The role of transmembrane chloride current in afterdepolarisations in canine ventricular cardiomyocytes.

Abstract

The physiological role of chloride currents (Icl) in cardiac cells is poorly understood. The aim of the present study was to reveal the role of Icl in the genesis of early and delayed afterdepolarisations (EADs and DADs, respectively). First we identified Icl under action potential voltage clamp conditions as the anthracene-9-carboxylic acid (ANTRA) (0.5 mmol/l)-sensitive current. The ANTRA-sensitive current was large and outwardly directed at the beginning, while it was moderate and inwardly directed at the end of the action potential. Application of ANTRA under current clamp conditions decreased the depth of the incisura, shifted the plateau upwards and lengthened the duration of action potentials. The effect of ANTRA was studied in three models of afterdepolarisations: the ouabain-induced DAD model, the caesium-induced EAD model, and in the presence of subthreshold concentration of isoproterenol. Preincubation of the cells with 0.5 mmol/l ANTRA failed to induce afterdepolarisations. Ouabain (200 nmol/l) alone caused DADs in 62.5% of the cells within 15 min. When ouabain was applied in the presence of ANTRA, 60% of the myocytes transiently displayed EADs before the development of DADs, and all cells developed DADs within 7 min. Isoproterenol (5 nmol/l) alone failed to induce afterdepolarisations. However, 75% of the cells produced DADs within 6 min when superfused with isoproterenol in the presence of ANTRA. Incubation of the myocytes with 3.6 mmol/l CsCl caused EADs in 71.4% of the cells within 30 min. Application of CsCl in the presence of ANTRA resulted in immediate depolarisation of the membrane from -79.6 +/- 0.4 to -54.2 +/- 3.5 mV. Summarizing our results we conclude that the ANTRA-sensitive current is an important mechanism of defence against afterdepolarisations. Suppression of Icl may thus increase the incidence and accelerate the rate of development of both EADs and DADs.

Full text

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Gen. Physiol. Biophys. (2003), 22, 341—353
341
The Role of Transmembrane Chloride Current in
Afterdepolarisations in Canine Ventricular Cardiomyocytes
L. Fülöp, E. Fiák, N. Szentandrássy, J. Magyar, P. P. Nánási
and T. Bányász
Department of Physiology, University Medical School of Debrecen,
Debrecen, Hungary
Abstract. The physiological role of chloride currents (ICl) in cardiac cells is poorly
understood. The aim of the present study was to reveal the role ofIClin the genesis
of early and delayed afterdepolarisations (EADs and DADs, respectively). First we
identified ICl under action potential voltage clamp conditions as the anthracene-
9-carboxylic acid (ANTRA) (0.5 mmol/l)-sensitive current. The ANTRA-sensitive
current was large and outwardly directed at the beginning, while it was moderate
and inwardly directed at the end of the action potential. Application of ANTRA
under current clamp conditions decreased the depth of the incisura, shifted the
plateau upwards and lengthened the duration of action potentials.
The effect of ANTRA was studied in three models of afterdepolarisations: the
ouabain-induced DAD model, the caesium-induced EAD model, and in the presence
of subthreshold concentration of isoproterenol. Preincubation of the cells with 0.5
mmol/l ANTRA failed to induce afterdepolarisations. Ouabain (200 nmol/l) alone
caused DADs in 62.5% of the cells within 15 min. When ouabain was applied in the
presence of ANTRA, 60% of the myocytes transiently displayed EADs before the
development of DADs, and all cells developed DADs within 7 min. Isoproterenol
(5 nmol/l) alone failed to induce afterdepolarisations. However, 75% of the cells
produced DADs within 6 min when superfused with isoproterenol in the presence of
ANTRA. Incubation of the myocytes with 3.6 mmol/l CsCl caused EADs in 71.4%
of the cells within 30 min. Application of CsCl in the presence of ANTRA resulted
in immediate depolarisation of the membrane from −79.6 ± 0.4 to −54.2 ± 3.5
mV. Summarizing our results we conclude that the ANTRA-sensitive current is an
important mechanism of defence against afterdepolarisations. Suppression of ICl
may thus increase the incidence and accelerate the rate of development of both
EADs and DADs.
Key words: Cardiomyocytes — Chloride currents — Afterdepolarisations — Iso-
proterenol — Ouabain
Correspondence to: Dr. Péter P. Nánási, Department of Physiology, University of
Debrecen, P.O.Box 22, H–4012 Debrecen, Hungary
E-mail: nanasi@phys.DOTE.hu

