Probing the Contribution of IKs to Canine Ventricular Repolarization
Key Role for β-Adrenergic Receptor Stimulation
Background— In large mammals and humans, the contribution of IKs to ventricular repolarization is still incompletely understood.
Methods and Results— In vivo and cellular electrophysiological experiments were conducted to study IKs in canine ventricular repolarization. In conscious dogs, administration of the selective IKs blocker HMR 1556 (3, 10, or 30 mg/kg PO) caused substantial dose-dependent QT prolongations with broad-based T waves. In isolated ventricular myocytes under baseline conditions, however, IKs block (chromanols HMR 1556 and 293B) did not significantly prolong action potential duration (APD) at fast or slow steady-state pacing rates. This was because of the limited activation of IKs in the voltage and time domains of the AP, although at seconds-long depolarizations, the current was substantial. Isoproterenol increased and accelerated IKs activation to promote APD95 shortening. This shortening was importantly reversed by HMR 1556 and 293B. Quantitatively similar effects were obtained in ventricular-tissue preparations. Finally, when cellular repolarization was impaired by IKr block, IKs block exaggerated repolarization instability with further prolongation of APD.
Conclusions— Ventricular repolarization in conscious dogs is importantly dependent on IKs. IKs function becomes prominent during β-adrenergic receptor stimulation, when it promotes AP shortening by increased activation, and during IKr block, when it limits repolarization instability by time-dependent activation. Unstimulated IKs does not contribute to cellular APD at baseline. These data highlight the importance of the synergism between an intact basal IKs and the sympathetic nervous system in vivo.
Received December 3, 2002; revision received March 4, 2003; accepted March 6, 2003.
In large mammals and humans, the contribution of IKs to the ventricular action potential (AP) is still unclear. In voltage-clamp studies, IKs appears as a large outward current during seconds-long depolarizing pulses. IKs deficiencies in the human congenital long-QT (LQT) syndromes 1 and 5 and in the Jervell and Lange-Nielsen syndrome are often associated with abnormally long QT intervals. Acquired QT prolongation (eg, in cardiac hypertrophy or failure) is often attended by a downregulation of IKs.1,2 Finally, some studies with the IKs-blocking drug chromanol 293B (293B) show AP prolongation in the dog3 and human.4
Other studies, however, indicate that pharmacological IKs block does not prolong canine ventricular AP durations (APDs) under baseline conditions.5,6 Also, in canine cardiac Purkinje cells, without β-adrenergic receptor stimulation, IKs contributes little to repolarization.7 Furthermore, in voltage-clamped canine ventricular myocytes, IKs is poorly activated during AP commands of normal duration.5 Discussions on these data and their implications are vivid.8,9
Given these paradoxes in the understanding of IKs, we combined in vivo and cellular electrophysiological experiments in dogs with the aim of better defining the importance of this K+ current for repolarization and to assess the usefulness of pharmacological IKs block for delaying repolarization in vivo.
Animal handling was in accordance with the Dutch Law on Animal Experimentation and the European Directive for the Protection of Vertebrate Animals Used for Experimental and Other Scientific Purposes (86/609/EU). Thirty-eight adult mongrel dogs (Maastricht University, Netherlands) and 6 beagle dogs (Center de Recherches Biologiques, Baugy, France) of either sex, weighing 12 to 37 kg, were used for the experiments.
In Vivo Studies With HMR 1556
For studies in conscious dogs (6 beagle dogs and 4 mongrel dogs), aliquots of the selective IKs blocker HMR 155610,11 were suspended in 0.5% hydroxyethylcellulose or packed in gelatin capsules and fed to the animals in the morning after overnight fasting. ECG monitoring was done with 24-hour Holter recordings or with telemetry transmitters and a computerized acquisition system (Data Science Inc). Concentrations of HMR 1556 in venous plasma were determined. (See Online Text Supplement 1.)
Cellular and Tissue Experiments
The procedure for isolating ventricular cells was the same as described earlier.12 (See Online Text Supplement 2.) Myocytes were obtained from most of the transmural wall, except from a rim (1.5 mm) of epicardial and endocardial tissue. In separate experiments, transmural needle biopsies (≈10×1×1 mm) were obtained from the left ventricular free wall for multicellular work. Experiments were performed at 37±1°C on a total of 83 cells and 4 biopsies from 35 dogs. (See Online Text Supplement 3.)
The data are expressed as mean±SEM. Intergroup comparisons were made with Student’s t test for unpaired and paired data groups after testing for the normality of distribution. Multiple groups were analyzed by 1-way ANOVA. Differences were considered significant at a value of P<0.05.
