PROJECT ABSTRACT/SUMMARY The gap junction (GJ) protein Connexin 43 (Cx43) has long been held to be obligate for conduction of action potential (AP) in the ventricular myocardium. However, accumulating evidence during the last two decades from non-mammals, Cx43 knockout mice and human Cx43 mutations suggests that Cx43 GJs do not provide a sufficient explanation for how AP propagates in the heart – and indeed, that Cx43 GJs may be dispensable for cardiac conduction. Based on its short half (~1.5 hours), we recently estimated that on average the human female heart produces more than 80 grams of Cx43 per year – a rate more than 30-times that of actin, the most abundant myocardial protein in steady state. My lab is focused on two questions that are raised by these data. First, what is the mechanism of AP propagation in the ventricle, if Cx43 GJ-based coupling alone cannot account for this phenomenon? Second, if not absolutely required for cardiac conduction, for what purpose does the heart produce Cx43 at such high rates? In recently reported studies, we have determined that: A) Trans- interacting complexes of voltage-gated sodium channels (trans-VGSCs) located at the GJ edge (the perinexus) contribute to cardiac conduction; and B) That the Cx43 carboxyl terminus (CT) has key roles in modulating the heart's response to ischemic injury. Building on these findings, the goals of this project are to: 1) Determine the contribution of trans-VGSCs to cardiac AP propagation and the extent to which this mechanism operates independent of Cx43 GJ-based coupling; 2) Develop a pharmacologic approach to targeting trans-VGSCs as a novel anti-arrhythmic strategy; 3) Determine the extent to which the high levels of Cx43 production by the heart may be accounted for by recent findings that Cx43 is exported in exosomes; 4) Determine whether circulating exosomal Cx43, including Cx43 CT isoforms, are part of a natural signaling mechanism that modulates ischemic injury severity; and 5) Develop a novel therapeutic approach to treat myocardial infarction based on exosomes carrying Cx43 CT mimetic peptides, including translational testing in a large animal model – the pig. The project will involve cross-disciplinary collaboration between biologists, medicinal chemists, biomedical engineers and clinicians – both human and veterinary. The PI has genuine translational bona-fides, with a drug targeting the Cx43 CT invented in his lab currently in multiple Phase III clinical trials. State-of-the-art approaches will be used including super resolution imaging, in silico drug design, genetically modified viruses, iPSCs and testing of exosome-based therapeutics on large animal models. We posit that the primary assignment of myocardial Cx43 may not be in AP propagation. Rather, it is our hypothesis that the high energetic commitment to Cx43 production made by the heart, and other tissues (e.g., skin, vasculature and brain), relates mainly to functions of Cx43 in an exosome-based signali...