© 2000 by European Society of Cardiology
Copyright © 2000, European Society of Cardiology
Injury current modulates afterdepolarizations in single human ventricular cells
aDepartment of Physiology, Academic Medical Center, University of Amsterdam, Amsterdam, The Netherlands
bDepartment of Clinical and Experimental Cardiology, Academic Medical Center, University of Amsterdam, Amsterdam, The Netherlands
cDepartment of Cardiology, Academic Medical Center, University Medical Center Utrecht, Utrecht, The Netherlands
dDepartment of Medical Physiology and Sports Medicine, University Medical Center Utrecht, Utrecht, The Netherlands
* Corresponding author. Tel.: +31-20-566-4670; fax: +31-20-691-9319 A.O.Verkerk{at}amc.uva.nl
Received 12 November 1999; accepted 25 February 2000
 |
Abstract |
|---|
 Top Abstract 1 Introduction2 Materials and methods3 Results4 Discussion5 ConclusionReferences |
|---|
Objective: Injury current (Iinjury) and afterdepolarizations are thought to play an important role in arrhythmias that occur during acute ischemia. However, little is known about the effects of Iinjury on afterdepolarizations. The present study was designed to study the effect of Iinjury on afterdepolarizations and action potentials in single human ventricular cells. Methods: The patch-clamp technique was used to record action potentials and to apply Iinjury to human ventricular cells. In these cells, early and delayed afterdepolarizations (EADs and DADs) were induced by 1 µM norepinephrine. Iinjury was simulated by coupling cells via a variable coupling resistance to a passive resistance circuit with a potential of 0, –20, or –40 mV, mimicking a depolarized ischemic region. Results: At all potentials, Iinjury induced depolarization of the resting membrane potential and action potential shortening. Flowing from 0 mV, Iinjury induced EADs by itself and aggravated the EADs and DADs that were induced by norepinephrine. Flowing from –40 mV, Iinjury abolished the noradrenaline-induced EADs and DADs. Conclusions: Our results demonstrate that Iinjury may either prevent or promote the occurrence of afterdepolarizations in human ventricle. The latter holds if conduction is slowed to such an extent that it permits flow of current from depolarized ischemic cells at plateau level to cells in phase 3 or phase 4.
KEYWORDS Arrhythmia (mechanisms); Cell communication; Impulse formation; Ischemia; Membrane potential
|
1 Introduction |
|---|
|
TopAbstract1 Introduction2 Materials and methods3 Results4 Discussion5 ConclusionReferences |
|---|
Injury current (Iinjury) and triggered activity are thought to play an important role in the initiation of arrhythmias that occur during acute myocardial ischemia. These ‘phase 1’ arrhythmias are subdivided in two distinct phases [1]. The first phase (phase 1A arrhythmias) usually occurs between 2 and 10 min after onset of ischemia, the second phase (phase 1B arrhythmias) between 12 and 30 min. Iinjury, which flows due to differences in membrane potential between ischemic and normoxic tissue, has long been postulated to be responsible for phase 1A arrhythmias [2–5]. However, the flow of Iinjury is not restricted to the first 10 min of ischemia only. During acute ischemia, Iinjury will stay present until electrical cell-to-cell uncoupling takes place [6], which occurs 15 to 20 min after onset of ischemia [7,8]. This implies that Iinjury is also present during phase 1B arrhythmias.
Triggered activity, induced by a massive catecholamine release, has long been postulated to be an important mechanism of phase 1B arrhythmias [5]. Moreover, induced by lysophoshoglyceride, triggered activity is also thought to play a role in phase 1A arrhythmias [9–11]. Iinjury and triggered activity may thus occur at the same moment. However, the effects of Iinjury on triggered activity have not systematically been studied. Kumar and Joyner [12] found that early afterdepolarizations (EADs) were abolished when guinea pig ventricular cells producing EADs were coupled to a model cell with a resting potential of –80 mV. Furthermore, they found that guinea pig ventricular cells producing delayed afterdepolarizations (DADs) became spontaneously active after coupling to a model cell with a resting potential of 0 mV.
In this study, we investigated the effects of Iinjury on afterdepolarizations and action potentials of human ventricular cells. Iinjury was simulated by coupling cells via a variable coupling resistance to a passive resistance circuit with a potential of 0, –20, or –40 mV. We found that Iinjury has either antiarrhythmogenic or proarrhythmogenic effects on triggered activity and action potentials, depending on the potential of the depolarized ischemic region. Part of this work has previously been published in abstract form [13].
