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JOURNAL OF PHYSIOLOGY AND PHARMACOLOGY 2016, 67, 3, 339-351 www.jpp.krakow.pl

Z. HORAKOVA1, P. MATEJOVIC1, M. PASEK1,2, J. HOSEK1,3, M. SIMURDOVA1, J. SIMURDA1, M. BEBAROVA1

EFFECT OF ETHANOL AND ACETALDEHYDE AT CLINICALLY RELEVANT CONCENTRATIONS ON ATRIAL INWARD RECTIFIER POTASSIUM CURRENT IK1: SEPARATE AND COMBINED EFFECT 1Department of Physiology, Faculty of Medicine, Masaryk University, Brno, Czech Republic; Institute of Thermomechanics - branch Brno, Academy of Sciences of the Czech Republic, Brno, Czech Republic; 3Department of Molecular Biology and Pharmaceutical Biotechnology, Faculty of Pharmacy, University of Veterinary and Pharmaceutical Sciences, Brno, Czech Republic

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Atrial fibrillation is the most common arrhythmia at alcohol consumption. Its pathogenesis is complex, at least partly related to changes of cardiac inward rectifier potassium currents including IK1. Both ethanol and acetaldehyde have been demonstrated to considerably modify IK1 in rat ventricular myocytes. However, analogical data on the atrial IK1 are lacking. The present study aimed to analyse IK1 changes induced by ethanol and acetyldehyde in atrial myocytes. The experiments were performed by the whole cell patch-clamp technique at 23 ± 1°C on enzymatically isolated rat and guinea-pig atrial myocytes as well as on expressed human Kir2.3 channels. Ethanol (8 – 80 mM) caused a dual effect on the atrial IK1 showing the steady-state activation in some cells but inhibition in others in agreement with the ventricular data; on average, the activation was observed (at 20 mM by 4.3 and 4.5% in rat and guinea-pig atrial myocytes, respectively). The effect slightly increased with depolarization above –60 mV. In contrast, the current through human Kir2.3 channels (prevailing atrial IK1 subunit) was inhibited in all measured cells. Unlike ethanol, acetaldehyde (3 µM) markedly inhibited the rat atrial IK1 (by 15.1%) in a voltage-independent manner, comparably to the rat ventricular IK1. The concurrent application of ethanol (20 mM) and acetaldehyde (3 µM) resulted in the steady-state IK1 activation by 2.1% on average. We conclude that ethanol and even more acetaldehyde affected IK1 at clinically relevant concentrations if applied separately. Their combined effect did not significantly differ from the effect of ethanol alone. K e y w o r d s : atrial arrhythmias, alcohol consumption, inward rectifier, potassium currents, ethanol, acetaldehyde, cardiomyocytes, action potential

INTRODUCTION Alcohol intoxication tends to alter the cardiac electrocardiographic parameters (1, 2) and even to induce arrhythmias, most often the atrial fibrillation (AF) (3-5). The pathogenesis of AF has been proved to result, among others, from modifications and/or heterogeneity of cardiac inward rectifier potassium currents including IK1 (6). The role of selective inhibitors of these currents in AF treatment has been analysed and discussed (7). Data concerning the changes of cardiac atrial IK1 in presence of ethanol and its primary metabolite acetaldehyde are quite limited. Sporadic studies, rather marginally related to IK1, showed a decrease of this current through expressed channels composed from Kir2.1 subunits, the main structural components of the ventricular IK1 channels (8, 9). In contrast, Laszlo et al. (10) observed an increase of IK1 by 25% in rabbit atrial myocytes isolated after 120-hours lasting intravenous alcohol infusion (the blood alcohol levels: 34 – 93 mM). However, the change was not statistically significant. As to the acetaldehyde, no changes of IK1 were observed by Chen et al. (11, 12) in bullfrog atrial myocytes

and guinea-pig ventricular myocytes even at high acetaldehyde concentrations. Our recent analysis showed a dual effect of ethanol on the rat ventricular IK1 at clinically relevant concentrations: inhibition (at a very low concentration of 0.8 mM) and activation (at concentrations ≥ 20 mM) (13). The sensitivity of ventricular IK1 to ethanol appeared to exceed that of the other currents examined so far (13, 14). We have also recently reported an inhibitory effect of clinically-relevant concentrations of acetaldehyde on IK1 in rat ventricular myocytes (15). However, it should be considered that the molecular structure of the atrial IK1 channels is different from the ventricular IK1 channels. Hence, the changes of the atrial IK1 induced by ethanol and acetaldehyde might differ from those observed in ventricular myocytes (for more details see Discussion). Considering the role of IK1 in the atrial electrophysiology, the participation of IK1 in AF pathogenesis, and the need of relevant data, we aimed to examine the effect of ethanol and acetaldehyde on the rat atrial IK1. The substances were applied separately and, for the first time in atrial cells, also in combination. Ethanol and acetaldehyde were applied at concentrations relevant to alcohol consumption in humans.

