The extracellular matrix regulates myoblast migration during wound healing.
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Date
2012
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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.
Description
Thesis (Ph.D.)-University of KwaZulu-Natal, Pietermaritzburg, 2012.
Keywords
Muscles--Regeneration., Muscles--Wounds and injuries., Myoblasts., Hepatocyte growth factor., Fibroblast growth factors., Theses--Biochemistry.