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-Opioid Receptor Antagonism Improves Recovery from Myocardial Stunning in Chronically Instrumented Dogs
From the *Department of Anaesthesiology and Intensive Care; and Institute of Clinical Chemistry and Laboratory Medicine, University Hospital Münster, Münster, Germany.
Address correspondence and reprint requests to Maike A. Grosse Hartlage, MD, Universitätsklinikum Münster, Klinik und Poliklinik für Anästhesiologie und operative Intensivmedizin, Albert-Schweitzer-Straße 33, 48149 Münster, Germany. Address e-mail to grosse.hartlage{at}anit.uni-muenster.de.
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Abstract |
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 Top Abstract IntroductionMETHODSRESULTSDISCUSSIONREFERENCES |
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-opioid receptor antagonist nor-binaltorphimine (nor-BNI) improves recovery from myocardial stunning. Ten dogs were chronically instrumented for measurement of heart rate, left atrial, aortic and left ventricular pressure (LVP), and the maximum rate of LVP increase (LV dP/dtmax) and decrease (LV dP/dtmax), coronary blood flow velocity and myocardial wall-thickening fraction. Regional myocardial blood flow was determined with fluorescent microspheres. Catecholamine plasma levels were measured by high-performance liquid chromatography, and ß-endorphin and dynorphin plasma levels by radioimmunoassay. An occluder around the left anterior descending artery (LAD) allowed induction of a reversible LAD-ischemia. Animals underwent two experiments in a randomized crossover fashion on separate days: (a) 10 min LAD-occlusion (control experiment), (b) second ischemic episode 24 h after nor-BNI (2.5 mg/kg IV) (intervention). Dogs receiving nor-BNI showed an increase in wall-thickening fraction, LV dP/dtmax and LV dP/dtmin before ischemia and during the whole reperfusion (P < 0.05 versus control experiment). After nor-BNI pretreatment, dynorphin levels increased after induction of ischemia to a peak level of 15.1 ± 3.6 pg/mL (P < 0.05 versus control experiment). The increase in plasma ß-endorphin during ischemia and early reperfusion was attenuated after nor-BNI. Compared with the control experiment, nor-BNI left global hemodynamics, regional myocardial blood flow, and catecholamine levels unchanged. In conclusion, nor-BNI improves recovery from myocardial stunning after regional myocardial ischemia in chronically instrumented dogs. |
Introduction |
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TopAbstractIntroductionMETHODSRESULTSDISCUSSIONREFERENCES |
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The endogenous opioid system is composed of three different peptides, endorphins, enkephalins, and dynorphins, each derived from a distinct precursor, proopiomelanocortin, proenkephalin, and prodynorphin, respectively. The endogenous opioid peptides act at three major types of opioid receptors, the µ, 
, and receptor, and they differ in their affinities for the individual receptor subtypes (4). The presence of the endogenous -opioid receptor agonist dynorphin, including the mRNA of its precursor prodynorphin, has been demonstrated in the heart (5). In addition, receptor binding studies revealed that -opioid binding sites are present in the myocardium (6). Besides physiological influences on cardiovascular functions (7), endogenous opioid peptides are also involved in the pathophysiology of shock, heart failure, and ischemic heart disease (8). Myocardial ischemia and reperfusion are critical stimuli for the systemic and local myocardial release of endogenous opioid peptides (9,10). There is substantial evidence for an activation of -opioid receptors during myocardial ischemia (8,11). -Opioid receptor stimulation causes detrimental effects like arrhythmias (11), bradycardia (12), negative inotropy (13), and impaired hemodynamics (14,15). Preischemic activation of -opioid receptors has been shown to increase infarct size (16).
