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Endothelin-1, endothelin-1 receptors and cardiac natriuretic peptides in failing human heart
Life Sciences 68 (2001) 2715–2730
Endothelin-1, endothelin-1 receptors and cardiac natriuretic peptides in failing human heart Silvia Del Ry, Maria Grazia Andreassi, Aldo Clerico, Andrea Biagini, Daniela Giannessi* CNR Institute of Clinical Physiology, Laboratory of Cardiovascular Biochemistry, Pisa, Italy Received 31 January 2000; accepted 7 November 2000
Abstract Endothelin (ET)-1 is a potent vasoconstrictor peptide produced in the myocardium that can exert important effects on cardiac myocyte growth and phenotype; cardiac natriuretic peptides (ANP and BNP) are known to act as physiological antagonists of ET-1. In this study a comparative determination of ET-1 receptors and of the local productions of ET-1 and of ANP and BNP was made in different sites of failing and nonfailing hearts. Tissue from right and left atrium, right and left ventricle and interventricular septum from seven adult heart transplant recipients with end-stage idiopathic dilated cardiomyopathy (functional class III and IV, with ejection fraction , 35%) and from four postmortem subjects without cardiac complications was analyzed. In failing hearts we observed a tendency to increase of density of binding sites, most evident in left ventricle (62.6 6 22.6 fmol/mg protein vs. 29.0 6 3.3, mean 6 SEM, p 5 ns). A prevalence of ET-A subclass, observed in all samples, resulted more pronounced in failing hearts where this increase, found in all the cardiac regions, was more evident in left ventricle (p 5 0.0007 vs nonfailing hearts). The local concentrations of ET-1, ANP and BNP resulted significantly increased in failing hearts with respect to controls in all sides of the heart. In failing hearts we have observed a tendency to increase in endothelin receptor density mainly due to a significant upregulation of ET-A subtype and a parallel increase of the tissue levels of ANP, BNP and ET-1 indicating an activation of these systems in heart failure. © 2001 Elsevier Science Inc. All rights reserved. Keywords: Endothelin; Cardiac natriuretic peptides; ANP; BNP; Heart failure; Endothelin receptors
Introduction Heart failure (HF) is the major cause of cardiovascular morbidity and mortality [1]; its complex haemodynamic and neuroendocrine mechanisms are not completely known, al* Corresponding author. Laboratory of Cardiovascular Biochemistry, CNR Institute of Clinical Physiology, Via Alfieri, 1 56010 Ghezzano (Pisa), Italy. Tel.: 39 050 3152664; fax: 39 050 553461. E-mail address: [email protected] (D. Giannessi) 0024-3205/01/$ – see front matter © 2001 Elsevier Science Inc. All rights reserved. PII: S 0 0 2 4 - 3 2 0 5 ( 0 1 )0 1 0 7 6 -1
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though an improved understanding of the pathophysiology is fundamental for the development of new therapeutic strategies for this disease [2]. Different neurohormonal systems (i.e. catecolamine and renin-angiotensin-aldosterone systems) are known to be progressively activated as the cardiac performance declines in patients with HF, in order to maintain circulatory homeostasis [3]. Endothelin (ET)-1, a peptide hormone with potent sustained vasoconstrictor properties which is synthesised predominantly by the vascular endothelium [4], has been implicated in the pathophysiological vasoconstriction in HF [5]: noteworthy increases in circulating ET-1 have been observed both in experimental models of HF and in humans with this disease, irrespective of aetiology [6–9], although it has been considered primarily a local paracrine factor. ET-1 has important direct cardiac effects and appears to be involved in the regulation of myocardial contractility; in human myocardium, ET-1 exerts clear positive inotropic actions, concentration-dependent, that seems to be less pronounced in failing hearts [10]. The various biological functions of ET-1 are mediated by two high affinity receptor subtypes, ET-A and ET-B, belonging to the G protein-coupled family that have been identified in various human tissues, including cardiac tissue [11]. ET-A receptors have selective affinity for ET-1 and are expressed principally on vascular smooth muscle cells and cardiac myocytes but not on endothelial cells [12]. ET-B receptors have equal affinity for all endothelin isoforms and are expressed mostly on endothelial cells [13], but also on vascular smooth muscle cells [14]. Stimulation of ET-A and ET-B receptors on vascular smooth muscle cells resulted in sustained vasoconstriction [15], by an increase of intracellular calcium, while stimulation of ET-B receptors on endothelial cells results in vasodilation, due to the release of nitric oxide and prostacyclin [16]. In different animal models of HF, alterations of ET receptor density have been described: in the heart, ET-1 binding site density has resulted significantly higher in the HF rats than in the control rats [17–19]. On the basis of these observations, ET receptor blockade was suggested as a further therapeutic advance in the anti-endothelin strategy in the treatment of this disease [2]. In fact, after the study of Sakai [20], that demonstrated that the selective ET-A receptor antagonist BQ-123 improves long term survival in rats with HF, a number of works showed the beneficial effects of the use of selective ET-A receptor antagonists both in animal models [21–24] and in humans [25, 26]. Also, treatment with a non selective ET antagonist, as bosentan, markedly increases survival in experimental models of HF [27–31] while short-term therapy with bosentan in patients with HF [32, 33] produces pulmonary and systemic vasodilation and may enhance conventional treatment with angiotensinconverting enzyme inhibitors [34]; however, it has not completely assessed whether selective ET-A antagonists or non-selective ET-A/ET-B blockers are the better therapeutic agents in heart failure treatment. Various endogenous vasodilators may act as physiological antagonists of ET-1; among those, atrial natriuretic peptide (ANP) and brain natriuretic peptide (BNP) constitute a vasoactive natriuretic peptide system that regulates circulatory homeostasis. Their circulating concentrations are elevated in HF and correlate positively with the severity of disease [35], indicating a regulatory function by balancing the actions of the vasoconstrictor antidiuretic neurohormones. A therapeutic use of these peptides in HF is largely suggested although the hemodynamic effects of the treatment with natriuretic peptides are not completely clarified owing to their complex potential role on myocardial systolic and diastolic functions, after-
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load and preload. ANP seems to have a negative inotropic action on the heart, mediated by reduction of calcium entry into the cardiac cell, as observed in cultured ventricular cells [36] and in ex-vivo experimental models [37]. A recent study on the effects of natriuretic peptides on load and myocardial function in normal and heart failure dogs has indicated that infusion of natriuretic peptides produces a mild positive inotropic effect in normal dogs; in HF dogs no inotropic effect was observed instead of the waited negative inotropic effect due to the higher myocardial levels of natriuretic peptides in this condition [38]. Moreover, endothelin and natriuretic peptide systems are also directly related: ET-1 can stimulate the ANP and BNP expression [39, 40], conversely, endogenous ANP directly inhibits endogenous ET-1 secretion through a cGMP-mediated pathway in severe HF [41]. In man, a characterization of ET receptors in failing and nonfailing myocardium has been made recently in right atrium [42] and left ventricle [10, 42–44] from patients with end-stage idiopathic dilated cardiomyopathy (DCM) or ischemic cardiomyopathy (ICM), but the results are not in close agreement. Moreover, a comparative determination of endothelin receptors and of tissue levels of ET-1 and natriuretic peptides in the various regions of human myocardium is yet lacking. Aim of this study was the assessment of endothelin status in human heart by the simultaneous determination of tissue ET-1 concentration in human myocardial tissue from multiple cardiac sites in patients with severe chronic HF and in controls and of endothelin binding sites and their distribution in the two main receptor subclasses. For a deeper knownledge of the local status of vasoactive peptides, the determination of the concentrations of the natriuretic hormones, ANP and BNP, in the same myocardial tissue has been also performed. These findings may be relevant in the evaluation of a possible paracrine action of these hormones in the regulation of cardiac function in both healthy and pathological conditions. Methods ET receptor determination on cardiac tissue The determination of binding parameters, affinity (Kd) and density (Bmax), was made by saturation experiments on cardiac membranes suspension by using mono-iodinated ET-1 as radioligand and following a previously described procedure [45]; the percent of ET-A and ET-B subtypes were obtained by competitive displacement experiments with the specific ligands, BQ-123 and IRL-1620. Tissue collection Patients. Human cardiac tissue was obtained from seven adult transplant recipients with idiopathic dilated cardiomyopathy (5 men and 2 women, mean age: 36 6 7.2 years), divided into two groups according to their functional class (NYHA class III, n 5 3 and NYHA class IV, n 5 4; left ventricular ejection fraction , 35%). Myocardial contractility, cardiac dimension and function were assessed by 2D echocardiography, radionuclide ventriculography, and hemodynamic study. Pharmacological treatment included vasodilators (generally an angiotensin converting enzyme and/or nitrates) and diuretics. Patients treated with cathecolamines or b-adrenoceptor antagonists were not included in the study.
