Skip Navigation

Cardiovascular Research 2001 51(1):30-40; doi:10.1016/S0008-6363(01)00246-2
© 2001 by European Society of Cardiology
This Article
Right arrow Abstract Freely available
Right arrow FREE Full Text (PDF) Freely available
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Add to My Personal Archive
Right arrow Download to citation manager
Right arrowRequest Permissions
Google Scholar
Right arrow Articles by Verkerk, A. O
Right arrow Articles by van Ginneken, A. C.G
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Verkerk, A. O
Right arrow Articles by van Ginneken, A. C.G
Social Bookmarking
Add to CiteULike   Add to Connotea   Add to Del.icio.us  
What's this?

Copyright © 2000, European Society of Cardiology

Effects of cell-to-cell uncoupling and catecholamines on Purkinje and ventricular action potentials: implications for phase-1b arrhythmias

Arie O Verkerka,*, Marieke W Veldkampb, Ruben Coronelb, Ronald Wildersa,c and Antoni C.G van Ginnekena,b

aDepartment of Physiology, Cardiovascular Research Institute Amsterdam, University of Amsterdam, Amsterdam, The Netherlands
bExperimental and Molecular Cardiology Group, Cardiovascular Research Institute Amsterdam, University of Amsterdam, Amsterdam, The Netherlands
cDepartment of Medical Physiology, University Medical Center Utrecht, Utrecht University, Utrecht, The Netherlands

* Corresponding author. Academic Medical Center, Department of Physiology, Room M01-09, Meibergdreef 15, 1105 AZ Amsterdam, The Netherlands. Tel.: +31-20-566-4670; fax: +31-20-691-9319 a.o.verkerk{at}amc.uva.nl

Received 2 November 2000; accepted 7 February 2001


   Abstract
Top
 Abstract
1. Introduction
 2. Methods
 3. Results
 4. Discussion
 References
 
Objective: The delayed phase of ventricular arrhythmias during acute ischemia (phase-1b arrhythmia) is associated with depletion of catecholamines and cell-to-cell uncoupling between depressed depolarized intramural ischemic region and surviving cells in subepicardium and subendocardium. In the present study we determined the effects of uncoupling and catecholamines on development of proarrhythmic afterdepolarizations. Methods: Depressed depolarized ischemic region was simulated by a passive electronic circuit with a potential of –73, –53, –33 or –13 mV. Using patch-clamp methodology, single sheep Purkinje and ventricular cells were coupled to the simulated ischemic region via a variable conductance. By varying coupling conductance, we were able to selectively study the effects of various degrees of uncoupling. Results: At strong coupling, cells were inexcitable and depolarized to potentials near those of the simulated ischemic region. Excitability, action potential duration and resting potential increased with progressive uncoupling. In a critical range of uncoupling, ventricular and ‘high-plateau’ Purkinje cells developed early afterdepolarizations when the potential of the simulated ischemic region was –13 mV. Norepinephrine (1 µM) frequently induced early and delayed afterdepolarizations in both ventricular and Purkinje cells, but these afterdepolarizations were only present during uncoupling when the potential of the simulated ischemic region was –33 mV or more positive. Conclusions: In a critical range of uncoupling, afterdepolarizations were present when the potential of the simulated ischemic region was –33 or –13 mV, suggesting that triggered activity plays a role in phase-1b arrhythmias when surviving layers uncouple from a highly depolarized intramural ischemic region.

KEYWORDS Adrenergic (ant)agonists; Arrhythmia (mechanisms); Cell communication; Gap junctions; Ischemia; Purkinje fiber; Ventricular arrhythmias

This article is referred to in the Editorial by Y. Rudy (pages 1–3) in this issue.


   1. Introduction
Top
 Abstract
 1. Introduction
2. Methods
 3. Results
 4. Discussion
 References
 
Acute regional myocardial ischemia results in a combination of ionic, metabolic, neurohumoral, and electrical alterations that can cause lethal ventricular arrhythmias (for reviews see Refs. [1,2]). These arrhythmias develop in two distinct phases, which are separated by a period of normal sinus rhythm [3]. The early phase (phase-1a) is closely related to altered K+ levels leading to a flow of ‘injury current’ between normal and ischemic myocardium [4–6]. The delayed phase of arrhythmias occurring between 12 and 30 min after onset of ischemia (phase-1b) is associated with cell-to-cell electrical uncoupling [7–9]. Smith et al. [10] demonstrated a close temporal association between occurrence of phase-1b ventricular fibrillation and the onset of cellular uncoupling. Moreover, De Groot et al. [11] recently demonstrated that the inducibility of ventricular fibrillation during ischemia was maximal during the initial phase of increasing tissue resistance, and decreased after resistance exceeded 50% of the final resistance. This suggests that a moderate cell-to-cell electrical uncoupling is important for the genesis of phase-1b arrhythmias.

Phase-1b arrhythmias are reduced by adrenergic blockade and depletion of catecholamines [12–14], which are normally released at between 15 and 20 min of ischemia [15]. This implies that the sympathetic nervous system also plays a prominent role in phase-1b arrhythmias. Application of β-receptor agonists has been shown to markedly increase intracellular resistance [16], probably due to the synergistic effect of cAMP on the Ca2+-induced decrease of gap-junction conductance [17,18]. In addition, it is well established that catecholamines induce proarrhythmic afterdepolarizations (for reviews see Refs. [19,20]).

