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(Circulation. 2000;101:2639.)
© 2000 American Heart Association, Inc.

Basic Science Reports

Calcium-Activated Cl^- Current Contributes to Delayed Afterdepolarizations in Single Purkinje and Ventricular Myocytes

Arie O. Verkerk, PhD; Marieke W. Veldkamp, PhD; Lennart N. Bouman, PhD; Antoni C. G. van Ginneken, PhD

From the Department of Physiology, Academic Medical Center, University of Amsterdam, The Netherlands.

Correspondence to Arie Verkerk, Department of Physiology, University of Amsterdam, Academic Medical Center, Meibergdreef 15, 1105 AZ Amsterdam, The Netherlands. E-mail a.o.verkerk{at}amc.uva.nl

	Abstract

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Background—The ionic mechanism underlying the transient inward current (I_ti), the current responsible for delayed afterdepolarizations (DADs), appears to be different in ventricular myocytes and Purkinje fibers. In ventricular myocytes, I_ti was ascribed to a Na⁺-Ca²⁺ exchange current, whereas in Purkinje fibers, it was additionally ascribed to a Cl^- current and a nonselective cation current. If Cl^- current contributes to I_ti and thus to DADs, Cl^- current blockade may be potentially antiarrhythmogenic. In this study, we investigated the ionic nature of I_ti in single sheep Purkinje and ventricular myocytes and the effects of Cl^- current blockade on DADs.

Methods and Results—In whole-cell patch-clamp experiments, I_ti was induced by repetitive depolarizations from -93 to +37 mV in the presence of 1 µmol/L norepinephrine. In both Purkinje and ventricular myocytes, I_ti was inward at negative potentials and outward at positive potentials. The anion blocker 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid (DIDS) blocked outward I_ti completely but inward I_ti only slightly. The DIDS-sensitive component of I_ti was outwardly rectifying, with a reversal close to the reversal potential of Cl^- currents. Blockade of Na⁺-Ca²⁺ exchange by substitution of extracellular Na⁺ by equimolar Li⁺ abolished the DIDS-insensitive component of I_ti. DIDS reduced both DAD amplitude and triggered activity based on DADs.

Conclusions—In both Purkinje and ventricular myocytes, I_ti consists of 2 ionic mechanisms: a Cl^- current and a Na⁺-Ca²⁺ exchange current. Blockade of the Cl^- current may be potentially antiarrhythmogenic by lowering DAD amplitude and triggered activity based on DADs.

Key Words: currents • sodium • calcium • arrhythmia

	Introduction

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Delayed afterdepolarizations (DADs) are oscillations in membrane potential occurring after completion of the action potential.¹ They depend on the preceding transmembrane activity and are provoked by high heart rates under various conditions in which [Ca²⁺]_i appears to be elevated.² The mechanism behind DADs is described as transient inward current (I_ti),³ activated by spontaneous Ca²⁺ release from the sarcoplasmic reticulum (SR).² Although DADs are an important mechanism for cardiac arrhythmias,⁴ the ionic nature of I_ti is still a subject of debate. Three ionic mechanisms have been proposed to contribute to I_ti: (1) an electrogenic Na⁺-Ca²⁺ exchange,^{5 6 7} (2) a nonselective cation current,^{8 9 10} and (3) a Cl^- current.^{11 12 13}

The contribution of electrogenic Na⁺-Ca²⁺ exchange to I_ti can be distinguished from the 2 channel mechanisms by the voltage-dependence of I_ti.^{6 14} I_ti will not reverse in sign when carried by Na⁺-Ca²⁺ exchange only, because the rise in [Ca²⁺]_i will shift the reversal potential of the exchanger toward a more positive potential. A reversal of I_ti was absent in several kinds of myocardial tissue and in several species, such as rabbit sinoatrial node,¹⁵ canine and guinea pig atrial myocytes,^{7 16} guinea pig and ferret ventricular myocytes.^{5 6 17 18 19 20} Channel mechanisms, on the other hand, will have a current-voltage relation with a distinct reversal of sign. Reversal of I_ti was found in Purkinje fibers of sheep^{9 11 12 13} and calf⁸ and in rabbit ventricular myocytes.¹⁰

