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Cardiovascular Research 2003 60(2):288-297; doi:10.1016/j.cardiores.2003.07.004
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Copyright © 2003, European Society of Cardiology

Conduction slowing by the gap junctional uncoupler carbenoxolone

Joris R. de Groota,1, Thijs Veenstrab,1, Arie O. Verkerka,b, Ronald Wildersb, Jeroen P.P. Smitsa, Francien J.G. Wilms-Schopmanc, Rob F. Wiegerincka, Jan Bourierb, Charly N.W. Beltermana, Ruben Coronela,d,1 and E.Etienne Verheijck*,b,1

aAcademic Medical Center, Task Force Heart Failure and Ageing, Experimental and Molecular Cardiology Group, Department of Experimental Cardiology, Amsterdam, The Netherlands
bAcademic Medical Center, Task Force Heart Failure and Ageing, Department of Physiology, University of Amsterdam, P.O. Box 22700, 1100 DE Amsterdam, The Netherlands
cInteruniversity Cardiology Institute the Netherlands, Utrecht, The Netherlands
dHeart Lung Center Utrecht, Department of Cardiology, University Medical Center Utrecht, The Netherlands

*Corresponding author. Tel.: +31-20-5664670; fax: +31-20-6919319. Email address: e.verheijck{at}amc.uva.nl

Received 17 December 2002; accepted 30 July 2003


   Abstract
Top
 Abstract
1. Introduction
 2. Methods
 3. Results
 4. Discussion
 Acknowledgments
 References
 
Background: Cellular electrical coupling is essential for normal propagation of the cardiac action potential, whereas reduced electrical coupling is associated with arrhythmias. Known cellular uncoupling agents have severe side effects on membrane ionic currents. We investigated the effect of carbenoxolone on cellular electrical coupling, membrane ionic currents, and atrial and ventricular conduction. Methods and Results: In isolated rabbit left ventricular and right atrial myocytes, carbenoxolone (50 µmol/l) had no effect on action potential characteristics. Calcium, potassium, and sodium currents remained unchanged. Dual current clamp experiments on poorly coupled cell pairs revealed a 21±3% decrease in coupling conductance by carbenoxolone (mean±S.E.M., n = 4, p<0.05). High-density activation mapping was performed in intact rabbit atrium and ventricle during Langendorff perfusion of the heart. The amplitude of the Laplacian of the electrograms, a measure of coupling current in intact hearts, decreased from 1.45±0.66 to 0.75±0.51 µA/mm3 (mean±SD, n = 32, p<0.05) after 15 min of carbenoxolone. Carbenoxolone reversibly decreased longitudinal and transversal conduction velocity from 66±15 to 49±16 cm/s and from 50±14 to 35±15 cm/s in ventricle, respectively (mean±SD, n = 5, both p<0.05). In atrium, longitudinal and transversal conduction velocity decreased from 80±29 to 60±16 cm/s and from 49±10 to 38±10 cm/s (mean±SD, n = 8, both p<0.05). Conclusions: Carbenoxolone-induced uncoupling causes atrial and ventricular conduction slowing without affecting cardiac membrane currents. Activation delay is larger in poorly coupled cells.

KEYWORDS Experimental; Heart; Organism; Cellular; Electrophysiology; Arrhythmias; Antiarrhythmic agents; Cellular communication; Conduction; Membrane potential


   1. Introduction
Top
 Abstract
 1. Introduction
2. Methods
 3. Results
 4. Discussion
 Acknowledgments
 References
 
Cellular electrical coupling plays an important role in normal and abnormal propagation of the cardiac action potential [1–3]. Various pathological states, such as acute [4,5] and chronic myocardial ischemia [6,7], cardiac hypertrophy and heart failure [7,8], are associated with reduced cellular coupling and arrhythmias [5,9]. Experimental studies on mice lacking connexin43 (Cx43) confirm the causative relationship between decreased cellular coupling and arrhythmogenesis [10,11]. However, when cellular uncoupling is complete, the arrhythmogenic substrate terminates [5].