340 MATERIALS AND METHODS The experiments were carried out with respect to recommendations of the European Community Guide for the Care and Use of Laboratory Animals; the experimental protocol (MSMT-29203/2012-30) was approved by the Local Committee for Animal Treatment at Masaryk University, Faculty of Medicine. Cell isolation Myocytes were isolated from either atria or ventricles of adult male Wistar rats (300 ± 20 g, n = 34), and from atria of adult male tricolour guinea pigs (370 ± 40 g, n = 9). The animals were anaesthetised by intramuscular administration of a mixture of tiletamin and zolazepam (65 mg/kg in rat, 40 mg/kg in guinea pig; Zoletil 100 inj., Virbac, France), and xylazine (20 mg/kg in rat, 5 mg/kg in guinea pig; Rometar inj., Spofa, Czech Republic). The procedure used for dissociation of rat ventricular myocytes was described in detail in our previous papers (16). Briefly, the extirpated heart was perfused via aorta with 0.9 mM CaCl2 Tyrode solution (3 – 5 min) and then with nominally Cafree Tyrode solution (4.5 min). During the first digestion step (2.75 min), the perfusion continued with nominally Ca-free Tyrode solution containing collagenase (type S, Yakult Pharmaceuticals; 0.16 mg/ml), protease (type XIV, SigmaAldrich; 0.053 mg/ml), and EGTA (Sigma-Aldrich; 34 µM). In the second digestion step (9 min), protease was omitted. The enzyme solution was then washed out in two steps by a perfusion with the low calcium Tyrode solutions (0.09 and 0.18 mM CaCl2). The right ventricular free wall was dissected from the heart, minced in 30 ml of 0.18 mM CaCl2 Tyrode solution, and filtered through a nylon mesh. After filtration, the suspension of cells was exposed to a gradually increasing external Ca2+ concentration (up to 0.9 mM within 20 min). All solutions were oxygenated by 100% O2 at 37°C. The dissociation procedure was similar in atrial cells, however, several modifications were required. To isolate the rat atrial cells, the time of the second digestion step was prolonged (16 – 18 min). During isolation of the guinea-pig atrial cells, the perfusion with the nominally Ca-free Tyrode solution was considerably longer (10 min). Moreover, a single digestion step lasting 30 min was performed using the Tyrode solution containing the same collagenase but in a lower concentration (0.08 mg/ml) and EGTA (34 µM); the protease was completely omitted. After the last perfusion step with 0.18 mM CaCl2 Tyrode solution, the right and left auricles of rats (and only the left auricle of guinea pigs) were dissected and separately minced in 3 ml of 0.18 mM CaCl2 Tyrode solution. After filtration through a nylon mesh, similarly as in ventricular myocytes, the suspension of cells was exposed to a gradually increasing external Ca2+ concentration (up to 0.9 mM within 20 min). Culture of freshly isolated rat cardiomyocytes Immediately after isolation, the rat cardiomyocytes were centrifuged and then resuspended in DMEM medium (Gibco) supplemented with 10% foetal calf serum (Sigma-Aldrich), 1% Lglutamine (Gibco), and 1% penicillin/streptomycin (Gibco). The cells were cultured for 24 hours at 37°C and 5% CO2 in the DMEM medium, either in the absence or in the presence of 20 mM ethanol/3 µM acetaldehyde. On the next day, electrophysiological analysis of the magnitude of IK1 (~ the current sensitive to 100 µm BaCl2) followed in the rat ventricular myocytes, already in absence of ethanol/acetaldehyde. Unfortunately, the rat atrial cells did not survive this procedure in a sufficient number (even for several hours) and could not be used for the analysis.

