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. 2013 Oct;27(10):4184-93.
doi: 10.1096/fj.13-228486. Epub 2013 Jun 27.

Autophagy is required for exercise training-induced skeletal muscle adaptation and improvement of physical performance

Vitor A Lira  1 , Mitsuharu Okutsu, Mei Zhang, Nicholas P Greene, Rhianna C Laker, David S Breen, Kyle L Hoehn, Zhen Yan
Affiliations

Autophagy is required for exercise training-induced skeletal muscle adaptation and improvement of physical performance

Vitor A Lira et al. FASEB J. 2013 Oct.

Abstract

Pathological and physiological stimuli, including acute exercise, activate autophagy; however, it is unknown whether exercise training alters basal levels of autophagy and whether autophagy is required for skeletal muscle adaptation to training. We observed greater autophagy flux (i.e., a combination of increased LC3-II/LC3-I ratio and LC3-II levels and reduced p62 protein content indicating a higher rate of initiation and resolution of autophagic events), autophagy protein expression (i.e., Atg6/Beclin1, Atg7, and Atg8/LC3) and mitophagy protein Bnip3 expression in tonic, oxidative muscle compared to muscles of either mixed fiber types or of predominant glycolytic fibers in mice. Long-term voluntary running (4 wk) resulted in increased basal autophagy flux and expression of autophagy proteins and Bnip3 in parallel to mitochondrial biogenesis in plantaris muscle with mixed fiber types. Conversely, exercise training promoted autophagy protein expression with no significant increases of autophagy flux and mitochondrial biogenesis in the oxidative soleus muscle. We also observed increased basal autophagy flux and Bnip3 content without increases in autophagy protein expression in the plantaris muscle of sedentary muscle-specific Pgc-1α transgenic mice, a genetic model of augmented mitochondrial biogenesis. These findings reveal that endurance exercise training-induced increases in basal autophagy, including mitophagy, only take place if an enhanced oxidative phenotype is achieved. However, autophagy protein expression is mainly dictated by contractile activity independently of enhancements in oxidative phenotype. Exercise-trained mice heterozygous for the critical autophagy protein Atg6 showed attenuated increases of basal autophagy flux, mitochondrial content, and angiogenesis in skeletal muscle, along with impaired improvement of endurance capacity. These results demonstrate that increased basal autophagy is required for endurance exercise training-induced skeletal muscle adaptation and improvement of physical performance.

Keywords: Bnip3; angiogenesis; mitochondrial biogenesis; mitophagy; voluntary wheel running.

