Regulation of Mitsugumin 29 expression in muscle physiology and diseases

 描述（由申请人提供）：Mitsugumin 29（MG 29）是突触素家族蛋白质的肌肉特异性成员，参与控制横管（TT）结构的成熟和发育以及维持骨骼肌中细胞内Ca 2+信号传导。mg 29的基因切除导致骨骼肌中有缺陷的TT结构，这与在肌营养不良症动物模型和人类患者中观察到的异常TT网络相似。该应用的主要研究者已经获得了证据，揭示了MG 29在肌肉生理学和疾病中控制TT膜网络的重要作用。免疫印迹显示，MG 29的表达在患有肌营养不良症的人和小鼠中均降低，表明MG 29可能参与肌营养不良症的发展。利用生物化学分析，我们鉴定了MG 29、dysferlin和Bin 1（在肌营养不良症中具有重要作用的其他TT驻留蛋白）之间的功能性相互作用。为了理解MG 29在控制TT生物发生中的生物学功能，我们使用了来源于人肌肉的HMCL-7304成肌细胞系。HMCL-7304细胞培养的肌管不表达MG 29，缺乏TT网络。在HMCL-7304肌管中拯救MG 29的初步研究显示TT膜的起始。建立一个控制TT膜的体外系统可能是一个有用的工具，为肌肉生理学研究。mg 29基因包含一个独特的3'非翻译区（UTR），具有与microRNA（miRNA）或RNA稳定因子结合的潜在位点。突变研究显示，miR-181 a可以靶向小鼠和人mg 29基因之间高度保守的3 'UTR区域，以显示对骨骼肌中MG 29表达的强控制。来自其他研究者的研究表明，miR-181在患有肌营养不良症的人类患者中显著升高。这些数据表明一种有趣的可能性，即升高的miR-181 a可能是肌营养不良条件下MG 29表达降低的促成因素。本提案中概述的实验将集中于测试“MG 29是骨骼肌中TT生物发生的组成部分，并且MG 29表达的miRNA介导的控制有助于肌营养不良症期间TT完整性的变化”的假设。首先，通过使用体外细胞培养和体内转基因动物方法，我们将辨别MG 29在控制TT完整性和肌肉生理学和营养不良中的重塑中的生物学功能。其次，我们将阐明骨骼肌中miRNA介导的MG 29表达控制的机制，并确定有助于营养不良肌肉中MG 29减少的机制。使用AAV介导的基因递送将Eschomir导入mdx小鼠，我们将测试抑制miRNA介导的MG 29表达下调是否可以改善TT结构并挽救营养不良肌肉的肌肉力量。

 DESCRIPTION (provided by applicant): Mitsugumin 29 (MG29) is a muscle-specific member of the synaptophysin family proteins that participates in controlling the maturation and development of the transverse-tubule (TT) structure and the maintenance of intracellular Ca2+ signaling in skeletal muscle. Genetic ablation of mg29 leads to defective TT structure in skeletal muscle, which show similarity to the abnormal TT network observed in animal models of, and human patients with, muscular dystrophy. The principal investigator of this application has obtained evidence that revealed an essential role for MG29 in controlling the TT membrane network in muscle physiology and diseases. Immunoblot showed that expression of MG29 was reduced in both human and mice with muscular dystrophy, indicating the possibility that MG29 may be involved in the development of muscular dystrophy. Using biochemical assay, we identified a functional interaction between MG29, dysferlin and Bin1, other TT- resident proteins with important roles in muscular dystrophy. Toward understanding the biological function for MG29 in control of TT biogenesis, we used the HMCL-7304 myoblast cell line derived from human muscle. Cultured myotubes derived from HMCL-7304 cells do not express MG29 and lack TT network. Preliminary study with rescue of MG29 in HMCL-7304 myotubes revealed the initiation of TT membranes. Establishing an in vitro system with control of TT membrane can potentially be a useful tool for muscle physiology research. The mg29 gene contains a unique 3' untranslated region (UTR) with potential binding sites for microRNA (miRNA) or RNA-stabilizing factors. Mutagenesis studies revealed that miR-181a could target a region in the 3'UTR that is highly conserved between mouse and human mg29 genes to exhibit a strong control of MG29 expression in skeletal muscle. Studies from other investigators have shown that miR-181 was significantly elevated in human patients with muscular dystrophy. These data suggest an intriguing possibility that elevated miR-181a may be a contributing factor for the reduced MG29 expression under muscular dystrophic conditions. Experiments outlined in this proposal will focus on testing the hypothesis that "MG29 is an integral component of TT biogenesis in skeletal muscle, and miRNA-mediated control of MG29 expression contributes to changes in TT integrity during muscular dystrophy". First, by using in vitro cell culture and in vivo transgenic animal approaches, we will discern the biological function for MG29 in control of TT integrity and remodeling in muscle physiology and dystrophy. Second, we will elucidate the mechanisms that underlie miRNA-mediated control of MG29 expression in skeletal muscle, and to identify the mechanisms that contribute to reduction of MG29 in dystrophic muscle. Using AAV-mediated gene delivery of antagomir into the mdx mice, we will test whether suppression of miRNA-mediated down-regulation of MG29 expression can improve TT structure and rescue muscle strength in dystrophic muscle.