Methadone is a μ-selective opioid receptor agonist and N-methyl-d-aspartate receptor antagonist frequently prescribed in doses ranging from 30 to 120 mg per day to patients with chronic pain or to subjects addicted to heroin. Methadone has an extended half-life (24–36 h) and relatively low cost compared with other opioids. In the United States, retail sales of methadone have increased 1177% from 1997 to 2006, the largest percentage change for grams of opiate medication sold.1 The heightened popularity of prescribing methadone for chronic pain warrants further investigation regarding its potential secondary effects, particularly on the cardiovascular system. An autopsy analysis from the Chief Medical Examiner of New York City revealed that long-term opiate exposure, predominantly with methadone, reduced the extent of age-, race-, and gender-adjusted coronary artery disease compared with nonopioid exposure.2
Our prior studies suggest that when morphine, a μ1- and δ-receptor agonist, is given before myocardial ischemia or reperfusion, the size of the myocardial infarction is reduced, predominately via a δ1-opioid receptor-mediated mechanism.3,4 It is unknown, however, whether methadone, which has low affinity for κ- or δ-opioid receptors, possesses similar cardioprotective properties.5 Furthermore, there are few data determining whether morphine’s and possibly methadone’s cardioprotective effects are dose dependent or contingent upon the duration of myocardial ischemia or the timing of administration.
Our aim was to compare whether methadone limits myocardial infarct size (IS) to a similar extent as morphine. We determined specifically what effects the dose and timing of administration of both opioids have on myocardial IS reduction compared with placebo. Because both the δ- and κ-opioid receptors, but not the μ-opioid receptor, are present in rat cardiomyocytes, we used a rat model of myocardial infarction to determine whether methadone-induced IS reduction is mediated by the δ-opioid receptor.6,7
The experimental protocol was approved by the Animal Care and Use Committee of the Medical College of Wisconsin, and it conformed to the National Institutes of Health Guide for the Care and Use of Laboratory Animals.
The experimental protocol is outlined in Figure 1. Eight-wk-old male Sprague-Dawley rats (Charles River Laboratories, Wilmington, MA) were anesthetized with thiobutabarbital sodium (Inactin, 100 mg/kg IP, Sigma, St. Louis, MO). A tracheotomy was performed, and the lungs were mechanically ventilated (model CV-101, Columbus Instruments, Columbus, OH) at 38–45 breaths/min using an air-oxygen mixture. The respiratory rate and tidal volume were adjusted on the basis of arterial pH, Pco2, and Po2 measurements (obtained before myocardial ischemia, during ischemia, and 2 h after myocardial reperfusion). Body temperature was kept between 36.5°C and 37.5°C with a heating pad and surgical lamps.
The left jugular vein was cannulated to administer drugs and the left common carotid artery to measure arterial blood pressure and heart rate via a PE23 pressure transducer (Gould, Cleveland, OH) connected to a polygraph monitored continuously throughout the experiment. Heart rate, mean arterial blood pressure, and rate pressure product were measured at baseline, after 15 min of myocardial ischemia, and 2 h after myocardial reperfusion.
A left-sided anterior thoracotomy was performed at the fifth intercostal space, the pericardium opened, and a silk ligature placed around the proximal left anterior descending coronary artery distal to the left atrial appendage. Myocardial ischemia was induced by tightening the ligature to occlude the left anterior descending coronary artery. Coronary occlusion was confirmed by the presence of myocardial dyskinesia and epicardial cyanosis distal to the occluded coronary artery. After 30 or 45 min, the ischemic myocardium was reperfused by loosening the ligature. After 2 h of reperfusion, the left anterior descending coronary artery was again occluded and patent blue dye was injected into the jugular vein, staining blue the myocardial tissue outside the area at risk (AAR). The heart was then excised, dissected into 4–5 slices (1–2 mm in thickness), and separated into the blue-stained normal zone and the myocardium AAR for injury. The myocardial pieces were then incubated in 1% 2,3,5-triphenyltetrazolium chloride for 15 min at 37°C staining viable tissue red, whereas nonviable infarct tissue failed to stain and remained white. The heart was then placed overnight in 10% formaldehyde. The next day the infarct tissue was dissected from the AAR under a microscope. IS and the AAR were assessed gravimetrically and expressed as a percent of AAR (IS/AAR%).
Methadone and Morphine Dose-Response Curves
Morphine sulfate and methadone (Sigma Biochemicals) were dissolved in water in varying concentrations to achieve a final volume between 0.2 and 0.3 mL. All agents and placebo were administered as a bolus via the right jugular vein. Eleven groups of rats (n = 6–8/group) were used, with 1 group administered placebo (water), 5 groups given methadone (doses of 0.03, 0.10, 0.30, 1, or 3 mg/kg), and 5 groups given morphine (doses of 0.03, 0.10, 0.30, 1, or 3 mg/kg) 30 min before coronary artery occlusion. Rats then underwent 30 min of myocardial ischemia and 2 h of reperfusion followed by determination of myocardial IS.