|
2 Materials and methods |
|---|
|
TopAbstract1 Introduction2 Materials and methods3 Results4 Discussion5 ConclusionReferences |
|---|
2.1 Cell isolation
Ventricular cells were isolated from explanted human hearts by an enzymatic dissociation procedure modified from Veldkamp et al. [14]. Hearts were obtained from patients with end-stage heart failure due to ischemic (n=6) or dilated cardiomyopathy (n=4), undergoing heart transplantation. Patient age was 54±3 years (mean±S.E.M.). All patients were in New York Heart Association class IV and had a mean ejection fraction of 20±4% (mean±S.E.M.). Informed consent was obtained before heart transplantation and the protocol complied with the ‘Declaration of Helsinki’ [15]. Directly after explantation, the heart was transported to the laboratory in oxygenated Tyrode solution I (4°C). A part of the left ventricular free wall was excised and perfused through a branch of the left anterior descending (LAD) coronary artery with the following solutions: (1) Tyrode solution I for 15 min at a constant pressure (50 mm Hg), (2) low Ca2+ Tyrode solution for 15 min (50 mm Hg), and (3) low Ca2+ Tyrode solution to which collagenase type B (0.15 mg/ml, Boehringer Mannheim), collagenase type P (0.05 mg/ml, Boehringer Mannheim), trypsine inhibitor (0.1 mg/ml, Boehringer Mannheim), and hyaluronidase (Sigma, St. Louis, MO, USA) were added. During this last period, the ventricular free wall was perfused at a constant flow in a recirculating manner. When perfusion pressure dropped from an initial value of 50 mm Hg to less than 2 mm Hg (usually after about 30 min), the left ventricular wall was cut into small pieces and was further fractionated using a standard shaking protocol [16].
No attempts were made to dissociate single myocytes from specific layers of the myocardium. In our dissociation procedure, however, the endocardial layer was not digested by the enzymes. Therefore, the experiments were exclusively performed on myocytes from midmyocardium and epicardium. Furthermore, the myocytes were not always isolated from exactly the same territory in the left ventricular wall. We always chose a branch of the LAD which perfused a part of the left ventricular wall which was not affected by infarcts. During the entire isolation procedure, all solutions were oxygenated and temperature was maintained at 36±1°C. Cells were stored at room temperature (21±1°C) in low Ca2+ Tyrode solution to which 1.3 mM CaCl2 and 1% albumin were added. The cells were used within 8 h after isolation.
2.2 Electrophysiological recording
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 after which continuous perfusion with Tyrode solution II (36±1°C) was started. Single rod-shaped cells having smooth surfaces were selected for electrophysiological measurements.
Action potentials were recorded in the whole-cell configuration of the patch-clamp technique [17] with a custom-made patch-clamp amplifier. Patch pipettes were pulled from borosilicate glass using a custom-made one-stage puller. The tips of the pipettes were heat-polished and had a resistance of 3 to 5 M
. The potential between pipette and the bath solution was adjusted to zero before a high-resistance seal was formed. The potentials were not corrected for the estimated 5 mV change in liquid junction potentials [18].
Membrane potentials recorded on tape were filtered off-line (1 kHz), digitized at 2 kHz, stored and analyzed using custom software. Action potentials were elicited at a frequency of 1.0 or 0.5 Hz by current pulses of 2 ms applied via the patch pipette. The following action potential parameters were measured: action potential duration at 20, 50, and 90% repolarization (APD20, APD50, and APD90, respectively), resting membrane potential (RMP), maximal upstroke velocity (dV/dtmax), and action potential amplitude (APA). The cell capacitance (Cm) was determined from the change in slope of the potential (
Vm) in response to 10-ms hyper- and depolarizing pulses of 100 pA (Im) applied during the plateau phase of the action potential. Cell capacitance was calculated using: Cm=Im/Vm.
2.3 Simulating injury current
The Iinjury flowing between a normal and a depolarized ischemic region was simulated by an experimental model system analogous to that used by Tan and Joyner [19] with minor modifications. In that study, isolated cells were coupled via a variable conductance to a passive resistor-capacitor (RC) circuit. Since we were interested in effects of Iinjury which occurred long after the flow of capacitative current, we left out the capacitor and used the passive resistance circuit diagrammed in Fig. 1A. This experimental model supplies Iinjury to the isolated cells with an amplitude and direction that depends on the coupling conductance (Gc) and the potential difference between model (Vmodel) and cell (Vreal cell). Iinjury is then given by the equation: Iinjury=Gc(Vmodel–Vreal cell).
|
Our Iinjury-model (Fig. 1A) is a passive electronic circuit. Downar et al. [20] demonstrated that ischemic tissue finally contained inexcitable cells which were depolarized to about –40 mV. However, during the first 8–10 min after onset of ischemia, the ischemic tissue was still able to produce action potentials. Sometimes, these action potentials were delayed to such an extent that, at the time when non-ischemic cells were in phase 3 repolarization or early in diastole, the ischemic cells were still in their plateau phase [3,4]. In such cases, Iinjury may flow from a membrane potential more positive to –40 mV. In this study, therefore, we used model potentials of 0, –20, and –40 mV and a wide range of coupling conductances.
