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Psychosomatic Medicine 67:168-171 (2005)
© 2005 American Psychosomatic Society

RAPID COMMUNICATION

Heart Rate Variability Is Associated With Polymorphic Variation in the Choline Transporter Gene

Serina A. Neumann, PhD, Elizabeth C. Lawrence, BA, J. Richard Jennings, PhD, Robert E. Ferrell, PhD and Stephen B. Manuck, PhD

From the Department of Psychology (S.A.N., S.B.M.), University of Pittsburgh, the Department of Human Genetics (E.C.L., R.E.F.), Graduate School of Public Health, University of Pittsburgh, and the Department of Psychiatry (J.R.J.), University of Pittsburgh Medical Center, Pittsburgh, PA.

Address correspondence and reprint requests to Serina A. Neumann, PhD, Behavioral Physiology Laboratory, 506 OEH, 4015 O’Hara Street, University of Pittsburgh, Pittsburgh, PA 15260. E-mail: neumannsa{at}msx.upmc.edu


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 METHODS
 RESULTS
 DISCUSSION
 NOTES
 REFERENCES
 
Objective: The objective of this study was to determine whether interindividual variation in parasympathetic (cholinergic) and sympathetic (adrenergic) regulation of heart rate (as estimated by frequency components of heart rate variability [HRV]) may be accounted for, in part, by genetic variation in the choline transporter, a component of acetylcholine neurotransmission.

Methods: Resting HRV estimates of high- (HF) and low-frequency (LF) power and LF/HF ratio were determined from electrocardiogram recordings collected continuously over 5 minutes in 413 white individuals of European ancestry (49% men; aged 30–54 years [mean, 44 years]). Subjects were genotyped for a single nucleotide polymorphism (SNP) located in the 3' untranslated region of the choline transporter gene (CHT1). Frequencies of the alternate CHT1 alleles, labeled G and T, were 76% and 24%.

Results: Compared with GG homozygotes, participants having any T allele had greater HF power (p <.02), lower LF power (p <.02), and lower LF/HF ratios (p <.005). Relative to men, women had lower LF power (p <.001) and lower LF/HF ratios (p <.005).

Conclusions: These findings show that polymorphic variation in the CHT1 gene is associated significantly with interindividual variability in HRV indices related to parasympathetic (cholinergic) activity.

Key Words: acetylcholine • choline transport • choline transporter gene • heart rate variability

Abbreviations: ACh = acetylcholine; CHT1 = choline transporter gene; UTR = untranslated region; HRV = heart rate variability; HF = high frequency; LF = low frequency; nu = normalized units; ln = natural log; BMI = body mass index.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
METHODS
 RESULTS
 DISCUSSION
 NOTES
 REFERENCES
 
Autonomic nervous system dysregulation, particularly low parasympathetic (vagal) activity, is associated with psychosocial risk factors for coronary heart disease (eg, hostility and depression), coronary atherosclerosis, and risk of clinical cardiac events (1–7). Aspects of parasympathetic and sympathetic nervous system function can be estimated from spectral analysis of variability in interbeat intervals of heart rate (ie, heart rate variability [HRV]). Fast Fourier transform, point process, and autoregressive procedures quantify components of HRV that are expressed in 2 main frequency regions or bands (Hz): high-frequency (HF) and low-frequency (LF) power. HF power primarily reflects respiratory-modulated parasympathetic outflow (3,8,9), whereas LF power is subject to both substantial sympathetic influence and varying amounts of parasympathetic contribution (3,9). The LF/HF ratio has been proposed, by some investigators, as an index of relative balance of sympathovagal influences on the heart, with higher LF/HF ratios reflecting increased sympathetic activity and/or decreased parasympathetic tone (4).

Biometric family and twin studies document significant genetic influence on HRV phenotypes indexing parasympathetic activity, with heritability estimates ranging from 29% to 65% (10–13). Therefore, it is conceivable that molecular variation in genes regulating components of autonomic function accounts for some portion of interindividual variability in autonomic neurocardiac control. It was reported recently, for instance, that the HF component of HRV is associated with allelic variation in a common polymorphism of the gene encoding angiotensin-converting enzyme (ACE) (14).

Variation in acetylcholine (ACh) neurotransmission (from the vagus nerve to the sinoatrial node on the heart) has been shown to modulate heart rate and to covary with HRV components, particularly HF power and the LF/HF ratio (15–17). High-affinity choline uptake and transport into ACh-releasing neurons is mediated by the choline transporter, which is rate-limiting for the biosynthesis of ACh (18). Variation in the choline transporter gene (CHT1), which encodes the choline transporter (18), may conceivably also account for some portion of interindividual variability in ACh transmission, as reflected in HRV phenotypes. We investigated whether a single nucleotide polymorphism (SNP) (ie, G to T nucleotide base pair substitution) located within the 3' untranslated region (3'UTR) of CHT1 would predict interindividual variability in HRV phenotypes.


