Cardiovascular Research

Cardiovascular Research 1998 38(2):441-450; doi:10.1016/S0008-6363(98)00021-2
© 1998 by European Society of Cardiology

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Copyright © 1998, European Society of Cardiology

Effects of the chromanol 293B, a selective blocker of the slow, component of the delayed rectifier K⁺ current, on repolarization in human and guinea pig ventricular myocytes

Ralph F. Bosch^a, Rania Gaspo^a, Andreas E. Busch^d, Hans J. Lang^e, Gui-Rong Li^a,b and Stanley Nattel^a,b,c,*

^aDepartment of Medicine and Research Center, Montreal Heart Institute, 5000 Bélanger Street E., Montreal, Quebec H1T 1C8, Canada
^bDepartment of Medicine, University of Montreal, Montreal, Quebec, Canada
^cDepartment of Pharmacology and Therapeutics, McGill University, Montreal, Quebec, Canada
^dThe Institute of Physiology, Eberhard-Karls-Universität Tübingen, Tübingen, Germany
^eHoechst AG, Frankfurt, Germany

^* Corresponding author. Tel.: +1 (514) 376 3330; Fax: +1 (514) 376 1355; E-mail: nattel@icm.umontreal.ca

Received 15 May 1997; accepted 15 December 1997

	Abstract

Top Abstract 1 Introduction 2 Methods 3 Results 4 Discussion 5 Conclusions References

 
Objectives: The slow component of the delayed rectifier K⁺ current (I_Ks) is believed to be important in cardiac repolarization, and may be a potential target for antiarrhythmic drugs, but its study has been limited by a lack of specific blockers. The chromanol derivate 293B blocks currents expressed by minK and not HERG in Xenopus oocytes, but little is known about its effects on native currents and action potentials. We aimed to establish the effects of 293B on K⁺, Na⁺ and Ca²⁺ currents and action potentials in human and guinea pig cardiomyocytes. Methods: Whole-cell patch clamp techniques were applied to assess the effects of 293B on isolated myocytes at 36°C. Results: Delayed rectifier current (I_K) elicited by pulses to +60 mV from a holding potential of –50 mV in guinea pig myocytes was strongly inhibited by 293B (maximum inhibition 96.9±0.8%; 50% inhibitory concentration, EC₅₀, 1.02 µM), but I_K during pulses to –10 mV was unaffected (3.9±8.4% inhibition at 50 µM). Half-activation voltages, current-voltage relations, and current densities of drug-resistant and drug-sensitive I_K correspond to those of I_Kr and I_Ks respectively. Inward rectifier K⁺ current, Na⁺ current and L-type Ca²⁺ current were unaffected by 293B. Transient outward current in human ventricular myocytes was inhibited by 293B at an EC₅₀ of 24 µM, less than one twentieth the potency for I_Ks inhibition in guinea pig myocytes. While dofetilide prolonged action potential duration (APD) with strong reverse use dependence, 293B prolonged guinea pig and human ventricular APD to a similar fractional extent at all frequencies. Conclusions: 293B is a selective I_Ks blocker, and the frequency dependence of APD prolongation caused by this I_Ks blocker is different from that caused by I_Kr blockade: 293B may be an interesting tool to study the physiologic role of I_Ks and the antiarrhythmic potential of I_Ks blockade.

KEYWORDS Potassium channels; Action potential; Biophysics; Cardiac arrhythmias; Antiarrhythmic drugs

	1 Introduction

Top Abstract 1 Introduction 2 Methods 3 Results 4 Discussion 5 Conclusions References

 
The pharmacological treatment of life-threatening ventricular arrhythmias has undergone striking changes in the last few years. As a consequence of the CAST results [1, 2], the focus has shifted from sodium channel blocking drugs to compounds that act by class III mechanisms to delay repolarization. d,l-Sotalol and amiodarone were the first drugs described to have class III actions [3, 4]and are still in wide clinical use; however, both compounds have mechanisms of action other than class III. Several recently-developed pure class III drugs strongly inhibit the fast component (I_Kr) of the delayed rectifier K⁺ current, and lengthen action potential duration (APD) in a reverse rate-dependent manner [5–7]. Such drugs have the least APD-prolonging effect during tachycardia and induce maximal APD prolongation during bradycardia or after a long diastolic interval, thus increasing the risk of Torsades de Pointes arrhythmias [8, 9]. The slow component (I_Ks) of delayed rectifier current has been shown to increase at fast stimulation rates in guinea pig ventricle because of slow deactivation kinetics at negative potentials [6]. It has therefore been hypothesized that an increased contribution of I_Ks at fast heart rates could be involved in the reverse rate-dependent APD prolongation caused by I_Kr-blocking drugs, and that selective I_Ks-blocking drugs may have a more favourable rate-dependent profile of APD-prolonging action [6].

