Atrial fibrillation (AF) is the most common sustained arrhythmia. Inflammation has been suggested to play a vital role in the pathogenesis. Previous studies have investigated expression of inflammatory markers in AF. Several studies have focused on the effects of toll-like receptors (TLRs) on heart in terms of capability of modulating inflammation. In this study, we aimed to investigate whether peripheral monocyte TLR expression was associated with the AF presence, and recurrence of AF after cryoablation, as a reflection of inflammatory status. Patients with AF who were scheduled for cryoballoon-based ablation for AF and age- and gender-matched subjects in sinus rhythm were included. Peripheral monocyte TLR-2 and TLR-4 expressions were evaluated by flow cytometric analysis in peripheral venous blood samples obtained during evaluation in outpatient clinics: 172 patients (56.5 ± 6.6 years, 52.3% men) were included in the study. Peripheral monocyte TLR-2 and TLR-4 expression levels were significantly higher in patients with AF (p <0.05). Among patients with AF, 12 patients (14.0%) developed AF recurrence at a follow- up of 17 months. Multivariate Cox regression analysis showed that left atrial volume index (hazard ratio 2.040, 95% CI 1.197 to 3.477, p = 0.009) and monocyte TLR-4 expression (hazard ratio 1.226, 95% CI 1.042 to 1.443, p = 0.014) were independent predictors of AF recurrence after blanking period following second-generation cryoballoon–based pulmonary vein isolation for paroxysmal AF. In conclusion, our study highlights the role of TLR-mediated inflammation in the pathogenesis of AF. This link may also constitute a therapeutic target in patients with AF.
Atrial fibrillation (AF) is the most common sustained arrhythmia. Inflammation has been suggested to play a vital role in the pathogenesis. The role of inflammation in the initiation of AF was first suggested on the basis of the observation that inflammatory states, such as myocarditis, pericarditis, and cardiac surgeries, were frequently associated with AF. The exact mechanism linking inflammation with non–operative-related AF is unknown, and it is unclear whether inflammation is an initiator or consequence of AF. Previous studies have investigated expression of inflammatory markers in AF. Toll-like receptors (TLRs) are of the pattern recognition receptors for recognition of pathogen-associated molecular patterns in the induction of innate immunity. They have been identified to play a crucial role in the induction and progression of chronic inflammatory disorders. Several studies have focused on the effects of TLRs on heart in terms of capability of modulating inflammation and tissue damage after non-infectious insults. In this study, we aimed to investigate whether peripheral monocyte TLR expression was associated with AF presence, and recurrence of AF after cryoablation, as a reflection of inflammatory status.
Methods
In this prospective study, 86 patients with AF who were scheduled for cryoablation with second-generation cryoballoon and age- and gender-matched 86 control subjects with sinus rhythm were enrolled from November 2013 to October 2014. Patients who had coronary artery disease, previous attempts of ablation, ejection fraction <50%, severe valvular heart disease, or chronic inflammatory disease were excluded. Subjects were confirmed to be infection-free for at least 1 month at the time of blood sampling and did not have fever within a week from the time the blood sample was drawn. The study was in compliance with the principles outlined in the Declaration of Helsinki and approved by the institutional ethics committee.
TLR-2 and TLR-4 expression analyses were performed by flow cytometry (Beckman Coulter Epics XL-MCL, Miami, Florida) in whole blood samples collected in ethylenediaminetetraacetic acid-anticoagulated tubes withdrawn from patients on admission. Monocytes, lymphocytes, and neutrophils were separated by their forward and side scattering signal characteristics on flow cytometry. Briefly, 100 μl of whole blood were incubated with 10 μl TLR-2 PE (CD282; cloneT2.5; Biolegend, San Diego, California), 10 μl TLR-4 PE (CD284; HTA 125; Biolegend), 10 μl CD14 FITC (clone M5E2; Biolegend) antibodies, and isotopic controls (MsIgG1 FITC; Biolegend; MsIgG1PE; Biolegend and MsIgG2FITC Biolegend) for 30 minutes at 4°C in the dark and then 10.000 events were acquired through a live gate drawn on forward light scatter and side light scatter.
All patients underwent transthoracic echocardiography within 1 week before ablation to assess intracavitary dimensions, left ventricular ejection fraction and to exclude valvular heart disease. Transoesophageal echocardiography was performed to rule out the presence of thrombus in the left atrial (LA) appendage, the day before procedure. Furthermore, patients underwent a preprocedural multidetector computed tomography scan with 3-dimensional (3D) construction of the LA to assess detailed LA anatomy, including evaluation of the pulmonary vein (PV) configuration. Anticoagulation was discontinued at least 48 to 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 before the procedure.
