A major limitation of pharmacological approaches to suppression of atrial fibrillation (AF) is the risk of induction of ventricular proarrhythmia. This liability can be reduced or eliminated with the use of agents that produce atrial-selective electrophysiological effects.1,2 The small conductance calcium-activated potassium (SK) channels have been suggested as prime targets for development of drugs for AF suppression because of atrial-selective expression of small-conductance Ca2+-activated K+ channel current carried by these channels.3,4 The relative contribution of the SK channel to atrial electrophysiology, however, remains poorly defined.4,5 Agents that block the SK channels can cause atrial-selective prolongation of the effective refractory period (ERP) and effectively suppress AF.6–9 However, the mechanism underlying atrial selective ERP prolongation by SK channel blockers is poorly understood. This study was designed to evaluate the electrophysiological effects of the SK channel blockers NS8593 and UCL16846–8,10,11 in canine coronary-perfused atrial and ventricular preparations and to assess their effectiveness in preventing the development of AF in an acetylcholine-mediated AF model. We also determined their ability to inhibit sodium channel activity in human embryonic kidney (HEK) cells as well as in native canine atrial and ventricular myocytes.
Adult mongrel dogs of either sex were used. This investigation conforms to the Guide for Care and Use of Laboratory Animals. Preparations consisting of right atrium with a rim of right ventricle (1.0–2.0 cm) were isolated from the canine hearts. The ostium of the right coronary artery was cannulated with polyethylene tubing (i.d., 1.75 mm; o.d., 2.1 mm) and the preparations were perfused with cold Tyrode's solution (12–15°C) containing 8.5 [K+]0. With continuous coronary perfusion, all ventricular branches of the right coronary artery were immediately clamped with metal clips (1.5 × 10 mm; Kent Scientific Corp, Torrington, CT). The total time from excision of the heart to cannulation and perfusion of the artery was <4 minutes. Ventricular and atrial coronary branches were ligated using silk thread. The preparations were placed in a temperature-controlled bath (8 × 6 × 4 cm) and perfused at a rate of 8–10 mL/min using Tyrode's solution. The composition of the Tyrode's solution was (in mM): NaCl 129, KCl 4, NaH2PO4 0.9, NaHCO3 20, CaCl2 1.8, MgSO4 0.5, and d-glucose 5.5, bubbled with 95% O2 and 5% CO2 (37 ± 0.5°C, pH = 7.35). The perfusate was delivered to the artery by a roller pump. An air trap was used to avoid bubbles in the perfusion line.
Transmembrane action potential (AP) recordings (sampling rate 41 kHz) were obtained using floating glass microelectrodes (2.7 M KCl, 10–25 MΩ DC resistance). A pseudoelectrocardiogram was recorded using 2 electrodes consisting of Ag/AgCl half cells placed in the Tyrode's solution bathing the preparation, 1.0–1.2 cm from opposite ends of the coronary-perfused preparations (Fig. 1). ERP was measured by delivering premature stimuli after every 10th basic beat at a pacing cycle length (CL) of 500 ms. The diastolic threshold of excitation (DTE) was determined by increasing stimulus intensity in 0.01-mA steps starting from 0.1 mA, until a steady 1:1 activation was achieved. The shortest S1-S1 pacing interval permitting a 1:1 activation was measured at a 2×DTE, with DTE being determined at a CL of 500 ms. Postrepolarization refractoriness (PRR) was recognized when ERP exceeded AP duration (APD90) in the ventricle