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1Department of Anesthesiology, Oregon Health and Sciences University, and Departments of 2Research and 3Anesthesiology Services, Veterans Affairs Medical Center, Portland, Oregon 97201; and 4Department of Anesthesiology and Resuscitology, Ehime University School of Medicine, Ehime, Japan 791-0295
Submitted 17 April 2003 ; accepted in final form 10 July 2003
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ABSTRACT |
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 TOP ABSTRACT METHODSRESULTSDISCUSSIONDISCLOSURESREFERENCES |
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60% compared with saline-treated controls. ME-induced protection was sensitive to opioid receptor blockade with naloxone [NAL 50 ± 2% vs. ME + NAL 39 ± 3%, P = not significant (NS)] and also to blockade of sarcolemmal and mitochondrial ATP-sensitive K+ (KATP) channels [5-hydroxydecanoate (5-HD) 33 ± 3% vs. ME + 5-HD 43 ± 8%, P = NS; and HMR-1098 60 ± 3% vs. ME + HMR-1098 54 ± 7%, P = NS]. We conclude that ME limits ischemic injury in vivo by an opioid receptor-mediated mechanism that involves both sarcolemmal and mitochondrial KATP channels.
ischemia; infarction; ischemic preconditioning; myocardial peptides; opioid
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METHODS |
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TOPABSTRACTMETHODSRESULTSDISCUSSIONDISCLOSURESREFERENCES |
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Surgical preparation. After the induction of anesthesia with 30 mg/kg iv pentobarbital sodium, male New Zealand White rabbits (1.9–3.3 kg) were intubated and mechanically ventilated using a ventilation rate of 30–35 breaths/min and a tidal volume of 15–20 ml. End-tidal PCO2 was continuously monitored. Ventilation was adjusted to maintain arterial pH between 7.36 and 7.44. A polyethylene (PE) catheter (PE-90) was advanced into the carotid artery for measurement of arterial pressure. Core body temperature was measured with an esophageal temperature probe and maintained at 38.5°C with a heating blanket. Electrocardiogram, heart rate (HR), and arterial pressure were recorded continuously.
A left thoracotomy was performed in the fourth intercostal space, and the pericardium was opened to expose the heart. A 4-0 suture on a curved taper needle was passed around the proximal segment of a dominant left coronary artery, and the ends of the suture were passed through a short segment of small diameter tubing to form a snare.
Induction of ischemia. Myocardial ischemia was elicited by tightening the snare around the coronary artery and was confirmed by ECG changes, regional myocardial cyanosis, and regional akinesis. Reperfusion was accomplished by release of the snare and was confirmed by visible epicardial hyperemia. All rabbits were subjected to 30-min regional myocardial ischemia and 3-h reperfusion. At the beginning of reperfusion, each rabbit received 1,000 IU heparin intravenously. At the end of the 3-h reperfusion period, hearts were excised, and infarct size was assessed as described below.
Measurement of the area at risk and infarct size. Upon completion of the reperfusion period, the heart was excised and transferred to a Langendorff apparatus and perfused with normal saline for 1 min at a pressure of 100 cmH2O to flush out intravascular blood. The coronary artery was then reoccluded, and fluorescent particles (3 ml of a 1.2 mg/ml suspension of zinc cadmium sulfide) were infused. These particles fluoresce bright yellow under ultraviolet light and thus delineate the area at risk as a negative image. The heart was then removed from the Langendorff apparatus, trimmed of atria and great vessels, and weighed. The heart was cut into transverse slices 2 mm thick and incubated in triphenyltetrazolium chloride for 20 min [1% (wt/vol) in sodium phosphate buffer at 37°C, pH 7.4]. Myocardium that did not stain red was presumed to be infarcted. The slices were then placed in 10% neutral buffered formalin for 10–15 min to increase the contrast between stained and unstained tissue. Risk and infarct areas (in cm2) for each slice were traced and digitized using computer-assisted planimetry (SigmaScan software, Jandel Scientific; Corte Madera, CA; model 2210-0.43.C digitizer, Numonics; Montgomeryville, PA). The volume (in cm3) of myocardium at risk and infarcted myocardium was calculated from the planimetered areas and slice thickness. Infarct size was normalized as a percentage of the area at risk. All infarct size analyses were performed in a blinded fashion.
