Skeletal Muscle. Ca Release Control Inside the Sarcoplasmic Reticulum.

描述(申请人提供)：CA2+信号是细胞用来反应和改变的通用语言。在骨骼肌中，它的兴趣模式涵盖多个时间尺度：毫秒--决定收缩和放松的钙运动--；几秒到几分钟--持续活动可能导致肌源性疲劳--以及几小时到几周--导致基因表达和长期适应变化的模式--。这项研究是关于它的细胞内存储，它的数量，它的浓度，[钙]SR，在每个时间尺度上调节钙信号。我们询问(1)[Ca~(2+)]SR是否以及如何控制细胞内的Ca~(2+)释放，以及(2)钙调素和Triadin这两个位于战略位置的SR蛋白是否以及如何参与这一控制。一个技术任务，我们称之为“目标0”，是成像和测量[Ca~(2+)]SR。这是在当前阶段完成的，并将在下一阶段继续，使用新型生物传感器-由细胞本身制造的分子-以及新的混合监测器，由放置在细胞中的高性能小型合成传感器组成，这些传感器被操纵以制造特殊的生物锚。为了回答问题1和2，我们将分别在测量的同时操纵[Ca~(2+)]SR(目标1)，并迫使细胞改变其钙固缩蛋白和Triadin的天赋(目标2)。这些目标现在在活着的动物身上是可行的，这要归功于一种DNA转基因方法，这种方法可以处理每一种蛋白质，也可以用来阻止它们的合成。我们认为，当肌肉疲劳时，[Ca~(2+)]SR会衰退，这是由SOCE维持的，SOCE是一种普遍的Ca~(2+)进入途径，对动员控制基因表达的转录因子至关重要。使用第一阶段开发的SOCE方法，我们提出目标3来定义新发现的SOCE分子在控制[Ca2+]SR中的作用。这些分子可能是抵抗疲劳的堡垒，并为在迭代方法中改变[Ca~(2+)]SR以解决主要问题提供强大的工具。在疾病、疲劳和衰老过程中，钙信号会恶化。快速钙信号在低PP、MH易感性、中央核心区和微核区等疾病以及老化的肌肉中都会失效。中期信号在疲劳和缺乏钙固醇素的MH样表型小鼠中受到影响。长期钙信号在肌肉和免疫系统中显示出惊人的相似之处的疾病包括SCID，这是一种家族性免疫缺陷，结合了淋巴细胞中SOCE的丢失和肌源性肌病。我们的工作将通过评估特定分子的作用及其相互作用来促进对这些缺陷的理解。虽然在本项目中将只具体讨论疲劳，但随着我们了解什么控制储存钙，以及储存的钙控制什么，关于功能缺陷、复杂的病理生理学和治疗矫正的合理设计之间的关系的问题将被更好地解决。与公共健康相关：这个项目和我们实验室的工作涉及肌肉中钙的运动。所谓的“钙信号”是细胞用来做出反应和改变的通用语言。在骨骼肌中，这些信号决定了休息和运动，从而影响了我们生活的方方面面，包括身体调节、新陈代谢，甚至体重。因为钙被用于许多任务，包括杀死细胞，信号是危险的，必须保持在控制之下。在这个项目中，我们问是什么控制了钙的运动，特别是关注细胞内钙存储区域的作用。我们还会问，店内钙是如何决定移动多少的，或者移动何时会停止。因此，我们的工作应该有助于理解信号失灵或失控的原因。这会发生在疾病中，包括肌肉营养不良、恶性体温过高等。信号在疲劳过程中也会衰减--它们的衰减实际上会导致疲劳--以及在老年时期。由于肌肉疲劳，商店里的东西都用完了。我们认为，两个新发现的分子是延缓这种耗尽的重要因素。已知这些分子在肌肉以外的器官和细胞中发挥作用，包括对抗感染的细胞。因此，它们的缺陷或失败将产生多重影响。因此，可以预见，我们的研究将产生肌肉和运动领域以外的影响。

DESCRIPTION (provided by applicant): Ca2+ signaling is a universal language used by cells to react and change. In skeletal muscle its patterns of interest cover multiple time scales: milliseconds -Ca2+ movements that determine contraction and relaxation-; seconds to minutes -when sustained activity may lead to myogenic fatigue- and hours to weeks -patterns that cause changes in gene expression and long-term adaptation-. This study is about inside its cellular store; its quantity, and its concentration, [Ca2+]SR, which conditions Ca2+ signals in every time scale. We ask (1) whether and how [Ca2+]SR controls Ca2+ release from the store, and (2) whether and how calsequestrin and triadin, two strategically located SR proteins, contribute to this control. A technical task, which we call "aim 0", is to image and measure [Ca2+]SR. This was accomplished in the current period and will continue in the next, using novel biosensors -molecules made by the cells themselves- and new hybrid monitors, consisting of high performance small synthetic sensors placed into cells manipulated to make special bio-anchors. To answer questions 1 and 2, we will respectively manipulate [Ca2+]SR while we measure it (aim 1) and force cells to change their endowment of calsequestrin and triadin (aim 2). These goals are now feasible in living animals thanks to a DNA transfection method that works with every protein and can be used also to prevent their synthesis. We propose that [Ca2+]SR -which decays when muscles fatigue- is sustained by SOCE, a universal Ca2+ entry pathway, crucial for mobilizing transcription factors that control gene expression. Using SOCE measures developed in the first period, we propose as aim 3 to define the role of newly discovered molecules of SOCE in the control of [Ca2+]SR. These molecules could be bulwarks against fatigue, and provide powerful tools for experimental alterations of [Ca2+]SR in iterative approaches to the main questions. Ca2+ signals deteriorate in disease, fatigue and aging. Fast Ca2+ signals fail in diseases like hypo-PP, MH susceptibility and central core and minicore, as well as in ageing muscle. Mid-range signaling is affected in fatigue and in an MH-like phenotype of mice lacking calsequestrin. Diseases of long term Ca2+ signals, which show striking parallels in muscle and the immune system, include SCID, a familial immune defect that combines loss of SOCE in lymphocytes and a myogenic myopathy. Our work will advance understanding of these deficits by evaluating roles of specific molecules and their interactions. While only fatigue will be specifically addressed in the present project, questions on the relationships among deficits of function, the intricate pathophysiology and the rational design of therapeutic corrections will be addressed better as we understand what controls stored calcium, and what the stored calcium controls. PUBLIC HEALTH RELEVANCE: This project and our lab's work deal with movements of calcium inside muscle. So-called "calcium signaling" is a universal language used by cells to react and change. In skeletal muscle these signals make the difference between rest and motion, thus influencing multiple aspects of our life, including physical conditioning, metabolism, and even body weight. Because calcium is used for many tasks, including killing cells, the signals are dangerous and must be kept under control. In this project we ask what controls the movements of calcium, in particular focusing on roles of calcium storage areas inside the cell. We also ask how the intra-store calcium determines how much will move, or when the movement will cease. Therefore, our work should help understand why signals fail, or get out of hand. This occurs in diseases, including muscular dystrophies, malignant hyperthermia, and others. Signals also decay during fatigue -their decay actually causing fatigue-- and in old age. In muscle fatigue, the stores are depleted. We propose that two newly discovered molecules are important factors that delay this depletion. These molecules are known to play roles in organs and cells other than muscle, including cells that combat infections. As a result there will be multiple repercussions to their defects or failures. It can be anticipated, therefore, that our research will have consequences outside its field of muscle and exercise.