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(Circulation. 2001;104:2728.)
© 2001 American Heart Association, Inc.

Basic Science Reports

Ionic Mechanism of Delayed Afterdepolarizations in Ventricular Cells Isolated From Human End-Stage Failing Hearts

Arie O. Verkerk, PhD; Marieke W. Veldkamp, PhD; Antonius Baartscheer, PhD; Cees A. Schumacher, BSc; Corinne Klöpping, MD; Antoni C.G. van Ginneken, PhD; Jan H. Ravesloot, PhD

From the Department of Physiology (A.O.V., A.C.G.v.G., J.H.R.) and the Experimental and Molecular Cardiology Group (M.W.V., A.B., C.A.S., A.C.G.v.G.), Academic Medical Center, University of Amsterdam, and the Department of Cardiac Transplantation, University Medical Center, Utrecht (C.K.), the Netherlands.

Correspondence to Arie Verkerk, Department of Physiology, 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— Animal studies have shown that the Ca²⁺-activated Cl^- current (I_Cl(Ca)) and the Na⁺/Ca²⁺ exchange current (I_Na/Ca) contribute to the transient inward current (I_ti). I_ti is responsible for the proarrhythmic delayed afterdepolarizations (DADs). We investigated the ionic mechanism of I_ti and DADs in human cardiac cells.

Methods and Results— Human ventricular cells were enzymatically isolated from explanted hearts of patients with end-stage heart failure and studied with patch-clamp methodology. I_tis were elicited in the presence of 1 µmol/L norepinephrine by trains of repetitive depolarizations from -80 to +50 mV. DADs were induced in the presence of 1 µmol/L norepinephrine at a stimulus frequency of 1 Hz. I_ti currents were inwardly directed over the voltage range between -110 and + 50 mV. Neither the Cl^- channel blocker 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid nor changes in [Cl^-]_i affected I_ti or DAD amplitude. This excludes an important role for I_Cl(Ca). Blockade of Na⁺/Ca²⁺ exchange by substitution of all extracellular Na⁺ by Li⁺, conversely, completely inhibited I_ti. In rabbit, I_Cl(Ca) density in ventricular cells isolated from control hearts did not differ significantly from that in ventricular cells isolated from failing hearts.

Conclusions— In contrast to many animal species, I_ti and DADs in human ventricular cells from failing hearts consist only of I_Na/Ca. In rabbits, heart failure per se does not alter I_Cl(Ca) density, suggesting that I_Cl(Ca) may also be absent during DADs in nonfailing human ventricular cells.

Key Words: arrhythmia • heart failure • ion channels • sodium • calcium

	Introduction

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Patients with congestive heart failure have a high incidence of ventricular arrhythmias.¹ They are at increased risk of sudden death resulting from ventricular tachycardia and ventricular fibrillation.² It was recently demonstrated that these potentially lethal arrhythmias in patients with end-stage idiopathic dilated cardiomyopathy arise in the subendocardium or subepicardium by a focal nonreentrant mechanism.³ Triggered activity developing from either early afterdepolarizations (EADs) or delayed afterdepolarizations (DADs) may play a pivotal role in the initiation of these rhythm disturbances.³

The afterdepolarizations are "oscillations" in membrane potential. EADs occur during the action potential, whereas DADs occur after completion of the action potential.⁴ In ventricular cells from end-stage failing human hearts, phase-2 EADs are due to reactivation of the L-type Ca²⁺ current.⁵ So far, the mechanism of DADs has been investigated in animal models only. These studies showed that DADs are provoked by high heart rates under conditions in which [Ca²⁺]_i is elevated.⁶ The current causing DADs is called the transient inward current (I_ti).⁷ I_ti is activated by spontaneous Ca²⁺ release from the sarcoplasmic reticulum (SR).⁶ I_ti proves to be a heterogeneous current. One component flows through nonselective cation channels.⁸ Current mediated by Na⁺/Ca²⁺ exchange (I_Na/Ca) constitutes a second component.⁹ More recently, a third component was identified. Evidence has accumulated that the Ca²⁺-activated Cl^- current (I_Cl(Ca)) also contributes to both I_ti^10–12 and DADs.^13–15 We recently demonstrated in sheep heart cells that blockade of I_Cl(Ca) reduced the DAD amplitude sufficiently to prevent 50% of the DADs from reaching the threshold for triggering action potentials.¹⁴ This observation suggests that I_Cl(Ca) blockade may be a promising therapeutic strategy in those instances in which DADs contribute to the genesis of arrhythmias; that is, of course, only when I_Cl(Ca) also plays a role in I_tis and DADs in human ventricular cells. In this article, we address this issue.