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Fülöp et al.
Introduction
Afterdepolarisations, defined as subthreshold depolarising afterpotentials of the cell
membrane following an action potential (AP), have been proposed as the mecha-
nism responsible for genesis of cardiac arrhythmias (Priori and Corr 1990; Fozzard
1992; Szabo et al. 1994). Afterdepolarisations are divided into two types according
to their relationship to the preceding AP. Early afterdepolarisations (EADs) de-
velop before the completion of repolarisation, whereas delayed afterdepolarisations
(DADs) arise from the resting potential level of the fully repolarised cell. Since
generation of both EADs and DADs requires a preceding AP, afterdepolarisations
are frequently referred to as triggered activities (Fozzard 1992). Both types of af-
terdepolarisations were studied extensively during the last two decades; however,
the underlying ionic mechanisms are still not fully elucidated. DADs are seen at
various conditions in both atrial and ventricular cells exposed to catecholamines,
digitalis or low extracellular sodium (Wit and Rosen 1992). It is generally be-
lieved that the prerequisite for DAD generation is an increased calcium load to
the cell, and consequently to the sarcoplasmic reticulum (SR), resulting in spon-
taneous oscillatory release of calcium from the overloaded SR. Transient elevation
of the myoplasmic calcium is responsible for activation of a current, called tran-
sient inward current (ITI) which, in turn, leads to membrane depolarisation. ITI
was characterised first as a non-selective cation current (Kass et al. 1978; Cannell
and Lederer 1986; Ehara et al. 1988; January and Fozzard 1988; Han and Ferrier
1992). Later, the involvement of the electrogenic Na+/Ca2+exchange current was
suggested by several investigators (Kimura et al. 1986; Mechmann and Pott 1986;
Callewaert et al. 1989; Šimurda et al. 1992; Janvier and Boyett 1996). Recently,
the calcium-activated chloride current was proposed as the charge carrier for ITI
in rabbit (Szigeti et al. 1998) and in dog (Zygmunt et al. 1998) myocardium.
EADs are typically observed when low pacing rates are associated with pro-
longed APs usually due to suppression of delayed rectifier potassium currents IKr
and IKs, or alternatively, due to increased density of ICaor INaduring the plateau
of the AP (January et al. 1988; January and Riddle 1990). The assumed mechanism
for generation of EADs is as follows. The initial depolarisation activates ICa, which
rapidly inactivates during the early plateau. The sustained plateau of a lengthened
AP or augmentation of ICaand INamight establish conditions necessary for reacti-
vation of ICa. Reactivation of ICais supposed to be responsible for interruption of
the normal repolarisation processes and generation of EADs (Fozzard 1992; Szabo
et al. 1994). Since ICl has been shown to contribute to repolarisation in canine
ventricular myocytes (Zygmunt 1994; Collier et al. 1996), one should attribute a
protective role to this current against EADs.
Beta-adrenergic activation or isoproterenol was shown to activate several ionic
currents, such as ICa, ICl, and IK in mammalian ventricular cells (Wang et al.
1997; Zygmunt et al. 1998; Wallis et al. 2001; Lei at al. 2002; Sah et al. 2002) in
addition to increasing the calcium load of the SR (Priori and Corr 1990; Zeng and
Rudy 1995). Therefore, submicromolar concentrations of isoproterenol are widely

