1Klinik und Poliklinik für Anästhesiologie und Operative Intensivmedizin, Westfälische Wilhelms-Universität Münster, Albert-Schweitzerstrasse 33, D-48149 Münster, Germany. 2Klinik und Poliklinik für Kardiologie und Angiologie, Innere Medizin C, Westfälische Wilhelms-Universität Münster, Münster, Germany*Corresponding author
Accepted for publication: August 31, 2001
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Abstract |
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Methods. Ten dogs were chronically instrumented for measurement of heart rate, left atrial, aortic, and left ventricular pressure (LVP), LV dP·dtmax/min1, and myocardial wall-thickening fraction. An occluder around the left anterior descending artery (LAD) allowed induction of reversible ischaemia in the LAD-perfused myocardium. Each dog underwent two ischaemic episodes (randomized crossover fashion; separate days): 10 min of LAD occlusion (1) after application of naloxone (63 µg kg1), and (2) without naloxone. ANP levels were measured at baseline (BL) and at predetermined time points until complete recovery of myocardial stunning occurred.
Results. LAD ischaemia-induced release of ANP (peak level: 182 (30) vs 27 (7) pg ml1 BL) only in the control group without naloxone. Between 1 and 180 min of reperfusion, ANP levels were significantly higher only in the control group (P<0.05).
Conclusion. Pre-ischaemic application of naloxone prevents this ischaemia-induced ANP-release in conscious dogs.
Br J Anaesth 2002; 88: 8793
Keywords: complications, myocardial stunning; antagonists opioid, naloxone; heart, atrial natriuretic peptide; brain, natriuretic peptide; heart, ischaemia
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Introduction |
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The present investigation in chronically instrumented conscious dogs tests the hypothesis that (1) ANP and BNP are released in response to myocardial regional ischaemia and stunning and (2) that naloxone prevents this ischaemia-induced release.
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Methods |
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Arterial blood samples for measurement of ANP and BNP were drawn from the aortic catheter, collected into chilled syringes and transferred to polypropylene tubes containing EDTA (1 mg ml1 of blood) and aprotinin (500 kIU ml1 of blood). Thereafter, the samples were centrifuged and the plasma stored at 70°C until analysis. ANP and BNP plasma concentrations were analysed by radioimmunoassays using a polyclonal rabbit IgG-antisera raised to the following peptides (Peninsula Laboratories, Belmont, CA, USA): -ANP 128 (human) and BNP-32 (human). Peptides were extracted from 3 ml plasma (Sep-Pak C18, Waters Associates, Milford, MA, USA) and eluted with 3 ml of a mixture of 60% acetonitrile, 0.1% trifluoroacetic acid, and 39% distilled water (by volume). All samples were assayed in triplicate. Standard curves were constructed with standard human ANP and BNP in radioimmunoassay buffer. The mean recovery of added natriuretic peptides from plasma was 6080%, and the lower detection limits as defined by 95% of the upper plateau of the standard curve were 0.1 nmol per tube for ANP and 0.5 nmol per tube for BNP. Cross reactivity between natriuretic peptides was less than 0.1%. The intra-assay and inter-assay coefficients of variation were 3.8 and 9.6% for ANP and 6.1 and 7.9% for BNP, respectively.
The experimental design was as follows: All 10 dogs were used in two ischaemic experiments. Each dog had one ischaemic episode without pre-treatment and the other ischaemic episode after pre-treatment with 63 µg kg1 naloxone (Curamed Pharma, GmbH, Ch.-B.: 0060697) i.v. 5 min before ischaemia.3 Five animals received their first coronary artery occlusion without pre-treatment. The other five received naloxone before their first ischaemia.
The following observations were made.
1. Measurement of baseline (BL) values in the awake state and induction of 10 min of LAD ischaemia, with follow-up of WTF, ANP, and BNP levels until complete recovery occurred.
2. Measurement of BL values in the awake state, application of 63 µg kg1 naloxone, induction of 10 min of LAD ischaemia, with follow-up of WTF, ANP, and BNP levels until complete recovery occurred.