QT Prolongation by IKs Block in Conscious Dogs
ECG changes on single oral administration of HMR 1556 at 3, 10, or 30 mg/kg were monitored in 10 conscious dogs. As shown in Figure 1, HMR 1556 caused a substantial dose-dependent prolongation of the QTc interval (Fridericia’s formula) within 3 hours of administration. T waves were broad-based and asymmetrical and had an unaltered polarity under these conditions (Figure 1B). P-wave and QRS-complex morphology and PQ and QRS intervals did not change. Although most dogs tended to develop a slight drop in heart rate during HMR 1556, this effect was not statistically significant at any dose. After 30 mg/kg, the maximal measured plasma HMR-1556 concentration was 2.68±0.55 μmol/L, providing full IKs block (see below). Plasma concentrations returned to zero within 24 hours.
IKs Block by HMR 1556 and 293B
The cellular basis for in vivo QT prolongation by IKs block was studied in isolated canine ventricular myocytes and multicellular preparations. HMR 1556 and 293B could fully block IKs (Figure 2A), because they inhibited all time-dependent activating and tail currents. Concentration-response studies on IKs tails (Figure 2B) confirmed that HMR 1556 is a more potent IKs blocker than 293B.10,11
During AP recordings with high-resistance microelectrodes under baseline conditions, AP configurations remained unaltered at 3 and 10 μmol/L 293B, but a slight loss of the notch occurred at 30 μmol/L, consistent with Ito inhibition.13 Complete loss of the notch, triangulation of the AP, and aspecific APD responses were observed at 100 μmol/L. No AP changes were observed with HMR 1556. In Figure 2C, the APD at 95% of repolarization (APD95) is plotted against pacing cycle length (CL) during 30 μmol/L 293B (ie, >3×IC50 for IKs block) and 500 nmol/L HMR 1556 (>7×IC50). Mean APDs remained unaltered at all CLs. Fast pacing–dependent shortening of the APD was maintained.
To directly compare IKs and IKr in individual cells, 293B-sensitive and almokalant-sensitive outward currents were examined during (APD-relevant) short depolarizations (Vtest) of 300 ms. At normal external K+ concentration ([K+]o), IKr activation reached maximal amplitudes within tens of milliseconds, whereas IKs became significant only at the end of 300-ms depolarizations. In Figure 3, voltage-and time-dependent activation of IKs is shown for 31 cells in 0 [K+]o. The arrow indicates that at 300-ms duration, no significant IKs is generated at voltage steps (Vtest) ≤10 mV. However, at 3000-ms duration, it was 0.4±0.1 pA/pF in these same cells. The time course of full activation of IKs could be measured during 5000-ms Vtest to 20 mV. Half-maximal activation time was 702±59 ms. Half-times for deactivation were voltage dependent and decreased from 333±27 ms on repolarization to −10 mV to only 40±5 ms at −80 mV, consistent with previous data.12,14
IKs Enhancement by β-Adrenergic Receptor Stimulation
IKs was markedly enhanced by isoproterenol, consistent with previous reports.7,15 Figure 4 shows time- and voltage-dependent activation during 100 nmol/L. Compared with baseline, half-maximal activation time decreased to 510±67 ms (P<0.05). Isoproterenol even enhanced IKs during very short pulses of 100 ms when hardly any current had been measurable at baseline. Enhancement and acceleration modified IKs such that for 300-ms pulses, significant current amplitudes were reached at Vtest equal to or more positive than −10 mV (Figure 4B). Enhanced IKs was completely inhibited by 100 μmol/L 293B (Figure 4C) or 500 nmol/L HMR 1556, indicating that the concentration-response curves of IKs inhibition at baseline were not shifted to the right by moderate β-adrenergic receptor stimulation.
In AP recordings in single myocytes, isoproterenol (20 or 40 nmol/L) increased plateau Vm by maximally 12 mV and prolonged APD at Vm >0 mV. APD95, however, was shortened at these concentrations. 293B (10 and 30 μmol/L) or HMR 1556 (100 and 500 nmol/L) partially reversed this shortening, most notably at slow pacing rates (Figure 5). Invariably, the AP-prolonging effects of IKs inhibition manifested at the end of the plateau, ie, >100 ms after the upstroke, resulting in prolongation of the APD95. During isoproterenol plus IKs block, fast pacing–dependent shortening of the APD was maintained. These data indicated that β-adrenergic receptor stimulation enhanced IKs directly and via favorable changes of the AP profile (increased plateau Vm and longer APD at Vm >0 mV).
IKs Block During β-Adrenergic Receptor Stimulation in Ventricular Tissue
Given the possibility that IKs channels of individual myocytes could have been damaged by the cell-isolation procedure, which could negatively influence the AP data, we repeated our experiments with isoproterenol plus HMR 1556 in ventricular multicellular preparations (n=4 dogs). Figure 6 shows that the AP data from a broad midmyocardial layer were very similar to the AP findings in myocytes (Figure 5), confirming the importance of β-adrenergic receptor stimulation for IKs in intact ventricular repolarization and ruling out a major negative influence of the enzymatic isolation procedure on IKs in single cells.
