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Impact of the 1-Adrenoceptor Arg389Gly Polymorphism on Heart-Rate Responses to Bisoprolol and Carvedilol in Heart-Failure Patients
T Rau1, H-D Dngen2, F Edelmann3, F Waagstein4, M Lainak5, S Dimkovi6, S Apostolovi7, ANNekovi8, W Haverkamp2, G Gelbrich9 and T Eschenhagen1
This pharmacogenetic substudy of the prospective, double-blind, randomized CIBIS-ELD trial determined the impact of the 1-adrenoceptor Arg189Gly polymorphism on heart-rate responses to bisoprolol or carvedilol in elderly patients with heart failure (421 with sinus rhythm, 107 with atrial fibrillation). Patients were randomized 1:1 to bisoprolol or carvedilol with a fortnightly dose-doubling scheme and guideline target doses. Patients with sinus rhythm responded essentially identically to bisoprolol and carvedilol, independent of genotype. Atrial fibrillation patients homozygous forArg389 had a much smaller response to carvedilol than carriers of at least one Gly389 allele (mean difference 12bpm, P < 0.00001). Carvedilol up to 2 12.5mg did not reduce heart rate in Arg389Arg homozygotes at all. Interestingly, the immediate response to carvedilol did not differ between genotypes. The Arg389Gly polymorphism has a major impact on the heart-rate response to carvedilol (but not bisoprolol) in patients with heart failure plus atrial fibrillation. Bisoprolol, carvedilol, and metoprolol have well-documented life-prolonging effects in patients with chronic heart failure and therefore constitute a cornerstone in the treatment of this frequent syndrome.1,2 The common molecular target of -blockersthe 1-adrenoceptorexhibits a frequent singlenucleotide polymorphism that causes a change of arginine (Arg) at position 389 to glycine (Gly).3,4 Approximately 40% of Caucasians are heterozygous and approximately 7% homozygous for the rarer Gly389 variant. This polymorphism has been intensely studied because, when it is overexpressed in heterologous cell systems, it exerts markedly less maximal stimulation of cyclic adenosine monophosphate (cAMP) synthesis than the Arg389 variant.3,5 The clinical settings in which the impact of this polymorphism was investigated and the reported outcomes differ widely, making it difficult to draw general conclusions as to the clinical relevance of the data (see the Discussion section). We therefore set out to investigate the impact of the Arg389Gly polymorphism on the heart ratelowering effects of -blockers in a prespecified pharmacogenetics trial accompanying theCardiac Insufficiency Bisoprolol Study in Elderly study (CIBIS-ELD). CIBIS-ELD was a randomized double-blind trial that compared the tolerability of bisoprolol and carvedilol in elderly patients with heart failure. The design and the main results have been published.6,7 The clinical effects of the randomized -blockers were examined at titration visits scheduled at 14-day intervals. At these visits, the dose of the -blocker was doubled when this was clinically possible. Thus, the data set generally contains sequential heart rate readings in the same patient at different doses. Of the 883 patients enrolled in CIBIS-ELD, 528 patients were studied in this accompanying pharmacogenetics trial. The three main findings of CIBIS-ELD were as follows: (i) the overall tolerability of the -blockers was relatively low, and only ~25% of the elderly patients with heart failure reached the primary end point (guideline target dose) with the specified titration schedule; (ii) bisoprolol caused a greater reduction in heart rate (P = 0.008); and (iii) bradycardia was more frequent in bisoprololtreated patients.7 In this accompanying pharmacogenetics study, we investigated whether the ADRB1-Arg389Gly polymorphism influences the heart ratelowering effects of the two -blockers and, if so, whether the effects differ between thetwo.

