Effects of Endothelin-1 and Endothelin-A Receptor Antagonist on Recovery After Hypothermic Cardioplegic Ischemia in Neonatal Lamb Hearts
Background Prior studies suggest an important role for coronary endothelium in ischemia/reperfusion (I/R) injury. Decreased endothelial release of the vasodilator nitric oxide occurs after I/R, but the role of the endothelium-derived vasoconstrictor endothelin-1 (ET-1) in I/R is unknown.
Methods and Results We measured plasma ET-1 concentrations by radioimmunoassay in isolated blood-perfused neonatal lamb hearts before and after 2 hours of 10°C cardioplegic ischemia and examined the effects of ET-1 and the endothelin-A (ET-A) receptor antagonist BE-18257B on the postischemic recovery of isolated hearts. ET-1 levels in coronary sinus blood before ischemia and at 0 and 30 minutes of reperfusion in 8 control hearts were constant (2.2±1.2 fmol/L, 2.2±1.3 fmol/L, and 2.5±1.0 fmol/L, respectively). In group 2 (n=6), 10 μmol/L of BE-18257B was given just before reperfusion. In group 3 (n=8), 10 pmol/L ET-1 was given just before the start of reperfusion. At 30 minutes of reperfusion, the ET-A antagonist hearts had significantly greater recovery of LV systolic (positive dP/dt and dP/dt at V10) and diastolic function (negative dP/dt), coronary blood flow (CBF), and MV̇o2 compared with controls (P<.05). The ET-1 hearts showed significantly reduced recovery of LV systolic (positive maximum and volume-normalized dP/dt) and diastolic (negative maximum dP/dt) function, CBF, and myocardial oxygen consumption compared with controls (P<.05).
Conclusions These results, combined with prior studies, suggest that I/R causes reduced production of endogenous vasodilators (eg, nitric oxide), leaving unopposed the vasoconstriction that is caused by the continued presence of ET-1. This imbalance may contribute to I/R injury. ET-A receptor antagonists may be useful therapeutic agents in reducing the injury that results from I/R.
Prior studies from our laboratory suggest an important role for the coronary endothelium in ischemia/reperfusion injury.1 2 The endothelium is a source of several relaxing (eg, EDRF or NO) and contracting factors (eg, ET), and it has been proposed that interactions between relaxing and contracting factors contribute to the normal physiology of blood flow regulation and play a role in cardiovascular disease.3 Our laboratory has previously provided evidence to suggest that decreased endothelial release of NO occurs after ischemia/reperfusion2 and that infusion of l-arginine, a precursor of NO, during early reperfusion increases CBF and improves the recovery of LV function and endothelial function after hypothermic ischemia/reperfusion.4 The role of ET, an endothelium-derived vasoconstrictor, in myocardial ischemia/reperfusion is unknown.
ETs are thought to be involved in the pathogenesis of hypertension, heart failure, bronchial asthma, renal failure, and vasospasm.5 There are three ET isoforms (ET-1, ET-2, and ET-3), and two types of ET receptors have been reported, designated ET-A (selective for ET-1) and ET-B (nonselective with respect to isopeptides of the ET family).5 ET-A receptors are abundant in cardiovascular tissues and are also found on vascular smooth muscle cells that respond to ET-1 by contraction.5
In the present study, we used ET-1 and an ET-A receptor antagonist (BE-18257B, a cyclic pentapeptide6 ) in neonatal lamb hearts in an attempt to determine whether endogenous ET-1 is released into the coronary circulation during hypothermic myocardial ischemia/reperfusion and whether ET-1 has an influence on recovery of postischemic ventricular function.
An isolated blood-perfused heart model described previously1 2 4 was used to study hearts from 22 neonatal lambs (weight, 2.3 to 5.4 kg; age, 2 to 5 days old). Briefly, coronary perfusion was established with a roller pump and oxygenator system, and then the heart was excised. A sampling catheter was placed in the coronary sinus via the hemiazygos vein for coronary venous blood analysis. Heparinized fresh homologous blood was used as the perfusate. Arterial pH was kept at 7.4 (corrected to perfusate temperature). Myocardial temperature was monitored by thermal probes, and perfusate and myocardial temperatures were maintained at 37°C except during the hypothermic phase. Coronary perfusion pressure was maintained at 60 mm Hg except during the hypothermic and initial reperfusion phases. A latex balloon containing a pressure transducer was placed inside the LV through the apex to measure LV function.