Until now, the contribution of triggered activity to arrhythmogenesis during acute ischemia is debated. Electrotonic influences between a normal and ischemic region strongly suppress the development of triggered activity [21]. Recently, however, it was shown in a heart cell model study that a moderate degree of uncoupling between normal myocardium and a depolarized ischemic region favors both initiation of early afterdepolarizations (EADs) and their spread to neighbouring tissue [22]. This suggests that EADs may be involved in arrhythmogenesis of phase-1b arrhythmias when a critical degree of cell-to-cell electrical uncoupling occurs.

The present study was designed to evaluate the effects of cell-to-cell uncoupling and catecholamines on amplitude and incidence of both EADs and delayed afterdepolarizations (DADs). Therefore, we coupled ventricular or Purkinje myocytes to a passive electronic circuit via a variable conductance, comparable to the method introduced by Tan and Joyner [23]. The real myocyte represents the surviving cells during acute ischemia in either the subendocardium or the subepicardium [24–27], and the electronic circuit represents the ischemic intramural region. The selected potentials of this circuit (–13, –33, –53, or –73 mV) correspond to the transmembrane voltages recorded in severely depressed intramural tissue [1,28]. By changing the coupling conductance, we were able to selectively study the effects of alterations in cell-to-cell electrical coupling between the ischemic and surviving layer of cells. Because catecholamines may leak out of the ischemic area to the surrounding surviving tissue [1,28], the effects of exposure to norepinephrine (1 µM) were also tested.


   2. Methods
Top
 Abstract
 1. Introduction
 2. Methods
3. Results
 4. Discussion
 References
 
2.1. Cell preparation
Purkinje and ventricular cells were isolated from sheep hearts by an enzymatic dissociation procedure [29]. Purkinje cells were isolated from hearts obtained from the slaughterhouse immediately after exsanguination of the animals and from hearts obtained from laboratory animals which were anaesthetized with intravenously injected Nesdonal® (10 mg/kg thiopental; Rhône-Mérieux, Lyon, France). Ventricular cells were isolated from hearts of anaesthetized sheep. The anaesthetized animals were handled in accordance with the institutional guidelines. Small aliquots of cell suspension were put in a recording chamber on the stage of an inverted microscope. The cells were allowed to adhere for 5 min before starting continuous perfusion with Tyrode's solution (35–37°C) containing (mM): 140 NaCl, 5.4 KCl, 1.8 CaCl2, 1.0 MgCl2, 5.5 glucose, and 5.0 HEPES; pH was adjusted to 7.4 with NaOH. Only rod-shaped cells with smooth surfaces were selected for electrophysiological measurements.

In intact Purkinje strands of sheep, dog, and baboon, two distinct types of action potential configuration are present [29–32]. Single sheep Purkinje cells share the propensity for having two distinct types of action potential configuration [29]. About 50% of the single cells showed action potentials (APs) with a prominent phase-1 repolarization and relatively negative plateau (‘low-plateau’ Purkinje cells), whereas the remaining single cells had little phase-1 repolarization and relatively positive plateau (‘high-plateau’ Purkinje cells). In the present study, both Purkinje cell types were used.

2.2. Electrophysiological recording
Membrane potentials and currents were recorded in the whole-cell configuration of the patch-clamp technique. Patch pipettes were pulled from borosilicate glass and their tips were heat polished. Pipettes had resistances of 3–5 M{Omega} when filled with the pipette solution which contained (mM): 125 K-gluconate, 20 KCl, 10 HEPES; pH was adjusted to 7.2 with KOH. All potentials were corrected for the estimated 13 mV change in liquid junction potential. Membrane currents and potentials were filtered on-line (1 kHz), digitized at 2 kHz, stored and analyzed by CUSTOM software.

Action potentials were elicited at 1.0 or 0.5 Hz by 2-ms current pulses (20–50% suprathreshold) applied via the patch pipette. Cell capacitance (Cm) was determined from the change in initial slope of the potential ({Delta}(dVm/dt)) in response to 10-ms hyper- and depolarizing pulses of 30 or 100 pA ({Delta}Im) applied during the action potential plateau. Cell capacitance was calculated as Cm={Delta}Im/{Delta}(dVm/dt).

2.3. Cell-to-cell coupling
Changes in cell-to-cell coupling between the intramural ischemic region and surviving cells in subendocardium or subepicardium was simulated by an experimental model system comparable to that introduced by Tan and Joyner [23] who coupled isolated cells to a passive resistor–capacitor circuit via a variable conductance. In our experiments, we were interested in effects of cell-to-cell uncoupling which occurred long after the massive capacitative load. Therefore, we left out the capacitor and used a passive resistance circuit (Fig. 1A). In our experiments, the real myocyte represents the electrophysiologically competent cells in either subendocardium or subepicardium, whereas the electronic circuit simulates the intramural ischemic region which has a depolarized resting membrane potential [1]. By changing the coupling conductance, we mimicked the alterations in cell-to-cell electrical coupling between the ischemic and surviving layer of cells as occur during the delayed phase of ischemia induced arrhythmias.


Figure 1
View larger version (22K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Fig. 1 (A) Electronic circuit for simulation of coupling current (Icoupling). (B) Icoupling (top) and action potential (bottom) of a ventricular cell when coupled to this circuit with a potential (Vmodel) of –13 mV (dashed line) at a coupling conductance (Gc=Rcoupling–1) of 0, 5 or 10 nS. Arrows indicate the points in time where Icoupling changes from repolarizing into depolarizing current.

