Introduction

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Early studies showed that endothelin-1 (ET-1) can directly affect the contractile state of cardiac tissues. Positive inotropic effects to ET-1 have consistently been observed in atrial tissues from many species, including humans (Davenport et al., 1989; Moravec et al., 1989; Meyer et al., 1996), that were greater than those observed in corresponding ventricular tissues, including those from humans (Davenport et al., 1989; Moravec et al., 1989). It is generally thought that ET-1 combines with specific cell surface endothelin receptors that mediate its effects. Radioligand binding, quantitative receptor autoradiography, polymerase chain reaction, and in situ hybridization studies showed the presence of two receptor subtypes, ET_A and ET_B, in human cardiac tissues (Bax et al., 1993; Molenaar et al., 1993). Since then, there have been increasing numbers of reports of ET receptors that do not fit the ET_A or ET_B receptor classification in a variety of tissues (Bax and Saxena, 1994). Using a single concentration of the ET_A selective antagonist BQ123 [cyclo(D-Trp-D-Asp-Pro-D-Val-Leu)] (200 nM), Meyer et al. (1996) suggested that ET_A receptors mediated the inotropic effects of ET-1 in human right atrium. The aim of our study was to use a wider range of ET receptor agonists and antagonists to more fully characterize the receptors that are responsible for the inotropic effects of agonists in human right atrium. We were interested to know whether in addition to the reported ET_A cardiac receptor (Meyer et al., 1996), ET_B and/or non-ET_A/ET_B receptors were also responsible for the cardiostimulant effects of ET receptor agonists.

There is evidence for a direct arrhythmogenic effect of ET-1. Arrhythmias following ischemia-reperfusion in rat hearts have been shown to be caused by ET-1 (Brunner and Kukovetz, 1996) and procedures that lower ET-1 levels (angiotensin-converting enzyme inhibition, bradykinin) or block the ET-1 receptor {SB 209670 [(+)-(1S,2R,3S)-5-propoxy-1-(3,4-methylenedioxyphenyl)-3-(2-carboxymethoxy-4-methoxyphenyl)indane-2-carboxylic acid disodium)]} prevent ischemia-reperfusion arrhythmias (Brunner and Kukovetz, 1996). In AT-1 cells, an atrial tumor myocyte cell line derived from transgenic mice, ET-1 caused the appearance of spontaneous diastolic calcium oscillations in both electrically driven and quiescent cells (Jiang et al., 1996). We were interested to know whether ET-1 was arrhythmogenic in human atrial tissue. For this purpose, we used a model previously described by Kaumann and colleagues (Kaumann and Sanders, 1993, 1994; Sanders et al., 1996) in human right atrial tissue in which it was shown that stimulation of Gs protein-coupled receptors, ₁- and ₂-adrenoceptors, 5-hydroxytryptamine (5-HT)₄- and H₂-receptors cause pacing frequency-dependent arrhythmic contractions.

	Materials and Methods

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Patients. Human right atrial appendages were obtained from patients undergoing coronary artery bypass grafting, aortic valve replacement, or combined aortic valve replacement-coronary artery bypass grafting at the Royal Melbourne Public and Private Hospitals and The Prince Charles Hospital. Human right and left atria and right ventricle from failing hearts were obtained from patients undergoing cardiac transplantation at the Alfred Hospital. Etiologies of heart failure were ischemic cardiomyopathy (n = 4), ventricular septal defects (n = 2), adriamycin-induced toxicity, hypertrophic cardiomyopathy, alcohol induced-cardiomyopathy, and Marfans syndrome (all n = 1) with New York Heart Association classification ranging from III to IV.

