| BackgroudAlcoholic cardiomyopathy(ACM)is a specific heart muscle disease found in individuals with a high daily level and duration of ethanol consumption.ACM is characterized by cardiac hypertrophy,reduced myocardial contractility(dilated left ventricle,reduced left ventricle ejection fraction),myocardial fibrosis and cardiomyocyte apoptosis.It was shown that angiotensin II(Ang II)participated in the pathogenesis of ACM,and decreasing Ang Ⅱ could decrease ethanol-induced cardiac damage.Ang Ⅱ can be synthesized by renin,angiotensin converting enzyme and chymase from angiotensinogen(AGT)in the local cardiac renin-angiotensin system(RAS).Previous evidence indicated that approximately 75%of Ang II in pig hearts was produced at tissue sites by conversion of in situ-synthesized angiotensin I(Ang I)rather than Ang I from blood.Local cardiac RAS plays a pivotal role in the pathological process of ACM.Ang II in mice hearts acted from an intracellular location,resulting in physiological responses including oxidative stress,cardiac remodeling and apoptosis in ACM.Despite these observations,how to decrease AGT and Ang II in the local cardiac RAS in ACM remains largely unexplored.Mitochondrial aldehyde dehydrogenase 2(ALDH2)has recently been identified as an important cardioprotective enzyme,which can markedly reduce cardiac injury induced by ischemia/reperfusion(I/R),diabetes,and alcohol.ALDH2-mediated detoxification of cytotoxic aldehydes including acetaldehyde represents a possible cytoprotective mechanism during ethanol consumption.Previous evidence showed that ALDH2 overexpression could effectively antagonize chronic ethanol intake-induced aldehyde accumulation,myocardial hypertrophy and contractile dysfunction.Previous study showed that protein kinase Cε-mediated ALDH2 activation in cardiac mast cells,decreased arrhythmia by preventing the dysfunctional consequences of local RAS activation in myocardial I/R injury.Despite the fact that the protective role of ALDH2 in ACM is found,the underlying mechanism,whether ALDH2 improves ethanol-induced myocardial injury through inhibiting the cardiac local RAS,remains elusive.P38 MAP kinase(p38 MAPK),a member of the mitogen-activated protein kinases(MAPK)family,is involved in a wide range of signaling pathways.Cyclic adenosine monophosphate response element-binding protein(CREB),a downstream signal molecule of p38 MAPK,is a transcription factor that interacts with DNA and regulates gene transcription.Long-term ethanol-feeding increased the phosphorylation of p38 MAPK in the cerebral cortex,which was reversed by ALDH2 overexpression.ALDH2 overexpression improved cardiac hypertrophy via inhibition of CREB phosphorylation in ACM.A previous study showed that p38 MAPK and CREB activation increased AGT gene expression leading to cell hypertrophy respectively while p38 MAPK could not inhibit CREB activation in hyperglycemia.However,whether ALDH2 decreases AGT and Ang Ⅱ in the local cardiac RAS via inhibition of the p38 MAPK/CREB pathway in ACM requires further investigation.Objective1.To explore whether ALDH2 improved cardiac damage by inhibiting the local RAS in ACM;2.To explore whether the p38 MAPK/CREB pathway was involved in the regulatory mechanism.Methods1.Animals and experimental proceduresMale C57BL/6J mice(8 weeks old)were fed using a Lieber-DeCarli liquid diet.The experiment consisted of 5 groups of mice(n=10):Control(Con)group,vehicle control(Vehicle)group,ethanol(ETOH)group,ETOH+Alda-1 group and ETOH+Daidzin group.Mice in the ETOH groups received the Lieber-DeCarli liquid diet containing 5%ethanol for 2 months.Ethanol provided approximately 36%energy.Mice in the Con group and the Vehicle group were pair-fed the same liquid diet without ethanol for 2 months.The ALDH2 activator Alda-1(10 mg/kg/day)or ALDH2 inhibitor