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A common, functionally significant polymorphism in GRK5 (Gln41Leu) encodes a gain-of-function enzyme that enhances desensitization of the β1-adrenergic receptor. GRK5 Leu41 has been postulated to confer endogenous ‘genetic β-blockade’ and contribute to an attenuated response to β-blockers in black subjects. The effects of this GRK5 variant on sensitivity to a β-blocker have not been studied in humans. We hypothesized that the GRK5 Gln41Leu variant contributes to interindividual variability in response to β-blockade and to the ethnic difference in sensitivity between black and Caucasian individuals.
We measured the heart rate at rest and during a graded incremental exercise in 154 healthy subjects (85 white and 69 black) before and after an oral administration of 25 mg atenolol. We determined the genotypes of GRK5 (Gln41Leu), β1-adrenergic receptor (ADRB1 Ser49Gly and Arg389Gly) genotypes and plasma atenolol concentrations. The effects of genotype and covariates on sensitivity to atenolol, measured as the reduction in exercise-induced tachycardia, were determined using multiple regression analyses.
The minor allele frequency of GRK5 Leu41 was 32.6% in blacks and 0% in whites. Black individuals were less sensitive to atenolol than white individuals (p ≤ 0.011) but this was not explained by the GRK5 genotype. The GRK5 genotype had no effect on resting heart rate before (p = 0.61) and after adjustment for age, sex, ethnicity, atenolol concentrations, BMI and ADRB1 genotypes (p = 0.81). The decrease in heart rate after atenolol administration did not differ significantly according to the GRK5 genotype at rest or after exercise, before (all p > 0.14) and after statistical adjustment for covariates (all p > 0.17).
The GRK5 Gln41Leu polymorphism does not affect sensitivity to the β1-adrenergic blocker, atenolol, during acute physiological adrenergic stimulation, nor does it contribute to the ethnic differences in sensitivity to atenolol among black and Caucasian individuals.
β-adrenergic receptor antagonists (β-blockers) are effective in the treatment of ischemic heart disease, heart failure and hypertension, and are among the most widely prescribed drugs [1,2]. There are marked interindividual and ethnic differences in responsiveness to β-blockers . Variation in the gene encoding the β1-adrenergic receptor (β1-AR), ADRB1, affects response to β-blockers in vivo [4–7]; however, we recently reported that such variation does not account for ethnic differences in sensitivity to a β1-blocker between black and white individuals .
Despite their widespread use, the mechanisms underlying the interindividual and ethnic differences in response to β-blockers are poorly understood . Recently, Liggett et al. characterized a functionally significant genetic polymorphism in GRK5, a member of a family of serine/threonine kinases that phosphorylate and thus desensitize G-protein-coupled receptors (GPCRs) . This GRK5 Gln41Leu polymorphism, present in approximately 40% of black and 2% of white individuals , encodes a gain-of-function enzyme that enhances desensitization of the β1-AR in vitro. Studies in transfected cells and transgenic mice expressing Grk5 Leu41 demonstrated that increased β1-AR desensitization resulted in attenuated responses after prolonged exposure to an adrenergic agonist and, similar to pharmacological β-blockade, protected against experimental catecholamine-induced cardiomyopathy, a phenotype described as ‘genetic endogenous β-blockade’ [10,11].
Since the GRK5 Leu41 variant is 20-fold more common in black than white subjects, it has been suggested as a mechanistic explanation for attenuated responses to β-blockade in black individuals . However, the effect of the GRK5 genotype on sensitivity to β-blockers has not been studied in humans.
Therefore, we examined the effect of the GRK5 genotype on response to a β1-blocker in a highly controlled setting, using a robust measure of sensitivity to β1-blockade – the reduction of exercise-induced tachycardia. Our hypothesis was that the GRK5 Gln41Leu variant attenuates responses to β-blockade and contributes to ethnic differences in sensitivity between black and white individuals.