Here, we report that I_ti and DAD in ventricular cells isolated from human end-stage failing hearts are caused by I_Na/Ca only. Furthermore, we show that in a rabbit model, heart failure does not lead to disappearance of I_Cl(Ca). Our results suggest that failing, and presumably also nonfailing, human ventricular cells do not possess I_Cl(Ca) channels. Our findings question the rationale behind I_Cl(Ca) blockade as a therapeutic approach in arrhythmias caused by triggered activity based on DADs.

	Methods

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Cell Preparation
Human Ventricular Cells
Hearts were obtained from patients with end-stage heart failure caused by either ischemic or dilated cardiomyopathy. Patient characteristics are shown in the Table. All patients were in NYHA functional class IV and received standard therapy for chronic heart failure. Informed consent was obtained before heart transplantation, and the protocol complied with institutional guidelines. Ventricular cells were isolated from the left ventricle by enzymatic dissociation.¹⁶

View this table: [in this window] [in a new window]   Table 1. Patient Data

Rabbit Ventricular Cells
Heart failure was induced in New Zealand White rabbits by volume and pressure overload.¹⁷ The heart failure index based on relative heart weight, relative lung weight, left ventricular end-diastolic pressure, third heart sound, and ascites was calculated as described previously.¹⁷ We used only hearts of rabbits in which 4 of the aforementioned 5 parameters were abnormal. Age-matched animals served as control group. Animal care was in accordance with institutional guidelines. Cells from the left ventricle were isolated by enzymatic dissociation.¹⁶ Cells isolated from failing hearts were significantly larger than control cells (301±53 pF [n=9] versus 110±37 pF [n=11]) and showed the well-known action potential prolongation.

Recording Procedures
Small aliquots of cell suspension were placed in a recording chamber on the stage of an inverted microscope and superfused with Tyrode’s solution (35°C to 37°C) containing (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).

Membrane potentials and currents were recorded in the ruptured-patch whole-cell configuration of the patch-clamp technique. Patch pipettes (3 to 5 M) were pulled from borosilicate glass and filled with solution containing either (mmol/L) potassium gluconate 125, KCl 20, and HEPES 10 (pH adjusted to 7.2 with KOH) or KCl 145 and HEPES 10 (pH adjusted to 7.2 with KOH). Potentials were corrected for the liquid junction potential. Membrane currents and potentials were low-pass filtered online with a cutoff frequency of 1 kHz and digitized at 2 kHz.

Stimulation Protocols
DADs and I_tis result from spontaneous SR Ca²⁺ release in Ca²⁺-overloaded myocytes.⁶ In our experiments, we induced sarcoplasmic Ca²⁺ overload by application of 1 µmol/L norepinephrine (Centrafarm). DADs were induced by eliciting action potentials (1 Hz) with current pulses applied through the patch pipette. I_ti was elicited by repeated trains of 15 to 20 200-ms voltage-clamp steps from -80 to +50 mV (Figure 1A). Time between the steps was 100 ms. Successive trains were 6 seconds apart.

View larger version (32K): [in this window] [in a new window]   Figure 1. A, Voltage-clamp protocol for eliciting Iti. B, Current recording made during this protocol in a human ventricular cell. Arrow, Iti. Inset, Protocol for measuring Iti amplitude. C, Effects of 0.2 mmol/L tetracaine on occurrence of Iti (left) and DAD (right).

During the first 5 to 6 successive voltage-clamp steps of a train, both the quasi steady-state current during the 200-ms depolarizing step and the inwardly directed "tail" current during the 100-ms repolarizing step increase until they reach a stable value (Figure 1B). Enhanced delayed rectifier current (I_K) and I_Na/Ca may underlie this phenomenon.¹⁸ Both I_K and I_Na/Ca increase during a rise in [Ca²⁺]_i,^18,19 as occurs during the train of voltage-clamp steps.^6,20 The latter results from repeated activation of Ca²⁺ current and the diminished Ca²⁺ extrusion by the Na⁺-Ca²⁺ exchanger.^6,20 Slow inactivation kinetics of I_K presumably also plays a role in the increase of the quasi steady-state current.

After cessation of a train, all cells develop 1 I_ti oscillations, typically within 3 seconds (Figure 1B, arrow). Every I_ti was accompanied by a visible aftercontraction of the cell. The amplitude of I_ti was measured as the difference between the peak of the transient current and mean current amplitude before and after the transient current (Figure 1B, inset). In case of successive I_ti oscillations, the first one was taken for analysis.

