Sacubitril/Valsartan Attenuates Right Ventricular Remodeling Induced By Pulmonary Artery Constriction In Mice

Background and ObjectiveHeart failure(HF)is the most common cause of hospitalization for patients with cardiovascular disease over 60 years old,and the incidence is increasing worldwide.Currently,effective treatment strategies have been developed for left heart failure,such as AECI(angiotensin converting enzyme inhibitor),ARB(angiotensin receptor blocker),?-adrenergic receptor blockers(?-blockers)and aldosterone antagonists.These drugs can effectively improve the symptoms and prognosis of left heart failure(LHF).Until recently,the right ventricle(RV)has often been viewed as less important than the left ventricle and in contemporary literature received the moniker"The Forgotten Ventricle".Clinical treatment of right heart failure(RHF)is very limited.Existing literature indicates that RV function is one of the most important predictors of prognosis in many cardiovascular diseases.However,many studies have confirmed that many targeted therapies for LHF do not appear to provide similar benefits for RHF.Developing effective targeted therapies for RHF has become an urgent worldwide problem.Sacubitril/Valsartan(LCZ696)is an angiotensin receptor neprilysin inhibitor(ARNI),which has a dual-target regulatory effect.Neprilysin inhibitors can increase the natriuretic peptides levels in the circulation and angiotensin receptor can inhibit the renin-angiotensin-aldosterone system(RAAS).ARNI have many effects like diuretic,vasodilating,anti-myocardial hypertrophy and fibrosis.A number of clinical trials have demonstrated that ARNI can significantly improve the prognosis of heart failure compared with ACEI/ARB,and has been recommended by the guidelines for heart failure with reduced ejection fraction(HFrEF).Other literatures have proven that sacubitril/valsartan can improve myocardial remodeling.But the effect of ARNI on right heart remodeling is unclear.The activation of natriuretic peptide and RAAS can be observed in RHF.So we hypothesized that ARNI could improve right ventricular function by reducing RV remodeling and fibrosis.To explore the effect of ARNI on right heart remodeling,we designed the following experiments.Method1.Establishment of right ventricular hypertrophy model induced by pulmonary artery constriction(PAC)Anesthetize the male C57BL/6 mice by intraperitoneal injection with a mixture of xylazine(5 mg/kg)and ketamine(100 mg/kg)for pain relief.Softly insert a lab made trachea cannula through the glottis and then connected to ventilator.Open the chest at the second intercostal space to fully expose the thymus and the pulmonary artery.Pass the 6-0 suture through the connective tissue between the pulmonary trunk and the ascending aorta with a latch needle.Place the padding needle on the pulmonary trunk and,then,ligate the pulmonary trunk together with the padding needle.The suture is passed through without ligation in Sham group.2.Treatment by oral gavage3 days after PAC,the pulmonary valve peak velocity(PV Peak Vel)of mice was measured by echocardiography,and the substandard mice were excluded(PV Peak vel<1500mm/s).Then the mice were randomly divided into 6 groups:(1)Sham+Vehicle;(2)Sham+Enalapril;(3)Sham+LCZ696;(4)PAC+Vehicle;(5)PAC+Enalapril;(6)PAC+LCZ696.Then treatment for 4 weeks with LCZ696(60mg/kg/d),Enalapril(8mg/kg/d)or Vehicle(ethanol-saline mixture)by oral gavage.3.Blood pressure and heart rate measurementAt 14d and 28d after gavage treatment,non-invasive blood pressure meter was used to measure blood pressure and heart rate of mice.4.EchocardiographyWe evaluated the right heart function by echocardiography and right heart catheterization in 28 days after the treatment.We measured the PV Peak Vel,right ventricular diastolic anterior wall(RVAWd),right ventricular diastolic internal diameters(RVIDd),and tricuspid annular plane systolic excursion(TAPSE).5.Histological staining and real-time PCRThe heart weight,right heart weight,liver weight,kidney weight,and tibia length were measured.The quantity of myocardial fibrosis and hypertrophy was evaluated in Masson and HE stained heart sections.Gene expression levels like matrix metalloproteinase(MMP),collagen and BNP were quantified by real-time PCR.Result1.Establishment of right ventricular hypertrophy modelCompared with Sham group,PV Peak vel increased significantly(613.5±78.0 vs.1897.6 ±342.87mm/s,p<0.01)at 3 days after PAC.28 days after PAC,RVAWd(0.42±0.09 vs 0.84 ±0.1,mm,p<0.001),the right heart weight/tibia length RHW/TL(0.99±0.16 vs 2.01±0.37,mg/mm,p<0.001)in PAC group were significantly increased.2.LCZ696 reduces right ventricular remodelingAt 28 days after treatment,RHW/TL(1.5±0.3 vs 2.1±0.4,mg/mm,p=0.001)and PV peak vel(2414.7±452.5 vs 2807.1±438.2mm/s,p<0.05)and RVAWd(0.7±0.1 vs 0.78±0.12,mm,p<0.05)in PAC+LCZ696 group were significantly reduced comparing with PAC+Enalapril group.Masson staining showed that the right ventricular fibrosis area of PAC+ LCZ696 group was smaller than PAC+Enalapril group(22.47%vs 42.94%,p=0.001).The systolic blood pressure of mice in PAC+Enalapril group and PAC+LCZ696 group decreased,but there was no statistical difference between the two groups.3.The effect of LCZ696 on cardiac markersAfter LCZ696 treatment in PAC group,the expression level of BNP in right ventricle was significantly lower than PAC+Enalapril group.While the expression level of MMP-2 was significantly lower than PAC+Vehicle group(p=0.03).There was no statistical differernce between PAC+Enalapril group and PAC+LCZ696 group.ConclusionSacubitril/Valsartan(LCZ696)attenuates right ventricular remodeling and improves right ventricular function induced by pulmonary artery constriction in mice.