Treatment groups. To determine whether sustained administration of ME limits myocardial infarct size and does so in an opioid receptor-mediated fashion that involves mitochondrial KATP channels, rabbits were randomly assigned to receive one of six treatments: control (CON/1; saline vehicle, 9.9 µl/h), ME (ME/1; 0.125 mg · kg–1 · h–1, 9.9 µl/h), saline vehicle plus naloxone (NAL; 3 mg/kg + 70 µg · kg–1 · min–1 iv), ME + NAL, saline vehicle plus 5-hydroxydecanoate (5-HD; 5 mg/kg iv), and ME + 5-HD. ME and saline vehicle were delivered for 24 h by subcutaneously implanted osmotic minipump (model 2ML1, Alzet; Cupertino, CA). NAL was administered for 60 min beginning 30 min before occlusion. 5-HD was given as a bolus 5 min before occlusion. These experiments are referred to as series 1.
Subsequently, to test the involvement of sarcolemmal KATP channels, rabbits were randomly assigned to receive one of four treatments: control (CON/2; saline vehicle, 9.9 µl/h), ME (ME/2; 0.125 mg · kg–1 · h–1, 9.9 µl/h), saline vehicle plus HMR-1098 (HMR; 5 mg/kg iv), or ME + HMR. HMR was given as a bolus 5 min before occlusion. These experiments are referred to as series 2.
To determine whether acute administration of ME limits myocardial infarct size, rabbits were randomly assigned to receive one of three treatments: control [saline vehicle (SAL group)], ME (10 mg/kg iv), and IP (IPx2; a positive control for the ability to observe infarct reduction). ME and saline vehicle were delivered over 5 min by a syringe pump beginning 10 min before coronary occlusion. IP was elicited with two cycles of 5-min ischemia beginning 20 min before coronary occlusion. These experiments are referred to as series 3. The experimental time lines for all three series are shown in Fig. 1.
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Statistics. Data analysis was performed with a personal computer statistical software package (Prism 3.0, GraphPad Software) and was done separately for each series. Hemodynamics were analyzed using two-way ANOVA with Bonferroni post hoc testing for multiple comparisons. Infarct size (expressed as a percentage of the area at risk) and the size of the area at risk were analyzed between groups using ANOVA with a Student-Newman-Keuls post hoc test for multiple comparisons. Because infarct size varies as a function of risk area, and this relationship has a nonzero x-intercept, infarct data were also analyzed using other techniques. Differences in infarct volume (in cm3) between groups were assessed using analysis of covariance (ANCOVA) with risk volume (in cm3) as the covariate. Additionally, linear regression was performed for pooled area at risk and infarct data of corresponding vehicle control and drug treatment groups. The vertical difference of the individual data points from the common regression line (residuals) was then calculated separately by group. The means of these differences were compared by ANOVA. The resulting P value indicated the probability that the distribution of the data was produced by chance. If this P value was lower than 0.05, we concluded that there was a significant difference between the compared groups. Data are expressed as means ± SE, and statistical significance was assumed for P < 0.05.
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RESULTS |
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TOPABSTRACTMETHODSRESULTSDISCUSSIONDISCLOSURESREFERENCES |
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HR tended to be lower during reperfusion compared with baseline; this was statistically significant in CON/1, ME/1, and NAL (series 1) and in SAL, ME, and IPx2(series 3). Similarly, MAP tended to decrease over time and was significantly lower compared with baseline in ME/1, NAL, and ME + NAL (series 1), CON/2, ME/2, and ME + HMR (series 2), and IPx2 (series 3). Differences in HR were observed at 180-min reperfusion between CON/1 and ME/1 (series 1) and at end occlusion between SAL and ME (series 3). Differences in MAP were observed at end occlusion between CON/1 and 5-HD and at 180-min reperfusion between CON/1 and ME/1 (both series 1).