 
Our model of the ischemic region had a potential of –73, –53, –33 or –13 mV. The potentials of –33 and –13 mV are too depolarized to be due to ischemia-induced changes in K+ equilibrium potential [1,2,10]. However, in the ischemic zone there is a delay in activation so that at the time non-ischemic cells are repolarized, the ischemic cells may still be in plateau phase [10,26]. Under such conditions, the potential of the ischemic tissue is positive to –50 mV. In our experiments, we simulated these conditions by our model with a fixed potential of –33 or –13 mV.

Our model simulating the ischemic tissue supplies a coupling current (Icoupling) to the real cell with an amplitude and direction that depended on the ohmic coupling conductance (Gc=Rcoupling–1) and potentials of the real cell (Vcell) and the model (Vmodel). Coupling current is then given as Icoupling=Gcx(Vmodel–Vcell). Fig. 1B shows a typical example of Icoupling (top) and APs of a ventricular cell (bottom) during uncoupled conditions (Gc=0 nS) and during coupling to the model with Vmodel= –13 mV. Coupling the cell to the model via a Gc of 5 or 10 nS, causes an Icoupling that is outwardly directed at membrane potentials positive to –13 mV and inwardly directed at membrane potentials negative to –13 mV (see arrows). Icoupling increases with increasing Gc. Although the aim of the present study was to elucidate the effects of cell-to-cell uncoupling, the threshold determination at the onset of the experiments forced us to commence in uncoupled conditions (Gc=0 nS) and subsequently increase the coupling conductance.

2.4. Statistics
Values are expressed as mean±S.E. and considered significantly different if P≤0.05 in Student's t-test. Action potential parameters were obtained from ten consecutive APs and averaged.


   3. Results
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
4. Discussion
 References
 
3.1. Effects of altered coupling conductance between surviving and ischemic tissue
First, we studied the effects of altered coupling between surviving and ischemic tissue in seven ventricular, four high-plateau Purkinje, and six low-plateau Purkinje cells at 0.5 Hz stimulation. In these experiments, no norepinephrine (NE) was added to the extracellular solution. Fig. 2 shows representative effects of changes in coupling conductance on APs of a ventricular cell. Coupling the cell to the model with a potential (Vmodel) of –73 mV (Fig. 2A) decreased plateau height and duration of the plateau phase (trace labeled 0.9 nS). Moreover, phase-3 repolarization was slightly decelerated. Also, the action potential was shortened while resting membrane potential (RMP) became less negative. These effects were enhanced with increasing Gc (traces labeled 2.5, 5 and 9 nS). Finally, the cell became inexcitable. Action potential parameters returned to control values on terminating coupling.


Figure 2
View larger version (26K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Fig. 2 Effects of altered coupling on the action potential of a ventricular cell. Vmodel was (A) –73, (B) –53, (C) –33 and (D) –13 mV. Numbers represent coupling conductance in nS.

 
Comparable effects were found when Vmodel was –53 (Fig. 2B) or –33 mV (Fig. 2C). At these more positive values of the model potential, however, changes in action potential configuration were less dramatic, apart from a more pronounced decrease of RMP. For example, coupling at 2.5 nS with a Vmodel of –73, –53 and –33 mV decreased action potential duration at 90% repolarization (APD90) from its control value of 600 to 150, 180, and 270 ms, respectively, whereas RMP decreased from its control value of –83 to –82, –80 and –78 mV, respectively.

An additional effect of altered coupling was found when Vmodel was –13 mV (Fig. 2D). At Gc≤10 nS, the plateau height, APD90, and RMP all decreased with increasing Gc as observed at Vmodel of –73, –53 and –33 mV. However, a further increase in Gc to 13 nS induced a prolongation of the action potential as compared to Gc=10 nS. At Gc≥13.5 nS, the cell developed EADs, thus establishing 13.5 nS as the ‘threshold conductance’ for EAD initiation. The number of EADs increased with increasing Gc. Finally at Gc≥21 nS, EADs disappeared and RMP remained {approx}–10 mV. These effects of altered coupling were consistently observed in both ventricular and high-plateau Purkinje cells. However, they differ from effects on low-plateau Purkinje cells (Fig. 3).


Figure 3
View larger version (23K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Fig. 3 Effects of altered coupling on the action potential of a low-plateau Purkinje cell. Vmodel was (A) –73, (B) –53, (C) –33 and (D) –13 mV. Numbers represent coupling conductance in nS.

 
Fig. 3 shows representative effects of changes in coupling on APs of a low-plateau Purkinje cell. Coupling the low-plateau Purkinje cell to the model with a Vmodel of –73 (Fig. 3A) or –53 mV (Fig. 3B) resulted in a decrease in plateau height, action potential duration and RMP. These effects progressed with increasing Gc, finally resulting in inexcitability of the cell. In contrast, coupling the cell to the model with a Vmodel of –33 (Fig. 3C) or –13 mV (Fig. 3D), resulted in action potential prolongation with only minor changes in plateau height. This prolongation was more pronounced at higher coupling conductances. The cell even failed to fully repolarize at Gc≥5 nS (Vmodel=–33 mV, Fig. 3C) or Gc≥3 nS (Vmodel=–13 mV, Fig. 3D), as long as coupling was maintained. During these prolonged APs, no EADs were observed. Similar effects of altered coupling were found in five other low-plateau Purkinje cells.

In summary, EADs were invariably induced as a result of coupling to the model with a potential of –13 mV in ventricular and high-plateau Purkinje cells, but not in low-plateau Purkinje cells. Threshold coupling conductance to induce EADs was 19±5 and 48±17 nS for ventricular (n=7) and high-plateau Purkinje cells (n=4), respectively. These EADs had a take-off potential — the potential where repolarization turns into depolarization — of –28±1 and –27±3 mV and an amplitude of 18±4 and 22±6 mV for ventricular and high-plateau Purkinje cells, respectively. The EADs disappeared when coupling conductance was higher than 54±18 and 80±29 nS for ventricular (n=7) and high-plateau Purkinje cells (n=4), respectively, indicating that in both cell types the EADs were present in a critical range of cell-to-cell coupling.

