1 Department of Anaesthesiology, University Hospital Hamburg Eppendorf, Martinistr. 52, D-20251 Hamburg, Germany. 2 Institute of Neural Signal Transduction, Center for Molecular Neurobiology, University of Hamburg, Germany
*Corresponding author. E-mail: patrick.friederich@zmnh.uni-hamburg.de
Accepted for publication: July 21, 2003
![]() |
Abstract |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Methods. Whole-cell patch-clamp recordings and site-directed mutagenesis were combined to compare local anaesthetic sensitivities of cloned and mutated human potassium channel subunits. The ion channels were activated by a protocol that approximated ventricular action potentials.
Results. The amide local anaesthetics bupivacaine, levobupivacaine and ropivacaine inhibited HERG channels at toxicologically relevant concentrations, with IC50 values of 20 (SEM 2) µM (n=29), 10 (1) µM (n=40) and 20 (2) µM (n=49), respectively. Hill coefficients were close to unity. There were no indications of qualitative differences in channel inhibition between the three anaesthetics. The putative subunit MiRP1 did not alter local anaesthetic sensitivity of HERG channels. The common single nucleotide polymorphism producing MiRP1T8A did not increase local anaesthetic sensitivity of HERG/MiRP1 channels.
Conclusions. Amide local anaesthetics target HERG and HERG/MiRP1 channels with identical potency. The effects on these ion currents may significantly contribute to local-anaesthetic-induced cardiac arrhythmia. MiRP1T8A does not seem to confer an increased risk of severe cardiac side-effects to carriers of this common polymorphism.
Br J Anaesth 2004; 92: 93101
Keywords: anaesthetics local; anaesthetics, toxicity; complications, arrhythmia
![]() |
Introduction |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Myocardial potassium channels are complexes formed by different subunits.15 The subunit composition may influence the effects of local anaesthetics on these membrane proteins.12 16 The effects of amide local anaesthetics on complexes formed by HERG and MiRP1 remain to be compared. It is therefore unclear if co-expression of HERG and MiRP1 alters the local anaesthetic sensitivity of HERG channels. If this were the case, MiRP1T8A might confer an increased sensitivity to HERG/MiRP1 channels, which may be indicative of an additional risk for carriers to develop local-anaesthetic-induced cardiac arrhythmia. Furthermore, it may suggest a role for genetic testing in the prevention of local-anaesthetic-induced cardiac arrhythmia. This study was designed to investigate if the local anaesthetic sensitivity of HERG channels is altered by the presence of MiRP1 or MiRP1T8A.
![]() |
Methods |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Cloning of ion channel subunits and cDNA-containing expression vectors and in vitro mutagenesis
Fragments encoding the complete open reading frames of the KCNH2 or KCNE2 genes were amplified from human cardiac cDNA by the reverse transcriptase polymerase chain reaction (PCR) employing primers specific for the particular gene.17 The amino acid exchange T8A was introduced into KCNE2 by PCR using the primer KCNE2-T8A (5'-ATGT CTACTTTATCCAATTTCGCACAGACGCTGGAAGACGTCTTCCGAAG-3') and the pcDNA6-BGH primer (Invitrogen, Groningen, The Netherlands). Successful mutagenesis and the MiRP1T8A cDNA sequence were verified by sequencing. The respective cDNAs were cloned into pcDNA6 expression vectors for transfection of CHO cells.
Transfection of CHO cells
CHO cells were seeded in 35 mm diameter monodishes 1 day before transfection. Transfection was performed using 3.5 µl of lipofectamine reagent (Gibco-BRL, Rockville, USA) according to the manufacturers protocol and 1.02.0 µg of the respective ion channel plasmid DNA. Co-expression of HERG channels with MiRP1 and MiRP1T8A within the cell membrane was verified using internal ribosomal entry site eGFP constructs of MiRP1 and MiRP1T8A, respectively. Only those cells transfected with HERG/MiRP1 or HERG/MiRP1T8A that emitted green fluorescence when stimulated by ultraviolet light were investigated.