1

Preparation of Tissues. After surgical removal, atrial tissues were placed immediately into ice-cold preoxygenated (95% O₂/5% CO₂) modified Krebs' solution containing (125 mM Na⁺, 5 mM K⁺, 2.25 mM Ca²⁺, 0.5 mM Mg²⁺, 98.5 mM Cl, 0.5 mM SO₄², 32 mM HCO₃, 1 mM HPO₄², 0.04 mM EDTA). The endomyocardial layer of the right ventricular free wall containing trabeculae was rapidly dissected in modified Krebs' solution at the surgical theater. Tissues were then transported to the laboratory where atrial strips containing intact trabeculae (<1 mm in diameter) and ventricular trabeculae (width 1.0 ± 0.1 mm; cross-sectional area 1.6 ± 0.3 mm²; n = 15) were dissected under continuous oxygenation. Atrial strips and ventricular trabeculae were often mounted in pairs in 50-ml tissue baths containing modified Krebs' solution at 37°C, attached to strain-gauge transducers, and driven with square-wave pulses (1 Hz, 5 ms-duration; just over threshold voltage). A length tension curve was constructed to determine the length at which maximal contractions occurred (L_max) and atrial strips were adjusted to 50% L_max, whereas ventricular trabeculae were maintained at L_max. The incubation medium was exchanged with modified Krebs' solution containing in addition 15 mM Na⁺, 5 mM fumarate, 5 mM pyruvate, 5 mM L-glutamate, and 10 mM glucose. Tension of atrial strips and ventricular trabeculae were recorded on eight-channel Watanabe recorders.

Effects on Human Atrial and Ventricular Contractile Force. Tissues were incubated with 300 nM CGP 20712A [2-hydroxy-5(2-((2-hydroxy-3-(4-((1-methyl-4-trifluoromethyl) 1H-imidazole-2-yl) -phenoxy) propyl) amino) ethoxy)-benzamide monomethane sulfonate] and 50 nM ICI 118,551 [erythro-D,L-1(7-methylindan-4-yloxy)-3-isopropylaminobutan-2-ol] for at least 60 min to block ₁- and ₂-adrenoceptors, respectively. In some experiments, ET receptor antagonists also were added and equilibrated with atrial tissues from nonfailing hearts for at least 60 min. Cumulative concentration-effect curves to ET receptor agonists in the absence or presence of antagonists were determined by sequential administration of agonist to the tissue bath in amounts that increased the total concentration by 1/2 log unit.

Human Coronary Arteries. After surgical removal of the heart from one patient with idiopathic dilated cardiomyopathy, large human epicardial coronary arteries were dissected, placed immediately into ice-cold preoxygenated modified Krebs' solution (described above), transported to the laboratory, cleared of fat and connective tissue, and set up in the organ bath as described in Kaumann et al. (1994). Briefly, the endothelium was removed by gently rubbing the lumen with paper towel. Helicoidal strips were mounted in the same apparatus used for cardiac muscle and resting force was adjusted to ~30 mN at the beginning of the experiment. The incubation medium was exchanged as described above. Tissues were allowed to stabilize for 3 h before addition of 90 mM KCl followed by washout and readdition of KCl 2 h later. Following washout, 10 µM BQ123 or 1 µM A-127722 [trans-trans-2-(4-methoxyphenyl)-4-(1,3-benzodioxol-5-yl)-1((N,N-dibutylamino)carbonylmethyl)pyrolidine-3-carboxylic acid] was added to some organ baths and incubated for 2 h before commencement of a cumulative concentration-effect curve to ET-1.

Arrhythmia Studies. The ability of ET-1 and sarafotoxin S6c to cause arrhythmic contractions in human right atrium was determined in a staircase model as described in detail for stimulation of human atrial ₁- and ₂-adrenoceptors (Kaumann and Sanders, 1993), 5-HT₄ receptors (Kaumann and Sanders, 1994), and H₂-receptors (Sanders et al., 1996). Briefly, atrial tissues of nonfailing hearts from patients undergoing coronary artery bypass grafting, aortic valve replacement or a combination of both procedures were set up as described above for the determination of cumulative concentration-effect curves and incubated with 300 nM CGP 20712A and 50 nM ICI 118,551 to block ₁- and ₂-adrenoceptors with or without ET receptor antagonists for at least 60 min. The pacing frequency was then set at 0.1 Hz and reset at 0.2, 0.5, 1, and 2 Hz at 2-min intervals (forward staircase). The staircase was then run backward (2-0.1 Hz), with 2-min intervals during which the stimulator was turned off (rest periods) between each 2-min stimulation period. The pacing rate was then set at 1 Hz and on stabilization, ET-1 or sarafotoxin S6c was added to the tissue bath. After equilibration, the backward staircase (2-0.1 Hz) was established with 2-min rest periods between each frequency repeated in the presence of ET-1 or sarafotoxin S6c.