Culture and transient transfections of Chinese hamster ovary cells Chinese hamster ovary (CHO) cells were cultured at 37°C / 5% CO2 in Ham’s F-12 medium (Sigma-Aldrich) supplemented with 10% foetal calf serum (Sigma-Aldrich) and 0.005% gentamycin (Sigma-Aldrich). The human Kir2.3 in a pXOOM vector (containing also the reporter enhanced green fluorescent protein EGFP) was kindly provided by Assoc. Prof. Morten B. Thomsen (University of Copenhagen, Copenhagen, Denmark). Plasmid DNA was cloned into One Shot chemically competent cells Escherichia coli Top10F’ (Invitrogen) by heat-shock technique (according to manufacture’s manual). After cultivation, plasmids were isolated from bacterial cells using an endotoxin-free QIAprep Spin Miniprep Kit (Qiagen). The quantity and purity of isolated plasmid were measured spectrophotometricaly by BioPhotometer (Eppendorf). TransFast Transfection Reagent (Promega) was used for transfection of the plasmid into CHO cells. One microgram of the plasmid DNA was blended with TransFast Reagent in the ratio 1:1.5 and used for transfection of CHO cells seeded on 35 mm Petri dish according to the manufacture’s manual. The whole cell patch clamp measurements were performed ~48 hours after the transfection. Solutions and chemicals Tyrode solution of the composition below was used during both the dissociation procedure and measurements (in mM): NaCl 135, KCl 5.4, MgCl2 0.9, HEPES 10, NaH2PO4 0.33, CaCl2 0.9, glucose 10 (pH was adjusted to 7.4 with NaOH). During the experiments on cardiomyocytes, the specific blockers were used to inhibit ionic currents potentially interacting with IK1 measurement. CoCl2 (2 mM) and tetraethylammonium chloride (TEA, 50 mM) were applied to inhibit calcium current ICa and delayed rectifier potassium current IK, respectively. During the AP recordings, TEA was omitted. Although it is unlikely to activate ATP-sensitive potassium current IK(ATP) under the given experimental conditions (i.e. 5 mM ATP in the pipette solution, isolated cells), it was inhibited by 10 µM glibenclamide. Atropine (1 µM) was added to avoid a contribution of changes of acetylcholine-sensitive inward rectifier potassium current IK(Ach). IK1 was evaluated as the current sensitive to 100 µM BaCl2. The patch electrode filling solution contained (in mM): Laspartic acid 120, KCl 15, MgCl2 1, K2ATP 5, EGTA 1, HEPES 5, GTP 0.1, Na2-phosphocreatine 3 (pH 7.25 adjusted with KOH). CoCl2 (Sigma-Aldrich), atropine (Sigma-Aldrich) and BaCl2 (Sigma-Aldrich) were prepared as 1 M, 1 mM and 10 mM stock solutions, respectively, in the deionized water. Glibenclamide (Sigma-Aldrich) was prepared as 100 mM stock solution in dimethyl sulfoxide (DMSO; AppliChem GmbH, Germany). The concentration of DMSO in the final control solution and test solutions was identical (0.01%). Moreover, it is unlikely that this concentration of DMSO had any effects on the cardiac IK1 (17, 18). To prepare the TEA-containing stock solution, NaCl in the used Tyrode solution (described above) was replaced equimolarly by TEACl (Sigma-Aldrich). Ethanol (99.5%; TAMDA, Czech Republic) and acetaldehyde (Sigma-Aldrich) were added to the Tyrode solution to obtain the final concentrations - between 2 and 80 mM (~0.009 and 0.37%) in the case of ethanol, and the concentration of 3 µM in the case of acetaldehyde. The solutions were applied in a close vicinity of the measured cell via a perfusion system; the time to change the solution was approximately 2 s.

341 Electrophysiological measurements and evaluation Single rod-shaped cells with well visible striations were used for the current and action potential (AP) recordings applying the whole cell patch-clamp technique in the voltage clamp and current-clamp modes, respectively (Axopatch 200B and pCLAMP 9.2 software). All the recordings were performed at room temperature (23 ± 1°C). The filled glass electrodes with a resistance below 1.5 MΩ were used. The series resistance was compensated up to 75%. The capacitance was not compensated because the contribution of capacity current to the measured current was negligible. The measured ionic current was digitally sampled at 5 kHz. IK1 was evaluated as the Ba2+-sensitive current (100 µM) at the end of 500-ms rectangular pulse to –110 mV preceeded by a prepulse to –50 mV to inactivate the sodium current INa. The duration of the prepulse was 15 ms and 100 ms during the analysis of ethanol and acetaldehyde effects, respectively. At the steady-state applications, 3-s ramp pulses between –110 and –10 mV were used to check the voltage dependence of the drug effects. In all voltage clamp experiments, the holding potential was –85 mV and the stimulation frequency 0.2 Hz. The results were corrected for the junction potential (–10 mV). APs in rat atrial cells were elicited by 0.5-ms suprathreshold current pulses of 4 – 10 nA applied at the stimulation frequency of 1 Hz. Statistical analysis The results are presented as means ± S.E.M from n cells (Origin, version 8.5.1; OriginLab Corporation). The curve fitting, paired and unpaired t-test, and one-way and repeated measures ANOVA with the Bonferroni post-test were performed using the GraphPad Prism, version 6.05 (GraphPad Software, Inc.); P < 0.05 was considered statistically significant. To test the significance of the Pearson’s correlation coefficient r, we used the t distribution as expressed below and comparison with the critical values according to the Student’s t-test.

t=r

(n − 2 ) / (1 − r 2 )

Mathematical simulations To simulate the impact of ethanol- or acetaldehyde-induced IK1 changes on rat atrial AP configuration, we have developed a model of rat atrial myocyte, based on our previous rat ventricular myocyte model (19, 20). The model incorporates detailed description of IK1 interaction with ethanol and acetaldehyde evolved on the basis of our experimental data. In simulations, all experimental conditions were preserved (namely intracellular and extracelular ion concentrations, presence of 1 mM EGTA in the cytosol, ICa inhibition). The numerical solution was performed using the computational software MATLAB v. 7.2 (MathWorks, Inc.).

