The Effect And Mechanism Of Bromodomain-containing Protein 7 On Cardiomyocyte Apoptosis In Diabetic Cardiomyopathy

BackgroundsDiabetic cardiomyopathy(DCM)is a diabetes-induced heart disease that is unrelated to coronary artery disease,valvular heart disease and hypertension.DCM increases the risk of heart failure and is the leading cause of diabetes-related morbidity and mortality.DCM causes extensive myocardial structural abnormalities in diabetic patients,such as myocardial interstitial fibrosis,cardiomyocyte hypertrophy and apoptosis,and eventually results in left ventricular hypertrophy and diastolic and systolic dysfunction or a combination of these.The pathogenesis of DCM was intensively investigated during the past decade,including impaired calcium homeostasis,myocardial insulin resistance,increased lipid uptake,glucose toxicity,active renin-angiotensin system(RAAS)and increased oxidative stress.In STZ-induced diabetic rats,cardiomyocyte apoptosis was significantly increased.As cardiomyocytes rarely proliferate,the loss of myocardial cells eventually leads to impaired cardiac function.Therefore,inhibition of myocardial apoptosis is an important strategy to prevent DCM.Endoplasmic reticulum stress(ERS)plays an important role in myocardial apoptosis during the development of DCM.However,the molecular mechanism of diabetes-induced myocardial apoptosis remains unclear.Bromodomain-containing protein 7(BRD7)is a pleiotropic and highly conserved protein that is ubiquitously expressed in human tissues,including brain,heart,lung,colon and breast.It is a member of the bromodomain-containing protein family that is known as a tumor suppressor.BRD7 serves as a transcriptional regulation factor which regulates many pathological processes such as cell growth,apoptosis,and cell cycle.Increasing studies have reported the role of BRD7 in the initiation of apoptosis.In addition,a recent study showed that BRD7 modulated ER stress through its ability to regulate XBP-1s nuclear translocation in the liver of obese mice.Although many studies have examined the effects of BRD7,little is known about the function of BRD7 in DCM.In the present study,we explored the expression and effect of BRD7 in DCM by using streptozotocin(STZ)-induced type 1 diabetic rats.Objectives1.To investigate the expression of myocardial BRD7 in DCM rats.2.To explore the effects of BRD7 on ventricular remodeling and myocardial dysfunction in DCM rats.3.To study the role of BRD7 on cardiac apoptosis and fibrosis in DCM rats.Methods1.Construction of lentivirus vector RNAShRNA-BRD7 plasmids were designed and efficiency of each plasmid was analyzed by western blotting.Plasmid with the highest efficiency was chosen to construct a lentiviral vector containing green fluorescent protein(GFP).The target sequence for BRD7 shRNA was 5’-GGACTCTGGAGATGCTGAA-3’ and negative control sequence 5’-TTCTCCGAACGTGTCACGT-3’.2.Establishment of type 1 diabetic rats and shRNA-BRD7 lentivirus interferenceSixty male Wistar rats(mean body weight 200 ± 20 g)were housed at 22℃with an alternating 12 hrs light/dark cycle.After 1 week of acclimatization,the animals were then randomly divided into four groups(n = 15 each):normal control,diabetes mellitus(DM),DM+shRNA-negative control(N.C)and DM+shRNA-BRD7.Diabetic rats received a single intraperitoneally injection of STZ(60mg/kg)dissolved in 0.1 ml of citrate buffer(pH 4.5)to induce diabetes.Normal rats were injected with citrate buffer only.One week after STZ injection,tail vein fasting glucose levels were measured using a glucometer.Rats with blood glucose levels ≥ 16.7 mmol/l were considered the type 1 diabetic rats.After induction of diabetes for 12 weeks,an amount of 1×108 UT/50 μl of lentivector with BRD7 shRNA or the same volume of lentivehicle was injected into the jugular vein.At 16 weeks after STZ injection,all rats were anaesthetized with sodium amobarbital(35 mg/kg of body weight intraperitoneally)and then killed.3.Basic biological evaluation of each groupBlood glucose detection:During the experiment,fasting blood glucose of all the experimental rats were measured at 0 week,1 week,12 weeks and 16 weeks after STZ injection.Blood