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J Thorac Cardiovasc Surg 1994;108:269-278
© 1994 Mosby, Inc.

CARDIOPULMONARY BYPASS,
MYOCARDIAL MANAGEMENT, AND SUPPORT TECHNIQUES

Nucleoside trapping during reperfusion prevents ventricular dysfunction, "stunning," in absence of adenosine: Possible separation between ischemic and reperfusion injury" in absence of adenosinePossible separation between ischemic and reperfusion injury

Anwar S. Abd-Elfattah, PhD, Michael E. Jessen, MD, Andrew S. Wechsler, MD


Richmond, Va.

Supported in part by the Natoinal Institute of Health Grant #HL-26302

Received for publication Feb. 5, 1993. Accepted for publication Jan. 28, 1994. Address for reprints: Anwar S. Abd-Elfattah, PhD, Department of Surgery, Medical College of Virginia, Virginia Commonwealth University, MCV Box 532, Richmond, VA 23298-0532.

Abstract

A previous study has shown that endogenous adenosine trapping during ischemia (by blocking adenine nucleoside transport and inhibiting adenosine breakdown) prevents myocardial stunning. In this study, we tested the hypothesis that delay of administration of inhibitors until reperfusion would similarly prevent myocardial stunning in the absence of entrapped adenosine. In both studies, a selective nucleoside transport blocker, p-nitrobenzyl-thioinosine, was used in combination with a potent adenosine deaminase inhibitor, erythro-9-(2-hydroxy-3-nonyl)adenine, to entrap adenosine (preischemic treatment) or inosine (postischemic treatment) in an in vivo canine model of reversible global ischemia. Twenty-five anesthetized adult dogs were instrumented (by sonomicrometry) to monitor left ventricular performance from the relationship between stroke work and end-diastolic length as a sensitive and load-independent index of contractility. Hearts of animals supported by cardiopulmonary bypass were subjected to 30 minutes of normothermic global ischemia and 60 minutes of reperfusion. Saline solution containing the pharmacologic agents were infused into the bypass circuit before ischemia (group 1) or during reperfusion (group 2). Control group (group 3) received saline before and after ischemia. Myocardial biopsy specimens were obtained before, during, and after ischemia, and levels of adenine nucleotides, nucleosides, oxypurines, and the oxidized form of nicotinamide-adenine dinucleotide were determined. Left ventricular contractility fully recovered within 30 minutes of reperfusion in the groups treated with erythro-9-(2-hydroxy-3-nonyl)adenine and p-nitrobenzyl-thioinosine (p < 0.05 versus control group). Myocardial adenosine triphosphate was depleted by 50% in all groups at the end of ischemia. Adenosine triphosphate recovered during reperfusion only in the group that was treated with inhibitors before ischemia (group 1). At the end of ischemia, adenosine levels were low (<10% of total nucleosides) in the control group (group 3) and in the group treated only after ischemia (group 2). A high level of adenosine (>90% of total nucleosides) was present in group 1. We infer that selective pharmacologic blockade of nucleoside transport, only after ischemic injury, accelerated functional recovery during reperfusion, even without trapping of endogenous adenosine during ischemia and without adenosine triphosphate recovery during reperfusion. Recovery of myocardial adenosine triphosphate required preischemic treatment and adenosine entrapment during ischemia and reperfusion. Therefore, nucleoside trapping may be used to prevent reperfusion-mediated injury after reversible ischemic injury. (J THORACCARDIOVASCSURG1994;108:269-78)

Patients referred for medical or surgical revascularization have variable degrees of ischemia and myocardial infarction. No intervention has been described that prevents reperfusion-mediated injury, ventricular arrhythmias, and dysfunction after reversible ischemic injury— "myocardial stunning." Myocardial injury to the heart during ischemia and reperfusion can be considered to consist of two interrelated components: (1) metabolic derangement induced by ischemic injury and (2) ventricular dysfunction mediated by reperfusion injury. Separating these two components of injury has been a challenge, in part because of the lack of a selective intervention that is capable of preventing reperfusion-mediated ventricular dysfunction. Recently, we have demonstrated that selective preischemic blockade of adenine nucleoside transport significantly accumulated endogenous adenosine and resulted in full recovery of ventricular function after moderate Go Go 1-3 or prolonged Go 4 ischemic periods. It is not known whether delaying administration of inhibitors until postischemic reperfusion would provide any protection against reperfusion injury after documented metabolic derangement induced by ischemia.

