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

基本信息

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
项目类别：
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

项目摘要

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.

缺血后的心脏保护作用以及缺血后的药理学预处理和后处理位于线粒体的离子通道在动物模型中的再灌注（IR）损伤非常成功。预计将成为保护心脏免受红外线损伤的遗传和药物干预的关键目标。支持线粒体离子通道的心脏保护作用，多个钾通道位于线粒体内部线粒体膜已被鉴定并证明直接参与 IR 的心脏保护然而，氯离子通道的分子特性定位于线粒体内膜，这是一个关键。线粒体中的阴离子及其在线粒体生理学中的直接作用尚未阐明。用茚满氧基乙酸 94 (IAA-94)（一种已知的氯离子细胞内通道蛋白）治疗心脏 (CLIC) 阻滞剂，在 IR 取消缺血预适应 (IPC) 的心脏保护作用之前环孢菌素 A (CsA) 涉及 CLIC 蛋白的心脏保护作用 CLIC 是离子通道的二态性。具有六个哺乳动物直向同源物 (CLIC1-6) 和一个果蝇同源物 (DmCLIC) 的蛋白质。使用 IAA-94 通过亲和纯化进行鉴定，并且 CLIC 的通道活性被 IAA-94 和亲和力阻断纯化的抗体，并通过低pH值促进我们发现IAA-94可以增加心肌梗死的发生。离体和体内动物模型，还调节线粒体活性氧 (ROS) 和我们使用 CLIC 无效突变果蝇的遗传方法表明，钙的消融能力。 DmCLIC 可保护心脏免受 IR 损伤，这与哺乳动物 CLIC (CLIC4) 的保护作用一致这种对比结果可能是由于 IAA-94 的非特异性和/或它无法做到这一点。我们发现 DmCLIC 和哺乳动物 CLIC4 和 CLIC5 定位于尽管我们的方法意味着 CLICs 用于心脏保护和线粒体。功能，缺乏哺乳动物 CLIC 作用的直接证据，阻碍了它们被提议作为目标因此，我们将检验以下假设：1) CLIC4 和 CLIC5 蛋白定位于心脏。 2) 线粒体通过被激活而在心脏免受 IR 损伤中发挥直接作用缺血期间细胞内 pH (pHi) 的变化；3) 通过调节有助于心脏保护线粒体体积、ROS、CRC 和通透性转换孔 (mPTP) 开放。使用心脏特异性条件敲除小鼠的方法将在以下特定目标中检验我们的假设目的：1) 识别心脏线粒体 CLIC 的分子相关性，2) 直接解决心脏线粒体的作用线粒体 CLIC 在心脏保护中的作用，3) 确定心脏 CLIC 在线粒体结构中的作用该计划的成果将为研究心脏氯离子通道提供机会。分子水平并通过以下方式推进心脏领域：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以及可能的线粒体介导的机制。