The role of alterations in mitochondrial dynamics and PGC-1α over-expression in fast muscle atrophy following hindlimb unloading

Abstract

Key points: Skeletal muscle atrophy occurs as a result of disuse. Although several studies have established that a decrease in protein synthesis and increase in protein degradation lead to muscle atrophy, little is known about the triggers underlying such processes. A growing body of evidence challenges oxidative stress as a trigger of disuse atrophy; furthermore, it is also becoming evident that mitochondrial dysfunction may play a causative role in determining muscle atrophy. Mitochondrial fusion and fission have emerged as important processes that govern mitochondrial function and PGC-1α may regulate fusion/fission events. Although most studies on mice have focused on the anti-gravitary slow soleus muscle as it is preferentially affected by disuse atrophy, several fast muscles (including gastrocnemius) go through a significant loss of mass following unloading. Here we found that in fast muscles an early down-regulation of pro-fusion proteins, through concomitant AMP-activated protein kinase (AMPK) activation, can activate catabolic systems, and ultimately cause muscle mass loss in disuse. Elevated muscle PGC-1α completely preserves muscle mass by preventing the fall in pro-fusion protein expression, AMPK and catabolic system activation, suggesting that compounds inducing PGC-1α expression could be useful to treat and prevent muscle atrophy.

Abstract: The mechanisms triggering disuse muscle atrophy remain of debate. It is becoming evident that mitochondrial dysfunction may regulate pathways controlling muscle mass. We have recently shown that mitochondrial dysfunction plays a major role in disuse atrophy of soleus, a slow, oxidative muscle. Here we tested the hypothesis that hindlimb unloading-induced atrophy could be due to mitochondrial dysfunction in fast muscles too, notwithstanding their much lower mitochondrial content. Gastrocnemius displayed atrophy following both 3 and 7 days of unloading. SOD1 and catalase up-regulation, no H2 O2 accumulation and no increase of protein carbonylation suggest the antioxidant defence system efficiently reacted to redox imbalance in the early phases of disuse. A defective mitochondrial fusion (Mfn1, Mfn2 and OPA1 down-regulation) occurred together with an impairment of OXPHOS capacity. Furthermore, at 3 days of unloading higher acetyl-CoA carboxylase (ACC) phosphorylation was found, suggesting AMP-activated protein kinase (AMPK) pathway activation. To test the role of mitochondrial alterations we used Tg-mice overexpressing PGC-1α because of the known effect of PGC-1α on stimulation of Mfn2 expression. PGC-α overexpression was sufficient to prevent (i) the decrease of pro-fusion proteins (Mfn1, Mfn2 and OPA1), (ii) activation of the AMPK pathway, (iii) the inducible expression of MuRF1 and atrogin1 and of authopagic factors, and (iv) any muscle mass loss in response to disuse. As the effects of increased PGC-1α activity were sustained throughout disuse, compounds inducing PGC-1α expression could be useful to treat and prevent muscle atrophy also in fast muscles.

Figure 1. CSA is reduced in gastrocnemius in the early phases of HU

A, quantification of average fibre area from haematoxylin and eosin-stained gastrocnemius muscle from control mice (C) and mice unloaded for 3 days (HU-3) and 7 days (HU-7). B, representative haematoxylin and eosin stainings of the gastrocnemius muscle of control mice (C), and mice unloaded for 3 days (HU-3) and 7 days (HU-7). *Significantly different from C, P < 0.05. Scale bar: 100 μm. Data are presented as means ± SEM.

Figure 2. The antioxidant defence system efficiently reacts to the initial ROS increase

A, determination of H₂O₂ concentration. B, quantification of protein carbonylation level. C, representative OxyBlot and Red Ponceau staining. D, quantification of protein levels of SOD1 and catalase by Western blot. E, representative Western blot of SOD1, catalase and α-tubulin. *Significantly different from C, P < 0.05; #significantly different from HU-3, P < 0.05, n = 6 for each group. Data are presented as means ± SEM.

