Patients undergoing PVI for documented paroxysmal/persistent AF at our Department were considered prospectively. The inclusion criteria were the following: (1) age 18–75 years, (2) failure of at least one antiarrhythmic drug and (3) willingness to sign a written informed consent. Exclusion criteria included: (1) long-standing persistent or permanent AF, (2) reversible cause of AF (e.g., hyperthyroidism), (3) presence of a LA thrombus, (4) previous heart surgery, (5) valvular heart disease, (6) left ventricular ejection fraction (LVEF) 50%, and clinical signs of HF, (7) unstable angina or myocardial infarction within the last 3 months, (8) acute inflammatory disorders, (9) pregnancy, (10) malignant disease and (11) previous AF ablation. The study design was in accordance with the guiding principles of the Declaration of Helsinki and was approved by the Regional and Institutional Ethics Committee of the University of Debrecen (DE RKEB/IKEB/4951-2018) and the National Institute of Pharmacy and Nutrition (OGYÉI/12743/2018). All patients signed a written informed consent form prior to inclusion.

All patients were subjected to a full baseline evaluation, including medical history, physical examination, 12-lead electrocardiogram (ECG), transthoracic echocardiographic (TTE) examination to measure LVEF and LA diameters before the procedure. Transoesophageal echocardiography (TEE) was performed in all patients within 24 hours prior to the procedure in order to exclude the presence of a cardiac thrombus. Blood samples for the measurement of biomarkers were taken from a peripheral vein within 24 hours prior to the procedure.

The procedures were performed under conscious sedation. The Seldinger technique was used for femoral vein cannulation. Multipolar electrode catheters were placed in the coronary sinus and in the right ventricle. A transseptal puncture was performed with a Brockenbrough needle under intracardiac echocardiography (ICE) guidance. After the transseptal puncture, 150-IU/kg body weight intravenous heparin bolus was given followed by continuous infusion to keep the Activated Clotting Time (ACT) level between 300–400 msec. All PVI procedures were performed with any of the following 3 ablation technologies at the operator’s discretion: (1) phased radiofrequency ablation (RFA) with the 2nd generation Pulmonary Vein Ablation Catheter (PVAC) Gold catheter (Medtronic Inc, 990078, Minneapolis, MN, USA); (2) cryoablation with the Arctic Front Advance catheter (Medtronic Inc, 2AF283, Minneapolis, MN, USA); (3) point-by-point PVI with focal irrigated RFA with contact force monitoring using a Thermocool, Smarttouch catheter (D133602, Biosense Webster Inc., Johnson & Johnson Medtech, Irvine, CA, USA). The procedural endpoint was the isolation of all PVs, which was verified with pacing manoeuvres. If necessary, SR was restored by cardioversion after the procedure. Each ablation protocol applied at our center is described in detail below [14, 15, 16].

Baseline blood samples were collected from the cubital vein within 24 hours prior to the procedure. Follow-up blood samples were collected from the cubital vein after the end of the 3-year follow-up period. Blood samples were collected into vacutainer tubes. Tubes containing ethylenediamine tetraacetic acid (EDTA) anticoagulant (3 mL Vacuette tube with K3EDTA, 454086, Greiner Bio-One GmbH Bad Haller Str. Kremsmünster, Austria) and tubes containing clot activator (serum tubes with polymer gel separator, 5 mL BD Vacutainer SST II Advance Plus Blood Collection Tubes, 367955, Becton, Dickinson and Company Franklin Lakes, NJ, USA) were used. Within two hours, the samples were centrifuged at 1500 g for 20 minutes at room temperature. From EDTA tubes the plasma phase was pipetted, from serum separator tubes the serum phase was pipetted into aliquots and these were stored at –70 °C until the analysis.

Serum CA-125 levels were determined by an electro-chemiluminescent microparticle immunoassay (Cobas® e602) (Roche Diagnostics, Mannheim, Germany). Plasma Caspase-3 concentration was measured with Human Caspase 3 ELISA kit (Thermo Fisher Scientific Inc., Carlsbad, CA, USA, Cat. No. BMS2012INST), while plasma Galectin-3 was measured with Human Galectin-3 DuoSet ELSA kit (R&D Systems, Inc., Minneapolis, MN, USA, Cat. No. DY1154) and plasma Cathepsin L with Human Cathepsin L DuoSet ELISA kit (R&D Systems, Inc., Minneapolis, MN, USA, Cat. No. DY952) according to the manufacturer’s instructions.

