the intercalated disc by applying Cx43 gene therapy in the novel context of PKP2 loss, and characterize the broad applicability of this therapeutic approach. My goal for this project is to provide a comprehensive characterization of desmosomal protein regulation across multiple platforms, leveraging predictive systems-level computational molecular models and state-of- the-art RNA binding protein pull-down techniques to identify essential mediators in this signaling network, while assessing the efficacy of Cx43 candidate gene therapy in ACM rescue using physiologically relevant tissue engineered models of human disease and in mice, to establish a generalizable mechanistic model to treat the array of ACM disease presentations in the clinic. Understanding common regulatory pathways controlling desmosomal protein expression at the intercalated disc could be critical to ...