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(Circulation. 2003;107:2753.)
© 2003 American Heart Association, Inc.

Basic Science Reports

Probing the Contribution of I_Ks to Canine Ventricular Repolarization

Key Role for ß-Adrenergic Receptor Stimulation

Paul G.A. Volders, MD, PhD; Milan Stengl, MD, PhD; Jurren M. van Opstal, MD, PhD; Uwe Gerlach, PhD; Roel L.H.M.G. Spätjens, BS; Jet D.M. Beekman; Karin R. Sipido, MD, PhD; Marc A. Vos, PhD

From the Department of Cardiology, Cardiovascular Research Institute Maastricht, Academic Hospital Maastricht, Netherlands; Aventis Pharma Deutschland GmbH, Medicinal Chemistry (U.G.), Frankfurt/Main, Germany; and the Laboratory of Experimental Cardiology (K.R.S.), University of Leuven, Belgium.

Correspondence to Paul G.A. Volders, MD, PhD, Department of Cardiology, Cardiovascular Research Institute Maastricht, Academic Hospital Maastricht, PO Box 5800, 6202 AZ, Maastricht, Netherlands. E-mail p.volders{at}cardio.azm.nl

	Abstract

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Background— In large mammals and humans, the contribution of I_Ks to ventricular repolarization is still incompletely understood.

Methods and Results— In vivo and cellular electrophysiological experiments were conducted to study I_Ks in canine ventricular repolarization. In conscious dogs, administration of the selective I_Ks 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, I_Ks 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 I_Ks in the voltage and time domains of the AP, although at seconds-long depolarizations, the current was substantial. Isoproterenol increased and accelerated I_Ks 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 I_Kr block, I_Ks block exaggerated repolarization instability with further prolongation of APD.

Conclusions— Ventricular repolarization in conscious dogs is importantly dependent on I_Ks. I_Ks function becomes prominent during ß-adrenergic receptor stimulation, when it promotes AP shortening by increased activation, and during I_Kr block, when it limits repolarization instability by time-dependent activation. Unstimulated I_Ks does not contribute to cellular APD at baseline. These data highlight the importance of the synergism between an intact basal I_Ks and the sympathetic nervous system in vivo.

Key Words: ion channels • action potentials • receptors, adrenergic, beta • long-QT syndrome • ventricles

	Introduction

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In large mammals and humans, the contribution of I_Ks to the ventricular action potential (AP) is still unclear. In voltage-clamp studies, I_Ks appears as a large outward current during seconds-long depolarizing pulses. I_Ks 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 I_Ks.^1,2 Finally, some studies with the I_Ks-blocking drug chromanol 293B (293B) show AP prolongation in the dog³ and human.⁴

Other studies, however, indicate that pharmacological I_Ks block does not prolong canine ventricular AP durations (APDs) under baseline conditions.^5,6 Also, in canine cardiac Purkinje cells, without ß-adrenergic receptor stimulation, I_Ks contributes little to repolarization.⁷ Furthermore, in voltage-clamped canine ventricular myocytes, I_Ks 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 I_Ks, 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 I_Ks block for delaying repolarization in vivo.

	Methods

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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 I_Ks blocker HMR 1556^10,11 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 (10x1x1 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.)

Statistics
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.

	Results

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QT Prolongation by I_Ks 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 QT_c 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 I_Ks block (see below). Plasma concentrations returned to zero within 24 hours.

View larger version (38K): [in this window] [in a new window]   Figure 1. Dose-dependent QT prolongation by IKs block in conscious dogs. A, Mean QTc responses after oral doses of 3, 10, and 30 mg/kg HMR 1556 versus placebo (0.5% hydroxyethylcellulose) in 6 animals. *P<0.05. B, Holter recording showing typical changes of QT interval and T-wave morphology at peak response (middle) and 21 hours after 30 mg/kg.

I_Ks Block by HMR 1556 and 293B
The cellular basis for in vivo QT prolongation by I_Ks block was studied in isolated canine ventricular myocytes and multicellular preparations. HMR 1556 and 293B could fully block I_Ks (Figure 2A), because they inhibited all time-dependent activating and tail currents. Concentration-response studies on I_Ks tails (Figure 2B) confirmed that HMR 1556 is a more potent I_Ks blocker than 293B.^10,11

View larger version (29K): [in this window] [in a new window]   Figure 2. IKs block by HMR 1556 and 293B fails to prolong APD in single canine ventricular myocytes at baseline. A, Full block of time-dependent IKs activation (-20 to 50 mV) and tail currents (-25 mV) by 100 µmol/L 293B in 0 [K+]o. B, Concentration-response curves for 293B (IC50=8 µmol/L) and HMR 1556 (IC50=65 nmol/L) on IKs tails. C, APD95/CL relations at baseline and during IKs block (high-resistance microelectrodes). Open bars indicate baseline; solid or hatched bars, IKs block. Values are mean±SEM of 12 myocytes.

