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

Abstract

Background The sodium channel, Na_v1.5, encoded by SCN 5A, undergoes developmentally regulated splicing from inclusion of exon 6A in the fetal heart to exon 6B in adults. These mutually exclusive exons differ in 7 amino acids altering the electrophysiological properties of the Na_v1.5 channel. In myotonic dystrophy type 1, SCN 5A is mis-spliced such that the fetal pattern of exon 6A inclusion is detected in adult hearts. Cardiac manifestations of myotonic dystrophy type 1 include conduction defects and arrhythmias and are the second-leading cause of death. Methods and Results This work aimed to determine the impact of SCN 5A mis-splicing on cardiac function. We used clustered regularly interspaced short palindromic repeat ( CRISPR) /CRISPR-associated protein 9 (Cas9) to delete Scn5a exon 6B in mice, thereby redirecting splicing toward exon 6A. These mice exhibit prolonged PR and QRS intervals, slowed conduction velocity, extended action potential duration, and are highly susceptible to arrhythmias. Conclusions Our findings highlight a nonmutational pathological mechanism of arrhythmias and conduction defects as a result of mis-splicing of the predominant cardiac sodium channel. Animals homozygous for the deleted exon express only the fetal isoform and have more-severe phenotypes than heterozygotes that also express the adult isoform. This observation is directly relevant to myotonic dystrophy type 1, and possibly pathological arrhythmias, in which individuals differ with regard to the ratios of the isoforms expressed.

Keywords: SCN5A; alternative splicing; arrhythmia; conduction; myotonic dystrophy cardiomyopathy.

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 distinguish exon 6A and 6B by RT‐PCR of heart tissue isolated from wild‐type (WT), heterozygotes (Scn5a ^Δe6B/+) and homozygotes (Scn5a ^Δe6B/Δe6B) mice with males and females showing no differences (n=3 for each group). Percent of exon 6A inclusion was calculated as in (B). RT‐PCR from an adult wild and postnatal day 1 (P1) type mice is shown to demonstrate that the Scn5a developmental splicing event is conserved in mouse and humans. **P<0.01. bp indicates base pair; CRISPR, clustered regularly interspaced short palindromic repeat; DM1, dystrophy type 1.

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.

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 ^Δe6B/+, and 6 Scn5a ^Δe6B/Δe6B except for inducibility of arrhythmias where 1 died. *P<0.05.

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.