(Circulation. 1995;91:1545-1551.)
© 1995 American Heart Association, Inc.
Articles |
Inhibition of Nitric Oxide Synthesis Reduces Infarct Size by an Adenosine-Dependent Mechanism
From the Department of Nephrology (R.G.W., G.H.N.), Institute of Urology and Nephrology; and The Hatter Institute for Cardiovascular Studies (V.C.P., D.M.Y.), Department of Academic Cardiology, University College London Medical School, London, UK.
Correspondence to Professor D.M. Yellon, The Hatter Institute for Cardiovascular Studies, Department of Academic Cardiology, University College London Medical School, Gower St, London, UK WC1E 6AU.
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Abstract |
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Background Nitric oxide (NO) is both a potent endogenous vasodilator with potential to attenuate ischemia-reperfusion injury and a mediator of tissue injury. The aim of the present study was to investigate the mechanism by which prior inhibition of NO synthesis can lessen ischemia-reperfusion injury in the isolated rabbit heart.
Methods and Results We examined the effects of inhibition of NO synthesis on infarct size using a model of coronary artery ligation in isolated rabbit hearts perfused at a constant flow rate of 35 mL/min. Infarct size averaged 65% of the zone at risk after 45 minutes of ischemia and 180 minutes of reperfusion. The addition of 30 µmol/L NG-nitro-L-arginine methyl ester (L-NAME), an inhibitor of NO synthesis, to the perfusate reduced the infarct-to-risk (I/R) ratio to an average of 41% (P<.05 versus control). This effect was abolished by pretreatment with 75.5 µmol/L 8-p-sulfophenyl theophylline (SPT), an adenosine receptor antagonist (I/R ratio, 63%). Ischemic preconditioning (5 minutes of ischemia and 10 minutes of reperfusion) before 45 minutes of ischemia and 3 hours of reperfusion reduced the I/R ratio to an average of 21%, and this was not augmented by pretreatment with L-NAME (I/R ratio, 20%). However, all protection due to preconditioning and L-NAME was lost in hearts pretreated with SPT (I/R ratio, 59%). In a separate set of experiments, adenosine concentration in the coronary perfusate and myocardial lactate concentrations were measured. Treatment with L-NAME increased the average adenosine concentration in the perfusate from 5.7 µmol/L per 100 g of heart (control) to a peak of 24.0 µmol/L per 100 g of heart; however, there was no effect on average myocardial lactate concentration (control, 4.6 µmol/g dry wt; L-NAME, 5.5 µmol/g dry wt). In contrast, after 5 minutes of global ischemia, the average adenosine concentration peaked at 139.0 µmol/L per 100 g of heart, and the average myocardial lactate concentration increased to 27.1 µmol/g dry wt.
Conclusions Infarct size limitation after inhibition of NO synthesis shares a common mechanism with that of ischemic preconditioning and is dependent on the release of adenosine. However, in this model, adenosine release after inhibition of NO synthesis is not secondary to myocardial ischemia. The protection of the heart against ischemic injury by adenosine appears to be concentration dependent.
Key Words: nitric oxide • adenosine • reperfusion • ischemia
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Introduction |
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TopAbstractIntroductionMethods Results Discussion References |
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Coronary artery occlusion followed by reperfusion leads to infarction of a proportion of the ischemic area. The extent of injury depends on the availability of collateral blood flow and the duration of ischemia as well as the accumulation of neutrophils and the generation of free radicals during reperfusion. Much of this injurious activity is mediated at the level of the endothelium.
