| Background: Non-hibernating animals experience extreme conditions such as waste,weight loss,hypoxia,ischemia-reperfusion,fasting,and so on.Skeletal muscle atrophy occurs significantly,and the function is greatly damaged.However,when hibernating animals experience the same extreme adverse conditions during hibernation,they can maintain the quality and function of skeletal muscle better than non-hibernating animals.By studying this natural anti-atrophy mechanism,in order to find strategies to prevent or treat skeletal muscle atrophy.Previous studies have shown that the fast contraction muscle and slow contraction muscle in mammalian skeletal muscle are non-homogeneous.The previous proteomics results in our laboratory also proved that the two kinds of skeletal muscle of Daurian Ground Squirrel have different coping strategies for long-term waste at the protein level,and the expression levels of some proteins have changed significantly during hibernation,but the upstream regulatory genes are still unclear.Therefore,this study took the small hibernating mammals Daurian Ground Squirrel as the experimental object to explore which genes were differentially expressed in the slow contraction muscle and fast contraction muscle of hibernating rats,and to understand the function of differential expression genes,and to explore the function of common differential expression genes and specific differential expression genes between different skeletal muscles.From a macro point of view to understand the gene expression in skeletal muscle during hibernation and differentially regulated genes and their functions.Methods :(1)RNA-seq technology was used to sequence different skeletal muscles(soleus muscle and extensor digitorum longus muscle)of Daurian Ground Squirrel(2)Gene functions were analyzed using Gene Ontology and KEGG pathway databases.(3)RT-q PCR was used to test the expression level of differential genes.(4)Trend analysis using STEM(Short Time-series Expression Miner).Results and Discussions(1)Screening of differentially expressed genes: Six differentially expressed genes,Gsc2,Nrip3,Mybph,Fosb,Il1 b and Egr1,which may play an important role in the maintenance of skeletal muscle quality and function during hibernation,were screened between the summer group and the hibernation group of soleus and extensor digitorum longus.Mybph was never reported in the skeletal muscle of hibernate animals.There were four differentially expressed genes in soleus muscle: Chac1,Drd2,Gdf6 and prkcb,and four differentially expressed genes in extensor digitorum muscle: angptl4,angptl5,Fndc11 and Mmp1,which proved that different skeletal muscle fiber types had different differentially regulated genes.(2)GO enrichment analysis2.1 Common differentially expressed genes: 323 GO entries were enriched,and the up-regulated genes were enriched in protein synthesis and degradation-related functions,suggesting that the synthesis and degradation of skeletal muscle-related proteins may occur during hibernation.The down-regulated genes were mainly enriched in the response to external stimuli,which was consistent with the decreased sensitivity of the external environment and cells to stimuli during hibernation.At the same time,the down-regulated genes were enriched in the functions related to skeletal muscle growth and development,suggesting that the body sacrificed some non-needed functions for survival during hibernation.2.2 Specific differential expression genes of soleus: 466 GO entries were enriched,and the up-regulated differentially expressed genes were mainly enriched to antioxidant-related and metabolic-related functional genes.The down-regulated genes were enriched into functional genes such as protein synthesis,protein degradation and phosphorylation,and apoptosis.The specific differential expression genes suggested that the soleus muscle mainly maintained the quality and function of skeletal muscle by simultaneously inhibiting protein synthesis,degradation,and apoptosis.At the same time,the up-and down-regulated differentially expressed genes are enriched in immune-related functions,suggesting that different components of the immune system may have different responses in the hibernation period,which will be studied in the future.2.3 Specific differential expression genes of extensor digitorum longus: 475 GO entries were enriched.The up-regulated differential genes were enriched in the functional entries of protein synthesis and degradation,ion transport and multicellular biological development,while the down-regulated genes were enriched to metabolism,autophagy,apoptosis and skeletal muscle structure-related functions.It is speculated that the long extensor digitorum during hibernation maintains the balance of protein synthesis and degradation rate at a high level,and maintains the quality and function of skeletal muscle by inhibiting autophagy and apoptosis.