Daidzin(50 mg/kg/day)were administered intraperitoneally using an Alzet osmotic pump for 2 months.2.Echocardiographic assessmentCardiac function was evaluated using the Vevo770 imaging system.Mice were anesthetized with 1.5%isoflurane.In M-mode images,left ventricular end-diastolic dimension(LVEDD)and left ventricular end-systolic dimension(LVESD)were obtained.Left ventricular ejection fraction(LVEF)was calculated.3.Histology and morphometric analysisAfter harvesting,myocardial tissues were fixed in 4%paraformaldehyde,embedded in paraffin and cut into 4 μm sections.Wheat germ agglutinin(WGA)staining was used to assess myocyte cross-sectional area.Masson staining and Sirius red staining were used to assess collagen deposition.4.Terminal deoxynucleotide nick-end labeling(TUNEL)assayAfter harvesting,myocardial tissues were fixed in 4%paraformaldehyde,embedded in paraffin and cut into 4 μm sections.Cardiomyocyte apoptosis was assessed by TUNEL assay.5.Isolation and culture of neonatal rat cardiomyocytesCardiomyocytes were isolated from 2-days-old Wistar rats by enzymatic digestion with 0.05%trypsin and 0.04%collagenase.Cardiomyocytes were exposed to different doses of ethanol(10-400 mmol/L)or Ang Ⅱ(1-1000 nmol/L)for 24 hours to measure ALDH2 expression and ALDH2 activity.In subsequent studies,we chose 400 mmol/L ethanol for 24 hours in the absence or presence of an ALDH2 activator(Alda-1,20 μmol/L),ALDH2 inhibitor(Daidzin,50 μmol/L),p38 MAPK inhibitor(SB203580,10 μmol/L),p38 MAPK activator(Anisomycin,10 μmol/L)or CREB inhibitor(KG-501,25 μmol/L)for further study.6.Adenoviral vector transfectionNeonatal rat cardiomyocytes were seeded in six-well plates at a density of 1-2×106 cells/well.The cardiomyocytes were then infected with Ad-ALDH2 virus at a multiplicity of infection of 60.Transfection efficiency was evaluated by GFP gene expression in cardiomyocytes under fluorescence microscopy and was confirmed by western blotting.7.Western blotting analysisProtein samples(30 μg)from myocardial tissue or neonatal cardiomyocytes were separated by SDS-PAGE and transferred to PVDF membranes,which were blocked with 5%milk and incubated overnight at 4 ℃ with primary antibodies.Then the membranes were incubated with secondary antibodies and chemiluminescence was implemented.The level of ALDH2,AGT,phospho-p38 MAPK,p38 MAPK,phospho-CREB and CREB were measured.All data were normalized by internal controls of GAPDH,β-tubulin,p38 MAPK or CREB.8.ALDH2 enzymatic activity measurementThe mitochondria isolated from myocardial tissue or neonatal cardiomyocytes were sonicated,centrifuged at 11,000×g for 10 minutes at 4℃ and the supernatant was used to measure ALDH2 activity.Enzymatic activity of ALDH2 was determined by measuring the conversion of NAD+ to NADH.9.Aldehydes determinationMyocardial tissue or neonatal rat cardiomyocytes were homogenized in phosphate buffered saline and centrifuged at 900×g for 10 minutes;50 μL of the supernatant or 0-1000 μmol/L aldehyde(standards)was mixed with 50 μL of AldeLightTM Blue reaction mixture.Following incubation in solid black 96-well plates for 15 minutes at room temperature,protected from light,25 μL of reaction buffer was added,and fluorescence was detected at Ex/Em = 365/435 nm.10.Detection of Ang Ⅱ by enzyme-linked immunosorbent assay(ELISA)Total proteins in myocardial tissue or neonatal cardiomyocyte homogenate supernatant were extracted and measured using the bicinchoninic acid protein assay kit.The levels of Ang II in myocardial tissue and cardiomyocytes were assessed by ELISA.Results1.Cardiac AGT and Ang II were increased in ACMIn vivo,AGT and Ang II were increased by 1.3-and 1.9-fold in the hearts of the ETOH group compared with the Con group.In vitro,AGT and Ang Ⅱ were gradually increased in response to ethanol,with a significant difference