Effects Of Combined Treatment With Simvastatin And Losartan On Cardiac Remodeling In Rats And Its Mechanisms

Objective:Congestive heart failure(CHF) is a complex clinical syndrome that features a failing heart together with advanced signs and symptoms arising from various cardiovascular diseases.Heart failure is a successive dynamic structure and function remodeling process,which is characterized by the heart responses to overloading and some other stimulation.Thus,target at cardiac remodeling is very important therapies in preventing the progress of heart failure.In this study,we aimed to investigate the effects of combined treatment with simvastatin and losartan on cardiac remodeling in Rats. Furthermore,the mechanisms of these effects were investigated focus on neuroendocrine hormonal activation,proinflammatory cytokines,apoptosis of myocardial cell and myocardial fibrosis.This maybe can lead to the notion that new drug therapy in clinical practice for heart failure.Methods:The pressure overload dependent heart failure model in rats was induced by ascending aortic constriction(AAC).After AAC,the animals were randomly separated 6 groups:normal control group,no operation and drug treatment;control sham group, no drug treatment;model group(AAC),no drug treatment,losartan treatment group(AAC+Los,Los,5mg/kg);simvastatin treatment group(AAC+Sim,Sim,2 mg/kg); combination of losartan and simvastatin(AAC+Los+Sim;Los,5mg/kg;Sim,2 mg/kg). Different drug was intragastric administrated to the animals daily from day 5 after operation to the end of experiment for 35 days.The control group was administered with the same volume of vehicle.The heart and lung weight was measured by transthoracic echocardiography.Cell morphology and myocardium collagen change was checked by light microscope and transmission electron microscope.These methods were used for investigating the effects of drug treatment on cardiac remodeling.The probability sum test(q test) was used to evaluate the synergism effects of simvastatin and losartan on pressure overload dependent heart failure in rats.The heart rate was calculated by electrocardiography and blood pressure was determined by carotid arterial cannula.The concentration of TCH,TG,LDL-C and HDL-C in serum was measured by the method of directly clearance.The concentration of Angâ…¡,ET and ANP in blood plasma was measured by the method of radioimmunity.The concentration of TNF-α, IL-6,BNP and CRP in serum was measured by ELISA.Myocardial cell apoptosis was checked by TUNEL method.In vitro experiment,Angâ…¡was used to induce apoptosis of myocardial cell.The activity of myocardial cell was checked by MTT method.Cell morphology of apoptotic cell was observed by fluorescence microscope.Double staining for FITC-Annexin V binding and for cellular DNA using propidium iodide(PI) was performed to check the ratio of apoptotic cell using flow cytometry.Bax and Bcl-2 protein expressions were measured by flow cytometry detection.The expression of caspase-3 was in myocardial cell was examined by immunohistochemistry.The protein expression of ERK1/2 and P- ERK1/2 in ventricular tissue was measured by Western blot.The mRNA expression of MMP2,MMP9,TIMP1,TIMP2,AT₂R,Collagen-1 and Collagen-3 in ventficular tissue was measured by real-time PCR.Results1.The evaluation of pressure overload dependent heart failure model in ratsThe results of TTE showed that the phenomenon of cardiac remodeling was emerged 3 weeks after AAC and more clear in 5 weeks after operation.The signs showed that left ventricular enlargement,ventricle wall thickening,the dysfunction of cardiac ejection and increasing of left ventricular mass or index of left ventricular mass. weight of heart,index of heart weight,left heart weight,index of left heart weight, weight of lung and index of lung weight of AAC group is significantly high than the group of control and sham.The concentration of BNP in serum significantly increased in AAC group.For AAC model group,the HE staining of heart and lung tissue showed as follows:The derangement structure of cardiac muscle fibers,bulk cloudy swelling degeneration of myocardial cell,fibronolysis in part of cardiac muscle fibers, coarctation of alveolar space,Congestion and edema in lung mesenchyma,small amounts bleeding and fluidify in alveolar space and inflammatory cell infiltration. Observed by projection electron microscope showed that in AAC group as follows:The derangement structure of cardiac muscle fibers,Partly disappearance in zone Z,unclear of crista mitochondriales,magnanimous vacuolar degeneration,disorder structrue of intercalary disc,apoptosis body and hyperplasy of collagen fibers.The results above showed that the pressure overload dependent heart failure model in rat was successful established by AAC.2.The assessment of cardiac remodelingThe results of TTE showed that combination of simvastatin and losartan significantly decreased the weight of heart and index of heart weight when compared with single drug treatment.There were no significant differences about systolic pressure and heart rates among all the groups with drug treatment for 5 weeks.Simvastatin and losartan alone relieved the increased of weight of heart,index of heart weight,weight of lung and index of lung weight induced by AAC.Moreover,the effects of combination of simvastatin and losartan on these indexs were much better.Using the probability sum test,the q value of LV Mass / Body Weight,HW / BW Ratio,LV Weight / BW Ratio and Lung Weight/BW Ratio were 1.17,1.17,1.16 and 1.26.All of the values were higher than 1.15.These results showed that synergism effects of simvastatin and losartan on cardiac remodeling. 