Although reperfusion is indispensable for the cell to survive under ischaemic stress, it paradoxically leads to cell death. In fact, during the early stage of reperfusion, calcium overload and generation of free radicals may lead to cardiomyocyte apoptosis and necrosis. This response is of clinical relevance and interventions that can reduce reperfusion injury in the setting of ischaemia–reperfusion have been extensively explored .
It is known that opioids may be part of the myocardium endogenous protective response to ischaemia–reperfusion injury . Bolling et al.  conducted a series of studies in which, using cardioplegia and hypothermia to simulate a hibernation-like state, they demonstrated that administration of opioid antagonists, and in particular δ-opioid antagonists, during ischaemia did indeed limit functional recovery when compared with untreated hearts.
Recently, much research in the ischaemia–reperfusion field has focused on delineating the temporal characteristics of acute pharmacological preconditioning, with evidence shifting the paradigm from preischaemic protection to reperfusion-mediated protection. Pharmacological preconditioning has had little clinical impact because patients with acute myocardial infarction usually present in the clinic or intensive care unit after the index ischaemia has begun. A more recent area that may have great clinical importance concerns the role of morphine administration at reperfusion targeting myocardial ischaemia–reperfusion injury [2–4]. Indeed, a beneficial effect has been shown with opioid treatment at reperfusion [5,6]. However, only a few studies have examined the effects of opioid agonist administration solely at the postischaemic period; hence, the mechanisms underlying the cardioprotective effect of opioids at reperfusion are still not fully understood.
It has long been recognized that stress response kinases are activated during ischaemia and reperfusion and a delicate balance between prodeath and survival pathways exists and seems to determine the fate of the stressed cell [7,8]. Thus, pharmacological interventions that can change the balance of activation of kinase signalling pathways towards increased activation of prosurvival signalling appear to limit myocardial injury. Agents such as insulin, erythropoietin, adenosine and natriuretic peptides are all shown to reduce myocardial infarct size either through the activation of the prosurvival signalling pathways, Akt (also known as ‘protein kinase B’) and ERK1/2 (extracellular signal-regulated kinases), or through the inactivation of prodeath pathways such as p38 MAPK (p38 mitogen-activated protein kinase) and JNK (c-jun N-terminal protein kinase) [7–9]. Furthermore, phosphorylation of small heat-shock proteins (HSPs) like HSP27 is shown to induce myocardial protection or adaptation to ischaemia . On the basis of this evidence, we investigated whether similar mechanisms are involved in the morphine-induced cardioprotection. Thus, we measured the activation pattern of these kinases after 45 min of reperfusion to identify whether differences exist between treated and nontreated hearts.
Previous studies [5,6] investigated the cardioprotective effect of morphine when administered only during the first minutes of reperfusion. However, this experimental design hardly resembles the clinical scenario, in which morphine is administered after an acute ischaemic insult during reperfusion without time restriction or postoperatively in cardiac surgery for pain management. Therefore, in the present study, we explored the effects of morphine administration during the whole period of reperfusion after an index ischaemia as well as potential molecular mechanisms underlying this response. This is of important clinical value, because morphine is used postoperatively in cardiovascular surgery  and in the emergency management of patients suffering from acute myocardial infarction [11,12].
Materials and methods
Twenty male Wistar rats (320–400 g) were used for this study. The rats were handled in accordance with the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH Pub. No. 83–23, Revised 1996).
Isolated heart preparation
A nonworking isolated rat heart preparation was perfused at a constant flow according to the Langendorff technique. An intraventricular balloon allowed measurement of contractility under isovolumic conditions. Left ventricular balloon volume was adjusted to produce an average initial left ventricular end-diastolic pressure (LVEDP) of 6–8 mmHg in all groups and was held constant thereafter throughout the experiment. As the balloon was not compressible, left ventricular contraction was isovolumic. As intraventricular volume was maintained at a constant value, diastolic fibre length, which represented preload, did not change. Thus, the left ventricular peak systolic pressure and the left ventricular developed pressure (LVDP), defined as the difference between left ventricular peak systolic pressure and LVEDP, represented contractility indexes obtained under isovolumetric conditions [13,14].
