Effect Of ACE2 Transfection On Cardiac Function In Diabetic Cardiomyopathy Rats

BackgroundThe prevalence of diabetes mellitus is increasing at an alarming rate,and with it the frequency of diabetes-related cardiovascular complication.In creasing amount of evidence has accumulated for the presence of myocardial dysfunction in diabetic patients in the absence of discernible coronary artery,valvular or hypertensive heart disease.In particular,it has been recently shown that diabetes is independently associated with nonischemic cardiomyopathie.Diabetic.cardiomyopahty(DCM) displays systolic and diastolic left ventricular dysfunction and commonly leads to heart failure.Several mechanisms for the pathogenesis of this diabetic cardiomyopathy have been proposed.These include the metabolic derangement,potentially adverse effects of hyperglycemia on endothelial function,autonomic dysfunction,myocardial fibrosis and myocyte hypertrophy.Myocardial fibrosis and myocyte hypertrophy are the most frequently proposed mechanisms to explain cardiac changes in diabetic cardiomyopathy.Renin-angiotensin system(RAS)are involved in the structural remodeling of the myocardial collagen matrix,resulting in fibrosis that disrupt the electrical and mechanical behavior of the hypertrophied myocardium,with subsequent systolic and diastolic cardiac failure.Angiotensin-converting enzyme2(ACE2),a newly discovered member of the renin-angiotensin system(RAS),sharing about 42%amino acid sequence identity with the catalytic domain of Angiotensin-converting enzyme(ACE).ACE2 is present in a wide variety of cells and tissues and has high expression in the cardiac and kidney tissue.ACE2 catalyses the cleavage of AngⅠto Ang(1-9)and AngⅡto Ang(1-7). Many studies have showed that AngⅡis a critical factor in LV remodeling,which involves hypertrophy,fibroblast proliferation,extracellular matrix production.We hypothesized that overexpression of ACE2 may reduce AngⅡlevels,and upregulate fibrosis,which provides a potential therapeutic target in the treatment of heart failure.Objective1.To establish a DCM animal model.2.To observe the expression of ACE2 after gene transfection.3.To study the therapy effect of adenoviral gene transfer of ACE2 on diabetic cardiomyopathy rats and explore the possible mechanism of the therapy effect.MethodsSeventy-two male Wistar rats were randomly divided into2 groups:control group (n=8)and model group(n=64).Diabetes was induced by a single intraperitoneal injection of STZ(65 mg/kg body wt and dissolved in 0.1 mol/1 citrate buffer,pH 4.2) in rats of model group.Control rats received citrate buffer alone.One week after injection of STZ,fasting plasma glucose levels were measured,and rats with plasma glucose at least two times higher than 16.7mmol/L were used.All rats were fed for 12 weeks after STZ or citrate buffer injections and had free access to standard rat diet and water.The DCM rats were randomly divided into3 groups:DCM group,EGFP group,and ACE2 group.Sterile saline,recombinant adenoviral carrying enhanced green fluorescent protein gene(AdV5.eGFP)and recombinant adenoviral carrying mouse ACE2 gene(AdV5.ACE2)were respectively injected to myocardia in different groups.10 days after gene transfection,myocardial ACE2 mRNA expression and ACE2 protein expression were detected.30 days after gene transfection,left ventricular function,myocardia collagen content and myocardial AngⅡlevel were measured.During the experiment,the rats were weighted and the contents of fast blood-glucose were detected every week.Results1.General features of the experimental ratsAt the end of the experiment,8 control rats,19 DCM rats,19 EGFP rats and 20 ACE2 rats were survived. 2.Body weight and fast blood-glucose measurementOne week after STZ injection,body weight in control group were insignificantly different from model group(P＞0.05),the level of fast blood-glucose of model group was significantly higher than that of the control group(P＜0.01).At the end of the experiment,compared with the control group,the body weight in others groups were significantly decrease(P＜0.01),while fast blood-glucose were significantly increase(P＜0.01).3.The result of Real-time RT-PCRThe expression of ACE2mRNA in ACE2group was higher than that of DCM group(P＜0.05).The expression of ACE2mRNA in ACE2group was higher than that of EGFP group(P＜0.05).The expression of ACE2mRNA in DCM group were insignificantly different from EGFP group(P＞0.05).4.The result of Western blotThe expression of ACE2 in ACE2group was higher than that of DCM group (P＜0.01).The expression of ACE2 in ACE2group was higher than that of EGFP group(P＜0.01).There were no significant difference between DCM group and EGFP group(P＞0.05).5.Echocardiographic examinationBefore gene transfection,compared with the control group,outstanding echocardiographic changes were detected in DCM rats,including increased LVESD, LVEDD and A peak velocity;decreased E peak velocity,ration of E/A,FS and EF. There is no significant differences of echocardiogram were found in three DCM groups.30 days after gene transfection,compared with DCM and EGFP rats, echocardiographic changes were detected in ACE2 rats.6.Pathological detectionThe myocytes from control group arranged regularly in HE-staining slice.The size of the nuclear was uniform.The staining cytoplasm was homogeneous.The myocytes from DCM and EGFP group arranged irregularly.The unclear was irregular and the interrupted myofibril arranged irregularly.The ACE2 group was obviously improved.The myocytes from ACE2 group arranged more regularly than DCM group. The interrupted myofibril was not common.The nuclear shape was more regular than DCM group.7.The content of collagen detection by Masson-stainingThe myocyte was red and collagen was green or blue.The collagen tissue was appropriately arranged among cardiomyocytes in control group.However,collagen tissue increased markedly,and disrupted in some area in the DCM and EGFP group. Compared with DCM group,the collagen tissue decreased and arranged regularly in ACE2 group.Quantitative analysis results:The collagen volume fraction(CVF)in DCM and EGFP group was higher significantly than that of control group (P＜0.01).The CVF in ACE2 group was lower significantly than that of DCM group(P＜0.01).8.The level of AngⅡCompared with control group,the level of AngⅡin DCM and EGFP group was increased(P＜0.01);Compared with DCM group,the level of AngⅡin ACE2 group was decreased(P＜0.01).Conclusions1.A DCM animal model was established by 12 weeks period of STZ-induced diabetes in male Wistar rats.2.ACE2 gene transfection was able to increase the expression of ACE2mRNA and protein.3.Overexpression of ACE2 may have therapeutic potential in improving cardiac function by decreased AngⅡlevel and myocardial fibrosis.