Relationship Between Genetic Polymorphisms of Angiotensin Converting Enzyme and the Degree of Electroanatomical Remodeling of Atrium in Patients with Non-valvular Atrial Fibrillation

International Journal of Arrhythmia 2014;15(3): 13-23.

ORIGINAL ARTICLES

Relationship between Genetic Polymorphisms of
Angiotensin-Converting Enzyme and the Degree
of Electroanatomical Remodeling of the Atrium
in Patients with Non-valvular Atrial Fibrillation

Introduction

Atrial fibrillation (AF) is the most common clinical arrhythmia. It is associated with cardiovascular morbidity and is related to increased disability.^1,2 The pathophysiology of AF is heterogeneous,³ and long-standing AF is associated with changes in left atrial (LA) morphology. AF alters the electrophysiological properties of atrial myocytes and causes alterations in the structure of the atrial myocardium.^4,5 The longer the duration of AF, the more persistent it becomes due to atrial remodeling. Both electrical remodeling and structural remodeling beget AF, and an increase in AF burden leads to more vulnerable substrates^.6 The structural remodeling is related to interstitial fibrosis, downregulation of gap junctions, and enlargement of atrial chamber size (critical mass).^7,8 The degree of structural remodeling as measured by LA size affects the clinical outcome of rhythm control strategies in patients with AF.⁹ LA scarring is also an independent predictor of procedure failure after radiofrequency catheter ablation (RFCA) of AF.¹⁰ The renin-angiotensin system (RAS) is involved in many cardiovascular diseases, including myocardial fibrosis and hypertrophy, and AF is associated with activation of the RAS in the atria.¹¹ Angiotensinconverting enzyme (ACE) stimulates fibroblast proliferation, collagen synthesis, and atrial structural remodeling in patients with AF.¹² Ravn et al.¹³ reported that the angiotensinogen (AGT) A-20C genotype in combination with the ACE I/D genotype predicts an increased risk of AF, but few studies have searched for a genetic predisposition to LA structural remodeling in patients with AF. Therefore, we hypothesized that ACE polymorphisms are associated with the degree of atrial structural remodeling in patients with AF. We investigated the relationship between ACE polymorphism and LA volume measured by a 3D-spiral computed tomography (CT) scan or LA voltage calculated by 3D-electroanatomical mapping in Korean AF patients who underwent RFCA.

Methods

Patient Selection

The study protocol was approved by the Institutional Review Board of our institute. All patients provided written informed consent. The study enrolled 351 patients with AF (male:female=282:69, mean age=54.2±11.1 years) who underwent RFCA. Among them, 235 patients had paroxysmal AF (PAF), and 116 had persistent AF (PeAF). The exclusion criteria were as follows: (1) permanent AF refractory to electrical cardioversion; (2) LA sizes ＞50 mm measured by echocardiogram; (3) AF with rheumatic valvular disease; (4) associated structural heart disease; (5) prior AF ablation; and (6) sinus rhythm not maintained for LA voltage mapping before RFCA. Patients with the presence of an LA thrombus were excluded by transesophageal echocardiography. We imaged all patients with a 3D-spiral CT (64 Channel, Light Speed Volume CT, Philips, Brilliance 63, Amsterdam, Netherlands) to visually characterize the anatomy of the LA and LVs. Transthoracic echocardiography was performed in all patients and the anterior-posterior (AP) diameter of the LA, left ventricular ejection fraction (LVEF), LV diastolic function measured by E/E' , LV end-systolic dimension (LVESD), and LV diastolic dimension (LVEDD) were measured.

Electrophysiological Mapping

Intracardiac electrograms were recorded using a Prucka CardioLab^TM Electrophysiology system (General Electric Health Care System Inc., Milwaukee, WI, USA). For AF RFCA (n=351), we used 5 mapping catheters and a deflectable 3.5-mm, 7 Fr open irrigation tip ablation catheter (Celsius, Johnson & Johnson Inc., Diamond Bar, CA, USA). The catheter ablation procedures were performed using 3D electroanatomical mapping (NavX system, St. Jude Medical Inc., Minneapolis, MN, USA) in all patients. Before the catheter ablation, we generated an LA 3D electroanatomical map and voltage map by obtaining contact bipolar electrograms from approximately 100-150 points throughout the LA endocardium of the high right atrium with pacing cycle lengths of 500 ms. The bipolar electrograms were filtered between 32-300 Hz. Color-coded voltage maps were generated by recording bipolar electrograms and measuring the peak-to-peak voltage.

