Minor allele of GJA1 gene polymorphism is associated with higher heart rate during atrial fibrillation

Atrial fibrillation (AF) tachycardia causes heart failure and requires more attention. The genetic background of individual heart rate (HR) variations during AF are unclear. We hypothesized that HR-associated single nucleotide polymorphisms (SNPs) reported in Genome-Wide Association Studies (GWAS) are also associated with HR during AF. We enrolled patients with persistent AF (311 for screening and 146 for replication) who underwent AF ablation and were genotyped for the 21 h-associated SNPs reported in GWAS. The patients underwent 24-h Holter monitoring before AF ablation and electrophysiological study after AF ablation during sinus rhythm. Only the GJA1 SNP rs1015451 (T>C) was significantly associated with total HR (TT 110,643 ± 17,542 beats/day, TC 116,350 ± 19,060 beats/day, CC 122,163 ± 25,684 beats/day, P = 8.5 × 10−4). We also confirmed this significant association in the replication set. The intra-atrial conduction was faster in AF patients with the GJA1 minor allele than in those without it. Multivariate analysis revealed the presence of a GJA1 SNP rs1015451 additive model, female gender, lower left ventricular ejection fraction, and higher 1:1 atrioventricular nodal conduction were independently associated with higher HR during AF. The GJA1 SNP might be a new genetic marker for AF tachycardia.

Relationship between the EPS parameters and the GJA1 SNP rs1015451 genotypes. The relationship between the electrophysiological study (EPS) parameters and GJA1 SNP rs1015451 genotypes is shown in Table 3. The GJA1 SNP rs1015451 genotypes were significantly associated with the intra-atrial conduction time. The intra-atrial conduction times from the high right atrium (HRA) to the His bundle electrogram (HBE) and from the HRA to the distal coronary sinus (CS) were shorter in the patients with the GJA1 SNP rs1015451 minor allele than in those without it (HRA to HBE: TT 39.7 ± 14.0 ms, TC 36.8 ± 15.1 ms, CC 29.6 ± 11.1 ms, P = 6.1 × 10 −4 , HRA to distal CS: TT 111.6 ± 23.1 ms, TC 108.6 ± 22.2 ms, CC 98.8 ± 18.6 ms, P = 6.4 × 10 −3 ). Furthermore, the AF cycle length was significantly shorter in the persistent AF patients with the GJA1 SNP rs1015451 minor allele than in those without it (TT 155 ± 21 ms, TC 149 ± 18 ms, CC 133 ± 16 ms, P = 2.2 × 10 −4 ). However, the maximum sinus node recovery time (SNRT), corrected SNRT (CSRT), atrial to the His bundle (AH) interval, His bundle to the first ventricular activation (HV) interval, 1:1 AV nodal conduction, and effective refractory period (ERP) of the AV node were similar among the 3 GJA1 SNP rs1015451 genotypes.
Multivariate analysis of the total HR during AF in patients with persistent AF. In the univariate analysis, the GJA1 SNP rs1015451 genotypes, female gender, lower LVEF, higher 1:1 AV nodal conduction, Table 1. Relationship between the HR associated SNPs and total HR in the screening set. AF atrial fibrillation, HR heart rate, PAF paroxysmal atrial fibrillation. *Uncorrected P value in additive model. # Adjusted by age, gender, and BMI. R 2 coefficient of determination. www.nature.com/scientificreports/ shorter AF cycle length, and shorter atrial conduction time were significantly associated with the higher total HR during AF. A multivariate analysis revealed that the presence of a GJA1 SNP rs1015451 minor allele C, female gender, lower LVEF, and higher 1:1 AV nodal conduction were independently associated with higher HR during AF (Table 4).

