Excitation-Contraction Coupling in Dystrophic Muscle

描述（由申请人提供）：将使用来自 mdx 小鼠的分离肌纤维来研究可能与贝克尔肌营养不良症（BMD）和杜氏肌营养不良症（DMD）中骨骼肌纤维变性有关的钙调节和兴奋收缩（EC）耦合机制的异常。 该动物模型的细胞与营养不良患者的细胞一样，在抗肌营养不良蛋白的表达方面存在缺陷。 尽管有大量生化证据证明肌营养不良蛋白-糖蛋白复合物与跨膜和膜结合肌肉蛋白的关联，但对其在肌纤维生理方面的具体作用知之甚少。 该提案的主要目标是获得关键的实验证据，将肌营养不良蛋白的缺失与横管系统和钙信号机制中电传播的特定改变联系起来。 将通过实验探索可以解释这些观察结果的几种可能性。将借助低亲和力钙敏感荧光指示剂记录由肌纤维的电活动触发的细胞内钙浓度的变化，并用电位指示剂监测横管中的膜电位变化。 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，而且可以在肌纤维的子区域内，甚至在单个肌节内评估 EC 耦合过程的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组进行这些测量，以便了解疾病随时间的进展。 我们还将测试来自缺乏肌营养不良蛋白/肌营养不良蛋白的双突变小鼠的肌肉纤维是否表现出更严重的病理学（类似于 DMD），在 EC 耦合中显示出更明显的缺陷迹象。 这些类型的实验对于揭示肌营养不良蛋白在骨骼肌钙代谢的正常调节中可能发挥的神秘作用是必要的。 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，迄今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。所提出的用于检测哺乳动物肌纤维局部亚显微区域内 EC 耦合机制缺陷步骤的增强方法可能成为未来评估单个肌细胞亚区域遗传治疗程序的最佳选择。

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.