It is not known whether cardiac protection provided by adenosine trapping is mediated by direct beneficial effects of adenosine Go Go 5-8 or by preventing release of purines and subsequent free radical generation during reperfusion. Go Go 9,10 It is well documented that administration of exogenous adenosine protects the heart by different mechanisms, including activation of A1-receptor and adenosine triphosphate (ATP)–sensitive potassium channels and attenuation of neutrophil and platelet activation. Go Go 11-14 Because of several side effects of exogenous adenosine administration, Go Go 15-19 its therapeutic potential may not be suitable for all patients. The present study was designed to determine whether postischemic nucleoside trapping by p-nitrobenzyl-thioinosine (NBMPR) and inhibition of adenosine deaminase by erythro-9-(2-hydroxy-3-nonyl)adenine (EHNA) is cardioprotective when these substances are administered only during reperfusion in the absence of entrapped endogenous adenosine.

MATERIALS AND METHODS

The following studies conform to the guiding principles of the American Physiological Society. All animals were treated humanely in accordance with the U.S. Public Health Service Standards as outlined in the "Principles of Laboratory Animal Care" formulated by the National Society for Medical Research and the "Guide for the Care and Use of Laboratory Animals" prepared by the National Academy of Sciences and published by the National Institutes of Health.

Biochemical reagents were purchased from Sigma Chemical Company (St. Louis, Mo.). EHNA was purchased from Burroughs Wellcome (Research Triangle Park, N.C.), and NBMPR was obtained from Aldrich (Milwaukee, Wis.).

Animal preparation
Twenty-five microfilaria-free adult dogs of either sex weighing 17 to 25 kg were anesthetized with sodium pentobarbital, 30 mg/kg, as an initial intravenous injection, followed by 10 mg/kg when needed (Nembutal, Abbott Laboratories, Chicago, Ill.). Dogs were intubated, ventilated, and prepared for cardiopulmonary bypass as previously described. Go Go 1-4 Mean perfusion pressure was maintained at 60 mm Hg. Arterial blood gases, pH, and hematocrit value were routinely determined and maintained at the following levels: oxygen tension = 100 to 140 torr, carbon dioxide tension = 30 to 40 torr, pH = 7.32 to 7.48, and hematocrit value = about 30%.

Assessment of left ventricular performance
Left ventricular performance was assessed (when the dog was weaned from bypass) from the relationship between stroke work and end-diastolic dimension as a sensitive and load-independent index of contractility. Go Go 20,21 In brief, all pressure measurements were obtained with intraventricular micromanometer-tipped catheters (Millar Instruments, Inc., Houston, Tex.). Left ventricular dimension data were obtained by pulse transit sonomicrometry (Triton Technology, San Diego, Calif.). One pair of LTZ-piezoelectric hemispheric crystals (Channel Industries, Santa Barbara, Calif.) was sutured to the front and back of the epicardial surface of the left ventricular wall in the minor axis. The spacing between the minor-axis crystals ranged from 40 to 66 mm. Analog data were digitized at 200 Hz and stored on magnetic disk with a microcomputer (DEC DPD 11/23, Digital Equipment Corp., Maynard, Mass.). Subsequent analysis was performed with interactive software developed in our laboratory. The venous line and left ventricular vent were clamped to create work loops, and the left ventricular and systemic pressures were allowed to rise to 100 to 120 mm Hg, with the bypass roller pump, after which the animal was separated from bypass. Left ventricular volume was gradually removed from the heart, which generated a family of progressively diminishing pressure-dimension work loops. The slope of the integrated work loops plotted against end-diastolic length has been established to be a load-independent index of contractility and represents an accurate measurement of ventricular function. Go Go 20,21 Left ventricular and arterial pressures and other hemodynamic parameters, including coronary flow, were also changing during the creation of progressively diminishing work loops.