Rats were anaesthetized with ketamine HCl (100 mg kg−1) and heparin 1000 IU was given intravenously before thoracotomy. The hearts were rapidly excised, placed in ice-cold Krebs–Henseleit buffer (composition in mmol l−1: sodium chloride 118.5, potassium chloride 4.7, potassium phosphate monobasic 1.2, magnesium sulphate 1.2, calcium chloride 1.4, sodium bicarbonate 25 and glucose 11) and mounted on the aortic cannula of the Langendorff perfusion system. Perfusion with oxygenated (95% O2 /5%CO2) Krebs–Henseleit buffer was established within 60 s after thoracotomy. The perfusion apparatus was heated to ensure a temperature of 37°C throughout the course of the experiment. In our experimental design, sinus node was removed and hearts were paced at 320 beats min−1 with a Harvard pacemaker. The pacemaker was turned off during ischaemia. The water-filled balloon, connected to a pressure transducer and coupled to a Gould RS 3400 recorder, was advanced into the left ventricle through an incision in the left atrium. Pressure signal was transferred to a computer using a data analysis software (IOX Emka Technologies, Paris, 75015, France), which allowed continuous monitoring and recording of heart function.
Hearts were removed, isolated and perfused in a Langendorff preparation. Isolated control hearts were subjected to 15 min of stabilization, 30 min of zero-flow global ischaemia and 45 min of reperfusion (CONT; n = 10). Additional hearts were subjected to 15 min of stabilization, 30 min of zero-flow global ischaemia and 45 min of reperfusion, whereas morphine hydrochloride (1 μ mol l−1) was added to the perfusion medium during reperfusion (MORPH; n = 10).
At the end of the experimental protocol, the left ventricle was isolated, frozen in liquid nitrogen and used for determination of kinase signalling activation.
Measurement of mechanical function
Left ventricular function was assessed by recording the LVDP, which was measured at the end of the stabilization period (LVDP, mmHg), and the positive and negative first derivative of LVDP (+dp dt−1and −dp dt−1 in mmHg s−1). Postischaemic myocardial function was assessed by the recovery of LVDP and expressed as percentage of the baseline value (LVDP%) as well as by LVDP, +dp dt−1 and −dp dt−1 at 15, 30 and 45 min of reperfusion. Diastolic function was assessed by monitoring isovolumic LVEDP (mmHg) as a measure of diastolic chamber distensibility. LVEDP was measured after 45 min of reperfusion (LVEDP45). Development of myocardial contracture during the first minutes of reperfusion was assessed by the maximal increase in LVEDP (LVEDPmax, mmHg).
Protein isolation, sodium dodecyl sulphate-protein polyacrylamide gel electrophoresis and immunodetection
Determination of protein expression was performed as previously described. Left ventricular tissue was homogenized in ice-cold buffer containing 10 mmol l−1 Tris-HCl; pH, 7.5; 3 mmol l−1 ethylenediaminetetraacetic acid; 1 mmol l−1 phenylmethanesulphonyl fluoride; 30 μmol l−1 leupeptin; 1 mmol l−1 Na3VO4 and Triton X-100 0.1% with a Polytron homogenizer. The resulting homogenate was centrifuged at 10000 g for 10 min at 4°C. The supernatant (Triton-soluble) corresponded to the cytosol-membrane fraction and was kept at −80°C for further processing. Protein concentrations were determined by the bicinchoninic acid (BCA) method, using bovine serum albumin as a standard [13,15].
