Abstract 520: Overexpression of a Non-muscle RBFOX2 Splice Isoform Induce Cardiac Arrhythmias in Myotonic Dystrophy

Abstract

Myotonic dystrophy type 1 (DM1) is a dominantly inherited neuromuscular disease caused by a CTG repeat expansion in 3’-UTR of DMPK gene. DM1 affects multiple tissues, but cardiac dysfunctions are the second leading cause of death which often include conduction defects and arrhythmias. The best characterized pathogenic mechanism of DM1 is toxic gain-of-function of expanded CUG repeat (CUG exp ) RNA that accumulates to form ribonuclear foci causing sequestration of MBNL and overexpression of CELF1 family of splicing factors. However, loss of MBNL or gain of CELF1 activity does not explain the cardiac phenotypes observed in DM1. Here we report that steady-state protein levels of RBFOX2, a critical splicing regulator, are drastically upregulated in DM1 heart tissue. This is accompanied by aberrant skipping of a muscle-specific 43bp exon in RBFOX2 transcript, resulting in selective up-regulation of the non-muscle RBFOX2 splice isoform in adult cardiomyocytes. We demonstrate that expression of CUG exp RNA in DM1 affects RBFOX2 in two distinct ways: reduced expression of a subset of microRNAs causes de-repression of RBFOX2 protein production; and CELF1 overexpression promotes skipping of the muscle-specific exon of RBFOX2 . Remarkably, tet -inducible overexpression of the non-muscle RBFOX2 isoform, or CRISPR/ Cas9 mediated deletion of the muscle-specific RBFOX2 exon in the mouse heart results in prolonged PR and QRS intervals, slower conduction velocity and spontaneous cardiac arrhythmias that mirror human DM1 pathology. Integration of RBFOX2-CLIP with RNA-seq data from cardiomyocytes of mice expressing the non-muscle RBFOX2 isoform identified a core network of mRNA splicing defects in genes encoding sodium, potassium, and calcium ion channels and key excitation-contraction coupling proteins that are similarly misspliced in hearts of DM1 patients. Thus, we have uncovered a central role for RBFOX2 isoform switching in DM1 cardiac pathogenesis and identified the molecular mechanisms that may alter electrophysiological properties and thereby induce cardiac arrhythmias in the DM1 heart.