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1Department of Internal Medicine, Carver College of Medicine, University of Iowa, and Veterans Affairs Medical Center, Iowa City, Iowa; and 2Zealand Pharma, Glostrup, Denmark
Submitted 19 July 2004 ; accepted in final form 4 October 2004
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ABSTRACT |
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-chloralose-anesthetized, open-chest dogs 1–4 h after coronary artery occlusion. Three-dimensional activation mapping was done using 6 bipolar electrograms on each of 23 multipolar needles in the risk zone. Dogs were randomly assigned to receive either saline or ZP123 cumulatively at three dose levels (an intravenous bolus followed by a 30-min infusion per dose). Attempts to induce VT were repeated in each dose. Mass spectrometry was used to measure plasma ZP123 concentrations. Standard microelectrode techniques were used for in vitro study of DADs and TA. Twenty-six dogs with focal VT were included. ZP123 did not affect the inducibility of focal VT at any plasma concentrations vs. saline (0.8 ± 0.1 nM, 77 vs. 75%; 7.8 ± 0.4 nM, 86 vs. 77%; and 78.8 ± 5.0 nM, 77 vs. 91%). In vitro, ZP123 did not affect the induction of DADs (12/12) and TAs (10/10) in ischemic tissues or tissue removed from the origin of focal VT (DADs, 8/8; TAs, 4/4). Therefore, although indirect, the data with the doses and concentrations used suggest that GJIC may not play a major role in the genesis of focal activity in the ischemic models studied.
gap junction intercellular communication; myocardial; delayed afterdepolarizations
We have developed a highly stable, antiarrhythmic peptide analog called ZP123 (Ac-D-Tyr-D-Pro-D-Hyp-Gly-D-Ala-Gly-NH2) that increases GJIC (17), prevents conduction slowing during acidosis (5), and reduces action potential (AP) dispersion (5, 8). Previously, we demonstrated that ZP123 prevents the induction of epicardial reentry VT during coronary artery occlusion by preventing unidirectional conduction block (17). In the present experiments, we used ZP123 as a tool to test the opposing hypothesis predicted from the simulation studies, that increased GJIC would 1) either prevent or promote the induction of focal VT during myocardial ischemia in intact dogs, and 2) suppress or facilitate induction of delayed afterdepolarizations (DADs) and triggered activities (TAs) in vitro in endocardial sites removed from ischemic areas.
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METHODS |
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Surgical preparation.
Dogs were pretreated with injections of 0.05 ml/kg im acepromazine maleate (10 mg/ml) mixed with 0.1 ml/kg im ketamine hydrochloride (100 mg/ml). Anesthesia was induced by administration of 0.4 ml/kg iv thiopental sodium (25 mg/ml) followed by an -chloralose (150 mg/kg) bolus. Anesthesia was maintained by constant infusion of -chloralose (8 mg·kg–1·h–1 dissolved in saline and polyethylene glycol, mol wt 200). The dogs were intubated and mechanically ventilated on a volume-cycled respirator (Harvard) to maintain a PO2 of 80–110 mmHg, a PCO2 of 35–45 mmHg, and a pH of 7.35–7.45 (Radiometer; Copenhagen, Denmark). The femoral vein and artery were cannulated for administration of fluid and drugs and for continuous measurement of mean arterial blood pressure (MAP), respectively.
The pericardium was incised through a midline sternotomy and sutured to the wound edges to form a pericardial support for the heart. A silk suture was passed under the left anterior descending (LAD) coronary artery 1 cm from the left atrial appendage. The suture was threaded in tubing to form a snare for coronary artery occlusion. As opposed to our previous study on the role of GJIC in reentry VT (17), no visible collateral vessels were ligated in this study. Epicardial temperature was maintained at 
37°C by an infrared heating lamp and a plastic sheet draped over the sternotomy. Warm saline was applied to the heart intermittently to prevent surface drying.
Electrophysiological preparation.
Surface ECG leads were recorded continuously. The sinus node was permanently clamped. The atria were stimulated at twice diastolic threshold with pulses of 2-ms duration with a bipolar electrode at a cycle length (CL) of 300 ms. To record transmural signals, 23 16-pole plunge-needle electrodes (J. Kassell; Fayetteville, NC) were inserted into the myocardium in and surrounding the risk zone of the LAD artery as previously reported (1–3). The interneedle distance was 1 cm depending on individual coronary artery anatomy.