   METHODS
TOP
 ABSTRACT
 INTRODUCTION
 METHODS
RESULTS
 DISCUSSION
 NOTES
 REFERENCES
 
Participants
Subjects were 413 whites of European ancestry aged 30 to 54 years (mean = 44 ± 7 years); 49% were men (see Table 1) and none had a history of myocardial infarction or coronary revascularization, chronic kidney or liver disease, cancer, or neurologic disorders. Current use of psychotropic, glucocorticoid, and antihypertensive medications was also exclusionary. These participants were derived from an ongoing study of cardiovascular disease risk factor covariation in a community sample recruited by mass-mail solicitation from Southwestern Pennsylvania (principally Allegheny County). Informed consent was acquired in compliance with the guidelines of the University of Pittsburgh Institutional Review Board.


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  		TABLE 1. Sample Characteristics Across and Within Choline Transporter Gene (G/T) Single Nucleotide Polymorphism Genotype

 

Heart Rate Variability Measures
Participants were seated in a temperature-controlled recording chamber at the Behavioral Physiology Laboratory, University of Pittsburgh. Heart rate was recorded continuously by a standard, 2-lead electrocardiogram (ECG) attached bilaterally to the wrists for a period of 5 minutes. ECG signals were amplified and filtered by Grass bioamplifiers and were acquired continuously using computerized analog to digital conversion at a rate of 1000 samples per second (19). Respiration rate was assessed concurrently by thoracic strain gauge. Before these measurements, participants were asked to refrain from caffeine for 4 hours, alcohol and exercise for 12 hours, and over-the-counter medications for 24 hours.

Frequency domain analyses were performed to estimate HRV using a program for spectral analysis of point events, including a test for stationarity called PSPAT (18). Spectral power analyses were performed on interbeat interval data to estimate HF power (respiration frequency [Hz] ± 0.015) and LF power (0.09–0.12 Hz) (8,20). Sympathovagal balance was calculated as the LF/HF ratio {ln [LF (ms2))/HF (ms2)]} (21).1 Normal units (nu%) for the spectral estimates were then computed {ie, ln [HF power/(total power – DC components)*100] and ln [LF power/(total power – DC components)]*100} (9). Data of 22 participants were missing as a result of cardiac ectopy, equipment malfunction, or noisy ECG signal.

DNA Extraction and Analysis
Blood samples were collected on a different day in 10 mmol/L EDTA and DNA was isolated from lymphocytes using a salting-out procedure (22). Genotyping of the CHT1 (G/T) SNP (position: chromosome 2 [107111468]; band 2q12.3; relative position: 26772; GenBank accession no.: AC009963:1; dbSNP: rs333229) was achieved through polymerase chain reaction (PCR) amplification and allele specific detection by fluorescence polarization (23). Amplification used primers F: 5'-GTAGGGACGAATGAAGGA-3' and R: 5'-GCTCTCTAGATACAATGG-3'. The reactions were performed using the following conditions: 95°C for 1 minute, 35 cycles of denaturation at 95°C for 30 seconds, annealing at 54°C for 30 seconds, extension at 72°C for 30 seconds, and then 72°C for 1 minute. The FP-TDI primer, 5'-TCACAAATCTATAGTGTGGGG-3', was used for detection.

By convention, the more common allele is designated "G" and the less common allele "T"; frequencies of the G and T alleles were 0.76 and 0.24, respectively. The resulting distribution of CHT1 (G/T) genotypes (GG = 236, GT = 138, and TT = 26) conformed to Hardy-Weinberg equilibrium (chi-square = 1.7, not significant [NS]) and did not differ between men and women (chi-square = 0.17, NS). As a result of the low number of TT homozygotes, the GT and TT genotype was combined for analysis. DNA amplification and genotyping was successful on 400 participants, 22 of whom had missing HRV data, yielding a final sample of 378 subjects (see Table 1 for sample characteristics).