The apparent differences in ionic mechanisms underlying I_ti between atrial/ventricular myocytes on one hand and Purkinje fibers on the other hand may reflect artifacts in voltage-clamp measurements and in control of the extracellular solution in the multicellular Purkinje fibers.¹⁹ Alternatively, the observed differences may be species-dependent. Purkinje fibers are generally obtained from sheep^{9 11 12 13} and calf,⁸ whereas ventricular and atrial myocytes are generally obtained from guinea pig,^{6 7 17 18 19 20} dog,¹⁶ and ferret.^{5 6} Knowledge of the exact ionic nature of I_ti may benefit treatment of arrhythmias due to DADs. Under physiological conditions, the reversal potential of Cl^- currents (E_Cl) is -50 mV.²¹ Thus, at resting membrane potential, activation of Cl^- current results in an inward current inducing a depolarization. If a Cl^- current contributes to DADs, blockade of this current will result in smaller afterdepolarizations. Thus, Cl^- current blockade may be potentially antiarrhythmogenic when it brings DAD amplitude to subthreshold level for triggered action potentials.²¹

Using the patch-clamp technique, we studied the current-voltage characteristics of I_ti and the effects of anion blockade and extracellular Na⁺ substitution on them in both single sheep Purkinje and ventricular myocytes. In addition, the effects of anion blockade on DADs and triggered activity based on DADs were investigated.

	Methods

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Cell Preparation
Purkinje and ventricular myocytes were isolated from sheep hearts by enzymatic dissociation.²² Small aliquots of cell suspension were put in a recording chamber on the stage of an inverted microscope. Cells were allowed to adhere for 5 minutes before starting continuous perfusion with Tyrode’s solution (36±1°C) containing (in mmol/L) NaCl 140, KCl 5.4, CaCl₂ 1.8, MgCl₂ 1.0, glucose 5.5, and HEPES 5.0 (pH adjusted to 7.4 with NaOH). Only rod-shaped cells with smooth surfaces were selected for electrophysiological measurements.

Electrophysiological Recording
Membrane potentials and currents were recorded by the whole-cell patch-clamp technique. Patch pipettes were pulled from borosilicate glass and heat-polished. The pipette solution contained (in mmol/L) K-gluconate 125, KCl 20, and HEPES 10 (pH adjusted to 7.2 with KOH). Series resistance was compensated up to 80%. All potentials were corrected for the estimated 13-mV change in liquid junction potential. Membrane currents and potentials were filtered online (1 kHz), digitized at 2 kHz, stored, and analyzed by custom software.

Action potentials were elicited at 1 Hz. Cell capacitance was calculated as C_m=I_m/V_m, where V_m is the change in slope of the potential on 10-ms hyperpolarizing and depolarizing pulses of 30 or 100 pA (I_m) applied during the action potential plateau.

I_ti was induced in the presence of 1 µmol/L norepinephrine (Centrafarm) by a train of 15 voltage steps of 200 ms from -93 to +37 mV at intervals of 100 ms (Figure 1A). The interval between 2 trains was 6 seconds. Such a train was invariably followed by an I_ti within 3 seconds (Figure 1B, arrow). The I_ti was accompanied by an aftercontraction of the myocyte, which was visible through the microscope. I_ti amplitude was measured as difference between the peak of the transient current and the mean of current just before and after the transient current (Figure 1B, inset). In case of successive I_tis, the first one was used for analysis. I_ti was normalized for cell size by dividing current amplitude by C_m. The current-voltage (I-V) relation of I_ti was determined by measuring I_ti amplitude during voltages between -113 and +57 mV. These potential steps (3-second duration) were applied with 10-mV increments directly after the train of voltage steps. I_ti was outward at positive potentials (see Figure 2), which makes the terminology "transient inward current" debatable. However, because this term has been used for many years, we choose to maintain it. Values are expressed as mean±SEM and are considered significantly different at P0.05 in a Student’s t test.

View larger version (15K): [in this window] [in a new window]   Figure 1. A, Voltage protocol for induction of Iti. B, Example of Iti (arrow) in ventricular myocyte. Inset, Protocol for measuring Iti amplitude.