Assessment of adenine nucleotide pool metabolism.
Transmural serial biopsy specimens (5 to 10 mg) were obtained with a Tru-Cut needle (Travenol Laboratories, Inc., Deerfield, Ill.) before ischemia, before and after drug administration, after 30 minutes of normothermic ischemia, and after 30 and 60 minutes of reperfusion. Biopsy specimens were immediately frozen and stored in liquid nitrogen. Each biopsy specimen was extracted by using 12% trichloroacetic acid (4° C) for 30 minutes with frequent homogenization. The soluble acid extract was separated from denatured protein by centrifugation and neutralized with 3:1 (vol/vol) tri-n-octylamine/fluorocarbon mixture (1:3 vol/vol) while the protein in the pellet was determined as previously described. Go 22 The neutralized extracts were stored at -70° C until analysis. Myocardial adenine nucleotide pool intermediates were eluted and quantified with high-performance liquid chromatography and external standards. Go Go 23,24 Results were expressed as nanomoles per milligram protein and presented as mean ± standard error of the mean.

Protocol
Dogs were randomly assigned to one of three groups: group 1, preischemic treatment group (n = 10); group 2, postischemic treatment (n = 7); and group 3, the control group (n = 8), in which dogs received saline before and after ischemia. Three boluses (500 ml each) of saline containing inhibitors (NBMPR 25 µmol/L and EHNA 100 µmol/L) were administered to the cardiopulmonary reservoir in group 1. The first bolus was infused before ischemia to assess the effect of inhibitors on ventricular contractility. Myocardial biopsy tissue was obtained to determine the adenine nucleotides and nucleosides of the normal heart before ischemia. The second bolus was infused immediately before crossclamping to ensure effective concentrations of these drugs in the myocardium during ischemia, and the third bolus was infused on reperfusion to ensure an adequate inhibitor concentration during reperfusion. In group 2, two boluses were infused. One bolus (1000 ml) was infused into the pump after 25 minutes of ischemia and 5 minutes before the aortic crossclamp was released. The second bolus was infused after 30 minutes of reperfusion. Group 3 received three boluses of saline without inhibitors, exactly as in group 1.

The half-life of EHNA is 1 to 2 hours (Dr. Donald Nelson, Burroughs Wellcome, Research Triangle Park, N.C.). Lamb and Nelson Go 25 have demonstrated that a single oral dose (50 mg/kg) totally inhibited the activity of adenosine deaminase in CBA mice. The half-life of NBMPR is about 120 minutes (unpublished observations of Drs. Wendy Gati and Alan R. P. Paterson, Department of Pharmacology, University of Alberta, Alberta, Canada). The respective inhibition constant values for these drugs are within the nanomolar ranges in vitro.

Statistical analysis
Data are presented as mean ± standard error of the mean. Sequential measurements were compared by repeated-measures analysis of variance using SAS (Statistical Analysis System Institute, Cary, N.C.). Differences were considered significant if the probability value for comparison of least squares means was less than 0.05.

RESULTS

LV performance
Fig. 1 depicts the effects of ischemia and reperfusion on left ventricular performance as a measurement of the systolic function (the slope of stroke work/end-diastolic length relationship). Administration of saline or drugs before ischemia did not affect left ventricular contractility of normal myocardium. Thirty minutes of normothermic global myocardial ischemia caused severe ventricular dysfunction, "stunning," during reperfusion in the control group. Left ventricular function in the control group returned to only 52% and 64% of preischemic function after 30 and 60 minutes of reperfusion, respectively (p < 0.05 versus preischemic function, 81.69 ± 5.1 dyne/cm 2 x10 3). Full recovery of left ventricular function was observed in groups 1 and 2. In group 1, cardiac function returned to 83% of normal after 30 minutes of reperfusion and recovered by 104% after 60 minutes of reperfusion (p = NS versus preischemic function). Ventricular function also completely recovered in group 2 within 30 minutes of reperfusion (p = NS versus preischemic function). It was also observed that the intercept of the linear relationship between stroke work and end-diastolic length was not significantly different between groups before or after ischemia and ranged between 49.02 and 51.6 mm (p = NS between groups).



