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Role of Endothelin in a Rabbit Model of Acute Myocardial Infarction: Effects of Receptor Antagonists

Vitola, João*; Forman, Mervyn*; Holsinger, John*; Kawana, Masatoshi*; Atkinson, James; Quertermous, Thomas*; Jackson, Edwin§; Murray, John*‡

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Journal of Cardiovascular Pharmacology: December 1996 - Volume 28 - Issue 6 - p 774-783
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Abstract

Myocardial infarction remains a leading cause of death in the United States and many other industrialized countries. Extensive experimental studies have been performed to further our understanding of the pathophysiology of irreversible myocardial damage during prolonged periods of myocardial ischemia followed by reperfusion. In addition, numerous strategies have been used in an attempt to reduce infarct size and improve ventricular function in models of regional ischemia (1).

Endothelin (ET) is a 21-amino acid peptide, first isolated from endothelial cells in 1988 (2). This peptide causes intense and prolonged vasoconstriction of numerous vascular beds, including the coronary vasculature (3), with a potency 10 times that of angiotensin II. Because myocardial ischemia and reperfusion result in an enhanced production or release (or both) of ET from the previously ischemic myocardium (4,5), there has been great interest in the investigation of the potential role of ET in myocardial ischemia and reperfusion. Whether or not ET induces or exacerbates myocardial necrosis during ischemia and reperfusion is unclear. ET theoretically may contribute to the microcirculatory failure seen after myocardial reperfusion, the so-called no-reflow phenomenon (6), which may play a part in the pathogenesis of reperfusion injury (7,8). ET may potentially have other unknown effects in the heart, which could alter cell survival during periods of myocardial ischemia and reperfusion.

The purpose of our study was to investigate the role of ET in the pathogenesis of myocardial infarction by using a rabbit model of regional myocardial ischemia and reperfusion. The ET family of peptides consists of several functionally and structurally related isopeptides named ET-1, ET-2, and ET-3. Moreover, two distinct ET-receptor subtypes, ETA and ETB, have been identified. ETA is selective for ET-1 and is found predominantly in cardiovascular tissue, whereas ETB is nonselective with regard to ET isopeptides and is found in many different sites including the heart (9,10). Therefore in our study, we examined the effects of a selective ETA-receptor antagonist, a nonselective ETA/ETB-receptor antagonist, and ET-1 itself on infarct size after ischemia and reperfusion. In addition, we determined the effects of myocardial ischemia/reperfusion on myocardial steady-state levels of ET-1 messenger RNA (mRNA).

MATERIAL AND METHODS

Myocardial blood flow and ET gene-expression protocol

Myocardial blood flow. The studies performed conform to the Position of the American Heart Association on Research and Animal Use adopted in 1984. New Zealand male rabbits (n = 14) weighing 3.5-4 kg were used. Animals were kept in their cages for 1 week before experiments to allow adaptation. A combination of ketamine (20 mg/kg) and xylazine (4 mg/kg) was used for preanesthesia. A catheter was placed in the marginal ear vein, and animals were anesthetized with sodium pentobarbital (10 mg/kg bolus followed by 3 mg/kg boluses) until adequate anesthesia was obtained. A tracheostomy was performed, and the animals were ventilated with a Harvard positive-pressure respirator (Harvard Apparatus, S. Natick, MA, U.S.A.). Supplemental anesthesia with pentobarbital was given as needed to maintain anesthesia during the procedure. One electrocardiographic lead (II or III) was monitored continuously throughout the protocol (system model VR-12; PG Biomedical Systems, Lenexa, KS, U.S.A.). The right and left femoral arteries and right femoral vein were cannulated for measuring arterial blood pressure, for arterial microsphere blood sampling, and for drug infusions, respectively. A left thoracotomy was performed at the fourth intercostal space. The pericardium was incised, and the left obtuse marginal branch of the circumflex artery was identified. A 4-0 silk ligature was placed around the artery just proximal to its branching near the atrial appendage. The ends of the ligature were then enclosed in a polyethylene tubing (PE90; Dow Corning, Midland, MI, U.S.A.) for later arterial occlusion. A catheter made of the same polyethylene tubing was placed in the left atrial appendage for microsphere injections. After the animals were allowed to stabilize, baseline hemodynamic measurements were obtained.