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 I_to 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, >3xIC₅₀ for I_Ks block) and 500 nmol/L HMR 1556 (>7xIC₅₀). Mean APDs remained unaltered at all CLs. Fast pacing–dependent shortening of the APD was maintained.

To directly compare I_Ks and I_Kr in individual cells, 293B-sensitive and almokalant-sensitive outward currents were examined during (APD-relevant) short depolarizations (V_test) of 300 ms. At normal external K⁺ concentration ([K⁺]_o), I_Kr activation reached maximal amplitudes within tens of milliseconds, whereas I_Ks became significant only at the end of 300-ms depolarizations. In Figure 3, voltage-and time-dependent activation of I_Ks is shown for 31 cells in 0 [K⁺]_o. The arrow indicates that at 300-ms duration, no significant I_Ks is generated at voltage steps (V_test) 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 I_Ks could be measured during 5000-ms V_test 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

View larger version (18K): [in this window] [in a new window]   Figure 3. Voltage- and time-dependent activation of IKs. IKs density–Vtest relations in 0 [K+]o. Time-dependent outward-current amplitudes were measured relative to their (close-to-)zero level at 100 ms. Values are mean±SEM of 31 cells. Arrow indicates that for clamp pulses of 300-ms duration, no significant IKs was generated if Vtest was 10 mV.

I_Ks Enhancement by ß-Adrenergic Receptor Stimulation
I_Ks 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 I_Ks during very short pulses of 100 ms when hardly any current had been measurable at baseline. Enhancement and acceleration modified I_Ks such that for 300-ms pulses, significant current amplitudes were reached at V_test equal to or more positive than -10 mV (Figure 4B). Enhanced I_Ks was completely inhibited by 100 µmol/L 293B (Figure 4C) or 500 nmol/L HMR 1556, indicating that the concentration-response curves of I_Ks inhibition at baseline were not shifted to the right by moderate ß-adrenergic receptor stimulation.

View larger version (28K): [in this window] [in a new window]   Figure 4. IKs enhancement during isoproterenol. A, Time-dependent activation during depolarizations of 100 to 2900 ms with 200-ms increments. B, Voltage-dependent activation in 11 cells. Protocol is same as in Figure 3. *P<0.05 vs baseline. C, Inhibition of isoproterenol-enhanced IKs, as evident from complete loss of tail currents, by 293B (100 µmol/L).

In AP recordings in single myocytes, isoproterenol (20 or 40 nmol/L) increased plateau V_m by maximally 12 mV and prolonged APD at V_m >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 I_Ks inhibition manifested at the end of the plateau, ie, >100 ms after the upstroke, resulting in prolongation of the APD₉₅. During isoproterenol plus I_Ks block, fast pacing–dependent shortening of the APD was maintained. These data indicated that ß-adrenergic receptor stimulation enhanced I_Ks directly and via favorable changes of the AP profile (increased plateau V_m and longer APD at V_m >0 mV).

View larger version (28K): [in this window] [in a new window]   Figure 5. ß-Adrenergic shortening of ventricular AP is partly reversed by IKs block. Data from isolated myocytes. A, Representative AP traces at baseline and during administration of isoproterenol (20 or 40 nmol/L), isoproterenol plus IKs blocker 293B, or isoproterenol plus HMR 1556. B, APD95/CL relations. Left, 293B (30 µmol/L). Right, HMR 1556 (500 nmol/L). *P<0.05 isoproterenol vs baseline; #P<0.05 isoproterenol vs isoproterenol plus IKs blocker.

I_Ks Block During ß-Adrenergic Receptor Stimulation in Ventricular Tissue
Given the possibility that I_Ks 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 ß-adrenergic receptor stimulation for I_Ks in intact ventricular repolarization and ruling out a major negative influence of the enzymatic isolation procedure on I_Ks in single cells.

View larger version (14K): [in this window] [in a new window]   Figure 6. ß-Adrenergic shortening of ventricular AP is partly reversed by IKs block. Data from multicellular preparations. Left, APD95/CL relations at baseline and during administration of isoproterenol (40 nmol/L) and isoproterenol plus IKs blocker HMR 1556 (500 nmol/L). *P<0.05 isoproterenol vs baseline; #P<0.05 isoproterenol vs isoproterenol plus IKs blocker. Right, transmembrane potential recordings. Bars=100 ms and 50 mV.