Samples were prepared for sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) by boiling for 5 min in Laemmli sample buffer containing 5% 2-mercaptoethanol. Aliquots (40 μg) were loaded onto 7.5 or 9% (w/v) acrylamide gels and subjected to SDS-PAGE in a Bio-Rad Mini Protean gel apparatus (Bio-Rad, Foster City, California, USA). Following SDS-PAGE, proteins were transferred electrophoretically to a nitrocellulose membrane (Hybond ECL; Amersham, Sunnyvale, California, USA) at 100 V and 4°C, for 1.5 h using Towbin buffer for western blotting analysis. Subsequently, filters were probed with specific antibodies against total p38 MAPK and dual phospho-p38 MAPK, total JNKs and dual phospho-JNKs, total ERK and dual phospho-ERK, total AKT and dual phospho-AKT (dilution 1: 1000; Cell Signaling Technology, Irvine, California, USA) overnight at 4°C, and total and phosphorylated HSP27 (Santa Cruz Biotechnology, SantaCruz, California, USA sc-1049, 1: 1000, 1 h at room temperature, Cell Signaling, #2401, dilution 1: 1000, overnight at 4°C). Filters were incubated with appropriate antimouse (Amersham) or antirabbit (Cell Signaling) HRP secondary antibodies and immunoreactivity was detected by enhanced chemiluminescence using Lumiglo reagents (New England Biolabs, San Diego, California, USA) and exposed to Hyperfilm paper (Amersham). For comparisons between groups, samples from each group were loaded on the same gel. Data were expressed as the ratio of phosphorylated to total protein for each case. Immunoblots and gels were quantified using the AlphaScan Imaging Densitometer (Alpha Innotech Corporation, San Leandro, California, USA).
Determination of oxidized actin
Samples were prepared for SDS-PAGE by boiling for 5 min in Laemmli sample buffer but without 2-mercaptoethanol, which is a reducing agent and brakes bisulphide bonds. Aliquots (40 μg) were loaded on 9% (w/v) acrylamide gels and subjected to SDS-PAGE in a Bio-Rad Mini Protean gel apparatus (Bio-Rad). Following SDS-PAGE, proteins were transferred electrophoretically to a nitrocellulose membrane (Hybond ECL) at 100 V and 4°C, for 1.5 h using Towbin buffer for western blotting analysis. Subsequently, filters were probed with specific antibody against α-actin (Sigma, San Diego, California, USA; 1: 1000, overnight, 4°C), incubated with appropriate antimouse (Amersham) HRP secondary antibodies and immunoreactivity was detected by enhanced chemiluminescence. Actin band is normally detected around 40 kDa. However, oxidized actin forms bisulphide cross-bridges and can be detected after western blot analysis only under nonreducing conditions as high-molecular mass bands . Two high-molecular mass bands (at around 70 and 95 kDa) of oxidized actin could be detected in hearts subjected to ischaemia–reperfusion and not in isolated perfused hearts. Immunoblots and gels were quantified using the AlphaScan Imaging Densitometer (Alpha Innotech Corporation) and data were expressed as the ratio of oxidized to total actin for each sample .
Measurement of lactate dehydrogenase release
Coronary effluent was collected during 45 min of reperfusion and was used for the measurement of lactate dehydrogenase (LDH) activity in IU l−1 spectrophotometrically (LDH UV Fluid, Rolf Greiner Biochemica, Flacht, Germany). The mean activity of LDH in IU l−1 was determined at the end of the experiment in the total perfusate collected from the heart during the 45 min of the reperfusion period as previously described . Given the total volume of the perfusate collected, the total mean LDH released through reperfusion was estimated and expressed per gram of tissue. LDH release was used to estimate the extent of myocardial injury.
Values are presented as mean ± SEM. Unpaired t-test and Mann–Whitney test were used for differences between the two groups. Correlations between LDH release and functional measurements were evaluated by the Pearson product-moment (Pearson r). Comparisons between groups in functional parameters at different time-points of reperfusion were assessed by multiple way analysis of variance (ANOVA). A P value less than 0.05 was considered significant.
