Excitation-Contraction Coupling in Dystrophic Muscle

描述（由申请人提供）：将使用mdx小鼠的分离肌纤维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）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。来自这个动物模型的细胞，就像那些营养不良患者的细胞一样，在表达营养不良蛋白方面存在缺陷。尽管有大量的生化证据表明肌营养不良蛋白-糖蛋白复合物与跨膜和膜结合肌肉蛋白有关，但对其在肌纤维生理方面的具体作用知之甚少。本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乏与横管系统和钙信号传导机制中电传播的特定改变联系起来。将通过实验探索几种可能解释这些观察结果的可能性。利用低亲和钙敏感荧光指示剂记录肌纤维电活动引发的细胞内钙浓度变化，利用电位指示剂监测横小管膜电位变化。研究将使用高分辨率光学方法进行，不仅在细胞水平上，而且在肌肉纤维的子区域内，甚至在单个肌节内，可以评估EC耦合过程中这些关键步骤的功能状态。我们将在mdx小鼠的三个不同年龄组中进行这些测量，以了解疾病随时间的进展。我们还将测试来自缺乏肌营养蛋白/肌营养不良蛋白的双突变小鼠的肌纤维，其表现出更严重的病理（类似于DMD），是否显示出更明显的EC偶联缺陷的迹象。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营养不良蛋白在哺乳动物骨骼肌中的功能作用，这是迄今为止肌营养不良研究中最基本和最难以捉摸的问题。在哺乳动物肌肉纤维局部亚显微区域检测EC偶联机制缺陷步骤的增强方法可能成为未来评估单个肌肉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.