Excitation-Contraction Coupling in Dystrophic Muscle

营养不良性肌肉的兴奋-收缩耦合

• 批准号: 6879652
• 负责人: Julio L Vergara
• 金额: $ 22.19万
• 依托单位: UNIVERSITY OF CALIFORNIA LOS ANGELES
• 依托单位国家: 美国
• 财政年份: 2003
• 资助国家: 美国
• 起止时间: 2003-04-01 至 2008-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6879652
• 关键词: action potentials; biological signal transduction; calcium flux; calcium metabolism; confocal scanning microscopy; dystrophin; electron microscopy; glycoproteins; laboratory mouse; muscle contraction; muscle proteins; muscular dystrophy; sarcoplasmic reticulum; striated muscles

DESCRIPTION (provided by applicant): Abnormalities in the mechanisms of calcium regulation and excitation-contraction (EC) coupling that may be linked to the degeneration of skeletal muscle fibers in Becker Muscular Dystrophy (BMD) and in Duchenne Muscular Dystrophy (DMD) will be investigated using isolated muscle fibers from mdx mice. Cells from this animal model, like those of dystrophic patients, have deficiencies in the expression of the protein dystrophin. Although there is substantial biochemical evidence demonstrating the association of the dystrophin-glycoprotein complex with transmembrane- and membrane-bound muscle proteins, little is known about its specific role in the physiological aspects of a muscle fiber. The main goal of this proposal is to obtain critical experimental evidence linking the absence of dystrophin with specific alterations in the electrical propagation in the transverse tubular system and calcium signaling machinery. Several possibilities that may explain these observations will be explored experimentally. Changes in intracellular calcium concentration triggered by electrical activity of the muscle fibers will be recorded with the aid of low affinity calcium sensitive fluorescent indicators and membrane potential changes in the transverse tubules will be monitored with potentiometric indicators. The investigations will be carried out using high-resolution optical methods that permit to assess the functional state of these critical steps of the EC coupling process, not only at the cellular level, but also within sub-regions of the muscle fiber and even within a single sarcomere. We will perform these measurements across three different age groups of the mdx mouse in order to understand the progression of the disease with time. We will also test if muscle fibers from a utrophin/dystrophin-lacking double mutant mouse, which exhibits a harsher pathology (similar to DMD), show signs of more pronounced defects in EC coupling. These types of experiments are necessary to unravel the mysterious role that dystrophin may play in the normal regulation of calcium metabolism in skeletal muscle. The knowledge gained in the proposed studies will help to elucidate the functional role of dystrophin in mammalian skeletal muscle, to this date the most fundamental and elusive problem in muscular dystrophy research. The enhanced methods proposed to detect defective steps in the EC coupling mechanisms within localized submicroscopic regions of mammalian muscle fibers may become the optimal choice for the future evaluation of genetic therapeutic procedures in sub-regions of a single muscle cell.

描述(由申请人提供)：将使用MDX小鼠的分离肌肉纤维来研究钙调节和兴奋-收缩(EC)耦合机制中的异常，这些异常可能与Becker肌营养不良症(BMD)和Duchenne肌营养不良症(DMD)的骨骼肌纤维退化有关。这种动物模型中的细胞，就像营养不良患者的细胞一样，在Dstrophin蛋白的表达上存在缺陷。虽然有大量的生化证据表明，肌营养不良蛋白-糖蛋白复合体与跨膜和膜结合的肌肉蛋白有关，但对其在肌肉纤维的生理方面的具体作用知之甚少。这项建议的主要目的是获得关键的实验证据，将dystrophin的缺失与横管系统和钙信号机制中电传播的特定变化联系起来。可能解释这些观察结果的几种可能性将通过实验进行探索。肌纤维电活动引发的细胞内钙离子浓度的变化将借助低亲和力钙敏感荧光指示器进行记录，横管内膜电位的变化将通过电位指示器进行监测。这些研究将使用高分辨率光学方法进行，这些方法不仅可以在细胞水平上评估EC偶联过程的这些关键步骤的功能状态，而且还可以在肌肉纤维的亚区域内甚至在单个肌节内评估这些关键步骤的功能状态。我们将对MDX小鼠的三个不同年龄段进行这些测量，以了解疾病随时间的发展。我们还将测试缺乏utroin/dystrophin的双突变小鼠的肌肉纤维是否显示出EC偶联更明显缺陷的迹象，该小鼠表现出更严重的病理(类似于DMD)。这些类型的实验对于揭开dystrophin在骨骼肌钙代谢的正常调节中可能扮演的神秘角色是必要的。在拟议的研究中获得的知识将有助于阐明肌营养不良蛋白在哺乳动物骨骼肌中的功能作用，到目前为止，这是肌营养不良症研究中最基本和最难以捉摸的问题。在哺乳动物肌肉纤维的局部亚微观区域内检测EC偶联机制缺陷步骤的改进方法可能成为未来评估单个肌肉细胞亚区域遗传治疗过程的最佳选择。