Chromanols inhibit delayed rectifier channels that may underlie the cAMP mediated epithelial K⁺ conductance in the rat colon [10]. The derivate 293B blocks currents resulting from minK expression in Xenopus oocytes and I_K in guinea pig myocardium in a concentration-dependent manner, without affecting HERG expressed in Xenopus oocytes [11]. The only studies of 293B in native myocytes to date suggest that 293B is more potent in blocking I_Ks than I_Kr [11], but the precise potency of I_Kr versus I_Ks block, effects on other currents, and effects on action potentials were not determined. The present study was designed to evaluate the effects of 293B on a variety of currents in guinea pig and human ventricular myocytes, in order to establish its selectivity of ionic action, and to assess its effects on repolarization in these tissues. The results suggest that 293B is a selective inhibitor of I_Ks that increases ventricular APD with a frequency dependence different from that of I_Kr blockers.

	2 Methods

Top Abstract 1 Introduction 2 Methods 3 Results 4 Discussion 5 Conclusions References

 
2.1 Cell isolation and solutions
Experiments were performed with guinea pig and human ventricular myocytes. Drug effects were initially characterized in detail in guinea pig cells because of the ready availability of disease-free tissue and the well-described nature of currents in the guinea pig heart. Selected studies were then performed with human ventricular myocytes, particularly to evaluate drug effects on currents (such as I_to) which are important in human ventricular myocytes [12]but absent in guinea pig hearts, and on APD. The explanted tissue samples available were from diseased human left ventricle, in which I_K has been reported to be small and difficult to detect [12], in agreement with our own findings. We therefore did not attempt to study the effects of 293B on I_K in the human heart.

The procedures for tissue procurement were approved by the Clinical Research and Animal Ethics Committees of the Montreal Heart Institute. Explanted hearts were obtained from five patients undergoing cardiac transplantation. Three had dilated cardiomyopathy and two ischemic heart disease. The hearts were initially placed in cold oxygenated (95% O₂, 5% CO₂) Kreb's solution containing (mM) NaC1 120; KC1 4; MgSO₄ 1.2; KH₂PO₄ 1.2; NaHCO₃ 25; CaC1₂ 1.25 and glucose 10. As previously described [13], a portion of the left ventricular free wall was removed along with the attached coronary artery and the coronary artery was cannulated in a cardioplegic solution (contents in mM: Na⁺ 118; K⁺ 33.5; Ca²⁺ 1.3; C1– 127.8; lactate 24; HCO₃^– 1.6; mannitol 87). The free wall was perfused with oxygenated (100% O₂, pH adjusted to 7.35 with NaOH) Tyrode's solution containing (mM) NaCl 136, KCl 5.4, MgCl 1.0; NaH₂PO₄ 0.33, HEPES 5 and glucose 10. After 20 to 30 min the solution was changed to one containing 200 to 300 U/ml collagenase (Type II, Worthington Biochemical, Freehold, NJ). Softened tissue was removed with a forceps, gently triturated and kept in a storage solution containing (mM) KCl 20, KH₂PO₄ 10, glucose 25, mannitol 40, L-glutamic acid 70, β-hydoxybutyric acid 10, taurine 20, EGTA 10, along with 1% albumin (pH adjusted to 7.35 with KOH).

Guinea pigs were sacrificed by cervical dislocation. The hearts were excised, mounted on a Langendorff apparatus and retrogradely perfused with oxygenated Tyrode's solution at 37°C. When hearts were clear of blood, the perfusate was changed to a nominally Ca²⁺-free Tyrode's solution until contraction ceased. Perfusion was continued with the same solution containing collagenase (120 U/ml, Type II, Worthington) and 1% bovine serum albumin (Sigma Chemical, St. Louis, MO) until left ventricular tissue softened. Small pieces of tissue were removed, mechanically dissociated by trituration, and isolated cells kept in the storage solution.

