Fig 2: Expression of Kcnn4 mRNA, KCa3.1 protein and current are differently affected by TGFß1 in neonatal rat and mouse microglia. (A) Transcript expression of the Ca2+-activated K+ channels (mean ± SEM; 4–6 individual cultures): Kcnma1 (KCa1.1/BK/maxi-K), Kcnn3 (KCa2.3/SK3), Kcnn4 (KCa3.1/SK4) and the regulatory molecule, calmodulin (Calm1). (B) Representative Western blot of KCa3.1 with both species on the same gel. Quantification resulting from normalization of each KCa3.1 band to total protein (Coomassie blue staining) in each lane. Fold changes were calculated with respect to unstimulated (control) microglia (mean ± SEM; 4–7 individual cultures for mouse, 5–6 for rat). For remaining figure, left panels represent neonatal rat microglia; right panels, neonatal mouse microglia. (C) Representative whole-cell recording showing KCa3.1 current evoked by the positive gating modulator, SKA-111 (1 µM), followed by inhibition by the blocker, 1 µM TRAM-34. From a holding potential of -90 mV, the voltage was stepped to +30 mV, followed by a ramp from -120 mV to +60 mV. (D) Examples of the time course, showing activation by the positive-gating modulator, SKA-111, and inhibition by the blocker, TRAM-34. (E) Summary of KCa3.1 current activation (i.e., the TRAM-34-sensitive component) in control cells vs. TGFß1-treated microglia. Significant differences are shown between control and TGFß1 treated cells (*), and between species (†). One symbol of either type indicates p < 0.05; 2, p < 0.01; and 4, p < 0.0001.

Fig 3: Canines electrophysiological examination by programmed stimulation and the expression of ion channel. (a) Canines electrocardiogram during programmed stimulation. In Sham group, the state at which atrial fibrillation (AF) was not reached. In Pacing group, the state at which AF was reached and the AF sustained more than 5 seconds. Compared with Pacing group, AF susceptibility attenuated by GW4869 injection because most irregular rhythm shortens than 5 seconds. (b) Effective refractory period of different parts of the atrium. GW4869 increased the ERP of RSPV and LIPV in Pacing group. (n =6). (c) Difference in AF inducibility, shown by the number of episodes. (n =6). (d) Difference in mean AF durations. (n =6). (e, f) Representative gel bands depicting KCa3.1 and Cav1.2 protein expression using specific antibodies. GAPDH was used as the loading control. (n =5). (g) Total iron level in atrial tissue. (n =3). (h) MDA level in atrial tissue. (n =3). Data are presented as the mean ± SD. Statistical significance was determined using one-way ANOVA with a post hoc Dunnett test. *P < 0.05, **P < 0.01, and ***P < 0.001 vs. Sham group; #P < 0.05, ##P < 0.01, and ###P < 0.001 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; dERP: dispersion effective refractory period.