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Control group. Cardiac tissue was also obtained from four patients died for an acute noncardiac disease (2 men and 2 women, mean age: 59 6 10.4 years), collected during the autopsy, 10–12 hours after death. The donor subjects did not present histories or clinical signs of heart disease and evident atherosclerosis of large blood vessels was not observed at autoptic examination. They do not received any drugs known to interfere with endothelin system, such as cathecolamines or b-adrenoceptor antagonists prior the death. In all subject right and left atrium, right and left ventricle and interventricular septum were analyzed. All specimens were processed for routine H&E and Mallory trichromic stain. Light microscopic analysis was carried out on sections of tissue for evaluation of the amount of connective tissue and myocardial cell damage, using a square counting grid of 25 points on 10 randomly sampled areas up to 1000 points for each sample. The connective tissue area was , 20% of total area and the cellular damage (due to tissue collection and storage) resulted , 10 % of tissue area. In all cases, tissue samples were frozen in liquid nitrogen immediatly after withdrawn and stored at 2808C until use; all samples were processed not later than 2 months of storage at 2808C after collection. Tissue was collected with the approval of the Ethical Committee of our institution, and the transplant recipient subjects gave informed consent. Membrane preparation The tissue (,50 mg ww) was homogenized in ice-cold Tris/HCl buffer (50 mmol/L Tris/ HCl, 0.25 mol/L sucrose, 1 mmol/L EDTA, 1 mmol/L MgCl2, 0.5 mmol/L phenylmethylsulfonyl fluoride, 0.5 mg/ml leupeptin, 0.5 mg/ml soybean trypsin inhibitor, pH 7.4) by using a glass Potter’s homogenizer; after centrifugation at 500 3 g for 15 min at 48C, supernatant was collected and centrifugated again at 50,000 3 g for 30 min at 48C. The pellet was resuspended in ice-cold Tris/HCl buffer and protein concentration was determined by Lowry’s method with bovine serum albumin (BSA) as standard [46]. Membrane suspension was stored frozen at 2808C at a concentration of 1–3 mg/ml. Radioligand preparation ET-1 (Novabiochem, Switzerland) was labelled with Na-[125I] (sp. a. 18 Ci/mg) (Sorin Biomedica, Saluggia, Italy) by lactoperoxidase technique. About 1 mCi of Na-[125 I], 5 mg of ET-1 and 5 ml of bovine lactoperoxidase (1 mg/ml) in a final volume of 60 ml of phosphate buffer 0.5 mol/L, pH 7.4 were added with 10 ml of H2O2 (10 vol, 1:12,000), and the mixture incubated for 29 300 at room temperature under mild agitation; this procedure was repeated three times. Radioiodinated ET-1 was purified by reverse-phase HPLC by using a Novapak C18 column (4 mm, 3.53300 mm, Waters Associates, Milford MA), eluted with a linear gradient, from 20% to 50%, of CH3CN in 0.1% trifluoracetic acid, for 60 min at a flow rate of 1 ml/ min. Only mono-iodinated endothelin was used for binding assay. Specific activity was determined by measuring the ET-1 concentration of serial dilutions of the radiolabelled preparation by an immunoenzymatic (EIA) system for ET-1, purchased from Cayman Chemical Company (Ann Arbor, MI). Scalar dilutions of radiolabelled peptide, from 2,000 to 60,000 cpm/ml, were assayed as unknown samples and the corresponding mass determined. The tracer was stored frozen at 2208C in the buffer used for binding assay.
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Binding assay For saturation experiments, [125 I]-ET-1 binding was performed in a total assay volume of 250 ml consisting of 100 ml radioligand (10–300 pmol/L), 50 ml of membrane preparation (25 mg/ml of protein), and 100 ml of Tris/HCl buffer; non-specific binding was determined by addition of unlabelled ET-1 (0.1 mmol/L). All tubes were incubated at 378C for 4 hrs under agitation. The reaction was stopped by rapid filtration through glass-fiber filters Whatman GF/B, prewet with Tris-HCl buffer, 0.3% BSA; the filters were then washed with 15 ml of cold buffer. Bound radioactivity was measured in a gamma scintillation counter (Cobra 2000, Packard Instrument Company, Meridien, CT), 80% counting efficiency. Competition studies were performed at 20 pmol/L of [125I]-ET-1 and 25 mg/ml of protein by using increasing concentrations of different ligands: BQ-123 (10 pmol/L – 50 mmol/L), ET-1 (15 pmol/L – 100 nmol/L) (Sigma, S. Louis, MO) and IRL-1620 (10 nmol/L – 10 mmol/L) (Alexis Biochemicals, San Diego CA). Data analysis The affinity constant (Kd), the density of binding sites (Bmax, expressed as fmol bound/ mg protein) and the Hill coefficient were obtained by analysis of the data using an iterative curve fitting programme (KaleidaGraph). Scatchard analysis was also performed. IC50 values for the various ligands from competition experiments were evaluated by an iterative curve fitting programme (KaleidaGraph); Ki values were derived by the method of Cheng and Prusoff [47]: Ki 5 IC50/(11L/Kd), where L is the concentration of the radioligand, IC50 is the concentration of competitor agent causing 50% inhibition of specific radioligand binding and the Kd the dissociation constant of the radioligand-receptor complex. Quantitation of peptides in cardiac tissue Vasoactive peptides were measured by specific immunometric assays after tissue extraction. Tissue extraction Cardiac tissue was homogenized in 10 vol of acetic acid 0.5 mol/L containing Triton X, 0.01% and then boiled for 15 min in a water bath. The homogenate was centrifuged at 48,000 3 g for 20 min and the concentrations of the different peptides were determined in the supernatant by specific immunometric assays. Immunometric assays Endothelin was measured by a competitive radioimmunological method (Biomedica Gruppe, Vienna, Austria; supplied by CIS Bio International, Gif-sur-Yvette, France) after solid-phase extraction on Sep-Pak C18 cartridge (Waters Associates, Milford, MA) showing 142 % cross-reaction with ET-2, 98% with ET-3, ,1% with ANP and Big-ET-1(1–38); the detection limit was about 2.0 pmol/L. Both ANP and BNP were measured by non competitive immunoradiometric (IRMA) methods using two monoclonal antibodies prepared against sterically remote epitopes of molecule (Shionoria ANP, Shionoria BNP, manufactured by Shionogi & Co, Ltd, Osaka, Japan; supplied by CIS Bio International). No important cross-reactions with the other peptides were observed (for ANP assay: , 0.01% of cross-reactions with BNP and ET-1; for BNP assay:
