Journal: PNAS Nexus

Article Title: The SK4 channel allosteric blocker, BA6b9, reduces atrial fibrillation substrate in rats with reduced ejection fraction

doi: 10.1093/pnasnexus/pgae192

Figure Lengend Snippet: BA6b9 treatment reduces the AF substrate of post-MI rats. A) Confirmatory in vitro recordings. Left: Representative trace of WT SK4 currents in the absence and presence of 20 µM BA6b9, displaying a degree of inhibition of ≈56%. Right: Representative traces of an inside-out macropatch from a CHO cell expressing WT SK4 channels in the absence and presence of 10 µM BA6b9 under internal saturating calcium concentrations. Currents were recorded by 10 repetitive 1 s duration voltage ramps from −100 mV to +100 mV from a holding potential of 0 mV. B) BA6b9 treatment significantly reduced AF induction and AF duration. C) Example of postburst AF episodes in the vehicle (left) and BA6b9 (right) groups. D) Mean power spectrum of the AF episodes following long-term BA6b9 vs. vehicle treatment. Notably, the vehicle group demonstrated greater amplitudes over a wide range of frequencies relative to the BA6b9 group. Notch (arrow) indicates the zone where a notch filter was applied due to power supply-related noise (50 Hz). E) Quantitative analysis of delta between the power spectrum integrals of arrhythmic episodes and preburst NSR. Note the significantly higher values in the vehicle group compared to the AF events among BA6b9-treated rats.

Article Snippet: Antigen retrieval was performed using BOND Epitope Retrieval Solution 1 (Citrate buffer, prediluted, pH 6.0) for 20 min at 100°C, followed by peroxide treatment for 10 min. For membranal staining, we primarily incubated the sections with WGA (29022, CF488 WGA, Biotium, 1:500 in PBS) conjugated with Alexa Fluor 488 for 30 min, followed by incubation with blocking buffer (10% normal goat serum, 0.1% Triton, and 10% bovine serum albumin) for 30 min. For analyses of SK4 expression and localization, we incubated the specimens with the primary antibody (ALM-051, Alomone Labs, mouse monoclonal antibody against the 3rd extracellular loop of human SK4, 1:50) for 90 min and then stained with a Cy3-conjugated secondary antibody (711-165-151, Jackson Immunoresearch Laboratories, Cy TM 3-conjugated AffiniPure Donkey Anti-Mouse, 1:100) for 90 min. For Cx43 expression and localization analyses, we incubated the specimens with the primary antibody (C6219, Sigma-Aldrich, rabbit polyclonal antibody against the C-terminus of human/rat Cx43, 1:400) for 90 min and stained with a Cy5-conjugated secondary antibody (711-175-152, Jackson Immunoresearch Laboratories, Cy TM 5-conjugated AffiniPure Donkey Anti-Rabbit, 1:200) for 90 min. At the end of the staining, we incubated the specimens with Vector TrueVIEW Autofluorescence Quenching kit (SP-8400-15, Vector laboratories) for 10 min to improve the signal-to-noise ratio and reduce autofluorescence.

Techniques: In Vitro, Inhibition, Expressing

Journal: PNAS Nexus

Article Title: The SK4 channel allosteric blocker, BA6b9, reduces atrial fibrillation substrate in rats with reduced ejection fraction

doi: 10.1093/pnasnexus/pgae192

Figure Lengend Snippet: Effect of BA6b9 on SK4 expression in the left atrium of rats with MI-induced HF. A) Statistical summary of overall left-atrial SK4 expression; vehicle- vs. BA6b9-treated rats, compared to control ( n = 7, n = 8, n = 4, respectively; one-way ANOVA, F(2, 16) = 14.27, Sidak's multiple comparisons test P = 0.0003). B1) Representative histological LA cross-section from a control rat (upper left), stained with DAB. B2, B3) Representative DAB-stained histological cross-sections of the LA from post-MI rats treated with vehicle (upper right) or BA6b9 (lower left) for 21 days. Brown staining intensity indicates the level of SK4 expression in the tissue, with darker brown indicating stronger expression. B4) Negative control SK4 staining. An inset in each photograph shows the full LA tissue in low resolution.

