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Am J Physiol Heart Circ Physiol 284: H2091-H2099, 2003. First published January 23, 2003; doi:10.1152/ajpheart.00843.2002
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Vol. 284, Issue 6, H2091-H2099, June 2003

Role of kappa -opioid receptor activation in pharmacological preconditioning of swine

James A. Coles Jr.1,3, Daniel C. Sigg1,2, and Paul A. Iaizzo1,2,3

Departments of 1 Surgery and 2 Physiology and 3 Biomedical Engineering Institute, University of Minnesota, Minneapolis, Minnesota 55455


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Pharmacological preconditioning with kappa -opioid receptor agonists is proarrhythmic and exerts antipreconditioning effects in rats. In swine, it is unknown whether kappa -opioid receptor stimulation plays a role in pharmacological preconditioning. Swine were preconditioned with 1) saline (controls), 2) [D-Ala2,D-Leu5]enkephalin (DADLE), 3) morphine, 4) pentazocine, 5) norbinaltorphimine (nor-BNI), 6) DADLE + nor-BNI, 7) morphine + nor-BNI, or 8) pentazocine + nor-BNI before occlusion (45 min) and reperfusion (180 min) of the left anterior descending coronary artery. Infarct size to area at risk (IS), regional (systolic shortening) and global (pressures and flows) myocardial function, and arrhythmia occurrence were assessed. Only DADLE + nor-BNI preconditioning significantly decreased infarct size compared with controls (47 ± 13 vs. 65 ± 5%, P < 0.05); morphine preconditioning was not cardioprotective with or without kappa -opioid receptor blockade (nor-BNI). DADLE preconditioning significantly increased ischemia-induced arrhythmias relative to controls, whereas pentazocine-preconditioned animals (n = 2) experienced intractable ventricular fibrillation during ischemia. kappa -Opioid receptor blockade with DADLE or pentazocine preconditioning alleviated proarrhythmic effects. These results suggest that kappa -opioid receptor activation during pharmacological preconditioning is proarrhythmic in swine.

morphine; norbinaltorphimine; pentazocine


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

ISCHEMIC PRECONDITIONING (IP) is a biological phenomenon whereby brief ischemic episodes followed by reperfusion protect tissue from a subsequent prolonged ischemic event (24). In the myocardium, IP has been shown to potentially be infarct limiting (24) and antiarrhythmic (17), although the latter of these effects has been disputed. It is also well established that endogenous opioid receptor activation participates in the myocardial IP (38, 40) and that preischemic administration of synthetic opioid agonists can mimic the benefits of IP in a variety of species (6, 37, 42), including isolated human atrial trabeculae (2). Although IP and opioid preconditioning share common signaling mechanisms, namely, activation of protein kinase C (PKC) (14, 19, 52), as well as other kinases (12, 13), and opening of mitochondrial ATP-dependent K+ channels (11, 27, 37, 39), species differences exist in the complex intracellular pathways that mediate preconditioning-induced cardioprotection (45): in rats and rabbits, inhibition of PKC abolished preconditioning-mediated cardioprotection (44); in swine, both PKC and tyrosine kinase must be inhibited (45).

Exogenous activation of the delta -opioid receptor subtype by highly specific agonists before ischemia has been shown to reduce infarct size in a number of species, including rats (36), rabbits (6), and swine (42). Additionally, administration of kappa - or µ-opioid receptor antagonists before IP did not lessen the infarct-sparing benefits of IP in the rat myocardium (36). However, the role of the kappa -opioid receptors in preconditioning has been a subject of much controversy. It has been reported that preischemic administration of selective kappa -agonists will reduce infarct size and ischemia-induced arrhythmias in the isolated rat heart (48). Conversely, specific activation of the kappa -opioid receptor before ischemia has also been shown to increase infarct size (1) and arrhythmias (49) and induce an "antipreconditioning"-like state in rats. More specifically, it has been proposed that the kappa -opioid receptor agonists, specifically U-50488H, exert a biphasic effect on the myocardium, producing pro- and antiarrhythmic effects in the rat (32, 53). Therefore, it has been unclear whether selective or nonselective activation of the kappa -opioid receptor subtype is beneficial during preconditioning, and although such conflicting information exists for the rat, the role of opioid receptor subtypes in IP and pharmacological preconditioning in other species is even more limited.

