Myocardial binding studies demonstrate that κ- and δ-opioid receptors (KORs and DORs, respectively) are present on adult ventricular myocytes of rats and humans, 1,2 while μ-opioid receptors are absent.
Schultz et al 3 first reported that activation of opioid receptors may induce acute cardioprotection. Following these initial studies, exogenous opioids have been shown to afford cardioprotection against both stunning and infarction. 4,5 Similarly, it has been reported that endogenous opioids have a role in the triggering and maintenance of both early and delayed preconditioning against myocardial infarction.
Opioid antagonists, both non-selective and selective for the DOR, have been demonstrated to attenuate the cardioprotective effects of ischemic preconditioning (IPC) in both rat and rabbit hearts. 6,7 Moreover, subtype selective δ1 opioid receptor (D1OR) agonists have been shown to provide pronounced cardioprotective actions in both intact animals and isolated myocytes via activation of Gi/o proteins, protein kinase C, and the mitochondrial KATP channel. 5,8 The cardioprotective effects provided by DOR activation manifest immediately following receptor activation and reappear 24 to 48 hours posttreatment in rats and rabbits. 9,10
While it is generally accepted that DOR activation affords cardioprotection, data regarding the KOR are conflicting with many papers reporting cardioprotection from KOR activation while others report no effect or protection after KOR blockade. While KOR activation appears not to be involved in acute IPC, recent data suggest that activation of the KOR is indeed protective in an isolated rat model. 11 Furthermore, treatment with the KOR agonist U50,488 leads to a preconditioning-like effect in isolated myocytes. 12 However, Huh et al 13 showed no protection via KOR in isolated chick myocytes while Aitchison et al 14 demonstrated that stimulation of KOR augmented infarct development. Indeed the data provided by Aitchison et al are supported by several earlier reports examining the arrhythmogenic effects of KOR agonists in which these compounds were shown to exacerbate ischemia/reperfusion-induced arrhythmias while KOR antagonists were found to attenuate arrhythmogenesis. Furthermore, it has been suggested that some of the actions of KOR agonists are due to direct effects on ion channels, independent of KOR activation.
Therefore, in light of the controversies surrounding the effects of KOR activation in the ischemic myocardium, the goal of this study was to examine and compare the effects of 3 selective KOR agonists, U50,488, ICI 204,448, and BRL 53527, on infarct size and the incidence of arrhythmias in an intact rat model of myocardial infarction.
This study was performed in accordance with the guidelines of the Animal Care Committee of the Medical College of Wisconsin, which is accredited by the American Association of Laboratory Animal Care.
General Surgical Preparation
Male Sprague-Dawley rats weighing 250 to 350 g were used throughout this study. The rats were anesthetized via intraperitoneal administration of thiobutabarbital sodium (Inactin; 100 mg/kg). A tracheotomy was performed, and the trachea was intubated with a cannula connected to a rodent ventilator (model CIV-101, Columbus Instruments: Columbus, OH, or model 683, Harvard Apparatus: Natick, MA). The rats were ventilated with room air supplemented with 100% O2. Atelectasis was prevented by maintaining a positive end-expiratory pressure of 5 to 10 mm H2O. Arterial pH, PCO2, and PO2 were monitored throughout the protocol by a blood gas analyzer (AVL 995, pH/Blood gas analyzer) and maintained within a normal physiological range (pH, 7.35–7.45; PCO2, 25–40 mm Hg; and PO2, 80–110 mm Hg) by adjusting the respiratory rate and/or tidal volume. Body temperature was maintained at 37°C by the use of a heating pad.