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Figures

Figure 1.
Figure 1.
Tonic, oxidative muscles have higher autophagy flux, autophagy, and mitophagy protein expression than phasic, glycolytic muscles. Immunoblot analyses were performed for homogenates of tonic, oxidative soleus (SO), plantaris (PL) with mixed fiber types, and phasic, glycolytic white vastus lateralis (WV) muscles. A) Representative immunoblot images of LC3, p62, Atg7, Atg6, Bnip3, Pgc-1α, Cyc, Cox4, and β-actin. B) Quantification of LC3-II, LC3-II/LC3-I ratio, and p62 protein collectively as an index of autophagy flux. C) Quantification of LC3 (LC3-I+LC3-II), Atg7, and Atg6 proteins. D) Quantification of the mitophagy protein Bnip3. E) Quantification of Pgc-1α, Cyc, and Cox4 proteins as indexes of mitochondrial biogenesis. Protein expression comparisons were performed after normalization to β-actin. Results are represented as means ± se (n=4/muscle). *P < 0.05 vs. WV; #P < 0.05 vs. PL.
Figure 2.
Figure 2.
Exercise training promotes basal autophagy, autophagy, and mitophagy protein expression in the plantaris muscle with mixed fiber types. Immunoblot analyses were performed for homogenates of plantaris muscles from mice that underwent 4 wk of voluntary wheel-running exercise (EX-4 wk) compared to the sedentary control (SED). A) Representative immunoblot images of LC3, p62, Atg7, Atg6, Bnip3, Pgc-1α, Cyc, Cox4, and β-actin in sedentary and trained mice. B) Quantification of LC3-II, LC3-II/LC3-I ratio, and p62 protein collectively as an index of autophagy flux. C) Quantification of LC3 (LC3-I+LC3-II), Atg7, and Atg6 proteins. D) Quantification of the mitophagy protein Bnip3. E) Quantification of Pgc-1α, Cyc, and Cox4 proteins as indexes of mitochondrial biogenesis. Protein expression comparisons were performed after normalization to β-actin. Results are represented as means ± se (n=10/group). *P < 0.05, **P < 0.01.
Figure 3.
Figure 3.
Exercise training promotes autophagy expression, but not basal autophagy and mitophagy protein expression in tonic, oxidative muscle. Immunoblot analyses were performed for homogenates of soleus muscle from mice that underwent 4 wk of voluntary wheel-running exercise (EX-4 wk) compared to the sedentary control (SED). A) Representative immunoblot images of LC3, p62, Atg7, Atg6, Bnip3, Pgc-1α, Cyc, Cox4, and Gapdh in sedentary and trained mice. B) Quantification of LC3-II, LC3-II/LC3-I ratio, and p62 protein collectively as an index of autophagy flux. C) Quantification of LC3 (LC3-I+LC3-II), Atg7, and Atg6 proteins. D) Quantification of the mitophagy protein Bnip3. E) Quantification of Pgc-1α, Cyc, and Cox4 proteins as indexes of mitochondrial biogenesis. Protein expression comparisons were performed after normalization to Gapdh. Results are represented as means ± se (n=10/group). *P < 0.05, ***P < 0.001.
Figure 4.
Figure 4.
Muscle-specific Pgc-1α overexpression promotes basal autophagy and the mitophagy protein Bnip3 expression, but not autophagy expression in skeletal muscle. Immunoblot analyses were performed for homogenates of plantaris muscles from MCK-Pgc-1α transgenic mice (TG) and WT littermates (WT). A) Representative immunoblot images of LC3, p62, Atg7, Atg6, Bnip3, Pgc-1α, Cyc, Cox4, and Gapdh. B) Quantification of LC3-II, LC3-II/LC3-I ratio, and p62 protein collectively as an index of autophagy flux. C) Quantification of LC3 (LC3-I+LC3-II), Atg7, and Atg6 proteins. D) Quantification of the mitophagy protein Bnip3. E) Quantification of Pgc-1α, Cyc, and Cox4 proteins as indexes of mitochondrial biogenesis. Protein expression comparisons were performed after normalization to Gapdh. Results are represented as mean ± se (n=9–12/group). *P < 0.05, ***P < 0.001.
Figure 5.
Figure 5.
Atg6+/− mice present deficient metabolic adaptations to exercise training in skeletal muscle and no improvement in endurance capacity. Immunoblot and immunofluorescence analyses were performed for plantaris muscles from Atg6+/− mice and WT littermates following 5 wk of voluntary wheel-running exercise (EX) or sedentary cage activity (SED). A) Representative immunoblot images of LC3, p62, Atg7, Bnip3, Pgc-1α, Cyc, Cox4, and β-actin. B) Quantification of LC3-II, LC3-II/LC3-I ratio, and p62 protein collectively as an index of autophagy flux. C) Quantification of LC3 (LC3-I+LC3-II) and Atg7 proteins. D) Quantification of the mitophagy protein Bnip3. E) Quantification of Pgc-1α, Cyc, and Cox4 proteins as indexes of mitochondrial biogenesis. F) Representative immunoblot images of MHCIIa and β-actin (top panel) and quantification of MHCIIa protein (bottom panel). G) Left panel: representative immunofluorescence images of plantaris cross-sections stained with antibodies for MHCIIa (green) and endothelial cell marker CD31 (red). Right panel: quantification of capillary density (right, n=6/group). H) Quantification of treadmill running distance during endurance test. Protein expression comparisons were performed after normalization to β-actin. Results are represented as means ± se (n=9–14/group, unless specified otherwise). *P < 0.05, **P < 0.01 vs. SED condition within the same genotype.
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