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Afterdepolarisations and Chloride Current
343
applied to induce both EADs and DADs (Priori and Corr 1990; Szabo et al. 1994;
Zeng and Rudy 1995), however, nanomolar concentrations of isoproterenol fail to
initiate afterdepolarisations. In spite of their apparent inefficacy, these very low
concentrations were found to facilitate the effects of other agents suitable to evoke
EADs or DADs (unpublished observation).
The present work was designed to study the role of the anthracene-9-carboxylic
acid (ANTRA)-sensitive component of ICl in the generation of different types of
afterdepolarisations. APs and ANTRA-sensitive currents were recorded in cardiac
myocytes enzymatically dissociated from canine left ventricle. Afterdepolarisations
were induced by isoproterenol, ouabain and caesium. We found that suppression of
IClby ANTRA may increase the incidence and accelerate the rate of development
of both EADs and DADs.
Materials and Methods
Isolation of single canine ventricular myocytes
Adult mongrel dogs of either sex were anaesthetised with intravenous injections of
10 mg/kg ketamine hydrochloride (Calypsolvet) plus 1 mg/kg xylazine hydrochlo-
ride (Rometar). The hearts were quickly removed in deep anaesthesia and placed
in Tyrode solution. The entire investigation conformed to the guidelines for the
care and use of laboratory animals published by the U.S. National Institutes of
Health, as well as to the principles outlined in the Declaration of Helsinki, and was
approved by the local ethical committee.
Single ventricular myocytes were obtained by enzymatic dispersion using the
segment perfusion technique (Bányász et al. 2001). Briefly, a wedge-shaped section
of the ventricular wall supplied by the left anterior descending coronary artery was
dissected, cannulated and perfused with oxygenated Tyrode solution containing (in
mmol/l): NaCl 144, KCl 5.6, CaCl22.5, MgCl21.2, HEPES 5, and glucose 11 at
pH = 7.4. Perfusion was maintained until the removal of blood from the coronary
system and then switched to a nominally Ca2+-free Joklik solution (Minimum
Essential Medium Eagle, Joklik Modification, Sigma Chemicals, St. Louis, MO,
USA) for 5 min. This was followed by 30 min perfusion with re-circulated Joklik
solution supplemented with 1 mg/ml collagenase (Type II, Worthington Chemical
Co. Lakewood, NJ, USA) and 0.2% bovine serum albumin (Fraction V, Sigma
Chemicals, St. Louis, MO, USA) containing 50 µmol/l Ca2+. Portions of the left
ventricular wall were cut into small pieces and the cell suspension obtained at the
end of the procedure was washed with Joklik solution and the Ca2+concentration
was gradually increased to 2.5 mmol/l. The cells were stored in Minimum Essential
Medium Eagle supplemented with (in mmol/l): taurine 20, pyruvic acid 2, ribose
5, allopurinol 0.1, NaHCO3 26, and NaH2PO4 1.5 at 14◦C until use (for periods
not longer than 24 hours).

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Fülöp et al.
Recording of APs and afterdepolarisations
APs were recorded from Ca2+-tolerant myocytes superfused with modified Krebs
solution containing (in mmol/l): NaCl 120, KCl 5.6, CaCl22.5, MgCl21.1, NaH2
PO41.1, NaHCO324 and glucose 11. The solution was equilibrated with carbogen
(5% CO2+ 95% O2) at a temperature of 37◦C and the pH was adjusted to 7.4.
Transmembrane potentials were recorded using glass microelectrodes filled with 3
mol/l KCl and having tip resistance between 20 and 40 MΩ. These electrodes were
connected to the input of an Axoclamp-2B amplifier (Axon Instruments, Union
City, CA, USA). The cells were continuously paced through the recording electrode
at a steady cycle length of 1000 ms using 1 ms wide rectangular current pulses
with 120% threshold amplitude. APs were digitised at 100 kHz using Digidata
1200 A/D card (Axon Instruments, Union City, CA, USA) and stored for later
analysis.
The effect of a 20 min superfusion with 0.5 mmol/l ANTRA on AP configura-
tion was studied in 8 myocytes. The effect of ANTRA pretreatment (0.5 mmol/l)
on the incidence of afterdepolarisations was investigated as follows. In the control
experiments the effects of isoproterenol (5 nmol/l, n = 6), ouabain (200 nmol/l,
n = 8) or CsCl (3.6 mmol/l with a concomitant reduction of KCl to 2 mmol/l,
n = 7) alone was tested on myocytes for 20–40 min. These protocols were repeated
in another sets of myocytes in the presence of ANTRA (n = 8, n = 5 and n = 5,
respectively). The application of ANTRA started 10 min before challenging the
cells with the above agents. APs were recorded continuously, the first appearance
of the EADs or DADs was monitored, and the fraction of cells displaying EADs
or DADs was plotted as a function of time generating thus a probability function
for the incidence of EAD or DAD (pEAD or pDAD, respectively). Finally, these
curves were fitted to a two-state Boltzmann function.
Recording of ANTRA-sensitive current using AP voltage clamp
The whole-cell configuration of the ruptured patch clamp technique was used
(Hamill et al. 1981). The cells were superfused with oxygenated Tyrode solution.
The patch electrodes were prepared from borosilicate glass, having tip resistance
of 2 MΩ when filled with the pipette solution, containing (in mmol/l): K-aspartate
100, KCl 45, MgCl21, EGTA 10, K-ATP 3, and HEPES 5 at pH = 7.4. Careful
suction was applied to help gigaseal formation and the subsequent disruption of
the membrane patch. Axoclamp-2B amplifier was used in current clamp or con-
tinuous single electrode voltage clamp mode. The output filter was set to 10 kHz.
Digidata 1200 A/D-D/A converter operated under a pClamp 6.0 software (Axon
Instruments, Union City, CA, USA) was used to collect data and to deliver com-
mand pulses. Ionic currents were normalised to cell capacitance (142 ± 5.4 pF on
average).
In these AP voltage clamp studies, the AP waveform was first recorded from
5 cells, undergoing steady-state stimulation at a cycle length of 1 s in the current
clamp mode, and stored on the hard disk. This record was transformed to a com-