Bipolar electrograms were recorded from 
6 different sites on each 16-pole electrode (1). To maximize the capability of recording from Purkinje fibers, noise-free bipolar signals were chosen by sequential recording on a storage oscilloscope of each successive bipole. Purkinje, endocardial, midwall, and epicardial electrograms were chosen as previously described (3). Electrograms were recorded simultaneously on two computers, and three-dimensional (3-D) activation maps were developed (1, 2).
Experimental protocol. Blood gas values and adequate anesthesia levels were confirmed before LAD artery occlusion. The effective refractory period (ERP) was determined, and VT was induced by programmed electrical stimulation using up to four premature stimuli (S2-S5; Refs. 1, 2, 17). Pacing protocols were started 1 h after occlusion. Previous studies (2, 17) using this model have demonstrated that VT may be reproducibly induced over this period. Sustained VTs were usually terminated by pacing or were cardioverted with epicardial shocks of 10–20 J. Repeat testing was done from apical, septal, or lateral left ventricular pacing sites every 20 min and was continued until the successful induction of two episodes of VT with similar surface-lead morphology. If VTs with similar morphology could not be induced twice within 3 h after occlusion, the experiment was terminated. In dogs in which VT was reproducibly induced, ZP123 or saline was given randomly and cumulatively as a bolus (11 x 10–7, 11 x 10–6, or 11 x 10 –5 g/kg iv) followed by a 30-min infusion (2.1 x 10–9, 2.1 x 10 –8, and 2.1 x 10–7 g·min–1·kg–1 iv, respectively) (17). At 20 min into each infusion, extrastimulus testing was performed from the same pacing site(s), which induced VT during the control period. A blood sample was taken at the end of each infusion period for later determination of plasma ZP123 concentration.
Intracellular recording techniques.
After 3-D activation mapping was performed, one focal origin of VT was located, and the heart was excised and placed in Tyrode solution with the following composition (in mM): 125 NaCl, 24 NaHCO3, 4.5 KCl, 1.8 CaCl2, 0.5 MgCl2, 0.25 NaHPO4, and 5.5 dextrose (pH 7.4). Endocardial focal tissue blocks (4 x 3 x 2 mm) were removed from the area of the mapping electrode that recorded the focus and were pinned in a 3-ml tissue bath (Warner Instruments). The tissue was superfused with Tyrode solution (37°C) at a rate of 9 ml/min. Fibers were stimulated with a bipolar electrode at twice diastolic threshold. AP measurements were performed during pacing at 1.5–5 Hz. The most superficial cells were impaled with 3 M KCl-filled glass capillary microelectrodes with tip resistances of 3–20 M
. Microelectrodes were connected to a high-input impedance preamplifier (Axoclamp-2A; Axon Instruments; Foster City, CA). The bath was grounded with an Ag-AgCl pellet. Potentials were recorded and stored on a computer with the use of commercial software (Axon Instruments) with data filtered at 1 kHz and sampled at 2 kHz. Zero offsets were recorded to correct for drift. Purkinje cells were identified by spontaneous phase 4 depolarization or automaticity at CL > 1 s. If there were no DADs or TAs inducible, isoproterenol (0.5 µM) was added. Tissues were first assessed for the presence or absence of DADs and TAs before and during isoproterenol superfusion. ZP123 was then added to the superfusate at 1, 10, and 100 nM concentrations to determine the effects of the drug on DADs and TAs. Washout of the drug and isoproterenol was also performed.
Definitions and analysis. Mapping of electrograms was done offline as previously described (1, 2, 17). VT was defined as at least five consecutive nonstimulated ventricular complexes, and sustained VT was defined as VT that did not self-terminate within 10 s; most episodes of VT were pace terminated. Ventricular fibrillation (VF) was defined as an irregular ventricular rhythm that resulted in hemodynamic collapse and required a shock to terminate. The CL of each VT episode was measured by averaging the first (up to 10) CLs. VT mechanisms were defined as follows: reentrant VT occurred when the electrode recording the earliest activity was immediately adjacent to the site of the latest activation from the previous complex, and diastolic activity was recorded between complexes. Focal VT occurred when the electrode recording the site of origin (SOO) was surrounded on six sides by other electrodes within 1–2 cm that recorded progressive and gradually later activity while moving away from the SOO. There was no late (>50% cycle) electrical activity on adjacent sites. Ischemia was defined by voltage reduction as in our prior report (1).
