Sarcolemmal Injury as an Upstream Trigger of the FSHD Molecular Phenotype


Translational Research

Poster Number: SC5


Adam Bittel, PT, DPT, PhD, Childrens National Medical Center, Surajit Bhattacharya, PhD, Children's National Research Institute (CNMC), Yi-Wen CHen, DVM, PhD, Children's National Research Institute (CNMC)

Background: Facioscapulohumeral muscular dystrophy (FSHD) is caused by transcriptional de-repression of the double homeobox 4 (DUX4) transcription factor. Muscle injury is associated with the onset or progression of muscle dysfunction in FSHD, and we previously reported that FSHD myoblasts have significant delays in plasmalemmal repair. We hypothesize that molecular stress from muscle injury and prolonged repair may negatively affect the muscle transcriptome, which could potentiate pathology downstream.

Approach: Two sibling pairs of FSHD and Healthy primary myoblasts were collected prior to, 6-hrs, and 24-hrs after cell scrape injury for single-cell RNA sequencing. Myoblasts were also injured following a 24-hr pre-treatment with 1 uM antisense oligonucleotide (AON) targeting DUX4 mRNA. Datasets from each pair were integrated and clustered at each timepoint. Single-cell proportion tests were used to identify clusters with consistent under-/over-representation of FSHD myoblasts (FDR p<0.05, log2FD>0.58). IPA and GSEA were used for functional annotation of cluster marker genes. qRT-PCR was used to detect DUX4, MBD3L2, and ZSCAN4 as well as validate mRNA differences identified by the single-cell study using four sibling pairs of FSHD and Healthy myoblasts.

Results: Scrape injury was associated with significant increases in DUX4 and MBD3L2 expression by 6-hrs post-injury (log2FC=3.07; log2FC=3.11, respectively, both p<0.05). scRNAseq identified myoblast subpopulations that consist predominantly of FSHD myoblasts (FSHD clusters). At 6-hrs post-injury, FSHD clusters expressed genes consistent with pro-inflammatory, mitochondrial dysfunction, and DNA damage signaling. At 24-hrs post-injury, FSHD clusters expressed well-known hallmarks of FSHD involved in extracellular-matrix and pro-inflammatory signaling. AON treatment significantly reduced the cumulative expression of these genes in both sets of FSHD myoblasts (p<0.05). Conclusion: Sarcolemmal injury serves as an upstream trigger of DUX4 expression in FSHD myoblasts, and activates genes previously reported to be upregulated in FSHD skeletal muscle biopsies. This activation was mitigated by AON treatment targeting DUX4. Sarcolemmal injury also increases expression of genes involved in inflammatory, DNA damage, and extracellular matrix signaling in FSHD myoblasts – providing a potential mechanistic link between muscle injury and the onset or progression of FSHD symptoms.