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  		Fig. 1. Effects of global ischemia and reperfusion on left ventricular performance in the presence or absence of EHNA/NBMPR. Slope of the relationship between the stroke-work/end-diastolic dimensions (SW/EDL) before and after 30 minutes of ischemia in the presence or absence of EHNA/NBMPR. Saline with or without inhibitors was infused into the bypass circuit before and after ischemia (groups 1 and 3, respectively). In group 2, drugs were infused into the bypass 5 minutes before release of the aortic crossclamp. Analysis of variance (ANOVA) demonstrated statistically significant differences between groups (n = 7 to 10). *p < 0.05 versus other groups. CPBP, Cardiopulmonary bypass.

 
Myocardial adenine nucleotide pool metabolism
Infusion of the vehicle, or EHNA/NBMPR, solution did not affect ATP levels in the normal myocardium before ischemia. All groups lost an equal amount of myocardial ATP (50% of baseline) at the end of 30 minutes of normothermic global ischemia (p < 0.05 versus preischemic levels). Myocardial ATP levels in the EHNA/NBMPR-pretreated group significantly recovered (26.0 ± 0.9 nmol/mg protein) at the end of 60 minutes of reperfusion (p = NS versus baseline after drug administration, 28.5 ± 1.4 nmol/mg protein). Myocardial ATP levels remained depressed (by about 50% of normal) in the control group (17.16 ± 1.0 and 16.54 ± 1.0 nmol/mg protein) and group 2 (14.86 ± 1.3 and 17.09 ± 1.2 nmol/mg protein), after 30 and 60 minutes of reperfusion, respectively (Fig. 2).



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  		Fig. 2. Effect of ischemia and reperfusion on myocardial ATP in the presence or absence of EHNA/NBMPR. Myocardial ATP levels were significantly depleted in all groups during ischemia. ATP levels recovered only in the group that was pretreated with EHNA/NBMPR. Analysis of variance (ANOVA) revealed significant differences between groups (n = 7 to 10).*p < 0.05 versus control. #p < 0.05 versus preischemic measurements. CPBP, Cardiopulmonary bypass.

 
Myocardial adenosine diphosphate (ADP) levels were similar in groups 1, 2, and 3 before ischemia, at the end of ischemia, and during 30 and 60 minutes of reperfusion (p = NS) Go(Table I). Myocardial ADP levels were significantly lower in all groups (by 40% to 50%) during postischemic reperfusion when compared with baseline levels (p < 0.05). Myocardial adenosine monophosphate (AMP) levels were not significantly different at baseline before ischemia in all groups (p = NS) Go(Table I). A transient accumulation of AMP was observed at the end of ischemia (1.9 ± 0.7, 1.4 ± 0.3, and 0.8 ± 0.3 nmol/ mg protein in groups 1, 2, and 3, respectively). The levels of myocardial AMP declined during reperfusion in all groups, compared with baseline. Analysis of variance revealed no significant differences between these groups with respect to myocardial ADP and AMP. Total adenine nucleotide levels were decreased by 50% at the end of 30 minutes of ischemia in all groups (p < 0.05 versus preischemic levels). During reperfusion, total myocardial adenine nucleotide levvels recovered only in group 1 and remained depressed in the other groups (p < 0.05). Among adenine nucleotides, ATP was the only metabolite that increased during reperfusion and only in group 1.


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  		Table I. Effects of selective entrapment of adenine nucleosides on myocardial ATP pool
 
Myocardial adenine nucleosides
Adenine nucleosides (adenosine and inosine) were not detectable in the normal myocardium before or after drug administration. In the control group (group 3), a transient increase in adenosine occurred, from undetectable to 0.81 ± 0.1 nmol/mg protein at the end of ischemia, and adenosine was washed out during reperfusion (Fig. 3). A tenfold increase in myocardial adenosine levels (9.91 ± 0.7 nmol/mg protein) was observed at the end of the ischemic period (p < 0.05 versus other groups) when a combination of EHNA/NBMPR was infused before ischemia. The ratio between myocardial adenosine and inosine was about 10:1 in group 1. Myocardial adenosine generated during ischemia in the control group (group 3) washed out within 30 minutes of reperfusion. A significant amount of adenosine and inosine remained detectable in group 1 during reperfusion. Inosine was the major (>90%) nucleoside accumulated during ischemia in groups 2 and 3 (11.03 ± 0.9 and 9.29 ± 1.2 nmol/mg protein, respectively) (Fig. 4). Inosine was the least accumulated nucleoside during ischemia in group 1. During reperfusion, the level of myocardial inosine rapidly declined only in the control group (group 3). Myocardial inosine accumulated during ischemia in group 2 and was detectable even after 60 minutes of reperfusion (Fig. 4). Total adenine nucleoside levels were not detectable before ischemia and were not elevated after the administration of EHNA/ NBMPR. The levels of total adenine nucleosides were similar in all groups (p = NS). Total adenine nucleosides washed out within 30 minutes of reperfusion in the control group but remained detectable in groups treated with EHNA/NBMPR (groups 1 and 2). The rate of ATP depletion during ischemia was not affected by significant accumulation of endogenous adenosine or inosine during ischemia in the treated groups.