View larger version (28K): [in this window] [in a new window]   Figure 2. A, Voltage protocol for studying voltage-dependence of Iti. Test potential (Vtest) was varied between -113 and +57 mV. B and C, Voltage-dependence of Iti in ventricular (B) and Purkinje (C) myocyte. Insets, Biphasic courses of Iti at +27 mV (B) and + 7 mV (C).

	Results

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Voltage-Dependence of I_ti
The contribution of Na⁺-Ca²⁺ exchange to I_ti is distinguished from channel mechanisms by the voltage-dependence of I_ti.^{6 14} I_ti will not reverse in sign if it reflects the Na⁺-Ca²⁺ exchanger. Channel mechanisms, on the other hand, do reverse in sign. Figure 2 shows examples of I-V relations of I_ti in ventricular (Figure 2B) and Purkinje myocytes (Figure 2C). Illustrated are I_tis at various test potentials (V_test) between -113 and +57 mV, recorded during the first 2 seconds directly after the trains of depolarizations (Figure 2A). At each potential, 1 I_tis were present. As in previous studies,^{2 8 9 13} the frequency of I_ti increased and the time to the first I_ti decreased with more depolarized potentials. These effects are most likely caused by the [Ca²⁺]_i increase at depolarized potentials, because of diminished Ca²⁺ extrusion by the Na⁺-Ca²⁺ exchanger.^{2 17}

In the Purkinje as well as in the ventricular myocyte, I_ti was inward at negative potentials and outward at very positive potentials. Between these 2, a voltage level could be found at which I_ti was biphasic in course, with outward current being followed by inward current. This biphasic course appeared at +27 and +7 mV for the ventricular and Purkinje myocytes, respectively (Figure 2B and 2C, insets). Figure 3 shows the mean I-V relation of I_ti for ventricular and Purkinje myocytes. The amplitude of inward I_ti was maximal at -73 mV and decreased at more negative and more positive membrane potentials. Outward I_ti progressively increased at more positive potentials. Amplitudes of I_ti in Purkinje and ventricular myocytes did not differ significantly.

View larger version (24K): [in this window] [in a new window]   Figure 3. Mean I-V relation of Iti in ventricular and Purkinje myocytes. Iti amplitude was not determined when Iti was biphasic.

These data demonstrate that I_ti reverses in both Purkinje and ventricular myocytes, which strongly argues for the involvement of a channel mechanism in the generation of I_ti in both cell types.

Effects of Blocking Cl^- Channels and Na⁺-Ca²⁺ Exchange on I_ti
The indication that in Purkinje and ventricular myocytes at least a channel mechanism contributes to I_ti does not eliminate the possibility that Na⁺-Ca²⁺ exchange is also involved. First, we investigated which channel mechanism contributes to I_ti. Candidates are a Cl^- current^{11 12 13} and a nonselective cation current.^{8 9 10} To test whether a Cl^- current is involved, I_ti was measured in both Purkinje and ventricular myocytes in the absence and presence of 0.5 mmol/L 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid (DIDS; Sigma Chemical Co; prepared as 0.5 mol/L stock solution in DMSO), an anion channel blocker. Figure 4A shows a typical example of this experiment in a ventricular myocyte. In the absence of DIDS, I_ti was outward at +47 mV, biphasic at +7 mV, and inward at -93 mV (Figure 4A, left). In the presence of DIDS, outward I_ti completely disappeared, only the inward current of the biphasic I_ti remained, and the amplitude of inward I_ti was slightly decreased (Figure 4A, middle). Thus, DIDS completely blocks outward I_ti but only slightly blocks inward I_ti. Washout of DIDS partly restored outward I_ti and biphasic I_ti (Figure 4A, right). Figure 4B and 4C shows mean I-V relations of I_ti in Purkinje (Figure 4B) and in ventricular (Figure 4C) myocytes in the absence and presence of DIDS and the DIDS-sensitive component of I_ti. For each myocyte, the DIDS-sensitive component of I_ti was calculated as amplitude of I_ti in the absence of DIDS minus the corresponding amplitude of I_ti in the presence of DIDS. In both Purkinje and ventricular myocytes, the DIDS-sensitive I_ti was outwardly rectifying and reversed at -40 mV (Figure 4D), which is close to the calculated E_Cl. Furthermore, outward rectification of the DIDS-sensitive component of I_ti is in agreement with the Goldman-Hodgkin-Katz equation for Cl^- currents with physiological transmembrane [Cl^-] gradients (146 mmol/L [Cl^-]_o and 20 mmol/L [Cl^-]_i).

