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Am J Physiol Heart Circ Physiol 283: H783-H791, 2002. First published May 9, 2002; doi:10.1152/ajpheart.00193.2002
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Vol. 283, Issue 2, H783-H791, August 2002

A highly potent peptide analgesic that protects against ischemia-reperfusion-induced myocardial stunning

Dunli Wu, Yi Soong, Guo-Min Zhao, and Hazel H. Szeto

Department of Pharmacology, Joan and Sanford I. Weill Medical College, Cornell University, New York, New York 10021


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

We recently discovered an opioid peptide analgesic, 2',6'-dimethyltyrosine (Dmt)-D-Arg-Phe-Lys-NH2 ([Dmt1]DALDA), that can protect against ischemia-induced myocardial stunning. In buffer-perfused hearts, 30-min global ischemia followed by reperfusion resulted in a significant increase in norepinephrine (NE) overflow immediately upon reperfusion and significant decline in contractile force (45%). Pretreatment with [Dmt1]DALDA before ischemia completely abolished myocardial stunning and significantly reduced NE overflow (68%). In contrast, pretreatment with morphine before ischemia only provided brief protection against myocardial stunning and no reduction in NE overflow. [Dmt1]DALDA inhibited [3H]NE uptake into cardiac synaptosomes in vitro (IC50 = 3.9 µM), whereas morphine had no effect. Surprisingly, protection against myocardial stunning was apparent even when hearts were perfused with [Dmt1]DALDA only upon reperfusion, whereas reperfusion with morphine had no effect. Binding studies with [3H][Dmt1]DALDA revealed no high-affinity specific binding in cardiac membranes, suggesting that the cardioprotective actions of [Dmt1]DALDA are not mediated via opioid receptors. These findings suggest that [Dmt1]DALDA is a potent analgesic that may be useful for myocardial stunning resulting from cardiac interventions or myocardial ischemia.

myocardial ischemia; norepinephrine uptake; µ-opioid; cardiac contractility; norepinephrine transporter


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

MYOCARDIAL ISCHEMIA leading to myocardial infarct is a major cause of morbidity and mortality, and treatment of myocardial ischemia-reperfusion injury remains a challenge. While surgical intervention and thrombolytic treatments allow restoration of blood flow to the cardiac tissue, reperfusion itself is known to be associated with a spectrum of events known as reperfusion injury including ventricular arrhythmias, stunning, and coronary vascular dysfunction (15). Myocardial stunning refers to the prolonged depression of contractile function of viable myocardium salvaged by timely reperfusion after a transient period of ischemia (2). Myocardial stunning has been observed in experimental models and in humans after coronary artery bypass surgery, angioplasty, and thombolysis.

Numerous studies have attempted to determine the mechanism(s) behind myocardial stunning with the goal toward rational design of pharmacotherapies to treat the "stunned" myocardium (5, 17). The primary mechanisms that have been proposed are oxygen-derived free radicals and disruption of Ca2+ homeostasis. While the free radical hypothesis is supported by overwhelming experimental evidence, the clinical application of oxygen radical scavengers to the treatment of the stunned myocardium has been disappointing (4). In terms of Ca2+ homeostasis, there is evidence of a calcium overload immediately upon reperfusion and decreased sensitivity of the myofilaments to Ca2+. Calcium sensitizers have been proposed for treatment of myocardial stunning, but there is concern that they may impair diastolic relaxation. It is now well recognized that Ca2+ overload occurs in part as a consequence of intracellular acidosis and activation of the Na+/H+ exchanger (NHE) and Na+/Ca2+ exchanger (10). NHE inhibitors have been found to be effective in experimental models and the favorable outcomes have led to clinical trials (19).

At the same time, it has become apparent that brief episodes of ischemia can protect the heart against the deleterious effect of more prolonged ischemia (ischemic preconditioning). The protective actions of ischemic preconditioning are believed to involve adenosine, alpha -adrenergic and opioid receptors, and ATP-sensitive K+ (KATP) channels, and it has been found that pretreatment with adenosine, opioid agonists, and KATP channel openers can mimic the protective action of ischemic preconditioning (8). While these agents have been shown to reduce infarct size, there is little evidence that they can improve contractile function in the stunned myocardium.