RESULTS Effect of ethanol on rat and guinea-pig atrial IK1 and on human Kir2.3 current Like in ventricular myocytes (13), the effect of ethanol on the rat atrial IK1 was dual: the current at the steady-state was either activated or inhibited as shown at 80 mM (~0.37%) ethanol in Fig. 1. The running changes of ionic current evaluated at the end of 500-ms hyperpolarizing pulse to –110 mV in the course of representative experiments are shown in Fig. 1A. The cell was exposed step by step to the control Tyrode solution

(con), 80 mM ethanol (eth), the control Tyrode solution (wash), 100 µM Ba2+, and a combination of ethanol and Ba2+ to distinguish IK1 from contaminating currents. The residual Ba2+insensitive current appeared to be insensitive to ethanol (for representative records see Fig. 1A; on average, –0.40 ± 0.16 pA/pF in control vs. –0.41 ± 0.16 pA/pF at ethanol; n = 11 for both, P > 0.05). The ethanol effect on the rat atrial IK1 always started with a significant transient inhibition of the current, by 10.5 ± 2.3 % at the steady-state IK1 activation (Fig. 1B, left panel; n = 9, P < 0.05), and by 18.3 ± 2.8% at the steady-state IK1 inhibition (Fig. 1B, right panel; n = 8, P < 0.001). At the steady-state, the average IK1 activation was 12.4 ± 2.7 % and IK1 inhibition 10.2 ± 2.9% (P < 0.05 for both). The effect of ethanol was fully reversible during the wash-out period (wash). Importantly, the change of IK1 induced by 80 mM ethanol appeared to significantly correlate with IK1 density in control; the cells with smaller IK1 in control conditions showed rather activation under the effect of ethanol and vice versa (Fig. 1D). The Pearson’s correlation coefficient (r = 0.47) was statistically significant (P < 0.05). The check measurements confirmed that IK1 at the steady-state of wash-out period returned to control value independently of IK1 density (Fig. 1C - the correlation coefficient was statisticaly insignificant). It follows that the response of IK1 to ethanol may be regarded as an intrinsic property of the measured cell isolated from heterogeneous tissue. The IK1 magnitude considerably varied among the measured cells (Fig. 1C) but this variation did not correlate with the value of resting membrane potential (RMP; not illustrated; the average RMP = –77.9 ± 1.3 mV, n = 15). The average current-voltage relationships of the rat atrial IK1 in control (con) and under 80 mM ethanol (eth) during 3-s ramp pulse between –110 and –10 mV are shown in Fig. 1E (for the experimental protocol see inset in the left panel). To evaluate voltage dependence of the ethanol effect from ratio of the IK1voltage (V) relationships recorded at the steady-state ethanol effect and in control conditions, noise had to be removed from the recordings. As shown in Fig. 2A, all the current-voltage relationships could be successfully approximated by 9th-order polynomial functions (the coefficient of determination R between 0.999 and 1). The reversal voltage (Vrev) was not affected by ethanol. On the other hand, the effect of ethanol estimated from the ratio of IK1-V relationships (Fig. 2B) increased moderately with depolarization within the relevant voltage range in both groups of cells. The voltage dependence of IK1 chord conductance (GK1) presented in Fig. 2C was obtained from the IK1-V relationships according to the equation GK1 = IK1 / (V – Vrev). The values of maximum chord conductance GK1max < –110 mV could not be estimated directly. However, after normalization to the values at –110 mV, the GK1–V curves nearly overlapped (not shown). It implies that Gmax appears to be the only parameter responsible for the effect of ethanol on IK1. The ethanol-induced IK1 changes evaluated from all measurements regardless of the occurrence of steady-state activation or inhibition are shown in Fig. 3. On average, the steady-state ethanol-induced IK1 activation by 3.9 ± 2.4% at 8 mM (~0.04%), by 4.3 ± 1.9% at 20 mM (~0.09%), and by 1.2 ± 3.5% at 80 mM (~0.37%) was present in rat atrial myocytes (Fig. 3A). The effect was clearly smaller in comparison with the ventricular IK1 data (13; the blue symbols in Fig. 3A). However, in agreement with the ventricular data, a marked dispersion of the ethanol effect including the dual effect was evident (for 20 mM ethanol illustrated also in Fig. 3B and 3C). Similarly like at 80 mM ethanol (Fig. 1A and 1B), the transient inhibition at the beginning of ethanol application was present also at the concentrations of 8 mM (by 9.4 ± 4.2%, n = 7) and 20 mM (by 9.9 ± 2.8%, n = 6; for representative

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Fig. 1. Dual effect of 80 mM ethanol on rat atrial IK1. (A): Time courses of representative experiments showing changes of the membrane current at –110 mV (I–110 mV) measured at the end of 500 ms pulses in the absence of ethanol (empty circles) and under the effect of 80 mM ethanol (full circles); 100 µM Ba2+ was applied both in the absence and presence of 80 mM ethanol; top panel - steady-state IK1 activation, bottom panel - steady-state IK1 inhibition; vertical arrows indicate the time of evaluation (con, control; eth-T, transient peak inhibition; eth-S, steady-state effect; wash, washout). The experimental protocol: 500-ms hyperpolarizing test pulse from the holding potential of –85 mV to –110 mV was preceded by 15-ms prepulse to –50 mV to inactivate the sodium current INa. (B): Averaged IK1 changes under 80 mM ethanol; left panel - steady-state IK1 activation: n = 9, right panel - steady-state IK1 inhibition: n = 8; * and *** - statistical significance at P < 0.05 and 0.001, respectively. (C): Magnitude of IK1 in control condition (IK1,con) was not considerably changed during the experiment (IK1,wash / IK1,con). (D): Correlation between magnitude of the current in control condition (IK1,con) and the effect of 80 mM ethanol on IK1 (IK1,eth / IK1,con); Pearson’s correlation coefficient = 0.47 was statistically significant (P < 0.05). (E): Averaged IK1 in control and at 80 mM ethanol during the ramp pulse between –110 and –10 mV (left panel - IK1 activation: n = 5; right panel - IK1 inhibition: n = 3). Inset in E, left panel: the used ramp protocol.