The first two authors contributed equally to this work. 1Department of Experimental Pharmacology and Toxicology and Cardiovascular Research Centre, University Medical Centre Hamburg Eppendorf, Hamburg,Germany; 2Charit, Campus Virchow, Department of Internal MedicineCardiology, Berlin, Germany; 3Department for Cardiology and Pneumonology, Georg-August-Universitt Gttingen, Gttingen, Germany; 4Sahlgrenska University Hospital, Wallenberg Laboratory, Gteborg, Sweden; 5University Clinic of Respiratory and Allergic Diseases, Department of Cardiology, Golnik, Slovenia; 6Cardiology Department, Zvezdara University Medical Center, Belgrade, Serbia; 7Clinical Centre Ni, Department of Cardiology, Ni, Serbia; 8University Clinical Hospital Zemun, Department of Cardiology, Belgrade, Serbia; 9University of Leipzig, Clinical Trial Center, Leipzig, Germany. Correspondence:T Eschenhagen (t.eschenhagen@uke.de) Received 28 October 2011; accepted 27 January 2012; advance online publication 23 May 2012. doi:10.1038/clpt.2012.18
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RESULTS Patient characteristics

CIBIS-ELD included 876 patients in the primary analysis (431 bisoprolol, 445 carvedilol) and 782 in the follow-up analysis (386/396).7 We report here on a random sample of 528 patients with the following inclusion criteria: (i) written informed consent was obtained, (ii) DNA samples of sufficient quality were available, (iii) at least one electrocardiogram (ECG) during randomized treatment was available, and (iv) the patient was in either sinus rhythm (n = 421) or atrial
100 Heart rate 1 SEM (1/min) 90 80 70 60 50
Bisoprolol Arg/Arg Bisoprolol Gly/X Carvedilol Arg/Arg Carvedilol Gly/X

fibrillation (n = 107) at baseline. On average, 4.4 ECGs were available per patient during randomized treatment in addition to the mandatory baseline ECG. Patient numbers varied over time (Figures 13). There were three reasons for this. First, patients who were pretreated with a -blocker at 25% of the target dose were, by study protocol, not treated with the first dosage level (12.5%), which explains the lower numbers at step 1 (Figure3). Second, a relevant fraction of patients did not reach the third or fourth titration step because of intolerance to the medication and therefore did not have a late
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Figure 1 Uncorrected heart rates at baseline and during randomized treatment categorized according to cardiac rhythm, -blocker, and ADRB1 genotype. Numbers in parentheses denote the numbers of patients. Arg, arginine; FU, follow-up; Gly, glycine.

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Figure 2 Model-derived estimated mean heart rates at the different clinical visits during randomized treatment. (a) Patients with sinus rhythm. (b) Patients with atrial fibrillation. In atrial fibrillation patients, carvedilol-treated patients with an Arg/Arg genotype differed from carvedilol-treated patients with a Gly/Xgenotype (P < 0.0001) and from bisoprolol-treated patients with an Arg/Arg genotype (P < 0.0001). Statistical significance at individual time points is indicated by asterisks for the comparison of carvedilol-treated Arg389-homozygous patients vs. carvedilol-treated patients with Gly/X genotype and by crosses for carvedilol-treated patients homozygous for Arg389 vs. bisoprolol-treated patients with an Arg/Arg genotype. Numbers in parentheses denote the numbers of patients. Arg, arginine; FU, follow-up; Gly, glycine.
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a
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Figure 3 Corrected mean heart rates at the dose levels reached during randomized treatment. (a) Patients with sinus rhythm. (b) Patients with atrial fibrillation. Patients in atrial fibrillation treated with carvedilol and homozygous for Arg389 had higher heart rates than carvedilol-treated patients with a Gly/X-genotype (P < 0.0001) or bisoprolol-treated patients with the same genotype (P < 0.0001). Statistical significance in patients with atrial fibrillation at the dose levels is indicated by asterisks (Arg/Arg vs. Gly/X patients treated with carvedilol) and crosses (carvedilol- vs. bisoprolol-treated Arg/Arg-genotype patients). Numbers in parentheses denote the number of observations and individual patients (n) at the different dose levels. Arg, arginine; Gly, glycine.