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, 0.01% of cross-reactions with BNP, CNP and ET-1). Sensitivity was 0.68 6 0.30 pmol/L for ANP and 0.57 6 0.19 pmol/L for BNP, respectively. The inter-assay variability of whole procedure (extraction 1 immunometric assay) was about 25%. Statistical analysis Multiple comparisons of the experimental data (binding parameters and vasoactive peptide concentrations in the various cardiac regions for the different groups of patients) were made by ANOVA. The difference of Hill coefficients from unit was checked by paired Student’s t-test. For linear regression, experimental data were analyzed by checking the null hypothesis by Student’s t-test. All values were reported as mean 6 SEM. Results Endothelin binding study The binding of endothelin to cardiac membranes was a saturable process and resulted in linear Scatchard plot (see Figure 1). The binding parameters of radiolabelled ET-1 for human cardiac membranes obtained in both HF patients and in controls are reported in table 1: the values of affinity, Kd, in both, controls and failing hearts, was in the range of picomol/L without any difference between the two groups (p 5 0.693 and p 5 0.912, by using the Scheffé test after ANOVA, in right and left ventricle, respectively). ET receptor density, Bmax, showed a tendency to be higher in failing hearts with respect to nonfailing hearts (this effect is most evident in left ventricle), but no correlation with the severity of disease was found. Hill coefficient values did not differ significantly from unit in all the cardiac regions, both in failing and nonfailing hearts (Hill coefficients were: right atrium, 1.8 6 0.31 and 1.4 6 0.2; left atrium, 1.4 6 0.29 and 1.3 6 0.23; right ventricle, 1.2 6 0.26 and 1.05 6 0.03; left ventricle, 1.2 6 0.15 and 1.4 6 0.15; septum, 1.4 6 0.29 and 1.5 6 0.15; respectively in failing and nonfailing hearts). As to competition experiments, the inhibition of the specific binding of the [125 I]-ET-1 observed for all the competitive agents used indicated the specificity of the binding assay. The non-selective agonist ET-1 inhibits the specific binding in a monophasic manner (see Figure 1), both in failing and nonfailing hearts, while the inhibition curves of the ET-A selective antagonist BQ-123 (see Figure 2) and of ET-B specific ligand IRL-1620 (data not shown) were biphasic indicating the presence of both receptor subtypes in these tissues, with a prevalence of ET-A subtype in all cases (Table 1). The percent of ET-A subtype results increased in failing hearts in all the cardiac regions and it is more evident in left ventricle (Table 1). An increase in the protein level of ET-A receptors in failing hearts without change in ET-B receptor density was observed in all the cardiac regions: Bmax values of the ET-A and ET-B receptors in the failing hearts and in controls, relative to the left ventricle, are reported in Figure 3. The affinity of ET-A subtype receptors for BQ-123 was in the range of nanomol/L while the ET-B subtypes were bound by BQ-123 with micromolar affinity, without significant differences between patients and controls (in failing hearts, KiET-A was 14.5 6 4.0 nmol/L in right atrium, 18.1 6 5.0 in left atrium, 13.5 6 0.4 in right ventricle, 10.7 6 2.9 in
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Fig. 1. Representative saturation curve (top), Scatchard analysis (middle) and ET-1 competition curve (bottom) for 125I-ET-1 specific binding to human cardiac membranes (right atrium of a failing heart sample).
left ventricle and 8.4 6 2.0 in septum, respectively; the corresponding KiET-B values ranged from 4.0 to 40 mmol/L). Peptide tissue concentrations The levels of endothelin are higher in failing hearts than in controls in all sides of the heart (see Figure 4) and increase as a function of the severity of disease; this effect, although not significant, is present in all the cardiac regions (right atrium: 602.5 6 185.8 vs. 1141.8 6
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Table 1 Binding parameters of endothelin receptors Kd, pmol/L Nonfailing hearts Failing hearts Bmax, fmol/mg protein Nonfailing hearts Failing hearts ET-A: ET-B Nonfailing hearts Failing hearts
Right atrium
Left atrium
Right ventricle
Left ventricle
Septum
10.5 6 1.4 15.0 6 3.7
13.6 6 2.3 13.3 6 2.8
8.6 6 0.96 20.1 6 3.9
9.1 6 2.8 18.4 6 2.9
7.6 6 0.78 9.5 6 0.5
56.5 6 12.9 40.5 6 5.9
39.4 6 7.6 61.4 6 25.9
23.5 6 11.8 42.0 6 6.3
29.0 6 3.3 62.6 6 22.6
43.5 6 3.9 34.7 6 11.6
73: 27 76: 24
63: 37 75: 25
50: 50 80: 20 *
47: 53 84: 16 **
55: 45 85: 15 §
* p 5 0.027; ** p 5 0.0007; § p 5 0.006 vs. nonfailing hearts values, Scheffé test after ANOVA.
370.1; left atrium: 414.8 6 58.7 vs. 819.6 6 496.5; right ventricle: 598.9 6 240.1 vs. 901.9 6 469.2; left ventricle: 331.5 6 123.0 vs. 795.0 6 213.1; septum: 267.7 6 43.0 vs. 715.06 146.2; NYHA class III vs. class IV, p 5 ns). The tissue concentrations of endothelin in failing hearts resulted higher in right atrium, but no significant differences were observed between the various cardiac regions analyzed. No direct relationship was observed between binding parameter values (Bmax) and peptide tissue levels. Figure 5 reports the levels of ANP and BNP in failing and nonfailing hearts. Both peptides were found mostly in atrium and they are increased in severe heart failure with respect to controls; in ventricle the increase of BNP production in failing hearts was higher than that of ANP. Discussion In our study we have made a parallel evaluation of the expression of endothelin and cardiac natriuretic peptides in human heart. We have found a tendency to increase in endothelin receptor density in failing hearts mainly due to a significant up-regulation of ET-A subtype and a parallel increase of endothelin tissue levels as well as of cardiac natriuretic peptides, ANP and BNP.
Fig. 2. Representative BQ-123 competition curves for 125I-ET-1 specific binding to human cardiac membranes (left ventricle): closed circles represent a failing heart sample and open circles a corresponding nonfailing sample.
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Fig. 3. Protein levels of ET-A and ET-B receptors in left ventricle of failing and nonfailing hearts. ** p 5 0.0064 Fisher’s test after ANOVA.
ET-1 receptors in human cardiac tissue The discrepancies among the various studies aimed at characterizing ET-1 receptors in both failing and nonfailing myocardium from patients with end-stage DCM [10, 42–44] or ICM [42, 44] could partially be explained by differences in the methodological procedure [48] as well as in the choice of the nonfailing control group. In fact, all the situations used to approach the healthy conditions — autoptic specimens, bioptic material from patients undergoing coronary artery by-pass grafting in NYHA functional class I and explanted donor hearts — present some potential intrinsic limitations [43]. Our results are in tune with the findings of Pieske et al [10] showing an increase of cardiac endothelin receptor densities in DCM, due to an up-regulation of ET-A receptors without any change of ET-B receptor density. An up-regulation of ET-A subtype receptors (associated to a
Fig. 4. Mean values (6 SEM) of endothelin levels in the different cardiac regions in controls and in failing hearts.
