Relationships between endurance exercise training-induced muscle fiber-type shifting and autophagy in slow- and fast-twitch skeletal muscles of mice

Abstract

Purpose: Endurance exercise induces muscle fiber-type shifting and autophagy; however, the potential role of autophagy in muscle fiber-type transformation remains unclear. This study examined the relationship between muscle fiber-type shifting and autophagy in the soleus (SOL) and extensor digitorum longus (EDL) muscles, which are metabolically discrete muscles.

Methods: Male C57BL/6J mice were randomly assigned to sedentary control (CON) and exercise (EXE) groups. After 1 week of acclimation to treadmill running, the mice in the EXE group ran at 12-15 m/min, 60 min/day, 5 days/week for 6 weeks. All mice were sacrificed 90 min after the last exercise session, and the targeted tissues were rapidly dissected. The right side of the tissues was used for western blot analysis, whereas the left side was subjected to immunohistochemical analysis.

Results: Endurance exercise resulted in muscle fiber-type shifting (from type IIa to type I) and autophagy (an increase in LC3-II) in the SOL muscle. However, muscle fiber-type transformation and autophagy were not correlated in the SOL and EDL muscles. Interestingly, in contrast to the canonical autophagy signaling pathways, our study showed that exercise-induced autophagy concurs with enhanced anabolic (increased p-AKTSer473/AKT and p-mTOR/mTORSer2448 ratios) and suppressed catabolic (reduced p-AMPKThr172/AMPK ratio) states.

Conclusion: Our findings demonstrate that chronic endurance exercise-induced muscle fiber-type transformation and autophagy occur in a muscle-specific manner (e.g., SOL). More importantly, our study suggests that endurance training-induced SOL muscle fiber-type transition may underlie metabolic modulations caused by the AMPK and AKT/mTOR signaling pathways rather than autophagy.

Keywords: AKT/mTOR; AMPK; LC3-II; exercise training adaptation; extensor digitorum longus; muscle fiber-type; soleus.

Figure 1.

The experimental design of this study includes an exercise training protocol.

Figure 2.

Muscle fiber type transformation and changes in the cross-section area (CSA) in response to 6 weeks of endurance exercise training. A. Representative images of immunohistochemical staining of soleus (SOL) muscle fibers: dystrophin (green), type I (blue), type IIa (red) muscle fibers, and merged images of type I and type IIa, wherein type IIx + type IIb fibers are in black, in the control (CON) and exercise (EXE) groups. B. Bar graph displaying percentages of each fiber type (type I: blue, type IIa: red, and type IIx+IIb: black) in the SOL muscle in the CON and EXE groups. C. The CSA of type I, type IIa, and type IIx+IIb fibers in the SOL muscle in the CON and EXE groups. D. Representative images of immunohistochemical staining of extensor digitorum longus (EDL) muscle fibers: dystrophin (green), type IIa (red), type IIb (blue), and merged image of type IIa and type IIb, wherein type IIx is shown in black. E. Bar graph displaying percentages of type IIa (red), type IIx (black), and type IIb (blue) fibers in the EDL muscle in the CON and EXE groups. F. The CSA of type IIa, type IIx, and type IIb fibers in the EDL muscle in the CON and EXE groups. Data are expressed as the mean ± SEM (n=6 per group). Statistically significant differences are denoted by asterisks: p < 0.05 (*), p < 0.01 (**), and p < 0.001 (***) compared with the CON group. Scale bar = 200 μm.

Figure 3.

Associations between endurance exercise-induced autophagy and muscle fiber-type shifting. A. Representative western blot images of LC3 proteins and Ponceau-stained total proteins as a loading control in soleus (SOL) and extensor digitorum longus (EDL) muscles of the CON and EXE groups. B. Protein quantification of LC3-I, LC3-II, and the LC3-II/I ratio in the SOL muscle in the CON and EXE groups. C. Protein quantification of LC3-I, LC3-II, and the LC3-II/I ratio in the EDL muscle in the CON and EXE groups. D. Correlations between LC3-II protein levels and type I fibers. E. Correlations between LC3-II protein levels and type IIa fibers. F. Correlations between LC3-II protein levels and type IIx+IIb fibers in the SOL muscle in the CON and EXE groups. G. Correlations between LC3-II protein levels and type IIa fibers. H. Correlations between LC3-II protein levels and type IIx fibers. I. Correlations between LC3-II protein levels and type IIb fibers in the EDL muscle in the CON and EXE groups. Data are expressed as the mean ± SEM (n=6-7 per group). Pearson’s correlation coefficient (r) and two-tailed p-value are presented in the bar graphs for the correlation analysis. Statistically significant differences are denoted by an asterisk: p < 0.05 (*) compared with the CON group. LC3, microtubule-associated proteins 1A/1B light chain 3.

Figure 4.

Changes in autophagy-related proteins in SOL and EDL muscles in response to endurance exercise training. A. Representative western blot images of autophagy-related proteins and Ponceau-stained total proteins as a loading control in the soleus (SOL) and extensor digitorum longus (EDL) muscles of the control (CON) and exercise (EXE) groups. B and C. Protein quantification of BCL2, p-BCL2^Ser87, and the p-BCL2^Ser87/BCL2 ratio in the SOL and EDL muscles in the CON and EXE groups, respectively. D-H. Protein quantification of BECN1, ATG7, p62/SQSTM1, LAMP2, and CTSL in the SOL and EDL muscles in the CON and EXE groups, respectively. Data are expressed as the mean ± SEM (n=6-7 per group). Statistically significant differences are denoted by asterisks: p < 0.05 (*), p < 0.01, (**), and p < 0.001 (***) compared with the CON group. BCL2, B-cell lymphoma 2; BECN1, Beclin 1; ATG7, autophagy-related protein 7; SQSTM1, sequestosome 1; LAMP2, lysosomal associated membrane protein 2; CTSL, cathepsin L.

Figure 5.

Alterations in autophagy regulatory proteins in response to endurance exercise training in SOL and EDL muscles. A. Representative western blot images of catabolic (AMPK), anabolic (AKT and mTOR), and autophagy initiation proteins (ULK1) in soleus (SOL) and extensor digitorum longus (EDL) muscles in the control (CON) and exercise (EXE) groups. B and C. Protein quantification of AMPK, p-AMPK^Thr172, and the p-AMPK^Thr172/AMPK ratio in the SOL and EDL muscles, respectively. D and E. Protein quantification of AKT, p-AKT^Ser473, and the p-AKT^Ser473/AKT ratio in the SOL and EDL muscles in the CON and EXE groups, respectively. F and G. Protein quantification of mTOR, p-mTOR^Ser2448, and the p-mTOR^Ser2448/mTOR ratio in the SOL and EDL muscles in the CON and EXE groups, respectively. H and I. Protein quantification of ULK1, p-ULK1^Ser555, and the p-ULK1^Ser555/ULK1 ratio in the SOL and EDL muscles in the CON and EXE groups, respectively. J and K. Protein quantification of ULK1, p-ULK1^Ser757, and the p-ULK1^Ser757/ULK1 ratio in the SOL and EDL muscles in the CON and EXE groups, respectively. Data are expressed as the mean ± SEM (n=6-7 per group). Statistically significant differences are denoted by asterisks: p < 0.01 (**) and p < 0.001 (***) compared with the CON group. AMPK, 5’adenosine monophosphate (AMP)-activated protein kinase; mTOR, mammalian target of rapamycin; ULK1, Unc-51-like autophagy-activating kinase 1.