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Cardiac Autoantibody Levels Predict Recurrence Following Cryoballoon-Based Pulmonary Vein Isolation in Paroxysmal Atrial Fibrillation Patients MUHAMMED ULVI YALCIN, M.D.,∗ KADRI MURAT GURSES, M.D.,† DUYGU KOCYIGIT, M.D.,∗ SACIT ALTUG KESIKLI, M.D.,‡ MUHAMMET DURAL, M.D.,∗ BANU EVRANOS, M.D.,∗ HIKMET YORGUN, M.D.,∗ LEVENT SAHINER, Ph.D.,∗ ERGUN BARIS KAYA, Ph.D.,∗ MEHMET ALI OTO, F.H.R.S., Ph.D.,∗ DICLE GUC, Ph.D.,† KUDRET AYTEMIR, Ph.D.,∗ and NECLA OZER, Ph.D.∗ From the ∗ Department of Cardiology, Hacettepe University Faculty of Medicine; †Department of Cardiology, Kars Harakani State Hospital, Kars, Turkey; and ‡Department of Basic Oncology, Hacettepe University Cancer Institute, Ankara, Turkey

Cardiac Autoantibodies and AF Recurrence. Introduction: Recent evidence has suggested that autoantibodies may play an important role in the development of atrial fibrillation (AF). The predictive value of preprocedural autoantibodies against beta-1 adrenergic receptor (anti-β1-R) and M2-muscarinic acetylcholine receptor (anti-M2-R) for AF recurrence following cryoballoon-based pulmonary vein isolation (PVI) is still unclear. We aimed to determine the predictive value of preprocedural anti-β1-R and anti-M2-R levels for AF recurrence. Methods: Eighty patients (mean age 54.25 ± 7.70 years; 40% female) with paroxysmal AF and preserved left ventricular function who underwent cryoballoon-based PVI were included in the study. Preprocedural anti-M2-R and anti-β1-R levels were measured with ELISA. Results: At 1-year follow-up after ablation, late AF recurrence was observed in 17 (21.25%) patients. In the Cox regression model, including number of antiarrhythmic drugs, early AF recurrence, anti-β1-R levels >159.88 ng/mL, anti-M2-R levels >277.65 ng/mL, AF duration, and left atrial volume index, only anti-β1-R levels >159.88 ng/mL (HR: 4.281, P = 0.039) and anti-M2-R levels >277.65 ng/mL (HR: 4.313, P = 0.030) were found to be independent predictors of late AF recurrence. Anti-β1-R level >159.88 ng/mL was shown to predict late AF recurrence with a sensitivity of 70.59% and specificity of 90.48%. A cut-off value of 277.65 ng/mL for anti-M2-R level predicted AF recurrence with a sensitivity of 70.59% and specificity of 95.24%. Conclusion: Preprocedural serum anti-β1-R and anti-M2-R levels are independent predictors of late AF recurrence following cryoballoon-based PVI in paroxysmal AF patients. Detection of preprocedural anti-β1-R and anti-M2-R levels may serve as a novel method for determination of paroxysmal AF patients who may not benefit from cryoballoon-based PVI. (J Cardiovasc Electrophysiol, Vol. 26, pp. 615-621, June 2015) atrial fibrillation, cardiac autoantibodies, catheter ablation, cryoballoon, recurrence, pulmonary vein isolation Introduction Atrial fibrillation (AF) is the most common sustained arrhythmia and is associated with decreased functional capacity, quality of life, and increased prevalence of stroke, thromboembolic events, and mortality.1 To date, mechanisms underlying AF have not been fully understood. Emerging evidence has indicated that autoimmunity may have an important role in atrial structural remodeling.2 Anti-myosin This study was supported by Hacettepe University Research Projects Coordination Unit (project number: 845). Disclosures: None. Address for correspondence: Kadri Murat Gurses, M.D., Department of Cardiology, Hacettepe University Faculty of Medicine, 06100, Sihhiye, Ankara, Turkey. Fax: 90-312-311-4058; E-mail: [email protected] Manuscript received 16 December 2014; Revised manuscript received 5 March 2015; Accepted for publication 9 March 2015. doi: 10.1111/jce.12665