LV function was measured during isovolumic contraction by stepwise inflation of the intraventricular balloon as described previously.1 2 4 The recovery of systolic function was evaluated by measuring the maximum (max) DP, positive (+) max LV dP/dt, peak DP at a constant balloon volume (V10), and peak dP/dt at V10. V10 was defined as the balloon volume required to produce an EDP of 10 mm Hg during baseline measurements. To assess diastolic function, negative (−) max dP/dt and EDP at V10 were measured. CBF was measured by use of an electromagnetic flowmeter, which was connected to the venous cannula. Arterial and venous blood were collected and MV̇o2 was calculated from the hemoglobin concentration, oxygen content, and saturation.1 2 4
Bioassay of Plasma ET-1
Coronary sinus blood samples were collected after 20 minutes of normothermic bypass (baseline), at the start of reperfusion (0 minutes), and again after 30 minutes of reperfusion in eight control hearts. Plasma ET-1 levels were quantitated using a commercially available ET-1, 21-specific radioimmunoassay system (Amersham). Briefly, we collected sheep blood into tubes containing 7.5 mmol/L EDTA, and the samples were centrifuged at 2000g for 10 minutes at 4°C. The plasma was immediately frozen at −20°C to minimize ET degradation. ET was extracted from 1 or 2 mL of acidified plasma using Amprep 500-mg C2 columns (Amersham). After removal of solvent by centrifugal evaporation, samples were reconstituted in 250-μL assay buffer (0.02 mol/L sodium borate, pH 7.4). Duplicate 100-μL sample aliquots plus 100-μL aliquots of synthetic ET-1 of known concentration (for standardization) were incubated with rabbit anti-endothelin antiserum for 4 hours at 4°C. A radioactive tracer, 125I ET-3, was added and incubated for 16 to 24 hours at 4°C. The antibody-bound ET was separated from ET by use of a donkey anti-rabbit antiserum coupled to magnetic beads followed by magnetic separation (Amerlex-M Separator, Amersham). The radioactive counts retained by the magnetic separator were measured in a gamma scintillation counter (Packard Cobra Auto-gamma). The concentration of ET-1 (in fmol/mL) was determined by plotting the average of duplicate sample counts against the standard curve of known ET-1 concentrations according to the manufacturer’s instructions.
Baseline measurements were made after a 20-minute equilibrium period. Then, the perfusate was cooled to 15°C. After 10 minutes of cooling (at myocardial temperature=15°C), coronary perfusion was stopped and 20 mL/kg of cardioplegic solution was given, followed by topical cooling (myocardial temperature was kept at 10°C). A second dose of cardioplegic solution (10 mL/kg) was given after 60 minutes. The venous efferent during cardioplegia administration was discarded. The cardioplegic solution consisted of 0.45% sodium chloride and 2.5% dextrose solution with 20 mEq/L of potassium chloride and 6 mEq/L of sodium bicarbonate (pH=7.4 at 37°C, osmolarity 360 mOsm/L). Reperfusion was begun with the perfusate at room temperature (25°C), and the heart was rewarmed by raising perfusate temperature to normothermia over a 25-minute period. Mean coronary perfusion pressure was maintained at 20 mm Hg for the first 5 minutes and raised to 40 mm Hg for the second 5 minutes, then kept at 60 mm Hg until the end of the experiment.1 2 High oxygen–content gas (95% O2, 5% CO2) was supplied to the oxygenator during the cooling phase and during the first 15 minutes of reperfusion to mimic conditions of clinical cardiopulmonary bypass. Thereafter, the gas was changed to normal oxygen levels (20% O2, 5% CO2, 75% N2).
Hearts were divided into three groups: (1) In the control group (n=8), there was no intervention during reperfusion; (2) In the ET-A receptor antagonist group (n=6), the ET-A receptor antagonist BE-18257B6 (purchased from Bachem) was given just before reperfusion at a concentration of 10 μmol/L; (3) In the ET-1 group (n=8), ET-1 (purchased from Bachem) was given just before reperfusion at a concentration of 10 pmol/L.
Animals in this study received humane care in compliance with “Principles of Laboratory Animal Care” formulated by the National Society for Medical Research and “Guide for the Care and Use of Laboratory Animals” prepared by the National Academy of Sciences and published by the National Institutes of Health.
All values are expressed as mean±SD and were analyzed by a statistical analysis system. One-way ANOVA and repeated measures two-way ANOVA were used to compare the differences in recovery between groups. Data were compared using Student-Newman-Keuls test if ANOVA was significant. A value of P<.05 was considered to be significant (NIH Publication No. 86-23, revised in 1985).
