Alteration And Mechanism Of Autophagy?Related Proteins During Endogenous Myocardial Protection Induced By Exercise Preconditioning

Objective: Exercise preconditioning(EP)is a way to produce strong endogenous myocardial protection through the repeated short-term highintensity intermittent aerobic exercise,which can improve the myocardial tolerance to ischemia and hypoxia,and reduce the subsequent long-term myocardial ischemia-hypoxia injury.It can be divided into the two stages:the induction of exercise precondition(IEP)and the protection of exercise precondition(PEP).Its protective effect on endogenous myocardia has been confirmed,but the mechanism is still under investigation.Besides,the relationship between EP and autophagy needs to be further explored.This study aimed at the exploration of the relationship between autophagy and EP-induced endogenous myocardial protection based on the study of four types of autophagy-related proteins Beclin1,LC3,Cathepsin D and p62,and at the examination of whether autophagy is involved in EP-induced myocardial protection through the use of the autophagy blocker Wormanmin,so as to provide an updated theoretical and experimental basis for other studies of EP-induced myocardial protection.Methods:(1)150 healthy male SD rats were used to establish models of EP protection of animal myocardial injury that was caused by high-intensity exercise.They were divided into six groups: Group C,the high-intensity exercise group(the HE group),the early EP group(the EEP group),the late EP group(the LEP group),the early EP protection group(the EEP + HE group)and the late EP protection group(the LEP + HE group).The EEP group and the LEP group were both given four times a 30 m / min running with 0 °grade for 10 minutes,followed by a 10-min rest to establish the EP model;a35 m / min treadmill exercise with 0 ° grade for 3 hours was used in HE group to establish the myocardial injury model;and the EEP + HE group and the LEP + HE group were given high-intensity exercise for 0.5 h and 24 h respectively after the EP exercise.The concentration of plasma cardiac troponin I(cTnI)and the C-2R BG staining of myocardial ischemia and hypoxia were examined to evaluate the degree of myocardial ischemiahypoxia injury and protection.Then the relationship between ischemiahypoxia and autophagy was discussed by comparing the myocardial C-2R BG staining and the adjacent sections of the four autophagy-related proteins Beclin1,LC3,Cathepsin D and p62 based on the immunohistochemistry technique.Immunohistochemistry and Western blotting were used to observe and detect the expressions of these four proteins.Finally,the relationship between autophagy and EP in the course of reducing myocardial injury induced by high intensity exercise was discussed.(2)Based on the establishment of the model of EP reducing myocardial ischemia-hypoxia injury induced by exhaustive exercise,the plasma concentration of cTnI and the ischemia-hypoxia staining of the myocardial tissue of 220 healthy male SD rats were detected,and ultrastructural myocardial cells were observed,to evaluate the degree of myocardial ischemia-hypoxia injury and cardio protection.At the same time,the hypothesis that autophagy is involved in the EP-induced endogenous myocardial protection was verified by the intraperitoneal injection of the autophagy inhibitor wormammin.The animals were divided into eight groups: Group C;the exhaustive exercise group(the EE group),given 30 m / min and 0 ° grade exhaustive exercise;the EEP group and the LEP group,also given 30 m / min 0 ° grade 10 min exercise four times,each time followed by a 10 min rest,to establish the EP model;the EEP + EE group and the LEP + EE group,both conducting exhaustive exercise after having had EP exercise for 0.5 h and 24 h respectively,to establish the model of EP reducing myocardial ischemiahypoxia injury induced by exhaustive exercise;and the W + EEP + EE group and the W + LEP + EE group,in both of which the myocardial ischemiahypoxia injury was induced by EP and wortmannin,an autophagy inhibitor,was injected intraperitoneally 0.5 h before the EP exercise.On the basis of the EP model,the autophagy-related proteins Beclin1,LC3 and Cathepsin D were observed and detected at different time points during the IEP stage in order to study their change and the relationship between them to clarify the role and mechanism of autophagy in myocardial protection of EP in Group C,the 0.5 h group,the 1 h group,the 2 h group,the 3 h group,the 5 h group and the 24 h group after EP.The expressions of Beclin1,LC3,Cathepsin D and p62 in myocardia were detected by immunofluorescence staining and WBResults:(1)Compared with Group C,the concentration of plasma cTnI and the IHA,IOD and MOD values of ischemia-hypoxia staining of the HE group were significantly higher,but there was no difference in the EEP group and the LEP group in those aspects.Compared with the HE group,those respects of the EEP + HE group and LEP + HE group were significantly decreased.The results of adjacent sections showed that the ischemia–hypoxia cardio myocytes were consistent in locat ion with the cardio-myocytes st ained by p62 immunohistochemistry.The results o f immunohistochemistry showed: compared with Group C,the MOD values of Beclin1,LC3 and Cathepsin D in the HE groups was significantly increased,but the MOD value of p62 had no change;the