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Fig. 5. ANP (left side) and BNP (right side) tissue levels in failing and nonfailing hearts (mean 6 SEM). Data reported are the mean of the concentrations found in the right and left side of atrium and ventricle, respectively (no significant differences in natriuretic peptide levels between right and left sides of heart were found).
decrease of ET-B receptors and no change of the total endothelin binding sites) has been also found by Zolk et al [43] in DCM hearts. Instead Ponicke et al [42] failed to find any significant variation of endothelin receptor density in a mixed group of patients with DCM and ICM with respect to control group. However, considering DCM and ICM patients as separate groups, a two-fold increase of receptor density with respect to nonfailing hearts was found in DCM group, suggesting a differential regulation of endothelin receptors in the two patient classes [42]. A limitation of the studies using cardiac membrane homogenates is that this preparation contains not only cardiomyocytes, but also non-myocyte cells such as fibroblasts, smooth muscle cells and endothelial cells, thus a direct evaluation of the respective contribution of myocytes or non-myocyte cells is not achievable. ET-A is the prevalent receptor subtype in cardiac membranes [10, 42–45, 49] and this prevalence is enhanced in myocytes where mRNA for ET-B is not expressed [50]. Although a differential regulation of ET-1 receptors in different kinds of cells cannot be excluded, these observations suggest that the ET-A upregulation found in myocardial membranes of failing patients could reflect mainly the contribution of myocytes, as previously indicated on the basis of indirect evidences [10]. This has been confirmed in a more recent study where the endothelin system has been evaluated in ventricle membrane suspensions as well as in isolated cardiomyocytes [44]: total ET-1 receptor density as well as ET-A and ET-B densities are significantly increased in failing hearts, both in isolated cardiomyocytes and in cardiac membranes, with respect to nonfailing hearts. So, because it has been demonstrated that ET-A is the only biologically active endothelin receptor in the human heart [42], myocytes appear to represent the target of biological actions of endothelin [50]. ET-1 peptide and ET-1 receptor relationship ET-1 binding sites on cardiocytes in culture are down-regulated by pre-treatment with ET-1 [51] and a blunted functional response to infusion of ET-1 has been observed in patients with
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elevated peripheral endothelin with respect to controls [52]. These observations indicated the possibility of a reduction of density of binding sites in patients with heart failure as a consequence of the high peripheral levels of endothelin in these subjects. Considering the paracrine action of ET-1, its tissue concentrations could be considered more relevant than plasma levels to reveal possible modulation of the peptide on endothelin cardiac binding sites [42]. Endothelin tissue concentration was found increased as a function of severity of disease in all the cardiac regions, in agreement with recent reports in left ventricles [10, 43]. The discrepancies found among the content of ET-1 in human heart could be mostly ascribed to accuracy of the assay methods: the best way to measure endothelin in biological samples is a separation on HPLC followed by specific immunometric assay of the elution fractions, a procedure that is expensive, time consuming and that needs a large amount of biological sample. The immunometric assays commercially available are non-selective for the isopeptides of endothelin and generally shown low, but non-negligible, cross-reaction with Big-ET-1. Big-ET-1 was found to represent about 60% of all circulating endothelins in HF patients while in controls this precursor was not found [9]. Possible overestimation of the endothelin content in our samples can be ruled out by the negligible extent of cross-reaction with Big-ET-1 of the immunometric assay used and by the observation that in cardiac tissue the mature ET-1 is the only molecular form present both in normal and in failing hearts [9]. As far as the mechanisms producing these elevated levels of ET-1 in HF is concerned, in failing heart of rat with heart failure due to myocardial infarction [53] the level of preproendothelin (prepro-ET-1) mRNA and the peptide level ET-1 markedly increased whereas the expression of endothelin-converting enzyme (ECE)-1 mRNA in the heart did not differ between HF and control rats suggesting that the increase in ET-1 peptide level derived from upregulation of prepro-ET-1 mRNA. On the other hand, in the left venticle of patients with dilated cardiomyopathy [43], prepro-ET-1 mRNA was found unchanged with respect to control hearts. Moreover a reduction of ET-1 clearance rate in the heart, mediated by ET-B subtype [54, 55], could contribute to the high local levels of ET-1 in HF patients. The lack of downregulation, observed also in this study, in face to the high local concentrations of endothelin seems to indicate the possibility that other mechanisms besides the agonistinduced downregulation could be involved in the modulation of cardiac endothelin receptors in heart failure. Angiotensin II [56], cAMP [57] and nitric oxide [58] have been suggested to have a role in ET receptor regulation inducing a heterologous upregulation of ET receptor expression. An up-regulation of ET-A subtype receptors in human failing hearts has been observed in left ventricle [10, 43]. In failing rat heart (left ventricle) ET-1 concentration was increased, protein level of both ET-A and ET-B receptors were upregulated and also mRNA levels of both subtypes were increased suggesting that both the ET-A and ET-B receptor systems are greatly accelerated in the failing heart of rats [53]. Similar data have been recently found in ICM patients — while DCM patients do not differ from nonfailing subjects — where the activation of endothelin system appears to contribute to the maintenance of cardiac function [44]. Our study demonstrates that a tendency to increase of ET receptor density, mainly due to ET-A subtype increase, is present to a different extent also in the other cardiac regions (the maximal effect being in the left ventricle). Although some more data need to confirm this observation and to explain this differential increase, the significant increase in this cardiac region of ET-A could be related to the left ventricular dysfunction of our failing patients.
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These findings could help to explain the observation that long-term treatment with an ET-A specific antagonist greatly improves the survival rate of rat with HF and that this effect is accompanied by significant amelioration of left ventricular dysfunction and prevention of unfavorable ventricular remodeling [20]. Cardiac natriuretic peptide production In failing hearts the activation of endothelin system is paralleled by the activation of cardiac natriuretic pepide production. In fact, ANP and BNP increase in failing hearts in all cardiac regions, a finding in close agreement with the concomitant increase of ET-1 that is a well known stimulus to their production [59]. A differential pattern of ANP and BNP production has been found in ventricles where the increase of BNP in failing hearts is more pronounced than that of ANP. This finding closely agrees with the recent observation that ET-1 directly induces BNP transcription in cultured ventricular myocytes [60]. Moreover our data seem to confirm the previous observation that in absence of ventricular dysfunction and during early experimental left ventricular dysfunction, atrial myocardium is the predominant site of BNP gene expression and production while in overt HF a further increase of BNP gene expression and production occurs in ventricular tissue [61]. The mechanisms that stimulate the ANP and BNP production are different in atrium and ventricle. In atrium, the main stimulus for both ANP and BNP production is the stretching (preload), while in the ventricle further mechanisms, besides the ventricle relaxation (afterload), contribute to natriuretic peptide production. With regard to BNP, it has been suggested that it plays a role in the local activation of substances such as angiotensin II and ET-1, known to induce transcription of early genes and stimulate myocyte growth. Finally the increase in BNP production is associated with the additional increase of the circulating levels of BNP in overt HF confirming a role for ventricle in the generation of BNP in severe HF [61]. Conclusion In our study we have found an activation of the endothelin system in failing heart in all cardiac regions, with more evident effects in left ventricle. Endothelin activation is associated to a parallel increase of myocardial concentrations of ANP and BNP. This latter, which is considered as the best neurohormonal diagnostic marker of altered myocardial function and structure [62, 63], in ventricle presents a notewhorthy differential pattern with respect to ANP. The activation of endothelin system, that in an early phase could be considered as a compensatory mechanism [18] in response to the reduction of positive inotropic effect observed in HF [10], on the time could produces negative effects as demonstrated by the beneficial action of receptor antagonists of endothelin. The up-regulation of ET-A subtype found in our as well as in other studies could suggest that a selective ET-A antagonist could be the preferred agent, reducing the noxious effects of ET-1, ET-A mediated, such as the induction of cardiac hypertrophy and remodeling. Cellular biology studies are pivotal to explain the mechanism of endothelin action as well as the endothelin-antagonism, however, for the complexity of the endothelin functions and for their possible alteration in heart failure, a therapeutic indication can only be derived by long-term clinical trials whose end-points are the morbidity/mortality evaluation as well as of the possible side-effects.
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The parallel activation of natriuretic peptide system, observed in this condition, that counteracts the actions of vasoconstrictor peptides, indicates the importance to evaluate, besides the inhibition of endothelin action, also the therapeutic efficacy in heart failure of pharmacological treatments that increase the cellular actions of natriuretic peptides or reduce their degradation.