A study from our laboratory demonstrated that the nonselective opioid receptor antagonist naloxone improves recovery from myocardial stunning in chronically instrumented conscious dogs (17). The use of receptor-selective antagonists allows delineation of the opioid receptor subtype that mediates the observed cardioprotective effect. The present study tested the hypothesis that the preischemic application of the selective -opioid receptor antagonist nor-binaltorphimine (nor-BNI) (18) improves recovery from myocardial stunning. Furthermore, we investigated the secretion patterns of ß-endorphin and dynorphin A during myocardial ischemia and reperfusion. To evaluate an interaction with the sympathetic nervous system, epinephrine and norepinephrine plasma levels were measured.
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METHODS |
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TopAbstractIntroductionMETHODSRESULTSDISCUSSIONREFERENCES |
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Surgical Preparation
Ten healthy, heartworm-free foxhounds of either sex, weighing 22–24 kg, were selected for the studies. After overnight fasting, but with free access to water, the dogs received IM premedication with piritramide 1 mg/kg and ketamine 5 mg/kg. Anesthesia was induced IV with propofol 7 mg/kg and fentanyl 20 µg/kg. Mechanical ventilation was adjusted to maintain arterial blood gas tension values in the physiologic range. Anesthesia was maintained with isoflurane in a mixture of oxygen and air, supplemented with intermittent application of 10 µg/kg fentanyl. To prevent hypovolemia, lactated Ringer’s solution was infused at a rate of 12 mL · kg–1 · h–1. Body temperature was monitored using a rectal temperature probe and maintained by use of a heating pad. Perioperative antibiotic prophylaxis was achieved with 30 mg/kg cefamandole for 3 days.
A left thoracotomy was performed in the fifth intercostal space under strictly aseptic conditions. The aorta and the left atrium were cannulated with 18-gauge Tygon catheters for measurement of pressures, injection of microspheres, and withdrawal of blood. For determination of the left ventricular pressure (LVP) and the rate of increase of LVP (LV dP/dt) a catheter-tip micromanometer (Janssen Pharmaceutica, Beerse, Belgium) was inserted into the left ventricle through an apical stab incision (19). Pulsed Doppler blood flow velocity probes (20 MHz; Baylor College of Medicine, Houston, TX) were placed around the left anterior descending coronary artery (LAD). Although this method does not measure true blood flow, there is an excellent correlation between Doppler frequency shift (kHz) and absolute blood flow (20). Proximal to the Doppler flowprobe, a pneumatic occluder was positioned around the LAD to induce reversible ischemic episodes in the LAD-perfused myocardium. To assess the regional myocardial wall-thickening, 10 MHz pulsed Doppler crystals (Baylor College of Medicine, Houston, TX) were sutured precisely perpendicular and flat to the epicardium of the LAD-perfused area. This atraumatic technique is based on principles of pulsed echo and Doppler ultrasound and has been described in detail (21,22). A validation for myocardial ischemia and reperfusion studies has been performed (21). At the end of the surgical instrumentation, the pericardial edges were approximated, and the thorax was closed in layers. All leads were tunneled subcutaneously and exteriorized between the scapulae. The dogs were fitted in a jacket to prevent damage to the instruments and catheters. Postoperative analgesia was provided with IM administered piritramide.
After instrumentation, the animals were trained daily to accustom them to the experimental environment. Experiments were performed after a postoperative recovery time of at least 12 days.
Experimental Protocol
The effect of the preischemic application of the selective -opioid receptor antagonist nor-BNI on the recovery from myocardial stunning was determined as follows. All animals were subjected to two experimental conditions-control experiment (without pretreatment) and intervention (with preischemic application of the -opioid receptor antagonist nor-BNI)—in a randomized crossover design on separate days (Fig. 1). Using a computer-generated random-number table, the animals were assigned to one of two groups (A and B), each consisting of five animals. The five dogs of group A were subjected to undergo at first the control experiment and then the intervention, whereas in group B, first the intervention was performed and then the control experiment.
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As the antagonistic effects of nor-BNI persist up to 4 wk (18), the time interval between the two experiments was 30 days.