and APD70 in atria. Under normal conditions, ventricular ERP coincided with APD90, whereas atrial ERP generally coincided with APD70-75.12,13 In the presence of acetylcholine, atrial ERP corresponds to APD90. Maximum rate of rise of the AP upstroke (Vmax): Stable AP recordings and Vmax measurements are difficult to obtain in vigorously contracting perfused cardiac preparations.14 The effect of NS8593 and UCL1684 on Vmax was therefore determined by selecting the largest Vmax in the absence and presence of these agents at a CL of 500 ms in each preparation. The effect of NS8593 and UCL1684 on Vmax was also evaluated as Vmax changes upon acceleration from a CL of 500 to 300 ms. In vigorously beating cardiac preparations, resting membrane potential (RMP) can only be approximated. The change in RMP was approximated by comparing the largest amplitude of phase 0 of AP recorded in control and after drug administration. Significant reduction of phase 0 AP amplitude and depolarization of RMP are generally correlated.15,16
Isolation of atrial and ventricular myocytes was prepared from canine hearts using techniques described previously.12 Briefly, left atrial and ventricular preparations were dissected and coronary perfused first with cardioplegic solution and then with a nominally Ca2+-free solution (mM): NaCl 129, KCl 5.4, MgSO4 2.0, NaH2PO4 0.9, glucose 5.5, and NaHCO3 20, bubbled with 95% O2/5% CO2 containing 0.1% Bovine serum albumin (BSA) for a period of about 5 minutes. The preparations were then subjected to enzyme digestion with the nominally Ca2+-free solution supplemented with 0.5 mg/mL collagenase, 0.1 mg/mL protease, and 1 mg/mL BSA for 8–12 minutes. Then, the tissues were minced and incubated in fresh buffer containing 0.5 mg/mL collagenase and 1 mg/mL BSA and agitated. The specific activities of the enzymes were as follows: protease (Sigma P5147, 3.5 U/mg) and collagenase (Worthington, type 2, 290 U/mg). The supernatant was filtered, centrifuged at 200 rpm for 2 minutes, and the myocyte-containing pellet was stored in 0.5 mM Ca2+ HEPES buffer at room temperature.
HEK cells were grown in Dullbecco's Modification of Eagle's Medium with Glutamax supplemented with 10% Fetal bovine serum in 35-mm culture dishes and placed in a 5% CO2 incubator at 37°C. To assess the effect on INa, HEK cells were cotransfected using FuGeneHD (Active Motif, Carlsbad, CA) with a 1:1 molar ratio of WT human SCN5A, and WT SCN1B ion channel genes cloned into the pcDNA3.1. In addition, enhanced green fluorescent protein cDNA-Nav1.5 was tagged for identification of transfected cells for electrophysiologic study 12–24 hours after transfection.
The equilibration period was 30 minutes. The electrophysiological parameters were recorded before (control) and after addition of NS8593 or UCL1684 to the perfusion solution. The concentration of NS8593 (3 and 10 µM) was increased in a step-wise manner, with at least 30 minutes at each concentration before the start of recording. Reported data with UCL1684 (0.5 µM) were obtained after >1 hour of exposure to the drug.
The anti-AF effect of NS8593 and UCL1684 was evaluated using an acetylcholine (ACh)-mediated AF model (1 µM). ACh permits electrical induction of persistent AF in 100% atrial preparations (by premature electrical stimulation or rapid pacing). After completion of the main protocol, ACh (1 µM) was added to the coronary perfusate in the presence of 10 µM of NS8593 and 0.5 µM of UCL1684, and induction of AF was attempted using premature electrical stimulation and rapid pacing.
