Roles Of HDAC3-Orchestrated Circadian Clock Gene Oscillations In Diabetic Rats Following Myocardial Ischemia/Reperfusion Injury

Part 1 Myocardial clock gene oscillation and HDAC3 expression in diabetes and myocardial ischemia/reperfusion injury ObjectiveTo investigate the extent of myocardial ischemic injury and its effect on clock gene oscillation and HDAC3 expression by establishing a model of myocardial ischemia/reperfusion injury(I/RI)in type 1 diabetic rats.MethodsSPF-grade healthy adult male sprague-dawley(SD)rats were used as experimental subjects.Streptozocin(STZ)60 mg/kg was injected intraperitoneally to establish type 1 diabetes.When the fasting blood glucose level≥16.7 mmol/L,and rats showed increased consumption of food and water with increased urination,the diabetes model was successfully prepared.After 8 weeks,the diabetic and non-diabetic rats at same age were randomly divided into four groups according to the random number table method at four time points of zeitgeber(ZT)0,6,12 and 18 respectively:non-diabetic sham operation group(NS group,n=6),non-diabetic ischemia/reperfusion group(NI/R group,n=12),diabetic sham operation group(DS group,n=6),diabetic ischemia/reperfusion group(DI/R group,n=12).A model of myocardial ischemia/reperfusion injury was established by ligating the left anterior descending coronary artery(LDA)with ischemia for 30 minutes and reperfusion for 120 minutes.TTC was used to detect myocardial infarction area;the levels of c Tn-I,CK-MB and LDH in serum were analysed by ELISA;the myocardial ultrastructure damage was detected by transmission electron microscope;the m RNA levels of Rev-erbα,BMAL1,CEBP/β in myocardial tissue were analysed by real-time quantitative PCR;the protein levels of HDAC3,Rev-erbα,BMAL1 and CEBP/β,BNIP3,Atg4 b,LC3Ⅱ/Ⅰ were detected by western blotting.Results1.The m RNA expression of clock genes Rev-erbα,BMAL1,CEBP/β in non-diabetic rats had significant circadian rhythm,while the m RNA oscillations of Rev-erbα,BMAL1,CEBP/β in diabetic rats were dysregulated.2.After myocardial I/R at ZT0,ZT6,ZT12 and ZT18 in non-diabetic rats,the degree of myocardial injury had a correlation with clock gene rhythm,and the injury degree reached a peak at ZT12 with lowest autophagy level.However,the myocardial injury in diabetic rats was more serious with decreased autophagy level,and there were no obvious fluctuations at different time points.3.The expression of HDAC3 and Rev-erbα in myocardial tissue was increased in diabetes and myocardial I/RI further augmented this changes with decreased BMAL1 expression and autophagy level.Compared with sham groups,the autophagy levels of non-diabetic and diabetic rats were increased after myocardial I/R insult.However,after myocardial I/RI in diabetic rats,autophagy levels was decreased with increased levels of autophagy proteins C/EBPβ and BNIP3 and decreased Atg4 b and LC3Ⅱ/Ⅰlevels compared with non-diabetic rats.ConclusionIn diabetes,the clock gene rhythm was oscillated and autophagy was down-regulated with increased myocardial I/RI,which may be related to the increased level of HDAC3 and clock gene oscillation imbalance.Part 2 Protective effect and mechanism of cardiac-specific HDAC3 knockout on myocardial ischemia/reperfusion injuryObjectiveTo investigate the role of cardiac-specific HDAC3 knockout in myocardial ischemia/reperfusion injury and its effect on clock gene-mediated mitochondrial autophagy by injecting adeno-associated virus in diabetic rats.MethodsStreptozocin(STZ)60 mg/kg was injected intraperitoneally to establish type 1diabetes.When the fasting blood glucose level≥16.7 mmol/L,the diabetes model was successfully prepared.HBAAV9-r-HDAC3 sh RNA1-GFP and HBAAV9-GFP NC were injected through the tail vein 3 weeks before myocardial I/R.After 8 weeks,the diabetic rats were divided into 3 groups according to the principle of random number table: diabetic sham operation group(DS group,n=6),diabetic ischemia/reperfusion group(DI/R group,n=12),diabetic ischemia/reperfusion+HBAAV9-r-HDAC3 sh RNA1-GFP group(DI/R+AAV9-HDAC3 group,n=12).The model of myocardial I/R was established by ligation of the LDA with ischemia for 30 min and reperfusion for 120 min.Transfection efficiency of adeno-associated virus was measured by frozen section;the area of myocardial infarction was detected by TTC;the serum levels of c Tn-I,CK-MB and LDH were detected by ELISA;the m RNA levels of Rev-erbα,BMAL1,CEBP/β were detected by real-time quantitative PCR level;western blotting was used to detect the expression of HDAC3,Rev-erbα,BMAL1,CEBP/β and