A small aliquot of the cell-containing solution was placed in a 1-ml perfusion chamber. After cell adhesion, the cells were perfused at 6 ml/min with a solution containing (mM) NaCl 136, KCl 5.4, CaCl₂ 2.0, MgCl 1.0; NaH₂PO₄ 0.33, HEPES 5 and glucose 10 (pH adjusted to 7.35 with NaOH) for the recording of action potentials, inward rectifier current (I_K1) and delayed rectifier current (I_K). To record I_Ca, we used a nominally Na⁺- and K⁺-free solution containing (mM) choline Cl 136, CsCl 5.6, CaCl₂ 2.0, MgCl₂ 1.0; NaH₂PO₄ 0.33, HEPES 5 and glucose 10 (pH adjusted to 7.35 with CsOH), and I_Na was recorded with a solution containing (mM) CsCl 132.5, NaCl 5.0, MgCl₂ 1.0, CaCl₂ 1.0, HEPES 20 and glucose 11 (pH adjusted to 7.35 with CsOH). To record I_K1 and I_K, Ca²⁺ current was blocked with 5 µM nifedipine (Sigma). For I_to recording, 200 µM Ba²⁺ was added to block I_K1 and 200 µM Cd²⁺ to block I_Ca. Bath temperature was maintained at the desired value (36°C for all studies other than I_Na measurement, 17°C for I_Na measurement) with a Peltier-effect device. The pipette solution used to record action potentials, I_to, I_K1 and I_K contained (mM) KCl 20, K-aspartate 110, MgCl 1.0, HEPES 10, EGTA 5, Mg₂ATP 5, GTP 0.1, Na₂-phosphocreatine 5 (pH adjusted to 7.2 with KOH). For I_Ca recordings, the pipette contained (mM) CsCl 20, Cs-aspartate 110, HEPES 10, EGTA 10, MgCl 1.0, Mg₂ATP 5, GTP 0.1, Na₂-phosphocreatine 5 (pH adjusted to 7.2 with CsOH), and for I_Na recording the pipette contained (mM) CsF 135, NaCl 5.0, HEPES 5.0, EGTA 10 and Mg₂-ATP 5.

2.2 Voltage-clamp technique
Ionic currents were recorded using the whole-cell configuration. Borosilicate glass electrodes (outer diameter 1.0 mm) had resistances around 1 M for the recording of I_Na and between 2.5 and 6 M for currents other than I_Na, and were connected to a patch clamp amplifier (Axopatch 200A, Axon Instruments, Foster City, CA). Data were sampled with an A/D converter (Digidata 1200, Axon Instruments) and stored for subsequent analysis. The sampling frequency depended on the recording interval, with a frequency of 10 kHz used for rapidly changing currents (such as I_Na, I_Ca and I_to), and frequencies as low as 0.4 kHz used for slowly changing currents like I_Ks.

Tip potentials were zeroed before formation of the pipette-membrane seal. After rupture of the cell membrane, pipette series resistance (R_s) was electrically compensated to minimize the capacitive surge on the current recording. R_s was calculated by dividing the capacitive time constant by the membrane capacitance (the time integral of the capacitive response to a 5 mV hyperpolarizing pulse from a holding potential of –60 mV, divided by the voltage drop). Membrane capacitance averaged 176.9±37.6 pF (n=9) in human and 125.2±18.2 pF (n=10) in guinea pig ventricular myocytes. Before compensation, R_s averaged 11.6±1.8 and 14.5±1.9 M in human and guinea pig cells, and the capacitive time constants were 1930±530 and 2001±264 µs, respectively. Corresponding values for R_s after compensation were 3.2±0.3 and 3.1±0.3 M, and for the capacitive time constants were 510±89 and 440±39 µs. Cells with significant leak current were rejected, and leakage compensation was not applied.

Action potentials were corrected for the junction potential. To calculate the junction potential, pipettes filled with pipette solution were immersed in pipette solution and then into the bath solution used for action potential recording. The average of 10 pipettes (11.5±0.2 mV) was subtracted from the measured resting membrane potential. Because action potential duration tended to decrease over time when recordings lasted over 10 min, drug effects on action potentials were studied by recording from several cells from each preparation in the presence or absence of drugs (i.e. each cell was studied under only one condition). All action potentials could thus be recorded within 5 min of membrane rupture and rundown avoided. A similar number of cells from each preparation was studied under all conditions.

2.3 Data analysis
Group data are expressed as the mean±SEM. A Student's paired t-test was used to compare means before and after treatment, a Student's non-paired t-test to compare results of two different groups, and a two-way analysis of variance (ANOVA) to compare group results with multiple means and frequency dependence of drug effects on APD. A two-tailed P value <0.05 was considered statistically significant. Nonlinear least-square curve-fitting was performed by CLAMPFIT in pCLAMP 6.0 or with Sigma Plot software.

