Beta-adrenoceptors in cardiac disease

Pharmac. Ther.Vol.60, pp. 405-430, 1993 Copyright© 1994ElsevierScienceLtd Printed in Great Britain.All rights reserved 0163-7258/93 $24.00

Pergamon

Associate Editor: D. G. McDEVITT

BETA-ADRENOCEPTORS

IN C A R D I A C DISEASE

OTTO-ERICH BRODDE Biochemisehes Forschungslabor, Medizinische Klinik & Poliklinik, Abtlg. Nieren- & Hochdruckkrankheiten, Universitiitsklinikum Essen, Hufelandstrasse 55, D-45122 Essen, Germany Abstract--The human heart contains both flj and fl:-adrenoceptors; both mediate positive inotropic and chronotropic effects. In chronic heart failure, fl-adrenoceptor number is reduced, presumably, by down-regulation by endogenous noradrenaline which is elevated due to increased sympathetic activity. Since the human heart contains only a few spare receptors for fl-adrenoceptor-mediated positive inotropic effects and the amount of spare receptors declines in chronic heart failure, it is not surprising that the reduced fl-adrenoceptor number is accompanied by decreased contractile responses to fl-adrenoceptor agonists (including endogenous catecholamines), and the extent of decrease in maximal inotropic response is more pronounced as the disease becomes more advanced. Moreover, in chronic heart failure myocardial Gi-protein, which inhibits cAMP formation, is increased, which might further contribute to the reduction in fl-adrenoceptor-mediated effects. It appears that, at present, the best therapy for severe heart failure is a successful heart transplant, since in the transplanted heart fl-adrenoceptor number and function seems to be normalized. Moreover, the data currently available do not suggest any development of super- or subsensitivity of postsynaptic cardiac fl-adrenoceptors in the transplanted human heart. Keywords--Human cardiac fl~-adrenoceptors, human cardiac fl2-adrenoceptors, chronic heart failure, G-proteins, fl-adrenoceptor desensitization. CONTENTS 1. Introduction 2. Molecular Biology of the Human fl-Adrenoceptors 3. ill- and fl2-Adrenoceptors in the Non-failing Human Heart 4. ill- and fl2-Adrenoceptor Changes in Chronic Heart Failure 5. Cardiac fl-Adrenoceptors in Acute Myocardial Ischemia 6. Cardiac fl-Adrenoceptors in the Transplanted Human Heart 7 Conclusion Acknowledgements References

405 406 409 414 419 419 422 423 423

1. I N T R O D U C T I O N In the human heart, contractility and/or heart rate are regulated by receptor systems acting via accumulation of intracellular c A M P (Gs-protein coupled), by receptor systems acting via inhibition of c A M P formation (G~-protein coupled) and by receptor systems acting independently of c A M P formation, possibly involving the phospholipase C/diacylglycerol/inositol-l,4,5-trisphosphatepathway (Fig. 1). Amongst all these receptors, the fl-adrenoceptor-G~-protein-adenylate cyclasec A M P system is, in the human heart, the most powerful physiological mechanism to regulate contractility and/or heart rate (cf. Fig. 1). This article, therefore, focuses on properties and functional importance of the human cardiac fl-adrenoceptors, their changes in chronic heart failure and the clinical implication of these alterations. Abbreviations--ACE, angiotensin-converting enzyme; flARK, fl-adrenoceptor kinase; PET, positron emission tomography; PKA, protein kinase A; PKC, protein kinase C.

405

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O.-E. BRODDE 2. M O L E C U L A R BIOLOGY OF THE H U M A N /?-ADRENOCEPTORS

The//-adrenoceptor(s) belong to the G-protein-coupled superfamily of receptors (Fig. 2) that have seven hydrophobic transmembrane-spanning regions and are proteins consisting of 402-560 amino acids (Dohlman et al., 1991). Molecular biology techniques have clearly demonstrated three distinct human genes (Fig. 3) encoding the ,8-adrenoceptor subtypes ,6'~ /~2 and ,83 (Chung et al., 1987; Dixon et al., 1987a,b; Frielle et al., 1987; Kobilka et al., 1987a,b; Emorine et al., 1989). The /~,-adrenoceptor shows 48.9% sequence similarity with the 32-adrenoceptor, and the /?3-adrenoceptor shows 50.7 and 45.5% sequence similarity with the /3,- and /?2-adrenoceptor, respectively (Emorine et al., 1989). All three receptors contain glycosylation sites at the amino terminus (Rands et al., 1990); in addition, /?r and /?2-adrenoceptors, but obviously not /?3-adrenoceptors, contain several phosphorylation sites in the third intracellular loop and the carboxy terminus region; these are potential phosphorylation sites for the cAMP-dependent protein kinase A (PKA), the protein kinase C (PKC) or the/?-adrenoceptor kinase (/?ARK; Benovic et al., 1988; Kobilka, 1992). While the genes for the/~,- and 32-adrenoceptors apparently lack introns in their coding sequences, introns have been detected in the human and rodent 33-adrenoceptor gene (Granneman et al., 1992). By the use of mutation deletions (Dixon et al., 1987a,b) and construction of chimeric receptors (Kobilka et al., 1988; Frielle et al., 1988), the functional domains of the/~-adrenoceptor have been explored. One important finding was that the ligand-binding site is contained in the transmembrane-spanning regions (see Tota et al., 1991). Many, or all, of the hydrophobic regions of the receptor contained in the transmembrane-spanning regions influence agonist-binding specificity, while antagonist-binding specificity is mainly influenced by the VI and VII transmembranespanning regions. On the other hand, the third intracellular hydrophilic loop is most important

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FIG. 1. Receptor systems and their signal-transduction mechanisms in the non-failing human heart. For details see text. Abbreviations: 3~,/32,:q, 31-, [32-and :q-adrenoceptors; H z, histamine H2-receptors; 5-HT4, 5-HT4-receptors; VIP, vasoactive intestinal peptide receptors; PGE~, prostaglandin Erreceptors; Glu, glucagon receptors; A], adenosine At-receptors; M 2, muscarinic M2-receptors; SS, somatostatin-receptors; A II, angiotensin lI-receptors; ET, endothelinreceptors; G~, stimulatory guanine nucleotide-binding protein; Gi, inhibitory guanine nucleotide-binding protein; C, catalytic unit of adenylate cyclase; PLC, phospholipase C; PIP,, phosphatidylinositol 4,5-bis-phosphate; DAG, 1,2-diacylglycerol; I P 3, inositol-l,4,5-trisphosphate; ISO, isoprenaline; O, activation; O, inhibition. Right atrium: positive inotropic effects were determined on isolated electrically driven right atria from patients without apparent heart failure undergoing coronary artery bypass grafting. Ventricular myocardium, positive inotropic effects were determined on isolated electrically driven right and left ventricular preparations obtained from would-be cardiac transplant donors. Modified from Brodde et al. (1992a).

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FIG. 2. (A) Generalized model for G-protein coupled receptors. The seven hydrophobic membrane-spanning helices are numbered by roman numerals. The amino terminus and hydrophilic sequences E l - E 3 project extracellularly, whereas the carboxy terminus and loops CI--C3 project into the cytoplasm of the cell. Potential glycosylation sites are indicated near the amino terminus. The breaks in loop C3 and carboxy terminus indicate variable changes in the length of these two segments among G-protein coupled receptors. Reprinted from Stadel and Nakada (1991) with permission of the authors and the copyright holder, Karger, Basel. (B) Primary structure of the human/32-adrenoceptor. Circles with letters represent amino acids identified by the single letter amino acid code. The proposed membrane spanning regions are numbered with roman numerals. Extracellular domains are designated by the prefix e-, intracellular domains by the prefix i-. Black circles with white letters indicate potential phosphorylation sites for PKA or/3ARK. Reprinted from Kobilka (1992), with permission of the author and the copyright holder, Annual Reviews Inc. Palo Alto, CA.

408

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for coupling the fl-adrenoceptor to Gs-protein (O'Dowd et al., 1988; Cheung et al., 1989; Strader et al., 1989; Raymond et al., 1990).