A recent study from our laboratory demonstrated that preconditioning of swine with specific delta -opioid receptor agonists ([D-Pen2,5]enkephalin and deltorphin-D) significantly reduced infarct size but not ischemia-induced arrhythmias (42). We also observed a significant increase in ischemia-induced arrhythmias with DADLE preconditioning compared with controls, and preliminary evidence suggested potential involvement of kappa -opioid receptors in this arrhythmogenic response. Furthermore, IP has been reported to be proarrhythmic in swine (15, 25, 42), and preischemic administration of naloxone failed to prevent ischemia-induced arrhythmias in this species (3). Therefore, the role of opioid preconditioning in preventing ischemia-induced arrhythmias in swine is unclear.

The aim of the present study was to evaluate the potential cardioprotective effects of pretreatment of swine with clinically relevant opioid agonists known to activate the kappa -opioid receptor, specifically, pentazocine (a kappa - and partial µ-opioid agonist) and morphine (a nonselective opioid agonist). Furthermore, we attempted to clarify the role of kappa -opioid receptor subtype activation in cardioprotection, when preconditioning with DADLE, morphine, or pentazocine, by administration of the specific kappa -opioid antagonist norbinaltorphimine (nor-BNI) before preconditioning. Using a swine acute coronary occlusion model, we determined the effects of these opioid agonists and antagonists on myocardial infarct size, regional and global myocardial functions, and the incidences of lethal and sublethal arrhythmias.


    METHODS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

This study was conducted in accordance with the Guide for the Care and Use of Laboratory Animals [Department of Health and Human Services Publication No. (NIH) 85-23, revised 1985], and the experimental protocol was approved by the Institutional Animal Care and Use Committee of the University of Minnesota.

Surgical preparation. Yorkshire, non-Pietrian swine [37 ± 5 (SE) kg] were sedated with midazolam (2 mg/kg im) and anesthetized with pentobarbital sodium (20 mg/kg iv), and then anesthesia was maintained with a continuous infusion (5-20 mg · kg-1 · h-1). After endotracheal intubation, ventilation (2:1 air-oxygen) was adjusted to maintain an arterial PCO2 of 40 ± 2 mmHg, and core temperature was maintained at 38 ± 0.5°C using convective air warming as needed (Bair Hugger, Augustine Medical, Eden Prairie, MN). Two Mikro-Tip catheter transducers (5-Fr, model MPC-500, Millar Instruments, Houston, TX) were placed via the right carotid artery: one into the ascending aorta and the other into the left ventricle. Bilateral femoral artery cannulas (superficial femoral artery) were inserted for blood pressure monitoring and blood sampling (blood gas analysis, myocardial blood flow). A medial sternotomy was performed, exposing the heart and the major vessels. A four-suture pericardial cradle was used to suspend the heart, and a myocardial thermocouple probe was fixed between the epicardium and pericardium. The left atrial appendage was cannulated for subsequent microsphere and patent blue dye injections. The aortic and left anterior descending (LAD) coronary artery flows were measured via flow probes (Transonic Systems, Ithaca, NY) placed on the ascending aorta and on the LAD coronary artery distal to the planned occlusion site. Two-millimeter ultrasound crystals (Sonometrics, London, ON, Canada) were placed on the end points of the two major axes of the left ventricles (4 crystals), which were used to determine left ventricular volumes and pressure-volume relations. Preload recruitable stroke work was assessed during temporary occlusion of the inferior vena cava. Additionally, regional left ventricular function was estimated by measuring systolic segmental shortening via crystals placed in a linear manner along the anterior surface of the left ventricle, forming four adjacent segments, ~1 cm apart, in the short axis. They were positioned in an array so that the first segment was always located in the center of the area at risk, and the most lateral segment was consistently in the non-area at risk. All data were acquired with Sonosoft software (Sonometrics), and postacquisition analysis was performed using Cardiosoft software (Sonometrics).

In each heart, a 2- to 3-mm segment of the LAD coronary artery was dissected distal to the first diagonal branch for occlusion and placement of the coronary flow probe (see above). The animals were fully heparinized after surgical preparation and throughout the subsequent experimental protocol; animals were given heparin as a bolus (300 IU/kg iv) followed by infusion (67 IU · kg-1 · h-1).