Atrial and ventricular arrhythmias present a large, and yet, unsolved problem and contribute importantly to mortality [12]. Blockade of ion currents reducing the number of arrhythmogenic triggers is ineffective in post-infarct patients and may even cause proarrhythmia and increased mortality [13,14]. Gap junctional uncoupling results in a nonlinear effect on conduction [2,15]. From theoretical studies, it follows that reduction of cellular coupling could preserve sufficiently rapid conduction in regions with initially normal coupling, whereas in regions where coupling already is compromised, conduction slowing may be converted into block [15], thereby reducing the arrhythmogenic substrate. Ideally, uncoupling drugs selectively affect gap junctions without affecting ion currents. However, long-chain n-alkanols and fatty acids [16] have potential arrhythmogenic effects by also reducing membrane ionic currents, including inward rectifier potassium current (IK1), delayed rectifier potassium current (IK), sodium current (INa), and L-type calcium current (ICa,L) [17–19].

Carbenoxolone, a known anti-ulcer drug, selectively blocks hemi-channel current in the retina of the gold fish [20] and uncouples glial and neuronal cells in rat brainstem slices [21]. Furthermore, carbenoxolone has been shown to dephosphorylate Cx43 in rat liver epithelial cells [22]. We tested the effect of carbenoxolone on conduction velocity in cardiac muscle, on cardiac membrane ionic currents (INa, ICa,L, IK1, IK, and Ito1) in isolated rabbit ventricular myocytes and on action potential transfer in isolated cell pairs.

We demonstrate that carbenoxolone is a selective uncoupling agent that does not affect ion currents. Action potential transfer is larger in poorly coupled than in well-coupled cell pairs [15,23].


   2. Methods
Top
 Abstract
 1. Introduction
 2. Methods
3. Results
 4. Discussion
 Acknowledgments
 References
 
Procedures conformed to institutional guidelines. New Zealand White rabbits of either sex weighing 2 to 3.5 kg were anaesthetized with fentanyl citrate 0.08 mg/kg, fluanisone 2.5 and 10 mg/kg midazolam i.v. and heparinized. Hearts were rapidly excised and immersed in Tyrode's solution (4 °C) containing (mmol/l): 130 NaCl, 5.6 KCl, 2.2 CaCl2, 0.55 MgCl2, 24.2 NaHCO3, 13.2 sucrose, 11.1 glucose, saturated with 95% O2 and 5% CO2. The aorta was cannulated and the heart was connected to a Langendorff perfusion system and perfused at 8.7 kPa.

Carbenoxolone slowed conduction in rabbit ventricle in concentrations between 20 and 100 µmol/l (unpublished observation). In all experiments described here, carbenoxolone was investigated at a concentration of 50 µmol/l, similar to concentrations described previously [20,24].

2.1. Cell isolation
Atrial and ventricular myocyte isolation was modified from Tytgat [25]. Hearts were retrogradely perfused with a solution containing (in mmol/l) 140 NaCl, 5.4 KCl, 1.8 CaCl2, 1.0 MgCl2, 5.0 HEPES, and 5.5 glucose, saturated with O2 (37 °C, pH 7.4 with NaOH) for 15 min, followed by perfusion with low Ca2+ solution containing (in mmol/l) 140 NaCl, 5.4 KCl, 0.01 CaCl2, 0.5 MgCl2, 1.2 KH2PO4, 5.0 HEPES and 5.5 glucose, saturated with O2 (37 °C, pH 7.4 with NaOH). After 15 min, collagenase B 30 U/l, collagenase P 62 U/l, trypsine inhibitor 50 mg/l, creatine 1.6 g/l (all Boehringer Mannheim), and hyalorunidase 100 mg/l (Sigma) were added.

After 20 min, the right atrium was removed and collected in a high potassium solution containing (in mmol/l) 85 KCl, 30 KH2PO4, 5.0 MgSO4, 20 glucose, 5.0 pyruvic acid, 5.0 creatine, 30 taurine, 0.5 EGTA, 5.0 β-hydroxybutyric acid, 5.0 succinic acid, 2.0 Na2ATP, and 50 g/l polyvinylpyrolidone (pH 6.9 with KOH). The atrium was cut into pieces (2 x 3 mm), and triturated through a pipette (tip diameter 1 mm) for 5 to 10 min. Single atrial cells were stored at room temperature in high potassium solution.

