Blockade of SK4 channels suppresses atrial fibrillation by attenuating atrial fibrosis in canines with prolonged atrial pacing

Figures

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  Figure 1. The results of electrophysiological measurements. The state that AF was not induced during S1S1 programmed stimulation. (B) The state that AF was induced during S1S1 programmed stimulation. (C) The state that intra-atrial CT was measured. (D) The differences in intra-atrial CT between the groups. (E) The differences in mean AF durations between the groups. ***P<0.001 versus the sham group. ##P<0.01 versus the pacing group. ▲▲P<0.01 versus the sham group. The data are presented as the mean ± SD, n=6, 7, and 7, respectively. Abbreviations: CT, conduction time.

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  Figure 2. The expression of SK4 channels in atria. (A) Double-labeling immunofluorescence staining of SK4 (green) and α-SMA (red) in right atria (400×). (B) SK4 AOD in atria (C) SK4 AOD in cardiac fibroblasts. AOD= IOD/area. (D) Representative Western blot images and the mean levels of SK4 channels in right atria. ***P<0.001 versus the sham group. ###P<0.001 versus the pacing group. ▲▲▲P<0.001 versus the sham group. The data are presented as the mean ± SD, n=6. Abbreviations: AOD, average optical density; IOD, integral optical density.

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  Figure 3. The expression of collagen and MMPs in atria. (A and B) Masson's trichrome staining of the atria and mean values of collagen area in the atria (200×). (C-E) Representative Western blotting images and mean levels of collagen I, collagen III, MMP-2 and MMP-9 in the atria. ***P<0.001 versus the sham group. ###P<0.001 versus the pacing group. ▲▲▲P<0.001 versus the sham group. The data are presented as the mean ± SD, n=6.

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  Figure 4. Colocalization of DDR-2 and α-SMA in atria. (A) Immunofluorescence staining of myofibroblast markers DDR-2 (green) and α-SMA (red) in the atria (400×). (B) The numbers of myofibroblasts in the atria. ***P<0.001 versus the sham group. ###P<0.001 versus the pacing group. ▲▲▲P<0.001 versus the sham group. The data are presented as the mean ± SD, n=6.

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  Figure 5. The role of SK4 channels in the activity of atrial fibroblasts from canines. (A) Immunofluorescence staining of SK4 (green) and α-SMA (red) in atrial fibroblasts in the Ang II-treated groups (400×). (B) Immunofluorescence staining of α-SMA (red) in atrial fibroblasts in the Ad-GFP (green)-treated groups (400×). (C) The values of SK4 AOD in the Ang II-treated groups. ***P<0.001 versus the control group. ##P<0.01 versus the Ang II group. ▲▲P<0.01 versus the control group. (D) The values of α-SMA AOD in all the groups of atrial fibroblasts. AOD= IOD/area. (E-I) Representative Western blot images and mean levels of SK4, α-SMA, collagen I and collagen III in all the groups of atrial fibroblasts. ***P<0.001; ##P<0.01; ###P<0.001; ▲▲P<0.01; ▲▲▲P<0.001. The data are presented as the mean ± SD, n=6.

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  Figure 6. TRAM-sensitive currents in atrial fibroblasts from canines detected by patch clamp. (A) Membrane currents recorded in isolated atrial fibroblasts from canines in different groups. (B) Current-voltage relationships of the mean values of TRAM-sensitive currents (pA) in the control group, the Ang II group and the Ang II + TRAM-34 group. (C) Current-voltage relationships of the mean values of TRAM-sensitive currents (pA) in the Ad-GFP group, the Ad-SK4 group and the Ad-SK4 + TRAM-34 group.

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  Figure 7. Effects of SK4 on the expression of CTGF, TGF-β1, Smad2, p-Smad2, Smad3 and p-Smad3 in atria. (A) Representative Western blot images of CTGF, TGF-β1, Smad2, p-Smad2, Smad3 and p-Smad3 expression levels in the atria. (B-G) The mean levels of CTGF, TGF-β1, Smad2, p-Smad2, Smad3 and p-Smad3 in the atria. ***P<0.001 versus the sham group. ###P<0.001 versus the pacing group. ▲▲P<0.01 versus the sham group. ▲▲▲P<0.001 versus the sham group. The data are presented as the mean ± SD, n=6.

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  Figure 8. Effects of SK4 on the expression of ERK1/2, p-ERK1/2, p38, p-p38, c-Fos and c-Jun in atria. (A) Representative Western blot images of ERK1/2, p-ERK1/2, p38, p-p38, c-Fos and c-Jun expression levels in atria. (B-G) The mean levels of ERK1/2, p-ERK1/2, p38, p-p38, c-Fos and c-Jun in atria. ***P<0.001 versus the sham group. ###P<0.001 versus the pacing group. ▲▲P<0.01 versus the sham group. ▲▲▲P<0.001 versus the sham group. The data are presented as the mean ± SD, n=6.

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  Figure 9. Effects of SK4 channels on the expression of ERK1/2, p-ERK1/2, p38, p-p38, c-Fos and c-Jun in atrial fibroblasts. (A) Representative Western blotting images of ERK1/2, p-ERK1/2, p38, p-p38, c-Fos and c-Jun expression levels in cardiac fibroblasts. (B-G) The mean levels of ERK1/2, p-ERK1/2, p38, p-p38, c-Fos and c-Jun in cardiac fibroblasts. ***P<0.001; ###P<0.001; ▲P<0.05; ▲▲P<0.01; ▲▲▲P<0.001. The data are presented as the mean ± SD, n=6.

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  Figure 10. Schematic diagram: the potential roles of SK4 channels in AF maintenance. Dotted arrow: negative effect. Solid arrow: positive effect