Figure 7. Elastase impairs myoblast proliferation, MyoD1 expression, myoblast fusion and myotube growth in a substrate-independent manner.

(a) C2C12 myoblasts were cultured in growth medium in the presence/absence of 0.75 U/mL elastase for 24 hours then fixed and immunostained as indicated (NT = non-treated). Arrowheads show examples of mitotic cells identified by strong Ki67 staining and condensed chromatin. (b,c) Quantification of (a) where myoblasts were cultured in the presence/absence of either 0.5 or 0.75 U/mL elastase. Plots in (b,c) are averages of 10 technical replicates for each one of 3 biological replicates (n = 30 images per data-point). Error bars are S.E.M., *p < 0.05, **p < 0.01. Scale bars: 30 μm. (d,e) The levels of MyoD1 are decreased in myoblasts treated with elastase. C2C12 myoblasts were cultured in growth medium for 1 day then for an additional 1 day in the presence/absence of 0.6 U/mL elastase as indicated (NT = non-treated) prior to fixation and immunostaining to detect MyoD1 and nuclei (DAPI) as indicated. (f) Myoblast fusion and myotube growth are impaired by elastase treatment. C2C12 myoblasts were cultured in growth medium until confluent then switched to differentiation medium in the presence/absence of 0.15 U/mL elastase as indicated and maintained for an additional 4 days prior to fixation and immunostaining to detect myosin heavy chain (MyHC) and nuclei (DAPI). (g,h) Quantification of (f) where the fusion index is the percentage of MyHC + cells in myotubes over the total number of MyHC + cells (g), while the myotube area (h) is calculated as total MyHC + area per image. Plots are averages of 10–15 technical replicates across 4 biological replicates (n > 40 per data-point). NT = non-treated, Elast = 0.15 U/mL elastase. Error bars are S.E.M. **p < 0.01. Scale bars are: 30 μm in (a,b) and 100 μm in (f).