Measurement of infarct size and risk area. On completion of the reperfusion period, the LAD coronary artery was reoccluded, and patent blue dye was injected via the left atrium to differentiate the ischemic area (area at risk) from the nonischemic area (non-area at risk). After being frozen at -20°C overnight, hearts were sliced into 4-mm transverse slices. The slices were then incubated with 1% triphenyltetrazolium chloride in phosphate buffer (pH 7.4) at 37°C for 10 min. Triphenyltetrazolium chloride forms a red formazan derivative on reaction with viable tissue, whereas necrotic tissue appears pale/white once the slices are fixed in 10% formalin. Areas at risk, non-areas at risk, and infarct sizes were assessed using computer-assisted planimetry (ImageTool software, University of Texas Health Science Center, San Antonio, TX); all areas were delineated by a trained individual who was blinded to the treatment protocols.

Regional myocardial blood flow. Regional myocardial blood flow (RMBF) to the area at risk and the non-area at risk was assessed to determine collateral blood flow during ischemia. Colored microspheres (15-µm-diameter blue ultraspheres; E-Z TRAC, Interactive Medical Technologies, Irvine, CA) were injected into the left atrium while a reference blood sample was simultaneously drawn to determine reference blood flow during 30 min of ischemia. Subsequently, the number of microspheres was assessed microscopically from the reference blood samples and the tissues from the areas at risk and at the non-areas at risk. Reference blood flow was calculated as the difference between syringe weights before and after withdrawal, corrected for blood density (1.05 g/ml), and divided by collection time. Routine tissue and blood processing was completed (according to instructions of Interactive Medical Technologies). RMBF was calculated using the following formula: RMBF = Qb × Ct/Cb, where Qb is reference blood flow, Ct is number of microspheres in tissue normalized per gram of wet weight, and Cb is number of microspheres of the blood reference sample (47).

Arrhythmia assessment. A standard peripheral lead electrocardiogram was used to monitor arrhythmias on reperfusion, and analysis was completed using Ponemah Physiology Platform software (version 3.1, Gould Instrument Systems, Valley View, OH). The following modified scoring system was used to quantify arrhythmias by a trained individual who was blinded to the experimental protocol: 0 for <10 premature ventricular contractions (PVCs) in 9 min, 1 for 10-50 PVCs in 9 min, 2 for >50 PVCs in 9 min, 3 for 1 episode of ventricular fibrillation (VF) in 9 min, 4 for 2-5 episodes of VF in 9 min, and 5 for >5 episodes of VF in 9 min (system modified from Refs. 9 and 11).

If and when VF occurred, it was treated by 50-J defibrillation shocks administered via internal paddles, and therapy was repeated until successful. If the animal did not recover a spontaneous atrioventricular rhythm after 1 min of continuous VF, it was considered intractable, and this animal was excluded from the study.

Experimental protocol. The experimental protocol is illustrated in Fig. 1. After completion of the surgery, animals were allowed to stabilize for >= 20 min. The animals were randomly assigned to the following eight groups, which differed only in their preconditioning protocol (preconditioning phase). The control group (n = 6) received an intravenous 0.9% saline injection (10 ml) during the preconditioning phase. The DADLE group (n = 6) received an intravenous injection of DADLE (1 mg/kg), an unspecific delta -opioid agonist, administered over two periods of 10 min (40 and 20 min before coronary occlusion). The morphine group (n = 4) received an injection of morphine sulfate (1 mg/kg iv; Abbott Laboratories, Chicago, IL), which is considered a nonselective opioid receptor agonist; the infusion protocol was the same as that used for the DADLE group. The pentazocine group (n = 2) received an injection of pentazocine lactate (5 mg/kg iv; Talwin, Abbott Laboratories), which is considered to be a kappa -opioid agonist and a partial µ-opioid agonist; the infusion protocol was the same as that used for the DADLE group. The nor-BNI group (n = 4) received an injection of nor-BNI dihydrochloride (1.5 mg/kg iv). This specific kappa -opioid antagonist was administered over a 10-min period, 120 min before saline preconditioning (as described for the control group). The DADLE + nor-BNI group (n = 6) received a 10-min infusion of nor-BNI (1.5 mg/kg iv) 120 min before DADLE (1 mg/kg) preconditioning (as described above). The morphine + nor-BNI group (n = 4) received a 10-min infusion of nor-BNI (1.5 mg/kg iv) 120 min before morphine (1 mg/kg) preconditioning (as described above). The pentazocine + nor-BNI group (n = 2) received a 10-min infusion of nor-BNI (1.5 mg/kg iv) 120 min before pentazocine (5 mg/kg) preconditioning (as described above).