We have recently discovered an opioid peptide analgesic that can protect against postischemic ventricular and coronary vascular dysfunction when given during reperfusion. 2',6'-Dimethyltyrosine (Dmt)-D-Arg-Phe-Lys-NH2 ([Dmt1]DALDA) is a synthetic analog of dermorphin, a heptapeptide found in skin secretions of the frog Phyllomedisa sauvagei (13). Intravenous administration of [Dmt1]DALDA produced a transient increase in blood pressure in sheep that was associated with an increase in cardiac output and no change in peripheral vascular resistance (27, 28). In the absence of any change in heart rate, we postulated that [Dmt1]DALDA might have a positive inotropic action on the heart to cause an increase in stroke volume. We have therefore investigated the effect of [Dmt1]DALDA on contractile function using the isolated perfused guinea pig heart. Our initial results confirmed that [Dmt1]DALDA did indeed exert a positive inotropic action on the heart; but, in addition, it also prolonged the survival of the in vitro perfused heart. This prompted us to explore the possibility that [Dmt1] DALDA might protect against myocardial ischemia-reperfusion injury. Our results show that [Dmt1] DALDA protects against postischemic myocardial stunning even when administered only during reperfusion.


    METHODS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Drugs and Chemicals

[Dmt1]DALDA and [3H][Dmt1]DALDA were provided by Dr. Peter W. Schiller (Clinical Research Institute of Montreal, Montreal, Quebec, Canada). [Dmt1]DALDA was synthesized according to the method described previously (18). For the preparation of [Dmt1]DALDA in tritiated form, a precursor peptide containing 2',6'-dimethyl-3',5'-diiodotyrosine [Tyr(2',6'-Me2,3',5'-I2)] needed to be synthesized. Fmoc-Dmt-OH was iodinated by treatment with I2 in the usual manner to yield Fmoc-Tyr(2',6'-Me2,3',5'-I2)-OH. This protected amino acid was then used in the solid phase synthesis of H-Tyr(2',6'-Me2,3',5'-I2)-D-Arg-Phe-Lys-NH2 according to a protocol published elsewhere (18). The peptide was purified by preparative reverse-phase chromatography and its structure was confirmed by fast atom bombardment mass spectrometry. Catalytic tritiation of this precursor peptide was performed at the Institute of Isotopes (Budapest, Hungary), resulting in a product with a specific radioactivity of 47.18 Ci/mmol. Morphine sulfate and naloxone hydrochloride were supplied by the National Institute on Drug Abuse (Rockville, MD). L-[7,8-3H]norepinephrine ([3H]NE; specific activity 1.37 TBq/mmol, radiochemical purity 93.2%) was purchased from Amersham Pharmacia Biotech (Buckingham, UK). All other drugs and chemicals were obtained from Sigma (St. Louis, MO).

Isolated Perfused Guinea Pig Heart

All experiments were conducted according to guidelines approved by the Institution for the Care and Use of Animals at Weill Medical College of Cornell University. Male Hartley guinea pigs (350-400 g) were euthanized by cervical dislocation while under light anesthesia with CO2 vapor. The heart was rapidly isolated, and the aorta was cannulated in situ and perfused in a retrograde fashion with an oxygenated Krebs-Henseleit solution (pH 7.4) at 34°C. The heart was then excised, mounted on a modified Langendorff perfusion apparatus, and perfused at constant pressure (40 cmH2O). Contractile force was measured with a small hook inserted into the apex of the left ventricle and the silk ligature tightly connected to a Grass force-displacement transducer. The heart was set at a predetermined tension at the beginning of the experiment, and the transducer was calibrated at the end of the experiment. Heart rate was determined with a pair of surface electrocardiogram electrodes. Total coronary flow was measured by timed collection of pulmonary artery effluent. Hearts were weighed at the end of the experiment.

Perfusion Experiments

Isolated hearts were perfused with either Krebs-Hensleleit solution or Krebs-Henseleit solution containing [Dmt1] DALDA (10-7 M) or morphine (10-6 M) for 3 h starting from the time of cannulation of the aorta. Contractile force, heart rate, and coronary flow were determined throughout the duration of the experiment.

Ischemia-Reperfusion Experiments

Drug treatment before and after ischemia. Isolated hearts were perfused continuously with either Krebs-Hensleleit solution or Krebs-Henseleit solution containing [Dmt1]DALDA (10-7 M) or morphine (10-6 M) and allowed to stabilize for 30 min. Global ischemia was then induced by complete interruption of coronary perfusion for 30 min. Reperfusion was carried out for 2 h using continuous perfusion with the same solution received before global ischemia.