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  		Fig. 3. Effect of ischemia and reperfusion on myocardial adenosine in the presence or absence of EHNA/NBMPR. Myocardial adenosine was not detectable before ischemia. However, adenosine was produced and was not deaminated during ischemia in the EHNA/NBMPR-pretreated group (group 1). Adenosine transiently accumulated in the control group (group 3) and in the group that was treated with EHNA/NBMPR (group 2) (p < 0.05 between groups, analysis of variance [ANOVA]). Adenosine washed out during reperfusion in the control group during reperfusion. However, some adenosine was detectable in the drug-pretreated group. *p < 0.05 versus other groups. #p < 0.05 versus preischemic measurements (n = 7 to 10). CPBP, Cardiopulmonary bypass.

 


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  		Fig. 4. Effect of ischemia and reperfusion on myocardial inosine in the presence or absence of EHNA/NBMPR. Myocardial inosine was not detectable before ischemia. However, inosine accumulated in the control group (group 3) and in the group that was treated with EHNA/NBMPR during ischemia (group 2). Inosine was detectable in group 2 during reperfusion. Minimal inosine levels transiently accumulated in the group that was preischemically treated (group 1). Analysis of variance (ANOVA) demonstrated statistically significant differences between groups (n = 7 to 10). *p < 0.05 versus other groups. #p < 0.05 versus preischemic measurements. CPBP, Cardiopulmonary bypass.

 
Myocardial oxypurines and the oxidized form of nicotinamide-adenine dinucleotide (NAD+)
Myocardial oxypurines (hypoxanthine and xanthine) were not detectable at baseline before ischemia. However, an increase in myocardial hypoxanthine was observed in all groups at the end of ischemia—1.23 ± 0.29, and 1.28 ± 0.36 and 2.12 ± 0.38 nmol/mg protein in groups 1, 2, and 3, respectively (p < 0.05 versus baseline) Go(Table I). Hypoxanthine was not detectable in the myocardium during reperfusion. The highest concentration of hypoxanthine was found in the control group at the end of ischemia. Myocardial xanthine also was not detected before ischemia but increased at the end of ischemia to 0.4 ± 0.1 and 0.7 ± 0.4, and 1.5 ± 0.1 nmol/mg protein in groups 1, 2, and 3, respectively (p < 0.05 versus baseline) Go(Table I). Myocardial xanthine levels were not detectable in the myocardium during reperfusion. The total oxypurine levels represent a minimal fraction of adenine nucleotides and nucleosides during the ischemic period, and all oxypurines washed out during reperfusion in all groups.

Myocardial NAD+ levels at baseline were 3.6 ± 0.2, 4.9 ± 0.2, and 4.3 ± 0.2 nmol/mg protein in groups 1, 2 and 3, respectively Go(Table I). NAD+ levels at the end of 30 minutes of ischemia were slightly reduced in all groups (p = NS). However, myocardial NAD+ levels returned to baseline during reperfusion (p = NS, GoTable I). No significant differences were found between groups with respect to the time of the experiment.

DISCUSSION

The rationale for the present study was to develop a pharmacologic strategy that selectively targets postischemic reperfusion injury of the stunned myocardium. No pharmacologic intervention prevents reperfusion-mediated injury in the ischemic heart (stunned or infarcted myocardium). Interventions capable of reducing the rate of ATP depletion during the ischemic period have proved to be cardioprotective when applied to normal hearts before ischemia. These strategies, however, failed to protect the myocardium when applied only during postischemic reperfusion.