3.2. Effects of altered coupling conductance between surviving and ischemic tissue in the presence of norepinephrine
The NE level is increased in ischemic tissue during the phase of 1b arrhythmias [15] and it is postulated that it may leak out of the ischemic area to the surrounding tissue [1,28]. Because cell-to-cell uncoupling and increase in NE level may thus occur at the same moment, we studied the effects of (1) NE on APs, and (2) altered coupling conductance in the presence of NE.

3.2.1. Effects of norepinephrine
First, we studied the effects of 1 µM NE on APs of 17 low-plateau Purkinje, 13 high-plateau Purkinje and 24 ventricular cells, which were stimulated at 1 Hz. In each cell type, NE induced a variety of effects within 3 min of the application (Fig. 4). Firstly, NE induced an elevation of the plateau phase and increased action potential duration in low-plateau Purkinje cells (Fig. 4A, left), but not in high-plateau Purkinje (Fig. 4A, middle) and ventricular cells (Fig. 4A, right). In the latter cell types, however, phase-1 repolarization was increased (see also Fig. 4A, inset). Secondly, in all cell types, NE induced EADs in ~10% of the cells (Fig. 4B). The EADs occurred as single rather than multiple afterdepolarizations and developed at potentials between –10 and –30 mV. The average take-off potential and amplitude of the EADs (n=6) was –21.8±3 and 10.1±2 mV, respectively. Thirdly, in all cell types, NE induced DADs in ~50% of the cells (Fig. 4C). In ~60% of the cells showing DADs, the development of these DADs was not the end-stage. After an initial 10—20 DADs, the amplitude of the DADs reached excitation threshold resulting in triggered APs. Table 1 summarizes the NE-induced effects on action potential parameters and the incidence of NE-induced EADs, DADs and triggered activity.


Figure 4
View larger version (25K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Fig. 4 Effects of 1 µM norepinephrine (NE) on APs of low-plateau (LP) Purkinje, high-plateau (HP) Purkinje, and ventricular cells. (A) Plateau elevation and action potential prolongation in low-plateau Purkinje cells (left), and increased phase-1 depolarization in high-plateau Purkinje (middle) and ventricular cells (right). (B) Early and (C) delayed afterdepolarizations in all three cell types. Note different time scales.

 

View this table:
[in this window]
[in a new window]

  		Table 1 Action potential parameters and incidence of afterdepolarizations of sheep Purkinje and ventricular cells upon exposure to 1 µM norepinephrinea

 
3.2.2. Effects of altered coupling conductance in cells lacking norepinephrine-induced afterdepolarizations
Some Purkinje and ventricular cells did not develop afterdepolarizations upon application of NE only (Table 1). In three ventricular, one high-plateau Purkinje, and three low-plateau Purkinje cells not displaying NE-induced afterdepolarizations, we studied the effects of altered coupling. The effects of various degrees of coupling in the presence of NE were similar to those in the absence of NE (Figs. 2 and 3Go). In ventricular myocytes, however, threshold coupling conductance to induce EADs was significantly decreased from 20.1±3 to 16.9±2 nS (n=3).

3.2.3. Effects of altered coupling conductance on norepinephrine-induced EADs and DADs
NE frequently induced EADs, DADs and triggered APs in Purkinje and ventricular cells (Table 1). Therefore, we also tested the effects of altered coupling on these NE-induced afterdepolarizations.

Fig. 5 shows effects of changes in coupling on NE-induced EADs. EADs were abolished when the cell was coupled at Gc≥1 nS to the model with a Vmodel of –73 (Fig. 5A) or –53 mV (Fig. 5B). However, when coupled to the model with a Vmodel of –33 (Fig. 5C) or –13 mV (Fig. 5D), the cell generated multiple EADs instead of a single EAD. The number of EADs increased with increasing Gc and finally, cells failed to repolarize (data not shown). These effects were consistently found in the three ventricular cells tested.


Figure 5
View larger version (21K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Fig. 5 Effects of altered coupling on NE-induced early afterdepolarizations in a ventricular cell. Vmodel was (A) –73, (B) –53, (C) –33 and (D) –13 mV. Numbers represent coupling conductance in nS.

 
Fig. 6 shows effects of changes in coupling on NE-induced DADs. DAD amplitude decreased upon coupling to the model with a Vmodel of –73 (Fig. 6A) or –53 mV (Fig. 6B). At relatively high coupling conductances DADs were almost completely abolished (traces labeled 10 and 15 nS in Fig. 6A and B, respectively). When Vmodel was –33 (Fig. 6C) or –13 mV (Fig. 6D), DAD amplitude was increased at relatively low coupling conductances but decreased when coupling conductance was further increased. Despite this decrease in DAD amplitude, DAD could reach excitation threshold resulting in trains of triggered APs (traces labeled 24 and 6 nS in Fig. 6C and D, respectively). These effects were consistently found in the three ventricular cells tested. In agreement with these observations, we found that NE-induced triggered APs due to DADs were abolished by coupling to the model with a Vmodel of –53 mV or more negative (data not shown). Coupling to the model with a Vmodel of –33 mV or more positive increased the frequency of triggered APs (data not shown).


