The Regulation Of Junctophilin 2 On The Expression Of Small-Conductance Ca²⁺-Activated K⁺Channels In Hypertrophic Heart

Background and ObjectiveAt present,cardiovascular diseases is still the main cause of death for Chinese residents.A lot of heart diseases,such as valvular disease,hypertension,and dilated cardiomyopathy,can be complicated with cardiac hypertrophy.Cardiac hypertrophy is initiated as an adaptive response to pressure or volume overload in order to maintain myocardial contractility and cardiac function.The sustained load will cause myocardial structural remodeling,ion channel remodeling（or electrical remodeling）and intracellular Ca²⁺remodeling,and ion channel remodeling is an important factor leading to the complication of with malignant arrhythmia and sudden death from pathological cardiac hypertrophy.Therefore,there is important academic value and scientific significance to reveal the molecular mechanism of myocardial hypertrophy electrical remodel and explore the interaction between various molecules,by doing which can effectively delay or reverse the pathological process of myocardial hypertrophy and reduce the occurrence of these cardiovascular adverse events.Small-conductance calcium-activated potassium channel subtype 2（SK2）is widely expressed in excitable tissues and is Ca²⁺-dependent rather than voltage-dependent.Some studies have shown that under physiological conditions,SK2channels are involved in the action potential（AP）repolarization of atrial myocytes,and are downregulated and remain dormant in ventricular myocytes.But the levels are elevated and functionally enhanced in certain heart diseases,such as cardiac hypertrophy,heart failure,and myocardial infarction.Our lab recently reported that Junctophilin 2（JP2）,which is located in the membrane,directly interacted with SK2 channels,and the recognition nexus（MORN motifs）in its N-terminus directly regulates the function and trafficking of SK2channels.JP2 expressed in cardiomyocytes provides a bridge for the signal transduction in excitable cells between plasma membrane ion channels and intracellular calcium release channels by anchoring the endo/sarcoplasmic reticulum（ER/SR）to the plasma membrane/T-tubules.Thus,it can improve the excitability of the myocardium,and facilitate the excitation-contraction coupling.There are the reduced JP2 expression in heart,structure destruction of the T-tube,a decreased Ca²⁺transient amplitude,and the weakened excitation-contraction coupling in pressure-overloaded myocardial hypertrophy,hypertrophic cardiomyopathy and heart failure patients.So,SK2 and JP2 remodeling can be present in certain cardiac pathological condition,such as cardiac hypertrophy.It has not been reported whether JP2 is involved in the expressed and functional remodeling of SK2 channel under pathological conditions.In this study,we plan to observe the expression of SK2channel and JP2 proteins in hypertrophic heart induced by transverse aortic constriction（TAC）mice and angiotensin II（Ang II）-induced hypertrophic cardiomyoblast in vivo and in vitro.The effect of JP2 expression on SK channel remodeling in cardiac hypertrophy will be investigated through knockdown or overexpression of JP2.These results are going to state the molecular mechanism of SK2 channel remodeling in cardiac hypertrophy theoretically,which will provide an important experimental basis for in-depth research of the occurrence and development of pathological cardiac hypertrophy mechanisms and prevention strategies.Methods1.To detect the expression and remodeling of SK2 channel and JP2 protein in H9c2 hypertrophic cardiomyoblastH9c2 cells were incubated with Ang II for 12,24,48 and 72 hours at different concentrations.The cell surface areas were measured using Image-Pro Plus 6.0software and natriuretic epeptide（BNP）protein expression was measured by western blot to determine concentration and time of Ang II.Western blot was used to test the expression of SK2 channel and JP2 protein in Ang II-induced H9c2 hypertrophic cardiomyoblast.2.To explore the effect of JP2 on the expression remodeling of SK2 channel in hypertrophic H9c2 cardiomyoblastWestern blot and RT-q PCR were used to detect the effects of knockdown and overexpression of JP2 on the expression levels of SK2 protein and m RNA in H9c2hypertrophic cardiomyocytes.H9c2 cells were divided into six groups:Control group（normal H9c2 cardiomyocytes）;AngⅡgroup（AngⅡinduced H9c2 hypertrophic cardiomyocytes）;AngⅡ+Ad-NC-si JP2 group（adenoviruses carrying negative control si RNA infected with H9c2 hypertrophic cardiomyocytes）;AngⅡ+Ad-NC-JP2OE group（adenoviruses carrying negative control JP2 overexpresing infected with H9c2 hypertrophic cardiomyocytes）;AngⅡ+Ad-si JP2（H9c2 hypertrophic cardiomyocytes were infected with adenovirus expressing knockdown JP2）;AngⅡ+Ad-JP2OE group（H9c2 hypertrophic cardiomyocytes were