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

描述（由申请人提供）：在心肌中，兴奋-收缩（EC）偶联期间线粒体对Ca 2+的摄取对于使ATP产生与收缩需求同步（兴奋-生物能量学（EB）偶联）非常重要。然而，一个综合的机制来描述EB耦合是失踪，主要是由于缺乏信息的分子身份的几个关键蛋白质参与这一过程。最近的突破性研究表明，线粒体融合蛋白2（Mfn 2）负责将内质网拴系到线粒体。此外，线粒体Ca ~（2+）单向转运体（mtCU）包括其孔单元（MCU）的几种组分已被发现。这些进展开辟了一个新的机会，应用分子工具来阐明的机制，线粒体-肌浆网（MITO-SR）的束缚在控制生物能量学和钙动力学。我们的实验室是第一个显示Ca 2+从SR到心肌细胞线粒体的特权转运的实验室，这是由于它们的并置，通过与Mfn 2家族蛋白的束缚来保证。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Ca 2+和活性氧（ROS）调节方面具有长期的专业知识，Csordas博士在使用生物化学和成像技术研究MITO-SR束缚和局部Ca 2+串扰方面具有良好的记录。总之，我们将联合收割机这些跨学科的方法来测试的假设，通过Mfn 2家族蛋白质的MITO-SR拴在EC耦合过程中创建一个高钙离子微域之间的这两个细胞器。此外，mtCU聚集在与SR接近的线粒体内膜（IMM）区域。这种并置的损失降低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）识别桥接MITO-SR关联的系留组件。假设：Mfn 2，可能是一种截短的形式，将SR与线粒体接触点对齐。2)确定mtCU在IMM中的分布。假设：线粒体CU优先定位于线粒体和SR接触的区域。3)阐明线粒体-SR结合破坏导致心力衰竭的机制。假设：MITO-SR结合的丧失导致EB偶联的无效性，其结果是电子传递链活性和基质NADPH水平降低，这导致ROS增加。ROS的增加和ATP的减少增加了线粒体通透性转换孔开放的敏感性，特别是在能量需求应激下，导致心肌损伤和衰竭。线粒体Ca 2+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，所述临床表型包括心脏病（例如HF）、神经退行性疾病、代谢疾病（糖尿病）和衰老。由于MITO-SR并置是控制线粒体Ca 2+动力学的关键因素，因此本提案将提出心脏MITO-SR束缚的分子机制，并将这种独特的结构转化为生物能量学中线粒体Ca 2+内流的生理调节以及HF中能量缺乏和氧化应激的病理意义，这具有科学重要性和临床意义。

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.