View larger version (29K): [in this window] [in a new window]   Figure 4. A, Current traces recorded at -93, +7, and +47 mV in control conditions (left), in presence of DIDS (middle), and after washout of DIDS (right) in ventricular myocyte. B and C, Mean I-V relation of Iti in absence and presence of DIDS and DIDS-sensitive current in Purkinje (B) and ventricular (C) myocytes. D, Mean I-V relation of DIDS-sensitive part of Iti in ventricular and Purkinje myocytes.

The DIDS-insensitive component of I_ti is inward, even at positive potentials (Figure 4B and 4C). This absence of reversal suggests that Na⁺-Ca²⁺ exchange underlies the DIDS-insensitive I_ti. To test this hypothesis, extracellular Na⁺ was replaced with equimolar Li⁺ to block Na⁺-Ca²⁺ exchange. Li⁺ permeates through Na⁺ channels and nonselective cation channels²³ but cannot replace Na⁺ on the Na⁺-Ca²⁺ exchanger.^{18 24} Figure 5 shows a typical example of an experiment in a ventricular myocyte in which the effects of Na⁺ substitution by Li⁺ on the DIDS-insensitive component of I_ti were studied. In the presence of 0.5 mmol/L DIDS (Figure 5A), only an inward I_ti was found, which completely disappeared after substitution of Na⁺ by Li⁺ (Figure 5B). Despite complete abolishment of I_ti, aftercontractions were still present, indicating that spontaneous Ca²⁺ release from the SR was not affected. Similar results were obtained in another 4 ventricular and in 3 Purkinje myocytes.

View larger version (13K): [in this window] [in a new window]   Figure 5. A and B, Current traces recorded at -93 and 47 mV in presence of DIDS (A) and in presence of DIDS after substitution of Na+ by equimolar Li+ (B) in single ventricular myocyte. Note abolishing of DIDS-insensitive Iti and absence of other transient currents.

From these data, we conclude that in both Purkinje and ventricular myocytes, I_ti has 2 components: a Cl^- current and a Na⁺-Ca²⁺ exchange current.

I_ti Induces Delayed Afterdepolarizations and Transient Repolarizations
Because I_ti was outward at positive potentials, one may expect that I_ti causes electrical abnormalities during the action potential plateau as well. To test this hypothesis, spontaneous Ca²⁺ release from the SR to activate I_ti was evoked in current-clamp by eliciting action potentials at 1 Hz in the presence of 1 µmol/L norepinephrine. This protocol induced DADs in 14 of 26 Purkinje and in 16 of 24 ventricular myocytes. In 4 Purkinje and 3 ventricular myocytes showing DADs, repolarization during the plateau of the action potential was transiently increased. Figure 6 shows a typical example of this phenomenon in a ventricular and a Purkinje myocyte. Figure 6A shows action potentials from a ventricular myocyte recorded during control (•) and 38 (1) and 45 seconds (2) after the application of norepinephrine. Norepinephrine elevated the plateau and prolonged the action potential (1), but 45 seconds after addition of norepinephrine (2), these effects were accompanied by a DAD and transient repolarizations during the plateau (arrows). Figure 6B shows action potentials from a Purkinje myocyte recorded during control (•) and 49 (1) and 52 seconds (2) after application of norepinephrine. Both action potentials recorded in the presence of norepinephrine were accompanied by DADs, but only the action potential recorded 52 seconds after application of norepinephrine (2) shows a transient repolarization (arrow) as well. In both cell types, transient repolarizations tend to shorten the action potential. The occurrence of transient repolarizations during the plateau and DADs during diastole complies with the observed outward and inward I_tis.

View larger version (21K): [in this window] [in a new window]   Figure 6. A and B, Ventricular (A) and Purkinje (B) action potentials recorded in absence and presence of norepinephrine. Arrows indicate transient repolarizations. C, Membrane currents in ventricular myocyte activated by voltage steps from -53 mV to potentials between -23 and +37 mV in presence of norepinephrine.