Depolarizations occurring after phase 3 of paced APs were defined as DADs. Nonpaced APs occurring at the peak of a DAD showing overdrive stimulation were defined as TAs.
ZP123 assay. The plasma concentration of ZP123 was measured by a combined liquid chromatography and mass spectrometry analysis of samples after solid-phase extraction as previously described (8).
Statistical analysis. The effect of ZP123 on the incidence of VT, DADs, and TAs was analyzed by a two-tailed Fisher's exact test at each dose level. The effects of ZP123 on VT CL, ERP, and MAP were analyzed by a two-way ANOVA with repeated measurements. P < 0.05 was considered statistically significant. All values are means ± SE.
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RESULTS |
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In vitro experiments in previously ischemic endocardium. ZP123 did not affect AP characteristics (Table 3), which is consistent with the lack of effect on membrane currents reported previously (5, 17). Also, ZP123 did not prevent the induction of DADs (10 of 12 dogs) and TAs (8 of 10 dogs) in ischemic tissues; in two preparations, DADs and repetitive TAs appeared to be reversibly blocked. Moreover, in those tissues, which did not have TAs, ZP123 did not facilitate the induction nor was abnormal automaticity exposed. Normal automaticity was uncovered in five preparations not observed before ZP123 was superfused, and the CLs ranged from 4.4 to 9 s; however, no early afterdepolarizations (EADs) were observed at these CLs. In eight experiments, we were able to remove tissue from the SOO of focal VT and record APs and responses to stimuli and isoproterenol. We saw no inhibition of induction of DADs (8 of 8 dogs), or TAs (4 of 4 dogs; Fig. 3). In addition, we also saw no facilitation of TAs in the four tissues with DADs alone.
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DISCUSSION |
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Our aim was to determine whether increased GJIC during ischemia would facilitate or suppress focal activity in an in vivo model of focal VT induced by programmed electrical stimulation in open-chest dogs 1–4 h after coronary artery occlusion and in Purkinje cells from ischemic myocardium. Computer simulations show that at very low levels of GJIC (i.e., high resistance), automaticity is facilitated but the impulse fails to propagate, whereas at high levels of GJIC, the loading effects of the surrounding cells suppress automaticity (6, 16, 18). However, simulations predict a window of intermediate GJIC where spontaneous ectopic activity can occur and may propagate to the surrounding cells and thereby initiate a focal arrhythmia (12, 16). Based on these findings, it is difficult to hypothesize what effect a compound that improves coupling during ischemia will have on focal activity. Moreover, impulse propagation is dependent on the degree of anisotropy with the chance of propagation being higher in anisotropic tissue, because the impulse is focused in a certain direction, which allows neighboring cells to reach threshold (16). According to this, conditions such as ischemia and infarction that may increase the anisotropic ratio would facilitate focal arrhythmias. Eloff et al. (5) recently showed that the anisotropic ratio is increased during acidosis in isolated, perfused guinea pig hearts due to a more severe slowing of conduction velocity in the transverse than the longitudinal direction. They also showed that 80 nM ZP123 was able to prevent the conduction velocity slowing and the increased anisotropy during acidosis, which suggests that ZP123 may prevent facilitation of impulse propagation by limiting anisotropy (5). However, the present study showed that ZP123 neither facilitated nor suppressed focal activity. There was no effect of ZP123 on the inducibility of focal VT or the number of ectopic foci during VT in dogs 1–4 h after LAD artery occlusion. However, although our conclusions are perhaps applicable to a clinical pathophysiological substrate, they do not exclude other models with different doses and concentrations that produce greater effects on GJIC from showing an effect.
Previously, we investigated the effects of ZP123 and another synthetic antiarrhythmic peptide analog, AAP10, on the time to VF during ouabain infusion (8). In this model, VF is likely caused by the formation of DADs and TAs associated with the high intracellular Ca2+ levels that result from ouabain infusion. ZP123 and AAP10 failed to dose dependently prevent VF, although both compounds significantly delayed the time until second-degree atrioventricular block, which is an indirect measure of prevention of Ca2+-induced uncoupling of gap junction channels. Thus the lack of effect of ZP123 on ouabain-induced VF in the murine study suggested a lack of effect of increased GJIC on DADs and focal VT. By looking directly at focal VT with 3-D mapping, we have in the present study confirmed this suggested lack of effect. Thus contrary to what may be predicted from computer simulation studies, ZP123 neither suppressed automaticity nor facilitated propagation. Previously we showed that ZP123 at the same doses as used in the present study prevented reentrant VT at all doses in the ischemic dog model of programmed electrical stimulation-induced VT (17). Therefore, the lack of effect of ZP123 on focal VT cannot be ascribed to the use of ineffective doses of the compound. Consistent with our finding that ZP123 does not promote focal arrhythmias during ischemia, we did not see an increase in focal activity after reentrant VT was blocked in our previous study (17).