Experimental Design and Analysis. Changes in contractile force above basal were calculated. Where two or more strips from one patient were used, mean changes in contractile force above basal values were calculated for each concentration. For agonists, pEC₅₀ (log concentration causing 50% of the maximal response) and maximal responses, expressed as a percentage of the response to 9.25 mM Ca²⁺, were measured.

Evaluation of Adsorption of ET-1, BQ123, and SB 209670 onto Components of Organ Bath-Tissue Holder Apparatus. We were concerned about the possibility of adsorption of peptides and SB 209670 onto components of our organ bath-tissue holder apparatus and therefore experiments were carried out to assess the extent of adsorption. ET-1 (6 nM), together with tracer ¹²⁵I-ET-1 (40 pM) were added to the organ bath-tissue holder apparatus in the absence of tissue. Samples were taken periodically up to 4 h after addition of ET-1 and counted in a gamma counter (Packard Model B5424). There was a 35 ± 12% (n = 4) loss after 2 h with no further loss after 4 h. In other experiments cumulative concentration-effect curves were established to ET-1 in right atrial trabeculae under conditions to reduce adsorption in which the glassware had been siliconized and 0.05% BSA added to the incubation solution. With trabeculae from the same patient, concentration-effect curves also were constructed in the absence of both siliconized glassware and BSA. There was no difference in ET-1 concentration-effect curves [pEC₅₀ (control, n = 3)  pEC₅₀ (siliconized glassware + BSA, n = 3) = 0.21 ± 0.13 log units, n = 3 hearts]. There was also no difference in pEC₅₀ values for ET-1 in the presence of 10 µM BQ123 with siliconized glassware and BSA, [pEC₅₀ (control, n = 5; Fig. 1)  pEC₅₀ (siliconized glassware + BSA, n = 2) = 0 log units]. Therefore, experiments were carried out in the absence of BSA and without siliconizing glassware. We also determined whether SB 209670 was adsorbed and used 30 nM SB 209670 together with tracer 1 nM [³H]SB 209670. Samples were counted in a liquid scintillation counter (Wallac System 1400). There was no loss of SB 209670 after 4 h.

View larger version (17K): [in this window] [in a new window]   Fig. 1.   Effects of ET receptor antagonists against the positive inotropic responses to ET-1 in human right atrium from nonfailing hearts. Shown are concentration-effect curves to ET-1 in the absence () or presence of 10 µM () or 100 µM bosentan () (a), 10 µM BQ123 () (b), 1 µM A-127722 (), 10 µM Ro-468443 (), 10 µM Ro-468443 + 1 µM A-127722 () (c). Positive inotropic effects were expressed as a percentage of the response obtained to 9.25 mM Ca2+. Values shown are means ± S.E. (vertical lines) where larger than symbol size (n = 4-17 patients).

Statistics. Comparisons of pEC₅₀ and maximal response values between groups of data were performed by Student's t test (unpaired). Values are expressed as means ± S.E. The significance of differences in the incidence of arrhythmic contractions between -blocked and non--blocked tissues was assessed with the Fisher's exact probability test. Student's t test and Fisher's exact probability test were performed with InStat (GraphPad Software, verson 2.0). P < .05 was used as the limit for statistical significance.