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  		Fig. 1.   Experimental protocol. Study was divided into preconditioning, ischemia, and reperfusion phases. Hemodynamic data were collected at time points indicated (see METHODS). Drugs were infused during preconditioning phase over 10 min, followed by a drug-free interval of 10 min and another infusion of drugs. Arrhythmias were assessed using continuous ECG data obtained during coronary occlusion (ischemia) and during first 45 min of reperfusion (90 min total). Microspheres were injected at 30 min of ischemia to assess regional myocardial blood flow (collateral blood flow) in area at risk. Areas at risk were assessed at the end of the protocol. nor-BNI, norbinaltorphimine; DADLE, [D-Ala2,D-Leu5]enkephalin.

Subsequent to the infusion period in all animals, the LAD coronary artery was occluded for 45 min (ischemia phase) with an arterial occluder (vascular size 2 single clamp, Sklar Instruments, West Chester, PA). Then the LAD coronary artery clamp was removed, and the ischemic myocardium was reperfused for 180 min (reperfusion phase). Hemodynamic data, electrocardiogram analysis, and RMBF were assessed at the indicated time points (Fig. 1).

Data analysis and statistics. Values are means ± SE. Data from all groups were analyzed using repeated-measures ANOVA and Fisher's protected least significant difference test as a post hoc test if significant time-dependent differences were detected within groups. Intragroup comparisons at specific time points and single measurements, such as infarct size, were analyzed using one-way ANOVA and Fisher's protected least significant difference test. All statistical analyses were performed using the Statview 5.0.1 program (SAS Institute, Cary, NC).


    RESULTS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Thirty-seven swine were enrolled in the study; five animals (14%) were excluded because of intractable VF during ischemia (1 control animal and 1 DADLE-, 2 pentazocine-, and 1 morphine-treated animals). No significant differences between animal weights or myocardial temperatures (average myocardial temperature 38.0 ± 0.05°C), total pentobarbital doses, or total fluid administrations were detected between any of the experimental groups.

Infarct size. Infarct size was significantly smaller in animals pretreated with DADLE + nor-BNI than in control animals and animals pretreated with DADLE, morphine, and nor-BNI (P < 0.05; Fig. 2A). The average area at risk of the left ventricle averaged 21.4 ± 0.8% (n = 32) and was not different between groups (Fig. 2B).


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  		Fig. 2.   A: relative infarct size in hearts of control animals and animals preconditioned with DADLE, morphine, nor-BNI, DADLE + nor-BNI, or morphine + nor-BNI. Infarct sizes were significantly reduced in DADLE + nor-BNI group compared with control animals and animals pretreated with DADLE, morphine, and nor-BNI. Protective response in DADLE + nor-BNI animals was varied and considered to be associated with low concentrations of nor-BNI. * P < 0.05 vs. control, DADLE, morphine, and nor-BNI. B: areas at risk in hearts of control animals and animals preconditioned with DADLE, morphine, nor-BNI, DADLE + nor-BNI, or morphine + nor-BNI. No differences were detected between groups. LV, left ventricle. , Mean values.

Hemodynamic findings. Hemodynamic findings are summarized in Table 1. Baseline heart rates were significantly greater in the nor-BNI group than in the control group. However, heart rates before (117 ± 11 beats/min) and 5 min after (120 ± 12 beats/min) nor-BNI infusion were not statistically different from respective control data. During early reperfusion, diastolic relaxation was significantly impaired (increased tau ) in the DADLE-treated animals relative to controls.

                              
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  		Table 1.   Systemic hemodynamics and blood flow in control and opioid-preconditioned groups

RMBF. The average blood flow to the non-area at risk at 30 min of ischemia for all animals (n = 32) was 1.4 ± 0.1 ml · min-1 · g-1, and no significant differences were identified between groups. Additionally, no significant collateral blood flows were detected in any of the animals; the average calculated transmural blood flow to the area at risk during ischemia was <0.02 ml · min-1 · g-1 (n = 32).