In general, there is good agreement between the pharmacologically defined and cloned fl-adrenoceptor subtypes (Fig. 3). Thus, the cloned fl~-adrenoceptor has similar affinity for noradrenaline and adrenaline, exhibits high affinity for fl~-selective antagonists, such as CGP 20712 A or betaxolol, and low affinity for the fl2-selective antagonist ICI 118,551 (Frielle et al., 1987; Marullo et al., 1990); the cloned fl2-adrenoceptor has higher affinity for adrenaline than for noradrenaline, high affinity for ICI 118,551, but very low affinity for CGP 20712 A (Marullo et al., 1990). Finally, the cloned fl3-adrenoceptor, which may or may not correspond to the previously described 'atypical' fi-adrenoceptor in the gastrointestinal tract, skeletal muscle and adipose tissue (Zaagsma and Nahorski, 1990), shows a higher affinity for noradrenaline than adrenaline, is sensitive to BRL 37344 and 28410, but rather insensitive to all classical fl-adrenoceptor antagonists (Emorine et al., 1989). Phosphorylation of the fl-adrenoceptor is involved in the desensitization phenomenon, i.e. the fact that the cell very rapidly loses its responsiveness upon agonist-stimulation. Recent evidence suggests that the mechanisms underlying rapid (i.e. within minutes) desensitization may be different from those that may be responsible for marked loss in fl-adrenoceptor responsiveness following long-term exposure (within hours) to agonists (for a review, see Hausdorff et al., 1990). Rapid desensitization appears to be caused by phosphorylation of the receptor by PKA (and possibly PKC) and flARK, leading to an uncoupling of the receptor from the Gs-protein without loss of cell-surface fl-adrenoceptors. PKA (and PKC) may be involved in heterologous desensitization by phosphorylating the fl-adrenoceptor following stimulation with low (nanomolar) agonist concentrations. On the other hand, flARK (and PKA) phosphorylates agonist-occupied receptors at higher agonist concentrations (i.e. when a large fraction of the fl-adrenoceptor is occupied by agonist) and is believed to be responsible for homologous desensitization (Sibley et al., 1987). In addition, flARK phosphorylation of the fl-adrenoceptor needs a co-factor, flarrestin, in order to inactivate the receptor (Lohse et al., 1990). In contrast to rapid desensitization following short-term agonist exposure, long-term desensitization caused by long-term exposure to agonists appears not to be primarily due to receptor phosphorylation, but seems to be due, in part, to a reduction in steady-state m R N A levels encoding the fl-adrenoceptor and to enhanced degradation of the fl-adrenoceptor. The reduction of steady-state m R N A levels upon prolonged agonist incubation appears to involve reduced message stability rather than reduced transcription (Hausdorff et al., 1990; Hadcock and Malbon, 1991; Collins et al., 1992). Finally, it is interesting to note that the three fl-adrenoceptor subtypes have different numbers of consensus sequences for phosphorylation by PKA (see above, Hausdorff et al., 1990; Emorine et al., 1991). Moreover, while the fl~- and fl2-adrenoceptor contain large carboxy-termini with many potential flARK phosphorylation sites, the fl3-adrenoceptor contains a much shorter carboxyterminus with only a very few potential flARK phosphorylation sites. Thus, it might be predicted that the pattern of desensitization and down-regulation could be different between the three fl-adrenoceptor subtypes. In fact, Thomas et al. (1992) have recently shown, in 3T3-F442A

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FIG. 4. Distribution of total fl-, fl~- and fl2-adrenoceptors in the non-failing human heart. Ordinate: Total fl -, ill- and fl2-adrenoceptordensity in fmol ( - )-[12sI]iodocyanopindolol(ICYP) specificallybound/mg protein. Given are means _+S.E.M.; number of experiments at the bottom of the columns. Reprinted from Brodde (1991b), with permission of the copyright holder, Academic Press Ltd., London. fibroblast cells expressing fl3-adrenoceptors, that during prolonged agonist exposure, fl3-adrenoceptors were not down-regulated.

3. fl~- AND fl2-ADRENOCEPTORS IN THE NON-FAILING H U M A N HEART It is now generally accepted that, in the human heart, fl~- and fl2-adrenoceptors coexist; on the other hand, at present, there is no evidence for the existence of the third fl-adrenoceptor subtype, the fl3-adrenoceptor, in the human heart (Kaumann, 1989). Initial evidence for the coexistence of fir and fl2-adrenoceptors was presented in 1974 by Ablad et al., who found, in isolated electrically driven human right atria, that the flradrenoceptor selective antagonist H93/26 (metoprolol) antagonized the positive inotropic effect of noradrenaline more potently than that of adrenaline, whereas the non-selective fl-adrenoceptor antagonist propranolol antagonized responses to both catecholamines to about the same degree. Subsequently, the coexistence of fl~- and fl2-adrenoceptors in the human heart has been confirmed, first, by radioligand-binding studies, thereafter, in functional experiments (for reviews, see Bristow, 1989; Jones et al., 1989; Bristow et al., 1990; Brodde, 1991a). The number of fl-adrenoceptors in the non-failing human heart is quite evenly distributed in right and left atrial and ventricular tissue (Fig. 4). This has been demonstrated by three different techniques: by radioligand-binding studies (resulting in an amount of about 80-90 fmol/mg protein in all four tissues; Brodde, 1991a,b; Steinfath et al., 1992b), by quantitative autoradiographic studies (Elnatan et al., 1992) and, very recently, in ~,it,o by positron emission tomography (PET) studies (De Silva et al., 1992). However, the proportion of fl2-adrenoceptors is somewhat higher in the atria (approximately 1/3 of the total fl-adrenoceptor population) than in ventricular myocardium (about 20% of the total fl-adrenoceptor population; see Brodde, 1991a,b; Steinfath et al., 1992b) and may be even higher (up to 50%) in the atrio-ventricular conducting system (Elnatan et al., 1991). Both fl~- and fl2-adrenoceptors couple to adenylate cyclase; this has been demonstrated in broken cell preparations (Brodde et al., 1984; Bristow et al., 1989; Kaumann et al., 1989a), as well as in intact human right atria (Ikezono et al., 1987). In the human heart, adenylate cyclase is preferentially activated by fl2-adrenoceptor stimulation, although flradrenoceptors predominate (Bristow et al., 1989; Kaumann et al., 1989a; Brodde, 1991a). Thus, in human right atrial membranes, fl2-adrenoceptor selective agonists, such as fenoterol, procaterol and terbutaline, caused activation of adenylate cyclase activity (Fig. 5) that amounted to about 50-70% of that of isoprenaline (Waelbroeck et al., 1983; Brodde et al., 1984; Bjornerheim et al., 1990; Schfifers et al., 1992), although only 30% of the total fl-adrenoceptor population is of the fl_,-subtype. Similarly, in ventricular membranes of the human heart, the fl2-adrenoceptor agonists terbutaline and zinterol