The number of ischemic episodes was restricted to two in each animal, because multiple stunning maneuvers may induce development of coronary collaterals and thus prevent the induction of postischemic dysfunction. The interindividual variability in canine coronary anatomy was considered, as the control experiment as well as the intervention were performed in the same animals and not in separate experimental groups. This is an established experimental model to study myocardial stunning (17,23).
The duration of each ischemic episode was 10 min to cause myocardial stunning of relevant severity (24) and to prevent, at the same time, the induction of myocardial infarction. Each dog had one 10-min LAD-ischemia without pretreatment (control experiment) and a second 10-min ischemic episode 24 h after IV application of the selective -opioid receptor antagonist nor-BNI (Sigma-Aldrich Chemie GmbH, Taufkirchen, Germany) (intervention). For the intervention, 2.5 mg/kg nor-BNI dissolved in 50 mL water was infused over the atrial catheter at a constant rate of 1.6 mL/min by use of a precalibrated pump.
Methods of Measurement
Hemodynamics and Wall-Thickening Fraction
Aortic and left atrial pressures (LAP) were measured using disposable pressure transducers.
The inserted solid-state catheter-tip micromanometer allows an in vivo calibration and displays a good zero-stability 1 wk after implantation. Zero pressure output was assessed by equating peak systolic left ventricular and aortic pressure. This calibration procedure can be used because the micromanometer system is practically linear, both in vitro and in vivo (19). The hysteresis of the device has been reported to be 1% of the full range (19). LVP measurements were performed under controlled normal body temperatures, whereby the micromanometer shows a negligible temperature drift of 0.5 mm Hg/°C. The LVP signal was electronically differentiated (Gould, Cleveland, OH) to obtain the maximum rate of LVP increase (LV dP/dtmax) and decrease (LV dP/dtmin).
The wall-thickening fraction (WTF) for a myocardial layer is the ratio between systolic thickening and thickness of that layer (21). Systolic thickening is defined as the maximal excursion of the myocardial wall recorded during systole. Using the pulsed Doppler method, WTF is calculated as
WTV (%) = (SE/R) x 100,
where SE is the systolic excursion of the myocardial wall through the Doppler range-gated sample volume (mm) and R is the range gate depth in the ventricular wall (mm) (21,22). To measure transmural thickening, the Doppler technique requires the range-gated sample volume to be positioned at a depth in the ventricular wall that is close to that of the endocardium at end-diastole.
Pressures, blood flow velocity and wall-thickening signals were processed using a six-channel pulsed Doppler system (Baylor College of Medicine, Houston, TX).
Data acquisition was performed at the following time points: under baseline (BL) conditions; 24 h after nor-BNI infusion; during ischemia and 1, 5, 10, 15, 20, 30, 45, 60, and 90 min and 2, 3, 6, 12, 24, and 48 h after beginning of reperfusion. At each of these time points, hemodynamic variables and wall-thickening data were recorded over a period of 30 s. Data presented are mean values of these recordings and not single values at specified time points.
Regional Myocardial Blood Flow Distribution
The transmural distribution of the regional myocardial blood flow was assessed by the use of fluorescent microspheres (NuFLOWTM microspheres, Interactive Medical Technologies, Irvine, CA), a technique that has been validated (23,25). The regional myocardial blood flow was measured three times in each experiment: (1) under BL conditions, respectively 24 h after nor-BNI infusion, (2) during ischemia, 3 min after LAD occlusion, and (3) after the third minute of reperfusion. Preparation and injection of the microspheres and the withdrawal of the reference blood sample have been described in detail (23). The administration of microspheres caused no changes in coronary blood flow, heart rate (HR), mean arterial blood pressure (MAP), LV dP/dtmax, and LV dP/dtmin.
