Autophagy plays a role in skeletal muscle mitochondrial biogenesis in an endurance exercise-trained condition

Abstract

Mitochondrial homeostasis is tightly regulated by two major processes: mitochondrial biogenesis and mitochondrial degradation by autophagy (mitophagy). Research in mitochondrial biogenesis in skeletal muscle in response to endurance exercise training has been well established, while the mechanisms regulating mitophagy and the interplay between mitochondrial biogenesis and degradation following endurance exercise training are not yet well defined. The purpose of this study was to examine the effects of a short-term inhibition of autophagy in response to acute endurance exercise on skeletal muscle mitochondrial biogenesis and dynamics in an exercise-trained condition. Male wild-type C57BL/6 mice performed five daily bouts of 1-h swimming per week for 8 weeks. In order to measure autophagy flux in mouse skeletal muscle, mice were treated with or without 2 days of 0.4 mg/kg/day intraperitoneal colchicine (blocking the degradation of autophagosomes) following swimming exercise training. The autophagic flux assay demonstrated that swimming training resulted in an increase in the autophagic flux (~100 % increase in LC3-II) in mouse skeletal muscle. Mitochondrial fusion proteins, Opa1 and MFN2, were significantly elevated, and mitochondrial fission protein, Drp1, was also increased in trained mouse skeletal muscle, suggesting that endurance exercise training promotes both mitochondrial fusion and fission processes. A mitochondrial receptor, Bnip3, was further increased in exercised muscle when treated with colchicine while Pink/Parkin protein levels were unchanged. The endurance exercise training induced increases in mitochondrial biogenesis marker proteins, SDH, COX IV, and a mitochondrial biogenesis promoting factor, PGC-1α but this effect was abolished in colchicine-treated mouse skeletal muscle. This suggests that autophagy plays an important role in mitochondrial biogenesis and this coordination between these opposing processes is involved in the cellular adaptation to endurance exercise training.

Keywords: Autophagy; Exercise; Mitochondrial biogenesis; Mitophagy; Skeletal muscle.

Fig. 1

Eight-week swimming exercise training increases expression of mitochondrial enzymes in triceps muscle. Representative blots are shown at the top of the figure (a). Average ALA synthase and cytochrome C protein values in muscles of control and exercised mice (b). Each bar represents the mean ± SE for muscles from eight mice. *p < 0.05 vs. sedentary controls

Fig. 2

Eight-week swimming exercise training increases autophagic flux in mouse skeletal muscle. Representative immunoblot images of LC3, p62, LAMP1 or actin (a). LC3 II/actin (b), LC3 I/actin (c), LC3 II/LC3 I ratio (d), p62/actin (e) and LAMP1/actin (f) ratios were quantitated via densitometry from 8 mice per treatment conditions. Example immunoblot of Beclin-1/Atg6, Atg7 or actin (h). Quantification of Beclin-1/actin (i) and Atg7/actin (j); values are means ± SE; (n = 8) * p < 0.05 vs. sed+sal, ^# p < 0.05 vs. sed+col. Expression of p62 mRNA in triceps muscles of mice was measured following 8-week of swimming exercise training. mRNA levels are relative to the GAPDH (n = 4 per group) (g)

Fig. 2

Eight-week swimming exercise training increases autophagic flux in mouse skeletal muscle. Representative immunoblot images of LC3, p62, LAMP1 or actin (a). LC3 II/actin (b), LC3 I/actin (c), LC3 II/LC3 I ratio (d), p62/actin (e) and LAMP1/actin (f) ratios were quantitated via densitometry from 8 mice per treatment conditions. Example immunoblot of Beclin-1/Atg6, Atg7 or actin (h). Quantification of Beclin-1/actin (i) and Atg7/actin (j); values are means ± SE; (n = 8) * p < 0.05 vs. sed+sal, ^# p < 0.05 vs. sed+col. Expression of p62 mRNA in triceps muscles of mice was measured following 8-week of swimming exercise training. mRNA levels are relative to the GAPDH (n = 4 per group) (g)

Fig. 3

Mitochondrial dynamics proteins are increased in muscle after 8-week swimming training. Muscle homogenates were subjected to immunoblotting with MFN2 (b), Opa1 (c), Drp1 (e), Fis1 (f), or actin. Each bar represents the mean ± SE for muscles from eight mice. (n = 8) * p < 0.05 vs. sed+sal, ^# p < 0.05 vs. sed+col, ^δ p < 0.05 vs. exe+sal

Fig. 4

Elevated mitophagy induced by exercise training may be mediated by Bnip3 in mouse skeletal muscle. Representative immunoblot images (a) and densitometric quantification of effects of 8-week swimming training on mitophagy signaling proteins PINK1/Parkin (b, d) and Bnip3 (f). Values are means ± SE; (n = 8) * p < 0.05 vs. sed+sal, ^# p < 0.05 vs. sed+col. ^δ p < 0.05 vs. exe+sal. Expression of PINK1 (c), Parkin (e) or Bnip3 (g) mRNA in triceps muscles of mice was measured following 8-week of swimming exercise training. mRNA levels are relative to the GAPDH (n = 4 per group)

Fig. 5

Increased mitochondrial biogenesis is abolished by inhibiting mitophagy in mouse skeletal muscle. Representative immunoblot images of PGC-1α, SDH and COX IV (a), which are indexes of mitochondrial biogenesis. Densitometric quantification of PGC-1α (b), SDH (c), COX IV (d), and CS enzyme activity (f). Values are means ± SE; (n = 8) * p < 0.05 vs. sed+sal, ^# p < 0.05 vs. sed+col, ^δ p < 0.05 vs. exe+sal. Relative mRNA expression of PGC-1α in triceps muscles of mice was measured following 8-week of swimming exercise training. mRNA levels are relative to the GAPDH (n = 4 per group) (e). Data represent mRNA levels are relative to the GAPDH (n = 4 per group). Amount of mitochondrial DNA (16s rRNA) adjusted by nuclear DNA (hexokinase 2) in triceps muscles isolated from the control and swimming trained mice (n = 4 per group) (g)