| Background:The high morbidity of diabetes mellitus(DM)and diverse associated complications have seriously threatened the health of the worldwide human beings.Diabetic myocardial injury(DMI)as one of the major cardiovascular complications of DM,is defined as a series of myocardial structural damage and dysfunction which occurred by the continuous hyperglycemia,even in absence of other cardiovascular diseases such as coronary atherosclerosis or hypertension,and finally will lead to heart failure,arrhythmia,even sudden death,with extremely high fatality and disability rate.The mainly typical clinical manifestations of DMI are: early cardiac diastolic dysfunction and myocardial fibrosis.The molecular mechanisms involved in the pathogenesis of DMI includes many signaling pathways are very complex,that have not been fully elucidated.O-GlcNAc modification is a classical post-translational protein modification which widely involved in the regulation of many cellular pathophysiological processes.Meanwhile,autophagy is a kind of programmed cell death,and its whole process called autophagic flux which consists of the autophagosomes formation,the extension of membranes and the degradation of autolysosome,that stabilizes homestasis of the cells themselves.Although both O-GlcNAc modification and autophagy have been confirmed to be closely related to the occurrence of DMI,little has been known about the mechanism of their interactive relationship in DMI.Recent studies have found that synaptosomalassociated protein 29(SNAP29)can bind with STX17 and VAMP8 to participate in the process of autophagic flux,meanwhile it as a substrate protein which also can be modified by O-GlcNAc.Our preliminary study found that an increased level of O-GlcNAc in streptozotocin(STZ)-induced DMI rat model was associated with inhibition of autophagic flux.However,whether O-Glc NAc-modified SNAP29 is involved in the regulation of autophagic flux,or the specific molecular mechanism of which has not been reported in the mammalian DM disease model.Objective:Previous studies have suggested that protein SNAP29 as a key link involved in both process of autophagy and O-GlcNAc modification is very important.The unknow relationship among O-GlcNAc modification,autophagy and DMI inspires us to explore: Is O-GlcNAc-modified SNAP29 involved in pathogenesis of DMI by regulating autophagy? For this purpose,we proposed the experiments in vivo and in vitro to verify the potential molecular mechanisms that O-GlcNAc glycosylation modification of SNAP29 was involved in the occurrence and development of DMI by regulating autophagic flux,and would offer possible new targets and opportunities for the future clinical treatment of DMI.Methods:The experimental methods in animal study include: blood glucose and body weight determination,echocardiography(Vevo 2100),Western-Blot,coimmunoprecipitation(Co-IP),histology Hematoxylin & Eosin(HE)staining,Masson staining,Transmission electron microscopy and the like.In vivo,Rats were divided randomly into 4 groups: Vehicle group,STZ+Nacl group,STZ+6-Diazoxan-5-oxo-Lnorleucine(STZ+Don)group and STZ+Thiamet G(STZ+TG)group.Experimental methods in vitro experiments include: isolation and culture of neonatal rat cardiomyocytes(NRCMs),treatment with high glucose(Glu:25mM/L),Western-Blot,coimmunoprecipitation,Plasmid isolation and purification.In vitro experiments were divided into sveral groups: normal glucose(Vehicle),high glucose(Glu),normal glucose+O-GlcNAc glycosylated agonist(TG),high glucose+O-GlcNAc glycosylated agonist group(Glu + TG),normal glucose+O-GlcNAc glycosylated inhibitor group(Don)and high glucose + O-GlcNAc glycosylation inhibitor group(Glu+Don),and normal glucose transfected control group(con+sh-ctrl group),normal glucose transfected interferential OGT group(con+sh-OGT),normal glucose transfected overexpressed OGA group(con+ad-OGA),high glucose transfected control group(Glu+sh-ctrl),high glucose transfected interference OGT group(Glu+sh-OGT),high glucose transfected overexpressed OGA group(Glu+ad-OGA).Results:1.A typical typeⅠDM performance was observed in STZ-induced 8-week rats(8-week STZ rats)with increased O-GlcNAc modification and inhibition of autophagic flux.Compared with the Vehicle group,increased blood glucose and dietary water intake,decreased body weight and cardiac hypertrophy(cardiac weight/body weight ratio