Drug treatment during reperfusion. All hearts were perfused with Krebs-Hensleleit solution and allowed to stabilize for 30 min. Global ischemia was then induced by complete interruption of coronary perfusion for 30 min. Reperfusion was carried out for 2 h using continuous perfusion with either Krebs-Hensleleit solution or Krebs-Henseleit solution containing [Dmt1]DALDA (10-7 M) or morphine (10-6 M).

Norepinephrine Assay

The coronary effluent was assayed for NE levels by HPLC coupled to electrochemical detection (7).

NE Uptake in Cardiac Synaptosomes

Cardiac synaptosomes were prepared according to the method described previously (1). Synaptosomes were preincubated in HEPES-buffered Krebs-Ringer solution [containing (in mM) 50 HEPES, 144 NaCl, 1.2 MgCl2, 5 KCl, 10 glucose, 1 ascorbic acid, and 0.0124 nialamide; pH 7.4] with varying concentrations of [Dmt1]DALDA or morphine for 10 min at 37°C. [3H]NE (10 nM) was then added, and the incubation continued for 6 min before being terminated by rapid cooling in ice-cold water for 3 min. The synaptosomal preparation was then centrifuged at 3,000 g for 15 min, and the pellet was resuspended in HEPES-buffered Krebs-Ringer solution. The process was repeated, the final pellet was resuspended in distilled deionized H2O, and an aliquot (0.15 ml) removed for liquid scintillation counting. [3H]NE accumulation at 37°C minus that at 0°C was taken as a measure of active uptake.

Radioligand Binding Assay

Binding of [3H][Dmt1]DALDA was determined using membrane homogenates from the guinea pig heart and mouse brain. Tissues were put in 30 volumes of ice-cold 50 mM Tris buffer (pH 7.4), homogenized for 10 s, and centrifuged at 40,000 g for 20 min at 4°C. After this process was repeated a second time, the pellet was resuspended in Tris buffer (pH 7.4) at a final concentration of 2 mg/ml. Protein concentrations were determined by the Bradford procedure (Bio-Rad; Hercules, CA). Aliquots of membrane homogenates (400 µg protein) were incubated with [3H][Dmt1]DALDA (10-800 pM) for 60 min at 25°C. Nonspecific binding was assessed by inclusion of 1 µM unlabeled [Dmt1]DALDA. Free radioligand was separated from bound radioligand by rapid filtration through Whatman GF/B filters with a Brandel cell harvester. Filters were washed three times with 10-ml Tris buffer, and radioactivity was determined by liquid scintillation counting. Binding affinities [dissociation constant (Kd)] and receptor number [maximal binding (Bmax)] were determined using nonlinear regression (Graphpad; San Diego, CA).

Data Analysis

All data are presented as means ± SE. A single-factor ANOVA with repeated measures (factor = time) was used to analyze the effects of the different drugs or buffer on various cardiac parameters. Post hoc analysis of significant differences was carried out using the Dunnett's test. Differences between drug treatments were analyzed using two-way ANOVA (factors = treatment, time). P < 0.05 was considered statistically significant.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Perfusion Experiments

Measurements of contractile force, heart rate, and coronary flow were obtained from all perfused guinea pig hearts within 10 min of cannulation. Isolated hearts were perfused with buffer (n = 5), [Dmt1]DALDA (10-7 M, n = 5), or morphine (10-6 M, n = 4). Two-way ANOVA revealed significant differences in contractile force (P < 0.001) and heart rate (P < 0.001), but not coronary flow, between the three treatment groups. In hearts perfused with Krebs-Henseleit solution, significant changes were observed in contractile force over the 3-h perfusion period (Table 1). There was a significant improvement in contractile force over the first hour of perfusion compared with the first measurement determined at 10 min (Fig. 1 and Table 1). Contractile function was maintained for at least 1.5 h before a significant decline in force was observed beyond 2 h. There was no significant change in heart rate or coronary flow over the 3-h period of perfusion with buffer (Table 1). Perfusion with [Dmt1]DALDA resulted in significantly higher contractile force and prolonged survival of the preparation (Fig. 1 and Table 1). Contractile force was significantly increased for 90 min, and force did not decline significantly below the initial value until 150 min. Perfusion with [Dmt1]DALDA was also associated with small but significant changes in heart rate and coronary flow, but these did not occur until later in the perfusion period when there was also a decline in contractile force (Table 1). As a result of the concurrent increase in contractile force and heart rate, the product of force × rate was significantly increased by [Dmt1]DALDA. In contrast, perfusion with morphine (10-6 M) resulted in a profound decrease in contractile force and shortened the survival of the preparation (Fig. 1 and Table 1). In addition to the decrease in contractile force, perfusion with morphine also resulted in a significant decrease in heart rate and coronary flow (Table 1). By 90 min after the onset of perfusion, contractile force, heart rate, and coronary flow in the morphine-perfused hearts were reduced to 37%, 46%, and 62% of the buffer controls, respectively. In contrast to [Dmt1]DALDA, the product of force × rate was significantly decreased by morphine.