The right carotid artery was cannulated with polyethylene tubing PE-50 to measure blood pressure and heart rate (HR) via a PE-23 (Gould) pressure transducer connected to a polygraph (model 7, Grass). The right jugular vein was cannulated for delivery of saline or drug infusion. A left thoracotomy was performed at the fifth intercostal space, followed by a pericardiotomy and adjustment of the left atrial appendage to reveal the location of the left coronary artery. A ligature (6-0 Prolene) was passed below the left descending vein and coronary artery from the area immediately below the left atrial appendage to the right portion of the LV. The ends of the suture were threaded through a propylene tube to form a snare. Occlusion of the coronary artery and subsequent regional LV ischemia was produced by pulling the ends of the suture taut and clamping the snare onto the epicardial surface with a hemostat. Epicardial cyanosis and a subsequent decrease in blood pressure verified coronary artery occlusion. Reperfusion of the heart was initiated via unclamping the hemostat and relieving tension from the snare and was confirmed by visualizing a marked epicardial hyperemic response. HR and blood pressure were allowed to stabilize before initiation of experimental protocols.
Determination of Infarct Size
On completion of the experimental protocols, the coronary artery was reoccluded, and the area at risk (AAR) was determined by negative staining. Patent blue dye was administered via the jugular vein to effectively stain the nonoccluded area of the LV. The heart was excised, and the LV was removed from the remaining tissue and subsequently cut into 5 thin cross-sectional pieces. This allowed for the delineation of the normal area, which stained blue, versus the AAR, which subsequently remained pink. The AAR was excised from the nonischemic area, and the tissues were placed in separate vials and incubated for 15 minutes in a 1% TTC solution in 100 mM phosphate buffer (pH 7.4) at 37°C. Tissues were stored in vials of 10% formaldehyde overnight and the infarcted myocardium was carefully dissected from the AAR by an independent observer under the illumination of a dissecting microscope (Cambridge Instruments). Infarct size (IS) and area at risk (AAR) were determined by gravimetric analysis. IS was expressed as a percentage of the AAR (IS/AAR).
Arrhythmic properties of various KOR agonists in the absence or presence of the KOR antagonist, nor-BNI, were also analyzed. Arrhythmias were quantitated via a modified scoring system previously validated by Curtis and Walker 15 in an in vivo model of ischemia. The scoring system employed assigned scores during ten 3-minute intervals of myocardial occlusion. Arrhythmias were scored as follows: 0 = < 10 premature ventricular contraction (PVCs)/3-minute period; 1 = 10 to 50 PCVs/3-minute period; 2 = > 50 PVCs/3-minute period; 3 = 1 episode of ventricular fibrillation (VF)/3-minute period; 4 = 2 to 5 episodes of VF/3-minute period, and 5 = > 4 episodes of VF/3-minute period.
Following equilibration of hemodynamic parameters, rats were divided into various experimental groups. Rats were either untreated, treated with agonist alone, antagonist alone, or treated with both agonists and antagonists. Agonists were given as a bolus dose 10 minutes before the onset of ischemia, whereas antagonists were given 20 minutes before ischemia. Following treatment, hearts were occluded for 30 minutes followed by 90 minutes of reperfusion before TTC staining. Since most previous studies concerning the cardioprotective effects of KOR stimulation were performed with U50,488, we included 2 additional KOR agonists to ascertain that other KOR agonists shared the cardioprotective effects previously demonstrated with U50,488. Thus, a peripherally active KOR agonist, ICI204,448, which does not cross the blood brain barrier, and a systemically active KOR agonist, BRL53527, which does cross the blood brain barrier, were used. In addition, the selective DOR agonist, BW373U86, was used for comparison purposes.
Rats were excluded from data analysis if they exhibited severe hypotension (<30 mm Hg systolic pressure) or if we were unable to maintain adequate blood gas values within a normal physiological range due to metabolic acidosis. Exclusion of animals from the present study was evenly distributed among protocol groups.
Statistical Analysis of Data
All values are expressed as mean ± SEM. One-way analysis of variance with Newman-Keuls post hoc test was used to determine whether any significant differences existed in any parameter between groups. Significant differences were determined at P < 0.05.
The hemodynamic data are summarized in Table 1. No differences from untreated rats were observed at baseline for any parameter. During the 30 minutes of coronary occlusion, both BW373U86 and ICI + nor-BNI treated groups displayed a significantly higher mean arterial pressure (MAP). At the completion of the reperfusion period, the BW373U86-treated rats still exhibited an elevated MAP, an effect that was not attenuated by the selective DOR-antagonist, BNTX.
