Exercise Promotes The Formation Of Exercise-induced Cardiac Hypertrophy By Activating Autophagy Through AMPK/ULK1 Signaling Pathway

Purpose:In this study,we established a mouse model of exercise-induced myocardial hypertrophy to analyze the role of AMPK/ULK1 signaling pathway in regulating autophagy system and promoting myocardial hypertrophy,so as to provide a reference for revealing the molecular mechanism of exercise-induced myocardial hypertrophy.Methods:1.Forty SPF male C57BL/6 mice aged 7 weeks were randomly divided into control group（group C,n=16）and swimming group（group E,n=24）.In this experiment,the model of myocardial hypertrophy in mice was established through8-week swimming exercise by referring to the exercise program of Kim and Zhao Yongcai et al.Body weight,cardiac coefficient and left ventricular coefficient of mice in group C and group E were compared and analyzed.Morphological changes of myocardial cells of mice in the two groups were observed by HE staining,and myocardial hypertrophy of mice in group E was confirmed.The gene expression levels of markers related to pathological myocardial hypertrophy were detected by real-time fluorescence quantitative PCR to confirm that the mice in group E did not have pathological myocardial hypertrophy,thus confirming the successful construction of exercise-induced myocardial hypertrophy model.2.The expression of autophagy genes LC3 and Beclin1 were determined by real-time fluorescence quantitative PCR and western blot.3.Real-time quantitative PCR and western blotting were used to analyze the role of autophagy in exercise-induced cardiac hypertrophy by regulating the signaling pathways related to cell growth and autophagy——ERK,AKT/mTOR and AMPK/mTOR/ULK1.Results:1.Establishment of mouse myocardial hypertrophy model:（1）After 8 weeks of swimming exercise,the body weight of mice in group E was lower than that in group C（P<0.05）.Compared with group C,the heart coefficient and left ventricular coefficient in group E were significantly increased after 8 weeks of swimming（P<0.01）.The results of two-dimensional echocardiography showed that compared with group C,the left ventricular cavity of group E was enlarged and the pumping function of the heart was significantly enhanced.Microscopic observation of HE staining tissue sections showed that myocardial fibers of mice in both groups were evenly stained and neatly arranged,and myocardial nuclei were oval.Compared with mice in group C,myocardial cell diameter of mice in group E was significantly increased（P<0.01）.The results showed that after 8 weeks of swimming,the myocardial hypertrophy was significantly changed,especially in the left ventricular hypertrophy.（2）After 8 weeks of swimming,the expression levels of ANP,α-actin and BNP in myocardium of mice in group E did not change significantly compared with group C（P>0.05）,nor did theα-MHC/β-MHC ratio change significantly（P>0.05）,suggesting that there was no pathological change in myocardium of mice in group E.The results showed that there were no pathological changes in the myocardium of group E mice,and the model of exercise-induced myocardial hypertrophy was established successfully.2.The level of autophagy in hypertrophic myocardium of mice increased:After 8weeks of swimming exercise,the results of observing autophagosomes by transmission electron microscopy showed that compared with mice in group C,the number of myocardial autophagosomes in group E mice was significantly increased;Real-time quantitative PCR showed that compared with group C,LC3B mRNA expression was increased in group E（P<0.05）,and Beclin1 mRNA expression was also significantly increased（P<0.01）.Western blotting results showed that compared with group C,the ratio of LC3 II/LC3 I and the expression level of Beclin1 protein in myocardial tissue of mice in group E were significantly increased（P<0.01）.The results showed that autophagy levels were increased in mice with exercise-induced cardiac hypertrophy after 8 weeks of swimming exercise.3.After 8 weeks of swimming,the real-time quantitative PCR results showed that compared with the mice in the C group,the AMPK mRNA expression in the myocardial tissue of the E group mice was significantly increased（P<0.01）,and the ULK1 mRNA expression level was also significantly increased（P<0.05）;Western blot results showed that compared with group C mice,the expression level of AMPK protein in myocardial tissue of mice in group E was increased（P<0.05）,the expression level of ULK1 protein was also significantly increased（P<0.01）,and the ratio of AMPKα^Thr172/AMPK was increased（P<0.05）.The ratio of ULK1^Ser555/ULK1was also significantly increased（P<0.01）.4.After 8 weeks of swimming,real-time quantitative PCR results showed that the mRNA expressions of Akt,ERK and mTOR in group E did not change significantly compared with group C（P>0.05）.Western blotting results showed that compared with group C,the protein expression levels of ERK,Akt and mTOR and the ratio of Akt ^Ser473/Akt,ERK^{Thr202/Tyr204}/ERK and mTOR^Ser2448/mTOR in myocardial tissue of group E mice did not change（P>0.05）.Conclusion:（1）Physiological hypertrophy of mouse myocardium occurred after 8 weeks of swimming exercise.（2）Autophagy level in hypertrophic myocardium of mice was significantly increased after 8 weeks of swimming exercise.（3）8-week swimming exercises regulate the cardiac autophagy system through AMPK/ULK1 signaling pathway,and the possible mechanism is that 8-week swimming exercises can activate and up-regulate AMPK expression level.The activated AMPK increases the phosphorylation level of ULK1^Ser555,activates ULK1,and up-regulates the autophagy level of hypertrophic myocardium,thus promoting the formation of exercise-induced cardiac hypertrophy.