Effects Of CaMKⅡ And L-type Ca²⁺channels On SK2Channels In Human Cardiomyocytes

Objective： Atrial fibrillation （AF） is the most frequentarrhythmia in the clinical setting. The basic mechanisms proposed tounderlie AF are rapid firing and multiple simultaneous functional reentrycircuits that alter atrial electrical properties. Evidence show that electricalremodeling has crucial links with ion channels intracellular Ca²⁺handlingabnormalities. Recently, research has emerged that Small conductanceCa²⁺-activated K+channels （SK） play an important role in cardiac actionpotention （AP） repolarization and atrial arrhythmogenesis. Besides, of thethree channel subtypes （SK1、 SK2and SK3）, SK2channels arepredominantly distributed in human and mouse atrial versus ventricularmyocytes. Thus, SK channels are considered as a new candidate in AFpathogenesis and a novel potential drug target for AF therapy.Thepore-forming subunits of SK2channels constitutively binds Calmodulin（CaM） that mediates Ca²⁺gating. CaM is a ubiquitously expressedCa²⁺-binding protein that transduces Ca²⁺signals by modulating theactivity of binding protein partners. Ca²⁺/calmodulin-dependent proteinkinase （CaMKⅡ）, as a target protein of CaM, its effect on SK2channelregulation is sarely reported. In AF, the alteration of SK2currents as wellas the mechanism of CaM and CaMKⅡ that regulates SK channels is incompletely understood. Discussion of these issues thoroughly will helpto reveal the mechanism of AF. In the present study, whole-cell patchclamp is used to detect the alteration of SK current and the regulation ofCaMK Ⅱ on SK channels. The study provided experimental data toelucidate the mechanism and provide the new drug target for AF therapy.Methods：Studies on SK2currents in atrial myocytes with whole-cellpatch clamp：Human right atrial appendages were obtained from patientsundergoing extracorporeal circulation cardiac surgery （patients with AFand14patients with SR）. Atrial samples were quickly immersed inoxygenated, nominally Ca²⁺-free cardioplegic solution and transported tothe laboratory quickly after excision. Then chopped the samples intosmall pieces （1×1mm3） in oxygenated cardioplegic solution. Singlemyocytes were enzymatically dissociated by modified procedure ofenzymatic dissociation with protease and collagenase. Choose the smooth,well-striated and rod-shaped myocytes for the experiments. Currents wererecorded with the whole-cell patch-clamp technique at room temperature.A voltage clamp protocol incuding preconditioning depolarizing clampsteps （-130to+60mV, duration of the pulse is200ms）. Conventionalpatch clamp experiments were performed with an Axopatch200Bamplifier. The amplified and filtered （1KHz） signals were sampled andstored on a computer. Clampex10.1software was used for dataacquisition and Clampfit10.1was used for data analysis. The whole-cell patch clamp was used to observe the regulation of SK2channels （AF andSR group） by KN-93（the CaMKII inhibitor） and verapamil (the L-typeCa²⁺channels inhibitor). Results:⑴The characteristics of SK2channelsin atrial myocytes:①We recorded macroscopic currents of atrialmyocytes in whole-cell patch, the macroscopic currents was inwardlyrectifying.②The macroscopic current density at-130mV was14.05±2.56pA/pF in SR （n=11） vs.43.05±28.93pA/pF in AF （n=6,P<0.05）.③The inward rectifier currents were inhibited by apamin（1×10-7mol/L） in20min. At-130mV, apamin reduecd current densityfrom11.92±6.21pA/pF to8.20±4.33pA/pF （n=6, P<0.01）. SK2currentswas recorded under the experiment condition and current density of SK2channels was the difference between dosing before and after. The I-Vcurve showed SK2channels were inwardly rectifying.④The SK2currentdensity at-130mV was3.67±0.37pA/pF in SR （n=9） vs.9.81±2.54pA/pF in AF （n=9, P<0.01）.⑵Effects of KN-93on inward rectifiercurrents of atrial myocytes:①In whole-cell patch, the inward rectifiercurrents was inhibited by KN-93（1×10-6mol/L） in30min. At-130mV,KN-93reduecd current density from17.25±8.06pA/pF to11.68±6.19pA/pF （n=4, P<0.01）.②At-130mV, the macroscopic currents of atrialmyocytes were blocked by apamin and produced2.83%±7.72inhibitionwhen there was KN-93in external solution（n=3）; apamin produced31.51%±4.98inhibition of currents without KN-93（n=6）. Inhibitory action was significnt difference in two groups （P<0.01）. The data showedthat KN-93decreased the sensibility of SK2channels to apamin.③At-130mV, KN-93produced33.6343%±13.8358inhibition of currents inSR （n=5） vs.44.73%±28.19inhibition of currents in AF （n=4, P<0.01）.⑶Effects of verapamil on SK2channels in atrial myocytes:①At-130mV, the macroscopic currents were blocked by apamin and produced2.37%±3.14inhibition （n=4） when there was verapamil in externalsolution; Apamin produced31.51%±4.98inhibition of currents withoutverapamil （n=6）. Inhibitory action was significnt difference in two groups（P<0.01）.②Atrial myocytes were put in solution with verapamil （10min）. The macroscopic currents density at-130mV was16.76±5.66pA/pF before applying apamin （n=4） vs.16.68±5.01（-130mV） afterapplying apamin （n=4, P>0.05）. The two results showed that verapamildecreased the sensibility of SK2channels to apamin. Conclusions：⑴SK2currents were detected in freshly isolated of atrial myocytes.Thecharacteristic was inwardly rectifying; Current density of SK2channelsin SR was smaller than in AF.⑵CaMKII plays a part in the regulation ofSK2channels and inhibition of CaMKII reduces the sensibility of SK2channels to apamin; the currents which were regulated by CaMKII in SRwere not so many as in AF.⑶The functional status of L-type Ca²⁺channel could regulate SK2channels, and L-type Ca²⁺channels’inhibition induce the sensibility of SK2channels to apamin reduced.