Improving myocardial preservation after the arrest

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1 Opioid Preconditioning: Myocardial Function and Energy Metabolism Daniel C. Sigg, MD, James A. Coles, Jr, MS, William J. Gallagher, BS, Peter R. Oeltgen, PhD, and Paul A. Iaizzo, PhD Departments of Anesthesiology and Physiology, and the Biomedical Engineering Institute, University of Minnesota, Minneapolis, Minnesota, and Department of Pathology, University of Kentucky, Lexington, Kentucky Background. Opioid receptor agonists are involved in ischemic preconditioning and natural hibernation. The aim of this study was to determine whether pretreatment with D-Ala2-Leu5-enkephalin or morphine confers cardioprotection in large mammalian hearts. We assessed myocardial functional recovery and global energy metabolism after ischemic cold storage. Methods. After pretreatment with D-Ala2-Leu5- enkephalin, morphine sulfate, or saline (n 6 each), swine hearts were excised and stored for 75 minutes at 4 C, then reperfused in a four-chamber isolated working heart apparatus. Serial myocardial biopsies were performed to assess cellular energy metabolism. Results. Improved systolic (cardiac output, contractility) and diastolic (tau) left ventricular functions were Improving myocardial preservation after the arrest storage reperfusion sequence could: (1) reduce acute allograft failure, which accounts for approximately 20% of early deaths after heart transplantations; and (2) lengthen the clinical limits of myocardial ischemia, which is currently only 4 to 6 hours. Consequently, a pharmacologic approach to preconditioning the heart during cardiac surgical procedures with opioid agonists would be of great clinical interest. Recently, a cardioprotective link between natural hibernation and ischemic preconditioning has been demonstrated [1]. -Opioid receptor stimulation and adenosine triphosphate (ATP)-dependent potassium-channel activation have been shown to be involved in ischemic preconditioning [2 5]. -Opioid stimulation mimics natural hibernation even in nonhibernating animals and is reported to enhance tissue survival when oxygen delivery to the tissue is minimal [6 8]. These findings are of clinical interest, as the altered cellular biology of coldstored hearts may parallel the one observed in natural hibernation. Promising reports on the use of opioid receptor agonists such as d-ala2-leu5-enkephalin (DADLE) [1, 9] and pentazocine [10] as myocardial protectants in a rabbit model of ischemic cold storage have been published. Additionally, nonselective opioid agonists morphine, buprenorphine, and pentazocine have also demonstrated myocardial protective effects after Accepted for publication June 28, Address reprint requests to Dr Iaizzo, Department of Anesthesiology, University of Minnesota, 420 Delaware St SE, MMC 294 UMHC, Minneapolis, MN 55455; iaizz001@tc.umn.edu. observed in hearts pretreated with D-Ala2-Leu5- enkephalin or morphine. These benefits were not correlated with changes in high-energy phosphate levels. Cardiac enzyme leakage (creatine kinase, troponin-i) was similar among treated and control groups. Lactate efflux increased significantly in controls, but not in opioidpretreated hearts (p < 0.01) at 75 minutes of reperfusion. Conclusions. D-Ala2-Leu5-enkephalin and morphine pretreatments improve postischemic function after cold storage of swine hearts. Postischemic lactate reduction, but not high-energy phosphate levels, may account for the observed cardioprotective effects. (Ann Thorac Surg 2001;72: ) 2001 by The Society of Thoracic Surgeons global ischemia and reperfusion in the same model [11]. The concept that hibernation can be induced in nonhibernators, such as humans, by the application of such -opioid agonists [7] has led to the belief that pharmacologic preconditioning has potential applications in humans. Interestingly, naloxone, an opioid receptor antagonist, has been shown to inhibit ischemic preconditioning in humans [12]. And very recently, -opioid receptor stimulation has been shown to mimic ischemic preconditioning in human heart muscle [13]. Although previous experimental studies with opioid agonists and myocardial protection have focused on small mammalian models (rats and rabbits) using the Langendorff perfused isolated heart model, recent evidence has suggested that a true working heart model is a more stringent and critical model for the evaluation of postischemic function [14]. Furthermore, species differences exist as to the intracellular pathways involved in ischemic preconditioning, and thus may distort the interpretation of such studies [15]. Therefore, we performed a large mammalian heart study to investigate pharmacologic preconditioning with opioids and their simultaneous effects on myocardial function and energy metabolism after cold ischemia. In preliminary studies, we tried to reproduce prolonged ( 18 hours) storage using DADLE pretreatment, and none of those hearts regained function. Therefore, we chose to investigate a relatively short period of ischemia to explore potential mechanisms, and possible limitations, of such treatment. More specifically, the present study was designed to answer the following questions: (1) Can pretreatment with the opioid agonists DADLE or morphine improve 2001 by The Society of Thoracic Surgeons /01/$20.00 Published by Elsevier Science Inc PII S (01)