Arrhythmia analysis. One control animal (1 of 7) and one DADLE (1 of 7)-, one morphine (1 of 5)-, and both pentazocine (2 of 2)-preconditioned animals were excluded because of intractable VF; it was not necessary to exclude any animals in the nor-BNI, DADLE + nor-BNI, and morphine + nor-BNI groups. The average cumulative arrhythmia scores during ischemia were significantly increased in the DADLE and nor-BNI groups compared with all other groups, except the morphine + nor-BNI group (Fig. 3). There were no detectable differences in arrhythmia scores during reperfusion. The incidence of ischemic PVCs was greatest in DADLE-preconditioned animals (Fig. 4A), whereas the average ischemic episodes of VF were most prevalent in the animals pretreated with nor-BNI alone (Fig. 4B).


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  		Fig. 3.   Cumulative arrhythmia scores during coronary occlusion (ischemia) and first 45 min of reperfusion in control animals and animals preconditioned with DADLE, morphine, nor-BNI, DADLE + nor-BNI, morphine + nor-BNI, and pentazocine (Pentaz) + nor-BNI. Cumulative scores from 2 sets (ischemia and reperfusion) of 5 consecutive 9-min intervals are shown (modified from Refs. 9 and 11). In animals treated with DADLE and nor-BNI, a significantly increased incidence of ventricular arrhythmias was observed during ischemia relative to all groups, except morphine + nor-BNI. Values are means ± SE. * P < 0.05 vs. control, morphine, DADLE + nor-BNI, and Pentaz + nor-BNI.



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  		Fig. 4.   A: incidence of ischemic premature ventricular contractions (PVCs) in control animals and animals preconditioned with DADLE, morphine, nor-BNI, DADLE + nor-BNI, morphine + nor-BNI, and Pentaz + nor-BNI. Animals preconditioned with DADLE exhibited significantly increased PVCs during ischemia; pretreatment of DADLE with prior exposure to nor-BNI (DADLE + nor-BNI) abrogated this proarrhythmic effect. Values are means ± SE. * P < 0.05 vs. control, morphine, DADLE + nor-BNI, and Pentaz + nor-BNI. B: incidence of ventricular fibrillation (VF) during ischemia in control animals and animals preconditioned with DADLE, morphine, nor-BNI, DADLE + nor-BNI, morphine + nor-BNI, and Pentaz + nor-BNI. Animals pretreated with nor-BNI alone displayed a significant increase in incidence of VF but not in occurrence of PVCs relative to control animals during ischemia. Pretreatment of animals with nor-BNI before administration of Pentaz completely removed profibrillatory effects of this kappa -agonist. Values are means ± SE. * P < 0.05 vs. morphine, DADLE + nor-BNI, morphine + nor-BNI, and Pentaz + nor-BNI.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

This study provides evidence that, at analgesic doses, preconditioning by the administration of morphine or pentazocine is not cardioprotective in swine. More specifically, preconditioning with pentazocine, a selective kappa - and partial µ-opioid agonist, exacerbated ischemia-induced arrhythmias, and animals preconditioned with this agent elicited intractable VF within the first 10 min of ischemia. Interestingly, preincubation of pentazocine-preconditioned animals with nor-BNI, a highly selective kappa -opioid antagonist, prevented the incidence of fatal arrhythmias during ischemia. In a previous study from our laboratory, we demonstrated that preconditioning with DADLE, a selective delta -opioid agonist, did not decrease infarct size and increased ischemia-induced arrhythmias (42). As the preliminary data from this previous study suggested, in the present study, kappa -opioid receptor blockade with DADLE preconditioning decreased infarct size and attenuated the proarrhythmic effects of DADLE. However, unspecific kappa -opioid receptor activation was not considered responsible for the lack of cardioprotection seen with morphine, because the coadministration of morphine and nor-BNI failed to decrease infarct size. Moreover, nor-BNI administration alone significantly increased heart rate and left ventricular systolic pressure before ischemia and increased the incidence of ischemia-induced arrhythmias.

Protocol limitations. The advantages and limitations of using the open-chest, anesthetized swine as a model of regional myocardial ischemia and reperfusion have been described previously (42, 46). One must be careful when extrapolating these results to humans, inasmuch as species differences exist in the opioid receptor subtype and intracellular mechanisms involved in pharmacological preconditioning (45). Additionally, there are fundamental differences in opioid receptor expression and functional binding affinities between species (21).

