Norbert Iost

The role of the delayed rectifier component IKs in dog ventricular muscle and Purkinje fibre repolarization

The relative contributions of the rapid and slow components of the delayed rectifier potassium current (IKr and IKs, respectively) to dog cardiac action potential configuration were compared in ventricular myocytes and in multicellular right ventricular papillary muscle and Purkinje fibre preparations. Whole‐cell patch‐clamp techniques, conventional microelectrode and in vivo ECG measurements were made at 37°C. Action potential duration (APD) was minimally increased (less than 7%) by chromanol 293B (10 μM) and L‐735,821 (100 nM), selective blockers of IKs, over a range of pacing cycle lengths (300–5000 ms) in both dog right ventricular papillary muscles and Purkinje fibre strands. D‐Sotalol (30 μM) and E‐4031 (1 μM), selective blockers of IKr, in the same preparations markedly (20–80%) lengthened APD in a reverse frequency‐dependent manner. In vivo ECG recordings in intact anaesthetized dogs indicated no significant chromanol 293B (1 mg kg−1 i.v.) effect on the QTc interval (332.9 ± 16.1 ms before versus 330.5 ± 11.2 ms, n= 6, after chromanol 293B), while D‐sotalol (1 mg kg−1 i.v.) significantly increased the QTc interval (323.9 ± 7.3 ms before versus 346.5 ± 6.4 ms, n= 5, after D‐sotalol, P < 0.05). The current density estimated during the normal ventricular muscle action potential (i.e. after a 200 ms square pulse to +30 mV or during a 250 ms long ‘action potential‐like’ test pulse) indicates that substantially more current is conducted through IKr channels than through IKs channels. However, if the duration of the square test pulse or the ‘action potential‐like’ test pulse was lengthened to 500 ms the relative contribution of IKs significantly increased. When APD was pharmacologically prolonged in papillary muscle (1 μM E‐4031 and 1 μg ml−1 veratrine), 100 nM L‐735,821 and 10 μM chromanol 293B lengthened repolarization substantially by 14.4 ± 3.4 and 18.0 ± 3.4% (n= 8), respectively. We conclude that in this study IKs plays little role in normal dog ventricular muscle and Purkinje fibre action potential repolarization and that IKr is the major source of outward current responsible for initiation of final action potential repolarization. Thus, when APD is abnormally increased, the role of IKs in final repolarization increases to provide an important safety mechanism that reduces arrhythmia risk.

Interaction of different potassium channels in cardiac repolarization in dog ventricular preparations: role of repolarization reserve.

The aim of this study was to investigate the possible role of the interaction of different potassium channels in dog ventricular muscle, by applying the conventional microelectrode and whole cell patch‐clamp techniques at 37°C. Complete block of IKr by 1 μM dofetilide lengthened action potential duration (APD) by 45.6±3.6% at 0.2 Hz (n=13). Chromanol 293B applied alone at 10 μM (a concentration which selectively blocks IKs) did not markedly lengthen APD (<7%), but when repolarization had already been prolonged by complete IKr block with 1 μM dofetilide, inhibition of IKs with 10 μM chromanol 293B substantially delayed repolarization by 38.5±8.2% at 0.2 Hz (n=6). BaCl2, at a concentration of 10 μM which blocks IKl without affecting other currents, lengthened APD by 33.0±3.1% (n=11), but when IKr was blocked with 1 μM dofetilide, 10 μM BaCl2 produced a more excessive rate dependent lengthening in APD, frequently (in three out of seven preparations) initiating early afterdepolarizations. These findings indicate that if only one type of potassium channels is inhibited in dog ventricular muscle, excessive APD lengthening is not likely to occur. Dog ventricular myocytes seem to repolarize with a strong safety margin (‘repolarization reserve’). However, when this normal ‘repolarization reserve’ is attenuated, otherwise minimal or moderate potassium current inhibition can result in excessive and potentially proarrhythmic prolongation of the ventricular APD. Therefore, application of drugs which are able to block more than one type of potassium channel is probably more hazardous than the use of a specific inhibitor of one given sort of potassium channel, and when simultaneous blockade of several kinds of potassium channel may be presumed, a detailed study is needed to define the determinants of ‘repolarization reserve’.

