Chloride intracellular channels in cardiac mitochondria and their direct role in cardioprotection

心脏线粒体中的氯离子细胞内通道及其在心脏保护中的直接作用

• 批准号: 9279247
• 负责人: Harpreet Singh
• 金额: $ 39.11万
• 依托单位: DREXEL UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2016
• 资助国家: 美国
• 起止时间: 2016-07-01 至 2021-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9279247
• 关键词: Ablation; Acids; Address; Affinity; Affinity Chromatography; Agreement; Animal Model; Anions; Antibodies; Biochemical; CASP3 gene; CLIC4 gene; Calcium; Cardiac; Cardiac Myocytes; Cations; Cell Death; Cell Nucleus; Cell membrane; Cells; Chloride Channels; Chlorides; Cleaved cell; Coronary artery; Cyclosporine; Data; Drosophila genus; Drosophila melanogaster; Electron Microscopy; Electrophysiology (science); Environment; Exhibits; Genetic; Geography; Glioma; Heart; Heart Injuries; Homologous Gene; Inner mitochondrial membrane; Intervention; Investigation; Ion Channel; Ion Channel Protein; Ischemia; Ischemic Preconditioning; Knock-out; Knockout Mice; Mammals; Mass Spectrum Analysis; Mediating; Medicine; Microscopy; Mitochondria; Mitochondrial Swelling; Modeling; Molecular; Mus; Myocardial Infarction; Myocardial Ischemia; Organ; Outcome; Oxygen; Permeability; Pharmacological Treatment; Pharmacology; Pharmacology Study; Physiology; Play; Potassium Channel; Production; Property; Proteins; Pulmonary Hypertension; Rattus; Reactive Oxygen Species; Regulation; Reperfusion Injury; Reperfusion Therapy; Resolution; Respiration; Rodent; Role; Shock; Signal Transduction; Structure; System; Testing; Translating; Tube; biophysical properties; conditioning; deprivation; design; experimental study; fly; genetic approach; in vivo; interdisciplinary approach; keratinocyte; macrophage; molecular targeted therapies; mouse model; mutant; paralogous gene; patch clamp; preconditioning; prevent; programs

Cardioprotection from ischemic as well as pharmacological pre- and post- conditioning after ischemia and reperfusion (IR) injury in animal models is highly successful. Ion channels located in the mitochondria are projected as key targets for genetic and pharmacological interventions to protect the heart from IR injury. Supporting the cardioprotective role of mitochondrial ion channels, several potassium channels located at inner mitochondrial membrane have been identified and shown to be directly involved in cardioprotection from IR injury. However the molecular identity of chloride channels localized to inner mitochondrial membrane, a key anion in mitochondria, and their direct roles in mitochondrial physiology are not elucidated. Pharmacological treatment of hearts with indanyloxyacetic acid 94 (IAA-94), a known chloride intracellular channel proteins (CLICs) blocker, before IR abolished the cardioprotective effect of ischemic-preconditioning (IPC) and cyclosporine A (CsA) implicating CLIC proteins in cardioprotection. CLICs are dimorphic class of ion channel proteins with six mammalian orthologues (CLIC1-6) and a single Drosophila homolog (DmCLIC). They were identified by affinity purification using IAA-94 and channel activity of CLICs was blocked by IAA-94 and affinity purified antibodies, and promoted by low pH. We have found that IAA-94 can increase myocardial infarction in ex vivo and in vivo animal models, and also modulate mitochondrial reactive oxygen species (ROS) and capacity for calcium (CRC). Our genetic approach using CLIC null mutant flies indicated that ablation of DmCLIC protect the heart from IR injury in agreement with protective role of mammalian CLICs (CLIC4) in pulmonary hypertension. This contrasting result could arise from non-specificity of IAA-94 and/or it’s in ability to differentiate between CLIC1-6. We discovered that DmCLIC and mammalian CLIC4 and CLIC5 localizes to the cardiac mitochondria. Even though our approaches implicate CLICs in cardioprotection and mitochondrial function, the lack of direct evidence of role of mammalian CLICs, prevent them from being proposed as targets for cardioprotection. Thus, we will test the hypothesis that: 1) CLIC4 and CLIC5 proteins localize to cardiac mitoplasts; 2) where they play a direct role in cardioprotection from IR injury by getting activated due to changes in intracellular pH (pHi) during ischemia; and 3) contribute to cardioprotection by regulating mitochondrial volume, ROS, CRC and permeability transition pore (mPTP) opening. A multidisciplinary approach with cardiac specific conditional knock out mice will test our hypothesis in the following specific aims to: 1) identify molecular correlates of cardiac mitochondrial CLICs, 2) directly address the role of cardiac mitochondrial CLICs in cardioprotection, and 3) establish the role of cardiac CLICs in mitochondrial structure and function. The outcomes of this program will open the opportunity to study cardiac chloride channels at the molecular level and advance the cardiac field by: providing identity of cardiac mitochondrial chloride channel(s), defining its role in cardioprotection and possibly a mitochondrial-mediated mechanism.

