HCN4 Gene Polymorphisms Are Associated With Occurrence of Tachycardia-Induced Cardiomyopathy in Patients With Atrial Fibrillation
Background: Tachycardia-induced cardiomyopathy (TIC) is a reversible cardiomyopathy induced by tachyarrhythmia, and the genetic background of the TIC is not well understood. The hyperpolarization-activated cyclic nucleotide-gated channel gene HCN4 is highly expressed in the conduction system where it is involved in heart rate control. We speculated that the HCN4 gene is associated with TIC.
Methods: We enrolled 930 Japanese patients with atrial fibrillation (AF) for screening, 350 Japanese patients with AF for replication, and 1635 non-AF controls. In the screening AF set, we compared HCN4 single-nucleotide polymorphism genotypes between AF subjects with TIC (TIC, n=73) and without TIC (non-TIC, n=857). Of 17 HCN4 gene-tag single-nucleotide polymorphisms, rs7172796, rs2680344, rs7164883, rs11631816, and rs12905211 were significantly associated with TIC. Among them, only rs7164883 was independently associated with TIC after conditional analysis (TIC versus non-TIC: minor allele frequency, 26.0% versus 9.7%; P=1.62×10–9; odds ratio=3.2).
Results: We confirmed this association of HCN4 single-nucleotide polymorphism rs7164883 with TIC in the replication set (TIC=41 and non-TIC=309; minor allele frequency, 28% versus 9.9%; P=1.94×10–6; odds ratio=3.6). The minor allele frequency of rs7164883 was similar in patients with AF and non-AF controls (11% versus 10.9%; P=0.908).
Conclusions: The HCN4 gene single-nucleotide polymorphism rs7164883 may be a new genetic marker for TIC in patients with AF.
See Editorial by Tsai
Tachycardia-induced cardiomyopathy (TIC) is a supraventricular or ventricular tachyarrhythmia that is reversible after control of the underlying arrhythmia. There are no established diagnostic criteria for TIC, but it should be suspected in patients with left ventricular (LV) dysfunction, tachycardia of >100 beats/min, and satisfying the following conditions: (1) no other identified cause of ischemic or nonischemic cardiomyopathy, (2) no enlargement of end-diastolic LV dimensions, and (3) recovery of LV function after control of tachycardia by rate control, cardioversion, or radiofrequency ablation.1,2
Many studies have examined alterations in cellular and neurohumoral regulation in TIC,3,4 but the pathophysiology of TIC is still not completely elucidated, and little is known about patient factors that increase vulnerability to TIC.5 In particular, no reliable genetic markers of TIC have been identified although one study reported that angiotensin-converting enzyme polymorphisms were associated with increased serum angiotensin-converting enzyme levels and a higher incidence of TIC.6
Atrial fibrillation (AF) is the most common cause of TIC in patients without a history of structural heart disease.7 Poorly controlled ventricular rates may worsen ventricular function, but only a fraction of patients with AF develop TIC. Fourteen genetic loci have been linked to AF in European ancestry groups, and 12 new genetic variants associated with AF have just been reported.8,9 Among these, only HCN4 (encoding the cardiac hyperpolarization-activated cyclic nucleotide-gated If channel) and KCNJ5 (encoding potassium voltage-gated channel subfamily J member 5) are cardiac ion channel genes.8,9 The HCN4 is strongly expressed in the impulse conduction system, including the sinus node (SN) and atrioventricular node, and is known to have a critical function in autonomic control of heart rate.10–12 Milanesi et al13 reported progressive development of severe bradycardia and fatal atrioventricular block in cardiac-specific HCN4 knockout mice. HCN4 loss-of-function mutations were also reported to promote SN dysfunction.14 However, an HCN4 gain-of-function mutation was reported to increase cAMP sensitivity and associate with familial inappropriate sinus tachycardia.15 Nakashima et al16 found that HCN4 overexpression in olfactory receptor neurons increased spontaneous firing rates by sensing basal cAMP levels and exacerbated firing induced by β-adrenergic stimulation. In addition, the If inhibitor ivabradine was shown to reduce ventricular rate during AF by inhibiting atrioventricular node conduction.17
Collectively, these studies indicate that the cardiac HCN4 channel is essential for normal heart impulse conduction and a critical mediator of heart rate control.13–17 We thus speculated that HCN4 is involved in TIC pathogenesis and investigated the association between HCN4 single-nucleotide polymorphisms (SNPs) and TIC.