pressure and heart rate were measured at the end of the experiment,heart rate(HR),tail artery systolic pressure(SBP),diastolic blood pressure(DBP)and mean blood pressure(MBP)were measured.Each animal was measured three times.4.Echocardiographic measurementAt 12 weeks and 16 weeks after STZ injection,cardiac function parameters of each group were obtained by M-ultrasonography and pulsed Doppler ultrasonography.Cardiac function was evaluated.5.HistopathologyRat hearts were dissected at the mid-ventricular level and immediately fixed in 4%paraformaldehyde.Tissue samples were paraffin embedded and cut into 5 μm sections.Cardiomyocyte width was determined by H&E-staining.To detect interstitial collagen deposition,heart sections were stained with Masson’s trichrome and Sirius red.Immunohistochemistry was performed to detect the expressions of BRD7.6.TUNEL stainingApoptotic cells in myocardium were detected by TUNEL staining.7.Western Blot analysisTotal protein was collected from freshly dissected rat hearts.Western blotting was used to detect the protein expression of BRD7、CHOP、cleaved caspase-3、full length caspase-3、Bax、Bcl-2 and β-actin.8.Statistical analysis SPSS 17.0 was used for statistical analysis.Data were presented as mean ± S.D.Differences were considered statistically significant at P<0.05.Results1.Basic situations of experimental animalsCompared to normal rats,diabetic rats showed more drinking,eating,polyuria and loss of weight.After inhibition of BRD7 for 4 weeks,the status of DM +shRNA-BRD7 group was slightly improved than vehicle-treated diabetic rats.2.Biological indicators of each groupAt the beginning of experiment,there was no significant difference in blood glucose between each group.The levels of blood glucose in the diabetic group were significantly higher at all time-points than at baseline,with BRD7 gene silencing,the blood glucose levels did not altered compared with vehicle group.At the end of experiment,there was no difference in blood pressure between each group.The ratio of heart weight to body weight was significantly higher in diabetic than normal rats,and shRNA-BRD7 treatment decreased the ratio of heart weight to body weight in diabetic rats.3.Diabetes increased myocardial BRD7 expressionDiabetic rats showed significantly increased BRD7 protein level in the heart as compared with normal controls,and the BRD7 expression was down-regulated in shRNA-BRD7 than in vehicle-treated diabetic rats as indicated by Western blot and immunohistochemistry.4.BRD7 inhibition attenuated diabetes-induced cardiac dysfunction in DCM ratsAt 12 weeks after the induction of diabetes,the diabetic rats showed a moderate decrease in LVEF,LVFS and E/A compared with controls.LVEDd,LVPWd and LV mass were higher than controls,and these data had no difference between three groups of diabetic rats.At the end of experiment,compared with the normal control group,LVEF,LVFS and E/A were significantly lower,while LVEDd,LVPWd and LV mass were significantly higher in the diabetic rats.Inhibition of BRD7 was associated with an improvement in LVEF,LVFS,E/A,LVEDd,LVPWd and LV mass compared with vehicle treatment.5.BRD7 inhibition prevented diabetes-induced myocardial remodeling and fibrosis in DCM ratsCompared with normal rats,cardiomyocyte width was significantly higher in diabetic than normal rats,and shRNA-BRD7 treatment decreased cardiomyocyte width.Masson’s trichrome and picrosirius red staining showed severe myocardial fibrosis in diabetic rats.Quantitative analysis of Masson’s trichrome staining revealed a significant increase of collagen deposition in diabetic as compared with controls,and shRNA-BRD7 treatment reduced collagen deposition as compared with vehicle treatment6.BRD7 inhibition alleviated ER stress-induced myocardial apoptosis in diabetic ratsDiabetes significantly increased myocardial CHOP expression,ratio of Bax/Bcl-2 and caspase-3 activity.In addition,the proportion of TUNEL-positive cells was significantly increased in diabetic hearts.BRD7 inhibition effectively ameliorated diabetes-induced CHOP expression,ratio of Bax/Bcl-2 and caspase-3 activity.Meanwhile,BRD7 inhibition decreased the proportion of