2 Ann Thorac Surg SIGG ET AL 2001;72: OPIOID PRECONDITIONING IN SWINE 1577 myocardial function after cold ischemia in a porcine ex vivo isolated four-chamber ejecting model? (2) What are the effects of these agents on ischemic and postischemic cellular energy metabolism (high-energy phosphates and glycolysis)? (3) Finally, are such opioid pretreatments associated with decreased myocardial damage (water content, cardiac enzyme release)? Material and Methods The following procedure was reviewed and approved by the University of Minnesota Institutional Animal Care and Use Committee, and ensured humane treatment of all animals as indicated by the Guide for the Care and Use of Laboratory Animals (NIH publication 85-23, revised 1985). Animal Preparation After initial sedation with intramuscular xylazine (5 mg/kg) and tiletamine/zolazepam (250 mg), swine (n 18, weight kg [mean standard deviation]) received intravenous thiopental (20 to 25 mg/kg) for intubation. The swine were then mechanically ventilated with a 65% N 2 O and 35% O 2 mixture, and anesthesia was maintained with halothane. Right and left ventricular pressures and derived measurements of positive maximal rate of increase of left ventricular pressure, relaxation time constant (tau), cardiac outputs, and arterial pressures were measured using standard methods. Isolated Heart Preparation After anticoagulation with heparin (300 IU/kg intravenously) a cardioplegia cannula (9F double lumen, Medtronic, Inc, Minneapolis, MN) was introduced into the aortic root. After cross-clamping the aorta and the superior and inferior venae cavae, cold (4 C) St. Thomas s cardioplegic solution (in mmol/l: NaCl, 110; KCl, 16; CaCl 2, 1.2; MgCl 2, 16; NaHCO 3, 10) was introduced by antegrade flow through the coronary vessels (1,000 ml during the entire period). After cardioplegia-induced cardiac arrest (mean time to arrest was 85 seconds, not different among groups), the excised heart was placed in an iced slurry of Krebs buffer and remained hypothermic throughout the 75-minute cold-ischemic period. Subsequently, excess tissue was removed, and the great vessels were isolated; next, an aortic cannula (24F) was directly inserted distal to the cardioplegia cannula. Likewise, cannulas were inserted into the pulmonary artery (28F), pulmonary vein (28F), and directly into the inferior vena cava (36F). Millar pressure catheters (Millar, Houston, TX) were placed in the right and left ventricles. Furthermore, Transonic flow probes (Transonic Systems Inc, Ithaca, NY) were placed at the following locations: aorta, pulmonary vein, and inferior vena cava. Left cardiac output was measured using the pulmonary vein flow, and the coronary flow was calculated as the difference between pulmonary vein flow and aortic flow. A pacing lead (Sprint 6932, Medtronic, Inc) was inserted through a superior vena cava port in the right atrium and placed into the apex of the right ventricle, and a grounding patch was placed epicardially on the lateral wall of the left ventricle. An electrocardiac programmer analyzer computer (9790C Vitatron, Medtronic, Inc) provided defibrillation as needed. After mounting the heart on the isolated heart apparatus, warm (38 C), oxygenated (95% O 2, 5% CO 2 ) modified Krebs-Henseleit buffer was supplied to the coronary circulation through a Langendorff constant-pressure perfusion [13, 14]. On the absence of spontaneous atrial-ventricular rhythm or ventricular fibrillation, the heart was defibrillated while in Langendorff mode (thereby maintaining constant-pressure perfusion of the coronary arteries retrograde through the aorta). Initially, the heart functioned in the Langendorff mode of perfusion for a period of 15 minutes to stabilize atrial-ventricular rhythm. Both sides of the heart were then worked by supplying fluid pressure heads into the preload and afterload chambers, permitting fluid ejection from the aorta and pulmonary artery cannulas. Preload was held constant throughout the experiment (mean atrial pressures, mm Hg at 15 minutes, and 14 1 mm Hg at 75 minutes of reperfusion, not significant using repeated measures analysis of variance), as well as resistance of ejection (afterload). Detailed methods have been described recently by our laboratory [16, 17]. Experimental Protocol After in vivo stabilization (arterial carbon dioxide partial pressure, 40 2 mm Hg; core temperatures, C), the swine were randomly assigned to receive an intravenous infusion of either 1 