The Long-term Inhibitory Effects Of Bepridil On Inward Rectifier Potassium Current

Objective:Atrial fibrillation is the most common persistent arrhythmia.The incidence of atrial fibrillation are continuously increasing with the growth of age,over the age of 75 it may up to 10%.I_K1 is the important regulators of myocardial excitability and arrhythmia,and can be used as the new approach of anti-arrhythmic.It has been clarified that I_K1was increased significantly in the patients with atrial fibrillation.With the increase of I_K1,the depolarization of membrane potential was accelerated,while the sodium current activity was increasing,and also shorten the process to the action potential,resulting in accelerated exhumation and stabilization.Bepridil is a calcium channel blocker,first as antiarrhythmic agent with anti angina effect.The current clinical studies show that long-term outcome of hydrochloride bepridilbepridil can effectively reverse chronic and refractory atrial fibrillation,and maintain sinus rhythm.Based on these ideas,we conclude that the long-term outcome of hydrochloride bepridil can reduce I_K1,prolong the action potential and promote the early termination of reentry,and achieve the therapeutic effect of effectively reversing atrial fibrillation and maintaining sinus rhythm.Methods:1 Isolate neonatal rat cardiomyocytesFirstly,1–3-day-old SD rats's heart was resected as described previously studies,the cardiomyocytes were obtained using collagenase II digestion technology[Wang Y.J Mol Cell Cardio 2007].Isolated cardiomyocytes were cultured in Dulbecco's modified Eagle's medium?DMEM??Sigma,USA?,at37°C in a 95%O₂-5%CO₂ incubator.Treatment group and control group?on time dependence?,patch-clamp recordings,molecular biology and biochemical studies were performed,respectively.2 Patch clamp techniqueWhole-cell patch clamp technique was used to record the isolated ventricular myocytes I_K1 current?voltage clamp mode?.75-mm plain capillary tubes preparation,and were achieve the pipette resistance at 2-4 M?when filled with the pipette solution.I_K1current was elicited from a holding potential of-40 mV to potentials ranging from-120 to+30 mV in 10-mV increments.All the current measurements were done at room temperature?20-24°C?.For the whole-cell I_K1current recording,the chamber was filled with bath solution contained?mM?:Na Cl 140,KCl 5.4,MgCl₂ 1,CaCl₂ 1.8,HEPES 10,HEPES 10,Glucose 10,4-AP 5,chromanol 293B?10?M?,CdCl₂0.3,blocked I_to,I_Ks,and I_Ca,L,respectively at pH 7.4.electrode internal solution?mM?:KCl 140,MgCl₂ 1,EGTA 10,HEPES 10,Mg-ATP 5,pH 7.2.3 Real-time PCR analysis2?l of cDNA as a template,repeat with reverse transcription as a sample,the transcripts were divided and then used.The real-time PCR was performed on SYBR green?Roche?.KCNJ2 primers,F 5'-TGCCCGATTGCTGTTTTC-3'and R5'-GGCTGTCTTCGTCTATTT-3';Caveolin-1,F 5'-GGGCATCTACTT TGCCATCCT-3'and R 5'-CGGCTGATGCACTGAATCTC-3';Caveolin-3,F5'-GAAGATGTGATTGCGGAGCC-3'and R 5'-TGGAGACGGTGAAAGTG GTG-3';GAPDH,F 5'-GCCATCAACGACCCCTTCAT-3'and R 5'-TTC AC ACCCATCACAAACAT-3',GAPDH as reference,Calculation of 2-Delta CT method.4 Statistical analysisAll data are expressed as mean±SD.Statistical analysis was performed by one-way ANOVA with Student–Newman–Keuls or Dunnett in Sigma plot software ver.10?SPSS,Chicago,IL,USA?.P<0.05 was considered signi-ficant.Results:The short-term effects of bepridil can reduce the inward rectifier potassium current in previously studies.Therefore,we directly evaluated the long-term effects of bepridil?24 hours?in neonatal rat cardiomyocytes.We first recorded that the long-term effect of bepridil can effectively inhibit inward rectifier potassium currents in neonatal rat cardiomyocytes.The long-term effects on inward rectifier potassium in control group and bepridil?1?M,5?M,and 10?M?group,clarified that bepridil had the long-term inhibitory effect on inwardly rectifying potassium currents?Fig.1 A?.The current-voltage curve reveals the long-term inhibitory effect of bepridil on inward potassium current in a dose-dependent manner?Fig.1 B?.The inward rectifier potassium currents were inhibited by 33.6%?1?M?,57.5%?5?M?and 82.7%?10?M?at-110 mV.Next,through real-time PCR,we found that the long-term effect of bepridil effectively increased the expression of KCNJ2-mRNA in neonatal rat cardiomyocytes.A dose-response relationship between the inward rectifier potassium current was inhibited by 33.6%?1?M?,57.5%?5?M?and 82.7%?10?M?.Long term effects of bepridil increased myocardial KCNJ2-mRNA level of neonatal rat expression by quantitative real-time PCR technology,the results of our study showed that the long-term effects of bepridil significantly increased the expression levels of KCNJ2-mR NA in a dose dependent manner?Fig.2A?.Considering that bepridil is also a calmodulin inhibitor.We found that both W-7 and KN93 increased the expression of KCNJ2-mRNA in neonatal rat cardiomyocytes after culture of neonatal rat cardiomyocytes for 24 hours in the presence of inhibitor of calcineurin?CaM?W-7 and KN93?Fig.2B?.As I mentioned earlier,the long-term effects of bepridil can reduce inward rectifier potassium current,and its KCNJ2-mRNA expression was significantly increased.This results suggested that it maybe present the possibility of post-transcriptional modification.Finally,we further investigated the inhibitory effect of bepridil on inward rectifier potassium currents and the relationship between caveolins.we also use CaM inhibitor and CaMKII inhibitor KN93 cultured neonatal rat cardiomyocytes?24 hours?.Further revealing the potential mechanism of bepridil to reduce I_K1.With the caveolin-3 primers,bepridil with inhibitor of W-7 and KN93 can increase the expression of KCNJ2-mRNA,also proved that caveolin-1 had the negative regulation on the inward rectifier potassium current.Caveolin-3 inhibits KCNJ2-mRNA expression and inhibits inward rectifier potassium currents,consistent with previous studies.Conclusion:The present study reveals that the long-term effects of bepridil can effectively inhibit the inward rectifier potassium current,further demonstrated that bepridil inhibited the inward rectifier potassium current mainly through the Ca2+-Ca/CaMKII signaling pathway.At the same time,the negative regulation of the inwardly rectifying potassium current in caveolin?CaV-1?was also demonstrated.Despite we found that inconsistency between the increase in KCNJ2-mRNA expression and the decrease in inward rectifier potassium current,we hypothesize that the expression of bevacizidine hydrochloride in the inward rectifier potassium channel may be post-transcriptional modified,which is also the main direction for our further study.Finally,the long-term inhibitory effect of bepridil on inward rectifier potassium currents is a potential mechanism for the treatment of atrial fibrillation,and it provides a new direction and target for further clinical guidance.