Association of Complex Fractionated Electrograms with Atrial Myocardial Thickness and Fibrosis

Background and Objectives: Although ablation of complex fractionated atrial electrograms (CFAE) in atrial fibrillation (AF) is one of the strategies for atrial substrate modification, the mechanism behind CFAE as an electrophysiological substrate remains unclear. We investigated structural differences between CFAE sites and their matched non-CFAE sites by comparing their histopathologic characteristics in canine AF models. Methods: Atrial electrograms of four dogs were obtained from the epicardial site. AF was induced through burst atrial pacing at 600 bpm for 30 min. CFAE sites were identified during AF according to patterns visualized on the electrograms, and their matched non-CFAE sites were selected in the adjacent region, within 5 mm of each CFAE site. Tissues were harvested from CFAE sites and their matched nonCFAE sites at various locations in both atria. Histopathologic differences were identified between CFAE and non-CFAE sites. Results: A total of 24 atrial tissues (12 with CFAE, 12 with non-CFAE) were evaluated. The atrial myocardium was significantly thicker at CFAE sites (1757.5±560.5 μm) than at non-CFAE sites (1279.5±337.2 μm) (p=0.036). At CFAE sites, it was filled with a significantly larger amount of fibrotic tissue than at non-CFAE sites (22.8±6.9% versus 7.2±4.7%, p<0.001). Results were consistent across various tissue locations. The distribution of autonomic nerve innervation was similar between CFAE and non-CFAE sites. Conclusion: This study provides a better understanding of histological characteristics of CFAE sites, namely a thicker wall and greater amount of fibrosis. These findings may be associated with the development of CFAE and its pathophysiological contribution to AF.


Introduction
Although radiofrequency ablation has been an important treatment strategy for persistent atrial fibrillation (AF), it is remains challenging and results are unsatisfactory. 1 Several additive substrate modification strategies have been attempted, to improve the recurrence rate and long-term outcomes of nonparoxysmal AF. Nademanee et al. reported that complex fractionated atrial electrograms (CFAEs) could be ideal target sites for ablation of refractory AF, and CFAE-guided ablation may show a higher success rate and survival benefit than pulmonary vein isolation alone. 2,3 However, the pathophysiologic mechanism of CFAE has not yet been clarified, and the structural relationship between CFAE sites and AF is also unknown. Since negative results of CFAE ablation have also been reported, characteristics of CFAE sites as an electrophysiological substrate should be more accurately identified and reflected in clinical practice. 4,5 Therefore, we sought to evaluate the structural differences between CFAE and non-CFAE sites by comparing their histopathologic features.

Animal Preparation
The study protocol was approved by the Seoul National University Hospital Institutional Animal Care and Use Committee (approval No. 16-0136-S1A0). A total of four adult mongrel dogs, weighing 20-25 kg, were involved in the present study. Following the standard and approved protocols, all dogs were anesthetized with thiopental (20 mg/kg IV) and intubated with cuffed endotracheal tubes for mechanical respiration followed by gaseous anesthesia (1-2% isoflurane/O 2). All measures were taken to ensure that discomfort, distress, pain, and injury were limited as much as possible. Standard surface electrocardiogram leads were monitored continuously throughout the entire study, and intermittent arterial blood gas analyses with ventilator adjustments were done to correct for any metabolic abnormalities. An electrical heating pad was used to maintain a body temperature of 36-37 . For the electrophysiological study and simultaneous tissue harvesting, a median sternotomy and pericardiectomy were performed to access the canine heart directly. The epicardial patch electrodes for electrophysiological studies were fixed to the atrial free walls by simple sutures.

Electrophysiological Study Protocol
Atrial electrograms were amplified and filtered from 0.05 to 500 Hz and were displayed and recorded on a Prucka Cardiolab

Atrial Fibrillation Induction and CFAE Detection Protocol
AF was induced with 600-bpm burst atrial pacing for 30 min, and AF that was sustained longer than 10 min was included in the evaluation. The CFAE sites were determined according to the patterns on the electrograms. 2 Electrograms with the following two characteristics simultaneously were considered a CFAE: 1) atrial electrograms that had fractionated electrograms composed of two deflections or more, and/or perturbation of the baseline with continuous deflection of a prolonged activation complex, over a 10-s recording period; 2) atrial electrograms with a very short cycle length (≤120 ms) averaged over a 10-s recording period. A 10-s duration was used for the assessment of the CFAE to avoid nonspecific sites and to confirm consistent fractionation. The matched non-CFAE sites were selected in the adjacent region within 5 mm from each CFAE site.