After 30 min enzyme perfusion, the left ventricle was removed and gently stirred in enzyme solution at 37 °C for 16 min. Albumin 10 mg/l was added during the last 6 min. Single cells were stored in HEPES buffered solution at room temperature.

2.2. Data acquisition in isolated cells and cell pairs
Transmembrane potentials and membrane currents were recorded at 35 °C (except INa) in the whole-cell configuration of the patch-clamp technique in single cells and in side–side coupled cell pairs. The HEPES buffered solution was used as bath solution. Pipettes pulled from borosilicate glass were filled with (in mmol/l) 125 K-gluconate, 20 KCl, 10 HEPES, pH 7.1 with KOH (resistance 3–5 M{Omega}). Series resistance of the pipette to the cell interior was 8–12 M{Omega} and compensated electronically up to ~80%. Membrane currents and potentials were filtered (cut-off frequency 1 kHz) and digitized (sampling frequency 2 kHz). Action potential recordings were filtered (cut-off 2 kHz) and sampled with 5 kHz. Ito,1 and INa recordings were filtered (cut-off 5 kHz) and sampled with 10 kHz.

INa was measured at room temperature with pipettes containing (in mmol/l): 3 NaCl, 133 CsCl, 2 MgCl2, 2 Na2ATP, 2 TEA, 5 HEPES, 10 EGTA, pH 7.3 with CsOH. The bath solution contained (in mmol/l): 7 NaCl, 133 CsCl, 1.8 CaCl2, 1.2 MgCl2, 5 HEPES, 11 glucose, 0.001 nifedipine (Sigma), pH 7.4 with CsOH. Action potentials and ion currents except INa were corrected for a liquid junction potential of 13 mV.

2.3. Test protocols and data analysis
Action potentials, elicited at 2 Hz (2 ms duration, 20% suprathreshold), were characterized by action potential duration at 20%, 50%, and 90% repolarization (APD20, APD50, and APD90), resting membrane potential (RMP), action potential amplitude (APA) and maximal upstroke velocity (dV/dtmax). Cell capacitance (Cm) was estimated as described previously [9]. Action potential transfer in cell pairs was studied with the dual current clamp technique [26].

ICa,L, IK, and IK1 were examined by 500-ms voltage-clamp steps every 2 s from a holding potential of –40 mV to membrane potentials from –120 to +50 mV. ICa,L was defined as the peak inward current, IK1 as the quasi-steady-state current at the beginning of hyperpolarizing voltage steps, and IK as the quasi-steady-state current at the end of depolarizing voltage steps (see Fig. 2). Ito1 was studied by depolarizing voltage-clamp steps (500 ms, every 4 s) from a holding potential of –80 mV to membrane potentials from –80 mV to +70 mV. Ito1 was defined as the difference between peak outward current and the current at the end of the voltage step (cf. Fig. 3). CdCl2 (1 mmol/l) was added to block calcium currents, thereby preventing activation of outward ICl(Ca) [27] and to substantially reduce INa [28]. INa was explored with voltage-clamp steps (50 ms, every 10 s) from a holding potential of –100 mV to membrane potentials from –100 mV to +30 mV and normalized to the peak current amplitude measured at –25 mV.


Figure 2
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  		Fig. 2 Carbenoxolone does not affect IK1, IK and ICa,L in ventricular myocytes. (A) Current traces and voltage clamp protocol during control (left) and carbenoxolone superfusion (right). (B) Current–voltage relationship of IK1 and IK. (C) Current–voltage relationship of ICa,L.

 

Figure 3
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  		Fig. 3 Carbenoxolone does not affect Ito1 and INa in ventricular myocytes. (A) Voltage clamp protocol for determination of Ito1 and current traces during control (left) and carbenoxolone superfusion (right). (B) Current–voltage relationship of Ito1. (C) Voltage clamp protocol for determination of INa and current traces during control (left) and carbenoxolone superfusion (right). (D) Current–voltage relationship of INa normalized to control current at –25 mV.

 
2.4. Recording and data acquisition in intact hearts
Hearts were perfused with Tyrode's solution (37 °C) containing carbenoxolone for 15 min, followed by a 25-min washout period with Tyrode's solution without carbenoxolone. Temperature remained constant during control and carbenoxolone perfusion.