Figure 6
View larger version (34K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Fig. 6 Effects of altered coupling on NE-induced delayed afterdepolarizations in a ventricular cell. Vmodel was (A) –73, (B) –53, (C) –33 and (D) –13 mV. Insets: superimposed delayed afterdepolarizations. Numbers represent coupling conductance in nS.

 

   4. Discussion
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
References
 
The aim of the present study was to investigate the role of cell-to-cell electrical uncoupling and catecholamines in the generation of phase-1b arrhythmias. Using the patch-clamp methodology, we studied the effects of NE on sheep ventricular cells and on the two electrophysiologically distinct sheep Purkinje cell types, i.e. the low-plateau and the high-plateau Purkinje cells. Moreover, the patch-clamp methodology enabled us to couple these three distinct cardiac cell types to a passive electronic circuit via a variable conductance, comparable to the method introduced by Tan and Joyner [23]. The real myocyte represented the surviving cells in either the subendocardium or subepicardium [24–27], whereas the electronic circuit simulated the depressed intramural ischemic region with various degrees of resting membrane depolarization [1]. Using this simplified representation of an ischemic myocardial region, the complex and inhomogeneous electrophysiological and biochemical changes during acute ischemia (for review, see Ref. [1]) were eliminated. By changing the coupling conductance, we were able to study selectively the effects of alterations in cell-to-cell coupling between a depolarized ischemic region and a normal region represented by either a ventricular cells or a low-plateau or high-plateau Purkinje cell. A wide variation of potentials of the simulated ischemic region, i.e. –13, –33, –53 or –73 mV, were used both in the absence and presence of NE. Our study extends thus the previous studies of Kumar and Joyner [33] and Huelsing et al. [34] to conditions more representing those of the delayed phase of acute regional ischemia. Kumar and Joyner [33] coupled guinea pig ventricular cells in both the absence and presence of isoproterenol to a normal polarized (–80 mV) region or to a completely depolarized (0 mV) region. Huelsing et al. [34] coupled rabbit Purkinje cells producing EADs to a normal polarized (–80 mV) region and to a region with moderately depolarized potentials (–60 and –50 mV).

The model we used can be scaled to represent the conduction between groups of cells under the conditions that, within each group, the cells of the group are themselves well coupled to each other and essentially isopotential. That this condition is met under certain ischemic conditions is indicated by the brief duration of each component of the fractionated signals which are characteristic of conduction in ischemic tissue, and also from direct microelectrode recordings of cells within the groups [35]. Thus, the conduction between groups of 100 cells connected by a conductance of 5 µS would have the same characteristics of electrical loading and also success or failure of conduction as one would observe between single cells connected by a conductance of 50 nS. In the case of the connections between large groups of cells, the interconnections might be a short bridge of cells, rather than a direct electrical connection between one cell of each group.

4.1. Effects of reducing coupling conductance between a real cell and simulated depolarized ischemic tissue
During acute ischemia, the intramural ischemic region depolarizes, due to altered K+ levels, leading to a flow of current between ischemic region and surrounding, normal cells [4–6]. We found that when the simulated ischemic region and the normal, real cell were well coupled, the electrotonic interaction resulted in depolarization of the real cells to values near the potential of the simulated ischemic region, and inexcitability. Upon cell-to-cell uncoupling, however, the electrotonic load decreased and the cells regained their excitability, showing APs of short duration. When uncoupling further progressed, APs prolonged and RMP increased. In a critical range of cell-to-cell uncoupling, these effects were accompanied by EAD formation in ventricular and high-plateau Purkinje cells when the simulated ischemic region had a potential of –13 mV.

The various mechanisms which have been suggested to underlie EAD formation, share the assumption of a reduction in net outward current during action potential repolarization. The primary mechanism may be a reduction in repolarizing K+ current and prolongation or reactivation of inward Ca2+ current [20,36–38]. In our experiments, when the real cell is coupled to a simulated ischemic region with a potential of –13 mV, the electrotonic load (Icoupling) will be inwardly directed in the potential range of the EADs. As a consequence, net outward current will be reduced, thus slowing repolarization and prolonging the time spent in the potential range of window Ca2+ current. Recovery from inactivation and reactivation of the Ca2+ channels may then occur, resulting in a transient depolarization [36,37]. Thus, the coupling of a myocyte to a very depolarized region (–13 mV) provides the substrate for induction of EADs. Nevertheless, in low-plateau Purkinje cells, EADs were not evoked by altered coupling, likely because L-type Ca2+ current in these cells is relatively small [29]. In addition, the potential range of the plateau phase in low-plateau Purkinje cells may be more negative than the potential range of maximum window Ca2+ current. The first hypothesis is supported by findings of Kumar and Joyner [33]. They were not able to induce EADs in guinea pig ventricular cells by coupling to a region of 0 mV unless 50 nM isoproterenol, which enhances the Ca2+ current, was applied [33].

4.2. Effects of reducing coupling conductance between a real cell and simulated depolarized ischemic tissue in the presence of norepinephrine
Although cell-to-cell electrical uncoupling is strongly involved in the development of phase-1b arrhythmias, the sensitivity of the arrhythmias to adrenergic blockade and depletion of catecholamines [12–14] implies that the sympathetic nervous system may also play an important role in arrhythmogenesis. It is well established that catecholamines frequently induce proarrhythmic afterdepolarizations (for reviews see Refs. [19,20]) but the role of triggered activity to arrhythmogenesis during acute ischemia is debated [21].