410

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caused 50% of maximal isoprenaline activation (Bristow et al., 1989; Sch/ifers et al., 1992), and isoprenaline, adrenaline and noradrenaline evoked their stimulatory effects on adenylate cyclase activity predominantly via fl2-adrenoceptor stimulation (Bristow et al., 1989a; Kaumann et al., 1989), although only 20% of the whole fl-adrenoceptor population is of the fl2-subtype (see above). The mechanism underlying the different coupling efficiencies of human cardiac fl~- and fl2-adrenoceptors to adenylate cyclase is not known at present. In this context, it is interesting to note that Green et al. (1992) recently showed that, in the mammalian fibroblast cell line CHW-I102 transfected with fl~- or fl2-adrenoceptor cDNAs, the fl2-adrenoceptor exhibited a much greater functional coupling to adenylate cyclase than did the flt-adrenoceptor. Thus, it might be a general phenomenon that fl2-adrenoceptors couple more efficiently to adenylate cyclase than do fl~-adrenoceptors. In the human heart, in vitro involvement of both fl~- and fl2-adrenoceptors in the positive inotropic effects of fl-adrenoceptor agonists has been convincingly demonstrated on isolated electrically driven atrial (Fig. 6) and ventricular preparations (see Jones et al., 1989; Kaumann et al., 1989a; Bristow et al., 1990; Feldman and Bristow, 1990a; Brodde, 1991a) and, very recently, also in single myocytes from human ventricle (Del Monte et al., 1993). Among the classical catecholamines, isoprenaline and adrenaline cause their positive inotropic effects on the human heart via stimulation of fl~- and fl2-adrenoceptors, while noradrenaline, tile main transmitter of the sympathetic nervous system, evokes its positive inotropic effect predominantly, if not exclusively, via fl~-adrenoceptor stimulation (Kaumann et al., 1989a; Motomura et al., 1990). In right and left atria, fl~- and fl2-adrenoceptor stimulation can evoke maximum positive inotropic effects, while on right and left ventricles only, fl~-adrenoceptor stimulation can evoke maximum positive inotropic effects, fl2-adrenoceptor stimulation only submaximal positive inotropic effects (Bristow, 1989; Kaumann et al., 1989a; Motomura et aL, 1990). In vivo experiments have, at least partially, confirmed that fl2-adrenoceptors can contribute to the positive chronotropic and inotropic effects of fl-adrenoceptor agonists. Several studies have shown that isoprenaline-induced tachycardia in humans is mediated by both fl~- and fl2-adrenoceptors to about the same degree, while exercise-induced tachycardia (which is mainly brought about by neuronally released noradrenaline) is mediated solely by fl~-adrenoceptor stimulation (see McDevitt, 1989; Brodde, 1991a), in close agreement with the in vitro data on isolated human right atria (see above). Moreover, in healthy volunteers, the positive chronotropic effect caused by i.v. infusions of terbutaline was not at all affected by the flradrenoceptor selective antagonists atenolol and bisoprolol (Fig. 7) given in doses that inhibited fl~-adrenoceptor mediated effects (Strauss et al., 1986; Levine and Leenen, 1989; Sch/ifers et al., 1992). Finally, Hall et al. (1989) demonstrated that the positive chronotropic effect of salbutamol induced by injections into the right coronary artery

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FIG. 6. (A-C) Effects of bisoprolol (upper panel) and ICI 118,551 (lower panel) on the positive inotropic effects of isoprenaline (A), noradrenaline (B) and procaterol (C) on the isolated electrically driven right atrium derived from patients undergoing coronary artery bypass grafting without apparent heart failure. Ordinates: positive inotropic effect in percent of maximal response. Abscissae: molar concentrations ofisoprenaline, noradrenaline and procaterol. Insets: Schild-plots of the antagonism of bisoprolol and ICI 118,551 against isoprenaline-, noradrenaline- and procaterol-induced positive inotropic effects. CR, concentration-ratio; [A], molar concentrations of the antagonists. Given are means _ SEM; n, number of experiments. Note that the slopes of the Schild-plots for antagonism of bisoprolol and ICI 118,551 against noradrenaline- and procaterol-evoked positive inotropic effects were not significantly different from unity, indicating that both agonists interact with a single class of fl-adrenoceptor subtypes. The pA2-values (bisoprolol against noradrenaline: 8.42, against procaterol 6.99; ICI 118,551 against noradrenaline: 6.62, against procaterol 9.49) revealed that noradrenaline acts at B~-, procaterol at fl2-adrenoceptors. In contrast, the slope of the Schild-plots of both antagonists against isoprenaline-evoked positive inotropic effects were significantly different from unity indicating that isoprenaline acts at both [~- and fl2-adrenoceptors. Modified from Zerkowski et aL (1986).

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of patients with chronic stable angina (thereby avoiding any systemic effects) was not affected by the flradrenoceptor selective antagonist practolol, but was significantly antagonized by propranoloi, indicating that it is mediated exclusively by (cardiac) fl2-adrenoceptor stimulation. A few studies have addressed the question of in vivo #2-adrenoceptor-mediated positive inotropic effects. Using echocardiographic assessment of the systolic blood pressure/end-systolic left ventricular volume ratio as the index of positive inotropic effects, Levine and Leenen (1989) showed that the positive inotropic effect brought about by terbutaline was much less antagonized by atenolol than could be expected, assuming that it is mediated by flradrenoceptors. Similarly, using the shortening of the pre-ejection period and heart rate-corrected QS2-time as a measure of positive inotropy, we recently showed, in healthy volunteers, that the flj-adrenoceptor selective antagonist bisoproloi (given in a dose that occupied about 75% of fl~-, but less than 5% of fl2-adrenoceptors) antagonized the positive inotropic effects brought about by i.v. infusion of terbutaline by far less than that caused by i.v. infusion of isoprenaline (Sch/ifers et al., 1992, 1993). Taken together, these data indicate that terbutaline evokes its positive inotropic effect in vivo predominantly via (cardiac) fl2-adrenoceptor stimulation. However, the fact that atenolol and bisoprolol, both given in flt-adrenoceptor selective doses, did slightly antagonize terbutaline-mediated positive inotropic effects suggests that a (small) fl~-adrenoceptor component might be included, possibly via endogenous noradrenaline released upon stimulation of presynaptic fl2-adrenoceptors. Thus, the human heart shows a unique feature when compared with the heart of commonly used laboratory animals: it contains a considerable number o f functional fl2-adrenoceptors that cause positive inotropic and chronotropic effects in vitro and in vivo. Moreover, in the human heart, catecholamines activate adenylate cyclase mainly via fl2-adrenoceptor stimulation, although #~-adrenoceptors predominate. And finally, the human heart contains only a few spare receptors for fl-adrenoceptor-mediated positive inotropic effects (Port and Bristow, 1988; Schwinger et al., 1990; Brown et al., 1992). The concept of spare receptors (receptor reserve) refers to the phenomenon that agonists can evoke maximal responses at only a submaximal receptor occupancy in many tissues. A linear relationship between receptor occupancy and response would indicate the absence of spare receptors, while a non-linear relationship (i.e. a 10% receptor occupancy yields a 50% response, etc.) would indicate the presence of spare receptors (receptor reserve; see Furchgott, 1972; Ruffolo, 1982; Kenakin, 1987). That the human heart might have only a few spare receptors for fl-adrenoceptor mediated positive inotropic effects was first suggested by Kaumann et al., (1982), who observed that, in ED50 % Vcttues f o r Increose in Heart Rate Isoprenaline "E 50-