increased)were found in the 8-week STZ group,ultrasonography showed that diastolic dysfunction(E/A ratio decreased significantly),increased left ventricular massand left ventricle end-diastolic/systolicvolume,while immunohistochemistry showed that myocardial disorder,fat accumulation,fibrosis and autophagosomal accumulation was detected by transmission electronmicriscope.Accompanied by the continuously increased blood glucose levels,the levels of O-GlcNAc modification and expression of N-acetylglucosamine transferase(OGT)were increased,however the level of N-acetylglucosaminidase(OGA)was decreased,and those all detected by western-blot in the 8-week STZ rats.Meanwhile,the expressions of autophagy-related marker protein LC3Ⅱ/Ⅰ and P62 was increased.2.Increasing O-GlcNAc modification in vivo aggravated myocardial injury and inhibit autophagic flux in typeⅠDM rats.The agonist Thiamet-G(TG)and the inhibitor6-diazo-5-oxo-l-norleucine(Don)of O-GlcNAc-modification were used to detect the effects of O-GlcNAc modification on the cardiac function and myocardium structure in typeⅠDM rat.The results showed that TG increased myocardial O-GlcNAc modification,decreased diastolic function(decreased E/A or E/A greater than 2),increased left ventricle end-diastolic/ systolic volume,meanwhile the pathological damage of myocardial structure was increased,and autophagic flow was further inhibited,However,Don reduced the level of O-GlcNAc modification,smoothed autophagic flux and improved diastolic function and myocardial abnormalities.3.Increasing O-GlcNAc modification inhibited the autophagic flux in NRCMs under high glucose conditionsIn agreement with in vivo experiments,TG and Don were administered to isolated NRCMs after stimulation with high glucose.The results showed that the expression of O-GlcNAc,LC3Ⅱ/Ⅰ,Beclin1,P62 and LAMP2 were increased,but autophagic flow was further inhibited,on the contrary,Don significantly reduced O-GlcNAc levels and promoted autophagic flux.The use of gene transfection technology also confirmed that interference OGT or overexpression OGA can reduce O-GlcNAc modification,and promote autophagic flux.4.High glucose inhibited the degradation stage of autophagy.To clarify the specific stage of autophagy which was affected by high glucose in NRCMs,autophagy inhibitor 3-MA and LAMP2-interference sequences were employed.Under high glucose conditions,the LAMP2 interference did not further increase the levels of Beclin1 or LC3Ⅱ/Ⅰ,the expression levels of LC3Ⅱ/Ⅰ and P62 decreased with 3-MA treatment compared to high glucose alone.The above results indicated that high glucose affected the degradation process of autophagic flux but was not involved in the initiation stage5.SNAP29 O-GlcNAc modification hindered SNAP29-STX17-VAMP8 complex formation and inhibited autophagy degradationO-GlcNAc modification of SNAP29 affected the degradation of autophagic flux by negatively regulating the formation of the SANP29-VAMP8-STX17 complex.When NRCMs were under continuous high glucose stimulation in the presence of TG,the O-GlcNAc modification of SNAP29 increased significantly,and the binding of SNAP29,VAMP8 and STX17 with each other decreased significantly.Treatment with Don,sh-OGT or ad-OGAdecreased the level of O-GlcNAc-modified SNAP29 but increased formation of the SNAP29-VAMP8-STX17 complex.Conclusion:1.8-week STZ rats had typical early typeⅠDMI manifestations: decreased cardiac diastolic function,increased left ventricular massand left ventricle enddiastolic/systolic volume andmyocardial disorder,fat accumulation,increased fibrosis.2.Increased myocardial O-GlcNAc modification,deteriorative DMI,and inhibited autophagic flux are found in 8-week STZ rats.3.Increasing level of O-GlcNAc modification further inhibitedautophagic flux,while reducing the O-GlcNAc modification could promote the autophagic flux,which were both confirmed in in vivo and in vitro experiments4.High glucose inhibited the degradation stage of autophagy flux.5.This is the first study to demostrate that the specific molecular mechanism which O-GlcNAc modification in typeⅠDMI inhibits the degradation of autophagic flux: O-GlcNAc-modified SNAP29 hinders formation of the SNAP29-STX17-VAMP8 complex,thereby reducing the degradation process of autophagic flux which mediated by the SNAP29-STX17-VAMP8 complex formation... |
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