Abstract:

Objective To explore differentially expressed proteins between tibialis anterior muscle and soleus muscle after denervation by proteomics, and to analyze the changes of tumor necrosis factor receptor-associated factor 6（TRAF6）expression and its significance between tibialis anterior muscle and soleus muscle after denervation. Methods Rat models were prepared by cutting sciatic nerve. The protein expression changes between tibialis anterior muscle and soleus muscle after denervation were analyzed by isobaric tags for relative and absolute quantitation (iTRAQ) quantitative proteomics methods. The differentially expressed proteins were compared between tibialis anterior muscle and soleus muscle to screen key proteins regulating muscle atrophy. The expression was verified by Western blotting, and the biological role of key proteins was further validated in vitro. Results The atrophy of slow-twitch soleus muscle was faster than fast-twitch tibialis anterior muscle after denervation（P<0.05，n=20）. There were 30 proteins which displayed different expression between tibialis anterior muscle and soleus muscle. These differentially expressed proteins included metabolic-related proteins, chaperones, contractile proteins and signaling molecules. Among the including signaling molecules, TRAF6 displayed a significant increase in slow-twitch soleus muscle compared to fast-twitch tibialis anterior muscle after denervation, which was confirmed by Western blotting（P<0.05，n=6）. Muscle ring-finger protein-1（MuRF1）and muscle atrophy F-box（MAFbx), target genes of TRAF6, also showed a markly increase in slow-twitch soleus muscle compared to fast-twitch tibialis anterior muscle after denervation（P<0.05，n=6）. In order to further investigate the impact of TRAF6 in muscle atrophy, TRAF6 siRNA and control siRNA were tranfected into myotubes, which was then treated by dexamethasone. The results showed that the diameter of myotubes transfected with TRAF6 siRNA was significantly larger than that in the myotubes transfected with control siRNA（P<0.05，n=6）. The expression of TRAF6, MuRF1 and MAFBx in TRAF6 siRNA transfection group were significantly lower than that in the control siRNA transfected group（P<0.05，n=6）, suggesting that TRAF6 siRNA effectively inhibited the expression of the target gene TRAF6, also inhibited the expression of downstream its target genes of MuRF1 and MAFBx. Conclusion TRAF6 displays a significant increase in slow-twitch soleus muscle compared to fast-twitch tibialis anterior muscle after denervation. Inhibition of TRAF6 alleviates myotube atrophy induced by dexamethasone. Therefore, we speculate that the atrophy of slow-twitch muscle is faster than fast-twitch muscle after denervation, which is possible caused by the higher expression of TRAF6 in slow-twitch muscle after denervation.

Key words: Nerve injury, Muscle atrophy, Tumor necrosis factor receptor-associated factor 6, Western blotting, Rat