Drugs. SB 209670 and [³H]SB 209670 were gifts from Dr. Eliot Ohlstein (SmithKline Beecham Pharmaceuticals, King of Prussia, PA). Bosentan {(4-tert-butyl-N-[6-(2-hydroxy)-ethoxy)-5-2(2-methoxyphenoxy)-2,2'-bipyrimidin-4-yl]-benzene sulfonamide} and Ro 46-8443 {(R)-4-tert-butyl-N-[6-(2,3-dihydroxy-propoxy)-5-(2-methoxy-phenoxy)-2-(4-methoxy-phenyl)-pyrimidin-4-yl]-benzenesulfonamide} were gifts from Dr. Martine Clozel (F. Hoffman-La Roche Ltd., Basel, Switzerland). A-127722 was a gift from Dr. Terry J. Opgenorth (Abbott Laboratories, Abbott Park, IL). ET-1 (human), sarafotoxin S6c, ET-2 (human), ET-3 (human), BQ123, and BQ788 [N-cis-2,6-dimethylpiperidinocarbonyl-L-Me-Leu-D-Trp(COOMe)- D-Nle.ONa] were purchased from AUSPEP, South Melbourne, Australia. CGP 20712A was a gift from Alexandra Sedlacek, Ciba-Geigy AG, Basel, Switzerland; ICI 118,551 from Zeneca, Wilmslow, Cheshire, UK; verapamil from Sigma Chemical Co., Castle Hill, NSW, Australia; and ¹²⁵I-ET-1 from Amersham, Baulkham Hills, NSW, Australia.

	Results

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Positive Inotropic Effects of ET Receptor Agonists. ET-1 caused concentration-dependent positive inotropic effects in human right atrial trabeculae (Fig. 2). ET-1 caused slowly developing, sustained positive inotropic effects that were usually preceded by small initial transient negative inotropic effects (data not shown). There was no difference in the potency or maximal positive inotropic effect of ET-1 in atrial tissues taken from patients treated with or without ₁-adrenoceptor antagonists before coronary artery bypass grafting surgery (CABG), aortic valve replacement (AVR), or combined CABG/AVR (P > .05, Table 2).

View larger version (22K): [in this window] [in a new window]   Fig. 2.   Modulation of contractile force (a) and time to reach 50% relaxation (b) by ET-1. Shown in (a) are the positive inotropic effects of ET-1 in right atrial strips taken from patients undergoing CABG, AVR, or combined CABG/AVR treated with () or without () -adrenoceptor antagonists before surgery or right atrial (), left atrial (), and right ventricular () trabeculae from hearts in terminal heart failure. Shown in (b) are the effects of ET-1 on the time to reach 50% relaxation in right atrial trabeculae from patients undergoing CABG () and right ventricular trabeculae from explanted hearts (). Positive inotropic effects were expressed as a percentage of the response obtained to 9.25 mM Ca2+. Values shown are means ± S.E. (vertical lines) where larger than symbol size (n = 3-28 patients).

Effects of ET-1 on Time Course of Contraction. ET-1 caused a concentration-dependent prolongation of the time to reach 50% relaxation (t_50%) in atrium (Fig. 2). In right ventricular trabeculae from explanted hearts, the cumulative addition of ET-1 caused no change in t_50% (Fig. 2).

Positive Inotropic Effects of ET-2, ET-3, and Sarafotoxin S6c in Human Right Atrium. ET-2, ET-3, and sarafotoxin S6c caused concentration-dependent positive inotropic effects in human right atrium (Fig. 3). The isoforms of ET-1, ET-2, and ET-3 had similar potencies and caused similar maximal positive inotropic effects (Table 3); however, it was noticeable that in comparision to ET-1 and ET-2, ET-3 caused smaller effects at concentrations up to 6 nM (Fig. 3). Sarafotoxin S6c was more potent than the ET isoforms but caused a smaller maximal positive inotropic effect (Fig. 3; Table 3).

View larger version (16K): [in this window] [in a new window]   Fig. 3.   Comparison of positive inotropic effects of ET-1 (), ET-2 (), ET-3 (), and sarafotoxin S6c () in human right atrium from patients undergoing CABG. Positive inotropic effects were expressed as a percentage of the response obtained by raising the Ca2+ concentration to 9.25 mM at the end of the experiment. Values shown are mean ± S.E. (vertical lines) where larger than symbol size (n = 6-53 patients).

