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(Circulation. 1996;93:656-659.)
© 1996 American Heart Association, Inc.

Articles

ATP-Dependent Potassium Channel in Rat Cardiomyocytes Is Blocked by Lidocaine

Possible Impact on the Antiarrhythmic Action of Lidocaine

A. Olschewski, MD; M.E. Bräu, MD; H. Olschewski, MD; G. Hempelmann, MD; W. Vogel, PhD

From the Departments of Anesthesiology and Intensive Care Medicine (A.O., M.E.B., G.H.), Physiology (A.O., W.V.), and Internal Medicine (H.O.), Justus-Liebig-University, Giessen, Germany.

Correspondence to Dr. A. Olschewski, Physiologisches Institut, Justus-Liebig-University, Aulweg 129, D-35392 Giessen, Germany.

	Abstract

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Background During myocardial ischemia, lidocaine has favorable antiarrhythmic properties. Malignant arrhythmias result from heterogeneity between ischemic and nonischemic regions in extracellular potassium concentration and action potential duration. These effects have been attributed to the activation of ATP-dependent potassium (K_ATP) channels. In this study, we investigated the action of lidocaine on the K_ATP channels to test the possible link between the antiarrhythmic properties of lidocaine and its action on the K_ATP channel.

Methods and Results The patch-clamp technique was employed on enzymatic dissociated cardiomyocytes of adult rats. Lidocaine was applied to the outer side of excised membrane patches by means of a multibarrel perfusion system. Lidocaine reversibly blocked the mean current of the K_ATP channels in a concentration-dependent manner (IC₅₀=43±4.7 µmol/L, E=0 mV, n=6), while the amplitude of the single-channel current remained unchanged. The half-maximum blocking concentration corresponds to the therapeutic range for the antiarrhythmic application of a lidocaine bolus in humans.

Conclusions The open probability but not the conductance of the K_ATP channel in the membrane of rat cardiomyocytes is blocked by lidocaine. This action may explain, in part, the favorable antiarrhythmic properties of lidocaine during acute myocardial ischemia.

Key Words: antiarrhythmia agents • electrophysiology • ischemia • myocardial infarction • potassium channel

	Introduction

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The local anesthetic lidocaine is one of the most frequently used drugs in the treatment of malignant ventricular arrhythmias.¹ Its pharmacological effects on electrical conduction and excitability are particularly pronounced during myocardial ischemia and reperfusion.^{2 3} Since lidocaine is a potent blocker of sodium channels,^{4 5} its antiarrhythmic properties are generally explained by its influence on sodium conductance. However, it is not clear whether some other mechanisms are involved in the lidocaine action, because local anesthetics may also exert strong blocking effects on potassium channels,⁶ and bupivacaine, which blocks sodium channels much more potently⁷ than lidocaine, reveals more proarrhythmic than antiarrhythmic properties.⁸

Myocardial ischemia is associated with a local increase in extracellular potassium concentration^{9 10} and with a shortening of the action potential duration¹¹ in ischemic regions of the heart. This leads to a regional heterogeneity between ischemic and nonischemic regions¹² that is supposed to be one of the main causes of malignant arrhythmia formation.¹³ Both the shortening of the action potential and the increase in extracellular potassium concentration during ischemia have recently been attributed to the activation of ATP-dependent potassium (K_ATP) channels^{14 15} that are present in the membrane of cardiomyocytes at a high density.¹⁶ Selective opening of these channels caused marked inhomogeneities of refractory period that provoked extrasystoles.¹⁷ Thus, it could be supposed that lidocaine exerts its antiarrhythmic properties by blocking of the ischemically activated K_ATP channels.

In the present study the patch-clamp technique was applied to freshly isolated rat cardiomyocytes to investigate the effect of lidocaine on K_ATP channels directly. Excised membrane patches in the absence of ATP were used as a model of the myocyte membrane under conditions of ATP loss during ischemia.

	Methods

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Preparation
Single ventricular myocytes were isolated from adult male Wistar rats using a modified method of enzymatic dissociation.¹⁸ In brief, rats (300 to 350 g body wt; n=11) were anesthetized with pentobarbital-sodium (30 to 50 mg/kg). Hearts were quickly removed and dipped in ice-cold Ca²⁺-free Tyrode's solution containing (mmol/L) NaCl 140.0, KCl 5.8, KH₂PO₄ 0.5, Na₂HPO₄ 0.4, MgCl₂ 5.0, and HEPES 10 (pH 7.3 with NaOH). Then the aorta was cannulated to the base of a Langendorff column and perfused retrogradely with Ca²⁺-free Tyrode's solution for 5 minutes at 37°C. Then 0.4 mg/mL collagenase (Worthington type CLS II, Biochrom) was added to the perfusing solution, and Ca²⁺ concentration was progressively increased from 0 to 120 µmol/L in four steps within 20 minutes. The perfusate was then washed out for 5 minutes in Tyrode's solution with 120 µmol/L CaCl₂. Perfusing solution was continuously bubbled with a 95% O₂/5% CO₂ gas mixture. After perfusion, the heart was removed from the column and the cells were dispersed by gentle mechanical agitation. Isolated ventricular cells were stored in Tyrode's solution containing 120 µmol/L Ca²⁺ at 4°C. This preparation could normally be used for up to 8 hours. Only rectangular ventricular cells with regular and clear striations were subjected to detailed investigation. All experiments were carried out at 20°C to 22°C.

