Role And Mechanism Of Hexokinase Ⅱ In The Damage Of Autophagic Flow In Cardiomyocytes Of Mice With Hypoxia

Background:Heart hypoxia caused by severe burns,trauma,high altitude hypoxic conditions,and other causes can cause myocardial damage.For example,after severe burns,myocardial hypoxia may occur due to insufficient local blood flow induced by strong stress and reduced circulating blood volume.As the heart acts as a highly energy-consuming circulating dynamic organ,myocardium is susceptible to hypoxia,resulting in serious damage which eventually induces or aggravates burn shock and increases the mortality rate of severe burns.Therefore,myocardial damage caused by hypoxia has become a problem that cannot be ignored in clinical treatment of patients with severe burns and trauma.It is of great clinical significance to elucidate the damage effect of hypoxia on cardiomyocytes and its mechanism.Autophagy is an evolutionarily conserved intracellular degradation pathway that relies on the lysosomal system.Under physiological conditions,autophagy maintains the normal function of cells by degrading long-lived proteins and aging damaged organelles in cells.However,when autophagy process,namely autophagic flow,is impaired,cells die due to accumulation of degrading substrates and insufficient supply of nutrient.Recent studies have shown that impaired autophagic flow is an important pathway for a variety of pathological factors leading to cell dysfunction and even death.Therefore,hypoxia after severe burns and trauma may cause the damage of cardiomyocytes through the damage of autophagic flow,but the specific effects and mechanisms are still unclear.Hexokinase is the rate-limiting enzyme in the first step of glycolysis.In mammals,hexokinase exists in different tissue cells in four subtypes,mainly hexokinase II in cardiomyocytes.Studies have shown that hypoxia can up-regulate the expression level of hexokinase II and cause damage to mouse nerve cells.Other studies have shown that hexokinase II is involved in the regulation of autophagy in rat cardiomyocytes during glucose-free starvation.Therefore,we hypothesized that hexokinase II may mediate the impairment of autophagic flow in cardiomyocytes caused by hypoxia.MTORC1 is a key regulator of cell growth and metabolism.Under starvation conditions,its activity can be rapidly inhibited to initiate autophagy,then its activity is reactivated by autophagy-derived nutrients to terminate autophagy.Thus,MTORC1 is an important "switch" that regulates autophagy.However,under hypoxic conditions,whether hexokinase II regulates autophagic flow in cardiomyocytes through MTORC1 has not been reported.Therefore,this study proposes a hypothesis that under hypoxic conditions,hexokinase II damages autophagic flow through MTORC1,thereby damaging cardiomyocytes.By constructing an hypoxic model in vitro and regulating the activity and expression of hexokinase II,this study aims to determine the role and mechanism of hexokinase II in the damage of autophagic flow in cardiomyocytes of mice with hypoxia.Methods:The hearts of totally 6 male and female C57BL/6 mice aged from 1 to 2 days were isolated to culture primary cardiomyocytes which were used for the following experiments:1.The cells were randomly divided into 6 groups,i.e.,normal control 3,6,and 9 h groups and hypoxia 3,6,and 9 h groups.After being regularly cultured for 48 h with DMEM/F12 medium,the cells in normal control groups were cultured with replaced fresh DMEM/F12 medium for 3,6,and 9 h,respectively,and those in hypoxia groups were cultured with replaced sugar-free serum-free medium in the low-oxygen incubator(1% oxygen、5% carbon dioxide)at 37°C for 3,6,and 9 h,respectively.Western blotting was used to detect the expressions of LC3Ⅰ,LC3Ⅱ,p62,and hexokinase Ⅱ,and cell viability was measured by the CCK-8 method.2.The cells were divided into normal control group,simple hypoxia 9 h group and hypoxia 9 h+2-DG group.After a regular culture for 48 h,the cells in normal control group were cultured with replaced fresh DMEM-F12 medium for 9 h,those in simple hypoxia 9 h group were replaced with