(Circulation. 2001;103:1048.)
© 2001 American Heart Association, Inc.
Brief Rapid Communications |
Blunted Cardiac Responses to Receptor Activation in Subjects With Thr164Ile ß2-Adrenoceptors
From the Departments of Pharmacology (O.-E.B., S.D.) and Anesthesiology (R.T., J.R.), University of Halle, Germany; the Department of Pediatric Nephrology (R.B.), University of Essen, Germany; and the Department of Pharmacology (P.A.I.), University of California at San Diego, La Jolla, Calif.
Correspondence to Paul A. Insel, MD, Department of Pharmacology, University of California at San Diego, 9500 Gilman Drive, MC 0636, La Jolla, CA 92093-0636. E-mail pinsel{at}ucsd.edu
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Abstract |
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Background—Recent evidence indicates that certain genotypes of ß2-adrenoceptors (AR) may indicate an increased risk of cardiovascular disease or an increased rate of disease progression. Of particular importance, the Thr164Ile polymorphism, which is found in
4% of humans, shows decreased
receptor signaling, blunted cardiac response when expressed in
transgenic mice, and is associated with a decreased survival rate in
patients with congestive heart failure. Methods and Results—In this study, we compared functional activity, ie, chronotropic (heart rate increases) and inotropic (duration of the electromechanical systole) responses to intravenously administered terbutaline, in 6 subjects (4 women and 2 men) who were heterozygous for Thr164Ile with the responses in 12 volunteers (6 women and 6 men) who were homozygous for wild-type (WT) ß2-AR (ie, Arg16, Gln27, and Thr164). The ß2AR polymorphism significantly affected the dose-response curves for terbutaline-induced inotropic and chronotropic responses: compared with WT individuals, subjects with the Thr164Ile receptor had substantial blunting in maximal increases in heart rate (WT, 29.7±3.9 beats/min; Ile164, 20.7±1.9 beats/min; P=0.016) and a shortening of the duration of electromechanical systole (WT, 51.9±4.5 ms; Ile164, 37.9±4.6 ms; P=0.02).
Conclusions—These data show that humans with the Ile164 genotype show blunted cardiac ß2-AR responsiveness, which may help explain the decreased survival of patients with this genotype in the setting of congestive heart failure.
Key Words: receptors, adrenergic, beta • myocardial contraction • heart rate • terbutaline • genetics
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Introduction |
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ß2-adrenoceptors (AR) are polymorphic, and there are 3 major coding sequence polymorphisms: Arg16Gly, Gln27Glu, and Thr164Ile.1 In vitro studies have shown that the functional (ligand binding and adenylyl cyclase stimulation) properties of the Arg16Gly and Gln27Glu polymorphisms do not differ from those of the wild-type (WT) ß2-AR (ie, Arg16 and Gln27); however, both polymorphisms differ from WT in their susceptibility to agonist-induced downregulation.2 In contrast, during in vitro studies, the Thr164Ile polymorphism exhibited a decreased affinity for ß2-AR agonists, a 50% reduction in agonist-stimulated adenylyl cyclase activation, and uncoupling of receptors from the Gs-protein.2 When expressed at high levels in transgenic mice, the Ile164 polymorphism shows evidence of uncoupling, decreased resting heart rate (HR), and impaired inotropic and chronotropic responses to isoproterenol.3
The aim of this study was to ascertain whether the Ile164 polymorphism of the ß2-AR endogenously expressed in humans exhibits reduced responsiveness to agonist-evoked stimulation. Therefore, we studied 18 healthy volunteers who were either homozygous for WT-ß2-AR or heterozygous for the Thr164Ile polymorphism and assessed the effect of an intravenous infusion of terbutaline on HR and systolic time intervals (as a measure of inotropy4 ). These terbutaline effects are mediated nearly exclusively by cardiac ß2-AR stimulation.5
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Methods |
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We screened 220 volunteers (aged 19 to 32 years; 113 women and 107 men) for the Arg16Gly, Gln27Glu, and Thr164Ile polymorphisms of the ß2-AR. All subjects were white and were recruited from the Halle area of Germany. To obtain human genomic DNA, 10 mL of blood were withdrawn into tubes containing EDTA, and DNA was isolated with a commercial DNA isolation kit (Pharmacia Biotech). Genotyping for the Arg16Gly and Gln27Glu polymorphism was performed as described previously6 7 ; genotyping for the Thr164Ile polymorphism was carried out as described by Aynacioglu et al8 with a few alterations: a 280-bp fragment of the coding sequence containing the Thr164Ile polymorphism of the ß2-AR gene (GenBank accession No. J02960) was amplified.
