Excitation-contraction Coupling in Normal and Dystrophic Mammalian Muscle

这项建议的总体目标是深入了解 肌营养不良蛋白糖蛋白复合体(DGC)的变化与钙(Ca~(2+))释放的损害 作为人类模型的不同营养不良动物肌肉纤维的机制观察 肌肉营养不良症。动作电位(AP)或电压钳脉冲引起的钙释放显著增加 成人MDX和高表达表型转基因蔗糖蛋白(SSPN)的肌纤维受损 (SSPN-TG)小鼠。出现的情况是，在营养不良的成年人中观察到的钙释放的局限性 肌肉纤维可能是潜在分子机制的次优分子组织造成的。 支持由于DGC缺陷而导致的激发-收缩耦合(ECC)过程。我们假设 DGC的适当组织不仅对维持肌肉纤维的肌膜完整性至关重要 在机械活动期间，而且还为横管提供结构和功能支撑 系统(TTS)和直接负责ECC过程的内膜室。这个 这项应用的具体目标将是首先研究导致 与Duchenne肌肉相同的动物模型MDX小鼠的钙释放障碍(Aim 1) 营养不良(DMD)指的是DGC中没有dystrophin，这是肌营养不良的最新进展 已经取得了研究成果。然而，由于MDX小鼠的表型变化模式导致了 相对良性的病理，可能是由于DGC中的utroin替代，也将进行实验 在表型与DMD相似的mdx/utroin(mdx/utr-/-)双基因敲除小鼠中缺失 患者(目标2)。我们还将使用其他遗传条件改变DGC的转基因动物模型 (SSPN-TG、UTR-TET和其他)，以便比较它们在控制方面在EC耦合方面的局限性 和mdx小鼠(目标2)。该提案的最后一个目标是研究活的肌肉纤维中是否存在 DGC的不同蛋白质组分的结合被归入肌膜或它们是否相互作用 对于TTS和肌浆网(SR)，DGC的变化可能最终解释 欧共体耦合进程的功能有限(目标3)。调查将使用以下方式进行 电生理方法、最先进的高分辨率光学方法(FRET和TIRFM)，允许 DGC等关键蛋白在活细胞中定位表达的研究 参与EC偶联的肌肉蛋白及其在细胞、单个肌节和肌肉功能中的作用 分子水平。

The overall goal of this proposal is to obtain an in depth understanding of the mechanistic links between alterations of the dystrophin glycoprotein complex (DGC) and the impairemnt in the calcium (Ca2+) release mechanisms observed in muscle fibers from various dystrophic animals which serve as models for human muscular dystrophies. Ca2+ release evoked by action potentials (APs) or voltage clamp pulses is significantly impaired in muscle fibers from adult mdx and transgenic sarcospan (SSPN) overexpressing phenotypic (SSPN-Tg) mice. The emerging picture is that the limitations in the Ca2+ release observed in adult dystrophic muscle fibers may result from a suboptimal molecular organization of the underlying molecular machinery supporting the excitation-contraction coupling (ECC) process due to a defective DGC. We hypothesize that the proper organization of the DGC is crucial not only to maintain the sarcolemmal integrity of the muscle fibers during mechanical activity, but also to provide structural and functional support for the transverse tubular system (TTS) and internal membrane compartments which are directly responsible for the ECC process. The specific goals of the application will be to first investigate the molecular mechanisms responsible for the impairment of Ca2+ release in mdx mice (Aim 1), an animal model that shares with Duchenne Muscular Dystrophy (DMD) the absence of dystrophin in the DGC in which most of the advances in muscular dystrophy research have been attained. However, since the pattern of phenotypic alterations in mdx mice results in a relatively benign pathology, possibly due to utrophin substitution in the DGC, experiments will be also carried out in double knockout mdx/utrophin (mdx/utr-/-) mice that display a phenotype comparable to that in DMD patients (Aim 2). We will also use transgenic animal models with other genetic conditions altering the DGC (SSPN-Tg, Utr-TET and others) in order to compare their limitations in the EC coupling with respect to control and mdx mice (aim 2). The last goal of the proposal is to investigate in live muscle fibers whether the association of the different protein components of the DGC is relegated to the sarcolemma or if they interact with the TTS and the sarcoplasmic reticulum (SR), such that alterations in the DGC may ultimately explain the limited functionality of the EC coupling process (Aim 3). The investigations will be carried out using electrophysiological methods, state-of-the-art high-resolution optical methods (FRET and TIRFM) that permit the assessment of the localized expression in live cells of protein components of the DGC and other key muscle proteins involved in EC coupling, and their role in muscle function at the cellular, single sarcomere, and molecular level.