Regulation of Mitsugumin 29 expression in muscle physiology and diseases

 描述(申请人提供)：Mitsugumin 29(MG29)是突触素家族蛋白中肌肉特有的成员，参与控制骨骼肌横管结构的成熟和发育以及维持细胞内钙信号。基因消融mg29导致骨骼肌TT结构缺陷，这与在肌营养不良动物模型和人类患者中观察到的异常TT网络相似。这一应用的主要研究者已经获得了证据，揭示了MG29在肌肉生理和疾病中控制TT膜网络的重要作用。免疫印迹显示MG29在人和肌营养不良症小鼠中的表达均降低，提示MG29可能参与了肌营养不良症的发生发展。通过生化分析，我们确定了MG29、迪弗林和BIN1之间的功能相互作用，这些蛋白在肌营养不良症中具有重要的作用。为了了解MG29在TT生物发生调控中的生物学功能，我们使用了来源于人肌肉的成肌细胞系HMCL-7304。培养的HMCL-7304细胞来源的肌管不表达MG29，也不表达TT网络。在HMCL-7304肌管中拯救MG29的初步研究揭示了TT膜的启动。建立一个控制TT膜的体外系统可能成为肌肉生理学研究的有用工具。Mg29基因含有一个独特的3‘非翻译区，具有与microRNA(MiRNA)或RNA稳定因子的潜在结合位点。诱变研究表明，miR-181a可以靶向MG29基因3‘端非编码区的一个高度保守的区域，从而显示出对MG29在骨骼肌中表达的强大控制。来自其他研究人员的研究表明，在患有肌肉营养不良的人类患者中，miR-181显著升高。这些数据暗示了一种有趣的可能性，即miR-181a的升高可能是肌肉营养不良条件下MG29表达减少的一个促成因素。这项提案中概述的实验将集中于验证这样的假设：“MG29是骨骼肌TT生物发生的一个组成部分，而miRNA介导的MG29表达控制有助于肌肉营养不良期间TT完整性的变化”。首先，通过体外细胞培养和体内转基因动物方法，我们将了解MG29在肌肉生理学和营养不良中控制TT完整性和重塑的生物学功能。其次，我们将阐明miRNA介导的MG29在骨骼肌中表达的调控机制，并确定有助于营养不良肌肉中MG29表达减少的机制。利用AAV介导的antagomir基因导入mdx小鼠，我们将测试抑制miRNA介导的MG29表达下调是否可以改善营养不良肌肉的TT结构和恢复肌肉力量。

 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.