| Sarcopenia is a phenomenon characterized by the age-related loss of skeletal muscle mass and function,of which the underlying mechanism and the therapeutic methods remain unclear.The present study aimed to reveal aging-related changes in the skeletal muscle of rats by comparing the global transcriptome using RNA-Seq technology following real-time PCR verification,integrated with the muscle mass and strength and aimed to test the effects of exercise training and resveratrol feeding on sarcopenia as well as on gene expression in skeletal muscle of old rats.In experiment 1,ten rats aged 25 months were set as the old group(OG)and ten rats aged 6 months were set as the young group(YG).After 6 weeks of feeding,the body mass,grip strength,and gastrocnemius muscle mass were determined,and the differentially expressed genes were analyzed by transcriptome sequencing,followed by GO enrichment analysis and KEGG analysis.The results showed that the muscle index and the relative grip strength were lower in OG rats than YG rats.We identified 525 DEGs in the muscle tissues between the OG rats and YG rats,within which 447 genes were down-regulated while 78 genes were up-regulated.The down-regulated genes in the OG rats included the genes of the AMPK subunit alpha-2(Prkaa-2),insulin-like growth factor-1(IGF-1),myocyte enhancer factor 2a(MEF2a),uncoupling protein 3(UCP-3),and forkhead box family genes(FOX03,FOXK1,FOXN1).Several ion channel related genes were also down-regulated,such as the calcium/calmodulin-dependent serine protein kinase(CASK),calcium voltage-gated channel auxiliary subunit alpha2delta 1(CACNA2d1),two pore calcium channel protein 1 isoform 1,potassium voltage-gated channel subfamily J member 2(KCNJ2),chloride voltage-gated channel 3(CLCN3),and piezo-type mechanosensitive ion channel component 2(PIEZ02).A large number of genes of the collagen family(COLlal,1a2,3a1,4a1,4a2,4a3,4a5,5a1,5a2,5a3,6a1,6a2,6a3,6a6,8a2,11a1,14a1)were also reduced in OG rats.Some inflammation related genes were reduced in the OG rats as well,including interleukin 1 receptor-like 1(IL1r11),interleukin 13 receptor subunit alpha 1(IL13ra1),interleukin 6 signal transducer(IL6ST).The up-regulated genes in the OG rats includes cholinergic receptor,nicotinic,alpha 1(CHRNa1),O-6-methylguanine-DNA methyltransferase gene(MGMT),fucose mutarotase(Fuom),sarcolipin(SLN),sodium voltage-gated channel alpha subunit 5(SCN5a),interleukin 3 receptor subunit alpha(IL3ra),and interleukin 15(IL15).The eight selected genes(Prkaa-2,FOX03,IGF-1,CASK,TRIM67,IL-6,MGMT,SCN5a)were verified by quantitative real-time PCR,within which five genes(Prkaa-2,IGF-1,CASK,MGMT,SCN5a)showed significant changes between OG and YG rats consistently to transcriptomic analysis,while three genes(FOX03,TRIM67,IL-6)showed no significant differences.The GO enrichment analysis revealed that 1199 terms were significantly overrepresented in the OG vs YG rats.The most enriched 20 GO terms were mainly associated with organ development,cellular response to stimulus,and the structure of extracellular matrix,of which most genes were down-regulated in the OG vs YG rats.The KEGG enriched analysis showed several significantly down-regulated pathways associated with muscle performance were identified in the OG vs YG rat,including focal adhesion,ECM-receptor interaction,PI3K/AKT signaling,protein digestion and absorption,and small cell lung cancer(corrected P<0.05).In experiment 2,rats aged 25 months were divided into the old control group(OC),the old exercise group(OE),and resveratrol feeding group(OR).The rat numbers was 10 for each group.The OC rats were fed for 6 week without any treating.The OE rats were trained by daily running for 6 week and the OR rats were treated by resveratrol feeding for 6 week.After 6 weeks,the body mass,grip strength,and gastrocnemius muscle mass were determined,and the differentially