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Ann Thorac Surg 1999;68:2089-2092
© 1999 The Society of Thoracic Surgeons

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Original Articles: Cardiovascular

Delta opioid receptors and low temperature myocardial protection

^a Section of Cardiac Surgery, University of Michigan Hospitals, Ann Arbor, Michigan, USA

Address reprint requests to Dr Bolling, Section of Cardiac Surgery, University of Michigan Hospitals, 1500 E Medical Center Dr, 2120D Taubman Center, Box 0344, Ann Arbor, MI 48109-0344;
e-mail: sbolling{at}umich.edu

Presented at the Thirty-fifth Annual Meeting of The Society of Thoracic Surgeons, San Antonio, TX, Jan 25–27, 1999.

	Abstract

Top Abstract Introduction Material and methods Results Comment References

 
Background. Cardiac surgery continues to be limited by an inability to achieve complete myocardial protection. This may result from the use of hypothermic cardioplegia. Interestingly, the subcellular changes of animal hibernation parallel the altered biology of induced hypothermic myocardial ischemia, but are well tolerated by hibernated mammalian myocardium. Evidence indicates this protection is mediated by activation of the delta opioid receptor, which elicits profound metabolic effects at the whole animal, organ, and cell level. In this study, we sought to determine if pentazocine, with agonist activity at the delta opioid receptor, could improve myocardial recovery following global ischemia over a wide range of temperatures.

Methods. Isolated rabbit hearts received either standard cardioplegia or were pretreated with racemic, d or l isomer pentazocine. Hearts were then subjected to 2 hours at 34°C, or 3.5 hours at 20°C, or 4 hours at 10°C of cardioplegic ischemia and reperfused. Functional recovery was compared to controls.

Results. Isovolumic developed pressure, coronary flow, oxygen consumption, and ultrastructural preservation were enhanced with pentazocine delta opioid mediated protection, which appears to be additive to standard cardioplegia, even at low temperatures.

Conclusions. Teleologically, delta opioid protection parallels animal hibernation, which occurs from 34° down to 0°C. The use of delta opioid receptor agonists may have important clinical implications for cardiac surgery and deserves further study.

	Introduction

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Presently cardioplegia and hypothermia provide considerable myocardial protection during induced ischemia for cardiac surgery, however perioperative infarction, stunning, and ventricular dysfunction remain significant problems, especially in high-risk patients with poor preoperative function, recent myocardial infarction, or left ventricular hypertrophy. Interestingly, mammalian hibernation biology closely parallels the altered cardiac cellular physiology noted with hypothermic cardioplegic arrest. However, while similar subcellular and molecular changes are seen, such as intracellular acidosis, hypoxia, hypothermia, energy store depletion, and volume shifts, these alterations are well tolerated for months by the hibernated mammalian myocardium, while the present limit of surgically induced ischemia is hours.

Hibernation occurs in only certain mammals: woodchucks, arctic and 13 lined ground squirrels, brown cave bats, and black bears [1]. The exact chemical responsible for the induction of hibernation is elusive, but a proto-opiate nature is well established as hibernation can be reversed or retarded by opiate antagonists [2]. Evidence indicates this action is mediated by the membrane delta opiate receptor, which is thought to be distinct from the nociceptive mu and kappa receptors [3]. Agonist compounds active at the delta opiate receptor elicit profound physiologic and metabolic effects at the whole animal, organ, and cell level. Furthermore, delta opioids are able to induce hibernation in nonhibernators [4]. In this study, we sought to determine if pentazocine and its isomers, with known activity at the delta opioid receptor, could improve myocardial recovery following global ischemia over a wide range of temperatures in a nonhibernating mammalian model.