occurring at 400 mmol/L.2.Cardiac ALDH2 activity was inhibited in ACMIn vivo,compared with Con group,heavy ethanol consumption did not changethe expression of ALDH2 in mouse hearts in ETOH group.However,ALDH2 activity was attenuated by 18.7%in mice hearts in the ETOH group compared with the Con group.The level of aldehydes in myocardial tissue was significantly increased in mice hearts in the ETOH group compared with the Con group.In vitro,different doses of ethanol did not change ALDH2 expression.However,ALDH2 activity was significantly attenuated in response to 100 mmol/L and 400 mmol/L ethanol.However,low doses of ethanol(10 mmol/L and 40 mmol/L)increased ALDH2 activity.In addition,the level of aldehydes in neonatal rat cardiomyocytes exposed to 400 mmol/L ethanol for 24 hours was also increased.3.ALDH2 improved ethanol-induced cardiac damage by decreasing AGT and Ang II in ACMIn vivo,it was found that LVEF was improved by 15.4%in the ETOH+Alda-1 group compared with the ETOH group.The cardiomyocyte cross-sectional area was markedly decreased in the ETOH+Alda-1 group compared with the ETOH group.Myocardial collagen content was significantly lower in the ETOH+Alda-1 group compared with the ETOH group.Myocardial apoptosis was decreased by 28.1%in the ETOH+Alda-1 group compared with the ETOH group.The myocardial aldehydes were decreased in the ETOH+Alda-1 group compared with the ETOH group.Furthermore,we found that AGT was decreased by 29.7%in the ETOH+Alda-1 group compared with the ETOH group.Ang II was decreased by 46.6%in the ETOH+Alda-1 group compared with the ETOH group.Conversely,ethanol-induced cardiac damage was aggravated in the ETOH+Daidzin group compared with the ETOH group.The myocardial aldehydes level was increased in the ETOH+Daidzin group compared with the ETOH group.AGT and Ang Ⅱ were increased in the ETOH+Daidzin group compared with the ETOH group.In vitro,AGT was decreased by 27.8%and Ang II was decreased by 26.9%in the ETOH+Alda-1 group compared with the ETOH group.AGT was increased by 16.6%and Ang II was increased by 37.3%in the ETOH+Daidzin group compared with the ETOH group.AGT and Ang Ⅱ were attenuated by 44.5%and 27.5%in the ETOH+Ad-ALDH2 group compared with the ETOH group.In addition,cardiomyocytes were exposed to 1-1000 nmol/L Ang Ⅱ for 24 hours.It was found that Ang II did not change ALDH2 expression or ALDH2 activity in cardiomyocytes.4.Ethanol increased cardiac AGT and Ang II via the p38 MAPK/CREB pathway in ACMIn vivo,the ratio of phospho-p38 MAPK/total p38 MAPK in hearts was increased by 1.0-fold in the ETOH group compared with the Con group.Phospho-CREB,a downstream signal molecule of p38 MAPK,was also markedly increased by 1.4-fold in the hearts of mice in the ETOH group compared with the Con group.In vitro,the ratio of phospho-p38 MAPK/total p38 MAPK and the ratio of phospho-CREB/total CREB were increased by 1.4-fold and 1.4-fold in neonatal rat cardiomyocytes in the ETOH group compared with the Con group.SB203580,a specific inhibitor of p38 MAPK,significantly inhibited the ethanol-increased ratio of phospho-CREB/total CREB by 48.3%.However,KG-501,a specific inhibitor of CREB,failed to decrease the ethanol-increased ratio of phospho-p38 MAPK/total p38 MAPK.AGT in cardiomyocytes was markedly decreased by 25.1%in the ETOH+SB203580 group compared with the ETOH group.The ethanol-increased AGT in cardiomyocytes was decreased by 25.5%in the ETOH+KG-501 group compared with the ETOH group.In addition,the ethanol-increased Ang Ⅱ in cardiomyocytes was also significantly decreased by SB203580 and KG-501.5-ALDH2 inhibited ethanol-induced activation of the p38 MAPK/CREB pathway in ACMIn vivo,the ratio of phospho-p38 MAPK/total p38 MAPK in cardiac tissue was decreased by 23.6%in the ETOH+Alda-1 group and increased by 36.3%in the ETOH+Daidzin