MG29 function in regulation of Store-Operated Calcium Entry during Muscle Aging

 描述（由申请人提供）：肌肉减少症是导致老年人虚弱、衰弱性损伤、丧失独立性和生活质量降低的主要因素，是衰老过程中骨骼肌质量的进行性损失。不幸的是，尽管采取了增加体力活动和改善饮食等干预措施，肌肉减少症仍在发展。在肌肉减少症期间，肌肉力量的年龄相关性降低是由肌肉质量损失（萎缩）和肌肉比力降低（即，每单位横截面积的肌肉力量）。越来越多的人认为，伴随肌肉减少症的肌肉无力，而不是肌肉大小本身的损失，是残疾的主要原因。我们最近发现，骨骼肌中产生收缩力的要素依赖于钙库操作的钙内流（SOCE），并且这种能力在衰老的骨骼肌中丧失。这种改变的SOCE与mitsugumin 29（MG 29）含量降低直接相关，MG 29是一种属于突触素（SYPL 2）家族的肌肉特异性蛋白，含有参与胆固醇结合和这些蛋白质寡聚体形成的MARVEL结构域。MG 29对于横小管（TT）系统的正确形成和有效的SOCE是必不可少的。这导致衰老肌肉中MG 29表达减少与肌肉减少症中肌肉功能障碍的发展之间的联系。来自年轻mg 29-/-小鼠的骨骼肌与来自老年野生型（WT）小鼠的骨骼肌相似，因为它们表现出降低的比收缩力、降低的SOCE、降低的从SR释放的Ca、降低的MG 29含量和改变的形态（即，增加缺失的三联体连接和SR聚集体的形成）。因此，我们的数据表明骨骼肌衰老的关键方面存在于mg 29-/-小鼠中，使该模型适用于这一系列研究。在这个提议中，我们特别假设MG 29是通过特定蛋白质结构域的作用在骨骼肌中适当的TT形成和SOCE功能所必需的。正常老化肌肉中MG 29含量的降低导致SOCE缺陷，这导致用于收缩性的Ca 2+的可用性降低和肌肉萎缩未解释的与年龄相关的肌肉力量损失。我们将使用两个特定的目标来测试这一假设，这两个目标都将检查MG 29在骨骼肌中功能的机制基础，并且还为肌肉减少症的治疗提供转化价值。目的1通过电穿孔MG 29 cDNA或RNAi构建体改变MG 29的表达，并同时评估衰老骨骼肌的SOCE功能、SR Ca释放和肌肉特异性收缩力，阐明MG 29对衰老骨骼肌的SOCE、SR Ca释放和收缩力的贡献。目标1中的其他研究将使用病毒基因递送来调节小鼠肌肉中的MG 29表达，并建立这种方法的治疗价值。在目标2中，我们将建立MG 29调节骨骼肌中SOCE的分子机制，以及这些机制如何在衰老中改变，使用分子生物学和生物化学方法来解析负责肌纤维中MG 29作用机制的蛋白质基序和特定残基。

 DESCRIPTION (provided by applicant): Sarcopenia is the progressive loss of skeletal muscle mass during aging leading factor that contributes to frailty, debilitating injuries, loss of independence, and reduced quality of life in the elderly. Unfortunately, sarcopenia progresses despite interventions such as increased physical activity and improved diet. During sarcopenia the age-related decreases in muscle strength result from a combination of loss of muscle mass (atrophy) and reduced muscle specific force (i.e., muscle force per unit of cross-sectional area). Increasingly, it is thought that the muscle weakness that accompanies sarcopenia, not the loss of muscle size per se, is the principal contributor to disability. We have recently found that elements of contractile force generation in skeletal muscle are dependent on store-operated Ca2+ entry (SOCE) and that this capacity is lost in aging skeletal muscle. This altered SOCE is directly associated with reduced content of mitsugumin 29 (MG29), a muscle-specific protein belonging to the synaptophysin (SYPL2) family containing a MARVEL domain involved in cholesterol binding and formation of oligomers of these proteins. MG29 is essential for the proper formation of the transverse-tubule (TT) system and efficient SOCE. This leads to a link between reduced MG29 expression in aged muscle and the development of muscle dysfunction in sarcopenia. Skeletal muscles from young mg29-/- mice are similar to that from aged wild-type (WT) mice in that they demonstrate decreased specific contractile force, reduced SOCE, reduced Ca release from the SR, reduced content of MG29, and altered morphology (i.e., increased missing triad junctions and formation of SR aggregates). Therefore, our data demonstrate that key aspects of skeletal muscle aging are present in mg29-/- mice, making this model applicable for this line of research. In this proposal we specifically hypothesize that MG29 is required for proper TT formation and SOCE function in skeletal muscle through the action of specific protein domains. Decreased MG29 content in normal aged muscle leads to defective SOCE, which results in decreased availability of Ca2+ for contractility and age-related loss of muscle strength not accounted for by muscle atrophy. We will test this hypothesis using two specific aims that will both examine the mechanistic basis for MG29 function in skeletal muscle and also provide translational value for the treatment of sarcopenia. Aim 1 will elucidate the contribution of MG29 to SOCE, SR Ca release and contractility in aged skeletal muscle by using electroporation of MG29 cDNA or RNAi constructs to alter the expression of MG29 and concomitantly evaluate SOCE function, SR Ca release, and muscle specific contractile force. Additional studies in Aim 1 will use viral gene delivery to modulate MG29 expression in mouse muscle and establish the therapeutic value of this approach. In Aim 2 we will establish the molecular mechanisms of MG29 regulation of SOCE in skeletal muscle and how these are altered in aging using molecular biology and biochemical approaches to resolve the protein motifs and specific residues responsible for the mechanism of action of MG29 in muscle fibers.