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Afterdepolarisations and Chloride Current
345
mand file using home-made software, then delivered as the command voltage in the
voltage clamp mode. In this case the current trace was a horizontal line at the zero
level (Doerr et al. 1990). Application of 0.5 mmol/l ANTRA for 2 min dissected the
ANTRA-sensitive current (presumably ICl) with an inverse polarity. In our graph
this ANTRA-sensitive current was inverted so as to appear as the current flows
normally, i.e., first as an outwardly, then as an inwardly directed current.
Statistics
Results are expressed as mean ± S.E.M. values. The statistical significance of dif-
ferences was evaluated using one-way ANOVA followed by Student’s t-test when
more than two groups were analysed. In the case of two groups, Student’s t-test for
paired or unpaired data was applied. Differences were considered significant when
p was less than 0.05.
Results
Effect of ANTRA on the AP configuration and transmembrane ion current
Superfusion of the myocytes with ANTRA (0.5 mmol/l) caused characteristic al-
terations in the AP configuration in Krebs solution: it decreased the depth of the
incisura, shifted the plateau towards more positive potentials, and lengthened AP
duration (APD) (Fig. 1A). This latter effect varied in magnitude when measured
at various levels of repolarisation, i.e. APD20was increased from 56±11 to 87±20
ms (31 ± 9 ms increase, 55 ± 9%), APD50from 152 ± 13 to 212 ± 16 ms (60 ± 12
ms increase, 40 ± 8%), and APD90 from 231 ± 16 to 308 ± 25 ms (77 ± 13 ms
increase, 33±5%). All these changes were statistically significant (p < 0.05) in the
8 myocytes studied. Although the absolute magnitude of the lengthening increased
from APD20 to APD90, the relative change was most pronounced at the level of
APD20, suggesting that the major effect of ANTRA developed during the early
plateau. No significant effect of ANTRA on the resting potential, AP amplitude,
or maximum rate of depolarisation was observed during the 20 min of exposure.
Since the effects of ANTRA on AP morphology reached their steady-state levels
by the 10thmin of superfusion (Fig. 1B), this period of pretreatment was applied
later in the experiments studying effects of ANTRA on afterdepolarisations.
A representative record of the ANTRA-sensitive current obtained under AP
voltage clamp conditions displays a transient positive peak of outward current
during the early plateau followed by a smaller negative peak of inward current
coinciding with the terminal repolarisation (Fig. 1C). The peak amplitude of the
outward current was significantly greater than that of the inward current (1.15 ±
0.39 pA/pF versus 0.53 ± 0.29 pA/pF). This difference was even larger when the
outward and inward change transfers were compared (23.2 ± 9.5 fC/pF versus
9.5 ± 5.8 fC/pF, p < 0.05, n = 5, Fig. 1D). These data are congruent with the
results obtained on APs, as suppression of a predominantly outward current is
expected to increase AP duration.