Animals underwent 30 min of coronary occlusion and 3 h of reperfusion. Hemodynamic measurements were obtained before each microsphere injection. Regional myocardial blood flow (MBF) was determined with a 2-ml bolus injection of 15 ± 3 μm microspheres (Du Pont, Biotechnology Systems, Wilmington, DE, U.S.A.) followed by a 3-ml saline flush. Microspheres were labeled with radioisotopes of increasing levels of photon energy and were injected at a dose of 10 μCi serially (≈200,000-300,000 per injection) in the following order: cerium (141Ce, 145 keV) at baseline, chromium (51Cr, 320 keV) at 15 min into occlusion, strontium (85Sr, 514 keV) at 1 min into reperfusion, niobium (95Nb, 765 keV) 1 h into reperfusion, and scandium (46Sc, 889 keV) at 3 h into reperfusion. Reference samples were drawn from the left femoral artery at a rate of 2.7 ml/min for 1.5 min, commencing 30 s before the injection of each microsphere bolus to allow calculation of MBF. The number of microspheres was kept at <300,000 per injection to prevent adverse systemic or cardiac effects (11). No hemodynamic effects were observed during blood withdrawal. After 3 h of reperfusion, the ligature was reoccluded, and Monastral blue dye (1 ml/kg) was administered into the left atrium over a 1-min period to define the AR. The heart was rapidly removed from the chest and washed. Myocardial samples from the central ischemic (CIZ) and nonischemic zones (NIZ) were obtained from the midventricular slices. Each slice was divided into endocardial and epicardial halves (total of 10 samples from each heart). These tissue samples and arterial reference samples were counted for 5 min in a multichannel autogamma scintillation spectrometer (model 5986; Packard Instrument Company, Downers Grove, IL, U.S.A.). Background contamination and overlapping radioactivity from other isotopes were accounted for by using a matrix correction method (Compusphere Software; Packard Instruments). MBF was calculated in milliliters per gram wet weight per minute, as previously described from our laboratory (7).

Quantitation of ET-1 mRNA. Six of the rabbits (n = 6) undergoing 30 min of circumflex occlusion and 3 h of reperfusion in the study described previously were randomly assigned for analysis of ET-1 mRNA expression in the heart. After the ischemic zones were defined, as described in detail previously, the heart was removed from the chest. Slices of the left ventricle were stained with triphenyltetrazolium chloride to define the area of necrosis. Tissue from the central ischemic zone, including necrotic and nonnecrotic tissue, and tissue from the nonischemic zone were isolated and stored in liquid nitrogen. These samples were then homogenized in 5 M guanidinium isothiocyanate and total cellular RNA isolated in a cesium chloride density gradient (12). Ten micrograms of each RNA was fractionated on a 1.3% formaldehyde-agarose gel and then transferred to a nitrocellulose filter. The filter was hybridized with a randomprimed 32P-labeled 1.5 kb EcoRI fragment of rabbit ET-1 cDNA (13,14). After stripping the blot of the ET-1 probe, it was hybridized to a β-actin probe. Hybridization signals were quantitated from the resulting autoradiographs by densitometry (Ultrascan XL, Pharmacia), and ET-1 signals were normalized to β-actin signals used as internal controls.

