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Ann Thorac Surg 2001;72:565-570
© 2001 The Society of Thoracic Surgeons

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Original article: cardiovascular

Endothelin receptor blockade reduces ventricular dysfunction and injury after reoxygenation

^a Division of Pediatric Cardiothoracic Surgery, Children’s Hospital Medical Center, Cincinnati, Ohio, USA

Accepted for publication April 19, 2001.

Address reprint requests to Dr Pearl, Division of Cardiothoracic Surgery, Children’s Hospital Medical Center, 3333 Burnet Ave, OSB 3, Cincinnati, OH 45229
e-mail: pearj0{at}chmcc.org

	Abstract

Top Abstract Introduction Material and methods Results Comment Acknowledgments References

 
Background. Reoxygenation of hypoxic myocardium during repair of congenital heart defects results in poor ventricular function and cellular injury. Endothelin-1 (ET-1), a potent vasoconstrictor that increases during hypoxia, may suppress myocardial function and activate leukocytes. The objective was to determine whether administration of an endothelin receptor antagonist could improve ventricular function and decrease cardiac injury after hypoxia and reoxygenation.

Methods. Fourteen piglets underwent 90 minutes of ventilator hypoxia, 1 hour of reoxygenation on cardiopulmonary bypass, and 2 hours of recovery (controls). Nine additional animals received an infusion of Bosentan, an ET(A/B) receptor antagonist, (5 mg/kg per hour) during hypoxia and reoxygenation.

Results. Right and left ventricular dP/dt in controls decreased to 78% and 52% of baseline, respectively, after recovery (p < 0.05). In contrast, Bosentan-treated animals had complete preservation of RV dP/dt and less depression of LV dP/dt. Bosentan reduced the hypoxia and reoxygenation–induced elevation of ET-1 and iNOS mRNA at the end of recovery (p < 0.05). Bosentan-treated animals had diminished myocardial myeloperoxidase activity and lipid peroxidation compared with controls (p < 0.05). Myocardial apoptotic index, elevated by hypoxia and reoxygenation, was lower in the Bosentan-treated animals (p < 0.05).

Conclusions. Endothelin-1 receptor antagonism improved functional recovery and decreased leukocyte-mediated injury after reoxygenation. The reduction in cardiac cell death might also improve long-term outcome after reoxygenation injury.

	Introduction

Top Abstract Introduction Material and methods Results Comment Acknowledgments References

 
Molecular and biochemical changes occurring during hypoxia increase the susceptibility of myocardium to reoxygenation injury [1]. Reoxygenation injury has been implicated in the significant myocardial dysfunction commonly seen after repair of cyanotic congenital heart defects during cardiopulmonary bypass [2]. This process would be anticipated to reverse upon recovery of metabolic activity during reoxygenation; however, molecular and biochemical changes that occur in response to hypoxia result in cardiopulmonary injury upon reoxygenation. Furthermore, cellular injury or death may occur from reoxygenation injury, resulting in progressive myocardial failure [3].

Reoxygenation injury manifests with poor ventricular systolic function, increases in myocardial stiffness (poor diastolic function), and myocardial edema [2]. Loss of coronary vasodilatory reserve also occurs, impairing myocardial blood flow. Myocardial histology after reoxygenation demonstrates interstitial and cellular edema, apoptosis (disruption of mitochondrial membranes and clumping of nuclear chromatin), and, if severe enough, necrosis [3]. In addition, marked neutrophil infiltration is evident along with increases in markers of leukocyte activity and free radical–mediated injury [4].

Reoxygenation injury can involve leukocyte adhesion and activation, direct free radical production, and coronary vasoconstriction [4], all of which can be promoted by endothelin-1 (ET-1). Endothelin-1, a potent vasoconstrictor, increases under conditions of hypoxia [5, 6]. Endothelin-1 depresses myocardial function and stimulates leukocyte adhesion and activation [7]. Furthermore, ET-1 inhibits production of nitric oxide (NO), which has important vasodilatory and antineutrophil activation properties [5]. Increased ET-1 levels, combined with the lower NO levels, might promote reoxygenation injury.

Bosentan, a nonpeptide ET(A/B) receptor antagonist, has been shown in previous experiments to increase plasma ET-1 levels and to decrease vascular resistance [8]. Therefore, we reasoned that administration of Bosentan could attenuate neutrophil adhesion and activation, preserve NO levels, and thereby improve cardiac function after hypoxia and reoxygenation.

