Integrative Physiology Methods and Results: We Identify the Endosome-based Eps15 Homology Domain 3 (ehd3) Pathway as Essential for Cardiac Physiology. Ehd3-deficient Hearts Display Structural and Functional Defects including Bradycardia and Rate Variability, Conduction Block, and Blunted Response to

H ighly evolved and differentiated for excitation–contraction coupling, cardiac myocytes express a specific profile of ion channels, pumps, and transporters that maintain cardiomyocyte electric excitability. Collectively, these membrane proteins mediate action potential (AP) formation and response, Ca-induced Ca release, and the secretion of natriuretic peptides. Equally important is the set of hormone receptors localized to the sarco-lemmal membrane that regulate the activity and response of ion channels and pumps through specific second messenger pathways. These highly evolved systems are tightly synchronized to tune cardiac output to meet the changing demands placed on the heart by variable stresses. Like other complex cells, cardiac Rationale: Cardiac function is dependent on the coordinate activities of membrane ion channels, transporters, pumps, and hormone receptors to tune the membrane electrochemical gradient dynamically in response to acute and chronic stress. Although our knowledge of membrane proteins has rapidly advanced during the past decade, our understanding of the subcellular pathways governing the trafficking and localization of integral membrane proteins is limited and essentially unstudied in vivo. In the heart, to our knowledge, there are no in vivo mechanistic studies that directly link endosome-based machinery with cardiac physiology. Objective: To define the in vivo roles of endosome-based cellular machinery for cardiac membrane protein trafficking, myocyte excitability, and cardiac physiology. Conclusions: These data provide new insight into the critical role of endosome-based pathways in membrane protein targeting and cardiac physiology. EHD3 is a critical component of protein trafficking in heart and is essential for the proper membrane targeting of select cellular proteins that maintain excitability.