Endothelin (ET) has been first characterized as a potent vasoconstrictor 21-amino-acid peptide (1), produced in vascular endothelial cells. Additional studies demonstrated that ET can also exert positive inotropic effects in the heart of several species (2), including human heart (3-7). Furthermore, it has been shown that ET is involved in the pathophysiology of chronic heart failure and that ET-receptor blockade improves cardiac function in experimental models of heart failure (8-10). Thus, ET-1 plasma levels were found to be elevated in clinical and in experimental heart failure (10-12). ET-1 has been shown to interact with the activation of the renin-angiotensin system and sympathetic nervous system in chronic heart failure (8,13). However, the functional role of ET and its crosstalk with other mediators in human cardiac tissue remains rather unclear.
The physiologic effects of ET-1 are mediated by two subtypes of ET receptors designated ETA and ETB receptors (14). Both ETA and ETB receptors coexist in the human heart (7,15-18). ET receptors in the heart of various species, including humans, couple via Gq/11 protein to the phospholipase C/inositol trisphosphate/diacylglycerol (PLC/IP3/DAG) system as the major signaling pathway (2). However, recent studies have demonstrated that, in rat cardiomyocytes (19) and human right atrium (17,20), ET-1 can also inhibit adenylylcyclase, presumably by coupling to Gi protein. Conversely, in human ventricular myocardium, ET-1 did not inhibit adenylylcyclase (17). The functional consequence of these differential effects of ET-1 on adenylylcyclase in atrium versus ventricle is not known at present. However, in human right atrium, it has been found that the positive inotropic effect of ET-1 is preceded by a transient negative inotropic effect (6) that could be due to the inhibition of adenylylcyclase. In that case, one should expect that, in ventricular myocardium, the positive inotropic effect of ET-1 is not preceded by a negative inotropic effect. Thus, the aim of the present study was to test this hypothesis. For this purpose, we studied in isolated electrically driven human right atrial and left ventricular preparations the time course of the effects of ET-1 on force of contraction in the presence or absence of the nonselective ETA- and ETB-receptor antagonist bosentan (21), the selective ETA-receptor antagonist BQ 123 (22), and the selective ETB-receptor antagonist BQ 788 (23). In addition, we wanted to find out whether the negative inotropic effect of ET-1 present in human right atrium might be altered in chronic heart failure.
All experiments were approved by the local ethics committee and are in accordance with the German laws. Right atrial and left ventricular tissues were obtained from the local department of cardiothoracic surgery. We investigated right atrial tissue from nonfailing hearts, which were obtained during coronary bypass grafting, and right atrial and left ventricular tissue from failing hearts with terminal heart failure (NYHA IV) during transplantation.
Patients and surgical procedure
We investigated right atrial appendages from 32 patients without apparent heart failure undergoing coronary bypass grafting (age, 62 ± 3 years; sex, 22 men, 10 women; body weight, 74.6 ± 2.7 kg). In addition, we examined right atrial appendages and left ventricular tissue from eight explanted hearts with terminal heart failure (NYHA IV; age, 52.3 ± 2.9 years; sex, six men, two women; body weight, 81 ± 5 kg).
None of the patients had been treated with sympathomimetics for ≥3 weeks before surgery. However, patients in the group without apparent heart failure were treated with nitrates, calcium antagonists, β-adrenoceptor antagonists, and angiotensin-converting enzyme inhibitors (alone or in combination); patients with terminal heart failure received treatment with angiotensin-converting enzyme inhibitors, digitalis, and diuretics.
Premedication typically consisted of 2 mg flunitrazepam given orally the evening before or on the morning of surgery. Anesthesia during operation was performed using modified neurolept anesthesia with flunitrazepam and fentanyl and controlled ventilation with a 1:1 mixture of oxygen and N2O with addition of up to 1.0% (vol/vol) isoflurane. Pancuronium was used for muscle relaxation. In all patients, right appendages were removed during installation of the cardiopulmonary bypass. Promptly after excision, all specimens were placed in ice-cold Tyrode's solution (composition later). Explanted hearts were placed in a cardioplegic solution (Bretschneider-HTK solution). Immediately after receiving the hearts, we removed right atrial appendages and ventricular strips and placed them in ice-cold Tyrode's solution.