Voltage Clamp Recordings of Peak INa
Peak INa was measured as previously described with minor modification.12 Experiments were performed using a MultiClamp 200B (Molecular Devices, Foster City, CA). Command voltages were delivered, and data acquired through a DigiData 1550B computer interface using pClamp9 (Molecular Devices) with data stored on a computer hard disk. Patch pipettes were pulled from borosilicate glass (1.5 mm o.d. and 1.1 mm i.d.) on a Model DMZ-Universal-Electrode-puller (Zeitz-Instruments, Germany). The electrode resistance was 1.5–2.5 MΩ when filled with the internal solution (see below). The membrane was ruptured by applying negative pressure and series resistance compensated by 75%–80%. Whole-cell current data were acquired at 20–50 kHz and filtered at 5 kHz. Cell capacitance was measured by integrating the transient charge after application of a −5 mV voltage clamp step. Currents were normalized to cell capacitance and expressed as density (pA/pF). External solution contained the following (in mM): choline Cl 120, NaCl 10, Na+ acetate 2.8, CaCl2 0.5, KCl 4, MgCl2 1.5, CoCl2 1, glucose10, HEPES10, NaOH5, and BaCl2 0.1, pH adjusted to 7.4 with NaOH/HCl. The pipette solution contained the following (mM): NaCl 15, CsF 120, MgCl2 1, KCl 5, HEPES 10, Na2ATP4, and EGTA 10, pH adjusted to 7.2 with CsOH. Recordings of INa were made at least 5 minutes after cell membrane rupture to minimize the effects of time-dependent negative shift of steady-state (SS) inactivation that occurs in conventional voltage clamp experiments. Whole-cell currents were analyzed using the Clampfit analysis program pClamp 9 (Molecular Devices).
NS8593 and UCL1684 (NeuroSearch, Denmark) were dissolved in 100% dimethyl sulfoxide as a stock of 10 and 1 mM, respectively.
Statistical analysis was performed using paired or unpaired t test and one-way repeated-measures analysis of variance followed by Bonferroni's or Student–Newman–Keuls test, as appropriate. All tissue data are expressed as mean ± SD and single cell data as mean ± SEM.
Electrophysiological Effects of NS8593 and UCL1684 in Atria and Ventricles
NS8593 (3–10 µM) caused marked depression of excitability in atria, but not in ventricles. At 10 µM and a CL of 500 ms, 2 of 8 atria but 0 of 5 ventricles were markedly depressed, failing to respond to stimulation in a 1:1 fashion, so that the electrophysiological parameter could not be reasonably measured in these 2 atria. NS8593 (3–10 µM) produced little to no change in APD70-90 in either atria or ventricles (Fig. 1). At a concentration of 3 µM, NS8593 prolonged ERP only in atria whereas at a concentration of 10 µM, ERP was prolonged in both atria and ventricles, but to a much greater extent in the atria (Fig. 1). The NS8593-mediated ERP prolongation was due to induction of PRR, which was much greater in atria versus ventricles (Fig. 1). NS8593 significantly decreased Vmax in an atrial-selective manner. However, a significant reduction of Vmax was evident in the ventricles as well (Fig. 2). DTE and the shortest S1—S1 pacing interval permitting 1:1 activation were also affected by NS8593 in an atrial-selective manner (Fig. 3). Of note, at an NS8593 concentration of 10 µM and CL of 300 ms, 0/8 atria but 5/5 ventricles displayed 1:1 activation (with a DTEx10 determined at a CL of 500 ms), demonstrating potent rate-dependent atrial-selective effect of NS8593 on excitability. Electrophysiological effects of UCL1684 in the cardiac preparations were similar to those observed with NS8593. APD70-90 was not significantly altered in both atrial and ventricular preparations, and ERP was prolonged in an atrial-selective manner due to development of PRR (Fig. 4). Vmax, DTE, and the shortest S1-S1 were also affected exclusively or primarily in atria versus ventricles (Fig. 4).
The maximum amplitude of atrial APs recorded in the presence of 3 and 10 µM of NS8593 and 0.5 µM of UCL1684 was 96 ± 7, 73 ± 13, and 94 ± 6%, respectively, of those recorded under control conditions (P = 0.14, P < 0.001, and P = 0.09, respectively; CL = 500 ms; n = 6–8), consistent with the effect of these agents to block INa and suggesting the possibility that 10-µM NS8593 depolarized RMP. The maximum amplitude of APs recorded from the ventricles was not statistically significantly affected by 3- or 10-µM NS8583 or 0.5-µM UCL1684 (98 ± 6, 94 ± 10, and 98 ± 5% of controls, respectively; n = 8–10, CL 500 ms).