2.4 Solutions
Tyrode solution I contained (mM): 128 NaCl, 4.7 KCl, 1.5 CaCl2, 0.6 MgCl2, 27 NaHCO3, 0.4 Na2HPO4, and 11 glucose; pH 7.4 by equilibration with 95% O2 and 5% CO2. Tyrode solution II contained (mM): 140 NaCl, 5.4 KCl, 1.8 CaCl2, 1.0 MgCl2, 5.5 glucose, and 5.0 HEPES; pH 7.4 with NaOH. Low Ca2+ Tyrode solution contained (mM): 155 NaCl, 0.02 CaCl2, 2.0 MgCl2, 3.3 KHCO3, 1.0 NaHCO3, 1.4 KH2PO4, 11 glucose, 10 creatine, and 16.8 HEPES; pH 7.3 with NaOH. Pipette solution contained (mM): 140 KCl, 5.0 K–ATP, and 10 HEPES; pH 7.2 with KOH.
2.5 Statistics
All data are presented as mean±S.E.M. Data on action potential parameters were obtained from 10 consecutive action potentials and averaged. For comparison of data between groups, an unpaired t-test with a significance level of P<0.05 was used.
|
3 Results |
|---|
|
TopAbstract1 Introduction2 Materials and methods3 Results4 Discussion5 ConclusionReferences |
|---|
3.1 Action potential configuration
We found significant differences in action potential duration between cells isolated from hearts with ischemic cardiomyopathy and cells isolated from hearts with dilated cardiomyopathy (Table 1). In ischemic cardiomyopathy, APD20, APD50, and APD90 were significantly shorter than in dilated cardiomyopathy, while other parameters did not differ significantly. Such a difference in action potential duration was also found by Koumi et al. [21] and was attributed to a difference in current density of the inward rectifier current. Furthermore, Koumi et al. [21] demonstrated that the action potential duration in ischemic cardiomyopathy hearts did not differ significantly from that in non-failing hearts. Thus, because action potentials in ischemic cardiomyopathy hearts seem more comparable to action potentials in non-failing myocardium than those in dilated cardiomyopathy hearts, we measured the effects of Iinjury in ventricular cells isolated from hearts with ischemic cardiomyopathy.
|
3.2 Effects of injury current on action potentials
The effects of Iinjury were measured in a total of eight ventricular cells isolated from four hearts with ischemic cardiomyopathy. Fig. 1 illustrates effects of Iinjury flowing from 0, –20, and –40 mV on action potentials of a human ventricular cell stimulated at a frequency of 1 Hz. Fig. 1B shows action potentials (top) and Iinjury (bottom) as a result of coupling to the model with a potential (Vmodel) of 0 mV. The action potential recorded under uncoupled conditions (0 nS) had a RMP of –80.5 mV and an APD90 of
430 ms. As expected, Iinjury during uncoupled conditions is zero. Coupling the cell to the model via a coupling conductance (Gc) of 10 nS caused an Iinjury that was outward at membrane potentials positive to 0 mV and inward at membrane potentials negative to 0 mV. Under this condition, Iinjury led to a decrease in plateau height from its control value of +38.8 to +32.6 mV and an acceleration of repolarization during the plateau phase. Furthermore, phase 3 repolarization was slightly decreased, APD90 was reduced to 305 ms and RMP was depolarized to –71.5 mV. Increasing Gc led to more pronounced effects. At a Gc of 20 nS, APD90 decreased to 275 ms and RMP depolarized to –55.6 mV. At a Gc of 25.8 nS, however, the phase 3 repolarization decreased to such a extent that the APD90 increased again to 340 ms. At a Gc of 26.3 nS, the cell developed an EAD. The EAD had a take-off potential of –15 mV and an amplitude of 17 mV. Uncoupling restored APD90 and RMP to control values (data not shown). These effects were observed in all cells tested. The lowest coupling conductance (normalized to Cm) to induce EADs was 43±7 pS/pF (n=8). The mean take-off potential and amplitude of these EADs was –16.6±1.9 and 15.3±2.8 mV (n=8), respectively. The generation of EADs is promoted at low stimulus frequency [11]. In four cells, we tested the effects of decrease in frequency on the coupling conductance to induce EADs. When the stimulus frequency was decreased from 1.0 to 0.5 Hz, the lowest value of coupling conductance required to induce EADs was significantly decreased from 49±5 pS/pF to 40±5 pS/pF (n=4), demonstrating that EADs are more easily induced by Iinjury at low stimulus frequency.
Fig. 1C and D show action potentials (top) and Iinjury (bottom) when coupled to the model with a Vmodel of –20 and –40 mV, respectively. Coupling conductances were identical to those used in Fig. 1B. The outwardly directed Iinjury was increased while inwardly directed Iinjury was decreased by a more negative potential of the model. This resulted in a larger decrease in APD90 but a less pronounced depolarization of RMP. No action potential prolongation or EADs were observed as a result of coupling to the model with a potential of –20, and –40 mV.