The dose and timing of nor-BNI administration were based on published observations in mice (5, 29) and from suggestions through personal communications (Dr. Philip Portoghese, University of Minnesota). An incubation period of 120 min was chosen because of the slow kinetics of nor-BNI binding to the kappa -opioid receptor (5, 29). Importantly, previous preconditioning studies utilizing nor-BNI have employed relatively short incubation periods (<15 min), which may have influenced reported results. However, 1.5 mg/kg iv is considered a relatively low dose of nor-BNI.

The dose of DADLE was based on published observations where it was found that 1 mg/kg iv induced hypoxic tolerance in rats (11), dogs (7), and swine (41). Although DADLE is considered a delta -opioid agonist, it has been shown to bind to delta - and kappa -opioid receptors at micromolar concentrations and to antagonize the kappa -opioid receptor at higher concentrations (>5 µM) (55). Additionally, the kappa -opioid-binding site is believed to be nonselectively activated when exposed to high concentrations of delta - or µ-selective ligands (28).

Infarct size. A significant reduction of infarct size relative to controls was observed only in animals preconditioned with DADLE + nor-BNI. The finding that neither morphine nor morphine + nor-BNI pretreatments elicited any infarct-limiting ability was unexpected. Previously, Aitchison et al. (1) demonstrated that administration of micromolar concentrations of DADLE to isolated rat hearts increased infarct size relative to lower doses (nanomolar) of DADLE and IP. Additionally, they observed that coadministration of DADLE and the delta -receptor antagonist naltrindole increased infarct size relative to controls, whereas animals treated with DADLE + nor-BNI exhibited decreased infarct size (1). In the same study, preconditioning with bremazocine, a kappa -opioid agonist, increased infarct size relative to controls (1). Similar to the study of Aitchison et al., the present study suggests that nonselective kappa -opioid receptor activation exerts an antipreconditioning-like effect in swine. However, in this study, because of the early onset of fatal arrhythmias in animals preconditioned with pentazocine, we are not able to discern whether direct kappa -receptor activation has an effect on infarct size in this model.

Although morphine is commonly described as mainly a µ-opioid receptor agonist, it can reportedly also interact with the delta - and kappa -opioid receptor subtypes (10, 28). Morphine has been shown to mimic preconditioning in isolated cardiomyocytes via opening of K+-dependent ATP channels (18, 22). Morphine preconditioning reduced infarct size in rabbits, but only when supraclinical doses were used (3 mg/kg) (23). Yet, when this same dose was administered to rats 10 min before permanent LAD coronary artery occlusion, infarct size increased relative to controls (20). Conversely, morphine preconditioning in rats at 0.3 mg/kg decreased infarct size (37). We speculated that this discrepancy in the rat might be explained by activation of the kappa -opioid receptor at higher concentrations of morphine. On the basis of this speculation and the positive results obtained with DADLE + nor-BNI, we hypothesized that the lack of cardioprotection found with morphine in swine was due to nonselective activation of kappa -opioid receptors. However, administration of morphine + nor-BNI was unsuccessful in reducing infarct size in swine. Because it was previously stated that a relatively low dose of nor-BNI was employed in this study, we are investigating the potential effects of pretreating morphine-preconditioned animals with a higher dose (5 mg/kg) of nor-BNI.

Hemodynamic effects. Although we previously demonstrated that preconditioning with specific delta -opioid agonists significantly decreased infarct size in swine, there was no difference in regional or global functional recovery between opioid-preconditioned and control animals after 180 min of reperfusion (42). Similarly, it was reported that swine subjected to IP demonstrated significant differences in infarct size, but not functional recovery, after regional ischemia and 90 min of reperfusion (35). Furthermore, a significant difference in functional recovery between rabbits subjected to IP and control rabbits was only observed after 72 h of reperfusion (8). Qiu et al. (34) reported a >50% recovery of regional function (wall thickening) during early reperfusion with IP in swine; however, it is unclear whether the unique preconditioning protocol they used (10 episodes of 2 min of ischemia followed by reperfusion) influenced these results. Previous studies (26), along with the data displayed in Table 1, suggest that neither IP nor pharmacological preconditioning prevents myocardial stunning after ischemia and reperfusion and that reperfusion-induced stunning may mask the functional benefits of preconditioning in acute coronary occlusion protocols.

Unfortunately, baseline regional systolic shortening data were not collected before nor-BNI administration (i.e., 120 min before baseline), inasmuch as previous preconditioning studies in rats suggested that the antagonist would not have a marked hemodynamic effect (36, 48). Therefore, we are unable to speculate whether the depressed regional contractilities at baseline (Table 1) in nor-BNI-treated animals were due to the location of the segment or the antagonist itself.

