Figure 4.

Treadmill exercise induces autophagy in wild-type muscle but not in Col6a1^–/– mice. (A) Western blot analysis for Akt phosphorylation and LC3 lipidation in TA (left) and diaphragm (right) muscles from 5-mo-old wild-type and Col6a1^–/– mice housed in standard conditions (no run) or after 1 h treadmill exercise (TM). (B) Densitometric quantifications of P-Akt levels and LC3-II vs LC3-I ratio, as determined by western blots of TA (left) and diaphragm (right) (*p < 0.05, n = 3). (C) Electron micrograph of double-membrane (arrowheads) autophagosome in TA of wild-type mice after treadmill exercise. Scale bar, 200 nm. (D) Fluorescence microscope analysis of cryosections of TA from wild-type;GFP-LC3 and Col6a1^–/–;GFP-LC3 mice after treadmill exercise. GFP-LC3 puncta (green fluorescence) are abundant in wild-type muscle fibers, but they are scarce and poorly detectable in Col6a1^–/– myofibers. Immunohistochemistry for laminin (red fluorescence) was performed to reveal myofiber outline, while Hoechst staining (blue) was used to label nuclei. The lower panels are higher magnifications of the squared areas, clearly showing GFP-LC3 puncta (arrowheads) in wild-type but not in Col6a1^–/– myofibers. Scale bar, 25 μm. TM, treadmill; WT, wild-type.