Antagonism of ET receptors in systemic and coronary vasculature protocol

The ET-1 used in all studies was obtained from Sigma Chemical; PD145065 was received as a gift from Parke-Davis Pharmaceutical (Ann Arbor, MI, U.S.A.); and FR139317 received as a gift from Fujisawa Research, Osaka, Japan. PD145065 is a hexapeptide, nonselective ETA-and ETB-receptor antagonist (15), whereas FR139317 is a linear tripeptide, selective ETA-receptor antagonist (16). The purpose of this portion of the study was to verify the efficacy of these receptor antagonists in blocking the cardiovascular effects of exogenous ET-1, with the final objective of testing the role of these antagonists and ET-1 itself on infarct size. In this protocol, New Zealand White rabbits (n = 14) were anesthetized and ventilated as described above. All animals had the right femoral artery and vein cannulated for measuring arterial blood pressure and for drug infusions, respectively. Animals underwent a left thoracotomy, performed at the fourth intercostal space. The pericardium was incised, and the left obtuse marginal branch of the circumflex artery was identified and dissected to allow placement of a 1-mm flow probe model 1 rb (Transonic Systems, Ithaca, NY, U.S.A.) around the vessel for blood-flow measurement. Flow was monitored by using a model T206 (Transonic Systems) flow meter. After a 1-h stabilization period, animals were randomized to receive saline (control group, n = 3) or FR139317 (n = 4). Animals randomized to the FR139317 group received it as an i.v. bolus of 3 mg/kg, followed by a continuous infusion of 0.1 mg/kg/min, whereas control animals received an equivalent volume of saline. The infusion of saline or FR139317 was begun 10 min before the administration of a series of ET-1 boluses starting at 10-9 mol/kg and increasing threefold every 20 min until the animal's death. Coronary blood flow, arterial blood pressure, and heart rate were continuously monitored throughout the protocol. Coronary vascular resistance was calculated by dividing the mean blood pressure value by the coronary blood-flow value. Other rabbits (n = 3) were cannulated for arterial blood pressure measurements only and treated with PD145065 as a bolus of 3 mg/kg, followed by a continuous infusion of 0.6 mg/kg/min, starting before the series of ET-1 boluses, as in the other groups.

Role of ET on infarct size and arrhythmogenesis protocol

The role of ET in the pathogenesis of myocardial reperfusion injury was evaluated by using the rabbit infarct model. Rabbits (n = 45) undergoing 30 min of circumflex occlusion and 48 h of reperfusion were prepared as described previously for those animals undergoing myocardial blood-flow measurements with radioactive microspheres, except that a second arterial line and the left atrial appendage catheter were not used for this protocol. Before coronary occlusion, animals were randomly assigned to receive PD145065 (n = 12), FR139317 (n = 10), ET-1 (n = 11), or saline (n = 12). Coronary occlusion was achieved by pressing the polyethylene tubing against the ventricular wall. The animals then underwent 30 min of ischemia confirmed by the appearance of epicardial cyanosis and ST-segment elevation. Animals did not receive prophylactic lidocaine, as this could decrease infarct size per se, as shown previously (17-19). Baseline hemodynamic parameters were measured before drug infusions and continued every 15 min throughout the reperfusion period. Reperfusion of the vessel was achieved by releasing the ligature. Successful reperfusion was confirmed by visualization of arterial blood flow through the artery, disappearance of epicardial cyanosis, and rapid resolution of the ST-segment changes. After 120 min of reperfusion, the loose ligature was secured, and the chest and tracheostomy were closed. Animals were taken to their cages and allowed to recover. Animals in the PD145065 group received a bolus (3 mg/kg) 10 min before occlusion followed by a continuous infusion of 0.6 mg/kg/min throughout the 30 min of occlusion and for an additional 120 min after reperfusion. Animals received FR139317 in the same fashion with a bolus of 3 mg/kg followed by infusion of 0.1 mg/kg/min. The doses of PD145065 and FR139317 were chosen as those that significantly prevented the hemodynamic changes and mortality caused by exogenously infused ET-1, as determined previously. Animals in the ET-1 group received a bolus of ET-1 (10-10 mol/kg) followed by a continuous infusion of 10-11 mol/kg/min. This dose of ET-1 was calculated to produce greater increases in plasma endothelin levels than previously shown to occur after myocardial reperfusion (4,5) while producing only minimal hemodynamic changes in our pilot studies. Animals in the control group received the same volume of saline. Those animals receiving saline, ET-1, or PD145065 had continuous monitoring and recording of one electrocardiographic lead (2 or 3) to determine ventricular arrhythmias by using a Macintosh computer and Software Acqknowledge Waveform Data Analysis Version 2.0 (Biopac Systems, Coleta, GA, U.S.A.). The episodes of ventricular fibrillation (VF) and premature ventricular contractions (PVCs) occurring during the 30 min of coronary occlusion and first 15 min of reperfusion were quantified.