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Fülöp et al.
0
5
10
15
20
25
0
100200 300400
APD20
APD50
APD90


0
1020 3040

Outward

0.0 0.40.81.21.6

Outward

0
200
400
-100
-50
0
50
0 mV
20 mV
10 ms


C
Time (ms)
B
A
Figure 1. Effect of 20 min superfusion with 0.5 mmol/l ANTRA on AP morphology
(A), and APD (B) measured at 20%, 50% and 90% levels of repolarization (APD20,
APD50 and APD90, respectively, n = 8) in canine ventricular myocytes paced at 1 Hz.
Events of the early plateau are enlarged in the inset. C. ANTRA-sensitive current (ICl)
recorded under action potential voltage clamp conditions, and displayed on the time scale
of the action potential. Dashed line indicates zero current. D. Comparison of peak current
amplitudes and the net charge carried by ICl into inward and outward directions during
an action potential (n = 5). Symbols and bars represent mean ± S.E.M. values, asterisks
denote the level of significance (p < 0.05).
Time (min)
D
Vm(mV)
APD (ms)
Krebs
Anthracene
o Krebs
? Anthracene
o
?
?
o
Charge movement (fC/pF)
Peak current (pA/pF)
Inward
Inward
*
*
Current (pA)




150100
50
0
-50
-100
0
100
200
300
-100
-50
0
50
Time (ms)
Vm(mV)

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Afterdepolarisations and Chloride Current
347
Effect of ANTRA pretreatment on the incidence of afterdepolarisations
The effect of ANTRA was studied in 3 models of afterdepolarisations: the ouabain-
induced DAD model, the caesium-induced EAD model, and in the presence of a
subthreshold concentration of isoproterenol. Preincubation of the cells with 0.5
mmol/l ANTRA failed to induce either type of afterdepolarisations during the 20
min of exposure (n = 8).
Application of ouabain (200 nmol/l) alone resulted in development of DADs in
5 of the 8 cells studied (62.5%) within 15 min (Fig. 2A). Action potential duration
was determined from records taken before superfusion with ouabain and also in
the presence of ouabain in each cell just prior to development of DADs. Ouabain
increased APD90from 240±25 to 264±19 ms (lengthening of 24±7 ms, p < 0.05,
n = 8). When ouabain was applied in the presence of ANTRA (ANTRA pre-
treatment started 10 min before addition of ouabain), 3 of the 5 myocytes (60%)
displayed transiently EADs at the 2ndmin of the ouabain treatment before the
development of DADs (Fig. 2B). However, the incidence of EADs fell back to zero
by the 5thmin. Independent of the transient appearance or absence of EADs, all
the 5 cells developed DADs within 7 min in the presence of ANTRA + ouabain.
The ouabain-induced prolongation of APD90(determined just before the first oc-
currence of the afterdepolarisation) was 20 ± 2 ms in the 2 cells failing to display
EADs, a value comparable to that obtained without ANTRA. In contrast, a much
greater prolongation of APD90(94±13 ms) was obtained in the 3 cells developing
first EADs and later DADs. The timing of the incidence of afterdepolarisations
is summarised in Fig. 2C. The transient appearance of EADs, obtained with the
conventional DAD-inducer ouabain in the presence of ANTRA, suggests that the
generation of EADs and DADs may share common mechanisms. Actually, we have
never observed both types of these afterdepolarisations simultaneously.
Application of CsCl (as equimolar replacement of KCl) is often used to study
EADs. Caesium is known to suppress IKcurrents, which results in extreme length-
ening of the AP (January and Riddle 1990). Indeed, superfusion of myocytes with
3.6 mmol/l CsCl alone increased APD90 significantly from 246 ± 11 to 389 ± 24
ms in our experiments (p > 0.05, n = 7), and evoked EADs in 5 of the 7 cells
(71.4%) within 30 min, whereas application of CsCl in the presence of ANTRA
resulted in immediate depolarisation of the membrane (within less than 2 min)
from −79.6 ± 0.4 to −54.2 ± 3.5 mV (p < 0.05, n = 5). Restoration of the normal
resting potential or development of afterdepolarisations was never observed from
this strongly depolarised membrane potential level (Fig. 3).
A subthreshold concentration of isoproterenol (5 nmol/l) – when applied alone
– shortened APD90significantly (from 230±6 to 209±6 ms, 21±4 ms of shortening,
p < 0.05, n = 6) and shifted the plateau potential markedly towards more positive
voltages. Afterdepolarisations were not observed at this very low concentration.
Application of isoproterenol following pretreatment with ANTRA failed to shorten
APD90 any more (237 ± 10 versus 236 ± 13 ms, not significant, n = 8), but the
elevation of plateau was still evident, and 6 of the 8 cells (75%) developed DADs