Hearts were paced (1-ms current pulses, twice diastolic stimulation threshold, cycle length 250 ms) from the central electrode terminal of a multi-electrode array (11 x 14 unipolar electrodes, distance 1 mm). Unipolar electrograms were simultaneously recorded from the right ventricular-free wall (n = 5 hearts). In a separate set of experiments (n = 8 hearts), electrograms were measured from the right atrial appendage below the crista terminalis (13 x 16 unipolar electrodes, interelectrode distance 0.5 mm) and digitized at a sampling frequency of 0.5 kHz.

The moment of steepest negative deflection of the electrogram defined local activation time. Activation maps were constructed and longitudinal and transversal conduction velocity were measured from the elliptic spread of activation as described previously [29]. Atrioventricular conduction time (AV time) was defined as the time between local activation of a central electrode of the multi-electrode positioned on the right atrium to the onset of the remote ventricular signal at the same site. The magnitude of cellular uncoupling was estimated from the decrease in amplitude of the Laplacian of electrograms. The Laplacian represents intercellular current [30] which is proportional to the coupling conductance when action potential shape and tissue impedance remain constant [31]. Total tissue impedance was measured with the four-electrode technique as described previously [5].

2.5. Statistics
Data for whole hearts were presented as mean±SD and for single cells and cell pairs as mean±S.E.M. Differences between groups were tested with a t-test, ANOVA or a Mann–Whitney test when appropriate. A p<0.05 indicated statistical significance.


   3. Results
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
4. Discussion
 Acknowledgments
 References
 
3.1. Unchanged action potentials and ion currents
Fig. 1 shows action potential recordings from an isolated ventricular myocyte (Fig. 1A) and an isolated atrial myocyte (Fig. 1B) before, during, and after the administration of carbenoxolone. Action potential parameters were unchanged by carbenoxolone (Tables 1 and 2)Go.


Figure 1
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  		Fig. 1 Carbenoxolone does not affect action potential configuration. Action potentials from a ventricular (A) and an atrial (B) myocyte before (solid line), during (dashed line) and after carbenoxolone (dash-dotted line).

 

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  		Table 1 Action potential characteristics of ventricular myocytes

 

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  		Table 2 Action potential characteristics of atrial myocytes

 
Fig. 2A shows current traces from the same ventricular myocyte as in Fig. 1 in the absence (left) and presence (right) of carbenoxolone upon voltage-clamp steps to determine IK, IK1, and ICa,L. Carbenoxolone did not affect the amplitude or time course of these currents nor the current–voltage relationships (n = 5, Fig. 2B and C). Similarly, no effects of carbenoxolone on Ito1 (n = 6) or INa (n = 6) were observed (Fig. 3).

3.2. Partial uncoupling of ventricular cell pairs
Next, we tested the effects of carbenoxolone on action potential transfer in isolated cell pairs [26]. In 23 out of 27 ventricular myocyte pairs tested, we observed virtually simultaneous activation of the paced cell and the follower cell. After administration of carbenoxolone, no increase in activation delay could be detected in these cell pairs at the 2-kHz sampling frequency (6 pairs tested). In 4 cell pairs, however, an initial activation delay between the paced and the follower cell was observed. In the representative example shown in Fig. 4A and B, carbenoxolone increased activation delay from 3.0 to 4.8 ms (horizontal arrows).


Figure 4
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  		Fig. 4 Action potential transfer in ventricular cell pairs. (A–C) Increased activation delay (horizontal arrows) and decreased capacitive loading of the non-stimulated ‘follower’ cell (voltage deflection {Delta}V2 versus {Delta}V1) reveal decreased intercellular coupling upon carbenoxolone superfusion in an isolated cell pair. (D–F) Increased activation delay (top), decreased intercellular coupling current (middle), and decreased voltage deflection {Delta}V of the follower cell (bottom) in a hybrid cell pair (model cell 1 coupled to real isolated cell 2 through coupling conductance Gc). Note differences in time scales. See text for details.

