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：
10392272
负责人：
Hua Linda Cai
金额：
$ 71.44万
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/10392272
关键词：
3-methyladenine Acute Angiotensin II Animal Model 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 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 Suggestion Systems Biology Telemetry Testing Therapeutic Time Tissues Transgenic Organisms Transplant Recipients Uncertainty Up-Regulation Zebrafish aged attenuation base calmodulin-dependent protein kinase II cohort confocal imaging embolic stroke experimental study genetic strain in vivo inhibitor innovation insight mitochondrial autophagy mitochondrial dysfunction 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.

项目概要心房颤动 (AF) 是最常见的心律失常，发生于 9% 的 65 岁以上人群，与发病率和死亡率增加有关，特别是栓塞性中风、猝死和心力衰竭。尽管氧化应激与 AF 的发病机制有关，但对氧化酶的详细机制了解激活及其下游效应器仍然难以捉摸。我们之前已经确定了之间的相关性心脏移植患者中的 NADPH 氧化酶同工型 4 (NOX4) 和 AF，以及 NOX4 在 AF 中的直接因果作用使用基于 RNA 的斑马鱼急性诱导 NOX4 进行开发。在初步研究中，我们已经表明 AF 是在一种新型的内部生成的心脏特异性 NOX4 转基因斑马鱼品系中开发的，该品系将用于目标 1 描述心脏特异性 NOX4 激活在 AF 发病机制中的因果作用以及一种新的机制内部建立 AF 小鼠模型（目标 1）。值得注意的是，这些小鼠表现出自发性 AF 发作（缺乏 P 瓣膜和不规则的不规则 RR 间隔），通过实时遥测心电图分析来表征。全球和心脏特异性基因敲除小鼠将用于检查心脏 NOX4 在 AF 发展中的特定作用（目的 1).在目标 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 基于大量的初步观察，在斑马鱼和小鼠模型中进行了开发 NOX4 过表达斑马鱼线粒体活性氧 (ROS) 的产生，并且显着在 AF 鼠模型中自噬标记物 LC3II 上调，但在 NOX4 中完全消除基因敲除小鼠。我们将使用自噬抑制剂和线粒体 ROS 清除剂来检查它们在通过减弱线粒体功能障碍-自噬耦合（目标 2）来预防 AF（目标 2 和 3）。变化将检查 MitoTempo 治疗下 LC3II、Atg7 和 Beclin-1 的自噬标记物（目标 2）。我们有创新性地表明，一氧化氮 (NO) 会减弱缺血/再灌注中 NOX4 的激活。确实，在初步实验表明，NO 供体治疗对于预防 NOX4 过表达斑马鱼的 AF 非常有效，并且心脏特异性 NOX4 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，以及 NO 抑制 NOX4 的新分子机制。在目标 3 中，我们将使用膜片钳、实时共焦成像和双电压/钙光学测绘，以检查电生理学和细胞内钙 (Ca) 处理老年小鼠 NOX4 表达的靶点，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 磷酸化增加，我们预计这会驱动肌浆网 (SR) Ca 渗漏、自发 SR Ca 释放和后除极增加。当一认为这些细胞变化发生在传导减慢的环境中，我们使用光学映射，这些变化是高度致心律失常的。综上所述，这些研究成果采用创新模式生物和新型遗传菌株的强大方法无疑会促进开发 AF 和术后 AF 的创新疗法。