Development of Slow Skeletal Muscle Fibers

描述(由申请人提供)：骨骼肌纤维类型之间的多样性由神经依赖和肌肉纤维类型特定的细胞信号和下游转录调节控制。已发现一条细胞信号通路，调节支配的快、慢二级禽肌纤维中慢肌球蛋白重链2(MyHC2)基因的表达。该信号转导通路由三磷酸肌醇受体1(IP3R1)介导，由神经支配引起的去极化反应启动。这项研究的目的是阐明IP3R1活性以一种纤维类型特异性的方式调节的机制，并阐明IP3R1活性调节慢速MyHC2基因表达从而调节肌纤维类型的机制。该提案的第一个目标是确定快肌纤维和慢肌纤维中IP3R1相关蛋白复合体的肌肉纤维类型特定蛋白质成分。IP3R1相关蛋白的鉴定将通过质谱学和蛋白质印迹分析来确定。该提案的第二个目的是确定IP3R1相关蛋白相互作用和磷酸化改变的功能效应。IP3R1介导的钙释放的测量将在快肌纤维和慢肌纤维中进行，这些肌纤维分别转染野生型和突变型IP3R1表达结构。此外，IP3R1介导的钙释放将在快肌纤维和慢肌纤维中进行测量，这些肌纤维的IP3R1相关细胞信号蛋白活性发生变化。该提案的第三个目的是确定IP3R1功能改变对慢速MyHC2基因转录调节的影响，并建立不同的肌肉纤维类型。快肌纤维和慢肌纤维中IP3R1活性的变化对慢速MyHC2启动子活性和慢速MyHC2启动子内转录激活位点占有率的影响将被确定。这些目标的结果将是从目标1获得的结构数据和从目标2获得的功能数据的合成，从而调节缓慢的MyHC2基因表达和肌肉纤维类型鉴定。钙释放机制的改变与许多疾病状态有关，包括细胞凋亡和骨骼肌、肌营养不良。所提出的IP3R1活性调节研究的意义对于了解骨骼肌和其他细胞类型的正常和疾病相关的细胞功能具有直接的相关性。

DESCRIPTION (provided by applicant): Diversity among skeletal muscle fiber types is controlled by innervation-dependent and muscle fiber type specific cell signaling and downstream transcriptional regulation. A cell signaling pathway has been revealed that regulates slow myosin heavy chain 2 (MyHC2) gene expression in innervated fast and slow secondary avian muscle fibers. This signaling cascade, initiated by innervation-induced depolarization, is mediated by the inositol 1,4, 5 triphosphate receptor 1 (IP3R1). The goal of the proposed research is to elucidate the mechanism by which IP3R1 activity is regulated in a fiber type specific manner and to elucidate the mechanism by which controlled IP3R1 activity regulates expression of the slow MyHC2 gene and thereby muscle fiber type. The first aim of the proposal is to determine the muscle fiber type specific protein components of the IP3R1-associated protein complexes in fast versus slow muscle fibers. The identity of IP3R1-associated proteins will be established by mass spectrometry and western blot analysis. The second aim of the proposal is to determine the functional effects of altered IP3R1-associated protein-protein interactions and phosphorylation. IP3R1-mediated calcium release measurements will be made in fast and slow muscle fibers transfected with wild type and mutated IP3R1 expression constructs. In addition, IP3R1- mediated calcium release measurements will be made in fast and slow muscle fibers with altered IP3R1-associated cell signaling protein activities. The third aim of the proposal is to determine the effects of altered IP3R1 function on the transcriptional regulation of the slow MyHC2 gene and establishment of diverse muscle fiber types. The effects of altered IP3R1 activities in fast versus slow muscle fiber types on slow MyHC2 promoter activity and occupancy of transcriptional activating sites within the slow MyHC2 promoter will be determined. The result of these aims will be a synthesis of the structural data obtained from aim 1 and the functional data derived from aim 2 with the resulting regulation of slow MyHC2 gene expression and muscle fiber type identity. Altered calcium release mechanisms are associated with many disease states involving apoptosis and in skeletal muscle, muscular dystrophy. The significance of the proposed studies of regulation of IP3R1 activity has immediate relevance to understanding normal and disease-associated cellular function in skeletal muscle and other cell types.