MOD values of Beclin1,LC3,Cathepsin D in the EEP group and the LEP group were significantly increased,and the MOD value of p62 was lower.Compared with the HE group,the MOD values of Beclin1 in the EEP + HE group did not change,but those of LC3 and Cathepsin D were significantly increased;the MOD values of Beclin1 and Cathepsin D in the LEP+HE group were significantly lower,the MOD value of LC3 was significantly increased,and that of p62 had no difference.Compared with the EEP group,the MOD values of Beclin1 and Cathepsin D in the LEP group were lower,but the MOD value of p62 was higher.Compared with the EEP + HE group,the MOD values of Beclin1 and Cathepsin D in the LEP group were lower,and the MOF value of p62 was higher.Western blot results showed: compared with Group C,the expressions of Beclin1,LC3 II and Cathepsin D in the HE group were increased,in the EEP group the expressions of Beclin1,LC3 II,LC3II/I and Cathepsin D was significantly increased and the expression of p62 was lower,and the expression of p62 in the LEP group was lower.Compared with the HE group,the expression of p62 was significantly decreased in the LEP group,and the expressions of Beclin1 and Cathepsin D in the LEP + HE group was lower.(2)Compared with Group C,the concentration of plasma cTnI and the IHA,IOD and MOD values of ischemia-hypoxia staining in the EE group was significantly increased,but there was no difference in the EEP group and LEP group in those aspects.Compared with the EE group,those aspects were significantly decreased(P<0.05)in the EEP + EE group and the LEP + EE group.Compared with the EEP + EE group,the concentration of plasma cTnI in the W + EEP + EE group was significantly lower,and the IHA,IOD and MOD values had a trend to increase but showed no significant difference.Compared with the LEP + EE group,the concentration of plasma cTnI,the IHA,IOD and MOD values of the W + LEP + EE group were significantly increased.The results of transmission electron microscopy showed:myocardial myofibrils were broken,the space between mitochondria was enlarged,the mitochondria were deformed,the outer membrane of mitochondria was swollen or even ruptured,the cristae of mitochondria were disordered,and the myocardial transverse tube dilation was occasionally seen in the EE group,the W + EEP + EE group and the W + LEP + EE group;and autophagy or the vacuole structure was observed in the EEP group,the LEP group and the EEP + EE group.Analysis of the immunofluorescence images of myocardia and the integral optical density(IOD)results shows:compared with Group C,Beclin1 was significantly increased in the PEP 2h group and the PEP 3h group(P<0.05);LC3 was significantly increased in the PEP 1h group(P<0.05);Cathepsin D was significantly increased in the PEP 1h group(P<0.05);and p62 was significantly decreased in the PEP 5h group and tge PEP 24 h group(P<0.05).Western blot results showed:compared with Group C,Beclin1 expressions were significantly increased in the PEP 2h group(P<0.05);there was were no difference in LC3I/GAPDH between these groups(P>0.05);LC3II/I significantly increased in the PEP1 h group;LC3II/GAPDH was significantly increased in the PEP1 h group(P<0.05);Cathepsin D expressions were significantly decreased in the PEP5 h group and the PEP 24 h group(P<0.05);and the expression of p62 was significantly decreased in the PEP 2h group,the PEP 3hr group,the PEP 5h group and the PEP 24 h group(P<0.05).Conclusions:(1)The detection of plasma cTnI and C-2R BG staining of myocardial ischemia-hypoxia confirms that EP can reduce myocardial ischemia-hypoxia injury caused by high-intensity exercise.By comparing the adjacent sections,it was found that hypoxia-ischemia induced autophagy.The observation and detection of the expressions of autophagy-related proteins in the rats' myocardium suggests that high-intensity exercise may induce cardiomyocyte autophagy through persistent ischemia-hypoxia,but the process of autophagy degradation can be blocked,which may be the factor to cause myocardial ischemia-hypoxia injury.EP-induced autophagy by means of intermittent ischemia-hypoxia may improve the blocking of autophagy induced by high intensity exercise,so autophagy may be involved in EP induced endogenous myocardial protection.(2)Through t he observation of plasma cTnI,myocardial ischemia-hypoxia staining and transmission electron microscopy,it was further confirmed that EP can reduce the myocardial ischemia-hypoxia injury caused by exhaustive exercise,and has protective effects in the early and late phases.After the use of the autophagy blocker wortmannin,the myocardial tissue damage caused by exhaustive exercise in LEP was found aggravated,which confirmed that autophagy was involved in the endogenous myocardial protection in LEP.There was no significant aggravation in EEP,suggesting that the involvement of autophagy in EEP protection needs to be confirmed.The expressions of autophagy-related proteins Beclin1,LC3,cathepsin D and p62 in rats' myocardia were observed and measured by the time-sharing method.The expressions of the autophagy-related proteins suggested that the level of autophagy increased first and then decreased after EP.The degradation process of autophagy was obvious during the first 2h of PEP and lasted for24 h in PEP.Autophagy may exist during the 24 h of PEP and participated in EP and endogenous myocardial protection.