MiR-29a Regulates Cardiomyocyte Apoptosis By Targeting Bak1 In Diabetic Cardiomyopathy

Diabetes mellitus(DM)is one of the biggest health problems in the world.Diabetic patients have more higher chance of developing heart disease than nondiabetic patients.Diabetic cardiomyopathy(DCM)is the main cause of morbidity and mortality in diabetic patients.DCM refers to diabetic myocardial injury that cannot be explained by coronary artery disease,hypertension,or valvular heart disease in diabetic patients.DCM is mainly characterized by myocardial structural and functional abnormalities,such as myocardial cell apoptosis,left ventricular diastolic and systolic dysfunction,and myocardial interstitial fibrosis.DCM patients are not easily detected early which can further result in heart failure.Therefore,there is an urgent need to further identify the specific causes of DCM and to identify an effective treatment method.The pathogenesis of DCM is complex,and various factors are found to be associated with the onset of DCM including,abnormal glucose metabolism,insulin resistance,remodeling of extracellular matrix(ECM),calcium dysregulation,increased oxidative stress,inflammatory response,altered metabolism and endothelial dysfunction.Some pathways as well as proteins were also found to be involved in the onset of DCM like Protein Kinase C(PKC),Phosphatidylinositol 3-kinase(PI3K),Nuclear factor-κB(NFκB),mitogen activated protein kinase(MAPK)signaling pathways.In addition,high glucose can lead to the production of mitochondrial Reactive Oxygen Species(ROS).ROS can induce cardiomyocyte apoptosis and mitochondrial dysfunction,which eventually lead to the occurrence of DCM.Cardiomyocyte apoptosis plays an important role in diabetic cardiomyopathy.Abnormal Cardiomyocyte apoptosis can be found at the early stage of DCM,which might be associated with abnormal gene expression.The classic apoptosis pathways of cardiomyocytes mainly include mitochondrial apoptosis pathway,endoplasmic reticulum stress apoptosis pathway and death receptor apoptosis pathway.The mitochondrial apoptosis pathway is mediated by mitochondria,which causes cytochrome C transfer to cytoplasm and leads to apoptosis.Besides,in the apoptotic regulatory genes,Bcl-2 and caspase families are the most concerned.The Bcl-2 family consists of multiple anti-apoptotic members and pro-apoptotic members.The former includes Bcl-2,Bcl-xl,Bcl-w and Mcl-1,while the latter includes Bax,Bim and Bak1.They all play important roles in the regulation of apoptosis.Cysteinyl aspartate specific protease-3(Caspase-3)is the most critical apoptotic protease in the process of both endogenous and exogenous apoptosis pathways.The main function of the anti-apoptotic Bcl-2 is to counteract the activation of Bax and Bak1.By forming a pore in the outer mitochondrial membrane,Bax and Bak1 have an essential role in mediating cytochrome C release.Previously,there were many studies on Bak1 in plants,but few in animals.Therefore,we focused on Bak1.Micro RNAs(mi RNAs)are noncoding single-stranded RNAs with a length of about 22 nucleotides,which can affect m RNA translation or degradation by binding to the 3’UTRs of their target m RNA.Studies have shown that mi RNAs can be involved in a variety of biological processes like cell proliferation,differentiation,metastasis,apoptosis,glucose metabolism,fat metabolism and insulin secretion.Cardiac mi RNAs are the recently discovered key modulators of gene expression in the heart.Both alterations in the synthesis of mi RNA and levels of specific mi RNA have been shown to play critical roles in cardiac remodeling and the development of heart failure.Under high glucose condition,mi RNA-mediated signals can be transferred to other cells or tissues.The stable levels of circulating mi RNAs appearing in the blood can also be altered under different phases of diabetic cardiomyopathy.Therefore,mi