Role And Molecular Mechanisms Of Myocardial Protection Effects Through AMPK-mTOR-ULK1 Pathway Activated Autophagy By Exercise Preconditioning

Objective:As a means of stimulating,the myocardial protective effects of exercise preconditioning（EP）has attracted much attention.Autophagy is an indispensable degradation pathway for myocardial health.Therefore,the myocardial protective effects which induced by exercise preconditioning may be related to myocardial autophagy.Recent studies have shown that AMPK-mTOR-ULK1 pathway plays an important role in autophagy regulation.In this study,we investigated the effects of early and late exercise preconditioning on AMPK-mTOR-ULK1 signaling pathway,and explored the mechanism of AMPK-mTOR-ULK1 signaling pathway which activated autophagy.We verified whether autophagy was involved in the exercise preconditioning-induced early and late myocardial protective effects by using autophagy inhibitor,wortmannim.Thus,the present study will further improve the theoretical system how exercise preconditioning participating in myocardial protective effect.Methods:Healthy male SD rats（aged 8 weeks,n=200）were randomly divided into 10groups:control（C）group;exhaustive exercise（EE）group,samples were harvested 0.5 hour after a single bout exhaustive treadmill running;EEP group,samples were harvested 0.5 hour after exercise;EEP+EE group,0.5hour after exercise preconditioning,an exhaustive running was added,samples were harvested 0.5 hour after exercise;W+EEP group,in which the rats were injected with wortmannin 0.5 hour before the exercise,which was performed the same way as for the LEP group,samples were harvested 0.5hour after exercise;W+EEP+EE group,in which the rats were injected with wortmannin 0.5 hour before the exercise,which was performed the same way as for the EEP+EE group did,samples were harvested 0.5 hour after exercise;LEP group,samples were harvested 24 hours after exercise;LEP+EE group:in which the exercise was performed the same way as for the LEP group,after which the rats ran to exhaustion after a 24-hour rest,samples were harvested 0.5 hour after exercise;W+LEP group:in which the rats were injected with wortmannin 0.5 hour before the exercise which was performed the same way as for the LEP group,samples were harvested 24hours after exercise;W+LEP+EE group:in which the rats were injected with wortmannin 0.5 hour before the exercise,which was performed the same way as for the LEP+EE group did,samples were harvested 0.5 hour after exercise.Model establishment:EP model was established with an intermittent high intensity treadmill running;and an exhaustive treadmill running was used to establish an acute myocardial injury model.The levels of cardic troponin I（cTnI）in the plasma was detected by immunoluminescence,which evaluated the degree of myocardial damage.Hematoxylin eosin（HE）staining was used to observe the myocardial morphological changes.Haematoxylin basie fuchsin picric acid（HBFP）staining was used to evaluate the myocardial ischemic-hypoxic injury and protection.Transmission electron microscopy（TEM）was used to observe the myocardial ultrastructure and to observe the autophagic structures.Western blot was used to detect the expression of AMPK、AMPKα^Thr172、mTOR、ULK1、ULK1^Ser757,and the ratio of ULK1^Ser757/ULK1 was calculated.These indicators were used to analyze the possible mechanism of exercise preconditioning affecting autophagy.The expression of autophagy-related proteins:LKB1,PI3KC3,Beclin 1,Bcl-2,LC3-I and LC3-II were detected by western blot,and the LC3-II/LC3-I ratio was calculated.By analyzing the changes of above indicators,we discussed the mechanism of AMPK-mTOR-ULK1 signaling pathway which activated autophagy,and that activated autophagy was partially involved in myocardial protection against EE-induced myocardial ischemic-hypoxic injury.Results:Compared with the C group,the plasma cTnI（marker of myocardial injury）levels in the EE group increased significantly and the degree of myocardial ischemia and hypoxia was serious（P<0.05）;HE staining showed:eosinophils increased,myocardial cell swelled,myocardial fiber partially dissolved,broke.After exhaustive exercise.Rats’myocardial ultrastructure changed significantly:myofibrils broke severely,many mitochondria vacuoles