Electrophysiology of potassium channels
Whole-cell currents were measured using the patch clamp method18 with an EPC-9 amplifier and Pulse software version 8.11 (HEKA Elektronik, Lambrecht, Germany). Patch electrodes with a pipette resistance of 2.04.0 M were pulled from borosilicate glass capillary tubes (World Precision Instruments, Saratoga, USA) and filled with a solution comprising 160 mM KCl, 0.5 mM MgCl2, 10 mM HEPES, 2 mM Na-ATP (all from Sigma, Deissenhofen, Germany), adjusted to pH 7.2 with KOH. The external solution applied to the cells was 135 mM NaCl, 5 mM KCl, 2 mM CaCl2, 2 mM MgCl2, 5 mM HEPES, 10 mM sucrose, phenol red 0.1 mg ml1 (all from Sigma, Deissenhofen, Germany) adjusted to pH 7.4 with NaOH. The capacitance of the cells was 18.2 (SD 5.7) pF (n=20). Series resistance was 2.57.5 M
and was actively compensated for by at least 80%. A leak subtraction protocol was not used in this study. The data were therefore only recorded from cells in which tight mega-ohm or giga-ohm seals had been formed. As leak currents tend to increase during the course of an experiment, we only analysed data that did not show more than a 6% difference in any tested parameter between control and wash-out conditions. The liquid junction potential was calculated according to the Henderson equation.19 It accounted for 3.86 mV and was not corrected for. Bupivacaine (Sigma, Deissenhofen, Germany), levobupivacaine (AstraZeneca, Södertalje, Sweden) and ropivacaine (AstraZeneca, Södertalje, Sweden) were dissolved in the extracellular solution. The drugs were superfused onto the cells by a hydrostatically driven perfusion system. We studied the activity of cardiac potassium channels using a pulse protocol that approximates a cardiac ventricular action potential.20 The potassium channels were activated from a holding potential of 80 mV by a step depolarization of the cell membrane to +60 mV and subsequent repolarization within 1000 ms back to the holding potential of 80 mV (ramp protocol). The ramp protocol was followed by a 30 ms depolarization of the membrane to a test potential of +40 mV.17 Repetitive ramps were applied to determine that steady-state inhibition was reached. The voltage dependence of local anaesthetic action on HERG channels was investigated after a 2 s depolarizing test pulse to +60 mV by stepping the membrane potential for 0.5 s to potentials of 120 mV, 40 mV and +40 mV. Experiments were performed at room temperature. The recorded signal was filtered at 2 kHz and stored with a sampling rate of 5 kHz on a personal computer for later analysis.
Data analysis
The currenttime relationship observed with the ramp protocol for cardiac potassium channel activation was used to quantify the charge crossing the membrane during the ramp protocol (Qramp). Similarly, the currenttime relationship observed during the step protocol for channel activation was used to quantify the charge crossing the membrane during the step protocol (Qstep). Inhibition of currents evoked by the ramp protocol was measured as inhibition of the maximal outward current and as reduction of Qramp. Inhibition of the current during the step protocol was analysed as a reduction of Qstep. The charge crossing the membrane is equivalent to the time integrals of current traces and was determined using Pulse software version 8.11 (HEKA Elektronik, Lambrecht, Germany). Inhibition was quantified by the ratio of time integrals of current traces in the presence of the drug to the mean of the time integrals before application and after wash out of the drug (inhibition=1Qdrug/[(Qcontrol+Qwash out)/2]) and by the ratio of the size of the current in the presence of the drug to the mean of the size of the current before application and after wash out of the drug (inhibition=1Imax drug/[(Imax control+Imax wash out)/2]). The concentrationresponse curves were fitted to the Hill equation (e/emax=c/[IC50
+c
], where e=effect, emax=maximal effect, c=anaesthetic concentration,
=Hill coefficient and IC50=concentration of half-maximal effect) using non-linear regression. Regression analysis was performed using Kaleidagraph (Syngery Software, Reading, Pennsylvania, USA). Data points are given as mean (SD) unless stated otherwise. Statistical analysis was performed with an unpaired Students t-test; n is the number of experiments.
