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

针对 AF 中 NOX4 依赖性线粒体功能障碍、自噬和钙处理缺陷

基本信息

批准号：
10540353
负责人：
Hua Linda Cai
金额：
$ 70.04万
依托单位：
UNIVERSITY OF CALIFORNIA LOS ANGELES
依托单位国家：
美国
项目类别：
财政年份：
2022
资助国家：
美国
起止时间：
2022-01-01 至 2025-12-31
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10540353
关键词：
3-methyladenine Acute Angiotensins Animals Arrhythmia Atrial Fibrillation Attenuated Autophagocytosis Calcium Calpain Cardiac Chloroquine Chronic Coupling Data Development Disease Disease model Electrocardiogram Electrophysiology (science)Environment Exhibits Fishes Heart Atrium Heart Injuries Heart Transplantation Heart failure Human Ischemia Knockout Mice Maps Mediating Mitochondria Modeling Molecular Morbidity - disease rate Mus NADPH Oxidase Nitric Oxide Nitric Oxide Donors Older Population Optics Oxidases Oxidative Stress Pathogenesis Pathway interactions Patients Peptide Hydrolases Phenotype Phosphorylation Postoperative Period Production Protein Isoforms Proteins RNA RNA Interference Reactive Oxygen Species Reperfusion Injury Reperfusion Therapy Role RyR2 Sarcoplasmic Reticulum Sudden Death Systems Biology Telemetry Testing Therapeutic Time Tissues Transgenic Organisms Transplant Recipients Uncertainty Up-Regulation Zebrafish aged attenuation calmodulin-dependent protein kinase II cohort confocal imaging embolic stroke experimental study genetic strain in vivo inhibitor innovation insight mitochondrial autophagy mitochondrial dysfunction model organism mortality mouse model multicatalytic endopeptidase complex novel novel therapeutics overexpression patch clamp prevent protein expression response voltage

项目摘要

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.

项目摘要心房颤动（AF）是最常见的心律失常，发生在65岁以上的人口中，IS 与发病率和死亡率的增加有关，特别是来自栓塞中风，猝死和心力衰竭。尽管氧化应激与AF的发病机理有关，但对氧化酶的详细机械见解激活及其下游效应子仍然难以捉摸。我们以前已经确定了心脏移植患者的NADPH氧化酶同工型4（NOX4）和AF，NOX4在AF中的直接因果作用使用基于RNA的急性诱导NOX4在斑马鱼中的开发。在初步研究中，我们已经表明 AF在一种新型内部生成的心脏特异性NOX4转基因斑马鱼线中发展，该系将用于目的1描绘NOX4在AF发病机理中的心脏特异性激活的因果作用以及一种新颖建立内部AF的鼠模型（AIM 1）。值得注意的是，这些小鼠表现出自发的AF发作（不存在P 阀门和不规则不规则的RR间隔），以实时遥测ECG分析为特征。全球和心脏特异性敲除小鼠将用于检查心脏NOX4在AF开发中的特定作用（AIM 1）。在AIM 2中，我们将检查NOX4依赖性线粒体功能障碍和自噬是否介导AF 基于实质性的初步观察，斑马鱼和小鼠模型的发展 NOX4过表达的斑马鱼中的线粒体活性氧（ROS）产生，显着在AF的鼠模型中自噬标记LC3II的上调，该模型完全废除了NOX4 淘汰老鼠。我们将采用自噬抑制剂和线粒体ROS清除剂来检查它们在防止AF（目标2和3），通过衰减线粒体功能障碍 - 自助体耦合（AIM 2）。变更将检查Mitotempo处理下LC3II，ATG7和Beclin-1的自噬标记（AIM 2）。我们有创新的表明，一氧化氮（NO）减弱了缺血/再灌注中的NOX4激活。确实，在初步实验没有任何供体治疗在防止NOX4过表达的斑马鱼中的AF和心脏特异性NOX4转基因斑马鱼。在AIM 2中，我们还将检查没有捐赠者对AF的逆转影响，并且 NOX4抑制的基础的新型分子机制。在AIM 3中，我们将使用斑块夹，现场共聚焦成像以及双电压/钙光学映射以检查电生理和细胞内钙（CA）在老年小鼠中处理NOX4表达的靶标，包括ROS和自噬的中间作用。我们的初步数据表明，这些动物表现出RYR2的磷酸化增加，我们希望这能驱动增加肌浆网（SR）Ca泄漏，自发的SR CA释放和倒置后。当一个认为这些细胞变化发生在传导缓慢的环境中，我们使用光学映射，这些变化是高度挑剔的。总的来说，这些研究的成就采用强大的创新模型生物和新型遗传菌株的方法会提示开发针对AF和术后AF的创新治疗剂。