Single-cell Transcriptional Responses to Plasma Membrane Injury in FSHD Myoblasts


Translational Research

Poster Number: Virtual


Adam Bittel, DPT, PhD, Children's Research Institute (CNMC), Surajit Bhattacharya, PhD, Children's National Hospital, Yi-Wen Chen, DVM, PhD, Children's National Hospital, George Washington University

Background: Facioscapulohumeral muscular dystrophy (FSHD) is caused by transcriptional de-repression of the double homeobox 4 (DUX4) transcription factor. FSHD myoblasts have significant delays in plasmalemmal repair compared to healthy myoblasts. Since rapid repair of membrane injury is essential for muscle health, we hypothesize molecular stress from membrane injury may exacerbate FSHD through transcriptional reprogramming.
Approach: A cell scrape approach was used to induce membrane injury in cultured myoblasts. FSHD and healthy myoblasts were collected at baseline (uninjured), and at 6- and 24-hours post-injury for single-cell sequencing. Myoblast subclusters were visualized with expression clustering. Differentially expressed genes (DEGs) among clusters and groups were identified using Seurat. DEGs were identified between FSHD and healthy myoblasts, and among different time points. Gene Ontology (GO) and KEGG analyses were performed for functional assessment.
Results: We identified 1,682 DEGs between uninjured FSHD and healthy myoblasts, 2,102 DEGs at 6-hours, and 1,732 DEGs at 24-hours post-injury (FDR p<0.05). At 6-hrs, upregulated GO-terms were related to muscle system development and organ growth in FSHD vs healthy myoblasts (FDR p<0.05). At 6-hours post injury, upregulated ontologies related to cell death in FSHD myoblasts. Likewise, we found 1,570 (uninjured vs. 6-hr), 1,492 (6-hr vs. 24-hr), and 1,817 (uninjured vs 24-hr) DEGs between FSHD myoblasts (FDR P<0.05). At 6-hours post-injury, ontologies related to muscle development/morphogenesis, and cell signaling are downregulated in injured compared to uninjured FSHD cells. By 24-hours, ontologies related to muscle development and cell proliferation are up-regulated vs. uninjured cells (FDR p<0.05). KEGG analysis of 24-hour FSHD samples indicated upregulation of PI3K/Akt, MAPK, and P53 signaling (FDR p<0.05).
Conclusion: Injury increased expression of genes involved in cell death in FSHD myoblasts, indicating they may have lower injury tolerance. Injury also upregulates pathways implicated in FSHD muscle pathology, including the MAPK signaling and apoptosis pathways, which could exacerbate disease.