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. 2014 Jan;35(1):135-42.
doi: 10.1038/aps.2013.144. Epub 2013 Dec 16.

Exercise ameliorates the detrimental effect of chloroquine on skeletal muscles in mice via restoring autophagy flux

Dan Jiang  1 , Kai Chen  2 , Xuan Lu  1 , Hong-jian Gao  3 , Zheng-hong Qin  1 , Fang Lin  1
Affiliations

Exercise ameliorates the detrimental effect of chloroquine on skeletal muscles in mice via restoring autophagy flux

Dan Jiang et al. Acta Pharmacol Sin. 2014 Jan.

Abstract

Aim: To study the roles of autophagy in muscle establishment during long-term exercise in mice.

Methods: Female ICR mice were submitted to exercise training with a wheel running regimen: 6 m/min, 15 min/time, 3 times/d (on 8:00, 14:00, and 20:00), 5 d/week for 2 months. The mice were treated with the autophagy activator trehalose (1% aqueous solution as a daily drinking water) or the autophagy inhibitor chloroquine (10 mg/kg, ip, 5 times a week) before the training. Western blotting analysis, TUNEL staining, H&E staining and transmission electron microscope were used to evaluate the activity of autophagy and the structure of the muscle fibers.

Results: The exercise training significantly stimulated the formation of autophagosomes, increased the LC3-II, cathepsin L and Bcl-2 levels, lowered the P62 level and increased the antioxidant capacity in the muscles. Meanwhile, the exercise training significantly improved the morphology of mitochondria, reduced the release of cytochrome c from mitochondria to cytoplasm, and slightly decreased the apoptosis rate in the muscles. Administration of trehalose increased the level of autophagy and protected the muscle fibers from apoptosis. Administration of chloroquine blocked autophagy flux and exerted detrimental effects on the muscles, which were ameliorated by the exercise training.

Conclusion: Long-term regular exercise activates autophagy process associated with muscle establishment, and ameliorates the detrimental effects of chloroquine on skeletal muscles via restoring autophagy flux.

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Figures

Figure 1
Figure 1
Exercise enhances muscle fibers damaged by chloroquine. All mice were assigned into six groups (n=8): untrained mice treated with saline (NS), untrained mice treated with trehalose (Tre), untrained mice treated with chloroquine (Chl), trained mice (Exe), trained mice treated with trehalose (Exe+Tre), and trained mice treated with chloroquine (Exe+Chl). Autophagy stimulator trehalose (1%) and autophagy inhibitor chloroquine (10 mg/kg) were the positive (+) and negative (−) controls, respectively. (A) The body weight. (B) The food consumption. (C) The fat/body ratio. Exercise decreased the body fat weight. (D) Exercise and autophagy inducer improved the morphology of muscle fibers. Muscle fibers were stained by H&E staining to expose the morphology, n=8. (E) Exercise and autophagy inducer improved the muscle fibers ultra-structure. The internal structure of the skeletal muscle fiber was observed under the lens of the electron microscope from 3 mice in each group. Scale bars: 1 μm. I: light band, A: dark band, M: M-line, H: H-band, Z: Z-line. Bar represents mean±SD, n=8. bP<0.05, cP<0.01 compared with the NS group or the trained group.
Figure 2
Figure 2
Exercise enhances autophagy activity in skeletal muscle and recovers autophagy level blocked by chloroquine (Chl) in lateralis muscle. (A) Protein expression and quantitative analysis of LC3 and P62. (B) The formation of autophagosomes were induced by exercise (Exe) and trehalose (Tre). White arrows indicate autophagosomes. Autophagosomes in the pictures were enlarged in order to observe the morphology clearly. For Western blotting, levels of GAPDH and Histone protein were used as the loading control. The data were from the same PVDF membrane, and were representative of at least three independent experiments. Quantitative analysis was performed with Sigma Scan Pro 5. Bar represents mean±SD from 6 mice in every group. bP<0.05, cP<0.01 compared with the NS group or the trained group.
Figure 3A-3H
Figure 3A-3H
Exercise enhances the mitochondria function and recovers the toxic effect of chloroquine on skeletal muscles. Quantitative analysis of (A) SOD levels. (B) GSH levels. (C) MDA levels. (D) The morphology of mitochondria. Scale bar=1 μm. White arrows indicate mitochondria. (E) The muscle tissue was stained by TUNEL (TdT-mediated dUTP nick end labeling), n=8. (F) Quantitative analysis of the number of apoptosis cells. (G) Protein levels and quantitative analysis of Bcl-2. (H) Immunoblotting and quantitative analysis of Bnip 3. Bar represents mean±SD from 6 mice in every group. bP<0.05, cP<0.01 compared with the NS group or the trained group.
Figure 3I
Figure 3I
(I) The level of cytochrome c in mitochondria and cytoplasm. Levels of GAPDH and Cox IV protein were used as the loading control. Quantitative analysis was performed with Sigma Scan Pro 5. Bar represents mean±SD from 6 mice in every group. bP<0.05, cP<0.01 compared with the NS group or the trained group.
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