The Role Of NEDD4 Regulating AMPK In Myocardial Adaptation To Chronic Hypoxia

Objectives: Chronic hypoxia is a main patho-physiological feature of many heart diseases, such as cyanotic congenital heart disease, coronary heart disease and so on. Hypoxia induces energy stress, oxidative stress and other stress, and promotes myocardial remodeling,mitochondrial biogenesis and internal environment changes. Previous studies showed that myocardial adaptation to chronic hypoxia has a protective effect on the heart,whereas the exact mechanism remains unclear.Energy metabolism regulation has been considered as a key point of myocardial adaptation to chronic hypoxia. AMP-activated protein kinase(AMPK) is an important regulator of energy metabolism. In the heart, phospho-AMPK(p-AMPK) can enhance energy generation and inhibite energy consumption by regulating various metabolic pathways. Neural precursor cell expressed developmentally down-regulated protein 4( NEDD4) is a E3 ubiquitin ligase. The domain of WW in NEDD4 can identify the phosphorylated threonine residues existing in the structure of activated AMPK. Then NEDD4 maybe participate in the regulation of AMPK activity. The purpose of this research is to explore the potential role of NEDD4 and AMPK in the myocardial adaptation to chronic hypoxia in cardiac tissue and embryonic rat-heart-derived H9c2 cells.Methods:1. Childrens with acyanotic( n = 14) or cyanotic( n = 18) cardiac defects were enrolled. Samples were taken from the right ventricular outflow tract. The expression of NEDD4 was detected by immunohistochemistry. The protein expression level of NEDD4, AMPK and p-AMPK were evaluated by western blot. We also analysed the relationship between the protein production of NEDD4 with the Sa O2.H9c2 cells were cultured and exposed to hypoxia(94% N2,5% CO2,1% O2) for 72 h. Detected the protein expression level of NEDD4, AMPK and p-AMPK by western blot. Then knockdowned the protein level of NEDD4 by small interfering RNA(si RNA) under chronic hypoxia and observed the proportion of cardiomyocyte apoptosis by flow cytometry.Results:In the experiments with the tissue of right ventricular outflow tract, immunohistochemistry revealed that NEDD4 was expressed in the cytoplasm mainly. Compared with the patients with acyanotic cardiac defects, the protein level of NEDD4 in patients with cyanotic was significantly lower(P < 0.05)which was positively correlated with the level of hypoxemia.The level of AMPK activation was higher(P < 0.05)in the tissue of right ventricular outflow tract from the patients with cyanotic compared with their acyanotic counterparts.In vitro experiments with H9c2 cells, western blot analysis suggested that protein level of NEDD4 stayed at a high levels initially and decreased significantly(P < 0.05) after 24 hours’ hypoxia. Meanwhile the ratio of p-AMPK/AMPK reached the maximum(P < 0.05)after 48 hours’ hypoxia.Compared with the normoxic group, the proportion of cardiomyocyte apoptosis in hypoxia group increased significantly(P < 0.05). However,a lower proportion was detected in the NEDD4 knockdown group(P < 0.05). The ratio of p-AMPK/AMPK in the knockdown group increased significantly(P < 0.05) than that of the control group.Conclusions:Compared with the patients with acyanotic cardiac defects, increased activation of AMPK and downregulation of NEDD4 protein expression was observed in the tissue of right ventricular outflow tract from the patients with cyanotic.The H9c2 cells were cultured under hypoxic condition. Compared with the normoxic group, The protein level of NEDD4 in hypoxic group decresed significantly. Meanwhile the level of AMPK activation in hypoxic group was higher than that in normoxic group.2. The proportion of cardiomyocyte apoptosis in hypoxia group was significantly increased as compared with normoxic group. However,a lower proportion was observed in the NEDD4 knockdown group. NEDD4 might participate in myocardial adaptation to chronic hypoxia by regulating the activation of AMPK.