When the regional myocardial function had completely recovered after the second ischemic episode, animals were killed during general anesthesia by infusion of potassium chloride, and the heart was excised. Transmural tissue samples of 1–2 g were obtained from the LAD-perfused area in the immediate vicinity of the implanted wall-thickness probes and further dissected into the endocardial and epicardial layer. Samples from the area that was normally perfused by the circumflex branch of the left coronary artery (LCX) served as controls. Extraction and measurement of microspheres in the samples were performed in an independent laboratory by flow cytometry analysis (Interactive Medical Technologies, Irvine, CA). For each heart, the induced ischemia was considered to be severe enough only if the tissue samples from the LAD-perfused myocardium had a regional myocardial blood flow less than or equal to 25% of the corresponding nonischemic regional myocardial blood flow under BL conditions. The endocardial-to-epicardial regional myocardial blood flow ratio was calculated for the LAD- and the LCX-perfused myocardium.
Plasma Catecholamine Assays
To evaluate an interaction with the sympathetic nervous system, arterial blood samples for measurement of plasma catecholamines were obtained at the following time points: under BL conditions, 24 h after nor-BNI infusion, during ischemia and 1, 5, 15, 30 min and 1, 3, and 6 h after reperfusion. Blood collection and plasma sample analysis were performed exactly as previously described (23). Epinephrine and norepinephrine were assayed using high-performance liquid chromatography technique.
The lower detection limit, as defined by 95% of the upper plateau of the standard curve, was 10 pg/mL per tube for epinephrine and norepinephrine. The intraassay coefficient of variation for epinephrine and norepinephrine was 5.4% and 5.8%, respectively, and the interassay coefficient of variation for epinephrine and norepinephrine was 10.6% and 9.4%, respectively.
ß-Endorphin and Dynorphin A Radioimmunoassay
Dynorphin A is an endogenous -opioid receptor agonist, whereas µ-opioid receptors are the preferential binding sites for endogenous ß-endorphin. Blood samples for ß-endorphin and dynorphin A analyses were withdrawn under BL conditions, 24 h after nor-BNI infusion, during ischemia, and 1, 5, 15, 30 min and 1, 3, and 6 h after reperfusion. To avoid an influence of the circadian rhythm, all experiments were started between 8:00 am and 9:00 am. Arterial blood 7.5 mL was collected into prechilled polypropylene syringes (S-Monovette®, Sarstedt, Nümbrecht, Germany) containing EDTA and 500 IU aprotinin (Trasylol®, Bayer Vital GmbH, Leverkusen, Germany) per mL blood as enzymatic inhibitor. The samples were centrifuged at 3000 rpm for 20 min at 4°C, and the plasma was separated and stored at (–70°C until analysis. Extraction and measurement of endogenous opioids in the samples were performed in an independent laboratory (Immundiagnostik AG, Bensheim, Germany). Plasma levels of ß-endorphin and dynorphin A were measured with commercially available radioimmunoassay (RIA) kits (Peninsula Laboratories, Division of Bachem, San Carlos, CA) according to the manufacturers’ directions. The ß-endorphin RIA used 125I-ß-endorphin as tracer. The lower detection limit for ß-endorphin was 3.1 pg/mL. The dynorphin A RIA used 125I-dynorphin A as tracer, and the lower detection limit was 8 pg/mL. The intraassay and interassay coefficients of variation were <5% for ß-endorphin and dynorphin A. All samples were assayed in duplicate, and the results were averaged.
Statistical Analysis
To analyze treatment effects by comparison of different points of measurement, statistical analysis was performed by using one-way analysis of variance for repeated measurements and Student’s t-test for dependent samples. Probability (P) values of <0.05 were considered significant. The reported data are normally distributed and presented as mean ± sd.