3.3 Effects of injury current on early and delayed afterdepolarizations
As emphasized above, Iinjury and afterdepolarization may occur at the same time during acute ischemia. Therefore, we studied the effects of Iinjury on afterdepolarizations by applying Iinjury to ventricular cells that already showed afterdepolarizations.
3.3.1 Induction of afterdepolarizations
EADs and DADs are often induced by norepinephrine [2,11]. In our study, we paced the cells at a frequency of 0.5 Hz and found that norepinephrine (1 µM) prolonged the human ventricular action potential without changing the RMP. In seven out of 11 cells the action potential prolongation was accompanied by EADs. These EADs occurred as single rather than multiple afterdepolarizations and EADs developed at voltages between 0 and –20 mV with a take-off potential of –7.0±1.9 mV (n=7) and an amplitude of 10.1±4.1 mV (n=7). Moreover, in five out of these 11 cells, we found that norepinephrine caused DADs. The DADs consisted of a single afterdepolarization, occurring at 242±31 ms (n=5) after final action potential repolarization. The DAD amplitude was 14±4.1 mV (n=5), which was never sufficient to induce triggered action potentials. We investigated the effects of Iinjury on norepinephrine-induced EADs and DADs.
3.3.2 Injury current and early afterdepolarizations
The effects of Iinjury on EADs were studied in a total of three ventricular cells. Fig. 2 illustrates effects of Iinjury flowing from 0, –20, and –40 mV on EADs of a human ventricular cell. When uncoupled (0 nS), the cell generated a single EAD with an amplitude of 12 mV. Iinjury flowing from 0 mV at Gc of 1.1 nS led to generation of multiple EADs with increasing amplitude (Fig. 2A). Upon uncoupling the EADs were restored to control values. Iinjury, simulated by coupling the cell to the model with a potential of –20 mV at a Gc of 1.3 nS, resulted in a shift of the take-off potential to more negative potentials and an increase in the EAD amplitude (Fig. 2B). However, Iinjury simulated at higher Gc (Gc of 1.8 nS) led to abolition of EADs. Uncoupling again restored the EAD. At all values of Gc, Iinjury flowing from –40 mV (Fig. 2C) led to abolition of EADs, which were restored after termination of the coupling. These effects were consistently observed in cells which produced EADs.
|
3.3.3 Injury current and delayed afterdepolarizations
The effects of Iinjury on DADs were studied in a total of another three ventricular cells. Fig. 3 illustrates effects of Iinjury flowing from 0, –20, and –40 mV on DADs of a human ventricular cell. When uncoupled, the cell generated a DAD with an amplitude of
18 mV. The DAD did not reach the threshold for excitation. At a Gc of 4.7 nS, Iinjury flowing from 0 mV caused an increase in the DAD amplitude to 30 mV (Fig. 3A, see also inset). At a Gc of 7.6 nS, the increase in DAD amplitude led, together with Iinjury-induced depolarization of RMP, to a triggered action potential. Termination of coupling ended the triggered action potentials and restored the DAD amplitude to control values. With Iinjury flowing from –20 mV (Fig. 3B) the proarrhythmogenic action of the combination of Iinjury and DADs was less prominent. Under this condition, the increase in DAD amplitude was smaller (from 19 to 22 mV by 5.6 nS) and triggered action potentials were only found at a Gc of 9.5 nS or higher. Iinjury flowing from –40 mV led to a decrease in DAD amplitude (Fig. 3C). At a Gc of 39.3 nS or more, DADs were completely abolished. The DADs were restored as soon as the coupling was terminated. These effects were consistently observed in cells which produced DADs.
|
3.4 Mechanisms of afterdepolarizations in human ventricular cells
Human ventricular cells invariably developed EADs in response to Iinjury flowing from 0 mV (Fig. 1B). In animal and model studies, EADs appeared to be due to reactivation of L-type calcium current [22,23] or to a current mechanism activated by Ca2+ release of the sarcoplasmic reticulum (SR) [24]. To determine the ionic nature of Iinjury-induced EADs in human ventricular cells, we tested in another six cells isolated from three hearts, whether the EADs could be blocked by 5 mM caffeine, an agent preventing spontaneous Ca2+ release of the SR, or by 1 mM CdCl2, a blocker of Ca2+ currents. The effects of caffeine and CdCl2 on EAD generation were measured 2–4 min after application.
