(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
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.
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Abstract |
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 Top Abstract IntroductionMethods Results Discussion References |
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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 (KATP) channels. In this study, we investigated the action of lidocaine on the KATP channels to test the possible link between the antiarrhythmic properties of lidocaine and its action on the KATP 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 KATP 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 KATP 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
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Introduction |
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TopAbstractIntroductionMethods Results Discussion References |
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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 concentration9 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 (KATP) channels14 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 KATP channels.
In the present study the patch-clamp technique was applied to freshly isolated rat cardiomyocytes to investigate the effect of lidocaine on KATP 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.
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Methods |
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TopAbstractIntroductionMethods Results Discussion References |
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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
KATP 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-Ko)
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 KATP 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 KATP current to calculate the relative block induced by lidocaine.
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Results |
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TopAbstractIntroductionMethods Results Discussion References |
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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 KATP channels. Original recordings
of the channels in external Tyrode's and high-Ko solutions
are shown in Fig 1A
. In high-Ko 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
KATP 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-Ko 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.
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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 KATP 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
KATP 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
KATP 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.
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) 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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TopAbstractIntroductionMethods Results Discussion References |
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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 KATP channels would be able to influence cardiac excitability. Furthermore, the application of the KATP 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 KATP channels.20 21 To the best of our knowledge, this is the first description of KATP channel block by a class 1b antiarrhythmic agent.
The KATP 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-Ko) 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 KATP 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 KATP channel in the membrane of rat cardiomyocytes at therapeutic concentrations used for antiarrhythmic treatment. If KATP 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 KATP channels.
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Acknowledgments |
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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.
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