AMPK?2 Regulating Autophagy To Participate In The Adaptive Response Of Myocardium Of Mice During Chronic Hypoxia

BackgroundMyocardial chronic hypoxia is a common pathophysiology of a series of clinical diseases like cyanotic congenital heart disease,pulmonary heart disease,high altitude heart disease and other systemic hypoxia diseases.It is also common in diseases with a state of regional myocardial ischemia and hypoxia caused by a variety of factors such as coronary artery atherosclerosis,cardiac hypertrophy and so on.To study the adaptive response of chronic hypoxic myocardium and its mechanism is of great significance for the treatment of such clinical diseases.Autophagy is a highly conserved catabolic process in cells.The basal level of autophagy contributes to the renewal of intracellular proteins and organelles and the maintenance of cellular homeostasis.Autophagy level will increase under the stimulations including nutrition deficiency,oxidative stress and other factors,thereby degrading excessive proteins to provide small-molecule metabolic substrates and removing damaged organelles to avoid cell damage.Previous studies have found that autophagy levels were elevated in the mice models of TAC-induced myocardial hypertrophy and in the myocardial ischemia/reperfusion models.Autophagy enhancement was also found in surgical specimens from patients with cyanotic congenital heart diseases.Since autophagy is closely related to the initiation and development of cardiovascular diseases,to study the changes and mechanisms of autophagy in chronic hypoxic myocardium may provide noval potential targets for the treatment of those diseases.AMPK,a conserved intracellular Ser/Thr kinase,is considered to be a key molecule in the regulation of intracellular energy homeostasis.AMPK is activated when the intracellular ATP content is reduced,it inhibits anabolism while promotes catabolism and ATP production.AMPK is also an upstream molecule of the autophag y pathway,and activation of AMPK is involved in the regulation of autophagy pathways in cells under the stress.Studies have found that AMPK activation can promote the adaptation and survival of cardiomyocytes under chronic hypoxic conditions,but its specific mechanism remains to be further elucidated.The AMPK molecule is consist of three subunits,namely ?,? and ?,and ? is the catalytic subunit,which is responsible for the implementation of the function of the activated AMPK molecule.There are ?1 and ?2 two subtypes,and the ?2 subunit distributes more widely in the heart.In this study,we created the mouse model of chronic hypoxia,and tested the effect of chronic hypoxia on myocardial autophag,and then observed the effects of normoxia or chronic hypoxia on myocardial autophagy in AMPK?2 knockout mice.Methods1.Establishment of mice model of chronic hypoxia: With reference to a patent,an atmospheric hypoxic chamber was produced.According to literatures,mice were placed in the hypoxic chamber with 10% O2 for 4 weeks to establish the chronic hypoxia model,and then the level of RBC and other related indicators were detected to evaludate the success of modeling.2.Effects of chronic hypoxia on myocardium autophagy in mice: Wild-type C57 mice were divided into normoxic group and hypoxic group randomly.The change of LC3-?/LC3-?ratio and p62 expression in myocardium was detected by Western blot.In addition,the expression of LC3 was also detected by immunofluorescence staining of frozen mouse heart sections.3.Effects of AMPK?2 knockout on myocardium autophagy in mice: The heterozygous mice were paired to reproduce the offsprings,and then the homozygotes were selected.The wild type and AMPK?2-/-mice were divided into normoxic group and hypoxic group,namely WT normoxic group,WT hypoxic group,KO normoxic group and KO hypoxic group,with 10 mice in each group.The change of LC3-?/LC3-?ratio was measured by Western blot and the expression of LC3 was also detected by immunofluorescence staining.And the survival curves of WT and KO mice under hypoxia were recorded.4.Statistical analysis: t test was used to compare the means of two groups while one-way ANOVA was used to compare the means of mutiple groups(?3)in SPSS 13.0.Results1.Establishment of chronic hypoxia model: Compared with normoxic group,red blood cells and hemoglobins were significantly increased in hypoxic group(P<0.05),the daily food intake and body weight were significantly decreased(P<0.05),heart / body weight ratio and lung / body weight ratio were significant declined(P<0.05).2.The effect of chronic hypoxia on myocardial autophagy level in mice: Compared with normoxic group,Western blot showed that the ratio of LC3-? /LC3-? in myocardium of hypoxic group was significantly increased(P<0.05)while the p62 level was significantly decreased(P<0.05).And immunofluorescence staining suggested an increase in the expression of LC3 in frozen sections from the hypoxic group.3.Effects of AMPK?2 knockout on myocardium autophagy in mice: There was no significant difference in LC3-?/LC3-?ratio in myocardium between WT and KO mice under normoxia(P>0.05),the ratio of LC3-?/LC3-? in myocardium of KO mice were decreased significantly under hypoxia compared with that of WT mice(P<0.05).And the median survival time of KO mice was significantly shorter than that of WT mice under hypoxia(P<0.05).Conclusion1.The level of autophagy in myocardium of mice increased under chronic hypoxia,indicating that autophagy might be a responsive mechanism of cardiomyocytes to hypoxia conditons.2.There was no significant difference in myocardial autophagy between the KO and WT mice in the normoxic condition.However,under the hypoxic condition,the level of myocardial autophagy in the KO mice was significantly lower than that in WT mice,suggesting that AMPK?2 might mediate chronic hypoxia-induced myocardial autophagy.The myocardial autophagy reduction in AMPK?2-/-mice might be a maladaptive response to chronic hypoxia in mice for they had a decreased median survival time.