group compared with the ETOH group.Similar trends were observed for the ratio of phospho-CREB/total CREB.In vitro,the ratio of phospho-p38 MAPK/total p38 MAPK was significantly decreased by 29.7%in the ETOH+Alda-1 group and increased by 27.3%in the ETOH+Daidzin group compared with the ETOH group.Concomitantly,the ratio of phospho-CREB/total CREB was decreased by 28.1%in the ETOH+Alda-1 group and increased by 18.5%in the ETON+Daidzin group compared with the ETOH group.The ratios of phospho-p38 MAPK/total p38 MAPK and phospho-CREB/total CREB were also decreased by 28.1%and 42.6%in the ETOH+Ad-ALDH2 group compared with the ETOH group.6.ALDH2 decreased AGT and Ang II via inhibition of the p38 MAPK/CREB pathway in ACMAGT and Ang Ⅱ were increased by 53.8%and 96.9%in the ETOH+Alda-1 +Anisomycin group compared with the ETOH+Alda-1 group.However,AGT and Ang Ⅱ were decreased by 34.7%and 49.2%in the ETOH+Alda-1 +Anisomycin+KG-501 group compared with the ETOH+Alda-1+Anisomycin group.These findings showed that Anisomycin,a specific activator of p38 MAPK,effectively mitigated Alda-1-provided protection against ethanol-increased AGT and Ang Ⅱ,the effect of which was reconciled by KG-501,indicating that ALDH2 decreased AGT and Ang II in the local cardiac RAS via the p38 MAPK/CREB pathway in ACM.Conclusion1.ALDH2 improved ethanol-induced cardiac damage by decreasing AGT and Ang II in the local cardiac RAS in ACM;2.ALDH2 decreased AGT and Ang II in the local cardiac RAS via inhibiting p38 MAPK/CREB pathway in ACM.BackgroudAcute myocardial infarction(AMI)is one of the leading causes of high morbidity and mortality worldwide.The prognosis of patients with AMI is conditioned by many risk factors.Among them,hyperglycemia in AMI patients with or without previous diabetes worsens the prognosis.Strong evidence demonstrates that elevation of plasma glucose independently exacerbates cardiac dysfunction,infarct size and cardiomyocyte apoptosis following myocardial ischemia/reperfusion(I/R).Despite these observations,the underlying mechanisms by which hyperglycemia worsens myocardial I/R injury are incompletely understood.Excessive elevation of plasma glucose could cause protein O-linked-N-acetylglucosamine(O-GlcNAc)modification through the hexosamine biosynthesis pathway which is a common post-translational modification that occurs on serine/threonine residues of proteins.There are numerous reports on O-GlcNAcylation of cytosolic,nuclear and mitochondrial proteins.The O-GlcNAcylation of specific proteins could modulate their structure and function and thus influence signaling cascades,antioxidant defenses,et al.However,whether O-GlcNAc modification of specific responsible proteins contributes to hyperglycemic exacerbation of myocardial I/R injury has not been investigated.Mitochondrial aldehyde dehydrogenase2(ALDH2)has recently been identified as an important cardioprotective enzyme,whose activity inversely correlates with infarct size.Enhancement of ALDH2 activity reduces infarct size induced by I/R,whereas inhibition of ALDH2 activity worsens infarct size.ALDH2-mediated detoxification of cytotoxic aldehydes,including 4-hydroxy-2-nonenal(4-HNE),represents a possible cytoprotective mechanism that enhances tissue survival in the heart during I/R.Studies have demonstrated that ALDH2 could be inactivated by hyperglycemia.However,whether O-GlcNAcylation of ALDH2 could be a mechanism for its activity down-regulation and resultant aggravation of myocardial I/R injury under hyperglycemia conditions has not been elucidated,despite candidates serine/threonine residues of ALDH2 protein exist.Objective1.To investigate whether ALDH2 is O-GIcNAcylated under hyperglycemic conditions;2.To determine the role of ALDH2 O-GlcNAcylation in the hyperglycemic exacerbation of myocardial I/R injury.Methods1.Animals