   RESULTS
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 ABSTRACT
 INTRODUCTION
 METHODS
 RESULTS
DISCUSSION
 NOTES
 REFERENCES
 
Analyses of covariance were performed to determine the effects of CHT1 genotype (GG vs. GT/TT) and sex on HF and LF power and LF/HF ratio (covarying for age and body mass index). Compared with GG homozygotes, participants having any T allele had greater HF power (F [1,368] = 5.97, p <.015), lower LF power (F [1,372] = 5.64, p <.018), and lower LF/HF ratio (F [1,368] = 8.07, p <.005) (see Fig. 1).2 Relative to men, women had significantly lower LF power (F [1,368] = 10.90, p <.001), lower LF/HF ratio (F [1,368] = 8.07, p <.005), and marginally greater HF power (F [1,368] = 3.10, p <.08) (see Table 2). No other significant main or interactive effects on the HRV components were observed.



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  		Figure 1. Association of CHT1 (G/T) genotype and components of heart rate variability. Note: high-frequency (HF) and low-frequency (LF) power estimates are scaled on the left Y axis, whereas LF/HF ratios are scaled on the right Y axis.

 

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  		TABLE 2. Heart Rate Variability Characteristics for All Participants and Within Gender

 


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 METHODS
 RESULTS
 DISCUSSION
NOTES
 REFERENCES
 
In this report, we present preliminary evidence that individual differences in resting cardiac autonomic control, as reflected in common HRV phenotypes, are associated with polymorphic variation in the CHT1 gene. Specifically, middle-aged adults (both men and women) who possess a CHT1 genotype containing any T allele exhibited greater HF power (greater parasympathetic activity), less LF power, and lower LF/HF ratio (sympathovagal balance) than subjects homozygous for the alternate G allele. Because the CHT1 (G/T) polymorphism is located in the gene’s 3'UTR, allelic differences at this site would not be expected to alter the amino acid sequence of the choline transporter molecule and, in fact, CHT1 (G/T) variation has no known functional significance. In principle, such variation could affect rates of protein synthesis by altering aspects of gene expression or, alternatively, vary in linkage disequilibrium with a functional polymorphism located elsewhere in the CHT1 gene. At present, only 1 other site of CHT1 variation has been identified (CHT1 I89V), a single nucleotide polymorphism causing a valine/isoleucine substitution in the protein’s third transmembrane domain (18). Although the CHT1 I89V variant encoding isoleucine reduces choline uptake by approximately 40% in an in vitro assay system, this mutation is quite rare and appears in homozygous form in less than 0.4% of individuals.

To our knowledge, this is the first report that polymorphic variation in the CHT1 gene may be associated with interindividual variability in parasympathetic activity and sympathovagal balance. If corroborated in subsequent investigation, this finding should contribute to our understanding of cholinergic–vagal modulation of heart rate and of HRV indices as indicators of altered ACh neurotransmission. By extension, this association may also prove relevant to certain behavioral phenotypes, such as depression, in which disruption of the ACh system has been hypothesized and to the increased risk of coronary disease events predicted by low vagal activity (1–7). In future studies, it would be of interest to examine the role of CHT1 variation in other cardiovascular disease risk factors, such as behaviorally evoked cardiovascular reactivity and recovery, baroreflex sensitivity, and blood pressure variability. The present findings should be interpreted cautiously, however, as a result of their basis in simple association analysis. As in all studies involving samples of unrelated individuals, spurious genetic association stemming from unknown sources of population substructure ("admixture’) remains a theoretical, if implausible, possibility (24,25). In this regard, confirmatory studies are warranted using more definitive genetic methodologies, such as family-based association designs (eg, transmission–disequilibrium analysis) (26,27) or statistical adjustment for population stratification by concurrent evaluation of multiple single-nucleotide polymorphisms ("genomic control") (28).


   NOTES
TOP
 ABSTRACT
 INTRODUCTION
 METHODS
 RESULTS
 DISCUSSION
 NOTES
REFERENCES
 
1As a result of the uncertainty regarding the mechanisms underlying the very low-frequency component (≤.04 Hz) of the power spectral density, we have focused on the better understood components of the heart rate variability spectrum associated with autonomic cardiac control (viz., high- and low-frequency spectral components). Back

2Because smoking is, in part, heritable (29,30), molecular variation in the nicotinic receptor alpha 2 gene was associated with smoking behavior (31), and smoking has been found to influence heart rate variability (HRV) measures (32), smoking status (smokers vs. non/exsmokers) was entered as an independent variable. No HRV component varied as a function of smoking status or its interaction with the CHT1 genotype. Back

Research Supported by NIH grants P01 HL 40962 (S.B.M.) and HL 07560.

Received for publication August 3, 2004; revision received October 18, 2004.

DOI:10.1097/01.psy.0000155671.90861.c2


   REFERENCES
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 INTRODUCTION
 METHODS
 RESULTS
 DISCUSSION
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 REFERENCES
 

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