heavy chain, anti-M2-muscarinic receptor (anti-M2-R), anti-β1-adrenergic receptor (anti-β1-R), and anti-heat shock protein autoantibodies have been suggested to play a part.2-4 M2-muscarinic receptor is a member of cardiac G-proteincoupled receptors.5 Circulating autoantibodies against the second extracellular loop of M2-muscarinic acetylcholine receptors (anti-M2-R) have been detected in several cardiac arrhythmias, including sinus node dysfunction, ventricular arrhythmias and AF.6 Anti-M2-R has been shown to be associated with higher prevalence of AF in patients with idiopathic dilated cardiomyopathy and Graves’ hyperthyroidism.7,8 The β1-adrenergic receptor is also a member of cardiac G-protein-coupled receptors.5 Circulating autoantibodies against the second extracellular loop of β1-adrenergic receptors (anti-β1-R) have been identified in several cardiac arrhythmias, including ventricular arrhythmias, conduction disturbances, and AF.7,9,10 Catheter ablation of AF with the primary aim of pulmonary vein (PV) isolation is regarded as an effective

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and safe therapeutic option for patients with symptomatic and drug-refractory AF.11,12 Despite improved procedural outcomes with advances in ablation techniques, postablation AF recurrence is still a major clinical problem, occurring in 25–50% of patients during follow-up.13,14 In the present study, we aimed to determine the predictive value of preprocedural anti-M2-R and anti-β1-R levels for AF recurrence following cryoballoon-based AF ablation. Methods Study Population Eighty patients with symptomatic AF despite ࣙ1 antiarrhythmic drug(s) who were scheduled for cryoballoon-based AF ablation procedure per the recent consensus recommendations between September 2011 and March 2013 were enrolled in this prospective study.14,15 Baseline demographic and clinical characteristics, including age, gender, body mass index, and co-morbidities including hypertension, diabetes mellitus, and habits such as active smoking, and alcohol consumption, were recorded for all patients. Data related to the diagnosis of AF, including date of first diagnosis, oral anticoagulation, rate control, and antiarrhythmic medications were also recorded. AF episodes that either lasted >7 days or required termination by cardioversion, either with drugs or by direct current cardioversion, were defined as persistent; whereas AF episodes self-terminating within 7 days were defined as paroxysmal AF.1 Symptomatic severity of the patients was evaluated according to the European Heart Rhythm Association (EHRA) score.15 Informed consent was obtained from each patient before enrollment. Patients older than 60 years and those with left ventricular ejection fraction (LVEF) < 50%, serum creatinine ࣙ 1.2 mg/dL, abnormal thyroid functions, history of coronary artery disease, congenital heart disease, rheumatic valvular heart disease, idiopathic dilated cardiomyopathy, autoimmune disease, or recent infections were not included in the study. Patients with hypertension or diabetes mellitus were included in this study, since any association between these comorbidities and autoantibody levels has not been clearly demonstrated yet. The study was in compliance with the principles outlined in the Declaration of Helsinki and approved by the Institutional Ethics Committee. Preprocedural Management All patients underwent transthoracic echocardiography (TTE) within 1 week prior to ablation to assess intracavitary dimensions (including left atrial volume index [LAVI]) and LVEF and to exclude valvular heart disease. Transesophageal echocardiography (TOE) was performed to rule out presence of thrombus in the left atrial (LA) appendage the day before the procedure. Furthermore, patients underwent a multidetector computed tomography (MDCT) scan with 3-dimensional (3D) construction of the LA and PVs to assess LA anatomy and PV configuration. Anticoagulation was discontinued at least 48–72 hours before the procedure, and the preprocedural interval was bridged with enoxaparin 1 mg/kg. Treatment with antiarrhythmic drugs was discontinued for at least 3 days prior to the procedure.

