Kenneth R. Wyse

Inhibition of the human ether-a-go-go-related gene (HERG) potassium channel by cisapride : affinity for open and inactivated states

Cisapride is a prokinetic agent which has been associated with QT prolongation, torsades de pointes and cardiac arrest. The cellular mechanism for these observations is high affinity blockade of IKr (encoded by HERG). In a chronic transfection model using CHO‐K1 cells, cisapride inhibited HERG tail currents after a step to +25 mV with similar potency at room and physiological temperatures (IC50 16.4 nM at 20–22°C and 23.6 nM at 37°C). Channel inhibition exhibited time‐, voltage‐ and frequency‐dependence. In an envelope of tails test, channel blockade increased from 27±8% after a 120 ms depolarizing step to 50±4% after a 1.0 s step. These findings suggested affinity for open and/or inactivated channel states. Inactivation was significantly accelerated by cisapride in a concentration‐dependent manner and there was a small (−7 mV) shift in the voltage dependence of steady state inactivation. Channel blockade by cisapride was modulated by [K+]o, with a 26% reduction in the potency of channel blockade when [K+]o was increased from 1 to 10 mM. In conclusion, HERG channel inhibition by cisapride exhibits features consistent with open and inactivated state binding and is sensitive to external potassium concentration. These features may have significant clinical implications with regard to the mechanism and treatment of cisapride‐induced proarrhythmia.

Inhibition of HERG potassium channels by the antimalarial agent halofantrine

Halofantrine is a widely used antimalarial agent which has been associated with prolongation of the ‘QT interval’ of the electrocardiogram (ECG), torsades de pointes and sudden death. Whilst QT prolongation is consistent with halofantrine‐induced increases in cardiac ventricular action potential duration, the cellular mechanism for these observations has not been previously reported. The delayed rectifier potassium channel, IKr, is a primary site of action of drugs causing QT prolongation and is encoded by the human‐ether‐a‐go‐go‐related gene (HERG). We examined the effects of halofantrine on HERG potassium channels stably expressed in Chinese hamster ovary (CHO‐K1) cells. Halofantrine blocked HERG tail currents elicited on repolarization to −60 mV from +30 mV with an IC50 of 196.9 nM. The therapeutic plasma concentration range for halofantrine is 1.67–2.98 μM. Channel inhibition by halofantrine exhibited time‐, voltage‐ and use‐dependence. Halofantrine did not alter the time course of channel activation or deactivation, but inactivation was accelerated and there was a 20 mV hyperpolarizing shift in the mid‐activation potential of steady‐state inactivation. Block was enhanced by pulses that render channels inactivated, and channel blockade increased with increasing duration of depolarizing pulses. We conclude that HERG channel inhibition by halofantrine is the likely underlying cellular mechanism for QT prolongation. Our data suggest preferential binding of halofantrine to the open and inactivated channel states.

Inhibition of HERG channels stably expressed in a mammalian cell line by the antianginal agent perhexiline maleate.

Perhexiline has been used as an anti‐anginal agent for over 25 years, and is known to cause QT prolongation and torsades de pointes. We hypothesized that the cellular basis for these effects was blockade of IKr. A stable transfection of HERG into a CHO‐K1 cell line produced a delayed rectifier, potassium channel with similar properties to those reported for transient expression in Xenopus oocytes. Perhexiline caused voltage‐ and frequency‐dependent block of HERG (IC50 7.8 μM). The rate of inactivation was increased and there was a 10 mV hyperpolarizing shift in the voltage‐dependence of steady‐state inactivation, suggestive of binding to the inactivated state. In conclusion, perhexiline potently inhibits transfected HERG channels and this is the probable mechanism for QT prolongation and torsades de pointes. Channel blockade shows greatest affinity for the inactivated state.

Action Potential Prolongation Exhibits Simple Dose-Dependence for Sotalol, but Reverse

Plasma drug concentrations in patients who develop torsade de pointes while receiving quinidine or disopyramide treatment have been reported to be usually in or below the therapeutic range, whereas patients developing the same complication during sotalol treatment usually have drug concentrations well above the therapeutic range. We wished to provide a cellular electrophysiologic rationale for this observation. Standard intracellular microelectrode techniques were used to record action potentials (APs) from canine Purkinje fibers at in-terstimulus intervals (ISI) of 1,000–6,000 ms, with and without three varying concentrations of quinidine, disopyramide, and sotalol. Cesium chloride 0.5 mM was added to reduce spontaneous diastolic depolarization. We observed a biphasic response in action potential duration (APD) to quinidine and disopyramide. Low concentrations tended to prolong APD, particularly at slower drive rates, whereas this effect tended to reverse as the concentration was increased. In contrast, sotalol produced a consistent, monophasic dose-dependent increase in APD across the therapeutic concentration range and well beyond it. We also observed an apparent increased likelihood of early afterdepolarizations (EADs), with or without triggered activity, at low concentrations of quinidine and disopyramide, with a trend toward reversal as the concentration was increased. We conclude that the biphasic dose response observed for APD with quinidine and disopyramide is due to the opposing effects of these agents on outward potassium and inward sodium currents and may cast some light on the clinical observation noted above. Sotalol on the other hand appears to produce EADs and triggered activity only at high concentrations.

