Author + information
- Received May 4, 2005
- Revision received August 11, 2005
- Accepted August 22, 2005
- Published online December 6, 2005.
- Heike Bruck, MD,
- Kirsten Leineweber, PhD,
- Thomas Temme,
- Melanie Weber,
- Gerd Heusch, MD, FACC,
- Thomas Philipp, MD and
- Otto-Erich Brodde, PhD⁎ ()
- ↵⁎Reprint requests and correspondence:
Dr. Otto-Erich Brodde, University of Essen Medical School, Departments of Nephrology and Pathophysiology, Hufelandstr. 55, D-45147 Essen, Germany.
Objectives The purpose of this research was to find out whether, in humans, dobutamine-induced hemodynamic effects and increase in plasma-renin activity (PRA) might be beta1-adrenoceptor (β1AR) genotype-dependent.
Background In vitro Arg389Gly-β1AR polymorphism exhibits decreased receptor signaling.
Methods We studied 10 male homozygous Arg389-β1AR subjects and 8 male homozygous Gly389β1AR subjects; to avoid influences of codon 49 polymorphism, all were homozygous Ser49-β1AR. Subjects were infused with dobutamine (1 to 6 μg/kg/min) with or without bisoprolol (10 mg orally) pretreatment, and PRA, heart rate, contractility, and blood pressure were assessed.
Results With regard to PRA, dobutamine increased PRA more potently in Arg389-β1AR versus Gly389-β1AR subjects. Bisoprolol markedly suppressed the dobutamine-induced PRA increase in Arg389- but only marginally in Gly389-β1AR subjects. With regard to hemodynamics, dobutamine caused larger heart rate and contractility increases and diastolic blood pressure decreases in Arg389- versus Gly389-β1AR subjects. Bisoprolol reduced dobutamine-induced heart rate and contractility increases and diastolic blood pressure decreases more potently in Arg389- versus Gly389-β1AR subjects.
Conclusions Codon 389 β1AR polymorphism is a determinant not only of hemodynamic effects but also of PRA. Thus, β1AR polymorphisms may be useful for predicting therapeutic responses to βAR-blocker treatment.
Beta1-adrenoceptors (β1ARs) play an important role in regulation of cardiac function in man; furthermore, they are involved in lipolysis and renin secretion (1). There are two functionally important single nucleotide polymorphisms (SNPs) in the β1ARgene: at position 49 serine is substituted by glycine (Ser49Gly), whereby in vitro Gly49-variant was more susceptible to agonist-mediated down-regulation than Ser49-variant. At position 389 arginine is substituted by glycine (Arg389Gly); in vitro Arg389-variant has basal and isoprenaline-activated adenylyl cyclase activity three- to four-fold higher than Gly389-variant; Gly389-variant, however, was initially considered the wild-type receptor but is the minor allele (2–5).
Controversial data exist on functional consequences of Arg389Gly-β1AR polymorphism in humans; in isolated human tissues, some studies found larger agonist-induced responses in Arg389- versus Gly389-β1AR, whereas others failed to find genotype-dependent differences (5,6). Similarly, in vivo several groups failed to find, in homozygous Arg389- or Gly389-β1AR subjects, differences in exercise-induced heart rate (HR) increases mediated by cardiac β1AR (6,7). La Rosee et al. (8), however, using a modified dobutamine stress echocardiography protocol, demonstrated recently that homozygous Arg389-β1AR subjects exhibited larger inotropic and blood pressure (BP) responses than subjects carrying one or two Gly389 alleles. In contrast to all exercise studies published so far, however, La Rosee et al. (8) administered dobutamine to subjects pretreated with atropine to exclude possible counter-regulatory parasympathetic effects.
In contrast to cardiac effects, little is known about the possible impact of β1AR polymorphisms on extracardiac effects such as renin secretion, that, in humans, is mediated by renal β1AR (1). We could not find genotype-dependent differences in exercise-induced increase in plasma-renin activity (PRA) in Arg389- versus Gly389-β1AR subjects (9). The renin-angiotensin-aldosterone system (RAAS) plays an important role in BP regulation and is certainly one target, out of several, for BP-lowering effects of AR-blockers (10). Thus, it could well be that antihypertensive effects of AR-blockers are modulated by β1AR polymorphisms (11).
