Protect Effects Of MCL-1/PINK1/PARKIN Pathway On High Glucose Induced Mitophagy In H9c2 Cell

With the onset of type Ⅰ and type Ⅱ diabetes is increasing today,diabetic cardiomyopathy is an important cause of diabetic cardiac failure.How to maintain diabetic myocytes at a normal level in metabolic disease and increase it survival under the variety of stress,has become an urgent problem to be solved.Autophagy plays an important role in maintaining normal cells functions and adapting to chronic stimuli.Studies have shown that autophagy plays a protective role in diabetic myocardium under chronic stress,while acute stress may directly lead to apoptosis and promote cell death.Mitochondria are intracellular energy synthesizers,and are particularly important for cardiomyocytes which are unable to utilize lactose as energy.Mitochondrial autophagy is an extremely important part of its quality control.Once damaged,it will lead to intracellular dysfunction and lead to a series of pathophysiological changes.In this study,we will establish cardiomyocytes cell line that can stably express green fluorescent protein coupled with LC3.Next,we will extract mitochondrial proteins and explore the changes of the expression of mitophagy proteins in myocardial cells under diabetic conditions.Gene silencing or overexpression intervention was used to study the role of MCL-1 on mitochondrial autophagy in diabetic myocardium.This study is divided into two parts:1.Establish a stable GFP-LC3 transfected H9c2 cell line and to observe the effect of high glucose on autophagy and mitophagy;2.Investigate the effect of silencing and overexpression of MCL-1 on mitochondrial autophagy in cardiomyocytes;This experiment is divided into two parts:Part One:To establish a stable GFP-LC3 transfected H9c2 cell line and observe the effect of high glucose on autophagy and mitophagyObjective:To establish a stable GFP-LC3-expressed H9c2 cell line,which laid the foundation for the study the changes of autophagy in cardiomyocytes under high glucose.To observe the changes of mitochondrial autophagy related proteins in H9c2 cells under high glucose conditionMethods:The MSCV-GFP-LC3 plasmid was constructed and transfected into H9c2 cells with transfection reagent.The stable transfectants were screened by G418.The expression of GFP-LC3 in vector and myocardial cells were detected by fluorescence microscopy,confocal microscopy and Western Blot.Autophagy of the stable expression myocardial cells in a high glucose environment was observed by confocal microscopy.H9c2 cells were divided into control group and high glucose treatment group.The cytosolic protein and mitochondrial protein were extracted,and the expression of MCL-1 and autophagy related protein was detected by Western Blot.Results:Transfected H9c2 cell line,screened repeatedly by G418,was successfully constructed.After the treatment of autophagy inducer,the expression rate of green fluorescence was above 95%,and the expression of GFP-LC3 fusion protein was confirmed by Western Blot.The observation from confocal microscopy demonstrated that autophagy decreased after 48 hours of high glucose treatment.In high glucose group,the expression of MCL-1 decreased,and the expression of PINK 1,Parkin and LC3II in mitochondrial protein decreased.Conclusion:A stable GFP-LC3-expressed H9c2 cell line was successfully constructed in this study.And the activation of autophagy and mitophagy were declined by high glucose treatment,probably due to the decrease of MCL-1 mediated translocation of PINK1/Parkin to mitochondria.Part Two:Effects of silencing and overexpression of MCL-1 on mitophagy in diabetic cardiomyocytesObjective:Observe the changes of mitophagy of cardiomyocytes which were silence or overexpression of MCL-1.Methods:H9c2 cells were transfected with silence-shRNA-MCL-1 and overexpression-shRNA-MCL-1 separately,and MCL-1-/-and MCL-1+/+ cells were treated by high glucose(30mmol/L).Western Blot texted the expression of MCL-1,PINK1,Parkin,LC3 from extracted mitochondrial proteins;GFP-LC3-H9c2 cells were cultured in normal or high glucose medium for 48 hours.The same sequence of the plasmid was transfected into GFP-LC3-H9c2 cells.After treated by HBSS for 4h,confocal microscopy was used for detecting the changes of autophagy..Results:In normal culture medium,compared with MCL-1+/-cells,the expression of MCL-1,PINK1,Parkin,LC3 were significantly decreased in MCL-1-/-cells(P<0.05),while that in MCL-1+/+just opposite.After treated with high glucose,abundant MCL-1-/-went dead,MCL-1+/+ cells showed increased expression of mitophagy related proteins.And no significant difference between normal and high glucose groups in MCL-1+/+ cells.The autophagy of MCL-1-/--GFP-LC3-H9c2 cells in normal glucose and high glucose group was attenuated,while autophagy was enhanced with MCL-1+/+-GFP-LC3-H9c2.Conclusion:In high glucose environment,MCL-1 activity was inhibited;overexpression of MCL-1 gene could increase mitophagy related proteins PINK1,Parkin and LC3,and partly reverse the damage caused by high glucose.