Role Of WDR26 In Mitophagy Induced By Hypoxia In H9c2 Cardiomyocytes Of Rats

Background Myocardial ischemic preconditioning is the most powerful phenomenonthat provides potent cardioprotection in mammalian hearts,in which the specific molecular mechanism now is still unclear.WDR26 is a new myocardial ischemic preconditioning induced upregulation gene cloned by c DNA microarray and suppression subtractive hybridization.This study researched the expression of WDR26 in hypoxia of H9c2 cardiomyocytes and the effects of WDR26 overexpression in mitophagy of H9c2 cardiomyocytes by western blot and immunofluorescence.To explore the mechanism of WDR26 in mitophagy induced by hypoxia,providing new ideas and experimental basis to the prevention and treatment of myocardial ischemia injury in clinical practice.Objectives This study researched the effects and mechanisms of WDR26 on mitophagy induced by hypoxia.The author also revealed the mechanisms of WDR26 on myocardial endogenesis protection.Furthermore,to provide new ideas and clues for the prevention and treatment of myocardial ischemia,Method 1.Detection of expression of WDR26 during cell hypoxia by western blot:The cell culture flasks placed in sealed anaerobic bag at 37?,5%CO2 incubator for 3,6and12 hours,which used N2 saturated low-sugar serum-free DMEM medium,to build hypoxia model.The experiment were divided into normal control group and hypoxia 3 hours,6 hours and 12 hours group,in which detected the expression of WDR26 in rat H9c2 cardiomyocytes hypoxia by Western Blot technology.2.To observe the effect of WDR26 overexpression when hypoxia in H9c2 cardiomyocytes: Culturing the H9c2 cardiomyocytes of WDR26 overexpressing.The open reading frame ORF of WDR26 was cloned into the eukaryotic expression vector pc DNA3.1.Then named the recombinant plasmid as pc DNA3.1-WDTR26.Finally,transfected the H9c2 cell lines by liposomein to promote the WDR26 overexpression,and building the empty vehicle group(Neo group).The experiment were divided into Neo control group,Neo hypoxia group,WDR26 control group and WDR26 hypoxia group,the cell model was constructed by hypoxia 6h to detect the content of LDH in cell supernatants and the cell viability by MTT colorimetric.3.To observe the effect of WDR26 overexpression on autophagy induced by hypoxia in H9c2 cardiomyocytes: The experiment were divided into Neo control group,Neo hypoxia group,WDR26 control group and WDR26 hypoxia group,and the cell model was constructed by hypoxia 6 h.To detect the change of LC3 and p62 by western blot and immunofluorescence.To detect the change of mitochondrial membrane potential by mitochondrial membrane potential assay kit JC-1,according to the operating instructions of JC-1,to observed the formation of JC-1 monomer or polymer fluorescent by microscope,and analyzed the optical density of red / green ratio with image J analysis software.4.To observe the change of the PINK1-Parkin pathway when hypoxia in H9c2 cardiomyocytes: The experiment were divided into Neo control group,Neo hypoxia group,WDR26 control group and WDR26 hypoxia group,and the cell model was constructed by hypoxia 6 h.To detect the change of PINK1 and Parkin during cell hypoxia by western blot.5.SPSS17.0 was used in statistical analysis of data.Results 1.Results from western blot showed that WDR26 protein expression increased in the rats H9c2 myocardial hypoxia,and peaked at 6h.2.Compared with the Neo control group and the WDR26 control group,the release of LDH in supernatant culture medium of Neo hypoxia group and WDR26 hypoxia group was significantly increased,while the cell viability decreased significantly;and WDR26 hypoxia group compared to the Neo hypoxia group,the release of LDH in supernatant culture medium decreased significantly,and cell viability has improved significantly.3.Compared with the Neo control group and the WDR26 control group,the ratio of LC3II/LC3 I of Neo hypoxia group and WDR26 hypoxia group was significantly increased,while p62 expression was significantly reduced;and WDR26 hypoxia group compared to the Neo hypoxia group,LC3II/LC3 I were significantly increased while the expression of p62 was significantly lower.Compared with the Neo control group and the WDR26 control group,the number of puncta per cell of LC3 mark in Neo hypoxia group and WDR26 hypoxia group was significant increased;and WDR26 hypoxia group compared to the Neo hypoxia group,the LC3 mark since the number of phages increased significantly.4.Compared with the Neo control group and the WDR26 control group,the ratio of red / green fluorescence in Neo hypoxia group and WDR26 hypoxia group was decreased significantly;and WDR26 hypoxia group compared to the Neo hypoxia group,the ratio of red/green fluorescence was significantly decreased.Compared with the Neo WDR26 control group and the control group,the expression of Parkin and PINK1 was increased significantly in Neo hypoxia group and WDR26 hypoxia group;and WDR26 hypoxia group compared to the Neo hypoxia group,the expression of Parkin and PINK1 was increased significantly.Conclusions 1.WDR26 can be induced by hypoxia in H9c2 cardiomyocytes.2.WDR26 promote mitophagy induced by by PINK1-Parkin transaction,playing a protective role on myocardial cells.