Opioid Receptor Antagonism with Naltrindole
Rats were administered the δ-opioid antagonist naltrindole (5 mg/kg) (Tocris, Ellisville, MO) 5 min before receiving methadone (0.3 mg/kg), morphine (0.3 mg/kg), or placebo given 30 min before coronary artery occlusion. Rats then underwent 30 min of myocardial ischemia and 2 h of reperfusion followed by determination of myocardial IS.
Timing of Methadone Administration
Rats were administered methadone (0.3 mg/kg) 5 min before reperfusion or 10 s after reperfusion. The rats underwent 30 min of myocardial ischemia and 2 h of reperfusion followed by determination of myocardial IS.
Length of Ischemia
A final set of rats subjected to 45 min of myocardial ischemia were administered methadone (0.3 mg/kg), morphine (0.3 mg/kg), or placebo 5 min before myocardial reperfusion. Myocardial IS was determined after 2 h of reperfusion.
An experimental group size of 6 animals was determined necessary to achieve at least a 15% minimal difference in myocardial IS (α <0.05, β <20%). All values were noted as mean ± sem. GraphPad Prism Software (GraphPad, La Jolla, CA), including the QuickCalcs online calculator, was used to perform statistical analysis and to calculate P values. To determine statistical significance for myocardial IS and hemodynamics, we performed a 1-way analysis of variance with Bonferroni correction comparing each group to the placebo-treated group. In addition, the rats receiving naltrindole + morphine or naltrindole + methadone were statistically compared using a 1-way analysis of variance with Bonferroni correction with groups treated with morphine (0.3 mg/kg given 30 min before ischemia) or methadone (0.3 mg/kg given 30 min before ischemia), respectively.
We used 159 animals to obtain 152 successful experiments. Five rats were excluded secondary to intractable ventricular fibrillation during myocardial occlusion or reperfusion (1 placebo undergoing 30 min of ischemia, 1 morphine at the 3.0 mg/kg dose, 1 naltrindole + morphine, 1 naltrindole + methadone, and 1 placebo with 45 min of ischemia). Two additional rats were excluded secondary to the suture breaking during patent blue staining to determine the AAR (1 morphine at the 3.0 mg/kg dose and 1 morphine at the 0.1 mg/kg dose).
Heart rate, mean arterial blood pressure, and rate pressure product results are shown in Table 1. The methadone groups receiving 1.0 or 3.0 mg/kg doses had significant differences in heart rate and rate pressure product during myocardial ischemia and reperfusion compared with the placebo group. Mean arterial blood pressure was lower in the methadone 3.0 mg/kg group than in the placebo group during myocardial ischemia. Mean arterial blood pressure and rate pressure product at reperfusion were different in the morphine 0.3 mg/kg group compared with the placebo group. A difference in rate pressure product occurred in the morphine + naltrindole group compared with the placebo group at reperfusion.
Myocardial IS Results
Methadone and Morphine Dose Response
Myocardial IS for the methadone, morphine, and control groups treated before induced myocardial ischemia are shown in Figure 2A. Several methadone doses reduced myocardial IS compared with the placebo group. The largest reduction in IS occurred with the 0.3 mg/kg methadone dose (versus placebo, P < 0.001). IS reduction for the morphine doses was similar to methadone doses, with maximal reduction of IS with the 0.3 mg/kg morphine dose (versus placebo, P < 0.001).
δ-Opioid Receptor Inhibition via Naltrindole
Myocardial IS/AAR% for the methadone, morphine, and placebo groups with or without naltrindole administration are shown in Figure 2B. The presence of naltrindole abrogated the reduction in myocardial IS from methadone (naltrindole + methadone, P < 0.001 versus methadone) and morphine (naltrindole + morphine, P < 0.001 versus morphine). Naltrindole given alone had no effect on myocardial IS (P = 0.1498 versus placebo).
Timing of Methadone Treatment
Myocardial IS results for animals given methadone (0.3 mg/kg) before myocardial ischemia, before reperfusion, or after reperfusion are shown in Figure 3A. Methadone reduced myocardial IS when administered 5 min before reperfusion compared with placebo; however, methadone did not abrogate IS when given 10 s after reperfusion (P < 0.001 and P = 0.675 versus placebo, respectively).