Article Snippet: Antigen retrieval was performed using BOND Epitope Retrieval Solution 1 (Citrate buffer, prediluted, pH 6.0) for 20 min at 100°C, followed by peroxide treatment for 10 min. For membranal staining, we primarily incubated the sections with WGA (29022, CF488 WGA, Biotium, 1:500 in PBS) conjugated with Alexa Fluor 488 for 30 min, followed by incubation with blocking buffer (10% normal goat serum, 0.1% Triton, and 10% bovine serum albumin) for 30 min. For analyses of SK4 expression and localization, we incubated the specimens with the primary antibody (ALM-051, Alomone Labs, mouse monoclonal antibody against the 3rd extracellular loop of human SK4, 1:50) for 90 min and then stained with a Cy3-conjugated secondary antibody (711-165-151, Jackson Immunoresearch Laboratories, Cy TM 3-conjugated AffiniPure Donkey Anti-Mouse, 1:100) for 90 min. For Cx43 expression and localization analyses, we incubated the specimens with the primary antibody (C6219, Sigma-Aldrich, rabbit polyclonal antibody against the C-terminus of human/rat Cx43, 1:400) for 90 min and stained with a Cy5-conjugated secondary antibody (711-175-152, Jackson Immunoresearch Laboratories, Cy TM 5-conjugated AffiniPure Donkey Anti-Rabbit, 1:200) for 90 min. At the end of the staining, we incubated the specimens with Vector TrueVIEW Autofluorescence Quenching kit (SP-8400-15, Vector laboratories) for 10 min to improve the signal-to-noise ratio and reduce autofluorescence.

Techniques: Expressing, Control, Staining, Negative Control

Journal: PNAS Nexus

Article Title: The SK4 channel allosteric blocker, BA6b9, reduces atrial fibrillation substrate in rats with reduced ejection fraction

doi: 10.1093/pnasnexus/pgae192

Figure Lengend Snippet: Effect of BA6b9 on collagen deposition, α-SMA expression, and SK4 expression in the LA epicardium of rats with MI-induced HF. A) Statistical summary of LA epicardial fibrosis (analysis of six randomized epicardial fields for each atrial section, total of 18 atrial-epicardium fields per animal); vehicle- vs. BA6b9-treated rats, compared to control ( n = 7, n = 8, n = 4, respectively; one-way ANOVA, F(2, 16) = 16.53, Sidak's multiple comparisons test P = 0.0001). A1) Representative histological cross-section of LA myocardium attached to the epicardial tissue from a control rat (left upper row). The analyzed area is marked by dashed lines. A2, A3) Representative histological cross-sections of the LA from post-MI rats treated with vehicle (left middle row) or BA6b9 (left lower row) for 21 days. Sections A1–A3 were stained with Masson's Trichrome. B) Statistical summary of LA epicardial α-SMA expression; (analysis: same as in A; vehicle vs. BA6b9-treated rats, compared to control ( n = 7, n = 8, n = 4, respectively; one-way ANOVA, F(2, 16) = 16.53, Sidak's multiple comparisons test P = 0.0001). B1) Representative histological cross-section of LA myocardium attached to the epicardial tissue from a control rat (middle upper row). B2, B3) Representative histological cross-sections of the LA in post-MI rats treated with vehicle (center) vs. BA6b9 (middle lower row) for 21 days. Sections B1–B3 were stained with Sirius Red. C) Statistical summary of LA epicardial α-SMA expression; (analysis: same as in A; vehicle- vs. BA6b9-treated rats, compared to control ( n = 7, n = 8, n = 4, respectively; one-way ANOVA, F(2, 16) = 10.21, Sidak's multiple comparisons test P = 0.0014). C1) Representative histological cross-section of LA myocardium attached to the epicardial tissue from a control rat (right upper row). C2, C3) Representative histological cross-sections of the LA in post-MI rats treated with vehicle (right middle row) or BA6b9 (right lower row) for 21 days. Sections C1–C3 were stained with DAB. Note the marked thickening of the atrial epicardium in MI rats compared to controls as well as the significant reductions in collagen deposition (A, A1–A3), α-SMA expression (B, B1–B3), and SK4 expression (C, C1–C3) in the BA6b9-treated rats compared to the vehicle group. An inset in each photograph shows the full LA tissue in low resolution.

