The Effect And Mechanism Of ARNi On Myocardial Hypertrophy And Heart Failure With Preserved Ejection Fraction

Background and Aim:Angiotensin receptor neprilysin inhibitors(ARNi),beyond blocking angiotensin Ⅱ(AngⅡ)signaling,augment natriuretic peptides by inhibiting their breakdown by neprilysin.The myocardial effects of ARNi have been little studied until recently.Ventricular hypertrophy significantly increases the risk of heart failure with preserved ejection fraction(HFpEF).HFpEF is characterized by impaired diastolic function.Therefore,dynamic observation to transverse aortic constriction model,which pathologic process simulated cardiac hypertrophy early in HFpEF.We evaluated the effects of sacubitril/valsartan(LCZ696)in HFpEF in characteristic of transverse aortic constriction(TAC)-induced ventricular hypertrophy mice and investigated the effects and mechanisms of LCZ696 on AngⅡ-dependent cardiomyocyte hypertrophy.We hypothesized that LCZ696 attenuates hypertrophy,reverses left ventricular(LV)remodeling and impaired diastolic function after TAC,and that these may be contributed to by inhibition of hypertrophy and fibrosis in cardiac cells.Methods: 1.20 male C57BL/6 mice,were randomly divided into sham group(n=10)and TAC group(n=10).The weight,tail artery pressure were recorded before and after operation.Ultrasound imaging and pressure-volume conductance catheter technique were used to analysis LV function,cavity and thickness changes at 4th and 8th week respectively.The morphology of cardiac myocyte and the degree of cardiac fibrillogenesis were observed by H-E/ IHC staining and masson staining.The protein expression levels of BNP,β-MHC and TGF-βwere assessed by Western Blotting analysis.2.Four weeks after TAC,male C57BL/6 mice were randomized to treatment for 4 weeks with LCZ696(60mg/kg body weight perorally,n=8)Valsartan(48mg/kg body weight perorally,n=8)or no treatment TAC models(n=8).We performed echocardiography after treatment,followed by pressure-volume conductance catheter technique.The morphology of cardiac myocyte and the degree of cardiac fibrillogenesis were observed by H-E/ IHC staining and masson staining.The protein expression levels of BNP,β-MHC and TGF-βwere assessed by Western Blotting analysis.qPCR was used to evaluate the mRNA level of BNP,β-MHC and TGF-β.Plasma level of BNP and AngⅡwere tested.3.Neonatal rat cardiomyocytes were prepared from 1-3 days old neonatal SD rats.Cardiomyocytes were randomly divided into four groups(control,AngⅡ,Valsartan and LCZ696).Intracellular resting free calcium was tested by laser confocal scanning analysis.The protein expression levels of calcineurin,NFATs,BNP,β-MHC were assessed by Western Blotting analysis.qPCR was used to evaluate the mRNA level of BNP and β-MHC.Results: 1.The blood flow peak velocity at ligation of TAC group was significantly increased compared with sham group.After the operation the blood pressure of TAC group had begin to increase.Compared with sham group,the heart weight to body weight ratio of TAC group were markedly higher.Ultrasound imaging and pressure-volume conductance catheter technique analysis showed that IVSTd,LVPWd,IVRT,EDT,LVEDP and Tau of TAC group significantly increased,E/A and-dp/dtmax of TAC group significantly decreased.H-E/IHC staining and masson staining showed that myocyte cross-sectional area and myofibrillogenesis of TAC group significantly increased.4 weeks after the operation the LVEF,LVFS and +dp/dtmax of TAC group were not significantly decreased.Compared with sham group,the protein expression of BNP,β-MHC and TGF-βof TAC group were markedly higher.2.4 weeks after the treatment the thickeness,IVRT and EDT of LCZ696 group significantly decreased than the other groups,as well as LVEDP、Tau were significantly lower,but E/A and-dp/dtmax were significantly increased.H-E/IHC staining and masson staining showed that myocyte cross-sectional area and myofibrillogenesis of LCZ696 group significantly decreased.Western Blotting and qPCR analysis revealed that the protein and mRNA expression level of β-MHC,TGF-βand BNP decreased obviously in LCZ696 group in comparison with other groups.3.LCZ696 significantly inhibited the hypertrophic response of cardiomyocytes to Ang Ⅱ.These effects were concomitant to the decrease in Ang Ⅱ induced up-regulation of the level of resting intracellular free calcium,as well as the expression levels of NFATs,BNP and β-MHC.Conclusions: TAC at 4 weeks is LV hypertrophy and impaired diastolic function.simulated the pathological process of cardiac hypertrophy in early HFpEF.The ARNi sacubitril/valsartan improved cardiac diastolic function with the reduction of ventricular hypertrophy and fibrosis in TAC-induced diastolic dysfunction model in mice,by suppressing BNP,β-MHC and TGF-β.There was superior inhibition of LCZ696 on cardiac hypertrophy and cardiac fibrosis than stand-alone angiotensin receptor blocker(ARB).This effect may be due to the specific inhibition of neprilysin,beyond the ARB effect of LCZ696.LCZ696 behaves as a potent suppressor of AngⅡ-induced cardiac hypertrophy and this effect is possibly medated by suppression of calcium-mediated calcineurin-NFAT signaling transduction pathways.