CRISPR‐Mediated Expression of the Fetal Scn5a Isoform in Adult Mice Causes Conduction Defects and Arrhythmias

Figures

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  Figure 1. Mis‐splicing of SCN5A in heart tissue from DM1 patients and in exon 6B–deleted mice. A, Schematic representation of SCN5A exon 6 splice variants with 3 primers (2 forward and 1 reverse) used for RT‐PCR of human heart samples. B, RT‐PCR of RNA from unaffected fetal and adult hearts and from DM1 adult human heart tissues using primers shown in (A). Percent of exon 6A inclusion was determined by [exon 6A/(exon 6A+6B)]×100 and represented in a graph. Levels of exon 6A inclusion in fetal and adult heart are consistent with previous studies.6, 7 A significant difference in Scn5a exon 6A expression was found in DM1 hearts compared to unaffected individuals (P=0.006), also consistent with previous studies.6, 7 C, Schematic representation with genome sequence of mouse Scn5a containing exons 6A and 6B demonstrating the CRISPR/Cas9 approach to delete exon 6B. Green scissors represent guide RNAs targeting the intronic regions flanking exon 6B. In the genomic sequence, exon 6A is highlighted in blue, exon 6B is highlighted in yellow, and the 2 guide RNAs are highlighted in gray. The underlined sequence indicates the deleted genomic sequence. D, Schematic representation of 3 primers to…

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  Figure 2. Electrocardiography and echocardiography of adult Scn5a Δe6B/Δe6B and Scn5a Δe6B/+ mice. Scn5a Δe6B/Δe6B and Scn5a Δe6B/+ mice compared with wild‐type (WT) littermates: (A) heart rate (P=0.002 and 0.143, respectively); (B) PR interval (P=0.008 and 0.384); (C) QRS interval (P=0.004 and 0.017); (D) corrected QT interval (P=0.389 and 0.114); (E) ejection fraction (P=0.112 and 0.759); (F) fractional shortening (P=0.139 and 0.845); (G) interventricular septal end diastole (P=0.141 and 0.254); and (H) left ventricular posterior wall end diastole (P=0.393 and 0.813). n=8 wild type, 8 Scn5a Δe6B/+, and 6 Scn5a Δe6B/Δe6B. *P<0.05, **P<0.01.

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  Figure 3. oElectrophysiological studies with programmed electrical stimulation in vivo. A, Schematic representation of electrical stimulation showing catheter with 4 electrodes inserted through the right jugular vein into the right atrium and right ventricle. Top electrode is used for atrial pace (“stimulating”) with the other 3 leads capture (“recording”) electrical activity in the heart. B, Duration of the sinoatrial node recovery time (SNRT) corrected for basic cycle length in Scn5a Δe6B/Δe6B and Scn5a Δe6B/+ mice compared with wild‐type (WT) littermates (P=0.013 and 0.465, respectively). C, Duration of the atrioventricular effective refractory period (AVERP) in Scn5a Δe6B/Δe6B and Scn5a Δe6B/+ mice compared with WT littermates (P=0.021 and 0.032, respectively). D, Representative intracardiac ECG tracing with premature atrial complex indicated by red arrow (4/5 Scn5a Δe6B/Δe6B, 0/8 WT). E, Representative intracardiac ECG tracing with sinus pauses indicated by red arrows (4/5 Scn5a Δe6B/Δe6B, 0/8 WT). F, Representative intracardiac ECG tracing with spontaneous reversion from bradycardia with the reversion indicated by the red arrow (3/5 Scn5a Δe6B/Δe6B, 0/8 WT). n=8 wild type, 8 Scn5a…

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  Figure 4. Optical mapping with programmed electrical stimulation ex vivo. Representative activation map from optical mapping in (A) wild‐type (WT), (B) Scn5a Δe6B/+ mice, and (C) Scn5a Δe6B/Δe6B mice, with color bar showing time from red to blue in milliseconds. D, Action potential duration in Scn5a Δe6B/Δe6B and Scn5a Δe6B/+ mice (P=0.028 and 0.069, respectively). E, Ventricular conduction velocity in Scn5a Δe6B/Δe6B and Scn5a Δe6B/+ mice (P=0.045 and 0.074, respectively). F, Representative tracing from ECG leads on hearts isolated ex vivo showing atrioventricular block (0/3 WT, 4/5 Scn5a Δe6B/Δe6B). Red arrows indicate the absence of a ventricular depolarization (large spikes) following atrial depolarization (small spikes). G, Representative tracing showing delayed afterdepolarizations (0/3 WT, 4/5 Scn5a Δe6B/Δe6B). Red arrows indicate ventricular afterdepolarizations. n=4 wild type, 4 Scn5a Δe6B/+, and 5 Scn5a Δe6B/Δe6B. *P<0.05.