LCZ696(Sacubitril/Valsartan) Inhibits Pulmonary Hypertension Induced Right Ventricular Remodeling By Targeting Pyruvate Dehydrogenase Kinase 4

Background and ObjectiveHeart failure(HF)is one of the main causes of hospitalization for patients over 60 years of age with cardiovascular disease,and its incidence is on the rise worldwide.HF can be classified as left heart failure(LHF),right heart failure(RHF),and end-stage HF.There are many effective treatment strategies for LHF,such asβ-blockers,SGLT2 inhibitors,angiotensin receptor neprilysin inhibitor(ARNI)and mineralcorticoid recept antagonist(MRA).But medicines targeting LHF are usually less effective or even ineffective in treating RHF.Pulmonary arterial hypertension(PAH),which is characterized by a marked increase in pressure overload of the right ventricle(RV),eventually leads to myocardial hypertrophy and RHF.PAH is a major cause of RHF,and RV function is an important predictor of prognosis in many cardiovascular diseases.However,the mechanism and treatment target of RHF are still unclear.It is of great significance to find therapeutic targets and drugs for RHF.Considering that RHF is mostly secondary to LHF mediated by PAH,modulating the similar phenotypic behaviors of RHF and LHF,e.g.,by inhibiting pathological hypertrophy induced by pressure overload of the left and right ventricles,should be able to prevent both types of heart failure.It is reasonable to speculate that targeting common differentially expressed genes(DEGs)in hearts with systemic or pulmonary pressure overload would identify potential targets for drugs that can treat both RHF and LHF.LCZ696(sacubitril/valsartan)significantly reduces hospitalization and mortality in patients with LHF and has been recommended by guidelines for the treatment of LHF with reduced left ventricular(LV)ejection fraction.However,its effect in patients with RHF or RV remodeling is unclear,and only a few reports have shown that LCZ696 can reduce RV remodeling and improve cardiac function in patients with RHF.Furthermore,the mechanisms by which LCZ696 improves RV remodeling are still poorly understood.In this study,we hypothesized that LCZ696 has common molecular targets in pressure overload-induced LV and RV remodeling and is able to ameliorate PAH-induced RV remodeling by a mechanism involving the Pdk4 and PDK4 protein.Methods1.Bioinformatics analysis to screen for target genesAfter total RNA was extracted from LV of transverse aortic constriction(TAC)mice and RV of pulmonary artery constriction(PAC)mice,mRNAs were reverse transcribed into cDNA with random primers.RNA sequencing was performed with an Illumina HiSeqTM 4000 system by a professional company.Bioinformatics analysis screened common differentially expressed genes(DEGs)between TAC and PAC.2.Molecular dockingThe receptor-based high-throughput virtual screen with 3 commercial compound databases(Selleck,APExBIO,Chemdiv)used to discover small molecule compounds capable of binding to PDK4 protein.3.TreatmentPAC mice with a peak blood velocity above 1800 mm/s at the banding site,as assessed by echocardiography,were included in the study.In the LCZ696 groups,LCZ696(60 mg/kg/day)was dissolved in normal saline and administered by oral gavage for 4 weeks,starting on day 3 after surgery.The vehicle groups were given the same volume of normal saline.4.Echocardiography and hemodynamic examinationWe performed echocardiography with a Vevo 2100 system.The RV outflow velocity was obtained from the parasternal short-axis view at the level of the pulmonary artery(PA)during end-diastole.PA peak velocity,PA pressure gradient,LV end-diastolic diameter(LVEDd),RV end-diastolic diameter(RVEDd),RV diastolic anterior wall thickness(RVAWd),and tricuspid annular plane systolic excursion(TAPSE)were measured by adjusting the measurement interface.Right heart catheterization was performed through the right jugular vein by using a 1.0F Millar catheter.The RV systolic blood pressure(RVSP)was recorded.5.Histological examination and qPCRFollowing euthanasia,the body weight,right ventricular weight and a tibia length were recorded.Then,hematoxylin-eosin(HE)and wheat germ agglutinin(WGA)were used to assess RV hypertrophy,and Azan-Masson staining was used to assess myocardial fibrosis.Myocardial fibrosis marker(Col1α1、Mmp2)and HF marker(Nppb)were measured by qRT-PCR.6.Generation of conditional Pdk4 deletion miceWe generated mice carrying Pdk4 flox/flox and inducible Myh6-MCM.Then,tamoxifen was administered to 8-to 10-week-old mice on alternate days(10 mg/mL,intraperitoneal injection)3 times to generate cardiac-specific Pdk4 conditional knockout mice(Pdk4-CKO).After the last injection,the mice were rested for 4 weeks before receiving PAC surgery.7.Overexpression of Pdk4 geneRecombinants with adeno-associated virus(AAV)with overexpressed mouse Pdk4 were produced by ObiO Technology Corp.,Ltd.pAAV-CMV-PDK4-P2A-EGFP-3×FLAG-WPRE(overexpression of Pdk4,Oe-Pdk4)and pAAV-CMV-EGFP-3×FLAG-WPRE(negative control,NC)virus particles(1×1012 viral genomes/mL)were administered by direct injection with a 30-gauge syringe needle into the RV free wall(two sites,10μL/site)in 8-week-old mice.Four weeks later,mice were subjected to PAC or sham surgery.Results1.Pdk4 is a common DEG of the pressure-overloaded LV and RVA total of 60 DEGs were shared by TAC and PAC mice,50 of which were upregulated and 10 downregulated.Pdk4 was significantly lower in the TAC and PAC groups than in the corresponding sham groups.GO analysis showed that Pdk4 was involved in a variety of processes,such as the cellular process of responding to organic substances and tissue remodeling.2.LCZ696 is predicted as a target of PDK4 protein with high docking scoreThe structure of PDK4 was obtained from the RCSB database,and the reliability of the docking method was verified by docking with the original ligand.We identified 47 compounds with docking scores larger than 7 and reasonable binding patterns.among these compounds,LCZ696 had high docking scores.3.LCZ696 attenuates RV remodeling and downregulates myocardial PDK4 of RV in PAC miceCompared with vehicle-treated PAC mice,LCZ696-treated mice had significantly smaller RV wall thickness and RV diameters,less myocardial fibrosis(less collagen deposition in the RV in the RV,and significantly lower mRNA levels of Collal and Mmp2),lower expression of PDK4 protein,and less phosphorylation of glycogen synthase kinase-3β(p-GSK3β).4.Overexpression of Pdk4 gene antagonizes the antihypertrophic effect of LCZ696Pdk4-CKO mice subjected to PAC showed smaller RV wall thickness and RV diameter,larger TAPSE,lower RVSP,smaller myocyte area of the RV,lower RVW:BW and RVW:TL ratios,less fibrosis in the RV myocardium,and lower expression of Nppb and Collal genes than MCM mice.In PAC mice,the inhibitory effects of LCZ696 on RV hypertrophy and dilation(RVAWd,RVEDd),RV dysfunction(TAPSE),compression of the LV due to RV dilation(LVEDd),myocyte area and myocardial fibrosis of the RV,RVW:BW and RVW:TL ratios,and Nppb and Collal were significantly antagonized by Oe-Pdk4.ConclusionsPdk4 is a common therapeutic target for pressure overload-induced LV and RV remodeling,and LCZ696 attenuates RV remodeling by downregulating Pdk4 and inhibiting PDK4/p-GSK3β signal.