Cardiomyopathy (CMY) is a worldwide problem associated with high morbidity and mortality. CMY patients may present with symptoms attributed to heart failure (HF), arrhythmias, cardiac conduction system abnormalities, and sudden cardiac death. Recently it has been increasingly recognized that genetic screening for etiologies of CMY is important, and that defects in cardiomyocyte (CM) proteins in cellular junctions can cause CMY. Mechanical and electrical coupling of CM occurs at the intercellular connection between CM, termed the intercalated disk (ID). The ID provides essential properties to allow the heart to function as a syncytium. Structural elements of the cardiac conduction system (CCS) such as the atrioventricular (AV) and sinoatrial (SA) nodes have cellular arrangements and intercellular connections that vary from working CMs in the atrium and ventricle. Understanding more about proteins essential for normal function of working CMs and CCS cells is critical to advance our knowledge of the basis of cardiac disease, and remains understudied. This proposal is focused on Vinculin (VCL) and Zonula Occludens (ZO) proteins, which bind directly to one another, are located in the ID and the lateral membrane of working CMs and have known links to human disease. Our global hypothesis here is that VCL, ZO-1 and ZO-2 have unique roles in preserving cardiac function, conduction, and rhythm, given their location in CMs and in the CCS. We propose two aims. AIM 1 will evaluate how loss of ZO and VCL proteins from CMs alters contractile function and the molecular phenotype of the working CM and adult heart. We hypothesize that with postnatal loss of CM ZO and VCL proteins, cell-cell communication and integrity of the working myocardium will be disturbed, resulting in contractile dysfunction. CM ZO loss is expected to be distinct from that caused by loss of CM VCL, despite these proteins directly binding one another. Unique mouse models in hand will be used to evaluate the effects of loss of ZO and VCL from mature CMs basally and after the heart is faced with stress. We will assess whole heart, tissue and single cells using physiological, biochemical, and microscopic evaluations, as well as a novel approach to single cell transcriptomics and single cell proteomics, to pursue the mechanistic basis for how ZO and VCL protein loss causes CMY. Differences by sex and in atria vs. ventricle will be considered. AIM 2 will determine the function of ZO-1 and VCL in cardiac conduction and rhythm. Deletion of VCL from CM caused ventricular arrhythmias and sudden death, while loss of CM ZO-1 produced CCS dysfunction. We hypothesize that these two proteins have unique roles in controlling cardiac rhythm and conduction, despite their direct binding, due to interactions with connexins and membrane ion channels. Using patch clamping, high-resolution confocal microscopy to detect Ca2+ transients, optical voltage/Ca2+ mapping, and single cell transcriptional and proteomic st...