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FIG. 8. Plot of receptor occupancy vs the positive inotropic effect of isoprenaline on isolated electrically driven left ventricular trabeculae from three non-failing hearts. For comparison, data on rat left papillary muscles are included (dotted lines). Ordinate: positive inotropic effect of isoprenaline in per cent of maximal response. Abscissa: receptor occupancy in percent, calculated from the pK:values of isoprenaline (assessed by (-)-[t25I]iodocyanopindolol binding). Dashed line: line of identity. isolated electrically driven human left papillary muscles, isoprenaline, adrenaline and noradrenaline exhibited a much lower potency than in the cat heart, a tissue known to have a large receptor reserve for positive inotropic effects of catecholamines (Kaumann et al., 1989b). Subsequently, Schwinger et al. (1990) demonstrated that, in left papillary muscles from three groups of patients with different degrees of heart failure [non-failing hearts, patients undergoing mitral valve replacement with mild-to-moderate heart failure (NYHA class II-III) and patients undergoing heart transplantation due to end-stage dilated cardiomyopathy (NYHA class IV)], a linear relationship between the total fl-adrenoceptor number and the maximal inotropic response to isoprenaline exists. In other words the maximal positive inotropic effect of isoprenaline declines with decreasing fl-adrenoceptor number. From these data, they concluded that there are no spare receptors for the full agonist isoprenaline in human ventricular myocardium. Using another approach, we have recently confirmed that the human heart contains only a very few, if any, spare receptors for fl-adrenoceptor-mediated inotropic effects (Brown et al., 1992). We determined, in isolated electrically driven human cardiac preparations, the positive inotropic effects of catecholamines and, simultaneously, their affinities to f l : and fl:adrenoceptors, and constructed plots of fractional receptor occupancy vs response from these data. The affinity of agonists was assessed by (-)-[~25I]iodocyanopindolol binding using as incubation medium phosphate-buffered Na+-rich Krebs-Henseleit solution to mimic the organ bath conditions as closely as possible, as originally suggested by McPherson et al. (1985). Under these conditions, we found that, in isolated electrically driven right atria from patients without apparent heart failure undergoing elective coronary artery bypass grafting, the catecholamines had to occupy about 8-10% of fl-adrenoceptors to cause 50% of maximal inotropic response and about 45-50% of fl-adrenoceptors to cause 90% of maximal response. With the same approach, we found that, in non-failing human left ventricular papillary muscles, isoprenaline has to occupy about 10% of fl-adrenoceptors to cause 5 0 o of maximal response (Fig. 8). In contrast, in rat left atrium, right and left papillary muscle (Brown et al., 1992) and in cat left ventricular papillary muscle (Kaumann et al., 1989b), isoprenaline has to occupy only 1.5-3% of fl-adrenoceptors to cause 50% of maximal response and about 20% to cause 90% of maximal response. These data suggest that, in the non-failing human heart, there is a only a small receptor reserve for the positive inotropic effects to catecholamines (at least to reach 50% of maximal response), and nearly all receptors have to be occupied to cause a maximal response. In this context, it is interesting to note that we recently observed, in isolated human right atria, a similar lack of receptor reserve for the positive inotropic effect evoked by serotonin (via 5-HT:receptors) and histamine (via H2-receptors; Zerkowski et al.,

414

O.-E. BRODDE

1993). Since both 5-HT4- and H2-receptors couple in the human heart to adenylate cyclase in an excitatory fashion (similar to the fl-adrenoceptor), these data raise the possibility that a lack of receptor reserve is a characteristic of human cardiac cAMP-coupled receptors. Because of this lack of a considerable receptor reserve, any decrease in fl-adrenoceptor number or reduction in coupling of the receptor to the adenylate cyclase will automatically lead to a reduction in functional responses to fl-adrenoceptor stimulation.

4. ill" AND fl2-ADRENOCEPTOR CHANGES IN CHRONIC HEART F A I L U R E It is now generally accepted that fl-adrenoceptors, rather than being static entities, are dynamically regulated by a wide variety of drugs, hormones, pathological and physiological conditions. One important clinically relevant consequence of fl-adrenoceptor regulation is the phenomenon of 'desensitization', i.e. the fact that after long-term exposure of a cell to a fl-adrenoceptor agonist, the subsequent response of the cell to that agonist is blunted ('desensitization'); this is often accompanied with a decrease in the number of available cell-surface fl-adrenoceptors ('down-regulation'; see Section 2 and for reviews, Hertel and Perkins, 1984; Lefkowitz and Caron, 1985; Hausdorff et al., 1990). This holds true also for the human fl-adrenoceptors. For example, we recently have shown that chronic treatment of healthy volunteers with ill- (xamoterol) or fl2-adrenoceptor agonists (procaterol, terbutaline) desensitized fl-adrenoceptor-mediated physiological in vivo effects, but in a subtype-selective manner: after xamoterol treatment, flradrenoceptor-mediated effects were reduced (Brodde et al., 1990) after procaterol or terbutaline treatment, fl2-adrenoceptor-mediated effects were reduced (Brodde et al., 1990, 1992c). In chronic heart failure, an increase in the activity of the sympathetic nervous system in compensation for the reduced cardiac output seems to be initially a mechanism of the organism to aid the failing heart, but will subsequently lead to a down-regulation of cardiac fl-adrenoceptors (Feldman and Bristow, 1990b). Increased sympathetic activity in patients with chronic heart failure has been directly demonstrated in studies with peroneal nerve recordings of sympathetic efferent nerve traffic (Leimbach et al., 1986). Furthermore, various authors have shown that, in patients with chronic heart failure, plasma noradrenaline levels are elevated (see Cohn, 1990), and it has been suggested that plasma noradrenaline may serve as a predictor of the prognosis of the patients (Cohn et al., 1984). In fact, in 21 patients with mild-to-moderate (NYHA class II) and severe chronic heart failure (NYHA class III-class IV), Bristow et al. 0988) found a significant inverse correlation between right ventricular fl-adrenoceptor density and coronary sinus noradrenaline concentrations. Similarly, we have recently observed, in a limited number of patients with mitral valve disease and different degrees of heart failure (NYHA class III-class IV), a weak, but significant, inverse correlation between left ventricular fl-adrenoceptor density and venous plasma catecholamine concentration (Brodde et al., 1989b). The mechanism underlying the increase in plasma catecholamines in chronic heart failure is not completely understood at present. It might be due to an increase in noradrenaline spillover from organs showing increased sympathetic drive, such as heart and kidney (Hasking et al., 1986; Davis et al., 1988) and a decrease in noradrenaline clearance (Davis et al., 1988). Moreover, several studies have shown that, in patients with chronic heart failure, neuronal uptake of noradrenaline (uptake0 is markedly impaired (Perch and Naylor, 1979; Sandoval et al., 1989; Merlet et al., 1992). This might well lead to the well-known finding that, in chronic heart failure, cardiac noradrenaline stores are (at least partly) depleted, whereas plasma noradrenaline levels are elevated (Chidsey and Braunwald, 1966; Anderson et al., 1992). Thus, an enhanced release of endogenous noradrenaline (at least locally in the heart; Bristow et al., 1992, see below) and, simultaneously a decreased cardiac neuronal uptake of noradrenaline may well lead to a prolonged increase in synaptic cleft noradrenaline concentrations. Since chronic exposure of the cell to agonists causes desensitization and finally down-regulation of cell-surface receptors (see above), it is conceivable that under these pathological conditions cardiac fl-adrenoceptors are down-regulated. In fact, at present there seems to be no doubt that a common feature of chronic heart failure is a reduced cardiac fl-adrenoceptor number and functional responsiveness, and the loss in cardiac

Beta-adrenoceptors in cardiac disease

415

fl-adrenoceptor function is related to the severity of the disease (Fig. 9; see Jones et al., 1989; Bristow et aL, 1990; Feldman and Bristow, 1990a; Brodde, 1991a). This has been first shown by Bristow et al. (1982), who demonstrated, by the use of radioligand-binding studies, that, in severe heart failure, cardiac fl-adrenoceptor number was markedly depressed when compared with non-failing hearts. Subsequently, many authors have confirmed these observations using different techniques: radioligand-binding studies (for reviews, see Jones et al., 1989; Feldman and Bristow, 1990a; Brodde, 1991a), quantitative autoradiographic studies (Summers et al., 1989) and, very recently, in vivo by PET studies (Merlet et al., 1993). In patients with biventricular failure, fl-adrenoceptors are uniformly decreased over the whole ventricular area (Steinfath et al., 1992b; Pitschner et al., 1993); this decrease appears to be a real loss in fl-adrenoceptors rather than due to increased internalization, since at least three groups did not find any differences in the amount of light vesicular ('internalized') fl-adrenoceptors between non-failing and severely failing human hearts (Denniss et al., 1989a; Murphree and Saffitz, 1989; Pitschner et al., 1993). Local, rather than systemic, changes in adrenergic neurotransmitters appear to be responsible for the down-regulation of cardiac fl-adrenoceptors in severe heart failure. This suggestion is based on recent findings of Bristow et al. (1992), who demonstrated that, in patients with end-stage primary pulmonary hypertension undergoing heart-lung transplantation, only in right ventricles fl-adrenoceptor number and tissue noradrenaline content was reduced (similar to the situation in right and left ventricles of patients with biventricular failure), whereas in the left ventricles of these patients, fl-adrenoceptor number and noradrenaline content was no different from those in non-failing hearts. In line with this suggestion are in vivo data of Merlet et al. (1992), who determined, in patients with idiopathic dilated cardiomyopathy, cardiac neuronal uptake by [~25I]metaiodobenzyl-guanidine scintigraphy and, simultaneously, the positive inotropic effect of intracoronary infused dobutamine. They found that both uptake~ and the positive inotropic effect of dobutamine were decreased when compared with healthy controls; moreover, there was 80-

L

c~60-

Q.