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Fülöp et al.
pEAD ( )
05101520
0.0
0.5
1.0
Ouabain
Anthracene + Ouabain

0.0
0.5
1.0

C
B
A
Time (min)
pDAD (
)






1

234






200 ms
50 mV
123
0 mV
0 mV
200 ms
50 mV

Figure 2. Effect of ANTRA pretreatment on the incidence of ouabain-induced af-
terdepolarisations. A. Effect of 200 nmol/l ouabain alone. Control record in Krebs
solution (1) followed by APs obtained before (2) and after (3) development of DAD
in the presence of ouabain. B. Effect of ouabain in the presence of ANTRA. Control
record in ANTRA (1) followed by an AP taken before development of ouabain-induced
afterdepolarisations in the presence of ANTRA (2). Representative EAD and DAD,
induced by ouabain in the presence of ANTRA, are displayed in records (3) and (4),
respectively. C. Probability of incidence of afterdepolarisations (pDAD and pEAD)
induced by ouabain in the absence (open symbols, n = 8) and presence of ANTRA
(filled symbols, n = 5). ANTRA was applied 10 min before addition of ouabain.
pDAD data were fitted to a two-state Boltzmann function yielding 50% probabilities
for DAD at 11.8 and 3.9 min, respectively, in the absence and presence of ANTRA.

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Afterdepolarisations and Chloride Current
349
010 203040
0,0
0,5
1,0
Caesium
Anthracene + Caesium


0,0
0,5
1,0





1




2
C
B
A
Time (min)
Fraction of depolarised
cells ( )
pEAD ( )
200 ms
50 mV
0 mV
3
200 ms
50 mV
1
0 mV
2
Figure 3. Effect of ANTRA pretreatment on the incidence of caesium-induced EADs.
A. Effect of 3.6 mmol/l caesium alone. Control record in Krebs (1) followed by APs
obtained before (2) and after (3) development of EAD in the presence of caesium. B.
Effect of caesium in the presence of ANTRA. Control record in ANTRA (1) followed
by an abortive slow-response type AP (2) arising from depolarised membrane potential
at the 2ndmin of caesium superfusion. C. Probability of incidence of EADs (caesium
alone, open symbols, n = 7) and depolarisation (caesium + ANTRA, filled symbols,
n = 5). ANTRA was applied 10 min before the addition of caesium. The 50% prob-
abilities were obtained at 20.6 and 2 min, respectively, in the absence and presence of
ANTRA.

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Fülöp et al.
0510 1520
0.0
0.5
1.0
Isoproterenol
Anthracene + Isoproterenol











2


C
Time (min)
pDAD
100 ms
50 mV
0 mV
13
100 ms
50 mV
B
A
0 mV
1
2
3
Figure 4. The enhancing effect of ANTRA pretreatment on the incidence of
DADs induced by isoproterenol. A. The absence of afterdepolarisations in the
presence of 5 nmol/l isoproterenol alone. Control record in Krebs (1) followed
by APs obtained after superfusion with isoproterenol (records 2 and 3). B.
Effect of isoproterenol in the presence of ANTRA. Control record in ANTRA
(1) followed by APs taken before (2) and after (3) development of isoproterenol-
induced DAD. C. Probability of incidence of DAD induced by isoproterenol in
the absence (open symbols, n = 6) and presence of ANTRA (filled symbols,
n = 8). ANTRA was applied 10 min before the addition of isoproterenol. The
50% probability for incidence of DADs was obtained at 1.6 min in the presence
of ANTRA.