 
Because there is no significant change in the excitation process of the paced cell, the amount of capacitive charging of the follower cell (Fig. 4C) is directly proportional to intercellular conductance. This approach allows us to measure the decrease in coupling conductance from the ratio {Delta}V1/{Delta}V2. This procedure was validated by comprehensive analysis of our previous coupling clamp data with known values of coupling conductance and coupling current [32]. We analyzed data from seven hybrid cell pairs (a real isolated ventricular cell and a paced real-time simulation of a Luo and Rudy model cell [33]). Fig. 4D–F, illustrates the action potential transfer in a hybrid cell pair at an (ohmic) coupling conductance (Gc) of 8.0, 7.0, and 6.5 nS, respectively (a minimum of 6.4 nS was required for successful action potential transfer). Activation delay increases with decreasing Gc (top panels). The computed intercellular coupling current and the capacitive charging of the real cell during stimulation of the model cell are shown in the middle and bottom panels. Charge transfer, calculated from the coupling current, decreases from 0.38 pC at Gc=8.0 to 0.34 nS and 0.31 pC at 7.0 and 6.5 nS, respectively. The decrease in the initial voltage deflection (‘{Delta}V’, bottom panels, 11% and 17%) correlates with the decrease in charge transfer (12% and 17%) and the decrease in coupling conductance (12% and 19%). Similar data were obtained from the other hybrid cell pairs studied.

We applied the above procedure to the cell pairs. In the example of Fig. 4C the initial voltage deflection resulting from capacitive charging of the follower cell decreased from 3.1 to 2.3 mV (labelled ‘{Delta}V1’ and ‘{Delta}V2’, respectively), i.e. by 26%. In the four poorly coupled cell pairs studied, the decrease in {Delta}V amounted 21±3% (p<0.05).

3.3. Conduction slowing in atrium and ventricle
Fig. 5 shows the time course of changes in ventricular longitudinal and transversal conduction velocity induced by carbenoxolone and by subsequent washout of the drug. Conduction slowing was not significantly different between 10 and 15 min of perfusion with carbenoxolone (n = 5). We therefore report effects of carbenoxolone on conduction under stable conditions at 15 min of perfusion.


Figure 5
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  		Fig. 5 Ventricular longitudinal (circles) and transversal (squares) conduction velocity during administration of carbenoxolone (dotted area) and washout. Bars indicate SD. Note that the effect of carbenoxolone levels of between 10 and 15 min and is fully reversible upon washout.

 
Fig. 6A shows representative extracellular electrograms from the right ventricle (Fig. 6B, asterisk) during control perfusion (left), carbenoxolone (middle), and after washout (right). Note the decrease in steepest negative slope of the electrogram and the decrease in amplitude, indicating slowing of conduction in the middle panel. After washout, the electrogram recovered to control. Fig. 6B shows corresponding epicardial activation maps. Ventricular longitudinal conduction velocity (five ventricles) decreased from 66±15 to 49±16 cm/s after carbenoxolone perfusion (74±16% of control, p<0.05), and recovered to 65±15 cm/s upon washout (p<0.05 vs. carbenoxolone, p = NS vs. control). Transversal conduction velocity decreased from 50±14 to 35±15 cm/s (69±14% of control, p<0.05) and increased again to 48±13 cm/s upon washout (p<0.05 vs. carbenoxolone, p = NS vs. control).


Figure 6
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  		Fig. 6 Carbenoxolone decreases conduction velocity in atrial and ventricular myocardium. (A) Representative unipolar ventricular electrograms before and during carbenoxolone, and after washout. Carbenoxolone causes smaller, slower complexes with decreased dV/dt. (B) Associated activation maps (4-ms isochrones). Asterisk indicates site of electrograms in panel A. (C) Unipolar atrial electrograms before, during and after carbenoxolone perfusion. (D) Associated activation maps (3-ms isochrones). Asterisk indicates site of electrogram in panel C.

 
Fig. 6C shows representative extracellular electrograms of one selected site under the multielectrode on the right atrium (Fig. 6D, asterisk) during control, 15 min of perfusion with carbenoxolone and washout. During carbenoxolone perfusion, the electrogram broadened, became less steep and its amplitude decreased. Upon washout of the drug, the electrogram configuration recovered to control. Atrial longitudinal conduction velocity (eight atria) decreased from 80±29 to 60±16 cm/s (77±12% of control, p<0.05) upon carbenoxolone perfusion, and recovered to 69±18 cm/s after washout (p<0.05 vs. carbenoxolone; p = NS vs. control). Similarly, transversal conduction velocity decreased from 49±10 to 38±10 cm/s (79±19% of control, p<0.05) and recovered to 49±14 cm/s after washout of the drug (p<0.05 vs. carbenoxolone; p = NS vs. control).