                              
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  		Table 1.   Effects of [Dmt1]DALDA and morphine perfusion on the isolated perfused guinea pig heart



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  		Fig. 1.   Effects of 2',6'-dimethyltyrosine (Dmt)-D-Arg-Phe-Lys-NH2 ([Dmt1]DALDA) and morphine perfusion on contractile force in the isolated perfused guinea pig heart. Hearts were perfused with buffer or buffer containing [Dmt1]DALDA (10-7 M) or morphine (10-6 M) for 3 h starting from the time of cannulation. Significant differences were found among the three treatment groups (2-way ANOVA, P < 0.001). The effect of each treatment over time was analyzed using one-way ANOVA with repeated measures (factor = time). Post hoc analysis was carried out using the Dunnett's test by comparing each time point to 10 min. *P < 0.05.

Ischemia-Reperfusion Experiments

Drug treatment before and after ischemia. Isolated hearts were perfused for 30 min with buffer (n = 6), [Dmt1]DALDA (10-7 M, n = 5), or morphine (10-6 M, n = 5) before the onset of 30-min global ischemia. Reperfusion was carried out with the same drug used before ischemia. Two-way ANOVA revealed significant differences in contractile force (P < 0.001), heart rate (P = 0.003), and coronary flow (P < 0.001) among the three treatment groups. In the buffer group, contractile force was significantly lower during reperfusion compared with before ischemia (Fig. 2). There were also significant changes in heart rate and coronary flow after ischemia-reperfusion with buffer (Table 2). Coronary flow was significantly increased during the first 2 min of reperfusion compared with before ischemia, but then became significantly lower after 60 min of reperfusion. The product of force × rate continued to decline during reperfusion. There was significant protection against ischemia-reperfusion injury in the [Dmt1]DALDA group, and contractile force remained near preischemia levels until late in the reperfusion period (Fig. 2 and Table 2). There was a significant increase in coronary flow during the first minute of reperfusion, and flow was maintained throughout the reperfusion period in the [Dmt1]DALDA group. Heart rate was not significantly affected by ischemia-reperfusion in the [Dmt1]DALDA group, and the product of force × rate was maintained at preischemic level until 60 min after onset of reperfusion. Perfusion with morphine provided some protection against ischemia during the early reperfusion period (Fig. 2 and Table 2). However, contractile force dropped precipitously between 0.5 and 1.0 h after onset of reperfusion. Heart rate was also significantly decreased 1 h after reperfusion, resulting in a decrease in the product of force × rate. The changes in coronary flow in the morphine group was similar to that in the [Dmt1]DALDA group, with a significant increase in flow immediately upon reperfusion.


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  		Fig. 2.   Effects of [Dmt1]DALDA and morphine pretreatment on contractile force during ischemia-reperfusion in the isolated perfused guinea pig heart. Hearts were perfused with buffer or buffer containing [Dmt1]DALDA (10-7 M) or morphine (10-6 M) for 30 min and then subjected to 30-min global ischemia. Reperfusion was carried out using the same solution. The hatched bar indicates perfusion with drug or buffer. Significant differences were found among the three treatment groups (2-way ANOVA, P < 0.001). The effect of each treatment over time was analyzed using one-way ANOVA with repeated measures (factor = time). Post hoc analysis was carried out using the Dunnett's test by comparing each time point to before ischemia. *P < 0.05.