I3-Adrenoceptor Density

I

/,0-

-~ 20E

~o ~100~

,80-

7

Maximal Positive Inotropic Effect I Evokedby Isoprenatine I

I

~60-

e~

i 5,0e~20-

".~

o

3

NYHA: NFH IHII IIHV

IV

FIG. 9. Left ventricular fl-adrenoceptor density (upper panel) and maximal positive inotropic effects evoked by isoprenaline on isolated electrically driven left ventricular preparations (lower panel) derived from patients with different degrees of heart failure. NFH, non-failing hearts; NYHA II-III and Ill-IV, patients with mitral valve disease; NYHA IV, patients with end-stage dilated and/or ischemic cardiomyopathy. Ordinate, upper panel: left ventricular fl-adrenoceptor density in fmoi (-)-[~25I]iodocyanopindolol (ICYP) specificallybound/mg protein. Lower panel: positive inotropic effect of isoprenaline in per cent of maximal Ca2+-response (that is not changed in end-stage heart failure; see Feldman and Bristow, 1990a; Brodde, 1991a). Given are means + SEM; number of experiments at the bottom of the columns. Modified from Brodde et al. (1989b) and unpublished data.

416

O.-E. BRODDE

a very strong correlation between the activity of uptake~ and the positive inotropic efficacy of dobutamine. However,/3~- and fl2-adrenoceptors are differentially changed in different forms of heart failure. In end-stage dilated cardiomyopathy, only /31-adrenoceptor number is down-regulated, while /32-adrenoceptor number is only marginally affected, if at all (Jones et al., 1989; Bristow et al., i 990; Feldman and Bristow, 1990a; Brodde, 1991a). While this might possibly also be true for patients with aortic valve disease (Michel et al., 1990; Steinfath et al., 1991), in patients with mitral valve disease (Brodde et al., 1989b; Steinfath et al., 1991) and in patients with tetralogy of Fallot (Brodde et al., 1989a), the decrease in cardiac fl-adrenoceptor number is due to a concomitant decrease in /3~- and/32-adrenoceptors. Divergent results have been reported on fl~- and/32-adrenoceptor changes in end-stage ischemic cardiomyopathy: while Brodde et al. (1989a) and Steinfath et al. (1991, 1992b) found a concomitant decrease in f i r and fl2-adrenoceptors in this disease, Bristow et al. (1991, 1992) and Ungerer et al. (1993) found the decrease in total fl-adrenoceptor number to be due selectively to/3~-adrenoceptors. Moreover, Ungerer et al. (1993) recently demonstrated that, in patients with end-stage dilated and ischemic cardiomyopathy, selectively steady-state levels of fl~-adrenoceptor mRNA are reduced, while fl2-adrenoceptor mRNA was not changed, when compared with non-failing hearts. The decrease in/3-adrenoceptor number is accompanied by a reduction in isoprenaline-activated adenylate cyclase (see Jones et al., 1989; Bristow et al., 1990; Brodde, 1991a). Since isoprenaline activates human cardiac adenylate cyclase mainly via/32-adrenoceptor stimulation (see Section 3), this indicates that even with an unchanged /32-adrenoceptor number, /32-adrenoceptor .function appears to be impaired. The reason for this might be that (a) recent data suggest that, in end-stage dilated and ischemic cardiomyopathy, steady-state mRNA levels of/3ARK and the activity of //ARK are increased (Ungerer et al., 1993), leading to enhanced phosphorylation of both/3~- and /32-adrenoceptors and by this, to an uncoupling from the adenylate cyclase and (b) several authors have convincingly demonstrated that, in end-stage heart failure, mRNA-levels (Feldman et al., 1989; Eschenhagen et al., 1992) and tissue-amount of the cardiac inhibitory guanine nucleotidebinding protein G~ is increased (see Feldman, 1991; Brodde, 1991a) thereby inhibiting cAMP formation. That the increase in G~, in fact, contributes to the reduction in/32- (and/3~-) adrenoceptor function recently has been directly demonstrated by Brown and Harding (1992). They could show that treatment of isolated cardiac myocytes from failing human myocardium with pertussis toxin, thereby inactivating Gi, increased the reduced maximal inotropic response to isoprenaline to values not significantly different from that obtained in myocytes from non-failing human myocardium. Interestingly, the increase in mRNA-levels (Eschenhagen et al., 1991) and tissue-amount of cardiac Gi-protein (Mende et al., 1992a) seen in end-stage human heart failure can be mimicked by chronic treatment of rats with isoprenaline, which supports the idea that, in end-stage heart failure, the increase in G~ might be due to the chronic exposure of the heart to noradrenaline. On the other hand, down-regulation of lymphocyte fi2-adrenoceptors by chronic terbutaline treatment is not accompanied by changes in G,-protein content (Brodde et al., 1992b) and lymphocyte G~ levels are unchanged in patients with end-stage heart failure (Maisel et al., 1990a). This indicates either that changes in the lymphocyte/3-adrenoceptor-G-protein(s) adenylate cyclase complex are not representative for changes occurring in the human heart (Brodde et al., 1989c) or that the increase in cardiac Gi following long-term/3-adrenoceptor agonist exposure is an organ (heart)-specific effect. In contrast to G~-protein, the amount of human cardiac G~-protein and its function (determined in a reconstitution assay using cyc cells) seems not be altered in chronic heart failure; the activity of the catalytic unit of the adenylate cyclase in end-stage heart failure is likewise obviously unchanged (Brodde, 1991a; Feldman, 1991). Since the human heart contains only a few spare receptors for/3-adrenoceptor mediated positive inotropic effects (see Section 3), and the amount of spare receptors declines in chronic heart failure (Fig. 10) (Brown et al., 1992), it is not surprising that the reduced/3-adrenoceptor number and the impaired ability of/3-adrenoceptor stimulation to activate adenylate cyclase are accompanied by decreased contractile responses to /3-adrenoceptor agonists. Numerous authors have shown that in isolated electrically driven human myocardial preparations (Jones et al., 1989; Feldman and Bristow, 1990a; Brodde, 1991a), in single human cardiac myocytes (Harding et al., 1990; Vescovo et al., 1992) and in vivo in patients with chronic heart failure (Gilbert et al., 1989b~ Feldman and

Beta-adrenoceptors in cardiac disease

417

f:J1- Adrenoceptors

,~/f..~"...........

--~"

80

6O c

/ /

.E x

~ / / / ~ / //p

/

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/ J / ~ ; ~ /Imp,',,

o

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,

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Receptor

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HumanLeft Papil.[ar7 Muscle .... PD2-Values • NYHA H-HI: 732 N N Y H A Ill-IV: 6.~

~ "'i ~:~

pKI -Value : 6.73 Rot Left Papittary Muscte pO . . . 2. .:.8.15 - pK[ '-" : 6. "74 ,

,

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60 80 Occupancy (°/o]

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100

FiG. 10. Plots of per cent receptor occupancy vs positive inotropic effect of isoprenaline (in the presence of 30 nM IC[ 118,551, i.e. acting solely at fl~-adrenoceptors) on isolated electrically driven left ventricular papillary muscles from patients with mitral valve disease and different degrees of heart failure (NYHA class II-III and III-IV) and from patients with end-stage dilated cardiomyopathy (NYHA class IV). For comparison, data on rat left papillary muscles are included (dotted line). Ordinate: positive inotropic effect of isoprenaline in percent of maximal response. For each group of patients, the maximal response was set to 100%. Abscissa: receptor occupancy in per cent, calculated from the pK~-value of isoprenaline (assessed by (-)-[12sI]iodocyanopindolol binding). Dashed line: line of identity. Reprinted from Brown et al. (1992), with permission of the copyright holder, Raven Press Ltd, New York.

