PROJECT SUMMARY/ABSTRACT Atrial fibrillation (AF) is the most common sustained adult arrhythmia, associated with an increased risk of stroke, heart failure and dementia. With increased longevity in chronic diseases, the prevalence of AF – currently estimated at 46 million worldwide – is dramatically rising. Catheter ablation – tissue destruction – is the most effective therapy but is fraught with procedural risks and suboptimal efficacy. The cardiac autonomic system (CANS) is known to be involved in the pathogenesis of AF, but no specific diagnostic or therapeutic approaches have evolved from this. Our long-term goal is to devise neuromodulatory AF treatment and preventive strategies and fill the knowledge gap on the mechanisms by which CANS neuronal and humoral paracrine output modulate atrial function in humans. We break through existing technical barriers that limited our understanding of intrinsic cardiac ganglia, capitalizing on the vein of Marshall as a vascular route to sample its electrophysiology and humoral responses, collecting atrial coronary circulation blood, and recording nerve activity from the ganglionated plexi (GP). Our extensive preliminary data in patients shows that apnea increases GP activity measured using novel percutaneous technology in AF patients undergoing ablation procedures. Remarkably, we found that Substance P (SP) collected from the coronary sinus is elevated compared to undetectable levels in peripheral blood of AF patients suggesting that GP activation via secreted SP may play a role in AF substrates. In large animal models, we have found that specific ablation of GP sensory neurons blunts the pro-fibrillatory response to apnea and that a crescendo GP response occurs after repeated consecutive apneas. Our data in human pluripotent stem cell-derived atrial cardiomyocytes (hiPSC-aCM) shows that chronic SP treatment affects cardiomyocyte electrophysiology and modifies gene expression of miR-21 targets. These exciting observations, innovative methods, and unique clinical and basic science expertise position our team to develop this project successfully. We propose the central hypothesis that CANS produces a substrate for AF through neural (nerve firing) and humoral effects (secretome), in which SP – released by GP sensory neurons – plays a major role in increasing susceptibility to AF through direct electrophysiological and genomic effects in atrial cardiomyocytes. The central hypothesis will be tested by pursuing studies 1) in humans with paroxysmal and persistent AF aiming to measure nerve activity and secretome of intrinsic ganglia through the vein of Marshall during ablation procedures, 2) in canine models of acute and persistent AF to determine whether ablation of GP or SP antagonism ameliorates AF, 3) in hiPSC-aCM and engineered atrial tissues to elucidate SP actions. The proposed research is significant because it is expected to provide a mechanistic understanding of the relationship between CANS and sleep a...