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RESULTS |
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TopAbstractIntroductionMETHODSRESULTSDISCUSSIONREFERENCES |
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The systolic WTF in the LAD-perfused myocardium increased significantly 24 h after nor-BNI infusion to a preischemic value of 157% ± 31%. Regional myocardial ischemia led to a significant reduction of the WTF to negative values ("wall thinning") in the control experiments (–58% ± 16%) as well as under nor-BNI pretreatment (–47% ± 20%). After nor-BNI application, reperfusion resulted in an immediate recovery of the WTF to the preischemic level, and this was sustained throughout the whole reperfusion. In the control experiments, WTF returned after reperfusion, on the average, only to 38% of the BL level, and preischemic WTF values were reached after 48 h. The positions of the wall-thickness probes in the LAD-perfused area were confirmed in the postmortem inspection of the heart to be located on the tissue samples included for analysis of LAD transmyocardial blood flow distribution.
Regional Myocardial Blood Flow
Compared with the control experiment, the administration of nor-BNI did not influence the regional myocardial blood flow in the LAD- and LCX-perfused myocardium at any time (Table 2). During coronary artery occlusion, transmural LAD-regional myocardial blood flow decreased significantly compared with preischemic values in the control experiment and in the intervention, indicating that there were no differences in collateral blood flow. Reperfusion caused a nonsignificant increase of LAD-regional myocardial blood flow under both experimental conditions. The endocardial-to-epicardial regional myocardial blood flow ratio of the LAD-perfused myocardium decreased after induction of LAD-ischemia in relation to preischemic values only in the control experiment (Table 3). There were no differences between the control experiment and nor-BNI pretreatment regarding the endocardial-to-epicardial regional myocardial blood flow ratio.
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Global Ventricular Function
LV dP/dtmax and LV dP/dtmin increased significantly 24 h after nor-BNI infusion compared with the preischemic values of the control experiment (Table 1). This difference between the two experimental conditions was significantly sustained throughout the entire protocol (Table 1). Neither induction of ischemia nor reperfusion had an influence on LV dP/dtmax and LV dP/dtmin.
Blood Flow Velocity in the LAD
Preischemic blood flow velocity BL values were similar under control conditions and after nor-BNI pretreatment (Table 1). During LAD-ischemia, blood flow velocity decreased to zero in the control experiment and in the intervention. After release of coronary artery occlusion, a pronounced reactive increase in blood flow velocity was observed. During the first 5 min of the reperfusion period, blood flow velocity values were significantly increased compared with preischemic records and then returned to BL values (Table 1). There were no differences between both experimental conditions regarding blood flow velocity.
MAP, LAP, and HR
Twenty-four hours after nor-BNI pretreatment, MAP, LAP, and HR remained unchanged compared with BL values (Table 1). In the control experiment and under the intervention, induction of ischemia did not change MAP and HR in comparison with the preischemic state, whereas LAP increased significantly. After reperfusion MAP, LAP, and HR showed no deviation from preischemic levels (Table 1).
Arterial Plasma Catecholamine Levels
BL levels of norepinephrine were 227 ± 32 pg/mL in the control experiment and 234 ± 35 pg/mL before nor-BNI pretreatment (Fig. 3a). BL plasma concentrations of epinephrine were 62 ± 12 pg/mL in the control experiment and 55 ± 13 pg/mL before nor-BNI application (Fig. 3b). Systemic plasma catecholamine levels were unchanged 24 h after nor-BNI pretreatment. Neither induction of ischemia nor reperfusion had a significant effect on norepinephrine and epinephrine values. Differences between experimental conditions were not observed at any time.
Arterial Plasma Levels of ß-Endorphin
BL plasma levels of ß-endorphin were 19.2 ± 5.1 pg/mL in the control experiment and 20.4 ± 5.9 pg/mL before the intervention (Fig. 4a). In the control experiment, induction of ischemia resulted in a significant increase in ß-endorphin levels to a peak value of 46.4 ± 6.7 pg/mL. After release of coronary artery occlusion, values decreased, but were still elevated after 1 min of reperfusion. They returned to BL levels after 5 min.
Pretreatment with nor-BNI had no influence on ß-endorphin levels after 24 h. Under the intervention, ß-endorphin values decreased significantly in response to regional ischemia to 7.3 ± 1.9 pg/mL. No deviation from BL measurements was observed during the initial 6 h of reperfusion. When compared with the values of the control experiment, ß-endorphin levels in dogs receiving nor-BNI were significantly lower during LAD-ischemia and 3 and 6 h after reperfusion.