Measurement of contractile force
Preparation of tissue usually started 15-20 min after surgical removal in oxygenated Tyrode's solution at room temperature. Atrial and ventricular preparations (strips of 1 mm diameter and 4-5 mm length) were placed into a 10-ml organ bath containing Tyrode's solution of the following composition (mM): NaCl 119.8, KCl 5.4, CaCl2 1.8, MgSO4 1.05, NaH2PO4 0.42, NaHCO3 22.6, EDTA 0.05, ascorbic acid 0.28, D-glucose 5.05, equilibrated with 95% O2 and 5% CO2, at 37°C. The preparations were electrically driven (1 Hz) with rectangular pulses (5 ms) 20% above threshold (3-12 V; mean, 8V) (Stimulator II; Hugo Sachs Elektronik, Hugstetten, Germany). After prestretching the preparation (4.9 mN), the developed force was recorded by a strain gauge on a Hellige recorder (Hellige, Freiburg, Germany). After 60 min of equilibration, preparations were incubated with 1 μM forskolin or vehicle for 30 min. Thereafter, ET-1 (0.1 μM) was added to the bath solution, and the response was recorded for 30 min. For antagonist experiments, preparations were incubated with 1 μM forskolin and the antagonist (1 μM BQ 123 or 1 μM BQ 788 or 1 μM bosentan) for 30 min. Thereafter, 0.1 μM ET-1 was added. In additional experiments on atrial tissue, the effect of ET-1 (0.1 μM) was assessed without pretreatment with forskolin. Furthermore, the positive inotropic effect of ET-1 was investigated more in detail in right atria from nonfailing hearts: without forskolin prestimulation, ET-1 (10−10 to 3 × 10−7M) was applied in the absence or presence of either BQ 123 (10 μM) or BQ 788 (10 μM). Force of contraction was measured 30 min after ET-1 application.
All values are presented as means ± SEM of n patients. Experimental data were fitted and analyzed by computer-supported iterative nonlinear regression analysis using the InPlot program (GraphPAD Software). Statistical significance of differences was analyzed by unpaired two-tailed Student's t test or, if appropriate, by repeated-measures analysis of variance (ANOVA) followed by t test using Bonferroni corrections for multiple comparisons. A value of p < 0.05 was considered to indicate a significant difference. The statistical evaluation was performed using the InStat Program (GraphPAD Software, San Diego, CA, U.S.A.).
Forskolin was purchased from Sigma (Deisenhofen, Germany); ET-1, BQ 123, and BQ 788 were obtained from Alexis (Grünberg, Germany). Bosentan (sodium salt) was a gift from Dr. M. Clozel (Hoffmann-La Roche Ltd., Basel, Switzerland). The other chemicals were from Merck (Darmstadt, Germany). All chemicals were of the purest commercially available grade.
Effects in atrium
Application of 1 μM forskolin led to an increase in contractile force from 2.7 ± 0.4 to 4.5 ± 0.5 mN in atria from nonfailing hearts (n = 15) and from 2.75 ± 0.7 to 4.9 ± 0.9 mN in atria from failing hearts (n = 8).
Exposure of the forskolin-prestimulated atria to 0.1 μM ET-1 resulted in a biphasic effect. In the first phase, a transient but distinct negative inotropic effect was observed, which was followed by a small, but sustained positive inotropic effect, as can be seen from the original registration in Fig. 1. This negative inotropic effect was significantly more pronounced in atria from failing hearts [decrease by 22.7 ± 3% (atria from failing hearts) vs. 14.3 ± 2.7% (atria from nonfailing hearts); p < 0.05; Fig. 2]. In addition, the negative inotropic effect of ET-1 was significantly prolonged to 9 ± 1 min in atria from failing hearts as compared with 5 ± 1 min in atria from nonfailing hearts (p < 0.05). The time to the peak negative inotropic effect, however, was not different between both groups [2 ± 1 min (atria from nonfailing) vs. 2 ± 1 min (atria from failing hearts, NS; Fig. 2]. The negative inotropic effect of ET-1 could be completely suppressed by 1 μM of the nonselective ET-receptor antagonist bosentan (Fig. 3). The selective ETB-receptor antagonist BQ 788 (1 μM) did not affect the negative inotropic ET effect. However, for technical reasons, unfortunately this could be evaluated only in a limited number of preparations (Fig. 3). In contrast, the selective ETA-receptor antagonist BQ 123 (1 μM) fully suppressed the transient negative inotropic effect of endothelin (see Fig. 3; for an original registration, see Fig. 1).
A possible dependence of the negative inotropic effect on prestimulation of adenylylcyclase was evaluated in an additional set of experiments in normal human right atrium (n = 17) without forskolin prestimulation. In these experiments, basal force was 4.1 ± 0.6 mN. In principle, we found the same transient negative inotropic effect, although it lasted a significantly shorter time (2 ± 0.2 min). The time to peak negative inotropic effect was also shortened (1 ± 0.3 min; see Fig. 4). Maximal negative inotropic effect was a reduction in contractile force by 18 ± 3%. Thus, prestimulation with forskolin accentuated the duration of the negative inotropic response to ET rather than the extent of the effect.