The previously prescribed antiarrhythmic drugs were continued after the procedure and stopped 3 months later. All patients were anticoagulated for a minimum of 3 months period after the procedure, then the decision on long-term treatment was based on the CHA${}_2$DS${}_2$-VASc score. All patients had follow-up visits every 6 months post-ablation, which included clinical evaluation, 12-lead ECG and 24-hour Holter recordings. Patients were encouraged to have ECG recordings in case of any symptom suggestive of an AF episode. In these cases, patients were also offered trans-telephonic monitoring for up to 6 weeks to facilitate the documentation of the rhythm during palpitation. Recurrence was defined as any documented AF episode lasting more than 30 seconds. At the 3-year follow-up LVEF and LA diameter was measured with TTE examination.

Statistical analysis was performed using GraphPad Prism version 8.0.0 for Windows (GraphPad Software, San Diego, CA, USA). Normality of data distribution was assessed with Kolmogorov-Smirnov test. Continuous variables are expressed as means $\pm$ standard deviation (SD) and variables not normally distributed as medians and interquartile range. Categorical data were presented as counts with percentages within brackets. Analyses were calculated using Student’s t test or the Mann–Whitney test for continuous variables. To compare baseline and follow-up measurements, a paired t-test or Wilcoxon test was applied. The differences between categorical variables were assessed by the Chi-square or Fisher’s exact test. Evaluation of correlation was analysed using the Spearman’s correlation coefficient. p 0.05 was considered statistically significant.

A total of 63 patients were included in the study (23 women; age 61.4 $\pm$ 8.8 years). Thirty-six (57.1%) patients had been on antiarrhythmic drug therapy before the procedure. PVI was performed with PVAC ablation in 11, with cryoballoon in 28, and with point-by-point focal irrigated RFA in 24 patients. The acute isolation of all PVs was achieved in all patients. There were no complications during the procedure. The baseline characteristics are presented in Table 1.

During a mean follow-up of 36.3 $\pm$ 6.3 months, AF recurrence was demonstrated in 26 (41.3%) patients. 23% of the patients with recurrence had persistent AF at baseline as compared to 8% in the no recurrence group. The only statistical difference between the groups with or without recurrence was the higher proportion of female patients with AF recurrence (Table 1). All patients with recurrence had paroxysmal AF after the ablation.

Baseline (pre-ablation) biomarker levels in patients free of arrhythmia versus in those with AF recurrence during follow-up were compared. No significant differences in CA-125, Galectin-3, Caspase-3 and Cathepsin L levels were demonstrated between the two groups. Similarly, post-ablation blood samples taken 36.3 $\pm$ 6.3 months after the ablation procedure demonstrated no differences between the 2 groups in the level of any of the 4 substances measured (Fig. 1).

Fig. 1.

Pre-ablation and post-ablation levels of the biomarkers. There was no significant difference in CA-125, Galectin-3, Caspase-3 and Cathepsin L pre-ablation and post-ablation levels in patients with or without AF recurrence. Results of Mann–Whitney U-test. AF, atrial fibrillation; CA-125, cancer antigen 125.

Biomarker levels obtained at baseline were also compared to those obtained at the end of follow-up after PVI in both groups. Serum CA-125 levels showed no significant change neither in the no AF recurrence group (9.7 (7.6–13.0) kU/L vs 8.7 (6.7–12.8) kU/L; p = 0.104) nor in the recurrence group (11.3 (7.8–15.1) kU/L vs 11.0 (7.2–14.0) kU/L; p = 0.681). The plasma Galectin-3 levels decreased significantly both in the arrhythmia free (4209.9 (3226.2–5662.4) pg/mL vs 3391.4 (2856.2–4182.5) pg/mL; p = 0.014) and in the AF recurrence group (3810.4 pg/mL (3156.8–5915.8) vs 3591.9 (2637.8–4786.8) pg/mL; p = 0.037). Similarly, plasma Caspase-3 levels decreased significantly during the follow-up period in both groups (564.1 (463.0–731.6) pg/mL vs 376.9 (310.9–467.6) pg/mL; p = 0.001) and (579.0 (523.9–735.6) pg/mL vs 413.5 (341.8–563.5) pg/mL; p = 0.032) as well as plasma Cathepsin L levels decreased in the no recurrence group (406.9 (200.4–763.3) pg/mL vs 309.2 (131.7–460.4) pg/mL; p = 0.025) and in the recurrence group (422.4 (248.3–600.4) pg/mL vs 294.4 (158.2–470.7) pg/mL; p = 0.036) (Fig. 2).

Fig. 2.

Changes in biomarker levels before and after ablation. The serum CA-125 levels were not changed significantly during the follow-up period in either group. The plasma Galectin-3, Caspase-3 and Cathepsin L levels decreased significantly both in the arrhythmia-free and the AF recurrence group. Results of paired t-test and Wilcoxon test. *p 0.05; **p 0.01. CA-125, cancer antigen 125; AF, atrial fibrillation.