The study protocol was approved by the Institutional Review Board of the Vanderbilt University Medical Center (TN, USA) and subjects gave written informed consent. All subjects contributed data to a previous study and details of the study design and methods have been published . Briefly, unrelated black and white American subjects were eligible to participate if they were between the ages of 18–50 years and had no clinically significant abnormality based on medical history, physical examination, electrocardiogram and routine laboratory testing. Subjects were free of medications for at least 1 week. They also received an alcohol- and caffeine-free diet for 6 days prior to the study, which provided 150 mmol of sodium, 70 mmol of potassium and 600 mmol of calcium daily.
Subjects performed graded incremental exercise on a supine bicycle ergometer (at 25, 50 and 75 W for 2 min each) before and 2.5 h after oral administration of 25 mg atenolol (Milan Pharmaceuticals Inc., WV, USA). A digitized electrocardiogram was recorded on a personal computer at 500 MHz using the Windaq software (v. 2.20; Data Instruments Inc., OH, USA).
Genotyping for GRK5 A122T (rs17098707), corresponding to the Gln41Leu amino acid change in the translated protein, was performed by allelic discrimination with an on-demand TaqMan® 5´-nuclease assay (C-15852506_10) on an ABI 7900 real-time PCR system (Applied Biosystems, CA, USA) using validated TaqMan probes (Applied Biosystems) and a 95% quality value threshold. Genotyping for the two ADRB1 SNPs, rs1801252 and rs1801253 (corresponding to Ser49Gly, Arg389Gly), was performed by TaqMan assay as previously described . The genotyping success rate was 94.5, 99.4 and 93.3% for Ser49Gly, ADRB1 Arg389Gly and GRK5 Leu41, respectively. Plasma atenolol concentrations were determined by high-performance liquid chromatography as previously described .
Heart rate was determined from the computerized ECG recordings as the mean heart rate during the second minute of each exercise step. To examine the effect of the GRK5 genotype and potential confounders on atenolol response, multiple linear-regression analyses were performed for the following response variables:
This analysis was adjusted for the corresponding heart rate (or heart-rate AUC) before atenolol, age, sex, ethnicity, BMI, S-atenolol plasma concentrations, and ADRB1 Ser49Gly and Arg389Gly genotypes. Analyses were performed with the statistical software R . Assuming a mean heart-rate reduction at 75 W of 13.6 ± 8.4 beats per minute (bpm)  and a GRK5 Gln41Leu carrier rate of 55% among black subjects (corresponding to a minor allele frequency of approximately 0.35), a sample size of 63 black subjects would provide 80 and 90% power at an α value of 0.05 to detect a difference of 6 and 7 bpm respectively, among GRK5 genotype groups.
We studied 154 healthy subjects (85 white individuals and 69 black individuals). The subject characteristics are shown in Table 1. There were proportionally more black than white female subjects (68.1% compared with 47.1%) and black subjects had a higher BMI (26.9 ± 6.3 kg/m2 compared with 24.3 ± 3.8 kg/m2; p = 0.004) than white subjects.
The minor allele frequencies for GRK5 (0 and 32.6% in white and black subjects, respectively; p < 0.001) were in the expected range and genotype distributions (85/0/0 and 29/35/5 for Gln/Gln, Gln/Leu and Leu/Leu in white and black subjects, respectively) conformed to the Hardy–Weinberg equilibrium (χ2 = 1.64; p = 0.20 for blacks).
The GRK5 genotype had no effect on baseline cardiovascular measures before (p = 0.61) and after adjustment for age, ethnicity, sex, BMI, and ADRB1 Arg389Gly and Ser49Gly genotypes (all p = 0.81). Similarly, the GRK5 genotype did not affect the increase in heart rate during exercise, as assessed by the heart-rate AUC, before (p = 0.12) and after (p = 0.80) adjustment for covariates.
