The Role of Germline Mutations in the Ras/MAPK Pathway on Skeletal Myogenesis

描述(申请人提供)：骨骼肌发生是一个动态的过程，在这个过程中，肌肉前体细胞首先增殖，然后融合形成多核肌管，最终成熟为骨骼肌纤维。Ras/丝裂原活化蛋白激酶(Ras/MAPK)通路是一条被广泛研究的肿瘤通路，它在肌肉发生的调控中起着关键作用，尤其是在成肌细胞从增殖向分化的转换过程中。早期的研究表明，高水平的RAS/MAPK通路激活扰乱了早期的肌肉发生。然而，对于RAS及其下游效应器级联如何调节和影响发育过程中肌肉发生的关键步骤，我们的理解存在着严重的差距。“Rasopathies”是一组新定义的医学遗传综合征，是已知的最大的多发性先天性异常综合征组之一，每1000人中就有1人受到影响。Rasopathies由编码RAS/MAPK途径组成部分的各种关键基因的胚系突变引起，具有先天性低张力或肌肉无力的共同表型特征。Costello综合征(CS)和心面部皮肤综合征(CFC)是两种肌肉表型最严重的Rasopathy。我们最近发现了一种新的肌病，定义为一种肌肉的内在异常，不能归因于神经功能障碍，在CS和CFC患者中。另外，我们初步的 研究支持我们的假设，即RAS/MAPK信号的失调通过抑制成肌细胞的分化来干扰早期的肌肉发生，并通过抑制肌肉的生长来扰乱分化后的肌肉发育的后期阶段。这项研究的目的是了解RAS/MAPK失调是如何影响肌肉发生的，以及这种影响的具体作用机制。我们将研究在Rasopathies中发现的新的种系突变，以帮助我们了解RAS失调如何影响肌肉发育。我们的具体目标是确定1)在CS和CFC中RAS/MAPK信号通路的失调是如何扰乱骨骼肌的；2)RAS/MAPK通路失调导致骨骼肌肌发生紊乱的机制；以及3)小分子抑制剂和小干扰RNA(SiRNA)是否能逆转调控不良的RAS/MAPK信号在肌发生中的作用。我们将使用CS和CFC的小鼠模型来阐明骨骼肌是如何被破坏的，通过区分肌病的哪个方面是由于成肌细胞分化的抑制，以及什么是由于肌肉纤维的形成和分化后对肌肉生长的抑制。我们将利用CS和CFC小鼠模型的原代成肌细胞阐明RAS/MAPK信号失调抑制肌肉生成的具体机制。这些实验的结果将被用来评估合理选择抑制剂的有效性，以纠正失调的RAS途径的发育效应，使用体外和体内的肌肉发生模型。 公共卫生相关性：骨骼肌发生是一个动态过程，在这个过程中，单核肌肉前体细胞增殖，然后融合形成多核肌管，最终成熟为骨骼肌纤维。有许多细胞内信号通路，包括RAS通路，参与正常的肌肉发育。拟议的实验使用两种遗传综合征的小鼠模型，Costello综合征和心面部皮肤综合征，解决了介导肌肉发生及其失调的细胞机制。这些研究的结果将为肌肉发育和疾病的机制提供新的见解，并为治疗干预提供新的靶点。

DESCRIPTION (provided by applicant): Skeletal myogenesis is a dynamic process in which muscle precursor cells first proliferate and then fuse to form multinucleated myotubes that ultimately mature into skeletal muscle fibers. The Ras/Mitogen-activated protein kinase (MAPK) pathway, a well-studied cancer pathway, plays a critical role in the regulation of myogenesis, particularly during the switch from myoblast proliferation to differentiation. Early studies have demonstrated that high levels of Ras/MAPK pathway activation disrupt early myogenesis. However, there are critical gaps in our understanding as to how Ras and its downstream effector cascades regulate and affect vital steps in myogenesis during development. The "RASopathies", a newly defined group of medical genetic syndromes, are one of the largest groups of multiple congenital anomaly syndromes known, affecting more than 1 in 1000 individuals. Caused by germline mutations in various key genes encoding components of the Ras/MAPK pathway, the RASopathies share a common phenotypic feature of congenital hypotonia, or weak muscles. Costello syndrome (CS) and cardio-facio-cutaneous (CFC) syndrome are two RASopathies with the most severe muscle phenotype. We have recently identified the presence of a novel myopathy, defined as an intrinsic abnormality of muscle that is not attributable to nerve dysfunction, in individuals with CS and CFC. In addition, our preliminary studies provide support for our hypothesis that dysregulation of Ras/MAPK signaling disrupts both early myogenesis by inhibiting myoblast differentiation, and later stages of muscle development following differentiation, by inhibiting muscle growth. The goal of the proposed research is to understand how myogenesis is affected by Ras/MAPK dysregulation, as well as the specific mechanism of action underlying this effect. We will examine novel germline mutations identified in the RASopathies to help us understand how Ras dysregulation affects muscle development. Our Specific Aims are designed to determine 1) how skeletal muscle is disrupted by dysregulation of Ras/MAPK pathway signaling in CS and CFC; 2) the mechanisms by which Ras/MAPK dysregulation causes disruption of skeletal muscle myogenesis, and 3) if small molecule inhibitors and small interfering RNAs (siRNA) can reverse the effects of dysregulated Ras/MAPK signaling during myogenesis. We will use mouse models of CS and CFC to elucidate how skeletal muscle is disrupted by distinguishing what aspect of the myopathy is due to inhibition of myoblast differentiation and what is due to muscle fiber formation from post-differentiation inhibition of muscle growth. We will elucidate the specific mechanisms by which Ras/MAPK signal dysregulation inhibits myogenesis using primary myoblasts derived from CS and CFC mouse models. Results derived from those experiments will be used to evaluate the effectiveness of rationally chosen inhibitors to correct the developmental effects of a dysregulated Ras pathway using in vitro and in vivo models of myogenesis. PUBLIC HEALTH RELEVANCE: Skeletal myogenesis is a dynamic process in which mononucleated muscle precursor cells proliferate, and then fuse to form multinucleated myotubes that ultimately mature into skeletal muscle fibers. There are numerous intracellular signaling pathways, including the Ras pathway, involved in normal muscle development. The proposed experiments address the cellular mechanisms that mediate myogenesis and its dysregulation using mouse models for two genetic syndromes, Costello and cardio-facio-cutaneous syndromes. The results of these studies should provide new insight into the mechanisms of muscle development and disease, and reveal new targets for therapeutic intervention.