Mitochondria and calcium signaling in skeletal muscle

描述（由申请人提供）：Ca2+控制骨骼肌中的许多细胞过程，Ca2+稳态的改变与杜氏肌营养不良（DMD）、恶性高热（MH）和中央核心疾病（CCD）等人类疾病有关。确定调节细胞内Ca2+信号的分子机制是在这些肌病中开发新的治疗干预措施的关键一步。钙离子通过钙离子释放通道（ryanodine receptor, RyRs）从肌浆网（SR）释放是骨骼肌兴奋-收缩耦合（ECC）的关键步骤。它是通过等离子体电压传感器与ryr的直接相互作用触发的，并且被认为是由Ca2+诱导的Ca2+释放（CICR）放大的，表现为Ca2+火花。然而，成熟的哺乳动物肌肉在生理ECC中不显示Ca2+火花，但在各种病理生理条件下会自发产生火花活动。导致哺乳动物肌肉中Ca2+火花产生的分子事件尚不清楚。了解这些机制是预防与许多人类肌肉疾病相关的Ca2+稳态变化的先决条件。我们的数据表明活性氧和氮物种（ROS/RNS）和线粒体是骨骼肌细胞内Ca2+信号传导的关键调节因子。它们使我们得出以下假设：1)。在生理条件下，电火花的出现受到胞质环境减少（维持RyR1的低活性）和线粒体Ca2+摄取的抑制。2）. 增加的细胞质Ca2+水平通过线粒体Ca2+超载和/或其他细胞来源刺激ROS/RNS产生促进ROS/RNS的产生。3)。ROS/RNS通过增强RyR1的Ca2+释放活性和/或抑制线粒体Ca2+摄取来刺激火花产生。4)。在MH中，由于SR Ca2+泄漏，细胞质Ca2+水平升高，而在DMD中，由于Ca2+内流增加。在这两种疾病中，细胞内Ca2+增加的结果是：a)增强ROS/RNS产生b)氧化修饰RyR1， c)增强修饰RyR1的Ca2+敏感性和d) Ca2+火花的出现。为了验证这些假设，我们将使用电生理学方法和最先进的成像技术（单光子和双光子共聚焦成像、数字光度测定、笼状化合物的紫外激光闪光光解）进行以下具体目标。我们建议：1)。确定生理条件下肌内胞质Ca2+信号、线粒体Ca2+摄取和ROS/RNS生成的机制。2）. 定义改变的ROS/RNS生成如何影响mh易感小鼠和mdx小鼠（DMD小鼠模型）肌肉中的细胞Ca2+稳态。公共卫生相关性：Ca2+从细胞内Ca2+储存释放是兴奋-收缩耦合的关键步骤。骨骼肌兴奋-收缩耦合的改变与人类疾病有关，如低钾性周期性麻痹、恶性高热和中央核心疾病，因此，确定调控ECC主要事件的分子机制对于开发针对这些疾病的新治疗干预措施至关重要。提出的实验将为我们提供关于骨骼肌中Ca2+信号的线粒体控制的新信息，并使我们更接近于理解与代谢和线粒体功能障碍以及Ca2+处理不当相关的一系列遗传性和获得性肌肉疾病。

DESCRIPTION (provided by applicant): Ca2+ controls numerous cellular processes in skeletal muscle and alterations in Ca2+ homeostasis are associated with human diseases such as Duchenne Muscular Dystrophy (DMD), Malignant Hyperthermia (MH) and Central Core Disease (CCD). Defining the molecular mechanisms regulating intracellular Ca2+ signaling is a crucial step for developing new therapeutic interventions in these myopathies. The release of Ca2+ from sarcoplasmic reticulum (SR) via Ca2+ release channels (ryanodine receptors, RyRs) is a key step in skeletal muscle excitation-contraction coupling (ECC). It is triggered through a direct interaction of the plasmalemmal voltage sensors with RyRs and it is thought to be amplified by Ca2+-induced Ca2+ release (CICR), manifest as Ca2+ sparks. However, mature mammalian muscle does not display Ca2+ sparks during physiological ECC but it develops spontaneous spark activity under various pathophysiological conditions. The molecular events that lead to Ca2+ spark generation in mammalian muscle are unknown. Understanding these mechanisms is a prerequisite to prevent changes in Ca2+ homeostasis associated with a number of human muscle diseases. Our data suggest that reactive oxygen and nitrogen species (ROS/RNS) and mitochondria are key regulators of intracellular Ca2+ signaling in skeletal muscle. They have led us to the following hypotheses: 1). Under physiological conditions, the appearance of sparks is suppressed by reduced cytosolic environment, which maintains a low activity of RyR1, and by mitochondrial Ca2+ uptake. 2). Increased cytosolic Ca2+ levels promote ROS/RNS production through mitochondrial Ca2+ overload and/or stimulation of ROS/RNS production by other cellular sources. 3). ROS/RNS stimulate spark production by enhancing the Ca2+ release activity of RyR1 and/or by inhibiting mitochondrial Ca2+ uptake. 4). Cytosolic Ca2+ levels are elevated in MH due to SR Ca2+ leak, and in DMD due to increased Ca2+ influx. In both disorders, the outcome of increased cytosolic Ca2+ is: a) enhanced ROS/RNS production b) oxidative modification of RyR1, c) enhanced Ca2+ sensitivity of the modified RyR1 and d) the appearance of Ca2+ sparks. To test these hypotheses, we will carry out the following Specific Aims using electrophysiological methods and state-of-the-art imaging techniques (single and two-photon confocal imaging, digital photometry, UV-laser flash photolysis of caged compounds). We propose to: 1). Determine the mechanisms connecting cytosolic Ca2+ signals, mitochondrial Ca2+ uptake and ROS/RNS generation in muscle under physiological conditions. 2). Define how altered ROS/RNS generation affect cellular Ca2+ homeostasis in muscle from MH-susceptible and mdx mice (a mice model of DMD). PUBLIC HEALTH RELEVANCE: Release of Ca2+ from intracellular Ca2+ stores is a key step of excitation-contraction coupling. Alterations in skeletal muscle excitation-contraction coupling are associated with human diseases such as Hypokalemic Periodic Paralysis, Malignant Hyperthermia and Central Core Disease and, hence, defining the molecular mechanisms regulating the primary event in ECC is crucial for developing new therapeutic interventions in these diseases. The proposed experiments will provide us with new information about the mitochondrial control of Ca2+ signaling in skeletal muscle, and bring us one step closer to the understanding of a whole range of hereditary and acquired muscle disorders associated with metabolic and mitochondrial dysfunctions and Ca2+ mishandling.