Bristow 1990a; Brodde, 1991a), the positive ino- and chronotropic effect to fl-adrenoceptor stimulation is decreased, while that to Ca 2+ is only marginally affected. In all these studies, it was consistently found that the more advanced the disease is (i.e. the more fl-adrenoceptor number is decreased, see above), the more pronounced is the extent of decrease in maximal positive inotropic effect (Fig. 9). This supports the idea that the reason for the reduction in positive inotropic response to fl-adrenoceptor agonists is, in fact, the decrease in fl-adrenoceptor number. A more striking line of evidence for this conclusion very recently has been presented by the in vivo study of Merlet et al. (1993). These authors determined in patients with idiopathic dilated cardiomyopathy, simultaneously cardiac fl-adrenoceptor number (by PET) and the positive inotropic potency of dobutamine (by intra-coronary infusion), and found that both the cardiac fl-adrenoceptor number and the positive inotropic effect of dobutamine were reduced. In addition, a highly significant correlation between cardiac fl-adrenoceptor number and the maximal positive inotropic effect of dobutamine existed. Another important clinically relevant finding was that the in vitro studies on isolated human myocardial preparations clearly demonstrated that, in all kinds of heart failure, not only were positive inotropic responses to flj-adrenoceptor stimulation reduced (as to be expected, since in all kinds of heart failure, fl~-adrenoceptor number is reduced), but also responses to fl2-adrenoceptor stimulation were diminished (presumably due to the uncoupling of the fl2-adrenoceptors from the Gs-adenylate cyclase complex; Bristow et al., 1991, 1992; Steinfath et al., 1992a) independently of whether fl2-adrenoceptor number was reduced or not (see above). This has the clinical implication that, if acutely fl-adrenoceptor-mediated positive inotropic support is needed, non-selective full agonists should be most effective. Partial agonists should be less effective, since their effects strongly depend on the number of fl-adrenoceptors available (Ruffolo, 1982; Kenakin, 1987), and the number of fl-adrenoceptors is reduced (see above). Indirect sympathomimetics will not be very effective, because cardiac noradrenaline stores are depleted (Chidsey and Braunwald, 1966; Anderson et al., 1992). Recently, we have tested this hypothesis (Brodde et al., 1993) and have determined, on isolated electrically driven left ventricular trabeculae from explanted hearts of patients undergoing heart-transplantation because of end-stage dilated or ischemic cardiomyopathy, the positive inotropic effect of various fl-adrenoceptors agonists that differentially activate JPT

6 0 / . ~

418

O.-E.

BRODDE

TABLE I. fl-Adrenoceptor Subtype(s) Involved in the Positive Inotropic Action o f 3-Adrenoceptor Agonists in the Human Heart

Noradrenaline Dopamine Denopamine Xamoterol Dobutamine Isoprenaline Adrenaline Epinine Dopexamine Procaterot Terbutaline

/3, /3, El 3J El > BE fll ~ 32 EL 32 fll = 32

Direct and indirect (via the release of endogenous noradrenaline) Partial agonist Weak partial agonist

:

Weak partial agonist + uptake~ -inhibitor Partial agonist Partial agonist

El ~ f12

32 3=

The fl-adrenoceptor subtype(s) involved in the positive inotropic action of the fl-adrenoceptor agonists were determined from concentration-response curves in the absence and presence of the selective flradrenoceptor antagonist CGP 20712 A and the selective fl2-adrenoceptor antagonist ICI 118,551. Data from: Brodde and Zerkowski 0989), Deighton et al. (1992), Motomura et al. (1990)' Sctifers et al. (1992), Zerkowski et al. 0986).

fit- and/or fl2-adrenoceptors (see Table 1). In both dilated and ischemic cardiomyopathy, the reduction in maximal positive inotropic effects of the non-selective fl-adrenoceptor agonists isoprenaline, adrenaline and epinine (about 40-50%) was significantly less (Fig. 1 l) than that of the agonists acting mainly via fll-adrenoceptor stimulation: dobutamine (about 70-80%) or dopamine (90%). These results support the idea that, in patients with end-stage heart failure, stimulation of (even reduced or uncoupled) cardiac flz-adrenoceptors causes positive inotropic effects additive to those via flt-adrenoceptor stimulation. Thus, in these patients, acute inotropic support can be brought about more efficiently by non-selective fl-adrenoceptor agonists than by agonists acting primarily at flcadrenoceptors.

100-

Oitoted Cordiomy.opathy

"/5-

*' 50-

3 -

T

5

EPI

DOB

2s-

ISO ~100"b

ADR

DA

Ischemic Cordiomyopathy

75-

9

~

50250

ISO

ADR

EPI

D0B

DA

FIG. 11. Maximal positive inotropic effects of fl-adrenoceptor agonists on isolated electrically driven left ventricular trabeculae from patients with end-stage chronic heart failure (NYHA class IV, idiopathic dilated cardiomyopathy [upper panel], ischemic cardiomyopathy [lower panel]) undergoing heart transplantation. Ordinate: positive inotropic effect in percent of maximal Ca2+-response (that is not changed in end-stage heart failure; see Feldman and Bristow, 1990a; Brodde, 1991a). ISO, isoprenaline; ADR, adrenaline; EPI, epinine (N-methyl-dopamine); DaB, dobutamine; DA, dopamine. Given are means _ SEM; number of experiments at the bottom of the columns.

Beta-adrenoceptors in cardiac disease

419

5. CARDIAC fl-ADRENOCEPTORS IN ACUTE MYOCARDIAL ISCHEMIA Acute myocardial ischemia produces dramatic effects on the heart, including arrhythmogenic and other effects of catecholamines (Corr et al., 1978; Curtis et al., 1987; Schwartz and Zuanetti, 1988). In acute myocardial ischemia, large amounts of catecholamines are released from myocardial sympathetic nerve terminals (see Schoemig et al., 1991); as discussed in Section 4, one should, therefore, expect that cardiac fl-adrenoceptors are desensitized. However, the opposite occurs. It has consistently been found that, in acute myocardial ischemia in dog, guinea-pig and rat heart, fl-adrenoceptor number is increased (Mukherjee et al., 1979, 1982; Maisel et al., 1985, 1987; Vatner et al., 1988; Strasser et al., 1990a,b); this appears to be an increase in functional fl-adrenoceptors, since coupling to adenylate cyclase is not impaired and responses to fl-adrenoceptor stimulation are enhanced. The mechanism underlying this increase in fl-adrenoceptor number in the face of elevated endogenous catecholamines (locally in the heart) is not completely understood; it might be due to enhanced externalization (Maisel et al., 1985, 1987) or an impaired internalization (Strasser et al., 1990a,b) of the receptor. In early myocardial ischemia (~< 30 min), fl-adrenoceptor sensitization is enhanced by an impairment of the inhibitory adenylate cyclase regulation (thereby suppressing tonic inhibition of adenylate cyclase; Niroomand et al., 1992) and an increased activity of adenylate cyclase; (Maisel et al., 1985; Strasser et al., 1990a,b, 1992), while during prolonged ischemia, fl-adrenoceptor responsiveness (despite the persisting increase in receptor number; Strasser et al., 1990b) and adenylate cyclase activity decreases (Vatner et al., 1988; Susanni et al., 1989; Strasser et al., 1990a,b, 1992). Recent studies suggest that the early sensitization of adenylate cyclase in acute ischemia is linked to a concomitantly occurring activation of PKC (Strasser et al., 1992), whereas the decrease in adenylate cyclase during prolonged ischemia appears to be accompanied by a decrease of the stimulatory G protein, Gs (Karliner et al., 1989; Susanni et al., 1989; Maisel et al., 1990b). Whether similar changes occur in humans is not known at present. In this context, it is, however, interesting to note that we recently observed, in children with acyanotic congenital heart disease undergoing open heart surgery, that 1 hr of cardiopulmonary bypass with cardioplegic cardiac arrest (i.e. a process known to be accompanied by vigorously increased catecholamines; Tan et al. 1976; Reves et al., 1982) led to a marked desensitization of fl-adrenoceptor-mediated right atrial adenylate cyclase activation (Fig. 12) without affecting right atrial fl-adrenoceptor number (Schranz et al., 1993). Very similar effects have been reported by Schwinn et al. (1991) for dog left ventricular fl-adrenoceptors during 155 min of cardiopulmonary bypass with cardiac arrest. Such a fl-adrenoceptor desensitization may be the reason why many patients need inotropic support after cardiopulmonary bypass, but do not sufficiently respond to fl-adrenoceptor agonists.