Electrophysiological effects of dronedarone (SR 33589), a noniodinated amiodarone derivative in the canine heart: comparison with amiodarone

The electrophysiological effects of dronedarone, a new nonionidated analogue of amiodarone were studied after chronic and acute administration in dog Purkinje fibres, papillary muscle and isolated ventricular myocytes, and compared with those of amiodarone by applying conventional microelectrode and patch‐clamp techniques. Chronic treatment with dronedarone (2×25u2003mg−1u2003kg−1 day p.o. for 4 weeks), unlike chronic administration of amiodarone (50u2003mg−1u2003kg−1 day p.o. for 4 weeks), did not lengthen significantly the QTc interval of the electrocardiogram or the action potential duration (APD) in papillary muscle. After chronic oral treatment with dronedarone a small, but significant use‐dependent Vmax block was noticed, while after chronic amiodarone administration a strong use‐dependent Vmax depression was observed. Acute superfusion of dronedarone (10u2003μM), similar to that of amiodarone (10u2003μM), moderately lengthened APD in papillary muscle (at 1u2003Hz from 239.6±5.3 to 248.6±5.3u2003ms, n=13, P<0.05), but shortened it in Purkinje fibres (at 1u2003Hz from 309.6±11.8 to 287.1±10.8u2003ms, n=7, P<0.05). Both dronedarone (10u2003μM) and amiodarone (10u2003μM) superfusion reduced the incidence of early and delayed afterdepolarizations evoked by 1u2003μM dofetilide and 0.2u2003μM strophantidine in Purkinje fibres. In patch‐clamp experiments 10u2003μM dronedarone markedly reduced the L‐type calcium current (76.5±0.7 %, n=6, P<0.05) and the rapid component of the delayed rectifier potassium current (97±1.2 %, n=5, P<0.05) in ventricular myocytes. It is concluded that after acute administration dronedarone exhibits effects on cardiac electrical activity similar to those of amiodarone, but it lacks the ‘amiodarone like’ chronic electrophysiological characteristics.

Pharmacological block of the slow component of the outward delayed rectifier current (IKs) fails to lengthen rabbit ventricular muscle QTc and action potential duration

The effects of IKs block by chromanol 293B and L‐735,821 on rabbit QT‐interval, action potential duration (APD), and membrane current were compared to those of E‐4031, a recognized IKr blocker. Measurements were made in rabbit Langendorff‐perfused whole hearts, isolated papillary muscle, and single isolated ventricular myocytes. Neither chromanol 293B (10u2003μM) nor L‐735,821 (100u2003nM) had a significant effect on QTc interval in Langendorff‐perfused hearts. E‐4031 (100u2003nM), on the other hand, significantly increased QTc interval (35.6±3.9%, n=8, P<0.05). Similarly both chromanol 293B (10u2003μM) and L‐735,821 (100u2003nM) produced little increase in papillary muscle APD (less than 7%) while pacing at cycle lengths between 300 and 5000u2003ms. In contrast, E‐4031 (100u2003nM) markedly increased (30u2003–u200360%) APD in a reverse frequency‐dependent manner. In ventricular myocytes, the same concentrations of chromanol 293B (10u2003μM), L‐735,821 (100u2003nM) and E‐4031 (1u2003μM) markedly or totally blocked IKs and IKr, respectively. IKs tail currents activated slowly (at +30u2003mV, τ=888.1±48.2u2003ms, n=21) and deactivated rapidly (at −40u2003mV, τ=157.1±4.7u2003ms, n=22), while IKr tail currents activated rapidly (at +30u2003mV, τ=35.5±3.1u2003ms, n=26) and deactivated slowly (at −40u2003mV, τ1=641.5±29.0u2003ms, τ2=6531±343, n=35). IKr was estimated to contribute substantially more to total current density during normal ventricular muscle action potentials (i.e., after a 150u2003ms square pulse to +30u2003mV) than does IKs. These findings indicate that block of IKs is not likely to provide antiarrhythmic benefit by lengthening normal ventricular muscle QTc, APD, and refractoriness over a wide range of frequencies.