343 experiments at 20 mM ethanol see Fig. 3B). The average values of IK1 density in control (con), under the effect of 20 mM ethanol (eth) and during the subsequent wash-out (wash) are illustrated in Fig. 3C, left panel. To summarize results presented in Fig. 1

and 3, ethanol caused transient inhibition of the rat atrial IK1 at the beginning of its application followed by dual changes of the current at the steady-state. The reaction was similar at all tested concentrations between 8 and 80 mM.

Fig. 2. Analysis of voltage dependence of ethanol effect on rat atrial IK1; left panel - IK1 activation, right panel - IK1 inhibition, both at steady-state effect of 80 mM ethanol. (A): IK1-voltage curves from Fig. 1E and results of their fitting by 9th-order polynomial functions (coefficient of determination R > 0.999). (B): Ratio of fitted curves presented in A (ethanol/control; expressed in %) as an indicator of voltage dependence of the ethanol effect on IK1. The dotted parts of the curves correspond to the voltage ranges where the current is close or equal to zero, thus, the ratio cannot be reliably determined. (C): The chord conductance (G) -voltage (V) relationships calculated as GK1 = IK1 / (V – Vrev) in control conditions and under the effect of ethanol. The dotted parts correspond to the voltage ranges around the reversal voltage (Vrev).

344 The described complex effect of ethanol on the rat atrial IK1 was also observed in experiments performed in isolated guineapig atrial myocytes with similar values of the average steadystate IK1 activation (at 20 mM ethanol: by 4.5 ± 2.8% in guinea pig vs. by 4.3 ± 1.9% in rat, n = 6 for both, P > 0.05; Fig. 3C, right panel). In contrast, the current carried by the human expressed Kir2.3 channels (the prevailing subunit forming IK1 channels in atria) was uniformly inhibited by ethanol (by 13.5 ±

3.6% at the steady-state effect of 80 mM ethanol, n = 5); no transient peak of the effect was present at the beginning of ethanol application (Fig. 3D). Effect of acetaldehyde on rat atrial IK1 Acetaldehyde at a clinically relevant concentration of 3 µM inhibited the rat atrial IK1 without considerable transient peak of

Fig. 3. Comparison of ethanol effect on IK1 in rat atrial and ventricular myocytes, in guinea-pig atrial myocytes, and in transiently expressed human Kir2.3 channels. (A): Pooled data in individual rat atrial cells (dots) and mean ± S.E.M. (crosses) are shown at 8, 20 and 80 mM ethanol (n = 7, 6 and 19). For comparison, analogical rat ventricular data are added in blue colour as recently reported under comparable experimental conditions (13). (B): Representative experiments illustrating the effect of 20 mM ethanol on rat atrial IK1 in a cell showing the steady-state activation (upper panel) and the steady-state inhibition (lower panel). (C): Average effect of 20 mM ethanol on IK1 in rat atrial myocytes (left panel, n = 6) and in guinea-pig atrial myocytes (right panel, n = 6). (D): Average changes of human Kir2.3 current under the effect of 80 mM ethanol (n = 5). Abbreviations: con, control; eth, ethanol; wash, wash-out; T, transient peak inhibition/current density at the time corresponding to transient peak inhibition (as shown in Fig. 1A and 1B), S, steady-state effect; * and ** - statistical significance at P < 0.05 and 0.01, respectively.

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Fig. 4. Inhibitory effect of 3 µM acetaldehyde on rat atrial IK1. (A): Time course of a representative experiment showing changes of the membrane current at –110 mV (I–110 mV) measured at the end of 500 ms pulses in the absence of acetaldehyde (empty circles) and under the effect of 3 µM acetaldehyde (full circles); 100 µM Ba2+ was applied both in the absence and presence of 3 µM acetaldehyde. Vertical arrows indicate the time of evaluation: con, control; acetald, acetaldehyde; T, current density at the time corresponding to the transient peak inhibition at the beginning of ethanol application (as shown in Fig. 1A and 1B), S, steady-state effect; wash, wash-out. For the experimental protocol see legend to Fig. 1A; the prepulse to –50 mV lasted 100 ms in this case. (B): Averaged effect of 3 µM acetaldehyde, n = 9; ** statistical significance at P < 0.01. (C): Averaged IK1 in control and at 3 µM acetaldehyde (n = 3) during the ramp pulse between –110 and –10 mV (for the protocol see inset in Fig. 1E, left panel). Inset: the acetaldehyde-induced relative IK1 changes at –110 and –50 mV (n = 9). (D): Comparison of acetaldehyde effect on IK1 in rat atrial myocytes (red dots at the concentration of 3 µM) and rat ventricular myocytes (black dots and line; 15).