TUNEL-positive cells in the diabetic rats.7.BRD7 inhibition restored the activity of AKT in diabetic ratsDiabetes significantly reduced myocardial phosphorylation of AKT(ser473)and AKT(thr 308),however,inhibited phosphorylation of AKT in diabetic group was restored after BRD7 silencing.Conclusion1.BRD7 protein level was increased in the myocardium of rats with type 1 diabetes2.BRD7 inhibition alleviated the diabetes-induced cardiac remodeling,fibrosis and dysfunction.3.BRD7 inhibition prevented myocardial apoptosis via ER stress pathway in diabetic rats.BackgroundsDiabetes mellitus is a serious and complex metabolic disease that affects about 4%of the population all over the world.Diabetic cardiomyopathy(DCM)is a diabetes-induced heart muscle specific disease in the absence of other vascular pathologies.The pathogenesis of DCM is a slow and complex process that is attributed to autonomic abnormalities,abnormal enzyme function,metabolic disorders and interstitial fibrosis.In addition,it is reported that myocardial cell apoptosis participates the development of DCM.The endoplasmic reticulum(ER)is an important organelle responsible for calcium homeostasis,lipid synthesis,protein folding and maturation.A variety of factors that cause ER dysfunction lead to accumulation of unfolded and/or incorrectly folded proteins,including oxidative stress,ischemia and disturbance of calcium homeostasis.This process is called endoplasmic reticulum stress(ERS).The ERS initially tries to restore the normal function of ER by suppressing protein translation and promoting the production of molecular chaperones participate in protein folding;however,if the stimulus is persisted or the disruption is prolonged,ERS initiates the apoptotic pathway,and eventually leds to apoptosis.Chronic hyperglycaemia increases the level of ER stress,such as glucose-regulated protein 78 kD(GRP78),spliced/active XBP-1s and CHOP.We have demonstrated that ERS was involved in the cardiac apoptosis in STZ-induced type 1 diabetic rats.CHOP can be induced at the transcript level by XBP-1s,and it is identified to play a prominent role in ER stress-induced apoptosis.Although ER stress participates in the pathogenesis of DCM,the molecular mechanisms underlying cardiac apoptosis still have not been well illustrated.Bromine-containing protein 7(BRD7)is a member of bromine-containing protein family as a tumor suppressor.It has been reported that activation of Ras/Raf/MEK/ERK pathway can induce the expression of BRD7 in hepatoma cells.ERK pathway is strongly activated by HG stimulation,ERK pathway increases activity of downstream factors and involves in the development of DCM.However,whether HG-induced activation of ERK1/2 facilitates the expression of BRD7 in H9c2 cardiomyoblasts has not been reported.Our previous study found that BRD7 mediated diabetes-induced cardiomyocyte apoptosis,while,the mechanism is not clear.A recent study showed that BRD7 regulated ERS pathway by binding to P85a to promote translocation of XBP-1s and enhance the activity of XBP-1s in the liver of obese mice.However,whether BRD7 mediated HG-induced myocardial apoptosis via endoplasmic reticulum stress signalling pathway is still not known.Objectives1.To explored the effect of BRD7 on myocardial apoptosis induced by high glucose.2.To investigate the mechanism of high glucose-induced protein expression of BRD7.3.To explored the underlying mechanism of BRD7 in high glucose-induced myocardial apoptosis.Methods1.Cell cultureIn this study,H9c2 cardiomyocytes exposed to DMEM containing 5.5 mmol/L glucose as normal control group(NG).The cells were stimulated with 33.3mmol/L glucose as high glucose group(HG),and then the myocardial cells were stimulated with HG for 6h,12h,24h and 48h respectively.5.5 mmol/L glucose + 27.5 mmol/L mannitol was used as osmotic control group(OC),and H9c2 cardiomyocytes were exposed to OC for 6h,12h,24h,48h respectively.2.Cell modelH9c2 cardiomyoblasts were infected with a lentivirus vector containing BRD7-shRNA to inhibit BRD7 expression,shRNA-N.C was used as negative control group.Moreover,a specific ERK1/2 inhibitor(U0126)was used to suppress the activation of ERK1/2,and dimethyl