	Material and methods

Top Abstract Introduction Material and methods Results Comment Acknowledgments References

 
An established model of acute hypoxia and reoxygenation was used for this study [6]. All animals received humane care in compliance with 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 Institute of Laboratory Animals Resources and published by the National Institutes of Health (NIH Publication no. 86-23, revised 1985). The protocol was approved by the Institutional Animal Care and Use Committee at Children’s Hospital Research Foundation.

Piglets (sus scrofa) weighing 4 to 6 kg were randomized to two groups undergoing hypoxia and reoxygenation with the administration of saline or Bosentan. Researchers were not blinded to the treatment groups. Animals were anesthetized with ketamine hydrochloride (40 mg/kg), intubated with a cuffed endotracheal tube, and mechanically ventilated. Initial fraction of inspired oxygen was 40% and arterial PCO₂ was maintained between 36 and 44 mm Hg. Deep sedation and paralysis were maintained by infusing sodium pentobarbital (20 mg/kg per hour), and intravenous injections of fentanyl citrate (10 µg/kg per hour) and pancuronium bromide (0.2 mg/kg per hour). Arterial blood gasses and co-oximetry were monitored continuously throughout the experiment (Chiron Diagnostics System 865; Chiron Diagnostics, East Walpole, MA).

Transducer-tipped catheters (Millar Instruments, Houston, TX) were placed in the pulmonary artery and right and left ventricles for determination of pulmonary artery pressure, RV end diastolic pressure (EDP), LVEDP, +dP/dt, and -dP/dt (Ponemah Physiology Platform, Gould Instrument Systems, Valley View, OH). Cardiac output was monitored with a Doppler flow probe placed around the pulmonary arterial trunk (Transonics Systems, Ithaca, NY). Systemic oxygen delivery was calculated as arterial oxygen content multiplied by cardiac output.

Baseline hemodynamic measurements along with aortic and pulmonary arterial blood samples were obtained after a 30-minute equilibration period. Hypoxia was induced by decreasing the inhaled fraction of inspired oxygen to 12%, resulting in systemic oxygen saturation of 65% to 70% (PaO₂ = 30 to 35 mm Hg). Hemodynamic measurements and arterial blood samples were taken 15, 30, 60, and 90 minutes after initiation of hypoxia. Bosentan-treated animals (n = 9) received a 10-mg/kg bolus of Bosentan (Ro 47-0203, Actelion, Allschwil, Switzerland) in distilled water 20 minutes before the induction of hypoxia, followed by a continuous infusion of 5 mg/kg per hour. This dosage has been shown to elevate ET-1 levels in a porcine model of endotoxin shock [8]. Bosentan or saline was infused throughout 90 minutes of hypoxia and the first 30 minutes of reoxygenation (140 minutes total infusion time) to ensure saturation of endothelin receptors during hypoxia and early reoxygenation. Control animals (n = 14) receiving saline infusion underwent the same protocol with equivalent time points.

After 90 minutes of hypoxia, animals were reoxygenated on cardiopulmonary bypass (CPB) with a minimum PO₂ of 450 mm Hg in the bypass circuit. The PO₂ in the CPB circuit was purposely kept high to exacerbate reoxygenation injury. Ionized calcium was maintained at 0.6 to 0.8 mmol/L during CPB. The CPB flow rate was maintained at 100 mL/kg with a minimum mean arterial pressure of 30 mm Hg. Animals were cooled to 32°C rectal temperature. Hearts were not subjected to ischemia by aortic cross clamping.

The animals were removed from CPB after 1 hour, warmed to 37°C, and returned to normoxic conditions (fraction of inspired oxygen = 40%). Hemodynamics were recorded and blood samples obtained at 15, 30, 60, and 120 minutes after removal from CPB. Animals were sacrificed by cardiectomy at the end of the recovery period. Tissue samples were collected from the same two areas of the outer wall of the left and right ventricles in each animal, immediately frozen in liquid nitrogen, and stored at -80°C or fixed in neutral-buffered formalin for sectioning. Previous studies by our laboratory have demonstrated no impairment in cardiovascular (unpublished data) or pulmonary function [9] in animals subjected to CPB without hypoxia and reoxygenation. Additional piglets (n = 6) were sacrificed immediately after sternotomy, with cardiac tissue and blood samples collected for baseline measurements.

Endothelin-1 assay
Blood samples were collected in ethylenediaminetetraacetic acid tubes containing indomethacin (10 µg/mL), immediately centrifuged at 4°C for 20 minutes, and the plasma frozen at -80°C for later analyses. A commercial ET-1 immunoassay kit (R&D Systems, Minneapolis, MN) was used to measure ET-1 protein concentration (pg/mL plasma). The assay did not cross-react with ET-3 or big ET-1.

