Enlargement of the left atrium is associated with increased infiltration of immune cells in patients with atrial fibrillation who had undergone surgery

Abstract

Background

Enlargement of the left atrium (LA) is a risk factor of atrial fibrillation (AF) recurrence after pharmacological and nonpharmacological interventions for AF. However, structural changes associated with LA enlargement have not been fully elucidated.

Methods

To examine inflammation in the structural changes associated with LA enlargement, human left appendages obtained from 27 patients who underwent cardiac surgery by using the maze procedure were subjected to immunohistochemical analysis.

Results

The extent of interstitial fibrosis increased according to the increase in LA dimension (LAD) as assessed by using ultrasound echocardiography. The extent of the infiltration of CD68-positive macrophages and CD3-positive T cells increased simultaneously according to the increments in LAD. The areas infiltrated by immune cells were positively and significantly correlated with LAD (r²=0.58, p<0.01 for CD68; r²=0.49, p<0.01 for CD3).

Conclusions

In the patients with AF, LA enlargement was associated not only with the increase in the extent of interstitial fibrosis but also with the changes in the LA component cells, including an increase in number of immune cells resident in tissues.

1. Introduction

Atrial fibrillation (AF), the most common type of sustained tachyarrhythmia, affecting approximately 0.9% of the population, is known to be associated with significant morbidity and mortality [1–4]. Recently, circumferential pulmonary vein isolation (CPVI) via catheter ablation has been established as a curative strategy for this type of arrhythmia, suggesting an important role of the thoracic veins in the mechanisms underlying AF. However, the success rate of CPVI ablation has been reported to decrease with increases in left atrial dimension (LAD) [5–7], similar to that of pharmacotherapy. This suggests potential pathological processes that invade the atrium from the pulmonary veins in a progressive manner in concert with the enlargement of the left atrium (LA).

A substantial body of evidence linking inflammation to the pathophysiology of a broad spectrum of cardiovascular diseases, including coronary artery disease, diabetes mellitus, and hypertension, has been developed [8–11]. Similarly, the hypothesis that inflammatory processes are involved in the pathogenesis of AF has attracted renewed attention. In previous studies, other researchers and we demonstrated the recruitment of immune cells across the atrial endocardium in patients with AF [12,13]; however, the degree of infiltration of immune cells widely varied between patients.

In the present study, we hypothesized that inflammatory processes occur and progress according to LA enlargement, which may contribute in part to the refractoriness to interventions for AF with an enlarged LA. Atrial appendage specimens from patients with AF who had undergone cardiac surgery were used for immunohistochemical analysis to assess potential relationships between LAD and the infiltration of immune cells.

2. Materials and methods

2.1. Patients

Left atrial appendage specimens were obtained from patients who had undergone cardiac surgery involving the maze procedure. LAD was evaluated based on transthoracic echocardiograms obtained within 1 week before the operation. All the patients had AF (persistent AF, 21; paroxysmal AF, 6). None of the patients had a previous myocardial infarction, febrile disorders, systemic inflammatory diseases, malignancy, or chronic renal failure. This investigation conformed to the principles outlined in the Declaration of Helsinki and was approved by an institutional review board (2005/7/13), and written informed consent was obtained from all the patients.

2.2. Histological examination and immunohistochemistry

Blocks of tissues were embedded in optimal cutting temperature compound and immediately frozen in liquid nitrogen after resection. Frozen cryostat sections (8 µm) were cut, air-dried, fixed in acetone, and thereafter evaluated with standard protocols for staining with hematoxylin–eosin and Masson trichrome stain. Immunostaining was performed in sequential sections by using Dako EnVision+Systems (Dako) with primary antibodies for CD68 (KT117, TransGenic, Inc.) and CD3 (Nichirei Bioscience 413591).

Infiltration of immune cells in the atrium was examined via light microscopy by using immunostained images at a magnification of 100×captured with a digital camera (Nikon). Using the Image-Pro Plus software (Media Cybernetics, Carlsbad, CA, USA), the extent (% area) of interstitial fibrosis and positive staining representing immunoreactivity was recorded and corrected to the total section areas. The mean value was obtained from 10 different fields selected blindly for each patient.

2.3. Statistical analysis

Data were expressed as mean±SD values. Correlations of LAD to interstitial fibrosis and immunoreactive areas were evaluated by using linear regression analysis. Statistical significance was set at a p<0.05.

3. Results

Table 1 shows the characteristics of the 27 patients in the present study from whom left atrial appendage specimens were obtained. Of the 27 patients, 17 were men, and the mean patient age was 59.9±10.1 years. The mean AF duration was 82.4±79.9 months. Cardiac surgeries were performed for mitral regurgitation with/without mitral stenosis in 19 patients, aortic regurgitation with/without stenosis in 3 patients, combined valvular diseases in 3 patients, and atrial septal defect in 2 patients. The ejection fraction as assessed by using echocardiography ranged from 50% to 80% (mean±SD, 63.4±9.2%), and the LAD ranged from 28 to 80 mm (mean±SD, 55.2±11.0 mm). Before the operations, all the patients received warfarin for anticoagulation. Loop diuretics were prescribed in 26 patients (96%), renin-angiotensin system blockers in 17 (63%), and beta-blockers in 16 (59%). Just before the operation, 6 patients with a history of paroxysmal AF were in sinus rhythm, and the remaining 21 patients were with persistent AF.

Table 1.