appeared,however,no nuclear pyknosis or disintegration were observed.Compared with the C group,there were no significant changes in plasma cTnI levels,degree of myocardial ischemia-hypoxia and ultrastructure in the EEP and LEP groups.Compared with the EE group,the levels of plasma cTnI caused by exhaustive exercise in the EEP+EE and LEP+EE groups decreased significantly,and the degree of myocardial ischemia-hypoxia decreased significantly（P<0.05）.HE staining showed that eosinophilic increased;some myofibrils showed wavy changes.Autophagy structure was observed in the LEP+EE group.Compared with the EEP+EE group,the plasma cTnI levels in the W+LEP+EE group decreased（P<0.05）,and there was no significant changes in ischemia and hypoxia,but HE staining showed widening of myocardial fiber spacing.TEM results showed that some mitochondria appeared vacuolation.Compared with the LEP+EE group,the cTnI levels and the degree of hypoxia and ischemia in the W+LEP+EE group increased significantly（P<0.05）,HE staining showed that eosinophilic increased.TEM showed some myofibrils were broken;the ultrastructure of myocardium changed obviously,and mitochondria vacuoles was serious.Compared with the C group,the expression of AMPK in AMPK-mTOR-ULK1 pathway increased significantly in the EEP group,but the expression of mTOR did not change significantly,and the ratio of ULK1^Ser757/ULK1 decreased significantly.Compared with the C group,the expression of AMPK and ULK1 in EEP+EE,LEP and LEP+EE groups increased significantly,the expression of mTOR did not change significantly,while the ratio of ULK1^Ser757/ULK1 decreased significantly;and the expression of AMPKα^Thr172 in the EEP+EE group also increased significantly.Compared with the EEP+EE group,the expression of AMPK,AMPKα^Thr172,mTOR,ULK1,ULK1^Ser757 in the W+EEP+EE group had no significant changed（P>0.05）.Compared with the LEP+EE group,the expression of AMPK,AMPKα^Thr172,mTOR,ULK1,ULK1^Ser757 in the W+LEP+EE group had no significant change（P>0.05）.In addition,compared with the C group,the expression of AMPK was significantly increased in the EE group,the expression of mTOR did not change significantly,while the expression of ULK1^Ser757 and the ULK1^Ser757/ULK1ratio were significantly decreased.Autophagy is involved in early and late exercise preconditioning-induced myocardial protection.Compared with the C group,PI3KC3 expression and the LC3-II/LC3-I ratio increased significantly in the EEP,EEP+EE,LEP and LEP+EE groups;in addition,the expression of LC3-I decreased significantly in the EEP group;Beclin 1 expression increased significantly in the EEP+EE group;and LC3-II expression increased in the EEP+EE and LEP groups.Compared with the EEP group,the expression of PI3KC3,Beclin 1 and LC3-II,and the LC3-II/LC3-I ratio in the W+EEP group showed a downward trend（P>0.05）.Compared with the EEP+EE group,the expression of PI3KC3,Beclin 1 and LC3-II in the W+EEP+EE group showed a downward trend（P>0.05）.Compared with the LEP group,PI3KC3and LC3-II expression,and the LC3-II/LC3-I ratio in the W+LEP group decreased significantly.Compared with the LEP+EE group,PI3KC3 and LC3-II expression in the W+LEP+EE group decreased significantly,and the LC3-II/LC3-I ratio showed a downward trend（P>0.05）.In addition,compared with the C group,PI3KC3 and LC3-II expression in the EE group increased significantly,and the LC3-II/LC3-I ratio increased significantly.Conclusion:A single bout of high-intensity exhaustive exercise can lead to severe ischemic-hypoxic and ultrastructural damage of myocardial cells,it is a reversible exercise-induced injury,which may be related to the reduction of autophagy clearance efficiency during exhaustive exercise.Early and late exercise preconditioning can reduce exhaustive exercise-induced myocardial ischemic-hypoxic injury.Exercise preconditioning activates autophagy through AMPK-mTOR-ULK1 signaling pathway.The main mechanism is that exercise preconditioning up-regulates and activates AMPK,decreases of mTOR-dependent phosphorylation of ULK1 at Ser757,and then activates ULK1 and up-regulates autophagy.Activated autophagy partially participates in the early and late myocardial protective effects induced by exercise preconditioning.Inhibition of autophagy aggravated myocardial ischemic-hypoxic injury.