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Figure Lengend Snippet: a – c , Reentrant and multifocal arrhythmias in ATS1 mouse hearts. a , Top, representative single camera pixel recording from an optical mapping experiment in an ATS1 mouse heart in the presence of 1 µM flecainide. Data show arrhythmic behavior induced by a brief tachypacing train (Stim). Three spontaneous APs are observed upon termination of the train (inset). Bottom, Phase maps reveal that those APs were generated by three sequential rotations of an unstable rotor. In each map, the center of rotation (singularity point) is indicated by a white circle; rotor direction is clockwise as indicated by the curved black arrows. b , Top, Single pixel recording shows AP amplitude alternans in a different ATS1 mouse heart perfused with 1 µM flecainide. Bottom, 4-ms color isochrone maps show distinctive epicardial breakthrough origin and wave propagation direction for each AP, revealing the multifocal origin of the arrhythmia. c , Quantification of epicardial activation time and APD (at 90% of repolarization) during sinus rhythm for non-infected, Kir2.1 WT and Kir2.1 ∆314-315 mouse hearts in the presence and absence of 1 µM flecainide. Each value is represented as mean ± s.e.m. One-way ANOVA test among basal conditions groups and two-tailed paired t -test between conditions. d – f , Electrophysiological defects in ATS1 mouse cardiomyocytes. d , e , I–V relationships of inward rectifying potassium current I K1 ( d ; P = 0.0112 at −100 mV; P = 0.0001 at −60 mV; and P = 0.0004 at −30 mV) and inward sodium current I Na ( e ; P = 0.0030 at −55 mV; P = 0.0002 at −25 mV; P = 0.0003 at −20 mV; and P = 0.0203 at −15 mV) densities in Kir2.1 ∆314-315 compared to WT ventricular cardiomyocytes. Statistical analyses were conducted using two-way ANOVA, followed by Tukey’s multiple comparisons. f , Representative AP recordings obtained by current-clamping show depolarized resting membrane potential, reduced excitability and prolonged AP duration in Kir2.1 ∆314-315 versus Kir2.1 WT cardiomyocytes. Graph shows the quantification of RMP. Different colors in the same group identify cells coming from one animal. Statistical analyses were conducted using two-level hierarchical one-way ANOVA analysis, followed by Bonferroni’s post test. Each value is represented as mean ± s.e.m. * P < 0.05; ** P < 0.01; *** P < 0.001; **** P < 0.0001.

Article Snippet: Primary antibodies were rabbit anti-Kir2.1 (1:200, APC-026, Alomone Labs), mouse anti-calnexin (1:200, MA3-027, Invitrogen), mouse anti-ATPase (1:2,500, ab7671, Abcam) and rabbit anti-Nav1.5 (1:500, AGP008, Alomone Labs).

Techniques: Generated, Activation Assay, Infection, Two Tailed Test, Membrane

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Figure Lengend Snippet: a , Optical mapping in hearts from non-infected control show normal and fast propagation. Representative single-pixel action potential recordings in a heart of a non-infected control mouse after 1 µM flecainide administration. The three corresponding wave propagation maps at bottom (isochrones are plotted every 0.5 ms), show sequential beat-to-beat epicardial breakthrough patterns emerging from the right ventricular apex (red) and propagating rapidly upward toward the basis, indicative of normal wave propagation during sinus rhythm. b , I K1 is significantly decreased after Kir2.1 ∆314-315 expression. Representative I K1 density traces in control conditions ( left ), after barium perfusion ( center ) and after subtraction of the barium-sensitive current ( right ) from non-infected control ( top ), Kir2.1 WT (middle) and Kir2.1 ∆314-315 (bottom) isolated cardiomyocytes. c , Cardiac expression of AAV-Kir2.1 Δ314-315 does not modify I Na voltage-dependence of activation or inactivation. Left , Na channel steady-state activation and inactivation curves for Non-infected, AAV-Kir2.1 WT and AAV-Kir2.1 ∆314-315 cardiomyocytes plotted according to the protocol detailed in the Methods section. Currents were plotted as a fraction of peak current and fitted according to a Boltzmann function. Right , table shows the activation and inactivation parameters for all experimental groups (Activation V 1/2 : P = 0.8790, Non-infected vs Kir2.1 WT ; P = 0.8516, n-infected vs Kir2.1 ∆314-315 ; and, P = 0.5371, Kir2.1 WT vs Kir2.1 ∆314-315 . Activation slope (s): P = 0.0030, Non-infected vs Kir2.1 WT ; P = 0.1514, n-infected vs Kir2.1 ∆314-315 ; and, P = 0.1037, Kir2.1 WT vs Kir2.1 ∆314-315 . Inactivation V 1/2 : P = 0.3521, Non-infected vs Kir2.1 WT ; P = 0.4322, n-infected vs Kir2.1 ∆314-315 ; and, P = 0.9555, Kir2.1 WT vs Kir2.1 ∆314-315 . Inactivation slope (s): P = 0.8386, Non-infected vs Kir2.1 WT ; P = 0.0410, n-infected vs Kir2.1 ∆314-315 ; and, P = 0.0186, Kir2.1 WT vs Kir2.1 ∆314-315 ). Each value is represented as the mean ± SEM. Statistical analyses were conducted using One-way ANOVA test followed by Tukey’s multiple comparisons posttest.

Article Snippet: Primary antibodies were rabbit anti-Kir2.1 (1:200, APC-026, Alomone Labs), mouse anti-calnexin (1:200, MA3-027, Invitrogen), mouse anti-ATPase (1:2,500, ab7671, Abcam) and rabbit anti-Nav1.5 (1:500, AGP008, Alomone Labs).

