The Determinant Role Of Autophagy In Delaying Sarcopenia Upon Exercise Intervention

Objective:Exercise training is one of the most effective interventional strategies for sarcopenia in aged people.Nevertheless,the underlying mechanisms are not well recognized.Numerous studies have actually discovered abnormal autophagy signals in aged skeletal muscle.Therefore,this study is to evaluate the inhibitory effect of exercise interventions on skeletal muscle atrophy of aged rats and explore the underlying mechanisms of exercise for regulating autophagy signal pathways to delay sarcopenia via an established natural aged rat model.Meanwhile,a natural aged mouse model with long-term drug(chloroquine)-induced autophagy inhibition and a mouse model with skeletal muscle-specific autophagy-related gene Beclin1 knockout were constructed,and the effect of exercise intervention on skeletal muscle of chloroquine-induced autophagy inhibition and Beclin1 conditional knockout mice was observed to explore the important role of autophagy in exercise intervention on the atrophy of skeletal muscle.Methods:To observe the inhibitory effect of exercise interventions on skeletal muscle atrophy of aged rats,fifty six-month-old male Wistar rats were reared to the age of 21 months old and then randomly divided into old control group(OC),treadmill exercise group(OE),ladder-climbing exercise group(OR),treadmill exercise combined with ladder-climbing exercise group(OM),and old voluntary wheel-running group(OV),with ten mice in each group.Another ten six-month-old Wistar rats were directly purchased as the youth control group(YC),after exercise intervention of the rats from other groups was completed.Treadmill running was performed at the speed of 12 m/min.The loaded ladder-climbing training was conducted as the resistance exercise.After adaptive training for 1 week,the rats were subjected to ladder-climbing training with the loaded weight of 20%of rat body weight as the initial loading amount,gradually increased by 20%of rat body weight per week to the maximum loading amount of 60%of rat body weight.The rats from OM group were subjected to treadmill running and ladder-climbing training with the same intensity as OE and OR groups alternately every other day.The rats from OV groups were reared in the cage supplied with a running wheel to ensure the voluntary wheel running of rats freely.The duration of all exercise interventions were 12 weeks.To observe the effect of exercise intervention on skeletal muscle of chloroquine-induced autophagy inhibition of aged mice,thirty-two three-month-old male ICR mice were randomly divided into old control group(OC),long-term resistance exercise group(LR),long-term chloroquine group(LCQ),long-term resistance exercise combined with chloroquine group(LCQR),with eight mice in each group.Another eight three-month-old ICR mice were directly purchased as the youth control group(YC),after exercise intervention of the rats from other groups was completed.The loaded ladder-climbing training was conducted as the resistance exercise.After adaptive training for 1 week,the rats were subjected to ladder-climbing training with the loaded weight of 20%of mouse body weight as the initial loading amount,gradually increased by 20%of mouse body weight per week to the maximum loading amount of 60%of mouse body weight.Mice in the chloroquine group and the resistance exercise group were intraperitoneally injected at a daily dose of 40 mg/kg(injection time at 9:00-11:00 am),five times a week,for 55 weeks.To observe the effect of exercise intervention on skeletal muscle of Beclin1 conditional knockout mice,twenty-four three-month-old male mice included 12 mice with identified Beclin1^flox/flox(BECN1^f/f)and 12 mice with identified skeletal muscle-specific Beclin1conditional knockout mice(BECN1^{c KO}).The BECN1^f/f mice were then randomly divided into non-exercise intervention and resistance ladder-climbing exercise groups(Q+BECN1^f/f,QB^f/fand R+BECN1^f/f,RB^f/f);and BECN1^{c KO} mice were also randomly divided into non-exercise intervention and resistance ladder-climbing exercise groups(Q+BECN1^{c KO},QB^{c KO} and R+BECN1^{c KO},RB^{c KO}),with six mice in each subgroup.The loaded ladder-climbing exercise was conducted as the resistance exercise,after adaptive training for 1 week,the rats were subjected to ladder-climbing training with the loaded weight of 20%of mouse body weight as the initial loading amount,gradually increased by 20%of mouse body weight per week to the maximum loading amount of 60%of mouse body weight,for 8 weeks.The GMW/BW ratio was calculated as the relative atrophic rate of skeletal muscle,and the cross-sectional area and ultrastructure of gastrocnemius muscle were examined by HE staining and transmission electron microscope.The protein expression levels of Atrogin-1,Mu RF1,Beclin1,p62,LC3,Bcl-2,Bax,Cyt-C,Akt,p-Akt^Ser473,Fox O3a,p-Fox O3a^Ser253,AMPK,p-AMPK^Thr172,PGC-1?,PINK1,Parkin and GAPDH were determined by Western blotting.Results:In the study on exercise intervention