Arterial Plasma Levels of Dynorphin
BL levels of dynorphin were 1.2 ± 0.8 pg/mL in the control experiment and 1.3 ± 0.7 pg/mL before the intervention (Fig. 4b). After coronary occlusion, dynorphin values increased significantly to 5.2 ± 1.7 pg/mL in the control experiment. They remained on this increased level during reperfusion and did not return to BL after 6 h.
Nor-BNI pretreatment induced a significant increase of dynorphin levels to 5.0 ± 2.0 pg/mL after 24 h. A further significant increase to 15.1 ± 3.6 pg/mL was observed in response to regional myocardial ischemia. With the beginning of reperfusion, dynorphin values decreased to BL levels. After 5 min of reperfusion, they again increased significantly to the preischemic level and did not return to predrug BL values after 6 h. In comparison with the values of the control experiment, nor-BNI pretreated dogs showed higher dynorphin levels during ischemia and lower values at the first minute of reperfusion.
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DISCUSSION |
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TopAbstractIntroductionMETHODSRESULTSDISCUSSIONREFERENCES |
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-opioid receptor antagonism on myocardial stunning continues our previous work, which has demonstrated improved recovery from stunning after pretreatment with the nonselective opioid receptor antagonist naloxone (17). The key findings of the present study are that the -opioid receptor antagonist nor-BNI increased regional and global myocardial contractility and improved recovery from myocardial stunning after transient regional myocardial ischemia.
Receptor binding studies have shown that -opioid binding sites are present in the heart (6). In agreement with this, functional studies showed that -opioid receptor agonists modulate cardiac functions when administered directly to isolated hearts (11) or cardiomyocytes (13). The presence of dynorphin and ß-endorphin, including the mRNA of their precursors prodynorphin (5) and proopiomelanocortin (26), has also been demonstrated in the heart. This indicates that -opioid peptides are synthesized in and released from the myocardium, suggesting that they may regulate the heart as autocrine or paracrine hormones (27). Data regarding the influence of -opioid receptor activation during ischemia/reperfusion are controversial. Exogenous -opioid receptor activation improved contractile recovery in isolated perfused mouse hearts undergoing 20 min global ischemia followed by 45 min reperfusion (28). Conversely, Aitchison et al. (16) demonstrated that -opioid receptor stimulation exacerbated infarct size in isolated rat hearts. The -opioid receptor agonists dynorphin and U50,488H increased the incidence and severity of ischemia-induced arrhythmias after coronary occlusion (8,11).
Nor-BNI acts as a reversible, selective -opioid receptor antagonist in vitro and in vivo. Experiments characterizing the time course of the antagonistic selectivity of nor-BNI revealed a partial µ antagonistic action that peaked after 30–60 min and disappeared completely within 4 h (18). In contrast, it has been demonstrated in several in vivo studies that 24 h after systemic application, nor-BNI has high affinity and selectivity exclusively for -opioid receptors (18,29). As we chose a 24 h pretreatment interval, the observed cardioprotection can be attributed to the selective antagonism of -opioid receptor-mediated effects. Nor-BNI sustains its peak antagonistic effects at -opioid receptors for up to 4 wk after administration (29). It has been suggested that this long duration of action may be due to a resistance to metabolism or an induction of conformational receptor changes. On the basis of this scientific background, we chose a time interval of 30 days between the two experiments.
The magnitude of WTF, LV dP/dtmax, and LV dP/dtmin increased significantly after nor-BNI pretreatment. In the literature, however, there is no indication for an intrinsic positive inotropic action of nor-BNI. The observation that the endogenous opioid peptide system is functionally not quiescent under physiologic BL conditions (7,15) is one explanation for the improved left ventricular contractility after nor-BNI administration.