	3 Results

Top Abstract 1 Introduction 2 Methods 3 Results 4 Discussion 5 Conclusions References

 
3.1 Properties of I_K block by 293B
Initial measurements of I_K were made 15 min after membrane rupture and the protocol was run at least three times at 5-min intervals to detect rundown. Only cells with stable I_K (<10% rundown over 15 min) were used (82% of cells studied). Initial measurements were followed by a 10-min drug equilibration period, and then study of drug action. In five cells, 293B was washed out for 15 min, and recovery from drug effect averaged 91±4%. Representative recordings are shown in Fig. 1A. A pulse to +60 mV caused a large instantaneous current shift due to inward rectification of I_K1, followed by a time-dependent current (upper left panel), which was strongly inhibited by 293B (50 µM, upper right). The drug effect was completely reversible upon washout (lower left). Drug-sensitive current (lower right, Fig. 1A) activated more slowly than the drug-resistant component. The current elicited by a step to –10 mV (Fig. 1B) was small and rapidly-activating (Fig. 1B, upper left panel). At –10 mV, 293B (50 µM) exerted no obvious effect on I_K step or tail currents (upper right, Fig. 1B). No drug-sensitive current was noted at –10 mV (lower right, Fig. 1B).

View larger version (17K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 1 Effects of 50 µmol/L 293B on delayed rectifier K+ current in guinea pig ventricle. Currents were elicited with 3-s pulses to voltages between –40 and +70 mV from a holding potential of –50 mV; tail currents were recorded on 2-s repolarization to –40 mV (protocol applied every 10 s). Step currents were measured as the difference between current immediately after the decay of the capacitive surge and the steady-state current at the end of the 3-s pulse. All IK recordings were at 36°C and the bath solution contained 5 µmol/L nifedipine to block calcium current. (A) Effect at a test potential of +60 mV. 293B effect was studied after 10 min of drug perfusion, and was completely reversible by 15 min of washout. (B) 293B effect at –10 mV. Drug-sensitive currents were obtained by digital subtraction.

 
Mean data for effects on I_K are shown in Fig. 2. 293B inhibited I_K at +60 mV in a concentration-dependent manner (Fig. 2A), with a 50%-effective inhibitory concentration (EC₅₀) of 1.02 µM. At –10 mV, where I_K is largely composed of I_Kr [5], the current was not significantly affected (Fig. 2B) by concentrations as high as 50 µM. We also tested the effect of 293B on tail currents after long (3 s) and brief (225 ms) activating pulses to +60 mV (Fig. 2C). At this voltage, I_Kr step current is negligible because of strong inward rectification, but deactivating I_Kr tail currents are maximally activated [5]. At short pulse durations the contribution of I_Ks to total I_K is minimized, so tail currents represent predominantly I_Kr. With 225-ms activating pulses, 293B decreased tail currents by 14±6% (n=4, P=NS), whereas with 3000-ms activating pulses, the drug reduced tail currents by 76±3% (n=6, P<0.001).

View larger version (19K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 2 Concentration and voltage dependence of delayed rectifier K+ current inhibition by 293B. The pulse protocol was the same as in Fig 1. (A) Inhibition of IKs step current elicited by 3-s depolarizations from –50 mV to +60 mV, plotted as a function of 293B concentration. Experimental data were fit with a Hill equation of the following form: f=100/(1+(x/a)b). The numbers in brackets indicate the number of cells studied at each concentration. (B) Block of IK step current (3-s pulses) at +60 and –10 mV at increasing 293B concentrations. Current amplitude in the presence of drug is expressed as a percentage of control values. (C) Block of IK tail current by 10 µmol/L 293B following short and long activating pulses. Tail current amplitudes were measured as the difference between peak outward current and steady state current at the end of a 2-s repolarizing pulse to –40 mV after a 225-ms or 3-s conditioning pulse to +60 mV from a holding potential of –50 mV. Results are from four and six cells for 225-ms and 3-s conditioning pulses respectively.

 
The voltage-dependent activation properties of 50 µM drug-sensitive and resistant I_K tails are illustrated in Fig. 3A. The half-activation voltage (V_h) for the drug-resistant component was –17.1±1.0 mV, with a slope factor (k) of 7.5±0.9 mV (n=7). For the drug-sensitive component, V_h averaged 30.9±0.4 mV and k was 11.9±0.4 mV. These values are similar to those reported for E-4031 sensitive I_Kr and E-4031 resistant I_Ks respectively [5]. Drug-sensitive current activated at voltages positive to –10 mV and displayed a linear I–V relation (Fig. 3B). In contrast, the drug-resistant component activated at more negative voltages (–30 mV), and current amplitude reached a peak at 0 mV, decreasing at more positive voltages.

