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

描述（由申请人提供）：线粒体生物能量学对细胞生存和死亡至关重要。生物能量维持主要依赖于线粒体膜的完整性。线粒体内膜的不渗透性为氧化还原反应生成ATP提供了条件。线粒体也通过快速Ca2+缓冲参与细胞质Ca2+表型。Ca2+对线粒体功能的影响有两个方面。生理条件下，Ca2+有利于线粒体功能刺激氧化磷酸化和ATP合成。当线粒体Ca2+ ([Ca2+]m)超载发生时，这些影响是否在病理条件下保持不变是值得怀疑的。虽然[Ca2+]m信号在生理和病理过程中都是至关重要的，但促进[Ca2+]m摄取的分子仍不清楚。[Ca2+]m缓冲是由线粒体膜内转运蛋白、交换蛋白和单转运蛋白精细控制的。一些蛋白参与[Ca2+]m的摄取，包括LETM1， MICU1和MCU。我们的目标RNAi筛选鉴定了一种线粒体内膜蛋白，线粒体Ca2+单转运调节因子1 (MCUR1)，可以增强[Ca2+]m的摄取。MCUR1沉默消除了正常线粒体膜电位下的[Ca2+]m摄取。我们的研究结果表明，MCUR1与线粒体单转运蛋白复合物线粒体Ca2+单转运蛋白（MCU）的Ru360敏感核心组分相互作用。基于我们最近的发现，我们假设MCUR1在I/R损伤期间促进mcu依赖的[Ca2+]m过载，触发线粒体膜去极化，导致生物能量崩溃和线粒体功能障碍。本研究应用RNAi技术、MCUR1诱变和MCU通道、生化、最先进的成像和动物模型系统来了解MCUR1如何诱导心肌细胞[Ca2+]m摄取。基于我们最近确定的MCUR1作为单转运复合物的调节因子，在Aim 1中，我们将描述MCUR1在心肌细胞[Ca2+]m摄取、MCUR1- mcu相互作用的关键区域和MCUR1的转录调节中的作用。在Aim 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Aim 3中，我们将应用心脏缺血/再灌注小鼠体内模型研究来证明MCUR1的敲低可以改善I/ r诱导的线粒体功能障碍和心肌细胞损伤。总的来说，这些研究结果将促进我们对病理生理条件下MCU活性如何增强的理解，并提出控制[Ca2+]m内流的新策略，作为心血管疾病的新治疗方法。

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.