Figure 1.
Relationship between the GJA1 single nucleotide polymorphism (SNP) rs1015451 genotypes and total heart rate (HR) in the patients with persistent atrial fibrillation (AF) in the screening set. The GJA1 SNP rs1015451 minor allele (C) was associated with a high total HR in the patients with persistent AF in the screening set (TT 110,643 ± 17,542 beats/day, TC 116,350 ± 19,060 beats/day, CC 122,163 ± 25,684 beats/day, P = 8.5 × 10 −4 ; TT vs CC: P = 2.5 × 10 −3 , TT vs TC: P = 1.5 × 10 −2 , TC vs CC: P = 0.12).  Fig. 2). The total HR during AF was higher in the persistent AF patients with the GJA1 SNP rs1015451 minor allele than in those without it and also in the replication set. (Fig. 2).
Relationship between the GJA1 SNP rs1015451 genotypes and serum MiRNA concentrations. After a quality check, we analyzed 353 miRNAs, in which there were no missing data. To normalize the signals across the different microarrays tested, Norm Finder software was used to assess the stability of the miRNAs. The hsa-miR-149-3p was selected as a normalizer. We investigated the relationship between the GJA1 SNP rs1015451 genotypes and plasma concentrations of the 353 miRNAs. Although only two miRNAs, miR-4708-3p (P = 4.6 × 10 −2 ) and miR-4448 (P = 4.6 × 10 −2 ), had Table 3. EPS parameters and the GJA1 SNP genotypes. AERP atrial effective refractory period, AF atrial fibrillation, AH atrial-His, AV atrioventricular, CS coronary sinus, CSRT corrected sinus node recovery time, EPS electrophysiological study, HBE His bundle electrogram, HRA high right atrium, HV His-ventricular, SNP single nucleotide polymorphism, SNRT sinus node recovery time.