mg/kg morphine sulfate (n 6), 1 mg/kg DADLE (n 6), or normal saline solution (control group; n 6). The drugs were administered during a 20-minute period, and the hearts were arrested 2 hours after infusion. In an additional two experiments, DADLE was administered similarly, but hearts were arrested 15 minutes after infusion. The investigators who performed the heart isolation and reanimation procedures were blinded as to the pretreatment used. Immediately before cardioplegia, hemodynamic data were obtained and a sample of the left atrium was taken using a freeze-clamp technique for analysis of highenergy phosphates (see below). Additionally, water content was determined by left atrial biopsy after lyophilization for 24 hours using the following formula: %H 2 O (wet weight dry weight)/wet weight 100). Before reperfusion in the isolated heart apparatus, another biopsy of the left atrium was performed, and the coronary sinus effluent was obtained for the determination of cardiac enzymes. After the initiation of four-chamber ejecting mode (t 0 hours, corresponding to 15 minutes reperfusion) and 1 hour after working in this mode (t 1 hour, 75 minutes of reperfusion), oxygen extraction as well as lactate and enzyme levels (creatine kinase, troponin I) were determined by collecting samples from the arterial and coronary sinus effluents. Myocardial oxygen consumption and lactate effluxes (Glucose-Lactate Analyzer, Yellow Springs Instruments, Yellow Springs, OH) were calculated. Additionally, hemodynamic data were acquired and left atrial biopsies were performed for high-energy phosphate analysis water content at these time points.

3 1578 SIGG ET AL Ann Thorac Surg OPIOID PRECONDITIONING IN SWINE 2001;72: Table 1. Functional Measurements of Isolated Working Hearts Time After Reperfusion (time working) Groups (n 6 in each group) LVSP (mm Hg) LVEDP (mm Hg) HR (beats/min) dp/dt max (mm Hg/s 10 3 ) Tau (s 10 3 ) MV o 2 ( mol/min) RPP (mm Hg/min 10 3 ) 15 min Saline (0 min) Morphine DADLE min Saline 67 7 a a a,b c d (60 min) Morphine DADLE Values are mean standard error of the mean. p 0.05 versus 15 min (analysis of variance). b p 0.05 versus morphine 75 min (analysis of variance). c not significant (analysis of variance), p 0.05 versus saline at 15 min (paired Student s t test, data from n 5 in saline group). d p 0.05 versus saline at 15 min (analysis of variance). DADLE d-ala2-leu5-enkephalin; dp/dt max maximal positive rate of increase of left ventricular pressure; HR heart rate; LVEDP left ventricular end-diastolic pressure; LVSP left ventricular systolic pressure; MV o 2 myocardial oxygen consumption; RPP rate pressure product (heart rate left ventricular systolic pressure/1000); Tau relaxation time constant. High-Energy Phosphates Freeze-clamped left atrial biopsy specimens were immediately cooled in liquid nitrogen, lyophilized, and kept frozen at 75 C until extraction. Phosphocreatine, adenosine, adenosine monophosphate, adenosine diphosphate, and ATP were extracted with acid, neutralized, and estimated using an isocratic high-performance liquid chromatography assay (MetaChem Inerstil ODS-2 column, Spectra-Physics high pressure pump, model 8810, Waters spectrophotometer and Waters Baseline software; Waters, Milford, MA). All the high-energy phosphate measurements were performed in duplicate, and mean values were calculated for each biopsy sample. Statistical Analysis Data are presented as mean and standard error of the mean. Cardiac outputs were normalized for body weight. Statistical analysis was performed using analysis of variance with Bonferroni post hoc test, either repeated measures for intragroup comparisons, or unpaired analysis of variance for intergroup comparisons. A p value of less than 0.05 was considered significant. Results The hemodynamic data are summarized in Table 1. Fifteen minutes after reperfusion, immediately at initiation of the isolated working heart mode, no significant differences among the groups were detected. However, at 75 minutes after reperfusion (60 minutes in working mode), groups treated with morphine and DADLE displayed significantly better-preserved systolic function, ie, left ventricular pressures, positive maximal rate of increase of left ventricular pressure, and cardiac outputs were higher compared with controls (Table 1, Fig 1). Similarly, morphine and DADLE preconditioning resulted in better diastolic function: in terms of lower time constants of left ventricular isovolumetric relaxation (tau; p 0.05; Table 1). No differences in left ventricular enddiastolic pressures were detected among groups (Table 1). The rate pressure product as a measure of myocardial oxygen consumption was maintained in opioidpreconditioned animals but not in controls (p 0.05, Table 1). Left ventricular cardiac outputs and tau values from 2 Fig 1. Shown are indexed cardiac outputs (per kilogram of body weight) of isolated hearts reperfused after 75 minutes of coldischemic storage. Hearts pretreated in vivo with either morphine sulfate or d-ala2, d-leu5-enkephalin (DADLE) showed higher cardiac outputs after 1 hour of functioning in working mode (controls, p 0.05 vs morphine, one-way analysis of variance; p 0.05 vs 0 hours, repeated measures analysis of variance), whereas immediately on initiation of working mode perfusion, no significant differences were detected among opioidpreconditioned and control hearts. The smaller figure shows that the in vivo preischemic cardiac outputs (per kilogram of body weight), obtained just before the ischemic period, were comparable among groups. (CO/kg cardiac output per kilogram of body weight.)