In the control experiment and the intervention, neither the induction of ischemia nor the beginning of reperfusion caused a significant decline of LV dP/dtmax and LV dP/dtmin compared with preischemic BL, because no global postischemic contractile dysfunction was induced. The WTF data, however, confirm the induction of a regional myocardial dysfunction.
Dogs receiving nor-BNI developed no postischemic contractile impairment. Our study design does not allow an analysis of the definite underlying mechanism, however, several possible explanations warrant consideration. Myocardial ischemia and reperfusion are critical stimuli for the release of endogenous opioid peptides (9,10). Correspondingly, induction of LAD-ischemia and reperfusion resulted in increased plasma levels of the -opioid receptor agonist dynorphin in the control experiment of our study (Fig. 4b). Dynorphin has been reported to enhance bradycardia and cardiogenic shock secondary to myocardial ischemia (8,14). In convincing in vitro (30) and in vivo (31) studies, negative inotropic effects of -opioid receptor agonists were demonstrated. In our study, nor-BNI pretreatment may have antagonized -opioid receptor mediated negative inotropy.
Severe ischemia was confirmed by a significant reduction of WTF to negative values and by a reduction of subendocardial perfusion to <20% of BL values. Thus, the cardioprotective effect of nor-BNI occurred without preventing the depression of systolic wall function that occurs during ischemia.
The two main theories regarding the pathogenesis of myocardial stunning are the oxyradical hypothesis and the calcium hypothesis (1). The oxyradical hypothesis postulates that myocardial stunning is caused by reactive oxygen species. In anesthetized cats undergoing LAD ischemia and reperfusion, Yang et al. (32) demonstrated that the nonselective opioid receptor antagonist naloxone significantly suppressed the ischemia-induced interstitial hydroxyl radical production in the left ventricular myocardium. Furthermore, perfusion of the left ventricular interstitium with the endogenous -opioid receptor agonist dynorphin increased hydroxyl radical formation (32). These observations suggest that the endogenous opioid peptide system, especially -opioid receptor agonists, may be related to the formation of oxygen-derived free radicals after myocardial ischemia.
The calcium hypothesis postulates that myocardial stunning is the result of an altered intracellular calcium (Ca2+) homeostasis with decreased myofilament responsiveness to Ca2+ and cytosolic Ca2+ overload (1). It has been demonstrated that the selective -opioid receptor agonist U50,488H increases the intracellular calcium concentration ([Ca2+]i) by depleting the sarcoplasmatic reticulum of Ca2+ via the phospholipase C/inositol 1,4,5-trisphosphate pathway (13,33). The resulting reduced availability of Ca2+ for release from the sarcoplasmatic reticulum in response to electrical stimulation is one explanation for the reduced contractility after -opioid receptor stimulation (13). Furthermore, -opioid receptor agonism induces increases in intracellular cytosolic sodium ([Na+]i) via activation of the protein kinase C/Na+-H+-exchange pathway. This, in turn, contributes to the increase in [Ca2+]i via Na+-Ca2+ exchange. Na+-H+-exchange gene expression in the heart is increased upon -opioid receptor stimulation (34). Another explanation for the increased [Ca2+]i is the decreased Ca2+ uptake into the sarcoplasmatic reticulum upon protein kinase C activation. Not only the negative inotropic effect, but also the proarrhythmic effect of -OR stimulation, has been explained by the altered Ca2+ homeostasis (35).