Plasma nitrite and nitrate assay
Plasma levels of NO were estimated by measuring both blood nitrate and nitrite using a two-part assay with nitrate converted to nitrite in the Griess reaction. The optical density at 550 nm, with correction at 650 nm, was measured. The data were analyzed by using GENESIS microplate software (Fisher Scientific, Pittsburgh, PA). Levels were reported as total nitrites (µmol/L). Although not a direct measure of systemic NO, nitrite/nitrate measurements have been established as an accepted methodology to estimate relative NO levels [10, 11].

Tissue myeloperoxidase
Myeloperoxidase (MPO) activity was determined as a marker of leukocyte activity, as previously described [6]. Myeloperoxidase activity was the quantity of enzyme degrading 1 µmol hydrogen peroxide per minute at 37°C expressed as mU/50 mg tissue.

Tissue lipid peroxidase
Frozen myocardial tissue was homogenized on ice in 10 volumes (1 mL/100 mg) of 10% (volume/volume) potassium chloride. Lipid peroxidation (LPO) was measured by a modified thiobarbituric acid reaction technique [12]. Concentrations were calculated using standard solutions of malondialdehyde-dimethylacetate (0.5 to 16 nmol/L) and were expressed in nmol/100 mg wet tissue. Myocardial MPO and LPO values at the end of recovery were compared with baseline values obtained from piglets sacrificed immediately after sternotomy.

Ribonuclease protection assay
Myocardial mRNA transcript levels were assessed by multiplex ribonuclease protection assays with a custom probe set (PharMingen, San Diego, CA) including templates for riboprobes to porcine inducible nitric oxide synthase (iNOS), ET-1, interleukin (IL)-1 and ß, IL-6, interferon ß and , and the housekeeping genes, L32 and glyceraldehyde-3-phosphate dehydrogenase. The [³²P]UTP-labeled probes were generated by in vitro transcription and hybridized to total mRNA (10 µg) isolated from the myocardium according to the assay manufacturer’s instructions (PharMingen). Each mRNA species was identified based on the appropriate size of the protected probe fragment and quantified by phosphorimaging analysis (Molecular Dynamics, Sunnyvale, CA). RNA levels were normalized by comparison with housekeeping genes L-32 and glyceraldehyde-3-phosphate dehydrogenase.

Apoptotic cell labeling
Myocardial tissue samples were collected from the same two areas in the outer wall of the LV of each animal. Tissues were fixed by overnight immersion in 10% neutral-buffered formalin. Apoptotic cells were identified by the incorporation of biotin into DNA fragments using the CardioTACS kit according to manufacturer’s directions (R&D Systems). The TUNEL assay generated a blue precipitate in the presence of DNA fragmentation with a streptavidin-conjugated horseradish peroxidase substrate (TACS Blue Label, R&D Systems). The samples were stained with Red Counterstain C, then examined under a light microscope. Positive controls were generated by incubating tissue sections with a nuclease and negative controls were incubated in buffer without TdT. The number of positive staining cells in each sample was counted in a grid pattern containing 20 fields of view (x 40) and covering most of the section. The apoptotic index reported is the mean number of positive cells per 20 fields of view from two LV tissue sections taken from each animal. Tissue samples from at least five separate animals per treatment were analyzed.

Apoptotic cells were also identified by immunohistochemical localization of activated poly(ADP-ribose) polymerase (PARP). Myocardial tissue sections were fixed, mounted, and deparaffinized as for the TUNEL assays. Tissue slices were incubated with 10 µg/mL rabbit anti-PARP cleavage site (214/215) specific antibody (Biosource International, Camarillo, CA) that recognizes the 85 kDa fragment of cleaved PARP. The secondary antibody was alkaline phosphatase–conjugated antirabbit IgG. The number of cells stained positive for activated PARP was determined as for the TUNEL assay.

Statistical analysis
Data are presented as the mean ± standard deviation. Repeated measures analysis of variance and two-tailed, paired Student t test were used when appropriate to compare values at different time periods. A p value less than or equal to 0.05 was considered significant (Statview 4.01 software, Abacus Concepts, Berkeley, CA).