RNAs may play potential diagnostic,prognostic and therapeutic roles in preventing and/or the treating diabetic cardiomyopathy.The mi R-29 family is mainly composed of mi R-29 a,mi R-29 b and mi R-29 c.mi R-29 b includes mi R-29b-1 and mi R-29b-2.mi R-29 family target genes mainly exist in biological processes like cell proliferation,cell differentiation,cell apoptosis and migration.In the past,mi R-29 family was mainly studied in liver fibrosis,tumor and other related diseases.Recent studies have shown that mi R-29 family can have regulating activity in various parts such as cardiovascular tissue and vascular endothelial cells,aortic tissue,myocardial tissue and cardiomyocytes and cardiac metabolism.Nevertheless,studies on the relationship between mi R-29 family and DM were focused on angiogenesis,insulin resistance,systemic inflammatory response,abnormal glucose metabolism and lipid metabolism.Recent studies have shown that mi R-29 b regulates the mitochondria-dependent apoptotic pathway by targeting Bax in doxorubicin cardiotoxicity.So far,there are no reports on whether mi R-29 a can participates in regulating the pathogenesis of DCM by the apoptosis pathway.In conclusion,we speculate that mi R-29 a changes in both cardiac tissue and peripheral blood in diabetic cardiomyopathy.In our study,patients with diabetic cardiomyopathy were recruited to detect the changes of mi R-29 a and N-terminal pro-brain natriuretic peptide(NT-pro BNP)levels.Their cardiac functions were detected by color Doppler echocardiography.Then,we investigated the relationship between mi R-29 a level in peripheral blood and cardiac function.To further explore the pathogenesis,DCM rat model was established by treating rats with streptozotocin(STZ).mi R-29 a expression was determined using Quantitative real-time PCR(QRT-PCR).High glucose(HG)-treated H9c2 cells were transfected with NC mimics and mi R-29a-3p mimics.The apoptosis rate of H9c2 cells was measured by flow cytometry.Western blot was utilized to evaluate the efficacy of mi R-29 a mimics on the expression of apoptosis-related proteins.Furthermore,we predicted target gene of mi R-29a-3p,Dual-luciferase reporter assay was carried out to determine whether mi R-29 a directly targets the gene.Finally,DCM rat myocardium were injected with NC or mi R-29a-3p mimics,so as to explore the possible pathogenesis of mi R-29 a in diabetic cardiomyopathy.The study mainly includes the following five parts.Part One The expression and value of mi R-29 a in peripheral blood of diabetic cardiomyopathyObjectives: 1.To observe the change of mi R-29 a level in peripheral blood of DCM patients.2.To investigate the relationship between mi R-29 a level and cardiac function in diabetic cardiomyopathy.Methods: 1.6 Diabetic cardiomyopathy patients(DCM)and 10 normal people(NC)were enrolled and general clinical data were collected.2.Elbow venous blood was collected from the two groups,followed by routine biochemistry test and NT-pro BNP test.3.Total RNAs were extracted by TRIzol LS regent to implement reverse transcription and q PCR determination.we obtained the relative expression of mi R-29 a in peripheral blood of the two groups.4.Echocardiography was performed to detect the following parameters of DCM and NC groups: left ventricular ejection fraction(LVEF),left ventricular fractional shortening(LVFS),interventricular septal thickness(IVST),left ventricular posterior wall thickness(LVPW),left ventricular internal dimensions at end systole(LVIDS),and left ventricular internal dimensions at end diastole(LVIDD).Results: 1.Comparison of general clinical data: there was no statistical significance in age,systolic blood pressure,diastolic blood pressure,cholesterol,triglyceride and low-density lipoprotein cholesterol levels between the two groups(P>0.05).Compared with normal control group,body mass index(BMI),urea