In our experiments, the effects of 1 µM NE alone on action potential duration differed for the various cell types. In low-plateau Purkinje cells the action potential duration was prolonged, whereas it was unaltered in high-plateau Purkinje and ventricular cells. We think that this different effect of NE on Purkinje APs is due to the differences in the ionic currents that contribute to electrical heterogeneity among the two Purkinje cell types, consistent with the effects of {alpha} 1-adrenergic agonists on different ventricular cell types [39]. Low-plateau Purkinje cells, but not high-plateau Purkinje cells, display a prominent transient outward K+ (Ito1) current, while density of L-type Ca2+ current (ICa,L) was lower in low-plateau Purkinje than in high-plateau Purkinje cells [29]. Independently of the response on action potential duration, NE frequently induced early and delayed afterdepolarizations in all cell types.

4.2.1. Effects of reducing coupling conductance on norepinephrine-induced early afterdepolarizations
When the real cell was coupled to a simulated ischemic region with a potential positive to –33 mV, the number of EADs was enhanced. EADs were abolished when the potential of the simulated ischemic region was negative to –53 mV. This latter finding agrees with the observation by Kumar and Joyner [33] that Icoupling abolishes EADs in guinea pig ventricular myocytes when it flows from a normal polarized (–80 mV) region. In our experiments, however, we additionally demonstrated that this also occurs when Icoupling flows from moderately depolarized resting potentials, as was recently also shown by Huelsing et al. [34] who coupled rabbit Purkinje cells producing EADs to an electronic circuit with potentials of –80, –60 and –50 mV.

The abolition of EADs can be explained by the fact that at a model potential of –53 mV or more negative, Icoupling is outwardly directed in the potential range of window Ca2+ current, thus increasing net outward current in this range and decreasing the propensity to transient depolarization. Additionally, the action potential shortening and the resulting reduction in time available for reactivation of L-type Ca2+ channels may also play a role [20,36–38]. The enhancement of EADs by a model potential of –33 mV or more positive can be explained by the fact that Icoupling is then inwardly directed with opposite effects on net outward current and action potential duration.

4.2.2. Effects of reducing coupling conductance on delayed afterdepolarizations
DADs were decreased in amplitude or even abolished completely when Icoupling was flowing from –53 mV or more negative. When flowing from –33 mV or more positive, it increased DAD amplitude, which, combined with the concomitant depolarization of resting membrane, resulted in triggered APs. This latter finding agrees with the observation by Kumar and Joyner [33] that abnormal spontaneous activity, induced by combined application of 50 nM isoproterenol and coupling to a model with a potential of 0 mV, was abolished when coupling was switched off. In our experiments, however, we additionally demonstrated that this also occurs when Icoupling flows from less depolarized resting potentials.

The generation of triggered APs can be explained by the fact that the DAD amplitude increases by small depolarizations of resting membrane, action potential prolongation, or increasing height of the plateau phase [19,20]. It may be hypothesized therefore that coupling to –33 or –13 mV, which depolarizes the resting membrane without much effect on duration and height of the action potential, results in an increased DAD amplitude, while, when coupling to –53 or –73 mV, the decreasing effect of action potential shortening on DAD amplitude dominates over the increasing effect of the depolarized resting membrane potential.

4.3. Implications for phase-1B arrhythmias
4.3.1. Role of Purkinje strands
Subendocardial Purkinje strands adjacent to an ischemic region are proposed to play an important role in the genesis of ventricular arrhythmias during acute myocardial ischemia [40,41]. Their relatively unstable resting membrane potential [29,42] and their relatively long APs [43] may predispose to the occurrence of cellular triggers for arrhythmogenesis, such as EADs, DADs and automaticity [19,20,37,38,44]. In the present study, however, we demonstrate that the incidence of afterdepolarizations in response to cell-to cell-uncoupling and elevated NE concentration was lower in typical Purkinje cells (low-plateau Purkinje cells) than in high-plateau Purkinje and ventricular cells. Thus, our data suggests that arrhythmogenesis is more likely to originate from ventricular and high-plateau Purkinje cells than from low-plateau Purkinje cells, which agrees with findings of Janse et al. [25], who demonstrated that destruction of the subendocardium, including the Purkinje system, does not completely abolish ectopic impulse formation during the acute phase of ischemia.

4.3.2. Uncoupling and afterdepolarizations
The occurrence of phase-1b ventricular fibrillation is closely temporally related to the onset of cellular uncoupling [10], and recently, it was demonstrated that the inducibility of ventricular fibrillation was restricted to the initial 40% of uncoupling [11]. In the present study, we found that at a critical degree of cell-to-cell uncoupling proarrhythmic EADs arise when the simulated ischemic region was depolarized to –13 mV. Moreover, we found that NE-induced afterdepolarizations are present during the process of cell-to-cell uncoupling when the simulated ischemic region was depolarized to –33 mV or more positive potentials. We conclude that afterdepolarizations formed in the surviving cells of the subendocardium and subepicardium may play a role in phase-1b arrhythmias when these cells are in the process of uncoupling from a strongly depolarized region. Such a very depolarized potential can be reached, when APs in the ischemic region are delayed to such extent that they occur during phase three repolarization of the normal region [10,28].

Time for primary review 27 days.


   Acknowledgements
 
The authors thank Jan Bourier and Berend de Jonge for excellent technical assistance, and Drs. Ronald W. Joyner and Joris R. de Groot for critical reading of the manuscript and valuable comments. This work was partly supported by the Research Council for Earth and Life Sciences (ALW) with financial aid from the Netherlands Organization for Scientific Research (NWO) through grants 805-06.154 and 805-06.155.