Polymerase chain reaction (PCR) conditions were as follows: 100 ng of genomic DNA was added to a solution containing 1 µmol/L forward primer (5'-GTGATCGCAGTGGATCGCTACT-3'), 1 µmol/L reverse primer (5'-AGAGCAAGACCATGATCACCAG-3'), 2.5 mmol/L MgCl2 (Perkin-Elmer, Roche Molecular Systems), 1xPCR buffer (50 mmol/L KCl, 10 mmol/L Tris-HCl, pH 8.3; Perkin-Elmer), 0.2 mmol/L deoxynucleotide triphosphate (Pharmacia Biotech), 5 U of AmpliTaq Gold (Perkin-Elmer), 5% DMSO (Sigma), and distilled H2O in a final volume of 100 µL. Temperature cycling proceeded as follows: 1 cycle at 95°C for 10 minutes to fully denature the genomic double strands for more efficient amplification; then 35 cycles, each consisting of 3 steps: 95°C for 30 s (denaturation), 58°C for 90 s (annealing), then 72°C for 90 s (extension); and finally 1 cycle of extension was performed at 72°C for 10 minutes. The PCR products, which were digested for 1 hour at 37°C with 10 U of MnlI (CCTC; MBI Fermentas) on a 2% agarose IE gel (Boehringer Mannheim), were visualized with ethidium bromide; the Thr164 variant is cut by MnlI into 3 fragments of 116, 114, and 50 bp, and Ile164 is cut into 2 fragments of 230 and 50 bp (data not shown). Direct sequencing verified the identity of the PCR products.
Seven volunteers (5 women and 2 men) were heterozygous for the Thr164Ile polymorphism (allele frequency, 3.2%; this is in good agreement with published data).1 9 Among these volunteers, 3 were homozygous for the WT-ß2-AR at position 16 (Arg) and 27 (Glu); 2 were homozygous for Arg16 and heterozygous for Gln27Glu; 1 was homozygous for Gln27 and heterozygous for Arg16Gly, and 1 was homozygous for Gly16 and Glu27. One female volunteer did not agree to participate in the study; thus, the study was performed in 6 volunteers (4 women and 2 men with a mean age of 24.7±1.3 years).
The effects of infused terbutaline were compared in the 6 volunteers who were heterozygous for the Thr164Ile polymorphism with those obtained in 12 randomly selected volunteers (6 women and 6 men with a mean age of 25.8±0.8 years) who were homozygous for WT-ß2-AR (ie, Arg16Arg, Gln27Gln, and Thr164Thr). Subjects were drug-free and of average physical fitness, and none exercised regularly. Normal health was established by medical history, physical examination, biochemical and hematological screening, and ECG to exclude asthma, diabetes mellitus, chronic pulmonary disease, hypertension, cardiac disease, and other symptoms pertaining to the cardiovascular system. All volunteers participated in the study after giving informed, written consent. The study protocol was approved by the Ethical Committee of the University Halle-Wittenberg. Volunteers and investigators were blinded regarding genotype.
After 1 hour of rest in a supine position, volunteers were
given an intravenous infusion of terbutaline
(Bricanyl, Pharmastern) at doses of 25, 50, 100,
and 150 ng · kg–1 ·
min–1 for 15 minutes each. Baseline
hemodynamics were assessed immediately before the start of infusion
(Table
).
The cardiovascular effects of intravenous terbutaline were assessed by
determining systolic (SBP) and diastolic blood pressure (DBP),
HR, and systolic time intervals exactly as recently
described.10 From the
systolic time intervals determined in this study, only data for
HR-corrected duration of electromechanical systole
(QS2c) are shown because this is the most
sensitive parameter for changes in
contractility.4
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All data given in text,
Figure,
and
Table
are expressed as mean±SEM of n experiments. For statistical analysis,
a multifactorial ANOVA for repeated measurements with a post hoc Tukey
HSD test for multiple comparisons was performed using the SYSTAT
statistical package software (Jandel Scientific);
ß2-AR genotype (2 steps) and sex (2 steps)
were factors. Dependent variables included
QS2c, HR, SBP, and DBP and used all
data from the dose-response curves, starting at the first dose, which
elicited a significant effect. The statistical procedure was corrected
for multiple measurements.
P<0.05 was considered
significant.
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Results |
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The basal values for HR, QS2c, SBP, and DBP did not differ between WT-ß2-AR and Ile164 volunteers (Table
).
