Effect Of Volume Sensitive Outwardly Rectifying Cl~- Channel On Cardiomyocyte Hypertrophy Induced By Angiotensin Ⅱ

Cardiac hypertrophy is recognized as an adaptive response to long-term of cardiac overload,characterized by myocyte hypertrophy,cardiac fibroblasts proliferation and matrix remodeling.Although initially,cardiac hypertrophy plays role as a compensatory response that tries to optimize biomechanical stress and normalize cardiac pump function,prolonged hypertrophy may eventually lead to cardiac systolic and diastolic dysfunction,heart failure and even sudden death.Therefore,it is of great clinical significance to study the mechanism of cardiac hypertrophy.A large number of studies have shown that the renin-angiotensin-aldosterone system(RAAS)plays an important role in the formation and development of cardiac hypertrophy.Angiotensin II(Ang II)is the major effector of RAAS,which can induce cardiac hypertrophy through a variety of ways,in which reactive oxygen species(ROS)is one of the key signaling pathways.Volume-sensitive outwardly rectifying chloride channel(VSOR Cl-channel)are widely expressed in various mammalian tissues and cells.Although VSOR Cl-channel has been implicated in numerous physiological and pathological processes,such as cell proliferation,migration,differentiation,apoptosis and so on.It’s most important physiological function is to regulate cell volume.The VSOR Cl-channel is activatied by cell swelling,so that the water is accompanied by chlorine and other osmotic substances discharged from the cell to maintain the volume of cells,which is called regulatory volume decrease(RVD).Since the primary cellular basis of cardiac hypertrophy is anirreversible enlargement of the cardiac myocyte,there are some assumptions.Is the VSOR Cl-channel involved in the process of cardiomyocyte hypertrophy induced by Ang II? What role does it play? What is the mechanism?Based on these hypothesis,our present experiment was to establish Ang II-induced cardiomyocyte hypertrophy model to observe the changes of VSOR Cl-channel.This study also inhibited the function of VSORCl-channel by chloride channel blockers DIDS and knocking dwon the expression of LRRC8 A which is the structural protein of VSOR Cl-channel to explore the effect of VSOR Cl-channel on cardiomyocyte hypertrophy induced by Ang II and the possible mechanism.Objectives 1.To establish Ang II-induced cardiomyocyte hypertrophy model 2.To observe the effect of Ang II on the expression of LRRC8 A and on the current of VSOR Cl-channel.3.To investigate the effect of VSOR Cl-channel on cardiomyocyte hypertrophy induced by Ang II,and to further explore the mechanisms.Methods 1.The experimental groups were divided into control group,Ang II group,Ang II+si RNA group,Ang II+DIDS group,si RNA group and DIDS group,respectively.The cell area,the m RNA expression of hypertrophic gene,and the level of ROS and the activity of NADPH oxidase were detected.2.The cultured rat cardiomyocyte cell line(H9C2)were stimulated by Ang II to induce cardiomyocyte hypertrophy.3.Phalloidin immunofluorescence staining for cell surface area detection.4.The m RNA levels of ANP、β-MHC and LRRC8 A were detected by RT-q PCR assay.5.The expression level of LRRC8 A protein was detected by the Western-blot technique.6.VSOR Cl-current of cardiiomyocytes was detected by whole-cell patch clamp recording technique.7.Adenovirus containing si RNA targeting to LRRC8 A was transfected to knockdown LRRC8 A expression.8.The intracellular ROS levelwas detected by DCFH-DA staining.9.NADPH oxidase activity was deteced by active reagent kit.Results1.According to the classic concentration,cardiomyocyte were stimulated by 10-6mol/L Ang II at 0h,12 h,24h,36 h,48h and 72 h respectively.The cell area and the m RNA level of hypertrophic gene ANP,β-MHC were elevated in a time-dependent manner.The cellarea reached the maximum at 48h(P<0.05,n=100),and the m RNA level of ANP and-MHC reached the maximum at 36h(P<0.05,n=6).2.Ang II can increase the m RNA and protein expression of LRRC8 A in a concentration and time dependent manner.The m RNA and protein expression of LRRC8 A reached the maximum after 10-6mol/L Ang II stimulating 36 h and 48 h respectively.(P<0.05,n=6;n=3).3.The m RNA and protein expression level of LRRC8 A could be effectively reduced by adenovirus transfection for 72 hours,and the most effective sequence from four candidate sequence was screened out.4.Under isotonic conditions,20μmol/L Ang II can activate VSOR Cl-current after stimulating 10 minutes,which can be inhibited by knockdown of LRRC8 A.(P < 0.05,n=4)5.Effects of DIDS and Knockdown of LRRC8 A on Ang II-induced hypertrophy:the results of immunofluorescence staining showed that compared with the control group,the cell area of Ang II group increased significantly(P < 0.05,n=100);compared with the Ang II group,the cell area of Ang II+DIDS group and Ang II+si RNA group was significantly reduced(P < 0.05,n=100).RT-q PCR results showed that compared with the control group,the expression of ANP and β-MHC mRNA of Ang II group were significantly increased(P< 0.05,n=6);compared with Ang II group,the expression of ANP and β-MHC m RNA of Ang II+DIDS group and Ang II+si RNA group was significantly reduced(P < 0.05,n=6).6.The DCFH-DA staining results showed that compared with the control group,the content of ROS of Ang II group was significantly increased(P<0.05,n=5);compared with the Ang II group,the content of ROS of Ang II+DIDS group and Ang II+si RNA group was significantly reduced(P< 0.05,n=5).The content of ROS in Ang II+si RNA group was lower than that in Ang II +DIDS group.7.The results of NADPH oxidase activity showed that compared with the control group,the NADPH oxidase activity of Ang II group was significantly increased(P = 0.05,n=5);compared with the Ang II group,the NADPH oxidase activity of Ang II+si RNA group was significantly reduced,and there was no difference in Ang II+DIDS group(P> 0.05,n=5).Conclusions 1.The Ang II-induced cardiomyocyte hypertrophy model was successfully established,which laid the foundation for further study.2.Ang II can increase the m RNA and protein expression of LRRC8 A in a concentration and time dependent manner,and Ang II can activate the VSOR Cl-current.3.Block of VSOR Cl-channel by DIDS and knockdown of LRRC8 A can inhibit the cardiomyocyte hypertrophy induced by Ang II 4.Knockdown of LRRC8 A can reduce ROS produced by NADPH oxidase,which may be the mechanism of inhibition of cardiomyocyte hypertrophy induced by Ang II.