Effects of Antagonists on Positive Inotropic Effects of ET-1 and Sarafotoxin S6c. The positive inotropic effects of ET-1 were resistant to antagonism by 10 µM bosentan, however a higher concentration, 100 µM, caused a rightward shift of the ET-1 concentration-effect curve (Fig. 1). The ET_A selective compounds BQ123 (10 µM) and A-127722 (1 µM) caused small shifts of the lower part of the concentration-effect curve of ET-1 (Fig. 1). The ET_B selective compounds Ro 46-8443 (10 µM; Fig. 1) and BQ788 (1 µM; n = 1; data not shown) did not block but actually caused leftward shifts of ET-1 concentration-effect curves. Coincubation of tissues with the ET_A and ET_B selective antagonists 1 µM A-127722 and 10 µM Ro 46-8443 had no additional effect on the cumulative concentration-effect curve to ET-1 compared with incubation with 1 µM A-127722 alone (Fig. 1).

View larger version (19K): [in this window] [in a new window]   Fig. 4.   Antagonism of the positive inotropic effects of ET-1 (a) and sarafotoxin S6c (b) by SB 209670 in right atrium from nonfailing hearts. Shown are concentration-effect curves to agonists in the absence () or presence of 30 nM (), 100 nM (), 300 nM (), 1 µM (), or 3 µM () SB 209670. A Schild plot (c) was constructed for SB 209670 with 100 nM, 300 nM, and 1 µM for experiments with ET-1 (), which gave a slope value of 0.80 ± 0.10 with a pKB value of 7.0 ± 0.1. The higher concentration, 3 µM SB 209670, was not used in the pKB calculation because it caused nonlinearity of the Schild plot (dotted line and ) and was not completely surmounted by 2 µM ET-1, the highest concentration used. In (a), the ET-1 concentration-effect curve in the absence of SB 209670 is a mean control curve. Concentration-ratios used for the Schild-plot (c) were obtained from individual experiments. A Schild plot also was constructed for SB 209670 against sarafotoxin S6c () with slope 0.94 ± 0.07 and pKB value of 7.9 ± 0.1. The mean sarafotoxin S6c concentration-effect curve in the absence of SB 209670 is a mean control curve. Concentration-ratios used for the Schild-plot were obtained from individual experiments. Positive inotropic effects were expressed as a percentage of the response obtained to 9.25 mM Ca2+. Values shown are means ± S.E. (vertical lines) where larger than symbol size (n = 5-17 individual experiments).

View larger version (15K): [in this window] [in a new window]   Fig. 5.   Lack of antagonism by sarafotoxin S6c against the positive inotropic effects of ET-1 in right atrium from nonfailing hearts. Shown are concentration-effect curves to ET-1 in the absence () or presence of 200 nM sarafotoxin S6c (). Positive inotropic effects were expressed as a percentage of the response obtained to 9.25 mM Ca2+. Values shown are means ± S.E. (vertical lines) (n = 4 individual experiments).

Blockade of Effects of ET-1 by BQ123 and A-127722 in Human Coronary Artery. In view of the small blocking effect of BQ123 and A-127722 in right atrium, we tested their ability to block ET-1-mediated contraction of human coronary arteries from one patient with identical tissue bath equipment as for right atrium. ET-1 caused concentration-dependent increases in contractile force that were blocked by 10 µM BQ123 (pK_B 6.50) and 1 µM A-127722 (pK_B 7.63) (Fig. 6).

View larger version (16K): [in this window] [in a new window]   Fig. 6.   Antagonism of the contractile effects of ET-1 by BQ123 and A-127722 in human epicardial coronary arteries. Shown are concentration-effect curves to ET-1 in the absence () or presence of 10 µM BQ123 () or 1 µM A-127722 (). Values shown are means ± S.E. (vertical lines) where larger than symbol size (n = 4-6 individual arterial helicoidal strips from one patient).

