Mitochondria and calcium signaling in skeletal muscle

描述（由申请人提供）：Ca 2+控制骨骼肌中的许多细胞过程，Ca 2+稳态的改变与人类疾病如杜氏肌营养不良症（DMD）、恶性高热（MH）和中央核心病（CCD）相关。确定调节细胞内Ca 2+信号传导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。肌浆网（SR）通过钙释放通道（Ryanodine receptors，RyRs）释放钙离子是骨骼肌兴奋-收缩偶联（ECC）的关键步骤。它是通过质膜电压传感器与RyR的直接相互作用触发的，并且被认为是通过Ca 2+诱导的Ca 2+释放（CICR）放大的，表现为Ca 2+火花。然而，成熟的哺乳动物肌肉不显示Ca 2+火花在生理ECC，但它开发自发火花活动在各种病理生理条件下。导致哺乳动物肌肉中产生Ca 2+火花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Ca 2+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Ca 2+信号转导的关键调节因子。这使我们得出以下假设：（1）。在生理条件下，火花的出现受到细胞质环境的抑制，细胞质环境保持低活性的RyR 1，并通过线粒体Ca 2+摄取。2）。增加的胞质Ca 2+水平通过线粒体Ca 2+过载和/或其他细胞来源刺激ROS/RNS产生来促进ROS/RNS产生。3）。ROS/RNS通过增强RyR 1的Ca 2+释放活性和/或通过抑制线粒体Ca 2+摄取来刺激火花产生。4）。细胞质Ca 2+水平升高，MH由于SR Ca 2+泄漏，DMD由于Ca 2+流入增加。在这两种疾病中，胞质Ca 2+增加的结果是：a）ROS/RNS产生增加B）RyR 1的氧化修饰，c）修饰的RyR 1的Ca 2+敏感性增加和d）Ca 2+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，数字测光，笼状化合物的紫外激光闪光光解）进行以下特定目标。我们建议：1）。确定生理条件下肌肉中细胞溶质Ca 2+信号、线粒体Ca 2+摄取和ROS/RNS产生的机制。2）。定义ROS/RNS产生的改变如何影响MH易感小鼠和mdx小鼠（DMD小鼠模型）肌肉中细胞Ca 2+稳态。

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).