The data, analytic methods, and study materials will be/have been made available to other researchers for purposes of reproducing the results or replicating the procedure. The Institutional Ethics Committee of the Graduate School of Biomedical Science at Hiroshima University approved all procedures that used human tissue. Written informed consent was obtained from all participants. Detailed methods are available in the Data Supplement.
Clinical Characteristics, Echocardiographic Parameters, and Electrophysiological Study Parameters in TIC and Non-TIC AF Patients of the Screening Set
We diagnosed 73 AF subjects with TIC (age, 61±9 years; 57 men). The ejection fraction improved in all TIC patients at 6 months after RFCA (34.5%±9.1% to 60.2%±6.2%; Figure 1). Recurrence of AF was detected in 5 patients, and they received rate control treatment.
Table 1 summarizes patient clinical characteristics, medications, and echocardiographic findings before RFCA in TIC (n=73) and non-TIC groups (n=857). The frequency of paroxysmal AF was lower in the TIC group than the non-TIC group (42.5% versus 64.6%; P=0.001). Left atrial diameter, left atrial volume index, and LV end-diastolic diameter were larger in the TIC group than the non-TIC group (41.8±8.0 versus 39.2±6.1 mm, P=0.001; 46.6±13.8 versus 41.5±12.7 mL/m2, P=0.003; 51.0±6.0 versus 47.9±5.0, P=0.0001) while ejection fraction was lower (34.5%±9.1% versus 60.3%±6.0%, P=3.52×10−13). Before RFCA, the usage rate of class I antiarrhythmic drugs was lower in the TIC group than the non-TIC group (11.0% versus 28.1%; P=0.0343) while usage rates of amiodarone (class III) and β-blockers were higher (38.4% versus 10.3%, P=0.0001; 63.1% versus 25.4%, P=2.8×10−12). All antiarrhythmic drugs other than β-blockers were stopped at least 1 half-lives before the procedure in all subjects. The β-blockers were uninterrupted before the procedure only in the TIC group. Other clinical characteristics and echocardiographic findings were similar in both groups.
Association of HCN4 Tag SNPs With TIC
To screen the entire HCN4 gene, 17 tag SNPs were genotyped in patients with AF from TIC and non-TIC groups (Table I in the Data Supplement). The SNP rs7172796 upstream of HCN4 as well as rs2680344, rs7164883, and rs11631816 in HCN4 intron 1 and rs12905211 in intron 2 were significantly associated with TIC after Bonferroni correction. Among these, HCN4 SNPs, rs7172796, rs2680344, rs7164883, and rs1163181 were closely linked. None of the haplotypes showed stronger associations than the single-marker association of rs7164883 (data not shown).
The genome-wide association study (GWAS) reported8 that AF-related HCN4 SNP rs7164883 was the most significantly associated with TIC. We performed conditional analysis accounting for rs7164883. Conditional analysis could not identify additional SNPs in this locus independently associated with TIC (Table II in the Data Supplement).
We downloaded Japanese genotype data (Hap Map JPT, n=104) of SNPs found in the 2-Mb region upstream and downstream of HCN4 from 1000 genome data and created a map of chromosomal position of each SNP against its r2 with rs7164883. The SNPs strongly linked with HCN4 SNP rs7164883 did not extend to other genes on chromosome 15 (Figure 2).
The genotype distributions of HCN4 SNP (rs7164883) in TIC and non-TIC AF patients of both screening and replication sets are shown in Table 2. In the screening set, the minor allele (G) frequency (MAF) of rs7164883 was higher in the TIC group than the non-TIC group (AA/AG/GG: TIC 42/24/7 versus non-TIC 698/151/8; MAF: 26.0% versus 9.7%; allele frequency model: P=1.6×10−9, odds ratio [OR]=3.2, 95% confidence interval, 2.2−4.9; dominant model: P=1.2×10−6, OR=3.2, 95% confidence interval, 2.0−5.3; recessive model: P=1.8×10−8, OR=11.3, 95% confidence interval, 3.9−32.0). We also confirmed this significant association of HCN4 SNP rs7164883 in the replication set (AA/AG/GG: TIC 20/19/2 versus non-TIC 249/59/81; MAF: 28.0% versus 9.9%; allele frequency model: P=1.69×10−6, OR=3.5; dominant model: P=5.7×10−6, OR=4.4; recessive model: P=1.14×10−5, OR=15.8).