Fig. 4A shows action potentials at a frequency of 0.5 Hz in absence (left panel) and presence (right panel) of 5 mM caffeine. In absence as well as in presence of caffeine, Iinjury flowing from 0 mV induced EADs. This was found in three out of three cells. Fig. 4B shows action potentials at a frequency of 0.5 Hz in absence (left panel) and presence (right panel) of 1 mM CdCl2. In absence of CdCl2, Iinjury flowing from 0 mV induced EADs. In presence of CdCl2, plateau height is reduced as expected from blockade of Ca2+ channels (trace labeled ‘0 nS’). In such cases, Iinjury led to very long action potentials (traces labeled ‘18, and 25.9 nS’). However, despite these long action potentials, no EADs were observed. This effect was found in three out of three cells. From these data we conclude that Iinjury-induced EADs in human ventricular cells are due to a calcium channel mechanism, rather than mechanisms involving spontaneous Ca2+ release from the SR.
|
|
4 Discussion |
|---|
|
TopAbstract1 Introduction2 Materials and methods3 Results4 Discussion5 ConclusionReferences |
|---|
During acute regional myocardial ischemia, the flow of Iinjury between ischemic and normoxic myocardium is an important potential arrhythmogenic factor [2–5]. Iinjury flows as a result of differences in membrane potential of cells in the ischemic and normoxic regions, and will stay present until electrical cell-to-cell uncoupling occurs at 15 to 20 min after onset of ischemia. During this period, triggered activity based on EADs and DADs may develop, which has also been postulated to be an important factor in arrhythmias during acute ischemia [5,9–11]. In this study, we investigated the changes in EADs, DADs, and action potentials in human ventricular cells as a result of Iinjury. We found that EADs and DADs are abolished when Iinjury is flowing from –40 mV, but are enhanced when it is flowing from 0 mV. Moreover, Iinjury induces action potential shortening and depolarization of RMP and when flowing from 0 mV, these effects are accompanied by EAD formation. Our data thus indicate that the arrhythmogenic effect of Iinjury on afterdepolarizations and action potentials depends on the potential of the depolarized ischemic region.
4.1 Effects of injury current on the action potential
4.1.1 Injury current induces action potential shortening and depolarization of RMP
Iinjury resulted in action potential shortening, depolarization of the RMP, and reduction of phase 3 repolarization rate (Fig. 1). The shape of an action potential results from a balanced ensemble of various inward and outward membrane currents [2]. Any change in the density of one of these membrane currents, or the introduction of a new current, disturbs the prevailing balance and will change the action potential configuration. Iinjury is an additional current which is outwardly directed when the potential of the real cell is more positive than that of our model circuit, while it is inwardly directed when the potential difference is reversed (Fig. 1). During the plateau phase of the action potential, Iinjury increases net outward current which complies with the observed abbreviation of the plateau phase. At the levels of RMP and final repolarization phase, Iinjury increases net inward current which complies with the observed depolarization and reduction of phase 3 repolarization rate. Moreover, the Iinjury-induced abbreviation of the plateau phase may also play a role in the decrease of phase 3 repolarization. The decrease in plateau height and abbreviation of the plateau phase altered activation and inactivation of IK and ICa, resulting in a decrease of net outward current (data not shown). During the plateau phase of the action potential, the net current flow and repolarization rate are low compared to phase 3 of the action potential. Therefore, inward Iinjury may reduce the repolarization rate at phase 3 of the action potential to some extent, but the increase in repolarization rate during plateau phase, due to outward Iinjury, is more pronounced. All together, this results in action potential shortening. Similar effects of Iinjury on ventricular action potentials were observed in experimental studies as well as computer stimulations using heart cell models [12,19,25,26].
4.1.2 Injury current induces early afterdepolarizations.
Iinjury flowing from a potential of 0 mV resulted invariably in EADs (Fig. 1B). Such EAD generation was also observed in a heart cell model study [25], and in ventricular cells of sheep [13], rabbit [A.O. Verkerk, unpublished observations], and guinea pig [12], although in the latter EADs were not produced unless 50 nM isoproterenol was applied [12]. In our study, the induction of EADs involves a Ca2+ channel mechanism (Fig. 4), which agrees with the suggestion that EADs are due to reactivation of Ca2+ channels when the membrane potential stays in the voltage range of the window Ca2+ current for a relatively long time [22,23]. We hypothesize that in our experiments the inwardly directed Iinjury keeps the membrane potential in the voltage range of the window Ca2+ current, i.e. between –40 and 0 mV [27], for such long time that Ca2+ channels recover from inactivation and reactivate, thus producing EADs.
4.2 Effects of injury current on afterdepolarizations
4.2.1 Effects of injury current on EADS
EADs were abolished when Iinjury was flowing from –20 mV or more negative potentials (Fig. 2B and C). This finding is comparable to what was found by Kumar and Joyner [12]. They demonstrated that Iinjury abolishes EADs when it flows from a normal polarized (–80 mV) region. In our experiments, we demonstrated that this also occurs when Iinjury is flowing from strongly depolarized potentials (up to –20 mV). This abolishing of EADs can be explained by the fact that at a model potential of –20 mV or more negative, Iinjury is outwardly directed in the voltage range of the window Ca2+ current. This increases the net outward current in this range and thereby prevents transient depolarizations. Also, due to the action potential shortening, less time remains for reactivation of the Ca2+ channels in the voltage range of window Ca2+ current.
