Expression profile of Wnt isoforms during differentiation of aging C2C12 myoblast cells.
Satellite cells are known as the definitive muscle stem cells and are responsible for skeletal muscle maintenance and repair. The capacity of these satellite cells to participate in myogenesis decreases with age and as a result, muscle repair and maintenance in an aging organism is characterized by fibrosis, lipid accumulation and atrophy, a process known as sarcopenia. Recent parabiotic studies have shown that satellite cells with reduced myogenic capability in aging muscle can be rejuvenated to undergo effective myogenesis when exposed to a young environment. Further analysis has suggested that the Wnt family of signaling proteins identified in serum is pivotal in regulating cell fate, proliferation and differentiation, during aging. Wnt3a is known to regulate fibrogenensis, Wnt10b adipogenesis and Wnt7 myogenesis. In the current study, we aim to determine the cytosolic and secreted expression profiles of the three Wnt isoforms, Wnt3a, 7 and 10b, during myogenesis of early and late passage C2C12 myoblasts. We then extend our analysis to determine whether conditioned media could improve the myogenic capacity of late passage cells. Late passage C2C12 cells had elevated Wnt3a cytosolic levels along with reduced differentiation capacity and a rapidly declining Wnt7 levels, in comparison to early passage cells. The elevated Wnt3a suggests an elevated fibrogenic predisposition, whereas the declining Wnt7 cytosolic levels, a decrease in myogenic capacity. Furthermore, analysis of the secreted vs. cytosolic ratio in Wnt7 levels revealed a more rapid decline in late vs. early passage cells during differentiation, supporting the observed decreased myogenic ability. Moreover, late passage cells also showed lower Wnt10b levels compared to early passage cells. This low level of Wnt10b is likely associated with an increase in adipogenic predisposition. The results obtained in the cross-over experiments indicated that conditioned media from early passage cells did not improve the differentiation of late passage cells by the low levels of Myogenin and MHC. However, early passage cells treated with conditioned media from late passage cells surprisingly showed a marginal increase in both Myogenin and MHC levels. Interestingly, cytosolic Wnt3a and 7 in late passage cells treated with ‘young media’ were increased compared to control whereas early passage cells treated with ‘old’ media showed significantly decreased levels of Wnt3a and 7. Furthermore, early passage cells acquired a declining expression when treated with ‘young’ media whereas late passage cells had an increasing level when treated with ‘old’ media. This indicates a possible improvement in differentiation in late passage cells. Taken together, our results support a role for Wnt7 and Wnt10b in promoting myogenesis while Wnt3a may decrease myogenesis. With the increase in passage numbers, the reduced myogenic predisposition is regulated by reduced Wnt10b, 7 and elevated Wnt3a levels, respectively. Moreover, we speculate that the lack of myogenic improvement in the cross-over experiment could be the presence of unknown secreted factors in ‘young’ media that impedes myogenesis. Finally, cell lines are known to be biologically different to primary myoblasts through the accumulation of mutations which could render the cells less sensitive to growth factors. Therefore, it is imperative that the current study is repeated with primary culture myoblasts.