A second ischaemic episode was only induced when there was complete recovery of regional myocardial function in the LAD-perfused area; the minimum time interval between the two experiments was 4 days.
Regional myocardial blood flow was measured using coloured microspheres (Triton Technology, San Diego, CA, USA). For each measurement, a total of 9x106 microspheres suspended in a volume of 3 ml 0.9% NaCl was injected into the left atrium. The reference blood sample was withdrawn from the aortic catheter at a rate of 10 ml min1. Animals were killed by injection of potassium chloride into the LA catheter during general anaesthesia when regional myocardial function had completely recovered after the last ischaemic episode. The heart was dissected and three tissue samples were obtained from the LAD-perfused left ventricle in each dog. LAD samples were taken from the immediate vicinity of the wall-thickening probes. Only samples from animals with severe ischaemic dysfunction, as determined by the wall-thickening probe, were included (no animal was excluded due to insufficient dysfunction). Samples were further dissected into the subendocardial, subepicardial, and mid-myocardial layers. Measurement of microspheres in the tissue samples was performed as described previously.7 Measurement of regional myocardial blood flow to the regions described was carried out four times during the experiment, as follows: (1) without naloxone and without ischaemia (control), (2) without naloxone during ischaemia, (3) after naloxone and without ischaemia, and (4) after naloxone and during ischaemia.
Experiments were conducted in chronically instrumented conscious dogs to avoid the effects of acute surgical trauma, anaesthesia, volume and ionic imbalances, and temperature on recovery from stunning. As multiple stun manoeuvres may induce extensive development of coronary collaterals thus precluding the induction of post-ischaemic dysfunction, the number of ischaemic episodes was restricted to two in each animal.
Statistical analysis
Data were analysed using repeated-measures two-way ANOVA followed by Bonferroni-corrected Students t-test as appropriate; P<0.05 was considered significant. Data are presented here as mean plus or minus standard error of the mean (SD).
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Results |
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ANP and BNP levels
Regional myocardial ischaemia in the LAD-perfused area caused a significant release of ANP (Fig. 023F1A) only in the control without naloxone (peak levels: 182 (SD 30) vs 29 (7) pg ml1 at BL). Between 1 and 180 min of reperfusion, ANP levels were significantly higher in the control (P<0.05) as compared with the experiment with naloxone pre-treatment. After naloxone pre-treatment, ANP levels remained unchanged during and after regional ischaemia. Myocardial BNP (Fig. 023F1B) release during regional ischaemia and reperfusion was not different between experiments with or without naloxone.
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Regional myocardial wall thickening
In all animals, severe regional myocardial dysfunction occurred during LAD occlusion. Induction of regional ischaemia led to a significant reduction in WTF to negative values (wall thinning) in both experimental conditions (Fig. 023F2). During ischaemia, WTF in the LAD-perfused area expressed as a percentage of the BL value was reduced to 56 (13)% in the experiment with naloxone and to 58 (14)% in the experiment without naloxone. During reperfusion, WTF in relation to pre-ischaemic BL recovered significantly faster with naloxone as compared without naloxone at time points between 1 and 30 min of reperfusion (94 (10) vs 23 (39)% at 1 min; 99 (7) vs 33 (48)% at 5 min; 98 (6) vs 40 (50)% at 15 min; 94 (12) vs 47 (36)% at 20 min; and 92 (9) vs 49 (29)% at 30 min). After 30 min of reperfusion no significant difference in WTF occurred. BL WTF values were reached after 48 h of reperfusion with and without naloxone.
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Discussion |
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Plasma concentrations of the cardiac natriuretic peptides ANP and BNP may be of prognostic value for risk stratification after myocardial infarction.1 ANP is released from granules in atrial myocytes during atrial stretch, whereas BNP is released from the ventricle. All effects mediated by natriuretic peptides, for example inhibition of sympathetic stimulation of the heart, serve to reduce arterial and venous pressures, and blood volume. They are similarly upregulated in heart failure, whereas BNP seems to be superior in detecting myocardial cell damage.1 In our investigation, ANP increased 6-fold during regional ischaemia and 7- to 8-fold in the first minute of reperfusion during myocardial stunning. Furthermore, the decreasing ANP levels over time correlated with the increased recovery of stunning. Peak ANP levels are, therefore, possibly useful in assessing the severity of myocardial stunning, especially in the absence of an elevated BNP release over time. They may also help to distinguish between stunning and post-infarction left ventricular dysfunction, because an elevated BNP release would be expected with myocardial infarction but not with myocardial stunning per se.8 9 Furthermore, in our investigation the 10 min of coronary artery occlusion did not induce an increase for myocardial BNP release, even after 3 days of reperfusion. This supports our stunning model, and the hypothesis that isolated increased ANP levels are strongly correlated with myocardial stunning in the absence of necrosis.
