Calcium Regulation in the Progression of Muscular Dystrophy

描述（由申请人提供）：杜氏肌营养不良症是一种常见的遗传性疾病，由肌营养不良蛋白基因内的突变引起。很可能营养不良表型不是直接由肌原纤维结构的改变引起的，而是肌膜完整性的破坏，其通常赋予细胞内Ca稳态的严格控制，这导致细胞内Ca升高和最终的肌肉变性。细胞内钙水平升高的病理生理机制尚不清楚，但我们最近的研究结果表明，与不受控制的钙火花活动相关的钙库操作的钙进入（SOCE）可能有助于在营养不良的肌肉中观察到的异常钙内流。虽然SOCE激活的机制仍然是一个深入研究的问题，但最近已经确定钙不敏感磷脂酶A2（iPLA 2）是SOCE的重要介质。该项目的重点是检验以下假设：异常的Ca火花活性作为SOCE的触发剂，从而通过涉及IPLA 2介导的信号传导的途径在哺乳动物骨骼肌中诱导营养不良级联反应。我们将通过三个具体目标来测试这一点：目标1：建立诱导的Ca火花作为健康和营养不良肌肉中SOCE的触发因素。我们将利用多种方法来测量SOCE，以检查营养不良纤维中Ca：entry的改变。膜片钳测量和调节钙内流将提供洞察SR钙释放和激活的SOCE在营养不良的纤维。目的2：确定肌营养不良症中IPLA 2活性对Ca内流的贡献。我们将使用各种体外分子和药理学方法研究营养不良肌肉中JPLA2功能的改变特征。目的3：阐明SOCE是否促进骨骼肌的营养不良级联反应。由于不能评估骨骼肌营养不良表型的变化，我们将调节营养不良动物的SOCE并测定营养不良表型的变化。这个项目将有助于我们了解杜氏肌营养不良症（DMD）是美国最常见的遗传病，肌肉损伤是如何发生的。我们的实验将确定钙如何进入肌肉细胞的缺陷导致DMD的发展。这些知识将提出治疗DMD的新方法，其中一些将在本研究的实验动物上进行测试。

DESCRIPTION (provided by applicant): Duchenne muscular dystrophy is a common genetic disorder resulting from mutations within the dystrophin gene. It is likely that the dystrophic phenotype does not result directly from alteration to the myofibrillar structures, rather it is a disruption of sarcolemmal membrane integrity that normally confers tight control of intracellular Ca homeostasis, which leads to elevated intracellular Ca and eventual muscle degeneration. The pathophysiological mechanism responsible for elevation of intracellular Ca levels is not clear, however our recent findings suggest that store-operated Ca entry (SOCE) linked to uncontrolled Ca spark activity may contribute to the aberrant Ca influx observed in dystrophic muscle. While the mechanism of SOCE activation is still a matter of intensive study, it has been recently determined that the Ca insensitive phospholipase A2 (iPLA2) is an important mediator of SOCE. The focus of this project is to test the hypothesis that aberrant Ca spark activity acts as a trigger for SOCE and thus induces a dystrophic cascade in mammalian skeletal muscle through a pathway that involves IPLA2 mediated signaling. We will test this with three specific aims: Aim 1: To establish induced Ca sparks as a trigger for SOCE in healthy and dystrophic muscle. We will utilize multiple methods for measurement of SOCE to examine alteration to Ca : entry in dystrophic fiber. Patch-clamp measurement and modulation of Ca influx will provide insight into SR Ca release and the activation of SOCE in dystrophic fibers. Aim 2: Determine the contribution of IPLA2 activity to Ca influx in muscular dystrophy. We will examine the altered characteristics of JPLA2 function in dystrophic muscle using various in vitro molecular and pharmacological methods. Aim 3: To elucidate if SOCE facilitates the dystrophic cascade in skeletal muscle. Due to the inability to assess changes in the dystrophic phenotype in skeletal muscle we will modulate SOCE in dystrophic animals and assay changes in dystrophic phenotypes. This project will contribute to our understanding of how muscle damage occurs in cases of Duchenne muscular dystrophy (DMD), the most common genetic disease in the United States. Our experiments will establish that defects in how calcium enters muscle cells result in the development of DMD. Such knowledge will suggest new methods to treat DMD, some of which will be tested on experimental animals in this study.