Exercise Preconditioning Exerts Cardioprotective Effect On Exhausted Rats By Regulating Mitochondrial Autophagy AMPK-mTOR-ULK1 Signaling Pathway

Objective:Exhaustive exercise can cause myocardial energy failure and myocardial damage.Exercise pre-adaptation can increase myocardial energy metabolism,make myocardial adaptability,and protect the heart.The mechanism may be closely related to mitochondrial autophagy.The mitochondrial autophagy AMPK-mTOR-ULK1 signaling pathway plays an important role in the mechanism of exercise cardioprotection.In this study,by establishing a pre-trained SD rat model and using one-time exhaustive exercise as a damaging factor,the effects and protective mechanisms of exercise-activated mitochondrial autophagy AMPK-mTOR-ULK1 signaling pathway on the heart of exhausted rats were investigated.Exercise Cardioprotection Research Provides New Reference.Methods:1.Eighteen healthy male SD rats were randomly divided into a quiet control group(Con group),exercise preconditioning + exhaustion group(EP + EE group),one-time exhaustion group(EE group),6 in each group only.According to the exercise load standards of Domenech and Lennon,exercise pre-adapted rat models were established.Except for the Con group,the other two groups performed treadmill exercise.In the EP + EE group,intermittent treadmill exercise was performed with a slope of 5 ° and a speed of 26 m / min,15 minutes of exercise,5 minutes of rest,3 repetitions,and 6 days / week of exercise for 3 weeks.On the following day after the end of the exercise training,the EE group and the EE group performed a continuous treadmill exercise with a slope of 0 ° and a speed of 30 m / min until the rats became exhausted.Con group was directly taken from myocardial tissue,EP + EE group and EE group were taken 30 min after exhaustive exercise.2.Observe the changes of myocardial microstructure under light microscope;The ultrastructure of mitochondria and autophagy were observed under transmission electron microscope.3.TUNEL method was used to detect the myocardial cell apoptosis and evaluate the degree of myocardial cell apoptosis.4.Real-time PCR was used to detect the expression of cardiomyocyte protein AMPK,ULK1,mTOR,LC3Ⅱ and Beclin1 in rats.5.Western blot detection of rat cardiomyocyte proteins AMPK,ULK1,mTOR,LC3Ⅱ,Beclin1,p-AMPK(Thr.172),p-ULK1(Ser757),p-ULK1(Ser555),p-mTOR(Ser2448)proteins Horizontal expression.Results :1.Myocardial histopathological observation of rats in each groupThe myocardial structure of the Con group was relatively complete under light microscope,the muscle fibers were arranged neatly,no breakage was found,and no edema was found in the interstitial tissue.The myocardial damage of the rats in the EE group and the EP + EE group was more significant than that in the Con group.Less than EE group.Under the electron microscope,the mitochondrial structure of the myocardium in the Con group was complete,and the mitochondrial crest was clearly visible and neatly arranged;the EE group and the EP + EE group showed obvious mitochondrial damage and the appearance of autophagosomes.Among them,the mitochondrial damage in the EP + EE group was reduced compared with the EE group,and the Autophagosomes are relatively reduced.2.Comparison of myocardial cell apoptosis index in each groupCompared with the Con group,the myocardial apoptosis index of the EE group and the EP + EE group was significantly increased,and the difference was statistically significant(P <0.05).The death index decreased,and the difference was statistically significant(P <0.05).3.Real-time PCR detection of cardiomyocyte protein AMPK,ULK1,mTOR,LC3Ⅱ,Beclin1 gene expression in rats3.1 Myocardial mitochondrial autophagy pathway related genes mRNA AMPK,ULK1,mTOR expression in each group of ratsCompared with the Con group,the mRNA expressions of autophagy-related proteins AMPK and ULK1 in the EE group and the EP + EE group were higher than those in the Con group,and the difference was statistically significant(P <0.05).The autophagy was related to the EE group and the EP + EE group.The mRNA expression of the protein mTOR was lower than that of the Con group,and the difference was statistically significant(P <0.05).Compared with the EE group,the mRNA expressions of autophagy-related proteins AMPK and ULK1 in the EP + EE group were lower than those in the EE group,and the difference was statistically significant(P <0.05).The mRNA