Effect of NS8593 and UCL1684 to Suppress Atrial Fibrillation
ACh alone (1 µM) significantly abbreviated atrial APD and ERP, permitting induction of persistent AF in 100% of atria (Fig. 5 and Table 1). Addition of ACh in the presence of NS8593 (10 µM) or UCL1684 (0.5 µM) resulted in a much smaller abbreviation of APD and ERP than observed with ACh alone (Table 1). Both drugs maintained their ability to induce PRR in the presence of ACh. Rate-dependent excitability was also significantly depressed, as evident by prolongation of the shortest S1-S1 interval, so that ERP at rapid activation rates was far longer than APD (Table 1). AF could not be induced in the presence of these agents in any of the preparations tested (Table 1). Potent anti-AF action of the drugs was associated with strong rate-dependent depression of excitability, which did not permit rapid activation of the atrium (Fig. 5 and Table 1).
Effects of NS8593 and UCL1684 on INa
Both agents significantly reduced INa in HEK cells, measured at holding potential (HP) of −80 mV (Fig. 6A). To test the hypothesis that the effect of SK channel blockers to produce atrial-selective depression of excitability is the result of inhibition of INa, we studied the direct effect of NS8593 on inhibition of INa in isolated canine atrial and ventricular myocytes at 2 HPs (−120 and −90 mV) using patch clamp techniques. NS8593 (10 µM) caused a minor nonsignificant reduction of INa in both atrial and ventricular myocytes at a HP of −120 mV but a potent atrial-predominant decrease of INa at a HP of—90 mV (Fig. 6B). SS inactivation of the sodium channels (h-curve) was not affected by NS8583 (10 µM) in the ventricular myocytes, but was shifted to negative potentials in the atrial myocytes (Fig. 6C). As we reported previously,12 the h-curve of atrial cells is typically more negative than that of ventricular cells in the absence of drug (Fig. 6C).
The principal findings of this study are that both SK channel inhibitors studied cause electrophysiological alterations in canine cardiac single cells and multicellular preparations (ie, reduction of INa density, negative shift of SS-inactivation, PRR induction, reduced Vmax, and increased DTE) consistent with potent atrial-selective inhibition of sodium channel activity. Both drugs have previously been shown to inhibit SK channels8,11 and these channels have previously been shown to be expressed in canine atria.8 However, the absence of APD prolongation in our atrial preparations with either NS8593 or UCL1684 suggests little to no functional contribution of SK channel inhibition in canine atria under these experimental conditions. Our data demonstrate an effect of both NS8593 and UCL1684 to prevent induction of vagally mediated AF, due to potent depression of excitability secondary to block of sodium channel activity. Collectively, our findings suggest that SK channel inhibition by NS8593 and UCL1684 does not contribute meaningfully to their potent anti-AF effects.
SK channels have been identified in the hearts of mouse, rat, rabbit, dog, and human, and the Ca+2-activated potassium current (ISK) carried by these channels is exclusively or largely expressed in the atrium.6,8,10,17 The selective expression of ISK in the atria is thought to make these channels potential targets for effecting atrial-selective APD and ERP prolongation, thus contributing to the management of AF without adverse consequences in the ventricles.3,4,8 Some agents that block the SK channels (such as NS8553, UCL1684, ICA, ICAGEN, AP14145, and AP30663) have been shown to prolong atrial ERP and suppress AF without prolongation of QT in experimental settings.6,7,9,18 There are, however, reports demonstrating a pro-AF effect induced by genetic ablation of SK channels3 or SK channel inhibition with apamin.19 The mechanism of atrial-selective ERP prolongation by SK channel inhibitors is not well defined. These inhibitors may or may not prolong atrial APD.5,8 Similar to the results of this study (Figs. 1–4), previous studies have reported that NS8593, UCL1684, and ICA produce atrial-selective alterations in sodium-channel-mediated parameters, that is, reducing Vmax, slowing conduction, and inducing PRR,10,20 indicating atrial-selective inhibition of the sodium channel. However, the effect of these agents to depress sodium channel parameters was attributed to an effect of ISK inhibition to effect depolarization of RMP. A significant NS8593-induced reduction of Vmax in the ventricles (where SK channel is not or poorly expressed) clearly points to a direct effect of NS8593 on INa (Fig. 2). Of note, the concentration of UCL1684 used in the current study was relatively low, resulting in minor depression of sodium-channel-mediated parameters in the ventricle (Fig. 4). In this study, we also demonstrate a direct effect of NS8593 and UCL1684 to inhibit INa using both isolated canine myocytes and HEK cells expressing the alpha and beta1 subunits of the cardiac sodium channel. Moreover, we demonstrate that INa inhibition and its atrial selectivity are critically dependent on HP. Inhibition of INa is clearly evident at an HP of −90 mV, but not at an HP of −120 mV (Fig. 6).