The slow component of the delayed rectifier potassium current in undiseased human ventricular myocytes

OBJECTIVEnThe purpose of this study was to investigate the properties of the slow component of the delayed rectifier potassium current (I(Ks)) in myocytes isolated from undiseased human left ventricles.nnnMETHODSnThe whole-cell configuration of the patch-clamp technique was applied in 58 left ventricular myocytes from 15 hearts at 37 degrees C. Nisoldipine (1 microM) was used to block inward calcium current (I(Ca)) and E-4031 (1-5 microM) was applied to inhibit the rapid component of the delayed rectifier potassium current (I(Kr)).nnnRESULTSnIn 31 myocytes, an E-4031 insensitive, but L-735,821 and chromanol 293B sensitive, tail current was identified which was attributed to the slow component of I(K) (I(Ks)). Activation of I(Ks) was slow (tau=903+/-101 ms at 50 mV, n=14), but deactivation of the current was relatively rapid (tau=122.4+/-11.7 ms at -40 mV, n=19). The activation of I(Ks) was voltage independent but its deactivation showed clear voltage dependence. The deactivation was faster at negative voltages (about 100 ms at -50 mV) and slower at depolarized potentials (about 300 ms at 0 mV). In six cells, the reversal potential was -81.6+/-2.8 mV on an average which is close to the K(+) equilibrium potential suggesting K(+) as the main charge carrier.nnnCONCLUSIONnIn undiseased human ventricular myocytes, I(Ks) exhibits slow activation and fast deactivation kinetics. Therefore, in humans I(Ks) differs from that reported in guinea pig, and it best resembles I(Ks) described in dog and rabbit ventricular myocytes.

Delayed rectifier potassium current in undiseased human ventricular myocytes

OBJECTIVEnThe purpose of the study was to investigate the properties of the delayed rectifier potassium current (IK) in myocytes isolated from undiseased human left ventricles.nnnMETHODSnThe whole-cell configuration of the patch-clamp technique was applied in 28 left ventricular myocytes from 13 hearts at 35 degrees C.nnnRESULTSnAn E-4031 sensitive tail current identified the rapid component of IK (IKr) in the myocytes, but there was no evidence for an E-4031 insensitive slow component of IK (IKs). When nifedipine (5 microM) was used to block the inward calcium current (ICa), IKr activation was fast (tau = 31.0 +/- 7.4 ms, at +30 mV, n = 5) and deactivation kinetics were biexponential and relatively slow (tau 1 = 600.0 +/- 53.9 ms and tau 2 = 6792.2 +/- 875.7 ms, at -40 mV, n = 7). Application of CdCl2 (250 microM) to block ICa altered the voltage dependence of the IKr considerably, slowing its activation (tau = 657.1 +/- 109.1 ms, at +30 mV, n = 5) and accelerating its deactivation (tau = 104.0 +/- 18.5 ms, at -40 mV, n = 8).nnnCONCLUSIONSnIn undiseased human ventricle at 35 degrees C IKr exists having fast activation and slow deactivation kinetics; however, there was no evidence found for an expressed IKs. IKr probably plays an important role in the frequency dependent modulation of repolarization in undiseased human ventricle, and is a target for many Class III antiarrhythmic drugs.