![]() |
Results |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
|
|
|
The inhibitory effects of the local anaesthetics on HERG currents evoked by the step depolarization following the ramp was also reversible and concentration dependent. The effects were quantified as a reduction of Qstep. Fitting Hill functions to the concentrationresponse data (Fig. 3C) yielded IC50 values for the reduction of Qstep 23 times higher than for the inhibition of Qramp. Hill coefficients were again close to unity (Table 1).
Local anaesthetic sensitivity of HERG/MiRP1 and HERG/MiRP1T8A
The influence of MiRP1 on local anaesthetic sensitivity of HERG channels and the influence of the T8A polymorphism of KCNE2 on local anaesthetic sensitivity of IKr were investigated at concentrations close to the IC50 values for inhibition of HERG channels (Qramp) of all three local anaesthetics. If MiRP1 or MiRP1T8A alter the local anaesthetic sensitivities of HERG channels, this effect can best be detected at the steepest part of the concentrationresponse curve for inhibition of HERG channels. All three anaesthetics reversibly inhibited both HERG/MiRP1 and HERG/MiRP1T8A channels (Fig. 4A). Inhibition of HERG/MiRP1 (Qramp, Fig. 4B) accounted for 55 (3)% (n=10) by bupivacaine 30 µM, 48 (6)% (n=7) by levobupivacaine 10 µM and 49 (8)% (n=12) by ropivacaine 25 µM. Inhibition of Qstep (Fig. 4C) accounted for 37 (6)% (n=5), 27 (8)% (n=4) and 29 (9)% (n=8) by bupivacaine, levobupivacaine and ropivacaine, respectively. The extent of inhibition of HERG/MiRP1 was not significantly different from that of HERG channels (P>0.05). Inhibition of HERG/MiRP1T8A (Qramp) accounted for 55 (6)% (n=7), 43 (5)% (n=14), and 50 (6)% (n=9) by bupivacaine, levobupivacaine and ropivacaine (Fig. 4B). The inhibitory effect on HERG/MiRP1T8A (Qstep) accounted for 33 (13)% (n=6), 26 (9)% (n=13) and 29 (5)% (n=9) by bupivacaine, levobupivacaine and ropivacaine, respectively (Fig. 4C). The degree of inhibition of HERG/MiRP1T8A was not significantly different from that seen for HERG/MiRP1 channels.
|
![]() |
Discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Establishing drug sensitivity of cardiac potassium channels is a common and widely accepted way of testing the cardiac safety of pharmacological agents.7 8 22 Impairment of repolarizing ventricular currents and suppression of ventricular currents that counterbalance sudden depolarizations may render the myocardium susceptible to severe arrhythmia.11 Possible modulating effects of genetic variations in ion channel subunits on local anaesthetic sensitivity have not been reported previously. The data presented here therefore provide information on the local anaesthetic sensitivity of an important cardiac potassium channel, the cardiac safety of clinically used local anaesthetics and the influence of a proposed arrhythmia susceptibility polymorphism14 on local anaesthetic sensitivity of a cardiac potassium channel complex that has repeatedly been associated with drug-induced ventricular arrhythmia.12 14
Bupivacaine, levobupivacaine and ropivacaine reduced the charge crossing the membrane through activated HERG channels in a concentration-dependent manner. The drugs exhibited two effects on Imax of HERG channels activated by the simulated action potential: they suppressed the maximal amplitude of the current and they delayed the occurrence of Imax. Both effects will decrease the repolarizing outward current during phases 3 and 4 of the cardiac action potential. Bupivacaine, levobupivacaine and ropivacaine also impaired the capability of HERG channels to conduct outward currents in response to a sudden depolarization. This effect was also concentration dependent and reversible. The difference in inhibition between 120 mV and 40 mV and between 120 mV and +40 mV would be compatible with a voltage-dependent block. However, inhibition of HERG by bupivacaine did not differ between 40 mV and +40 mV. It may thus be postulated that the increased driving force acting on the drug molecule at higher membrane depolarizations may be offset by conformational changes of the ion channel protein such as that occurring during channel inactivation. The direction of HERG current flux also differs between currents measured at 120 mV and 40 mV. It may therefore be hypothesized that the potassium ions passing through the ion channel pore may interfere with the action of the local anaesthetic on the HERG channel protein. In any case the magnitude of the inhibitory effect is largest at potentials where HERG channels pass the largest currents during the ventricular action potential. Both the prolongation of phases 3 and 4 of the ventricular action potential and a reduced ability to counteract sudden depolarizations are likely to facilitate the occurrence of clinically observed ventricular arrhythmia during local anaesthetic intoxication. Further study is needed to characterize the precise molecular mechanism of the drugchannel interaction.