In this study we determined, in male homozygous Arg389- or Gly389-β1AR subjects, the effects of dobutamine on PRA and its attenuation by the β1AR selective blocker bisoprolol to find out whether there are genotype-dependent differences. Studies were performed in the absence of atropine to find out whether atropine is necessary for demonstration of β1AR genotype-dependence of dobutamine-evoked effects (8). Codon 49 SNP can modulate functional responsiveness of codon 389 SNP (12); therefore, all subjects participating in this study were homozygous Ser49-β1AR.
To obtain human genomic deoxyribonucleic acid (DNA), 10 ml of blood was withdrawn in ethylenediaminetetraacetic acid (EDTA) tubes, and DNA was extracted with the GenomicPrep Blood DNA Isolation Kit (Amersham Biosciences, Buckinghamshire, United Kingdom); β1AR genotypes at codon 49 and 389 were determined by polymerase chain reaction and subsequent restriction length polymorphism (restriction enzymes Sau96I and BstNI, respectively) with modified protocols from previously described methods (3,4).
We genotyped 201 subjects for β1AR codon 49 and 389 SNPs (Ser49Gly and Arg389Gly); 18 male subjects participated in the study (Table 1).Ten were homozygous Arg389 (mean age 25 ± 3 years), eight were homozygous Gly389 (mean age 25 ± 3 years), and all were homozygous Ser49.
All study participants gave written informed consent. The ethical committee of the University of Essen Medical School approved the study protocol. Subjects were in normal health, based on cardiovascular and other medical history, physical examination, and biochemical, hematologic, and electrocardiographic screening. No subjects took any medication, and all were nonsmokers. Subjects and investigators were blinded for β1AR haplotype. All subjects were studied in the morning in supine position after an overnight fast. Subjects were advised to avoid caffeine, alcohol, and physical exercise before each study. Room temperature was kept stable between 24°C to 26°C.
Subjects were studied on two separate days with at least a one-week interval; after arrival at the clinical laboratory at 7:30 amand after affixment of instruments, indwelling polythene catheters were positioned in right and left antecubital veins. Blood samples were drawn from the right arm; drugs were separately administered in the left arm.
After 1 h of rest in the supine position, subjects were intravenously infused with dobutamine (DobutaminSolvay/Solvay Arzneimittel, Hannover, Germany) in doses of 1, 2, 4, and 6 μg/kg/min for 15 min each (13). For bisoprolol (Concor, Merck, Darmstadt, Germany) experiments, subjects received a 10-mg tablet orally 150 min before the infusion started.
We assessed PRA and cardiovascular effects of dobutamine immediately before bisoprolol intake, immediately before start of infusion, and during the last 5 min of each dobutamine dose. Cardiovascular effects were assessed by determining systolic (SBP) and diastolic BP (DBP), HR, and systolic time intervals exactly as described elsewhere (9). From systolic time intervals, only data for HR-corrected duration of electromechanical systole (QS2c) are shown, which is the most sensitive parameter for contractility changes (14).
For PRA and plasma bisoprolol determination, 10-ml ice-cold EDTA blood was withdrawn at the given time points; PRA was assessed by radioimmunoassay (DiaSorin, Saluggia, Italy), and bisoprolol plasma levels were assessed by the high-pressure liquid chromatography method.
Data given in text and figures are mean values ± SEM of the number of experiments. Differences between baseline values of HR, QS2c, SBP, DBP, and PRA were assessed by the unpaired two-tailed Student ttest.
Dose-response curves of dobutamine-induced changes in HR, contractility, BP, and PRA were analyzed by factorial analysis of variance (ANOVA) (factors: dobutamine dose and bisoprolol pretreatment or haplotype) with Bonferroni’s post-test for multiple comparisons.
Effects of maximum dobutamine dose or bisoprolol on resting parameters were analyzed by a paired two-tailed Student ttest.
