Premature senescence of cardiac fibroblasts and atrial fibrosis in patients with atrial fibrillation

Abstract

Premature senescence is associated with atrial fibrosis and has an antifibrotic effect in mice. However, the role of senescence in atrial fibrillation (AF) remains unclear. Here, we investigated the association of premature senescence with fibrosis and also determined the role of senescence in the recurrence of AF after surgery ablation. Western blot, Sirius red staining, SA-β-gal staining and immunohistochemistry were performed to detect the degree of atrial fibrosis ,the expression of TGF-β and collagens, and also the senescence markers in 72 tissue specimens of left atrial appendage in this study. Then the patients undergoing successful surgical ablation were followed up for 12 months. The expression of collagens and TGF-β was paralleled by a high level of atrial fibrosis and were increased in AF group, especially in the persistent AF group. Western blotting of P16 and SA-β-gal staining showed an increased premature senescence in the sinus rhythm, paroxysmal AF and persistent AF groups. In addition, positive area of senescence markers, SA-β-gal and P16, was correlated positively with fibrotic lesions. We also found a lower ratio of P16/TGF-β in patients with recurrence of AF than in patients without recurrent AF. In conclusion, premature senescence is associated with atrial fibrosis in AF, and may have an antifibrotic role in AF.

Keywords: Gerotarget; P16; atrial fibrillation; cardiac fibroblast; fibrosis; premature senescence.

Figure 1. Up-regulation of atrial fibrosis in AF

A. & B., The expression of Col I, Col III and TGF-β were increased in the AF groups. C.&D., Statistical analyses of the ratio of Col I/Col III and TGF-β (n = 3 per group). E., Sirius red staining showed a gradient increase of atrial fibrosis in SR, PaAF and PeAF groups; bar = 200μm. F., Quantification of Sirius red positive area in the three groups (n = 5 per group). Values were expressed as mean ± SD. *P < 0.05, **P < 0.01, ***P < 0.001.

Figure 2. Accumulation of senescence accompanied by fibrosis in AF

A., The expression of P21 and P16 was significantly increased in PeAF group, followed by PaAF and SR groups. B., Statistical analyses of P21 and P16 (n = 3 per group). C., Representative images of SA-β-gal staining in SR, PaAF and PeAF groups. D., Quantification of SA-β-gal positive area in the three groups (n = 5 per group). E., Left, representative images of Sirius red staining and immunohistochemical staining for P16; bar = 50μm. Right, linear analysis of P16- and Sirius red-positive area (n = 26). F., Left, representative images of Sirius red staining and immunohistochemical staining for P21; bar = 50μm. Right, linear analysis of P21-postive cells and Sirius red-positive area (n = 21). G., Left, representative images of Sirius red staining and SA-β-gal staining; bar = 100μm. Right, linear analysis of SA-β-gal- and Sirius red-positive area (n = 24). Values were expressed as mean ± SD. *P < 0.05, **P < 0.01, ***P < 0.001.

Figure 3. Correlation of P16 with clinical parameters

A., P16 was positively associated with TGF-β, n = 72. B., P16 was positively associated with age, n = 72. C., P16 was positively associated with left atrial diameter (LAD), n = 72. D., P16 was positively associated with pulmonary artery systolic pressure (PASP), n = 72.

Figure 4. Cardiac fibroblasts are the predominant cells experiencing premature senescence in human heart

A., Representative image of Sirius red staining for perivascular fibrosis (bar = 50μm). B., Quantification of P16-positive cells in perivascular area that express vimentin, α-smooth muscle actin (SMA), troponin T and CD31. C., Representative image of co-stained for P16 (red) and vimentin (green) (bar = 50μm). D., Representative image of co-stained for P16 (red) and α-SMA (green) (bar = 50μm). E., Representative image of co-stained for P16 (red) and troponin T (green) (bar = 50μm). F., Representative image of co-stained for P16 (red) and CD31 (green) (bar = 50μm).