After 48 h of reperfusion, the animals were reanesthetized with 50 mg of pentobarbital sodium and were reintubated through the tracheostomy. The thoracotomy site was reopened, and the ligature was tightened. Monastral blue dye (1 ml/kg) was administered via the marginal ear vein over a 1-min period to define the AR. The heart was rapidly removed from the chest, washed to prevent counterstaining, and fixed in 10% phosphate-buffered formaldehyde. The hearts were sectioned starting from the apex and parallel to the posterior atrioventricular groove in four to five slices at 3- to 4-mm intervals. The slices were photographed on their basal surface for later confirmation of the AR. The right ventricle was removed, and the left ventricular slices were weighed. Tissue sections were dehydrated and embedded in paraffin. Microscopic sections (4 μm) were then cut and stained with hematoxylin-eosin and Masson's trichrome stain. Five microscopic sections from each heart were used for measurements of area of necrosis (AN) and AR. The paraffin blocks were superimposed on the histologic sections mounted on glass slides and the AR marked and confirmed from the photographs of the gross sections. The microscopic slices were enlarged (×10) by using a microscopic projector. The percentage of the left ventricular necrosis (AN/LV, stained in blue) and the percentage of left ventricular area at risk (AR/LV), were computed by using computer planimetry, as previously described (20). Measurements of the AR and AN were determined by an observer unaware of the treatment groups.

Statistical analysis

All animals in the blood-flow and hemodynamic studies were included in the analysis. In the infarct study, animals were excluded from analysis by prestudy criteria including an AR <25% or >85% of the left ventricle. Our criteria also excluded animals with hemodynamically unstable arrhythmias for >60 s, although none of the animals in the study met this criteria. One animal in the control group, one in the PD145065, one in the FR139317, and two in the ET-1 group died in their cages in the first 24 h of reperfusion. One animal in the control group, one in the FR139317 group, and one in the PD145065 group were excluded from the study because the AR did not meet our criteria previously established. The remainder of the animals were used for infarctsize calculations. Data were expressed as mean ± SEM. Analyses were performed by one-factor analysis of variance (ANOVA) with repeated measures and Student's t test as appropriate. Comparison of linear regression curves were analyzed for differences by examining the slopes of the data of the various groups (21). Differences in the incidence of VF between groups were determined by χ2 analysis, excluding animals not meeting criteria for size of AR described previously. A p ≤ 0.05 was considered significant.

RESULTS

Myocardial blood flow and ET gene expression

There was a significant decrease in the MBF during coronary occlusion in comparison with the baseline value and with the flow in the nonischemic zone (Fig. 1). Such a marked decrease in regional flow during ischemia is consistent with the fact that rabbits have a very poorly collateralized myocardial circulation (22). During the first minute of reperfusion, there was a significant increase in MBF to the CIZ (p ≤ 0.03 from baseline). These results demonstrate the existence of reactive hyperemia in the rabbit model after 30 min of myocardial ischemia.

After 3 h of reperfusion, there was a 37% decrease in the MBF to the CIZ compared with baseline (p ≤ 0.05), and this was also significantly decreased in comparison with the flow in the NIZ. This finding demonstrates in the rabbit model the existence of a progressive microcirculatory failure (no-reflow phenomenon) to the reperfused myocardium. As we previously found that ET levels increased during this same period (4), we examined whether this was the result of increased release or enhanced ET-1 gene expression. Northern blood analysis of ET-1 mRNA levels, at 3 h into reperfusion, indicated that the ET-1 mRNA level in the CIZ was significantly (p ≤ 0.05) increased, an average of 2.6-fold, when compared with the NIZ and relative to β-actin mRNA, which is constitutively expressed (Table 1 and Fig. 2).