Arrhythmogenic Effects of ET-1 and Sarafotoxin S6c in Right Atrium from Nonfailing Hearts. ET-1 (20 nM) caused arrhythmic contractions in right atrial trabeculae that were consistently prevented (13/13 trabeculae from 10 patients) by preincubation of tissues with 10 µM SB 209670 (Fig. 7) but not by 10 µM BQ123 (four trabeculae from three patients) or 1 µM A-127722 (four trabeculae from four patients) (Fig. 8). Sarafotoxin S6c (20 nM) also caused arrhythmic contractions that were prevented (7/7 trabeculae from 4 patients) by preincubation with 10 µM SB 209670 (Fig. 9). ET-1-induced arrhythmic contractions in atria from non--blocker-treated and -blocker-treated patients were concentration and pacing-frequency dependent (Fig. 10). The incidence of ET-1-induced arrhythmic contractions in tissues taken from patients pretreated with or without -adrenoceptor antagonists before surgery was investigated further with 6 nM ET-1. There was a higher incidence of ET-1-induced arrhythmic contractions in tissues taken from patients pretreated with -adrenoceptor antagonists at all pacing rates except at 2 Hz than in atria from non--blocker-treated patients (Fig. 11). We also investigated whether spontaneous contractions could be induced by 100 nM ET-1 in nonstimulated right atrial tissue. Trabeculae were set up and set at 50% L_max and the stimulator turned off. Spontaneous contractions were observed in seven of nine trabeculae from three patients undergoing coronary artery bypass surgery (Fig. 11). Verapamil (100 nM) reduced, but did not completely reverse, spontaneous contractions in four of four trabeculae from two patients (Fig. 12).

View larger version (72K): [in this window] [in a new window]   Fig. 7.   ET-1 (20 nM) -evoked arrhythmic contractions and prevention by 10 µM SB 209670. Shown are two atrial strips from a 72-year-old female patient undergoing CABG treated with frusemide, glyceryl trinitrate, simvastatin, lisinopril, amiodarone, and prednisolone before surgery. Top and bottom strips were treated identically except that the bottom strip was preincubated with 10 µM SB 209670 for 60 min before commencement of the experimental protocol. A forward staircase (0.1-2 Hz) was run (a) followed by a backward staircase (2-0.1 Hz) with 2-min intervals between changes in frequency (b). ET-1 was added to both tissues (arrows) (c) and after equilibration another backward staircase with 2-min intervals was run (d). ET-1 induced arrhythmic contractions in the upper strip were not observed in the presence of SB 209670 (bottom strip). There were no arrhythmic contractions in another time-matched strip that was not treated with ET-1 or SB 209670 (data not shown). Solid bars indicate 2-min periods in which the stimulator was turned off.

View larger version (90K): [in this window] [in a new window]   Fig. 8.   Persistence of ET-1-induced arrhythmic contractions in the presence of ETA receptor blockade with BQ123 or A-127722. Shown are four right arial strips from a 70-year-old female patient undergoing coronary artery bypass surgery treated with amlodipine, glyceryl trinitrate, isosorbide mononitrate, and atenolol. Tissue (b) was incubated with 10 µM BQ123 and tissue (c) was incubated with 1 µM A-127722. Forward (0.1-2 Hz) and backward staircases (2-0.1 Hz) were carried out as in Fig. 7 during which time no arrhythmic contractions were observed (data not shown). Shown is the reverse staircase (2-0.1 Hz) with 2-min intervals between changes in frequency of stimulation following equilibration with 20 nM ET-1 (tissues a-c) in the absence (a) or presence of BQ123 (b) or A-127722 (c). Tissue (d) was a time-matched control. ET-1-induced arrhythmic contractions were maintained in the presence of BQ123 or A-127722. Solid bars show time periods when the stimulator was turned off.

View larger version (14K): [in this window] [in a new window]   Fig. 9.   Sarafotoxin S6c-evoked arrhythmic contractions and prevention by SB 209670. Shown are two atrial strips from a 78-year-old male patient undergoing CABG treated with atenolol, amlodipine, quinapril, and isosorbide mononitrate before surgery. Top and bottom strips were treated identically except that the bottom strip was preincubated with 10 µM SB 209670 for 60 min before commencement of the experimental protocol. The experiment was carried out as in Fig. 7 with forward and backward staircases in the absence of sarafotoxin S6c (data not shown). Shown is the backward staircase in the presence of sarafotoxin S6c (arrows) with 2-min intervals between changes in frequency (solid bars) when the stimulator was turned off. SB 209670 (10 µM) prevented 20 nM Sarafoxin S6c-induced arrhythmic contractions.

