The Role Of The Mechanosensitive Ion Channel Piezo1 In Calcific Aortic Valve Disease And Related Mechanisms

Background:Calcific aortic valve disease(CAVD)is a common heart valve disease in cardiovascular surgery.With the aging population and increasing life expectancy,the incidence of CAVD will continue to rise.CAVD can be classified into stages of aortic valve sclerosis and calcification based on the progression of the disease.When the disease advances to the stenosis stage,severe patients require surgical aortic valve replacement(SAVR)or transcatheter aortic valve implantation(TAVI).Increasing evidence suggests that the development of CAVD is related to various factors,including pathological processes similar to atherosclerosis and bone formation.These factors collectively lead to aortic valve calcification and subsequently aortic stenosis(AS).If not diagnosed and treated promptly,AS can result in heart failure and even death.Thus,it is essential to investigate the molecular and cellular biological mechanisms influencing aortic valve calcification to identify potential therapeutic targets,providing new insights and approaches for the prevention and treatment of CAVD.Under physiological conditions,the aortic valve is subjected to cyclic bending stress,pressure,and cyclic tensile stress,while being exposed to a complex environment containing inflammatory cytokines and macrophages in the blood.Existing evidence indicates that mechanical stress can cause endothelial damage in the aortic valve,which in turn triggers lipid deposition,inflammatory cell infiltration,and interstitial cell transformation,ultimately leading to valve sclerosis and calcification.After valve sclerosis,the mechanical forces acting on the aortic valve,such as bending stress,pressure,and cyclic tensile stress,change,and the influence of pathological mechanical forces accelerates the process of valve calcification.However,the specific mechanism of how mechanical signals are converted into biological signals remains unclear.The mechanically sensitive ion channel Piezo1 can sense various mechanical forces,including stretch,membrane tension,cyclic pressure,shear stress,and others,subsequently affecting multiple signaling pathways and leading to changes in cellular function and phenotype,gene expression,and cellular behaviors such as proliferation,migration,apoptosis,and remodeling.In atherosclerosis,studies have shown that Piezo1 is associated with calcification deposition:additionally.Piezo1 also plays a crucial role in bone formation.Whether fluid dynamics can affect the occurrence and progression of calcific aortic valve disease(CAVD)through Piezo1 remains unexplored.Therefore,investigating this issue is of great significance for understanding the pathogenesis of CAVD in depth.Objectives:1.To construct a three-dimensional model and fluid dynamics simulation model of the aortic root using computed tomography(CT)to study the fluid dynamics changes around the aortic valve in patients with different types of CAVD.2.To explore the correlation between the mechanically sensitive ion channel Piezo1 and human CAVD valve calcification through the analysis of clinical specimens.3.To study the relationship between Piezo1 and the calcification process in human aortic valve interstitial cells using cell biology methods.Methods:1.Choose five AS patients scheduled for TAVI surgery with tricuspid aortic valve stenosis(TAV)and five AS patients with bicuspid aortic valve stenosis(BAV),and use healthy patients undergoing coronary CT angiography(CTA)as the control group(N=5).Gather their CTA photos,which should include optimum systolic and diastolic images.To create a three-dimensional model,import the obtained original CT medical pictures into Mimics Medical 21.0 program.The three-dimensional model should next be imported into 3-matic software for detail optimization processing.Then,with the ICEM CFD 19.2 software,mesh the model.Consistently apply the same AS inlet velocity boundary condition.For fluid dynamics simulation computations,use the finite volume approach CFD with ANSYS Fluent 19.2 software.For steady-state and transient simulations,use the k-epsilon turbulence model.Finally,import the results of the fluid dynamics simulation calculations into CFD Post 19.2 software for additional processing,obtaining distribution maps of vascular wall pressure and wall shear stress(WSS)during systole and