It is important to recognize that the ability of the other SK channel blockers (ie, ICA, ICAGEN, AP14145, AP30663, etc.) to inhibit the sodium channel has either not been tested or has been tested at an HP of −120 mV. Our data emphasize the need to test such agents under physiologically relevant conditions (ie, HPs and pacing rates) to avoid missing their atrial-selective actions to inhibit INa. Our observation regarding the SK channel inhibitors is reminiscent of the recent experience with IKur blockers, including vernakalant, AVE0118, and AZD1305, which upon close examination proved to exert their actions to prolong ERP solely or largely through atrial-selective inhibition of sodium channel current and not as a result of inhibition of IKur.14,21–23 Our findings provide yet another example of potassium channel blockers exerting their antiarrhythmic effect principally through inhibition of sodium channel activity.
INa blockers are known to abbreviate atrial APD, through inhibition of late INa24 and it is possible that a similar action on the part of NS8593 and UCL1684 might have negated the effect of SK channel inhibition to prolong atrial APD.
As discussed above, the depression of INa produced by SK channel blockers has in previous studies been attributed to drug-induced depolarization of RMP in atria.20 This suggestion is based largely on the observation that RMP is slightly depolarized in the presence of high concentrations of NS8553, ICAGEN, and ICA in human and rat atrial cells and tissues (by 2–4 mV).20 The contribution of such RMP depolarization is unlikely to provide an explanation for the strong reduction in sodium channel activity because a 2–4-mV depolarization cannot account for the 40%–50% reduction of Vmax recorded.16,20 Our demonstration using patch clamp techniques of a direct effect of NS8593 or UCL1684 to significantly produce an atrial-selective inhibition of INa (Fig. 6) can account for the atrial-selective induction of PRR and reduction of Vmax. It stands to reason that concomitant depolarization of RMP by whatever means is likely to accentuate the effect of the SK channel blockers to inhibit INa. Several studies have reported that inhibition of the SK channels alone does not alter RMP.3,5,8,25 Also relevant to this issue is the observation that relatively high concentrations of INa blockers are associated with depolarization of RMP, suggesting that depolarization of RMP observed in some settings may be secondary to INa inhibition rather than SK channel inhibition.15,26,27
Moreover, ICA at10 µM has been shown to be effective against AF but to only depolarize atrial RMP by 1–2 mV (nonsignificantly) in rat cardiac preparations.20,28 Further studies are needed to assess the contribution of SK channel inhibitors to indirect inhibition of INa through depolarization of RMP as well as the contribution of this mechanism to their antiarrhythmic efficacy.
