Study On The Mechanism Of Soluble Guanylate Cyclase Agonist Vericiguat Targeting AMPK/mTOR And Autophagy Pathway To Inhibit Pathological Cardiac Hypertrophy

Objective:Cardiac hypertrophy is an important risk factor for heart failure.The NO-sGC-cGMP signaling pathway was significantly impaired in heart failure and myocardial hypertrophy,resulting in the significant decrease in cGMP production.In recent years,the vericiguat(a novel long-acting oral soluble guanylate cyclase stimulator),has emerged as a promising therapeutic drug for cardiovascular disease,such as heart failure.The NO-sGC-cGMP pathway is expected to be the promising therapeutic target for myocardial hypertrophy in the future.This study aims to elucidate the cardiovascular effects and molecular mechanisms of vericiguat in cardiac hypertrophy.The study may also provide a theoretical basis and new ideas for the prevention and treatment of heart failure,hypertrophic cardiomyopathy,and pathological myocardial remodeling.This study was conducted in vitro and in vivo.Methods:(1)In vitro study,we employed the cardiac hypertrophy model with H9c2 cells induced by 1μM AngⅡ for 48h.CCK-8 assay was used to identify the optimal concentration of vericiguat.The effects of vericiguat on cardiomyocyte hypertrophy,apoptosis,reactive oxygen species production and autophagy were evaluated by flow cytometry,confocal laser scanning microscopy.RT-PCR,and western blot.Further,computational virtual screening based on reverse docking was performed to assess the potential target.(2)In the present study,we used a mouse model of cardiac hypertrophy induced by continuous subcutaneous AngⅡ infusion using Alzet(?)Osmotic Pump for 28 days to represent cardiac remodeling progression associated with heart failure.Healthy male C57BL/6 mice aged 8 to 10 weeks were randomly divided into 4 groups,saline group(saline in pump),Vericiguat group(Ver group,intragastric administration 30mg/kg),AngⅡ infusion group(1.44mg/kg/d),AngⅡ+Ver group.Enzyme-linked immunosorbent assay(ELISA),immunofluorescence staining,confocal laser scanning microscopy,histopathological staining,echocardiography and hemodynamic testing,RT-PCR and western blot were used to investigate the effect of vericiguat on cardiac remodeling,including oxidative stress,fibrosis,cardiac hypertrophy,autophagy,and other signaling pathways.Additionally,RNA-seq was used to analyze the gene expression difference in mouse ventricular tissue before and after drug treatment.Results:(1)Vericiguat prevented AngⅡ-mediated increases in reactive oxygen species(ROS),apoptosis and cardiomyocyte hypertrophy of H9c2 cells.Vericiguat(10μM)regulated the autophagy signaling pathways and the expression of HO-1/SOD proteins during cardiac hypertrophy in vitro to protect cardiomyocytes.Scintillation proximity assay revealed that vericiguat could inhibit PDE4D(IC50,6.184μM)and PDE5A(IC50,2.584μM).(2)In the mouse model of cardiac hypertrophy induced by Ang II,vericiguat could significantly reduce the systolic and diastolic blood pressure,decrease cardiac interstitial and perivascular fibrosis,ROS production,and attenuated cardiac hypertrophy.Vericiguat reversed the AngⅡ-induced increase in VASP,AMPK and mTOR phosphorylation;autophagy related proteins(Atg3,Atg5,Beclin-1,LC3Ⅱ/Ⅰ)and HO-1/SOD proteins were also increased.Furthermore,bioinformatics analysis of RNA-seq and GSEA datasets revealed that vericiguat mainly regulate oxidative stress,extracellular matrix degradation,oxidative phosphorylation,cell proliferation,cell cycle and AMPK/mTOR signaling pathways.Conclusion:In vivo and in vitro experiments showed that vericiguat could attenuate myocardial remodeling process by targeting AMPK/mTOR,autophagy pathway and oxidative stress to improve cardiac function in cardiac hypertrophy.This research has shed new light on the function and mechanisms of NO-cGMP-SGC signaling pathway in myocardial hypertrophy.