induced autophagy to delay sarcopenia in aged rats,the aged rats revealed the significantly decreased GMB/BW ratio(P<0.001).However,exercise interventions,especially ladder-climbing exercise,can significantly increase the GMB/BW ratio of aged rats and rescue aging-induced atrophy of skeletal muscle(P<0.05,P<0.01,P<0.001).The gastrocnemius muscle of the aged rats revealed disorderly arranged fiber structure with swollen and vacuolated mitochondria.Meanwhile,the cross-sectional area of gastrocnemius muscle was significantly decreased(P<0.01).In contrast,the rats from exercise intervention group,especially ladder-climbing exercise group exhibited clear and ordered fiber arrangement and normal mitochondrial morphology in skeletal muscle,as well as remarkably increased cross-sectional area of skeletal muscle(P<0.05,P<0.01),at the same time,the autophagosomes were observed.Western blot showed that exercise intervention suppressed E3 ubiquitin ligase in skeletal muscle of aged rats,as well as inhibited protein ubiquitination.And also exercise intervention up-regulated the autophagy level of aging rats,improved the autophagy flux of skeletal muscle cells,promoted the degradation of metabolic wastes to reduce the accumulation of damaged cells,and alleviated the senescence process of skeletal muscle cells,as well as inhibited apoptosis of excessive skeletal muscle cells and promoted the survival of skeletal muscle cells in aged rats.Meanwhile,exercise intervention inhibited the Akt/Fox O3a signaling pathway in the gastrocnemius of aged rats by decreasing the phosphorylation levels of Akt in Ser473 site and Fox O3a in Ser253 site,and promoted the transcription activity of Fox O3a.In addition,exercise intervention improved the energy metabolism level of gastrocnemius of aged rats by increasing the phosphorylation level of AMPK at Thr172 site and the expression of PGC-1?protein,activating the AMPK/PGC-1?signaling pathway.In the meantime,exercise intervention promoted mitochondrial autophagy mediated by PINK1/Parkin signaling pathway and maintained the mitochondrial homeostasis in gastrocnemius of aged rats.In the study on the effect of long-term exercise intervention on sarcopenia under the condition of chloroquine-induced autophagy inhibition,we found that long-term ladder-climbing exercise could significantly delay the decline of gastrocnemius muscle mass in natural aged mice,while long-term chloroquine administration increased the mass loss of gastrocnemius muscle(P<0.001),and exercise could partially alleviated the atrophy of skeletal muscle due to the chloroquine-inhibited autophagy.Meanwhile,long-term resistance ladder-climbing exercise improved the structure of gastrocnemius muscle fibers,increased the cross-sectional area of skeletal muscle fibers of aged mice(P<0.01),and also autophagosomes were observed.As the same time,long-term resistance ladder-climbing exercise improved the aggravation of muscle fiber damage caused by chloroquine-mediated autophagy inhibition in aged mice.Western blot showed that long-term resistance ladder-climbing exercise significantly reduced the protein expressions of Atrogin-1 and Mu RF1 in the gastrocnemius of aged mice,and also could significantly suppressed overexpression of E3 ubiquitin ligase,Atrogin-1 and Mu RF1 protein in skeletal muscle of aged mice induced by chloroquine-mediated deficient autophagy,and inhibit the protein ubiquitination.And also long-term resistance ladder-climbing exercise could increase autophagy level of aged mice,enhance autophagy flux of skeletal muscle,accelerate the degradation of metabolic wastes to reduce the accumulation of damaged cells,alleviate the aging process of skeletal muscle,and partially rescue chloroquine-mediated impaired autophagy of mouse skeletal muscle to alleviate the atrophy of skeletal during aging process,as well as inhibited the excessive apoptosis of skeletal muscle in aged mice and the increase of apoptosis induced by chloroquine.Also,long-term ladder-climbing exercise deceased the phosphorylation levels of Akt Ser473 sites and Fox O3a Ser253 in gastrocnemius muscle of aged mice,inhibited Akt/Fox O3a signaling pathway,promoted the Fox O3a nuclear translocation,and then improved the Fox O3a transcriptional activity.While long-term chloroquine administration enhanced the activation of Fox O3a in the gastrocnemius muscle of mice and promoted the transcriptional regulation of Fox O3a,and exercise reduced the detrimental effect of chloroquine.in addition,long-term ladder-climbing exercise can activate the AMPK/PGC-1?signaling pathway and promote energy metabolism by increasing the phosphorylation level of AMPK at Thr172 site and the expression of PGC-1?protein in gastrocnemius muscle of aged mice,while long-term chloroquine administration can suppress AMPK/PGC-1?signaling pathway,and exercise can alleviate the detrimental effect of chloroquine.as the same time,long-term ladder-climbing exercise induced mitophagy of gastrocnemius muscle in aged mice,and while long-term chloroquine administration