The present study investigated whether the contractile responses to nor-BNI were related to changes in circulating catecholamine plasma levels. Systemic epinephrine and norepinephrine levels, however, provided no explanation, as they remained unchanged in the control experiment and in the intervention, throughout the entire protocol. Endogenous -opioid peptides are stored in sympathetic nerve fibers in myocardium and vasculature. After sympathetic stimulation, opioid peptides and catecholamines are co-released from neuronal terminals (7). Noradrenergic neurons within the heart and vasculature possess presynaptic -opioid receptors, which mediate inhibition of excitatory neurotransmitter release (12,36). In anesthetized dogs, intracoronary administration of dynorphin significantly reduced coronary overflow of norepinephrine during left cardiac nerve stimulation (37). Correspondingly, -opioid receptor antagonism by nor-BNI may result in a disinhibition of norepinephrine release from sympathetic nerve endings within the ventricular wall. A local release of norepinephrine does not necessarily need to be reflected in the systemic catecholamine levels, but it may contribute to a regional increase in contractile force.
The absence of a chronotropic response of dogs to nor-BNI in the present study, as well as to dynorphin pretreatment (37), may indicate that -opioid receptors have in this species only a minor influence on the pacemaker and conduction system of the heart. In rabbits (12), -opioid receptor agonism resulted in a negative chronotropic effect. We did not observe an influence of nor-BNI on MAP. In most previous studies, -opioid receptor activation is consistently associated with decreases in MAP (15).
Blood flow velocity in the LAD and regional myocardial blood flow were unaffected by nor-BNI pretreatment. The endocardial/epicardial regional myocardial blood flow ratio during LAD-ischemia was insofar improved as the decrease relative to BL was smaller than that in the control experiment. Specific -opioid receptor-mediated effects on myocardial perfusion have not been described. It is notable that the increase in WTF and contractile force was not reflected in an increased coronary blood flow. The suggested local myocardial release of norepinephrine from autonomic nerve endings after -opioid receptor antagonism may explain this discrepancy. Acting on coronary adrenergic receptors, norepinephrine may influence arterial tonus and prevent significant increases in regional myocardial blood flow.
To our knowledge, this is the first report of a chronically instrumented animal model detailing relative changes in plasma levels of ß-endorphin and dynorphin during regional myocardial ischemia and stunning. In patients with acute myocardial ischemia, Oldroyd et al. (10) observed a negative correlation between the duration of ischemia and ß-endorphin plasma levels, and suggested that ß-endorphin is released as a single pulse at the onset of myocardial ischemia. This hypothesis is supported by our results. In the cited investigation, the highest ß-endorphin concentrations were seen in patients whose clinical course was complicated by the development of heart failure (10). The decrease in ß-endorphin levels during LAD-ischemia under the intervention may be explained by a -opioid receptor-mediated influence on ß-endorphin release. Dynorphin levels were significantly increased after nor-BNI pretreatment and showed a further pronounced increase during myocardial ischemia. It is anticipated that a negative feedback control of dynorphin release by dynorphin plasma levels was inhibited through the -opioid receptor antagonist. The observed decrease in dynorphin levels in early reperfusion can be attributed to metabolism by plasma enzymes (38) paralleled with protracted new processing of prodynorphin after the extensive dynorphin release during ischemia. In the control experiment, myocardial ischemia and reperfusion resulted in constantly increased dynorphin levels, which may be explained by a positive feedback regulation (39).
In summary, the endogenous opioid peptide system is activated during regional myocardial ischemia and reperfusion causing impaired ventricular contractility in chronically instrumented conscious dogs. Especially -opioid receptor agonists enhance fatal complications secondary to myocardial ischemia such as arrhythmias, bradycardia, and negative inotropy. The IV application of nor-BNI improves recovery from myocardial stunning via an inhibitory action on -opioid receptors. Opiates are used for µ agonist induced analgesia and anesthesia in diseased myocardial states. The demonstration that opioid receptors not only mediate analgesia, but also influence myocardial ischemia-reperfusion injury, may have profound clinical implications.
The use of selective -opioid receptor antagonists that are ineffective against µ agonist-induced antinociception may provide perioperative myocardial protection, for example, in patients undergoing cardiac surgical interventions.
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Footnotes |
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Accepted for publication June 20, 2006.
Supported in part by the Medical Faculty of the Westfälische Wilhelms-Universität, Münster, Germany IMF grant WE 220013.
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