The results of our study demonstrate that all three drugs are potent inhibitors of human ventricular potassium channels at concentrations that induce polymorphic ventricular arrhythmia in vivo.2 Our results obtained with a stimulation protocol that approximates ventricular action potentials support the notion11 that HERG channels constitute cardiac targets relevant for the induction of ventricular arrhythmia by local anaesthetics. As inhibition of repolarizing potassium channel complexes in human myocardium will prolong the duration of ventricular action potentials, the effects of local anaesthetics on these potassium channels will also influence local anaesthetic action on cardiac sodium channels. Prolongation of the action potential will reduce sodium channel availability by increasing the fraction of inactivated sodium channels. This increased inactivation will add to the direct inhibitory effect of local anaesthetics on human heart sodium channels.23
The concentration of local anaesthetic needed to half-maximally inhibit HERG channels was used to compare the sensitivity of HERG, HERG/MiRP1 and HERG/MiRP1T8A to local anaesthetics. Co-expression of HERG with MiRP1 did not alter the sensitivity of HERG channels. The interaction of the auxiliary subunit MiRP1 with HERG channels consequently does not perturb the binding of amide local anaesthetics to HERG channels. The same lack of difference in local anaesthetic sensitivity between HERG and HERG/MiRP1 also holds true for the comparison of HERG/MiRP1 with HERG/MiRP1T8A. The T8A single nucleotide polymorphism (SNP) occurs in 1.6% of healthy Caucasians14 and has been suggested to constitute an important risk factor for drug-induced ventricular arrhythmia.14 However, our results do not provide any evidence that this SNP may render HERG channels more susceptible to the pharmacological action of local anaesthetics. Our experimental results do not suggest a role for genetic testing of this common MiRP1 gene polymorphism in the prevention of local-anaesthetic-induced cardiac arrhythmia.
In summary, the amide local anaesthetics bupivacaine, levobupivacaine and ropivacaine inhibited cloned repolarizing ventricular potassium channels from human heart at toxicologically relevant concentrations. All three anaesthetics reduced outward currents in response to a simulated action potential and impaired the capability of these cardiac ion channels to respond to a sudden depolarization. There were no indications of qualitative differences in channel inhibition between bupivacaine, levobupivacaine and ropivacaine. The sensitivity of HERG channels was not altered by co-expression with MiRP1, and the common SNP producing MiRP1T8A did not influence local anaesthetic sensitivity of HERG/MiRP1 channels. As drug-induced inhibition of repolarizing potassium channels from cardiac ventricles is a well-known cause of severe cardiac arrhythmia and sudden death, our results support the notion that none of the local anaesthetics investigated may be considered as safe with regard to cardiotoxic side-effects. MiRP1T8A seems not to confer an increased risk of these severe cardiac side-effects to carriers of this common polymorphism.