Measurement of Autoantibody Levels Peripheral venous serum samples were obtained from patients within 24 hours preprocedurally. Samples were centrifuged at 1000 x g for 10 minutes. Anti-M2-R levels were measured using the “Human muscarinic acetylcholine receptor M2 (mAChRM2) autoantibody Enzyme-Linked Immunosorbent Assay (ELISA) kit (EASTBIOPHARM, Hangzhou, People’s Republic of China, Catalogue No: CK-E90715) following the manufacturer’s instructions. Spectrophotometric optical density (OD) measurements were followed by secondary calculations using the SOFTmaxPro version 2.6.1 (Molecular Devices Corp., Sunnyvale, CA, USA) software. Anti-M2-R concentrations were calculated using a 4-parameter standard curve and the OD measured for each sample. The equation that represented the standard curve was calculated by using 5 standard values: 320 ng/mL, 160 ng/mL, 80 ng/mL, 40 ng/mL and 20 ng/mL. The lower limit of detection was stated as 3.21 ng/mL in the manufacturer’s instructions. Levels of anti-β1-R were measured using the “Human anti-beta1-adrenergic receptor (B1AR) autoantibody ELISA kit (EASTBIOPHARM, Hangzhou, People’s Republic of China, Catalogue No: CK-E90714)” following the manufacturer’s instructions. Spectrophotometric optical density (OD) measurements were followed by secondary calculations using the SOFTmaxPro version 2.6.1 (Molecular Devices Corp., Sunnyvale, CA, USA) software. Anti-β1-R concentrations were calculated using a 4-parameter standard curve and the OD measured for each sample. The equation that represented the standard curve was calculated by using 5 standard values: 240 ng/mL, 120 ng/mL, 60 ng/mL, 30 ng/mL and 15 ng/mL. The lower limit of detection was stated as 2.12 ng/mL in the manufacturer’s instructions. None of the serum samples that were used had been prediluted. Each OD value was determined as the average of 2 OD values obtained from 2 wells belonging to the same sample. The coefficient of variation (CV) between 2 wells was always 159.88 (ng/mL) Anti-M2-R (ng/mL) Anti-M2-R > 277.65 (ng/mL) AF duration (months) LAVI (mL/m2 ) Current smoking (%)

Multivariate Model

HR (95% CI)

P Value

HR (95% CI)

P Value

2.165 (1.063–4.410) 5.787 (2.134–15.963) 1.080 (1.040–1.130) 7.770 (2.732–22.098) 1.040 (1.010–1.070) 6.719 (2.331–19.363) 1.005 (0.989–1.021) 1.297 (1.101–1.528) 1.964 (0.751–5.136)

0.033*

0.838 (0.319–2.206) 3.058 (0.624–14.982) – 4.281 (1.108–18.175) – 4.313 (1.151–16.164) 0.983 (0.959–1.007) 0.989 (0.959–1.007) –

0.721 0.168 – 0.039* – 0.030* 0.167 0.714 –

0.001* 0.001* 0.001* 0.007* 0.001* 0.047* 0.002* 0.169

AF = atrial fibrillation; anti-β1-R = antibodies against beta1-adrenergic receptors; anti-M2-R = antibodies against M2-muscarinic receptors; CI = confidence interval; HR = hazard ratio; LAVI = left atrial volume index. *P < 0.05.

Figure 3. Spearman’s correlation analysis revealing the correlation between LAVI and anti-β1-R (A) and anti-M2-R levels (B).

of 95.24% and 70.59%, respectively (AUC: 0.793, 95% CI: 0.688–0.875, P = 0.001) (Fig. 2). A cut-off level of anti-β1-R level as 159.88 ng/mL predicted late AF recurrence with a sensitivity and specificity of 90.48% and 70.59%, respectively (AUC: 0.748, 95% CI: 0.638–0.838, P < 0.001) (Fig. 1). Univariate Cox regression analysis for late AF recurrence showed that active smoking; number of antiarrhythmic medications; duration of AF; LAVI; early AF recurrence; anti-M2-R and anti-β1-R levels were associated with increased late AF recurrence in the follow-up (P < 0.05) (Table 2). In multivariate Cox proportional hazard regression analysis, only anti-M2-R levels >277.65 ng/mL (HR: 4.313, 95% CI: 1.151–16.164, P = 0.03) and anti-β1-R levels > 159.88 ng/mL (HR: 4,281, 95% CI: 1,108–18,175, P = 0.039) were found to be independent predictors of late AF recurrence (Table 2). Of baseline characteristics, Spearman’s correlation analysis revealed a positive correlation between LAVI and anti-β1R (r = 0.342, P = 0.003) (Fig. 3A) and anti-M2-R levels (r = 0.254, P = 0.023) (Fig. 3B). There was no significant correlation between autoantibody levels and other parameters related to structural remodeling including age, diabetes mel-