(3)KEGG enrichment analysis3.1 Common differentially expressed genes: 59 signaling pathways were enriched,up-regulated genes were enriched in protein synthesis and degradation-related pathways,down-regulated genes were enriched in NF-?B and apoptosis-related pathways.KEGG analysis also suggested that protein synthesis and degradation-related pathways in soleus and extensor digitorum longus were enriched during hibernation,which was consistent with GO enrichment analysis.3.2 Muscle-specific differentially expressed genes in soleus: 62 signaling pathways were enriched,and up-regulated genes were enriched in HIF-1 signaling pathways.HIF-1 can respond to hypoxia conditions,up-regulate the transcription of various genes that enhance hypoxia tolerance,up-regulate HIF-1 can protect skeletal muscle from hypoxia stress and avoid injury,down-regulate genes enriched in NF-?B,Ras and apoptosis signaling pathways,and maintain skeletal muscle quality and function by inhibiting protein synthesis and degradation.3.3 Differentially expressed genes in extensor digitorum longus: 37 signaling pathways were enriched,up-regulated genes were enriched in PI3K/Akt,proteasome and peroxidase pathways,and down-regulated genes were enriched in metabolism-related,MAPK and calcium signaling pathways.PI3K/Akt pathway is the most important protein synthesis pathway.Peroxidase pathway can promote the conversion of muscle fiber type II to type I,which is consistent with our previous research results.At the same time,the conversion of muscle fiber type to type I can improve its oxidation ability to adapt to hypoxia stress during hibernation.Up-regulated genes are also enriched in the main protein degradation pathway proteasome signaling pathway.The down-regulated genes are enriched in metabolism,which is consistent with the low metabolic physiological state during hibernation.At the same time,the down-regulated genes are also enriched in the calcium signaling pathway.It is speculated that the down-regulated protein degradation level of extensor digitorum longus during hibernation is due to the inhibition of the calcium protease signaling pathway activated by calcium overload.(4)Results of qRT-PCR: Mybph,Mustn1,Nrip3 and Egr1 were verified in common differentially expressed genes,Chac1 and prkcb were verified in soleus-specific differentially expressed genes,Angptl5 and Stc1 were verified in extensor digitorum longus-specific differentially expressed genes.The expression trends of these eight genes were consistent with RNA-seq results,indicating that our RNA-seq results are true and reliable.(5)Trend analysis5.1 Trend analysis of differential genes: 9 and 11 significant enrichment trends were obtained by trend analysis of soleus muscle and extensor digitorum longus muscle,respectively.The profile11,a significant trend module that continuously up-regulates genes during hibernation,was analyzed by Wayne,and some genes that may be related to maintaining skeletal muscle quality and function were obtained: Clcf1,Kng1,S1pr5,Map6 and Ihh.5.2 GO analysis: GO analysis was performed on the genes in profile11,a significant trend module in two muscles,and the results showed that the genes were enriched in energy metabolism,immunity,protein synthesis and degradation.The soleus muscle is enriched in the function of inhibiting apoptosis and protein hydrolysis.The extensor digitorum longus is enriched in skeletal muscle remodeling-related functions.5.3 KEGG pathway analysis: Both soleus muscle and extensor digitorum longus muscle are enriched in the active ligand receptor interaction pathway,which mainly plays a basic regulatory role.The differentially expressed genes in this pathway are analyzed in detail.Sphingosine-1-phosphate receptor 5(S1pr5)gene is expressed in both muscles,which can regulate embryonic development and promote vascular development.Some studies have shown that S1P can regulate the number of lymphocytes in small mammals during hibernation.We speculate that the expression of this gene is beneficial to the development of blood vessels in skeletal muscle during hibernation,promote vascular regeneration,and is related to the improvement of autoimmunity,thereby protecting skeletal muscle in many ways.In summary,first of all,this study found that the gene Mybph,which has never been reported in the skeletal muscle of rats during hibernation,is related to muscle growth and affects the actin-myosin effect,which may play an important role in maintaining the quality and function of skeletal muscle during hibernation.Secondly,during hibernation,the anti-atrophy mechanisms of the two skeletal muscles are not the same.According to the analysis of differential expression genes,the genes related to protein synthesis and degradation in soleus and extensor digitorum were up-regulated during hibernation.However,the difference was that the genes related to protein phosphorylation and enzyme activity were inhibited in the analysis of specific differential expression genes in soleus,but not in extensor digitorum.The genes differentially expressed in soleus muscle mainly include anti-oxidation and tumor suppressor genes,and inhibit protein synthesis mediated by Ras signaling pathway and maintain skeletal muscle quality and function by inhibiting NF-?B mediated protein degradation and inhibiting phosphorylation-related genes.Among the differentially expressed genes in extensor digitorum longus,PI3K/Akt signaling pathway was up-regulated to promote protein synthesis,while proteasome signaling pathway was up-regulated to improve protein degradation rate.At the same time,peroxidase signaling pathway was up-regulated to realize muscle fiber type conversion in extensor digitorum longus and improve its antioxidant capacity.Two typical skeletal muscles maintain the quality and function of skeletal muscle by regulating different genes and pathways. |
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