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.
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.
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