AV time increased by 58% from 81±14 to 128±30 ms (n = 6, p<0.05). In two experiments, AV time did not recover; in the other experiments, full recovery was observed.

3.4. Decrease in coupling current
Total tissue impedance did not change after administration of carbenoxolone (287±74 vs. 273±83 {Omega}cm; n = 3, p = NS). Because action potential characteristics are not affected by carbenoxolone, a change in the amplitude of the Laplacian electrogram, therefore, can be used as a measure of cellular coupling. We quantified uncoupling in intact hearts with ventricular Laplacian electrograms (32 Laplacians from five hearts). Fig. 7A shows a representative example of a Laplacian electrogram, of which, the amplitude decreased during carbenoxolone and recovered to control after washout. Fig. 7B summarizes data from all experiments: amplitude decreased from 1.45±0.66 to 0.75±0.51 µA/mm3 after carbenoxolone (p<0.05) and recovers to 1.25±0.91 µA/mm3 after washout (n = 32; p = NS vs. control).


Figure 7
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  		Fig. 7 Uncoupling by carbenoxolone in the intact heart quantified by the amplitude of Laplacian electrograms. (A) Representative example of ventricular Laplacian electrogram. Note decreased amplitude upon carbenoxolone perfusion. (B) Mean Laplacian amplitude (n = 32, ±SD, asterisk p<0.05).

 

   4. Discussion
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
Acknowledgments
 References
 
Our results show that carbenoxolone causes moderate cellular electrical uncoupling and slowing of conduction in intact atrial and ventricular myocardium in a reversible manner. Unlike commonly used uncoupling drugs, it does not affect action potential characteristics or the underlying major ion currents. Furthermore, our study demonstrates that carbenoxolone-induced increase in activation delay is, as expected from a theoretical point of view [15,23], more prominent when cells are initially poorly coupled.

4.1. Cellular uncoupling and propagation
Computer simulations show that a decrease in gap junctional conductance only modestly affects conduction velocity in regions of high conductance. However, in regions of initially lower gap junctional conductance, a decrease in conductance may lead to significant conduction slowing [2,15]. It is this characteristic that potentially makes gap junctional uncoupling an antiarrhythmic target. However, this speculation requires further investigation.

The decreased conduction velocity in atrial and ventricular myocardium by carbenoxolone can be due to either changes in excitability or reduced gap junctional coupling [34]. However, we demonstrated that action potential upstroke velocity, and INa and ICa remained unchanged, which excludes changed excitability. Furthermore, we provide direct evidence that carbenoxolone decreases coupling conductance in isolated ventricular cell pairs. Moreover, we observed a decrease in Laplacian amplitude in intact tissue, which is proportional to coupling conductance, provided that transmembrane potential and tissue impedance remain constant [30,31], which was the case in our experiments. The lack of rise in total tissue impedance can be explained by subtle changes in, for example, the volumes of the intra- and extracellular space. Therefore, we conclude that the observed increase in activation time can be fully attributed to reduced gap junctional coupling.

Here, we did not investigate the exact mechanism and site of action of carbenoxolone. However, from our data in can be inferred that this drug is not specific for Cx43 since also atrioventricular conduction was slowed in our experiments. Hence, most likely Cx40 conductance is decreased as well, and it has been suggested that carbenoxolone also exerts effects on Cx32 [35]. Goldberg et al. [36] showed that carbenoxolone-induced uncoupling in C6 glioma cells transfected with Cx43 was associated with altered connexon configuration within the gap junction plaque and not with obvious changes in protein synthesis or phosphorylation. In the intact rabbit heart, 50-µmol/l carbenoxolone did not alter the phosphorylation status compared to saline perfused hearts (data not shown). The molecular mechanism of carbenoxolone-induced gap junctional uncoupling in the heart thus needs to be elucidated.

