The extracellular matrix regulates myoblast migration during wound healing.

Abstract

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.