The present work provides a unique pharmacologic strategy (with EHNA/NBMPR) that selectively targets postischemic reperfusion-mediated injury without affecting ischemic injury. Despite metabolic ischemic injury, full recovery of left ventricular performance was achieved by selective blockade of nucleoside transport after ischemia (nucleoside trapping). These data implicate purine efflux and subsequent formation of free radicals during reperfusion in postischemic ventricular dysfunction (myocardial stunning). Indeed, purine release has been known to be a sensitive index of prior ischemic injury. Go Go 26-29

Administration of EHNA and NBMPR into the cardiopulmonary bypass circuit did not have inotropic effects and did not alter the levels of adenine nucleotide or nucleoside metabolism in normal canine myocardium. Pretreatment with EHNA/NBMPR neither prevented ischemia-induced ATP depletion nor limited stoichiometric accumulation of endogenous adenine nucleosides (adenosine and inosine) during ischemia. These drugs, however, changed the ratio between adenosine and inosine when administered before ischemia. It is well established that adenosine is transiently produced in the adult myocardium by AMP dephosphorylation, catalyzed by the cytosolic as well as the ecto 5'-nucleotidase. Go 30 Rapid deamination of adenosine to inosine occurs either inside or outside rat cardiomyocyte. Go 31 The present results also demonstrate that phosphorylation of entrapped adenosine to AMP does not occur during ischemia as evidenced by lack of recovery of AMP, ADP, and ATP. However, adenosine may have been incorporated into ATP during reperfusion only when endogenous adenosine was trapped in the group that was treated with EHNA/NBMPR before ischemia. These findings suggest that most of the endogenous adenosine entrapped by EHNA/NBMPR is compartmentalized inside cells. However, some nucleosides are also identified outside the cell after ATP depletion induced by metabolic inhibitors. Indeed, Walsh and associates Go 32 have demonstrated that EHNA/NBMPR markedly reduced adenosine release (by 85%) from isolated perfused anoxic rabbit hearts, compared to the untreated control group. Go 32 In the present study, the ratio of adenosine/inosine at the end of ischemia was 1:10 in the control group and in the group that was reperfused with warm blood containing EHNA/NBMPR (group 2). Because inosine is not a salvageable precursor, ATP recovery was not expected with inosine trapping. Recovery of left ventricular function in group 2 was not associated with ATP replenishment. Nucleoside washout has been demonstrated during the first minute of reperfusion in animal models Go Go 26,27 and in clinical events of postischemic reperfusion such as angioplasty and coronary artery bypass grafting. Go Go 28,29 Oxypurine (hypoxanthine and xanthine) and uric acid are produced during a single passage of inosine and adenosine through the interstitial space and the endothelium, as demonstrated in coronary sinus effluent. Therefore, it is plausible that administration of EHNA/NBMPR, before or after ischemia, prevented coronary vascular damage induced by a burst of free radicals on reperfusion. Recovery of myocardial NAD+ during reperfusion suggests that 30 minutes of ischemia caused reversible injury. Significant reduction in NAD+ is indicative of irreversible injury and cell death. Go 33 The levels of nucleosides and purines in the interstitium are not known from the present study.

Although myocardial ATP levels long have been considered an important index of functional and metabolic recovery after ischemic injury, Go 34 lack of correlation between ATP and ventricular function also has been demonstrated. Go Go 35,36 These discrepancies may be attributed to (1) the lack of separation between ischemic and reperfusion injury and (2) the fact that the normal myocardium contains at least five times more ATP than the critical levels below which the heart undergoes rigor. Go Go Go 4,37,38 Previous reports demonstrated that pretreatment with EHNA/NBMPR resulted in complete recovery of ventricular function after severe normothermic global ischemia (60 minutes) in dogs. Go 4 This prolonged ischemic period caused 80% loss of ATP and about 80% loss of left ventricular function during reperfusion in the control group. Morphologic evidence of myocardial cell death was observed in a similar model of 60 minutes of normothermic global ischemia in vitro and in vivo. Go Go 33,39 In the latter studies, left ventricular function was not assessed. Therefore it seems that despite reduced ATP levels the heart can function adequately when nucleosides, generated during ischemia, are trapped by EHNA/ NBMPR. Go Go Go Go 1-4,37,38

In the present study, complete functional recovery was observed in the group that was treated with EHNA/ NBMPR after ischemia in the absence of entrapped adenosine or ATP repletion. On the basis of these observations, it is conceivable that nucleoside efflux during reperfusion is playing an important part in reperfusion-mediated ventricular dysfunction and fibrillation.