![]() |
Acknowledgements |
---|
![]() |
References |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
2 Chang DH, Ladd LA, Copeland S, Iglesias MA, Plummer JL, Mather LE. Direct cardiac effects of intracoronary bupivacaine, levobupivacaine and ropivacaine in the sheep. Br J Pharmacol 2001; 132: 64958
3 Roden DM, Lazzara R, Rosen M, Schwartz PJ, Towbin J, Vincent GM. Multiple mechanisms in the long-QT syndrome. Current knowledge, gaps, and future directions. The SADS Foundation Task Force on LQTS. Circulation 1996; 94: 19962012
4 Sanguinetti MC, Jiang C, Curran ME, Keating MT. A mechanistic link between an inherited and an acquired cardiac arrhythmia: HERG encodes the IKr potassium channel. Cell 1995; 81: 299307[ISI][Medline]
5 Splawski I, Shen J, Timothy KW, Vincent GM, Lehmann MH, Keating MT. Genomic structure of three long QT syndrome genes: KVLQT1, HERG, and KCNE1. Genomics 1998; 51: 8697[CrossRef][ISI][Medline]
6 Trudeau MC, Warmke JW, Ganetzky B, Robertson GA. HERG, a human inward rectifier in the voltage-gated potassium channel family. Science 1995; 269: 925[ISI][Medline]
7 Keating MT, Sanguinetti MC. Molecular and cellular mechanisms of cardiac arrhythmias. Cell 2001; 104: 56980[ISI][Medline]
8 Mitcheson JS, Chen J, Lin M, Culberson C, Sanguinetti MC. A structural basis for drug-induced long QT syndrome. Proc Natl Acad Sci USA 2000; 97: 1232933
9 Lipka LJ, Jiang M, Tseng GN. Differential effects of bupivacaine on cardiac K channels: role of channel inactivation and subunit composition in drug-channel interaction. J Cardiovasc Electrophysiol 1998; 9: 72742[ISI][Medline]
10 Gonzalez T, Longobardo M, Caballero R, Delpon E, Tamargo J, Valenzuela C. Effects of bupivacaine and a novel local anesthetic, IQB-9302, on human cardiac K+ channels. J Pharmacol Exp Ther 2001; 296: 57383
11 Gonzalez T, Arias C, Caballero R, et al. Effects of levobupivacaine, ropivacaine and bupivacaine on HERG channels: stereoselective bupivacaine block. Br J Pharmacol 2002; 137: 126979
12 Abbott GW, Sesti F, Splawski I, et al. MiRP1 forms IKr potassium channels with HERG and is associated with cardiac arrhythmia. Cell 1999; 97: 17587[ISI][Medline]
13 Mazhari R, Greenstein JL, Winslow RL, Marban E, Nuss HB. Molecular interactions between two long-QT syndrome gene products, HERG and KCNE2, rationalized by in vitro and in silico analysis. Circ Res 2001; 89: 338
14 Sesti F, Abbott GW, Wei J, et al. A common polymorphism associated with antibiotic-induced cardiac arrhythmia. Proc Natl Acad Sci USA 2000; 97: 1061318
15 Roden DM, Balser JR, George AL Jr, Anderson ME. Cardiac ion channels. Annu Rev Physiol 2002; 64: 43175[CrossRef][ISI][Medline]
16 Gonzalez T, Navarro-Polanco R, Arias C, et al. Assembly with the Kvbeta1.3 subunit modulates drug block of hKv1.5 channels. Mol Pharmacol 2002; 62: 145663
17 Isbrandt D, Friederich P, Solth A, et al. A novel mutation in the KCNE2 (MiRP1) gene that alters HERG channel kinetics. J Mol Med 2002; 80: 52432[CrossRef][ISI][Medline]
18 Hamill OP, Marty A, Neher E, Sakmann B, Sigworth FJ. Improved patch-clamp techniques for high-resolution current recording from cells and cell-free membrane patches. Pflugers Arch 1981; 391: 85100[ISI][Medline]
19 Barry PH, Lynch JW. Liquid junction potentials and small cell effects in patch-clamp analysis. J Membr Biol 1991; 121: 10117[ISI][Medline]
20 Hancox JC, Levi AJ, Witchel HJ. Time course and voltage dependence of expressed HERG current compared with native rapid delayed rectifier K current during the cardiac ventricular action potential. Pflugers Arch 1998; 436: 84353[CrossRef][ISI][Medline]
21 Smith PL, Baukrowitz T, Yellen G. The inward rectification mechanism of the HERG cardiac potassium channel. Nature 1996; 379: 8336[CrossRef][ISI][Medline]
22 Weerapura M, Nattel S, Chartier D, Caballero R, Hebert TE. A comparison of currents carried by HERG, with and without coexpression of MiRP1, and the native rapid delayed rectifier current. Is MiRP1 the missing link? J Physiol 2002; 540: 1527
23 Nau C, Wang SY, Strichartz GR, Wang GK. Block of human heart hH1 sodium channels by the enantiomers of bupivacaine. Anesthesiology 2000; 93: 102233[CrossRef][ISI][Medline]