                              
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  		Table 2.   Effects of [Dmt1]DALDA and morphine pretreatment on ischemia-reperfusion in the isolated guinea pig heart

Drug treatment during reperfusion. Isolated hearts were perfused for 30 min with buffer (n = 16) before the onset of 30-min global ischemia. Reperfusion was carried out with buffer (n = 6), [Dmt1]DALDA (10-7 M, n = 5), or morphine (10-6 M, n = 5). Two-way ANOVA revealed significant differences in contractile force (P < 0.001), heart rate (P = 0.007), and coronary flow (P < 0.001) among the three treatment groups. The buffer group was the same as presented above. Reperfusion with morphine did not result in any significant differences in contractile force or coronary flow compared with reperfusion with buffer (Fig. 3 and Table 3). However, reperfusion with [Dmt1]DALDA provided significant protection against ischemia-reperfusion injury. Contractile force was not significantly different from before ischemia with the exception of a small decrease by the end of the reperfusion period, and the product of force × rate was also maintained at near preischemic level (Table 3). Interestingly, the normal increase in coronary flow in the immediate reperfusion period was not found in the [Dmt1]DALDA group. Heart rate response in the [Dmt1]DALDA group was not different from the buffer group.


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  		Fig. 3.   Effects of [Dmt1]DALDA and morphine during reperfusion on contractile force on ischemia-reperfusion in the isolated perfused guinea pig heart. Hearts were perfused with buffer for 30 min and then subjected to 30-min global ischemia. Reperfusion was carried out using buffer or buffer containing [Dmt1]DALDA (10-7 M) or morphine (10-6 M). The hatched bar indicates perfusion with drug or buffer. Significant differences were found among the three treatment groups (2-way ANOVA, P < 0.001). The effect of each treatment over time was analyzed using one-way ANOVA with repeated measures (factor = time). Post hoc analysis was carried out using the Dunnett's test by comparing each time point to before ischemia. *P < 0.05.


                              
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  		Table 3.   Effects of [Dmt1]DALDA and morphine during reperfusion on ischemia-reperfusion in the isolated guinea pig heart

NE Release Associated With Reperfusion After 30-min Global Ischemia

When guinea pig hearts were subjected to 30-min global ischemia followed by reperfusion with buffer, NE overflow into the coronary effluent was significantly increased over preischemic levels (Table 4). Perfusion of the heart with [Dmt1]DALDA before global ischemia and followed by reperfusion with [Dmt1]DALDA significantly reduced NE overflow over the first minute after reperfusion. However, if the hearts were only treated with [Dmt1]DALDA during reperfusion, NE overflow was not significantly different from the buffer group. Pretreatment with morphine before ischemia had no effect on NE overflow during the first minute upon reperfusion.

                              
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  		Table 4.   NE overflow into the coronary effluent in isolated guinea pig hearts during the first minute of reperfusion after 30-min global ischemia

[3H]NE Uptake in Cardiac Synaptosomes

Incubation with [Dmt1]DALDA inhibited [3H]NE uptake in a concentration-dependent manner (Fig. 4A). The IC50 for inhibition of [3H]NE uptake was 3.9 (2.6-6.2) µM. Morphine did not inhibit [3H]NE uptake even at 10-4 M (data not shown), and naloxone did not block the action of [Dmt1]DALDA (Fig. 4B).


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  		Fig. 4.   A: effects of [Dmt1]DALDA on [3H]norepinephrine (NE) uptake in cardiac symaptosomes. Each value represents the mean ± SE determined from 3 to 5 experiments. The IC50 value for [Dmt1]DALDA was 3.9 (2.6-6.2) µM. Morphine did not inhibit NE uptake even at 10-4 M. B: inability of naloxone (10-5 M) to block inhibition of [3H]NE uptake by [Dmt1]DALDA (10-5 M) in cardiac synaptosomes.

[3H][Dmt1]DALDA Binding

Figure 5 illustrates the saturation binding curves for [3H][Dmt1]DALDA in mouse brain membrane homogenates. High-affinity specific binding was observed with a Kd = 123 ± 9 pM. The binding of [3H][Dmt1] DALDA to brain membranes was completely displaced by morphine [inhibitory constant (Ki) = 5.64 ± 0.24 nM, n = 4] and H-Tyr-D-Ala-Gly-MePhe-Gly-ol, a selective µ-opioid ligand (Ki = 2.80 ± 0.14 nM, n = 4). Binding studies with guinea pig heart membranes revealed no specific binding.