Article Snippet: Antigen retrieval was performed using BOND Epitope Retrieval Solution 1 (Citrate buffer, prediluted, pH 6.0) for 20 min at 100°C, followed by peroxide treatment for 10 min. For membranal staining, we primarily incubated the sections with WGA (29022, CF488 WGA, Biotium, 1:500 in PBS) conjugated with Alexa Fluor 488 for 30 min, followed by incubation with blocking buffer (10% normal goat serum, 0.1% Triton, and 10% bovine serum albumin) for 30 min. For analyses of SK4 expression and localization, we incubated the specimens with the primary antibody (ALM-051, Alomone Labs, mouse monoclonal antibody against the 3rd extracellular loop of human SK4, 1:50) for 90 min and then stained with a Cy3-conjugated secondary antibody (711-165-151, Jackson Immunoresearch Laboratories, Cy TM 3-conjugated AffiniPure Donkey Anti-Mouse, 1:100) for 90 min. For Cx43 expression and localization analyses, we incubated the specimens with the primary antibody (C6219, Sigma-Aldrich, rabbit polyclonal antibody against the C-terminus of human/rat Cx43, 1:400) for 90 min and stained with a Cy5-conjugated secondary antibody (711-175-152, Jackson Immunoresearch Laboratories, Cy TM 5-conjugated AffiniPure Donkey Anti-Rabbit, 1:200) for 90 min. At the end of the staining, we incubated the specimens with Vector TrueVIEW Autofluorescence Quenching kit (SP-8400-15, Vector laboratories) for 10 min to improve the signal-to-noise ratio and reduce autofluorescence.

Techniques: Expressing, Control, Staining

Journal: International Journal of Molecular Sciences

Article Title: The Gárdos Channel and Piezo1 Revisited: Comparison between Reticulocytes and Mature Red Blood Cells

doi: 10.3390/ijms25031416

Figure Lengend Snippet: Transcriptome and protein analysis. Panel ( A ) shows the transcriptomic analysis for the Gárdos channel (KCNN4), Piezo1 and VDAC2 based on RNA isolated from circulating reticulocytes. The columns present the mean values from 4 donors with the error bars being the standard error of mean (SEM). Gárdos channel (KCNN4) is slightly lower in the reads compared to Piezo1 but the difference is not significant whereas both of them are significantly smaller than VDAC2. Comparisons of the means were tested depicting a p > 0.05 for not significant (ns), * for p < 0.05 and ** for p < 0.01. Panel ( B ) shows the result of the proteomic analysis performed on highly enriched reticulocyte (retics) and reticulocyte-depleted mature RBCs (erys) lysates from 4 different donors with the error bars being SEM. In reticulocytes, the transferrin receptor, Piezo1 and VDAC2 could be detected, whereas the Gárdos channel was below the detection limit. In mature RBCs only Piezo1 could be detected. Comparisons of the means were tested depicting a p > 0.05 for not significant (ns). Panel ( Ca ) shows a dot plot of Gárdos channel antibody (KCNN4, FITC) vs. transferrin receptor (CD71, pacific blue) for proerythroblasts presenting a ‘positive control’ for the isotype vs. antibody stains. Panel ( Cb ) shows the same measurements (but different gain settings) for RBCs presenting the reticulocytes in sectors Q2 and Q3.

Article Snippet: Antibodies used were CD71-PB (1:100 dilution; Miltenyi Biotec, Germany) and KCa3.1 (SK4) (1:100 dilution; Abgent, San Diego, CA, USA).

Techniques: Isolation, Positive Control