Development of Slow Skeletal Muscle Fibers

描述（由申请人提供）：不同骨骼肌的发育 纤维类型由细胞间相互作用的复杂机制控制， 导致收缩蛋白基因表达的信号转导事件 肌纤维类型的特征。在脊椎动物骨骼肌 发育，胎儿肌纤维的神经支配导致表达缓慢， 肌球蛋白重链（MyHC）基因在不同的肌纤维亚群 从而建立和维持肌纤维表型差异。 直到最近，还没有一个合适的，容易操纵的模型系统 探讨神经支配诱导纤维型发育的机制。的 这项研究的目的是利用一种新的肌纤维-运动神经元 共培养系统，以阐明胎儿纤维型发育的机制。 第一个目标是定义和表征细胞内信号传导 调节慢MyHC基因表达和肌纤维类型的机制。这 将通过使用肌纤维运动神经元的相互作用，在一个新的 共培养系统，并通过使用显性阳性和阴性突变， 体外骨骼肌纤维内信号级联的已知成员。 重点将放在鸟嘌呤核苷酸结合蛋白（G 蛋白质）和蛋白激酶C（PKC）信号中的其它信号分子 转导级联研究还将集中在钙调神经磷酸酶信号 这两个信号转导级联之间的通路和串扰。 肌源性调节因子（MRF）和其他特异性 转录因子通过这些信号级联和随后的慢MyHC 2 基因表达将通过非磷酸化 转录因子突变第二个目标是确定监管 参与慢MyHC 2的外源性神经依赖性调节的组分 基因在与运动神经元共培养的胎儿肌纤维中的表达。MyHC 2基因 获得了其启动子。特异性顺式元件的鉴定 通过由神经支配启动的机制调节纤维类型身份将 通过转染转录驱动的报告基因构建体来完成， 慢MyHC 2基因调控序列。慢MyHC 2基因调控区将 通过缺失和诱变改变。提案的具体目的是 强调细胞和分子生物学方法来阐明外在的 神经支配对离体骨骼肌肌纤维类型影响 第一次真正模拟肌肉纤维类型的调节， vivo.

DESCRIPTION (provided by applicant): Development of diverse skeletal muscle fiber types is controlled by a complex mechanism of cell-cell interactions and signal transduction events leading to expression of contractile protein genes characteristic of the muscle fiber type. During vertebrate skeletal muscle development, innervation of fetal muscle fibers causes expression of slow myosin heavy chain (MyHC) genes in a distinct subpopulation of muscle fibers and thereby establishes and maintains muscle fiber phenotypic differences. Until recently, there has been no appropriate, easily manipulated model system to investigate the mechanism of innervation-induced fiber type development. The goal of the proposed research is to use a novel muscle fiber - motor neuron co-culture system to elucidate the mechanism of fetal fiber type development. The first aim is to define and characterize the intracellular signaling mechanism that regulates slow MyHC gene expression and muscle fiber type. This will be done by use of muscle fiber motor neuron interactions in a new co-culture system and by use of dominant positive and negative mutations of known members of signaling cascades within skeletal muscle fibers in vitro. Emphasis will be placed on the role of guanine nucleotide binding proteins (G proteins) and other signaling molecules in the protein kinase C (PKC) signal transduction cascade. Studies will also focus on the calcineurin signaling pathway and cross-talk between these two signal transduction cascades. Phosphorylation of myogenic regulatory factors (MRFs) and other specific transcription factors by these signaling cascades and subsequent slow MyHC2 gene expression will be examined by expression of non-phosphorylatable transcription factor mutations. The second aim is to identify the regulatory components involved in extrinsic, nerve-dependent regulation of the slow MyHC2 gene in fetal muscle fibers co-cultured with motor neurons. The slow MyHC2 gene with its promoter has been obtained. Identification of specific cis-elements regulating fiber type identity through mechanisms initiated by innervation will be done by transfection of reporter gene constructs transcriptionally driven by slow MyHC2 gene regulatory sequences. Slow MyHC2 gene regulatory regions will be altered by deletion and mutagenesis. The specific aims of the proposal place emphasis on cell and molecular biological approaches to elucidate the extrinsic innervation-induced regulation of skeletal muscle fiber type within in vitro settings that for the first time truly mimic regulation of muscle fiber type in vivo.