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  		Fig. 5.   Saturation binding curve for [3H][Dmt1]DALDA using mouse brain membranes (open circle ) and guinea pig cardiac membranes (). Membrane homogenates were incubated with [3H][Dmt1]DALDA (10-800 pM) for 60 min at 25°C. Nonspecific binding was assessed in the presence of 1 µM unlabeled [Dmt1]DALDA. Saturation binding was found with mouse brain membranes, with a dissociation constant = 123 ± 9 pM. No saturation binding was detected with cardiac membranes.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Previous findings in the intact animal suggested that [Dmt1]DALDA may increase cardiac output because of a direct inotropic action on the heart. The results of the present study clearly show that [Dmt1]DALDA increased contractile force in the isolated perfused guinea pig heart without significantly affecting heart rate or coronary flow and prolonged contractile function of the isolated heart preparation. As a result, oxygen consumption, as reflected by the product of force × rate, was significantly increased. Interestingly, despite the increase in oxygen consumption, survival of the heart was greatly prolonged when perfused with [Dmt1]DALDA. This inotropic action does not appear to be mediated via opioid receptors. Radioligand binding studies revealed no specific binding of [Dmt1]DALDA to cardiac membranes, whereas [3H][Dmt1]DALDA bound with very high affinity to mouse brain membranes. Previous studies have reported the presence of delta - and kappa -, but not µ-, opioid receptors in the rat heart (31, 32, 36). [Dmt1]DALDA is highly selective, being 14,700-fold and 100-fold more selective for µ- than delta - or kappa -receptors, respectively (18). Furthermore, morphine perfusion significantly reduced contractile force and shortened survival of the perfused heart despite a significant reduction in oxygen consumption. The negative inotropic and chronotropic actions of morphine and other opioids have been reported previously (11, 12, 29, 33), and they are thought to be mediated by delta - or kappa -opioid receptors (3) or nonopioid receptors (33). Reports of positive inotropic action of morphine in intact animals are thought to be the result of sympathoadrenal discharge (30).

When the isolated perfused guinea pig heart was subjected to 30-min global ischemia, contractile force declined by 45% and continued to decrease during reperfusion, indicative of myocardial stunning. The later postischemic stunning was associated with a decrease in heart rate but no significant change in coronary flow. Pretreatment with either morphine or [Dmt1]DALDA offered significant protection against contractile dysfunction during the early phase of reperfusion.

Opioid receptors have been implicated in ischemic preconditioning (22), and morphine can mimic the cardioprotective effect of ischemic preconditioning (21). Morphine-induced preconditioning significantly reduced infarct size, and this effect was abolished by naloxone. However, morphine has not been shown to enhance ventricular function in the postischemic stunned myocardium. Our results show that pretreatment of the perfused heart with morphine before ischemia significantly improved contractile function for 30-45 min of reperfusion before a rapid decline to the same level as the untreated group. The preconditioning action of morphine appears to be mediated by delta -opioid receptors (24) and involves KATP channels (23). Subsequent studies have shown that preconditioning with selective delta 1-selective opioid agonists can reduce infarct size (23), but it is not known if they enhance ventricular function.

Pretreatment of the heart with [Dmt1]DALDA provided significantly longer protection against postischemic myocardial stunning compared with morphine, and the enhancement of contractile function was not associated with any significant change in heart rate or coronary flow. This cardioprotective action of [Dmt1]DALDA is unlikely to be mediated by delta -opioid receptors because of its low affinity for the delta -receptor (Ki = 2.1 µM) (18) and maximal protection was observed with only 100 nM.

The ability of [Dmt1]DALDA to reduce NE overflow may contribute to its cardioprotective action. Postischemic reperfusion is associated with high levels of NE release into the coronary perfusate caused by reversal of the NE transporter (NET) under ischemic conditions (carrier-mediated NE release) (9, 20). The experimental protocol utilized in the present study (30-min global ischemia) has been shown to result in carrier-mediated NE release (6). Exaggerated NE release is a major cause of reperfusion arrhythmias, and pharmacological agents that reduce this carrier-mediated NE release, including adenosine A1 agonists and histamine H3 agonists, reduce the incidence of postischemic ventricular fibrillation (6). Pretreatment with [Dmt1]DALDA decreased NE release into the coronary perfusate by 66% at a concentration (0.1 µM) that is similar to that reported for H3 agonists and A1 agonists. The ability of [Dmt1]DALDA to inhibit carrier-mediated NE release is supported by our finding that it can dose dependently inhibit NE uptake into cardiac synaptosomes. The IC50 for [Dmt1]DALDA in the synaptosomal preparation is higher than the concentration used in the perfused heart, but the synaptosomal preparation may be less "physiological" than the isolated heart, and the NE uptake studies were not carried out under ischemic conditions. Importantly, morphine had no effect on NE overflow during reperfusion and did not inhibit NE uptake into cardiac synaptosomes. The mechanism by which [Dmt1]DALDA inhibits NET is not known, but opioid receptors are not involved because it was not blocked by naloxone.