EADs were enhanced when Iinjury was flowing from 0 mV (Fig. 2A). This may be explained by the fact that Iinjury is now inwardly directed at the potential of the window Ca2+ current. This increases net inward current and thereby increases the propensity to transient depolarization.
4.2.2 Effects of injury current on DADs
The amplitude of DADs decreased when Iinjury was flowing from –40 mV (Fig. 3C), but increased when Iinjury was flowing from 0 mV (Fig. 3A). The increased DAD amplitude, combined with the concomitant depolarization of the resting membrane potential, resulted in triggered action potentials (Fig. 3A and B). This latter finding agrees with findings of Kumar and Joyner [12]. They found that spontaneous activity, due to a combined effect of isoproterenol and coupling to a model with a potential of 0 mV, was abolished when the cell was uncoupled. Several variables may modify the amplitude of DADs, including stimulus frequency, the potential range at which the DADs occur, and the duration and height of the preceding action potential. DAD amplitude is increased by depolarization of RMP, increase of plateau height, and action potential prolongation [for review, see Ref. [11]]. It can be hypothesized that coupling to 0 mV, which resulted in a depolarized membrane potential without affecting duration and height of the action potential much (Fig. 1B and 3A
), results in an increased amplitude, whereas, when coupled to –40 mV, the decreasing effect of the action potential shortening (Figs. 1D and 3C) on DAD amplitude dominates over the increasing effect of the depolarized membrane potential.
4.2.3 Values of coupling conductance.
In our experiments, Iinjury was induced by coupling ventricular cells with variable conductances to an electronic circuit with a potential of 0, –20, or –40 mV. It is important to note that the coupling conductances (<40 nS) we used are low compared to that present in normal heart tissue, in which coupling conductance between cells may vary between 250 and 2500 nS [2]. In ischemic tissue, however, coupling conductance decreases [6–9]. Although the exact conductance of cell-to-cell coupling in ischemic tissue is unknown, the minimum value of coupling conductance required for propagation of action potentials during ischemia is 8 nS [28]. Thus Iinjury will exert its effect on afterdepolarizations at conductances comparable to that necessary for impulse conduction.
4.3 Relevance for arrhythmogenesis during acute ischemia
Iinjury is thought to play an important role in arrhythmias during acute ischemia [2–5]. Our experiments demonstrate that Iinjury caused depolarization of the RMP and action potential shortening in human ventricular cells, which are indeed important triggers for re-entrant arrhythmias [5]. Furthermore, Iinjury resulted in potentially arrhythmogenic EADs when it was flowing from 0 mV. Such a very depolarized potential can be reached during acute ischemia. When action potentials in the ischemic region are delayed to such an extent that they occur during phase 3 repolarization of the normal region, Iinjury may flow from potentials of around 0 mV [3,4].
Triggered activity based on EADs and DADs is thought to be an important mechanism for initiation of arrhythmias during acute ischemia [9–11]. Our results, however, demonstrate that during acute ischemia concomitant Iinjury may either prevent or promote the occurrence of afterdepolarizations in human ventricle, depending on the potential of the ischemic region. According to our results, EADs and DADs may play a role in arrhythmogenesis when Iinjury is flowing from 0 mV, thus by sufficient slowing of conduction.
4.4 Limitations of this study
4.4.1 Ventricular cells isolated from failing human hearts
Our experiments were performed on cells from failing human hearts. A prominent feature of human ventricular myocardium from patients with severe heart failure is action potential prolongation [29–31], which is thought to be proarrhythmogenic because it predisposes to the development of EADs [23]. We found indeed that in the human cells Iinjury flowing from 0 mV could evoke EADs. However, the finding that this also occurs in non-failing ventricular cells from rabbit, sheep, and guinea pig, as well as in a model study suggests that such EAD generation is a generic feature. Furthermore, our experiments were performed on cells isolated from hearts with ischemic cardiomyopathy for which action potential prolongation is less pronounced than for cells isolated from hearts with dilated cardiomyopathy (Table 1).
