| Objective: Exercise preconditioning(EP)refers to a phenomenon that high intensity intermittent aerobic exercise induces organism to produce endogenous protection,so that it can protect the myocardium in the following prolonged ischemia and hypoxia injury.Previous studies found that EP can induce autophagy to participate in the early cardioprotection,but researches on EP and autophagy mostly focus on the induction of autophagy,and the relationship between EP and the formation of autophagosome has been rarely reported.The formation of autophagosome involves ATG12-ATG5 ubiquitination system and LC3-PE system.As the common E1 like enzyme of these two systems,ATG7 is an important factor in autophagosome assembly.LC3-Ⅱ,mediated by ATG7,is positively related to the quantity of autophagosome formation.p62 is another indicator of autophagy,representing the clearance level of autophagic substrate.Autophagy is one of the protective mechanisms of EP,and ATG7 plays a key role in autophagy.However,the changes and roles of ATG7 in EP haven’t been reported.Therefore,by detecting the expression of ATG7,LC3 and p62,we explored the role of ATG7 in inductive phase and early protective phase of EP,to provide a new theoretical and experimental basis for studying the relationship between autophagy and EP.Methods: 80 healthy male SD rats were randomly divided into control group(C group),early exercise preconditioning group(EEP group),exhaustive exercise group(EE group),and early exercise preconditioning + exhaustive exercise group(EEP+EE group),20 rats in each group.(1)The content of cardiac troponin I(cTnI)in plasma was detected by chemiluminescent immunoassay,and the degree of myocardial ischemia and hypoxia was examined by hematoxylin basic fuchsin picric acid(HBFP)staining,to comprehensively evaluate exercise-induced injury and protection in myocardium.(2)Distribution and expression of ATG7 in myocardium were observed by immunofluorescence and western blot experiments.(3)The changes of LC3 and p62 expression were detected by western blot to assess the level of autophagy expression.Results:(1)Compared with C group,cTnI level had no significant changes in EEP group(P>0.05);the cardiomyocyte structure of EEP group was similar to C group,but a few red spot stains were observed in cardiomyocytes,and average optical density(MOD)had an increasing trend in EEP group in comparison with C group,but had no significant changes(P>0.05).Compared with C group,cTnI level was significantly increased in EE group(P<0.05);the cardiomyocyte structure of EE group was damaged,large and densely stained red areas were observed,and MOD was significantly increased in EE group in comparison with C group(P<0.05).Compared with EE group,cTnI level was significantly decreased in EEP+EE group(P<0.05);EEP+EE group displayed relatively well cardiomyocyte structure,and compared with EE group,the areas of ischemia–hypoxia and MOD were significantly decreased in the EEP+EE group(P<0.05),suggesting that EP produced the early cardioprotection by alleviating the exhaustive exercise-induced myocardial injury.(2)Immunofluorescence results of ATG7 showed that ATG7 was marked as red fluorescence,scattered in the cytoplasm of cardiomyocytes,and compared with C group,the red fluorescence intensity of EEP group,EE group and EEP+EE group were increased to different degrees.The trend of ATG7 fluorescence intensity was similar to that of ATG7 expression in western blot.Compared with C group,ATG7 fluorescence intensity and ATG7 expression were significantly increased in EEP group,EE group and EEP+EE group(P<0.05);compared with EE group,ATG7 fluorescence intensity and ATG7 expression had no significant changes(P>0.05).(3)Western blot results of LC3 showed that compared with C group,LC3-Ⅰ expression was significantly increased in EEP group,EE group and EEP+EE group(P<0.05);compared with EE group,LC3-Ⅰ expression had no significant changes in EEP+EE group(P>0.05).Compared with C group,LC3-Ⅱ expression was significantly increased in EEP group(P<0.05),while there were no significant changes in EE group(P>0.05);compared with EE group,LC3-Ⅱ expression was significantly increased in EEP+EE group(P<0.05).Compared with C group,the LC3-Ⅱ/LC3-Ⅰ ratio was significantly higher in EEP group(P<0.05),and there were no significant changes in EE group(P>0.05);compared with EE group,the LC3-Ⅱ/LC3-Ⅰ ratio was significantly higher in EEP+EE group(P<0.05).The western blot result of p62 showed that compared with C group,p62 expression had no significant changes in EEP group(P>0.05),and p62 expression was significantly increased in EE group(P<0.05);compared with EE group,p62 expression was significantly decreased in EEP+EE group(P<0.05).Conclusion: EP could produce the early cardioprotection by alleviating the exhaustive exercise-induced myocardial injury.Inductive phase of EP promoted the increase of ATG7 expression and the autophagy level of cardiomyocytes,increasing the formation of autophagosome mediated by ATG7,the esterification of LC3 and the degradation of p62 protein during exhaustive exercise,so as to maintain the high level of autophagic expression during exhaustive exercise,and reduce the exhaustive exercise-induced myocardial injury in the early protective phase of EP. |