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Afterdepolarisations and Chloride Current
351
within 6 min (Fig. 4). EADs were not observed with 5 nmol/l isoproterenol even
in the presence of ANTRA, in contrast to the well-known EAD-inducing effect of
higher, submicromolar concentrations of isoproterenol (Priori and Corr 1990; Szabo
et al. 1994; Zeng and Rudy 1995).
Discussion
In this study we were first to visualise the ANTRA-sensitive current (which is me-
diated predominantly by chloride ions) flowing during the AP in canine ventricular
cells. The current was large and outward at the time of the early plateau but in-
wardly directed around terminal repolarisation. The outward peak of this current
clearly contributes to formation of the incisura of the AP, (an effect analogous
to that of the transient outward potassium current), since 0.5 mmol/l ANTRA
halved the depth of the incisura. These results are congruent with those of others
obtained with conventional voltage clamp protocols (Zygmunt 1994; Szigeti et al.
1998). Since the charge carried by the outward component of the ANTRA-sensitive
current was more than twice as large as the inward component, the net effect of
the current is to facilitate repolarisation – primarily during the incisura and the
crest of the dome of APs. This is a very critical period of time regarding determi-
nation of AP configuration. It is now generally believed that EADs result from the
imbalance of inward and outward ionic currents during the AP plateau. In these
terms, inhibition of a repolarising current (by ANTRA in our case) can promote
the generation of EADs. Indeed, our results show that EADs transiently appeared
in ouabain-treated cells in the presence of ANTRA in spite of the fact that neither
ANTRA nor ouabain alone were able to evoke EADs at concentrations applied by
us. This effect of ANTRA was even more pronounced in the experiments with cae-
sium, where the simultaneous suppression of two repolarising currents (IKby Cs,
and IClby ANTRA) caused a rapid and irreversible depolarisation of the membrane
to a second stable level (Gadsby and Cranefield 1977).
The enhancing effect of ANTRA pretreatment on the incidence of DADs re-
quires more explanation since the calcium-dependent ICl was implicated to con-
tribute to development of DADs as a charge carrier in mammalian cardiac prepara-
tions (Szigeti et al. 1998; Zygmunt et al. 1998). Our results obtained with ouabain
and isoproterenol clearly show that the incidence of DADs was increased in the
presence of ANTRA, probably due to suppression of a predominantly outward cur-
rent. This, in turn, suggests that IClmay provide a mechanism of defence against
both types of afterdepolarisations. However, the question arises, what is the pos-
sible mechanism of defence against DADs, which are known to be due to calcium
overload (Priori and Corr 1990; Wit and Rosen 1992; Szabo et al. 1994). The
ANTRA-induced changes in AP configuration, namely the prolongation of APs
and elevation of their plateau may increase calcium entry, and what is more impor-
tant, may limit the efflux of calcium through the Na+/Ca2+exchanger, resulting
thus in calcium overload of the SR, which is a prerequisite for generation of DADs.

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Fülöp et al.
It is also worthy of speculation why the appearance of EADs was transient in
myocytes exposed to ouabain in the presence of ANTRA. Following from the nature
of afterdepolarisations, once an EAD or DAD has developed, its incidence should
continuously increase; in other words, spontaneous disappearance of an afterdepo-
larisation (either EAD or DAD) has never been reported previously. Furthermore,
EADs and DADs have never been observed simultaneously in ouabain-treated my-
ocytes. These interesting observationscan be interpreted if one assumes that genesis
of EADs and DADs share common mechanisms (e.g., both can be modulated by
the calcium content of the SR), but their appearance during or after the AP de-
pends on the actual balance of inward and outward membrane currents (Szabo et
al. 1994). Further studies are required to elucidate this point.
Acknowledgements. Financial support for the studies was obtained from grants from
the Hungarian Research Found (OTKA-T037332, OTKA-T037334), Hungarian Ministry
of Health (ETT-244/2000, ETT-06031/2003), and Hungarian Ministry of Education
(FKFP-0243/2000). Further support was obtained from the National Research and De-
velopment Programs (NKFP-1A/0011/2002).
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Final version accepted: March 12, 2003