Surprisingly, protection against myocardial stunning was apparent even when [Dmt1]DALDA was only given upon reperfusion. Reperfusion with morphine offered no protection at all, again highlighting the different mechanisms of action of the two agents. This is a significant advantage of [Dmt1]DALDA compared with many other cardioprotective agents. Interestingly, inhibitors of the cardiac NHE (NHE1) can enhance postischemic ventricular function when given during reperfusion (34). Protons accumulate in the heart during ischemia leading to activation of the NHE resulting in Na+ influx. Increase in intracellular Na+ in turn activates the Na+/Ca2+ exchanger, and an increase in intracellular Ca2+ is thought to contribute to ischemic and/or reperfusion injury. Recent studies suggest that the NHE is actually activated on reperfusion rather than during ischemia (16), and postischemic contractile performance was significantly improved when the NHE inhibitor HOE-642 was given only during reperfusion (34).

Our results suggest that [Dmt1]DALDA may be beneficial for the stunned myocardium and may be given after ischemic damage. It should be pointed out that the isolated heart is not an ideal model of stunning because full functional recovery cannot be studied in an in vitro model with limited viability. Our data clearly show the progressive deterioration of the perfused heart over 1-2 h even without ischemia. The effect of [Dmt1]DALDA on the ischemic heart needs to be studied in a whole animal model. It is encouraging though that [Dmt1]DALDA clearly prolonged the survival of the in vitro perfused heart. If these findings can be confirmed in an in vivo model, [Dmt1]DALDA may offer several advantages over other pharmacological interventions. Unlike adrenergic agonists, [Dmt1]DALDA can enhance ventricular function without increasing heart rate and myocardial demand, and, unlike adenosine, [Dmt1]DALDA does not reduce coronary blood flow or heart rate. An added advantage of [Dmt1]DALDA is that it is a highly potent and long-acting analgesic that is relatively free of adverse effects (14, 25, 27, 28, 35), making it an attractive agent for cardiac interventions that may put the patient at risk for cardiac ischemia. Finally, [Dmt1]DALDA is metabolically stable, and its elimination half-life has been found to be six times longer compared with morphine (26). Taken together, [Dmt1]DALDA holds much promise as a pharmacological treatment for myocardial stunning. However, whether the stunned myocardium should be treated remains debatable. There is concern to some that stunning represents a protective mechanism for the ischemic heart, and increased contractile work may further increase oxygen demand in the compromised heart. However, myocardial stunning remains a significant cause of morbidity and mortality in patients with profound late ventricular dysfunction after acute myocardial ischemia or coronary bypass surgery, and, in these cases, [Dmt1]DALDA may represent a novel therapeutic approach.


    ACKNOWLEDGEMENTS

We thank Dr. Peter W. Schiller (Clinical Research Institute of Montreal, Montreal, Quebec, Canada) for the synthesis of [Dmt1]DALDA and [3H][Dmt1]DALDA. We also thank Dr. Roberto Levi (Weill Medical College of Cornell University) for advice and helpful discussions, and members of his laboratory for help in setting up the in vitro cardiac perfusion system. Abimbola Omniyi and Michelle Arndt both helped in the early phase of this project.


    FOOTNOTES

This work was supported in part by a multicenter program project grant (P01 DA08924) from the National Institute on Drug Abuse.

Address for reprint requests and other correspondence: H. H. Szeto, Dept. of Pharmacology, Weill Medical College, Cornell Univ., 1300 York Ave., New York, NY 10021 (E-mail: hhszeto{at}med.cornell.edu).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

10.1152/ajpheart.00193.2002

Received 6 March 2002; accepted in final form 30 April 2002.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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Am J Physiol Heart Circ Physiol 283(2):H783-H791
0363-6135/02 $5.00 Copyright © 2002 the American Physiological Society


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