Terbutaline infusion dose-dependently increased HR, shortened
QS2c
(Figure),
increased SBP, and decreased DBP (data not shown). The dose-response
curves for the terbutaline-induced HR increase and
QS2c shortening were significantly affected by
ß2AR polymorphism: maximal HR increases (WT,
29.7±3.9 beats/min; Ile164, 20.7±1.9 beats/min;
P=0.016) and
QS2c shortening (WT, 51.9±4.5 ms; Ile164,
37.9±4.6 ms; P=0.022) were
significantly lower in Ile164 volunteers than in WT volunteers. SBP
increases (WT, 15.2±1.7 mm Hg; Ile164, 9.9±1.4 mm Hg;
P=0.1) showed a similar
tendency, but DBP decreases (WT, -16.7±1.8 mm Hg; Ile164,
-17.6±1.9 mm Hg) were nearly identical in both groups. Because both groups (WT and Ile164 volunteers) consisted of male and female volunteers, we also analyzed the data with respect to possible sex differences. Sex significantly affected the dose-response curves for terbutaline-induced HR increases (P=0.001) and QS2c shortening (P=0.006), and it tended to do so for SBP increases (P=0.1). However, the differences between the 164 genotypes persisted, even among individuals of the same sex. Thus, the effects of terbutaline infusion in the 4 women with Ile164 were blunted compared with those in the 6 women with Thr164, as shown when comparing increases in HR (WT, 41.1±3.1 beats/min; Ile164, 23.5±4.3 beats/min; P<0.02) and SBP (WT, 15.8±2.1 mm Hg; Ile164, 8.1±1.7 mm Hg; P=0.1) and QS2c shortening (WT, -63.3±5.3 ms; Ile164, -40.5±10.1 ms; P=0.02). Such responses in women tended to be greater than those in men. Thus, for the 6 male WT volunteers, HR response was 18.3±2.6 beats/min, SBP was 14.2±2.9 mm Hg, and QS2c was -40.4±3.1 ms. By comparison, in the 2 men with Ile164, values for HR, SBP, and QS2c were 15.0±3.9 beats/min, 13.4±2.1 mm Hg, and 27.2±8.9 ms, respectively. The interaction between sex and polymorphism was not significant (P>0.165).
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Discussion |
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TopAbstractIntroductionMethodsResultsDiscussionReferences |
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The present data show that a reduced functional responsiveness of the Ile164 polymorphism of the ß2-AR can be demonstrated in humans in vivo, at least in the heart; these results are akin to those observed with transgenic mice that have cardiac overexpression of the Ile164 receptor.3 Thus, terbutaline-induced increases in HR and contractility were significantly lower in the Ile164 versus the WT volunteers; the same held true for terbutaline-induced SBP increases. It should be noted that we could only study volunteers who were heterozygous for the Ile164 polymorphism. The reduction in responsiveness would presumably be even greater if subjects were homozygous for polymorphism. However, such a homozygosity may be lethal because no such individuals have been identified. In this context, it is interesting to note that patients with congestive heart failure who are heterozygous for the Ile164 polymorphism have a significantly reduced survival rate compared with patients homozygous for Thr16411 ; Ile164 patients also exhibit reduced exercise capacity,12 although exercise-induced cardiac effects are predominantly mediated by ß1-AR.5
Because of the small sample size, we cannot unequivocally exclude the possibility that a sex effect might contribute to the different cardiac responses in Ile164 versus WT volunteers. Furthermore, terbutaline-induced decreases in DBP (an index of vascular ß2-AR responsiveness) were not different between the Ile164 and WT volunteers. The small number of subjects that we studied did not allow us to determine whether the Ile164 polymorphism affects vascular ß2-AR function. Recent data implicate the Arg16Gly and Gln27Glu polymorphisms in altered vascular response to infused isoproterenol.13
In conclusion, these studies show that humans with the Ile164 polymorphism of the ß2-AR show substantial blunting in cardiac response to ß2-AR activation. Such differences in response may occur physiologically and with pharmacological treatment and may also relate to observed differences in survival after the onset of congestive heart failure.
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Acknowledgments |
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Supported by the Deutsche Herzstiftung (O.-E. B.), Deutsche Forschungsgemeinschaft (grant DFG BR 526/7-1), and IPHORES (R.B.) and by a grant from the National Institutes of Health (HL58120).
Received November 10, 2000; revision received December 31, 2000; accepted January 4, 2001.
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References |
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1. Liggett SB. Beta(2)-adrenergic receptor pharmacogenetics. Am J Respir Crit Care Med. 2000;161:S197–S201.
2. Green SA, Turki J, Innis M, et al. Amino-terminal polymorphisms of the human beta 2-adrenergic receptor impart distinct agonist-promoted regulatory properties. Biochemistry. 1994;33:9414–9419.[Medline] [Order article via Infotrieve]
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Turki J, Lorenz JN,
Green SA, et al. Myocardial signaling defects and impaired cardiac
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4. Belz GG. Systolic time intervals: a method to assess cardiovascular drug effects in humans. Eur J Clin Invest. 1995;25(suppl 1):35–41.
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6. Martinez FD, Graves PE, Baldini M, et al. Association between genetic polymorphisms of the ß2-adrenoceptor and response to albuterol in children with and without a history of wheezing. J Clin Invest. 1997;100:3184–3188.[Medline] [Order article via Infotrieve]
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8. Aynacioglu AS, Cascorbi I, Gungor K, et al. Population frequency, mutation linkage and analytical methodology for the Arg16Gly, Gln27Glu and Thr164Ile polymorphisms in the beta2-adrenergic receptor among Turks. Br J Clin Pharmacol. 1999;48:761–764.[Medline] [Order article via Infotrieve]
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