Figure 4
β2-AR agonist, clenbuterol treatment increased GC muscle mass and protein expression of MHC even with IS treatment, but could not reverse muscle weakness. (A) The ratio of mouse body weight change before and after one-week treatment of indoxyl sulfate (IS), and IS plus clenbuterol is shown. (B) The ratio of gastrocnemius (GC) muscle wet weight over the total body weight of mice after one-week treatment is shown. (C) Immunohistochemical analysis of fast (top, green) and slow (bottom, green) myosin heavy chain (MHC) isoforms counterstained with laminin (red) was performed for mouse GC muscle cryosections obtained from those 2 groups. The scale bar is 50 μm. The cross-sectional area and size distribution of fast and slow myofibers was quantitated (right). As a statistical analysis, a Cochran-Armitage trend test for fiber size distribution was performed. N = 5–6 (fast twitch) and 3 (slow twitch) mice per group. Error bars denote SEM. (D) Western blotting analysis of the fast and slow MHC isoforms in GC muscle obtained from those 2 groups was performed (top). Ponceau S staining was examined as a loading control (bottom). A full-length view of the membrane is shown in the supplementary Fig. 4. (E) Maximal running velocity (Vmax) measured in treadmill running exhaustion tests (left) and grip strength measured in four limbs grip tests (right) are shown. (F) 4-HNE staining on mouse GC muscle cryosections after one week of treatment with vehicle or clenbuterol and IS (left). The same cryosections without adding the first anti-4-HNE antibody were also examined as a negative control. The scale bar is 50 μm. Signal intensity was quantitated (right). N = 3–6 mice per group. Error bars indicate SEM. ** p < 0.01, *** p < 0.001. #; p < 0.05 compared to vehicle (VEH) without IS treatment. Clen; clenbuterol. N.S.; no significance.