The small sustained positive inotropic effect of ET-1 in atria, which followed the transient negative inotropic effect, was not different between atria from failing and nonfailing hearts (see Fig. 2). The rate of change during the positive inotropic effect also was not significantly different between the groups.
Further to characterize the positive inotropic effect of ET-1, concentration-response curves were assessed in right atria from nonfailing hearts. ET-1 (10−10 to 3 × 10−7M) caused a concentration-dependent increase in force: maximal effect was obtained at 10−7M; the pD2 value was 7.7 ± 0.2 (n = 21; Fig. 5). The ETB-receptor antagonist BQ 788 used in the high concentration of 10 μM did not significantly affect the ET-1-induced positive inotropic effect, whereas the ETA-receptor antagonist BQ 123 (10 μM) caused a parallel rightward shift of the ET-1 concentration-response curve (Fig. 5). It should be mentioned, however, that lower concentrations of BQ 123 (0.1 or 1 μM) caused only marginal rightward shifts of the ET-1 concentration-response curve.
Effects in ventricle
Forskolin (1 μM) led to a positive inotropic effect in ventricular tissue from failing hearts (NYHA IV; from 5.3 ± 1.2 to 6.9 ± 1.4 mN). Application of 0.1 μM ET-1 resulted in a monophasic, slow-onset positive inotropic effect reaching its maximum of 12 ± 2% after 10 min (n = 8), without an initial negative inotropic effect (Fig. 6). This positive inotropic ET effect could be antagonized by BQ 123 (1 μM;Fig. 6).
Because of limited availability and for ethical reasons, there are no data on ventricular tissue from nonfailing hearts.
In rat (19) and in human heart (17,20), ET receptors couple to formation of inositol phosphates and to inhibition of adenylylcyclase. However, we have recently shown that, in the human heart, endothelin inhibits adenylylcyclase only in right atrium, but not in left ventricles (17). The present study clearly demonstrates that the pattern of effects of ET-1 on contractile force also differs between human right atrium and left ventricle: in right atria, ET-1 first caused a transient negative inotropic effect followed by a positive inotropic effect, whereas in left ventricle, ET-1 lacked the transient negative inotropic response and caused only positive inotropic effects.
In previous studies (17,20), we had shown that in right atria of the human heart, ET-1 was about 100 times more potent than ET-3 in increasing inositol phosphate formation and inhibition of adenylylcyclase, indicating that both effects are mediated by ETA receptors. The results of the present study indicate that-in parallel to these findings-the negative and positive inotropic responses to ET-1 may be also mediated by ETA receptors. Thus, the transient negative inotropic effect was abolished in the presence of the selective ETA-receptor antagonist BQ 123 but was not affected by pretreatment with the selective ETB-receptor antagonist BQ 788. In addition, the positive inotropic effect of ET-1 was not affected by BQ 788, but was antagonized by (high concentrations of) BQ 123. Similar results have been described by Meyer et al. (6), who demonstrated that in human right atrium, the positive inotropic effect of ET-1 is antagonized by BQ 123. Thus, although binding studies demonstrated the existence of both ETA and ETB receptors in human right atrium (15-17), among these, only ETA receptors appear to be of functional importance. Similarly, Pieske et al. (7) have recently shown that, in human left ventricle, the positive inotropic effect of ET-1 is induced by ETA-receptor stimulation because it was antagonized by BQ 123. Furthermore, the selective ETB-receptor agonist sarafotoxin S6c (Sf6c) was without any effect on force of contraction (7).
However, it should be noted that, in right atria, the rightward shift of the ET concentration-response curve induced by 10 μM BQ 123 was by far less than could be expected, considering the affinity of BQ 123 to the ETA receptor [∼20-300 nM (22)]. Recently, similar results have been obtained by Burrell et al. (24), showing that in right atrial appendages from nonfailing and failing human hearts, BQ 123 was only a weak inhibitor of ET-1-induced positive inotropic effects; ETB-receptor antagonists were without any effect. Conversely, these authors found that the ETB-receptor agonist Sf6c was more potent than ET-1 in inducing positive inotropic effects, although causing smaller maximal effects. From these results, these authors concluded that the positive inotropic effect of ET-1 in the human atrium might be mediated by a non-ETA, non-ETB receptor. According to these data and our findings of an only weak inhibitory effect of BQ 123, we cannot rule out that also in our study, the positive inotropic effect of ET-1 is mediated by a non-ETA, non-ETB receptor. This, however, would be not in agreement with our recent data, that in human right atrium, ET-receptor agonists caused inositol phosphate formation with an order of potency of ET-1 ≥ Sf6b ≫ ET-3 ≥ Sf6c (17), which is typical for an ETA receptor (14).
