Blunted Cardiac Responses to Receptor Activation in Subjects With Thr164Ile β2-Adrenoceptors
Background—Recent evidence indicates that certain genotypes of β2-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) β2-AR (ie, Arg16, Gln27, and Thr164). The β2AR 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 β2-AR responsiveness, which may help explain the decreased survival of patients with this genotype in the setting of congestive heart failure.
β2-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) β2-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 β2-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 β2-AR endogenously expressed in humans exhibits reduced responsiveness to agonist-evoked stimulation. Therefore, we studied 18 healthy volunteers who were either homozygous for WT-β2-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 β2-AR stimulation.5
We screened 220 volunteers (aged 19 to 32 years; 113 women and 107 men) for the Arg16Gly, Gln27Glu, and Thr164Ile polymorphisms of the β2-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 β2-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), 1×PCR 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-β2-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-β2-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
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); β2-AR genotype (2 steps) and sex (2 steps) were factors. Dependent variables included ΔQS2c, ΔHR, ΔSBP, and ΔDBP 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.
The basal values for HR, QS2c, SBP, and DBP did not differ between WT-β2-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 β2AR 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).
The present data show that a reduced functional responsiveness of the Ile164 polymorphism of the β2-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 β1-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 β2-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 β2-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 β2-AR show substantial blunting in cardiac response to β2-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.
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.
- Copyright © 2001 by American Heart Association
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