Blockade by N-3 polyunsaturated fatty acid of the Kv4.3 current stably expressed in Chinese hamster ovary cells.

The Kv4.3 gene is believed to encode a large proportion of the transient outward current (Ito), responsible for the early phase of repolarization of the human cardiac action potential. There is evidence that this current is involved in the dispersion of refractoriness which develops during myocardial ischaemia and which predisposes to the development of potentially fatal ventricular tachyarrhythmias. Epidemiological, clinical, animal, and cellular studies indicate that these arrhythmias may be ameliorated in myocardial ischaemia by n‐3 polyunsaturated fatty acids (n‐3 PUFA) present in fish oils. We describe stable transfection of the Kv4.3 gene into a mammalian cell line (Chinese hamster ovary cells), and using patch clamp techniques have shown that the resulting current closely resembles human Ito. The current is rapidly activating and inactivating, with both processes being well fit by double exponential functions (time constants of 3.8±0.2 and 5.3±0.4 ms for activation and 20.0±1.2 and 96.6±6.7 ms for inactivation at +45 mV at 23°C). Activation and steady state inactivation both show voltage dependence (V1/2 of activation=−6.7±2.5 mV, V1/2 of steady state inactivation=−51.3±0.2 mV at 23°C). Current inactivation and recovery from inactivation are faster at physiologic temperature (37°C) compared to room temperature (23°C). The n‐3 PUFA docosahexaenoic acid blocks the Kv4.3 current with an IC50 of 3.6 μmol L−1. Blockade of the transient outward current may be an important mechanism by which n‐3 PUFA provide protection against the development of ventricular fibrillation during myocardial ischaemia.

Effects of disopyramide and flecainide on the kinetics of inward rectifier potassium channels in rabbit heart muscle

1 Standard patch‐clamp techniques were used to study the interaction of therapeutic concentrations of flecainide and disopyramide with single inwardly‐rectifying potassium channels in cell‐attached membrane patches from rabbit ventricular myocytes. 2 Under drug‐free conditions, the potassium channels had a conductance of 31 ± 2 pS (n = 13), a mean open time of 230 ± 6 ms (n = 11) recorded at the resting cell potential, and an open probability of 0.66 ± 0.20 (n = 39). The resting potential of the cells studied was − 68.5 ± 3.6 mV (n = 32). 3 Disopyramide did not reduce the open probability of the channel when the cell was voltage‐clamped at the resting cell potential. However, disopyramide increased the mean open time of the channel, recorded at the resting cell potential, by 15% at 5 μm and by 29% at 20 μm. The action potential prolonging actions of disopyramide in therapeutic concentrations appear not to be due to blocking the inward rectifier K+ channel. 4 Flecainide (3.0 μm, but not at 0.5 μm) decreased the open probability without changing the conductance of the channel, at 3 μm (51.0 ± 7.2%, n = 6, P = 0.03) at the resting cell potential. Flecainide increased the mean open time of the channel, recorded at the resting cell potential, by 12% at 3.0 μm. 5 We propose that flecainide stabilized the inward rectifier K+ channel in an inactivated state, without plugging the conducting pore. In addition, it appeared to bind to an open conformation of the channel, since some of the reduction in open probability could be accounted for by the lengthening of the mean open time. The changes in open‐state kinetics suggest that this binding may be in the region of the activation gate.

Effects of Hyperkalemia, Acidosis, and Hypoxia on the Depression of Maximum Rate of Depolarization by Class I Antiarrhythmic Drugs in Guinea Pig Myocardium: Differential Actions of Class Ib and Ic Agents

Standard microelectrode methods were used to record intracellular action potentials from strips of guinea pig right ventricular myocardium superfused with either standard physiological saline (pH 7.3; PO2 > 650 mm Hg; [K+] = 5.6 mM) or the same solution modified to produce either hyperkalemia ([K+] = 11.2 mM), acidosis (pH = 6.3), or hypoxia (PO2 = 60 mm Hg). The effects on action potential parameters of three therapeutic concentration of lidocaine, flecainide, and encainide were studied under all four conditions at four different drive rates (interstimulus interval = 2.400, 1.200, 600, and 300 ms). Hyperkalemia in the absence of drugs produced reductions in resting potential (-87.9 ± 3.8 to −74.6 ± 3.3 mV), maximum rate of depolarization (316 ± 68 to 240 ± 12 V/s). and action potential duration (178 ± 21 to 165 ± 27 ms). All three drugs produced increased depression of Vmax in hyperkalemia compared to control conditions but, at all three concentrations and all four rates, this enhancement of effect was greater for lidocaine than for either of the other two agents (which did not differ significantly from each other; p < 0.001). Similar though less marked effects were produced by acidosis (3.5 mV depolarization and 19% reduction in Vmax), and once again the depression of Vmax by lidocaine was enhanced more by this intervention than were the actions of encainide or flecainide (p < 0.01). Hypoxia had no effect on action potential parameters other than duration and no significant modulation of drug actions was seen for this intervention. It is concluded that of the electrophysiological changes induced by ischemia, those due to hyperkalemia are the most important in causing enhanced depression of Vmax by class I antiarrhythmic agents and that lidocaine (subclass Ib) is significantly more selective under such conditions than encainide or flecainide (subclass lc).