	Results

Top Abstract Introduction Material and methods Results Comment Acknowledgments References

 
Mean RV dP/dt in the control group increased to 148% of baseline (370 ± 45 vs 533 ± 46 mm Hg/s) at the end of hypoxia (Fig 1), but fell to 78% of baseline after reoxygenation (281 ± 34 mm Hg/s, p < 0.05). Left ventricle dP/dt in control animals decreased to 80% of baseline by end-hypoxia (1242 ± 37 vs 994 ± 54 mm Hg/s, p < 0.05) and after reoxygenation decreased to 52% of baseline (646 ± 36 mm Hg/s, p < 0.01). In contrast, RV dP/dt was preserved throughout hypoxia, reoxygenation, and recovery in Bosentan-treated animals. Bosentan administration also improved LV dP/dt through recovery when compared with controls (74% vs 52% of baseline; p < 0.05). Left ventricle -dP/dt was significantly less depressed at end-hypoxia and end-recovery in treated animals (p < 0.05; Fig 1). Right ventricle -dP/dt was unchanged at end-recovery in treated animals. Although there was a trend toward lower RV -dP/dt in controls, the difference was not significant (data not shown). Systemic oxygen delivery in the control group decreased from 74.5 ± 8.2 mL/min at baseline to 45.2 ± 13.3 mL/min at the end of recovery (p < 0.05; Table 1). In contrast, systemic oxygen delivery in Bosentan-treated animals was steady throughout recovery and unchanged from baseline. Arterial ET-1 levels in the control animals increased during hypoxia (0.55 ± 0.3 pg/mL at baseline vs 0.90 ± 0.6 pg/mL at end-hypoxia, p < 0.05) and more markedly after reoxygenation to 3.1 ± 1.8 pg/mL (591% of baseline, p < 0.05). Endothelin-1 levels increased sharply after infusion of Bosentan, reflecting displacement of bound ET-1 (Table 1). Arterial nitrite and nitrate levels decreased at the end of hypoxia in controls (p < 0.05), but were preserved during hypoxia and recovery in Bosentan-treated animals (Table 1). Nitrite levels in control animals returned to baseline levels at the end of recovery.

View larger version (23K): [in this window] [in a new window]   Fig 1. Cardiac function in piglets undergoing hypoxia and reoxygenation. (Top panel) Right ventricle dP/dt; (center panel) left ventricle dP/dt; (lower panel) left ventricle -dP/dt during hypoxia and recovery in controls and Bosentan-treated animals. *p < 0.05, time point compared with baseline; p < 0.05, controls compared with Bosentan-treated animals at indicated time point.

View larger version (29K): [in this window] [in a new window]   Fig 2. Myocardial lipid peroxidase levels and myeloperoxidase activity. (A) Myocardial lipid peroxidase levels and (B) myeloperoxidase activity in controls, baseline, and Bosentan-treated tissues. *p < 0.05, time point compared with baseline tissues; p < 0.05, controls compared with Bosentan-treated animals at end of recovery. (LPO = lipid peroxidase; MPO = myeloperoxidase.)

View larger version (156K): [in this window] [in a new window]   Fig 3. Photomicrograph of apoptotic cell markers in myocardium from control and Bosentan-treated animals. Cells stained blue during the TUNEL assay indicate DNA fragmentation and apoptosis (arrow). Sections were counterstained with Red Counterstain C. (A) Myocardium collected at baseline; (B) myocardium from control animals after hypoxia and reoxygenation; (C) myocardium from Bosentan-treated animals after hypoxia and reoxygenation; (D) DNAse-treated positive control tissue. (Magnification x40).

View larger version (17K): [in this window] [in a new window]   Fig 4. Cytokine and adhesion molecule mRNA levels in control and Bosentan-treated animals. *p < 0.05, controls compared with Bosentan-treated animals. (ET-1 = endothelin-1; IL = interleukin; iNOS = inducible nitric oxide synthase.)

	Comment

Top Abstract Introduction Material and methods Results Comment Acknowledgments References

Depressed RV and LV systolic function after hypoxia and reoxygenation was associated with increased myocardial iNOS, ET-1, IL-6, and IL-1ß mRNA expression and elevated plasma ET-1 levels at the end of recovery. Previous reports have demonstrated increased ET-1 after hypoxia in cardiomyocytes [13] and in circulation [14]. Endothelin-1, signaling through the ET(A) receptor, has been shown to result in the adhesion, migration, and activation of neutrophils [15, 16] by increasing CD18 and CD11b expression [7] and inducing ICAM-1 exposure on endothelium [17]. Increased plasma ET-1 has also previously been correlated with neutrophil accumulation in the heart [7].

Furthermore, alterations in NOS activity and depletion of L-arginine, the substrate for NO production, decrease NO levels and vasodilatation during hypoxia and reoxygenation [18]. The multifactorial regulation of leukocyte activity by both NO and ET-1, playing opposing roles on neutrophil adhesion and activation [19], might be key to alleviating myocardial dysfunction after reoxygenation. The increased ET-1 and decreased NO levels during acute hypoxia, detected in this and previous studies [6], implicate the balance between these two opposing mediators as a possible regulator of reoxygenation injury.