Safety-Factor Role of I_Ks When Repolarization Is Impaired by I_Kr Block
Finally, we evaluated the effects of I_Ks block after AP preprolongation with the I_Kr blocker almokalant (1 µmol/L; Figure 7 for CL 2000 ms) in single cells, assuming that during a longer AP, more I_Ks 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.

View larger version (27K): [in this window] [in a new window]   Figure 7. Exaggerated repolarization instability by IKs block after AP preprolongation with IKr blocker almokalant. A, APs at CL 2000 ms. B, Beat-to-beat APD95 at baseline and during superfusion with 293B (30 µmol/L; left black bar), almokalant (1 µmol/L; gray bar), and almokalant plus 293B (right black bar), with washout periods to show reversibility of effects. C, Poincaré plots of APD95 data depicted in B.

	Discussion

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The results of the present study indicate that (1) I_Ks is a major contributor to ventricular repolarization in conscious dogs; (2) I_Ks is most prominent during ß-adrenergic receptor stimulation, when it promotes AP shortening by increased and accelerated activation, and during I_Kr block, when it limits repolarization prolongation and instability by time-dependent activation; and (3) under baseline conditions in ventricular myocytes, adrenergically unstimulated I_Ks does not contribute significantly to repolarization, because its activation is restrained by the voltage and time domains of the AP.

I_Ks Is a Major Contributor to Ventricular Repolarization in Conscious Dogs
I_Ks-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 I_Ks. Thomas et al¹¹ found an IC₅₀ for I_Ks block of 11 nmol/L, whereas I_Kr, I_CaL, and I_to were only half-maximally inhibited at 13, 28, and 34 µmol/L, indicating >1000-fold selectivity. I_K1 was unaffected at concentrations as large as 50 µmol/L. In our hands, HMR 1556 blocked I_Ks 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 I_Ks 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 I_Ks block, although a small contribution of I_Kr block¹¹ cannot be excluded. At the lower dosages (3 and 10 mg/kg), the effects must be fully ascribed to the inhibition of I_Ks with QT_c prolongations of 11±1% and 34±5%, respectively.

Other investigators administered the benzodiazepine-derived I_Ks 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.

I_Ks Becomes Prominent During I_Kr Block and ß-Adrenergic Receptor Stimulation
In Poincaré plots of prolonged APDs during I_Kr block with almokalant, we noted an exaggeration of repolarization instability when 293B was also added. This strongly suggested that time-dependent activation of I_Ks is recruited when other influences cause excessive AP prolongation.^5,20 Such a protective role would be enhanced during naturally stimulated or drug-induced I_Ks increases. ß-Adrenergic receptor stimulation increased and accelerated I_Ks activation, so that it contributed significantly to AP shortening.

Shimizu and Antzelevitch³ provided the important insight that I_Ks block combined with ß-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, I_Ks is inhibited in wedge preparations.

Kinetics of I_Ks Activation Preclude a Significant Contribution to Cellular Repolarization at Baseline
Among the known sarcolemmal K⁺ currents, I_Ks has distinctively slow activation kinetics and a unique pharmacological responsiveness, ß-adrenergic sensitivity, and regional myocardial distribution. In our recordings, I_Ks 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 I_Ks 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.

Clinical Implications
Pharmacological inhibition of I_Ks 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 ß-blocking therapy, sympathetic ganglionectomy, or other modalities could be tested.

On the basis of the results of this study, the antiarrhythmic properties of I_Ks 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 QT_c 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 I_Ks.

Finally, the present data indicate that the inhibition of I_Ks under circumstances in which I_Kr is already blocked or downregulated can cause an exaggeration of repolarization instability and AP prolongation, possibly leading to arrhythmias.

Limitations
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 I_Ks does not contribute significantly to cellular APD under baseline conditions. However, the fact that I_Ks 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 I_Ks 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 I_Ks and that this would bias the conclusion that I_Ks 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, I_CaL) were not influenced by EGTA.²⁶ This would also apply to I_Ks function; and (2) our AP recordings were obtained with high-resistance microelectrodes with no Ca²⁺ buffer inside.

Conclusions
Both our in vivo and cellular data highlight the prominent role of I_Ks in canine ventricular repolarization. I_Ks limits excessive AP prolongation by time-dependent activation during I_Kr block. ß-Adrenergic receptor stimulation increases and accelerates I_Ks activation to promote APD₉₅ shortening. The synergism between an intact basal I_Ks and a balanced sympathetic nervous function promotes a short and homogeneous repolarization of the ventricles.

	Acknowledgments

 
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.

	Footnotes

 
Additional information about Methods is available in the online-only Data Supplement at http://www.circulationaha.org.

Received December 3, 2002; revision received March 4, 2003; accepted March 6, 2003.

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