Development of Slow Skeletal Muscle Fibers

描述（由申请人提供）：骨骼肌纤维类型的多样性受神经依赖性和肌肉纤维类型特异性细胞信号传导和下游转录调控的控制。在神经支配的禽类快、慢次生肌纤维中发现了一条调节慢肌球蛋白重链2 （MyHC2）基因表达的细胞信号通路。这种信号级联由神经诱导的去极化启动，由肌醇1,4,5三磷酸受体1 （IP3R1）介导。本研究的目的是阐明IP3R1活性以纤维类型特异性方式调控的机制，并阐明受控IP3R1活性调控MyHC2基因表达从而调控肌纤维类型的机制。该提案的第一个目的是确定快肌纤维与慢肌纤维中ip3r1相关蛋白复合物的肌肉纤维类型特异性蛋白质成分。ip3r1相关蛋白的身份将通过质谱和western blot分析确定。该提案的第二个目的是确定改变的ip3r1相关蛋白-蛋白相互作用和磷酸化的功能影响。IP3R1介导的钙释放测量将在转染野生型和突变IP3R1表达构建体的快肌纤维和慢肌纤维中进行。此外，IP3R1介导的钙释放测量将在IP3R1相关细胞信号蛋白活性改变的快、慢肌纤维中进行。该提案的第三个目的是确定IP3R1功能改变对MyHC2基因转录调控和多种肌纤维类型建立的影响。将确定快肌纤维与慢肌纤维类型中IP3R1活性的改变对慢MyHC2启动子活性和慢MyHC2启动子内转录激活位点占用的影响。这些目标的结果将是综合从aim 1获得的结构数据和从aim 2获得的功能数据，以及由此产生的MyHC2基因缓慢表达和肌纤维类型识别的调节。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、肌肉萎缩。拟议的IP3R1活性调控研究的意义与理解骨骼肌和其他细胞类型的正常和疾病相关的细胞功能直接相关。

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.