autophagy protein in myocardial tissue.Results1.The frozen section showed that the GFP signal was significantly expressed after tail vein injection of HBAAV9-r-HDAC3 sh RNA1-GFP or HBAAV9-GFP NC,indicating that AAV-GFP-HDAC3 and AAV-GFP-NC were successfully transfected and expressed in myocardial tissue.Compared with the control group,the level of HDAC3 protein in AAV-HDAC3 transfected group was significantly reduced.This indicates that tail vein injection of HBAAV9-r-HDAC3 successfully knocked down HDAC3 expression in myocardium.2.Compared with DI/R group,the area of myocardial infarction,myocardial injury and serum c Tn-I,CK-MB and LDH levels in DI/R+AAV9-HDAC3 group were significantly reduced.At the same time,HDAC3 and Rev-erbα expressions were sinificantly decreased,and BMAL1 level was significantly up-regulated with increased mitophagy level.ConclusionThe results confirm that the adeno-associated virus AAV9-HDAC3 plays protective effect on diabetic myocardial I/RI by down-regulating the level of HDAC3.AAV9-HDAC3 treatment can significantly increase the level of mitophagy and effectively reduce myocardial cell damage,and this effect may be related to the activation of mitophagy regulated by HDAC3-mediated Rev-erbα/BMAL1 signaling pathway.Part 3 Mechanism of HDAC3-regulated clock gene mediating mitophagy in hypoxia/reoxygenation injury of primary cardiomyocytesObjectiveTo investigate the mechanism of HDAC3-mediated clock gene mediating mitophagy in hypoxia/reoxygenation(H/R)injury of primary cardiomyocytes.MethodsPrimary neonatal rat cardiomyocytes were obtained from one-to three-day-old SD rats.The cardiomyocytes were plated in 6-well plates or 96-well plates and divided into 4 groups according to the random number table method:(1)low glucose group(LG,5.5m M glucose concentration);(2)low glucose+hypoxia/reoxygenation group(LG+H/R);(3)high glucose group(HG,30 m M glucose concentration);(4)high glucose+hypoxia/reoxygenation group(HG+H/R).In further experiments,in order to verify the effects of HDAC3 or Rev-erbα deletion on H/R injury under high glucose condition of myocardial primary cells and the role of BMAL1-mediated autophagy in this process,cells were divided into the following 4 groups:(1)high glucose+hypoxia/reoxygenation group(HG+H/R);(2)high glucose+hypoxia/reoxygenation+si RNA normal control group(HG+H/R+si NC);(3)high glucose+hypoxia/reoxygenation+si RNA HDAC3 group(HG+H/R+si HDAC3);(4)high glucose+hypoxia/reoxygenation+si RNA Rev-erbα group(HG+H/R+si Rev).Cells in the control group were cultured at 37°C under normoxic(5% CO2,95% air)condition;cells in the hypoxia/reoxygenation groups were cultured under hypoxia(5%CO2+0.9% O2+94.1% N2)for 6 hours and then reoxygenation for 2 h in normoxic environment.CCK-8 kit was used to detect cell viability;ELISA kit was used to detect LDH release level of the cell supernatant;Mito Tracker Red CMXRos kit was used to detect mitochondrial ROS level;JC-1 kit was used to detect cell mitochondrial membrane potential;autophagy dual standard adenovirus m RFP-GFP-LC3 was used to detect cell autophagy flow level.The m RNA levels of HDAC3,Rev-erbα,BMAL1,CEBP/β were detected by q PCR.Western blotting was used to detect the expression levels of HDAC3,Rev-erbα,BMAL1 and autophagy proteins CEBP/β,BNIP3,p62 and LC3Ⅱ/Ⅰ.Results1.Compared with LG group,the expression of HDAC3 and Rev-erbα was increased in HG group,and the expression of BMAL1 and autophagy level were decreased.After H/R stimulation,the cell survival rate was decreased significantly both in low glucose and high glucose condition with increased LDH activity,HDAC3 and Rev-erbα expression and decreased BMAL1 expression,and cell autophagy level was also increased.However,compared with LG+H/R group,the cell viability was significantly reduced in HG+H/R group with increased LDH activity,increased expression of HDAC3 and Rev-erbα and decreased expression of BMAL1,and the cell autophagy level was also reduced.2.HDAC3 and Rev-erbα si RNA transfection significantly reduced high glucose H/R injury,shown in increased cell activity and mitochondrial membrane potential,reduced LDH levels,up-regulated BMAL1 expression and increased mitophagy levels in primary myocardial cells.ConclusionThe results confirm that HDAC3 plays an important role in primary myocardial cell injury induced by H/R.Si RNA to silence HDAC3 or Rev-erbα can significantly effectively reduce high glucose H/R injury of primary neonatal rat cardiomyocytes by increasing mitophagy level which may be related the Rev-erbα/BMAL1-mediated autophagy signaling pathway.