Figure 3

Effect of exercise on muscle fibrosis in mdx mice. (A) Sirius Red and H&E staining of gastrocnemius muscle of mdx mice that were exercised three times weekly, for 30 minutes at a speed of 12 meters per minute with a rest of 5 minutes each 10 minutes of exercise, for one, two and three months, compared to sections of muscle from unexercised mdx mice. All the samples were collected when the animals were six months old (see the Methods section). (B) Representative immunofluorescence for collagen I and fibronectin in muscle sections of control or exercised mice as shown in (A). (C) Quantitative RT-PCR of connective tissue growth factor (CTGF) and transforming growth factor beta 1 (TGFβ1) mRNA levels after exercising for the indicated period as compared to unexercised age-matched mdx mice. Data correspond to the mean ± SEM; n = 4 sedentary and 4 exercised mdx mice for each exercise time point. One-way analysis of variance with Tukey’s post hoc multiple comparison test; *P <0.05, **P <0.01, ***P <0.001 versus control. (D) Biochemical quantification of collagen protein content in mdx gastrocnemius muscle after exercising for the indicated periods, as compared to unexercised age-matched mdx mice. Data correspond to the mean ± SEM; n = 4 sedentary and 4 exercised mdx mice for each exercise time point. One-way analysis of variance with Tukey’s post hoc multiple comparison test; *P <0.05, **P <0.01, ***P <0.001 versus control. (E) Immunofluorescence for phosphorylated-Smad2/3 proteins on sections from gastrocnemius muscle of six-month-old mdx mice after three months of exercise, as evidence for TGFβ activation, compared to unexercised age-matched control mdx mice. (F) Ex vivo maximum isometric force (tetanic force) of gastrocnemius muscle of age-matched unexercised and three-month-trained mdx mice. Values as mean ± SEM; n = 7 on each group. Non-parametric Mann–Whitney U test; **P <0.01 versus non-exercised. Scale bars = 50 μm.