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

Abstract

Aims: Atrial fibrillation (AF) is the most common cardiac arrhythmia. Pathogenic variants in genes encoding ion channels are associated with familial AF. The point mutation M1875T in the SCN5A gene, which encodes the α-subunit of the cardiac sodium channel Nav1.5, has been associated with increased atrial excitability and familial AF in patients.

Methods and results: We designed a new murine model carrying the Scn5a-M1875T mutation enabling us to study the effects of the Nav1.5 mutation in detail in vivo and in vitro using patch clamp and microelectrode recording of atrial cardiomyocytes, optical mapping, electrocardiogram, echocardiography, gravimetry, histology, and biochemistry. Atrial cardiomyocytes from newly generated adult Scn5a-M1875T+/- mice showed a selective increase in the early (peak) cardiac sodium current, larger action potential amplitude, and a faster peak upstroke velocity. Conduction slowing caused by the sodium channel blocker flecainide was less pronounced in Scn5a-M1875T+/- compared to wildtype atria. Overt hypertrophy or heart failure in Scn5a-M1875T+/- mice could be excluded.

Conclusion: The Scn5a-M1875T point mutation causes gain-of-function of the cardiac sodium channel. Our results suggest increased atrial peak sodium current as a potential trigger for increased atrial excitability.

Keywords: Atrial electrophysiology; Channelopathy; Sodium channel; Sodium channel blocker.

Graphical Abstract

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 kb. DNA samples 1, 2, 4, 5, 7, 8, 10, 11, and 13 contain correctly targeted Scn5a gene (Scn5a-M1875T^+/−). Positions of the size marker (in bp) are shown on the right. The WT control animal is labeled ‘wt’. (c) Sequencing analysis of the Scn5a gene region containing the mutation site in back-crossed adult mice (on pure genetic background). The T-C mutation on one allele causing the methionine-threonine exchange at position 1875 (1877) is indicated by an arrow.

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.

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 (I_Na) 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 I_Na in LA cardiomyocytes over test potentials ranging from −100 to +40 mV. (C) At the peak I_Na test potential of −30 mV, Scn5a-M1875T^+/− cardiomyocytes had a significantly larger I_Na 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.

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.

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 under experiment conditions for the same time period (35 min from baseline recording). n = 12 per group at 330–100 ms PCL. Atria were excluded from further analysis at shorter PCLs if 1:1 capture was lost, only data in steady state was used. n = 5 for time control experiments. *P < 0.05, **P < 0.01, WT vs. Scn5a-M1875T^+/−; ANOVA statistics. Data are represented as box-whiskers with box limits denoting the interquartile range (IQR) and the whiskers projecting to min and max values (B) or as the mean ± SEM (D).