4 Ann Thorac Surg SIGG ET AL 2001;72: OPIOID PRECONDITIONING IN SWINE 1579 groups (Table 3). Lactate production was preserved in opioid-preconditioned animals, whereas in the control groups it increased significantly at 1 hour of working mode (p 0.01, Table 3). Fig 2. Left ventricular relaxation time constant (tau) and left ventricular cardiac outputs (per kilogram of body weight) of isolated working hearts pretreated in vivo with 1 mg/kg d-ala2, d-leu5- enkephalin are shown. The interval between drug infusion and hypothermic ischemia (75 minutes) was only 15 minutes in these two additional animals (as opposed to 2 hours in the main treatment groups). animals receiving DADLE 15 minutes before ischemiainduced arrest are shown in Figure 2. High-energy phosphates and adenosine data are summarized in Table 2. Morphine- and DADLE-pretreated animals elicited better preservation of end-ischemic phosphocreatine levels (saline-treated declined significantly; p 0.05; Table 2) as well as energy charge of the adenylate pool (Fig 3). However, on reperfusion, no differences in high-energy phosphate levels were detected among groups (Table 2). Although the ATP levels were not significantly better preserved at the end of ischemia, higher adenosine levels were observed in the control group at the end of ischemia, possibly because of increased ATP hydrolysis (p 0.05). Importantly, the better mechanical performance was not explained by reduced tissue injury or myocardial necrosis, as neither total creatine phosphate kinase nor troponin I levels were significantly different among Comment The present study demonstrated that in vivo pretreatment with either morphine or -opioid agonist DADLE improved postischemic systolic and diastolic function of the large mammalian heart. Furthermore, morphine and DADLE pretreatment preserved end-ischemic phosphocreatine levels and energy charge of the adenylate pool better than saline solution in controls. However, postischemic high-energy phosphates were not better preserved when swine were pretreated with DADLE or morphine, and therefore may not be the ultimate effector of such therapy. Decreased lactate efflux from such preconditioned hearts may explain better preserved function, inasmuch as myocardial necrosis determined by enzyme leakage was not different among the groups. Hypothermic cardioplegia normally provides adequate myocellular protection for cardiac surgical procedures, yet postischemic dysfunction remains a significant problem. Hypothermic storage of the heart induces similar changes to those associated with natural mammalian hibernation, such as intracellular acidosis, hypoxia, hypothermia, ATP depletion, and volume shifts. However, in hibernators, these alterations are well tolerated up to 5 to 6 months. Opioids may exert myocardial protective effects through mechanisms similar to ischemic preconditioning, most likely by means of the -opioid receptor pathway [2 5]. A common cardioprotective link between ischemic preconditioning and natural mammalian hibernation has been reported [1], and pharmacologic ischemic preconditioning by the synthetic -opioid DADLE was found to be myocardially protective in isolated rat heart experiments. This effect was reported to be mediated by ATP-dependent potassium channels, which are Table 2. Myocardial High-Energy Phosphates In Vivo and In Vitro a Experimental Time Groups (n 6 in each) ATP ADP AMP Phosphocreatine Adenosine In vivo Saline Morphine DADLE End-ischemia Saline g d f f Morphine DADLE min reperfusion Saline b (0 min) Morphine DADLE c f 75 min reperfusion Saline c (60 min) Morphine e Values are mean standard error of the mean. DADLE d e a High-energy phosphates were determined by high-performance liquid chromatography in duplicate (see Material and Methods). b p versus saline in vivo (repeated measures analysis of variance with Bonferroni correction). c p versus DADLE in vivo and DADLE endischemia. d p 0.01 versus DADLE end-ischemia. e p 0.05 versus saline in vivo, versus saline 15 min, versus saline 75 minutes. f p 0.05 versus saline in vivo, p 0.01 versus saline 75 minutes. g Not significant. ADP adenosine diphosphate; AMP adenosine monophosphate; ATP adenosine triphosphate; DADLE d-ala2, d-leu5-enkephalin.