View larger version (15K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 3 (A) Voltage dependence of activation of 50 µmol/L 293B sensitive and resistant IK. The activation curves were obtained by normalizing tail currents following 3-s pulses from –50 mV to the test potentials (TP) indicated to the tail currents at the most positive TP (+70 mV). Experimental data were fit with a Boltzmann function of the following form: A=1/{1+exp [Vh – Vm]/k), where A is the activation variable (tail current at test potential Vm divided by tail current at +70 mV), Vm is the test potential (TP), Vh and k are the half-activation voltage and the slope factor, respectively. (Results are mean±SEM, n=7). (B) I–V relations for 50 µmol/L 293B sensitive and resistant delayed rectifier K+ current. Step currents were measured with 3-s steps from –50 mV as described in Fig 1. (Results are mean±SEM, n=7).

 
The kinetics of 50 µM 293B-sensitive and resistant I_K were studied in five cells. At +50 mV, 293B-sensitive current had a fast activation time constant (_fast) of 417±46 ms and a slow time constant (_slow) of 1649±201 ms. Deactivation _fast at –40 mV was 216±9 ms and _slow 479±36 ms. 293B-resistant current activated and deactivated more rapidly. For example, for a test potential (TP) of 0 mV, the time constants of 293B-resistant current averaged 81±17 ms and 161±26 ms for activation and deactivation respectively. These values were compared with those of 1 µM dofetilide-resistant I_Ks and dofetilide-sensitive I_Kr in five cells. The activation time constants for I_Ks at +50 mV were 399±28 ms and 1269±188 ms for _fast and _slow respectively (P=NS versus 293B-sensitive I_K), whereas deactivation _fast and _slow averaged 257±78 ms and 630±70 ms (P=NS versus 293B-sensitive I_K). Dofetilide-sensitive I_Kr had activation time constants of 94±27 ms at 0 mV and 152±20 ms for deactivating tails (P=NS versus 293B-resistant I_K). The current density of 293B-resistant I_K at +50 mV (0.30±0.06 pA/pF, n=7) was very similar to that of dofetilide-sensitive I_K (0.31±0.04 pA/pF, n=9), and 293B-sensitive current density (2.6±0.7 pA/pF) was similar to dofetilide-resistant current density (2.5±0.4 pA/pF).

3.2 Effect of 293B on other potassium currents
To assess the specificity of 293B block of I_Ks, we studied its effects on the inward rectifier K⁺ current (I_K1) and the transient outward current (I_to). Exposure to high concentrations of 293B (50 µM) did not significantly alter I_K1 in human or guinea pig ventricle (Fig. 4). After perfusion with 50 µM 293B, steady-state current at the end of the pulse was 99.0±7.0% of the control value at –90 mV, and 97.1±3.4% at –40 mV (P=NS for both versus control) in human myocytes (n=5). For guinea pig I_K1, current amplitudes after drug exposure averaged 90.7±8.4 and 98.2±8.4% of control (n=6, P=NS versus control) at –90 and –40 mV. In contrast, 50 µM 293B significantly decreased I_to in human ventricular cells (Fig. 5A). I_to was measured with a 30-ms prepulse to –40 mV to inactivate I_Na, in order to use a HP (–80 mV) at which no I_to inactivation was present. The prepulse was too short to produce significant I_to inactivation at this voltage. I_to inhibition occurred at high concentrations, with an EC₅₀ for I_to inhibition of 24.0 µM (Fig. 5B), more than 20 times the value for I_Ks in guinea pig. Maximal inhibition was observed at 100 µM, and averaged 75.0±4.3% at +50 mV. Higher drug concentrations (up to 1 mM) did not further decrease I_to amplitude.

View larger version (22K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 4 293B (50 µmol/L) does not affect inward rectifier K+ current (IK1) in human (Panel A) and guinea pig (Panel B) ventricular myocytes. Currents were elicited at 36°C with 200-ms pulses to the voltages shown before and after perfusion with 50 µmol/L 293B. In human myocytes, 200 µmol/L CdCl2 was added to block ICa; in guinea pig cells, 5 µmol/L nifedipine was used.