346 the effect (Fig. 4A and 4B). The effect was similar at all tested voltages between –110 and –10 mV (n = 3; Fig. 4C; inset: comparison of the acetaldehyde-induced IK1 changes at –110 and –50 mV as evaluated from the rectangular pulses lasting 500 and 100 ms, respectively, n = 9). Fig. 4D shows that the steady-state IK1 inhibition at 3 µM acetaldehyde in atrial cells (by 15.1 ± 3.2%; n = 9) was similar to that in ventricular myocytes (13.1 ± 3.0%, n = 16, (15); P > 0.05, ventricular vs. atrial data). The Ba2+-insensitive current was not affected by acetaldehyde (for a representative record see Fig. 4A; on average, –0.99 ± 0.09 pA/pF in control vs. –0.98 ± 0.09 pA/pF at acetaldehyde; n = 4 for both, P > 0.05). Combined effect of ethanol and acetaldehyde on rat atrial IK1 Ethanol and its primary metabolite acetaldehyde are simultaneously present in the blood for a considerable period of time during and after alcohol consumption. Hence, we selected a clinically-relevant concentration of ethanol and acetaldehyde to investigate their combined effect on the rat atrial IK1. The rat atrial IK1 showed changes not essentially different from those

produced by the ethanol alone if ethanol (20 mM) and acetaldehyde (3 µM) were applied concurrently (Fig. 5A). The average transient inhibition of the current was similar (9.9 ± 2.8% for the ethanol alone, n = 6 vs. 9.5 ± 1.5% for the combination of both substances, n = 9; P > 0.05) despite the higher inhibitory effect of acetaldehyde alone (16.1 ± 3.0% measured at the same time when the transient effect of ethanol was evaluated, n = 9). However, at the steady-state, the average IK1 activation in the presence of both ethanol and acetaldehyde was minor (2.1 ± 1.4%, n = 9, vs. 4.3 ± 1.9% for the ethanol alone, n = 6; P > 0.05). These results may be tentatively interpreted in terms of the biophysically based quantitative model described in our previous work dealing with the effect of ethanol on IK1 in ventricular myocytes (13). The model was able to reproduce the experimental results precisely if the values of its parameters were set accordingly (cf. Figs. 5B and Fig. 5A). Fig. 5C illustrates the model simulations of mean time course of IK1 changes during the application of ethanol, acetaldehyde, and their combination in a relative scale (see the bar graph shown in Fig. 5B). More information concerning the model is presented in the Discussion.

Fig. 5. Transient and steady-state changes of rat atrial IK1 induced by separate and combined effects of 20 mM ethanol and 3 µM acetaldehyde. (A): The effect of separately applied ethanol (eth; n = 6), acetaldehyde (acetald; n = 9), and combined ethanol and acetaldehyde (n = 9). (B) and (C): Results of simulations from the biophysically based mathematical model.

347

Fig. 6. Steady-state effect of 20 mM ethanol (eth; A) and 3 µM acetaldehyde (acetald; B) on rat atrial action potential (AP) configuration (0.5-ms suprathreshold current pulses of 4 – 10 nA were applied at the stimulation frequency of 1 Hz). Upper panels: Representative AP waveforms as recorded in rat atrial myocytes. Insets: Significant changes of APD90 in comparison with the respective control values (con); * and ** - statistical significance at P < 0.05 and 0.01, respectively. Lower panels: The mathematically simulated rat atrial AP waveforms including the experimentally acquired ethanol or acetaldehyde effects on IK1; insets: respective changes of APD90.

Changes of action potential configuration at clinically relevant concentrations of ethanol and acetaldehyde Ethanol at the concentration of 20 mM induced either shortening or prolongation of AP duration at 90% repolarization (APD90) in individual cells. The shortening was observed in most of the cells, at maximum by 10.7%, whereas the prolongation was present rarely, at maximum by 3.2%. On average (Fig. 6A, upper panel), a slight but significant shortening of APD90 was revealed, by 2.3 ± 1.0 % (n = 11, P < 0.05). On the contrary, acetaldehyde at 3 µM significantly prolonged APD90 and also AP duration at 50% repolarization (APD50), on average by 12.0 ± 3.2 % and 4.0 ± 1.3 %, respectively (n = 7, P < 0.01 and 0.05, respectively; Fig. 6B, upper panel). All other AP parameters including RMP remained unaltered by both substances. Mathematical modelling of rat atrial cell APs showed qualitatively the same results if the mean effect of each substance on IK1 was implemented into the model (Fig. 6A and

6B, lower panels). APD90 was slightly shortened by ethanol (20 mM) but prolonged by acetaldehyde (3 µM). In contrast to the experimental AP data, no changes were observed in other parameters including APD50. The changes of APD90 were considerably less pronounced (at ethanol: APD90 shortening by 1.5%; at acetaldehyde: APD90 prolongation by 6.0%). These quantitative differences between the model and experiments are probably related to the effect of ethanol and acetaldehyde on other components of the total repolarizing current in rat atrial cells (see Discussion). Changes of IK1 at prolonged exposure to clinically relevant concentrations of ethanol and acetaldehyde To investigate changes of the rat IK1 caused by a prolonged exposure to ethanol or acetaldehyde (imitation of alcohol intoxication), the freshly isolated rat ventricular myocytes were cultured either in control culture medium, or in the medium