Patient characteristics.

	n (%)
Male/female	17/10
Age (years), mean±SD	59.9±10.1
EF (%), mean±SD	63.4±9.2
LAD (mm), mean±SD	55.2±11.0
Paroxysmal (%)	6 (22.2)
AF duration (months), mean±SD	82.4±79.9
Concomitant heart diseases
MR	16 (59)
MR+TR	3 (11)
MR+AR/AS	3 (11)
AR/AS	3 (11)
ASD	2 (7)
Medications
VKA	27 (100)
Diuretics	26 (96)
RAS inhibitor	17 (63)
Beta-blocker	16 (59)

EF, ejection fraction; LAD, left atrial dimension; VKA, vitamin K antagonist; RAS, renin-angiotensin system; MR, mitral regurgitation; TR, tricuspid regurgitation; AR, aortic regurgitation; AS, aortic stenosis; and ASD, atrial septal defect.

The extent of interstitial fibrosis, including subendocardial and perivascular fibroses, determined on Masson-trichrome staining, varied widely in these patients. The interstitial fibrotic area increased progressively according to the increments of LAD (Fig. 1A). A statistically significant positive correlation was observed between LAD and the % area of interstitial fibrosis (Fig. 1B, r²=0.44, p<0.01).

Fig. 1.

(A) Masson-trichrome staining of left atrial appendage specimens from patients with left atrial dimensions (LADs) of 43 mm (left), 59 mm (middle), and 67 mm (right), respectively. The extent of interstitial fibrosis (blue area) increased as the LAD increased. (B) Correlation between LAD (mm) and fibrosis area (% of the total section area). A statistically significant positive correlation was observed.

As observed in our previous study [12], immunohistochemical analysis with the macrophage-specific CD68 antibody revealed the infiltration of inflammatory immune cells, with dominant distribution in the subendocardium (Fig. 2A). The degree of infiltration varied among patients; however, when we aligned the images from the patients with smaller to larger LADs, the magnitude of the macrophage infiltration showed a parallel increase (Fig. 2A). The CD68 immunostaining area increased according to the increments of LAD, and a significant positive correlation between LAD and the CD68 immunostaining area was detected (Fig. 2B, r²=0.58, p<0.01). CD3-positive T cells were observed to infiltrate into the atrium, although to a lesser extent than macrophages (Fig. 3A). A significant positive correlation between the LAD and the CD3 immunostaining area was also detected (Fig. 3B, r²=0.49, p<0.01).

Fig. 2.

(A) CD68 immunostaining of the left atrium from the same patients presented in Fig. 1. High number of immunostained macrophages (brown area) were observed as left atrial dimension (LAD) increased. (B) Correlation between LAD (mm) and the CD68-immunostained area (% of the total section area). A statistically significant positive correlation was observed.

Fig. 3.

(A) CD3 immunostaining of the atrium from the same patients presented in Fig. 1. More immunostained T cells (brown area) were observed as left atrial dimension (LAD) increased. (B) Correlation between LAD (mm) and the CD3-immunostained area (% of the total section area). A statistically significant positive correlation was observed.

4. Discussion

The major findings of the present study were that the extent of interstitial fibrosis increased as LA enlarged (as shown in previous studies [12–14]) and that the infiltration of immune cells, including macrophages and T cells, also increased in concert with LA enlargement.

Proinflammatory macrophages and T cells are known to contribute substantially to the progression of various cardiovascular diseases [8–11]. They include atherosclerosis, myocardial infarction, congestive heart failure, and recently, AF [12,13]. In atherosclerosis, activated macrophages are known to transmigrate across the endothelium and make critical contributions to lesion formation by secreting a variety of proinflammatory cytokines [8]. However, the mechanism by which the inflammatory cells participate chronically in the progression of other cardiac diseases such as AF remains unclear. Descriptive results from our previous study [12] demonstrated the recruitment of immune cells across the endocardium of the LA in patients with AF, suggesting the existence of chronic inflammation in the atrium. Despite these results, the cause-and-effect relationships in AF remain uncertain.

A recent meta-analysis has shown that an enlarged LA increases the risk of AF recurrence after a single CPVI, regardless of the follow-up duration [5]. This finding can be applied to paroxysmal and persistent AF [6,7]. In clinical practice, measurement of LAD can provide a window into the status of the structural remodeling of LA and may influence the ablation strategy. However, the pathophysiological mechanisms connecting a dilated LA to postablation AF recurrence are not fully understood.

One plausible mechanism involves atrial interstitial fibrosis, as it could slow the intra-atrial conduction inhomogeneously, leading to reentry in LA [14]. By demonstrating that LAD was correlated positively with the interstitial fibrosis deposits in the LA, the results of the present study support this hypothesis. A second plausible mechanism involves C-reactive protein (CRP) [15], as a study revealed that CRP level was an independent risk factor of AF recurrences after ablation and was also associated with LA enlargement. In the present study, we observed immunohistochemical evidence that supports the results of the CRP clinical study on the basis that the infiltration of immune cells into LA represented a local inflammatory response. However, it should be noticed that the present results cannot be extrapolated directly to patients with AF without structural heart diseases; therefore, future investigations are required.

A potential limitation of the present study was the lack of information owing to ethical issues regarding patients without structural heart diseases. Therefore, it may be argued that the observations regarding local inflammation in the atrium could be applied to patients with AF without valvular diseases. Second, only left atrial appendage samples were available; therefore, our findings may not represent other areas of the atrium. Lastly, the number of patients examined was relatively small. Although limited for these reasons, the results of the present study provide new data regarding the pathophysiological significance of enlarged LA in patients with AF by demonstrating the relationships between LAD and the infiltration of immune cells. However, elucidation of the precise roles of the immune cells infiltrating into the enlarged atrium will require further investigations.

Conflict of interest

None.