The emphasis of recent work has been on devising strategies to mitigate against ischemia-reperfusion injury. In 1986, Murry et al1 showed that a brief period of ischemia followed by reperfusion leads to increased tolerance to a subsequent and sustained ischemic insult as determined by infarct size. This phenomenon, termed "ischemic preconditioning" (IP) has been demonstrated by many other groups and is associated with both preservation of function and an antiarrhythmic action, using both in vivo and in vitro models of myocardial ischemia (see Reference 2 for a review). The intracellular mechanism responsible for IP may involve activation of ATP-dependent potassium channels or protein kinase C. Cogent evidence appears to support an important primary role for adenosine released by ischemic cells during the preconditioning period.3 The final effector pathway of adenosine-induced protection remains unclear, but it is mediated by A1 (and not A2) receptors on the cardiac myocytes. This emphasis on adenosine is supported by other studies that have identified a cardioprotective role for adenosine in ischemia-reperfusion injury.4 5
Nitric oxide (NO), synthesized by a constitutive enzyme from L-arginine,6 is a highly reactive species that is released continuously by endothelial cells.7 NO maintains the coronary microcirculation in a state of active vasodilatation,8 9 prevents platelet and leukocyte adherence,10 11 and preserves physiological vascular impermeability.12 Although other endothelium-derived autocoids, such as adenosine and prostacyclin, have similar properties,13 14 they are less important physiological determinants of coronary tone. Inhibition of NO synthesis in the coronary vasculature by L-arginine analogues, such as NG-nitro-L-arginine methyl ester (L-NAME),15 leads to vasoconstriction of the microcirculation8 9 and can cause myocardial ischemia.16 17 In response, there is increased release of adenosine and prostacyclin, which vasodilate and partially compensate for the myocardial hypoperfusion that results from diminished NO production.18 19 20
Continuing controversy surrounds the involvement of NO in ischemia-reperfusion injury. Ischemia-reperfusion injury leads to the loss of endothelium-dependent NO-mediated relaxation due to inactivation of NO by oxygen-derived free radicals produced by adherent leukocytes21 22 23 and injured endothelial cells.21 Given its antiadhesion and vasodilatation properties, NO deficiency during reperfusion would be expected to contribute to reperfusion injury. This contention is supported by several studies that have shown that augmentation of NO levels during reperfusion preserves endothelial function, reduces neutrophil accumulation, and decreases markers of myocardial injury.24 25 26 However, there is other evidence that supports an injurious role for NO if it is present in substantial excess. This could be relevant during early reactive hyperemia that is mediated by a substantial increase in NO release18 20 or later when activated neutrophils capable of expressing inducible NO synthase7 have accumulated. NO-dependent toxicity results from the formation of NO-derived free radical species, such as the peroxynitrite anion,27 and the inactivation of iron-sulfur–centered enzymes involved in essential cellular activity, such as mitochondrial respiration.28 Evidence to support a noxious role for NO when overproduced comes from experimental models of myocardial injury after hypoxemia,29 postischemic cerebral reperfusion injury,30 and neutrophil-mediated tissue injury.31 We have recently found that prior inhibition of NO synthesis reduces infarct size after regional coronary artery occlusion and reperfusion in the in situ rabbit heart.16 However, we did not determine whether the mechanism of this protection depended on either inhibition of physiological production of NO before ischemia or inhibition of overproduction of NO during reperfusion. The present study was designed to elucidate the mechanism by which inhibition of NO synthesis by L-NAME protects against ischemia-reperfusion injury using a model of coronary artery occlusion in the isolated, buffer-perfused rabbit heart free of circulating neutrophils.
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Methods |
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Isolated Heart Model
Male New Zealand White rabbits (weight, 2.0 to 3.0 kg) were anesthetized with sodium pentobarbital (40 mg/kg IV) after cannulation of a marginal ear vein. A tracheostomy was performed with the rabbit under local anesthetic (3 mL 2% lidocaine), and the rabbit was ventilated mechanically with 100% oxygen. The chest was opened through a left thoracotomy, and the pericardium was incised to expose the heart. A 3-0 silk suture on a small round-bodied needle was passed through the myocardium underneath a proximal branch of the left coronary artery, and the ends were threaded through a small vinyl tube to form a snare, which was used to occlude the artery as required. The rabbits were given 1000 U heparin IV; then, the heart was rapidly excised by cutting the great vessels, placed in room-temperature saline, and mounted on the Langendorff apparatus within 1 minute.
The hearts were perfused retrogradely at a constant flow rate of 35 mL/min in a nonrecirculating system using a roller pump. The perfusate was Krebs-Henseleit buffer equilibrated with 95% O2–5% CO2 at 37°C that contained (in mmol/L) NaCl 118.0, KCl 4.7, MgSO4 · 7H2O 1.2, CaCl2 · 2H2O 1.8, NaH2PO4 14.6, KH2PO4 1.2, and glucose 11.0, pH 7.4. All hearts were electrically paced at 180 beats per minute via the right atrium. Left ventricular pressures were measured by means of a fluid-filled latex balloon, connected by polyethylene tubing to a pressure transducer, and inserted into the left ventricle via an incision in the left atrial appendage. The balloon volume was adjusted to give an initial diastolic pressure of 10 mm Hg. Coronary perfusion pressure (CPP) was measured via a side arm in the aortic cannula using a pressure transducer. The heart was allowed to stabilize for at least 30 minutes before the experiment was begun.