Arrhythmias. A biphasic cardiovascular response to exogenous kappa -opioid administration has been previously described (30). Specifically, in rats, U-50488H, a kappa -opioid agonist, was reported to be proarrhythmic at a low dose (49) and antiarrhythmic at a high dose (31). This phenomenon may be attributed to the ability of micromolar concentrations of U-50488H to block cardiac Na+ and/or K+ channels (30, 33) and, hence, increase VF thresholds (31). It has been proposed that the antiarrhythmic effects of IP may be due to a decreased binding of endogenous kappa -opioid peptides, thereby increasing the threshold for VF (50). However, higher doses of kappa -opioid agonists (40-50 µM) also induced arrhythmias in rats, possibly via increased myocardial intracellular calcium concentrations and oscillations (54). Nevertheless, although many of these studies have examined the antiarrhythmic effects of kappa -opioid agonists in rats, the electrophysiological response of rats to antiarrhythmic therapies may differ from that of swine (3, 4).

In swine, phase 1b arrhythmias, which are associated with a high mortality, occur 15-60 min after LAD coronary artery occlusion (43). Although this may be due to catecholamine release from nerve endings in the ischemic area at risk (16), it also has been proposed that, during ischemia, endogenous kappa -opioid peptides are released, thereby inhibiting beta -adrenoceptor stimulation, decreasing arrhythmias, and increasing the threshold for VF (51, 54). We observed an increase in mean arrhythmia scores in animals preconditioned with nor-BNI, a kappa -opioid antagonist (Fig. 3), that was attributed to an increase in the mean incidences of VF during ischemia (Fig. 4B). Therefore, it is possible that the proarrhythmic effects of nor-BNI may be due to inhibition of endogenous kappa -peptide binding and, hence, increased beta -adrenergic stimulation during ischemia, resulting in increased phase 1b arrhythmias and VF.

Finally, Wang et al. (48) suggested that kappa - and not delta -opioids are involved in antiarrhythmic benefits of IP in rats. In the present study, we observed intractable VF in animals preconditioned with a kappa -opioid agonist (pentazocine), whereas animals treated with nor-BNI + pentazocine before ischemia did not exhibit any episodes of VF (Fig. 4B). Additionally, inhibition of kappa -opioid receptor binding with DADLE preconditioning decreased arrhythmia scores and total PVCs during ischemia (Fig. 4A), further suggesting a proarrhythmic role of exogenous kappa -opioids in swine.

In summary, this study demonstrated that neither pentazocine nor morphine was cardioprotective in swine. Furthermore, an increase in fatal arrhythmias was observed with pentazocine, and this proarrhythmic effect was abolished with kappa -opioid receptor blockade. Also important was the observation that blockade of endogenous kappa -opioid binding (with nor-BNI) was proarrhythmic during ischemia. Additionally, although nor-BNI administration decreased infarct size in DADLE-preconditioned animals, kappa -opioid receptor activation was not the explanation for the lack of cardioprotection observed with morphine. Collectively, these results suggest that the exogenous activation of kappa -opioid receptors before ischemia exerts an antipreconditioning effect in swine. Finally, with a limited availability of specific opioid receptor subtype agonists and the potential nonselective coactivation of multiple opioid receptor subtypes with commonly used opioid anesthetics (28), it is important to continue to strive for better understanding and delineation of the coactivation of multiple opioid receptor subtypes and their roles in pharmacological preconditioning.


    ACKNOWLEDGEMENTS

The authors thank Kristy Schaffer, Grant Beckstrand, Anna Lindlief, Charles Soule, and William Gallagher for technical support and Monica Mahre for editorial assistance.


    FOOTNOTES

This research was supported by funding from the Lillehei Heart Institute and Medtronic.

Address for reprint requests and other correspondence: P. A. Iaizzo, Dept. of Surgery, University of Minnesota, 420 Delaware St. SE, MMC 107 UMHC, Minneapolis, MN 55455 (E-mail: iaizz001{at}tc.umn.edu).

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.

First published January 23, 2003;10.1152/ajpheart.00843.2002

Received 27 September 2002; accepted in final form 20 January 2003.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
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Am J Physiol Heart Circ Physiol 284(6):H2091-H2099
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