infected with JP2-overexpressing adenovirus vector）.3.To evaluate the changes of JP2 and SK2 in pressure overload-induced cardiac hypertrophyC57BL mice were used to construct a pressure-overloaded cardiomyocyte hypertrophy mouse model by TAC surgery.M-mode echocardiography,HE staining and Masson staining were used to detect the cardiac function,structure and myocardial tissue changes at 4 weeks post-TAC.To analyze SK2 and JP2 expressions in mouse myocardial tissue after aortic constriction,Western blotting was used.4.To determine the effects of JP2 protein on cardiac structure,function and SK2 channel expression in cardiac hypertrophy miceAAV9-mediated JP2 overexpression vector（AAV9-JP2OE）or negative control virus（AAV9-con）were delivered to the mice through tail vein injection,2w after TAC.M-mode echocardiography,HE and Masson staining were performed on the mice after two weeks to evaluate cardiac structure and function,and the levels of JP2and SK2 proteins were detected by Western blot.Result1.JP2 expression was decreased and SK2 channel protein expression was increased in Ang II-induced H9c2 hypertrophic cardiomyoblastThe areas of H9c2 cells treated with AngⅡfor 10^-8,10^-7and 10^-6mol/L were significantly increased,but the effect of 10^-7mol/L AngⅡwas the most significant after 48 hours.Compared with the control group,the expression level of BNP in Ang II-induced H9c2 cells increased significantly,P value was 0.0142.In subsequent experiments,H9c2 hypertrophic cardiomyocytes were prepared with 10^-7mol/L Ang II for 48 hours.The expression level of SK2 increased（P=0.0282）,but the expression level of JP2 decreased（P=0.0204）in hypertrophic cardiomyoblasts compared with the control group.2.JP2 may regulate the expression of SK2 in H9c2 hypertrophic cardiomyocytes by affecting synthesisThe levels of JP2 in H9c2 cells transfected with Ad-si JP2 and Ad-NC-JP2OE were successfully interfered.Knockdown of JP2 expression in H9c2 hypertrophic cardiomyocytes can significantly up-regulate the SK2 protein levels,compared with AngⅡgroup,P value is 0.0038,compared with AngⅡ+Ad-NC-si JP2 group,P value is 0.0033.Compared with AngⅡgroup and AngⅡ+Ad-NC-JP2OE group,JP2 overexpression decreased the expression level of the SK2 channel in H9c2hypertrophic cells（P=0.0134,P=0.0398）.At the same time,the changes trend of SK2 m RNA levels in H9c2 hypertrophic cells with JP2 overexpression and knockdown was also similar to the changes of protein levels.These results reveal that JP2 can affect the SK2 protein expression by affecting m RNA synthesis.3.Pressure-overload induced cardiac hypertrophy increased SK2expression and decreased JP2 expression levelsRepresentative results of the M-mode echocardiography,the heart weight/body weight（HW/BW）,lung weight/body weight（LW/BW）and heart weight/tibial length（HW/TL）ratios were shown that ejection fraction（EF）and fractional shortening（FS）suggested that the systolic function,ejection,wall thickening and weight of heart and lung increased 4 weeks after TAC,suggesting that the heart hypertrophy appeared,and the model of pressure overload myocardial hypertrophy mice was successfully prepared.The level of JP2 protein significantly reduced in TAC group,and the level of SK2 protein increased significantly.The expression of JP2 and SK2 are remodeling,which is consistent with the results of H9c2hypertrophic cardiomyocytes in vitro.5.AAV9-mediated JP2 overexpression improved cardiac function and decreases SK2 expression in cardiac hypertrophy miceTAC mice injected with AAV9-JP2OE were detected increased EF and FS compared with the control group（TAC+AAV9-con）,and the P values were 0.0003and 0.0001,respectively.In addition,the internal dimension at end-diastole and left ventricular posterior wall thicknes were also significantly shorter compared to AAV9-con group（P=0.0416,P=0.0482）.The degree of cardiac fibrosis in TAC mice was reduced,and the collagen volume fraction reduced by about 46%compared with the TAC+AAV9-con group.The results suggest that JP2 overexpression can alleviate heart expansion,weaken the degree of fibrosisand improve the left ventricular function.The relative expression of SK2 in mice with myocardial hypertrophy after injection with AAV9-JP2OE were significantly lower than that of the mice injected with saline（P=0.0367）and with the AAV9-con（P=0.0035）.These results show that JP2 regulate the expression of SK2 channel in the pathological process of myocardial hypertrophy in mice in vivo.ConclusionThe expression levels of SK2 and JP2 are changed in hypertrophic heart induced by TAC and Ang II-induced H9c2 hypertrophic cardiomyoblast.JP2 expression can regulate the remodeling of SK2 channel protein expression in cardiac hypertrophy by affecting the synthesis of protein.AAV9-mediated JP2 overexpression is found to improve cardiac remodeling and function in cardiac hypertrophy mice.