nitrogen,creatinine,uric acid,fasting blood glucose and NT-pro BNP level were increased in DCM group,while high density lipoprotein cholesterol level was lower than normal control group,the differences were statistically significant(P<0.05).2.mi R-29 a level in peripheral blood: mi R-29 a expression level in DCM group was significantly higher than that in healthy people,and the difference was statistically significant(P<0.05).3.Comparison of echocardiography: Compared with healthy people,the left ventricular ejection fraction(LVEF)and left ventricular short axial constriction rate(LVFS)in DCM group were significantly decreased,while left ventricular posterior wall thickness(LVPWT),left ventricular end contractile diameter(LVIDS)and left ventricular end diastolic diameter(LVIDD)values were significantly increased,and the differences were statistically significant(P<0.05).4 Correlation analysis: mi R-29 a was negatively correlated with LVEF and LVFS(P<0.0001),but positively correlated with LVPWT,LVIDS,LVIDD and the NT-pro BNP level(P< 0.05).Conclusions: 1.The expression of mi R-29 a in peripheral blood of DCM patients was significantly increased.2.The expression of mi R-29 a in peripheral blood was negatively correlated with the cardiac function of DCM patients.Part Two Establishment of diabetic cardiomyopathy rat model and expression of mi R-29 a in rat myocardial tissueObjectives: 1.To observe the cardiac function and morphological changes of myocardial tissue in DCM rat model.2.To observe the expression of mi R-29 a in myocardial tissue of DCM rats.Methods: 1.Adult male Sprague-Dawley(SD)rats were randomly split into control group and DCM group.The rats in DCM group were fed with high-glucose and high-fat diet,and the rats in control group were fed with normal diet.On he 4th weeks of feeding,rats in DCM group were intraperitoneally injected with 1% streptozotocin(STZ)30 mg/kg twice,and rats in control group were intraperitoneally injected with the same amount of citric acid buffer.All rats were fed to 14 weeks.The successful modeling of type 2 diabetes mellitus was determined by blood glucose,M-mode echocardiography was performed to detect the cardiac function,and we performed hematoxylin and eosin(H&E)staining for the detection of histological morphology of myocardium to confirm the successful modeling of DCM.2.The expression of mi R-29 a in the DCM group and the normal group was detected by q RT-PCR.Results: 1.Establishment of DCM rat model 1)Animal model: Finally,7 rats were included in the DCM group.10 rats in normal group,all of which survived.Compared with normal group,blood glucose level in DCM group was significantly higher(P<0.05),while the body weight decreased(P<0.05).2)M-mode echocardiography: Left ventricular ejection fraction(LVEF),left ventricular fractional shortening(LVFS),maximum velocity of early diastolic ventricular filling(E),maximum velocity of early diastolic ventricular filling/maximum velocity of late diastolic ventricular filling(E/A ratio)were significantly decreased in DCM rats(P<0.05),while the maximum velocity of ventricular filling(A),Left ventricular internal dimensions at end systole(LVIDS)and Left ventricular internal dimensions at end diastole(LVIDD)were significantly increased(P<0.05).3)Morphological changes of myocardial tissue: HE staining results of myocardial cells showed that,myocardial cells were of different sizes,chaotic arrangement,sparse nucleus,degeneration and necrosis of interstitial structure in DCM group.2.Expression of mi R-29 a in rat cardiomyocytes of the two groups The expression of mi R-29 a in DCM group was significantly downregulated(P<0.05).Conclusion: The expression of mi R-29 a was significantly downregulated in DCM group,suggesting that mi R-29 a may be involved in the pathogenesis of DCM.Part Three Changes of mi R-29 a in HG-induced cardiomyocytes and its