Infarct Size Experiments
Experimental Protocols
There were seven experimental groups with rabbits assigned
sequentially to each. In each experiment, ischemia lasted 45 minutes
and was followed by 180 minutes of reperfusion.
Group 1, the control group, was monitored for an additional 15 minutes before being subjected to ischemia-reperfusion. In group 2, 30 µmol/L L-NAME was added to the perfusate 10 minutes before ischemia and continued for 15 minutes into the reperfusion period. In group 3, 75.5 µmol/L 8-p-sulfophenyl theophylline (SPT) was added to the perfusate 5 minutes before the addition of 30 µmol/L L-NAME; SPT was continued until after the start of ischemia and the L-NAME continued until 15 minutes into reperfusion. In group 4, 75.7 µmol/L SPT was present in the perfusate until after the start of ischemia. Group 5, the preconditioned group, received 5 minutes of coronary branch occlusion followed by 10 minutes of reperfusion before the 45-minute occlusion and 180 minutes of reperfusion. Group 6 received 30 µmol/L L-NAME 10 minutes before IP. Group 7 received 75.5 µmol/L SPT, followed by 30 µmol/L L-NAME before IP.
Measurement of Infarct and Risk Area
At the end of
each experiment, the heart was flushed with
room-temperature saline for 1 minute. The coronary branch was then
reoccluded, and 1- to 10-µm fluorescent zinc cadmium sulfide
particles were infused into the perfusate until the risk area could be
visualized with UV light. The ligature was then released; 2 to 3 mL of
1% triphenyl tetrazolium chloride (TTC) was injected through the side
arm; and the heart was immersed in Krebs-Henseleit buffer at 37°C for
2 minutes. (TTC stains viable tissue deep red.) The heart was weighed
and frozen. While frozen, the heart was cut into 2-mm transverse
slices. Tracings were made of the risk zones (lacking fluorescence
under UV light) and the infarct zones (TTC-negative tissue), and the
areas were determined by planimetry of the tracings. This experimental
method is well established.3 5
Biochemical Assays
Adenosine Assay
In a
separate group of hearts, coronary effluent samples were
assayed for adenosine by reverse-phase high-performance liquid
chromatography (HPLC) according to the method used by Jenkins and
Bellardinelli.32 The HPLC system included a Waters 710B
injector, an LKB2150 pump, and a Beckman 160 absorbance detector.
Effluent was collected in 5-mL aliquots, freeze-dried, and
reconstituted with deionized water to a volume of 0.5 mL. Samples were
passed through a Waters 8NVC18 (4-µm) column containing 20 mmol/L
KH2PO4 (pH 5.6) with 60% vol/vol methanol. The
adenosine peaks were detected at 254 nm, recorded on a BBC Goerz
metrawatt chart recorder, integrated using planimetry, and referenced
to a standard curve. Adenosine concentration in the perfusate was
expressed in micromoles per liter per 100 g of heart tissue.
Lactate Assay
When coronary perfusate collection
was complete, the same hearts
were freeze-clamped with precooled (-180°C) tissue forceps and
stored in liquid nitrogen to permit subsequent measurement of
myocardial lactate concentration. Tissue extraction was performed using
350 µL ice-cold perchloric acid (6%) followed by neutralization to
pH 5.5 to 6.0. Quantitative lactate estimation was performed using the
enzymatic reaction of lactate dehydrogenase linked to nicotinamide
adenine dinucleotide in glycine-hydrazine buffer medium.33
Myocardial lactate concentration was expressed in micromoles per gram
of dry weight.
Experimental Protocol
For the
adenosine assays, a 5-mL aliquot of coronary effluent
was collected after stabilization of the isolated, perfused rabbit
heart. Additional aliquots were collected at 1, 2, 5, and 10 minutes
after continuous perfusion (control) or the addition of 30 µmol/L
L-NAME to the perfusate or 5 minutes of global ischemia. At the
conclusion of the experiment, the hearts were freeze-clamped, and the
myocardial lactate concentration was measured.