Effects of endothelin-1 on calcium and potassium currents in undiseased human ventricular myocytes

Abstract. Endothelins have been reported to exert a wide range of electrophysiological effects in mammalian cardiac cells. These results are controversial and human data are not available. Our aim was to study the effects of endothelin-1 (ET-1, 8xa0nmol/l) on the L-type calcium current (ICa-L) and various potassium currents (rapid component of the delayed rectifier, IKr; transient outward current, Ito; and the inward rectifier K current, IK1) in isolated human ventricular cardiomyocytes. Cells were obtained from undiseased donor hearts using collagenase digestion via the segment perfusion technique. The whole-cell configuration of the patch-clamp technique was applied to measure ionic currents at 37°C. ET-1 significantly decreased peak ICa-L from 10.2±0.6 to 6.8±0.8xa0pA/pF at +5xa0mV (66.7% of control, P<0.05, n=5). This reduction of peak current was accompanied by a lengthening of inactivation. The voltage dependence of steady-state activation and inactivation was not altered by ET-1. IKr, measured as tail current amplitudes at –40xa0mV, decreased from 0.31±0.02 to 0.06±0.02xa0pA/pF (20.3% of control, P<0.05, n=4) after exposure to ET-1. ET-1 failed to change the peak amplitude of Ito, measured at +50xa0mV (9.3±4.6 and 9.0±4.4xa0pA/pF before and after ET-1, respectively), or steady-state IK1 amplitude, measured at the end of a 400-ms hyperpolarization to –100xa0mV (3.6±1.4 and 3.7±1.4xa0pA/pF, n=4). The present results indicate that in undiseased human ventricular myocytes ET-1 inhibits both ICa-L and IKr; however, the degree of suppression of the two currents is different.

Effect of a neuroprotective drug, eliprodil on cardiac repolarisation: importance of the decreased repolarisation reserve in the development of proarrhythmic risk

The aim of this study was to analyse the effects of eliprodil, a noncardiac drug with neuroprotective properties, on the cardiac repolarisation under in vitro circumstances, under normal conditions and after the attenuation of the ‘repolarisation reserve’ by blocking the inward rectifier potassium current (IK1) current with BaCl2. In canine right ventricular papillary muscle by applying the conventional microelectrode technique, under normal conditions, eliprodil (1 μM) produced a moderate reverse rate‐dependent prolongation of the action potential duration (7.4±1.5, 8.9±2.1 and 9.9±1.8% at cycle lengths of 300, 1000 and 5000 ms, respectively; n=9). This effect was augmented in preparations where IK1 was previously blocked by BaCl2 (10 μM). BaCl2 alone lengthened APD in a reverse frequency‐dependent manner (7.0±1.3, 14.2±1.6 and 28.1±2.1% at cycle lengths of 300, 1000 and 5000 ms, respectively; n=8). When eliprodil (1 μM) was administered to these preparations, the drug induced a marked further lengthening relative to the APD values measured after the administration of BaCl2 (12.5±1.0, 17.6±1.5 and 20.5±0.9% at cycle lengths of 300, 1000 and 5000 ms, respectively; n=8). In the normal Langendorff‐perfused rabbit heart, eliprodil (1 μM) produced a significant QTc prolongation at 1 Hz stimulation frequency (12.7±1.8%, n=9). After the attenuation of the ‘repolarisation reserve’ by the IK1 blocker BaCl2 (10 μM), the eliprodil‐evoked QTc prolongation was greatly enhanced (28.5±7.9%, n=6). In two out of six Langendorff preparations, this QTc lengthening degenerated into torsade de pointes ventricular tachycardia. Eliprodil significantly decreased the amplitude of rapid component of the delayed rectifier potassium current (IKr), but slow component (IKs), transient outward current (Ito) and IK1 were not considerably affected by the drug when measured in dog ventricular myocytes by applying the whole‐cell configuration of the patch‐clamp technique. The results indicate that eliprodil, under normal conditions, moderately lengthens cardiac repolarisation by inhibition of IKr. However, after the attenuation of the normal ‘repolarisation reserve’, this drug can induce marked QT interval prolongation, which may result in proarrhythmic action.

