Author + information
- Received December 28, 2001
- Revision received April 8, 2002
- Accepted April 30, 2002
- Published online August 7, 2002.
- Eeva Pietilä, BM*,
- Heidi Fodstad, MSc†,
- Elmo Niskasaari, BM*,
- P.äivi J Laitinen, MSc†,
- Heikki Swan, MD†,
- Markku Savolainen, MD*,
- Y.Antero Kesäniemi, MD‡,
- Kimmo Kontula, MD† and
- Heikki V Huikuri, MD, FACC*,* ()
- ↵*Reprint requests and correspondence:
Dr. Heikki Huikuri, Division of Cardiology, Department of Internal Medicine, University of Oulu. P.O. Box 5000, FIN-90014 University of Oulu, Finland.
Objectives The aim of this study was to test whether a recently reported polymorphism in the HERG gene coding for the rapidly activating delayed rectifier K+ channel has influence on myocardial repolarization.
Background The length of myocardial repolarization, measured as the QT interval, has a hereditary component, but no genes that would explain the variability of repolarization have been identified in healthy subjects.
Methods QT intervals were measured from the 12-lead electrocardiogram in a random middle-aged population (226 men/187 women). The longest QT interval at any of the 12 leads (QTmax), QTV2, and the Tpeak-Tend interval were used as measures of repolarization. Deoxyribonucleic acid samples were genotyped for the nucleotide 2690A>C variation of the HERG gene, corresponding to the HERG K(lysine)897T(threonine) amino acid polymorphism.
Results The allele frequencies were 0.84 (A) and 0.16 (C). Females with the genotype AC or CC had longer QTcmax (477 ± 99 ms) and Tpeak-Tend intervals (143 ± 95 ms) than females with the genotype AA (441 ± 69 ms and 116 ± 65 ms, p = 0.005 and p = 0.025, respectively). In males, the QTcmax and the Tpeak-Tend intervals did not differ between the genotypes. After adjustment for echocardiographic and various laboratory variables, the HERG K897T polymorphism remained as an independent predictor of QTcmax (p = 0.009) and the Tpeak-Tend intervals (p = 0.026) in females.
Conclusions The common K897T polymorphism of the HERG channel is associated with the maximal duration and transmural dispersion of ventricular repolarization in middle-aged females.
The length of myocardial repolarization, most often measured as the QT interval, varies widely in healthy populations (1). The duration of the QT interval in an individual is affected by several factors, such as gender (2) and age (3). Recent studies suggest that there is a hereditary component that partly determines the length of the QT interval and explains some of the interindividual variation in the length of the repolarization of the myocardium (4–6). However, no genes that would explain the interindividual variation in the QT interval duration in the healthy population have been found to date.
Mutations in the HERG gene, which encodes for the major subunit of the rapidly activating delayed rectifier K+ channel (Ikr) (7), cause the long QT syndrome (LQTS) type 2 (8). Recently, an amino acid polymorphism K897T of the HERG gene, corresponding to the nucleotide variation 2690A>C was found (9). As a cause of the nucleotide change, a basic lysine is expected to change into a neutral threonine, possibly influencing the electric properties of the HERG channel. The HERG K897T polymorphism was associated with the length of the QT interval in Finnish long QT syndrome type 1 (LQT1) patients (9). We hypothesized that the polymorphism might associate with the repolarization in a randomly selected, healthy middle-aged population.
The study population was randomly selected from the Social Insurance Register covering all the inhabitants of the city of Oulu in northern Finland, which has been used for a population-based epidemiologic study of cardiovascular risk factors, the Oulu Project Elucidating Risk of Atherosclerosis. Three hundred women and 300 men between 40 and 60 years of age were invited to take part and 88% participated (267 women, 259 men). The details of this population have been previously described (10–12). Past and current medical history, smoking habits, alcohol consumption, and physical activity were assessed by a standardized health questionnaire. A standard 12-lead electrocardiogram (ECG) and a peripheral blood sample were obtained from each subject. Routine blood tests and deoxyribonucleic acid extraction were performed on each blood sample. M-mode and two-dimensional echocardiographic examinations were also performed. Subjects on any medication were excluded from the study. The study was approved by the Ethics Committee of the University of Oulu, and the subjects gave informed consent.
The HERG K897T polymorphism was genotyped from each subject, and the 12-lead ECG was analyzed for each measurable lead. After the exclusion of subjects on any medication, the ECG analysis and the HERG K897T genotype were available for 226 men and 187 women (mean age 50.5 ± 5.9 years). Because gender is a significant determinant of repolarization (2,3), the data were compared separately among women and men.