5 1580 SIGG ET AL Ann Thorac Surg OPIOID PRECONDITIONING IN SWINE 2001;72: altered tissue metabolism, reduced or eliminated platelet or leukocyte aggregation, and improved microcirculation through vasodilation [20]. An immediate goal of reducing ischemic damage is to improve postischemic function, either after extracorporeal bypass or after hypothermic storage. The studies cited above have shown beneficial effects using isolated heart models. However, an isolated four-chamber working heart model may be a more critical and stringent model for inherent cardiac function than a Langendorff perfused model [14]. Such a four-chamber working heart model was developed and is used in our laboratory and allows reanimation of large mammalian hearts (human, porcine, canine). Fig 3. High-energy phosphates from left atrial biopsy specimens were determined by high-performance liquid chromatography to calculate the energy charge (EC) of the adenylate pool, which was calculated as follows: ATP (0.5 adenosine diphosphate)/(atp adenosine diphosphate adenosine monophosphate). The energy charge was better preserved at the end of cold ischemia in hearts preconditioned with morphine sulfate or d-ala2, d-leu5-enkephalin (DADLE), whereas in controls there was a significant decrease (p 0.05, repeated measures analysis of variance, Bonferroni post hoc test). Furthermore, by direct group comparison, the energy charge was significantly higher in the DADLE-treated group compared with the saline-treated group at end-ischemia (p 0.05, one-way analysis of variance, Bonferroni). However, on reperfusion, no significant differences were detected among groups, except that the DADLEtreated group had significantly lower energy charge on reperfusion at 15 minutes compared with in vivo and with end-ischemia (repeated measures analysis of variance, Bonferroni). considered to open in ischemic preconditioning as well [3]. However, pathways of classic ischemic preconditioning may not be the only mechanism by which these opioids exert their myocardial protective effects. Other mechanisms mediated by opioid receptors have been implicated, such as enhanced protection against oxygen free radicals, alteration of intracellular calcium handling, or ion channel gating [18, 19]. Enhanced ubiquitin levels in the infarcted region of left ventricle of rats pretreated with DADLE suggest another protective mechanism (personal communication, Joan Smith-Sonneborn, University of Wyoming, Laramie, WY). Hibernating induction trigger protein, and possibly also DADLE, may increase tissue survival time by reduced or Comparison With Other Studies In isolated Langendorff perfused rabbit hearts, the -opioid and synthetic hibernation induction trigger DADLE, administered before cardioplegia, have been reported to improve postischemic myocardial function after 2 hours of cold (4 C) ischemia [21]. In a follow-up study, the same group demonstrated significantly improved myocardial function (left ventricular pressures, coronary flows, and myocardial oxygen consumption) after 18 hours of ischemia using the same rabbit model [9]. In vivo pretreatment with serum from hibernating animals also has been reported to improve postischemic function in isolated rabbit hearts after 2 hours of global ischemia at 34 C [22]. It should be noted that exceedingly high doses of DADLE were used in these studies, up to 2 mmol/l. Nevertheless, Chien and colleagues [8, 23] performed similar experiments using a canine autoperfusion multiorgan preparation, and demonstrated an extension of organ survival time of up to 2 days using hibernation induction trigger or 1 mg/kg DADLE, administered intermittently. Other opioids have been suggested to be myocardially protective. In recent studies, pentazocine, morphine, buprenorphine, all opioids with agonist activity on the -opioid receptor were shown to be myocardially protective in an isolated rabbit heart model [10, 11]. The positive benefits attributed to serum from hibernators or DADLE were more dramatic in these previous studies than those we have observed here. In fact, in pilot studies using 24-hour and 1-hour pretreatment with DADLE, we were unable to resuscitate isolated swine Table 3. Measures of Myocardial Injury of Isolated Working Hearts Time After Reperfusion (time working) b Groups (n 6 in each) Lactate Efflux (mg/dl) Troponin I a (ng/ml) Total CK (U/L) Myocardial Water Content (%) 15 min (0 min) Saline Morphine DADLE min (60 min) Saline 8 3 b Morphine DADLE c Values are mean standard error of the mean. The hearts were reperfused in Langendorff mode at time 0 minutes, and from time 15 to 75 min in working mode. a Saline (n 4), morphine and DADLE (each n 5). b p 0.01 versus 15 minutes. c not significant. CK creatine kinase; DADLE d-ala2, d-leu5-enkephalin.