Atrial-selective peak INa block is a typical feature of many INa blockers displaying relatively rapid unbinding kinetics, including ranolazine, amiodarone, vernakalant, dronedarone, and AZD1305.12,21–23,29–31 Previous work from our group has shown that atrial selectivity of INa blockers is due to several factors including: (1) the presence of a more depolarized RMP in atria versus ventricles; (2) a more negative half-inactivation voltage for the sodium channel in atrial versus ventricular cells32–34 (both of these factors reduce the availability of sodium channels in atria more than ventricles), and (3) a more gradual phase 3 repolarization in atrial cells; this characteristic of the atrial AP leads to progressive diminution or disappearance of the diastolic interval at rapid rates of activation, which reduces the ability of sodium channel blockers to dissociate from the sodium channel, thus leading to accumulation of sodium channel block. These distinctions between atrial and ventricular cell electrophysiology led us to suggest a novel strategy for AF suppression, namely atrial-selective sodium channel block.12,32,33 The ability of NS8593 to preferentially shift atrial V0.5 to more negative potentials contributes to further accentuation of the atrial-selective inhibition of INa (Fig. 6).
Both NS8593 and UCL1684 stunted the effect of ACh to abbreviate atrial APD. This would seem to be consistent with SK channel inhibition, but because ACh dramatically reduces intracellular calcium activity,35 it is expected to reduce SK channel activity (assuming that calcium activity is reduced in the vicinity of the SK channels). Accordingly, inhibition of SK channel is unlikely to prolong APD owing to the dramatically reduced levels of intracellular calcium. The effect NS8593 and UCL1684 to blunt the ability of ACh to abbreviate APD is similar to that reported with other sodium channel blockers. Previous studies have shown that all sodium channel blockers tested at therapeutically relevant concentrations in the coronary perfused canine right atrial preparations, including ranolazine, lidocaine, propafenone, amiodarone, AZD1305, and Wenxin Keli, blunt the effect ACh to abbreviate atrial APD.12,22,29,36,37 Similar data have been reported regarding the effect of flecainide and ICA to blunt the ability of carbachol to abbreviate repolarization in rat atrial tissues.28 These effects may be due to the ability of INa blockers to directly inhibit IK-ACh as previously reported for flecainide, propafenone, disopyramide, and pilsicainide.38,39 Because the extent of PRR prolongation greatly exceeded that of APD prolongation in the presence of ACh (Table 1), it can be argued that sodium channel block by NS8593 and UCL1684 is the most important driver underlying the observed antiarrhythmic effect of these agents.
The electrophysiological actions of NS8593 and UCL1684 were studied acutely in isolated normal nonremodeled Tyrode's-perfused atrial preparations and acutely dissociated atrial and ventricular myocytes. SK channel expression is reported to be augmented in diseased ventricles (eg, in heart failure),40 thus reducing the predominance of SK channels in atria. This, however, is unlikely to alter the principal conclusion of our study, that is, that the electrophysiological and anti-AF effects of NS8593 and UCL1684 are due to atrial selective INa inhibition. We studied the efficacy of NS8593 and UCL1684 to prevent but not to terminate AF. It is possible that SK channel inhibition by these agents may play more of a role in suppressing AF because AF is known to increase intracellular Ca2+ activity (including during ACh-mediated AF35), which may increase the influence of SK channels on atrial repolarization and thus the efficacy of SK channel blockers to prolong atrial ERP during AF. This hypothesis remains to be tested with specific SK channel blockers. The presence of autonomic influences and other factors present in vivo may modulate the effect of the drugs, resulting in outcomes different from those observed in this study.
NS8593 and UCL1684 are very effective in preventing the development of AF in our ACh models of AF. The electrophysiological alterations induced by NS8593 and UCL1684 in canine cardiac preparations and isolated cells suggest that these compounds exert their electrophysiological and anti-AF effects predominantly or exclusively through atrial-selective inhibition of the sodium channel. These conclusions pertain to the specific drugs and conditions studied and cannot be directly applied to other conditions or other SK channel blockers. Our findings, coupled with available data, suggest that NS8593 and UCL1684 are effective for the management of AF and lack any proarrhythmic liability in the ventricle because of their atrial selectivity.
The authors thank Robert Goodrow for expert technical support.
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