QT interval analysis
The standard 12-lead ECG was recorded at a paper speed of 50 mm/s. The QT interval was measured at two points of every measurable lead from the beginning of the QRS complex to the end of the T-wave. The end of the T-wave was defined as the point where the descending T-wave crossed the isoelectric TP baseline (the baseline drawn from the T-wave to the P-wave). When separate U waves were present, the QT interval was measured to the nadir of the curve between the T and U waves. The QT intervals were corrected for heart rate by Bazett’s formula (QTc intervals), and the mean of the two measurements of every lead was considered the QTc of that lead. The longest QTc, measured at any of the leads, was taken to represent total ventricular repolarization (QTcmax). In addition to QTcmax, the QTc at lead V2 (QTcV2), which has been commonly used in previous studies to indicate the duration of ventricular repolarization, was determined and used in the analyses.
Because the Tpeak-Tend interval has been suggested to provide an index of the transmural dispersion of repolarization (13), the Tpeak-Tend interval (from the apex of the T wave to the point where the descending T wave crossed the baseline) was also measured from the two complexes of every lead. The maximum value of Tpeak-Tend was used in the analyses.
The measurements were made manually by two persons blinded to the genotypes and the other data of the subjects. Interobserver variability for QTcmax was 5.0 ± 4.6 ms (95% confidence interval from 3.4 to 6.6 ms) in males and 6.6 ± 6.2 ms (95% confidence interval from 4.4 to 8.8 ms) in females.
Deoxyribonucleic acid was extracted from peripheral blood nucleated cells by standard methods (14). The analysis of the HERG K897T polymorphism was performed by primer-induced restriction analysis as previously described (9,15). The site of this polymorphism is in exon 11 of the HERG gene. A nucleotide transition (2690A>C) is predicted to substitute threonine for lysine at codon 897. In the previous study the coding region of the HERG gene was sequenced in 39 Finnish patients with LQTS. Apart from the HERG K897T polymorphism, which was found in 5 patients, no other common polymorphisms of the HERG gene were reported (9).
Statistical analyses were performed with SPSS for Windows software package (SPSS 10.0.1, SPSS Inc., Chicago, Illinois). Student t test and chi-square test were used to compare the differences between continuous and categorical data. Analysis of variance followed by Bonferroni’s post hoc analysis was used to compare the differences in the ECG variables between the HERG genotypes. Because only five women and five men belonged to the genotype CC, the genotypes AC and CC were combined for the statistical analyses. Pearson’s correlation coefficients were calculated in the analysis of mutual correlations between two variables. Multiple linear regression analysis was used in the assessment of multiple correlations. A value of p < 0.05 was considered significant.
Gender differences in cardiac repolarization
The QTc measured from the lead V2 was longer in females than in males (411 ± 25 ms and 399 ± 28 ms respectively, p < 0.001). However, QTcmax was not significantly different between the two genders. The Tpeak-Tend interval was longer in males than in females. The values for these ECG variables with the corresponding p values are presented in Table 1.
Effects of HERG K897T polymorphism on QT interval
The allele frequencies of the HERG K897T polymorphism were 0.84 (A) and 0.16 (C), and the distribution of the genotypes was similar in both genders. The whole study group was genotyped, and the exclusion of subjects with medications did not affect the allelic distribution. Similar allele frequencies were also recently reported for another Finnish study population (9).
In females, the HERG K897T polymorphism was associated with the QTcmax and the Tpeak-Tend intervals. The QTcmax interval was shorter in females with the genotype AA (441 ± 69 ms) than in females with the genotype AC or CC (477 ± 99 ms, p = 0.005). A similar trend was seen when comparing the HERG genotypes with the length of QTc at lead V2, but the association did not reach statistical significance. The Tpeak-Tend interval also followed the same trend, being shorter in the genotype AA (116 ± 65 ms) than in the genotypes AC or CC (143 ± 95 ms, p = 0.025). No significant differences were observed in the QTc or the Tpeak-Tend intervals between the genotypes in males (Table 2). Heart rate was not associated with the HERG K897T polymorphism in either of the two genders.
Relations of QT interval and Tpeak-Tend interval to other variables
In univariate analysis, QTcmax was associated with age in both genders (r = −0.255, p < 0.001 for females and r = −0.348, p < 0.001 for males). The Tpeak-Tend interval was also associated with age in both genders (r = −0.214, p = 0.003 for females and r = −0.330, p < 0.001 for males). Other, weaker associations emerged between cholesterol and the QTc interval in males and between body mass index and the Tpeak-Tend interval in males. None of the other clinical or echocardiographic variables were associated with either the QTc interval or the Tpeak-Tend interval.
After adjustment for the demographic, laboratory, and echocardiographic variables, the HERG K897T polymorphism remained as an independent predictor of the QTcmax (p = 0.009) and the Tpeak-Tend intervals (p = 0.026) in multivariate analysis in females.
The present study demonstrates the association between the common amino acid polymorphism K897T of the HERG gene and the duration of repolarization measured from a standard 12-lead ECG in healthy middle-aged Finnish females. The female subjects with the genotype AC or CC had longer QTcmax and Tpeak-Tend intervals than the females with the genotype AA. In males, the HERG K897T genotype had no effects on the QTcmax or the Tpeak-Tend intervals.