Effect of morphine administration on left ventricular function
Baseline contractile indices were not different between CONT and MORPH (Table 1). Values of LVDP, +dp dt−1 and −dp dt−1 after 30 min of ischaemia and 15, 30 and 45 min of reperfusion are shown in Table 1. There was a small trend towards increased contractile function in morphine-treated hearts; however, this difference never reached statistical significance at any time-point of reperfusion (P = NS; Fig. 1). LVEDPmax (mmHg), usually reached during the first 3–4 min of reperfusion, was found to be 125.1 ± 5.7 in CONT and 116.8 (8.3) in MORPH hearts (P = NS). Furthermore, LVEDP at 45 min of reperfusion (mmHg) was 61 ± 4.2 for CONT and 58 ± 6.4 for MORPH hearts (P = NS).
Effect of morphine administration on lactate dehydrogenase release and correlation with recovery of function
LDH release (IU g−1) was shown to be significantly reduced in MORPH as compared with CONT hearts (7.2 ± 0.3 vs. 8.8 ± 0.6, P < 0.05 respectively), demonstrating reduced myocardial injury after morphine administration at reperfusion (Fig. 2a). However, the recovery of function at 45 min of reperfusion was 50.1% ± 6.8 in MORPH and 44.07% ± 5.5 in CONT hearts (P = NS). Thus, in control hearts, myocardial injury seemed to match with recovery of left ventricular function, whereas dissociation between these two variables was shown in morphine-treated hearts. In fact, recovery of function was shown to have a strong negative correlation with LDH release in CONT hearts (r = −0.8, P = 0.006), whereas in MORPH hearts, no correlation was found (Fig. 2b and 2c).
Effect of morphine administration on intracellular kinase signalling
Phosphorylated levels of the proapoptotic p38 MAPK and JNKs at 45 min of reperfusion between groups are shown (Fig. 3a and 3b). The levels of phosphorylated p38 MAPK and JNKs in response to ischaemia and reperfusion were similar in both groups (P = NS).
Phosphorylated levels of the prosurvival ERK and Akt at 45 min of reperfusion between groups are shown in Fig. 4a and 4b. The levels of phosphorylated ERKs and Akt in response to ischaemia and reperfusion were also similar in both groups (P = NS).
The ratio of phosphorylated HSP27 to total HSP27 was found to be increased 1.5-fold in MORPH hearts as compared with CONT hearts (P < 0.05). No difference was found in total HSP27 levels between the two groups (Fig. 5).
Effect of morphine administration on oxidized actin levels
As shown in Fig. 6, oxidized actin bands could be detected only in hearts subjected to ischaemia–reperfusion and not in hearts only perfused in Langendorff apparatus. Furthermore, the ratio of oxidized actin to total actin was found to be 1.9-fold more in MORPH as compared with CONT hearts (P < 0.05; Fig. 6).
Postischaemic recovery vs. myocardial injury
Morphine administration at reperfusion resulted in a significantly lower LDH release in the perfusate, indicating that morphine treatment can limit the extent of ischaemic injury. This finding is in accordance with previous reports. Recently, Chen et al. examined the effects of three different doses of morphine (0.3, 3 and 30 μmol l−1) upon reperfusion in rats. These authors showed that morphine protects the heart against reperfusion injury in a dose-independent manner by mimicking postconditioning in the isolated rat hearts, and both the mitochondrial-Katp channel and κ-opioid receptors mediated this cardioprotective response. Jang et al. also advocated that 1 μmol l−1 of morphine administration at reperfusion in an isolated rat heart model resulted in reduced infarct size, though recovery of function was not evaluated. In addition, in an in-vivo experimental rat model, Gross et al. showed considerable reduction in infarct development following activation of opioid receptors at reperfusion with either morphine or the selective δ-opioid receptor ligand, BW373U86. The same authors  also demonstrated that the cardioprotective effects of opioid stimulation at reperfusion are mediated via glycogen synthase kinase beta (GSK-β) and the phosphatidyl-inositol-3 kinase (PI3K) pathway.