DIFFERENTIAL EFFECTS ON ACTION POTENTIAL DURATION OF CLASS IA, B AND C ANTIARRHYTHMIC DRUGS: MODULATION BY STIMULATION RATE AND EXTRACELLULAR K+ CONCENTRATION

1. Standard microelectrode techniques were used to study the effects on the action potential duration (APD) of canine Purkinje fibres of a therapeutic concentration of nine Class I antiarrhythmic drugs. At an extracellular K+ concentration of 5.6 mmol/L all nine agents reduced APD at all drive rates studied (range of interstimulus intervals = 200–1000 ms). At lower levels of K+, quinidine (5 μmol/L) and disopyramide (10 μmol/L) (Class Ia agents) revealed dual effects on APD. At the lowest levels of K+ (2 mmol/L) and the longest interstimulus interval used (2000 ms), both agents significantly prolonged APD. Under all other conditions, APD was either unchanged or reduced. Lignocaine, 15 μmol/L (Class Ib agent) reduced APD at all rates and all K+ concentrations and this effect was greatest at the slowest rates.

Effects of hyperkalaemia on the depression of maximum rate of depolarization by class I antiarrhythmic agents in guinea-pig myocardium.

1 Standard microeletrode methods were used to record intracellular action potentials from strips of guinea‐pig right ventricular myocardium superfused with either standard physiological saline ([K+] = 5.6 mm) or the same solution modified to contain [K+] = 11.2 mm. 2 The effects on action potential parameters of three therapeutic concentrations of mexiletine, quinidine and disopyramide were studied under both conditions at four different drive rates (interstimulus intervals = 2400, 1200, 600 and 300 ms). 3 Hyperkalaemia in the absence of drugs produced reductions in resting potential (−86.7 ± 2.5 mV to −71.8 ± 3.7 mV; n = 30; P < 0.001), maximum rate of depolarization (300 ± 46.5 Vs−1 to 205.6 ± 37.6 Vs−1; P < 0.0001), and action potential duration (205 ± 26 ms to 188 ± 32 ms; P < 0.05). 4 All three drugs produced increased depression of maximum rate of depolarization in hyperkalaemia compared to control conditions, but at all three concentrations this enhancement of effect was greater for mexiletine than for quinidine, with disopyramide exhibiting intermediate behaviour. 5 Mexiletine behaved very similarly to therapeutic concentrations of lignocaine as described in previous reports from this laboratory. 6 Quinidine behaved very similarly to Class Ic agents. 7 It is concluded that mexiletine demonstrated significantly greater selectivity for depolarized myocardium than quinidine and that this may have implications in terms of proarrhythmic potential. 8 Disopyramide exhibited intermediate selectivity for depolarized myocardium between mexiletine and quinidine.

Comparative Study of the Effects of Erythromycin and Roxithromycin on Action Potential Duration and Potassium Currents in Canine Purkinje Fibers and Rabbit Myocardium

Background: Erythromycin and roxithromycin are macrolide antibiotics in common clini cal use. Erythromycin occasionally produces life-threatening arrhythmias (torsades de pointes) by blocking the outward potassium current responsible for repolarization of the cardiac action potential. Methods and Results: We used standard cellular electrophysiological and whole-cell patch- clamping techniques to compare the relative efficacy of erythromycin and roxithromycin in prolonging cardiac action potential in canine Purkinje fibers and in blocking individual out ward potassium currents in isolated rabbit ventricular myocytes. We demonstrated signifi cant prolongation of action potential duration in canine Purkinje fibers by erythromycin but not roxithromycin at a concentration of 100 μM. The delayed rectifier, the outward potas sium current thought to be most sensitive to modulation by drugs, was significantly depressed by both agents at concentrations of ≥30 μM in isolated rabbit ventricular myo cytes. Both drugs had similar potencies (26% and 21 % reduction by 30 μM erythromycin and roxithromycin, respectively, and 50% and 36% reduction by 100 μM erythromycin and roxithromycin). Neither agent significantly blocked other potassium currents (including the transient outward current). Conclusions: Taking into account normally observed peak blood concentrations of these agents in clinical use and the fact that roxithromycin is not normally administered intrave nously, we conclude that the risk of proarrhythmia during normal clinical use of oral roxithromycin is extremely remote.