Increased areas of myocyte apoptosis have also previously been reported after hypoxia and reoxygenation [3]. Furthermore, elevated LPO levels and MPO activity detected in the current study agree with other in vivo studies demonstrating hypoxia and reoxygenation induced markers of lipid peroxidation [20] and leukocyte activation [21].

Bosentan is a nonpeptide competitive antagonist of ET(A/B) receptors with an affinity of 4.7 nmol/L for ET(A) and 95 nmol/L for ET(B) and a terminal half-life of approximately 4 hours [22]. Bosentan is currently in the late stages of development as an oral treatment for pulmonary arterial hypertension and chronic heart failure (for review see Roux and associates [22]). In randomized clinical studies Bosentan was well tolerated with few side effects, and was associated with decreased blood pressure [23], marked improvement in cardiac performance, and decreased pulmonary resistance [24]. Our laboratory recently reported the effectiveness of Bosentan in preventing pulmonary hypertension after hypoxia and reoxygenation in a piglet model [6].

In the current study, Bosentan significantly improved hemodynamic function and attenuated the increased myocardial MPO activity after reoxygenation. Presumably, effective blockade of ET(A) receptors with Bosentan prevented ET-1-mediated upregulation of neutrophil CD18 and CD11b [7] and ICAM-1 expression on vascular endothelium [17]. In addition, lower myocardial LPO levels in Bosentan-treated animals suggested reduced lipid peroxidation and oxidative stress.

The complex interaction of ET-1 and NO during hypoxia and reoxygenation is not clear at this time. The depressed systemic nitrite levels during hypoxia in this study are supported by previous reports of decreased eNOS activity during hypoxia [25]. However, induction of pulmonary or systemic iNOS by hypoxia may be responsible for the return of plasma nitrite levels to baseline by the end of recovery in control animals. Because of the limitations of collecting cardiac tissue only at the end of the experiments, mRNA levels were not measured in this study during hypoxia. However, hypoxia has previously been shown to induce cardiac iNOS mRNA expression [26]. The induction of cardiac iNOS mRNA, measured at the end of recovery in this study, was reduced in the Bosentan-treated compared with control animals, and previous studies have shown that iNOS inhibition protected hearts from ischemia-reperfusion injury [27]. Restoring the balance between ET-1 and NO production during reoxygenation of hypoxic myocardium might contribute to the improvement in ventricular recovery with Bosentan treatment.

Interleukin-1ß is generally thought of as an inflammatory cytokine and induces iNOS production during hypoxia [26]. Therefore, it is interesting that ventricular IL-1ß mRNA was elevated with Bosentan treatment in the current study, whereas iNOS mRNA expression was reduced. There have been recent reports of cell-specific patterns of iNOS regulation by IL-1ß [26] and differential regulation dependent upon timing of IL-1ß exposure [28].

Bosentan also prevented areas of increased DNA fragmentation and PARP activation detected in the control animals after hypoxia and reoxygenation. The higher number of cells detected with the DNA fragmentation assay compared with PARP activation could indicate that cell death is occurring by both apoptosis and necrosis in injured myocardium. Both forms of cell death have been detected by electron microscopy after hypoxia and reoxygenation in cultured myocytes [29]. Bosentan treatment decreased markers of cell death in both assays, and might prevent the loss of myocytes. In addition to the immediate postoperative benefit of alleviating cellular damage, improved long-term preservation of myocardial function might also be realized. It has been postulated that repeated episodes of hypoxia and reoxygenation, as well as cold ischemia during heart surgery, could result in progressive cell death. Although complete neonatal repair of congenital heart defects has become commonplace and early results continue to improve, some children continue to endure poor long-term myocardial performance. The long-term decline in cardiac function could result from repeated induction of leukocyte-mediated injury and myocyte death. The ability to attenuate this process would have significant clinical benefits. Bosentan, recently submitted to the FDA as an oral treatment for pulmonary hypertension (Tracleer, Actelion Ltd), might eventually contribute to improving the outcome for children undergoing cardiopulmonary bypass during repair of congenital heart disease.

	Acknowledgments

Top Abstract Introduction Material and methods Results Comment Acknowledgments References

 
This study was supported in part by a Trustee Grant from the Children’s Hospital Research Foundation, Cincinnati, OH.

	References

Top Abstract Introduction Material and methods Results Comment Acknowledgments References

 

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