Mechanistic Basis And Therapeutic Potential Of Targeting The Non-muscle Rbfox2 Isoform In Myotonic Dystrophy

Myotonic Dystrophy type 1 (DM1), the most prevalent form of adult-onset muscular dystrophy, is caused by CTG trinucleotide repeat expansion in the 3’-UTR of the DMPK gene. Heart dysfunctions occur in nearly 80% of DM1 patients, and cardiac arrhythmias or conduction abnormalities are a prominent cause of mortality in affected individuals. Yet, the underlying mechanisms causing such abnormalities are not well understood. We recently demonstrated that aberrant expression of a non-muscle splice isoform of RNA-binding protein RBFOX2 triggers cardiac conduction delay, atrioventricular heart blocks, and spontaneous arrhythmogenesis in DM1 hearts. Here we studied the mechanism(s) by which non-muscle RBFOX2 induces mis-splicing of cardiac conduction genes and tested new therapeutic strategies for treating the lethal cardiac symptoms of this disease. By performing eCLIP and high-resolution RNA-sequencing studies on cardiomyocytes isolated from wild type (expressing the normal muscle-specific RBFOX2 43 isoform), Rbfox2 Δ43/Δ43 (expressing the non-muscle RBFOX2 40 isoform), and RBFOX2 40 overexpressing (OE) mice, we deconstructed the splicing regulatory networks of RBFOX2 43 and RBFOX2 40 isoforms, characterized their respective RNA binding landscapes, and determined the RBFOX2 40 -driven transcriptome alterations in DM1 heart tissue. We acquired induced pluripotent stem cells (iPSC) from healthy, moderate (238 CTG repeats) and severely (1001 CTG repeats) affected DM1 individuals and differentiated them into cardiomyocytes (iPSC-CMs) to generate a human cardiac cell culture model of DM1. Utilizing anti-sense oligonucleotides and RNAi-based approaches, we restored the muscle-specific Rbfox2 splicing pattern and depleted the non-muscle RBFOX2 isoform in the DM1 IPS-CMs. We are currently analyzing the spontaneous electrical phenotypes of normal and DM1 iPSC-CMs. Collectively, our studies provide an in-depth understanding of the molecular basis for DM1-related electrophysiological abnormalities and offer an avenue to test the potential therapeutic utility of targeting the non-muscle RBFOX2 40 isoform in treating cardiac features of DM1.