Discussion
In our study, the GJA1 SNP rs1015451 (T>C) minor allele was associated with a higher HR, shorter atrial conduction time, and shorter AF cycle length in the patients with persistent AF. AF is the most common cardiac arrhythmia that causes tachycardia and heart failure 1-3 . The HR during AF differs from person to person, and many factors are involved in the AF HR. The determinants of the AF HR have not been completely identified. Also, the genetic background of individual HR variations during AF has not been examined. The 21 SNPs associated with the HR during sinus rhythm have been reported by a previous GWAS 11 . We investigated whether those SNPs were also associated with the HR during AF in patients with persistent AF. We initially clarified that only the GJA1 SNP rs1015451 (T>C) minor allele was associated with a higher HR during AF in patients with persistent AF. As a pertinent issue, the GJA1 SNPs including rs1015451 have recently been reported to be associated with heart failure and a reduced ejection fraction 12 .
Cardiac conduction is mediated by gap junction channels formed by connexin protein subunits. The GJA1 gene encodes connexin-43 (Cx43), which is one of the main connexin proteins 13,14 . Three types of connexins, Cx40, 43, and 45, are expressed differently depending on the site of the human cardiomyocytes 15,16 . Cx43 is the most expressed connexin in the atrial working myocardium, and it is also abundantly expressed in the working ventricular myocardium. Cx40 is abundantly expressed in the intraventricular conduction systems that control fast conduction, such as in the His bundle and Purkinje fibers. Cx45 is mainly expressed in the sinoatrial node and AV node, which have slow conduction properties. On the other hand, the expression level of Cx43 at the sinus node and AV node is very low 16,17 . In our study, the GJA1 SNP rs1015451 was associated with the conduction time of the atria, but it was not associated with the AV node conduction or SNRT. Those results were consistent with the localization of Cx43.
The GJA1 SNP rs1015451 minor allele (C) was also associated with a short AF cycle. Many studies on the AF cycle length have used an F-wave frequency analysis of the surface electrocardiography (ECG), and studies using an AF cycle length measurement from the intracardiac ECG have been few 18,19 . These two methods are reported to be correlated 20,21 , but the intracardiac ECG is more accurate. We analyzed the intracardiac ECG to evaluate the AF cycle length accurately. The HR during AF is influenced by many factors, such as the conduction characteristics of the AV node and autonomic tone. In our study, a shorter AF cycle was also associated with a higher HR during AF. The GJA1 SNP rs1015451 minor allele was associated with a higher HR during AF through a faster atrial conduction and shorter AF cycles, independent of the AV node conduction.
The SNP rs1015451 is called the GJA1 gene SNP 11 because the GJA1 gene is the closest gene. However, the GJA1 SNP rs1015451 is located outside of the GJA1 gene, approximately 370 kB away from the GJA1 gene. Variants that are in a strong linkage disequilibrium (LD) with the GJA1 SNP rs1015451 (r 2 > 0.8), are located in www.nature.com/scientificreports/ uncharacterized LOC105377979 and do not spread to the GJA1 gene ( Supplementary Fig. 1). Previous studies have suggested that variations in the intergenic regions might regulate transcription factor binding and chromatin modification 22 . We investigated the expression quantitative trait locus (eQTL) data acquired from 429 human LA appendage samples, 432 human left ventricle samples and 532 human peripheral nerves available from the Genotype-Tissue Expression (GTEx) website (http://gtexp ortal .org; V7 release) for the cis-eQTL effects of GJA1 SNP rs1015451. We analyzed the genes located within 1 Mb upstream and downstream to GJA1 SNP rs1015451, but we found no genes, including GJA1, for which the expression was significantly associated with the GJA1 SNP rs101545. In addition, there were no LD SNPs around the GJA1 SNP rs101545 associated with the GJA1 expression ( Supplementary Fig. 2-1,2). On the other hand, the expression level of the GJA1 gene in the peripheral nerves was significantly higher in patients with the GJA1 SNP rs1015451 minor C allele ( Supplementary Fig. 2-3), suggesting that epigenetic regulation could be involved in this process. We investigated the relationship between the GJA1 SNP rs1015451 genotypes and plasma concentrations of 353 miRNAs, and they were not significantly associated with the GJA1 SNP rs1015451 genotypes. More functional studies focused on this intergenic region on chromosome 6q22 will be required to understand its potential effect on GJA1.
There have been some negative reports about the association of the genetic SNPs with AF rate control. Barret et al. reported that no SNP was associated with acute HR control after the administration of diltiazem 9 . Kolek et al. reported that no SNP was significantly associated with a poor AF HR control 10 . In our study, the GJA1 gene SNP was associated with the HR during AF in patients with persistent AF in screening and replication sets.
To the best of our knowledge, this is the first report to demonstrate that for the GJA1 gene, encoding the gap junction Cx43, the SNP is associated with the HR during AF. This finding has a clinical implication in that early detection of high-risk AF patients with AF tachycardias who are prone to develop heart failure, and early intervention in these patients are needed to avoid heart failure.
This study had some study limitations. It was a retrospective study conducted at a single center, and the total number of cases was small, and therefore the statistical power of this study was inadequate. Therefore, we must investigate the association between the HR during AF and these SNPs using a larger sample of cases in future research.

Conclusion
The GJA1 gene, encoding the gap junction protein (Cx43), SNP rs1015451 minor allele is associated with a high HR in AF patients. The GJA1 SNP rs1015451 might be a new genetic marker for AF tachycardia.

Methods
Participants. This was a single-center retrospective study. We enrolled 311 patients (243 men and 68 women, mean age 63 ± 10 years) with persistent AF who underwent radiofrequency catheter ablation (RFCA) at the Hiroshima University Hospital from November 2009 to March 2016 for screening. We also enrolled 146 consecutive Japanese persistent AF patients (114 men, 32 women; mean age, 61 ± 10 years) who underwent RFCA at the Hiroshima University Hospital from April 2016 to July 2018 for replication. Persistent AF was defined as AF that lasted longer than 7 days. All procedures involving the human genome use were approved by the Institutional Ethics Committee of the Graduate School of Biomedical Science at the Hiroshima University. Written informed consent was obtained from all participants prior to participation in the study. All methods were performed in accordance with the relevant guidelines and regulations. Echocardiography. Transthoracic echocardiography was performed at our institution using a commercially-available system (Vivid E9, GE Healthcare, Milwaukee, WI, USA; or iE33, Philips Medical Systems, Andover, MA, USA) before the RFCA. Experienced echocardiographers who were blinded to the genotyping results conducted all echocardiographic examinations and analyzed the echocardiographic parameters.

