The Changes And Mechanism Of HSP 70-mediated Selective Autophagy In High-intensity Interval Training-induced Skeletal Muscle Protection

Objectives: The purpose of this study was to investigate the effect and mechanism of chaperone-assisted selective autophagy(CASA)on protein homeostasis in exhaustive exercise animal models,and to obtain exercise adaptation of muscles through high-intensity interval training(HIIT),revealing the changes of CASA in HIIT-induced skeletal muscle injury.Methods: Forty-five male SD rats were selected and fed adaptively for one week,during which adaptive treadmill training for three days.Then,the rats were randomly divided into three groups,fifteen rats in each group:(1)control group(group C,n=fifteen),without any intervention;(2)Exhaustive exercise group(EE group,n=fifteen);(3)High-intensity interval exercise + exhaustive exercise group(HIIT+EE group,n= fifteen).The treadmill slope was 0°,and the EE group exercised on the treadmill at a speed of fifteen twenty-five ～ twenty-eight m/min until exhaustion.The HIIT + EE group performed intermittent treadmill exercise for 3 consecutive days at a speed of 28 m / min,with 10 min exercise and 10 min rest as a group,repeated 4 groups.The model of the EE group was reproduced twenty-four hours later.After exercise,blood glucose was detected immediately.The movement distance of the rats was recorded during movement.The exercise capacity of skeletal muscle and muscle fiber injury were evaluated by detecting plasma CK,HE staining and transmission electron microscopy(TEM).The expression levels of Bip,HSP 70,Beclin 1,p62,ubiquitin,LC3 and Cathepsin D were detected by Western Blot to evaluate the levels of endoplasmic reticulum stress(ERS)and autophagy.The expression of Bip and Cathepsin D was further detected by immunofluorescence staining and immunohistochemical staining to evaluate endoplasmic reticulum stress and autophagosome degradation.The colocalization of CASA-related factors was analyzed by immunofluorescence colocalization staining,including HSP 70 + BAG 3,BAG 3 + LC3,p62 + ubiquitin,LC3 + p62,LC3 + LAMP2,to detect the occurrence and degradation of CASA.Results: 1.Exhaustive exercise induced an increase in plasma CK,significantly decreased in intramuscular ATP level,muscle fiber swelling,muscle membrane tortuosity,and dissolution of Z-line in rats,which were improved after HIIT intervention.These results suggest that exhaustive exercise induces skeletal muscle injury and HIIT improves muscle fiber injury.2.Exhaustive exercise promoted the expression of endoplasmic reticulum stress marker protein Bip,and the expression levels of autophagy-related proteins Beclin 1,p62,LC3 II,LC3-II/LC3-I,mature Cathepsin D and HSP 70 were significantly increased;the colocalization of HSP 70 + BAG 3,p62 + ubiquitin,LC3 + p62,BAG 3 + LC3,and LC3 + LAMP-2 was abundantly stored in the gastrocnemius muscle,suggesting that exhaustive exercise activation induces CASA to degrade damaged,unfolded/ misfolded proteins.3.After HIIT intervention,Bip level decreased,CASA was down-regulated,HSP 70 level was still high,but there was a downward trend,suggesting that HIIT alleviated CASA by inhibiting endoplasmic reticulum stress.Conclusion: Exhaustive exercise induces skeletal muscle injury and activates endoplasmic reticulum stress,and maintains protein homeostasis in muscle fibers by activating CASA to remove damaged proteins and unfolded/misfolded proteins.HIIT enables muscles to acquire exercise adaptation,and inhibits exhaustive exercise to induce CASA enhancement by improving myofibril damage and ERS.