sulfoxide(DMSO)treatment was acted as negative control.3.TUNEL assayTUNEL staining was used to detect the apoptosis of cardiomyocytes under different conditions.4.Western blottingCells were collected after different stimulation,and total protein and nucleoprotein were extracted.Western blotting was used to detect the expression of BRD7,Bax,Bcl-2,cleaved caspase-3,full length caspase-3,CHOP,XBP-1s,p-ERK,t-ERK,p-AKT and t-AKT.5.Laser scanning confocal microscopyImmunofluorescence was used to detect the distribution of BRD7 and the nuclear translocation of XBP-1s in cardiomyocytes underwent different stimulations.6.Statistical analysisSPSS 17.0 was used for statistical analysis.Data were presented as mean±S.D.Results1.High glucose induced apoptosis of H9c2 cardiomyoblasts and increased the expression of BRD7 in a time-dependent mannerExposure of H9c2 cardiomyoblasts to HG for 6,12,24 and 48 hrs.The ratio of Bax to Bcl-2 was increased with HG treatment at 24 and 48 hrs.The expression of cleaved caspase-3 was higher with HG than NG treatment at 24 and 48 hrs,while Bax/Bcl-2 ratio and levels of cleaved caspase-3 did not alter over time with OC as compared with NG Meanwhile,the BRD7 protein level started to increase during the first 24 hrs and reached its peak at 48 hrs with HG.Similarly,these effects were not observed with OC treatment.2.BRD7 was required for HG-induced H9c2 cardiomyoblasts apoptosisThe expression of BRD7 was significantly decreased after transfection with BRD7-specific shRNA as compared with vehicle treatment.HG stimulation significantly increased H9c2 cardiomyoblasts apoptosis,while inhibition of BRD7 attenuated Bax/Bcl-2 ratio,the level of cleaved caspase-3 and as well as number of TUNEL-positive cells induced by HG.3.Inhibition of ERK pathway reduced HG-induced expression of BRD7 and apoptosisStimulation of H9c2 cardiomyoblasts with HG increased the phosphorylation of ERK1/2 level as compared with NG treatment.Pre-treating H9c2 cardiomyoblasts with U0126 decreased phospho-ERK1/2 level and the expression of BRD7 as compared with DMSO treatment.In addition,immunofluorescence analysis revealed that HG significantly increased the accumulation of BRD7 in the nucleus,which was attenuated by U0126.We further explored the effects of ERK 1/2 on HG induced apoptosis,HG stimulation markedly decreased AKT phosphorylation and p-AKT level was enhanced by U0126 treatment.Moreover,HG-induced apoptosis was decreased in U0126 treatment group as indicated by Bax/Bcl-2 ratio and the level of cleaved caspase-3.4.BRD7 mediated HG-induced apoptosis via ER stress pathway in H9c2 cardiomyoblasts4.1 BRD7 mediated the nuclear translocation of XBP-1s in H9c2 cardiomyoblasts exposed to HGWestern blotting showed that HG promoted the nuclear translocation of XBP-ls,while its translocation was decreased by inhibition of BRD7.Total expression of XBP-1s was increased following exposure to HG as compared with NQ and BRD7 inhibition had no effect on the total expression of XBP-1s.Immunofluorescence analysis was also performed to monitor the localization of BRD7 and XBP-1s.BRD7 was mainly located in the nucleus,and XBP-1s was predominately in the cytosol of NG-treated H9c2 cardiomyoblasts.After stimulation with HQ XBP-1s was mainly located in the nucleus with coexpression of BRD7,while the nuclear XBP-1s immunofluorescence signals were reduced upon BRD7-shRNA treatment compared to vehicle treatment.4.2 Inhibition of BRD7 reduced the HG-induced expression of CHOPWestern blotting was used to detect the effect of BRD7 on the expression of CHOP.After HG treatment for 24 hrs,the expression of CHOP was significantly increased and lasted to 48 hrs.At the same time,the protein level of CHOP did not altered with OC treatment.In addition,Western blot showed that inhibition of BRD7 effectively decreased HG-induced expression of CHOP.Conclusion1.High glucose up-regulated BRD7 expression via ERK pathway in H9c2 cardiomyoblasts.2.High glucose-induced H9c2 cardiomyoblasts apoptosis was reduced following the inhibition of BRD7.3.BRD7 gene silencing reduced the high glucose-induced cardiomyocyte apoptosis by inhibiting XBP-1s/CHOP signalling pathway.