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.

缺血前后的缺血前后，缺血后的药物保护以及 动物模型中的再灌注（IR）损伤非常成功。线粒体中的离子通道是 预计是保护心脏免受IR损伤的遗传和药物干预措施的关键目标。 支持线粒体离子通道的心脏保护作用，位于内部的几个钾通道 线粒体膜已被鉴定，并显示出直接参与IR的心脏保护 受伤。然而 线粒体的阴离子及其在线粒体生理学中的直接作用未阐明。药理 用吲哚美乙酸94（IAA-94）的心脏处理，一种已知的氯化物内通道蛋白 （CLICS）BLOCKER，在IR废除缺血性前提的心脏保护作用（IPC）和 在心脏保护中，环孢菌素A（CSA）隐式clic蛋白。库是离子通道的二态类 具有六个哺乳动物直系同源物（CLIC1-6）和一个果蝇同源物（DMCLIC）的蛋白质。他们是 通过使用IAA-94的亲和力纯化识别，CLICS的通道活动被IAA-94和亲和力阻止 纯化的抗体，并通过低pH促进。我们发现IAA-94可以增加心肌梗塞 离体和体内动物模型，还调节线粒体活性氧（ROS）和 钙的能力（CRC）。我们使用Clic Null突变蝇的遗传方法表明 DMCLIC保护心脏免受IR损伤，同意哺乳动物插曲（CLIC4）在 肺动脉高压。这种对比结果可能是由IAA-94的非特异性和/或具有能力引起的 区分Clic1-6。我们发现dmclic和哺乳动物clic4和clic5定位于 心脏线粒体。即使我们的方法牵涉到心脏保护和线粒体中的策略 功能，缺乏直接证据表明哺乳动物库的作用，阻止它们被提出为目标 用于心脏保护。那就是我们将测试以下假设：1）CLIC4和CLIC5蛋白本地化为心脏 线粒体； 2）由于由于因IR损伤而受到激活而在心脏保护中发挥直接作用 缺血期间细胞内pH（PHI）的变化； 3）通过调节为心脏保护做出贡献 线粒体体积，ROS，CRC和渗透率过渡孔（MPTP）开口。多学科 与心脏特异性有条件敲除小鼠的方法将在以下特定目的中检验我们的假设 至：1）确定心脏线粒体插曲的分子相关性，2）直接解决心脏的作用 线粒体在心脏保护方面，3）确定心脏库在线粒体结构中的作用 和功能。该计划的结果将为研究心脏氯化物通道的机会 分子水平并通过：提供心脏线粒体氯化物的身份。 通道，定义其在心脏保护中的作用，并可能是线粒体介导的机制。