Safety-Factor Role of IKs When Repolarization Is Impaired by IKr Block
Finally, we evaluated the effects of IKs block after AP preprolongation with the IKr blocker almokalant (1 μmol/L; Figure 7 for CL 2000 ms) in single cells, assuming that during a longer AP, more IKs is activated. Whereas 293B did not change the APD, almokalant increased it significantly, from 293±2 to 374±4 ms (+28%) at CL 500 ms and from 353±2 to 813±21 ms (+130%) at CL 2000 ms. The addition of 293B exaggerated the repolarization instability and further increased the APD from 374 to 398±19 ms and from 813 to 1120±100 ms at the same CLs (P<0.05 for both). Early afterdepolarizations were frequently seen under these circumstances. Similar observations were made at CL 1000 ms. Poincaré plots16 of APD95 (beat n/n−1; Figure 7C) showed narrow clustering of the data points at baseline, increased deviations from the line y=x during almokalant, and more complex polygons during almokalant plus 293B. These effects were reversible on washout.
The results of the present study indicate that (1) IKs is a major contributor to ventricular repolarization in conscious dogs; (2) IKs is most prominent during β-adrenergic receptor stimulation, when it promotes AP shortening by increased and accelerated activation, and during IKr block, when it limits repolarization prolongation and instability by time-dependent activation; and (3) under baseline conditions in ventricular myocytes, adrenergically unstimulated IKs does not contribute significantly to repolarization, because its activation is restrained by the voltage and time domains of the AP.
IKs Is a Major Contributor to Ventricular Repolarization in Conscious Dogs
IKs-blocking agents have become available only since 1995 (modified chromanols), 1996 (modified benzodiazepines), and 2000 (modified benzamides). Of these agents, the chromanol HMR 1556 is probably the most selective for IKs. Thomas et al11 found an IC50 for IKs block of 11 nmol/L, whereas IKr, ICaL, and Ito were only half-maximally inhibited at 13, 28, and 34 μmol/L, indicating >1000-fold selectivity. IK1 was unaffected at concentrations as large as 50 μmol/L. In our hands, HMR 1556 blocked IKs of canine ventricular myocytes half-maximally at 65 nmol/L.
Our in vivo results with HMR 1556 indicate that the QT interval can prolong quite dramatically by IKs inhibition. QT responses were dose-dependent at 3, 10, and 30 mg/kg (Figure 1). According to the selectivity data, at the highest oral dose of 30 mg/kg with a measured plasma concentration of 2.68±0.55 μmol/L, the observed repolarization effects were largely a result of IKs block, although a small contribution of IKr block11 cannot be excluded. At the lower dosages (3 and 10 mg/kg), the effects must be fully ascribed to the inhibition of IKs with QTc prolongations of 11±1% and 34±5%, respectively.
Other investigators administered the benzodiazepine-derived IKs blocker L-768,673 (IC50=6 nmol/L17) to conscious dogs and found a dose-dependent QT prolongation of 5% to 15% at 0.03 to 1.0 mg/kg.17 The oral bioavailability of L-768,673 in Methocell suspension was only 27%, and plasma concentrations were not reported. Therefore, we cannot compare these QT responses with our own findings. Bauer et al18 reported on the effects of 10 mg/kg 293B in anesthetized dogs with acute complete atrioventricular block, demonstrating that ventricular effective refractory periods were prolonged at fast (CL 300 ms) and slow (CL 850 ms) pacing rates. In dogs with subacute myocardial infarction, 293B prolonged local effective refractory periods more in the infarct zone than in normal areas.19 Unfortunately, no quantitative QT-interval or monophasic-APD data were provided.
IKs Becomes Prominent During IKr Block and β-Adrenergic Receptor Stimulation
In Poincaré plots of prolonged APDs during IKr block with almokalant, we noted an exaggeration of repolarization instability when 293B was also added. This strongly suggested that time-dependent activation of IKs is recruited when other influences cause excessive AP prolongation.5,20 Such a protective role would be enhanced during naturally stimulated or drug-induced IKs increases. β-Adrenergic receptor stimulation increased and accelerated IKs activation, so that it contributed significantly to AP shortening.
Shimizu and Antzelevitch3 provided the important insight that IKs block combined with β-adrenergic receptor stimulation in canine left ventricular wedge preparations increases transmural dispersion of repolarization and can induce polymorphic tachyarrhythmias. These investigators reported homogeneous prolongations of transmural APDs by 293B,3 which appears to be in contrast to our present finding that cellular APs were not prolonged at baseline (Figure 2). We assume that in ventricular wedge preparations, residual adrenergic activity remains present for a considerable time after excision of the tissue. For example, norepinephrine is progressively released after ≥5 to 10 minutes of no-flow ischemia and reperfusion in excised cardiac tissue of various species, including human21 and dog.22 This could mean that adrenergically stimulated, not basal, IKs is inhibited in wedge preparations.
