KCa3.1 Promotes Proinflammatory Exosome Secretion by Activating AKT/Rab27a in Atrial Myocytes during Rapid Pacing

Figures

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  Figure 1. Canine electrophysiological examination during programmed stimulation. (a) The state of atrial fibrillation (AF) could not be induced in the sham group, and the state of AF was induced in the pacing group, while the state of AF was no more than 5 seconds in the pacing+TRAM-34 group. (b) Effective refractory period in different parts of the atrium. TRAM-34 increased the ERP of RIPV, LSPV, and LIPV after rapid atrial pacing. (c) Difference in AF inducibility, as shown by the number of episodes. (d) Difference in mean AF durations. (e) Difference in the dispersed effective refractory period. The data are presented as the mean ± SD of six biological replicates. ∗p < 0.05, ∗∗p < 0.01, and ∗∗∗p < 0.001 vs. sham group; #p < 0.05 and ##p < 0.01 vs. pacing group. Abbreviations: RA: right atrium; RSPV: right superior pulmonary vein; RIPV: right inferior pulmonary vein; LA: left atrium; LSPV: left superior pulmonary vein; LIPV: left inferior pulmonary vein; ERP: effective refractory period.

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  Figure 2. TRAM-34 attenuates inflammation in atrial tissue during rapid pacing. (a) Representative images of inflammation and fibrosis, as reflected by the H&E staining and Masson staining. Black arrows indicate the infiltration of inflammatory cells. (b, c) Representative images of CD68, as reflected by immunofluorescence analysis. White arrows indicate CD68-positive macrophages. (d–g) RT-PCR analysis of Arg-1, iNOS, IL-1β, and IL-6 expression normalized to GAPDH in atrial tissue during rapid pacing. The data are presented as the mean ± SD of three to six biological replicates. ∗, ∗∗, and ∗∗∗ indicate p < 0.05, 0.01, and 0.001, respectively.

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  Figure 3. TRAM-34 attenuates the secretion of exosomes in atrial tissue during rapid pacing. (a) Representative images of KCa3.1 and Rab27a, as reflected by immunohistochemistry. (b, c) Representative images of CD81, as reflected by immunofluorescence analysis. (d) Representative gel bands showing the effects of TRAM-34 administration on atrial tissue during rapid pacing. (e–i) Levels of KCa3.1, p-AKT/AKT, Rab27a, TSG101, and CD81. The data are presented as the mean ± SD of three to six biological replicates. ∗, ∗∗, and ∗∗∗ indicate p < 0.05, 0.01, and 0.001, respectively.

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  Figure 4. Rapid pacing stimulates HL-1 cell secretion. (a, b) Representative images of KCa3.1, as reflected by immunofluorescence analysis. (c) The characteristic markers CD81 and TSG101 in isolated exosomes were detected by immunoblotting. (d) Morphology of isolated exosomes from HL-1 cells, as examined by transmission electron microscopy. (e, f) Mean exosome diameter and concentration, as shown by the Zeta View System. (g–j) TNF-α, IL-β, IL-6, and TGF-β1 concentrations in HL-1 cell supernatants. The exosomes were extracted from the same volume of supernatant from cells of equal density. The data are presented as the mean ± SD of three biological replicates. ∗p < 0.05 and ∗∗∗p < 0.001 vs. control group; #p < 0.05, ##p < 0.01, and ###p < 0.001 vs. pacing group.

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  Figure 5. Exosomes derived from rapid-pacing HL-1 cells promote macrophage polarization. (a–c) Representative images of CD68 and iNOS, as reflected by immunofluorescence analysis. (d, e) RT-PCR analysis of iNOS and IL-6 expression normalized to GAPDH in macrophages. The data are presented as the mean ± SD of three biological replicates. ∗, ∗∗, and ∗∗∗ indicate p < 0.05, 0.01, and 0.001, respectively.

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  Figure 6. Rapid pacing activates the KCa3.1/AKT/Rab27a signaling pathway. (a) Representative gel bands showing the effects of BAPTA, GSK690693, and si-Rab27a on rapid-pacing HL-1 cells. (b) Representative gel bands showing the effects of different concentrations of SC79. (c–g) The levels of KCa3.1, p-AKT/AKT, Rab27a, TSG101, and CD81 after treatment with BAPTA, GSK690693, and si-Rab27a. (h–j, l) The levels of p-AKT/AKT, Rab27a, TSG101, and CD81 after treatment with different concentrations of SC79. (k, m) Representative images of Rab27a after treatment with BAPTA, GSK690693, and si-Rab27a, as reflected by immunofluorescence analysis. The data are presented as the mean ± SD of three biological replicates. ∗p < 0.05, ∗∗p < 0.01, and ∗∗∗p < 0.001 vs. control group; #p < 0.05 and ##p < 0.01 vs. pacing group.

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  Figure 7. KCNN4 overexpression promotes exosome secretion. (a) Representative gel bands showing the effects of TRAM-34 administration on KCNN4-overexpressing HL-1 cell. (b) Representative gel bands showing the effects of BAPTA, GSK690693, and si-Rab27a on KCNN4-overexpressing HL-1 cell. (c–g). The levels of KCa3.1, p-AKT/AKT, Rab27a, TSG101, and CD81 in KCNN4-overexpressing HL-1 cell after TRAM-34 administration. (h, i) Mean exosome diameter and concentration, as shown by the Zeta View System. The exosomes were extracted from the same volume of supernatant from cells of equal density. (j–n) The levels of KCa3.1, p-AKT/AKT, Rab27a, TSG101, and CD81 in KCNN4-overexpressing HL-1 cell after treatment with BAPTA, GSK690693, and si-Rab27a. The data are presented as the mean ± SD of three biological replicates. ∗, ∗∗, and ∗∗∗ indicate p < 0.05, 0.01, and 0.001, respectively.