James T. Milnes

Investigating dynamic protocol-dependence of hERG potassium channel inhibition at 37 °C: Cisapride versus dofetilide

INTRODUCTION Pharmacological inhibition of cardiac potassium channels encoded by hERG (human ether-à-go-go-related gene) is associated with QT interval prolongation and torsades de pointes arrhythmia. Electrophysiological assays of hERG channel inhibition are integral to the safety testing of novel drug candidates. This study was conducted to compare, for the high affinity hERG inhibitors dofetilide and cisapride, hERG blockade between action potential (AP) and conventional (step and step-ramp) screening waveforms. Furthermore, it evaluated dynamic (pulse-by-pulse) protocol-dependence of hERG channel inhibition by these drugs. METHODS Whole-cell patch-clamp recordings were made at 37 degrees C from hERG-expressing HEK 293 cells. Half-maximal inhibitory concentrations (IC(50) values) for I(hERG) blockade were obtained using conventional voltage clamp and action potential clamp, using previously digitised ventricular and Purkinje fibre (PF) AP waveforms. RESULTS A more marked variation in IC(50) values with different command waveforms was observed for cisapride (ranging from 7 to 72 nM) than for dofetilide (ranging from 4 to 15 nM), with higher IC(50)s obtained with AP than step or step-ramp commands. The two drugs differed little from one another in effects on voltage-dependent activation; however, I(hERG) blockade by each drug was initially voltage-dependent, but at steady-state was only voltage-dependent for cisapride. There was comparatively little difference between the two drugs in effects on I(hERG) availability or time constants of development of inactivation. Features of time-dependence of blockade and the use of protocols employing varying rest periods in drug or commands of alternating duration highlighted a pronounced ability of cisapride, but not dofetilide, to dissociate and reassociate from hERG on a pulse-by-pulse basis. DISCUSSION Protocols described here that demonstrated dynamic variation (drug dissociation/reassociation) in hERG channel current blockade at 37 degrees C for cisapride may have future value for investigating drug interactions with the hERG channel. Downloadable digitised ventricular and PF AP waveforms that can be used in AP clamp experiments also accompany this article.

Inhibition of the HERG K+ channel by the antifungal drug ketoconazole depends on channel gating and involves the S6 residue F656

The mechanism of human ether‐à‐go‐go‐related gene (HERG) K+ channel blockade by the antifungal agent ketoconazole was investigated using patch‐clamp recording from mammalian cell lines. Ketoconazole inhibited whole‐cell HERG current (I HERG) with a clinically relevant half‐maximal inhibitory drug concentration (IC50) value of 1.7 μM. The voltage‐ and time‐dependent characteristics of I HERG blockade by ketoconazole indicated dependence of block on channel gating, ruling out a significant role for closed‐state channel inhibition. The S6 HERG mutations Y652A and F656A produced ∼4‐fold and ∼21‐fold increases in IC50 for I HERG blockade, respectively. Thus, ketoconazole accesses the HERG channel pore‐cavity on channel gating, and the S6 residue F656 is an important determinant of ketoconazole binding.

The positive frequency-dependent electrophysiological effects of the IKur inhibitor XEN-D0103 are desirable for the treatment of atrial fibrillation

Background Selective inhibitors of Kv1.5 channels are being developed for the treatment of atrial fibrillation (AF). Objectives The purpose of this study was to investigate the effects of the highly selective Kv1.5 inhibitor XEN-D0103 on human atrial action potentials (APs) at high excitation rates and to assess safety. Methods Intracellular APs (stimulation rates 1–5 Hz) were measured in right atrial trabeculae from patients in sinus rhythm (SR), chronic AF (cAF; AF of >6 months duration), and paroxysmal AF (pAF). The safety and tolerability of XEN-D0103 were tested in a double-blind, randomized, placebo-controlled phase 1 study. Results Depending on its concentration, XEN-D0103 elevated the plateau potential. At 1 Hz, XEN-D0103 (3 µM) shortened action potential duration at 90% repolarization (APD90) and effective refractory period (ERP) in SR preparations, but prolonged these parameters in cAF preparations. In SR and pAF preparations, the shortening effects on APD90 and ERP turned into prolongation at high rates. In cAF trabeculae, XEN-D0103 prolonged APD90 and ERP at 2 and 3 Hz. At high rates, more SR and pAF preparations failed to capture excitation in the presence of the drug than in its absence. XEN-D0103 (10 µM) did not significantly affect human ventricular APs. Even with plasma concentrations reaching 7000 ng/mL, XEN-D0103 did not increase ∆∆QTcF (QT interval corrected by the Fridericia formula) in the analysis of electrocardiograms of healthy volunteers, and no subjects receiving an active treatment had a QT or QTcF interval >450 ms, or increase in QTcF from baseline >30 ms. Conclusion APD prolongation and suppression of APs by XEN-D0103 at high stimulation rates in SR and pAF tissue, but not cAF, could be of therapeutic benefit for reducing AF burden. This concept needs to be confirmed in clinical trials.