Analyses of LA Remodeling: 3D-Spiral CT and Electroanatomical Voltage Map

The 3D-spiral CT images of the LA were analyzed on an imaging processing workstation (Aquarius, Terarecon, Inc, Concord, MA, USA). The curvilinear lengths of the LA were measured at the following linear ablation sites: the bilateral antral ablation line, roof line, posterior inferior line, left lateral isthmus line, and anterior line. Each LA image was divided into the following parts according to embryological origin: the venous LA (posterior LA including the antrum and posterior wall), LA appendage (LAA), and anterior LA (excluding the LAA and venous LA).¹⁴ We also measured the curvilinear lengths of circumferential pulmonary vein ablation, the roof line, posterior inferior line, anterior linear line, and left lateral isthmus line as described in a previous study.¹⁵
We analyzed the color-coded LA electroanatomical voltage maps in the AP and posterior-anterior (PA) views. The low voltage areas ≤0.2 mV were coded with a gray area and the high voltage areas ＞5.0 mV were colored purple. The reference distance was measured by the inter-electrode distances of coronary sinus catheters (Duodecapolar Catheter, St. Jude Medical Inc. Minnetonka, MN, USA). The LA was divided into 4 quadrants in each of the views. To quantify the mean voltage of the LA, the percent area represented by each color was calculated using customized software (Image-Pro) with reference to a color scale bar.¹⁵

Genetic Polymorphism Analyses

We selected haplotype-tagging single nucleotide polymorphisms (SNPs) of the ACE gene using the HapMap Japanese (JPT) data bank (http://www.hapmap.org) and NCBI SNP database (http://www.ncbi.nlm.nih.gov/projects/SNP/). To identify eligible tag SNPs in our population, we carried out a pilot study by genotyping for 16 selected SNPs in 48 Korean subjects. We identified 7 candidate SNPs (C-3927T, A-262T, P405P, T776A, ACE I/D, F1129F, and C2359). Genomic DNA was extracted from whole blood samples using a commercially available kit (Qiagen, Valencia, CA, USA). Genotyping for 6 of the SNPs was conducted by a single-base extension method using the SNaPShot^TM Assay kit (Applied Biosystems, Foster City, CA, USA), and genotyping of the I/D polymorphism was performed with polymerase chain reaction as described previously.¹⁶

Data Analyses

We selected ACE variants that were related to the degree of structural remodeling, as indicated by the entire LA volume, regional LA volume, regional curvilinear LA lengths, mean and regional LA voltage, and the LA/LVEDD ratio measured by echocardiography.

Statistical analyses were performed using the SPSS statistical package release 17.0.1 (SPSS, Inc., Chicago, IL, USA). Data were expressed as means ± standard deviations (SDs). Between-group data for baseline characteristics were compared with the Student’s unpaired t-test for continuous data and the χ² test for categorical data. For statistical analyses, we defined the cutoff as the median rounded to 0.1 decimal places and validated it by a receiver-operating characteristic (ROC) curve analysis. All genotype frequencies were in Hardy-Weinberg equilibrium (HWE) (p＞0.05). HWE of the genotype frequencies was evaluated using a χ² test. In single-locus analyses, we first compared the allele and genotype frequencies between the cases and controls with the χ² test or Fisher’s exact test. Statistical significance was defined as p＜0.05.

Results

ACE Variants Associated with LA Structural Remodeling in Patients with AF

Figure 1 shows representative examples of highly remodeled (Figures 1A and 1C) and less remodeled LAs (Figures 1B and 1E) in patients with AF, and their ACE genotypes (Figure 1D). The patients with significant electroanatomical remodeling of the LA show an enlarged LA volume, a low endocardial voltage (Figure 1A), and a high LA/LVEDD ratio (Figure 1C). In contrast, the patients with a less remodeled LA had a relatively small LA volume with a high endocardial voltage (Figure 1B) and a low LA/ LVEDD ratio (Figure 1E).

Among the 7 SNPs evaluated, 5 polymorphisms of the ACE gene were associated with structural remodeling of LA in the 351 patients with nonfamilial non-valvular AF. Table 1 summarizes the relationships between the ACE variants and the degree of structural remodeling of the LA. The F1129F C (p=0.024), P405P T (p=0.030), T-3927C T (p=0.030), and A-262T A (p=0.027) ACE alleles were associated with an enlargement of LA volume. LA enlargement relative to LV size (LA/LVEDD) measured by echocardiography was significantly higher in patients with the ACE F1129F C allele (p=0.039) and the P405P T allele (p=0.026) than in other patients. The ACE F1129F T allele (p=0.021) and ACE D carriers (DD+ID) allele (p=0.021) were the predominant genotypes in patients with low mean LA voltage.

ACE Variants Related to LA Structural Remodeling Measured by LA Volume

Table 2 summarizes the segmental volume and segmental curvilinear length of the LA adjusted for body surface area (BSA) with respect to the F1129F genotype. We also compared the mean and regional LA voltage and echocardiography parameters. Generally, patients with the F1129F C allele had larger total and regional LA volumes (p＜0.01) and longer regional curvilinear lengths of the LA (p＜0.05) than those with the F1129F TT genotype. In contrast, patients with the F1129F T allele had a lower LA voltage than those with the F1129F CC genotype (p＜0.05). The characteristics of LA remodeling in patients with the ACE P405P allele, T-3927 T allele, and A-262T allele are listed in Table 3.

ACE Variants and Clinical Outcomes after Catheter Ablation of AF

The clinical recurrence rate of AF after a 3-month blanking period was 20.45% during the 28.29±5.83 month follow-up. We did not find ACE-related polymorphisms associated with long-term clinical recurrence after catheter ablation. However, the ACE F1129F T allele, which was related to a low endocardial LA voltage, was associated with a higher early recurrence rate (within 3 months) (44.9%) after RFCA than the ACE F1129F CC genotype (27.5%, p=0.0217).