The mechanisms underlying the positive and negative inotropic effects of ET-1 in the human right atrium are not completely understood. As mentioned earlier, ET receptors in human right atria and left ventricles couple to formation of inositol phosphates (17,20); this subsequently increases [Ca2+]i(25), which might be involved in the positive inotropic effect of ET-1. Moreover, ET-1 has been shown to increase the Ca2+ sensitivity of the myofilaments through activation of the Na+/H+ exchanger, and it has been suggested that these effects are (at least partly) due to DAG-induced PKC activation (6,7,26).
Conversely, in human right atria, ET-1 inhibits adenylylcyclase activation (17,20) and decreases force of contraction (this study), whereas in human left ventricles, it did not inhibit adenylylcyclase (17) and did not cause negative inotropic effects (this study). Thus, it is tempting to speculate that the negative inotropic effect of ET-1 is linked to inhibition of adenylylcyclase. Our finding that the negative inotropic response was significantly prolonged in preparations prestimulated with forskolin is in support of this hypothesis. Because the inhibition of adenylylcyclase in the heart is linked to coupling to Gi, it might be that ET receptors in the human right atrium also use this pathway. This assumption is further supported by the fact that in the present study, ET-1-very similar to the classic cardiac receptor coupled to Gi, the muscarinic M2 receptor [for a recent review, see (27)]-can inhibit both basal force of contraction and force of contraction that has been enhanced by cyclic adenosine monophosphate (cAMP) elevation by forskolin. Coupling of ET receptors to Gi has been demonstrated in adult rat ventricular cardiomyocytes (19) and in SK-N-MC-cells (28). However, it is presently unknown why ET receptors couple to formation of inositol phosphates and to inhibition of adenylylcyclase only in human atrium, whereas in left ventricle, they only couple to inositol phosphate formation but not to adenylylcyclase inhibition.
It is interesting to note that in the present study in right atria obtained from terminally failing human hearts, the negative inotropic effect of ET was significantly larger and more prolonged than in atria from nonfailing hearts. This is in contrast to the positive inotropic effect that was found to be unchanged (in right atria, present study; see also 24) or even decreased (left ventricle, (7). However, a general finding in terminally failing human hearts is an increased amount and/or activity of Gi protein (29-31). If our assumption is correct that the negative inotropic effect of endothelin-1 is due to the coupling of the ET receptors to Gi proteins (see earlier), it can be well understood that in a situation of an increased activity of Gi proteins, the negative inotropic effect of ET-1 is enhanced. However, this explanation might be an oversimplification, because the negative inotropic effects of classic Gi-coupled receptors such as M2 muscarinic receptors (27,32) and A1-adenosine receptors (32) are not changed in failing human hearts.
In human right atrium, dual coupling of ETA receptors to inositol phosphate formation and to inhibition of adenylylcyclase is accompanied by a biphasic inotropic response to ET-1: a transient negative inotropic effect followed by a sustained positive inotropic response. In human left ventricle, however, ETA receptors couple only to formation of inositol phosphates; accordingly ET-1 evoked only positive, but no negative, inotropic effects. There was no evidence for a functional role of ETB receptors in the human heart; however, it remains to be elucidated whether the positive inotropic effect of ET-1 is mediated by ETA or by a non-ETA, non-ETB receptor.
Acknowledgment: We thank Dr. M. Clozel (Hoffmann-La Roche Ltd., Basel, Switzerland) for the generous gift of bosentan. This work was supported by the Deutsche Forschungsge-meinschaft (DFG Br 526/6-1) and by the BMBF.