The incidence of transient repolarizations was much lower than that of DADs. To clarify this difference, we studied the moment of occurrence of outward I_ti in ventricular myocytes during depolarizing voltage steps (500 ms) between -53 and +37 mV with 10-mV increments (frequency was 0.5 Hz). Norepinephrine 1 µmol/L was present to induce I_ti. Figure 6C shows that depolarization to -23 mV induced a large Ca²⁺ current. Superimposed on this current is a transient outward current, which became more pronounced with stronger depolarizations. The vast majority of this current was sensitive to DIDS (data not shown) and probably reflects Ca²⁺-activated Cl^- current [I_Cl(Ca)].²¹ Depolarization to +17, +27, and +37 mV showed, in addition, a second transient outward current occurring toward the end of the 500-ms depolarization and a transient outward current at the holding potential. Similar results were obtained in another 8 ventricular myocytes. The mean time of occurrence of the second outward I_ti during depolarization to +37 mV was 362±35 ms (n=9). This indicates that transient repolarizations caused by outward I_ti can be found only in action potentials with a sufficient long plateau.

Effects of Cl^- Channel Blockade on DADs
Our experiments demonstrate that in both Purkinje and ventricular myocytes, a Cl^- current contributes to I_ti. Because E_Cl is -50 mV under physiological conditions, activation of a Cl^- current during diastole results in an inward depolarizing current. In our experiments with physiological [Cl^-] gradients across the membrane, DIDS reduced I_ti at -73 mV in 8 ventricular and 6 Purkinje myocytes by 19.2±4% and 22.1±6%, respectively. This suggests that the Cl^- current component contributes by 20% to DADs. In a concluding series of experiments, we determined the effects of Cl^- current blockade on DADs and on the development of triggered activity due to DADs.

Figure 7A clearly demonstrates that 0.5 mmol/L DIDS decreased DAD amplitude. Mean reductions of DAD amplitude in 4 ventricular and 5 Purkinje myocytes were 17.5±5% and 19.8±4%, respectively. Figure 7B shows that the DIDS-induced decrease of DAD amplitude may be sufficient to abolish triggered activity. This effect was found in 3 of 5 Purkinje and in 1 of 2 ventricular cells. In the other cells, triggered activity remained despite the presence of DIDS.

View larger version (11K): [in this window] [in a new window]   Figure 7. A, Action potentials recorded during exposure of 1 µmol/L norepinephrine in Purkinje myocyte in absence and presence of DIDS. DIDS decreased DAD amplitude. B, Action potentials recorded during exposure of 1 µmol/L norepinephrine in Purkinje myocyte in absence and presence of DIDS. DIDS abolished triggered activity.

These data demonstrate that blockade of Cl^- currents diminishes DAD amplitude by 20% and reduces the occurrence of triggered activity by 50%. The latter finding suggests an antiarrhythmogenic action of Cl^- current blockade.

	Discussion

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Ionic Nature of I_ti
The aim of this study was to elucidate, in both sheep Purkinje and ventricular myocytes, the ionic composition of I_ti, which is activated by spontaneous Ca²⁺ release from the SR and which is responsible for the development of DADs.^{2 3} In both cell types, we found that I_ti was inward at negative potentials and outward at positive potentials. The anion blocker DIDS completely blocked outward I_ti and only slightly blocked inward I_ti. The DIDS-sensitive I_ti component was outwardly rectifying, with a reversal close to E_Cl. Substitution of extracellular Na⁺ by equimolar Li⁺ abolished the DIDS-insensitive component of I_ti. From these findings, we conclude that I_ti in Purkinje and ventricular myocytes is composed of a Cl^- current and a Na⁺-Ca²⁺ exchange current. Other ionic currents that could contribute to I_ti are the Ca²⁺-activated K⁺ current and the Ca²⁺-activated nonselective cation current. However, because the combined blockade of anion currents and Na⁺-Ca²⁺ exchange abolished all transient currents, a contribution of these currents to I_ti in our experiments can be excluded. The continued presence of aftercontractions under these conditions indicated that spontaneous Ca²⁺ release from the SR was not affected.