expression of the autophagy-related proteins mTOR in the EP + EE group was higher than that in the EE group.In the EE group,the difference was statistically significant(P <0.05).3.2 Expression of LC3Ⅱ and Beclin1 in myocardial autophagy related genes of rats in each groupCompared with the Con group,the mRNA expression of autophagy-related proteins LC3Ⅱ and Beclin1 in the EE group and the EP + EE group were higher than those in the Con group,and the difference was statistically significant(P <0.05).Compared with the EE group,the EP + EE group The mRNA expression of LC3Ⅱ and Beclin1 decreased,and the difference was statistically significant(P <0.05).4.Western blot to detect rat cardiac protein AMPK,ULK1,mTOR,LC3Ⅱ,Beclin1,p-AMPK(Thr.172),p-ULK1(Ser757),p-ULK1(Ser555),p-mTOR(Ser2448)proteins Horizontal expression4.1 Myocardial mitochondrial autophagy pathway related proteins AMPK,ULK1,mTOR,p-AMPK(Thr.172),p-ULK1(Ser757),p-ULK1(Ser555),p-mTOR(Ser2448)expression levels in each group Comparison4.1.1 Comparison of the contents of AMPK,ULK1 and mTOR of myocardial mitochondrial autophagy pathway related rats in each groupCompared with the Con group,the relative content of myocardial protein AMPK in rats in the EE group and the EP + EE group increased significantly,the difference between the Con group and the EE group was statistically significant(P <0.05),and there was no significant difference between the EP + EE group(P <0.05).> 0.05);Compared with the EE group,the content of AMPK in myocardial proteins of rats in the EP + EE group was reduced,and the difference was statistically significant(P <0.05).There were no significant differences in myocardial mTOR and ULK1 protein content between the Con group,EE group and EP + EE group(P> 0.05).4.1.2 Comparison of the contents of myocardial mitochondrial autophagy pathway-related proteins p-AMPK(Thr.172),p-ULK1(Ser757),p-ULK1(Ser555),and p-mTOR(Ser2448)in each group of ratsCompared with the Con group,the myocardial protein p-AMPK(Thr.172)and p-ULK1(Ser555)in the EE group and the EP + EE group were significantly increased,and the difference was statistically significant(P <0.05).Compared with the control group,the relative content of myocardial protein p-ULK1(Ser555)in the EP + EE group was reduced,and the difference was statistically significant(P <0.05).The relative content of p-AMPK(Thr.172)was also reduced,but the difference was not statistically significant.(P> 0.05).Compared with the Con group,the expressions of myocardial proteins p-mTOR(Ser2448)and p-ULK1(Ser757)in the EE group and EP + EE group were reduced,which were significantly different from those in the EE group(P <0.05).There was no significant difference in the EP + EE group(P> 0.05).Compared with the EE group,the relative content of p-mTOR(Ser2448)and p-ULK1(Ser757)in the EP + EE group increased,and the difference was statistically significant(P <0.05).4.2 Comparison of the content of mitochondrial autophagy-related proteins Beclin1 and LC3Ⅱ between groupsCompared with the Con group,the content of Beclin1 and LC3Ⅱ in the myocardium of the rats in the EE group was significantly increased,and the difference was statistically significant(P <0.05).The relative content of Beclin1 and LC3Ⅱ in the myocardium of the EE + EP group was higher,and the difference was not statistically significant.Significance(P> 0.05);Compared with the EE group,the relative content of Beclin1 and LC3Ⅱ in the myocardium of the EE + EP group decreased,and the difference was statistically significant(P <0.05).4.3 Correlation analysis between mitochondrial autophagy-related proteinsAMPK-mTOR-ULK1 signaling pathway protein correlates with Beclin1 and LC3Ⅱ.Conclusion:1.Exhaustive exercise can cause myocardial injury.The mechanism may be related to mitochondrial autophagy AMPK-mTOR-ULK1 signaling pathway activated by exhaustive exercise.By causing overexpression of AMPK-mTOR-ULK1 signaling pathway,mitochondrial autophagy is excessive and autophagy decline Perish.2.Exercise preconditioning can reduce exhaustive heart injury.The mechanism may be through regulating the mitochondrial autophagy AMPK-mTOR-ULK1 signaling pathway,inhibiting the overexpression of AMPK-mTOR-ULK1 signaling pathway,reducing apoptosis and exerting a protective effect on the heart.3.Excessive mitochondrial autophagy will aggravate exercise-induced heart injury,and moderate mitochondrial autophagy has a protective effect on the heart.