expressed genes were analyzed by transcriptome sequencing,followed by GO enrichment analysis and KEGG analysis.We identified 21 DEGs in the muscle tissues between the OE rats and OC rats,within which 14 genes were down-regulated while 7 genes were up-regulated in OE rats.The down-regulated genes includes ribosomal RNA processing 7 homolog A(Rrp7a),rRNA promoter binding protein(LOC257642),YIF1B protein(LOC1 03689986),and potassium sodium-activated channel subfamily T member 1(Kcntl).The up-regulated genes included slingshot protein phosphatase(SSH2),polypeptide N-acetylgalactosaminyltransferase 5(GALNT5),RGD1562339(RGD1562339),zinc finger protein 474(ZFP474),mitochondrial carrier triple repeat 1(MCART1),and cytidine deaminase-like(LOC100909857).The eight selected genes(Prkaa-2,FOX03,IGF-1,CASK,TRIM67,IL-6,MGMT,and SCN5a)were verified by quantitative real-time PCR and all genes showed no significant expression differences between OE and OC rats.The GO enrichment analysis revealed that 70 terms were significantly overrepresented in the OE vs OC rats.The most enriched 20 GO terms were mainly associated with cell response to stimulation and cytidine deamination.KEGG analysis identified several significantly up-regulated pathways in the OE rats vs OR rats,including mucin type O-Glycan biosynthesis,pyrimidine metabolism,and drug metabolism(corrected P<0.05).Only 12 DEGs were identified between the OR rats and OC rats(Table 4),within which 2 genes were down-regulated and 10 genes(aldehyde dehydrogenase 1 family,member A1(ALDHlal),tripartite motif-containing 67(TRIM67),synaptotagmin 1(SYT1),syntaxin-7-like(LOC1 00910446),synaptosomal-associated protein 25(SNAP25),transthyretin(TTR),solute carrier family 17 member 7(SLC17a7),neural EGFL like 2(NELL2),and apolipoprotein C-1-like(LOC100910181))were up-regulated in the OR vs OC.The eight selected genes(Prkaa-2,FOX03,IGF-1,CASK,TRIM67,IL-6,MGMT,and SCN5a)were verified by quantitative real-time PCR and all genes showed no significant expression differences between OR and OC rats.We found 219 GO terms that were enriched in the OR vs the OC and the most enriched GO terms were mainly associated with neurotransmitter transport,synaptic vesicle,and alcohol metabolic process.KEGG analysis identified several significantly up-regulated pathways in the OR rats vs OC rats,including synaptic vesicle cycle,nicotine addiction,retinol metabolism,insulin secretion,retrograde endocannabinoid signaling,and glutamatergic synapse(corrected P<0.05).The results suggest that the reduced expressions of AMPK and UCP3 in the muscle of old rats may reduce the resistance to inflammation and oxidative stress.The reduced expression of IGF-1 may suppress the synthesis of muscle myofibrillar protein and reduce muscle contraction.The reduce expressions of CASK and calcium channel-associated genes may reduce muscle excitation-contraction.The up-regulated expressions of CHRNal may induce dysfunction of neuromuscular junction through de-repression of extra-synaptic gene inhibition.Those above expression changes of genes can explain the losses of muscle mass and function in the aged rats.Neither exercise training nor resveratrol feeding has remarkable effects on the skeletal muscle function and gene expression(e.g.AMPK,IGF-1,MGMT,and CASK)of old rats.It suggests that exercise training and resveratrol feeding have only limited effects on the expression of genes in the skeletal muscle of aged rats and therefore on addressing aging-related sarcopenia.However,both exercise training and resveratrol feeding have remarkable effects on weight loss.Therefore,the potential disturbance of treatment on body mass should be considered when study the therapeutic methods of sarcopenia.Interestingly,resveratrol feeding may have positive effects on synaptic vesicle cycle in the old rats. |
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