inhibited the mitophagy of skeletal muscle in aged mice.Therefore,exercise reduced the toxic effect of chloroquine and maintained mitochondrial homeostasis.In the study on the effect of exercise intervention on sarcopenia under the condition of skeletal muscle-specific Beclin1 knockout,it is found that under the condition of normal autophagy function,the resistance ladder-climbing exercise significantly increased the GMB/BW ratio in the gastrocnemius muscle of mice(P<0.01),promoted the gastrocnemius hypertrophy and increased the muscle mass of the mice.On the contrary,the defect autophagy induced the decrease in the mass of gastrocnemius muscle in mice(P<0.001),and the resistance ladder-climbing exercise aggravated the mass loss of gastrocnemius muscle in skeletal muscle-specific Beclin1 conditional knockout mice(P<0.01).And also,the resistance ladder-climbing exercise enhanced the stability of the fiber structure,increased the cross-sectional area of muscle fiber(P<0.001)and promoted mitochondrial enlargement to enhance mitochondrial function under the condition of normal autophagy function.In contrast,the defect autophagy induced the decrease in cross-sectional area of gastrocnemius muscle(P<0.001)and disorderly arranged fiber structure with swollen and vacuolated mitochondria in mice,and the resistance ladder-climbing exercise aggravated the atrophy of skeletal muscle of the mice with Beclin1 conditional knockout.Western blot showed that the resistance ladder-climbing exercise reduced the protein expressions of Atrogin-1 and Mu RF1 in the gastrocnemius of mice under the condition of normal autophagy function,however,the deficiency of autophagy induced the activation of E3 ubiquitin ligase,thereby promoting the atrophy of skeletal muscle,meanwhile,the resistance ladder-climbing exercise aggravated its atrophy under the impaired functional status of autophagy.As the same time,the resistance ladder-climbing exercise decreased the apoptosis of gastrocnemius muscle in mice under the condition of normal autophagy function,while autophagy deficiency activated apoptotic signaling pathway of skeletal muscle in mice,and resistance ladder-climbing exercise further can promote the apoptosis due to the non-suppressive function of autophagy.In the mean time,the resistance ladder-climbing exercise activate the Akt/Fox O3a signal pathway by increasing the phosphorylation levels of Akt at Ser473 site and Fox O3a at Ser253 site in the gastrocnemius muscle of mice under the condition of normal autophagy function,while autophagy deficiency activated Fox O3a and promoted the transcriptional regulation of Fox O3a by inhibiting the Akt/Fox O3a signaling pathway in the gastrocnemius muscle,as well as resistance ladder-climbing exercise further promoted the activation of Fox O3a in the gastrocnemius muscle of Beclin1 conditional knockout mice.as the same time,the resistance ladder-climbing exercise activated the AMPK/PGC-1?signaling pathway and promote energy metabolism by increasing the phosphorylation level of AMPK at Thr172 site and the expression of PGC-1?protein in gastrocnemius muscle of mice under the condition of normal autophagy function,while autophagy deficiency reduced energy metabolism by inhibiting AMPK/PGC-1?signaling pathway in gastrocnemius muscle,and resistance ladder-climbing exercise further aggravated the decrease of energy metabolism in gastrocnemius muscle in Beclin1 conditional knockout mice.And also,the resistance ladder-climbing exercise activated the PINK1/Parkin-mediated mitophagy in gastrocnemius muscle of mice under the condition of normal autophagy function,while the autophagy deficiency inhibited the PINK1/Parkin-mediated mitophagy,and resistance ladder-climbing exercise promoted the suppression of PINK1/Parkin-mediated mitophagy,thereby leading to the mitochondrial dysfunction.Conclusion:The aged Wistar rats at the age of 21 months old appeared significant skeletal muscle atrophy,but exercise interventions for 12 weeks can inhibit Akt/Fox O3a and activate AMPK/PGC-1?signal pathways in the skeletal muscle of aged rats,effectively induce autophagy,reduce excessive apoptosis,inhibit protein ubiquitination,improve skeletal muscle energy metabolism and maintain mitochondrial quality control,thereby delaying sarcopenia,moreover,resistance ladder-climbing exercise exhibits the best interventional efficiency.Long-term chloroquine intervention can inhibit autophagy flux to result in the accumulation of metabolic wastes and damaged cells,thereby aggravating the atrophy of skeletal muscle in aged mice,while long-term ladder-climbing exercise rescue the impaired autophagy flux of skeletal muscle to alleviate the atrophy of skeletal in aging mice.The inhibition of autophagy caused by Beclin1 defect can lead to the atrophy of skeletal muscle in mice,but the ladder-climbing exercise can aggravate the atrophy in skeletal muscle-specific Beclin1 knockout mice,suggesting the determinant regulatory role of autophagy in the atrophy of skeletal muscle during exercise intervention.