The effect of Wnt isoforms on myogenesis.
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.