 

View larger version (17K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 5 Effect of 293B on transient outward current (Ito) in human ventricular myocytes. (A) Original recordings obtained at 36°C under control conditions, in the presence of 50 µmol/L 293B, and upon washout with the use of 400-ms depolarizing pulses to the voltages indicated. Transient outward current was measured as the difference between peak outward current and the steady state current at the end of the voltage step. 200 µmol/L CdCl2 and 200 µmol/L BaCl2 were added to avoid contamination by ICa and IK1, respectively. (B) Percentage of Ito inhibition, plotted as a function of 293B concentration. Experimental data were fit with a Hill function of the following form f=75/(1+(x/a)b). Maximal inhibition was 75% at 100 µmol/L: increased drug concentrations had no further effect on Ito. The EC50 was 24.0 µmol/L.

 
3.3 Effects on other ionic currents
To pursue the specificity of drug action, we studied the effects of 293B on I_Na and I_Ca (Fig. 6). Neither I_Na nor L-type I_Ca were significantly affected by 293B in concentrations sufficient to block completely I_Ks. Overall, I_Na at –30 mV averaged 15.9±3.1 pA/pF under control conditions and 17.9±3.7 pA/pF in the presence of 50 µM 293B in human cells (n=8, P=NS), and 25.2±6.2 pA/pF (control) and 25.5±6.2 pA/pF (50 µM 293B) in guinea pig cells (n=5, P=NS). I_Ca at +10 mV averaged 4.9±0.4 pA/pF (control) and 4.6±0.5 pA/pF (50 µM 293B) in human cells (n=2, P=NS), and 9.0±0.7 pA/pF (control) and 8.6±0.72 pA/pF (50 µM 293B) in guinea pig cells (n=4, P=NS).

View larger version (11K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 6 Calcium and sodium currents are not affected by 293B. Representative calcium current tracings at 36°C from human (Panel A) and guinea pig (Panel B) ventricle were elicited by 400 ms depolarizing pulses to +10 mV from a holding potential of –80 mV before and after 50 µmol/L 293B. INa in human (Panel C) and guinea pig (Panel D) ventricle were recorded upon 40 ms depolarizations to –30 mV from –120 mV before and after 50 µmol/L 293B. Neither current was inhibited by 293B at concentrations up to 50 µmol/L.

 
We further assessed potential effects of 50 µM 293B on I_Ca voltage dependence and kinetics in guinea pig myocytes. Activation V_1/2 averaged –8.9±1.4 mV before and –9.9±0.9 mV after 293B, and k averaged 8.1±0.9 and 8.8±0.7 mV respectively (P=NS, n=4). Inactivation was studied with 1-s prepulses to various voltages, and had a V_1/2 of –36.5±3.8 mV before and –40.4±3.9 mV after 293B, and k of 10.2±1.2 and 12.9±1.9 mV respectively (P=NS, n=5). Recovery of I_Ca inactivation was tasted with a two-pulse protocol from a HP of –50 mV, and proceeded with time constants of 188±38 and 1070±385 ms before versus 155±38 and 1041±398 ms after 293B (P=NS, n=4).

3.4 Effects of 293B and dofetilide on action potentials
To determine the effects of 293B on action potentials, isolated myocytes were perfused with 1 µM 293B, and action potential properties were compared with those of control myocytes and (for guinea pig cells) with those of cells perfused with 1 µM dofetilide, which has been shown to inhibit completely I_Kr [6]. Typical recordings from guinea pig ventricular myocytes are illustrated in Fig. 7A. Both dofetilide and 293B prolonged action potential duration; however, action potentials were longer at a frequency of 0.1 Hz in the presence of dofetilide, while they were longer in the presence of 293B at 4 Hz, as shown by the mean values in 8, 7 and 6 myocytes under control conditions, in the presence of 293B, and in the presence of dofetilide in Fig. 7B. Fig. 7C illustrates the frequency dependence of the APD-prolonging effect of each compound. The frequency dependence of prolongation was assessed with ANOVA, and revealed that dofetilide's effect showed significant reverse use dependence (P<0.001), while 293B delayed repolarization to a similar extent over the whole frequency range (P=NS for frequency dependence). Representative action potential recordings from human ventricular myocytes are shown in Fig. 8A. As in guinea pig cells, APD was longer in the presence of 293B at both slower and faster rates. Mean data for five control cells and five cells in the presence of 293B are shown in Fig. 8B. In human cells, APD₉₀ was prolonged by 33.3% at a frequency of 0.1 Hz and by 33.5% at 4Hz, and as in guinea pig there was no significant interaction between frequency and the effect of 293B on APD.