and experimental proceduresAdult male Wistar rats were randomly divided into six groups(n=10):Sham group,normal saline(NS)+I/R group,acute hyperglycemia(AHG)+I/R group,diabetes(DM)+I/R group,DM group and DM+I/R+Alda-1 group.Rats were anesthetized intraperitoneally(i.p.)with pentobarbital sodium(30 mg/kg)and ventilated via a tracheostomy on a rodent respirator.A left thoracotomy was performed,and a reversible coronary artery snare was placed around the left anterior descending(LAD)coronary artery.Myocardial I/R was performed by tightening the snare for 30min and then loosening it for 90 minutes.Sham group:sham rats were received saline by intravenous infusion at a rate of 4mL/kg/h throughout the experimental period.NS+I/R group:rats were treated with saline by intravenous infusion at a rate of 4mL/kg/h throughout I/R procedures.AHG+I/R group:rats were treated with glucose by a bolus intravenous injection(1.5 mg/kg)and then maintained with intravenous infusion at a rate of 4 mL/kg/h throughout I/R procedures.For AHG+I/R group rats,plasma glucose increased to more than 20 mmol/L instantly after the bolus injection of glucose,and maintained over 20 mmol/L during myocardial I/R procedure.DM+I/R group:diabetic rats were treated with I/R procedures.Diabetic rats were given a single injection(i.p.)of 60mg/kg streptozotocin and studied 4 weeks later.For Alda-1 treatment,rats were administered with intravenous injection of Alda-1(8 mg/kg)10 minutes before ligation.Serum glucose was measured by glucose analyzer every 10 minutes during the experimental procedures.2.Cardiac function measurementAt the end of reperfusion,a micro catheter was inserted into left ventricle through right carotid artery to measure left ventricular end diastolic pressure(LVEDP),left ventricular end systolic pressure(LVESP),left ventricular maximal rate of pressure increase(+LVdp/dtmax)and left ventricular maximal rate of pressure decrease(-LVdp/dtmax).Then,the cardiac ventricles were excised rapidly,stored at-80℃ or fixed in 4%paraformaldehyde until analysis.3.2,3,5-triphenyltetrazolium chloride(TTC)staining Myocardial infarct area was determined by staining with 1%TTC.4.Cell culture and hypoxia-reoxygenation(H/R)proceduresRat cardiomyoblasts cell line(H9c2 SV40)was cultured in Dulbecco’s Modified Eagle Medium with 10%of Fetal Bovine Serum and 1%of penicillin/streptomycin,followed by either normal glucose(5.0 mmol/L plus 25.0 mmol/L mannitol)or high glucose(30.0 mmol/L)conditions.In the glucose dose-dependent experiments,the glucose concentrations ranged from 5.0 mmol/L to 35.0mmol/L.H9c2 cells were exposed to 1%O2,94%N2 and 5%CO2 for 12 hours using a modular incubator and then reoxygenation(95%air,5%CO2)lasted for 2 hours.H9c2 cells were exposed to 100 μmol/L PUGNAc to increase O-GlcNAc modification,or 50 μmol/L DON to decrease O-GlcNAc modification.For separate experiments,Alda-1(10 μmol/L)was given to H9c2 cells.5.Human heart specimens collectionHeart tissues were obtained from left ventricles of body donors included three patients with diabetes and three controls without diabetes.Inclusion in tissue-based studies was not restricted on the basis of age,sex,race or ethnic status.Diabetes was confirmed by medical history.The causes of death in those subjects were not heart disease.The informed contents were obtained from their relatives before body donation.6.Histological examinationLeft ventricular tissues which were below ligature point were harvested.After treated by 4%buffered paraformaldehyde and embedded in paraffin,heart tissues were cut into sections of 4 μm in thickness and stained with hematoxylin&eosin(H&E).7.Terminal deoxynucleotide nick-end labeling(TUNEL)assayIn vivo,myocardium apoptosis was assessed with an in situ apoptosis detection kit through the immunohistochemical