| Objective:Hypoxia-induced skeletal muscle atrophy not only affects the athletes’altitude training effect,but also affects the physical fitness of the people living in the plains after reaching the altitude.Myostatin/Smad3 signaling pathway plays an important regulatory role in skeletal muscle atrophy.However,it is unclear whether this pathway participates in the regulation process under the influence of hypoxia and resistance training.Therefore,this study explored the changes of Myostatin/Smad3 signaling pathway and its downstream factors in resistance training to relieve hypoxia-induced skeletal muscle atrophy through animal experiments.Methods:Forty male 8-week-old Sprague-Dawley rats were randomly divided into 4 groups of 10 rats,the normoxic control group（C）,the normoxic exercise group（R）,the hypoxic control group（H）,and the hypoxic exercise group（HR）.The hypoxic group lived in a hypoxic environment（12.4%O2,24 h/day）.The exercise group carried out weight-bearing ladder resistance exercise every other day,3sets/session and 5 repeats/set.After 4 weeks of hypoxia and resistance exercise,the soleus muscle（SOL）,extensor digitorum longus muscle（EDL）,and biceps brachii muscle（BRA）were separated and weighed.After HE staining,the cross-sectional area of muscle fibers（FCSA）was observed.RT-qPCR was used to detect the transcription levels of Myostatin,Follistatin,Act R2B,Smad3,Smad7,MyoD,Myogenin,MyHC subtypes.Western Blotting was used to detect the protein expression levels of Myostatin,Follistatin,ActR2B,Smad3,P-Smad3,Smad7,MyoD and Myogenin.By immunohistochemistry,the main positions of Myostatin protein expression were observed.The STRING 11.0 protein interaction software was used to analyze the interaction network relationships between genes and proteins that showed significant changes after hypoxia and hypoxic resistance exercise.Results:1)After hypoxic exposure,the relative wet weight of SOL and the relative FCSA of EDL and BRA were decreased（P<0.05）.The relative wet weight of EDL and BRA,and the relative FCSA of BRA were significantly higher in group HR than in group H（P<0.05）.2)MyHC1 and MyHC2A mRNA expressions in SOL and BRA of group H（both P<0.01）,and MyHC2B mRNA expressions（both P<0.05）in EDL and BRA were significantly lower than those in group C.The expressions of MyHC1 and MyHC2B mRNA in the BRA and MyHC2X and MyHC2B mRNA in the EDL were significantly higher in group HR than in group H（BRA MyHC1:P<0.01;all other P<0.05）.3)The EDL and BRA MyoD mRNA expression（P<0.05,P<0.01）,and SOL Myogenin mRNA expression（P<0.05）in group H were significantly lower than those in group C.The expression of MyoD mRNA of the EDL and BRA（both P<0.01）,and Myogenin mRNA of the BRA（P<0.05）in the HR group were significantly higher than those in the H group.The expression of MyoD protein and the expression of Myogenin of SOL and BRA in group H were significantly lower than those in group C（all P<0.05）.The expressions of EDL and BRA MyoD protein and BRA Myogenin protein in the R and HR groups were significantly higher than those in the C and H groups（all P<0.05）.4)Myostatin mRNA expression（P<0.05）of SOL,Myostatin mRNA（both P<0.01）,and protein expression（P<0.05 and P<0.01,respectively）of the EDL and BRA were significantly higher in group H than in group C.Myostatin mRNA expression（P<0.05 and P<0.001,respectively）and protein expression（P<0.05 and P<0.01,respectively）of EDL and BRA were significantly lower in the HR group than in the H group.The expression of Follistatin mRNA in SOL,EDL,and BRA（P<0.01,P<0.01,and P<0.05,respectively）and the SOL Follistatin protein expression（P<0.05）of group H were significantly lower than those in group C.The expression of Follistatin mRNA（P<0.01,P<0.001）and protein expression（both P<0.05）of the BRA in the R and HR groups were significantly higher than those in the C and H groups,respectively.The expression of P-Smad3 in the EDL of group H was significantly higher than that of group C（P<0.05）,and the expression of P-Smad3 of the BRA in HR group was significantly lower than that of group H（P<0.01）.The EDL Smad7 protein expression（P<0.05）and BRA Smad7mRNA and protein expression（P<0.01）were significantly higher in the HR group than in the H group.It was observed by immunohistochemistry that the positive reaction site of Myostatin protein in the EDL of group C was on the muscle fiber membrane and between the muscle fibers,but in group H was located around the muscle fiber membrane.STRING 11.0 software analysis showed that five proteins,Myostatin,Follistatin,Smad3,ActR2B and MyoD,have degree of confidence higher than 0.9 in their interactions.Conclusions:1)Hypoxia exposure upregulates Myostatin expression in rat skeletal muscle to promote Smad3 phosphorylation,and down-regulates mRNA expression of MyoD,Myogenin and My HC subtypes,resulting in atrophy of rat skeletal muscle.2)Hypoxic resistance training reduces Myostatin overexpression and Smad3 phosphorylation,up-regulates Follistatin,Smad7,MyoD,Myogenin,and MyHC subtype mRNA expressions,and slows hypoxic-induced skeletal muscle atrophy.3)The effect of hypoxic resistance training on Myostatin,Follistatin,MyoD,Smad7 and MyHC subtype gene expression is greater than resistance training under normoxia.4)The effects of hypoxic resistance training on MyoD,MyHC2X and MyHC2B are mainly manifested in EDL and BRA,and the effects on Myogenin,MyHC1 and MyHC2A are mainly manifested in SOL and BRA. |