View larger version (21K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 7 Action potential prolongation by 293B (1 µmol/L) and dofetilide (1 µmol/L) in guinea pig ventricular myocytes. (A) Typical action potentials recorded at 0.1 and 4 Hz under control conditions (left), in the presence of 293B (middle) and in the presence of dofetilide (right). (B) Action potential duration to 90% repolarization (APD90) as a function of frequency under control conditions and in the presence of 293B or dofetilide. (n=8, 7, and 6 in controls, 293B, and dofetilide treated cells, respectively). (C) Percentage of APD90 increase as a function of stimulation frequency. *P<0.05, **P<0.01, ***P<0.001 for difference between drug effect at each frequency versus effect at 4 Hz (ANOVA).

 

View larger version (18K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 8 Effects of 293B (1 µmol/L) on action potentials recorded in human ventricular myocytes. (A) Action potentials recorded at 36°C from left ventricular midmyocardial cells from an explanted heart of a patient with dilated cardiomyopathy under control (top) and 293B (middle) conditions. Resting membrane potential is not corrected for the junction potential. (B) Frequency dependence of differences in action potential duration. APD90 was significantly prolonged at all frequencies and the effect was not rate-dependent. *P<0.05 versus control, n=5 for each group.

 

	4 Discussion

Top Abstract 1 Introduction 2 Methods 3 Results 4 Discussion 5 Conclusions References

 
We have demonstrated that the 293B is a selective blocker of I_Ks, with no effect on I_K1, I_Kr, I_Ca or I_Na and an inhibitory effect on I_to at concentrations 20-fold higher than those needed to inhibit I_Ks. 293B prolonged repolarization in human and guinea pig ventricular myocytes to the same fractional extent at all frequencies, which contrasted with the reverse use-dependent action potential prolongation caused by the I_Kr blocker dofetilide.

4.1 Comparison between the ionic effects of 293B and those of other class III drugs
d,l-Sotalol and amiodarone were the first drugs whose principle antiarrhythmic mechanism was ascribed to delayed repolarization [3, 4]. d,l-Sotalol is also a β-blocker [3], while amiodarone has sodium- [14]and calcium- [15, 16]channel blocking and non-competitive anti-adrenergic properties [17]. Amiodarone may prolong APD by a variety of ionic mechanisms, including inhibition of I_K1 [18, 19], I_to [20], I_Kr [19]and I_Ks [19, 21]. In recent years, several pure class III drugs have been developed. E-4031, d-sotalol and dofetilide act by blocking I_Kr [5, 6]. Ibutilide may act by enhancing a plateau Na⁺ current [22], but is also a potent I_Kr blocker [23].

Our extensive characterization of I_K inhibition by 293B shows that the properties of 293B-resistant and sensitive current correspond closely to those previously described for I_Kr and I_Ks respectively with the use of the pharmacological probes E-4031 and dofetilide [5, 6, 24, 25]. Furthermore, our data indicate that 293B is highly selective for I_Ks over I_Kr, with the latter unaffected at concentrations 50 times the EC₅₀ of I_Ks. These findings confirm and extend the preliminary observations of Busch et al. [11].

4.2 Potential significance
A major limitation of current class III antiarrhythmic drugs is their rate-dependent profile of action potential prolongation, which results in maximal effects at slow rates and minimal effects during tachycardia [8]. This property limits efficacy in terminating rapid arrhythmias [26, 27], while maximizing the risk of Torsades de Pointes arrhythmias associated with bradycardia-dependent early afterdepolarizations [28]. Reverse use-dependent effects on repolarization have been reported for dofetilide in vitro [6, 29, 30], and in vivo [31], and in healthy humans dofetilide-induced QT-prolongation during exercise shows reverse use dependence [32]. Other I_Kr-blocking class III antiarrhythmics (e.g. E-4031, sotalol and UK-66,914) also prolong repolarization preferentially at slow heart rates in vitro [33–35]and in man [36, 37]. It has been suggested that I_Ks accumulation at increased frequencies decreases the relative importance of I_Kr, reducing the impact of I_Kr blockade and contributing to reverse use-dependent APD prolongation [6]. I_Ks blockers might thus be expected to have a more favourable rate-dependent profile of APD prolongation. APD and refractoriness prolongation by amiodarone and ambasilide show less reverse use dependence than those of I_Kr blockers [27, 38], and this has been attributed to the ability of these compounds to block I_Ks [21, 39]. However, amiodarone [14–16]and ambasilide [40, 41]also block other currents. The present study is significant in showing that concentrations of 293B that inhibit only I_Ks appear to have rate-independent effects on APD in human and guinea pig myocytes.