Electrophysiological Techniques
Ionic channels were investigated by means of the standard patch-clamp method.¹⁹ Pipettes were pulled in two stages from a borosilicate glass tube (GC150F-7.5, Clark Electromedical Instruments), coated with Sylgard 184 (Dow Corning), and fire polished directly before the experiment. Pipette resistance was 7 to 9 M. Membrane currents were recorded using an EPC-7 patch-clamp amplifier (List), low-pass filtered at 10 kHz, and stored on videotape via a modified PCM-501ES (Sony) pulse-code modulation unit. For analysis the data were filtered with a 4-pole low-pass Bessel filter, digitized with a Labmaster TM-40 AD/DA board (Scientific Solutions), and recorded on a personal computer with PCLAMP 5.0 software. Most of the single channel recordings were performed with outside-out membrane patches. Inside-out patches were used only in experiments where the identity of K_ATP channels was confirmed by their sensitivity to internally applied ATP. Commercially available software (PCLAMP 5.0) was used to calculate the channel open probability. The channel was considered open if its amplitude exceeded 50% of its mean amplitude. Values are given as mean±SEM.

Solutions
The external solution contained (mmol/L) NaCl 140.0, KCl 5.6, KH₂PO₄ 0.5, Na₂HPO₄ 0.4, MgSO₄ 0.9, CaCl₂ 1.8, and HEPES 10 (pH 7.4 with NaOH); the high potassium external (high-K_o) solution contained (mmol/L) KCl 145, CaCl₂ 1.8, MgCl₂ 1.0, and HEPES 5.0 (pH 7.4 with KOH). The internal solution contained (mmol/L) KCl 145, EGTA 10, and HEPES 5 (pH 7.2 with KOH); the inside-out solution contained (mmol/L) KCl 145, MgCl₂ 1.0, EGTA 5, and HEPES 10 (pH 7.4 with KOH).

Lidocaine-HCl, K₂ATP, and glibenclamide were purchased from Sigma Chemical Co; K₂ATP and glibenclamide were directly added to internal and external solutions, respectively. Stock solution of lidocaine (100 mmol/L) was first prepared in distilled water and then diluted in external Tyrode's solution directly before the experiment. The drug was applied to excised patches by means of a multibarrel perfusion system. The time of the solution exchange did not exceed 5 seconds. Because of well-known run-down of K_ATP channels, the patches were washed out in control solution after application of each lidocaine concentration. The mean currents recorded in control solutions directly before and after each lidocaine application were averaged. This value was used as the baseline K_ATP current to calculate the relative block induced by lidocaine.

	Results

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ATP-dependent potassium channels were observed in 80% of outside-out membrane patches obtained with 7 to 9 M pipettes with the use of ATP-free internal solution. Such patches normally contained 2 to 6 K_ATP channels. Original recordings of the channels in external Tyrode's and high-K_o solutions are shown in Fig 1A. In high-K_o solution, the single-channel conductance was 81.2 pS for inward currents and 48.9 pS for outward currents. Current-voltage (I-E) curves for the K_ATP channel crossed the voltage axis at 0 mV and showed a pronounced inward rectification (Fig 1B). The reversal potential was shifted to values of -80 mV after substitution of external high-K_o solution with Tyrode's solution in which the calculated reversal potential for K⁺ ions was -81 mV. This shift of reversal potential is in good agreement with the value predicted from the Nernst equation and implies a high selectivity of the channel for K⁺ ions. In external Tyrode's solution, the channel conductance was 23.1 pS. The channel open probability was independent of membrane potential (not shown). The channels demonstrated a typical rundown within 10 minutes.

View larger version (21K): [in this window] [in a new window]   Figure 1. Effect of lidocaine in external solution on KATP channels. A, Currents through KATP channels in the absence of lidocaine (control), in the presence of 300 µmol/L lidocaine, and after washout with external solution, measured in an outside-out patch at a holding potential of 0 mV and filter frequency of 200 Hz. The moments of solution changes are indicated by arrows. B, Amplitude histograms obtained in control solution (left), during application of 300 µmol/L lidocaine (middle), and after washout with control solution (right) at a filter frequency of 600 Hz, sampling rate of 3.33 kHz, and a bin width of 0.2 pA. N is the number of points in each interval. Histograms were fitted with the sum of three gaussian curves to give a sufficient fit. The peaks at 0 pA represent the channel closed states. The single-channel current is measured as the difference between two peaks. C, Concentration dependence of block (fractional block, fb) of mean KATP current by lidocaine at 0 mV. Mean±SEM from six outside-out patches. The curve represents the nonlinear least-squares fit of the equation fb=c/(c+IC50) to the data points assuming a one-to-one reaction of lidocaine with the KATP channel if Hill coefficient=1.