litus, hypertension, smoking, alcohol consumption, duration of AF, left ventricular end-diastolic diameter, LVEF (Supporting Table S1). Discussion In the present study, we have demonstrated the association between serum anti-M2-R and anti-β1-R levels measured by ELISA and AF recurrence following cryoablation. To the best of our knowledge, this is the first study evaluating this relationship. Autoantibodies against the second extracellular loop of M2-muscarinic acetylcholine receptors (anti-M2-R) have been proposed to play a role in pathogenesis of AF. Anti-M2R, which has been found to be present in 23% of patients with idiopathic AF and 8% of healthy subjects,5 has also been reported to be an independent predictor for development of AF in patients with idiopathic dilated cardiomyopathy and Graves’ disease.6,8 Addressing the pathophysiological link between cardiac autoantibodies and development of AF, Hong et al.16 have demonstrated that anti-M2-R-positive rabbits had longer intra-atrial activation times, shorter atrial effective refractory periods (AERP) and significantly increased

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atrial arrhythmogenicity compared to control rabbits. Electrophysiological changes were suggested to be secondary to activation of acetylcholine-gated potassium channel IK-ACh . The authors have also reported that anti-M2-R-positive rabbits had increased atrial fibrotic deposition and this finding was considered to make up the underlying structural substrate for the atrial arrhythmogenicity. Similarly, in a previous study from our group, we demonstrated that serum anti-M2R levels were associated with the severity of LA fibrosis in paroxysmal AF patients.17 In a study conducted by Zou et al.,18 anti-M2-R has been found to be significantly higher in AF patients when compared to subjects in sinus rhythm. In the same study, anti-M2-R levels were reported to be associated with AF recurrence following radiofrequency catheter ablation. In our study, anti-M2-R level greater than 277.65 ng/mL was found to be an independent predictor for late AF recurrence following cryoballoon-based AF ablation. In previous studies, circulating autoantibodies against the second extracellular loop of β1-adrenergic receptors (antiβ1-R) have been observed in patients with idiopathic dilated cardiomyopathy, Chagas disease, ventricular arrhythmias, conduction disturbances, or AF.9,10,19-21 Stavrakis et al.8 have shown that seropositivity for anti-β1-R differed significantly between patients with AF and sinus rhythm in Graves’ hyperthyroidism (94 vs. 38%, P < 0.001). In a small study by Novikova et al.,22 anti-β1-R seropositivity was detected in 41.6% of patients with AF and 10% of patients in the healty control group. Based on the observation of agonistic β1-adrenergic receptor autoantibodies stimulating apoptosis23,24 and stress response of the endoplasmic reticulum,25 it has been suggested that the pathophysiological mechanism may involve direct cardiac cytotoxicity. In animal studies, anti-β1-R autoantibodies have been found to promote the passage of calcium, whose progressive increase in amount is known to lead to myocyte destruction, fibrotic repair, and electrical instability of the heart, through L-type calcium channels by increased production of cAMP and protein kinase A.26-28 These changes have all been shown to be important in the development and maintenance of AF.29 In the present study, we demonstrated for the first time that anti-β1-R level greater than 159.88 ng/mL was an independent predictor of AF recurrence following cryoablation. LAVI is an echocardiographic parameter reflecting atrial structural remodeling.30 LAVI has been associated with17 and found to be an independent correlate of AF recurrence30,31 following catheter ablation in previous studies. In our study, a positive correlation between LAVI and autoantibody levels was detected. This correlation may be due to the fibrotic changes associated with anti-β1-R28 and anti-M2-R16 levels demonstrated in animal studies. Patients with some of the other well-known correlates of atrial structural remodeling, such as older age and low EF, have not been included in the study owing to their demonstrated relationship with autoantibody levels. Liu et al.32 have demonstrated that autoantibodies to β1-adrenergic and M2muscarinic receptors in the sera of healthy subjects increased significantly with age, and this rise was apparent following the 6th decade. Furthermore, Yoshikawa et al.3 have reported that various autoantibodies were detected in patients with dilated cardiomyopathy.

