The Effect Of Chrna1 On Sarcopenia And Its Mechanism

Objectives:1.To observe the changes of skeletal muscle mass and function during the growth of mice and establish a model of primary sarcopenia.2.To explore the effect of nicotinic acetylcholine receptor ?1(CHRNA1)on the skeletal muscle mass and function in mice.3.To explore the effect of CHRNA1 on neuromuscular junctions(NMJs)and compound muscle action potentials(CMAPs)and its mechanism in mice.4.To explore the effect of CHRNA1 on skeletal muscle fibrosis and its mechanism in mice.Methods:1.20 4-month-old male C57BL/6 mice were used as the young mice group,33 14-month-old male C57BL/6 mice were routinely raised to 24 months old as the old mice group,every 2 months the dual-energy X-ray absorptiometry(DEXA)was used to detect the changes of the skeletal muscle mass and fat mass with age in mice,and every 2 months the forelimb grip strength were measured to observe the changes of the muscle function with age in mice.2.To observe the difference in gastrocnemius mass and function between the young control group and the old sarcopenia group,the gastrocnemius muscle contractility were measured through the biological information collection system,the gastrocnemius muscle mass were detected by analytical balances,the cross-sectional area(CSA)were measured by H&E staining,the ultrastructure of myofibrils were observed under an electron microscope.To observe the difference in the expression of CHRNA1,CHRNA1 was detected by western blot,q-PCR and immunohistochemistry.To observe the effect of CHRNA1 on the gastrocnemius muscle mass,the CSA and the muscle contractility of the young control group and the old sarcopenia group,the left hindlimb were injected with adeno-associated virus 9(AAV9)that overexpress or knock down CHRNA1 for two months,the right hindlimb were injected with empty virus for two months.3.After overexpression of CHRNA1 in the young control group and the old sarcopenia group,the innervation rate of gastrocnemius NMJs were detected by immunofluorescence,the gastrocnemius CMAPs were measured by the biological information collection system,the activity of calpain1 was detected by Suc-Leu-Tyr-AMC,the protein level of CHRNA1,calpain1 and its substrate p25 were measured by western blot,the expression of calpain1 was also detected by immunohistochemistry.C2C12 myotube cells were divided into negative control group,negative control + carbachol group,overexpression CHRNA1 group and overexpression CHRNA1+ carbachol group by transfecting empty plasmids or overexpressing CHRNA1 plasmids with or without carbachol.The activity of calpain1 was detected by Suc-Leu-Tyr-AMC,the expression of calpain1 was detected by western blot,and the changes of the cytoplasm calcium ion concentration were detected by the cell membrane permeable calcium ion fluorescent probe.4.After knockdown of CHRNA1 in the old sarcopenia group,the expression of CHRNA1,macrophages marker CD68,myofibroblasts marker ?-SMA and calpain1 were detected by immunohistochemistry.Sirius red staining was used to detect the expression of collagen fibers,western blot was used to detect the expression of calpain1,Collagen1,Collagen3,?-SMA,MMP2,MMP9 and TGF?1,and Suc-Leu-Tyr-AMC was used to detect the activity of calpain1.C2C12 myotube cells were divided into negative control group,negative control + carbachol group and knockdown CHRNA1 + carbachol group by transfecting the empty virus or knockdown CHRNA1 virus with or without carbachol.The activity of calpain1 was detected by Suc-Leu-Tyr-AMC,the expression of calpain1 was detected by western blot,and the changes of the cytoplasm calcium ion concentration were detected by the cell membrane permeable calcium ion fluorescent probe.Results:1.The body weight and the fat mass of mice were gradually increased with age,the average hindlimb skeletal muscle SMI and forelimb grip strength were gradually decreased with age.Compared with the young control group,the body weight and the fat mass of the old sarcopenia group were significantly increased,the forelimb grip strength and the average hindlimb SMI of the old sarcopenia group were significantly decreased.2.The muscle contractility,the gastrocnemius SMI and the CSA of gastrocnemius in the old sarcopenia group were lower than that in the young control group.Under the electron microscope,the old sarcopenia group showed obviously myofibril structure disorder and mitochondrial swelling.Compared with the young control group,the expression of CHRNA1 was significantly increased in the old sarcopenia group.After overexpression of CHRNA1,the SMI,the muscle contractility and the CSA of gastrocnemius in the old sarcopenia group were significantly decreased,however,there was no significant difference in the young control group.After knockdown of CHRNA1,the SMI,muscle contractility and CSA of gastrocnemius in the old sarcopenia group were significantly increased.3.After overexpression of CHRNA1 in mice,the CMAPs and innervation rate of the young control group and the old sarcopenia group were both decreased significantly.CHRNA1 activated calpain1,which were manifested by up-regulation of protein expression and increased substrate.After CHRNA1 was overexpressed in C2C12 myotube cells,calpain1 was activated and the calcium ion concentration were increased.4.After knocking down of CHRNA1 in mice,the degree of skeletal muscle fibrosis in the old sarcopenia group was improved,which were manifested by down-regulation of collagen fibers and the expression of CD68,Collagen1,Collagen3,?-SMA,MMP2,MMP9 and TGF?1.Knockdown of CHRNA1 inhibits calpain1 in the old sarcopenia group.Knockdown of CHRNA1 in C2C12 myotube cells inhibits calpain1,and the calcium ion concentration were decreased.Conclusion:Mice began to experience decline in skeletal muscle mass and function from 18 months,and gradually get worsed with age.The expression of CHRNA1 in the skeletal muscle of old sarcopenia group was increased,overexpression of CHRNA1 aggravated the decline in skeletal muscle mass and strength in old sarcopenia mice,while knocking down CHRNA1 alleviated the decline in skeletal muscle mass and strength in old sarcopenia mice,CHRNA1 participates in the occurrence and development of sarcopenia.CHRNA1 cause structural dysfunction of NMJs and aggravate skeletal muscle fibrosis by activating calpain1.These are possible mechanisms by which CHRNA1 participates in the occurrence and development of sarcopenia.