Effects Of Intracellular Ca²⁺ Alteration On SK Currents In Human Atrial Cardiomyocytes

Abstract：Objective：Atrial fibrillation （AF） is the most frequent andsevere arrhythmia. It is very rare in children, but it becomes extremelyfrequent in the elderly with incidence rate a19.2‰in patients who are morethan65years old..AF is associated with a wide range of potentialcomplications （stroke, heart failure et al.） and contributes significantly tomorbidity and mortality. However, the mechanisms of AF are incompletelyunderstood recently. The basic mechanisms of AF include multiple waveletreentry hypothesis and pulmonary veins elicited conduction accompanied withfibrillation as well as the AF begets AF theory. Evidence suggests that theoccurrence of AF has links with the shortening of action potential’s effectiverefractory period. Besides, the action potential duration is regulated bydifferent ion channels in the cardiac, so the functional change of ion channelsmay contribute to AF. Small conductance Ca²⁺-activated K⁺channels are thesubset of the Ca²⁺-activated K⁺channel family. Electrophysiological propertiesof SK channels are voltage-independent, and solely activated by changes inintracellular Ca²⁺([Ca²⁺]i). Recently, some papers report that SK channels areexisted in hearts of mouse, rat, and human. Biochemical evidence indicatesthat SK2channels are predominantly distributed in human and mouse atrialversus ventricular myocytes. SK channels are highly sensitive to [Ca²⁺]ithat couple the membrane potential to fluctuations in intracellular Ca²⁺concention.Thus, we hypothesis that the overload of [Ca²⁺]imay alter the function of SKchannels which is an interesting target in AF. In the present study, performedwhole-cell patch clamp was used to detect the Ca²⁺sensitivity of SK currentsbetween SR and AF. L-type calcium channel activator Bay-K8644andinhibitor verapamil were also used to observe the regulation of SK current. Itprovided experimental data to elucidate the mechanism and provide the newdrug target for AF therapy. Methods：42patients undergoing ExtracorporealCirculation cardiac surgery were divided into2groups:31patients in sinusrhythm（SR） and11patients with AF. Cardiomyocytes obtaintion: Humanright atrial appendages were quickly immersed in oxygenated, nominallyCa2+-free cardioplegic solution and transported to the laboratory quickly afterexcision. Then the samples were chopped into small pieces （1×1mm3） inoxygenated cardioplegic solution. Single myocytes were enzymaticallydissociated by modified procedure of enzymatic dissociation with proteaseXXIV and collagenase V. The smooth, well-striated and rod-shaped myocyteswere chosen for the experiments. SK2current recording: Currents wererecorded with the performed whole-cell patch-clamp technique at roomtemperature. Patch clamp experiments were performed with Axopatch200Bamplifier. The amplified and filtered （1KHz） signals were sampled and storedon a computer. Clampex10.1software was used for data acquisition andClampfit10.1as well as OriginPro8.0software was used for data analysis.（1） Amphotericin B and β-escin were used as performed solution to record SK2currents in human atria cardiomyocytes, and fluorescence scan technologywas used to detect the [Ca²⁺]ialteration before and after the perforationformation.（2） SK2currents were recorded in both SR and AF groups withperforated whole-cell patch clamp to detect the alteration of SK currents.（3）Effects of different [Ca²⁺]ion the SK2currents in both SR and AF groups.（4）The whole-cell performed patch clamp was used to detect the influence ofL-type Ca²⁺channel activator Bay-K8644and inhibitor verapamil on SK2currents. Results:⑴Couple use of Amphotericin B and β-escin can formthe state performed patch clamp recording model. The intracellular freecalcium ions increase after the formation of the perforation with amphotericinB plus β-escin as perforated fluid （F340/380enhance） and SK2currents wererecorded after preformed patch clamp model was formed.（2） The functionalcharacteristics of SK2currents in atrial cardiomyocytes:①We recorded theintegrated inward currents before and after application of apamin; theapamin-sensitive SK2currents are obtained with digital subtraction.The SK2currents recorded in this experiment are voltage-independent and inwardlyrectifying.②In the whole-cell performed patch clamp,100nM apamin blocksintegrated inward currents partly, and the integrated inward currents canrecovery to the basic state when apamin was washed out.（2） The difference ofSK2currents between SR and AF groups: The SK2current density at-130mVwas-2.92±0.35pA/pF in SR group vs.-6.83±0.19pA/pF in AF group （nSR=6,nAF=3, p＜0.05）.（3）Effect of different [Ca²⁺]ion the SK2currents：In SR group, The SK2current densities in different [Ca²⁺]iare-1.43±0.33pA/pF、-2.92±0.35pA/pF、-10.11±2.15pA/pF （n0=7，n5×10－7=6，n10－6=8，p＜0.05）; In AF group,the SK2current densities are-2.17±0.40pA/pF、-6.83±0.19pA/pF、-14.47±2.89pA/pF (n0=4，n_5×10－7=3，n_10－6=4, p＜0.05).（4）The relative between L-type calcium channels and SK2currents:①Effect ofL-type calcium channel activator BayK8644on SK2currents: BayK8644canincrease the integrated inward currents when the [Ca2+]－7iis5×10mol/L.②Effect of L-type calcium channel inhibitor verapamil on SK2currents: Theintegrated inward current didn’t change under the condition that verapamil andapamil were added into the bath solution when the [Ca²⁺]iis5×10－7.Conclusions：⑴Couple use of Amphotericin B and β-escin can form the stateperformed patch clamp recording model, Ca²⁺can go into the intracellularwhich cause the increase of [Ca²⁺]ithat is suitable to detect the regulation ofSK2currents by different [Ca²⁺]i.（2） When the [Ca²⁺]iis5×10^－7mol/L, SK2current density in AF were significantly larger than that in SR whichsuggested that SK currents involve the cardiomyocytes electric remodeling inAF.（3） In AF, the SK2currents are more sensitive to [Ca²⁺]I. It showed thatSK2currents contribute to the occurrence and maintenance of AF.（4） L-typeCa²⁺channels activator Bay-K8644cause the increase of integrated inwardcurrents, and inhibitor verapamil induce the sensibility of SK2channels toapamin reduced. The two results suggested that SK2currents have crucial links with the L-type Ca²⁺channel state.