View larger version (12K): [in this window] [in a new window]   Fig. 10.   Concentration (0.6 , 2.0 , 6.0 , and 20 nM ) and frequency (2, 1, 0.5, 0.2, and 0.1 Hz) dependence of ET-1-induced arrhythmic contractions in human right atrial strips from patients treated without (a) or with -adrenoceptor antagonists (b) before CABG surgery. ET-1-induced arrhythmic contractions depended both on concentration and pacing frequency. Values shown were obtained from n = 4 to 39 tissues from 4 to 23 patients.

View larger version (16K): [in this window] [in a new window]   Fig. 11.   Incidence of arrhythmic contractions induced by 6 nM ET-1 in atrial tissues taken from patients undergoing CABG surgery. Open columns show the incidence in tissues taken from patients not receiving -adrenoceptor antagonists (23 strips from 12 patients); open plus closed columns show the incidence in tissues from patients receiving -adrenoceptor antagonists before surgery (35 strips from 19 patients). Values beneath the columns indicate pacing frequency (Hz) immediately before the rest period in which arrhythmic contractions were observed. Values above the columns are P values obtained with the Fisher's exact probability test to compare the incidence of arrhythmic contractions in -blocked and non--blocked tissues.

View larger version (22K): [in this window] [in a new window]   Fig. 12.   ET-1-induced arrhythmic contractions in a quiescent atrial trabeculum taken from a 63-year-old male patient undergoing CABG. Drug therapy before surgery included atenolol, enalapril, simvastatin, and glyceryl trinitrate. The trabeculum was set up at 50% Lmax and the stimulator turned off. ET-1 (100 nM) was added at time zero min, first arrow. Spontaneous contractions appeared after a 7-min ET-1 incubation. Verapamil (100 nM) was added (second arrow, time 60 min), which reduced, but did not abolish spontaneous contractions that were still evident at time 120 min.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

This study addresses mainly the question of which ET receptors mediate the positive inotropic effects of several agonists in human atrial tissues. We also show that ET-1 causes arrhythmic contractions in right atrial tissues. Only a minor component of the atrial positive inotropic effects of ET-1 was mediated through ET_A receptors but mostly through non-ET_A and non-ET_B receptors. Sarafotoxin S6c elicited positive inotropic effects in atrium through a second distinct receptor population. The ET-1-evoked arrhythmias were not mediated through ET_A receptors.

Characteristics of Positive Inotropic Effect of ET-1. ET-1 caused concentration-dependent positive inotropic effects in human cardiac tissues with the maximal effect in right atrium > left atrium = right ventricle. Both Davenport et al. (1989) and Moravec et al. (1989) showed that ET-1 was less effective at causing increases in contractile force in human right ventricle compared with right atrium. Davenport et al. (1989) also reported right ventricular trabeculae that did not respond to ET-1. Interestingly, there was little or no evidence of regulation of the mechanisms responsible for causing positive inotropic effects in human right atrium because ET-1 had similar potencies and maximal effects in atria from patients chronically treated with or without -adrenoceptor antagonists and patients with terminal heart failure. This is unlike the responses caused by stimulation of some human right atrial Gs protein-coupled receptors, ₂-adrenoceptors (Hall et al., 1990), 5-HT₄- (Sanders et al., 1995), and H₂-receptors (Sanders et al., 1996), and to a minor extent ₁-adrenoceptors (Molenaar et al., 1997) that are sensitized in atrial tissues taken from patients treated with -adrenoceptor antagonists. Furthermore, maintenance of positive inotropic effects of ET-1 in terminal heart failure is unlike ₂-adrenoceptor-mediated responses for ()-epinephrine (in the presence of 300 nM CGP 20712A) in human right atrium (pEC₅₀ heart failure 6.7 ± 0.2, n = 6; coronary artery bypass grafting 7.2 ± 0.1, n = 12; P = .01; unpublished data) and ₁-adrenoceptor responses in ventricle (Bristow et al., 1986).

