Mitochondria-SR Tethering: Its Role in Cardiac Bioenergetics and Ca2+ Dynamics

描述（由申请人提供）：在心肌中，线粒体在兴奋-收缩（EC）偶联期间对Ca2+的摄取对于同步ATP生产和收缩需求（兴奋-生物能量学（EB）偶联）是重要的。然而，描述EB偶联的综合机制主要是由于缺乏有关该过程中涉及的几个关键蛋白质的分子特性的信息。最近的突破性研究表明，有丝分裂蛋白2 （Mfn2）负责将内质网系在线粒体上。此外，线粒体Ca2+单转运体（mtCU）的几个组成部分，包括其孔单元（MCU）已经被发现。这些进展为应用分子工具阐明线粒体-肌浆网（MITO-SR）拴系在控制生物能量学和Ca2+动力学中的机制开辟了新的机会。我们的实验室首次展示了Ca2+在心肌细胞中从SR到线粒体的特权运输，这是由于它们与Mfn2家族蛋白拴在一起并并而获得的。Sheu博士在使用遗传和生理工具研究体内和体外心脏线粒体Ca2+和活性氧（ROS）调节方面有着长期的专业知识，Csordas博士在使用生化和成像技术研究MITO-SR系结和局部Ca2+串扰方面有着良好的记录。总之，我们将结合这些跨学科的方法来测试假设，通过Mfn2家族蛋白的MITO-SR系固在EC偶联过程中在这两个细胞器之间创建了一个高Ca2+的微结构域。此外，mtcu聚集在靠近sr的线粒体内膜（IMM）区域。这种并置性的丧失降低了EB偶联效率，导致能量缺乏和氧化应激以及随后的心力衰竭（HF）。三个具体目标是：1)确定连接MITO-SR关联的系固组件。假设：Mfn2，可能是截断的形式，将SR与线粒体接触点对齐。2)确定mtCU在IMM中的分布。假设：mtCU优先定位于线粒体和SR接触的区域。3)阐明MITO-SR关联被破坏导致HF的机制。假设：MITO-SR关联缺失导致EB偶联效率低下，导致电子传递链活性和基质NADPH水平降低，从而导致ROS升高。ROS的增加和ATP的减少增加了线粒体通透性过渡孔打开的易感性，特别是在高能量应激下，导致心脏损伤和衰竭。线粒体Ca2+稳态的破坏是导致线粒体功能障碍相关临床表型的关键因素，包括心脏病（如HF）、神经退行性疾病、代谢性疾病（糖尿病）和衰老。由于MITO-SR并立是控制线粒体Ca2+动力学的关键因素，因此，本研究将揭示心肌MITO-SR系立的分子机制，并将这种独特的结构转化为生物能量学中线粒体Ca2+内流的生理调节，以及HF中能量缺乏和氧化应激的病理含义，具有重要的科学意义和临床意义。

DESCRIPTION (provided by applicant): In cardiac muscle, uptake of Ca2+ by mitochondria during the excitation-contraction (EC) coupling is important for synchronizing ATP production with the needs of contraction (excitation-bioenergetics (EB) coupling). However, an integrative mechanism to describe the EB coupling is missing mainly due to the lack of information about the molecular identities of several key proteins involved in this process. Recent ground-breaking studies have shown that mitofusin 2 (Mfn2) is responsible for tethering endoplasmic reticulum to mitochondria. Moreover, several components of the mitochondrial Ca2+ uniporter (mtCU) including its pore unit (MCU) have been uncovered. These progresses open up a new opportunity for applying molecular tools to elucidate the mechanisms of mitochondria-sarcoplasmic reticulum (MITO-SR) tethering in controlling bioenergetics and Ca2+ dynamics. Our labs were the first to show a privileged transport of Ca2+ from SR to mitochondria in cardiomyocytes due to their juxtaposition, secured by tethering with Mfn2 family proteins. Dr. Sheu has a long standing expertise in using genetic and physiological tools to study in and ex vivo the cardiac mitochondrial Ca2+ and reactive oxygen species (ROS) regulation and Dr. Csordas has a strong track record in using biochemical and imaging techniques to investigate MITO-SR tethering and local Ca2+ crosstalk. Together, we will combine these interdisciplinary approaches to test the hypothesis that MITO-SR tethering via Mfn2 family proteins creates a micro-domain of high Ca2+ between these two organelles during EC coupling. Moreover, mtCUs are clustered in the region of inner mitochondrial membrane (IMM) that is in proximity with SR. Losses of this juxtaposition decrease EB coupling efficiency that leads to energy deficiency and oxidative stress and subsequent heart failure (HF). Three specific aims are: 1) to identify the tethering components that bridge MITO-SR associations. Hypothesis: Mfn2, possibly a truncated form, aligns SR with mitochondrial contact points. 2) To determine the distribution of mtCU in the IMM. Hypothesis: mtCU is preferentially localized in the areas where mitochondria and SR are in contact. 3) To elucidate the mechanisms by which the disrupted MITO-SR association leads to HF. Hypothesis: The loss of MITO-SR association leads to the inefficiency of EB coupling, as a result, electron transport chain activities and matrix NADPH levels decrease, which cause ROS to increase. The increase in ROS together with the decrease in ATP enhances the susceptibility of mitochondrial permeability transition pore for opening, especially under the energy-demanding stresses, which leads to cardiac injury and failure. The destruction of mitochondrial Ca2+ homeostasis is a key element for leading to mitochondrial dysfunction-associated clinical phenotypes including heart diseases (e.g. HF), neurodegenerative diseases, metabolic diseases (diabetes), and aging. Because MITO-SR juxtaposition is a critical factor in controlling mitochondrial Ca2+ dynamics, it is of scientific importance and clinical relevance that the present proposal will bring forth the molecular mechanism underlying the cardiac MITO-SR tethering and translate this unique structure to the physiological regulation of mitochondrial Ca2+ influx in bioenergetics and to the pathological implication of energy deficiency and oxidative stress in HF.