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

• 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
• 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

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.

项目总结 房颤(房颤)是最常见的心律失常，发生在9%的65岁以上的人口和 与发病率和死亡率增加有关，尤其是由于栓塞性中风、猝死和心力衰竭。 尽管氧化应激与房颤的发病机制有关，但对氧化酶的详细机制洞察 激活及其下游效应器仍然难以捉摸。我们之前已经确定了 心脏移植患者NADPH氧化酶4亚型(NOX4)与房颤的关系及其在房颤中的直接作用 基于RNA的斑马鱼NOX4急性诱导研究进展。在初步研究中，我们已经表明 AF是在一种新型的内部培育的心脏特异的NOX4转基因斑马鱼品系中培育出来的，该转基因斑马鱼将用于 目的1探讨心脏特异性激活NOX4在房颤发病机制中的作用。 建立房颤小鼠模型(目标1)。值得注意的是，这些小鼠表现出自发的房颤发作(无P 瓣膜和不规则的RR间期)，如实时遥测心电分析。全球和 心脏特异性基因敲除小鼠将被用来研究心脏NOX4在房颤发生中的特定作用(AIM 1)。在目标2中，我们将研究依赖于NOX4的线粒体功能障碍和自噬是否介导了房颤 斑马鱼和小鼠模型的发展，基于对大量 在NOX4过表达的斑马鱼中产生线粒体活性氧物种(ROS)，并显著 自噬标记物LC3II在房颤小鼠模型中的上调，该基因在NOX4中被完全取消 基因敲除老鼠。我们将使用自噬抑制剂和线粒体ROS清除剂来检测它们在 通过减弱线粒体功能障碍-自噬偶联来预防房颤(目标2和3)。中的更改 将检测在MitoTEMPO处理下的LC3II、ATG7和Beclin-1的自噬标记(目标2)。我们有 创新地表明，一氧化氮(NO)可减轻缺血/再灌流中NOX4的激活。事实上，在初步阶段 没有供体处理的实验在防止NOX4过度表达的斑马鱼的房颤方面非常有效，并且 心脏特异的NOX4转基因斑马鱼。在目标2中，我们还将研究NO供体对房颤的逆转作用，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，实时共聚焦 成像和双电压/钙光学标测以检测电生理和细胞内钙(Ca) 老年小鼠中NOX4表达的处理靶点，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的磷酸化增加，我们预计这将推动 肌浆网钙渗漏、自发性肌浆网钙释放和后除极增加。当一个人 认为这些细胞变化发生在传导减慢的环境中，我们使用 光学标测，这些变化是高度诱发心律失常的。总而言之，这些研究的成就 采用强有力的创新模式生物和新基因菌株的方法无疑将促进 房颤和术后房颤的创新疗法的发展。