Discussion

This study demonstrated the association between ACE polymorphisms and structural remodeling of the LA measured by LA volume and endocardial voltage. ACE polymorphism also affected early recurrence after catheter ablation of AF. A genetic predisposition of specific ACE genotypes predicts atrial remodeling and may provide the basis for a treatment strategy.

The Mechanisms of Electroanatomical Remodeling of AF

AF begets AF. Wijffels et al.⁶ reported that the higher the AF burden, the more persistent it becomes owing to atrial remodeling. There are two kinds of atrial remodeling. Electrical remodeling is a process of ion channel adaptation to tachyarrhythmia, ^4,6 and structural remodeling is the change in LA volume, voltage, and conduction velocity by matrix remodeling.^15,17 The former is reversible by maintaining a sinus rhythm, while the latter is irreversible.¹⁸ Because structural remodeling changes the morphology and endocardial voltage of the atrium, clinicians call it electroanatomical remodeling. Electroanatomical remodeling is provoked by mechanical stretch-related extracellular matrix genes.^19,20 Profibrotic signals including angiotensin II,²¹ TGF-β,²² platelet-derived growth factor (PDGF),²³ or connective tissue growth factor (CTGF)²⁴ are known to proceed extracellular matrix remodeling. Those profibrotic signals also induce the proliferation of myofibroblasts.²⁵ Myofibroblasts contribute to collagen deposition with apoptosis or necrosis of cardiomyocytes,²⁰ electroanatomical remodeling,^15,17 and the non-reentrant mechanism of AF by automaticity.^20,26 Recently, we reported a higher LA volume, slower conduction velocity, lower endocardial voltage, and poorer clinical outcome after catheter ablation in patients with significant electroanatomical remodeling than those with a less remodeled LA.^15,17 However, there are individual differences in the degree and rate of electroanatomical remodeling of the LA in patients with AF. Therefore, we determined the ACE polymorphisms related to angiotensin II, one of the profibrotic signals, and their association with the degree of electroanatomical remodeling of AF.

Genetic Polymorphisms of Renin-Angiotensin System and Matrix Remodeling

The RAS is involved in many cardiovascular diseases, including heart failure and myocardial infarction related to oxidative stress, inflammation, or mechanical overload.^27,28 The ACE D allele (DD+ID) is more common in patients with significant LV remodeling after myocardial infarction.^29,30 The ACE DD genotype and the AT1R A1166C (AC+CC) genotype are associated with the LV mass index and diastolic heart failure.³¹ However, genetic studies of the RAS related to AF or atrial remodeling are limited. Recently, Tsai et al.³² reported that the ACE I/D polymorphism and several variants in the angiotensinogen and angiotensin II type I receptor are associated with nonfamilial structural AF. Watanabe et al.³³ reported that the ACE D allele is associated with a longer PR interval in patients with lone AF. In this study, we reported several ACE polymorphisms associated with electroanatomical remodeling of the LA in patients with AF. Specifically, it was associated with LA enlargement, reduced endocardial voltage, and early recurrence after catheter ablation. These ACE polymorphisms might be useful for the detection of patients with AF who are susceptible to structural remodeling. Although we found an association of these genes with LA remodeling, they were not significantly associated with LV size or LV systolic and diastolic function in this highly selected and relatively homogeneous patient group with non-valvular AF.

Clinical Implications

ACE polymorphisms associated with electroanatomical remodeling of the LA might be useful for the early detection of susceptible patients and prevention of the progression to chronic permanent AF with electroanatomical remodeling. Upstream therapy with an ACE inhibitor or angiotensin II receptor blocker prevents LA remodeling and is used as a tailored management.^34,35 Those variants also may justify the early intervention with catheter ablation and improve the prognostic value and clinical outcome.

Study Limitations

The patients included in this study were a highly selected group referred for rhythm control, and the number of patients was limited. The exclusion of patients with large atria (greater than 50 mm) may influence the results and clinical outcomes. Because we acquired voltage maps by point-by-point contact mapping, they did not reflect a spatiotemporally homogeneous distribution. We analyzed 3D voltage maps using 2D measurements.

Conclusion

We demonstrated the association between ACE polymorphisms and structural remodeling of the LA as measured by LA volume and endocardial voltage. Individuals with specific ACE genotypes are predisposed to atrial remodeling and these genotypes may provide the foundation for a therapeutic strategy.

Acknowledgements

This work was supported by grants from the Korea Health 21 R&D Project, the Ministry of Health and Welfare (A085136) and the National Research Foundation of Korea (NRF) funded by the Ministry of Science, ICT & Future Planning (MSIP; 7-2013- 0362).

Conflicts of Interest

The authors have no conflict of interest disclosures.

References

Korean Heart Rhythm Society A-1604, Centreville Asterium Seoul, 372, Hangang-daero, Yongsan-gu, Seoul, Korea TEL: +82-2-318-5416 FAX: +82-2-318-5417 E-mail: khrs4@k-hrs.org, khrs6@k-hrs.org
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