method.In vitro,H9c2 cell apoptosis was assessed with another apoptosis detection kit using an immunofluorescence method.8.Immunoprecipitation and western blotting analysisAbout 500μg myocardial tissues and H9c2 cells protein were incubated overnight with 2-10 μg of ALDH2,COX I,or NDUFA9 primary antibody rotating at 4 ℃,followed by incubation with 20 μL of protein A/G PLUS agarose for 2 hours at 4 ℃.Immunoprecipitates were extensively washed,resuspended in 1 x sample buffer,boiled for 5 minutes and analyzed by western blotting analysis.30 μg or 20 pL of protein was separated by 10%SDS-PAGE gel and transferred to nitrocellulose membranes,and blots were probed with antibodies against β-actin,O-GlcNAc,ALDH2,COX I,NDUFA9,4-HNE.Membranes were washed 3 times and then incubated with horseradish peroxidase-conjugated secondary antibodies,and detected by using an ECL detection method.9.ALDH2 enzymatic activity measurementThe mitochondria isolated from myocardial tissue or H9c2 cells were sonicated,centrifuged at 11,000 ×g for 10 minutes at 4℃ and the supernatant was used to measure ALDH2 activity.Enzymatic activity of ALDH2 was determined by measuring the conversion of NAD+ to NADH.10.Protein carbonyl formation determinationProtein from myocardial tissue or H9c2 cells was extracted.The levels of protein carbonyl formation were determined.11.Aldehydes determinationMyocardial tissue or H9c2 cells was homogenized in phosphate buffered saline and centrifuged at 900 xg for 10 minutes;50 μL of the supernatant or 0-1000 μmol/L aldehyde(standards)was mixed with 50 μL of AldeLightTM blue reaction mixture.Following incubation in solid black 96-well plates for 15 minutes at room temperature,protected from light,25 μL of reaction buffer was added,and fluorescence was detected at Ex/Em = 365/435nm.Results1.Hyperglycemia exacerbated myocardial I/R injury in ratsLVEDP was significantly increased and LVESP,+LVdp/dtmax and-LVdp/dtmax were significantly decreased in AHG+I/R group and DM+I/R group compared with NS+I/R group.Infarct size area and apoptosis index were exacerbated in AHG+I/R group and DM+I/R group compared with NS+I/R group.2.ALDH2 O-GlcNAc modification was increased after myocardial I/R in rats with hyperglycemiaMyocardial I/R per se did not increase overall O-GlcNAc modification of proteins,compared with Sham controls.However,the total protein O-GlcNAcylation was apparently increased in AHG+I/R and DM+I/R groups,compared with Sham and NS+I/R groups.ALDH2 O-GIcNAc modification was slightly increased in NS+I/R group compared with Sham controls,but the difference did not reach significant level.However,ALDH2 O-GlcNAc modification was increased by 2.29 fold in AHG+I/Rgroup and it was enhanced by 2.67 fold in DM+I/R group compared with NS+I/R group.Compared with Sham group,ALDH2 expression did not change in NS+I/R group,AHG+I/R group or DM+I/R group.ALDH2 activity was similar between Sham and NS+I/R groups,whereas it was decreased by 17.2%in AHG+I/R rats and by 19.2%in DM+I/R rats in comparison with NS+I/R rats.3.Hyperglycemia increased ALDH2 O-GIcNAc modificationHuman studies showed that ALDH2 O-GlcNAc modification was significantly increased by 28.3%in the diabetic patients compared with non-diabetic controls.A similar tendency was observed with the comparison between the diabetic rats and wild-type rats.High glucose treated H9c2 cells for 48 hours markedly increased ALDH2 O-GlcNAc modification compared with normal controls.ALDH2 O-GlcNAc modification was increased gradually with significance occurred at 30 mmol/L and 35 mmol/L as the concentrations of glucose were increased from 5 mmol/L to 35 mmol/L.Myocardial ALDH2 activity was decreased by 15.9%and 19.6%in diabetic patients and animals compared with their controls.The decreased ALDH2 activity was also observed as the