Effect Of JPH2 On A Calcium-activated Potassium Current In Mouse Cardiac Myocytes

1 Backgrounds and Objectives:Small-conductance calcium-activated potassium channels(SK, Kca2) expresse in almost all of the excitable cell’s membrane. The SK2 channel is an important kind of ion channels which exist in the mammal’s hearts and plays a key role on the repolarization of cardiac action potentials(AP), especially in the late phrase of repolarization. Evidences have shown that, SK2 channel is activated by intracellular Ca2+ linked with calmodulin, which results in the channels conformation of the SK channels. Both Ca2+ influx through voltage-gated Ca2+ channels VGCC) on the cell membrane and Ca2+ release from endplasmic/saroplasmic reticulum(ER/SR) through ryanodine receptors(Ry Rs) can modulat the function of the SK2 channel.The function crosstalk of ion channels between SR and surface membranes is an important feature of the excitable cells, and the junctional membrane complexes(JMCs) between them is a structure foundation of effective crosstalk between surface and SR membranes. Junctophilin(JP,JPH)is a protein involved in the formation of JMCs. Its main function is set a relatively fixed distance between cytoplasmic membrane and SR membrane and provides a basic structure of signal transduction between them. In the mammals, JP proteins were encoded by at least four kind of gene: JPH1, JPH2, JPH3 and JPH4. JPH2 protein plays a potential crucial role of adjusting the excitation-contraction coupling of the myocardial cells. Evidences have showed that JPH2 protein can be decisive of affecting the spared distance between L-VGCC on the cytoplasmic membrane and Ry Rs on SR.Our previous study confirmed that both JPH2 and the SK2 protein are expressed in mouse cardiac tissue. An immuno complex between JPH2 and the SK2 channel was present in the mouse cardiac muscle by co-immunoprecipitation,suggesting that there may be a interaction between the JPH2 protein and the SK2 channel in the native cardiac tissue. Nest, we reported that the levels of JPH2 m RNA and protein in the neonatal mouse cardiomyocytes(NCMCs) treated with Lentivirus-mediated sh RNA targeted against JPH2 gene depressed. Moreover, our data showed that knockdown of JPH2 decreased the level of the SK2 protein in the NCMCs infected with Lentivirus-mediated specific sh RNA targeted against JPH2. We wonder that if JPH2 knockdown affects the functional role of the SK2 channel.In this present study we tried to interfeirn the expression of JPH2 in the NCMCs using RNAi technique and documented the role of JPH2 knockdown on the SK2 channel mediated K+ current in the NCMCs using whole cell patch clamp technique.2 Materials and Methods: 1) 1~3 day male and female C57BL/6 mice were obtained from Beijing Weitonglihua experiment limited company(No. SCXK(Jing)2012-0001). 2) The targeting sequences(19nt sh RNA fragment) specific for JPH2 m RNA used in our laboratory was insert into psi AD-h U6-EGFP vector to constructe the recombinant adenovirus vectors expressed JPH2-sh RNA. The PCR and positive cloning sequencing were used to identify the recombinat vectors The recombinant adenovirus vectors were packaged and amplified in the 293 AT cells to generate p AD-JPH2-si RNA. 3) Culture cardiac myocytes isolated from neonatal mice were divided into non-specific control(AD-NT), nontransfected control(Ctrl-NC)and the adenovirus infection groups(AD-JPH2-si RNA). After the infection of 48 hours, the infective efficiency with green fluorescent cells was surveied by flow cytometry analysis. 4) The expression of the JPH2 protein and SK2 protein and the recording of the calcium-activated potassure current( Ik,Ca) in the neonatal mouse cardiomyocytes transduced with the adenovirus mediated sh RNA targeted against JPH2 were determined by Western blot and whole cell patch clamp techniques,separetely.5) Statistical analysis: the data were performed using SPSS17.0 software and the difference among different groups was measured with ANOVA analyses. α=0.05 was considered statistically significant. 3 Results: 1) The recombinant adenovirus vectors carried sh RNA targeting against JPH2 were successfully constructed by PCR and sequencing. 2) When MOI valu of infection is 100, the infection efficiency of the neonatal mouse cardiac myocytes infected with AD-NC and AD-JPH2-si RNA vectors is 76.9%、75.4% by Flow cytometry analysis. 3) Expression of SK2 channel in the AD-JPH2-si RNA cells was obvious inhibited compared with the AD-NC cells by Western blot analysis. 4) The SK2 channel mediated Ik,Ca in the neonatal mouse cardiac myocytes infected with the AD-JPH2-si RNA vectors was decreased compared with the AD-NC cells using whole cell patch clamp technique. 4 Conclusion:Culture the neonatal mouse cardiac myocytes were infected the recombinant of the adenovirus mediated sh RNA specific for JPH2. Knockdown of JPH2 in the neonatal mouse cardiac myocytes infected with the recombinant adenovirus vectors suppressed the expression of the SK2 protein and the SK2 channel mediated Ik,Ca.