We did demonstrate an effect of carbenoxolone in intact normal myocardium but not in well-coupled cell pairs. This discrepancy may in part be due to limitations in the maximum rate of data acquisition (2 kHz) in the experiments on cell pairs that necessitated the simultaneous recording of 2 action potentials. In addition, the data on activation delay in ventricular cell pairs cannot be unequivocally extrapolated to the intact heart: (1) the myocardial interstitium with all its components may reduce the effective coupling as compared to coupled cell pairs, and (2) in intact tissue cells have multiple connections with neighbouring cells, which may influence source–sink relations. Our observation that uncoupling has more effect on action potential transfer time in poorly compared to well-coupled tissue corroborates the experimental work of Zampighi et al. [23] and the predictions of simulation studies by Shaw and Rudy [2], and Jongsma and Wilders [15]. From these simulation studies, it can also be predicted that homogeneous gap junction uncoupling results in a larger reduction in transversal than longitudinal conduction velocity. However, in our study, we did not find a significant difference in the reduction of transversal versus longitudinal conduction velocity.

4.2. Methodological considerations
We measured right ventricular transversal conduction velocities that are somewhat higher than values from left ventricular-free wall [37] or left ventricular epicardium [38] reported in the literature. Consequently, we measured a lower anisotropic ratio. In experiments from our laboratory in a similar rabbit model as described here, we measured anisotropic ratios between 2 and 3 in the rabbit left ventricle (R.F. Wiegerinck, unpublished data), which is in concordance with previously published values. To the best of our knowledge, we present for the first time data on transversal conduction velocity and anisotropy in the rabbit right ventricular myocardium.

Carbenoxolone has been reported to increase vascular tonus, which could potentially influence coronary flow [39]. Occasionally, we observed a decrease in perfusion flow at prolonged or repetitive carbenoxolone exposure (data not shown). Such adverse effects are not likely to have influenced the current experiments because we investigated only short perfusion periods during which coronary flow remained within physiological limits. This study aimed at demonstrating that carbenoxolone causes conduction slowing at a concentration of 50 µmol/l. We therefore did not systematically study the drug at other concentrations. Whether uncoupling occurs at higher concentrations needs to be determined.

4.3. Clinical implications
Various observations support the importance of gap junctional uncoupling in arrhythmogenesis, especially in regions where cellular coupling is reduced. Such regions include acute ischemia [4] and the border zone of a previous infarction [6]. Also, degenerative changes associated with hypertrophy [8], ischemic heart disease, and heart failure [7] might create conditions with reduced coupling. In addition, electrical remodelling that occurs after chronic atrial fibrillation alters gap junction distribution [40].

Since the publication of large trials that showed proarrhythmic effects of class 1C antiarrhythmic agents and sotalol [13,14], the effort to develop new antiarrhythmic drugs directed at specific ion channels has decreased dramatically. Antiarrhythmic interventions that are applicable on a large scale are still desperately needed. Changing the properties of intercellular electrical communication might be a powerful alternative tool to change the arrhythmogenic substrate. The conditions for reentrant arrhythmias and the occurrence of arrhythmogenic after depolarizations are confined to a restricted range of critically reduced electrical coupling [5,9]. Moreover, there is evidence that cell injury after reperfusion expands via a gap junction dependent mechanism [41,42]. Contrary to the application of class 1C drugs that may induce an arrhythmogenic substrate, reduction of cellular coupling might potentially convert an arrhythmogenic substrate to a less vulnerable situation (while having no effect on normal tissue) and might become a new target in the study of atrial and ventricular arrhythmias. However, its potential antiarrhythmic properties need to be further investigated.


   Acknowledgments
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
 Acknowledgments
References
 
Supported by Netherlands Heart Foundation grants 97-198 and 2000T020, and EU projects IST-1999-13047 (MicroTrans) and Esprit IV 33485 (MicroCard).


   Notes
 
1 Both authors contributed equally. Back

Time for primary review 20 days


   References
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
 Acknowledgments
 References
 

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J. R de Groot and R. Coronel
Acute ischemia-induced gap junctional uncoupling and arrhythmogenesis
Cardiovasc Res, May 1, 2004; 62(2): 323 - 334.
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