Effects Of JP2 On SK2 Channel Currents In Angiotensin Ⅱ-induced H9c2 Hypertrophic Cardiomyocyte

BachgroundMyocardial hypertrophy is an adaptive response of the heart to myocardial contractility and cardiac function under pressure overload,including physiological and pathological cardiac hypertrophy.The physiological hypertrophy refers to the heart’s physiological stimulation caused by long-term exercise,pregnancy and other activities;the pathological cardiac hypertrophy is an adaptability of myocardial contractility and cardiac function when chronic pathological stimulation such as hypertension,myocardial infarction,gene polymorphism,diabetes,etc.As the progresses of pathological myocardial hypertrophy,ventricular muscles can undergo structural remodeling,ion channels remodeling（electrical remodeling）,and Ca²⁺remodeling.The cardiac function gradually becomes decompensated,leading to cardiovascular adverse events.Among them,electrical remodeling of myocardium makes hypertrophic myocardium prone to early and late post-depolarization,leading to the occurrence of malignant arrhythmia and sudden death.Therefore,it is urgent to explore the electrical remodeling of pathological myocardial hypertrophy and the interaction between various factors,and to find effective prevention and treatment strategies.The small conductance calcium-activated potassium channel（SK）is is widely expressed in excitable cells of mammals,and it has the characteristics of high K⁺selectivity,high sensitivity to Ca²⁺,and non-voltage dependence.According to different coding genes,SK channels can be divided into four subtypes:SK1,SK2,SK3,and SK4,SK2 and SK3 can be detected in myocardial tissue.Studies have shown that the SK2 channels are mainly expressed in atrial myocytes under physiological conditions,participate in the repolarization of action potential.SK2channels are up-regulated in myocardial hypertrophy and heart failure,especially in the ventricular,but the mechanism of upregulation of SK2 expression in the ventricle is not clear.In the preliminary work of our laboratory,it has been proved by immunoprecipitation and pull-down experiments that mouse cardiomyocytes Junctophilin-2（JPH2,JP2）interacts with SK2.Cell immunofluorescence experiments also show that JP2 and SK2 are co-localization in atrial myocyte,ventricular myocyte and transfected HEK293 cells.Knocking down JP2 gene induced a significant decrease in the SK2 channel currents,and the peak value of calcium transients was also significantly reduced,indicating that JP2 may affect the SK2 channel currents by intracellular Ca²⁺levels.Junctophilins（JPs） are the protein expressed in the coupled membrane complex of excitable cells such as striated muscle and neurons,bridging the subcellular space between the plasma membrane and the endoplasmic reticulum/sarcoplasmic reticulum（ER/SR）.JPs can be divided into four subtypes:JP-1,JP-2,JP-3,and JP-4.JP2,which is mainly located in the heart muscle and skeletal muscle,can regulate the intracellular Ca²⁺homeostasis and maintain the horizontal tube structure.Studies have shown that the expression of JP2 in the heart is down-regulated in patients with myocardial hypertrophy and heart failure.The animal models of cardiac hypertrophy also show a decrease in JP2 expression,the destruction of ordered T-tube structure,a decrease in Ca²⁺transients,and excitement-contraction coupling weakened;and specific knockdown of JP2 in mouse hearts can lead to myocardial hypertrophy and acute heart failure;in addition,studies have identified four JP2 mutants susceptible to hypertrophic cardiomyopathy,and the mutation sites are S101R,Y141H,S165F and A405S.Therefore,studies have shown that the expressions of SK2 and JP2 are changed during myocardial hypertrophy,so whether there are relationships between the reconstruction of the two has not yet been studied.ObjectiveIn the study,we observed whether there is any change in the SK2 channel currents and JP2 knockdown and overexpression affected the SK2 channel current in H9c2 cardiomyocyte hypertrophic induced by angiotensin Ⅱ.To detect the effect of JP2 on the function of SK2 channel in myocardial hypertrophy in vitro,which was going to provide a theoretical basis for exploring the molecular mechanism of pathological cardiac hypertrophy and electrical remodeling.Methods1.The target sequence of JP2 m RNA was designed to prepare JP2 overexpressed recombinant adenovirus expression vector;si RNA targeting JP2 knockdown has been used and survived freezing in the laboratory for a long time.2.JP2 protein expressions in H9c2 cells transfected with adenovirus vector were detected by Western bloting.3.Passaged for 24 hours,H9c2 cells were added angiotensin Ⅱ to build a cell model of ventricular myocyte hypertrophy.After 24 hours of culture,the cells were observed and measured cell area under an inverted microscope,then the levels of B-type natriuretic epeptide（BNP）expressions were determined by Western bloting.4.The experiment was divided into six groups:A:Control group:normal H9c2 cells served as blank control group;B:Ang Ⅱ hypertrophic group:angiotensin Ⅱ induced hypertrophic H9c2 cell group;C:Ang Ⅱ+Ad-NC-si JP2 group:knockdown negative control group,hypertrophic group cells transfected with JP2 knockdown negative adenovirus vector;D:Ang Ⅱ+Ad-si JP2 group:hypertrophic group cells transfected with JP2knockdown adenovirus vector;E:Ang Ⅱ+Ad-NC-JP2OE group:hypertrophic group cells were transfected with JP2 overexpression negative adenovirus vector,as overexpression negative control group;F:Ang Ⅱ+Ad-JP2OE group:hypertrophic group cells transfected with JP2overexpressing adenovirus vector;5.SK2 channel currents of the transfected cells observed the green fluorescence were recorded using whole-cell patch clamp.Results1.PCR and positive clone sequencing confirmed the successful construction of JP2 overexpressing adenovirus vector.2.The levels of JP2 expression were no significant difference in Control group,Ad-NC-si JP2 and Ad-NC-JP2OE group,and the expressions of JP2 protein in Ad-si JP2 group were significantly lower than that in Ad-NC-si JP2 group（P=0.0289）.The JP2 protein expressions in the Ad-JP2OE group were significantly higher than that in the Ad-NC-JP2OE group（P=0.0001）.These results suggested that the adenovirus vector was successfully transfected into H9c2 cells,and it effectively knocked down and overexpressed JP2 in H9c2 cells.3.The H9c2 cells treated with 10^-7mol/L angiotensin Ⅱ were significantly larger under inverted microscope,and the BNP protein expressions in hypertrophic cells were significantly increased（P=0.0161）,indicated that the hypertrophic H9c2 cell model was successfully constructed.4.The results of all current-voltage relationship curves showed the apamin-sensitive SK2 channel currents possessed inward rectification characteristics.Apamin-sensitive SK2 channel currents in Ang Ⅱ-induced hypertrophy group H9c2cells was significantly greater than in control group;knockdown of JP2 expression could significantly increase apamin-sensitive SK2 channel currents in hypertrophic H9c2 cell;and overexpression of JP2 significantly reduced apamin-sensitive SK2channel currents in H9c2 cells.ConclusionSK2 channel currents were significantly increased,and JP2 expression levels could affect the SK2 channel currents in Ang Ⅱ-induced hypertrophic H9c2 cells.