4.4.2 Injury current
In this paper, we used an experimental model analogous to that introduced by Tan and Joyner [19] who coupled isolated cells to a passive resistor-capacitor circuit via a variable conductance. Results obtained by such a technique, however, have some limitations. Firstly, it should be noted that Iinjury was simulated by coupling a real cell to a simple electronic circuit with a potential of 0, –20, or –40 mV. The real cell either represents a normal cell producing action potentials or an abnormal cell producing EADs or DADs. The electronic circuit represents an ischemic region with a depolarized RMP. We are using this experimental setup as a simplification of the complex, inhomogeneous distributions of electrophysiological properties, cell-to-cell coupling conductances, and biochemical changes that have been observed in cardiac tissue at the ‘borderzone’ of regions of myocardial ischemia [for review, see Ref. [5]]. Thus, in our experiments, the structural complexities during myocardial ischemia were eliminated, allowing us to focus on the general phenomenon of how Iinjury affects afterdepolarizations.
Secondly, our electronic circuit was purely passive. Although it was demonstrated that ischemic tissue finally contained inexcitable cells which were depolarized to about –40 mV [20], the ischemic tissue is still able to produce action potentials during the first 8–10 min after onset of ischemia. These action potentials may be delayed so that at the time the non-ischemic cells are repolarized, the ischemic cells are still in plateau phase. Under such conditions, Iinjury may flow from a membrane potential more positive to –40 mV. In our experiments, we simulated these conditions by the electronic circuit with a fixed potential of –20 or 0 mV. Under our experimental conditions, the circuit thus provided inward, depolarizing current for a longer period than would occur by an ischemic action potential. Although the effects of Iinjury simulated by a Vmodel of 0 or –20 mV may thus be exaggerated, we feel that the passive and depolarized electronic circuit is a useful experimental model to study systematically the general phenomenon of how Iinjury affects afterdepolarizations in human ventricular cells, especially late during the acute phase of ischemia when ischemic tissue is inexcitable and depolarized up to –40 mV.
|
5 Conclusion |
|---|
|
TopAbstract1 Introduction2 Materials and methods3 Results4 Discussion5 ConclusionReferences |
|---|
From our results, we conclude that triggered activity based on EADs and DADs does not play a role in phase 1 arrhythmias when Iinjury is flowing from potentials negative to –20 mV. Only when conduction slowing is so prominent that it permits flow of Iinjury from cells at plateau level to cells in phase 3 repolarization it may enhance or induce proarrhythmogenic triggered activity.
Time for primary review 26 days.
|
Acknowledgements |
|---|
We thank Jacques de Bakker, Jessica Vermeulen, Ton Baartscheer, and Cees Schumacher for preparation of single cells and Tobias Opthof for critical reading of the manuscript and valuable comments. This work was supported by grants from the Netherlands Organization for Scientific Research (805-06.154 and 805-06.155).
|
References |
|---|
|
TopAbstract1 Introduction2 Materials and methods3 Results4 Discussion5 ConclusionReferences |
|---|
- 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] - Carmeliet E. Cardiac ionic currents and acute ischemia: from channels to arrhythmias. Physiol Rev (1999) 79:917–1017.
[Abstract/Free Full Text] - Coronel R., Opthof T., Taggart P., Tytgat J., Veldkamp M. Differential electrophysiology of repolarization from clone to clinic. Cardiovasc Res (1997) 33:503–517.
[Free Full Text] - Janse M.J. The premature beat. Cardiovasc Res (1992) 26:89–100.
[Free Full Text] - 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] - 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] - Dekker L.R., Rademaker H., Vermeulen J.T., et al. Cellular uncoupling during ischemia in hypertrophied and failing rabbit ventricular myocardium: effects of preconditioning. Circulation (1998) 97:1724–1730.
[Abstract/Free Full Text] - Smith W.T., Johnson T.A., Engle C.L., Cascio W.E. The Ib 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] - Pogwizd S.M., Corr P.B. Reentrant and nonreentrant mechanisms contribute to arrhythmogenesis during early myocardial ischemia: results using three-dimensional mapping. Circ Res (1987) 61:352–371.
[Abstract/Free Full Text] - Pogwiz 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] - 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.
- Kumar R., Joyner R.W. An experimental model of the production of early after depolarization by injury current from an ischemic region. Pflügers Arch (1994) 428:425–432.[CrossRef][Web of Science][Medline]
- van Ginneken A.C.G., Verkerk A.O., Veldkamp M.W., Bouman L.N. Cellular mechanisms of arrhythmias during acute ischaemia studied in single human and sheep myocytes. Eur Heart J (1998) 19:3330. (Abstract).
- Veldkamp M.W., van Ginneken A.C.G., Opthof T., Bouman L.N. Delayed rectifier channels in human ventricular myocytes. Circulation (1995) 92:3497–3504.
[Abstract/Free Full Text] - World Medical Association. World medical association declaration of Helsinki: recommendations guiding physicians in biomedical research involving human subjects. Cardiovasc Res (1997) 35:2–3.
