The Role And Mechanism Of Activatory AMPK Promotes Autophagic Response Involved In The Myocardial Chronic Hypoxia Adaption

Objectives:Congenital heart disease(congenital heart disease, CHD) is one of the common diseases in infants. Nowadays, the cure rate of CHD has improved significantly than before, but the operative mortality of cyanotic congenital heart disease(CCHD)remains high level. Chronic hypoxia is the common pathophysiological process of CCHD. Currently, the molecular mechanism of myocardial chronic hypoxia adaptation in CCHD remains unclear. Therefore, further study of the molecular mechanism in myocardial chronic hypoxia adaptation could provide new myocardial protection strategies to improve the clinical treatment of CCHD.AMPK is Ser/Thr kinase in eukaryotic cells. Studies have confirmed that the activation of AMPK is involved in myocardial energy metabolism in chronic hypoxia. Recent s tudies have shown that the activation of AMPK is closely related to autophagic response in tumor cells. We speculate that AMPK may have protective effects by modulating autophagy in the chronic hypoxia adaptation on myocardial cells. This study will verify our speculation on three aspects: 1.To detect the expression levels of activated AMPK in chronic hypoxia cardiac tissue; 2.To study the effect of activated of AMPK on chronic hypoxia cardiomyocytes; 3.To explore the influence of activated AMPK on the autophagic response of chronic hyoxia cardiomyocytes.Methods:1. Chose 24 male SD rats of 4-6 weeks in clean level, and divided them into normoxia group and hypoxia group randomly. Put the hypoxia group into low pressure tank(simulated altitude of 5000 m, 405 mmHg), and fed the normoxia groups in normal condition contrastively. After feeding 28 days at the same time, executed the rats, and immediately took out the rat heart and isolated right ventricular part as samples; Selected 24 cases with congenital heart disease were going to undergo the correction of cardiac defects in 2013. Among them, there were 12 cases with noncyanotic defects and the remaining 12 cases with cyanotic ones.Removed the myocardial tissue from right ventricular outflow tract as specimens during surgery.Using Western blot to detect the expreesion level of the activated AMPK in hypoxia and nomoxia myocardial tissue of rats and patients respectively.2.Chose H9c2 cell lines and devided them into normoxia group, hypoxia group, blocking group and activating group. Put hypoxia group, blocking group and the activating group in hypoxia incubator 3d(94% N2,5% CO2,1% O2), set up H9c2 chronic hypoxia model. Put normoxia group in cell culture box 3d(74% N2,5% CO2, 21% O2) as control simultaneously. Added Compound C and AICAR in blocking group and the activating group respectively.Using Western blot to test the expression of p-AMPK,adopted Hoechst staining to observe the nuclear shape,and LDH to test the release rate of lactic acid in myocardial cell; Adopted Flow cytometry and TUNEL to detect the level of myocardial cell apoptosis.3. Used Western blot to detect the expression levels of LC3 in normoxia group, hypoxia group, blocking group and activating group respectively.Results:1. Activated AMPK levels in normoxia and hypoxia myocardial tissues.Western blot results show that compared with the normal myocardial tissue, the ratio of p-AMPK/AMPK increased significantly in chronic myocardial tissue. In the aspect of myocardial tissue in patients, compared with noncyanotic defects, AMPK protein activated levels increased significantly in cyanotic ones( P<0.005). In the aspect of the myocardial tissue in SD rats, compared with normoxia group, AMPK protein activation levels increased significantly in hypoxia group.( P<0.005).2. The protection of activated AMPK to chronic hypoxia myocardial cell.a: Western blot results show that AMPK activated level reduced in blocking group compared with the hypoxia group;(P<0.01) At the same time, AMPK activated levels increased in the activating group.(P<0.005).b: Compared with hypoxia group, the level of apoptosis in cardiomyocytes increased in AMPK blocking group, at the same time, and decreased in AMPK activating group.Flow cytometry: Compared with hypoxia group, apoptosis cell percentage increased obviously in AMPK blocking group, at the same time, and decreased obviously in AMPK activating group.TUNEL experiment: Compared with hypoxia group, proportion of TUNEL positive cells increased obviously in AMPK blocking group,(P<0.05), at the same time, and decreased obviously in AMPK activating group.(P<0.05).c: Compared with hypoxia control group, the degree of necrosis in cardiomyocytes increased in AMPK blocking group, at the same time, and decreased in AMPK activating group.Hoechst staining: Compared with hypoxia group, abnormal nucleus cell percentage increased obviously in the AMPK blocking group,(P<0.01), at the same time, and decreased obviously in AMPK activating group.(P<0.01).LDH experiment: Compared with hypoxia control group, release rate of lactic acid increased obviously in AMPK blocking group(P<0.01), at the same time,and decreased obviously in AMPK activating group.(P<0.01)3.The effection of AMPK activation on autophagic response in chronic hypoxia cardiomyocytes.Western blot results show that compared with the hypoxia group, autophagic response in AMPK blocking group attenuated obviously(LC3Ⅱ/LC3Ⅰ),(P<0.01), and in AMPK activation group enhanced obviously(LC3Ⅱ/LC3Ⅰ),(P<0.01).Conclusions:1.Compared the p-AMPK/AMPK ratio in myocardial tissue from cyanotic congenital heart disease and chronic hypoxia SD rats with noncyanotic congenital heart disease and normoxia SD rats,suggesting that the level of activated AMPK increased significantly in myocardial tissue.2. Cultivating H9c2 cells 72 h in chronic hypoxia condition, added Compound C to block the activation of AMPK, and the levels of apoptosis and necrosis were increased; Added AICAR to promote the activation of AMPK, and the levels of apoptosis and necrosis were decreased.Suggested that p-AMPK has certain protective effect on H9c2 cells in myocardial chronic hypoxia adaptation.3. Cultivating H9c2 cells 72 h in chronic hypoxia condition, added Compound C to block the activation of AMPK,and the LC3-Ⅱ/LC3-Ⅰ ratio was decreased; Added AICAR to promote the activation of AMPK, and the LC3-Ⅱ/LC3-Ⅰ ratio was increased.Suggested that p-AMPK promoting autophagic response in H9c2 cells might be one of the adaptative mechanism in myocardial chronic hypoxia adaptation.