Effects Of Junctophilin-2 On The Coupling Of SK2 Channel And RyR2 Function In Mouse Myo Cardium Cells

1 Background and objectives:Junctophilin-2,encoded by the JPH2 gene,is a member of heart Junctophilins?JPs,JPHs?protein family which involves in the formation of membrane-coupled complexes,and it is necessary for intracellular Ca²⁺signal transduction,which is physically close to the plasma membrane L-type calcium channels and sarcoplasmic reticulum.JPH2 knockdown mice can cause death during their embryogenesis,irregular calcium treatment,heterotopias and random contractions which have nothing to do Ca²⁺ release from sarcoplasmic reticulum,suggesting that JPH2 plays a key role between promoting SR calcium release and cardiac contraction.Further studies have shown that JPH2 plays a decisive role in setting the spatial distance between the voltage-gated calcium channels of cell membrane and the ryanodine receptors of the sarcoplasmic reticulum.In myocardial hypertrophy models,the down-regulation of JPH2 is associated with a coupling defect between L-type Ca²⁺channels and RyR2 receptors.Small-conductance calcium-activated potassium channel?SK,Kca2?is non-voltage-dependent,which is almost expressible in all excitable cells.According to sensitivity to apamin,a specific blocking agent,Kca2 can be divided into four subtypes: SK1,SK2,SK3 and SK4.In most time,SK1 is only present in the nerve tissue,SK2 and SK3 exist in both the nerve tissue and the peripheral tissues.As a calcium-activated potassium channel,in cardiomyocytes,the SK2 channel is highly sensitive to calcium in the cytoplasm,and it can be activated by low concentration of calcium ions in cells.Thus,the SK2 channel plays an important role in the combination of Ca²⁺ concentration and the membrane potential changes,as well as the regulation of signaling pathways which contains Ca²⁺.The intracellular Ca²⁺ is mainly originated from two ways: depolarization-activated voltage-gated Ca²⁺channels?VGCCs?,and releasing from the sarcoplasmic / reticulum?SR / ER?by ryanodine receptors?RyRs?.Some studies on our laboratory have demonstrated thatthe RyR2-sensitive Ca²⁺release channel can regulate calcium-activated potassium currents in the cardiomyocytes,but the specific molecular mechanism of functional coupling between RyR2 and SK2 channels is not entirely clear.In cardiomyocytes,it is unclear whether JPH2 is a key molecule when coupling between the SK2 channels and the RyR2 receptors.In this study,JPH2 gene knockdown is transducted by adenovirus mediated RNA interference technology.After neonatal mouse's cardiomyocytes infected with recombinant adenovirus,whole-cell patch clamp technique is used to record the SK2 channel current in the voltage range of-120 mv to +60mv,then RyR2 agonist caffeine,inhibitors ryanodine and thapsigargin?TG?were added,so as to observe the effect of JPH2 on the functional coupling of RyR2 calcium release channels and SK2 channels in cardiomyocytes.Through these processes,we can provide a reliable experimental basis,so as to reveal the molecular mechanisms of the SK channels,and propose the prevention strategies of some cardiac diseases.2 Materials and Method:1)The mice used in the experiment were 1-3 days C57 BL / 6 male and female mice?certificate number: SCXX?Beijing?2012-0001?,and they were purchased in Beijing Weitong Lihua Experimental Animal Technology Co.,Ltd.2)The cultured neonatal mouse cardiomyocytes?NMCMs?were classified as:non infected normal control cardiomyocytes?Ctrl-NC?,cardiomyocytes infected with scrambled adenovirus?Ad-scramble siRNA?,and cardiomyocytes infected with adenovirus mediated JPH2 si RNA?Ad-JPH2-SiRNA?.3)Real-time PCR was used to detect the relative expressions of JPH2 mRNA in each group of cDNA samples.4)Calcium-activated potassium currents?Ik,Ca?in the NMCMs were recorded by whole-cell patch clamp.The cells were divided into three groups: non infected normal control,Ad-scramble siRNA,and Ad-JPH2-siRNA groups.To probe the role of JPH2 on the coupling of the SK2 to RyR2 in the cardiac myocytes,Ik,Ca in the NMCMs was also recorded using whole-cell patch clamp technique.The NMCMs were dividedinto 4 groups.They are Ad-scramble siRNA,Ad-JPH2-siRNA,Ad-JPH2-si RNA +ryanodine + TG,and Ad-JPH2-siRNA + caffeine groups.5)Statistical analysis: Statistical software SPSS 17.0 was used to analyze the data.The significant differences among the groups were compared by one-way ANOVA.Significant level is set as ?=0.05,and sample number is represented as “n”.3 Results1)The relative JPH2 mRNA in the cDNA samples of each group of cardiomyocytes was detected by Real-time PCR.There is no significant difference in the levels of JPH2 mRNA between Ctrl-NC and ad-scramble siRNA groups.Compared with the ad-scramble siRNA cells,the relative level of JPH2 mRNA in the NMCMs of Ad-JPH2-siRNA group was significantly decreased,and the interference efficiency was about 90%.2)The Ik,Ca currents were recorded by whole-cell patch clamp techanique,which showed the Ik,Ca in the Ad-JPH2-siRNA cardiomyocytes was significantly decreased compared with the Ctrl-NC and Ad-scramble siRNA groups.Compared with the Ad-scramble-siRNAgroup,the Ik,Ca in the Ad-JPH2-siRNA+ group decreased after administration of Ryanodine + TG.Although the Ik,Ca was tending downwards,no significant difference between Ad-JPH2-siRNA and Ad-JPH2-si RNA +Ryanodine +TG groups.The Ik,Ca in the Ad-JPH2-siRNA cellls after administration of Caffeine,a RyR2 receptor agonist,was decreased compared with Ad-scramble si RNA group,but the Ik,Ca in the Ad-JPH2-siRNA cellls by administration of Caffeine was increased compared with the NMCMs transduced with Ad-JPH2-siRNA.4 SummarysiRNA knockdown of JPH2 in mouse cardiomyocytes can significantly suppresed the expression of JPH2 mRNA in the cardiomyocytes and reduce the SK2 channel current through the regulation of RyR2 Ca²⁺ release channels,suggesting that JPH2 might mediate the functional coupling between SK2 and RyR2 in the cardiac myocytes.