In our study, morphine administration after the ischaemic event led to dissociation between postischaemic recovery of function and the extent of ischaemic injury. In fact, a dose of 1 μmol l−1 morphine limited the LDH release while having no effect on postischaemic recovery of LVDP. This response is different from that observed in previous studies demonstrating that opioid administration before the ischaemic event resulted in improved cardiac function. In fact, a dose of 40 mg given prior to extracorporeal circulation (ischaemic event) has been shown to improve cardiac function in patients undergoing coronary artery by-pass graft . Similarly, in an experimental model of ischaemia–reperfusion, Benedict et al. also showed that morphine administration before ischaemia led to myocardial protection and also improved postischaemic contractile function.
In our study, morphine administration at reperfusion resulted in different haemodynamic effects from those occurring when the compound had been given in the preischaemic period. In our study, morphine administered solely at reperfusion was found to significantly increase the oxidation of actin. It is well known that oxidative modifications of actin are very likely to hamper the contractile function of the heart [21,22]; hence, morphine administration at reperfusion may reduce cardiac contractility via actin oxidation
Potential underlying mechanisms of morphine limiting effect on myocardial injury
Opioids are implicated in both early [20,23] and delayed cardioprotection , a response-mediated via δ1-opioid receptor . Opioid-induced cardioprotection appears to involve downstream signalling pathways, including JAK2 (janus kinase 2), STAT3 (signal transducers and activator of transcription protein 3), PI3K-dependent proteins and Akt . Opioids can also act through the Gi-linked pathway, which in turn may stimulate Ras-Raf signalling pathway and ERK activity . In fact, blockade of ERK or p38 MAPK by specific inhibitors given before administration of δ-opioid receptor agonist abolished late cardioprotection . On the basis of this evidence, we investigated the possible involvement of this kinase signalling in morphine-mediated cardioprotection. Interestingly, our study showed no difference in the phosphorylated levels of all kinases measured between the untreated and morphine-treated hearts at 45 min of reperfusion. However, Gross et al. previously showed that, in an in-vivo model of rat coronary occlusion and reperfusion, morphine administration increased activation of Akt and ERK1 after 5 min of reperfusion. As the activation pattern of kinase signalling has not been studied at earlier time-points of reperfusion in the present study, a transient effect of morphine on these kinases cannot be excluded. Further studies are needed to clarify whether this discrepancy is due to different time-points studied or due to differences in experimental models used.
One of the most well studied molecules in regard to cardioprotection is the small HSP27. HSP27 is antiapoptotic and overexpression of HSP27 is shown to protect the integrity of microtubules and actin cytoskeleton in cardiac myocytes and endothelial cells exposed to ischaemia [1,29]. Phosphorylation of this protein is induced by several stressful stimuli and is required for its protective function . Interestingly, stimulation of δ-opioid receptor by opioids administered before ischaemia was shown to increase phosphorylation of HSP27 and conferred cardioprotection . Similarly, in this study, morphine administration at reperfusion increased the phosphorylation of HSP27 and limited LDH release. Taken together, it seems that morphine administration at reperfusion limits myocardial injury and this could at least in part be attributed to increased phosphorylation of HSP27. It is known that phosphorylation of HSP27 can be mediated through p38 MAPK/MAPKAP2 or can be p38-independent [31,32]. In the present study, activation of p38 MAPK was not found to be different between treated and nontreated hearts at 45 min of reperfusion, implying either an earlier activation of p38 during the first minutes of reperfusion or a direct effect of morphine on HSP27 phosphorylation state.
In conclusion, morphine administration at reperfusion fails to improve postischaemic cardiac function probably due to increased actin oxidation. However, morphine limits the extent of myocardial injury and increased HSP27 phosphorylation might be at least in part involved in this response.
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Keywords:© 2009 European Society of Anaesthesiology
HSP27; ischaemia–reperfusion; ischemia; MAP kinases; morphine