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

  1. Janse M.J, Wit A.L. Electrophysiological mechanisms of ventricular arrhythmias resulting from myocardial ischemia and infarction. Physiol Rev (1989) 69:1049–1168.[Free Full Text]
  2. Cascio W.E, Johnson T.A, Gettes L.S. Electrophysiologic changes in ischemic ventricular myocardium: I influence of ionic, metabolic, and energetic changes. J Cardiovasc Electrophysiol (1995) 6:1039–1062.[ISI][Medline]
  3. Kaplinsky E, Ogawa S, Balke C.W, Dreifus L.S. Two periods of early ventricular arrhythmias in the canine acute infarction model. Circulation (1979) 60:397–403.[Abstract/Free Full Text]
  4. Janse M.J, Capelle F.J.L, van Morsink H, et al. Flow of ‘injury’ current and patterns of excitation during early ventricular arrhythmias in acute regional myocardial ischemia in isolated porcine and canine hearts. Circ Res (1980) 47:151–165.[Free Full Text]
  5. Coronel R, Fiolet J.W, Wilms-Schopman F.J, et al. Distribution of extracellular potassium and its relation to electrophysiologic changes during acute myocardial ischemia in the isolated perfused porcine heart. Circulation (1988) 77:1125–1138.[Abstract/Free Full Text]
  6. Coronel R, Wilms-Schopman F.J, Dekker L.R, Janse M.J. Heterogeneities in [K+]o and TQ potential and the inducibility of ventricular fibrillation during acute regional ischemia in the isolated perfused porcine heart. Circulation (1995) 92:120–129.[Abstract/Free Full Text]
  7. Kléber A.G, Riegger C.B, Janse M.J. Electrical uncoupling and increase of extracellular resistance after induction of ischemia in isolated, arterially perfused rabbit papillary muscle. Circ Res (1987) 61:271–279.[Abstract/Free Full Text]
  8. Dekker L.R, Fiolet J.W, Van Bavel E, et al. Intracellular Ca2+, intercellular electrical coupling, and mechanical activity in ischemic rabbit papillary muscle. Effects of preconditioning and metabolic blockade. Circ Res (1996) 79:237–246.[Abstract/Free Full Text]
  9. Lerner D.L, Yamada K.A, Schuessler R.B, Saffitz J.E. Accelerated onset and increased incidence of ventricular arrhythmias induced by ischemia in Cx43-deficient mice. Circulation (2000) 101:547–552.[Abstract/Free Full Text]
  10. Smith W.T, Fleet W.F, Johnson T.A, Engle C.L, Cascio W.E. The 1B phase of ventricular arrhythmias in ischemic in situ porcine heart is related to changes in cell-to-cell electrical coupling. Circulation (1995) 92:3051–3060.[Abstract/Free Full Text]
  11. de Groot J.R, Coronel R, Wilms-Schopman F.J.G, Remme C.A, Janse M.J. Critical cellular coupling required for 1B ventricular fibrillation in the isolated regionally ischemic porcine heart. Circulation (1999) 100:4427. Abstract.
  12. Sheridan D.J, Penkoske P.A, Sobel B.E, Corr P.B. {alpha}-Adrenergic contributions to dysrhythmia during myocardial ischemia and reperfusion in cats. J Clin Invest (1980) 65:161–171.[ISI][Medline]
  13. Penny W.J. The deleterious effects of myocardial catecholamines on cellular electrophysiology and arrhythmias during ischemia and reperfusion. Eur Heart J (1984) 5:960–973.[Abstract/Free Full Text]
  14. Podrid P.J, Fuchs T, Candinas R. Role of sympathetic nervous system in the genesis of ventricular arrhythmia. Circulation (1990) 82:I103–I113.[Medline]
  15. Schömig A, Dart A.M, Dietz R, Mayer E, Kübler W. Release of endogenous catecholamines in the ischemic myocardium of the rat. Part A: locally mediated release. Circ Res (1984) 55:689–701.[Abstract/Free Full Text]
  16. Wojtczak J. Influence of cyclic nucleotides on the internal longitudinal resistance and contractures in the normal and hypoxic mammalian cardiac muscle. J Mol Cell Cardiol (1982) 14:259–265.[CrossRef][ISI][Medline]
  17. Burt J.M, Spray D.C. Inotropic agents modulate gap junctional conductance between cardiac myocytes. Am J Physiol (1988) 254:H1206–H1210.[ISI][Medline]
  18. Xiao R.P, De Mello W.C. Intracellular resistance in rat papillary muscle: interaction between cyclic AMP and calcium. J Cardiovasc Pharmacol (1991) 17:754–760.[ISI][Medline]
  19. Wit A.L, Rosen M.R. The heart and cardiovascular system. Fozzard H.A, Haber E, Jennings R.B, Katz A.M, Morgan H.E, eds. (1992) 2nd ed. New York: Raven Press. 2113–2163.
  20. Antzelevitch C, Sicouri S. Clinical relevance of cardiac arrhythmias generated by afterdepolarizations. Role of M cells in the generation of U waves, triggered activity and torsade de pointes. J Am Coll Cardiol (1994) 23:259–277.[Abstract]
  21. Méndez C, Delmar M. Cardiac electrophysiology and arrhythmias. Zipes D.P, Jalife J, eds. (1985) Orlando, FL: Grune and Stratton. 311–313.
  22. Saiz J, Ferrero J.M Jr., Monserrat M, Ferrero J.M, Thakor N.V. Influence of electrical coupling on early afterdepolarizations in ventricular myocytes. IEEE Trans Biomed Eng (1999) 46:138–147.[CrossRef][ISI][Medline]
