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European Journal of Heart Failure 2008 10(1):55-59; doi:10.1016/j.ejheart.2007.10.011
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© 2008 European Society of Cardiology

Ile164 variant of β2-adrenoceptor does not influence outcome in heart failure but may interact with β blocker treatment

Mathew D. Littlejohna,*, Barry R. Palmerb, A. Mark Richardsb, Chris M. Framptonb, Anna P. Pilbrowb, Richard W. Troughtonb, Vicky A. Cameronb and Martin A. Kennedya

a Department of Pathology and Carney Centre for Pharmacogenomics, University of Otago Christchurch, PO Box 4345, Christchurch, New Zealand
b Department of Medicine, University of Otago Christchurch, PO Box 4345, Christchurch, New Zealand

* Corresponding author. Dexcel, c/o ViaLactia Biosciences (NZ) Ltd, P O Box 109-185, Newmarket, Auckland, New Zealand. E-mail address: littlem{at}vialactia.com (M. D. Littlejohn).


   Abstract
Top
 Abstract
1. Introduction
 2. Methods
 3. Results
 4. Discussion
 References
 
Background: The Ile164 variant of the β2-adrenoceptor has been shown to alter cardiovascular phenotypes and adversely affect survival in heart failure patients.

Aims: We aimed to replicate this observation by genotyping a cohort of 451 heart failure patients for the Ile164 polymorphism.

Methods: Patient outcome was recorded over a median follow-up period of 3.09 years, and genotypes were derived by multiplex amplification refractory mutation system PCR.

Results: Genotypes were obtained for 443 patients, and 3.2% of these (14 patients) were heterozygous for the Ile164 SNP. Demographic data, cardiac function and neurohormonal profiles did not differ between genotype groups. Ile164 genotype did not significantly affect survival in this cohort (Thr164 homozygotes 48.9%, Ile164 heterozygous 42.9%, p=0.66), although multivariate analysis suggested that β-blocker treatment may negatively impact survival in the heterozygote group.

Conclusion: This study suggests that the Ile164 polymorphism of the β2-adrenoceptor does not have a major impact on outcome in individuals with heart failure, although it's potential interaction with β-blockers requires further examination.

Key Words: β2-adrenoceptor • Genetic polymorphism • β-blockers • Heart failure

Received February 15, 2007; Revised September 13, 2007; Accepted October 29, 2007


   1. Introduction
Top
 Abstract
 1. Introduction
2. Methods
 3. Results
 4. Discussion
 References
 
During the progression of heart failure (HF), there is an increased activation of the sympathetic nervous system including enhancement and alteration of adrenergic signalling pathways. This increased adrenergic drive is thought to be an over-compensatory mechanism that may lead to a further decline of the failing myocardium, where the β1 and β2 adrenergic receptors (β1AR and β2AR) play a key role in mediating this endogenous sympathetic traffic. The ratio of β1ARs to β2ARs shifts from ~80:20 in healthy heart tissue to approximately 60:40 in failing ventricles [1], as a result of a 60% reduction in β1-receptor density. In contrast, β2AR density is unchanged, but responses to β2-agonists are reduced by approximately 30%, due to β2-receptor uncoupling [2]. The reduction in β1-receptor density and β2-receptor coupling results in a 50% decrease in the response to exogenously administered β-agonists. These changes may theoretically serve a cardioprotective role given that the β1AR and β2AR are the primary receptor subtypes targeted for antagonism in HF; however, they may also compromise the ability of the receptor G-protein-adenylate cyclase complex to support cardiac function at times when increased contractility is desirable [2]. The recognition of adrenergic receptors as important molecules in both the pathology and treatment of HF has led to their extensive study within these contexts.

Of the numerous variants in the β2 adrenergic receptor gene (ADRB2), an isoleucine substitution at position 164 of the receptor (Thr164Ile) has been associated with altered cardiovascular function and outcomes in HF. This markedly dysfunctional variant displays reduced ligand binding and functional uncoupling of the receptor with its effector molecule adenylyl cyclase in vitro and in transgenic mice [3,4]. Furthermore, a study examining the survival of 259 patients with HF showed that individuals carrying the Ile164 polymorphism were at a significantly increased risk of rapid progression towards the endpoints of death or transplant [5]. Given this demonstrated dysfunction, we performed a retrospective analysis of the Ile164 variant in a group of HF patients, and examined the association of this polymorphism on a variety of clinical measures and outcomes.