Previously, we demonstrated that naloxone improved functional recovery of myocardial stunning and the ischaemic and post-ischaemic systolic left ventricular performance in conscious dogs through its action on the central nervous system.3 EOPs are found in central areas near the cardiovascular centres of the brain, in the hypothalamus, and peripherally in the myocardium.10 Naloxone exerts central and peripheral effects on the heart tissue itself.11 However, the above described protective effects of naloxone3 are probably, in the main, mediated by its antagonistic action at central opioid receptors.12 The opioid receptor antagonist naloxone methylbromide, which unlike naloxone does not cross the bloodbrain barrier, did not improve myocardial stunning in conscious dogs.3 Central effects of naloxone are, therefore, predominant, mainly on the hypothalamus.13 However, the central sites at which naloxone exerts this action have not been determined. EOPs, released upon myocardial ischaemia,4 activate all central opioid receptors (µ, ,
). This activation leads to sympathoinhibition and contributes to negative inotropic effects on the myocardium. Experimental data suggest that the increased endogenous opioids during heart failure act mainly on the
-receptors13 and
-receptors.15 Activation of these receptors decreases myocardial mechanical performance and alters regional blood flow distribution. Suggestions exist that the effects of non-opioid sigma receptor ligands are mediated via two receptor subtypes, of which one is positive and the other is negative inotropic.16 From current knowledge it is unclear which effect is predominant. Pharmacological studies led to the proposal that myocardial
receptors do not play an important role, at least on cardiac rhythm.17 In contrast, anti-arrhythmic effects mediated by
-,18 µ-,
-receptor agonists, and
-receptor mediated proarrhythmic effects are described.17 The
opioid receptor has been demonstrated to play a major role in cardiac protection in acute instrumented animals19 20 or isolated organs and in relation to ischaemic preconditioning.21 In our experiments, performed in chronically instrumented dogs, the antagonistic effects of naloxone dominate. Also, we did not use ischaemic preconditioning before the ischaemic episode. Therefore, the different results may only reflect the different experimental settings. Acute instrumentation induces myocardial damage due to massive sympathetic stimulation and
opioid agonism mediates cardioprotective effects during acute surgical stress. This is not inevitably at variance to our results. Nevertheless, selective opioid receptor block might provide a starting-point for effectively treating the negative inotropic effects of myocardial stunning. However, further investigations in this area will be needed.
Naloxone not only improved systolic function, but also left ventricular diastolic function during regional ischaemia and myocardial stunning. Naloxone also prevented a significant left atrial pressure increase and improved endocardial blood flow during ischaemia. These improvements explain, at least in part, the absence of an increased ANP release during ischaemia and myocardial stunning with naloxone pre-treatment. The absent ANP release, therefore, reflects the positive effects of naloxone on ischaemic and post-ischaemic myocardial dysfunction. No cardiovascular effects of naloxone alone, or a direct inhibitory effect on ANP secretion were found under BL conditions.
This study has some limitations. First, the results obtained are restricted to dogs; there may be relevant species differences in severity and duration of the ischaemic response and its modulation by naloxone. Secondly, the study design does not allow any conclusion of possible mechanisms of the observed effect. Thirdly, a dose response relationship regarding the protective effect of naloxone was not established.
In conclusion, ANP release, in contrast to BNP release, is enhanced during regional myocardial ischaemia and stunning in conscious dogs. Naloxone completely prevents this increased ANP release. This study is the first description showing that ANP is part of the neurohumoral profile that occurs during myocardial stunning.
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