titration visit (Figures 13). Finally, some patients did not attend all titration visits but participated in the follow-up visit (Figures 1 and 2). The pertinent characteristics of the patients allocated to bisoprolol and carvedilol are listed in Table1. In the bisoprolol group, 117 patients were -blocker-naive, 87 were on a selective -blocker, and 60 were on carvedilol before entry into the study. The corresponding numbers for carvedilol were 104, 96, and 64, respectively. Overall, the genotype frequencies and pertinent clinical characteristics were well balanced between the treatment arms. The frequencies of Arg/Arg ( n = 284), Arg/Gly (n = 200), and Gly/Gly (n = 44) genotypes were 53.8, 37.9, and 8.3%, respectively, which is in accordance with published data for Caucasians. The patients were also genotyped for the hepatic CYP2D6 gene (nine most frequent alleles) and the Ser49Gly polymorphism of the 1-adrenoceptor. No association was observed between these polymorphisms and either heart rate or response to bisoprolol or carvedilol (data not shown).
Baseline heart rates

Heart-rate responses to randomized treatment

Baseline heart rates were significantly lower in patients with sinus rhythm than in those with atrial fibrillation and in patients pretreated with a -blocker than in those who were -blocker-naive (P < 0.0001 for both comparisons). Before enrollment, 308 patients (58.3%) were treated with a -blocker (one-fourth target dose, n = 226, 42.8%; one-eighth target dose, n = 82, 15.5%). At baseline, the ADRB1 genotype was not significantly associated with differences in heart rates in patients subsequently randomized to carvedilol or bisoprolol (Table1).

Figure1a and b show uncorrected heart rates at baseline and the different titration time points (2, 4, 6, and 8 weeks after start of randomized treatment and ~12 weeks for follow-up) categorized according to -blocker allocated, genotype, and cardiac rhythm. In patients with sinus rhythm, bisoprolol and carvedilol lowered heart rates from ~70 to ~64bpm with no genotype-related effects and no apparent differences between the effects of the two -blockers. In patients with atrial fibrillation, the overall heart ratelowering response to -blockers (from ~85 to ~72bpm) was larger than in those with sinus rhythm (from ~70 to ~64bpm) and showed clear differences in effects between the two -blockers and also between the two genotypes. Patients homozygous for the Arg389 allele responded less well than patients carrying at least one Gly389-allele, and this difference was restricted to treatment with carvedilol. Genotype had no apparent effect on heart-rate lowering by bisoprolol in patients with atrial fibrillation. Given the structure of the data set, with differing numbers of study visits available for individual patients and variable progression of dosing over time, the data were analyzed using mixed linear modeling, with correction for the individual heart rates at baseline. In addition to the expected highly significant impact of the individual baseline heart rate, pretreatment with a -blocker and the underlying rhythm (all P < 0.0001), the analyses revealed a highly significant interaction between the ADRB1 genotype and the -blocker allocated (P < 0.0001). The response was further modified by the type of rhythm present in an individual patient (P < 0.0036). Figure2a and b depict the model-derived mean heart rates during treatment categorized according to -blocker allocated, genotype, and underlying
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Table 1 Pertinent patient characteristics according to randomized -blocker or ADRB1 genotype
Randomized to Bisoprolol (n = 264) Age mean SD Male sex: n (%) Sinus rhythm (%) Atrial fibrillation (%) Heart rate -Blocker dose at baseline None (%) 1/8 of target dose (%) 1/4 of target dose (%) Predominant cause of heart failure Coronary artery disease Hypertension Cardiomyopathy Other/indeterminate NYHA class I II III IV ADRB1 genotype Arg/Arg Gly/Arg Gly/Gly 140 (53.0) 101 (38.3) 23 (8.7) 144 (54.5) 99 (37.5) 21 (8.0) 10 (3.8) 176 (66.7) 77 (29.2) 1 (0.4) 13 (4.9) 173 (65.5) 78 (29.5) 0 (0) 9 (3.2) 193 (64.4) 91 (32.0) 1 (0.4) 14 (5.7) 166 (68.0) 64 (26.2) 0 (0) 0.20 111 (42.0) 95 (36.0) 20 (7.6) 38 (14.4) 113 (42.8) 104 (39.4) 17 (6.4) 30 (11.4) 112 (39.4) 106 (37.7 25 (8.8) 41 (14.4) 112 (45.9) 90 (45.9) 12 (4.9) 30 (12.3) 0.20 116 (43.9) 42 (15.9) 106 (40.2) 104 (39.4) 40 (15.2) 120 (45.5) 111 (39.1) 47 (16.5) 126 (44.4) 109 (44.7) 35 (14.3) 100 (41.0) 0.26a 72.3 5.7 158 (59.8) 211 (79.9) 53 (20.1) 74.3 15.5 Carvedilol (n = 264) 72.2 5.5 155 (58.7) 210 (79.5) 54 (20.5) 72.9 13.5 Arg/Arg (n = 284) 72.3 5.6 166 (58.5) 221 (77.8) 63 (22.2) 74.4 14.4 Genotype Gly/X (n = 244) 72.2 5.6 147 (60.2) 200 (82.0) 44 (18.0) 72.8 14.7 P value 0.79 0.72 0.28 0.20