LA diameter demonstrated a slight decrease from baseline to the end of follow-up in patients with no recurrence (41.8 $\pm$ 4.1 mm vs 40.5 $\pm$ 4.6 mm; p = 0.231) and a minimal increase in patients with AF recurrence (41.3 $\pm$ 5.2 mm vs 42.7 $\pm$ 4.9 mm; p = 0.087), however, none of these changes reached the level of statistical significance (Fig. 3). The LVEF was not changed significantly in the two groups (58.49 $\pm$ 5.3% vs 57.16 $\pm$ 4.3%; p = 0.248; no recurrence group) (57.04 $\pm$ 7.7% vs 56.5 $\pm$ 5.5%; p = 0.786; AF recurrence group).

Fig. 3.

Left atrial diameter changing during the 3-year follow-up period in patients with and without AF recurrence. LA diameters did not change significantly in either group. Results of paired t-test. AF, atrial fibrillation; LA, left atrial.

We found positive correlation between Caspase-3 levels and LA diameters in the AF recurrence group both before (r = 0.477; p = 0.018) and after the procedure (r = 0.533; p = 0.019; Fig. 4). There was no correlation between levels of the other biomarkers and LA diameters either pre- or post-ablation (Figs. 5,6,7).

Fig. 4.

Correlation of Caspase-3 levels with LA diameters before and after the procedure. We compared the no recurrence group with the AF recurrence group. We found correlation between the Caspase-3 levels and LA diameters in the AF recurrence group before and after the procedure. There was no correlation in the arrhythmia-free group. Results of Spearman’s correlations. AF, atrial fibrillation; LA, left atrial.

Fig. 5.

Correlation of CA-125 levels with LA diameters before and after the procedure. We compared the no recurrence group with the AF recurrence group. There was no correlation between the pre-ablation and post-ablation levels of CA-125 with the LA diameters. Results of Spearman’s correlations. AF, atrial fibrillation; LA, left atrial; CA-125, cancer antigen 125.

Fig. 6.

Correlation of Galectin-3 levels with LA diameters before and after the procedure. We compared the no recurrence group with the AF recurrence group. There was no correlation between the pre-ablation and post-ablation levels of Galectin-3 with the LA diameters. Results of Spearman’s correlations. AF, atrial fibrillation; LA, left atrial.

Fig. 7.

Correlation of Cathepsin L levels with LA diameters before and after the procedure. We compared the no recurrence group with the AF recurrence group. There was no correlation between the pre-ablation and post-ablation levels of Cathepsin L with the LA diameters. Results of Spearman’s correlations. AF, atrial fibrillation; LA, left atrial.

A 3-year clinical success after the successful acute isolation of all PVs with any of 3 different ablation technologies was demonstrated in 37 out of 63 patients (58.7%) with female sex being the only significant difference between the two groups. None of the biomarkers measured at baseline were associated with the arrhythmia outcome in our study. Levels of CA-125 demonstrated no significant change before versus 3 years after AF ablation. On the contrary, all the other biomarkers (Galectin-3, Cathepsin-L and Caspase-3) demonstrated a significant decline in all patients regardless of the arrhythmia outcome during long-term follow-up. The mean diameters of the LA and LVEF demonstrated no statistically significant change in any group. Positive correlation was found between Caspase-3 levels and LA diameters in the AF recurrence group both before and after the procedure.

The serum/plasma concentrations of these potential biomarkers in patients with versus without AF have been investigated by several studies.

CA-125 is a known tumor marker for the neoplasm of the ovarium and other organs. CA-125 has also been demonstrated strong predictor of mortality in patients with decompensated HF. Dudink et al. [5] investigated over 90 cardiovascular blood biomarkers in 60 AF patients without overt forms of cardiovascular diseases, and compared with 120 matched individuals without known AF. Higher CA-125 levels were demonstrated to be independent predictors of idiopathic AF. In a meta-analysis [6], 7 out of 9 studies reported significantly higher CA-125 levels in patients with AF as compared with those in SR using a cut-off level at 35 U/L. These studies included heterogeneous patient cohorts with significant comorbidities like HF and inflammatory conditions. Neither Dudink’s nor Cheung’s report contained data on the relationship between CA-125 and AF ablation results or on the long-term changes in CA-125 levels. In our study including patients free of clinical and echocardiographic signs of HF, the levels of CA-125 were lower (10 U/L before the ablation and 9 U/L at the end of follow-up) than the 35 U/L cut-off proposed by this meta-analysis and no statistical difference was demonstrated between our patients with versus without AF recurrence. Of note, CA-125 was the only biomarker among the 4 substances we studied which demonstrated no change during a 3-year follow-up. In context with these published data, our results may suggest, that CA-125 concentrations may rather be related to HF or other comorbidities with significant inflammatory response than to AF per se and may not be used to predict ablation success in patients with idiopathic AF.