6. CARDIAC fl-ADRENOCEPTORS IN THE TRANSPLANTED H U M A N HEART The opposite of desensitization (and down-regulation) is sensitization (and 'up-regulation') of fl-adrenoceptors, i.e. the phenomenon that after long-term withdrawal of endogenous catecholamines from the fl-adrenoceptor (either by denervation or by long-term receptor blockade), the subsequent response to agonists is increased (see Trendelenburg, 1963, 1966; Stiles et al., 1984; Lefkowitz and Caron, 1985; Brodde, 1989). The transplanted human heart is a denervated organ showing no evidence for re-innervation up to 12 years post-transplant (Cannom et al., 1973; Mason et al., 1976; Rowan and Billingham, 1988; Bristow 1990; Jessup and Brozena, 1990). As a denervated organ containing a markedly reduced amount of tissue noradrenaline (Bristow, 1990; Port et al., 1990a; Regitz et al., 1990), it could be expected that it might develop up-regulation of fl-adrenoceptors and/or supersensitivity of the fl-adrenoceptors to fl-adrenergic stimulation. Studies in animal models of cardiac transplantation or experimentally induced denervation have, in fact, shown an increase in myocardial fl-adrenoceptors (Lurie et al., 1983; Vatner et al., 1985). In addition, in two studies with patients after transplantation, an increased chronotropic response to isoprenaline infusion was observed (Borow et al., 1985; Yusuf et al., 1987). However, in these studies, it was not considered that the vagus influences isoprenaline effects in healthy controls (i.e. increases during isoprenaline infusion thus blunting the effects of isoprenaline, see Arnold and McDevitt, 1984) but not in heart transplant recipients, because the transplanted heart is denervated

420

O.-E. BRODDE A.13-Adrenoceptor

BI31:FJ2 -Adrenoceptor Rotio

c

cL >-

C.Aden),late C~tase Activity 800 [] PRE-CPB{10) 700 • POST-[PB(]0) i T *~aozs~s PRE'CPB o 120 ~90

.

0

ill g~sot

gTP ISO NaF FORS Mn2÷ (10pH) (100pM) (10raM} (1001aN) (10raN)

Fto. 12. Upper panel: Effect of cardiopulmonary bypass (CPB) with cardioplegic cardiac arrest on/3-adrenoceptor density (A) and//j:/32-adrenoceptor ratio (B) in right atria from children undergoing open heart surgery. Left ordinates: right atrial /3-adrenoceptor density in fmol (-)-[~2sI]iodocyanopindolol (ICYP) specifically bound/mg protein. Right ordinate: right atrial /3~:/~2-adrenoceptor ratio in per cent of total ~-adrenoceptors. Given are means _+SEM of 12 experiments. Lower panel: Effects of cardiopulmonary bypass (CPB) with cardioplegic cardiac arrest on adenylate cyclase activity in right atria from children undergoing open heart surgery. Ordinate: right atrial adenylate cyclase activity in pmol cAMP formed/mg protein/min. Given are means 4-SEM of 10 experiments. ISO, isoprenaline; FORS, forskolin. Reprinted from Schranz et al. (1993), with permission of the copyright holder, American Heart Association, La Jolla, CA.

(see above). Thus, the difference between cardiac transplant recipients and healthy controls in the isoprenaline response could be due to the fact that in healthy volunteers, but not in the transplant patients, the isoprenaline effect is attenuated by the increased vagal tone (Quigg et al., 1989). In fact, when heart transplant recipients are pretreated with atropine, the chronotropic effect of isoprenaline was not different between the native (innervated) and transplanted (denervated) atrium (Gilbert et al., 1989a). On the other hand, even in the presence of atropine, the chronotropic response to adrenaline was more pronounced in the transplanted (denervated) than in the native (innervated) atrium (Gilbert et al., 1989a). This difference may be due to the fact that adrenaline is taken up into sympathetic nerve terminals in the normal, but not in the transplanted, heart. Hence, at each dose, the concentration of adrenaline in the synaptic cleft and at the receptor is higher than in normal hearts, leading to an enhanced response. In fact, von Scheidt et al. (1992) recently demonstrated identical positive chronotropic and inotropic responses to adrenaline infusion in patients after heart transplantation and healthy controls, when the control subjects had been pretreated with desipramine, thus preventing neuronal uptake. A few data on/~-adrenoceptor number in the transplanted human heart are available at present. A general finding of these studies was that total/3-adrenoceptor number in the transplanted human heart is not significantly different from that of normal hearts, but it is significantly higher than in the explanted diseased hearts of these patients. This was demonstrated in left ventricles from previously transplanted hearts from patients with normal cardiac function who were undergoing retransplantation because of graft atherosclerosis (Port et al., 1990a) and in right ventricular endomyocardial biopsies (Denniss et al., 1989b). Three recent studies performing serial measure-

Beta-adrenoceptors in cardiac disease

421

ments of the development of fl-adrenoceptors in the transplanted human heart are in agreement with these findings. In the first study, in l0 patients, fl-adrenoceptor number and adenylate cyclase response to 10-#M isoprenaline was assessed in right ventricular endomyocardial biopsies in weekly intervals for 12 weeks after transplantation. No significant change in either parameter was observed (Cruz Caturla et al., 1992). In the second study, in 8 patients fl-adrenoceptor density had been followed up in right ventricular endomyocardial biopsies in monthly intervals over a period of 6-18 months. No consistent significant changes in fl-adrenoceptor density were observed in these patients. However, with increasing posttransplant time, fl~:fl2-adrenoceptor ratio was shifted towards fl2-adrenoceptors (Fig. 13): while immediately after transplantation, it was 80:20%, after 18 months, it was 65:35% (Brodde et al., 1991). A similar shift in the ventricular fl~:fl2-adrenoceptar ratio towards flz-adrenoceptors was also observed by Port et al. (1990b) in explanted, previously transplanted human hearts (mean posttransplant time: 21 months). These findings have been confirmed and extended by a recent study of Steinfath et al. (1992c), who determined fl-adrenoceptor number and subtype distribution in right ventricular endomyocardial biopsies taken from 100 patients 1-60 months after transplantation. Again, over the whole period, fl-adrenoceptor number was not significantly changed, although it showed after 36 months a tendency of declining. On the other hand, in agreement with our own study (see above) fl,:fl2-adrenoceptor ratio significantly changed from initially 80:20% to about 60:40% with increasing posttransplant time. The reason for this shift in the fll:fl2-adrenoceptor ratio towards fl2-adrenoceptors remains to be elucidated. However, the increase in fl2-adrenoceptors appears to be associated with an increased response to fl2-adrenoceptor stimulation. Thus, activation of adenylate cyclase by the fl2-adrenoceptor agonist terbutaline and by isoprenaline (which, in the human heart, causes adenylate cyclase 17) |

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FIG. 13. fl-Adrenoceptor development in right ventricular endomyocardial biopsy samples from eight heart transplant recipients. Upper panel: fl-adrenoceptor density in fmol (-)-[~25I]iodocyanopindolol (ICYP) specifically bound/mg protein. Lower panel: relative amount of fl~- and fl2-adrenoceptors in per cent of total fl-adrenoceptors. Abscissa: time after transplantation in weeks. The time point 0 weeks refers to biopsies taken from the donor hearts immediately prior to transplantation ('donor-biopsies'). Black square: mean + SEM of left ventricular [3-adrenoceptor density in the explanted hearts of the heart transplant recipients. Given are means + SEM; number of experiments performed in different biopsies in parentheses (upper panel) and at the bottom of the columns (lower panel), respectively. **P < 0.01, *P < 0.05 (paired Student's t-test) when compared with the same patients' mean fl~:fl_~-adrenoceptor ratio during the first nine posttransplant weeks. Reprinted from Brodde et al. (1991), with permission of the copyright holder, Springer, Heidelberg.