Arg, arginine; Gly, glycine; NYHA, New York Heart Association. Counts (percentages) are given for categorical variables. Continuous variables are given by mean and SD. P values were calculated by 2 or Fishers exact test and t-test for the association of the genotypes with categorical and continuous variables, respectively. No P values were calculated for the association between the -blocker and baseline characteristics because the allocation of the drug was random.
aMannWhitney test.

rhythm. An impact of genotype was evident only in patients with atrial fibrillation who were receiving carvedilol. In these patients, heart rates during titration were significantly higher in those homozygous for Arg389 than in those carrying at least one Gly389-allele (P < 0.0001). Also, in patients with atrial fibrillation, the heart rates in the Arg389-homozygous group were significantly higher with carvedilol treatment than with bisoprolol treatment (P < 0.0001). The differences in heart rates between the groups leveled off over the duration of the titration period; however, in carvedilol-treated patients with atrial fibrillation the impact of the genotype remained significant during the entire study period. The lowering of heart rate with time in the former analysis is caused mainly by the study protocol of progressive dose increase. The dose levels of -blockers achieved at the different time points did not differ between bisoprolol- and carvedilol-treated patients (P > 0.2 for all time points). To address the impact of the dose levels more directly, we used the actual dosing levels reached and reanalyzed the data using a mixed linear model. The derived values for mean heart rates are shown in Figure3a and b. The
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results of this reanalysis fully supported those obtained from the earlier analysis. In patients with atrial fibrillation, the mean difference between the ADRB1 genotypes in carvedilol-treated patients during randomized treatment was estimated at 12bpm, and the difference between carvedilol- and bisoprolol-treated patients was estimated at 8bpm (P = 0.00029). Once more, there was no discernible effect of genotype in bisoprolol-treated patients, regardless of the underlying rhythm (all P > 0.08). In addition, we addressed the temporal emergence of genotype-associated differences. To this end, we analyzed the heartrate effects evoked acutely by administration of the first dose of the allocated -blocker (Figure4a and b). The effect of the first dose was monitored by pulse-counting with the patient in a seated position, immediately after administration and again 2h later. Pulse data immediately after administration of the randomized dose were available for 524 (99.2%) patients, data after 1h for 518 (98.1%) patients, and data after 2h for 455 (86.2%) patients. As expected, the two -blockers acutely lowered heart rates. Unexpectedly, however, the magnitude of this immediate effect was independent of the ADRB1 genotype and the type
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a
Heart rate 1 SEM (1/min) Sinus rhythm
Bisoprolol Arg/Arg (89) Bisoprolol Gly/X (79) Carvedilol Arg/Arg (76) Carvedilol Gly/X (79)

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100 Heart rate 1 SEM (1/min)

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Figure 4 Mean heart rates immediately before and after initiation of the randomized treatment and at titration visits T1 and T2 in patients for whom pulse data were available from the phase immediately after treatment start. (a) Patients with sinus rhythm. (b) Patients with atrial fibrillation. In all patient subgroups, the start of the randomized treatment was associated with heart-rate lowering. However, in contrast to all other subgroups, the effect was not sustained in atrial fibrillation patients with an Arg/Arg-genotype who were treated with carvedilol. Arg, arginine; Gly, glycine.