6 Ann Thorac Surg SIGG ET AL 2001;72: OPIOID PRECONDITIONING IN SWINE 1581 Table 4. Preliminary Results From Swine Hearts Pretreated With Either Hibernation Induction Trigger or d-ala2, d-leu5-enkephalin Before Prolonged Ischemic Storage Pretreatment 12 ml woodchuck plasma (hibernation induction trigger) iv 2 hours before cardioplegia, ischemic storage of the heart at 4 C for 19 hours 1 mg/kg DADLE iv 2 hours before cardioplegia; storage in Krebs solution for 18 hours at 4 C 1 mg/kg DADLE iv 24 hours and 2 hours before cardioplegia; storage for 18 hours at 4 C 2 mg/kg DADLE iv 24 hours before cardioplegia; stored in Krebs solution at 4 C for 18 hours Results No right or left ventricular function No right or left ventricular function Some right ventricular and minimal left ventricular function (LVSP 30 mm Hg) No right or left ventricular function DADLE d-ala2, d-leu5-enkephalin; LVSP left ventricular systolic pressure. hearts after 18 hours of cold storage (Table 4). Hence, we took a more conservative approach and investigated potential effects of such opioids using a relatively short ischemia time. It is suggested that such differences may also be species-dependent, which should be of interest for those working in the area of xenotransplantation. Furthermore, as previously mentioned, the working heart model is a much more stringent model to examine effects on postischemic function compared with the Langendorff model used in rabbit heart studies [14, 24]. Interestingly, no information was available on highenergy phosphate metabolism when using opioid preconditioning, although it was suggested that ATP levels may be preserved by Bolling and colleagues [9]. Hence, the correlated data we provided here between performance and myocardial metabolism were unique. Critique of Methods An altered delivery protocol of the drug administration immediately before cardioplegia may have resulted in more dramatic functional and even metabolic effects as suggested by ischemic preconditioning studies. To address that issue, DADLE was administered 15 minutes before cardioplegia in two pilot studies; very good left ventricular systolic and diastolic functional recovery (Fig 2), but not reduced cardiac enzyme leakage, was observed when compared with controls even at 2 and 3 hours after reperfusion (results not shown). The biopsy specimens for analysis of high-energy phosphates were taken from the left atrium and not the ventricle. We chose transmural left atrial biopsy samples to be able to perform serial biopsies in the same animal with minimal impairment to overall cardiac performance. However, differences in the atrial high-energy phosphate content may reflect overall changes in global myocardial high-energy phosphate content. In preliminary studies (n 9), in vivo left ventricular ATP content was mol/g dry weight compared with mol/g dry weight in the control groups of the current study; likewise, phosphocreatine contents were higher in ventricular biopsy specimens ( mol/g dry weight compared with mol/g dry weight in controls). In four pilot studies, we performed parallel biopsies of left atrium and left ventricle in isolated hearts: average atrial ATP levels were mol/g dry weight versus mol/g dry weight in ventricles, and average phosphocreatine levels were mol/g dry weight versus mol/g dry weight in the ventricles. However, these in vitro pilot study biopsies were performed at the end of the experiments ( 3 hours of reperfusion) compared with 0 and 1 hour in the current study. In spite of accurate measurement of changes of high-energy phosphate changes, assessment of total high-energy phosphate contents would not detect shifts of ATP within the cell (ATP compartmentalization). Noteworthy was the fact that improved myocardial performance was not associated with reduced myocardial injury (creatine kinase, troponin I). This was somewhat surprising and raises several new questions as to whether pharmacologic preconditioning may improve postischemic dysfunction by mechanisms other than delaying myocardial cell death. Whereas classic ischemic preconditioning clearly delays myocardial cell death across species, the effects on other aspects on reperfusion injury such as arrhythmias and stunning are less uniform. In the case of myocardial stunning, this