To assess bisoprolol effects on dobutamine-induced changes in HR, contractility, BP, and PRA for each dobutamine dose, individual differences in hemodynamic or PRA changes measured in the presence and absence of bisoprolol were calculated and analyzed by factorial analysis of variance (factors: dobutamine dose and haplotype) with Bonferroni’s post-test for multiple comparisons.
Power calculations for the primary end point, a delta maximal PRA increase of 1.0 ng angiotensin I formed/ml/h, revealed with the given number of 10 Arg389-β1AR and 8 Gly389-β1AR subjects a power of 75%. Statistical calculations were performed with GraphPad Prism 4.0 and GraphPad StatMate 2.0 programs (GraphPad Software, San Diego, California). A p value <0.05 was considered statistically significant.
From 201 subjects genotyped, 53% were homozygous Arg389, 10% homozygous Gly389, and 37% heterozygous Arg389Gly; thus, allele frequencies with Gly as a minor allele (f(−) = 0.28) were in agreement with previous reports and in Hardy-Weinberg equilibrium (5,6,8,9).
Basal hemodynamic parameters and PRA were not significantly different between Ser49Arg389-β1AR and Ser49Gly389-β1AR subjects (Table 1).
Effects of dobutamine
Dobutamine infusion caused dose-dependent PRA increases that were significantly larger in Arg389- versus Gly389-β1AR subjects (Fig. 1).Maximal PRA increase was 1.56 ± 0.32 ng angiotensin I formed/ml/h in Arg389- and 0.60 ± 0.20 ng/ml/h in Gly389-β1AR subjects (p < 0.05, Bonferroni adjustment with four comparisons) (Fig. 1).
HR, contractility, and BP
Maximal increases in HR (12.9 ± 2.0 beats/min vs. 5.2 ± 1.5 beats/min) and contractility (−97.0 ± 4.6 ms vs. −79.1 ± 6.1 ms) were significantly larger in Arg389- versus Gly389-β1AR subjects. A similar tendency of larger effects in Arg389- versus Gly389-β1AR subjects was also found for dobutamine-induced maximal DBP decreases, but this did not reach statistical significance. Maximal SBP increases, however, showed no genotype-dependent difference (Fig. 3).
Effects of bisoprolol on dobutamine effects
A total of 150 min after 10 mg bisoprolol orally, plasma bisoprolol levels were 48.5 ± 5.3 ng/ml in Arg389- and 49.7 ± 1.9 ng/ml in Gly389-β1AR subjects.
Bisoprolol significantly decreased basal PRA, HR, and SBP, slightly prolonged QS2c, but only marginally affected DBP (Table 1).
Bisoprolol markedly suppressed the dobutamine infusion-induced PRA increase in Arg389-β1AR subjects but did not significantly affect the dobutamine-induced (weak) PRA increase in Gly389-β1AR subjects (Fig. 1).
Moreover, bisoprolol markedly suppressed the dobutamine infusion-induced HR increase in Arg389-β1AR subjects, but did not significantly affect the (weak) dobutamine-induced HR increase in Gly389-β1AR subjects (Fig. 2).
Similarly, bisoprolol reduced the dobutamine-induced contractility increase and DBP decrease at most dobutamine doses more pronounced in Arg389- versus Gly389-β1AR subjects, although this was not statistically significant at all dobutamine doses (Figs. 2 and 3). Attenuation of the dobutamine-induced SBP increase by bisoprolol, however, was not genotype-dependent (Fig. 3).
The new finding of this study is that the β1AR agonist dobutamine evoked, via renal β1AR stimulation (1), significantly larger PRA increases in homozygous Arg389-β1AR subjects than in homozygous Gly389-β1AR subjects. Moreover, the β1AR-blocker bisoprolol inhibited dobutamine-evoked PRA increases significantly more potently in Arg389- versus Gly389-β1AR subjects. This might have important clinical implications: AR-blockers are first-line drugs for treatment of hypertension. Although the experience of antihypertensive treatment with AR-blockers has existed for nearly 40 years, the mechanism of their BP-lowering effects is still not completely understood (10). Among various hypotheses, RAAS inhibition might play an important role. Thus, various authors showed that BP-lowering effects of AR-blockers were better the higher PRA was (10). From the present results it can be predicted that Arg389-β1AR patients should have a good response to AR-blocker treatment, whereas Gly389-β1AR patients should be poor or non-responders.