Antagonism of ET receptors in systemic and coronary vasculature

This portion of the study was designed to establish the efficacy of the antagonists being tested, as well as the dose used, in blocking the cardiovascular effects of ET-1, with the final objective of selecting appropriate doses for the infarct protocol. The administration of ET-1 (10-9 mol/kg) alone produced a dramatic and significant increase in mean blood pressure (≈60 mm Hg in the first 5 min after infusion compared with baseline, p < 0.02). These changes in blood pressure were accompanied by a significant decrease in heart rate 5 min after infusion (≈50 beats/min from baseline; p ≤ 0.01), and decrease in coronary blood flow leading to elevation in the coronary vascular resistance (Fig. 3) Administration of ET-1 was followed by a high incidence of PVCs. Early after the second bolus of ET-1 (3 × 10-9 mol/kg), all control animals died. All animals pretreated with FR139317 were protected from the hemodynamic changes after ET infusion that led to death in the untreated group (Fig. 3). FR139317 was also effective in blocking the effects of ET-1 in the coronary vasculature. Animals treated with PD145065 also survived the second bolus of ET-1 (Fig. 3). Infusion of PD145065 before administration of ET-1 significantly decreased the mean blood pressure (p ≤ 0.02 from control) and prevented changes induced by ET-1 (p < 0.001) from control; Fig. 3).

We observed that the administration of ET-1 in rabbits was associated with an intense production of a milky secretion in the animals' eye mucosa, apparently originating from the lacrimal system. Interestingly, this production was nearly abolished by treating the animals with either PD145065 or FR139317, suggesting that this phenomenon also was induced by ETA-receptor activation.

Role of ET on infarct size and arrhythmogenesis

The rate-pressure product, an indirect measurement of myocardial oxygen consumption, was similar for all groups, without significant difference between the treatment and control groups throughout the whole protocol (Table 2). The dose of ET-1 used, a bolus of 10-10 mol/kg followed by a continuous infusion of 10-11 mol/kg/min, caused a slight but significant increase in mean blood pressure and decrease in heart rate compared with the baseline value and with the control values during the reperfusion period (Table 2).

The infusion of ET-1 did not alter infarct size compared with that of controls, 46 ± 8% versus 47 ± 7%, respectively (Fig. 4A). PD145065 significantly reduced infarct size expressed as a percentage of the area at risk (AN/AR), 22 ± 7% in comparison with control (p ≤ 0.02; Fig. 4A). There was no statistically significant difference between the infarct size of control and FR139317 animals, although there was a tendency for the treatment with the ETA antagonist to increase infarct size (55 ± 9%; Fig. 4A). There was no statistically significant difference in the size of the AR/LV in any group compared with control. Linear regression analysis demonstrated a positive correlation between the size of the AR and the size of the AN for all groups (Fig. 4B). Animals treated with PD145065 demonstrated much lower infarct size for similar AR/LV compared with the other groups (Fig. 4B). Comparison of the slopes of the linear regression co-efficient of the PD145065 group compared with the untreated group showed a significant reduction in the AN (p < 0.05), whereas the slope of the ET-1-treated group was significantly increased (p < 0.0001). The slope of the FR139317-treated group was not different from that of the control group.

Intravenous infusion of ET-1 caused a marked increase in the incidence of PVCs during the 30 min of occlusion and first 15 min of reperfusion compared with control (Fig. 5A). Infusion of PD145065 significantly decreased the incidence of PVCs compared with controls and ET-1. There was, in addition, statistically significant (p ≤ 0.05) difference in the total number of episodes of VF between the FR139317-and the PD145065-treated groups (Fig. 5B).

DISCUSSION

Since its discovery, ET has drawn significant attention from the scientific community, and recent reports have described its pathophysiologic role not only in the cardiovascular system but also in the pulmonary, renal, and central nervous systems (23). The results of our study support the hypothesis that endogenous production of ET during myocardial ischemia and reperfusion is detrimental to the reperfused myocardium. These findings included the demonstration of a 2.6-fold increase in the levels of ET-1 mRNA in the ischemic zone after 3 h of reperfusion, significant hemodynamic changes leading to death after intravenous administration of exogenous ET-1, and finally, the demonstration of significant myocardial salvage after ischemia and reperfusion in animals treated with the nonselective ET-receptor antagonist PD145065. Furthermore, this cardioprotection seen with PD145065 was associated with decreased incidence of ventricular arrhythmias during the periods of ischemia and early reperfusion.

