Cellular regulators of myoblast migration and myogenesis

Abstract

Migration of myogenic cells is an important step in myogenesis and skeletal muscle repair. Migration is required for the cells to reach the site of damage, for their alignment and subsequent fusion. Limited migration is also one of the limitations of proposed therapies of diseases, such as Duchenne Muscular Dystrophy (DMD). Therefore, revealing the regulators of myogenic cell migration is important for improving our knowledge of myogenesis, but could also be applied in therapies for conditions, associated with loss of muscle mass and muscle weakness. In this thesis, extracellular and intracellular regulation of C2C12 myoblast migration was investigated. It was demonstrated that medium conditioned by myotube cultures in vitro, is capable of inducing the migration and chemotaxis of myoblasts. A model of serially passaged myoblasts was used to reveal potential changes in the migratory behaviour of these cells, in the context of skeletal muscle ageing. PI3K/AKT and MAPK/ERK pathways were investigated and their requirement for the process of myoblast migration was revealed. Further activation of these pathways with phospho-tyrsoine phosphatase and PTEN inhibitor Bpv(Hopic) was associated with larger increases in myoblast migration. Silencing of either PI3K/AKT or MAPK/ERK signalling pathways, in a situation where the other pathway remained activated, resulted in a significant inhibition of myoblast migration. Similarly, inhibition of FAK signalling, using the PF-228 inhibitor did not significantly affect PI3K/AKT and MAPK/ERK pathways, but resulted in reduced myoblast migration, suggesting the indispensability of individual signalling pathways for myoblast migration in response to myotube CM, regardless of the activity of other signalling pathways. Finally, considering the link between myoblast fusion and migration and in an attempt to propose genetic targets for future research, an investigation was made on the expression of Spire and Formin genes, involved in actin polymerisation and intracellular trafficking, in myoblasts undergoing differentiation and fusion. The expression of these genes was revealed in C2C12 myoblasts and it was demonstrated that the expression levels of two of these genes (Spire1 and Formin1) are altered following inhibition of myoblast differentiation/fusion by both 10μM Bpv(Hopic) and serial passaging, suggesting their potential association with these processes. Further investigations to reveal the function of Spire and Formin genes and their protein products in skeletal muscle are proposed