We analyzed the relationships among TIC, total heart rate in 24-hour Holter ECG, and HCN4 SNP (rs7164883) to rule out spurious associations and found that total heart rate and HCN4 SNP rs7164883 were independently associated with TIC (total heart rate: OR=1.0, P=5.05×10−4; rs7164883: OR=3.31, P=2.50×10−8; Table 3).
Multivariate analysis for TIC using significant factors by univariate analysis, age, and sex was performed and revealed that nonparoxysmal AF and HCN4 SNP rs7164883 minor allele were independently associated with TIC after adjusting for these confounders (Table 4; OR, 2.15, P=2.86×10−3 and OR, 3.05, P=2.16×10−7, respectively).
Finally, we compared these results on rs7164883 in patients with AF (n=930) to non-AF controls (n=1635) and found that the MAF of rs7164883 was similar (MAF, 11% versus 10.9%; P=0.908). Nagelkerke pseudo R2 of Rs7164883 was calculated in the logistic regression model. This SNP explained 7.9% of the variance for TIC.
Various structural and hemodynamic changes have been reported in TIC, including elevated LV filling pressure, impaired LV systolic and diastolic functions, LV cavity dilation, and reduced cardiac output.18 Depletion of myocardial energy stores, reduced myocardial blood flow, increased oxidative stress, decreased β-adrenoceptor responsiveness, and abnormal calcium handling have been reported to be involved in TIC pathogenesis.19 AF is the most common cause of TIC while rapid heart rate, loss of atrial contraction, and rhythm irregularity may contribute to progression. However, not all patients with AF tachycardia develop TIC.3,4,6 The mechanisms underlying development of cardiomyopathy in patients with AF have not been clearly elucidated, and little is known about patient factors that increase vulnerability to TIC. In addition, genetic markers for TIC have not been sufficiently studied.
The HCN-gated channels are responsible for the If pacemaker current activated by hyperpolarization and are thought to influence SN function in cardiac impulse generation.19 This channel family comprises 4 members (HCN1-4), each composed of 6 transmembrane domains (S1–S6) and a linking site for cAMP in the C-terminal region of the peptide. Of these channel isoforms, HCN4 is known to have the strongest influence on heart rate.20 Molecular studies of the conduction system have found that HCN4 is expressed not only in the SN but also the atrioventricular node.21,22 Multiple studies have reported that loss-of-function HCN4 mutations induce SN dysfunction23,24 and that HCN4 overexpression induces sinus tachycardia, suggesting that HCN4 plays a crucial role in heart rate control.13–16 Heart rate reduction or prevention of AF after cardiac surgery with combined administration of the If inhibitor ivabradine and the β-blocker metoprolol was reported to be more effective than treatment with metoprolol alone.25 In another report, ivabradine significantly decreased ventricular rate during AF compared with placebo in nonparoxysmal AF patients.26
We conducted typing of 17 HCN4 tag SNPs in patients with AF (TIC=73 and non-TIC=857). The some closely linked HCN4 SNPs on upstream, intron 1, or intron 2 were significantly associated with TIC. Among them, only the HCN4 SNP rs7164883, G>A was independently associated with TIC by conditional analysis. We then confirmed this significant association in a replication set.
However, the HCN4 SNP did not reach significance association between Japanese non-AF controls and Japanese AF patients also in our study. A GWAS in European ancestry populations reported that the HCN4 SNP rs7164883 was associated with AF, but in the Japanese Bio Bank, study showed a negative replication for SNPs in HCN4.8 More recently, another GWAS replication in the Japanese population reported that the HCN4 SNP rs7164883 was not statistically significantly associated with AF in the Japanese population, indicating population heterogeneity in genetics of AF.27 In our study, the minor (G) allele frequency of rs7164883 was 10.9% in non-AF controls and 11.0% in total AF patient cohort, which was consistent with the frequencies reported in the Hap Map Project (11.6% in Japanese and 8.1% in East Asian populations). The HCN4 SNP rs7164883 may be a genetic marker of TIC rather than AF in Japanese.
A 2013 GWAS reported that the minor allele of rs4489968 in HCN4 increased heart rate.28 We genotyped 21 heart rate-associated SNPs, but none were associated with TIC after Bonferroni correction (data not shown). In our study, total heart rate/d and HCN4 SNP rs7164883 were independently associated with TIC.
