Familial atrial fibrillation mutation M1875T-SCN5A increases early sodium current and dampens the effect of flecainide

Figures

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  Figure 1. Targeting of exon 28 of the mouse Scn5a gene in order to introduce the M1875T coding mutation and sequencing confirmation of its presence. (A) The intronic and intergenic regions are shown as lines, and exons are shown as filled boxes. The empty box corresponds to the neomycin resistance cassette (neo) flanked by the Locus of x-over, P1 (LoxP) sites (vertical arrows). Exon numeration is shown above. The arrows below are corresponding to Bacillus amyloli (BamHI) restriction endonuclease sites (B). The black box corresponds to Southern probe sequences (So3). The expected sizes of restriction DNA fragments are indicated below in kb. (AI) Wildtype (WT) locus. (AII) Targeted vector structure (without negative selection marker and plasmid backbone). (AIII) Genomic locus after the homologous recombination. The neomycin cassette is present in intron 27 and flanked by two LoxP sites. (AIV) Genomic locus after the Cyclic recombinase-mediated neo cassette deletion. (B) Southern blot analysis of DNA isolated from mouse tail biopsy of the F1 offspring (1–13) and hybridized with the So3 probe. With help of the BamHI enzymatic digestion, we detect the WT allele 7.6 kb and targeted allele 5.8…

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  Figure 2. Unaltered atrial amount of extracellular matrix content and myocyte diameter. (A) Hematoxylin and eosin-stained sections of wildtype (WT) and Scn5a-M1875T+/− litter pair hearts (ventricles, upper panel; left atria, lower panel). Scale bars represent 1 mm. There were no obvious differences between genotypes. (B) Exemplary immunofluorescence images from atrial regions of interest (ROI) of wheat germ agglutinin (WGA) staining and corresponding binary images for quantification in left atrial (LA) appendage and LA posterior wall from WT and Scn5a-M1875T+/− hearts. Scale bars represent 10 µm. (C) Quantification of WGA-stained atrial area and atrial cell diameters from transverse sections as depicted in (B). Neither parameter was affected by the point mutation (WGA-stained atrial area quantified from LA appendage: n = 6 hearts and 65/67 individual ROI per group; LA posterior wall n = 6 hearts and 26/29 ROI per group. Atrial cell diameter quantified from LA appendage: n = 6 hearts and 14 563/13 368 individual cells per group; LA posterior wall and 8283/8384 individual cells per group). Data are presented as mean ± SD.

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  Figure 3. Isolated atrial cardiomyocytes with the Scn5a-M1875T+/− mutation have a larger sodium current than wildtypes when measured with whole-cell patch clamp electrophysiology. (A) Representative sodium current (INa) traces from whole-cell voltage clamp recording of isolated left atrial (LA) cardiomyocytes from wildtype (WT) (left, dark turquoise) and Scn5a-M1875T+/− (light red, right) hearts. (B) Normalized grouped data revealed that the Scn5a-M1875T+/− mutation increases peak INa in LA cardiomyocytes over test potentials ranging from −100 to +40 mV. (C) At the peak INa test potential of −30 mV, Scn5a-M1875T+/− cardiomyocytes had a significantly larger INa than WTs, both when comparing individual cells (n = 109 WT, n = 107 Scn5a-M1875T+/−) (CI) and hearts (n = 15 WT, n = 14 Scn5a-M1875T+/−) (CII). Data are presented as the mean ± SEM.

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  Figure 4. Action potentials from atria with the Scn5a-M1875T+/− mutation have a larger action potential amplitude and a faster peak upstroke velocity when measured with the sharp microelectrode technique. (A) Scn5a-M1875T+/− left atria (LA) had a significantly larger action potential (AP) amplitude at shorter pacing cycle lengths (PCLs) of 80–120 ms (P < 0.05) and (B) a significantly faster peak upstroke velocity (dV/dt). (C) Representative AP traces from WT (dark turquoise) and Scn5a-M1875T+/− (light red) LA stimulated at 100 ms PCL. (D–F) When paced at 1000 ms PCL, Scn5a-M1875T+/− LA had a significantly shorter AP duration (APD) when measured at 50% (APD50) (D) and 90% (APD90) (E) repolarization. *P < 0.05, **P < 0.01, wildtype (WT) vs. Scn5a-M1875T+/−, n = 8 per group, n = 24 WT, n = 25 Scn5a-M1875T+/−; ANOVA statistics. (F) Representative AP traces from WT (dark turquoise) and Scn5a-M1875T+/− (light red) LA stimulated at 1000 ms PCL. Data in (A), (B), (D), and (E) are presented as box-whiskers with box limits denoting the interquartile range (IQR) and the whiskers projecting to the outliers.

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  Figure 5. The response to the anti-arrhythmic agent flecainide is reduced in Scn5a-M1875T+/− atria in optical mapping. (A) Example left atrial (LA) activation maps from wildtype (WT, top panels) and Scn5a-M1875T+/− (bottom panels) mice. Left panels show activation maps at baseline, right panels show activation of the same atria following exposure to 1 µM flecainide for 20 min. (B) Grouped data showing change in conduction velocity (ΔCV) following exposure to 1 µM flecainide in WT (dark turquoise) and Scn5a-M1875T+/− (light red) LA for 20 min at different pacing cycle lengths. (C) Example traces of optical action potentials (APs) recorded following further treatment of WT (top, dark turquoise) and Scn5a-M1875T+/− (bottom, light red) LA with 5 µM flecainide for 15 min while reducing pacing cycle length (increasing pacing frequency). (D) Grouped data showing minimum pacing frequency in beats per minute (bpm) at which 1:1 stimulus capture was maintained in WT (dark turquoise) and Scn5a-M1875T+/− (light red) LA following exposure to 5 µM flecainide. Time control data (green) shows minimum PCL at which 1:1 stimulus capture was maintained in atria that were not exposed to flecainide but had been…