Conclusion This is the first study demonstrating an association between serum anti-M2-R and anti-β1-R levels measured by ELISA and AF recurrence following cryoablation. However, due to the complex relationship between AF and autoimmunity, it is still not possible to differentiate if the forementioned autoantibodies are actors, biomarkers, or bystanders,33 and this merits further studies. Study Limitations Our study has some limitations. First, this is a singlecenter study and the small number of study population has limited several statistical analyses to be performed. Second, none of the patients involved in the study were implanted with an internal loop recorder. Therefore, asymptomatic episodes might have occurred unnoticed and success rate may have been overestimated. Third, these results do not reveal a causal relationship, but only demonstrate an association. References 1. Camm AJ, Lip GY, De Caterina R, Savelieva I, Atar D, Hohnloser SH, Hindricks G, Kirchhof P, Guidelines-CPG ESCCfP, Document R: 2012 focused update of the ESC Guidelines for the management of atrial fibrillation: An update of the 2010 ESC Guidelines for the management of atrial fibrillation—developed with the special contribution of the European Heart Rhythm Association. Europace 2012;14:1385-1413. 2. Baba A, Fu M: Autoantibodies in atrial fibrillation: Actor, biomaker or bystander? Autoimmunity 2008;41:470-472. 3. Yoshikawa T, Baba A, Nagatomo Y: Autoimmune mechanisms underlying dilated cardiomyopathy. Circ J 2009;73:602-607. 4. Lazzerini PE, Capecchi PL, Guideri F, Acampa M, Selvi E, Bisogno S, Galeazzi M, Laghi-Pasini F: Autoantibody-mediated cardiac arrhythmias: Mechanisms and clinical implications. Bas Res Cardiol 2008;103:1-11. 5. Herda LR, Felix SB, Boege F: Drug-like actions of autoantibodies against receptors of the autonomous nervous system and their impact on human heart function. Br J Pharmacol 2012;166:847-857. 6. Baba A, Yoshikawa T, Fukuda Y, Sugiyama T, Shimada M, Akaishi M, Tsuchimoto K, Ogawa S, Fu M: Autoantibodies against M2-muscarinic acetylcholine receptors: New upstream targets in atrial fibrillation in patients with dilated cardiomyopathy. Eur Heart J 2004;25:1108-1115. 7. Yalcin MU, Gurses KM, Kocyigit D, Kesikli SA, Ates AH, Evranos B, Yorgun H, Sahiner ML, Kaya EB, Oto MA, Guc D, Ozer N, Aytemir K: Elevated M2-muscarinic and beta1-adrenergic receptor autoantibody levels are associated with paroxysmal atrial fibrillation. Clin Res Cardiol 2015;104:226-233. 8. Stavrakis S, Yu X, Patterson E, Huang S, Hamlett SR, Chalmers L, Pappy R, Cunningham MW, Morshed SA, Davies TF, Lazzara R, Kem DC: Activating autoantibodies to the beta-1 adrenergic and m2 muscarinic receptors facilitate atrial fibrillation in patients with Graves’ hyperthyroidism. J Am Coll Cardiol 2009;54:1309-1316. 9. Chiale PA, Ferrari I, Mahler E, Vallazza MA, Elizari MV, Rosenbaum MB, Levin MJ: Differential profile and biochemical effects of antiautonomic membrane receptor antibodies in ventricular arrhythmias and sinus node dysfunction. Circulation 2001;103:1765-1771. 10. Brisinda D, Sorbo AR, Venuti A, Ruggieri MP, Manna R, Fenici P, Wallukat G, Hoebeke J, Frustaci A, Fenici R: Anti-beta-adrenoceptors autoimmunity causing ‘idiopathic’ arrhythmias and cardiomyopathy. Circ J 2012;76:1345-1353. 11. Calkins H, Reynolds MR, Spector P, Sondhi M, Xu Y, Martin A, Williams CJ, Sledge I: Treatment of atrial fibrillation with antiarrhythmic drugs or radiofrequency ablation: Two systematic literature reviews and meta-analyses. Circ Arrhythm Electrophysiol 2009;2:349-361. 12. Wazni OM, Marrouche NF, Martin DO, Verma A, Bhargava M, Saliba W, Bash D, Schweikert R, Brachmann J, Gunther J, Gutleben K, Pisano E, Potenza D, Fanelli R, Raviele A, Themistoclakis S, Rossillo A, Bonso A, Natale A: Radiofrequency ablation vs antiarrhythmic drugs as first-line treatment of symptomatic atrial fibrillation: A randomized trial. JAMA 2005;293:2634-2640.

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Supporting Information Additional supporting information may be found in the online version of this article at the publisher’s website: Table S1. Correlation analysis between autoantibody levels and parameters of structural remodeling.