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Endothelin-1, endothelin-1 receptors and cardiac natriuretic peptides in failing human heart Silvia Del Ry, Maria Grazia Andreassi, Aldo Clerico, Andrea Biagini, Daniela Giannessi* CNR Institute of Clinical Physiology, Laboratory of Cardiovascular Biochemistry, Pisa, Italy Received 31 January 2000; accepted 7 November 2000
Abstract Endothelin (ET)-1 is a potent vasoconstrictor peptide produced in the myocardium that can exert important effects on cardiac myocyte growth and phenotype; cardiac natriuretic peptides (ANP and BNP) are known to act as physiological antagonists of ET-1. In this study a comparative determination of ET-1 receptors and of the local productions of ET-1 and of ANP and BNP was made in different sites of failing and nonfailing hearts. Tissue from right and left atrium, right and left ventricle and interventricular septum from seven adult heart transplant recipients with end-stage idiopathic dilated cardiomyopathy (functional class III and IV, with ejection fraction , 35%) and from four postmortem subjects without cardiac complications was analyzed. In failing hearts we observed a tendency to increase of density of binding sites, most evident in left ventricle (62.6 6 22.6 fmol/mg protein vs. 29.0 6 3.3, mean 6 SEM, p 5 ns). A prevalence of ET-A subclass, observed in all samples, resulted more pronounced in failing hearts where this increase, found in all the cardiac regions, was more evident in left ventricle (p 5 0.0007 vs nonfailing hearts). The local concentrations of ET-1, ANP and BNP resulted significantly increased in failing hearts with respect to controls in all sides of the heart. In failing hearts we have observed a tendency to increase in endothelin receptor density mainly due to a significant upregulation of ET-A subtype and a parallel increase of the tissue levels of ANP, BNP and ET-1 indicating an activation of these systems in heart failure. © 2001 Elsevier Science Inc. All rights reserved. Keywords: Endothelin; Cardiac natriuretic peptides; ANP; BNP; Heart failure; Endothelin receptors
Introduction Heart failure (HF) is the major cause of cardiovascular morbidity and mortality [1]; its complex haemodynamic and neuroendocrine mechanisms are not completely known, al* Corresponding author. Laboratory of Cardiovascular Biochemistry, CNR Institute of Clinical Physiology, Via Alfieri, 1 56010 Ghezzano (Pisa), Italy. Tel.: 39 050 3152664; fax: 39 050 553461. E-mail address: [email protected] (D. Giannessi) 0024-3205/01/$ – see front matter © 2001 Elsevier Science Inc. All rights reserved. PII: S 0 0 2 4 - 3 2 0 5 ( 0 1 )0 1 0 7 6 -1
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though an improved understanding of the pathophysiology is fundamental for the development of new therapeutic strategies for this disease [2]. Different neurohormonal systems (i.e. catecolamine and renin-angiotensin-aldosterone systems) are known to be progressively activated as the cardiac performance declines in patients with HF, in order to maintain circulatory homeostasis [3]. Endothelin (ET)-1, a peptide hormone with potent sustained vasoconstrictor properties which is synthesised predominantly by the vascular endothelium [4], has been implicated in the pathophysiological vasoconstriction in HF [5]: noteworthy increases in circulating ET-1 have been observed both in experimental models of HF and in humans with this disease, irrespective of aetiology [6–9], although it has been considered primarily a local paracrine factor. ET-1 has important direct cardiac effects and appears to be involved in the regulation of myocardial contractility; in human myocardium, ET-1 exerts clear positive inotropic actions, concentration-dependent, that seems to be less pronounced in failing hearts [10]. The various biological functions of ET-1 are mediated by two high affinity receptor subtypes, ET-A and ET-B, belonging to the G protein-coupled family that have been identified in various human tissues, including cardiac tissue [11]. ET-A receptors have selective affinity for ET-1 and are expressed principally on vascular smooth muscle cells and cardiac myocytes but not on endothelial cells [12]. ET-B receptors have equal affinity for all endothelin isoforms and are expressed mostly on endothelial cells [13], but also on vascular smooth muscle cells [14]. Stimulation of ET-A and ET-B receptors on vascular smooth muscle cells resulted in sustained vasoconstriction [15], by an increase of intracellular calcium, while stimulation of ET-B receptors on endothelial cells results in vasodilation, due to the release of nitric oxide and prostacyclin [16]. In different animal models of HF, alterations of ET receptor density have been described: in the heart, ET-1 binding site density has resulted significantly higher in the HF rats than in the control rats [17–19]. On the basis of these observations, ET receptor blockade was suggested as a further therapeutic advance in the anti-endothelin strategy in the treatment of this disease [2]. In fact, after the study of Sakai [20], that demonstrated that the selective ET-A receptor antagonist BQ-123 improves long term survival in rats with HF, a number of works showed the beneficial effects of the use of selective ET-A receptor antagonists both in animal models [21–24] and in humans [25, 26]. Also, treatment with a non selective ET antagonist, as bosentan, markedly increases survival in experimental models of HF [27–31] while short-term therapy with bosentan in patients with HF [32, 33] produces pulmonary and systemic vasodilation and may enhance conventional treatment with angiotensinconverting enzyme inhibitors [34]; however, it has not completely assessed whether selective ET-A antagonists or non-selective ET-A/ET-B blockers are the better therapeutic agents in heart failure treatment. Various endogenous vasodilators may act as physiological antagonists of ET-1; among those, atrial natriuretic peptide (ANP) and brain natriuretic peptide (BNP) constitute a vasoactive natriuretic peptide system that regulates circulatory homeostasis. Their circulating concentrations are elevated in HF and correlate positively with the severity of disease [35], indicating a regulatory function by balancing the actions of the vasoconstrictor antidiuretic neurohormones. A therapeutic use of these peptides in HF is largely suggested although the hemodynamic effects of the treatment with natriuretic peptides are not completely clarified owing to their complex potential role on myocardial systolic and diastolic functions, after-
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load and preload. ANP seems to have a negative inotropic action on the heart, mediated by reduction of calcium entry into the cardiac cell, as observed in cultured ventricular cells [36] and in ex-vivo experimental models [37]. A recent study on the effects of natriuretic peptides on load and myocardial function in normal and heart failure dogs has indicated that infusion of natriuretic peptides produces a mild positive inotropic effect in normal dogs; in HF dogs no inotropic effect was observed instead of the waited negative inotropic effect due to the higher myocardial levels of natriuretic peptides in this condition [38]. Moreover, endothelin and natriuretic peptide systems are also directly related: ET-1 can stimulate the ANP and BNP expression [39, 40], conversely, endogenous ANP directly inhibits endogenous ET-1 secretion through a cGMP-mediated pathway in severe HF [41]. In man, a characterization of ET receptors in failing and nonfailing myocardium has been made recently in right atrium [42] and left ventricle [10, 42–44] from patients with end-stage idiopathic dilated cardiomyopathy (DCM) or ischemic cardiomyopathy (ICM), but the results are not in close agreement. Moreover, a comparative determination of endothelin receptors and of tissue levels of ET-1 and natriuretic peptides in the various regions of human myocardium is yet lacking. Aim of this study was the assessment of endothelin status in human heart by the simultaneous determination of tissue ET-1 concentration in human myocardial tissue from multiple cardiac sites in patients with severe chronic HF and in controls and of endothelin binding sites and their distribution in the two main receptor subclasses. For a deeper knownledge of the local status of vasoactive peptides, the determination of the concentrations of the natriuretic hormones, ANP and BNP, in the same myocardial tissue has been also performed. These findings may be relevant in the evaluation of a possible paracrine action of these hormones in the regulation of cardiac function in both healthy and pathological conditions. Methods ET receptor determination on cardiac tissue The determination of binding parameters, affinity (Kd) and density (Bmax), was made by saturation experiments on cardiac membranes suspension by using mono-iodinated ET-1 as radioligand and following a previously described procedure [45]; the percent of ET-A and ET-B subtypes were obtained by competitive displacement experiments with the specific ligands, BQ-123 and IRL-1620. Tissue collection Patients. Human cardiac tissue was obtained from seven adult transplant recipients with idiopathic dilated cardiomyopathy (5 men and 2 women, mean age: 36 6 7.2 years), divided into two groups according to their functional class (NYHA class III, n 5 3 and NYHA class IV, n 5 4; left ventricular ejection fraction , 35%). Myocardial contractility, cardiac dimension and function were assessed by 2D echocardiography, radionuclide ventriculography, and hemodynamic study. Pharmacological treatment included vasodilators (generally an angiotensin converting enzyme and/or nitrates) and diuretics. Patients treated with cathecolamines or b-adrenoceptor antagonists were not included in the study.