Genotyping.
Twenty-four-hour Holter monitoring. Twenty-four-hour Holter monitoring was performed one day before the RFCA. Antiarrhythmic drugs, including β-blockers and calcium channel blockers, were stopped at least five half-lives before performing the 24-h Holter monitoring. Only amiodarone was routinely discontinued at least 2 weeks prior to performing the 24-h Holter monitoring. We confirmed that all the subject were AF all the time. The total HR during the AF was recorded in all of the subjects.
Electrophysiological study and RFCA. We also confirmed that all the patients were AF at the beginning of their AF ablation.
The AF cycle length was recorded on the LA posterior wall and four pulmonary veins. An average cycle length of 5 s before the pulmonary vein isolation (PVI) was calculated. We defined the shortest cycle length of them all as the AF cycle length.
A continuous PVI was performed in all subjects using an open-irrigation 3.5-mm tip deflectable catheter (THERMOCOOL SMARTOUCH; Biosense Webster) under the guidance of a 3-dimensional electro-anatomical mapping system (CARTO3, Biosense Webster) with computed tomography integration (CARTOMERGE, www.nature.com/scientificreports/ Biosense Webster) to achieve electrical isolation of the left-and right-sided pulmonary veins. We confirmed the PVI entrance and exit block and then rechecked these under the infusion of isoproterenol plus adenosine triphosphate 23 . After the PVI, an EPS was performed during stable SR. Four 5-French-gauge catheters were used: one catheter was a 10-polar electrode catheter positioned in the CS, and three catheters were quadripolar electrode catheters positioned in the HRA, His bundle, and right ventricle, all with a 5-mm interelectrode distance. The AH and HV intervals were measured during the baseline intracardiac ECG. The SNRT was measured as the recovery interval after a 30-s stimulation from the HRA. The CSRT was defined as the recovery interval in excess of the sinus cycle (i.e., CSRT = maximum SNRT − sinus cycle length) 23 . The ERP was measured using extrastimulus pacing with varied coupling intervals at a basic cycle length of 600 ms. The ERP was defined as the longest coupling interval that failed to propagate through that tissue. The maximal rate of 1:1 AV nodal conduction was determined using incremental atrial pacing. miRNA. We investigate the relationships between the GJA1 SNP rs1015451 and serum concentrations of 2555 miRNAs. The total RNA was extracted from individual serum samples using the 3D-Gene RNA Extraction Reagent from a liquid sample kit (Toray Industries, Inc., Kanagawa, Japan). A total of 2555 miRNA sequences were detected using the 3D-Gene miRNA Labeling kit and 3D-Gene Human miRNA Oligo Chip (Toray Industries, Inc). We analyzed the relationships between the GJA1 SNP rs1015451 genotype and serum concentrations of the miRNAs in the AF patients 24 .
Statistical analysis. Continuous are presented as the mean ± standard deviation. The deviation from the Hardy-Weinberg equilibrium was tested among the cases and controls using an ordinary χ 2 test. An additive mode of inheritance was assumed where the SNPs were coded as 0, 1, and 2 in the linear regression model. The association between the GJA1 SNP rs1015451 genotype and serum concentrations of the miRNAs was also analyzed by the linear regression model where the SNPs were coded as 0, 1, and 2. A multivariate analysis was performed by means of multiple regression analysis. The Bonferroni-corrected P-value threshold was P < 0.002 (0.05/21 SNPs) for relationships between the 21 h-associated SNPs and HR in the AF patients. Values of P < 0.05 were considered significant for the other analyses. The statistical analyses were conducted using the R3.3.1 and the JMP statistical package (version 13, SAS Institute, Cary, NC).