	Material and methods

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Preparation of isolated heart
Rabbits (male or female, 2.2 to 2.7 kg body weight) were anesthetized with sodium pentobarbital (45 mg/kg, intravenously) and heparinized (700 U/kg, intravenously). The heart was rapidly excised and immersed in ice-cold physiologic salt solution (PSS), pH 7.4, containing 118.0 mM NaCl, 4.0 mM KCl, 22.3 mM NaHCO₃, 11.1 mM glucose, 0.66 mM KH₂PO₄, 1.23 mM MgCl₂, and 2.38 mM CaCl₂. The aorta was cannulated in the Langendorff mode and the heart was perfused with PSS that was equilibrated with 95% O₂, 5% CO₂ at 37°C and passed twice through filters with 3.0 µm pore size. Perfusion pressure was maintained at 90 mm Hg. An incision was made in the left atrium, and a fluid-filled latex balloon was passed through the mitral orifice and placed in the left ventricle. A Millar pressure transducer was used for continuous measurement of left ventricular pressure (LVP) and the first derivative of LVP (dP/dt). The caudal vena cava, the left and right cranial vena cava, and the azygous vein were ligated. The pulmonary artery was cannulated to enable timed collection measurements of coronary flow, and the cannula was connected to an oxygen meter (Diamond Electro-Tech, Inc, Ann Arbor, MI) for continuous measurement of the oxygen partial pressure. The analog signals were continuously recorded and digitized to an online computer (AST Premium/386, AST Research Inc, Irvine, CA). To characterize cardiac function, developed pressure (DP) is defined as peak systolic pressure (PSP) minus end-diastolic pressure (EDP). Myocardial oxygen consumption (MVO₂) was calculated as MVO₂ = CF x [(PaO₂–PvO₂) x (c/760)], where CF is coronary flow (ml/min/g), (PaO₂–PvO₂) is the difference in the partial pressure of oxygen (PO₂ mm Hg) between perfusate and coronary effluent flow, and c is the Bunsen solubility coefficient of O₂ in perfusate at 37°C (22.7 µl O₂ · atm^-1 · ml^-1 perfusate). The PO₂ of the perfusate was 665 mm Hg. Coronary flow was measured by performing timed collections of the pulmonary effluent flow with a graduated cylinder. Oxygen extraction (O₂ EXT) was calculated as O₂ EXT = MVO₂/oxygen content in the perfusate. Wet weight of the heart was determined at the conclusion of each experiment after trimming the great vessels and fat and blot drying with nine-layer cotton gauze. The left ventricular wall was weighed, desiccated for 48 hours at 65°C and reweighed. Water content was determined using the formula (1-dry weight/wet weight)/100%. A section of the LV was prepared for histopathology.

After completing instrumentation and performing calibrations, left ventricular balloon volumes were varied over a range of values to construct modified left ventricular function curves. In this manner, it is possible to define a specific balloon volume that is associated with a developed pressure from 100 to 140 mm Hg. This volume was maintained the same during baseline and reperfusion conditions. Baseline data were obtained after an equilibration period of 30 minutes. During the baseline period, data were obtained with hearts maintained at 37°C by a water-jacketed organ bath. To induce ischemia, the PSS infusion was stopped and 60 ml of 4°C solution was injected into the aorta at a rate of 1 ml/sec to begin ischemia.

Hearts were randomly assigned to groups. To determine if the delta opioid agonist pentazocine could improve myocardial recovery following global ischemia, hearts received either standard cardioplegia (CP, controls) or were pretreated 10 minutes prior to ischemia with 10 mg of racemic, d or l isomer pentazocine. Hearts were then subjected to 2 hours at 34°C, 3.5 hours at 20°C, or 4 hours at 10°C of cardioplegic ischemia. These time periods were chosen to represent equivalent depression of control myocardial postischemic function. The cardioplegia contained 109.0 mM NaCl, 25.0 mM KCl, 21.9 mM NaHCO₃, 16.0 mM MgCl₂, and 0.8 mM CaCl₂. After ischemia, hearts were reperfused with oxygenated PSS at 37°C. Hemodynamic data were recorded for 45 minutes to compare with baseline data and to determine the degree of functional recovery. The Statview 5.01 Program (Abacus Concepts, Inc, Berkeley, CA) was used for statistical analysis. Data were evaluated with analysis of variance (Scheffe’s test). Differences were considered significant when p was less than 0.05. All animals received humane care in compliance with the "Principles of Laboratory Animal Care" formulated by the National Society for Medical Research. All work was performed in the section of Cardiac Surgery, University of Michigan Medical Center, Ann Arbor, MI.

	Results

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There were no differences in any functional or metabolic indices during preischemia between groups. Table 1 summarizes the postischemic metabolic and functional recovery results. At all temperatures, racemic pentazocine had enhanced recovery of function. This was also noted at 34°C with the l isomer of pentazocine, but not with the r isomer, which was not significantly different than controls. Ultrastructural preservation was significantly improved with pentazocine pretreatment, as compared to controls at each temperature. There were no significant differences in heart weight or water content between groups.