   2. Methods
Top
 Abstract
 1. Introduction
 2. Methods
3. Results
 4. Discussion
 References
 
Patients were from a genetic sub-study (n=451) of the Christchurch HF Registry which has been described elsewhere [6]. All patients were NYHA class IV at time of hospital admission and those who survived were NYHA class II-III at time of discharge. There were no significant differences in demographics, symptomatic status, clinical history, neurohormone levels, echocardiography or renal function between those sampled and the larger HF group. The study was approved by the Canterbury Ethics Committee and conformed to the principles outlined in the Declaration of Helsinki. All patients gave written, informed consent.

Demographic characteristics and treatment with diuretics, ACE inhibitors and/or β-blockers on discharge were recorded. Clinical events were determined from consulting the patient notes, the National Health Information Services and Christchurch Hospital Patient Management System databases.

Circulating levels of angiotensin II and N-terminal pro-brain natriuretic peptide (NT-proBNP) were assayed as previously described [7]. Glomerular filtration rate (GFR) was estimated by the Modification of Diet in Renal Diseases method [8]. Left ventricular internal diastolic (LVIDD) and systolic dimensions (LVISD), and mass index (LVMI) were assessed by M-mode echocardiography using a Sonos 5500 Echocardiograph (Philips, Andover, MA). Left ventricular ejection fraction (LVEF) was measured from M-mode (Teicholz formula) and by the modified Simpson's method.

Genomic DNA was extracted from whole blood as previously described [9]. β2-adrenergic receptor genotyping was performed by multiplex amplification refractory mutation system polymerase chain reaction (ARMS-PCR). Two separate PCR reactions were carried out for each sample, each containing two allele-specific primers and a single common primer. These primers were: Common Primer 5'-CAGCCAGCAGAGGGTGAAAGTGCCCATGAT-3', +491C 5'-TCTGATGGTGTGGATTGTGTCAGGCCTTGC-3', +491T 5'-TCTGATGGTGTGGATTGTGTCAGGCCTTGT-3', +523C 5'-CCTTCTTGCCCATTCAGATGCACTGGTAGA-3' and +523A 5'-CCTTCTTGCCCATTCAGATGCACTGGTAGA-3'. The SNP number denotes the nucleotide position relative to the first A of the start codon with the Ile164 SNP being +491. The ADRB2+523 SNP is a synonymous SNP and was included in the assay to aid as an internal control for the reactions. Both reactions were performed under the same thermal cycling conditions: initial template denaturation of 94 °C for 3 min, followed by 30 cycles of 94 °C for 30 s, 64 °C for 30 s and 65 °C for 30 s, with a final elongation step of 65 °C for 5 min. The 10 µl PCR reactions contained the following: 1xHotMasterTM Taq buffer, 200 µM dNTPs, 0.4 U HotMasterTM Taq DNA Polymerase (Eppendorf, Hamburg, Germany), and 1 µl (approximately 25-50 ng) of genomic DNA. Primer concentrations were: Common Primer, +523A, +523C 0.5 µM; +491C, +491 T 0.05 µM. The resultant 399 bp (+491) and 367 bp (+523) products were then visualized by electrophoresis in adjacent lanes of a 3% agarose gel and scored for the presence or absence of bands. All Ile164 genotypes were subsequently validated by restriction digest analysis with Mnl1 as described elsewhere [10].

Univariate analysis, comparing ADRB2 genotype status with other variables, was performed using {chi}2 and ANOVA tests. Skewed data (notably plasma hormones) were log transformed. Survival was assessed using Kaplan-Meier survival curves and log-rank tests. Cox proportional hazard models were used to investigate independent risk factors for all cause mortality. Ethnicity of patients was self-declared. The patient group was 90.8% European, 3.7% Maori, 4.4% non-Maori/non-European and 1.1% Not-Stated. The Thr164Ile polymorphism was only detected in the European sub-group. All analyses were performed using SPSS version 10.


   3. Results
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
4. Discussion
 References
 
The mean age of HF patients on admission was 74.2 years and 51% of patients were male (Table 1). Nearly 60% of patients had HT documented prior to admission, 23% had type 2 diabetes, and approximately one third had a previous history of HF or MI. None of the patients were treated with device therapies or received a heart transplant during the study.


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  		Table 1 Baseline characteristics and drug treatment data for the HF cohort

 
Genotypes were obtained for 443 out of 451 patient samples in the HF cohort. A total of 14 patients were identified as Ile164 heterozygotes, giving a frequency of 3.2%. No Ile164 homozygotes were found, and these genotype data were in Hardy-Weinberg equilibrium. There were no significant differences in baseline characteristics between the genotype groups (Table 1).