1. Yanagisawa M, Kurihara H, Kimura S, et al. A novel potent vasoconstrictor peptide produced by vascular endothelial cells. Nature
2. Rubanyi G, Polokoff MA. Endothelins: molecular biology, biochemistry, pharmacology, physiology and pathophysiology. Pharmacol Rev
3. Moravec CS, Rexnolds EE, Stewart RW, et al. Endothelin
is a positive inotropic agent in human and rat heart in vitro. Biochem Biophys Res Commun
4. Zerkowski H-R, Broede A, Kunde K, et al. Comparison of the positive inotropic effects of serotonin, histamine, angiotensin II, endothelin
and isoprenaline in the isolated human right atrium. Naunyn Schmiedebergs Arch Pharmacol
5. Moody CJ, Dashwood MR, Sykes RM, et al. Functional and autoradiographic evidence for endothelin
-1 receptors on human and rat heart cardiac myocytes. Circ Res
6. Meyer M, Lehnart S, Pieske B, et al. Influence of endothelin
-1 on human atrial myocardium-myocardial function and subcellular pathways. Basic Res Cardiol
7. Pieske B, Beyermann B, Breu V, et al. Functional effects of endothelin
and regulation of endothelin
receptors in isolated human nonfailing and failing myocardium. Circulation
8. Kiowski W, Sutsch G, Hunziker P, et al. Evidence of endothelin
-1-mediated vasoconstriction in severe chronic heart failure. Lancet
9. Sakai S, Miyauchi T, Kobayashi M, et al. Inhibition of myocardial endothelin
pathway improves long-term survival in heart failure. Nature
10. Onishi K, Ohno M, Little WC, et al. Endogenous endothelin
-1 depresses left ventricular systolic and diastolic performance in congestive heart failure. J Pharmacol Exp Ther
11. Love MP, McMurray JJV. Endothelin
in congestive heart failure. Basic Res Cardiol
12. Cohn JN. Is there a role for endothelin
in the natural history of heart failure? Circulation
13. Miller WL, Redfield MM, Burnett JC Jr. Integrated cardiac, renal, and endocrine actions of endothelin
. J Clin Invest
14. Masaki T, Vane JR, Vanhoutte PM. International Union of Pharmacology nomenclature of endothelin
receptors. Pharmacol Rev
15. Molenaar P, O'Reilly G, Sharkey A, et al. Characterization and localization of endothelin
receptor subtypes in human atrioventricular conducting system and myocardium. Circ Res
16. Bax WA, Bruinnvels AT, van Suylen R-J, et al. Endothelin
receptors in the human coronary artery, ventricle and atrium. Naunyn Schmiedebergs Arch Pharmacol
17. Pönicke K, Vogelsang M, Heinroth M, et al. Endothelin
receptors in the failing and non-failing human heart
18. Zolk O, Quattek J, Sitzler G, et al. Expression of endothelin
-converting enzyme, and endothelin
receptors in chronic heart failure. Circulation
19. Hilal-Dandan P, Merck ST, Lujan JP, et al. Coupling of the type A endothelin
receptor to multiple responses in adult rat cardiac myocytes. Mol Pharmacol
20. Vogelsang M, Broede-Sitz A, Schäfer E, et al. Endothelin
-receptors couple to inositol phosphate formation and inhibition of adenylate cyclase in human right atrium. J Cardiovasc Pharmacol
21. Clozel M, Breu V, Gray GA, et al. Pharmacological characterization of bosentan, a new potent orally active nonpeptide endothelin
receptor antagonist. J Pharmacol Exp Ther
22. Moreland S. BQ-123, a selective endothelin
-receptor antagonist. Cardiovasc Drug Rev
23. Ishikawa K, Ihara M, Noguchi K, et al. Biochemical and pharmacological profile of a potent and selective endothelin
B receptor antagonist, BQ-788. Proc Natl Acad Sci U S A
24. Burrell KM, Molenaar P, Dawson PJ, et al. Contractile and arrhythmic effects of endothelin
receptor agonists in human heart
in vitro: blockade with SB 209670. J Pharmacol Exp Ther
25. Berridge MJ, Irvine RF. Inositol phosphates and cell signalling. Nature
26. Krämer BK, Smith TW, Kelly RA. Endothelin
and increased contractility in adult rat ventricular myocytes: role of intracellular alkalosis induced by activation of protein kinase C-dependent Na+
-exchanger. Circ Res
27. Giessler C, Dhein S, Pönicke K, et al. Muscarinic receptors in the failing human heart
. Eur J Pharmacol
28. Heinroth-Hoffmann I, Vogelsang M, et al. Mechanism of ETA
-receptor stimulation-induced increases in intracellular Ca2+
in SK-N-MC cells. Br J Pharmacol
29. Brodde O-E, Michel MC, Zerkowski H-R. Signal transduction mechanisms controlling cardiac contractility and their alterations in chronic heart failure. Cardiovasc Res
30. Böhm M. Alterations of β-adrenoceptor-G-protein-regulated adenylylcyclase in heart failure. Mol Cell Biochem
31. Feldman AM. Modulation of adrenergic receptors and G-transduction proteins in failing human ventricular myocardium. Circulation
32. Böhm M, Gierschik P, Jakobs KH, et al. Increase of Gi
alpha in human hearts with dilated but not ischemic cardiomyopathy. Circulation