Previously, an I_Cl(Ca) was observed in a variety of cardiac tissues in rabbit and dog (for review, see Reference ²¹ ). This I_Cl(Ca) is activated by Ca²⁺-induced Ca²⁺ release from the SR and is ligand (Ca²⁺)–gated. It seems very likely that the DIDS-sensitive I_ti in our experiments and I_Cl(Ca) are identical currents.

A contribution of both a Na⁺-Ca²⁺ exchange current and a Cl^- current to I_ti in Purkinje and ventricular myocytes can explain the observed biphasic course of I_ti. Both currents are activated by the transient rise in [Ca²⁺]_i caused by spontaneous Ca²⁺ release from the SR. The Na⁺-Ca²⁺ exchange follows the time course of this transient rise,²⁵ but I_Cl(Ca) declines early, before [Ca²⁺]_i reaches its peak.²⁶ Consequently, during the transient rise of [Ca²⁺]_i, initially both Na⁺-Ca²⁺ exchange and Cl^- current will be active, whereas later, only Na⁺-Ca²⁺ exchange activity will follow. When current flows of the 2 ionic mechanisms are of opposite direction, ie, positive of E_Cl, and when the amplitude of the I_Cl(Ca) is larger than that of the Na⁺-Ca²⁺ exchange current, I_ti will be first outward and later inward. This biphasic course was recently also shown by Trafford et al.²⁷

Comparison With Previous Studies
Our results indicate that in both Purkinje and ventricular myocytes, a Cl^- current and the Na⁺-Ca²⁺ exchange current contribute to I_ti. These findings agree with those in sheep Purkinje fibers^{11 12 13} and rabbit ventricular myocytes (A.O. Verkerk, PhD; unpublished data, 1997) but are in contrast with findings in rabbit sinoatrial node,¹⁵ atrial myocytes of dog¹⁶ and guinea pig,⁷ and ventricular myocytes of guinea pig^{6 17 18 19 20} and ferret.^{5 6} In the latter studies, I_ti consisted of only Na⁺-Ca²⁺ exchange current. Species differences in current densities may well account for these contrasting findings. I_Cl(Ca) appears to be absent in ventricular myocytes of guinea pig,²⁰ a species often used for studying the ionic mechanisms underlying I_ti. The Na⁺-Ca²⁺ exchange current density, on the other hand, is higher in guinea pig ventricular and human atrial myocytes than in rat ventricular myocytes.²⁸ Larger Na⁺-Ca²⁺ exchange current may mask the channel mechanisms more easily. An additional explanation is that intracellular and extracellular Cl^- conditions may vary strongly among the different studies, yielding distinct Cl^- reversal potentials and thus Cl^- current amplitudes. Finally, in our experiments, we used 1 µmol/L norepinephrine to induce Ca²⁺ overload. It is possible that norepinephrine, a well-known - and ß-adrenergic receptor stimulator, affects the DIDS-sensitive part of I_ti. Indeed, I_Cl(Ca) amplitude is doubled by 1 µmol/L norepinephrine (A.O. Verkerk, PhD; unpublished data, 1998), suggesting that norepinephrine also doubles the amplitude of the Cl^- component of I_ti. Whether this increase is due to an increase of Ca²⁺ release or a direct effect on I_Cl(Ca) channels remains to be elucidated.

Extrapolation of our findings to humans must be made with caution, because the presence of I_Cl(Ca) appears to be species-dependent. Thus far, the presence of I_Cl(Ca) in humans is unclear. A Ca²⁺-activated transient outward current insensitive to 4-aminopyridine, most likely the same current as I_Cl(Ca), was observed in human atrial cells.^{29 30} Conversely, Li et al³¹ were not able to demonstrate I_Cl(Ca) in human atrial myocytes. In human ventricular myocytes, despite several studies on the 4-aminopyridine–sensitive transient outward current no studies related to the Ca²⁺-activated transient outward current were performed.

Conclusions
The present work demonstrates that in isolated Purkinje as well as ventricular myocytes of sheep, I_ti is carried by both an electrogenic Na⁺-Ca²⁺ exchange and a Cl^- current. At resting membrane potential, both currents generate inward current, resulting in DADs. During the plateau of action potentials, however, the 2 current mechanisms are of opposite directions. Because at these potentials the Cl^- current is larger than the Na⁺-Ca²⁺ exchange current, activation of I_ti leads to transient repolarizations.