Mortality was recorded over a median follow-up period of 3.09 years (range 2 days-5.17 years). Survival (proportion of deaths during follow-up) did not differ between genotypes (Thr164 0.489 vs. Ile164 heterozygous 0.421, p=0.66). However, survival of Ile164 heterozygotes was different when β-blocker treatment status was also considered. The Thr164 homozygotes demonstrated the expected univariate association between β-blocker treatment and significantly improved survival during the follow-up period (p=0.002). However, this relationship appeared to be reversed in those carrying the Ile164 genotype, with only three of seven patients treated with β-blockers surviving compared to five of seven patients who were not receiving β-blockers (Fig. 1a). The interaction of ADRB2 genotype and β-blocker treatment was a significant independent predictor of survival (p=0.019, Table 2), when multivariate analysis of these survival data was performed using a Cox proportional hazards model which included the covariates of age at admission, NT-proBNP and angiotensin II levels, diabetic status, glomerular filtration rate and history of cardiac ischaemic events (previous MI) (Table 2).


Figure 01
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  		Fig. 1 Kaplan-Meier Survival Analysis of HF cohort patients a) ADRB2 Ile164 genotype subgroup and b) ADRB2 Thr164 genotype subgroup stratified by β-blocker treatment after adjustment for age, sex, log-transformed NT-proBNP and angiotensin levels, diabetic status and GFR.

 


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  		Table 2 Cox's proportional hazards model analysis of factors predictive of mortality for the HF cohort

 

   4. Discussion
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
References
 
In terms of absolute survival, our results contrast with those of Liggett and colleagues who found that CHF patients carrying the Ile164 allele had a significantly increased chance of rapid progression to the endpoints of death or transplant compared to those homozygous for the Thr164 genotype [5]. We found no such association. Given that Liggett's finding was based on 10 Ile164 patients only (out of a cohort of 259), these results need to be interpreted with a certain amount of caution. Our finding must also be interpreted with care given that it is based on 14 heterozygous patients only, however this is inherent in any analysis of a rare polymorphism, and it is interesting to compare further the findings of the Liggett et al. study and those of our own. Liggett reported that "the survival of those with the Ile164 polymorphism was not due to the use of β-blockers, as in fact none of the survivors were taking these agents" [5]. That observation is consistent with our findings given that β-blockers may have negatively impacted on the survival of these individuals in our cohort.

The finding that unfavourable Ile164 phenotype expression may be dependent on concurrent use of β-blockers must also be regarded with caution due to limited numbers of individuals within the β-blocker treated and untreated groups. Given the potential pharmacogenetic relevance of this observation, however, it is worthwhile to consider the way in which genotype and β-blocker use might combine to affect patient survival in HF. Given the reduced sensitivity of the heart to β-agonists in HF [2], the imposition of pharmacological blockade combined with reduced adrenergic responsiveness due to a dysfunctional receptor might result in excessive impairment of cardiac function. This combination of inhibitory influences on the β-receptor G-protein-adenylate cyclase complex might compromise cardiac function when increased contractility is required, and could therefore reasonably be expected to impact on patient survival.

Interestingly, six out of the seven Ile164 heterozygotes being treated with β-blockers were receiving the β1-selective compounds metoprolol and atenolol, and this was representative of β-blocker use in the HF group as a whole. An expansion of our proposed mechanism might take into consideration the alternative roles of the β2AR, and its relationship to β1AR signalling. It is well established that both β1ARs and β2ARs couple to Gs and activate adenylyl cyclase to form cAMP. However, β2ARs can also activate more recently discovered non-classical signalling pathways such as the Gi signalling cascade [11]. Gi signalling through β2ARs has been shown to have antiapoptotic effects [12,13], and although it is unknown whether the Ile164 polymorphism depresses Gi coupling in the same manner that it depresses Gs coupling, a reduction in the beneficial signalling elicited by Gi activation, in addition to β1AR antagonism, might therefore lead to an accelerated decline in heart function.

In conclusion, we found that HF patients carrying the β2AR Ile164 polymorphism did not have a significantly different clinical outcome to homozygous Thr164 individuals, although β-blocker treatment status may have negatively influenced survival in the heterozygote group. This raises the possibility that there is an identifiable group of HF patients for whom β-blocker treatment would not be indicated, although prospective replication in a larger cohort is essential to build upon these preliminary findings.


