Browsing by Author "Goetsch, Kyle Peter."

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The extracellular matrix regulates myoblast migration during wound healing.
(2012) Goetsch, Kyle Peter.; Niesler, Carola Ulrike.; Myburgh, Kathryn Helen.
Mammalian skeletal muscle can regenerate after injury and this response is primarily mediated by the satellite cell, a muscle stem cell. Following injury, satellite cells are activated to myoblasts, undergo rapid proliferation, migrate towards the injury site, and subsequently differentiate into myotubes in order to facilitate functional muscle repair. Fibrosis, caused by the secretion of structural extracellular matrix (ECM) proteins such as collagen I and fibronectin, by fibroblasts, impairs complete functional repair of the muscle. In this study, the role of the microenvironment during wound conditions was assessed by analysing the effect of specific extracellular matrix and growth factors on myoblast migration. The role of the Rho/ROCK pathway as a possible mechanism in mediating the effects seen was investigated. In order to analyse wound repair in an in vitro setting, we optimised and improved a wound healing model specifically designed for skeletal muscle repair. To this end we also developed a co-culture assay using primary myoblasts and fibroblasts isolated from the same animal. The studies showed that collagen I and fibronectin both increased myoblast migration in a dose-dependent manner. Decorin displayed opposing effects on cellular movement, significantly increasing collagen I-stimulated, but not fibronectin-stimulated, migration of myoblasts. ROCK inhibitor studies revealed a significant increase in migration on uncoated plates following inhibition with Y-27632 compared to untreated control. When cells were cultured on ECM components (Matrigel, collagen I, or fibronectin), the inhibitory effect of Y-27632 on migration was reduced. Analysis of ROCK and vinculin expression, and localization at the leading front, showed that ROCK inhibition resulted in loosely packed focal adhesion complexes (matrix dependent). A reduced adhesion to the ECM could explain the increased migration rates observed upon inhibition with Y-27632. We also investigated the role of TGF-β and decorin during wound repair, as TGF-β is a known pro-fibrotic agent. TGF-β treatment decreased wound closure rates; however, the addition of decorin with TGF-β significantly increased wound closure. The addition of ECM components, Matrigel and collagen I enhanced the effect seen in response to TGF-β and decorin; however, fibronectin negated this effect, with no increase in migration seen compared to the controls. In conclusion, the importance of extracellular matrix components in regulating myoblast migration and therefore skeletal muscle wound repair was demonstrated. We emphasize that, in order to gain a better understanding of skeletal muscle wound repair, the combination of ECM and growth factors released during wounding need to be utilised in assays which mimic the in vivo environment more closely.