A second-generation 28-mm cryoballoon catheter (Arctic Front Advance, Medtronic CryoCath LP, Qubec, Canada) was used for pulmonary vein isolation (PVI). A minimum of 2 consecutive freezing cycles were performed. The procedure systematically began with the left superior PV, followed by the left inferior, right superior, and right inferior PVs, respectively. At the end of the procedure, PV conduction was re-evaluated with the Achieve catheter. Successful PVI was defined as the elimination of all PV potentials.
Transthoracic echocardiographic examination was performed immediately after the procedure and 24 hours after the procedure to exclude the presence of pericardial effusion. All patients were followed up for at least 48 hours in the telemetry unit. Oral anticoagulation was initiated 12 hours after the procedure. Enoxaparin 1 mg/kg was administered until target levels of warfarin was maintained. Bridging with enoxaparin was not applied in case novel oral anticoagulant was initiated. The patients remained on antiarrhythmic drug regimen for a period of 3 months. After discharge from the hospital, enrolled patients were scheduled for visits in the outpatient clinics at 1, 3, 6, and 12 months after ablation, or earlier, if symptoms consistent with recurrent AF developed after the ablation. At each visit, patients were evaluated for the recurrence of arrhythmias with physical examination, questioning for arrhythmia-related symptoms (palpitations, chest discomfort, fatigue, and dizziness) and a 12-lead electrocardiogram (ECG). A 24- hour ambulatory ECG monitorization was conducted in 1, 3, 6, and 12 months for all patients. Patients were anticoagulated for at least 3 months post-procedurally and the need for further oral anticoagulation was re-evaluated in the third month, based on the CHA 2 DS 2 -VASc score. Any episodes of AF, atrial flutter, or atrial tachycardia lasting at least 30 seconds and occurring during and after the 90-day blanking period were classified as early and late AF recurrence, respectively.
SPSS 21.0 software (SPSS Incorporated, Chicago, Illinois) was used for statistical analysis. Descriptive characteristics of continuous variables are expressed as mean ± SD. Categorical data were compared by the chi-square test. Cox regression analyses were used to assess the predictors of recurrent AF. Optimal cut-off values were determined by the analysis of the sensitivity and specificity values derived from receiver operating characteristic (ROC) curve data. A p value <0.05 was considered statistically significant in all analyses.
Results
A total of 172 patients (56.5 ± 6.6 years, 52.3% men) were involved in the study. Baseline characteristics of the study population, including demographic, clinical, laboratory, and transthoracic echocardiographic parameters, are listed in Table 1 . Patients with AF had higher serum C-reactive protein (CRP) levels; larger LV end-diastolic diameter, and LA diameter compared with the control group (p <0.05). Peripheral monocyte TLR-2 and TLR-4 expression levels were also significantly higher in patients with AF (p <0.05). Monocyte TLR-2 and TLR-4 expression levels did not differ between paroxysmal and persistent AF patient groups (p = 0.399, p = 0.814). A total of 335 PVs were attempted for PVI. The number of cryo applications per PV was 2 (1 to 5). Acute procedural success rate was 334/335 (99.7%).
| Parameters | Study Population (n= 172) | Atrial fibrillation | p value | |
|---|---|---|---|---|
| No (n= 86) | Yes (n= 86) | |||
| Age (years) | 56.5± 6.6 | 56.4± 5.5 | 56.6± 7.5 | 0.835 |
| Men | 90 (52.3%) | 46 (53.5%) | 44 (51.2%) | 0.879 |
| Hypertension | 89 (51.7%) | 43 (50.0%) | 46 (53.5%) | 0.760 |
| Diabetes mellitus | 24 (14.0%) | 11(12.8%) | 13 (15.1%) | 0.826 |
| Hyperlipidemia | 68 (39.5%) | 32 (37.2%) | 36 (41.9%) | 0.640 |
| White blood cell count (x10 3 /μL) | 7.3± 2.4 | 7.1± 0.9 | 7.6± 3.3 | 0.148 |
| C- reactive protein (mg/dL) | 0.2 (0.1- 7.1) | 0.2 (0.1- 0.2) | 1.1 (0.2- 7.1) | <0.001 ∗ |
| Left ventricular end- diastolic diameter (mm) | 4.8± 0.4 | 4.7± 0.4 | 4.9± 0.4 | <0.001 ∗ |
| Left ventricular ejection fraction (%) | 64.2± 5.2 | 65.7± 3.0 | 62.4± 6.5 | <0.001 ∗ |
| Left atrial diameter (mm) | 3.6± 0.5 | 3.3± 0.3 | 3.8± 0.5 | <0.001 ∗ |
| Left atrial volume index (mL/m 2 ) | 26.3± 3.7 | 23.4± 2.1 | 29.2± 2.6 | <0.001 ∗ |
| Peripheral monocyte TLR- 2 expression (%) | 9.5 (2.0- 56.0) | 4.0 (2.0- 9.0) | 21.0 (10.0- 56.0) | <0.001 ∗ |
| Peripheral monocyte TLR- 4 expression (%) | 8.5 (2.0- 59.0) | 4.0 (2.0- 8.0) | 22.0 (9.0- 59.0) | <0.001 ∗ |
All patients with AF underwent cryoablation and were followed up for 17 (10 to 21) months. Antiarrhythmic drug therapy prescribed during the first three months of the follow-up is listed in Table 2 . When blanking period of 3 months was considered, freedom from AF after a single ablation procedure was 86.1%.