The finding that DADs are carried by 2 current mechanisms in both Purkinje and ventricular myocytes may be of special importance for treatment and prevention of arrhythmias due to DADs. Blockade of Na⁺-Ca²⁺ exchange current would be very effective in reducing DAD amplitude, because it takes 80% of I_ti. However, this would elevate [Ca²⁺]_i drastically, ultimately resulting in cell death.³² In this study, we demonstrate that blockade of the Cl^- current reduces DAD amplitude sufficiently to prevent DADs from reaching the threshold for triggering of action potentials. Thus, Cl^- current blockade may be potentially antiarrhythmogenic.

	Acknowledgments

 
This work was supported by the Dutch Organization for Scientific Research (grant 900–516-093). The authors thank Jan Bourier and Berend de Jonge, Department of Physiology, Academic Medical Center, University of Amsterdam, for their excellent technical assistance.

Received September 1, 1999; revision received December 16, 1999; accepted December 24, 1999.

	References

Top Abstract Introduction Methods Results Discussion References

 
1. Cranefield PF. Action potentials, afterpotentials, and arrhythmias. Circ Res. 1977;41:415–423.[Abstract/Free Full Text]

2. Wit AL, Rosen MR. Afterdepolarizations and triggered activity: distinction from automaticity as an arrhythmogenic mechanism. In: Fozzard HA, Haber E, Jennings RB, et al. The Heart and Cardiovascular System. 2nd ed. New York, NY: Raven Press; 1992:2113–2163.

3. Lederer WJ, Tsien RW. Transient inward current underlying arrhythmogenic effects of cardiotonic steroids in Purkinje fibres. J Physiol. 1976;263:73–100.[Abstract/Free Full Text]

4. Janse MJ, Wit AL. Electrophysiological mechanisms of ventricular arrhythmias resulting from myocardial ischemia and infarction. Physiol Rev. 1989;69:1049–1168.[Free Full Text]

5. Karagueuzian HS, Katzung BG. Voltage-clamp studies of transient inward current and mechanical oscillations induced by ouabain in ferret papillary muscle. J Physiol. 1982;327:255–271.[Abstract/Free Full Text]

6. Arlock R, Katzung BG. Effects of sodium substitutes on transient inward current and tension in guinea-pig and ferret papillary muscle. J Physiol. 1985;360:105–120.[Abstract/Free Full Text]

7. Lipp P, Pott L. Transient inward current in guinea-pig atrial myocytes reflects a change of sodium-calcium exchange current. J Physiol. 1988;397:601–630.[Abstract/Free Full Text]

8. Kass RS, Lederer WJ, Tsien RW, et al. Role of calcium ions in transient inward currents and aftercontractions induced by strophanthidin in cardiac Purkinje fibres. J Physiol. 1978;281:187–208.[Abstract/Free Full Text]

9. Cannell MB, Lederer WJ. The arrhythmogenic current I_TI in the absence of electrogenic sodium calcium exchange in sheep cardiac Purkinje fibres. J Physiol. 1986;374:201–219.[Abstract/Free Full Text]

10. Giles W, Shimoni Y. Comparison of sodium-calcium exchanger and transient inward currents in single cells from rabbit ventricle. J Physiol. 1989;417:465–481.[Abstract/Free Full Text]

11. Han X, Ferrier GR. Ionic mechanisms underlying the arrhythmogenic transient inward current in the absence of Na⁺-Ca²⁺ exchange in rabbit Purkinje fibres. J Physiol. 1992;456:19–38.[Abstract/Free Full Text]

12. Han X, Ferrier GR. Contribution of Na⁺-Ca²⁺ exchange to stimulation of transient inward current by isoproterenol in rabbit cardiac Purkinje fibres. Circ Res. 1995;76:664–674.[Abstract/Free Full Text]

13. Han X, Ferrier GR. Transient inward current is conducted through two types of channels in cardiac Purkinje fibres. J Mol Cell Cardiol. 1996;28:2069–2084.[Medline] [Order article via Infotrieve]

14. Noble D. The surprising heart: a review of recent progress in cardiac electrophysiology. J Physiol. 1984;353:1–50.[Free Full Text]