Plasma ET-1 bioassay (Table 1⇓). ET-1 concentrations from coronary sinus blood in the control group before ischemia and at 0 and 30 minutes of reperfusion were 2.2±1.2 fmol/L (5.32 pg/mL), 2.2±1.3 fmol/L, and 2.5±1.0 fmol/L (6.05 pg/mL), respectively. There were no significant differences between these values.
Baseline measurements (Table 2⇓). There were no significant differences among the three groups in any of the baseline hemodynamic data.
LV function (Table 3⇓). The ET-A receptor antagonist group had a significantly greater recovery of +max dP/dt, dP/dt at V10, and −max dP/dt compared with both the control group and the ET-1 group. The ET-1–treated hearts had significantly reduced recovery of LV systolic function indexes including +max dP/dt and dP/dt at V10. The LV diastolic function index (−max dP/dt) at 30 minutes of reperfusion was worse in the ET-1 hearts compared with the control group.
Coronary blood flow (Table 4⇓). The ET-A receptor antagonist group had significantly higher CBF than both the control group and the ET-1 group at 10 minutes of reperfusion and thereafter. The ET-1 group had significantly lower CBF than the control group at 25 minutes of reperfusion and thereafter.
Oxygen consumption (MV̇o2) (Table 5⇓). The ET-A receptor antagonist group had significantly higher MV̇o2 than the control group and the ET-1 group at 15 minutes of reperfusion. At 30 minutes of reperfusion, the ET-1 hearts showed significantly lower MV̇o2 than the control group.
Effects of ET-1 at normothermia without ischemia (Table 6⇓). To assess the effect of ET-1 and the ET-A receptor antagonist at normothermia without ischemia, we infused 10 pmol/L ET-1 (n=4) and 10 μmol/L ET-A receptor antagonist (n=2) in the same isolated heart model without ischemia. The changes in LV function, CBF, and MV̇o2 were examined 30 minutes later. Data were compared with those in the control group at normothermia. ET-1 (n=4) caused no significant change in LV function indexes except that −max dP/dt declined. CBF and MV̇o2 declined, but not significantly. Administration of the ET-A receptor antagonist at normothermia (n=2) resulted in no significant change in LV function and caused an insignificant (P>.05) increase in CBF and MV̇o2.
The present study shows that in the neonatal lamb heart, ET-1 levels were constant before and after 2 hours of hypothermic ischemia and reperfusion and that administration of additional ET-1 reduced the recovery of systolic and diastolic LV function, CBF, and MV̇o2. Administration of the ET-A receptor antagonist improved LV systolic and diastolic function, CBF, and MV̇o2. These data strongly suggest that ET-1 is involved in the pathogenesis of ischemia/reperfusion injury in neonatal hearts, but also make it likely that other factors are involved.
ET-1 is a potent vasoconstrictive peptide, containing 21 amino acid residues, that is produced by endothelium and has multiple biological actions.7 Three ET isoforms (ET-1, ET-2, and ET-3) have been identified, and there appear to be few interspecies differences in these three ET isoforms.6 Two types of ET receptors have been reported. The ET-A receptor is selective for ET-1 and is located predominantly on vascular smooth muscle cells,2 whereas the ET-B receptor is nonselective with respect to isopeptides of the ET family. ET-A receptors are abundant in cardiovascular tissues including the kidney, adrenal glands, and the central nervous system.8 9 10 ET-B receptors are also found on the endothelium.2
Few reports have addressed the effects of ischemia and reperfusion on ET release. Increases in circulating levels of ET have been documented in states of severe cardiovascular stress, including cardiogenic shock,10 acute myocardial infarction,11 12 congestive heart failure,12 13 14 and essential hypertension.14 15 Brunner et al15 have reported that ET release was decreased during low-flow ischemia and then transiently increased during early reperfusion in isolated crystalloid-perfused rat hearts before returning to control levels. Our finding that postischemic ET-1 levels did not change significantly from preischemic levels suggests that little postischemic ET-1 release occurred, because most of the clearance of ET-1 is reported to occur in the kidney and lung.5 The plasma levels of ET-1 that we measured before ischemia (5.3 pg/mL) are on the same order of magnitude (0.3 to 1.7 pg/mL) as for human plasma in reports summarized by Nayler,5 but they are lower than reported in normal adult sheep.5
ET-1 administration before reperfusion resulted in significantly worse recovery of ventricular function and reduced CBF. ET-1 has been shown to induce dose-dependent coronary constriction and impaired diastolic relaxation in isolated rabbit hearts.16 Brunner et al15 found that administration of ET-2 at the onset of reperfusion initially resulted in improved aortic output (first 20 minutes), but thereafter aortic output was reduced compared with controls.