The effect of heart failure on I_Cl(Ca) was studied in rabbit ventricular cells. I_Cl(Ca) was elicited by depolarizing 500-ms voltage-clamp steps from a holding potential of -50 mV. The steps were applied once every 2 seconds and were incremented by 10 mV. I_Cl(Ca) was defined as the transient outward current sensitive to the Cl^- channel blocker 4,4'diisothiocyanatostilbene-2,2'-disulfonic acid (DIDS; Sigma Chemical Co). DIDS was prepared as 0.5-mol/L stock solution in DMSO (Merck) and diluted before use in Tyrode’s solution to a final concentration of 0.5 mmol/L. All currents were normalized for cell size as described previously.¹⁴

Statistics
Action potential characteristics were derived from 10 consecutive action potentials and averaged. Microsoft Excel software was used for statistical analysis of the data. Values are expressed as mean±SEM and considered significantly different at a value of P<0.05 in ANOVA or Student’s t test.

	Results

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In Human Ventricular Cells, I_ti and DADs Are Caused by Spontaneous SR Ca²⁺ Release
We first asked the question whether in human ventricular cells I_ti and DADs are activated by spontaneous SR Ca²⁺ release. We tested the effects of 0.2 mmol/L tetracaine and 5 mmol/L caffeine, drugs known to inhibit spontaneous SR Ca²⁺ release.^6,21 Both tetracaine (Figure 1C, n=3) and caffeine (n=4, data not shown) completely abolished I_ti and DAD. From these experiments, we conclude that as in animal cells, in human ventricular cells I_ti and DADs also are caused by spontaneous SR Ca²⁺ release.

No Role for a Cl^- Current in I_ti and DAD in Human Ventricular Cells
Next, we investigated the currents contributing to I_ti and DAD in human ventricular cells. We determined the current-voltage (I-V) relationship of I_ti and its sensitivity to intracellular Cl^- substitution. We reasoned that if I_Cl(Ca) contributes to I_ti, then a change in [Cl^-]_i has to alter the I_ti I-V relationship. We applied the aforementioned train protocol, but now directly followed by 17 voltage-clamp steps of 3-second duration and ranging from -110 to +50 mV with 10-mV increments (Figure 2A, inset). Figure 2A shows typical current traces recorded at the membrane potentials indicated. In this and 4 other experiments, the low-Cl^- pipette solution was used. The corresponding Cl^- reversal potential (E_Cl) was calculated at -50 mV, which is the physiological E_Cl value in cardiac cells.²² For 4 other cells, the high-Cl^- pipette solution was used, for which E_Cl was 0 mV. Figure 2B shows the I-V relationships of I_ti measured with low and high [Cl^-]_i. Irrespective of [Cl^-]_i, I_ti was inwardly directed at all potentials tested and had a maximal amplitude at approximately -70 mV. The 2 I_ti I-V relationships did not differ significantly. In accordance with this observation, we also found no significant effect of [Cl^-]_i on DAD amplitude (Figure 2C). In fact, resting membrane potential and the duration and amplitude of the action potential also were not affected. From these experiments, we conclude that it is not likely that I_Cl(Ca) contributes much to I_tis and DADs in failing human ventricular cells.

View larger version (24K): [in this window] [in a new window]   Figure 2. A, Typical example of voltage-dependence of Iti in human ventricular cells. Inset, Applied voltage-clamp protocol. Iti was measured at potentials between -110 and +50 mV. B, I-V relationship of Iti in human ventricular cells recorded with pipette solution containing high (n=4; ECl=0 mV) and low (n=5; ECl=-50 mV) Cl-. C, Action potential and DAD parameters recorded with pipette solution containing high (n=4; ECl=0 mV) and low (n=5; ECl=-50 mV) Cl-. APD50 and APD90 indicate action potential duration at 50% and 90% repolarization; APA, action potential amplitude; and Vm, resting membrane potential.

To substantiate this conclusion, we studied the effects of I_Cl(Ca) blockade on the I_ti I-V relationship and DAD amplitude. We applied DIDS, a potent inhibitor of anion transport proteins, including I_Cl(Ca) channels.²³ In these experiments, we used the low-Cl^- pipette solution (E_Cl=-50 mV). Figure 3A shows typical examples of I_tis measured at -80 and +50 mV in the absence and presence of DIDS. The drug had no effect. Figure 3B shows the lack of effect of the stilbene on the mean I_ti I-V relationship of 4 cells. Figure 3C and 3D shows that DIDS also had no effect on DAD amplitude or other action potential characteristics (n=4).