Multiple cellular electrophysiological effects of azimilide in canine cardiac preparations

The cellular electrophysiological effect of azimilide (0.1-30 microM) was analyzed in canine ventricular preparations by applying the standard microelectrode and patch-clamp techniques at 37 degrees C. In papillary muscle, the drug prolonged the action potential duration (APD) in a concentration-dependent manner at a cycle length (CL) of 1000 ms. In Purkinje fibers, at the same CL, the concentration-dependent lengthening of the APD was observed in the presence of up to 3 microM azimilide (at 3.0 microM: 24.1+/-4.2%, n=9); at higher drug concentration, no further APD prolongation was observed. Azimilide lengthened APD in a reverse frequency-dependent manner in papillary muscle and Purkinje fibers alike. Azimilide (10 microM) caused a rate-dependent depression in the maximal upstroke velocity of the action potential (V(max)) in papillary muscle. The time and rate constants of the offset and onset kinetics of this V(max) block were 1754+/-267 ms (n=6) and 5.1+/-0.4 beats (n=6), respectively. Azimilide did not prevent the APD shortening effect of 10 microM pinacidil in papillary muscle, suggesting that the drug does not influence the ATP-sensitive K(+) current. Azimilide inhibited the rapid (I(Kr)) and slow component (I(Ks)) of the delayed rectifier K(+) current and the L-type Ca(2+) current (I(Ca)). The estimated EC(50) value of the drug was 0.59 microM for I(Ks), 0.39 microM for I(Kr) and 7.5 microM for I(Ca). The transient outward (I(to)) and the inward rectifier (I(k1)) K(+) currents were not influenced by the drug. It is concluded that the site of action of azimilide is multiple, it inhibits not only K(+) (I(Kr), I(Ks)) currents but, in higher concentrations, it also exerts calcium- and use-dependent sodium channel block.

Antiarrhythmic and electrophysiological effects of GYKI-16638, a novel / N- phenoxyalkyl -N-phenylalkylamine, in rabbits

The effect of N-[4-[2-N-methyl-N-[1-methyl-2-(2, 6-dimethylphenoxy)ethylamino]-ethyl]-phenyl]-methanesulfonamide. hydrochloride (GYKI-16638; 0.03 and 0.1 mg/kg, i.v.), a novel antiarrhythmic compound, was assessed and compared to that of D-sotalol (1 and 3 mg/kg, i.v.) on arrhythmias induced by 10 min of coronary artery occlusion and 10 min of reperfusion in anaesthetized rabbits. Also, its cellular electrophysiological effects were studied in rabbit right ventricular papillary muscle preparations and in rabbit single isolated ventricular myocytes. In anaesthetized rabbits, intravenous administration of 0.03 and 0.1 mg/kg GYKI-16638 and 1 and 3 mg/kg D-sotalol significantly increased survival during reperfusion (GYKI-16638: 82% and 77%, D-sotalol: 75% and 83% vs. 18% in controls, P<0.05, respectively). GYKI-16638 (0.1 mg/kg) significantly increased the number of animals that did not develop arrhythmias during reperfusion (46% vs. 0% in controls, P<0.05). In isolated rabbit right ventricular papillary muscle, 2 microM GYKI-16638, at 1 Hz stimulation frequency, lengthened the action potential duration at 50% and 90% repolarization (APD(50-90)) without influencing the resting membrane potential and action potential amplitude (APA). It decreased the maximal rate of depolarization (V(max)) in a use-dependent manner. This effect was statistically significant only at stimulation cycle lengths shorter than 700 ms. The offset kinetics of this V(max) block were relatively rapid, the corresponding time constant for recovery of V(max) was 328.2+/-65.0 ms. In patch-clamp experiments, performed in rabbit ventricular myocytes, 2 microM GYKI-16638 markedly depressed the rapid component of the delayed rectifier outward and moderately decreased the inward rectifier K(+) current without significantly altering the slow component of the delayed rectifier and transient outward K(+) currents. These results suggest that in rabbits, GYKI-16638 has an in vivo antiarrhythmic effect, comparable to that of D-sotalol, which can be best explained by its combined Class I/B and Class III actions.