of -blocker used, both in patients with sinus rhythm and in those with atrial fibrillation. By contrast, there were significant and large (~13bpm) genotype-related differences in carvediloltreated patients with atrial fibrillation at the first and second titration visits (as similarly shown in Figures 13). Under continuous exposure to carvedilol, Arg/Arg-patients with atrial fibrillation experienced a marked (~12bpm) increase in heart rate. In other words, the genotype-dependent difference in these patients emerged subacutely within the time between the starting visit and the first titration visit.
DISCUSSION

In this prespecified pharmacogenetics substudy of the ran domized CIBIS-ELD trial, we investigated whether the wellknown and frequent polymorphism of the 1-adrenoceptor (Arg389Gly) modulates heart-rate responses in elderly patients with heart failure during the period when dosages of carvedilol or bisoprolol are being titrated. The first main finding was that the Arg389Gly polymorphism markedly affected the heart rate lowering effect of carvedilol, with patients homozygous for Arg389 responding less than Gly389 carriers. This effect was restricted to patients with atrial fibrillation. Those who were Arg389-homozygous among the group with atrial fibrillation showed almost no heart-rate lowering from baseline when treated with carvedilol, even at doses reaching guideline target doses. Second, this genotype-specific difference in -blocker effects emerged subacutely (judging from the fact that the immediate heart-rate lowering effect of the first dose administered did not differ among patients with different genotypes and -blockers). Finally, the ADRB1 polymorphism did not affect the heart ratelowering effect of bisoprolol, irrespective of whether the patient had atrial fibrillation or sinus rhythm. The genotype-based differences as well as the -blockerrelated differences were large in patients with atrial fibrillation.