The GRK5 genotype did not affect the atenolol-induced decrease in heart rate at rest or at maximal exercise before adjustment (p = 0.14 and p = 0.31 at rest and at 75 W, respectively; Figure 1A) or after adjustment for covariates (p = 0.44 and p = 0.80 at rest and at 75 W, respectively; Table 2). Similarly, the summary measure of atenolol effect during the entire exercise period, the AUC of the atenolol-induced reduction in heart rate, was not associated with the GRK5 genotype before (p = 0.29) or after adjustment for covariates (p = 0.17). When only black subjects were considered, there was again no effect of the GRK5 genotype at rest or at maximal exercise before adjustment (p = 0.43 and p = 0.71 at rest and at 75 W, respectively; Figure 1B) or after adjustment for covariates (p = 0.65 and p = 0.91 at rest and at 75 W, respectively). The AUC of the atenolol-induced reduction in heart rate when only black subjects were considered was not associated with the GRK5 genotype before (p = 0.49) or after (p = 0.35) adjustment for covariates.
As shown in our previous analysis , atenolol reduced heart rates to a greater extent in white than in black subjects at rest (mean adjusted difference: 4.4 bpm; 95% CI: 1.7–7.2 bpm, p = 0.002; Table 2) and at maximal exercise (mean adjusted difference: 4.0 mmHg; 95% CI: 0.9–7.0, p = 0.011; Table 2), corresponding to a 1.4–1.8-fold greater decrease in heart rate in whites compared with blacks. Similarly, we confirmed our previous findings that S-atenolol plasma concentrations, male gender and ADRB1 Arg389Gly genotype were associated with a greater heart-rate reduction after atenolol consumption (Table 2).
This is the first study to examine the effects of a functional genetic variant in GRK5 on β-blocker sensitivity in humans. Our main findings are that the presence of the GRK5 Leu41 allele does not affect the response to a β-blocker during acute adrenergic stimulation in healthy subjects, nor does it contribute to the differences in sensitivity observed between black and white individuals.
Since there are no selective antagonists for specific GRK subtypes, the functional role of GRK subtypes in healthy humans can only be extrapolated from animal studies (mainly in knock-out mice) and quantitative data on protein and mRNA expression in human hearts. Both GRK2 and GRK5 are abundantly expressed in the normal human heart, in approximately similar amounts, depending on the location within the four heart chambers [12,13]. Both mediate β1-adrenoceptor phosphorylation and β-arrestin recruitment, resulting in the uncoupling from G-proteins, and therefore are believed to act as physiological regulators of β-AR activity . β1-AR desensitization also occurs in normal subjects after short-term physiological or pharmacological sympathetic stimulation . Thus, GRK2 and GRK5 are likely to contribute to the physiological desensitization of β1-ARs in humans.
The GRK5 Gln41Leu variant encodes a gain-of-function enzyme resulting in enhanced in vitro desensitization of the β1-AR . Transfected cells expressing the Leu41 variant show enhanced desensitization and a 33% lower maximal response after prolonged agonist stimulation than Gln41-expressing cells . Similarly, in vivo phenotypic differences are observed following stimulation with an agonist. The dose response to isoproterenol is shifted to the right in transgenic mice expressing Grk5 Leu41, and after prolonged infusion of isoproterenol there is enhanced desensitization and a faster decline in myocardial contractility . In keeping with this enhanced desensitization of adrenergic responses, Grk5 Leu41 transgenic mice are protected from chronic catecholamine-induced cardiomyopathy , a phenotype that mimics pharmacological β-blockade.
There is little information regarding the effects of the GRK5 Gln41Leu variant in humans. In patients with heart failure, GRK5 Leu41 is associated with improved survival. Moreover, in contrast with GRK5 Gln41 homozygotes, β-blocker therapy does not improve the outcome (death or heart transplantation) in carriers of the Leu41 allele . The observations in transgenic mice and in patients with heart failure are compatible with the concept of endogenous genetic β-blockade mediated by enhanced desensitization to adrenergic stimulation as a result of the variant GRK5 Leu41 . Thus, we hypothesized that in individuals carrying the Leu41 allele, the increase in heart rate in response to exercise would be attenuated (analogous to the shifted dose response to isoproterenol in the animal model) and that responses to atenolol would be smaller (since there would already be a phenotype analogous to endogenous β-blockade). The latter hypothesis was based on the fact that the Leu41 allele is much more common in African–Americans, a group in whom attenuated responses to β-blockers have been reported .
