Excitation-Contraction Coupling in Normal and Dystrophic Mammalian Muscle

描述（由申请人提供）：该提案的总体目标是深入了解哺乳动物骨骼肌中肌营养不良蛋白糖蛋白复合物（DGC）的改变与兴奋收缩耦合（ECC）过程受损之间的机制联系。这种功能表征将在来自人类肌营养不良症的各种动物模型的肌纤维中实现。我们发现，成年 mdx 小鼠和表型肌跨 (SSPN) 过表达小鼠 (SSPN-Tg) 两种模型的肌纤维中动作电位 (AP)（或电压钳脉冲）引起的 Ca2 释放明显小于野生型纤维。我们假设 DGC 的破坏破坏了横管系统 (TTS) 和肌浆网 (SR) 的结构和功能支持，从而减弱了 ECC 过程。我们将首先研究 mdx 小鼠 Ca2 释放受损的机制（目标 1），mdx 小鼠是杜氏肌营养不良症 (DMD) 最常用的动物模型，其 DGC 中缺乏肌营养不良蛋白。然而，由于 mdx 小鼠的表型改变相对良性，可能是由于 DGC 中 utropin 替代所致，因此还将在双敲除 mdx/utropin (mdx/utr-/-) 小鼠中进行实验，这些小鼠表现出与 DMD 患者更相似的表型（目标 2）。为了进一步表征 DGC 完整性和功能齐全的 ECC 之间的联系，我们将利用我们通过体内电穿孔表达 DGC 蛋白的能力，并使用具有改变 DGC 的其他遗传条件（例如 SSPN-Tg 和 Utr-TET）的转基因动物模型。该提案的最后一个目标是使用 2 光子激光扫描显微镜 (TPLSM) 研究代表性 DGC 蛋白成分的亚细胞分布，以评估它们是否仅与肌膜相关，或者是否具有与 Z 线和 TTS 相关的更普遍的分布。这一表征将帮助我们了解 DGC 在 ECC 和肌膜完整性方面的功能（目标 3）。这些研究将通过使用电生理学和最先进的光学方法进行，例如 F'ster 共振能量转移 (FRET) 和全内反射荧光显微镜 (TIRFM)，以评估 DGC 和 ECC 蛋白相对于表面和 TTS 膜的内部和外部小叶的纳米级定位。 公共健康相关性：在杜氏肌营养不良症 (DMD) 中，肌肉缺乏肌营养不良蛋白，这是一种不可或缺的成分我们发现，mdx 小鼠（一种广泛使用的 DMD 动物模型）的肌纤维中缺乏肌营养不良蛋白，会损害其响应电刺激而从肌浆网释放 Ca2+ 的能力，从而解释在 DGC 病理学中观察到的肌肉无力的机制，这些结果将显着拓宽我们对肌肉疾病机制的理解。潜在地提供被认为是基因治疗进一步发展所必需的治疗分子工具。

DESCRIPTION (provided by applicant): 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 impairment of the excitation-contraction coupling (ECC) process in mammalian skeletal muscle. This functional characterization will be achieved in muscle fibers from various animal models of human muscular dystrophies. We have found that Ca2+ release evoked by action potentials (APs) (or voltage-clamp pulses) in muscle fibers from two of such models, the adult mdx mouse and the phenotypic sarcospan (SSPN) overexpressing mouse (SSPN-Tg), is significantly smaller than in wild type fibers. We hypothesize that disruption of the DGC undermines the structural and functional support for the transverse tubular system (TTS) and the sarcoplasmic reticulum (SR), thus attenuating the ECC process. We will first investigate the mechanisms responsible for the impairment of Ca2+ release in mdx mice (Aim 1), the most prevalently used animal model for Duchenne Muscular Dystrophy (DMD), which lacks dystrophin in the DGC. However, since the phenotypic alterations in mdx mice are relatively benign, 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 more comparable to that in DMD patients (Aim 2). To further characterize the link between the DGC integrity and a fully functional ECC, we will take advantage of our ability to express DGC proteins by in vivo electroporation and use transgenic animal models with other genetic conditions altering the DGC (e.g. SSPN-Tg, and Utr-TET). The last goal of the proposal is to investigate, using 2-photon laser scanning microscopy (TPLSM) the subcellular distribution of representative DGC protein components in order to assess if they are associated exclusively with the sarcolemma or if they have a more ubiquitous distribution in association with the Z-line and the TTS. This characterization will help us understand the function of the DGC in terms of ECC and sarcolemmal integrity (Aim 3). These investigations will be carried out by using electrophysiological and state-of-the-art optical methods, such as F"ster resonance energy transfer (FRET) and total internal reflection fluorescence microscopy (TIRFM), to also assess the nanoscale localization of DGC and ECC proteins with respect to the internal and external leaflets of the surface and TTS membranes. PUBLIC HEALTH RELEVANCE: In Duchenne Muscular Dystrophy (DMD) the muscles lack the protein dystrophin, an integral component of a dystrophin-glycoprotein complex (DGC). We have discovered that the absence of dystrophin in the muscle fibers of the mdx mouse, a widely used animal model of DMD, impairs their ability to release Ca2+ from the sarcoplasmic reticulum in response to electrical stimulation, thus explaining the muscle weakness observed in the DGC pathology. We now propose to investigate the mechanisms that link DGC alterations with a deficient Ca2+ release. The results will significantly broaden our understanding of muscle disease mechanisms and will potentially provide therapeutic molecular tools which are deemed necessary for further advances in gene therapy.