PROJECT SUMMARY Critical electrogenic proteins responsible for maintaining cardiac excitability and conduction, including sodium channels (NaV1.5), inward-rectifying potassium channels (Kir2.1), L-type calcium channels (Cav1.2), sodium-potassium ATPase (NKA), and sodium-calcium exchanger (NCX) have been identified to reside in distinct ion channel ‘pools,’ with localization at the cell-cell junction, the intercalated disk (ID). These distinct ion channel pools suggest regulation via both ‘global’ and ‘local’ control mechanisms. Within the ID, heterogeneous nanoscale structure results in channels concentrating around gap junctions and mechanical junctions, forming specialized nanodomains. ID nanodomains perturbation can induce proarrhythmic conduction defects, and disruption of these nanodomains has been identified in human arrhythmia patients, suggesting that these sites are key determinants of conduction. However, ID nanoscale structure and molecular organization and their implications for functional electrophysiology have yet to be systematically investigated in health or disease. In this project, we will undertake the first-ever comprehensive and granular quantification of ID structure and molecular organization using cutting-edge light and electron microscopy techniques and computational analysis. Further, we will develop a novel computational modeling framework to incorporate experimental measurements of these distinct ion channel pools (lateral membrane and ID) and ID nanoscale structure to assess regulation of tissue-scale cardiac conduction, for direct comparison with optical mapping of murine myocardium. Simulations will extend predictions to conduction in human ventricles and predict how both chronic and acute ID perturbations impact conduction in conjunction with additional functional defects, including non-ischemic heart failure. Upon successful completion of these aims, we will produce a new theoretical underpinning for which distinct ion channel pools and intercalated disk nanoscale structure confer a ‘global/local control’ of cardiac conduction and suggest new therapeutic approaches to preserve conduction during disease progression.