Chemicals
L-NAME was purchased from Sigma Chemicals Ltd, and
SPT was
purchased from Semats Technical Ltd. All studies were performed in
accordance with the United Kingdom Home Office regulations for the care
and use of laboratory animals (project license no. 70/01759).
Statistical Analysis
All results are given as
mean±SEM. Statistical analysis
between groups was performed using ANOVA followed by Scheffe's
F test.
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Results |
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Infarct-to-Risk Ratios
Prior inhibition of NO synthesis by L-NAME significantly reduced the ratios of infarct to risk area after 45 minutes of branch coronary artery occlusion and 180 minutes of reperfusion from 64.6±4.3% (control, n=7) to 41.4±5.1% (L-NAME, n=8) (P<.05). This protection was prevented by pretreatment with SPT (ie, L-NAME plus SPT), which alone had no effect on the infarct-to-risk ratio (Fig 1
and Table). After IP,
the infarct-to-risk ratio was 21.4±1.2% (n=6), which was
significantly less than both control (P<.001) and
L-NAME–treated (P<.05) hearts. The combination of L-NAME
and IP (L-NAME plus IP) conferred no additional benefit. However,
pretreatment with SPT abolished the protection conferred by
preconditioning and L-NAME (ie, IP plus L-NAME plus SPT) (see Fig
1 and
Table). Mean risk areas did not significantly differ between
groups
(see Table).
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Hemodynamic Data
Hemodynamic data relating to ventricular
performance for
all experiments are presented in the Table
. There were no
differences between groups in the hemodynamic variables measured after
the period of stabilization. In all groups, the left ventricular
developed pressure (LVDP) decreased and diastolic pressure increased
over the duration of the experiments. At the end of reperfusion, LVDP
was significantly higher in L-NAME–treated and IP hearts in comparison
to control (P<.05). Diastolic pressures were lower in
hearts treated with L-NAME, IP, or both (L-NAME plus IP) in comparison
to control (P<.05). There were no differences between
groups in coronary perfusion pressure (CPP) measured at the start and
the conclusion of the experiment. However, CPP was significantly
increased 10 minutes after the administration of L-NAME in all treated
hearts. SPT alone increased CPP, but this was not significantly greater
than control. After 30 minutes of reperfusion, CPP was significantly
higher than control in every group of hearts except those assigned to
IP (see Table and Fig 2).
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Biochemical Assays
Adenosine Assay
Mean
adenosine concentration in the coronary effluent was
the same in each group before treatment and did not increase after 10
minutes of perfusion in the control group (3.4±0.7 versus
4.8±0.4
µmol/L per 100 g of heart, n=5, P=NS). However, the
addition of 30 µmol/L L-NAME to the coronary perfusate increased the
adenosine concentration progressively to a peak of 24.0±3.7 µmol/L
per 100 g of heart (n=5) after 10 minutes (P<.06 versus
control). After 5 minutes of global ischemia, the adenosine
concentration in the perfusate peaked at 1 minute of reperfusion
(139.0±17.9 µmol/L per 100 g of heart, n=6) and remained
significantly higher than control hearts at 10 minutes (83.1±7.2
µmol/L per 100 g of heart, n=6; P<.001 versus control;
see Fig 3).
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Lactate Assay
Myocardial
lactate concentration was the same after 10 minutes of
perfusion with either 30 µmol/L L-NAME (5.5±1.2 µmol/g dry wt,
n=5) or control (4.6±0.5 µmol/g dry wt, n=5;
P=NS). In
contrast, after 5 minutes of ischemia and 10 minutes of reperfusion,
myocardial lactate had risen to 27.1±3.4 µmol/g dry wt, n=6;
P<.01 versus control; see Fig 4).
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Discussion |
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The present study demonstrates clearly that inhibition of NO synthesis by L-NAME reduces infarct size after coronary artery occlusion and reperfusion in the rabbit isolated, perfused heart. This result supports an earlier study in which we observed a similar cardioprotective effect after NO inhibition in the in situ rabbit heart.16 Because the isolated, perfused heart is free from circulating leukocytes, we can now confidently conclude that the observed protection does not result from either altered neutrophil accumulation or overproduction of NO by activated neutrophils.