No differences were noted in the ratio of left-ventricle mass to area at risk between groups. Activation of the DOR following pretreatment with 1 mg/kg BW373U86 markedly reduced infarct size from 52.4 ± 2.7% for untreated rats to 37.2 ± 1.8% (Fig. 1). This protection was completely reversed by 1 mg/kg of the selective δ1 opioid receptor antagonist, BNTX. BNTX alone had no effect on infarct size.
KOR activation with U50,488 (0.1 mg/kg), ICI 204,448 (0.3 mg/kg), and BRL 52537 (0.5 mg/kg) all significantly reduced infarct development to 38.5 ± 2.6%, 34.9 ± 3.1%, and 41.6 ± 1.7%, respectively (Fig. 2). Pretreatment with the selective KOR antagonist, nor-BNI (0.1 mg/kg), abolished the infarct-reducing effects of U50,488 and ICI 204,448; however, it failed to attenuate the infarct-sparing effect of BRL (37.3 ± 3.9%). Nor-BNI had no effect on infarct size when administered alone.
Incidence of Arrhythmias
Arrhythmia scores for the 30-minute occlusion period are presented in Figure 3. U50,488 and BRL 52537 significantly reduced the incidence of arrhythmias, while ICI 204,448 failed to exhibit an effect. Interestingly, nor-BNI failed to inhibit the anti-arrhythmic effect of either U50,488 or BRL 52537, which suggests that this effect of these two agonists is not mediated by the KOR.
The results of this study provide clear evidence that exogenous KOR activation provides protection against infarct development in an in vivo rat model. Indeed, the infarct-sparing ability of KOR activation was equal to that of DOR activation. Furthermore, our data demonstrate that U50,488 and BRL 52537 also produce a pronounced anti-arrhythmic effect. While previous studies have demonstrated anti-arrhythmic and infarct-reducing properties of KOR activation, 11,13,16,17 this is the first study to examine the effects of exogenous KOR activation by several structurally different KOR agonists on arrhythmias and infarct size in the same model.
In a previous study from our laboratory, we demonstrated a lack of involvement of the KOR in mediating ischemic preconditioning (IPC) in the intact rat. 18 While KOR activation may not be involved in the genesis of IPC, the disparity of the findings may have been due to the length of drug exposure to the KOR antagonist, nor-BNI. Nor-BNI exhibits slow binding kinetics to the KOR 19,20; thus the 15-minute incubation time used by Schultz et al may have been too short to allow thorough binding. Regardless, nor-BNI used at a dose similar to that which did not block IPC, did abolish the infarct-sparing effects of U50,488 and ICI 204,448 in this study. Interestingly, KOR blockade failed to inhibit the protection provided by BRL 52537. BRL 52537 is considered a highly selective KOR agonist without any preferential binding to putative KOR subtypes, 21 while nor-BNI may be a K1OR-selective antagonist. 19,22 Furthermore, nor-BNI failed to attenuate the anti-arrhythmic effect produced by BRL 52537. Thus, it appears that the effects of BRL 52537 may be independent of the KOR, or at least, insensitive to nor-BNI. Zhang et al 22 have provided evidence for 2 KOR subtypes in cardiac tissue, whereby one subtype is U50,488-sensitive and DADLE-insensitive, and one is U50488-insensitive and DADLE-sensitive. Thus, it is possible that BRL 52537 may be acting via the latter subtype that may be insensitive to nor-BNI.