The increased levels of ET-1 mRNA found in areas of no-reflow phenomenon suggest a possible cause-effect relation between ET production and the progressive microcirculatory failure seen in these animals. Future studies will be necessary to determine whether the ET antagonist PD145065 directly decreases the no-reflow phenomenon and whether this is the mechanism of the cardioprotective effects found with this antagonist, or to determine if this beneficial effect is through another, presently unknown, mechanism. In support of the microcirculatory-failure hypothesis is the preliminary data from other investigators demonstrating that blockade of both ETA and ETB receptors with a nonpeptide antagonist, bosentan, significantly decreased the no-reflow phenomenon in pigs. This was associated with a decrease in infarct size (24).

We previously demonstrated that plasma ET-1 levels were markedly increased 3 h after reperfusion in dogs (4), and that the administration of drugs that blunt the cardiac release of ET significantly reduces infarct size in the canine model (5). Increased plasma levels of ET-1 after an acute myocardial infarction have been confirmed in other animals and also demonstrated in human beings by other investigators (25). Our finding of increased ET-1 mRNA in the reperfused myocardium of rabbits further supports the production of ET-1 in the heart after regional ischemia. Regional myocardial ischemia in pigs with or without reperfusion is associated with increased ET-1 mRNA in the ischemic myocardium (26). These results support our findings that the ischemic challenge during an acute myocardial infarction triggers ET-1 gene expression in the myocardium, which will result in increased, continuous production of ET-1 during the reperfusion period.

Infusion of intravenous boluses of ET-1 significantly increased mean blood pressure and coronary vascular resistance in our acute protocol. The ET antagonist FR139317, selective for ETA receptors, significantly prevented these hemodynamic changes induced by exogenous ET-1 but did not change significantly the infract size in our survival protocol. A similar negative finding with FR139317 was recently described by other investigators also by using the rabbit infarct model, although the reperfusion time was only 2 h (27). Our results demonstrated that the ET antagonist PD145065, which antagonizes both ETA and ETB receptors nonselectively, significantly reduced infarct size, similar to the findings described by other investigators with another nonselective nonpeptide ET antagonist in a pig infarct model (24). These findings raise important questions about the pathophysiology of the detrimental effects of ET in the heart.

A conclusion from our study is that ET mediates its detrimental effects through the ETB receptor. An additional explanation derives from the fact that different forms of ET have different affinities for the ET receptors (16). The ETA receptor is highly specific for the isoform ET-1, whereas the ETB receptor is nonspecific and allows binding of the other forms of ET as well. Experiments performed in the isolated rat heart showed that both ET-1 and ET-3 have vasoconstrictor actions in the coronary artery and that this is mediated by both the ETA and the ETB receptors (28). Therefore our results may suggest that forms of ET other than ET-1 may be responsible for the deleterious effect of ET, in addition to the fact that this deleterious effect may be mediated by the activation of the ETB receptors. Although our findings suggest that ETB activation is deleterious to the reperfused myocardium, it is conceivable that only simultaneous blockade of both ETA and ETB receptors, such as obtained with PD145065, will result in significant myocardial protection. The use of a highly selective ETB-receptor antagonist, when available, will help to clarify this issue.

Infusion of ET-1 intravenously before, during, and after coronary occlusion, at a dose calculated to increase the ET-1 level above those found after reperfusion of the ischemic myocardium, did not demonstrate an increase in infarct size (Fig. 4A). This finding would suggest, therefore, that if ET is deleterious based on the findings with PD145065, either another of the isoforms of ET, such as ET-2 or ET-3, may also contribute to infarct size or that a high concentration of locally produced ET-1 is required for the deleterious effects to become more clearly evident. Alternatively, the effects caused by ET-1 may be already nearly maximized by the endogenous local production of this peptide during ischemia and reperfusion or that there may be a complexity of actions of ET. In animals with greater ARs, there appears to be a detrimental effect of ET in infarct size, whereas at lower ARs, the infusion of ET appears to be beneficial in reducing infarct size (Fig. 4B).