Examination of morphometric data for series 1 and 2 showed that the area at risk was slightly larger in the NAL group; this difference was statistically significant for NAL vs. ME + NAL and NAL vs. 5-HD (P < 0.05). These data are shown in Table 3. The area at risk for series 3 was not different between groups. These data are shown in Table 4. Infarct data for series 1 and 2 are shown in Figs. 2, 3, 4 and for series 3 in Figs. 5 and 6.
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When infarct data were normalized as a percentage of the area at risk, we found that 24-h administration of ME in vivo resulted in an 60% reduction of infarct size compared with saline-treated rabbits (CON/1 40.7 ± 4.8% vs. ME/1 14.7 ± 4.5%, P < 0.01; and CON/2 69.2 ± 3.1% vs. ME/2 29.2 ± 3.7%, P < 0.001). ME-induced protection was reversed by treatment with the nonselective opioid receptor antagonist NAL [NAL 49.9 ± 2.1% vs. ME + NAL 39.2 ± 2.8%, P = not significant (NS)]. NAL treatment by itself did not alter infarct size compared with control. Furthermore, treatment of the animals with 5-HD, a blocker of mitochondrial KATP channels, did not increase infarct size (CON/1 40.7 ± 4.8% vs. 5-HD 33.2 ± 3.2%, P = NS) but did abolish ME-induced infarct limitation (5-HD 33.2 ± 3.2% vs. ME + 5-HD 42.6 ± 8.3%, P = NS). Likewise, administration of HMR, a selective blocker of sarcolemmal KATP channels, did not alter infarct size (CON/2 69.2 ± 3.1% vs. HMR 60.0 ± 3.3%, P = NS) but did eliminate the infarct limitation seen with ME (HMR 60.0 ± 3.3% vs. ME + HMR 54.4 ± 7.1%, P = NS). These data are shown in Fig. 2.
To evaluate the effect of area at risk size on infarct size, infarct volume (in cm3) was also analyzed as a function of risk volume (in cm3). ANCOVA testing showed that infarct volume was significantly reduced by ME treatment compared with vehicle controls in both series 1 and 2 (CON/1 vs. ME/1, P = 0.005; and CON/2 vs. ME/2, P < 0.0001). When the data were analyzed by linear regression, we found that the data points from ME-treated groups (ME/1 and ME/2) predominantly fell below the regression line of their respective controls (CON/1 and CON/2), which is indicative of infarct limitation (Fig. 3). Opposed to this, data points from NAL-, 5-HD-, and HMR-treated animals were interspersed with their respective controls, indicating a lack of protection. ANOVA of the residuals of the data points from the pooled linear regressions (Fig. 4) demonstrated a highly significant effect of ME treatment in both series 1 and 2 (CON/1 0.04 ± 0.04 vs. ME/1 –0.26 ± 0.06, P < 0.001; and CON/2 0.15 ± 0.03 vs. ME/2 –0.17 ± 0.03, P < 0.001). The distribution of ME-treated data points below the pooled regression line was reversed by the opioid receptor antagonist NAL (NAL 0.15 ± 0.03 vs. ME + NAL 0.03 ± 0.03, P = NS) and the mitochondrial and sarcolemmal KATP blockers 5-HD (5-HD –0.02 ± 0.03 vs. ME + 5-HD 0.04 ± 0.08, P = NS) and HMR (HMR 0.04 ± 0.01 vs. ME + HMR 0.07 ± 0.05, P = NS), indicating a blockade of ME-induced protection.
In contrast, we found that 5-min administration of ME in vivo, beginning 10 min before coronary occlusion, did not result in infarct limitation (SAL 61.2 ± 5.5% vs. ME 56.7 ± 6.3%, P = NS). IP did result in profound early protection (SAL 61.2 ± 5.5% vs. IPx2 2.0 ± 1.0%, P < 0.001). Evaluation of the effect of area at risk size on infarct size, by analyzing the infarct volume (in cm3) as a function of risk volume (in cm3), showed that the infarct volume was not reduced by ME treatment compared with the saline control, whereas IP resulted in a marked reduction of infarct volume (ANCOVA; SAL vs. IPx2, P < 0.0001). When the data were analyzed by linear regression, we found that the data points from ME-treated animals (ME group) were interspersed with those from saline-treated controls (SAL group), which is indicative of a lack of protection. Data points from preconditioned hearts (IPx2 group) fell below the regression line of the saline controls (SAL group), which is indicative of infarct limitation (Fig. 6, top). ANOVA of the residuals of the data points from the pooled linear regressions (Fig. 6, bottom) demonstrated a highly significant effect of IP (SAL 0.18 ± 0.06 vs. IPx2 –0.40 ± 0.04, P < 0.001) but no effect of acute ME treatment (SAL 0.18 ± 0.06 vs. ME 0.11 ± 0.05, P = NS). These data are shown in Figs. 5 and 6.