In isolated atria and ventricle preparation, ET does exert a positive inotropic effect,5 17 18 19 but we did not find a significant effect of either ET-1 or the ETA receptor antagonist on CBF or systolic ventricular function 30 minutes after administration to isolated hearts that were not subjected to ischemia. Diastolic function was reduced, however (Table 6⇑). Thus, in our experiments, whatever positive inotropic effects of ET-1 existed seemed to have been outweighed by its other effects.
In contrast to the effects of ET-1, the administration of the ET-A receptor antagonist BE-18257B resulted in improved recovery of ventricular function and CBF in our experiments. To our knowledge, no prior experiments have been carried out in a model of hypothermic ischemia using this ET receptor antagonist. Watanabe et al20 found that an ET-A antibody did reduce myocardial infarct size in a rat model of normothermic myocardial ischemia and reperfusion. Thus, it appears that even if ET-1 levels are not elevated during reperfusion, blockade of ET-1 action is likely to be beneficial.
The results of our current experiments and prior experiments from our laboratory lead us to hypothesize that an imbalance of endothelium-derived vasodilators and vasoconstrictors exists after hypothermic ischemia and reperfusion, and this imbalance likely plays a significant role in ischemia/reperfusion injury. This hypothesis is based on several observations. We have demonstrated a decreased vasodilator response to intra-arterial acetylcholine1 2 in the isolated lamb heart model of hypothermic ischemia and reperfusion, a finding that implies a reduced ability of the endothelium to release EDRF (NO). We have also shown that provision of supplemental amounts of the EDRF precursor l-arginine results in improved postischemic ventricular function4 and partially restores the defect in the ability to release NO. Other investigators have provided evidence of increased sensitivity to ET after ischemia. Brunner et al15 have reported that ET-2 had a 30-times-greater potency after ischemia, and Nayler and coworkers5 21 have reported upregulation of ET receptors after ischemic or hypoxic insults. Our finding that postischemic CBF was reduced below preischemic levels also suggests that a relative vasoconstrictive influence was present. Finally, the finding that the ET-A receptor antagonist exerted beneficial effects in the absence of elevated ET-1 levels but did not have significant effects in the absence of ischemia suggests that after ischemia, naturally occurring antagonists to ET-1 are missing or are present in lower concentrations compared with preischemia. It is also noteworthy that EDRF normally inhibits the release of ET-122 23 and that ET normally stimulates the release of EDRF.24 It seems reasonable to hypothesize that the unopposed vasoconstrictor effect of ET-1 in a vascular bed, which may be more sensitive to its effects, could result in reduced postischemic CBF, which would in turn limit the postischemic recovery of the heart.
An alternative mechanism by which an ET-1/EDRF imbalance could reduce postischemic recovery is suggested by the recent observation that ET-1 increases neutrophil adhesion to endothelial cells by inducing leukocyte integrin expression.25 Since EDRF has been shown to inhibit the expression of endothelial adhesion molecules26 and to inhibit neutrophil-endothelial adhesion,27 an imbalance of ET-1 and EDRF could also enhance myocardial injury post–ischemia/reperfusion through accumulation of activated neutrophils in the coronary circulation.
These conclusions are limited by the fact that experiments were carried out in the isolated heart, although this model has the advantage of being free of the neural and hormonal influences that would be present in a whole-animal preparation. In addition, this model does have the advantage of being similar to the conditions of an extracorporeal bypass circuit and hypothermia, under which ischemia is usually induced during cardiac surgery. Additional studies in the whole animal will be necessary and are planned.
In summary, the results of the present study combined with findings in prior studies suggest that ischemia/reperfusion causes reduced production of endogenous vasodilators (eg, EDRF or NO), which consequently do not offset the vasoconstriction caused by the continued presence of ET-1. This imbalance may contribute to ischemia/reperfusion injury. Therefore, ET-A receptor antagonists may be useful therapeutic agents in ischemia/reperfusion.
Selected Abbreviations and Acronyms
|CBF||=||coronary blood flow|
|EDRF||=||endothelium-derived relaxing factor|
|MV̇o2||=||myocardial oxygen consumption|
We sincerely thank Pascal Gebeyan, Mikael E. Roy, Tara E. Hamond, Sigrid H. Wolfram, BS, and Shayla S. Rose for their technical assistance.
Reprint requests to John E. Mayer, Jr, MD, Department of Cardiovascular Surgery, Children’s Hospital, 300 Longwood Ave, Boston, MA 02115.
Presented in part at the 67th Scientific Sessions of the American Heart Association, Dallas, Tex, November 14-17, 1994, and published in abstract form (Circulation. 1994;90[pt 2]:I-422).
- Copyright © 1995 by American Heart Association
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