Because inhibition of nucleoside transport before ischemia resulted in a significant trapping of endogenous adenosine, it is presumed that excellent myocardial protection with EHNA/NBMPR could be mediated by direct cardioprotective effects of adenosine, similar to that described for exogenous adenosine. Go Go Go 8-10,40 It has been postulated that infusion of exogenous adenosine reduces the rate of ATP depletion during ischemia. Go Go Go 8,40,41 Our results demonstrate that despite significant accumulation of adenosine when animals were pretreated with EHNA/ NBMPR, the rate of ATP depletion during ischemia was not reduced by endogenous adenosine accumulation. In this particular group, it is also possible that myocardial protection provided by EHNA/NBMPR could be mediated in part by A1-receptor activation. This specific question was not the focus of this study. However, cardioprotective effects of exogenous adenosine have been demonstrated to be mediated by A1-receptors. Go 42 A brief episode of ischemia and reperfusion followed by a lethal episode of ischemia and reperfusion increases myocardial tolerance (preconditioning) against repetitive or lethal ischemia. Adenosine receptor agonists (adenosine or R-N Go 6 phenylisopropyladenosine) markedly limited the infarct size similar to that seen after ischemic preconditioning in rabbit hearts. Go Go 43,44 Cardiac function after ischemic preconditioning is poor, despite significant reduction in the infarct size. Administration of A1-receptor antagonists abolish ischemic preconditioning in rabbits. Go Go 43,44 In isolated perfused rabbit hearts, improved functional recovery in the preconditioned myocardium was found not to be mediated by A1-receptor. Go Go 45,46 It has been hypothesized that mechanisms of ventricular dysfunction and myocardial infarction are different. The present results demonstrate that ventricular function completely recovers when EHNA/NBMPR is administered only during reperfusion in the absence of entrapped endogenous adenosine, suggesting that protection by these agents may not be related solely to activation of an A1-receptor. Intracoronary infusion of exogenous oxypurines (hypoxanthine and xanthine) in a group of dogs pretreated with EHNA/NBMPR, and accumulated endogenous adenosine during ischemia, resulted in ventricular dysfunction similar to that of the control group. Go 3 These observations suggest that purine efflux via nucleoside transport plays a significant role in postischemic reperfusion-mediated injury, without affecting metabolic derangement associated with ischemia. On the basis of these observations, a correlation between myocardial ATP and ventricular function could be established at reduced ATP levels when reperfusion injury is prevented by EHNA/NBMPR. Although our results support the hypothesis that interruption of oxypurine (hypoxanthine and xanthine) formation provides excellent functional recovery, other cardioprotective mechanisms may be involved in EHNA/NBMPR therapy and deserve further investigation.

SUMMARY

Selective blockade of adenosine nucleoside transport was an effective pharmacologic strategy in preventing postischemic ventricular dysfunction, "stunning," after reversible global ischemic injury. Furthermore, the heart can function adequately with reduced ATP levels (50% of normal) if reperfusion injury is managed. Restoration of myocardial ATP, after severe ischemic injury, is dependent on the availability of entrapped adenosine, but not inosine, and when reperfusion injury is attenuated. We report here that pharmacologic intervention with EHNA/NBMPR could be used to separate metabolic injury induced by ischemia from ventricular dysfunction, "stunning," mediated by reperfusion. Myocardial protection with EHNA/NBMPR may have wide potential therapeutic benefits in preventing reperfusion injury associated with medical or surgical procedures such as balloon angioplasty, thrombolytic therapy, coronary artery bypass grafting, and organ transplantation.

Footnotes

J THORAC CARDIOVASC SURG 1994;108:269-78 Back

*NS = Not significant. Back

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