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DISCUSSION |
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TOPABSTRACTMETHODSRESULTSDISCUSSIONDISCLOSURESREFERENCES |
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In 1995, Schultz et al. (20) showed that endogenous opioid peptides participate in IP-induced infarct limitation in rats. We (3) subsequently reported a similar result in rabbits and extended the finding to show that NAL blockade of cardioprotection is stereoselective, supporting the concept of a true opioid receptor-mediated effect. Participation of endogenous opioids in IP in swine has also been reported (21). Exogenous administration of the nonselective opioid agonist morphine also protects the heart against ischemic damage (17, 19), as do the opioid anesthetic fentanyl (12) and the synthetic opioid peptides DADLE and DPDPE (24, 28).
In the present study, we found that sustained administration of the endogenous opioid peptide ME also protects against ischemic damage when administered in vivo. These results are consistent with our previous findings in isolated rabbit cardiomyocytes, in which we demonstrated that the naturally occurring opioid peptides ME, Leu5-enkephalin, and Met5-enkephalin-Arg-Phe (MEAP) protected against simulated ischemia (25). Because ME is rapidly degraded by amino- and carboxypeptidases in vivo (1, 16, 23), it is difficult to compare the concentration of ME in the present study to that used in our previous in vitro study. However, the dose of ME used (0.125 mg · kg–1 · h–1 for 24 h) resulted in a comparable extent of infarct limitation (60% reduction in ischemia-reperfusion), as previously reported by other investigators for synthetic opioid agonists (17, 19), and did so without alterations in systemic hemodynamics. ME-induced infarct limitation was completely blocked by NAL, confirming that the ME-induced cardioprotection is mediated via opioid receptors.
In the present study, we observed infarct limitation after chronic administration of ME for 24 h by continuous subcutaneous infusion with osmotic minipumps. This is in contrast to studies with adenosine by Downey and colleagues (10, 27), in which chronic (72 h) adenosine receptor agonist infusion resulted in tachyphylaxis and a loss of cardioprotection. In those studies, ischemia-reperfusion was instituted immediately after drug administration. However, Dana et al. (6) showed that when repetitive administration of an A1 adenosine receptor agonist over 48 h was followed by ischemia-reperfusion 48 h later, cardioprotection was preserved. Although continuous infusion of an adenosine agonist may not be fully analogous to repetitive bolus administration of an adenosine agonist, it appears that chronic infusion of adenosine may result in a loss of acute cardioprotection but a preservation of delayed protection. In the present study, the 24-h infusion of ME was followed immediately by ischemia-reperfusion; whether preservation of protection despite sustained administration is a result of negligible receptor downregulation/desensitization or a result of a delayed type of cardioprotection is not known. However, transient intravenous infusion of 10 mg/kg ME (5 min beginning 10 min before occlusion) failed to elicit infarct limitation. Robust early-phase cardioprotection was observed in IP hearts. The absence of protection in the acute (series 3) experiments may have been due to the rapid metabolism of the peptide so that there was never a cardioprotective dose present at the cardiomyocytes; however, the dose given (10 mg/kg iv over 5 min) was much higher than that used for the sustained administration protocols (3 mg/kg over 24 h). Alternatively, ME may be able to induce delayed cardioprotection but not acute cardioprotection. Because opioid receptor stimulation was maintained for 24 h by continuous ME administration, the cardioprotection observed could have been a combination of acute and delayed phase protection. Regardless, the ability of a sustained ME infusion to elicit protection suggests that opioid agonists may have the potential to be used as a prophylaxis against ischemic injury.