The mechanisms by which the intron SNPs of HCN4 regulate HCN4 function or expression are not clear. The presence of a myocyte enhancer factor 2C–binding site has been reported in intron 1 of Rat HCN4.29,30 The myocyte enhancer factor 2C–binding site (Ch15:73658006-73658022) also exists in intron 1 of human HCN4 gene, but it is not perfectly matched with the rs7164883 site (Ch15: 73652174). According to the JASPER2018 (http://jaspar.genereg.net/) CORE, upstream transcription factor (USF-1, Ch15:73652295-73652584) 1 and USF-2 (Ch15:73652441-73652640) exist in intron 1 of HCN4 and the rs7164883 site is included in the USF site. USF is a family of transcription factors characterized by a highly conserved basic-helix-loop-helix leucine zipper (bHLH-zip) DNA-binding domain. The USF-1 and USF-2 were reported as transcriptional repressor and negative regulator of proliferation.31,32 The binding affinity of transcription factors may be modulated by rs7164883. Further functional analyses are desirable to reveal that allelic change of the SNP modulate binding affinity between the SNP and some transcription factors.
The HCN4 (If channel) mediates protein kinase A–dependent phosphorylation of sarcoplasmic reticular, mitochondrial, and ion channel proteins, which in turn regulates Ca2+ cycling and drives generation of spontaneous rhythmic action potentials.33 Recently, Mueller et al34 reported that TIC is characterized by changes in cardiomyocyte and mitochondrial morphology. Milano et al,35 who first found that mutations in HCN4 are associated with structural abnormalities of the myocardium, linked HCN4 mutations to LV noncompanction cardiomyopathy.36
We queried expression quantitative trait locus data acquired from 264 human left atrial appendage samples available in the Genotype-Tissue Expression website (http://gtexportal.org; V7 release) for cis-expression quantitative trait locus effects of HCN4 rs7164883. We analyzed genes located within 1 Mb upstream and downstream of HCN4 rs7164883 but found no genes, including HCN4, for which expression was significantly associated with rs7164883. However, these expression quantitative trait locus expression data were not from the target tissue of interest, and we could not find any publicly available expression quantitative trait locus-database using optimal tissue, therefore we further have to evaluate its possible association with other gene.
We think that HCN4 SNPs are potential genetic risk markers for TIC in patients with AF. Stratification of TIC risk is important because it can facilitate early therapeutic intervention (eg, stricter heart rate control or early rhythm control) to prevent heart failure in patients with an HCN4 minor allele. Differential diagnosis of TIC and dilated cardiomyopathy with AF tachycardia is also difficult but critical for selection of patient treatment strategy. Greater attention should be paid to recurrence of heart failure in patients with the HCN4 minor allele. Ivabradine may be an effect treatment for TIC.
In conclusion, the HCN4 SNP rs7164883 independently increases risk of TIC. Furthermore, HCN4 SNP may be a reliable genetic marker for TIC risk. Persistent AF patients with diabetes mellitus and the HCN4 SNP rs7164883 minor allele may be at particularly high risk and so should be considered for early therapeutic intervention.
Diagnosis of TIC is difficult because LV ejection fraction is dependent on heart rate during AF. Thus, patients susceptible to TIC may have been selected as control patients if persistent AF and ventricular rate were well controlled. In addition, enrollment of patients with AF treated by RFCA may introduce selection bias as this population does not represent the entire AF population. Specifically, the proportion of AF patients with TIC may be higher than in the general AF population because of selection bias. We did not perform GWAS analysis and so could not elucidate precise relationship with nearby genes. Also, mechanisms by which rs7164883 regulates HCN4 function or expression are not clear. TIC accounted for ≈10% of the total subjects, so the total number of cases was small. We, therefore, must validate the association between TIC and HCN4 SNP rs7164883 in a larger sample of cases and controls. Nonetheless, HCN4 SNPs are promising genetic markers for TIC and may be useful for selecting the most appropriate therapeutic intervention.
We thank the members of the clerical and medical staff at Hiroshima University Hospital for their assistance. We thank ENAGO Group (English editing system) for editing a draft of this manuscript.
Sources of Funding
Dr Nakano was supported by Japan Society of the Promotion of Science, Grant-in-Aid for Scientific Research (JSPS KAKENHI) grant number 17K09501.
The Data Supplement is available at http://circgenetics.ahajournals.org/lookup/suppl/doi:10.1161/CIRCGEN.117.001980/-/DC1.
- Received October 8, 2017.
- Accepted June 8, 2018.
- © 2018 American Heart Association, Inc.
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