   Acknowledgements
 
We thank the participants in the HF study, Endolab and Cardiology Outpatient staff. This work was supported by grants from the Health Research Council of New Zealand, National Heart Foundation, and Canterbury Medical Research Foundation. ML was funded by a PhD scholarship from the Lottery Grants Board (NZ), APP held a Foundation of Research Science and Technology Postdoctoral Fellowship and AMR held the National Heart Foundation of New Zealand (NHF) Chair of Cardiovascular Studies.


   References
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
 References
 

  1. Bristow M.R., Ginsburg R., Umans V., et al. Beta 1- and beta 2-adrenergic-receptor subpopulations in nonfailing and failing human ventricular myocardium: coupling of both receptor subtypes to muscle contraction and selective beta 1-receptor down-regulation in heart failure. Circ Res (1986) 59:297–309.[Abstract/Free Full Text]
  2. Gilbert E.M., Olsen S.L., Renlund D.G., Bristow M.R. Beta-adrenergic receptor regulation and left ventricular function in idiopathic dilated cardiomyopathy. Am J Cardiol (1993) 71:23C–29C.[CrossRef][Medline]
  3. Green S.A., Cole G., Jacinto M., Innis M., Liggett S.B. A polymorphism of the human beta 2-adrenergic receptor within the fourth transmembrane domain alters ligand binding and functional properties of the receptor. J Biol Chem (1993) 268:23116–23121.[Abstract/Free Full Text]
  4. Turki J., Lorenz J.N., Green S.A., Donnelly E.T., Jacinto M., Liggett S.B. Myocardial signaling defects and impaired cardiac function of a human beta 2-adrenergic receptor polymorphism expressed in transgenic mice. Proc Natl Acad Sci U S A (1996) 93:10483–10488.[Abstract/Free Full Text]
  5. Liggett S.B., Wagoner L.E., Craft L.L., et al. The Ile164 beta2-adrenergic receptor polymorphism adversely affects the outcome of congestive heart failure. J Clin Invest (1998) 102:1534–1539.[Web of Science][Medline]
  6. Pilbrow A.P., Palmer B.R., Frampton C.M., et al. Angiotensinogen M235T and T174M gene polymorphisms in combination doubles the risk of mortality in heart failure. Hypertension (2007) 49(2):322–327.[Abstract/Free Full Text]
  7. Collins R.P., Palmer B.R. Evaluation of AMPD1 C34T genotype as a predictor of mortality in heart failure and post-myocardial infarction patients. Am Heart J (2006) 152:312–320.[CrossRef][Web of Science][Medline]
  8. Levey A.S., Bosch J.P., Lewis J.B., Greene T., Rogers N., Roth D. A more accurate method to estimate glomerular filtration rate from serum creatinine: a new prediction equation. Modification of Diet in Renal Disease Study Group. Ann Intern Med (1999) 130:461–470.[Abstract/Free Full Text]
  9. Palmer B., Pilbrow A., Frampton C., et al. ACE gene polymorphism interacts with LVEF and BNP levels to predict mortality following myocardial infarction. J Am Coll Cardiol (2003) 41:729–736.[Abstract/Free Full Text]
  10. Littlejohn M.D., Taylor D.R., Miller A.L., Kennedy M.A. Determination of beta2-adrenergic receptor (ADRB2) haplotypes by a multiplexed polymerase chain reaction assay. Hum Mutat (2002) 20:479.[Medline]
  11. Xiao R.P., Ji X., Lakatta E.G. Functional coupling of the beta 2-adrenoceptor to a pertussis toxin-sensitive G protein in cardiac myocytes. Mol Pharmacol (1995) 47(2):322–329.[Abstract]
  12. Zhu W.Z., Zheng M., Koch W.J., Lefkowitz R.J., Kobilka B.K., Xiao R.P. Dual modulation of cell survival and cell death by beta(2)-adrenergic signaling in adult mouse cardiac myocytes. Proc Natl Acad Sci U S A (2001) 98(4):1607–1612.[Abstract/Free Full Text]
  13. Communal C., Singh K., Sawyer D.B., Colucci W.S. Opposing effects of beta(1)- and beta(2)-adrenergic receptors on cardiac myocyte apoptosis: role of a pertussis toxin-sensitive G protein. Circulation (1999) 100(22):2210–2212.[Abstract/Free Full Text]

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