effect on apoptosis of cardiomyocytesObjectives: 1.To observe the apoptosis of HG-induced cardiomyocytes in rats.2.To observe the expression of mi R-29 a in HG-induced cardiomyocytes.3.To observe the effect of overexpression of mi R-29 a on apoptosis of HG-induced cardiomyocytes.Methods: 1.Rat cardiomyocyte cell(H9c2)were cultured and transfected with mi R-29 a mimics or NC mimics using Lipofectamine2000 reagent.The cells were divided into Normal Glucose group(NG,5.5mm),High Glucose group(HG,33 m M),High glucose+ mi R-29 a mimics group(HG + mi R-29 a mimics),and High glucose + negative control mimics group(HG + NC mimics).Flow cytometry was used to detect the cardiomyocyte apoptosis in the four groups.2.The protein expressions of Bcl-2,Mcl-1,Bax,caspase-3 and Bak1 were determined by western blot in each group.3.q RT-PCR was used to detect the expression of mi R-29 a in H9c2 of the four groups.Results: 1.High glucose can aggravate cardiomyocyte apoptotic index,Upregulation of mi R-29 a reduces the apoptosis index of HG-induced cardiomyocytes: The apoptosis rate of myocardial cells in rats increased with time in high glucose condition.The apoptosis rate of cardiomyocyte in HG group was significantly higher than that in NG group(P<0.05),compared with HG group and HG+NC mimics group,the apoptosis of cardiomyocyte in HG+mi R-29 a mimics group was significantly decreased(P<0.05).2.Effects of high glucose on apoptosis-associated proteins expression in rat cardiomyocytes and the effects of Upregulation of mi R-29 a on the expression of apoptosis-associated protein in HG-induced cardiomyocytes: Compared with NG group,the expressions of Bax,Caspase-3 and Bak1 were increased(P<0.05)in HG group.Especially,the expressions of caspase-3 and Bak1 were significantly increased(P<0.001),while the expression of Bcl-2 and Mcl-1 were significantly decreased(P<0.05),Compared with HG group and HG+NC mimics group,the expressions of Bax,Caspase-3 and Bak1 were significantly decreased in HG+mi R-29 a mimics group(P<0.05),while the expression of Bcl-2 and Mcl-1 were significantly increased(P<0.05).3.High glucose can downregulate the expression of mi R-29 a in cardiomyocytes: Compared with NG group,the expression of mi R-29 a in cardiomyocytes was significantly decreased in HG group(P<0.05),compared with HG group and HG+NC mimics group,the expression of mi R-29 a was significantly increased in HG+mi R-29 a mimics group(P<0.05).Conclusions: 1.The apoptosis of HG-induced cardiomyocytes increased,and the expression of mi R-29 a in HG-induced cardiomyocytes decreased.2.High glucose can promote cardiomyocyte apoptosis by increasing the expression of Bax,Caspase-3 and Bak1 and decreasing the expression of Bcl-2 and Mcl-1.3.mi R-29 a can reduce the apoptosis of HG-induced cardiomyocytes by regulating the expression of apoptosis-associated proteins.Part Four mi R-29 a participates in the regulation of HG-induced cardiomyocyte apoptosis by targeting Bak1Objective: To investigate the mechanism of mi R-29 a regulating HG-induced cardiomyocyte apoptosis by target gene Bak1.Methods: 1.The sequence of the 3’untranslated region(3’UTR)of the potential target gene of mi R-29 a was analyzed by Target Scan,mi Randa and Pic Tar atabase,which predict mi R-29 a binding to the Bak1 gene.2.The correlation of mi R-29 a with Bak1 was verified by dual-luciferasereporter gene experiments.3.Western blot was carried out to further determine the correlationbetween Bak1 and mi R-29 a in H9c2 cells.Results: 1.Bioinformatics prediction of mi R-29 a target genes: Target scan7.2 bioinformatics software(http://www.targetscan.org/)predicted the mi R-29a-3p binding to the Bak1 gene.2.mi R-29 a directly targeted Bak1: Bak1-3’UTR-WT and Bak1-3’UTR-MUT were constructed into the luciferase gene plasmids,and the above luciferase reporter plasmid was co-transfected into H9c2 cells with mi R-29 a