In our study dobutamine evoked larger HR and contractility increases in Arg389- than in Gly389-β1AR subjects, in accordance with recent findings in chronic heart failure that exercise capacity was significantly better in Arg389- than in Gly389-β1AR patients (15). Similarly, La Rosee et al. (8) found, in subjects pretreated with atropine, that those homozygous Arg389-β1AR exhibited larger SBP and contractility responses to dobutamine than those carrying one or two Gly389 alleles. Our data now clearly show that atropine pretreatment is not necessary because also in the absence of atropinedobutamine caused significantly larger cardiac effects in Arg389- versus Gly389-β1AR subjects.
In contrast to β1AR genotype-dependent dobutamine effects (, present data), in several studies exercise-induced HR and contractility increases were not different between Arg389- and Gly389-β1AR subjects (6). Preliminary data from our group indicate that this also holds true for subjects treated with atropine.
The reason for this discrepancy in cardiac responses to exercise versus dobutamine is not completely understood. However, it should be considered that: 1) responses to exercise are strongly dependent on the physical fitness of test subjects, and it is extremely difficult to precisely control for that. Hence, subjects participating in exercise studies may be of different physical fitness, and that would evoke unpredictable results. 2) Exercise may induce more physiologic responses, whereas dobutamine infusion may induce more pharmacologic responses. 3) In all exercise studies published so far, subjects were not controlled for codon 49 SNP. Codon 49 SNP, however, can modulate functional responsiveness of codon 389 SNP; accordingly, β1AR haplotype analysis could be more important than single SNP analysis (12,16). Thus, differences obtained in exercise versus dobutamine studies could also be due to β1AR haplotype inhomogeneity of study groups. In our study, however, all subjects were homozygous Ser49-β1AR.
Recent evidence suggested that subjects exhibited larger BP and HR responses to β1AR-blockers if they carried Arg389-β1AR, and this could be modulated by codon 49 SNP (11,16). Similarly, in chronic heart failure, long-term AR-blocker treatment improved left ventricular ejection fraction significantly better in Arg389- than in Gly389-β1AR patients (17,18). However, no genotype-dependent differences during AR-blocker treatment were also published (19,20). In our study bisoprolol inhibited all dobutamine-induced hemodynamic changes, with the exception of SBP changes, in Arg389-β1AR subjects more potently than in Gly389-β1AR subjects. Thus, our data might be taken as a further indication that β1AR polymorphisms might predict hemodynamic responses to AR-blocker treatment (11).
Dobutamine infusion causes larger PRA, HR, and contractility increases in Arg389- than in Gly389-β1AR subjects. In addition, the β1AR-blocker bisoprolol inhibited PRA and cardiac responses to dobutamine more potently in Arg389- versus Gly389-β1AR subjects. Thus, β1AR polymorphisms may be useful to predict therapeutic responses to AR-blocker treatment.
The authors thank Klaus Pönicke, PhD, University of Halle, Germany, for assessment of plasma bisoprolol levels.
This work was supported by a grant of the Deutsche Forschungsgemeinschaft (DFG BR526/8-1), Bonn, Germany.
- Abbreviations and Acronyms
- blood pressure
- diastolic blood pressure
- heart rate
- plasma-renin activity
- heart rate-corrected duration of the electromechanical systole
- renin-angiotensin-aldosterone system
- systolic blood pressure
- single nucleotide polymorphism
- Received May 4, 2005.
- Revision received August 11, 2005.
- Accepted August 22, 2005.
- American College of Cardiology Foundation
- Hoffman B.B.,
- Taylor P.
- Frielle T.,
- Collins S.,
- Daniel K.W.,
- Caron M.G.,
- Lefkowitz R.J.,
- Kobilka B.K.
- Mason D.A.,
- Moore J.D.,
- Green S.A.,
- Liggett S.B.
- Cruickshank J.M.,
- Prichard B.N.C.
- Daul A.,
- Hermes U.,
- Schäfers R.F.,
- Wenzel R.,
- Von Birgelen C.,
- Brodde O.-E.