Effects Of Autophagy On H9c2 Cardiomyocytes Apoptosis Induced By High Glucose/High Lipids

Objective:The role of autophagy in the pathogenesis of diabetic cardiomyopathy remains unknown.In order to establish the myocardial hypertrophy model of diabetic cardiomyopathy,H9c2 cardiomyocytes were incubated by high glucose/high lipids(HGHL).We observed the effect of high glucose/high lipids on cell injury including cell apoptosis.To investigate the effect of autophagy on cardiomyocyte hypertrophy induced by high glucose/high lipids in H9c2 cardiomyocytes.Methods:The experiment is divided into three parts:(1)H9c2 cardiomyocytes were grouped by the stochastic indicator method: control group,different concentrations of high glucose/high lipids(250 μM,500 μM,1 000 μM)were used to treat H9c2 cardiomyocytes in different time(12 h,24 h,36 h,48 h)groups,HE staining was used to observe the effect of high glucose/high lipids on hypertrophy of H9c2 cardiomyocytes by measuring the surface area of cardiomyocytes.The expression of cardiac hypertrophy markers ANP and α-SKA were detected by RT-PCR.(2)The H9c2 cardiomyocytes were grouped by the stochastic indicator method(as the first part).The degree of cell injury was assessed by lactate dehydrogenase(LDH)activity.Cell viability and apoptotic rate were measured by flow cytometry(FCM).(3)Based on the above experimental results,the cultured H9c2 cardiomyocytes were grouped by the stochastic indicator method: control group,500 μM HGHL treatment of H9c2 cardiomyocytes 36 h group(HGHL group),autophagy inhibitor chloroquinepretreatment HGHL intervention group(CQ+HGHL group).In order to observe the effect of autophagy on cardiomyocyte hypertrophy in diabetic cardiomyopathy,Western Blot was used to detect the autophagy marker LC3 Ⅱ and the apoptotic marker protein cleaved csapase-3.To investigate the effect of autophagy flux in HGHL on cardiomyocyte apoptosis,Western Blot was used to detect the expressions of autophagy marker LC3 Ⅱ and autophagy signaing pathway p62/SQSTM1(p62),Beclin-1,lysosomal-associated membrane proteins LAMP1 and LAMP2 in the control group and the 500 μM group.Results:1.High glucose/high lipids treatment can significantly induce hypertrophy of H9c2 cardiomyocytes in a concentration and time-dependent manner.Compared with the control group,the cell surface area increased significantly(P<0.05)at 36 h after treatment HGHL(500 μM),and the expressions of ANP and α-SKA were significantly increased(P<0.01).2.High glucose/high lipids treatment can damage the cardiomyocytes,leading to the degree of cell injury and the apoptosis rate were significantly increased in a concentration and time-dependent manner,and the apoptosis rate was about50%(P<0.05)at 36 h.3.Autophagy flux plays an important role in HGHL induced cardiomyocyte apoptosis.Compared with the control group,the expression of cleaved csapase-3protein in HGHL group was increased(P<0.05).Compared with the control group and the HGHL group,pretreatment with autophagy inhibitor chloroquine for 1 h significantly increased the rate of apoptosis(P<0.05).The level of autophagy flux changed significantly under the HGHL intervention,compared with the control group,the expression of LC3 Ⅱ was very significantly increased(P<0.01),and the expressions of Beclin-1,LAMP1 and LAMP2 were significantly decreased(P<0.05),but the expression of p62 was significantly increased(P<0.01)of the 500 μM group.Conclusions:1.High glucose/high lipids(HGHL)induced H9c2 cardiomyocytes hypertrophy in a concentration and time-dependent manner,suggesting that HGHL in the induction of diabetic cardiomyopathy myocardial injury and disease development process plays an important role.2.High glucose/high lipids(HGHL)can induce myocardial injury by inducing H9c2 cardiomyocyte apoptosis.It is suggested that the effect of HGHL on myocardial injury is related to myocardial apoptosis.3.High glucose/high lipids(HGHL)can inhibit autophagy flux,leading to abnormal accumulation of autophagy and promote apoptosis of cardiomyocytes.It is suggested that apoptosis is associated with autophagic abnormalities caused by autophagic abnormal accumulation.