PROJECT SUMMARY Atrial fibrillation (AF) is the most common cardiac arrhythmia occurring in 9% of population older than 65 and is associated with increased morbidity and mortality, notably from embolic stroke, sudden death and heart failure. Although oxidative stress has been implicated in the pathogenesis of AF, detailed mechanistic insights into oxidase activation and its downstream effectors have remained elusive. We have previously identified a correlation between NADPH oxidase isoform 4 (NOX4) and AF in cardiac transplant patients, and a direct causal role of NOX4 in AF development using RNA based acute induction of NOX4 in zebrafish. In preliminary studies, we have shown that AF develops in a novel in-house generated, cardiac-specific NOX4 transgenic zebrafish line, which will be used in Aim 1 to delineate a causal role of cardiac-specific activation of NOX4 in AF pathogenesis together with a novel murine model of AF established in-house (Aim 1). Notably, these mice exhibit spontaneous AF episodes (absent P valves and irregularly irregular RR intervals), as characterized by real time telemetry ECG analyses. Global and cardiac specific knockout mice will be employed to examine a specific role of cardiac NOX4 in AF development (Aim 1). In Aim 2, we will examine whether NOX4-dependent mitochondrial dysfunction and autophagy mediate AF development in both the zebrafish and mouse models, based on preliminary observations of substantial mitochondrial reactive oxygen species (ROS) production in NOX4 overexpressed zebrafish, and significant upregulation of autophagy marker LC3II in the murine model of AF, which was completely abrogated in NOX4 knockout mice. We will employ autophagy inhibitors and mitochondrial ROS scavengers to examine their effects in preventing AF (Aims 2 & 3), via attenuation of mitochondrial dysfunction-autophagy coupling (Aim 2). Changes in autophagy markers of LC3II, Atg7 and Beclin-1 under MitoTempo treatment will be examined (Aim 2). We have innovatively shown that nitric oxide (NO) attenuates NOX4 activation in ischemia/reperfusion. Indeed, in preliminary experiments NO donor treatment was robustly effective in preventing AF in NOX4 overexpressed zebrafish, and the cardiac specific NOX4 transgenic zebrafish. In Aim 2 we will also examine reversal effects of NO donors on AF, and novel molecular mechanisms underlying NO inhibition of NOX4. In Aim 3 we will use patch clamp, live confocal imaging, and dual voltage/calcium optical mapping to examine the electrophysiological and intracellular calcium (Ca) handling targets of NOX4 expression in aged mice, including the intermediate roles of ROS and autophagy. Our preliminary data indicate that these animals exhibit increased phosphorylation of RyR2, which we expect to drive increased sarcoplasmic reticulum (SR) Ca leak, spontaneous SR Ca release and afterdepolarizations. When one considers that these cellular changes occur in the environment of slowed conduction, which we identi...