抗缺血保护以及缺血后的药理学预处理和后处理， 在动物模型中的再灌注（IR）损伤是非常成功的。位于线粒体中的离子通道 预计作为遗传和药物干预的关键目标，以保护心脏免受IR损伤。 支持线粒体离子通道的心脏保护作用，几个钾通道位于内部 线粒体膜已被鉴定并显示直接参与IR的心脏保护 损伤然而，氯离子通道的分子身份定位于线粒体内膜，这是一个关键。 线粒体中的阴离子，以及它们在线粒体生理学中的直接作用尚未阐明。药理 用茚满基氧基乙酸94（IAA-94）治疗心脏，所述茚满基氧基乙酸94（IAA-94）是已知的氯离子细胞内通道蛋白 （CLIC）阻断剂，在IR之前取消缺血预处理（IPC）的心脏保护作用， 环孢菌素A（CsA）涉及CLIC蛋白在心脏保护。CLIC是一类二型的离子通道 蛋白质与六种哺乳动物直系同源物（CLIC 1 -6）和一种果蝇同源物（DmCLIC）。他们 通过使用IAA-94的亲和纯化来鉴定CLIC，并且CLIC的通道活性被IAA-94阻断，并且亲和纯化的CLIC的通道活性被IAA-94阻断。 我们已经发现，IAA-94可以增加心肌梗死， 离体和体内动物模型，并且还调节线粒体活性氧（ROS）， 钙容量（CRC）。我们使用CLIC无效突变果蝇的遗传方法表明， DmCLIC保护心脏免受IR损伤，这与哺乳动物CLIC（CLIC 4）的保护作用一致。 肺动脉高压。这种相反的结果可能是由于IAA-94的非特异性和/或它能够 CLIC 1 -6之间的区别。我们发现DmCLIC和哺乳动物CLIC 4和CLIC 5定位于 心肌线粒体尽管我们的方法涉及CLIC在心脏保护和线粒体 功能，缺乏哺乳动物CLIC作用的直接证据，阻止它们被提议作为靶点 保护心脏因此，我们将检验以下假设：1）CLIC 4和CLIC 5蛋白定位于心脏， 线粒体; 2）它们在心脏保护中起直接作用，通过由于 缺血期间细胞内pH（pHi）的变化;以及3）通过调节 线粒体体积、ROS、CRC和渗透性转换孔（mPTP）开放。一个多学科 采用心脏特异性条件性敲除小鼠的方法将在以下特定目的中检验我们的假设 目的：1）鉴定心脏线粒体CLIC的分子相关性，2）直接解决心脏线粒体CLIC的作用， 线粒体CLIC在心脏保护中的作用，以及3）建立心脏CLIC在线粒体结构中的作用 和功能该计划的结果将打开机会，研究心脏氯离子通道， 分子水平和推进心脏领域：提供心肌线粒体氯化物的身份 通道，确定其在心脏保护中的作用，并可能是一种神经介导的机制。