Project Summary With every heartbeat, the junctional complexes that couple cardiomyocytes must transmit the mechanical forces of contraction while maintaining adhesive homeostasis. Cardiomyocytes are linked end-to-end by the intercalated disc (ICD), a specialized junction that coordinates cell signaling, electrical and mechanical operations. The ICD comprises adherens junctions (AJs) and desmosomes that connect the actin and intermediate filament cytoskeletons, respectively, of adjoining cells. ICD function requires multiple adhesion and cytoskeletal proteins and mutations in these proteins are linked to cardiomyopathies. However, relatively little is known about how adhesion complexes are regulated to establish and maintain heart tissue integrity. The core of the adherens junction is the cadherin-catenin complex, which is connected to the actin cytoskeleton through a-catenin. a-Catenin is a multifunctional, mechanoresponsive actin-binding protein that localizes to the cardiomyocyte ICD. However, a-catenin functions in cell-cell adhesion in cardiomyocytes are poorly understood. Our objective in this application is to determine how mechanical and molecular inputs are transduced through the two a-catenins expressed in the human heart – aE(Epithelial)-catenin and aT(Testes)- catenin – to regulate cardiomyocyte adhesion. Our rationale is that defining the individual and collective roles of a-catenin at cardiomyocyte AJs will provide fundamental insight into how cardiomyocyte AJs balance mechanical and signaling functions. We hypothesize that aE-catenin and aT-catenin form unique cadherin- catenin complexes in response to cellular cues to function as mechanical checkpoints for ICD assembly and cardiomyocyte organization. In this proposal, we seek to: 1) define the allosteric mechanisms that regulate a- catenin ligand binding at the cardiomyocyte AJ; 2) determine how ligand recruitment to a-catenin promotes AJ assembly and cytoskeletal attachment; and 3) identify how external mechanical cues regulate AJ organization. Knowledge to be gained through our studies include a detailed, mechanistic understanding of how a-catenin molecular properties are tuned for adhesion in cardiomyocytes; how unique molecular complexes are built along the cardiomyocyte AJ to establish and maintain adhesion; and how external mechanical cues feed into the cardiomyocyte AJ to drive organization. This comprehensive, multiscale analysis will develop new tools and acquire new knowledge about the molecular mechanisms of cell adhesion in cardiomyocytes. Understanding the fundamental mechanisms of cell adhesion in cardiomyocytes is necessary for determining how mutations in ICD proteins lead to heart disease and inform the development of new strategies for the treatment of cardiomyopathies.