Myocardial IS After 30 or 45 min of Induced Ischemia
Myocardial IS results after administration of methadone (0.3 mg/kg) or morphine (0.3 mg/kg) given 5 min before reperfusion after either 30 or 45 min of myocardial ischemia are shown in Figure 3B. We observed no difference in myocardial IS between rats given placebo that underwent 30 or 45 min of ischemia (P = 0.069). However, administration of either methadone or morphine failed to salvage the myocardium after 45 min of ischemia (P = 0.867 and P = 0.836 versus placebo with 45 min of ischemia, respectively).
Our results demonstrated that methadone, similar to morphine, reduces myocardial IS in an in vivo rat model in a dose-dependent manner. The δ-opioid receptor mediates myocardial protection observed with methadone when given either before or during myocardial ischemia. Neither methadone nor morphine reduced myocardial IS when the myocardial ischemic time was lengthened to 45 min.
We found that both methadone and morphine demonstrated maximal myocardial IS-sparing effects at a dose of 0.3 mg/kg. The opioid-induced myocardial salvage effect was blocked by the δ-opioid antagonist naltrindole, which has a reported affinity approximately 200 times greater for δ-opioid receptors compared with μ- or κ-opioid receptors.8 Our findings are consistent with previous studies evaluating morphine in intact rats and in isolated cardiomyocytes that have demonstrated cardioprotection via δ-opioid receptor activation.3,4,9 In addition, other studies have shown that the μ-opioid receptor agonists remifentanil and fentanyl provide myocardial IS reduction by activation of the δ-opioid receptor.10,11
We noted reduced myocardial protection for methadone and morphine at doses larger than 0.3 mg/kg. These findings are consistent with other studies using the selective δ-opioid receptor agonist fentanyl isothiocyanate and the μ-selective agonist remifentanil, which both showed a lesser degree of myocardial salvage when doses larger than the optimal IS reduction dose were given.12,13 The mechanisms causing an attenuated myocardial protection for opioids at a dose larger than the optimal are not known.
In our study, methadone failed to salvage myocardium when given immediately after reperfusion. Our previous studies showed that both the κ-opioid agonist, U-54086, and morphine failed to salvage myocardium when given immediately after reperfusion.14,15 However, BW373U86 (a δ1-specific opioid agonist that has a 10-fold greater binding affinity for δ-opioid receptors than μ-opioid receptors), unlike μ-selective and κ-selective opioid agonists, can salvage the myocardium when given immediately after reperfusion.15,16 Hence, a more selective δ-opioid receptor agonist such as BW373U86 may provide a longer window of myocardial salvage compared with morphine and methadone (which both have approximately 1000 times lower affinity for the δ-opioid receptor compared with the μ-opioid receptor5).
The consequence of the duration of myocardial ischemia on pharmacological or ischemic myocardial protection is largely understudied. Our results showed that lengthening myocardial ischemia from 30 to 45 min duration inhibited the cardioprotective effects of morphine and methadone. A previous study in canines showed a preconditioning stimulus of either 1 or 4 cycles of 5 min myocardial ischemia and 5 min reperfusion failed to reduce myocardial IS when the ischemia time was lengthened from 60 to 90 min.17 Taken together, these data suggest that both opioids and ischemic preconditioning-induced myocardial protection are limited by the duration of myocardial ischemia. Additional myocardial salvage modalities, such as ischemic postconditioning or other pharmacological agents, need to be evaluated.
Our study has several potential limitations that need to be considered while interpreting the data. Naltrindole is a nonspecific δ1- and δ2-opioid receptor antagonist; therefore, the specific opioid receptor subtype that elicits myocardial IS reduction cannot be concluded from this study. Previous data from isolated cardiomyocytes and intact rat hearts suggest that the δ1-opioid receptor is primarily involved in myocardial IS reduction.4,14,15 Furthermore, the κ-opioid receptor was not investigated in this study; however, the κ-opioid receptor antagonist, norbinaltorphimine, partially abrogated remifentanil-induced IS reduction.10 We did not investigate the role of various molecular signaling cascades activated by methadone in myocardial ischemic protection. Nonetheless, signaling pathways of myocardial IS reduction via δ-opioid receptor activation have been studied, indicating that the mechanism involves inhibition of glycogen synthase kinase 3β and the mitochondrial permeability transition pore.18,19 Different animal species and humans could cause variability for the dose-response curves of morphine and methadone-induced myocardial IS reduction as observed in our study.20
Presuming that daily methadone or morphine does not downregulate cardiac δ-opioid receptors, our findings indicate that myocardial protection occurs even with repeated methadone or morphine doses. A previous study in mice with implanted morphine tablets showed that myocardial ischemic protection is enhanced with continuous drug infusion, an effect that may be independent of opioid tolerance.21 Therefore, our data support future studies as to whether daily opioid users have improved myocardial salvage after myocardial infarction compared with nonopioid users.
The authors thank Patricia Rohrs for proofreading the manuscript.
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