The selectivity of 293B as an I_Ks blocker makes it a new and potentially valuable tool in studying the physiological role of I_Ks. The importance of I_Ks in repolarizing guinea pig ventricular myocytes has been suggested by in vitro studies [42, 43]and supported by modelling work [44]. E-4031-sensitive and -resistant components of I_K have been demonstrated in human ventricle [13]and atrium [45], suggesting that both I_Kr and I_Ks are present in the human heart. E-4031 prolongs human ventricular APD, indicating a role for I_Kr in human ventricular repolarization [13]. The effects of 293B in the present study provide evidence for a physiological role of I_Ks in guinea pig and human ventricular repolarization.

It has become evident that there is marked electrical heterogeneity within the ventricular myocardium [46]. Epicardial cells have characteristic differences in AP morphology compared to endocardial cells because of differences in I_to density [46–48]. The most striking difference, however, is the much greater action potential duration of a population of cells (termed M cells) in the deep subepicardial and midmyocardial regions [46]. M cells have been found in intact human ventricle [49]and in single cells from human hearts [50]. The ionic basis of the longer APD in M cells may be a smaller I_Ks [51, 52]. I_Kr appears to be similar in all layers, with the lack of I_Ks potentially explaining the susceptibility of M cells to excessive APD prolongation and early afterdepolarizations with I_Kr blockers [53]. A selective I_Ks blocker like 293B may be useful for testing these ideas about the ionic mechanisms of transmural heterogeneity and arrhythmogenic afterdepolarizations. If reduced I_Ks in M cells is important, exposure to 293B should reduce transmural heterogeneities in APD. Furthermore, I_Ks blockade should be much less likely than I_Kr blockade to cause early afterdepolarizations in M cells, and might also be less likely to induce the acquired long QT syndrome and Torsades de Pointes in vivo.

4.3 Potential limitations
Rundown can be a problem when studying I_K and I_Ca. We therefore conducted serial control measurements of current amplitudes and only cells with stable currents for at least 15 min were studied. Washout of drug effects was obtained whenever possible. All action potential recordings were obtained within 3 to 4 min of membrane rupture to avoid contamination by time-dependent changes.

Human ventricular cells were obtained from explanted recipient hearts of cardiac transplant patients. We did not attempt to study the direct effects of 293B on I_K in human ventricular cells, because of the small amplitude of such currents in diseased human tissues which makes them technically very difficult to record. Possible explanations of the difficulty in recording I_Ks during step voltage clamps with the tight-seal patch technique include rapid current run-down and the difficulty of suppressing multiple overlapping currents in the native tissue without interfering with I_Ks. Even in relatively normal human ventricular tissue, I_Ks is not large [13]. Performance of AP recording within several minutes of cell rupture (minimizing time for cell dialysis and rundown) may have been important in observing the repolarization-delaying effect of 293B. In addition, the possibility must be considered that 293 B affects human ventricular action potentials by mechanisms additional to, or other than, its I_Ks-blocking properties.

We studied 293B effects on I_K, I_K1, I_Na and I_Ca. Drug effects on other ion transport mechanisms (such as the Na⁺, K⁺-ATP'ase, Na⁺–Ca²⁺ exchange and C1^– currents) cannot be excluded based on the present work. Prior to evaluating 293B effects on such components, they would first have to be characterized carefully in human ventricle, a significant undertaking that goes beyond the scope of the present manuscript.

	5 Conclusions

Top Abstract 1 Introduction 2 Methods 3 Results 4 Discussion 5 Conclusions References

 
The chromanol 293B is a selective blocker of I_Ks, with a different rate-dependent profile of APD prolongation compared to the I_Kr blocker dofetilide. This compound may be a very useful tool for assessing the physiological and pathophysiological role of I_Ks.

Time for primary review 22 days.

	Acknowledgements

 
This work was supported by grants from the Medical Research Council of Canada, the Quebec Heart Foundation and the Fonds de Recherche de l'Institut de Cardiologie de Montreal. Dr. Bosch is a fellow of the Deutsche Forschungsgemeinschaft, Dr. Gaspo holds a fellowship of the Medical Research Council of Canada. Dr. Busch is a Heisenberg fellow of the Deutsche Forschungsgemeinschaft. 293B was a friendly gift from Hoechst AG/Frankfurt, Germany. The authors thank Johanne Doucet, Mirie Levi and Natalie Talbot for expert technical assistance, and Luce Bégin and Caroll Boyer for secretarial help with the manuscript.

	References

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