Alternative Splicing of SORBS1 Affects Neuromuscular Junction Integrity in Myotonic Dystrophy Type 1

Myotonic dystrophy type 1 (DM1) is a multisystemic neuromuscular disorder characterized by CTG repeat expansion in the 3' untranslated region of the dystrophia myotonica protein kinase coding gene. The presence of expanded CTG repeats in DMPK mRNAs leads to the sequestration of RNA-binding factors such as the Muscleblind-like (MBNL) proteins, resulting in widespread splicing defects that contribute to progressive muscle weakness and myotonia. Previously, we identified misregulation of SORBS1 exon 25 splicing in both DM1 and MBNL1/2 double-knockout human-induced pluripotent stem cells (hiPSC)-derived skeletal muscle cells, suggesting a potential role in DM1 physiopathology.

We investigated SORBS1 exon 25 splicing misregulation in human skeletal muscle biopsies from DM1 patients and healthy controls. The functional consequence of SORBS1 exon 25 exclusion was assessed in mice, zebrafish and hiPSC-derived skeletal muscle cells using an antisense oligonucleotide-mediated exon-skipping strategy.

In human congenital DM1 fetal skeletal muscle biopsies, SORBS1 exon 25 inclusion was reduced by 52.6 ± 10% compared to controls (p < 0.001). Analysis of RNA sequencing data from the DMseq database further revealed significant misregulation in tibialis anterior biopsies from 40 adult DM1 patients, with a 15.8 ± 3.7% decrease in splice inclusion (p < 0.0001). In mice, forced exclusion of Sorbs1 exon 25 led to neuromuscular junction degeneration, with increased denervation (10.5% ± 3.4%, p < 0.01) and postsynaptic destabilization (5.7% ± 2.5%, p < 0.05). In zebrafish, sorbs1 exon 25 misregulation significantly impaired locomotion, reducing trajectory, distance (57.9% ± 12%, p < 0.0001) and velocity (14% ± 0.5%, p < 0.05), while also disrupting acetylcholine receptor cluster morphology. Similarly, forced SORBS1 exon 25 exclusion in hiPSC-derived skeletal muscle cells diminished the formation of large acetylcholine receptor clusters upon agrin stimulation by 34% ± 4.5% (p < 0.0001).

Our study identifies SORBS1 alternative splicing as an essential MBNL-regulated event during skeletal muscle development, potentially involved in neuromuscular junction formation and maintenance. The aberrant splicing of SORBS1 exon 25 in DM1 expands our understanding of how splicing dysregulation compromises neuromuscular system communication, shedding light on the broader impact of mRNA splicing regulation on NMJ integrity.