[Free Full Text] - Ter Welle H.F., Baartscheer A., Fiolet J.W.T., Schumacher C.A. The cytoplasmic free energy of ATP hydrolysis in isolated rod-shaped rat ventricular myocytes. J Mol Cell Cardiol (1988) 20:435–441.[CrossRef][Web of Science][Medline]
- Hamill O., Marty A., Neher E., Sakmann B. Improved patch-clamp techniques for high-resolution current recording from cells and cell-free membrane patches. Pflügers Arch (1981) 391:85–100.[CrossRef][Web of Science][Medline]
- Barry P.H., Lynch J.W. Liquid junction potentials and small cell effects in patch clamp analysis. J Membr Biol (1991) 121:101–117.[CrossRef][Web of Science][Medline]
- 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] - Downar E., Janse M.J., Durrer D. The effect of acute coronary artery occlusion on subepicardial transmembrane potentials in the intact porcine heart. Circulation (1977) 56:217–224.
[Abstract/Free Full Text] - Koumi S.I., Backer C.L., Arentzen C.E. Characterization of inwardly rectifying K+ channel in human cardiac myocytes: alternations in channel behaviour in myocytes isolated from patients with idiopathic dilated cardiomyopathy. Circulation (1995) 92:164–174.
[Abstract/Free Full Text] - January C.T., Riddle J.M. Early afterdepolarizations: mechanism of induction and block: a role for L-type Ca2+ current. Circ Res (1989) 64:563–571.
[Abstract/Free Full Text] - Zeng J., Rudy Y. Early afterdepolarizations in cardiac myocytes: mechanism and rate dependence. Biophys J (1995) 68:949–964.[Web of Science][Medline]
- Priori S.G., Corr P.B. Mechanisms underlying early and delayed afterdepolarizations induced by catecholamines. Am J Physiol (1990) 256:H1796–H1805.
- Riemer T.L., Sobie E.A., Tung L. Stretch-induced changes in arrhythmogenesis and excitability in experimentally based heart cell models. Am J Physiol (1998) 275:H431–H442.[Web of Science][Medline]
- Tan R.C., Osaka T., Joyner R.W. Experimental model of effects on normal tissue of injury current from ischemic region. Circ Res (1991) 69:965–974.
[Abstract/Free Full Text] - Bénitah J.P., Bailly P., D’Agrossa M.C., Da Ponte J.P., Delgrado C., Lorente P. Slow inward current in single cells isolated from adult human ventricles. Pflügers Arch (1992) 421:176–187.[CrossRef][Web of Science][Medline]
- Wilders R., Verheijck E.E., Joyner R.W., et al. Effects of ischemia on discontinuous action potential conduction in hybrid pairs of ventricular cells. Circulation (1999) 99:1623–1629.
[Abstract/Free Full Text] - Beuckelmann D.J., Näbauer M., Erdmann E. Intracellular calcium handling in ventricular myocytes from patients with terminal heart failure. Circulation (1992) 85:1046–1055.
[Abstract/Free Full Text] - Beuckelmann D.J., Näbauer M., Erdmann E. Alternations of K+ currents in isolated human ventricular myocytes from patients with terminal heart failure. Circ Res (1993) 73:379–385.
[Abstract/Free Full Text] - Näbauer M., Kääb S. Potassium channel down-regulation in heart failure. Cardiovasc Res (1998) 37:324–334.
[Abstract/Free Full Text]
CiteULike 
Connotea 
Del.icio.us What's this?
This article has been cited by other articles:
|
|
 |
|
  H. M. Den Ruijter, G. Berecki, A. O. Verkerk, D. Bakker, A. Baartscheer, C. A. Schumacher, C. N.W. Belterman, N. de Jonge, J. W.T. Fiolet, I. A. Brouwer, et al. Acute Administration of Fish Oil Inhibits Triggered Activity in Isolated Myocytes From Rabbits and Patients With Heart Failure Circulation, January 29, 2008; 117(4): 536 - 544. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 R. Wilders Dynamic clamp: a powerful tool in cardiac electrophysiology J. Physiol., October 15, 2006; 576(2): 349 - 359. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 D. J. Huelsing, K. W. Spitzer, and A. E. Pollard Spontaneous activity induced in rabbit Purkinje myocytes during coupling to a depolarized model cell Cardiovasc Res, September 1, 2003; 59(3): 620 - 627. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
K. R Sipido, P. G.A Volders, M. A Vos, and F. Verdonck Altered Na/Ca exchange activity in cardiac hypertrophy and heart failure: a new target for therapy? Cardiovasc Res, March 1, 2002; 53(4): 782 - 805. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M.W Veldkamp, A.O Verkerk, A.C.G van Ginneken, A Baartscheer, C Schumacher, N de Jonge, J.M.T de Bakker, and T Opthof Norepinephrine induces action potential prolongation and early afterdepolarizations in ventricular myocytes isolated from human end-stage failing hearts Eur. Heart J., June 1, 2001; 22(11): 955 - 963. [Abstract] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||