Kinetics of IKs Activation Preclude a Significant Contribution to Cellular Repolarization at Baseline
Among the known sarcolemmal K+ currents, IKs has distinctively slow activation kinetics and a unique pharmacological responsiveness, β-adrenergic sensitivity, and regional myocardial distribution. In our recordings, IKs appeared as a robust outward current during seconds-long depolarizations. Slow activation and rapid deactivation kinetics typically characterized it. At pulses to 20 mV, half-maximal activation was reached at ≈700 ms, with little current generated in the first 100 ms of depolarization. Accordingly, drug-induced IKs inhibition did not prolong the cellular APD at baseline. Half-times for complete deactivation were as short as 40 ms at −80 mV (in line with Reference 14). The recent demonstration of similar kinetics in human ventricular myocytes23 underscores the clinical relevance of our data.
Pharmacological inhibition of IKs by HMR 1556 in conscious dogs could be suitable as an in vivo model of the human congenital LQT 1 syndrome. Future studies could focus on the potential arrhythmogenic consequences of adrenergic challenges during exercise, excitement, or arousal from sleep in this model. Alternatively, the antiarrhythmic properties of β-blocking therapy, sympathetic ganglionectomy, or other modalities could be tested.
On the basis of the results of this study, the antiarrhythmic properties of IKs blockers warrant further evaluation. Previous experiments with L-768,673 in exercising dogs with healed anterior-wall myocardial infarction and superimposed ischemia (transient occlusion of left circumflex coronary artery) demonstrated the efficacy of the drug against ventricular fibrillation, even at a modest 7% increase in QTc at baseline.24 It is tempting to speculate that cardiac sympathetic activity was intense but dispersed during exercise and regional ischemia and that L-768,673 prevented arrhythmogenic dispersion of repolarization by attenuating adrenergically sensitive IKs.
Finally, the present data indicate that the inhibition of IKs under circumstances in which IKr is already blocked or downregulated can cause an exaggeration of repolarization instability and AP prolongation, possibly leading to arrhythmias.
For our cellular experiments, we used myocytes from the transmural left ventricular free wall, except from a rim of epicardial and endocardial tissue. Thus, the layer from which the cells were isolated constitutes most of the transmural mass (≥75%), so its contribution to global repolarization would be dominant. It might be argued that the exclusion of thin layers of epicardial and endocardial myocytes could bias our conclusion that IKs does not contribute significantly to cellular APD under baseline conditions. However, the fact that IKs shows a limited activation within the normal AP is primarily because of its kinetic characteristics (this study). These characteristics are similar in epicardial, midmyocardial, and endocardial myocytes,25 indicating that even for the largest IKs amplitudes that we found (often as large as the amplitudes of epicardial and endocardial myocytes reported by others25), the contribution to repolarization is still limited.
The internal pipette solution for voltage-clamp experiments contained EGTA. One could argue that intracellular Ca2+ buffering by EGTA influences Ca2+-dependent activation of IKs and that this would bias the conclusion that IKs does not contribute significantly to cellular APD under baseline conditions. However, (1) although EGTA buffers the bulk cytoplasmic Ca2+ well, it fails to do so in the subsarcolemmal space.26 Previously, it was shown that ionic currents dependent on subsarcolemmal Ca2+ (eg, ICaL) were not influenced by EGTA.26 This would also apply to IKs function; and (2) our AP recordings were obtained with high-resistance microelectrodes with no Ca2+ buffer inside.
Both our in vivo and cellular data highlight the prominent role of IKs in canine ventricular repolarization. IKs limits excessive AP prolongation by time-dependent activation during IKr block. β-Adrenergic receptor stimulation increases and accelerates IKs activation to promote APD95 shortening. The synergism between an intact basal IKs and a balanced sympathetic nervous function promotes a short and homogeneous repolarization of the ventricles.
Dr Volders was supported by The Netherlands Organization for Health Research and Development (ZonMw 906-02-068). The contributions of Dr Eric Martel (Center de Recherches Biologiques, Baugy, France) are gratefully acknowledged. The authors wish to thank Dr Alexander Bauer and Professors Edward Carmeliet, André Kleber, and Michael Rosen for helpful discussions. Drs Wolfgang Ulmer and Dietmar Schmidt (Aventis Pharma, Frankfurt/Main, Germany) performed the HMR-1556-plasma analyses.
Additional information about Methods is available in the online-only Data Supplement at http://www.circulationaha.org.
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