Skeletal Muscle. Ca Release Control Inside the Sarcoplasmic Reticulum.

描述（由申请人提供）：Ca 2+信号传导是细胞用于反应和变化的通用语言。在骨骼肌中，其感兴趣的模式涵盖多个时间尺度：毫秒-决定收缩和放松的Ca 2+运动-;秒到分钟-持续活动可能导致肌源性疲劳-以及数小时到数周-导致基因表达和长期适应变化的模式。本研究是关于它的细胞内储存，它的数量，和它的浓度，[Ca 2 +]SR，它在每个时间尺度上的条件Ca 2+信号。我们问（1）是否和如何[Ca 2 +]SR控制Ca 2+从存储的释放，和（2）是否和如何calsequestrin和triadin，两个战略定位SR蛋白，有助于这种控制。我们称之为“aim 0”的技术任务是成像和测量[Ca 2 +] SR。这一任务在本期完成，并将在下一个时期继续，使用新型生物传感器-由细胞本身制造的分子-和新的混合监测器，包括放置在细胞中的高性能小型合成传感器，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[Ca 2 +]SR时对其进行操作（目的1），并迫使细胞改变其钙螯合蛋白和三聚体的禀赋（目的2）。这些目标现在在活体动物中是可行的，这要归功于一种DNA转染方法，这种方法对每种蛋白质都有效，也可以用来阻止它们的合成。我们提出，[Ca 2 +]SR -肌肉疲劳时衰减-是由SOCE，一个通用的Ca 2+进入途径，动员控制基因表达的转录因子至关重要。使用SOCE措施开发的第一个时期，我们提出的目标3定义的作用，新发现的SOCE分子的控制[Ca 2 +] SR。这些分子可以抵御疲劳，并提供强大的工具，实验改变[Ca 2 +]SR的迭代方法的主要问题。Ca 2+信号在疾病、疲劳和衰老中恶化。快速Ca 2+信号在诸如低PP、MH易感性和中央核心和微核心以及老化肌肉的疾病中失效。中等范围信号传导在疲劳和缺乏钙螯合蛋白的小鼠的MH样表型中受到影响。长期Ca 2+信号的疾病，在肌肉和免疫系统中显示出惊人的相似之处，包括SCID，一种家族性免疫缺陷，结合了淋巴细胞中SOCE的丧失和肌源性肌病。我们的工作将通过评估特定分子及其相互作用的作用来促进对这些缺陷的理解。虽然只有疲劳将在本项目中具体解决，功能缺陷，复杂的病理生理学和合理设计的治疗校正之间的关系的问题将得到更好的解决，因为我们了解什么控制储存钙，什么储存钙控制。公共卫生相关性：这个项目和我们实验室的工作涉及肌肉内钙的运动。所谓的“钙信号”是细胞用来反应和改变的通用语言。在骨骼肌中，这些信号在休息和运动之间产生差异，从而影响我们生活的多个方面，包括身体状况，新陈代谢，甚至体重。由于钙被用于许多任务，包括杀死细胞，因此这些信号是危险的，必须加以控制。在这个项目中，我们问是什么控制钙的运动，特别是关注细胞内钙储存区域的作用。我们还想知道，钙库内的钙是如何决定运动量的，或者运动何时停止的。因此，我们的工作应该有助于理解为什么信号失败或失控。这发生在疾病中，包括肌肉萎缩症、恶性高热等。信号在疲劳时也会衰减--它们的衰减实际上会导致疲劳--在老年时也会衰减。在肌肉疲劳中，储存被耗尽。我们认为，两个新发现的分子是延迟这种消耗的重要因素。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，他们的缺陷或失败将产生多重影响。因此，可以预见，我们的研究将在肌肉和运动领域之外产生影响。

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.