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

Abstract

Background The sodium channel, Na v 1.5, 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 v 1.5 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.

Overexpression of a non-muscle RBFOX2 isoform triggers cardiac conduction defects in myotonic dystrophy

“…6G, H). Frequent transitions observed between a-and 310-helix further indicate that the activated state of S4 in the 6A isoform is destabilized and it may deactivate more readily due to a reduction in energy barrier, which is consistent with a depolarized shift in the voltage-dependence of channel activation for the pro-arrhythmic 6A isoform 52,54 . Additionally, we note that although D211 in 6B and K211 in 6A are both positioned close to the lipid headgroups, unlike D211, which preferably interacts with the choline moiety at a slightly higher position with respect to the lipid bilayer, K211-due to its longer side-chain-can reach down into the headgroup region and coordinate closely with the nearby phosphate group of POPC lipids (Fig.…”

“…We further propose that compared to the muscle isoform, RBFOX240 stimulates the higher-order assembly of LASR complex 46,47 , which boosts its splicing activity, resulting in altered isoform expression of proteins that can disrupt the normal cardiac rhythm and function. Indeed, we demonstrated that RBFOX240 promotes the generation of pathogenic splice variants of voltage-gated sodium and potassium channels that are known to exhibit slower conduction velocity and increased susceptibility to arrhythmias 52,54,68,69 . Our MD simulations showed that a RBFOX240-mediated splicing switch in Nav1.5 transcript changes the amino acid composition of the voltage sensing element within domain I that promotes persistent lipid-interactions and stabilizes the 310-helical conformation of S4 helix, which reduces the energy barrier and allows for rapid deactivation of the channel.…”

“…6A). Of these, we elected to focus on SCN5A, CACNA1C, and KCND3 because they encode the major voltage-gated sodium, calcium, and potassium channels in the heart respectively; and they switch to producing their pro-arrhythmic splice isoforms [52][53][54][55] in DM1 patients. Additionally, multiple genome-wide association studies have identified variants of SCN5A and KCND3 among genetic determinants of prolonged PR interval and QRS duration 56,57 , which are distinctive features of cardiac-conduction delay in DM1 patients.…”

“…6C). Because a shift from exon 6B to 6A is linked to DM1associated cardiac-conduction delay and heart arrhythmias 52,54 , we investigated functionally relevant structural and dynamical differences between the two alternatively spliced Nav1.5 isoforms using molecular dynamics (MD) simulations. Given that the three-dimensional structure of Nav1.5 channel is unavailable, we built homology models of the exon 6A or exon 6B containing human Nav1.5 isoforms using existing structures of the activated sodium channels Nav1.4 and NavPas 62,63 .…”

“…We propose that the observed persistent lipidinteraction of K211 in 6A isoform is crucial for stabilizing a 310-helical conformation near the top portion of S4 to allow rapid deactivation that would slow the activation kinetics (Supplementary video 1, 2). Indeed, forced expression of the exon 6A containing NaV1.5 isoform in adult mouse heart reduces excitability, slows conduction velocity, prolongs PR and QRS intervals, and increases susceptibility to arrhythmias 52,54 .…”