  23. Tan R.C, Joyner R.W. Electrotonic influences on action potentials from isolated ventricular cells. Circ Res (1990) 67:1071–1081.[Abstract/Free Full Text]
  24. Ideker R.E, Klein G.J, Harrison L, et al. The transition to ventricular fibrillation induced by reperfusion after acute ischemia in dog: a period of organized epicardial activation. Circulation (1981) 63:1371–1379.[Abstract/Free Full Text]
  25. Janse M.J, Wilms-Schopman F, Wilensky R.J, Tranum-Jensen J. Cardiac electrophysiology and arrhythmias. Zipes D.P, Jalife J, eds. (1985) Orlando: Grune and Stratton. 353–362.
  26. Wilensky R.L, Tranum-Jensen J, Coronel R, et al. The subendocardial border zone during acute ischemia of the rabbit heart: an electrophysiologic, metabolic, and morphologic correlative study. Circulation (1986) 74:1137–1146.[Abstract/Free Full Text]
  27. Pogwizd S.M, Corr P.B. Mechanisms underlying the development of ventricular fibrillation during early myocardial ischemia. Circ Res (1990) 66:672–695.[Abstract/Free Full Text]
  28. Janse M.J. The premature beat. Cardiovasc Res (1992) 26:89–100.[Free Full Text]
  29. Verkerk A.O, Veldkamp M.W, Abbate F, et al. Two types of action potential configuration in single cardiac Purkinje cells of sheep. Am J Physiol (1999) 277:H1299–H1310.[ISI][Medline]
  30. Draper M.H, Weidmann S. Cardiac resting and action potentials recorded with an intracellular electrode. J Physiol (1951) 115:74–94.[Free Full Text]
  31. Hauswirth O, Noble D, Tsien R.W. The dependence of plateau currents in cardiac Purkinje fibres on the interval between action potentials. J Physiol (1972) 222:27–49.[Abstract/Free Full Text]
  32. Dangman K.H, Dresdner K.P, Michler R.E. Transmembrane action potentials and intracellular potassium activity of baboon cardiac tissues. Cardiovasc Res (1988) 22:202–212.
  33. Kumar R, Joyner R.W. An experimental model of the production of early afterdepolarizations by injury current from an ischemic region. Pflügers Arch (1994) 428:425–432.[CrossRef][ISI][Medline]
  34. Huelsing D.J, Spitzer K.W, Pollard A.E. Electrotonic suppression of early afterdepolarizations in isolated rabbit Purkinje myocytes. Am J Physiol (2000) 279:H250–H259.[ISI]
  35. Kienzle M.G, Tan R.C, Ramza B.M, Young M.L, Joyner R.W. Alterations in endocardial activation of the canine papillary muscle early and late after myocardial infarction. Circulation (1987) 76:860–874.[Abstract/Free Full Text]
  36. January C.T, Riddle L.M. Early afterdepolarizations: Mechanism of induction and block: A role for L-type Ca2+ current. Circ Res (1989) 64:977–990.[Abstract/Free Full Text]
  37. Zeng J, Rudy Y. Early afterdepolarizations in cardiac myocytes: Mechanism and rate dependence. Biophys J (1995) 68:949–964.[Abstract/Free Full Text]
  38. Roden D.M. Early after-depolarizations and torsade de pointes: implications for the control of cardiac arrhythmias by prolonging repolarization. Eur Heart J (1993) 14(Suppl_H):56–61.[ISI][Medline]
  39. Burashnikov A, Antzelevitch C. Differences in the electrophysiologic response of four canine ventricular cell types to {alpha} 1-adrenergic agonists. Cardiovasc Res (1999) 43:901–908.[Abstract/Free Full Text]
  40. Arnar D.O, Bullinga J.R, Martins J.B. Role of the Purkinje system in spontaneous ventricular tachycardia during acute ischemia in a canine model. Circulation (1997) 96:2421–2429.[Abstract/Free Full Text]
  41. Bagdonas A.A, Stuckey J.H, Piera J, Amer N.S, Hoffman B.F. Effects of ischemia and hypoxia on the specialized conducting system of the canine heart. Am Heart J (1961) 61:206–218.[CrossRef][ISI][Medline]
  42. Cordeiro J.M, Spitzer K.W, Giles W.R. Repolarizing K+ currents in rabbit Purkinje cells and ventricular cells. J Physiol (1998) 503:811–823.
  43. Surawicz B. Role of potassium channels in cycle length dependent regulation of action potential duration in mammalian cardiac Purkinje and ventricular muscle fibres. Cardiovasc Res (1992) 26:1021–1029.[Abstract/Free Full Text]
  44. Gilmour R.F, Zipes D.P. The heart and cardiovascular system. Fozzard H.A, Haber E, Jennings R.B, Katz A.M, Morgan H.E, eds. (1986) New York: Raven Press. 1239–1257.

Add to CiteULike CiteULike   Add to Connotea Connotea   Add to Del.icio.us Del.icio.us    What's this?



This Article
Right arrow Abstract Freely available
Right arrow FREE Full Text (PDF) Freely available
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Add to My Personal Archive
Right arrow Download to citation manager
Right arrowRequest Permissions
Google Scholar
Right arrow Articles by Verkerk, A. O
Right arrow Articles by van Ginneken, A. C.G
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Verkerk, A. O
Right arrow Articles by van Ginneken, A. C.G
Social Bookmarking
Add to CiteULike   Add to Connotea   Add to Del.icio.us  
What's this?