Agonist Effects of ET Isoforms in Human Right Atrium. ET-1 (pEC₅₀ = 8.0), ET-2 (pEC₅₀ = 8.1), and ET-3 (pEC₅₀ = 7.7) had similar potencies in human right atrium from nonfailing hearts; however, unlike ET-1 and ET-2, ET-3 had little effect at lower (up to 6 nM) concentrations. Sarafotoxin S6c was more potent (pEC₅₀ = 8.6). These agonist potencies are not consistent with involvement of only an ET_A receptor for which characteristically, the rank order of potency is ET-1 = ET-2 ET-3 sarafotoxin S6c or ET_B receptor where ET-1 = ET-2 = ET-3 = sarafotoxin S6c (Panek et al., 1992; Davenport and Masaki, 1998) or putative ET_C receptor where ET-3 > ET-1 (Douglas et al., 1995).

ET Receptor Heterogeneity: Minor Role of ET_A Receptors. Several ET receptor antagonists were used to characterize the receptors responsible for mediating the cardiostimulant effects of ET-1 and sarafotoxin S6c in right atria from nonfailing hearts. In previous functional studies (Clozel et al., 1994), the nonpeptide antagonist bosentan (formally Ro 47-0203) was reported to competitively antagonize the effects of ET-1 in rat aorta with a pA₂ value of 7.3, rabbit superior mesenteric artery (ET_B, pA₂ = 6.7) and rat trachea (ET_B, pA₂ = 5.9). A concentration of bosentan (10 µM) that would have been expected to block the effects of ET-1 in human right atrium if the receptors were identical with those described in the study of Clozel et al. (1994) was ineffective. Furthermore, it has recently been shown that 3 µM bosentan causes a 1 log rightward shift of the concentration-positive effect curve to ET-1 in human left ventricular strips (Pieske et al. 1999), suggesting that human atrial ET receptors differ from human ventricular receptors. We observed only a 1/2 log rightward shift of the ET-1 concentration-effect curve with 100 µM bosentan. Because bosentan is a relatively nonselective blocker of ET_A and ET_B receptors it appears that the human atrial receptors that mediate positive inotropic effects of ET-1 are mostly neither of ET_A nor ET_B nature.

Arrhythmic Contractions Induced by ET-1. ET-1 and sarafotoxin S6c caused frequency-dependent arrhythmic contractions in our model of human right atrium and also in nonstimulated right atrium. Arrhythmic contractions were prevented by the ET receptor antagonist SB 209670.

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Possible Clinical Relevance. It has been shown and argued that atenolol is likely to be washed out of cardiac tissues in our protocol (Hall et al., 1990), as is metoprolol (A.J.K. and P.M., unpublished data). Therefore, the ET-1-evoked arrhythmias in our tissues obtained from patients treated with -adrenoceptor antagonists may be clinically relevant to the -adrenoceptor blockade withdrawal syndrome (Prichard et al., 1983). It is possible that ET-1 may contribute to transient postcardiac surgical supraventricular arrhythmias together with other arrhythmic agents. Plasma levels of ET-1 are increased as a result of open-chested cardiac surgery (Knothe et al., 1996; Te Velthuis et al., 1996) and remain high for at least 1 day postoperatively (Knothe et al., 1996). This may be relevant in terms of the onset of atrial fibrillation that can occur on the day of surgery, but the peak incidence is 2 days after surgery (Fuller et al., 1989; Kalman et al., 1995). Although plasma levels of ET-1 are not high enough to directly stimulate human cardiac muscle, it would be more likely that locally synthesized and abluminally released ET-1 (Wagner et al., 1992) would directly stimulate cardiac muscle. It remains to be determined whether cardiac ET receptor antagonists will be of value for this disorder. It is interesting to note that SB 209670 prevented exogenous ET-1-induced fatal ventricular arrhythmias in an in vivo canine model, suggesting a broader therapeutic spectrum of antiarrhythmic activity (Douglas et al., 1998).

Conclusions. The following conclusions can be drawn from the present study. First, ET-1 increases contractile force in both human atrium and ventricle. Sarafotoxin S6c causes positive inotropic effects in atrium but not in ventricle. Most atrial ET receptors activated by ET-1 are of non-ET_A nature and differ from atrial receptors activated by sarafotoxin S6c. Second, ventricular ET receptors are different from atrial receptors. Third, ET-1 and sarafotoxin S6c mediate pacing frequency-dependent atrial arrhythmias that are prevented by SB 209670. And fourth, ET-1-induced arrhythmias were mediated through non-ET_A receptors.