We propose that prior inhibition of NO synthesis by L-NAME protects the myocardium of the isolated rabbit heart by an adenosine-dependent mechanism that is similar to the mechanism described in IP.3 Our evidence is that, first, in the present study, we have shown that the protection induced by L-NAME depends on the release of adenosine because it can be abrogated by SPT, an adenosine receptor antagonist. Second, L-NAME does not confer additional protection on hearts that are subsequently preconditioned. Third, SPT prevents all of the protection induced by the combination of L-NAME and IP, and, fourth, L-NAME and brief global ischemia both give rise to an increase in the adenosine concentration in the coronary effluent, although the increment was less marked after L-NAME than after ischemia. These observations support a common mechanism for the protection induced by pretreatment with L-NAME and IP. However, in contrast to IP, we propose that the trigger for adenosine release after inhibition of NO synthesis is an alteration in coronary hemodynamics rather than myocardial ischemia.
Liu et al3 have previously observed that the reduction in infarct size that follows IP in the rabbit heart is dependent on adenosine release and A1 receptor stimulation, which can be abolished by SPT. SPT is a methylxanthine derivative with an added sulfophenyl group that prevents the molecule from entering the cell; therefore, intracellular phosphodiesterases are not affected. SPT is an unselective adenosine receptor antagonist with a Ki of 4.5 µmol/L and 6.3 µmol/L for the A1 and A2 receptors, respectively34 ; therefore the concentration used in our study (75.5 µmol/L) would have been sufficient to block adenosine receptors. In view of the cardioprotective properties of adenosine,4 5 it is perhaps surprising that pretreatment with SPT (group 4) did not increase infarct size; however, this finding is consistent with other reports. Interestingly, Zhao et al5 recently showed that SPT can worsen myocardial injury either if additional doses are administered during ischemia and early reperfusion or if SPT is administered just before reperfusion. Thus, it appears that the release of adenosine during and after any ischemic period will mitigate against the reperfusion injury that follows.
Treatment with either L-NAME or SPT increased CPP, although the
increment reached statistical significance only after L-NAME (see Fig
2). These data support an important role for NO but not
adenosine as a
determinant of coronary artery tone in the unstressed isolated,
perfused heart.8 9 However, during the reperfusion
period
that followed 45 minutes of regional ischemia, CPP was higher in both
L-NAME– and SPT-treated groups compared with control (see Fig
2),
which indicates that both NO and adenosine contribute to vasomotor tone
during reperfusion. Therefore, the concentration of adenosine
present in the coronary perfusate during reperfusion may have been
increased in L-NAME–treated hearts, which could have further
contributed to myocardial protection.5
There is basal release of ATP from the isolated, perfused heart that is metabolized by extracellular ectophosphatases to adenosine, which vasodilates the coronary circulation35 36 (see previous discussion). The release of adenine nucleotides, principally from endothelial cells but also from cardiac myocytes and purinergic nerves, increases substantially during ischemia.36 37 In the present study, in which L-NAME–treated isolated hearts were perfused at a constant rate of 35 mL/min, the increased concentration of adenosine in the coronary perfusate was not associated with increased myocardial lactate levels, which suggests that these hearts were not ischemic. Although it is possible that we failed to detect localized ischemia using this whole-heart assay or that the assay was inadequately sensitive to detect mild ischemia, our result is consistent with a recent report from Pohl et al,17 who measured lactate concentration in the coronary perfusate of isolated rabbit hearts. These reseachers showed that inhibition of NO synthesis causes myocardial ischemia only if perfusion is pressure controlled, not if the coronary flow rate is maintained in the face of increased CPP. Their report is consistent with our previous finding that inhibition of NO synthesis in the in situ rabbit heart causes myocardial ischemia16 ; in that situation, raised myocardial lactate concentration presumably reflected a combination of myocardial hypoperfusion due to coronary vasoconstriction and increased ventricular work due to increased systemic vascular resistance.