	Comment

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Hibernation in mammals is a unique adaptation allowing certain animals to conserve up to 90% of the energy required if they remained active during winter [9]. In differing hibernators, body temperatures range from 34°C (bears) down to 2°C below freezing (Arctic ground squirrels) [10]. The opiate nature of the chemical responsible for hibernation has been established as the behavioral and physiological changes noted are blocked or retarded by the opiate antagonists, naloxone and naltrexone. The exact molecule may be either an opiate or a neuropeptide hormone with agonist activity at the delta opioid receptor [3, 4]. As evidence, only the delta opioid D-Ala2-Leu5-Enkephalin, DADLE induces hibernation in summer-active ground squirrels [4]. Furthermore, not only has it has been shown that the opioid antagonist naloxone [11] can block hibernation, but the potent synthetic kappa agonist U69593 [12], the mu agonists, morphine and morphiceptin, as well as the natural kappa brain opioid agonist, dynorphin, all of which can occupy the delta opioid receptor site, also block hibernation [13, 14]. That kappa and mu opioids reverse or block hibernation may reflect induced conformational, binding, or stereotactic changes, as delta opioids may be displaced by either antagonists or other nondelta agonists.

Evidence that delta opioids can induce cardiac metabolic changes in nonhibernating species was demonstrated by Swan and Schatte [15], who noted suppression of metabolic rate, and particularly myocardial oxygen consumption, which decreased to 65% of control at 30 minutes. Other investigators have also noted this alteration in MVO₂. The delta opioid DADLE, which mimics natural hibernation, has also been used to extend survival time of transplant organs [16–18]. Finally, a recent study from this laboratory demonstrated markedly enhanced return of function following 18 hours of storage in a cardiac transplant model utilizing delta opioids [19].

The exact role that delta opioids play in ischemic tolerance is unknown. Delta opioid receptors are known to be present in many peripheral organs. A previous report using [³H]DPDPE has demonstrated delta opioid receptors in the heart [20]. Our results in the present study confirm that delta opioid receptors exist in the heart and that opioids act directly on the myocardium. Further investigators have demonstrated that the opioid mechanism involved in cardiac preservation during hypoxia or ischemia is specifically mediated via the delta opioid receptor. Reports have provided indirect and direct evidence that delta opioid receptors may be involved in ischemic preconditioning in rat myocardium [21, 22]. Other authors showed that both ischemic preconditioning and morphine-induced cardioprotection are actually mediated via the delta opioid receptor in the rat, as delta opioid blockade completely abolished the cardioprotective effect induced by preconditioning ischemia or morphine [23].

The specific cardioprotective action of the delta opioids remains hypothetical and may be through multiple pathways. Delta opioid receptors belong to the 7-trans-membrane, guanine nucleotide binding protein (G-protein)-linked superfamily of receptors. Although both opioid receptors and endogenous opioids are found in cardiac tissues, the signal transduction pathways of delta opioids in cardiac membranes have yet to be exactly identified. However, binding of opioid receptor antagonists and effects of mu, delta, and kappa agonists on cardiac GTPase and adenylyl cyclase have been measured [24]. The mu opioid receptor agonists have no effect on either adenylyl cyclase or GTPase activities. These experiments suggested, however, that in cardiac sarcolemma, delta opioid receptors are coupled to pertussis toxin sensitive G-proteins and mediate inhibition of adenylyl cyclase activity. Additionally, delta opioid linked cardiac GTPgS binding has been noted to alter intracellular signal transduction pathways to open ATP linked potassium channels, which can have cardioprotective effects [25].

In the present study, pentazocine, an opioid of the benzomorphan family, was utilized because it is a known potent delta opioid receptor agonist. The beneficial action of pentazocine appears to be via the l isomer, as the r isomer does not have an effect. Pentazocine may also have other mechanisms of cardioprotection, as it is active at the sigma opioid receptor site and has 1/50 the opioid antagonist activity as nalorphine, which may alter its binding in the opioid super-receptor.

In conclusion, the delta opioid pentazocine, produces profound myocardial preservation effects, even at the lowest temperatures. This parallels hibernations’ beneficial action over a wide range of temperatures and mirrors the spectrum of temperatures utilized in clinical cardiac surgery. This "natural" approach to cardiac preservation is interesting and may have clinical application.

	Acknowledgments

 
This work was supported in part by grant 95008730 from the American Heart Association.

	References

Top Abstract Introduction Material and methods Results Comment References

 

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