Techniques: Infection, Control, Expressing, Isolation, Activation Assay

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Figure Lengend Snippet: a , Top, confocal images of Kir2.1, Na V 1.5 and cardiomyocyte membrane (WGA). Bottom, fluorescence profiles (right) and Na V 1.5–Kir2.1 fluorescence correlation in isolated control (left). b , Top, confocal images of Kir2.1, NaV1.5 and cardiomyocyte membrane (WGA) after formamide-mediated detubulation of cardiomyocytes reveals the intracellular Kir2.1 band alone. Bottom, Fluorescence profiles show absence of Nav1.5 staining at the t-tubules (right), with absence of Na V 1.5–Kir2.1 fluorescence correlation (left). c , Left, Kir2.1, calnexin, Na + /K + ATPase and Na V 1.5 analysis by western blot after ventricle membrane fractionation in 10–25% of iodixanol. Right, Graph shows quantification of protein analysis shown in left. d , Representative confocal image ( n = 10 from three animals) of Kir2.1 and SERCA in the SR network connected with the envelope of an isolated nucleus. a.u., arbitrary units.

Article Snippet: Primary antibodies were rabbit anti-Kir2.1 (1:200, APC-026, Alomone Labs), mouse anti-calnexin (1:200, MA3-027, Invitrogen), mouse anti-ATPase (1:2,500, ab7671, Abcam) and rabbit anti-Nav1.5 (1:500, AGP008, Alomone Labs).

Techniques: Membrane, Fluorescence, Isolation, Control, Staining, Western Blot, Fractionation

Journal: European journal of pharmacology

Article Title: Dapagliflozin: A sodium-glucose cotransporter 2 inhibitor, attenuates angiotensin II-induced atrial fibrillation by regulating atrial electrical and structural remodeling.

doi: 10.1016/j.ejphar.2024.176712

Figure Lengend Snippet: Fig. 6. DAPA alleviated ion channel disorders and atrial fibrosis caused by Ang II. A. Immunoblotting analysis of the protein expression of CAMKII, ox-CAMKII, Nav1.5, Kv4.3, Kv4.2, Kchip2, Kir2.1, Cx40, smad2/3, p-smad2/3, TGF-β1 in atrial tissue. B, C. Quantification of CAMKII, ox-CAMKII expression level in atrial tissue. n = 4. *p < 0.05 vs. CTL group and #p < 0.05 vs. Ang II group. D-I. Quantification of atrial electrical activity associated protein expression level. n = 4. *p < 0.05 vs. CTL group and #p < 0.05 vs. Ang II group. J, K. Quantification of atrial fibrosis associated protein expression level. n = 4. *p < 0.05 vs. CTL group and #p < 0.05 vs. Ang II group. Statistical significance was assessed by one-way ANOVA analysis followed by Tukey tests or Kruskal-Wallis post hoc multicomparison test (B–K). Data are presented as means ± SEM.

Article Snippet: Protein lysates of tissue or cells were separated by 8% SDS-PAGE and transferred to NC membrane, and blocked for 2 h using 5% non-fat milk and probed with primary polyclonal antibodies against CaMKII (Abclonal), oxCaMKII (GeneTex), Nav1.5 (Abmart), Kv4.3 (Abclonal), Kv4.2 (Cell Signaling Technology), Kchip2 (Abclonal), Kir2.1(Zenbio), Cx40 (GeneTex), TGF-β1(Santa cruz), smad (Zenbio), p-smad (Zenbio) at 4 ◦C overnight.

Techniques: Western Blot, Expressing, Activity Assay

Journal: European journal of pharmacology

Article Title: Dapagliflozin: A sodium-glucose cotransporter 2 inhibitor, attenuates angiotensin II-induced atrial fibrillation by regulating atrial electrical and structural remodeling.

doi: 10.1016/j.ejphar.2024.176712

Figure Lengend Snippet: Fig. 7. SGLT2i alleviated ion channel disorders and atrial fibrosis caused by activation of CAMKII. A-I Immunoblotting analysis and quantitation of the protein expression of CAMKII, ox-CAMKII, Nav1.5, Kv4.3, Kv4.2, Kchip2, Kir2.1, Cx40 in HL-1 atrial myocytes. n = 4. *p < 0.05 vs. CTL group and #p < 0.05 vs. Ang II group. J-N. Immunoblotting analysis and quantitation of the protein expression of CAMKII, ox-CAMKII, smad2/3, p-smad2/3, TGF-β1 in primary mouse fibroblast. n = 4. *p < 0.05 vs. CTL group and #p < 0.05 vs. Ang II group. Statistical significance was assessed by one-way ANOVA analysis followed by Tukey tests or Kruskal- Wallis post hoc multicomparison test (B–I and K–N). Data are presented as means ± SEM.

Article Snippet: Protein lysates of tissue or cells were separated by 8% SDS-PAGE and transferred to NC membrane, and blocked for 2 h using 5% non-fat milk and probed with primary polyclonal antibodies against CaMKII (Abclonal), oxCaMKII (GeneTex), Nav1.5 (Abmart), Kv4.3 (Abclonal), Kv4.2 (Cell Signaling Technology), Kchip2 (Abclonal), Kir2.1(Zenbio), Cx40 (GeneTex), TGF-β1(Santa cruz), smad (Zenbio), p-smad (Zenbio) at 4 ◦C overnight.

Techniques: Activation Assay, Western Blot, Quantitation Assay, Expressing