The difference in heart-rate lowering by carvedilol between Arg/ Arg patients and Gly/X patients amounted to 12bpm. There was a similar level of difference when comparing the heart rate lowering effects of carvedilol vs. bisopropol in Arg/Arg genotype patients with atrial fibrillation. These numbers need to be compared with the overall heart ratelowering effect of the two -blockers in the entire cohort, which amounted to ~6bpm in patients with sinus rhythm and ~13bpm in patients with atrial fibrillation. Approximately 50% of patients in the total cohort were homozygous for Arg/Arg, and 20% had atrial fibrillation. Consequently the group affected comprises 10% of the total cohort with heart failure. These calculations suggest that the marked genotype-specific differences in patients with atrial fibrillation contributed to the small difference (2.1bpm) between the heart-rate effects of bisoprolol and carvedilol reported in the main study.7 At present, we are not able to explain the molecular mechanism underlying the interactions among the Arg389Gly genotype, the -blocker allocated, and the time point during treatment; however, we can exclude a few candidates. For example, the smaller heart ratelowering effect of carvedilol observed in the main trial had initially been attributed to a putative mechanism involving the 1-adrenoceptor-blocking effect of carvedilol, giving rise to a counteracting sympathetic nervous activation. However, at least three observations argue against this hypothesis. The difference between the effects of carvedilol and bisoprolol was restricted to patients who were Arg389 homozygotes, to patients in atrial fibrillation, and to the chronic phase of treatment. The 1-adrenoceptor activity of carvedilol, if relevant, should have been seen in all these groups and, indeed, in the acute phase of treatment, particularly because 1-adrenoceptor antagonists are well known for first-dose phenomena and tachyphylaxis of their blood pressurelowering effect. It is interesting that the nonresponse of Arg389-homozygous carriers to carvedilol
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is the opposite of what would be expected on the basis of the higher activity of this variant. It also contradicts published data suggesting, if anything, that stronger responses to -blockers would be expected for this genotype. Although direct experimental evidence is lacking, some ideas regarding the mechanisms involved may be put forward to explain this discrepancy. Bisoprolol is characterized as a pure 1-preferential antagonist that has no major effects on other receptor types. By contrast, carvedilol is a biased agonist at the 1- and 2-adrenoceptors.8,9 This term has been coined to describe a divergence in the activation of intracellular signaling pathways evoked by binding to the same receptor. Carvedilol is an antagonist with respect to classic 1- and 2-pathways (e.g., no increase in cAMP production). However, when carvedilol binds to the receptor, it activates arrestin-dependent, nonclassic, 1-dependent signal pathways, for instance, activation and internalization of EGF receptors with subsequent phosphorylation of mitogen-activated protein kinases.8,9 Activation of the latter pathways is shared by agonists such as isoprenaline and noradrenaline, hence the terms biased agonism and collateral efficacy.10 The onset of these signaling processes is rapid, but the consequences may become apparent only with time. The functional consequences of the activation of these nonclassic -adrenergic pathways in atrial and ventricular myocytes are largely unknown, although epidermal growth factor receptor activation has been viewed as being beneficial.11 When overexpressed at high levels in animal models, different ADRB1 receptor variants lead to different phenotypes. Arg389-overexpressing mice developed ventricular dysfunction and fibrosis whereas Gly389 mice did not show a similarly severe phenotype despite the fact that they also had increased cardiac cAMP levels.12 Gene expression patterns in these models suggested an important contribution of cAMP-independent effects to the phenotype.13 When these observations are integrated with our findings, the hypothesis may be put forward that the nonclassic effects of carvedilol are modulated by the ADRB1 genotype. The biased agonism of carvedilol could modulate the phenotype of the atrioventricular node and/or of the atrial myocardium in Arg389 homozygotes, resulting in a reduced AV-nodal filter function and/or increased atrial wavelet frequency in patients with atrial fibrillation. These changes would abrogate the acute effects of -blockade after administration of the first dose. As stated earlier, direct experimental evidence supporting this hypothesis is currently lacking. The impact of the Arg389Gly polymorphism in humans on classic -adrenergic signaling pathways (cAMP-production, inotropic, or chronotropic responses) and on clinical effects during -blocker treatment remains incompletely resolved. On the one hand, overexpression studies have repeatedly shown that the Gly389 variant gives rise to much less (80%) stimulation of cAMP production than the Arg389 variant.3,5 The affinities of these two variants toward antagonists (including bisoprolol and carvedilol) are similar.5 However, only the Arg389 variant appears to have high-affinity states for agonists. The lower efficacy of the Gly389 variant has been attributed to diminished
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Gs coupling.3 On the other hand, despite this difference in receptor properties after overexpression, human in vitro and in vivo data are inconsistent. Some reports show no effects of the Arg389Gly polymorphism on contractile force and its -adrenergic stimulation in isolated human myocardium,14 whereas others show a lower agonist potency of noradrenaline in preparations made from Gly389 homozygotes15 or a lower efficacy (maximum inotropic effect) of isoprenaline without change in the half maximal effective concentration.16 There is similar variability in data on in vivo effects (e.g., during dobutamine stress echo).17,18 Importantly, however, where differences were reported, the results pointed toward stronger activity of the Arg389 variant. Studies of the impact of the Arg389Gly polymorphism on the clinical efficacy of -blockers in patients with heart failure also yielded mixed results.19 The only large, prespecified, pharmacogenetics survival trial (BEST) reported improved survival and reduced hospitalization rates in Arg389 homozygotes treated with bucindolol. By contrast, Gly389 carriers did not profit from bucindolol, and survival in placebo-treated patients was not significantly influenced by genotype.16 Post hoc analyses of other heart-failure studies (randomized and registers) with end points on prognosis or functional improvement yielded conflicting, mostly negative, results.1921 Our study now provides evidence that the ADRB1 genotype has an important impact on the heart ratelowering effect of carvedilol but has no such effect on bisoprolol. Although these findings were initially surprising, the smaller heart rate lowering effect of carvedilol in patients with atrial fibrillation is in good accordance with data from two studies. One small randomized trial (47 patients) showed a formal increase in heart rate after monotherapy with carvedilol in patients with heart failure plus atrial fibrillation.22 We observed the same effect in patients with atrial fibrillation who were randomized to carvedilol (Figures 1 and 4). We can explain the increase in the first phase of titration by the fact that 60% of the patients (160/264) had been pretreated with a -blocker that, in the majority of cases, was not carvedilol (carvedilol was used in 96/160 patients, 36% of the total). Therefore, the increase in heart rate was very likely the consequence of the washout of a heart ratelowering -blocker. A register study in Japanese patients with heart failure showed a significantly lower heart ratelowering effect by carvedilol than by bisoprolol in the entire cohort and also within the subgroup with atrial fibrillation.23 Ours is, to our knowledge, the first prospective trial that specifically addressed the impact of the genotype on rate responses in patients with heart failure randomized to two different -blockers with different pharmacologic profiles. During the review process of this study, a paper was published that demonstrated, in a cohort of patients with atrial fibrillation, that the heart ratelowering response rate was higher in Gly389 carriers.24 Interestingly, the most striking difference between carriers of Arg/Arg and Gly/x with respect to the drug dose necessary to achieve adequate rate control was seen in those receiving carvedilol (greater than twofold). Bisoprolol was not studied. Together, these studies provide strong evidence for the
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relevance of the Arg389Gly genotype in heart-rate control in patients with atrial fibrillation. Our study is limited by the lack of data on outcome. The smaller extent of heart-rate lowering by carvedilol in patients with atrial fibrillation and an Arg/Arg genotype does not necessarily translate into differences in prognosis. In fact, evidence exists that carvedilol is effective in patients with heart failure plus atrial fibrillation.25 By contrast, subgroup analyses of data from CIBIS II26 and MERIT-HF27 did not show beneficial effects of bisoprolol or metoprolol in these patients. However, the confidence intervals of the effect sizes are wide, precluding any categorical conclusions; further studies are needed to address this important question. Finally, the mechanisms underlying the genotype-dependent difference between the effects of carvedilol and bisoprolol in patients with atrial fibrillation need further evaluation, as does the question of whether this difference would also be found in patients with atrial fibrillation in the absence of heart failure (i.e., lone atrial fibrillation). A second potential confounder is that 60% of the patients had been pretreated with -blockers at the time of entry into the study, and an unknown number may have received -blocker treatment and found they could not tolerate the drug. In this respect, the pretreated subgroup may represent a relatively resistant sample. However, we believe that this issue is of minor, if any, relevance, given that hypersensitivity to bisoprolol or carvedilol was an exclusion criterion of the CIBIS-ELD study.6 In conclusion, our data show that the heart ratelowering effects of carvedilol in patients with heart failure plus atrial fibrillation are modulated by the Arg389Gly polymorphism of the 1-adrenoceptor. In essence, ~50% of patients with heart failure plus atrial fibrillationthose homozygous for the ADRB1 Arg389 alleleshow little or no sustained heart-rate lowering in response to the commonly used doses of carvedilol.
CIBIS-ELD trial, patients, and genotyping. We undertook this inves-

