Effects And Mechanism Of LRRC8A On Cardiac Hypertrophy Induced By Angiotension Ⅱ

Cardiac hypertrophy is an adaptive response of the heart to the overload caused by physiological or pathological stimulation,which is mainly manifested in the increase of myocyte volume to enhance myocardial contractility.However,long-term cardiac hypertrophy can lead to myocardial ischemia,cardiac systolic and diastolic dysfunction,and eventually develop into heart failure.Therefore,it is of great significance to explore the new mechanism of cardiac hypertrophy and find new targets for intervention.Volume regulated anion channel(VRAC)is widely distributed in various mammalian cells.Its main function is to regulate the volume of cells,and it also participates in the physiological proliferation of various cells and the pathological hypertrophic process of adipocytes.However,due to the unclear molecular structure and lack of specific blockers,the relationship between VRAC and cardiac hypertrophy has not been clarified.In recent years,it has been found that leucine rich repeat containing protein 8A(LRRC8A)is the main structural component of VRAC,which lays a foundation for the study of the relationship between VRAC and cardiac hypertrophy.What role does LRRC8A play in cardiac hypertrophy?Angiotensin Ⅱ(AngⅡ)is an important humoral factor that promotes the development of cardiac hypertrophy.It can induce cardiac hypertrophy through many ways,among which the nicotinamide adenine dinucleotide phosphate(NADPH)oxidase and reactive oxygen species(ROS)are the key signaling pathways.It is reported that NADPH oxidase and ROS are also involved in the activation of VRAC,but the mechanism is still unclear.So,what is the relationship between LRRC8A and NADPH oxidase and ROS in cardiomyocytes?Is LRRC8A involved in AngⅡ-induced cardiac hypertrophy by regulating NADPH oxidaseROS pathway?Based on the above scientific problems,this study intends to observe the effect of LRRC8A on AngⅡ-induced cardiac hypertrophy animal and cell models,and further explore the role of NADPH oxidase ROS pathway in them.Objectives1.To investigate the role of LRRC8A in AngⅡ-induced cardiac hypertrophy.2.To explore the relationship between LRRC8A and NADPH oxidase-ROS pathway.Methods1.Experimental group:cell experimental group:NC+PBS group,NC+AngⅡ group,shLRRC8A+PBS group,shLRRC8A+AngⅡ group.Animal experimental groups:NC+saline group,NC+AngⅡ group,shLRRC8A+saline group,shLRRC8A+AngⅡgroup.2.Primary cardiomyocytes were cultured and the model of cardiomyocyte hypertrophy was constructed by AngⅡ.3.The mouse model of cardiac hypertrophy was established by continuous perfusion of AngⅡ with a micro pump.4.Cardiomyocytes were infected adenovirus carrying shLRRC8A to knock down LRRC8A.5.The adeno-associated virus carrying shLRRC8A was injected into the myocardium of mice to knockdown LRRC8A.6.The area of cytoskeleton was detected by immunofluorescence staining.7.The mRNA levels of ANP,β-MHC and LRRC8A were detected by real-time quantitative PCR.8.HE staining and WGA staining were used to detect the cross-sectional area of myocardial cells.9.Masson staining was used to detect the area of myocardial collagen fibers.10.Echocardiography was used to detect the changes of cardiac function in mice.11.ROS level was detected by DCFH-DA staining.12.The activity of NADPH oxidase was determined by a lucigenin-enhanced chemiluminescence assay.13.Whole cell patch clamp technique was used to detect the changes of VRAC current in cardiomyocytes.14.The membrane protein and cytoplasmic protein of cardiomyocytes were extracted by the technology of cell membrane separation and extraction.15.Western blot was used to detect the expression of LRRC8A,Nox2,Nox4,p22phox,p67phox and p47phox.16.The localization of LRRC8A,Nox2,Nox4,p22phox was detected by immunofluorescence staining.17.Immunocoprecipitation was used to detect the coprecipitation of LRRC8A with Nox2,Nox4,p22phox.18.Lentivirus carrying normal LRRC8A and mutant LRRD(LRRC8A Δ91/+35)infected cardiomyocytes to overexpress LRRC8A and LRRC8A α91/+35.Results1.Established models of cardiomyocyte hypertrophy induced by AngⅡ:after 1μM AngⅡstimulated primary cardiomyocytes for 48h,the area of cardiomyocytes and the mRNA expression of ANP and β-MHC were increased significantly(P<0.05).2.AngⅡ increased the expression of LRRC8 A in cardiomyocytes:different concentrations of AngⅡ stimulated primary cardiomyocytes at different times,LRRC8A protein and mRNA expression increased in concentration and time-dependent manner(P<0.05,n=6).3.Established the animal model of cardiac hypertrophy induced by AngⅡ::after continuous perfusion of AngⅡ micropump in mice for 28 days,the cross-sectional area of myocardial cells and the mRNA expression of ANP and β-MHC increased significantly(P<0.05).4.The expression of LRRC8A increased in