3.Results of lipoprotein cholesterol and triglyceride in bloodThere were no significant differences about TCH,LDL-C,HDL-C and TG among all the groups with drug treatment for 5 weeks.4.The concentration of Angâ…¡,ET-1,ANP,BNP,CRP,IL-6 and TNF-αin bloodIn AAC group,the concentration of Angâ…¡,ET-1,ANP,BNP,CRP,IL-6 and TNF-αin blood significantly increased.Treatment with simvastatin and losartan alone and in combination decreased the level of ET-1,ANP,BNP,CRP,IL-6 and TNF-α,but had no effect on Angâ…¡.Combination of simvastatin and losartan significantly decreased the level of ET-1,ANP,BNP and TNF-αin blood when compared with single drug treatment.5.The mRNA expression of AT₁ R and AT₂RThe mRNA expression of AT₁ R in ventricular tissue significantly increased In AAC group.Treatment with losartan had no effect on the expression of AT₁R.Simvastatin or combination of simvastatin and losartan both significantly decreased the high level of AT₁R mRNA expression by AAC.There was no significant difference of mRNA AT₂R expression among the normal and model rats.Treatment with losartan had no effect on the expression of AT₂R. Losartan or combination of simvastatin and losartan both significantly increased the AT₂ R mRNA expression.6.ERK and P-ERK proteinIn pressure overload dependent heart failure rats,the degree of phosphorylation of ERK1/2 increased,Treatment with simvastatin,losartan or combination of simvastatin and losartan all partial inhibited the phosphorylation ERK1/2.The degree of inhibition of phosphorylation ERK1/2 was much higher when combination of simvastatin and losartan compared with single drug treatment.7.Cardiomyoeyte apoptosisCardiomyocyte apoptosis was checked by TUNEL method and the results showed that drug treatment decreased the cardiomyocyte apoptosis induced by AAC.The degree of anti-apoptotic effect was much higher in the combination of two drugs when compared with single drug treatment.In vitro experiment,incubation with 10^-7 mol/L Angâ…¡for 48 h increased cardiomyocyte apoptosis and decreased cell viability.Losartan(10^-5 mol/L) and simvastatin(10^-5 mol/L),either alone or in combination,significantly decreased Angâ…¡-induced cardiomyocyte apoptosis and increased cell viability.The q-values calculated by the probability sum test were 1.31 and 1.21 for cardiomyocyte apoptosis and cell viability,respectively.These demonstrated that there was a synergistic effect between losartan and simvastatin in inhibiting Angâ…¡-induced cardiomyocyte apoptosis and increasing cell viability.Angâ…¡induced a significant increase in Bax protein expression but decreased those of Bcl-2.Losartan decresed Bax expression and increased Bcl-2 expression while simvastatin had no such effect.Angâ…¡induced the protein expression of caspase-3 in cardiomyocyte.Simvastatin,losartan or combination of simvastatin and losartan significantly decreased the protein expression of caspase-3.The degree of inhibition in expression of caspase-3 was much higher when combination of simvastatin and losartan compared with single drug treatment.8.Collagen production and the mRNA expression of MMPs and TIMPsIn AAC group,the mRNA expression of collagenâ… andâ…¢significantly increased. Treated with simvastatin and losartan alone and in combination,the collagen and the ratio of mRNA expression of collagenâ… andâ…¢significantly decreased.The degree of inhibition in the ratio of collagenâ… andâ…¢mRNA expression was much higher when combination of simvastatin and losartan compared with single drug treatment. In AAC group,the mRNA expression of MMP2 and MMP9 significantly increased. Treatment with simvastatin and losartan alone and in combination had no effects on the mRNA expression of MMP2 and MMP9.Also,the mRNA expression of TIMP1 and TIMP2 significantly increased In AAC group.Treatment Simvastatin and losartan alone and in combination significantly decreased the TIMP1 and TIMP2 mRNA expression. The degree of inhibition in the ratio of TIMP1 and TIMP2 mRNA expression,was much higher when combination of simvastatin and losartan compared with single drug treatment.Conclusions1.The pressure overload dependent heart failure model in rat was successful established by ascending aortic constriction.2.Combination of simvastatin and losartan significantly inhibited cardiac remodeling induced by AAC when compared with single drug treatment.3.Some mechanisms of effects of combination simvastatin and losartan on cardiac remodeling were investigated as follows.a,Combination of simvastatin and losartan significantly decreased the protein expression of caspase-3 when compared with single drug treatment.b,Combination of simvastatin and losartan significantly increased the activity of cells and inhibited the apoptosis of myocardial cell induced by Angâ…¡when compared with single drug treatment.c,Combination of simvastatin and losartan significantly inhibited myocardium collagen production induced by AAC when compared with single drug treatment.More interesting,Combination treatment significantly decreased the ratio of mRNA expression of collagenâ… and collagenâ…¢.d,Combination of simvastatin and losartan significantly decreased the mRNA expression of TIMP 1 and TIMP2 when compared with single drug treatment.e,Simvastatin significantly decreased the high level mRNA expression of AT₁R by AAC;losartan significantly increased the mRNA expression of AT₂ R.f,Combination of simvastatin and losartan significantly inhibited the phosphorylation ERK1/2 when compared with single drug treatment.g,Combination of simvastatin and losartan significantly decreased the concentration of ET-1,ANP,BNP and TNF-αin blood when compared with single drug treatment.