Histopathologic Examination
Myocardial tissue from the CFAE and non-CFAE sites,

Statistical Analysis
Continuous variables were expressed as mean ± standard deviation and were analyzed using the paired t test. All probability values were two-sided, and a p-value <0.05 was considered statistically significant. Statistical analyses were performed using SPSS statistical package version 19.0 (IBM Corp, Armonk, NY, USA).

Baseline Distribution of CFAE in Various Atrial Tissues
A total of 24 atrial myocardial tissues (12 from CFAE sites and 12 from their matched non-CFAE sites) were evaluated. Table 1 shows the anatomical location and distribution of the tissues used in the analysis. Of the 12 sites, 7 were located in the right atrium         (Table 3).

Major Findings
In the present study, CFAE was observed at various locations throughout the left and right atrium. Compared with matched non-CFAE sites, a thicker atrial myocardium and a larger amount of fibrosis was observed at all CFAE sites, in all locations.
However, distribution of the autonomic nervous system did not differ significantly between the CFAE sites and the matched non-CFAE sites.

Histopathologic Features of CFAE Sites
CFAE sites are known to exhibit low voltage and slow conduction. 6 In addition, focal discharge, wave break and fusion, and pivoting activation observed in CFAE are known to contribute to the maintenance of AF through wave propagation. 7 CFAE areas could indicate the re-entry of fibrillation waves or the overlap of other wavelets superimposed in the same area at different times. 1 CFAE sites may also have rotor boundaries or be associated with migrating rotors and wave breaks rather than the center of a stable rotor itself. 8 In our previous study, CFAE sites have been identified as AF substrates different from the AF nest. 9 One computational study suggested that CFAE is more strongly related to the tissue characteristics of the atrial substrate than to the electrical activation system. 10    procedural success of catheter ablation. 12 Furthermore, left atrial wall thickness measured by cardiac computed tomography was also found to be more in the CFAE area (3.0±1.0 mm) than in the non-CFAE area (2.2±0.9 mm), which is consistent with our results. 13 These results suggest that atrial myocardial thickness is closely related to the CFAE sites. This also suggests the possibility of heterogeneous tissue organization and arrangement at CFAE sites, which may be an important factor associated with AF and the procedural success of ablating atrial substrates.
Second, we found that a large amount of atrial myocardial fibrosis was identified as another histopathologic characteristic of CFAE. It has already been suggested that fibrosis is an important component of the AF substrate in several human and animal AF model studies. 14 In addition, structural heterogeneities, such as random microstructural alterations, have been reported to be the major pathophysiological mechanisms of CFAE and persistent AF. 15 Particularly in the CFAE sites, increased fibrosis with decreased connexin 43 has been suggested to underlie structural abnormalities. 16 Jacquement et al. suggested in their computational microstructural modeling study, that microfibrosis could also contribute to CFAE genesis. 17 In addition to simple collagen deposition, fibroblast proliferation in the atria has also been shown to affect CFAE generation during AF. 18 Since this study has confirmed that increased atrial fibrosis is one of the microscopic characteristics of CFAE sites, we may now better understand the underlying mechanism of CFAE-guided AF ablation. However, in studies analyzing late gadolinium enhancement cardiac magnetic resonance imaging of left atrial wall composition, it has been reported that normal substrate without fibrosis is even more associated with CFAE than that with fibrotic sites. 19 Although inconsistent, it is clear that fibrosis is closely related to the CFAE sites, which should be clarified in future studies.
The autonomic basis for CFAE formation and effects of the autonomic nervous system on CFAE are well known. 20

Conclusions
A thicker atrial myocardium and a larger amount of fibrosis were identified as the most important histopathologic characteristics of CFAE sites compared to their matched non-CFAE sites. These results may help elucidate the underlying mechanism of CFAE and the implications for catheter ablation.