View larger version (20K): [in this window] [in a new window]   Figure 3. A, Typical current traces recorded at -80 and +50 mV in absence and presence of 0.5 mmol/L DIDS. B, I-V relationship of Iti in absence and presence of 0.5 mmol/L DIDS (n=4). C, Typical action potential and DAD configuration in absence and presence of 0.5 mmol/L DIDS. D, Action potential and DAD parameters in absence and presence of 0.5 mmol/L DIDS (n=4). Abbreviations as in Figure 2.

From these experiments, we conclude that there is no or only a very limited role for a Cl^- current in DADs and I_ti in failing human ventricular cells.

Dominant Role for I_Na/Ca in I_ti and DAD Formation in Human Ventricular Cells
A salient feature of the I_ti I-V relationship is the absence of a reversal potential (Figures 2B and 3B). This suggests that not a channel mechanism but rather an electrogenic Na⁺/Ca²⁺ exchange underlies I_ti. Na⁺/Ca²⁺ exchange can support inwardly and outwardly directed currents.²⁴ During spontaneous SR Ca²⁺ release under voltage-clamp conditions, however, I_Na/Ca does not reverse sign and remains inwardly directed, because any rise in sarcoplasmic Ca²⁺ concentration will shift the reversal potential of the exchanger toward more positive membrane potentials.^25,26 The I_ti in human ventricular cells was small at positive membrane potentials and increased at more hyperpolarized potentials, with a maximum at -70 mV. At potentials negative to -70 mV, I_ti decreases (Figures 2B and 3B). This behavior is most likely related to voltage-dependency of I_Na/Ca itself, but also to the amount of Ca²⁺ released by the SR.^24,27 Spontaneous [Ca²⁺]_i oscillations seem to decrease at more hyperpolarized potentials,²⁰ which will result in a smaller I_ti.

Next, we tested the hypothesis that I_Na/Ca contributes to I_ti. We inhibited Na⁺/Ca²⁺ exchange by replacing extracellular Na⁺ with equimolar amounts of Li⁺. Li⁺ permeates through Na⁺ channels and nonselective cation channels²⁸ but cannot replace Na⁺ on the Na⁺/Ca²⁺ exchanger.^29,30 Throughout these experiments, 0.5-mmol/L DIDS was present, and the low-Cl^- pipette solution (E_Cl=-50 mV) was used. Figure 4A shows a typical example of the effects of Li⁺ on I_ti. Both at -80 and at +50 mV, an inwardly directed I_ti was found (left), which completely disappeared after substitution of Li⁺ for Na⁺ (right). Aftercontractions were still visible, however, indicating that spontaneous SR Ca²⁺ release was not disrupted. Figure 4B shows the mean I_ti I-V relationship of 4 cells before and after the maneuver. After substitution of Li⁺ for Na⁺, I_ti was abolished at all potentials. Inhibition of Na⁺/Ca²⁺ exchange and the consequent further loading of the sarcoplasm with Ca²⁺ prevents stable current-clamp recordings. For this reason, we could not evaluate the effect of Li⁺ on DAD formation. Nevertheless, from these data, we conclude that I_Na/Ca plays a dominant role in I_ti and DAD formation in human ventricular cells of failing hearts.

View larger version (22K): [in this window] [in a new window]   Figure 4. A, Typical current traces recorded before (left) and after (right) substitution of all extracellular Na+ by Li+. B, I-V relationship of Iti before and after substitution of all extracellular Na+ by Li+ (n=4).

In a Rabbit Model, I_Cl(Ca) Density Is Not Affected by Heart Failure
Thus far, we found that in human ventricular cells, DAD and I_ti are carried predominantly by I_Na/Ca but not by I_Cl(Ca). This is at odds with observations made in a number of animal models. In our experiments, ventricular cells were isolated from explanted hearts of patients with end-stage heart failure. We cannot exclude the possibility that heart failure affects I_Cl(Ca) density, the more so because heart failure is associated with downregulation of a number of cationic currents^31,32 and alterations in Ca²⁺ metabolism.³³

In a concluding series of experiments, we determined whether in an animal model, heart failure influences I_Cl(Ca) density. We studied I_Cl(Ca) in cells isolated from control and failing rabbit hearts using the low-Cl^- pipette solution (E_Cl=-50 mV). Moreover, the results obtained in rabbit cells were compared with those obtained with failing human cells. To measure I_Cl(Ca), we applied voltage-clamp steps (see Methods) in the absence and presence of DIDS. Figure 5A shows typical examples of the current traces recorded at +50 mV in a control rabbit (left), a failing rabbit (middle), and a failing human (right) cell. By subtraction of the appropriate traces, the DIDS-sensitive I_Cl(Ca) was obtained (Figure 5B). Figure 5C shows mean I-V relationships of I_Cl(Ca) of 11 control rabbit, 9 failing rabbit, and 6 failing human cells. In both groups of rabbit cells, the typical bell-shaped I_Cl(Ca) I-V relationship was found. I_Cl(Ca) was not observed in human ventricular cells. Moreover, the I_Cl(Ca) density in failing rabbit cells did not differ significantly from that in control rabbit cells. From these data, we conclude that in rabbit, heart failure per se does not necessarily lead to downregulation of I_Cl(Ca).