were used. In patients with atrial fibrillation, a 12-beat average was used to determine heart rate. In patients deemed intolerant to a further dose increase, an increase to the extent prescribed would either be put off to a subsequent titration visit or to the follow-up visit (scheduled 2 weeks after the last titration visit). The prespecified pharmacogenetics substudy of CIBIS-ELD was intended to determine the impact of common gene polymorphisms that had been thought to be associated with variable responses to -blockers: CYP2D6 (ref. 6) with its nine major alleles (for its impact on hepatic drug metabolism) and the two most frequent 1-adrenoceptor polymorphisms, Arg389Gly and Ser49Gly (specified in a grant proposal for the German Research Foundation, DFG Es 88/10-1). We report here on a random sample of 528 patients who had provided consent, for whom DNA samples of sufficient quality were available, who had had at least one ECG during randomized treatment, and who were in sinus rhythm or atrial fibrillation at baseline (i.e., excluding patients with pacemakers). The data set comprises heart rates from 2,851 ECGs. A multiplex PCR with subsequent primer extension and mass-spectroscopic analysis was used for genotyping. Genotyping for ADRB1 (rs1801253) was performed by a licensed company (BioGlobe, Hamburg, Germany). Ten percent of the samples were additionally genotyped by restriction-fragment-length polymorphism analysis, using standard techniques.4
Data handling and statistics. Data were prospectively collected in standardized case-report questionnaires. No imputation of missing values was carried out the analyses presented here. Heart rates were analyzed as appropriate, using mixed linear modeling. The modeling process started with a saturated model containing explanatory variables and all possible interactions. Subsequently, nonsignificant interactions were deleted stepwise from the model. This backward elimination was continued until all P values were 0.1. The baseline heart rate was used as a covariate to correct for the individual heart rate. A heteroscedastic autoregressive covariance matrix for repeated determinations was used, and random effects were added to the covariate. As explanatory factors, we considered the type of -blocker to which the patient had been randomized (bisoprolol vs. carvedilol), pretreatment with a -blocker before enrollment (yes vs. no), the type of rhythm (sinus rhythm vs. atrial fibrillation), ADRB1Arg389Gly genotype (Arg/Arg vs. Gly/X), and the time point or the dose reached. Patients homozygous for Arg389 (Arg/Arg) were compared with patients carrying at least one Gly389-allele (Gly/X). A separate consideration of the Gly/Gly subgroup would not have yielded meaningful results, given the very small sample size. Modeling was performed using SPSS (version 19; SPSS, Chicago, IL). All P values are double-sided. P values <0.05 were considered statistically significant. ACKNOWLEdGmENTS CIBIS-ELD was an investigator-initiated trial supported by a grant from the German Ministry for Research and Education (BMBF, Competence Network Heart Failure). The study medication was a gift from Merck, Darmstadt, Germany. The sponsor had no role in data acquisition, analysis, or manuscript drafting. The pharmacogenetics substudy reported in this article was supported by a grant from the German Research Foundation (DFG Es 88/10-1 to T.E., T.R., and H.-D.D.). We are indebted to K. Wegscheider and G. Schn (both from the Department of Biostatistics and Biometry, University Medical Centre Hamburg-Eppendorf, Germany) for advice regarding the setup for mixed linear modeling. We thank June Uebeler and Melanie Quast, Hamburg, for technical assistance. AUTHOR CONTRIBUTIONS T.R. wrote the manuscript, designed research, performed research, and analyzed data. H.-D.D. designed research and performed research. F.E. performed research. F.W. designed research. M.L. performed research. S.D. performed research. S.A. performed research. A.N.N. performed research. W.H. designed research. G.G. analyzed data. T.E. wrote the manuscript and designed research. CONfLICT Of INTErEST The authors declared no conflict of interest.
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METhOdS