sugar-free serum-free medium,and those in hypoxia 9 h+2-DG group were replaced with sugar-free serum-free medium in which 2-DG was dissolved(a concentration of 10 mmol/L),and then they were cultured with hypoxia for 9 h.Western blotting was used to detect the expressions of LC3Ⅰ,LC3Ⅱ,and p62,transmission electron microscopy was used to observe autophagosomes/autolysosomes,and cell viability was measured by the CCK-8 method.3.The cells were divided into normal control group,simple hypoxia 9 h group,hypoxia 9 h+ HK-ⅡsiRNA1 group and hypoxia 9 h+HK-ⅡsiRNA2 group.The cells in normal control group and simple hypoxia 9 h group were regularly cultured for 48 h,those in the other 2 groups were respectively transfected with 200 nmol/L HK-ⅡsiRNA1 and HK-ⅡsiRNA2 and then cultured for 48 h.The cells in normal control group were cultured with replaced fresh DMEM-F12 medium for 9 h,and those in the other 3 groups were cultured with replaced fresh sugar-free serum-free medium and hypoxia for 9 h.Western blotting was used to detect the expressions of LC3Ⅰ,LC3Ⅱ,p62,and hexokinase Ⅱ,and cell viability was measured by the CCK-8 method.4.The cells were grouped and treated the same as those in method 1,Western blotting was used to detect the expressions of phosphorylated p70S6 K and 4E-BP1 reflecting MTORC1 activity;The cells were grouped and treated the same as those in method 2,Western blotting was used to detect the expressions of phosphorylated p70S6 K and 4E-BP1;The cells were grouped and treated the same as those in method 3,Western blotting was used to detect the expressions of phosphorylated p70S6 K and 4E-BP1.Results:1.Compared with those of normal control 3,6,and 9 h groups,the LC3Ⅱ/Ⅰratio and expression level of p62,the two indicators reflecting autophagic flow,were significantly increased,cell viability was significantly decreased and the expression level of hexokinase Ⅱ was significantly increased in cardiomyocytes at 3,6 and 9 h groups following hypoxia.2.Compared with those of normal control group,the LC3Ⅱ/Ⅰratio and expression level of p62 were obviously increased,autophagosome/autolysosome accumulated,and cell viability was obviously decreased in cardiomyocytes of simple hypoxia 9 h group;Compared with those of simple hypoxia 9 h group,the LC3Ⅱ/Ⅰratio and expression level of p62 were obviously decreased,autophagosome/autolysosome accumulation was obviously improved,and cell viability was obviously increased in cardiomyocytes of hypoxia 9 h+2-DG group.3.Compared with those of normal control group,the LC3Ⅱ/Ⅰratio and expression levels of p62 and hexokinaseⅡ were significantly increased,cell viability was significantly decreased in cardiomyocytes of simple hypoxia 9 h group;Compared with those of simple hypoxia 9 h group,the LC3Ⅱ/Ⅰratio and expression levels of p62 and hexokinaseⅡ were significantly decreased,cell viability was significantly increased in cardiomyocytes of hypoxia 9 h+HK-ⅡsiRNA1 group and hypoxia 9 h+HK-ⅡsiRNA2 group.4.Compared with those of normal control groups,the expression levels of phosphorylated p70S6 K and 4E-BP1 were obviously decreased in cardiomyocytes of hypoxia groups;Compared with those of simple hypoxia 9 h group,the expression levels of phosphorylated p70S6 K and 4E-BP1 were obviously increased in cardiomyocytes of hypoxia 9 h+2-DG group;Compared with those of simple hypoxia 9 h group,the expression levels of phosphorylated p70S6 K and 4E-BP1 were obviously increased in cardiomyocytes of hypoxia 9 h+HK-ⅡsiRNA1 group and hypoxia 9 h+HK-ⅡsiRNA2 group.Conclusions:After hypoxia,the expression of hexokinase II in cardiomyocytes is increased,which damages autophagic flow by inhibiting the activity of MTORC1,eventually leading to decreased cell viability.To Inhibit the activity of hexokinase II or its expression can attenuate the hypoxia damage of cardiomyocytes.This conclusion provides a new idea for clinical prevention and treatment of cardiomyocyte damage caused by hypoxia after severe burns,and also provides a reference for the prevention and treatment of cardiac hypoxia damage caused by trauma,high altitude hypoxia environment and other causes.