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Control group. Cardiac tissue was also obtained from four patients died for an acute noncardiac disease (2 men and 2 women, mean age: 59 6 10.4 years), collected during the autopsy, 10–12 hours after death. The donor subjects did not present histories or clinical signs of heart disease and evident atherosclerosis of large blood vessels was not observed at autoptic examination. They do not received any drugs known to interfere with endothelin system, such as cathecolamines or b-adrenoceptor antagonists prior the death. In all subject right and left atrium, right and left ventricle and interventricular septum were analyzed. All specimens were processed for routine H&E and Mallory trichromic stain. Light microscopic analysis was carried out on sections of tissue for evaluation of the amount of connective tissue and myocardial cell damage, using a square counting grid of 25 points on 10 randomly sampled areas up to 1000 points for each sample. The connective tissue area was , 20% of total area and the cellular damage (due to tissue collection and storage) resulted , 10 % of tissue area. In all cases, tissue samples were frozen in liquid nitrogen immediatly after withdrawn and stored at 2808C until use; all samples were processed not later than 2 months of storage at 2808C after collection. Tissue was collected with the approval of the Ethical Committee of our institution, and the transplant recipient subjects gave informed consent. Membrane preparation The tissue (,50 mg ww) was homogenized in ice-cold Tris/HCl buffer (50 mmol/L Tris/ HCl, 0.25 mol/L sucrose, 1 mmol/L EDTA, 1 mmol/L MgCl2, 0.5 mmol/L phenylmethylsulfonyl fluoride, 0.5 mg/ml leupeptin, 0.5 mg/ml soybean trypsin inhibitor, pH 7.4) by using a glass Potter’s homogenizer; after centrifugation at 500 3 g for 15 min at 48C, supernatant was collected and centrifugated again at 50,000 3 g for 30 min at 48C. The pellet was resuspended in ice-cold Tris/HCl buffer and protein concentration was determined by Lowry’s method with bovine serum albumin (BSA) as standard [46]. Membrane suspension was stored frozen at 2808C at a concentration of 1–3 mg/ml. Radioligand preparation ET-1 (Novabiochem, Switzerland) was labelled with Na-[125I] (sp. a. 18 Ci/mg) (Sorin Biomedica, Saluggia, Italy) by lactoperoxidase technique. About 1 mCi of Na-[125 I], 5 mg of ET-1 and 5 ml of bovine lactoperoxidase (1 mg/ml) in a final volume of 60 ml of phosphate buffer 0.5 mol/L, pH 7.4 were added with 10 ml of H2O2 (10 vol, 1:12,000), and the mixture incubated for 29 300 at room temperature under mild agitation; this procedure was repeated three times. Radioiodinated ET-1 was purified by reverse-phase HPLC by using a Novapak C18 column (4 mm, 3.53300 mm, Waters Associates, Milford MA), eluted with a linear gradient, from 20% to 50%, of CH3CN in 0.1% trifluoracetic acid, for 60 min at a flow rate of 1 ml/ min. Only mono-iodinated endothelin was used for binding assay. Specific activity was determined by measuring the ET-1 concentration of serial dilutions of the radiolabelled preparation by an immunoenzymatic (EIA) system for ET-1, purchased from Cayman Chemical Company (Ann Arbor, MI). Scalar dilutions of radiolabelled peptide, from 2,000 to 60,000 cpm/ml, were assayed as unknown samples and the corresponding mass determined. The tracer was stored frozen at 2208C in the buffer used for binding assay.
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Binding assay For saturation experiments, [125 I]-ET-1 binding was performed in a total assay volume of 250 ml consisting of 100 ml radioligand (10–300 pmol/L), 50 ml of membrane preparation (25 mg/ml of protein), and 100 ml of Tris/HCl buffer; non-specific binding was determined by addition of unlabelled ET-1 (0.1 mmol/L). All tubes were incubated at 378C for 4 hrs under agitation. The reaction was stopped by rapid filtration through glass-fiber filters Whatman GF/B, prewet with Tris-HCl buffer, 0.3% BSA; the filters were then washed with 15 ml of cold buffer. Bound radioactivity was measured in a gamma scintillation counter (Cobra 2000, Packard Instrument Company, Meridien, CT), 80% counting efficiency. Competition studies were performed at 20 pmol/L of [125I]-ET-1 and 25 mg/ml of protein by using increasing concentrations of different ligands: BQ-123 (10 pmol/L – 50 mmol/L), ET-1 (15 pmol/L – 100 nmol/L) (Sigma, S. Louis, MO) and IRL-1620 (10 nmol/L – 10 mmol/L) (Alexis Biochemicals, San Diego CA). Data analysis The affinity constant (Kd), the density of binding sites (Bmax, expressed as fmol bound/ mg protein) and the Hill coefficient were obtained by analysis of the data using an iterative curve fitting programme (KaleidaGraph). Scatchard analysis was also performed. IC50 values for the various ligands from competition experiments were evaluated by an iterative curve fitting programme (KaleidaGraph); Ki values were derived by the method of Cheng and Prusoff [47]: Ki 5 IC50/(11L/Kd), where L is the concentration of the radioligand, IC50 is the concentration of competitor agent causing 50% inhibition of specific radioligand binding and the Kd the dissociation constant of the radioligand-receptor complex. Quantitation of peptides in cardiac tissue Vasoactive peptides were measured by specific immunometric assays after tissue extraction. Tissue extraction Cardiac tissue was homogenized in 10 vol of acetic acid 0.5 mol/L containing Triton X, 0.01% and then boiled for 15 min in a water bath. The homogenate was centrifuged at 48,000 3 g for 20 min and the concentrations of the different peptides were determined in the supernatant by specific immunometric assays. Immunometric assays Endothelin was measured by a competitive radioimmunological method (Biomedica Gruppe, Vienna, Austria; supplied by CIS Bio International, Gif-sur-Yvette, France) after solid-phase extraction on Sep-Pak C18 cartridge (Waters Associates, Milford, MA) showing 142 % cross-reaction with ET-2, 98% with ET-3, ,1% with ANP and Big-ET-1(1–38); the detection limit was about 2.0 pmol/L. Both ANP and BNP were measured by non competitive immunoradiometric (IRMA) methods using two monoclonal antibodies prepared against sterically remote epitopes of molecule (Shionoria ANP, Shionoria BNP, manufactured by Shionogi & Co, Ltd, Osaka, Japan; supplied by CIS Bio International). No important cross-reactions with the other peptides were observed (for ANP assay: , 0.01% of cross-reactions with BNP and ET-1; for BNP assay:
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, 0.01% of cross-reactions with BNP, CNP and ET-1). Sensitivity was 0.68 6 0.30 pmol/L for ANP and 0.57 6 0.19 pmol/L for BNP, respectively. The inter-assay variability of whole procedure (extraction 1 immunometric assay) was about 25%. Statistical analysis Multiple comparisons of the experimental data (binding parameters and vasoactive peptide concentrations in the various cardiac regions for the different groups of patients) were made by ANOVA. The difference of Hill coefficients from unit was checked by paired Student’s t-test. For linear regression, experimental data were analyzed by checking the null hypothesis by Student’s t-test. All values were reported as mean 6 SEM. Results Endothelin binding study The binding of endothelin to cardiac membranes was a saturable process and resulted in linear Scatchard plot (see Figure 1). The binding parameters of radiolabelled ET-1 for human cardiac membranes obtained in both HF patients and in controls are reported in table 1: the values of affinity, Kd, in both, controls and failing hearts, was in the range of picomol/L without any difference between the two groups (p 5 0.693 and p 5 0.912, by using the Scheffé test after ANOVA, in right and left ventricle, respectively). ET receptor density, Bmax, showed a tendency to be higher in failing hearts with respect to nonfailing hearts (this effect is most evident in left ventricle), but no correlation with the severity of disease was found. Hill coefficient values did not differ significantly from unit in all the cardiac regions, both in failing and nonfailing hearts (Hill coefficients were: right atrium, 1.8 6 0.31 and 1.4 6 0.2; left atrium, 1.4 6 0.29 and 1.3 6 0.23; right ventricle, 1.2 6 0.26 and 1.05 6 0.03; left ventricle, 1.2 6 0.15 and 1.4 6 0.15; septum, 1.4 6 0.29 and 1.5 6 0.15; respectively in failing and nonfailing hearts). As to competition experiments, the inhibition of the specific binding of the [125 I]-ET-1 observed for all the competitive agents used indicated the specificity of the binding assay. The non-selective agonist ET-1 inhibits the specific binding in a monophasic manner (see Figure 1), both in failing and nonfailing hearts, while the inhibition curves of the ET-A selective antagonist BQ-123 (see Figure 2) and of ET-B specific ligand IRL-1620 (data not shown) were biphasic indicating the presence of both receptor subtypes in these tissues, with a prevalence of ET-A subtype in all cases (Table 1). The percent of ET-A subtype results increased in failing hearts in all the cardiac regions and it is more evident in left ventricle (Table 1). An increase in the protein level of ET-A receptors in failing hearts without change in ET-B receptor density was observed in all the cardiac regions: Bmax values of the ET-A and ET-B receptors in the failing hearts and in controls, relative to the left ventricle, are reported in Figure 3. The affinity of ET-A subtype receptors for BQ-123 was in the range of nanomol/L while the ET-B subtypes were bound by BQ-123 with micromolar affinity, without significant differences between patients and controls (in failing hearts, KiET-A was 14.5 6 4.0 nmol/L in right atrium, 18.1 6 5.0 in left atrium, 13.5 6 0.4 in right ventricle, 10.7 6 2.9 in
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Fig. 1. Representative saturation curve (top), Scatchard analysis (middle) and ET-1 competition curve (bottom) for 125I-ET-1 specific binding to human cardiac membranes (right atrium of a failing heart sample).
left ventricle and 8.4 6 2.0 in septum, respectively; the corresponding KiET-B values ranged from 4.0 to 40 mmol/L). Peptide tissue concentrations The levels of endothelin are higher in failing hearts than in controls in all sides of the heart (see Figure 4) and increase as a function of the severity of disease; this effect, although not significant, is present in all the cardiac regions (right atrium: 602.5 6 185.8 vs. 1141.8 6
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Table 1 Binding parameters of endothelin receptors Kd, pmol/L Nonfailing hearts Failing hearts Bmax, fmol/mg protein Nonfailing hearts Failing hearts ET-A: ET-B Nonfailing hearts Failing hearts
Right atrium
Left atrium
Right ventricle
Left ventricle
Septum
10.5 6 1.4 15.0 6 3.7
13.6 6 2.3 13.3 6 2.8
8.6 6 0.96 20.1 6 3.9
9.1 6 2.8 18.4 6 2.9
7.6 6 0.78 9.5 6 0.5
56.5 6 12.9 40.5 6 5.9
39.4 6 7.6 61.4 6 25.9
23.5 6 11.8 42.0 6 6.3
29.0 6 3.3 62.6 6 22.6
43.5 6 3.9 34.7 6 11.6
73: 27 76: 24
63: 37 75: 25
50: 50 80: 20 *
47: 53 84: 16 **
55: 45 85: 15 §
* p 5 0.027; ** p 5 0.0007; § p 5 0.006 vs. nonfailing hearts values, Scheffé test after ANOVA.
370.1; left atrium: 414.8 6 58.7 vs. 819.6 6 496.5; right ventricle: 598.9 6 240.1 vs. 901.9 6 469.2; left ventricle: 331.5 6 123.0 vs. 795.0 6 213.1; septum: 267.7 6 43.0 vs. 715.06 146.2; NYHA class III vs. class IV, p 5 ns). The tissue concentrations of endothelin in failing hearts resulted higher in right atrium, but no significant differences were observed between the various cardiac regions analyzed. No direct relationship was observed between binding parameter values (Bmax) and peptide tissue levels. Figure 5 reports the levels of ANP and BNP in failing and nonfailing hearts. Both peptides were found mostly in atrium and they are increased in severe heart failure with respect to controls; in ventricle the increase of BNP production in failing hearts was higher than that of ANP. Discussion In our study we have made a parallel evaluation of the expression of endothelin and cardiac natriuretic peptides in human heart. We have found a tendency to increase in endothelin receptor density in failing hearts mainly due to a significant up-regulation of ET-A subtype and a parallel increase of endothelin tissue levels as well as of cardiac natriuretic peptides, ANP and BNP.
Fig. 2. Representative BQ-123 competition curves for 125I-ET-1 specific binding to human cardiac membranes (left ventricle): closed circles represent a failing heart sample and open circles a corresponding nonfailing sample.
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Fig. 3. Protein levels of ET-A and ET-B receptors in left ventricle of failing and nonfailing hearts. ** p 5 0.0064 Fisher’s test after ANOVA.
ET-1 receptors in human cardiac tissue The discrepancies among the various studies aimed at characterizing ET-1 receptors in both failing and nonfailing myocardium from patients with end-stage DCM [10, 42–44] or ICM [42, 44] could partially be explained by differences in the methodological procedure [48] as well as in the choice of the nonfailing control group. In fact, all the situations used to approach the healthy conditions — autoptic specimens, bioptic material from patients undergoing coronary artery by-pass grafting in NYHA functional class I and explanted donor hearts — present some potential intrinsic limitations [43]. Our results are in tune with the findings of Pieske et al [10] showing an increase of cardiac endothelin receptor densities in DCM, due to an up-regulation of ET-A receptors without any change of ET-B receptor density. An up-regulation of ET-A subtype receptors (associated to a
Fig. 4. Mean values (6 SEM) of endothelin levels in the different cardiac regions in controls and in failing hearts.
