Inhibition of MCUR1-MCU mediated mitochondrial Ca2+ uptake prevents I/R injury

抑制 MCUR1-MCU 介导的线粒体 Ca2 摄取可预防 I/R 损伤

• 批准号: 8824559
• 负责人: MADESH MUNISWAMY
• 金额: $ 38.42万
• 依托单位: TEMPLE UNIV OF THE COMMONWEALTH
• 依托单位国家: 美国
• 财政年份: 2014
• 资助国家: 美国
• 起止时间: 2014-04-01 至 2018-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8824559
• 关键词: ATP Hydrolysis; ATP Synthesis Pathway; Address; Animal Model; Autophagocytosis; Biochemical; Biochemistry; Bioenergetics; Biological Models; Buffers; Cardiac; Cardiac Myocytes; Cardiovascular Diseases; Cell Death; Cell Membrane Permeability; Cell Survival; Cell membrane; Cells; Cellular biology; Charge; Complex; Coupling; Disease; Down-Regulation; Health; Heart; Homeostasis; Hypoxia; Image; Inner mitochondrial membrane; Ion Channel; Ions; Ischemia; Learning; Maintenance; Mediating; Membrane Potentials; Membrane Transport Proteins; Mica; Mitochondria; Mitochondrial Matrix; Mitochondrial Membrane Protein; Molecular; Mus; Mutagenesis; Myocardial Ischemia; Nature; Outcome; Oxidation-Reduction; Oxidative Stress; Pathologic Processes; Pathway interactions; Pattern; Phenotype; Phosphorylation; Physiological; Physiological Processes; Play; Production; Proteins; RNA Interference; Reaction; Reactive Oxygen Species; Regulation; Reperfusion Injury; Reperfusion Therapy; Rest; Role; Shapes; Side; Signal Transduction; Staging; Study models; Technology; Therapeutic Intervention; Tissues; Transcriptional Regulation; Translating; base; calcium uniporter; cell type; driving force; in vivo; in vivo Model; mitochondrial dysfunction; mitochondrial membrane; mitochondrial permeability transition pore; oxidation; prevent; small hairpin RNA; spatiotemporal; uptake

DESCRIPTION (provided by applicant): Mitochondrial bioenergetics is crucial for cell survival and death. The bioenergetic maintenance primarily depends on the integrity of mitochondrial membranes. The impermeable nature of the mitochondrial inner membrane sets the stage for redox reactions to generate ATP. Mitochondria also participate in cytosolic Ca2+ phenotype via rapid Ca2+ buffering. There are two sides to the effects of Ca2+ on mitochondrial function. Under physiological conditions, Ca2+ is beneficial for mitochondrial function to stimulate oxidation-phosphorylation and ATP synthesis. It is questionable whether these effects remain the same under pathological conditions when mitochondrial Ca2+ ([Ca2+]m) overload occurs. While [Ca2+]m signaling is crucial for both physiological and pathological processes, molecules that facilitate [Ca2+]m uptake remain unclear. [Ca2+]m buffering is exquisitely controlled by inner mitochondrial membrane transporters, exchangers and uniporter. Several proteins have been implicated to participate in [Ca2+]m uptake, including LETM1, MICU1 and MCU. Our targeted RNAi screen identified a mitochondrial inner membrane protein, Mitochondrial Ca2+ Uniporter Regulator 1 (MCUR1) that augments [Ca2+]m uptake. MCUR1 silencing abrogates [Ca2+]m uptake under normal mitochondrial membrane potential. Our results demonstrate that MCUR1 interacts with the Ru360 sensitive core component of the mitochondrial uniporter complex, Mitochondrial Ca2+ Uniporter (MCU). Based on our recent discovery, we hypothesize that MCUR1 promotes MCU-dependent [Ca2+]m overload during I/R injury, triggering mitochondrial membrane depolarization, that results in bioenergetic collapse and mitochondrial dysfunction. This proposal applies RNAi technology, mutagenesis of MCUR1 and MCU channel, biochemical, state-of-the-art imaging and an animal model system to understand how MCUR1 elicits cardiomyocyte [Ca2+]m uptake. Based on our recent identification of MCUR1 as a regulator of the uniporter complex, here in Aim 1, we will characterize the MCUR1 role in cardiomyocyte [Ca2+]m uptake, critical regions of MCUR1-MCU interaction and transcriptional regulation of MCUR1. In Aim 2 we will investigate how MCUR1 controls mitochondrial bioenergetics, ROS production and autophagy. Finally, in Aim 3 we will apply cardiac ischemia/reperfusion in vivo murine model studies to show that knockdown of MCUR1 ameliorates I/R-induced mitochondrial dysfunction and cardiomyocyte damage. Overall, the results of these studies will advance our understanding of how MCU activity is augmented under pathophysiological conditions, and suggest new strategies for controlling [Ca2+]m influx as a new treatment for cardiovascular diseases.

描述(申请人提供)：线粒体生物能量学对细胞的生存和死亡至关重要。生物能量的维持主要依赖于线粒体膜的完整性。线粒体内膜的不透性为氧化还原反应生成三磷酸腺苷奠定了基础。线粒体也通过快速的钙缓冲参与细胞内的钙表型。钙离子对线粒体功能的影响有两个方面。在生理条件下，Ca~(2+)有利于线粒体的氧化-磷酸化和ATP合成。在病理条件下，当线粒体钙超载时，这些效应是否保持不变是值得怀疑的。虽然[Ca~(2+)]_m信号在生理和病理过程中都是至关重要的，但促进[Ca~(2+)]_m摄取的分子仍不清楚。线粒体内膜转运体、交换器和单转运体精确地控制着[Ca~(2+)]m的缓冲。有几种蛋白质参与了钙离子的摄取，包括LETM1、MICU1和MCU。我们的靶向RNAi筛选发现了一种线粒体内膜蛋白，线粒体钙统一转运体调节器1(MCUR1)，它能增加[Ca+]m的摄取。在正常线粒体膜电位下，MCUR1沉默可抑制[Ca~(2+)]_m的摄取。我们的结果表明，MCUR1与线粒体单一转运蛋白复合体中Ru360敏感的核心成分--线粒体钙统一转运蛋白(MCU)相互作用。根据我们最近的发现，我们假设MCUR1在I/R损伤过程中促进MCU依赖的[Ca~(2+)]m超载，触发线粒体膜去极化，导致生物能崩溃和线粒体功能障碍。这项建议应用RNAi技术、MCUR1和MCU通道的突变、生化、最先进的成像技术和动物模型系统来了解MCUR1如何诱导心肌细胞[Ca~(2+)]m摄取。根据我们最近发现的MCUR1作为单转运蛋白复合体的调节因子，在目标1中，我们将描述MCUR1在心肌细胞[Ca2+]m摄取中的作用，MCUR1-MCU相互作用的关键区域以及MCUR1的转录调控。在目标2中，我们将研究MCUR1如何控制线粒体生物能量学、ROS产生和自噬。最后，在目标3中，我们将应用在体小鼠心脏缺血/再灌注模型研究表明，敲除MCUR1可以改善I/R诱导的线粒体功能障碍和心肌细胞损伤。总体而言，这些研究的结果将促进我们对MCU活性在病理生理条件下如何增强的理解，并为控制[Ca+]m内流作为心血管疾病的新治疗方法提供新的策略。