Association of Autoantibodies against M2-Muscarinic Acetylcholine Receptor with Atrial Fibrosis in Atrial Fibrillation Patients

Abstract

Objectives: To investigate the association of serum autoantibodies against M2-muscarinic acetylcholine receptor (anti-M2-R) with atrial fibrosis in long-standing persistent atrial fibrillation (AF) patients.

Methods: Twenty-four long-standing persistent AF patients, scheduled to undergo hybrid ablation surgery, were enrolled in the study. Twenty-six patients with sinus rhythm, scheduled to undergo coronary artery bypass grafting surgery, were enrolled into the non-AF group. We detected serum anti-M2-R levels. Left atrial appendages were subjected to histological and molecular biological assays. Patients in the AF group received follow-up for two years.

Results: The AF group showed significantly higher serum anti-M2-R levels compared to the non-AF group (496.2 ± 232.5 vs. 86.3 ± 25.7 pmol/L, p < 0.001). The AF group exhibited severe fibrosis in the left atrial appendages, as indicated by increased collagen volume fraction (45.2 ± 4.7% vs. 27.6 ± 8.3%, p < 0.001), and higher levels of collagen I (0.52 ± 0.04 vs. 0.24 ± 0.06, p < 0.001) and collagen III (0.51 ± 0.07 vs. 0.36 ± 0.09, p < 0.001). TGF-β1 and CTGF were also upregulated in the AF group. A positive correlation between serum anti-M2-R levels and fibrosis of the left atrial appendage and fibrogenic indexes was observed.

Conclusions: Serum anti-M2-R levels are higher in AF patients and are associated with the severity of atrial fibrosis. In addition, serum anti-M2-R levels are positively correlated to TGF-β1 and CTGF expression in the left atrial appendage.

Figure 1

Comparison in serum anti-M2-R levels among three groups. The serum anti-M2-R level in the AF group was 496.2 ± 232.5 pmol/L, which was significantly higher than 86.3 ± 25.7 pmol/L in the non-AF group, p < 0.001. No difference in serum anti-M2-R levels between the non-AF and control group was observed (86.3 ± 25.7 vs. 82.4 ± 34.9 pmol/L, p=0.654).

Figure 2

Immunohistochemical analysis of M2 receptor, TGF-β1, and CTGF expression. Representative sections of the immunohistochemistry- (IHC-) stained left atrial appendage tissue of patients in the non-AF and AF group. (a) M2 receptor; (b) TGF-β1; (c) CTGF.

Figure 3

Western blotting analysis. Western blotting examined the left atrial appendage of patients in the non-AF group and the AF group. Left, representative western blot analysis the expression in the non-AF group and the AF group; right, quantitative results of the protein levels. ^∗∗∗p < 0.001. GAPDH, glyceraldehyde-3-phosphate dehydrogenase.

Figure 4

Correlation between serum anti-M2-R levels and fibrosis-related indexes. Pearson correlation analysis showed that M2 receptor, CVF, and collagen I and collagen III were all correlated with serum anti-M2-R levels (M2 receptor: r = 0.90, p < 0.001; CVF: r = 0.84, p < 0.001; collagen I: r = 0.87, p < 0.001; collagen III: r = 0.78, p < 0.001). Fibrogenic indexes were also correlated with serum anti-M2-R levels (TGF-β1: r = 0.92, p < 0.001; CTGF: r = 0.89, p < 0.001).

Figure 5

Histological analysis of the left atrial appendage. (a) Hematoxylin and eosin (H&E) staining of the left atrium appendage in the non-AF and the AF groups. Left, representative image; right, statistical results for the cell surface area. (b) Masson's trichrome staining of the left atrium appendage in the non-AF and AF groups. Left, representative image; right, quantification of the collagen volume fraction. ^∗∗p < 0.01; ^∗∗∗p < 0.001.

Figure 6

Immunofluorescence analysis the expression of collagen I and collagen III. Immunofluorescence staining of the left atrial appendage of patients in the two groups. Left, representative image; right, statistical results for the collagen (a) I and (b) III. ^∗∗∗p < 0.001; magnification, 400×.