concentrations of glucose were increased in H9c2 cells.The significant decrease of ALDH2 activity occurred at the glucose concentration of 30 mmol/L and 35 mmol/L.4.ALDH2 O-GlcNAc modification modulated its activityIn vitro,ALDH2 O-GlcNAc modification was increased by 62.9%in PUGNAc group compared with Con group,whereas decreased by 44.1%in DON group compared with Con group.Accordingly,ALDH2 activity was decreased by 18.1%in PUGNAc group in comparison with Con group,whereas increased by 17.9%in DON group in comparison with Con group.ALDH2 O-GlcNAc modification was increased by 1.16 fold in the HG+PUGNAc group compared with basal conditions,whereas hyperglycemia per se increased ALDH2 O-GlcNAc modification by 66.2%,indicating that PUGNAc could further increase ALDH2 O-GlcNAc modification under high glucose conditions.ALDH2 O-GlcNAc modification was decreased to the levels that similar to basal conditions in the HG+DON group.ALDH2 activity was significantly decreased in the presence of high glucose compared with basal conditions.HG+PUGNAc group further decreased ALDH2 activity,whereas ALDH2 activity in HG+DON group was enhanced to the levels in basal conditions.We further confirmed whether PUGNAc and DON could modulate ALDH2 activity in the H/R conditions.H9c2 cells were randomly devided in Con+H/R group,Con+PU GNAc+H/R group,Con+DON+H/R group,HG+H/R group,HG+PUGNAc+H/R group and HG+DON+H/R group.Similar tendency of alteration for ALDH2 O-GlcNAc modification and ALDH2 acticity among these groups was observed under H/R conditions compared with those under normal oxygen conditions.5.ALDH2 O-GlcNAc modification promoted 4-HNE,aldehydes,protein carbonyl accumulation and apoptosisIn vivo,the levels of 4-HNE were slightly increased in NS+I/R rat group compared with Sham group and were significantly increased in AHG+I/R group and DM+I/R group.Similar trends were observed for aldehydes and protein carbonyl formation.In vitro studies showed that 4-HNE was detectable in control group,whose levels were remarkably increased under HG conditions and they further increased in the HG+H/R group.However,4-HNE was prominently decreased in the HG+H/R+DON group,compared with HG and HG+H/R groups,respectively.Similar trends were observed for aldehydes and protein carbonyl formation.Compared with Con group,apoptosis index was significantly increased in HG and HG+H/R groups.Apoptosis index was significantly decreased in HG+H/R+DON group compared with HG and HG+H/R groups.6.Alda-1 removed ALDH2 O-GlcNAc modification and improved myocardial I/R injuryWe examined ALDH2 activity in Con,HG,HG+H/R and HG+H/R+Alda-1 conditions in H9c2 cells.ALDH2 activity was significantly decreased in HG group and HG+H/R group comparied with Con group.However,ALDH2 activity was significantly improved in HG+H/R+Alda-1 group compared with HG and HG+H/R groups.O-GIcNAcylated ALDH2 levels were significantly decreased in HG+H/R+Alda-1 group,compared with HG group and HG+H/R group,respectively,suggesting Alda-1 prominently inhibited ALDH2 O-GlcNAc modification.In vivo,O-GlcNAcylated ALDH2 was significantly decreased in DM+I/R+Alda-1 group,compared with DM and DM+I/R groups.We next examined the effect of Alda-1 on cardiac function,infarct size and cardiac apoptosis.Alda-1 significantly decreased LVEDP and increased LVESP,+LVdp/dtmax and-LVdp/dtmax compared with DM+I/R group.Infarct size and cardiac apoptosis were also markedly decreased in DM+I/R+Alda-1 group.Conclusion1.ALDH2 is O-GlcNAcylated excessively under hyperglycemic conditions.2,ALDH2 O-GlcNAc modification decreasing ALDH2 activity contributes to the hyperglycemic exacerbation of myocardial I/R injury.3.Alda-1 inhibiting ALDH2 O-GlcNAcylation improves the hyperglycemic exacerbation of myocardial I/R injury. |
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