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Fig. 5. ANP (left side) and BNP (right side) tissue levels in failing and nonfailing hearts (mean 6 SEM). Data reported are the mean of the concentrations found in the right and left side of atrium and ventricle, respectively (no significant differences in natriuretic peptide levels between right and left sides of heart were found).
decrease of ET-B receptors and no change of the total endothelin binding sites) has been also found by Zolk et al [43] in DCM hearts. Instead Ponicke et al [42] failed to find any significant variation of endothelin receptor density in a mixed group of patients with DCM and ICM with respect to control group. However, considering DCM and ICM patients as separate groups, a two-fold increase of receptor density with respect to nonfailing hearts was found in DCM group, suggesting a differential regulation of endothelin receptors in the two patient classes [42]. A limitation of the studies using cardiac membrane homogenates is that this preparation contains not only cardiomyocytes, but also non-myocyte cells such as fibroblasts, smooth muscle cells and endothelial cells, thus a direct evaluation of the respective contribution of myocytes or non-myocyte cells is not achievable. ET-A is the prevalent receptor subtype in cardiac membranes [10, 42–45, 49] and this prevalence is enhanced in myocytes where mRNA for ET-B is not expressed [50]. Although a differential regulation of ET-1 receptors in different kinds of cells cannot be excluded, these observations suggest that the ET-A upregulation found in myocardial membranes of failing patients could reflect mainly the contribution of myocytes, as previously indicated on the basis of indirect evidences [10]. This has been confirmed in a more recent study where the endothelin system has been evaluated in ventricle membrane suspensions as well as in isolated cardiomyocytes [44]: total ET-1 receptor density as well as ET-A and ET-B densities are significantly increased in failing hearts, both in isolated cardiomyocytes and in cardiac membranes, with respect to nonfailing hearts. So, because it has been demonstrated that ET-A is the only biologically active endothelin receptor in the human heart [42], myocytes appear to represent the target of biological actions of endothelin [50]. ET-1 peptide and ET-1 receptor relationship ET-1 binding sites on cardiocytes in culture are down-regulated by pre-treatment with ET-1 [51] and a blunted functional response to infusion of ET-1 has been observed in patients with
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elevated peripheral endothelin with respect to controls [52]. These observations indicated the possibility of a reduction of density of binding sites in patients with heart failure as a consequence of the high peripheral levels of endothelin in these subjects. Considering the paracrine action of ET-1, its tissue concentrations could be considered more relevant than plasma levels to reveal possible modulation of the peptide on endothelin cardiac binding sites [42]. Endothelin tissue concentration was found increased as a function of severity of disease in all the cardiac regions, in agreement with recent reports in left ventricles [10, 43]. The discrepancies found among the content of ET-1 in human heart could be mostly ascribed to accuracy of the assay methods: the best way to measure endothelin in biological samples is a separation on HPLC followed by specific immunometric assay of the elution fractions, a procedure that is expensive, time consuming and that needs a large amount of biological sample. The immunometric assays commercially available are non-selective for the isopeptides of endothelin and generally shown low, but non-negligible, cross-reaction with Big-ET-1. Big-ET-1 was found to represent about 60% of all circulating endothelins in HF patients while in controls this precursor was not found [9]. Possible overestimation of the endothelin content in our samples can be ruled out by the negligible extent of cross-reaction with Big-ET-1 of the immunometric assay used and by the observation that in cardiac tissue the mature ET-1 is the only molecular form present both in normal and in failing hearts [9]. As far as the mechanisms producing these elevated levels of ET-1 in HF is concerned, in failing heart of rat with heart failure due to myocardial infarction [53] the level of preproendothelin (prepro-ET-1) mRNA and the peptide level ET-1 markedly increased whereas the expression of endothelin-converting enzyme (ECE)-1 mRNA in the heart did not differ between HF and control rats suggesting that the increase in ET-1 peptide level derived from upregulation of prepro-ET-1 mRNA. On the other hand, in the left venticle of patients with dilated cardiomyopathy [43], prepro-ET-1 mRNA was found unchanged with respect to control hearts. Moreover a reduction of ET-1 clearance rate in the heart, mediated by ET-B subtype [54, 55], could contribute to the high local levels of ET-1 in HF patients. The lack of downregulation, observed also in this study, in face to the high local concentrations of endothelin seems to indicate the possibility that other mechanisms besides the agonistinduced downregulation could be involved in the modulation of cardiac endothelin receptors in heart failure. Angiotensin II [56], cAMP [57] and nitric oxide [58] have been suggested to have a role in ET receptor regulation inducing a heterologous upregulation of ET receptor expression. An up-regulation of ET-A subtype receptors in human failing hearts has been observed in left ventricle [10, 43]. In failing rat heart (left ventricle) ET-1 concentration was increased, protein level of both ET-A and ET-B receptors were upregulated and also mRNA levels of both subtypes were increased suggesting that both the ET-A and ET-B receptor systems are greatly accelerated in the failing heart of rats [53]. Similar data have been recently found in ICM patients — while DCM patients do not differ from nonfailing subjects — where the activation of endothelin system appears to contribute to the maintenance of cardiac function [44]. Our study demonstrates that a tendency to increase of ET receptor density, mainly due to ET-A subtype increase, is present to a different extent also in the other cardiac regions (the maximal effect being in the left ventricle). Although some more data need to confirm this observation and to explain this differential increase, the significant increase in this cardiac region of ET-A could be related to the left ventricular dysfunction of our failing patients.
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These findings could help to explain the observation that long-term treatment with an ET-A specific antagonist greatly improves the survival rate of rat with HF and that this effect is accompanied by significant amelioration of left ventricular dysfunction and prevention of unfavorable ventricular remodeling [20]. Cardiac natriuretic peptide production In failing hearts the activation of endothelin system is paralleled by the activation of cardiac natriuretic pepide production. In fact, ANP and BNP increase in failing hearts in all cardiac regions, a finding in close agreement with the concomitant increase of ET-1 that is a well known stimulus to their production [59]. A differential pattern of ANP and BNP production has been found in ventricles where the increase of BNP in failing hearts is more pronounced than that of ANP. This finding closely agrees with the recent observation that ET-1 directly induces BNP transcription in cultured ventricular myocytes [60]. Moreover our data seem to confirm the previous observation that in absence of ventricular dysfunction and during early experimental left ventricular dysfunction, atrial myocardium is the predominant site of BNP gene expression and production while in overt HF a further increase of BNP gene expression and production occurs in ventricular tissue [61]. The mechanisms that stimulate the ANP and BNP production are different in atrium and ventricle. In atrium, the main stimulus for both ANP and BNP production is the stretching (preload), while in the ventricle further mechanisms, besides the ventricle relaxation (afterload), contribute to natriuretic peptide production. With regard to BNP, it has been suggested that it plays a role in the local activation of substances such as angiotensin II and ET-1, known to induce transcription of early genes and stimulate myocyte growth. Finally the increase in BNP production is associated with the additional increase of the circulating levels of BNP in overt HF confirming a role for ventricle in the generation of BNP in severe HF [61]. Conclusion In our study we have found an activation of the endothelin system in failing heart in all cardiac regions, with more evident effects in left ventricle. Endothelin activation is associated to a parallel increase of myocardial concentrations of ANP and BNP. This latter, which is considered as the best neurohormonal diagnostic marker of altered myocardial function and structure [62, 63], in ventricle presents a notewhorthy differential pattern with respect to ANP. The activation of endothelin system, that in an early phase could be considered as a compensatory mechanism [18] in response to the reduction of positive inotropic effect observed in HF [10], on the time could produces negative effects as demonstrated by the beneficial action of receptor antagonists of endothelin. The up-regulation of ET-A subtype found in our as well as in other studies could suggest that a selective ET-A antagonist could be the preferred agent, reducing the noxious effects of ET-1, ET-A mediated, such as the induction of cardiac hypertrophy and remodeling. Cellular biology studies are pivotal to explain the mechanism of endothelin action as well as the endothelin-antagonism, however, for the complexity of the endothelin functions and for their possible alteration in heart failure, a therapeutic indication can only be derived by long-term clinical trials whose end-points are the morbidity/mortality evaluation as well as of the possible side-effects.
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The parallel activation of natriuretic peptide system, observed in this condition, that counteracts the actions of vasoconstrictor peptides, indicates the importance to evaluate, besides the inhibition of endothelin action, also the therapeutic efficacy in heart failure of pharmacological treatments that increase the cellular actions of natriuretic peptides or reduce their degradation.
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