View larger version (23K): [in this window] [in a new window]   Figure 5. A, Superimposed current traces elicited by voltage-clamp steps from -50 to +50 mV in absence and presence of DIDS in a control rabbit cell (91 pF), failing rabbit cell (290 pF), and failing human cell (265 pF). B, DIDS-sensitive currents. C, I-V relationship of ICl(Ca) in ventricular cells from control rabbit (n=11), failing rabbit (n=9), and failing human (n=6) hearts.

	Discussion

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Overview
The aim of this study was to elucidate the ionic mechanism of DADs and their underlying current, I_ti, in failing human ventricular cells. We found that the I_ti I-V relationship was inwardly directed between -110 and +50 mV (Figures 2 and 3). Neither changes in [Cl^-]_i nor application of DIDS (Figure 3) affected the DADs or I_tis (Figure 2). I_ti was completely abolished, however, by substitution of Li⁺ for extracellular Na⁺ (Figure 4). Because this maneuver blocks I_Na/Ca, we conclude that I_Na/Ca is the principal ionic mechanism of DADs and I_ti in failing human ventricular cells.

Species-Dependency of Ionic Nature of DADs
Our results indicate that in failing human ventricular cells, only I_Na/Ca contributes to I_ti. This is similar to findings in ventricular cells of guinea pig^25,29 but contrasts with observations made in ventricular cells of dog,¹¹ rabbit,¹² sheep,¹⁴ and ferret.³⁰ In these species, I_ti consisted of both I_Cl(Ca) and I_Na/Ca. In our experiments, norepinephrine was present. Adrenoceptor stimulation enhances I_Cl(Ca) amplitude,¹¹ thereby potentially favoring I_Cl(Ca) detection. Furthermore, we found that the density of I_Cl(Ca) in rabbit heart cells is not affected by heart failure (Figure 5). Thus, neither our experimental conditions nor heart failure per se can clarify why I_Cl(Ca) cannot be found in our failing human ventricular cell preparation. The simplest explanation is that our failing human heart cell preparation does not possess I_Cl(Ca). Indeed, Köster et al³⁴ were not able to detect I_Cl(Ca) currents in human ventricular cells when they applied caffeine to induce SR Ca²⁺ release, whereas in rabbit Purkinje cells, that same maneuver does activate I_Cl(Ca).³⁵ Finally, the lack of effect of DIDS application and/or intracellular Cl^- substitution on steady-state membrane currents and action potentials of our failing human ventricular cells (Figures 2 and 3) also implies that in our preparation, both the heart failure–induced, persistently active swelling-induced Cl^- current³⁶ and cAMP-dependent Cl^- current (I_Cl(cAMP))³⁷ are absent. The latter agrees with findings of Oz and Sorota,³⁸ who were also not able to detect I_Cl(cAMP) in human ventricular cells.

Limitations of Our Study
Animal models contribute much to our understanding of the electrophysiology of failing hearts. They can never completely substitute, however, for experiments with normal human cells. We found that heart failure in rabbits does not significantly alter I_Cl(Ca). We take that as evidence that heart failure per se cannot account for our inability to detect I_Cl(Ca) in human ventricular cells of failing hearts. Final proof for the notion that human heart cells do not express I_Cl(Ca), of course, lies in experiments with normal human heart cells.

Implications of Our Study
The present study demonstrates that in failing human ventricular cells, DADs and their underlying current, I_ti, are composed virtually exclusively of I_Na/Ca. I_Na/Ca is activated by spontaneous Ca² release from SR. This contrasts with observations made in ventricular cells of a number of animal species in which I_Cl(Ca) also contributes to the DADs and I_ti. Our findings question the rationale behind I_Cl(Ca) blockade as a therapeutic approach in arrhythmias caused by triggered activity.

	Acknowledgments

 
This work was supported by the Research Council for Earth and Life Sciences with financial aid from the Netherlands Organization for Scientific Research (grant 805-06.155).

Received July 23, 2001; revision received September 17, 2001; accepted September 18, 2001.

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