348

Fig. 7. Effect of 24-hours exposure to 20 mM ethanol (eth) or 3 µM acetaldehyde (acetald) on rat IK1; n = 13, 9, and 11 for the groups of cells cultured in the control culture medium, and in the medium supplemented with 20 mM ethanol, or 3 µM acetaldehyde, respectively; IK1 was measured as Ba2+ sensitive current in control Tyrode solution.

supplemented with 20 mM ethanol or 3 µM acetaldehyde for 24 hours. After wash-out of ethanol/acetaldehyde, IK1 magnitude was measured (for details see Methods). No differences of IK1 density were apparent among all the cell groups at –110 and –50 mV (Fig. 7).

DISCUSSION The present study demonstrates the effect of clinicallyrelevant concentrations of ethanol and its primary metabolite acetaldehyde on inward rectifier potassium current IK1 in atrial cardiomyocytes. For the first time, ethanol and acetaldehyde were applied both separately and in combination. Different effect of ethanol on atrial IK1 and expressed human Kir2.3 channels IK1 channels in atria and ventricles are tetramers composed of Kir2.1, Kir2.2, and Kir2.3 subunits (e.g. 21). The Kir2.3 subunits are dominant in atria whereas Kir2.1 in ventricles (21, 22-24); the level of Kir2.1 subunits in atria was estimated to be only 25% of that in ventricles (24, 25). Ethanol affects the rat atrial IK1 in a dual manner showing steady-state activation in one group of the cells and inhibition in another one (Fig. 1, 3A and 3B) similarly as in rat ventricular cell (13). The observed high variance in IK1 density (Fig. 1C and 1D) may be regarded as a random error, or as a manifestation of real diversity of individual cells due to heterogeneity of the atrial tissue. Significant correlation between the ethanol effect and the control IK1 density (Fig. 1D) provides a strong argument supporting interpretation of the observed dual effect of ethanol on IK1 as resulting from real differences of the measured (incidentally chosen) cells isolated from heterogeneous tissue. Significant differences between IK1 density in myocytes from left and right atria of adult mouse have been well documented (26, 27). The characteristic feature of the ethanol effect in both groups of cells (i.e. cells showing steady-state activation or inhibition) was a noticeable transient inhibition of IK1 at the beginning of ethanol application (Figs. 1A, 1B, 3B, and 3C). These atrial IK1 data are qualitatively in a good agreement with our recently published results in ventricles (13). The initial transient

inhibition may be interpreted as a consequence of the faster kinetics of the ethanol-induced inhibition in comparison with the kinetics of ethanol-induced activation. In guinea-pig atrial myocytes, similar changes of the atrial IK1 were found (Fig. 3C, right panel). On the contrary, the current through expressed homomeric Kir2.3 channels was lacking any transiently increased inhibition at the beginning of ethanol application. The steady-state inhibition was obvious in all measured cells (Fig. 3D). Similarly, the expressed homomeric Kir2.1 channels were shown to be only inhibited by ethanol (9); it was explained by the strong affinity of Kir2.1 channels to phosphatidylinositol-4,5bisphosphate (PIP2) which disables further increase of the current under the effect of ethanol. In addition, several recent studies have shown that heteromeric assemblies of Kir2.x subunits exhibit quite different properties from those of their homomers (28-30). Different drug-induced action in native cardiac channels and their expressed single pore-forming channel subunit has been also described (31). Hence, the dual effect of ethanol on cardiomyocytes may be regarded as a manifestation of various homo- and heterotetrameric channels which may differ in both the mode of action (activation/inhibition) and its kinetics. The steady-state effect depends on the features of the channels that predominate in the given cell. Possible explanation of the correlation between the effect of ethanol on IK1 and its magnitude in control (Fig. 1D) might be given under assumption that the channels inhibited by ethanol have greater unitary conductance and/or mean open time in control than those activated. Aryal et al. (9) reached a similar conclusion comparing the effect of ethanol on IRK1 and GIRK channels (a different interaction between the channel and PIP2). The dual effect of ethanol was also observed in neuronal nicotinic acetylcholine receptors and pentameric ligand-gated ion channels, and was consistent with a two-site model of the ethanol-induced inhibition and activation (32, 33). Inhibition of atrial IK1 by acetaldehyde In agreement with our previous study on rat ventricular myocytes (15), acetaldehyde exerted an inhibitory effect on the rat atrial IK1 in the current study (Fig. 4); the inhibition at 3 µM acetaldehyde matched the ventricular data. The former studies analysing the effect of acetaldehyde in cardiac cells, namely in guinea pig ventricular cells (11) and in bullfrog atrial cells (12), did not reveal any changes of IK1 in comparison with our present study even at much higher concentrations of acetaldehyde (500 and 1000 µM). We have no obvious explanation of this discrepancy; at any rate, the differences in species and tissue types have to be considered. As the cardiac IK1 channels are homo- or heteromeric tetramers composed of different Kir2.x, isoforms with various proportions, the varying arrangement of IK1 channel subunits may lead to uneven drug sensitivity, even in atria and ventricles of the same heart (e.g. 21, 25, 34). Changes of atrial IK1 under separate and combined effect of ethanol and acetaldehyde Remarkably, the marked acetaldehyde-induced inhibition of IK1 (by 15.1%) did not manifest itself when applied in combination with ethanol. The transient inhibition due to both ethanol and acetaldehyde was similar to that induced by ethanol alone; a slight average IK1 activation, less than that observed in the presence of ethanol alone, was apparent at the steady-state. We tried to formulate a possible explanation using our previously described model (13) which we slightly modified for