We previously demonstrated that attenuated responses to atenolol in black compared with white subjects were not explained by common ADRB1 variants, although these variants did affect responses to atenolol . Thus, in view of its functional characteristics and ethnic distribution, the GRK5 Gln41Leu variant was considered a potential explanation for the ethnic difference in sensitivity to β-blockade. However, we found that GRK5 Leu41 did not affect the attenuation of exercise-induced tachycardia by atenolol, nor did it contribute to ethnic differences in sensitivity to atenolol – depending on the exercise stage, white subjects had a 1.4–1.8-fold greater reduction in heart rate, a finding that was not affected after adjusting for the GRK5 genotype. Specific components of the study design warrant consideration in the interpretation of the results.
Our study had a number of strengths that maximized our ability to detect a genetic signal, as indeed we did for ADRB1 variants. These include the assessment of sensitivity to β-blockade using a robust measure (attenuation of exercise-induced tachycardia ), a carefully controlled setting, a physiological stimulus, statistical adjustment for potential genetic and environmental confounding variables, and the selection of healthy individuals to minimize the confounding effect of disease and concomitant medications on the outcome. The adrenergic stimulus (6 min of incremental exercise) represents acute, intensive β1-AR stimulation, and it could be argued that more prolonged adrenergic stimulation would be more likely to elucidate an effect on desensitization. However, desensitization can occur rapidly during adrenergic stimulation , and even at rest there is some chronic downregulation of β-receptors in humans . Nevertheless, we cannot exclude the possibility that the GRK5 Gln41Leu polymorphism could have different effects on sensitivity to β-blockers under conditions of chronic excessive sympathetic stimulation, as occurs in patients with heart failure, for example.
In human myocytes from failing hearts, GRKs are upregulated and contribute to chronic β1-AR desensitization. Upregulation of the different GRK subtypes is dynamic, complex and affected by the exact localization within the heart, concomitant drug therapy and disease stage. In heart failure, the GRK2 subtype appears to be upregulated to a greater extent than GRK5 [12,13], suggesting that the relative contribution of GRK5 to total GRK activity is lower than in the nonfailing myocardium. Thus, since the precise physiological role and regulation of the GRK5 subtype in human hearts in health and disease is poorly characterized, we cannot extrapolate our findings to subjects substantially different from our study group, such as elderly subjects, cardiac patients or patients on chronic β-blocker therapy, or disease states such as heart failure.
Our study was sufficiently powered to detect differences of approximately 40% in maximal heart-rate reduction among the GRK5 genotype groups, a magnitude similar to the ethnic difference and is potentially clinically meaningful. Larger cohorts will be necessary to exclude smaller genotype effects; however, if present, the clinical relevance of such smaller genotype effects would be uncertain.
Our results suggest that the GRK5 Gln41Leu polymorphism does not affect sensitivity to the β1-adrenergic blocker atenolol during acute physiological adrenergic stimulation, nor does it contribute to the ethnic differences observed in sensitivity to atenolol.
Studies examining the effect of the GRK5 genotype on adrenergic stimulation and β-blockade in models of chronic, moderate-intensity adrenergic stimulation will be of interest. In addition, ethnic differences in response to acute β1-blockade remain unexplained. Therefore, future studies could focus on other candidate genes in the β1-AR signal-transduction pathway with polymorphisms whose prevalence differs significantly among ethnicities.
Financial & competing interests disclosure
This study was supported by the following grants: P01 HL56693, a Pharmacogenetics Research Network Grant (U01 HL65962) and the Vanderbilt CTSA grant 1 UL 1 RR024975. The authors have no other relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript apart from those disclosed.
No writing assistance was utilized in the production of this manuscript.
Ethical conduct of research
The authors state that they have obtained appropriate institutional review board approval or have followed the principles outlined in the Declaration of Helsinki for all human or animal experimental investigations. In addition, for investigations involving human subjects, informed consent has been obtained from the participants involved.