In the present study, we also investigated the effects of ZP123 on DADs and TAs in vitro in tissues removed from ischemic endocardial sites, some of which were at the SOO of a focal VT in vivo. We found that these events were neither facilitated nor clearly blocked by ZP123, which thus explains the lack of effect of ZP123 on ouabain-induced VF that was previously seen (8). Only very slow Purkinje automaticity occurring at normal resting membrane potentials appeared with ZP123 superfusion.
Several studies have shown that when normal, isolated ventricular cardiomyocytes or Purkinje cells are coupled to a passive resistance circuit to simulate ischemic cells with depolarized resting membrane potentials, EADs form in the real cell (6, 14). When the coupling conductance is increased, the EADs disappear. In cells in which EADs were induced by norepinephrine, the EADs could be suppressed by coupling the cell to a simulated cell with nearly normal resting membrane potential; however, when the cell was coupled to a partly depolarized simulated cell, the cell generated multiple EADs instead of a single EAD (14). From these simulations, it seems that the effect of coupling is highly related to the state of the coupled cells. In the present study, although the tissue was obtained from ischemic endocardial sites, the resting membrane potentials were nearly normal during measurements, and EADs were not observed. The reason that these cells were able to withstand the prolonged ischemia may be related to their location in close proximity to the cavity, where they received some oxygen and nutrients from the cavity blood. Also, the tissue was allowed to equilibrate in Tyrode solution for at least 30 min before recordings were obtained.
As expected from previous studies with ZP123 and other antiarrhythmic peptides (4, 5, 7–10) and in line with its selective effect on GJIC, there was no effect of ZP123 on any of the AP parameters measured during the in vitro study, and there was no effect on ERP or MAP in vivo. However, we cannot exclude that changing parameters of measurement such as the interval of electrodes could show significantly different effects on GJIC.
In summary, the present study showed that ZP123 did not affect automaticity or propagation of focal activity either in vivo in dogs 1–4 h after LAD artery occlusion or in vitro in tissue from ischemic endocardium. New gap junction-modifying compounds like ZP123, which show promising potential for the prevention of reentrant VT, may not prevent focal VT. Whether this difference was due to improved conduction caused by increased GJIC is speculative as it is based in indirect observations with limited apparent effect produced. Because the relative importance of focal and reentrant mechanisms in ischemia-induced life-threatening ventricular tachyarrhythmias is unknown, clinical trials with ZP123 may provide important information to address this fundamental question.
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FOOTNOTES |
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The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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REFERENCES |
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2-adrenoceptors in Purkinje? Am J Physiol Heart Circ Physiol 280: H1182–H1190, 2001.-- and 2-adrenergic influences. Am J Physiol Heart Circ Physiol 267: H376–H382, 1994.This article has been cited by other articles:
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 J.-q. Zhong, G. Laurent, P. P.-S. So, Xudong Hu, J. K. Hennan, and P. Dorian Effects of Rotigaptide, a Gap Junction Modifier, on Defibrillation Energy and Resuscitation From Cardiac Arrest in Rabbits Journal of Cardiovascular Pharmacology and Therapeutics, March 1, 2007; 12(1): 69 - 77. [Abstract] [PDF]
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J. M. Guerra, T. H. Everett IV, K. W. Lee, E. Wilson, and J. E. Olgin Effects of the Gap Junction Modifier Rotigaptide (ZP123) on Atrial Conduction and Vulnerability to Atrial Fibrillation Circulation, July 11, 2006; 114(2): 110 - 118. [Abstract] [Full Text] [PDF]
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H. V.M. van Rijen, J. M.T. de Bakker, and T. A.B. van Veen Hypoxia, electrical uncoupling, and conduction slowing: Role of conduction reserve Cardiovasc Res, April 1, 2005; 66(1): 9 - 11. [Full Text] [PDF]
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