mimics or NC mimics.Our data clarified that the relative luciferase activity was reduced in Bak1-3’UTR-WT by mi R-29 a mimics,but remained unchanged in Bak1-3’UTR-MUT.3.Effect of upregulation of mi R-29 a on the expression of target gene Bak1 in H9c2 cells: Bak1 protein expression was downregulated after transfection with mi R-29 a mimic in H9c2 cells.Conclusion: mi R-29 a directly targeted Bak1,mi R-29 a participates in the regulation of HG-induced cardiomyocyte apoptosis by targeting Bak1.Part Five The role of mi R-29 a in the occurrence and development of diabetic cardiomyopathy.Objectives: 1.To observe the effect on cardiac function and morphological changes of myocardial tissue in diabetic cardiomyopathy rat which overexpression of mi R-29 a.2.To observe the effect on myocardial cell apoptosis in diabetic cardiomyopathy rat which overexpression of mi R-29 a.Methods: 1.Adult male SD rats were divided into control group(n=3)and DCM roup.DCM model was successfully established as described above.successfully modeled DCM rats were randomly subdivided into DCM group,DCM+NC mimics group(myocardial injection of mi R-29a-3p-NC mimics)and DCM+mi R-29a-3p mimics group(myocardial injection of mi R-29a-3p mimics),with 3 rats in each group,M-mode echocardiography was performed to detect the cardiac function,and we performed hematoxylin and eosin(H&E)staining and Masson’s trichrome staining for the detection of cardiomyocyte size and fibrosis,respectively.2.Flow cytometer was utilized to determine the apoptosis in the cardiomyocytes.3.The protein expressions of Bcl-2,Mcl-1,Bax,caspase-3 and Bak1 in each group were determined by western blot.Results: 1.The effect of mi R-29 a on cardiac function of DCM rats: Compared with control group,LVEF,LVFS,E value,and E/A ratio of DCM rats were significantly decreased(P<0.05),while the A value,LVIDS and LVIDD were significantly increased(P<0.05).After injection of mi R-29a-3p mimics into the myocardium of DCM rats,the levels of LVEF and LVFS in the DCM+mi R-29 a mimics group were significantly higher than those in the DCM group(both P<0.01),A value,LVIDS and LVIDD levels were dramatically reduced(P<0.05,P<0.0001 and P<0.0001,respectively).2.The effects of mi R-29 a on myocardial histomorphology in DCM rats: H&E staining results showed that cardiomyocytes in DCM group were varied in size,with chaotic cell arrangement,sparse nucleus,degeneration and necrosis of interstitial structure,indicating severe DCM-related pathological features.However,the degrees of restructuring,fibrosis and necrosis of cardiomyocytes were relatively mild in the DCM+mi R-29 mimics group(P<0.01).Masson’s trichrome staining results showed that,in the control group,the myocardial fibers were arranged uniformly,without collagen,while in the DCM group,myocardial fibrosis and collagen composition were enhanced,which were significantly relieved by the upregulated mi R-29a xpression(P<0.01).3.The effect of mi R-29 a on cardiomyocyte apoptosis in DCM rats: Compared with the control group,the apoptosis rate of cardiomyocyte was significantly increased(P<0.001)in DCM group and DCM+NC mimics group.However,the apoptosis rate was decreased after injection of mi R-29a-3p mimics(P<0.05).4.The effects of mi R-29 a on the expression of apoptotic proteins in DCM rats: Compared with the control group,the expressions of Bax,Caspase-3 and Bak1 were increased(P<0.05)in DCM group and DCM+NC mimics group,while the expression of Bcl-2 and Mcl-1 decreased(P<0.0001).Upregulation of mi R-29 a inhibited Bax,cleaved-caspase3 and Bak1 levels(P<0.001,P<0.01 and P<0.05,respectively)and promoted Bcl-2 and Mcl-1 levels in cardiomyocytes of rats(both P<0.001).Conclusion: mi R-29 a is associated with the pathogenesis of DCM by regulating the apoptosis pathway.Conclusion: mi R-29 a regulates cardiomyocyte apoptosis by targeting Bak1 in diabetic cardiomyopathy.