In inside-out membrane patches, the channels were reversibly blocked by 2 mmol/L ATP applied from the internal side of the membrane (Fig 1C). The single-channel currents were reversibly blocked by 10 µmol/L glibenclamide, which is a specific blocker of channels (Fig 1D).

The effects of lidocaine on the K_ATP channnel were examined using outside-out patches with ATP-free internal solution in the patch pipette. Lidocaine concentrations of 10, 30, 100, 300, and 1000 µmol/L were externally applied to every patch (Fig 2C). Application of lidocaine resulted in a concentration-dependent reduction of the mean current of the K_ATP channels (IC₅₀=43±4.7 µmol/L, E=0 mV, n=6). The best fit was obtained assuming a Hill coefficient of 1, suggesting a one-to-one reaction of lidocaine with the K_ATP channel. The amplitudes of the single-channel currents remained unchanged. This is obvious from the histograms shown in Fig 2B, in which the current amplitude of the single channel as obtained from the distance of peaks of the gaussian curves was 2.0 pA in control solution, during application of 300 µmol/L lidocaine, and after washout with control solution. Thus, the binding of lidocaine interferes with the gating of this channel, but it does not reduce the conductance of the open channel.

View larger version (17K): [in this window] [in a new window]   Figure 2. Single ATP-dependent potassium (KATP) channel currents in membranes of rat cardiomyocytes. A, Original recordings of single-channel currents in external Tyrode's (5.6 mmol/L Ko and 145 mmol/L Ki; left) and in high-Ko (145 mmol/L Ko and 145 mmol/L Ki; right) solutions at different potentials, with outside-out patch, filter frequency 500 Hz, temperature 20°C to 22°C. The dotted line represents the current level of closed channels. B, Current-voltage relations of single-channel currents in Tyrode's () and high-Ko () solutions. C, Traces of two KATP channels (left) and their sensitivity to 2 mmol/L internal ATP (right) at 0 mV. Conditions are as in A. D, Block of KATP channel by externally applied 10 µmol/L glibenclamide in external solution, with a holding potential of 0 mV; other conditions are as in Fig 2A.

	Discussion

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Until recently it was unclear whether ATP-dependent potassium channels can play an important role in the formation of malignant arrhythmias. However, according to Faivre and Findlay,¹⁴ even the activation of a very small proportion of K_ATP channels would be able to influence cardiac excitability. Furthermore, the application of the K_ATP channel blocker glibenclamide to ischemic mammalian heart prevented the hypoxia-induced shortening of the action potential duration and decreased the rate of extracellular potassium accumulation.¹¹ These findings suggest a significant contribution of the ATP-sensitive potassium conductance to the formation of cardiac arrhythmias during ischemia.¹⁵ Recently some class 1a antiarrhythmic drugs were found to block K_ATP channels.^{20 21} To the best of our knowledge, this is the first description of K_ATP channel block by a class 1b antiarrhythmic agent.

The K_ATP channels were identified on the basis of their sensitivity to internally applied ATP and externally applied glibenclamide.²² The single-channel conductance for high symmetrical potassium ion (high-K_o) solutions and Tyrode's solution and the observed inward rectification are in good agreement with the original description by Noma.¹⁶ In the present study, lidocaine was applied to outside-out patches with the bath solution. This external application of lidocaine corresponds to the situation of an intravenous lidocaine bolus that is usually applied for the treatment of a ventricular tachycardia. After application of a 100-mg bolus in a human, the peak plasma concentration of the drug reaches about 100 µmol/L, decreasing to 15 to 20 µmol/L within 2 to 3 minutes.²³ These values correspond to the IC₅₀ of 43 µmol/L obtained in the present study for the blocking action of lidocaine on the K_ATP channel. However, this action at the same time may be deleterious for the survival of ischemic cells²⁴ and may explain why lidocaine increased rather than decreased the incidence of ventricular fibrillation during ischemia in dogs²⁵ and lidocaine prophylaxis did not reduce the overall mortality among patients with myocardial infarction.²⁶

In conclusion, lidocaine blocks the K_ATP channel in the membrane of rat cardiomyocytes at therapeutic concentrations used for antiarrhythmic treatment. If K_ATP channels are involved in cardiac susceptibility to the formation of malignant arrhythmias, the action of lidocaine on these channels would antagonize this process. Thus, our data suggest that the antiarrhythmic action of lidocaine during myocardial ischemia may be explained, in part, by its blocking action on K_ATP channels.

	Acknowledgments

 
This work was supported by the Förderverein für Anästhesie Giessen. We thank Dr B. Safronov and C. Nau for revision of the manuscript and figures and Dr A. Scholz for his critical and valuable comments.

Received September 12, 1995; revision received December 7, 1995; accepted December 10, 1995.

	References

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