| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
Ann Thorac Surg 2001;72:1576-1582
© 2001 The Society of Thoracic Surgeons
a Department of Anesthesiology, University of Minnesota, Minneapolis, Minnesota, USA
b Department of Physiology, University of Minnesota, Minneapolis, Minnesota, USA
c Biomedical Engineering Institute, University of Minnesota, Minneapolis, Minnesota, USA
d Department of Pathology, University of Kentucky, Lexington, Kentucky, USA
Accepted for publication June 28, 2001.
* Address reprint requests to Dr Iaizzo, Department of Anesthesiology, University of Minnesota, 420 Delaware St SE, MMC 294 UMHC, Minneapolis, MN 55455, USA
e-mail: iaizz001{at}tc.umn.edu
 |
Abstract |
|---|
 Top Abstract IntroductionMaterial and methodsResultsCommentReferences |
|---|
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 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 opioid-pretreated 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.
|
Introduction |
|---|
|
TopAbstractIntroductionMaterial and methodsResultsCommentReferences |
|---|
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 cold-stored 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 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 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 |
|---|
|
TopAbstractIntroductionMaterial and methodsResultsCommentReferences |
|---|
Animal preparation
After initial sedation with intramuscular xylazine (5 mg/kg) and tiletamine/zolazepam (250 mg), swine (n = 18, weight 73 ± 4.5 kg [mean ± standard deviation]) received intravenous thiopental (20 to 25 mg/kg) for intubation. The swine were then mechanically ventilated with a 65% N2O and 35% O2 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; CaCl2, 1.2; MgCl2, 16; NaHCO3, 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% O2, 5% CO2) 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, 14.1 ± 1 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, 38.0° ± 0.5°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 high-energy phosphates (see below). Additionally, water content was determined by left atrial biopsy after lyophilization for 24 hours using the following formula: 
.
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.
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 |
|---|
|
TopAbstractIntroductionMaterial and methodsResultsCommentReferences |
|---|
|
|
|
|
|
|
|
Comment |
|---|
|
TopAbstractIntroductionMaterial and methodsResultsCommentReferences |
|---|
-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 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 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).
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 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].
|
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 25.3 ± 0.5 µmol/g dry weight compared with 19 ± 2.1 µmol/g dry weight in the control groups of the current study; likewise, phosphocreatine contents were higher in ventricular biopsy specimens (45.3 ± 5.8 µmol/g dry weight compared with 29.3 ± 3.6 µmol/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 5.6 ± 3.3 µmol/g dry weight versus 13.3 ± 1 µmol/g dry weight in ventricles, and average phosphocreatine levels were 6.1 ± 2.4 µmol/g dry weight versus 41.9 ± 7 µmol/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 is widely used. Fentanyl is an unspecific µ-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 |
|---|
|
TopAbstractIntroductionMaterial and methodsResultsCommentReferences |
|---|
This article has been cited by other articles:
|
|
 |
|
  J. G. Bovill Intravenous Anesthesia for the Patient with Left Ventricular Dysfunction Seminars in Cardiothoracic and Vascular Anesthesia, March 1, 2006; 10(1): 43 - 48. [Abstract] [PDF]
|
|
|
|
 |
|
 S. Pepe, O. W.V van den Brink, E. G Lakatta, and R.-P. Xiao Cross-talk of opioid peptide receptor and {beta}-adrenergic receptor signalling in the heart Cardiovasc Res, August 15, 2004; 63(3): 414 - 422. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 H. V. CAREY, M. T. ANDREWS, and S. L. MARTIN Mammalian Hibernation: Cellular and Molecular Responses to Depressed Metabolism and Low Temperature Physiol Rev, October 1, 2003; 83(4): 1153 - 1181. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J. A. Coles Jr., D. C. Sigg, and P. A. Iaizzo Role of kappa -opioid receptor activation in pharmacological preconditioning of swine Am J Physiol Heart Circ Physiol, June 1, 2003; 284(6): H2091 - H2099. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. G. Bovill Anesthesia for Patients with Impaired Ventricular Function Seminars in Cardiothoracic and Vascular Anesthesia, March 1, 2003; 7(1): 49 - 54. [PDF]
|
|
|
|
|
|
D. C. Sigg, J. A. Coles Jr., P. R. Oeltgen, and P. A. Iaizzo Role of delta -opioid receptor agonists on infarct size reduction in swine Am J Physiol Heart Circ Physiol, June 1, 2002; 282(6): H1953 - H1960. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
| ANN THORAC SURG | ASIAN CARDIOVASC THORAC ANN | EUR J CARDIOTHORAC SURG |
| J THORAC CARDIOVASC SURG | ICVTS | ALL CTSNet JOURNALS |
. 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 DADLE-treated 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).