The effect of Wnt isoforms on myogenesis.
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Date
2014
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Abstract
Satellite cells are muscle stem cells that are responsible for the growth and repair of
skeletal muscle tissue. Satellite cells typically exist in a quiescent state in their niche
between the sarcolemma and basal lamina. In response to muscle tissue injury, activated
satellite cells, otherwise known as myoblasts, migrate to the site of injury where they
proliferate and subsequently differentiate and fuse to repair damaged myofibers. The
success of muscle growth and repair is highly dependent on the speed and degree to which
these myoblasts migrate, proliferate and differentiate. This overall process, referred to as
myogenesis, is largely controlled by the myogenic regulatory factors, a group of basic helixloop-
helix transcription factors including MyoD, Myf5, myogenin and Mrf4. It has
recently been found that the Wnt family of secreted signalling proteins are highly involved
in the regulation of developmental processes such as myogenesis. Wnt proteins are a
family of 21 highly-conserved, secreted, cysteine-rich signalling molecules which are found
in all multi-cellular organisms. Wnt signalling is highly versatile and is initiated by the
binding of extracellular Wnt to cell-surface Frizzled receptors (Fz). It is highly dependent
on both the Wnt isoform and Fz type and may initiate one of three known signalling
pathways. Wnt3A and Wnt7A are of particular interest as they have previously been
linked with myogenesis. C2C12 myoblasts over-expressing Wnt3A have been seen to have
reduced levels of motility and terminal differentiation. Wnt7A is suspected to maintain a
healthy satellite cell pool by regulating self-renewal; injection of recombinant Wnt7A into
mouse leg muscle resulted in increased satellite cell numbers. In vitro Wnt studies have
typically involved the treatment of mouse cells with conditioned medium containing Wnt,
often at unknown concentrations. In our study we wished to test the effects of known
concentrations of recombinant Wnt3A and Wnt7A on mouse C2C12 and donor-derived
human skeletal muscle myoblasts (HSkM) in vitro. Wnt3A and Wnt7A were seen to
increase the rate of C2C12 migration in a dose dependent manner. HSkM cells treated
with 10 ng/ml Wnt3A also displayed increased motility. Neither Wnt3A nor Wnt7A were
seen to have any significant effects on the proliferation of C2C12 or HSkM cells. Wnt3A
(10ng/ml and 100 ng/ml) but not Wnt7A was seen to decrease C2C12 terminal
differentiation as measured by expression of myosin heavy chain (MyHC). Subsequent
confocal microscopy revealed that Wnt3A significantly reduced the percentage of MyoD+
C2C12 nuclei during differentiation. A reduction in nuclear MyoD would support the
observed impaired commitment to differentiation. However, donor-derived human
skeletal muscle myoblasts treated with 10 ng/ml Wnt3A were not seen to have significantly
reduced nuclear MyoD levels or terminal differentiation; the reason for this is unclear but
may relate to a number of factors including the concentration of Wnt, Fz and co-receptor
profiles and the presence of specific extracellular matrix and serum factors. These studies
provide new insight into the role of Wnts in myogenesis and lay the foundation for future
work on Wnt3A and Wnt7A.
Description
Thesis (M.Sc.)-University of KwaZulu-Natal, Pietermaritzburg, 2014.
Keywords
Satellite cells., Myoblasts., Muscle cells., Wnt proteins., Theses--Biochemistry.