PROJECT SUMMARY Arrhythmogenic Cardiomyopathy (ACM) is a heritable disease that bridges the gap between the cardiomyopathies and the inherited arrhythmia syndromes. In its early “concealed” phase, ACM promotes the incidence of ventricular arrhythmias in the absence of overt structural or mechanical remodeling. As the disease progresses, myocyte loss, inflammation, and fibrofatty infiltration emerge, culminating in biventricular failure and further risk of sudden cardiac death (SCD). The pathophysiological significance of the disease is underscored by the fact that ACM is a leading cause of SCD in young individuals < 35 years of age. Mutations in desmosomal proteins account for the majority (approx. 60%) of ACM cases, and in this project we focus on a form of ACM known as Desmoplakin (DSP) cardiomyopathy (DSP-CM). DSP-CM has recently emerged as a unique clinical entity that engenders a severe left-dominant form of the disease. DSP-CM is now well recognized to be a heritable disease that is transmitted in an autosomal dominant pattern, albeit with incomplete and highly variable penetrance. Indeed, a major challenge in the field has been the lack of ability to distinguish whom amongst carriers of pathogenic DSP variants are truly at risk of SCD and whom will go on to live healthy and symptom- free lives. This issue takes on added urgency given that the prevention strategy for SCD in DSP-CM is exercise restriction, a rather draconian measure for young healthy individuals, often athletes. The highly variable penetrance associated with DSP-CM as well as the typical mode of SCD that these patients exhibit highlight the importance of gene-environment interactions in unmasking disease pathogenicity. Our own recent work has identified calpain-mediated desmoplakin degradation as a key factor linking DSP mutations with the development of ACM and its exacerbation by exercise. Our central hypothesis is that: 1) calpain-mediated loss of myocyte DSP protein is a key molecular event that is unmasked by exercise and β-adrenergic stimulation, and 2) the pathogenic effects of DSP degradation at the intercalated disc (ID) are exacerbated by abnormal stretch-related mechanotransduction leading to arrhythmias and heart failure. We will address this dual hypothesis using a multi- scale approach encompassing complementary studies in human engineered heart tissues (hEHT) and innovative genetic and surgical mouse models that are designed to address the complex interactions between external stressors (increased preload) and genetic predisposition (DSP mutations) in the manifestation of DSP- CM. Our studies will enable us to tease out contributions of separate aspects of endurance exercise to myocyte dysfunction and expose pathophysiological mechanisms by which calpain vulnerability is unmasked by external stressors to promote early onset arrhythmias and heart failure progression. Finally, we will test novel gene and small molecule-based approaches to inhibit exercise-relate...