15. Brown HF, Noble D, Noble S, et al. Relationship between the transient inward current and slow inward currents in the sino-atrial node of the rabbit. J Physiol. 1986;370:299–315.[Abstract/Free Full Text]

16. Tseng GN, Wit AL. Characteristics of a transient inward current that causes delayed afterdepolarizations in atrial cells of the canine coronary sinus. J Mol Cell Cardiol. 1987;19:1105–1119.[Medline] [Order article via Infotrieve]

17. Fedida D, Noble D, Rankin AC, et al. The arrhythmogenic transient outward current I_TI and related contraction in isolated guinea-pig ventricular myocytes. J Physiol. 1987;392:523–542.[Abstract/Free Full Text]

18. Kimura J, Miyamae S, Noma A. Identification of sodium-calcium exchange current in single ventricular cells of guinea-pig. J Physiol. 1987;384:199–222.[Abstract/Free Full Text]

19. Benndorf K, Biskup C, Friedrich M. Voltage-dependent kinetics of Na-Ca exchange current in Ca²⁺-loaded guinea pig heart cells. Am J Physiol. 1993;265:C1258–C1265.[Abstract/Free Full Text]

20. Sipido KR, Callewaert G, Porciatti F, et al. [Ca²⁺]_i-dependent membrane currents in guinea-pig ventricular cells in the absence of Na/Ca exchange. Pflugers Arch. 1995;430:871–878.[Medline] [Order article via Infotrieve]

21. Sorota S. Insights into the structure, distribution and function of the cardiac chloride channels. Cardiovasc Res. 1999;42:361–376.[Abstract/Free Full Text]

22. Verkerk AO, Veldkamp MW, Abbate F, et al. Two types of action potential configuration in single cardiac Purkinje cells of sheep. Am J Physiol. 1999;277:H1299–H1310.[Abstract/Free Full Text]

23. Ehara T, Noma A, Ono K. Calcium-activated non-selective cation channel in ventricular cells isolated from adult guinea-pig hearts. J Physiol. 1988;403:117–133.[Abstract/Free Full Text]

24. Ponce-Hornos JE, Langer GA. Sodium-calcium exchange in mammalian myocardium: the effects of lithium. J Mol Cell Cardiol. 1980;12:1367–1382.[Medline] [Order article via Infotrieve]

25. Lipp P, Pott L, Callewaert G, et al. Calcium transients caused by calcium entry are influenced by the sarcoplasmic reticulum in guinea-pig atrial myocytes. J Physiol. 1992;454:321–338.[Abstract/Free Full Text]

26. Sipido KR, Callewaert G, Carmeliet E. [Ca²⁺]_i transients and [Ca²⁺]_i-dependent chloride currents in single Purkinje cells from rabbit heart. J Physiol. 1993;468:641–667.[Abstract/Free Full Text]

27. Trafford AW, Díaz ME, Eisner DA. Ca-activated chloride current and Na-Ca exchange have different timecourses during sarcoplasmic reticulum Ca release in ferret ventricular myocytes. Pflugers Arch. 1998;435:743–745.[Medline] [Order article via Infotrieve]

28. Sham JS, Hatem SN, Morad M. Species differences in the activity of the Na⁺-Ca²⁺ exchanger in mammalian cardiac myocytes. J Physiol. 1995;488:623–631.[Abstract/Free Full Text]

29. Escande D, Coulombe A, Faivre J-F, et al. Two types of transient outward currents in adult human atrial cells. Am J Physiol. 1987;252:H142–H148.[Abstract/Free Full Text]

30. Wang Z, Fermini B, Nattel S. Delayed rectifier outward current and repolarization in human atrial myocytes. Circ Res. 1993;73:276–285.[Abstract/Free Full Text]

31. Li G-R, Feng J, Wang Z, et al. Comparative mechanisms of 4-aminopyridine-resistant Ito in human and rabbit atrial myocytes. Am J Physiol. 1995;269:H463–H472.[Abstract/Free Full Text]

32. Sheu S-S, Blaustein MP. Sodium/calcium exchange and control of cell calcium and contractility in cardiac and vascular muscles. In: Fozzard HA, Haber E, Jennings RB, et al. The Heart and Cardiovascular System. 2nd ed. New York, NY: Raven Press; 1992:903–943.

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