ATP activates P2Y receptors on the endothelium, leading to the release of NO36 ; therefore, it is conceivable that inhibition of NO might affect endothelial release of ATP. However, we believe that the linkage between release of ATP and NO from the endothelium is hemodynamic rather than metabolic. Coronary flow exerts shear stress on the endothelium, leading to ATP release, which can be enhanced by increased shear stress due to, for example, adrenergic vasoconstriction.35 38 We propose that inhibition of NO synthesis led to vasoconstriction that under conditions of constant flow increased luminal shear stress and resulted in increased release of ATP, which was subsequently degraded to adenosine. Under physiological conditions, NO is probably the major determinant of flow-mediated coronary vasodilatation and eclipses the contributions of other autocoids such as ATP, adenosine, bradykinin, substance P, and prostaglandins.39 However, the contribution from these autocoids is likely to be greater in certain diseases, such as atherosclerosis, diabetes mellitus, and hyperlipidemia, where there is evidence of impaired endothelium-dependent NO-mediated vasodilatation. Thus, it is possible that in hearts affected by these diseases, the concentration of adenosine in the coronary perfusate is higher than that in healthy hearts. Because adenosine preserves myocardial blood flow during reperfusion5 and inhibits neutrophil accumulation,13 we hypothesize that increased adenosine release could explain a recent observation that streptozotocin-induced non–insulin-dependent diabetes mellitus protects the rabbit heart from ischemia-reperfusion,40 although this observation was not repeated in the diabetic dog.41
Reperfusion after 5 minutes of global ischemia results in a
substantially greater amount of adenosine being washed out into the
coronary perfusate than does treatment of the whole heart with L-NAME.
(Branch coronary artery occlusion, which affects a smaller portion of
the myocardium than global ischemia, would obviously lead to the
washout of less adenosine into the perfusate, although the affected
myocardium would have been exposed to a similar interstitial
concentration.) In view of this observation, we suggest that a relation
may exist between the amount of adenosine washed out into the coronary
effluent and the extent of cardioprotection. The data do not permit us
to comment as to whether it is the peak concentration of adenosine
achieved or the total released (ie, either the peak or the area under
the curve on Fig 3) that determines the extent of protection.
One aim of the present study was to investigate whether
overproduction of NO during reperfusion might contribute to myocardial
injury. This question would not have been properly addressed if L-NAME
had been added to the perfusate at the beginning of reperfusion since
NO release from the occluded coronary circulation would not have been
immediately inhibited. Therefore, we chose to include L-NAME in the
perfusate from before ischemia until 15 minutes after the start of
reperfusion. As a result, L-NAME caused vasoconstriction that persisted
for the first 30 minutes of reperfusion (see Fig 2), but
despite this,
infarct sizes were smaller in L-NAME–treated hearts. We believe that
the adverse vasoconstrictive effects of L-NAME during reperfusion were
mitigated by the constant flow of coronary perfusate and the
compensatory release of adenosine. It remains possible that the
benefits conferred by these additional factors obscured a minor degree
of myocardial protection that resulted from inhibition of
overproduction of NO during the first 15 to 30 minutes of
reperfusion.
Progressive loss of myocardial function with decreasing LVDP and increasing ventricular diastolic pressure is a feature of the isolated, perfused heart preparation. However, the decreases in LVDP were more marked in control hearts and in the three groups of hearts pretreated with SPT, which reflects the larger infarcts sustained by the hearts in those groups in comparison to those treated with L-NAME, IP, or both (ie, IP plus L-NAME). Similarly, increases in diastolic pressure were more marked in those hearts that had sustained larger infarcts (ie, control, L-NAME plus SPT, SPT, IP plus L-NAME plus SPT) in contrast to the smaller-infarct groups (ie, L-NAME, IP, IP plus L-NAME).
Our results contrast with those of Vegh et al,42 who have demonstrated that NO generation contributes to the antiarrhythmic effects of IP in the in situ canine heart. This may reflect either a disparity between arrhythmogenesis and infarct size as measures of myocardial injury or that L-NAME may inhibit the opening of preformed collaterals in the canine coronary circulation43 that are absent in the rabbit heart.
In conclusion, results of the present study support our previous report that inhibition of NO synthesis protects against ischemia-reperfusion injury in the rabbit heart.16 We propose that the mechanism responsible for this cardioprotection is an increase in release of adenosine, which has cardioprotective properties. This adenosine-mediated mechanism is similar to that implicated in IP except that the stimulus for adenosine release after inhibition of NO synthesis is not myocardial ischemia. Our results do not exclude the possibility that some myocardial protection results from inhibition of NO release during early reperfusion; however, we conclude that most of the benefit is derived from the release of adenosine. Finally, we suggest that a concentration-response relation exists between adenosine concentration in the coronary perfusate and the extent of protection against subsequent injury.
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Acknowledgments |
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This work was supported by grants from the British Heart Foundation and the Hatter Foundation.
Received June 1, 1994; revision received August 17, 1994; accepted October 27, 1994.
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