may also be because of the fact that ischemic preconditioning itself can cause stunning. However, it is not established whether pharmacologic preconditioning can reduce myocardial stunning. The possibility that myocardial stunning was indeed reduced by our study protocol remains to be investigated. Furthermore, to determine whether myocardial stunning is indeed responsible, its reversibility has to be proven. Stunning is an ischemic syndrome that may last from hours up to days, and the study of myocardial stunning in isolated heart models is complicated by the fact that such models are typically deteriorating as a function of time. Surprisingly, the described better ventricular function was not correlated with reduced myocardial necrosis (enzyme leakage). Although there was a trend for lower troponin I levels in the DADLE group, in two pilot studies using preconditioning with DADLE 15 minutes before ischemia, troponin I levels were either similar or in one case much higher than in the morphine group (50 and 500 ng/ml), whereas function was similar to the morphine and DADLE study groups (Fig 3). Morphine and possibly also DADLE act on -opioid receptors, therefore leading to a potential antipreconditioned state [25]. However, this theory (which is based on an isolated rat heart study) has recently been challenged as -opioid receptor stimulation was found to reduce infarct size in a rat model [26]. In today s surgical practice, fentanyl-based anesthesia

7 1582 SIGG ET AL Ann Thorac Surg OPIOID PRECONDITIONING IN SWINE 2001;72: is widely used. Fentanyl is an unspecific -opioid receptor agonist, and in high doses, there may be stimulation of -opioid receptors. Thus, an additional stimulation of -opioid receptors may limit the use of specific -opioid agonists for pharmacologic preconditioning in routine cardiac surgical procedures. However, fentanyl did not confer significant cardioprotection in a recent report [11]. Another report, however, reported cardioprotective effects of fentanyl in an isolated rat heart model; unfortunately, this group used supraclinical dosages [27]. Whether the synthetic opioids such as fentanyl, sufentanil, and remifentanil stimulate the -opioid receptor at clinical dosages, and therefore may already confer cardioprotection, remains to be determined. Conclusions In vivo pretreatment with unspecific opioid agonist morphine and -opioid receptor specific agonist DADLE improves immediate postischemic systolic and diastolic function in isolated swine hearts after short-term cold storage. These effects were not associated with reduced myocardial necrosis, but with altered glycolytic pathways (reduced lactate levels). This suggests that DADLE or morphine may improve ventricular performance by reducing postischemic dysfunction that is not caused by necrosis (for example, stunning). More research on the optimal timing, dosage, and agent for pharmacologic preconditioning with opioids is warranted. And many questions regarding preconditioning with opioids still remain unanswered, such as receptor types and subtype involvement in different species, role of other clinically used opioids such as fentanyl, other mechanisms of actions, and the role of -opioid receptors, to mention just a few. Finally, the interesting link between these agents and mammalian hibernation may make them good candidates for pharmacologic preconditioning in myocardial protection that goes beyond mechanisms involving ischemic preconditioning. Potential clinical application may include extension of heart survival time (transplantation), as well as for pharmacologic preconditioning for patients undergoing surgical procedures using extracorporeal bypass. References 1. Kevelaitis E, Peynet J, Mouas C, et al. Opening of potassium channels: the common cardioprotective link between preconditioning and natural hibernation? Circulation 1999;99: Schultz JE, Rose E, Yao Z, Gross GJ. Evidence for involvement of opioid receptors in ischemic preconditioning in rat hearts. Am J Physiol 1995;268:H Schultz JE, Hsu AK, Gross GJ. Morphine mimics the cardioprotective effect of ischemic preconditioning via a glibenclamide-sensitive mechanism in the rat heart. Circ Res 1996; 78: Schultz JE, Hsu AK, Gross GJ. 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