The potential role of ET in extending myocardial injury after reperfusion has been addressed in a few studies. In support of our findings, Whatanabe et al. (29) reported that infusion of a monoclonal antibody to ET-1, either during the initial period of occlusion or immediately before reperfusion, significantly reduced the infarct size in rats at 24 and 48 h after reperfusion. A similar effect also was seen in rabbits treated with this same monoclonal antibody (30). This group has also demonstrated significant reduction on infarct size in rats by using an ET antagonist that, similar to PD145065, blocks both the ETA and ETB receptors (31). The administration of the ETA-receptor antagonist BQ123 reduced infract size by 40% in the dog infarct model after 5 h of reperfusion (32), and the use of this and another ET antagonist was recently shown also to have cardioprotective effects in the isolated rat heart (33). In contrast, Velasco and Yanagisawa (34) demonstrated in preliminary data that one of the receptor antagonists used in the current study, PD145065, at a dose 360 times lower than the current dose and infused intracoronary rather than systemically, significantly increased infarct size in the dog model, after 5 h of reperfusion (34). Velasco et al. (35) also demonstrated that FR139317 increased infarct size when administered in the same fashion, which again contrasts to the results obtained in our infarct model in which treatment with FR139317 failed to alter infarct size. It is possible that specific blockade of ETA with FR139317 causes ET to become more available to stimulate the ETB receptors, which may further enhance the detrimental effect of ET, explaining the increase in infarct size seen with FR139317 in the study done by Velasco et al. An important distinction between the canine infarct model used for these latter studies and ours, the rabbit, is the degree of collateral blood flow, which is well described as an important determinant of infarct size (22). The dog has a highly collateralized myocardium, whereas the rabbit is poorly collateralized. What role the ETB receptor has in blood flow during ischemia/reperfusion or other effects, including systemic effects of ETB-receptor activation, remains to be elucidated.

The detrimental effect of ET on the reperfused myocardium may involve mechanisms other than alteration in blood flow. This is supported by studies demonstrating that ET produces intracellular alkalinization, inositol phosphate production, and increase in intracellular calcium levels, all of which can be potentially detrimental to the survival of reperfused cells (36). Furthermore, ET may have an important role in the inflammatory response because it seems to mediate the effects of cytokines (37). An additional effect of ET may be associated with its effects on ion fluxes or membrane potential changes, such as through its effects in a calcium-activated potassium channel (38). Administration of ET-1, in our study, caused significant increase in the incidence of PVCs during the 30 min of ischemia and first 15 min of reperfusion in the infarct protocol. This effect of ET-1 also was observed after boluses of ET-1 during the acute protocol. This proarrhythmogenic effect was prevented by treating animals with the nonselective ET antagonist PD145065. Comparison of the incidence of VF between groups revealed a significant difference between animals receiving PD145065, which had a lower incidence of VF, in comparison with animals receiving FR139317 (9 vs. 44%, p ≤ 0.05). This finding suggest that ETB-receptor activation may be responsible for this arrhythmogenic effect and confirms the proarrhythmic effect of ET observed by other investigators in the canine model (39,40).

CONCLUSIONS

ET is produced in the heart after myocardial ischemia and reperfusion. This increased production might contribute to the progressive myocardial microcirculatory failure seen in these animals after reperfusion. Antagonism of both the ETA and the ETB receptors, but not ETA alone, during myocardial ischemia and reperfusion, significantly reduced infarct size and the incidence of ventricular arrhythmias in rabbits, suggesting that ET is detrimental to the survival of the ischemic and subsequently reperfused tissue. Further studies with protocols designed to examine the role of ET antagonism only during the period of ischemia or reperfusion appear warranted to define the timing of the detrimental effect of ET. The mechanism by which ET damages myocardial cells may be mediated through ETB receptors or by forms of ET other than ET-1 activating the ETB receptor. The development of a specific antagonist for the ETB receptor may clarify the mechanism by which PD145065 reduces infarct size in the rabbit. Plasma levels of ET are increased in patients with acute myocardial infarction, and this is associated with a poor prognosis in these patients (41-43). Recent investigations have demonstrated elevated levels of ET-1 in patients with chronic heart failure, with higher ET-1 levels being highly predictive of cardiac death (44). These investigations suggest that ET also may be detrimental to human hearts. If the beneficial effects of PD145065 can be reproduced in human beings, these findings may represent a novel therapy for acute myocardial infarction.