Although the intracellular signal transduction cascade of IP and pharmacological preconditioning is incompletely described, it is well recognized that KATP channels play a pivotal role. Infarct limitation induced by ME in the present study was blocked by both HMR (the water-soluble derivative of HMR-1883) and 5-HD, suggesting that both sarcolemmal and mitochondrial KATP channel opening participates in the infarct-limiting effect of ME. In contrast, the infarct-limiting effect of classical IP is not blocked by HMR or HMR-1883 (2, 11). In these studies, Birincioglu et al. (2) and Jung et al. (11) used an in vivo rabbit model and administered a dose of HMR-1883 (3 mg/kg iv) comparable to that used in the present study. Despite the failure of HMR-1883 to block infarct limitation, both groups concluded that the dose of HMR-1883 was adequate to block sarcolemmal KATP channels, based on changes in the monophasic action potential duration (11) or ST elevation on the epicardial ECG (2). Similarly, Fryer et al. (8) examined the role of KATP channels on acute infarct limitation induced by the 
1-selective opioid agonist TAN-67 and found that HMR (3 mg/kg iv) was ineffective in eliminating TAN-67-induced infarct limitation, whereas 5-HD abolished the cardioprotection conferred by this synthetic opioid agonist. Thus the results of this study suggest that sarcolemmal KATP channel activation is not involved in acute opioid cardioprotection. However, Patel et al. (18) recently reported that HMR blocked the infarct-limiting effect of transiently administered SNC121 (a synthetic nonpeptide opioid agonist) given 24 h previously, suggesting that sarcolemmal KATP channels can trigger delayed opioid cardioprotection. While our experimental paradigm was somewhat different due to the sustained 24-h infusion of ME, together the aforementioned studies suggest that the HMR-sensitive ME-induced protection may be analogous to delayed opioid-induced cardioprotection.
Series 1–3 were not performed concurrently, and therefore a separate control group was used for each series. This is important, given that infarct size may be affected by the season, animal health/development, temperature, and experimenter (4, 13, 32). Indeed, control (saline vehicle-treated) group infarct size did vary between the experimental series (series 1 40.7 ± 4.8%, series 2 69.2 ± 3.1%, and series 3 61.2 ± 5.5%). Although the precise reason for the dissimilar control infarct sizes is not known, it is notable that series 1 was conducted predominantly in the autumn to winter months, whereas series 2 and 3 were conducted during the spring months.
We believe that the dose of HMR used in the present study (5 mg/kg iv) was adequate given that 3 mg/kg results in ECG changes indicative of sarcolemmal KATP channel blockade (2, 11). A much larger dose would have increased the possibility of nonselectivity of effect (i.e., inhibition of mitochondrial KATP as well as sarcolemmal KATP) (14). We do not believe the dose administered was high enough to be nonselective given that a comparable dose of 6 mg/kg failed to block IP, whereas 10 mg/kg 5-HD did block cardioprotection (7, 8).
In summary, the present data demonstrate that 24-h continuous infusion of the opioid peptide ME reduces infarct size after regional myocardial ischemia-reperfusion in vivo, and this cardioprotective effect is blocked by HMR and 5-HD, indicating participation of both sarcolemmal and mitochondrial KATP channels.
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DISCLOSURES |
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TOPABSTRACTMETHODSRESULTSDISCUSSIONDISCLOSURESREFERENCES |
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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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.
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REFERENCES |
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TOPABSTRACTMETHODSRESULTSDISCUSSIONDISCLOSURESREFERENCES |
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-opioid receptors and protein kinase C in rats. Br J Anaesth 84: 608–614, 2000.-opioid receptor agonists on infarct size reduction in swine. Am J Physiol Heart Circ Physiol 282: H1953–H1960, 2002.-opioid receptors. Am J Physiol Heart Circ Physiol 277: H2442–H2450, 1999.)- and kappa (
)-receptors in ischemic preconditioning in a rat model of myocardial infarction. Methods Find Exp Clin Pharmacol 24: 139–144, 2002.[Web of Science][Medline]
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