Activation of Glibenclamide-Sensitive ATP-Sensitive K + Channels During β-Adrenergically Induced Metabolic Stress Produces a Substrate for Atrial Tachyarrhythmia

Background— Cardiac ATP-sensitive K+ channels have been suggested to contribute to the adaptive physiological response to metabolic challenge after &bgr;-adrenoceptor stimulation. However, an increased atrial K+-conductance might be expected to be proarrhythmic. We investigated the effect of ATP-sensitive K+ channel blockade on the electrophysiological responses to &bgr;-adrenoceptor-induced metabolic challenge in intact atria. Methods and Results— Atrial electrograms were recorded from the left atrial epicardial surface of Langendorff-perfused rat hearts using a 5×5 electrode array. Atrial effective refractory period and conduction velocity were measured using an S1–S2 protocol. The proportion of hearts in which atrial tachyarrhythmia was produced by burst-pacing was used as an index of atrial tachyarrhythmia-inducibility. Atrial nucleotide concentrations were measured by high performance liquid chromatography. Perfusion with ≥10–9 mol/L of the &bgr;-adrenoceptor agonist, isoproterenol (ISO), resulted in a concentration-dependent reduction of atrial effective refractory period and conduction velocity. The ISO-induced changes produced a proarrhythmic substrate such that atrial tachyarrhythmia could be induced by burst-pacing. Atrial [ATP] was significantly reduced by ISO (10–6 mol/L). Perfusion with either of the ATP-sensitive K+ channel blockers, glibenclamide (10–5 mol/L) or tolbutamide (10–3 mol/L), in the absence of ISO had no effect on basal atrial electrophysiology. On the other hand, the proarrhythmic substrate induced by 10–6 mol/L ISO was abolished by either of the sulfonylureas, which prevented induction of atrial tachyarrhythmia. Conclusions— Atrial ATP-sensitive K+ channels activate in response to &bgr;-adrenergic metabolic stress in Langendorff-perfused rat hearts, resulting in a proarrhythmic substrate.

Activation of Glibenclamide-Sensitive KATP Channels during β-Adrenergically-Induced Metabolic Stress Produces a Substrate for Atrial Tachyarrhythmia

Background— Cardiac ATP-sensitive K+ channels have been suggested to contribute to the adaptive physiological response to metabolic challenge after &bgr;-adrenoceptor stimulation. However, an increased atrial K+-conductance might be expected to be proarrhythmic. We investigated the effect of ATP-sensitive K+ channel blockade on the electrophysiological responses to &bgr;-adrenoceptor-induced metabolic challenge in intact atria. Methods and Results— Atrial electrograms were recorded from the left atrial epicardial surface of Langendorff-perfused rat hearts using a 5×5 electrode array. Atrial effective refractory period and conduction velocity were measured using an S1–S2 protocol. The proportion of hearts in which atrial tachyarrhythmia was produced by burst-pacing was used as an index of atrial tachyarrhythmia-inducibility. Atrial nucleotide concentrations were measured by high performance liquid chromatography. Perfusion with ≥10–9 mol/L of the &bgr;-adrenoceptor agonist, isoproterenol (ISO), resulted in a concentration-dependent reduction of atrial effective refractory period and conduction velocity. The ISO-induced changes produced a proarrhythmic substrate such that atrial tachyarrhythmia could be induced by burst-pacing. Atrial [ATP] was significantly reduced by ISO (10–6 mol/L). Perfusion with either of the ATP-sensitive K+ channel blockers, glibenclamide (10–5 mol/L) or tolbutamide (10–3 mol/L), in the absence of ISO had no effect on basal atrial electrophysiology. On the other hand, the proarrhythmic substrate induced by 10–6 mol/L ISO was abolished by either of the sulfonylureas, which prevented induction of atrial tachyarrhythmia. Conclusions— Atrial ATP-sensitive K+ channels activate in response to &bgr;-adrenergic metabolic stress in Langendorff-perfused rat hearts, resulting in a proarrhythmic substrate.

The role of L-type calcium current in the generation of repolarization-induced contraction in cardiac myocytes

OBJECTIVE Early experiments into the arrhythmogenic transient inward current frequently showed apparent coupling of this current to repolarization from a depolarizing voltage clamp step. Calcium transients have subsequently been shown to couple to such repolarization and are the result of calcium release from the sarcoplasmic reticulum. We have investigated whether this phenomenon is due to calcium entry via non-inactivated calcium channels or to voltage-activated SR release. METHODS Voltage clamp steps were imposed on isolated guinea pig and rabbit cardiac myocytes. Calcium release was monitored by tracking cell contraction. L-type calcium current at the moment of repolarization was manipulated by the rapid application of 2 mM cadmium or 10 mM calcium. RESULTS Repolarization-induced contraction was abolished by the rapid application of 2 mM cadmium immediately prior to repolarization, and was augmented by the rapid change of extracellular calcium concentration from 2 mM to 10 mM immediately prior to repolarization. There is no evidence of coupling of drive train-induced aftercontractions to repolarization from the final action potential of the drive train and 2 mM cadmium does not alter the appearance or timing of these aftercontractions. Simulation of phase 1 repolarization in the mammalian cardiac action potential decreases rather than increases twitch amplitude. CONCLUSION Repolarization-induced contraction results from calcium entry through non-inactivated calcium channels, not from voltage-activated release. It plays no physiological role in contributing to the stimulated twitch and no pathological role in generating drive train-induced aftercontractions.