Effects of HERG gene on repolarization
Mutations in the HERG gene cause the LQTS type 2 (8). As a result of a mutation in the HERG gene and a decrease in the Ikr current, long QT syndrome type 2 (LQT2) most often manifests as a prolonged QT interval. Cellular models show that the HERG mutations in LQT2 produce preferential prolongation of the M cell action potential duration (16), and thus mainly affect the terminal phase of repolarization. Depending on the type of the mutation, Ikr blockade may also produce notched T waves and/or increase the propensity to cardiac arrhythmias such as torsade de pointes (TdP) (17,18). Evidence for linkage to the T-wave axis was also recently shown for the LQT2 locus (19).
In the HERG K897T polymorphism, the adenosine nucleotide at position 2690 is changed into a cytosine, which probably alters the electrical charge of the protein product and interferes with the function of the HERG channel. Recent functional in vitro analyses have shown that the T897 channel generates Ikr, but with altered properties: the current amplitude is reduced, and deactivation is markedly slowed compared with the K897 channel (20). This is in accordance with our results, which indicate that the HERG K897T polymorphism has effects on cardiac repolarization, probably by its actions on the Ikr current. However, these actions seemed to be gender-related.
Gender-related differences in repolarization
The effect of the HERG K897T polymorphism on the QT interval was only observed in females. In males, this polymorphism had no influence on the duration of the QT interval. The reasons for this gender-related difference are not evident, but may be explained by the interaction between the sex hormones and the Ikr current.
Animal studies indicate that the Ikr current densities are lower in females than males (21), which may partly explain why Ikr-blocking drugs or mutations in the HERG gene produce a more pronounced effect on the repolarization process in females with less functional Ikr channels. Women are also more vulnerable to TdP caused by Ikr-blocking drugs (22,23). In LQT2, females have more prolonged QT intervals and experience more often the symptoms of the disease than male patients do (24–26). It has also been shown that the QT interval and the response to ibutilide of the QTc varies with the menstrual cycle, and the protective effect of progesterone has been suggested (27).
In molecular studies, testosterone, but not estrogen, has been shown to decrease the effects of drugs that block the HERG channel (28). Thus, testosterone may protect the changes in the late ventricular repolarization phase against various factors influencing the Ikr channel function. Females seem to lack this protection, and the ventricular repolarization phase in females is more vulnerable to harmful effects influencing the Ikr current. These gender-related differences may well explain the present finding that the changes in the terminal phase of the QTc interval in the AC and CC genotypes were observed only in females.
The method of measurement of the QT interval is a key issue for any interpretations of repolarization abnormalities. The selection of the lead for the QT interval measurement, the method of defining the end of the T-wave as well as the formulas for heart rate correction are of importance. We measured here the QT interval to the end of the T-wave and defined the maximum QT interval at any lead to indicate the total ventricular repolarization. This definition was based on the recent recommendations to include all the repolarization signals originating from the ventricular myocardium into the T-wave (29). Recent studies also suggest that many of the ECG leads actually underestimate the true length of ventricular repolarization because the axis of the terminal part of the T loop and the axis of the lead are perpendicular to each other (30,31). Furthermore, recent experimental data based on an analysis of repolarization in various cell types of the myocardium also suggest that all repolarization currents should be included in the T wave (13). The present study showed that the HERG gene polymorphism had major influences only on the terminal phase of repolarization, which were more evident in the QTcmax and Tpeak-Tend measurements than in QTcV2.
Bazett’s formula may also have some bias in the correction of the QT interval for heart rate. However, the average heart rate did not differ between the different HERG genotypes. Thus, the correction formula cannot explain the present observations.
This study demonstrates the association between the HERG K897T polymorphism and the duration and transmural dispersion of ventricular repolarization in healthy, middle-aged Finnish females. In females, the less common genotype of the HERG K897T polymorphism resulted in a prolonged QTcmax interval, possibly because of a decrease in the Ikr current due to the amino acid polymorphism. Because females are more vulnerable to acquired TdP, which also results from an Ikr block, it will be of importance for future studies to assess whether the HERG K897T polymorphism explains the subject-specific response to TdP or other clinically important arrhythmias.
☆ Supported by a grant from the Medical Council of the Academy of Finland, the Sigrid Juselius Foundation and the Finnish Foundation for Cardiovascular Research.
- rapidly activating delayed rectifier K+ channel
- long QT syndrome
- heart rate corrected QT interval
- heart rate corrected QTmax
- heart rate corrected QT interval measured from lead V2
- torsade de pointes
- Received December 28, 2001.
- Revision received April 8, 2002.
- Accepted April 30, 2002.
- American College of Cardiology Foundation
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