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activation predominantly through //2-adrenoceptor stimulation, see Section 3) was found to be markedly enhanced in ventricular membranes from transplanted human hearts (mean posttransplant time: 4-21 months; Bjornerheim et al., 1987; Port et aL, 1990b). Somewhat different results were recently reported by Chester et al. (1992). They assessed //-adrenoceptors in right ventricular endomyocardial biopsies in 43 patients with different posttransplantation times (1-13 months) and found that after 3~4 months total//- and flradrenoceptor number was increased; however, both parameters returned to control values after 5-6 months. Taken together, however, the few data available, at present, strongly indicate that //-adrenoceptor number is not up-regulated with time after cardiac transplantation, and there is no evidence for a postsynaptic//-adrenoceptor supersensitivity. However, the shift in the //l://2-adrenoceptor ratio may have (patho) physiological and clinical implications. Regulation of contractility and heart rate of the denervated human heart is obviously dependent on circulating catecholamines (Pope et al., 1980). Noradrenaline is a rather selective//~-adrenoceptor agonist (Lands et al., 1967) acting in the human heart nearly exclusively at //radrenoceptors (see Section 3), while adrenaline has virtually identical affinity to ill- and//2-adrenoceptors, (Lands et al., 1967). Thus, with increasing posttransplant time and a concomitant shift in the cardiac fll://2-adrenoceptor ratio towards fl2-adrenoceptors the transplanted heart will become more and more dependent on circulating (or possibly extraneuronal; Kennedy and Ziegler, 1991) adrenaline. Since cardiac effects of adrenaline are exaggerated in the transplanted heart (because of lack of neuronal uptake, see above), the net effect of these changes is to increase the ability of adrenaline to support the transplanted human heart. In addition, the increase in the relative amount of fl2-adrenoceptors in the transplanted human heart with increasing posttransplant time has the clinical implication that, if inotropic support is needed, non-selective and/or //2-adrenoceptor selective agonists will be superior to fl~-adrenoceptor selective agonists.

7. CONCLUSION The human heart is endowed with many receptor systems regulating heart rate and contractility. Among these the //-adrenoceptor-G-protein(s) adenylate cyclase-cAMP pathway is the most powerful mechanism for acutely increasing contractility and heart rate. Compared with the hearts of commonly used laboratory animals, the human heart shows a unique feature: it contains fl~- and //2-adrenoceptors that can mediate both positive chronotropic and inotropic effects; fl2-adrenoceptors are much more efficiently coupled to adenylate cyclase than are fl~-adrenoceptors; and the human heart contains only a few spare receptors for fl-adrenoceptor-mediated positive inotropic effects, and nearly all receptors must be occupied to reach maximal increases in contractile force. Thus, any decrease in fl-adrenoceptor number (for example, in chronic heart failure) or any situation where fl-adrenoceptors uncouple from the adenylate cyclase (for example, during cardiopulmonary bypass with cardioplegic cardiac arrest) will automatically lead to a reduced inotropic response. In chronic heart failure, human cardiac //~-adrenoceptor number (and, hence, inotropic responsiveness) is decreased presumably due to down-regulation by the (locally in the heart) enhanced release of endogenous noradrenaline, which is a rather selective fll-adrenoceptor agonist (Lands et al., 1967). Cardiac fl2-adrenoceptor number may or may not decrease; however, fl2-adrenoceptor functional responsiveness is reduced, possibly due to the fact that, in end-stage heart failure, the amount and mRNA levels of the inhibitory G-protein G. is increased, or due to enhanced phosphorylation by //ARK and, by this, uncoupling from the adenylate cyclase. Because in the human heart the//-adrenoceptor-Gs-protein-adenylate cyclase pathway is such a powerful physiological mechanism to increase heart rate and contractility (see above), these biochemical abnormalities have therapeutic implications: to give the failing heart acutely inotropic support fl-adrenoceptor agonists are still useful, whereby agonists acting at //~- and//2-adrenoceptors are superior to //-adrenoceptor agonists acting primarily at flj-adrenoceptors. For long-term treatment of patients with chronic heart failure, it is conceivable that one therapeutic goal should be to normalize the cardiac fl-adrenoceptor-G-protein adenylate cyclase system. This

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could be achieved by any intervention that prevents the chronic (over)stimulation of the system, i.e. either by blocking the receptor (with fl-adrenoceptor antagonists) or by decreasing sympathetic drive (for example, with ACE-inhibitors). Recent trials have shown beneficial effects of fl-adrenoceptor antagonists (mainly metoprolol) in patients with chronic heart failure (for a recent review, see Eichhorn, 1992), presumably because they can protect the heart from the deleterious effects of chronic exposure to high concentrations of noradrenaline. In fact, at least in the rat, propranoloi can prevent down-regulation of cardiac fl-adrenoceptors and (the simultaneous) up-regulation of G~ caused by chronic isoprenaline treatment (Eschenhagen et al., 1991; Mende et al., 1992a). Moreover, fl-adrenoceptor antagonists can up-regulate cardiac fl-adrenoceptor number in the non-failing, as well as in the severely failing, human heart (see Brodde, 1991a), thereby restoring receptor function. It should be noted, however, that it is not at all clear whether the up-regulation of fl-adrenoceptors during fl-adrenoceptor antagonist treatment is responsible for their beneficial effects in chronic heart failure, especially since it has been recently demonstrated that long-term treatment of patients with idiopathic dilated cardiomyopathy with the fl-adrenoceptor antagonist carvedilol caused improvement of left ventricular function very similar to that induced by metoprolol, but did not lead to an up-regulation of ventricular fl-adrenoceptors (Gilbert et al., 1991). Angiotensin II can facilitate noradrenaline release (Starke, 1977) and, by this, can further increase the (already increased) sympathetic tone in patients with chronic heart failure. Thus, angiontensin-converting enzyme (ACE)-inhibitors, by inhibiting the formation of angiotensin II, could decrease sympathetic tone, which may partly explain their beneficial effect in treatment of patients with chronic heart failure (Packer, 1992a,b). In favor of this idea is the observation that, in two large recent trials, ACE-inhibitors have been shown to reduce mortality mainly in those patients who had the most pronounced neurohumoral activation (Packer 1992a,b). As discussed above, a decrease in sympathetic tone should lead to a 'restoration' of cardiac fl-adrenoceptor function. The experimental data available at present support this idea. Thus, Gilbert et aL (1993) demonstrated that treatment of heart failure patients with the ACE-inhibitor lisinopril was associated with an increase in ventricular fl-adrenoceptors, but only in those patients with elevated sympathetic activity. Similarly, Mende et aL (1992b) recently showed that, in patients with idiopathic dilated cardiomyopathy, captopril significantly enhanced fll-adrenoceptor density. It appears, however, that, at present, ultimately the best therapy for severe heart failure is a successful heart transplant, since in the transplanted heart fl-adrenoceptor number and function seems to be normalized. Moreover, the data currently available do not suggest any development of super- or subsensitivity of postsynaptic cardiac fl-adrenoceptors in the transplanted human heart. However, with increasing time after heart transplantation, the relative proportion of cardiac ill- and flE-adrenoceptors is shifted towards fl2-adrenoceptors. The functional importance of this flj- and fl2-adrenoceptor redistribution is not known at present, but might indicate that, with increasing time after transplantation, the transplanted human heart will become more and more dependent on circulating adrenaline. A cknowledgements--Part of the author's work cited in this article was supported by the Deutsche Gesellschaft

ffir Herz- und Kreislaufforschung and the Deutsche Forschungsgemeinschaft(DFG Br 526/3-1).

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