hypertrophic myocardium:the expression of LRRC8A protein and mRNA in myocardium increased significantly after continuous perfusion of AngⅡ micropump for 28 days(P<0.05,n=6).5.Knockdown of LRRC8A inhibited AngⅡ-induced cardiomyocyte hypertrophy:compared with NC+PBS group,the area and the mRNA expression of ANP and β-MHC of cardiomyocytes in NC+AngⅡ group increased significantly(P<0.05,n=6).Compared with NC+AngⅡ group,the area and the mRNA expression of ANP andβ-MHC of cardiomyocytes decreased significantly(P<0.05,n=6).6.Knockdown of LRRC8A inhibited AngⅡ-induced cardiac hypertrophy in mice:compared with NC+saline group,the cardiac volume,HW/BW,HW/TL,the mRNA expression of ANP and β-MHC,and the cross-sectional area of myocardium of NC+AngⅡ group was significantly increased(P<0.05,n=6).Compared with NC+AngⅡgroup,the cardiac volume,HW/BW,HW/TL,the mRNA expression of ANP and βMHC,and the cross-sectional area of myocardium of shLRRC8A+AngII group were significantly reduced(P<0.05,n=6).7.Knockdown of LRRC8A inhibited AngⅡ-induced myocardial fibrosis in mice:compared with NC+saline group,the area of myocardial fibrosis in NC+AngⅡ group increased significantly(P<0.05,n=6).,while compared with NC+AngⅡ group,the area of myocardial fibrosis in shLRRC8A+AngII group decreased significantly(P<0.05,n=6).8.The effect of LRRC8A knockdown on mouse cardiac function:there was no significant difference in EF%and FS%between the experimental groups(P>0.05,n=6);compared with NC+saline group,ⅣSd in NC+AngⅡ group increased significantly(P<0.05,n=6),while compared with NC+AngⅡ group,IVSd in shLRRC8A+AngII group decreased significantly(P<0.05,n=6).9.Knockdown of LRRC8A inhibited AngⅡ-induced ROS production:AngⅡ stimulation significantly increased the ROS level of cardiomyocytes and tissues(P<0.05),while compared with NC+AngⅡ group,the ROS level of shLRRC8A+AngII group significantly decreased(P<0.05).10.Knockdown of LRRC8A inhibited AngⅡ-induced NADPH oxidase activity:the activity of NADPH oxidase in cardiac myocytes and tissues was significantly increased by AngⅡ stimulation(P<0.05),while compared with NC+AngⅡ group,the activity of NADPH oxidase in shLRRC8A+AngII group decreased significantly(P<0.05).11.Knockdown of LRRC8A inhibited AngⅡ-induced the expression of NADPH oxidase membrane subunits:compared with NC saline group,the expression of Nox2,Nox4 and p22phox subunits in NC+AngⅡ group was significantly increased(P<0.05,n=6);compared with NC+AngⅡ group,the expression of Nox2,Nox4 and p22phox subunits in shLRRC8A+AngII group was significantly decreased(P<0.05,n=6).However,there was no significant difference in the expression of p67phox and p47phox subunits.12.Knockdown of LRRC8A inhibited AngⅡ-induced the translocation of NADPH oxidase cytoplasmic subunits:compared with NC+PBS group,the expression of p67phox and p47phox protein in cell membrane of NC+AngⅡ group increased significantly(P<0.05,n=6),the expression of p67phox and p47phox protein in the cytoplasm NC+AngⅡgroup decreased significantly(P<0.05,n=3);while compared with NC+AngⅡ group,the expression of p67phox and p47phox protein in cell membrane of shLRRC8A+AngII group decreased significantly(P<0.05,n=3),the expression of p67phox and p47phox in the cytoplasm of shLRRC8A+AngII group increased significantly(P<0.05,n=3).13.AngⅡ-activated VRAC current was dependent on LRRC8A and was regulated by NADPH oxidases and ROS production:200nM AngⅡ stimulated cardiomyocytes for 10 minutes producing a typical VRAC current,which can be inhibited by the VRAC inhibitor DIDS,ROS scavenger NAC,NADPH oxidase inhibitor apocynin(P<0.05,n=4).In addition,when the cardiomyocytes were infected with the adenovirus carrying shLRRC8A in advance to knock down LRRC8A,the typical VRAC current could not be generated by AngⅡ stimulation(P<0.05,n=4).14.Co-localization of LRRC8A and NADPH oxidase membrane subunits:immunofluorescence staining results showed that LRRC8A and Nox2,Nox4,p22phox were co-located.15.Co-precipitation of LRRC8A and NADPH oxidase membrane subunits:immunocoprecipitation showed that LRRC8A co-precipitated with NADPH oxidase membrane subunits Nox2,Nox4,p22phox.16.LRRC8A directly interacted with Nox2,Nox4,and p22phox via the LRRD:When wildtype LRRC8A were overexpressed in cardiomyocytes,it co-immunoprecipitated with Nox2,Nox4,and p22phox more efficiently compared to that in the control group.However,when LRRC8Aα91/+35 was overexpressed in cardiomyocytes,LRRC8A coimmunoprecipitated with Nox2,Nox4,and p22phox less efficiently than that in the control group.Conclusions1.The expression of LRRC8A is enhanced during AngⅡ-induced cardiac hypertrophy.2.Knockdown of LRRC8A inhibits AngⅡ-induced cardiac hypertrophy3.LRRC8A contributes to AngⅡ-induced cardiac hypertrophy by regulating NADPH activity and ROS production through interacting with NADPH oxidases via LRRD.