| Parameters | Atrial fibrillation recurrence | p value | |
|---|---|---|---|
| No (n= 74) | Yes (n= 12) | ||
| Age (years) | 56.1± 9.1 | 57.50± 8.17 | 0.612 |
| Men | 36 (48.7%) | 8 (66.7%) | 0.354 |
| Hypertension | 41 (55.4%) | 5 (41.7%) | 0.535 |
| Diabetes mellitus | 11 (14.9%) | 2 (16.7%) | 1.000 |
| Hyperlipidemia | 32 (43.2%) | 4 (33.3%) | 0.754 |
| Duration of AF (months) | 27 (1- 300) | 12 (2- 120) | 0.676 |
| Type of AF: persistent | 20 (27.0%) | 7 (58.3%) | 0.076 |
| Statin | 12 (16.2%) | 2 (16.7%) | 1.000 |
| ACEi/ ARB | 19 (25.7%) | 2 (16.7%) | 0.717 |
| White blood cell count (x10 3 /μL) | 7.6± 3.2 | 7.5± 3.9 | 0.878 |
| C- reactive protein (mg/dL) | 1.1 (0.2- 6.9) | 4.3 (0.3- 7.1) | 0.031 ∗ |
| Left ventricular end- diastolic diameter (mm) | 4.9± 0.4 | 5.0± 0.3 | 0.345 |
| Left ventricular ejection fraction (%) | 62.7± 6.2 | 60.9± 8.0 | 0.387 |
| Left atrial diameter (mm) | 3.7± 0.5 | 4.3± 0.5 | <0.001 ∗ |
| Left atrial volume index (mL/m 2 ) | 28.6± 2.2 | 32.7± 2.5 | <0.001 ∗ |
| Peripheral monocyte TLR- 2 expression | 20.0 (10.0- 35.0)% | 29.5 (25.0- 56.0)% | <0.001 ∗ |
| Peripheral monocyte TLR- 4 expression | 21.0 (9.0- 53.0)% | 35.5 (29.0- 59.0)% | <0.001 ∗ |
| Amiodarone | 40 (54.1%) | 6 (50.0%) | 1.000 |
| Sotalol | 5 (6.8%) | 1 (8.3%) | 1.000 |
| Propafenone | 29 (39.2%) | 5 (41.7%) | 1.000 |
| Early recurrence | 9 (12.2%) | 2 (16.7%) | 0.648 |
Baseline demographic parameters, co-morbidities, and post-procedural antiarrhythmic drug use did not differ between patients with and without AF recurrence (p >0.05; Table 2 ). Patients with AF recurrence had a significantly larger LA diameter and LA volume index (LAVI). Serum CRP levels and peripheral monocyte TLR-2 and TLR-4 levels were also found to be significantly higher in patients with AF recurrence (p <0.05).
Multivariate Cox regression analysis showed that LAVI (hazard ratio 2.040, 95% CI 1.197 to 3.477, p = 0.009) and monocyte TLR-4 expression (hazard ratio 1.226, 95% CI 1.042 to 1.443, p = 0.014) were independent predictors of AF recurrence after blanking period following second-generation cryoballoon–based PVI for paroxysmal AF ( Table 3 ). A cut-off value of 29.5 for TLR-4 expression level was demonstrated to be associated with a sensitivity and specificity of 91.7% and 77.0% (area under curve = 0.840; 95% CI 0.758 to 0.922, p <0.001) for predicting AF recurrence in the ROC curve analysis.