Acknowledgment: This study was supported in part by grants HL40892 and GM15431 from the National Institutes of Health.

FIG. 1.
FIG. 1.:
Effects of ischemia and reperfusion on myocardial blood flow in the rabbit. Rabbits have poorly collateralized myocardium and coronary occlusion (OCCL) results in significant and marked decrease in blood flow to the central ischemic zone (CIZ) compared with the baseline (BAS) flow or with the nonischemic zone (NIZ) flow. Reperfusion (REP) results in markedly increased myocardial blood flow in the previously ischemic area, known as reactive hyperemia. At 3 h into reperfusion, the no-reflow phenomenon becomes evident in the CIZ.
FIG. 2.
FIG. 2.:
Effects of ischemia reperfusion on myocardial endothelin-1 (ET-1) mRNA. Northern blot analysis of myocardial tissue from two representative rabbits undergoing 30 min of coronary occlusion and 3 h of reperfusion. Note the marked increased in ET-1 mRNA (360 and 560%) in tissue from the central ischemic zone (CIZ) compared with the nonischemic zone (NIZ). β-Actin cDNA labeled with 32P was used as the internal control to normalize the data of ET-1 mRNA. Data for all rabbits are summarized in Table 1.
FIG. 3.
FIG. 3.:
Hemodynamic effects of antagonism of endothelin receptors in systemic and coronary vasculature. Animals were pretreated with saline, FR139317, or PD145065 for 10 min as described in Methods, before the administration of endothelin-1 (ET-1) as an intravenous bolus (10-9 mol/kg) at time 0 and a second bolus with a 3 times higher dose 20 min later. In animals pretreated with saline alone, ET-1 infusion caused a marked increase in mean blood pressure and coronary vascular resistance and induced a decrease in heart rate. Injection of the second dose of ET-1 (3 × 10-9 mol/kg) led to death in all control animals. Pretreatment with the ET-receptor antagonists (FR139317 or PD145065) prevented the hemodynamic changes induced by ET-1 and protected animals from death.
FIG. 4.
FIG. 4.:
Effect of endothelin-1 (ET-1) and ET antagonists on infarct size. A: Treatment of rabbits with ET-1 or FR139317 before, during, and after myocardial ischemia did not alter infarct size compared with control, whereas treatment with PD145065 significantly reduced infarct size. Treatment was started 10 min before onset of myocardial ischemia and given throughout the 30 min of ischemia and the first 2 h of reperfusion. AR, area at risk; LV, left ventricle; AN, area of necrosis. B: Linear regression analysis demonstrated a positive correlation between size of the area at risk and infarct size for all groups. Similar area at risk was associated with markedly reduced infarct size in animals treated with PD145065 compared to the untreated or ET-1- and FR139317-treated animals. The slopes of the regression coefficients of the various groups showed a significant increase in infarct size in the ET-1-treated group compared with control (p < 0.001), whereas the group treated with PD145065 was significantly reduced (p < 0.05) compared with the untreated group. The slope of the group treated with FR139317 was not different from that of the control group.
FIG. 5.
FIG. 5.:
Effects of endothelin-1 (ET-1) and ET antagonists on ventricular arrhythmias. A: ET-1 infusion induced a marked increase in the number of premature ventricular contractions (PVCs) during the 30 min of ischemia and the first 15 min of reperfusion. Treatment with PD145065 significantly reduced the incidence of PVCs during the same period. B: The full open bars represent the total number of animals included in the analysis, and the solid bars represent the incidence of ventricular fibrillation (VF) for each group. Animals not meeting the preestablished criteria for size of the area at risk of the left ventricle (≥25% or ≤85%) were excluded. Animals treated with PD145065 had a significantly lower total number of episodes of VF than animals treated with FR139317 (9 vs. 44%; p < 0.05). None of the animals had VF after myocardial reperfusion.

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Keywords:

Endothelin; Endothelin-receptor antagonist; Reperfusion injury; Myocardium; Hemodynamics; Infarct size

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