Very limited human data have been reported on Cathepsin-L as a biomarker of cardiovascular diseases. Mehra et al. [7] described a correlation between the increased expression of Cathepsin-L in peripheral blood mononuclear cells with the severity of left ventricular dysfunction in patients with dilated cardiomyopathy. In the investigation of Dudink et al. [5], Cathepsin L concentrations were higher in AF patients as compared with controls in SR; however, this correlation was not found to be an independent predictor of the arrhythmia. The expression of Caspase-3 was investigated by Chen et al. [8] in patients with rheumatic heart disease undergoing valve replacement. Caspase-3 expression was increased in permanent atrial fibrillation as compared with the expression in SR. Further, Caspase-3 levels demonstrated a positive correlation with both LA dimensions and AF durations. In our study, the baseline levels of neither Cathepsin L nor Caspase-3 demonstrated an association with AF recurrence. However, a significant decrease in the concentrations of both substances was demonstrated at the end of the 3-year follow-up after the ablation. In addition, Caspase-3 was the only biomarker in our study which demonstrated a positive correlation with LA diameters in the AF recurrence group. Of note, 23% of our patients with recurrence had persistent AF as compared to only 8% in the no recurrence group. Although Caspase-3 levels were not associated with AF recurrence in our investigation, the most significant decrease after the ablation was demonstrated with this substance. Based on these observations it might be speculated that Caspase-3 levels are sensitive markers of fibrosis mainly in patients with more persistent forms of AF or with high AF burden.

Higher Galectin-3 levels were consistently demonstrated in patients with versus without AF [9, 10]. The majority of the patients in these studies had persistent or long-standing persistent AF. Furthermore, Galectin-3 has also been tested as a potential predictor of AF recurrence after AF ablation. Based on the measurements in 160 patients (55% had paroxysmal AF; mean LA diameter 42 mm) before and 12 months after a successful PVI. Clementy et al. [11] reported a 1-year arrhythmia-free survival rate of 91% in those with Galectin-3 level 15000 pg/mL and LA diameter 40 mm. The predictive value of Galectin-3 for AF recurrence demonstrated in this, but not in our study could be explained by the longer follow-up in our examination as well as the differences in baseline patient characteristics. Patients with recurrence were older (64 $\pm$ 10 years vs 59.9 $\pm$ 8.5 years), had larger LA (45 mm vs 41 mm) and more comorbidities including HF (33% vs none) and higher proportion of patients with persistent AF (64% vs 23%) in the report of Clementy [11] versus in our cohort, respectively. Again, these differences might suggest that Galectin-3 concentrations may rather be related to HF or other comorbidities as well as to more persistent forms of AF. This speculation is further supported by another investigation from the same group [12] including patients with persistent AF and systolic HF (LVEF 40%), who underwent PVI. In this study the mean LA volume was 48 $\pm$ 16 mL/m${}^2$ and over 21,000 pg/mL serum Galectin-3 levels were measured. In another study [13] including patients with long standing persistent AF, enlarged LA (mean LA diameter: 47.8 $\pm$ 1.4 mm) and structural heart disease higher Galectin-3 levels were measured in blood samples obtained from the LA cavity and from the coronary sinus in patients with persistent, than in those with paroxysmal AF. In addition, Galectin-3 levels $>$13,050 pg/mL in CS and $>$11,900 pg/mL in LA were predictive of recurrences after a single ablation. Importantly, markedly lower levels of Galectin-3 were measured (4209 pg/mL in patients with no recurrence and 3810 pg/mL in the AF recurrence cohort) in our study.

Based on our results in view of published data, markers of atrial fibrosis may predict recurrence after AF ablation in patients with more advanced state of LA disease, mostly in those with significant comorbidities and longer-term AF duration but not with a structurally normal heart, normal size LA and mainly paroxysmal form of the arrhythmia. Lower initial concentrations of biomarkers, especially of Galectin-3 measured in our cohort are in line with this explanation. The shorter follow-up durations and the uncertainties in the evaluation of recurrence in the absence of continuous long-term monitoring provide further explanations of the discrepancies between our results and the findings of other investigators.

With the exception of CA-125, all the other biomarkers included in our research demonstrated a significant decline in their concentration by the end of the 3-year follow-up. This is a novel observation, as no data have been reported on long-term changes after AF ablation; therefore, our findings await confirmation by others. A possible explanation we propose is that AF ablation might reverse fibrotic alterations by reducing the AF burden post-ablation even in those patients who have AF recurrence. Recent studies demonstrated a significant decrease in AF burden in patients with recurrence, which possibly explains the significant improvement in outcome especially in HF patients [17], an observation leading to a paradigm shift in the assessment of the long-term clinical value of AF ablation. Our findings might provide further evidence supporting this concept. Prospective studies including continuous monitoring and serial measurements of fibrosis markers could provide solid evidence to answer these questions.