Figure 3. Mitochondrial fusion alteration is established early during HU in gastrocnemius muscle

A, quantification of mRNA levels of PGC-1α by RT-PCR. B, quantification of protein levels of PGC-1α by Western blot. C, quantification of protein levels of DRP1 involved in fission machinery by Western blot. D, quantification of protein levels of mitochondrial complexes by Western blot. E, quantification of protein levels of citrate synthase by Western blot. F, determination of citrate synthase activity in skeletal muscle. G, determination of OXPHOS capacity, normalized per CS activity. H, quantification of mRNA levels of pro-fusion proteins by RT-PCR. C, control; HU-3, 3 days of hindlimb unloading; HU-7, 7 days of hindlimb unloading. *Significantly different from C, P < 0.05; #significantly different from HU-3, P < 0.05. Data are presented as means ± SEM.

Figure 4. The AMPK pathway is activated early during HU in gastrocnemius muscle

Determination of the activation level of AMP-kinase (A) and of ACC (B) by Western blot measuring the ratio between the phosphorylated (p) and total forms. C, control; HU-3, 3 days of hindlimb unloading; HU-7, 7 days of hindlimb unloading. *Significantly different from C, P < 0.05, n = 6 for each group. Data are presented as means ± SEM.

Figure 5. In transgenic mice, PGC-1α levels are maintained during HU

A, quantification of mRNA levels of PGC-1α in WT and transgenic mice. B, quantification of protein levels of PGC-1α by Western blot in WT and transgenic mice. C, representative Western blot of PGC-1α. *Significantly different from C; #significantly different from C and HU-14 at P < 0.05, n = 5 for each group. Data are presented as means ± SEM.

Figure 6. Increased PGC-1α expression in muscle prevents muscle atrophy during HU

A, CSA of gastrocnemius fibres stained with haematoxylin and eosin. B, Scale bar: 100 μm. C-TgPGC-1α, control, transgenic PGC-1α; HU3-TgPGC-1α, 3 days of hindlimb unloading, transgenic PGC-1α; HU14-TgPGC-1α, 14 days of hindlimb unloading, transgenic PGC-1α. *Significantly different from C, P < 0.05. Data are presented as means ± SEM.

Figure 7. Increased PGC-1α expression in muscle prevents activation of the catabolic systems

A, quantification of mRNA levels of MuRF-1 and atrogin-1 (ubiquitin proteasome system) and of Beclin1 and p62 (autophagy system) by RT-PCR. B, quantification of protein levels of LC3-II based on the ratio between the content in forms II and I of LC3 by Western blotting. C, determination of activity levels of AKT, S6R and 4EBP1 by Western blot analysis of the ratio between the content in the phosphorylated (p) and total forms. D, representative Western blot of synthetic factors. *Significantly different from C, P < 0.05; #significantly different from C-TgPGC-1α, P < 0.05. Data are presented as means ± SEM.

Figure 8. Increased PGC-1α expression in muscle prevents the reduction of pro-fusion proteins

A, quantification of mRNA levels of pro-fusion proteins (Mfn-1, Mfn-2 and OPA-1) by RT-PCR. B, determination of the activation level of ACC by Western blot measuring the ratio between the phosphorylated (p) and total forms. *Significantly different from C, P < 0.05. Data are presented as means ± SEM.

Figure 9. Scheme representing the effects of the early stages of disuse on gastrocnemius muscle

Skeletal muscle disuse would cause down-regulation of pro-fusion proteins leading to mitochondrial dysfunction and AMPK activation. AMPK activation leads to the activation of FoxO, which induces the transcription of genes encoding proteins involved in protein degradation via both the autophagy and the ubiquitin proteasome pathways. The increase in the rate of protein degradation causes gastrocnemius atrophy. AMPK might also prevent the down-regulation of PGC-1α in gastrocnemius in the early stages of HU. Changes of protein synthesis would not be responsible for gastrocnemius atrophy at this stage of disuse.