diastole,as well as streamline diagrams of blood flow within the vascular lumen.2.AS patients undergoing SAVR surgery were chosen as the AS group(N=64),while organ donors were chosen as the control group(N=7).The aortic valve is harvested and immediately stored in formaldehyde solution and liquid nitrogen for histological and molecular biology studies.Simultaneously,split the same patient’s AS valve into valve calcification and non-calcification groups based on calcification distribution(N=14).Examine the variations in Piezo1,BMP2(Bone morphogenetic protein 2).BMP4(Bone morphogenetic protein 4).and TGF-β1(Transforming growth factor-β1)expression in tissue samples using qRT-PCR,Western blotting,and immunohistochemistry studies.3.Culture immortalized human aortic valve interstitial cells in calcification media based on-glycerophosphate.While setting up a blank control group,add Piezo1 agonist Yoda1 at doses of 1.5,10.and 100 μmol/L.and Piezo1 inhibitor GsMTx4 at concentrations of 1.5,and 10 μmol/L.Collect the cells after 7 days.Using qRT-PCR and Western blotting,examine the differences in Piezol,BMP2,BMP4,and TGF-β1 expression between groups.Results:1.The pressure at the aortic valve and ascending aorta steadily increased in the direction of blood flow during cardiac systole.The pressure was greater in the BAV and TAV groups than in the control group(14kPa>13.5kPa and 14kPa>13.5kPa,respectively),as was the pressure area range.Higher WSS appeared at higher places in the BAV and TAV groups compared to the control group,and the range of high WSS was greater.In addition,the BAV and TAV groups displayed greater turbulence and increased flow velocities.During cardiac diastole,the pressure along the direction of blood flow decreased progressively.The pressure was somewhat lower in the BAV and TAV groups than in the control group(1.06kPa<1.08kPa and 1.06kPa<1.08kPa.respectively),but the pressure distribution and range were comparable among the three groups.There were no significant differences between the three groups in terms of WSS at the aortic valve and ascending aorta.Nevertheless,compared to the control group,the BAV and TAV groups exhibited greater blood flow velocity and disordered blood flow streamlines.2.The relative mRNA expression and protein expression of BMP2,BMP4,and TGF-β1 in the aortic valves of AS group patients were substantially higher than in the control group(P<0.05).Similarly,the relative mRNA expression and protein levels of Piezo 1 were higher in the AS group’s aortic valves than in the control group(P<0.05),which was consistent with the expression of BMP2,BMP4,and TGF-β1.In the same AS patients’ aortic valve specimens,the relative mRNA expression and protein expression of BMP2,BMP4,and TGF-β1 were higher in the calcified area than in the non-calcified area(P<0.05).Similar to the expression of BMP2,BMP4,and TGF-β1,the relative mRNA expression and protein expression of Piezo 1 in the calcified area were higher than in the non-calcified area(P<0.05).3.mRNA and protein were isolated from the interstitial cells of each group’s aortic valve.Piezo 1 mRNA relative expression and protein expression in groups treated with various dosages of Yodal agonist and GsMTx4 inhibitor did not differ significantly from the control group(P>0.05).In contrast,when the concentration of Yodal agonist was 1,5,or 10 μmol/L,the relative mRNA expression and protein expression of BMP2,BMP4,and TGF-β1 rose significantly(P<0.05),with the tendency being more apparent with increasing concentration.However,when the concentration of Yoda1 agonist reached 100 μmol/L,there were no significant alterations in the relative expression of mRNA and protein compared to the control group(P>0.05).The relative mRNA expression and protein expression of BMP2,BMP4,and TGF-β1 in groups treated with various dosages of GsMTx4 inhibitor did not differ significantly from the control group(P>0.05).Conclusion:1.Hemodynamic changes occur around the aortic valve in CAVD patients,characterized by increased flow velocity,WSS,and pressure.2.Through human specimen studies,the mechanical-sensitive ion channel Piezo 1 is confirmed to be correlated with human aortic valve calcification.3.From a cellular perspective,Piezo 1 is involved in the calcification process of human aortic valve interstitial cells,possibly by affecting the BMP/TGFβ signaling pathway in aortic valve interstitial cells,leading to CAVD.