349 the current purpose. Three populations of channels underlying the ventricular IK1 were incorporated into the model, one responding to ethanol by activation, the other two by inhibition. Both ethanol and acetaldehyde were assumed to reach the steady-state binding on channel structures quickly. On the other hand, the link to channel gating (very likely mediated by PIP2) is supposed to be responsible for the observed slow responses of IK1 to the application of substances (Fig. 5C). The transient inhibition preceding the steady-state activation indicated that the time constant of inhibition (set to 20 s) was faster than the time constant of activation (55 s) when using either ethanol or the mixture of 20 mM ethanol and 3 µM acetaldehyde. Simulations of the effect of acetaldehyde alone showed a tiny overshoot of increased inhibition (in accordance with Fig. 5A). Weak interaction of acetaldehyde with the activation binding site had to be assumed (beside inhibition as the main effect). However, the acetaldehyde-induced overshoot of the inhibition was not significant (see also Fig. 4B). Thus, the assumption of the exclusive inhibitory effect of acetaldehyde is also plausible. A strong counteracting effect of the channels showing activation and those showing inhibition had to be introduced into the model to simulate the time course of the ethanol-induced IK1 changes (Fig. 5C). According to the model, the steady-state IK1 change under ethanol results from ~36% increase due to the activation counteracted by ~32% decrease due to the inhibition. It implies that the ethanol-induced inhibition is considerably stronger than the acetaldehyde-induced inhibition (the decrease by ~15%), thus, the effect of ethanol prevails in presence of both substances. It should be emphasized that the model with the same setting of numeric values of parameters was able to satisfactorily simulate the experimentally obtained effect of ethanol at different concentrations (Fig. 3A). It is necessary to take into account that the blood concentrations of ethanol and acetaldehyde and, consequently, the ratio of their blood concentrations change in time which makes it difficult to study their combined effect properly. The combined effect will change with the changing ratio, first being more or even exclusively related to the ethanol effect, but finally to be related more or even exclusively to the effect of acetaldehyde. Effect of ethanol and acetaldehyde on action potentials of atrial cells The quantitative differences between recorded and simulated APs (Fig. 6) are very likely related to an effect of ethanol and/or acetaldehyde on other components of the total repolarizing current in rat atrial cells. There are several candidates among potassium channels that may be affected, particularly the abundantly expressed rapid component of delayed rectifier current IKr and ultrarapid delayed rectifier current IKur (35-38), acetylcholinesensitive inward rectifier current IK(Ach) (39), and calcium-activated currents IKCa playing an important role in electrophysiology of various cell types including atrial cells (40-44). Clinical relevance Our study demonstrates that ethanol even at relatively low concentrations affected the atrial IK1 at physiological voltage range in two different ways with great variability in reactions of individual cells. Variable response of cardiomyocytes from different ventricular layers to changes of ionic channel properties is known to be highly dependent on the natural heterogeneity of electrical properties of the cells which may result in the formation of arrhythmogenic substrate (45).

Similarly, the natural heterogeneity of electrical properties of cardiac cells in the atrial tissue (46, 47) may be also proarrhythmogenic if modified by ethanol. Acetaldehyde, the primary ethanol metabolite, at a clinically relevant concentration significantly inhibited this current in all the measured cells. Moreover, genetic variants of acetaldehyde dehydrogenase may prolong and increase accumulation of acetaldehyde in some individuals (48, 49). Our findings suggest that the ethanol- and acetyldehydeinduced regional modification of electrical heterogeneity throughout the atrial tissue might increase the risk of atrial arrhythmias, particularly in the presence of various cardiac and non-cardiac co-factors that are also known to increase susceptibility to AF (50). Acknowledgements: The authors thank to Assoc. Prof. Morten B. Thomsen (University of Copenhagen, Copenhagen, Denmark) for kindly providing the human Kir2.3 in a pXOOM vector. We thank to Prof. P. Bravený for reading the manuscript and valuable comments, and to Mrs. B. Vyoralova for excellent technical assistance. This work was supported by the grant project NT14301-3/2013 from the Departmental Program for Research and Development of the Ministry of Health of the Czech Republic, and partially by the institutional support RVO: 61388998. Conflict of interests: None declared.

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