Mitochondria and calcium signaling in skeletal muscle

描述(申请人提供)：钙离子控制骨骼肌中的许多细胞过程，钙离子稳态的变化与人类疾病有关，如杜氏肌营养不良症(DMD)、恶性高热(MH)和中央核心疾病(CCD)。确定调控细胞内钙信号的分子机制是开发这些肌病新的治疗干预措施的关键一步。肌浆网(SR)通过钙离子释放通道(RyRs)释放钙是骨骼肌兴奋收缩偶联(ECC)的关键步骤。它是通过质膜电压传感器与RyRs的直接相互作用而被触发的，被认为是通过钙离子诱导的钙释放(CICR)而被放大，表现为钙火花。然而，成熟的哺乳动物肌肉在生理ECC过程中不显示钙火花，但它在各种病理生理条件下产生自发的火花活动。导致哺乳动物肌肉中钙离子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钙稳态变化的先决条件。我们的数据表明，活性氧和氮(ROS/RNS)和线粒体是骨骼肌细胞内钙信号的关键调节因子。他们把我们引向了以下假设：1)。在生理条件下，火花的出现受到维持RyR1活性的胞浆环境的减少和线粒体钙摄取的抑制。2)。细胞内钙离子水平的升高通过线粒体钙超载和/或其他细胞来源刺激ROS/RNS的产生来促进ROS/RNS的产生。3)。ROS/RNS通过增强RyR1的钙释放活性和/或通过抑制线粒体的钙摄取来刺激火花的产生。4)。MH细胞内钙水平升高是由于SR钙泄漏，DMD细胞内钙水平升高是由于细胞内钙内流增加。在这两种疾病中，细胞内钙离子增加的结果是：a)ROS/RNS的产生增加；b)RyR1的氧化修饰；c)修改后的RyR1对钙的敏感性增强；d)出现钙火花。为了验证这些假设，我们将使用电生理方法和最先进的成像技术(单光子和双光子共焦成像、数字测光、笼状化合物的紫外激光闪光光解)来实现以下具体目标。我们建议：1)。确定生理条件下肌肉细胞内钙信号、线粒体钙摄取和ROS/RNS生成的机制。2)。明确改变的ROS/RNS生成如何影响MH易感和MDX小鼠(DMD的小鼠模型)肌肉中的细胞钙稳态。与公共健康相关：从细胞内钙库释放钙是兴奋-收缩偶联的关键步骤。骨骼肌兴奋收缩偶联的改变与低血钾性周期性麻痹、恶性高热和中枢性疾病等人类疾病有关，因此，明确ECC中主要事件的分子调控机制对于开发针对这些疾病的新的治疗措施至关重要。这些实验将为我们提供有关骨骼肌中线粒体对钙信号的控制的新信息，并使我们更进一步地了解与代谢和线粒体功能障碍以及钙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.