tigator-initiated, randomized, double-blind, parallel-group trial in patients with heart failure aged >65 years in 21 centers in Germany, 1 in Montenegro, 15 in Serbia, and 4 in Slovenia. The CIBIS-ELD protocol was approved by all relevant federal institutes for drugs and medical devices as well as by national and local ethics committees. Patients provided written informed consent, and the trial conformed to the principles outlined in the Declaration of Helsinki. Details of the CIBIS-ELD trial design have been published elsewhere.6 The study is registered with controlled-trials.com (ISRCTN 34827306). The study was a longitudinal double-blind, double-dummy trial with a 1:1 bisoprolol carvedilol randomization. The patients enrolled were those with clinical heart failure due to systolic or diastolic dysfunction. Before enrollment, patients were given a maximum of 25% of the target dose of a -blocker. -Blocker-naive patients were started at one-eighth of the guideline target doses. Patients who were not -blocker-naive at baseline were switched from the nonrandomized -blocker to the equivalent dose of the randomized -blocker. After starting the allocated treatment, dose doubling was attempted at 14-day intervals. For this purpose, clinical visits were scheduled at intervals of 2 weeks (titration visits, T1T4). That is, a patient who was -blocker-naive at study entry and tolerated the dose increases would be on the target dose at the fourth titration visit. At each titration visit, tolerability was assessed through oral interviews, clinical investigations, and ECG recordings. The standard procedure was to record a 12-lead ECG after 3min of rest for the patient in a supine position; a paper speed of 25mm/s and standardization of 10mm/1 mV

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