Interestingly, the peripherally active KOR agonist ICI 204,448, 23,24 while displaying a pronounced infarct-limiting effect, failed to possess any effect upon arrhythmogenesis. This has been previously demonstrated in a similar model of coronary ligation, 25 even in the presence of a much higher dose of ICI 204,448 (5 mg/kg). Furthermore, McIntosh et al 25 demonstrated an anti-arrhythmic effect of nor-BNI at a dose of 2.5 mg/kg, but not at 0.5 mg/kg. Indeed, many previous studies have suggested a role for endogenous opioids in arrhythmogenesis during/following ischemia-reperfusion 26,27; however, this appears to occur at higher doses of the agonist/antagonist. At the dose of nor-BNI used in the present study (0.1 mg/kg) we failed to demonstrate an anti-arrhythmic effect of nor-BNI. Recently, a study by Yu et al 28 described pro- and anti-arrhythmic effects of the KOR agonist U50,488 at high and low concentrations respectively. Our findings show that U50,488 did indeed attenuate arrhythmogenesis; however, similar to results obtained with BRL 52537, nor-BNI failed to inhibit these antiarrhythmic effects. These data are supported by those of Pugsley et al 17 who also demonstrated that the antiarrhythmic effect of U50,488 was independent of opioid receptors since the non-selective opioid antagonist, naloxone, failed to antagonize the antiarrhythmic effect of U50,488 in a similar anesthetized model. Interestingly, it has been suggested that the primary antiarrhythmic effect of KOR agonists is not the result of KOR activation but from a direct effect on ion channels. Several lines of evidence support an ion channel-blocking property of KOR agonists that appears to be independent of KOR activation. U50,488, along with other KOR agonists, has been shown to prolong the P-R interval and QRS duration, along with inhibiting contractile function in isolated rat hearts, 29 suggesting that these agents may block Na+ and K+ channels. These effects were not blocked by naloxone. 29,30 KOR agonists may also antagonize l-type Ca2+ channels. 31 Indeed, the apparent receptor-independent antiarrhythmic actions of KOR agonists may be due to direct blockade of cardiac Na+, K+, or Ca2+ channels. 29,30,32
However, there is evidence to suggest that the KOR agonists may, indeed, elicit their actions on ion channels via the KOR. Alterations in the [Ca2+]i transient produced by U50,488 appears to be mediated via pertussis toxin-sensitive G proteins. 33,34 Analgesia induced by U50,488 appears to be mediated by pertussis-sensitive G proteins coupled to l-type Ca2+ channels. 35 Furthermore, the arrhythmogenic action of U50,488 coupled to a rise in [Na+]i and [Ca2+]i may be due to a protein kinase C/ Na+-H+ exchange pathway 36 and involves phospholipase C and inositol 1,4,5 triphosphate. 33,34 Indeed, a report by Zhang et al 37 suggests that Ca2+ channel blockers compete with KOR agonists in binding to the KOR in a dose-dependent manner.
Regardless, KOR blockade failed to inhibit the anti-arrhythmic actions of KOR agonists in our intact rat model. While nor-BNI failed to block the anti-arrhythmic effects of U50,488, blockade of the KOR negated the infarct-sparing actions of U50,488. These data are supported by those of Wang et al 11 as well as others. 38 However, a recent study by Aitchison et al 14 reported that KOR activation increased infarct size while DOR activation reduced infarct size in isolated rat hearts. The increase in infarct size observed by Aitchison was reversed by nor-BNI. However, the KOR agonist employed by Aitchinson, bremazocine, was shown to inhibit cyclic AMP accumulation, which was not attenuated by nor-BNI. 39 Furthermore, Coles et al 40 reported that the DOR agonist DADLE only provided protection against infarct development in swine when administered in the presence of nor-BNI. The variation between results reported by Coles et al and our findings may be explained by species and model differences. Indeed, the swine exhibit a lower metabolic rate than the rat and protection afforded by DADLE was not associated with alterations in the levels of high-energy phosphates in swine. 41
In summary, we provide evidence demonstrating the anti-arrhythmic and/or infarct-sparing actions of 3 KOR agonists. The infarct-limiting effect of the KOR agonists was as great as that provided by exogenous DOR activation. KOR blockade failed to modify the marked anti-arrhythmic actions of U50,488 and BRL 53527, which suggests that the anti-arrhythmic effect may be independent of KOR activation. However, these data provide evidence that use of KOR agonists may have beneficial clinical effects.
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