Targeting NOX4-dependent mitochondrial dysfunction, autophagy and defective calcium handling in AF

项目摘要 心房颤动（AF）是最常见的心律失常，发生在9%的65岁以上人群中， 与发病率和死亡率增加相关，尤其是栓塞性卒中、猝死和心力衰竭。 虽然氧化应激与房颤的发病机制有关，但对氧化酶的详细机制认识 激活及其下游效应物仍然难以捉摸。我们之前已经发现了 心脏移植患者中NADPH氧化酶亚型4（NOX 4）和AF，以及NOX 4在AF中的直接因果作用 在斑马鱼中使用基于RNA的N 0X 4急性诱导的开发。在初步研究中，我们已经表明， AF在一种新的内部产生的心脏特异性NOX 4转基因斑马鱼系中发展，该转基因斑马鱼系将用于 目的1描述心脏特异性激活NOX 4在AF发病机制中的因果作用， 内部建立的AF小鼠模型（目的1）。值得注意的是，这些小鼠表现出自发性AF发作（P 瓣膜和不规则的不规则RR间期），如通过真实的时间遥测ECG分析表征的。全球和 心脏特异性基因敲除小鼠将被用于检查心脏NOX 4在AF发展中的特定作用（Aim 1）。在目标2中，我们将研究NOX 4依赖性线粒体功能障碍和自噬是否介导AF 在斑马鱼和小鼠模型中的发展，基于对大量 线粒体活性氧（ROS）的产生在NOX 4过表达的斑马鱼，和显着 自噬标志物LC 3 II在AF的鼠模型中的上调，其在NOX 4中被完全废除。 敲除小鼠我们将使用自噬抑制剂和线粒体ROS清除剂来检查它们在细胞凋亡中的作用。 通过减弱线粒体功能障碍-自噬偶联（Aim 2）预防AF（Aim 2和3）。变化 将检查在MitoTempo处理下的LC 3 II、Atg 7和Beclin-1的自噬标志物（目的2）。我们有 创新性地显示一氧化氮（NO）减弱缺血/再灌注中的NOX 4活化。事实上，在初步 NO供体处理在NOX 4过表达的斑马鱼中预防AF是稳健有效的， 心脏特异性NOX 4转基因斑马鱼。在目标2中，我们还将研究NO供体对AF的逆转作用， NO抑制NOX 4的新分子机制。在目标3中，我们将使用膜片钳，实时共聚焦 成像和双电压/钙光学标测，以检查电生理和细胞内钙（Ca） 处理老年小鼠中NOX 4表达的靶点，包括ROS和自噬的中间作用。我们 初步数据表明，这些动物表现出增加的RyR 2磷酸化，我们预计这将驱动 增加肌浆网（SR）钙泄漏，自发SR钙释放和后去极化。当一个 认为这些细胞变化发生在传导减慢的环境中，我们使用 光学测绘，这些变化是高度programmic。这些研究的成果， 采用创新模式生物和新的遗传菌株的强大方法无疑将促进 开发AF和术后AF的创新疗法。

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 identified using optical mapping, these changes are highly proarrhythmic. Taken together, accomplishments of these studies employing powerful approaches of innovative model organisms and novel genetic strains will no doubt prompt development of innovative therapeutics for AF and postoperative AF.