Transcriptomic analysis of paired healthy human skeletal muscles to identify modulators of disease severity in DMD

Shirley Nieves-Rodriguez; Florian Barthélémy; Jeremy D Woods; Emilie D Douine; Richard T Wang; Deirdre D Scripture-Adams; Kevin N Chesmore; Francesca Galasso; M Carrie Miceli; Stanley F Nelson

doi:10.3389/fgene.2023.1216066

Transcriptomic analysis of paired healthy human skeletal muscles to identify modulators of disease severity in DMD

Front Genet. 2023 Jul 27:14:1216066. doi: 10.3389/fgene.2023.1216066. eCollection 2023.

Authors

Shirley Nieves-Rodriguez^{1

2}, Florian Barthélémy^{2

3}, Jeremy D Woods⁴, Emilie D Douine^{2

5}, Richard T Wang^{1

2}, Deirdre D Scripture-Adams^{2

3}, Kevin N Chesmore^{1

2}, Francesca Galasso¹, M Carrie Miceli^{2

3}, Stanley F Nelson^{1

2

5

6}

Affiliations

¹ Department of Human Genetics, David Geffen School of Medicine, University of California, Los Angeles, Los Angeles, CA, United States.
² Center for Duchenne Muscular Dystrophy at UCLA, Los Angeles, CA, United States.
³ Department of Microbiology, David Geffen School of Medicine and College of Letters and Sciences, University of California, Los Angeles, Los Angeles, CA, United States.
⁴ Department of Pediatrics, David Geffen School of Medicine, University of California, Los Angeles, Los Angeles, CA, United States.
⁵ Department of Neurology, David Geffen School of Medicine, University of California, Los Angeles, Los Angeles, CA, United States.
⁶ Department of Pathology and Laboratory Medicine, David Geffen School of Medicine, University of California, Los Angeles, Los Angeles, CA, United States.

Abstract

Muscle damage and fibro-fatty replacement of skeletal muscles is a main pathologic feature of Duchenne muscular dystrophy (DMD) with more proximal muscles affected earlier and more distal affected later in the disease course, suggesting that different skeletal muscle groups possess distinctive characteristics that influence their susceptibility to disease. To explore transcriptomic factors driving differential gene expression and modulating DMD skeletal muscle severity, we characterized the transcriptome of vastus lateralis (VL), a more proximal and susceptible muscle, relative to tibialis anterior (TA), a more distal and protected muscle, in 15 healthy individuals using bulk RNA sequencing to identify gene expression differences that may mediate their relative susceptibility to damage with loss of dystrophin. Matching single nuclei RNA sequencing data was generated for 3 of the healthy individuals, to infer cell composition in the bulk RNA sequencing dataset and to improve mapping of differentially expressed genes to their cell source of expression. A total of 3,410 differentially expressed genes were identified and mapped to cell type using single nuclei RNA sequencing of muscle, including long non-coding RNAs and protein coding genes. There was an enrichment of genes involved in calcium release from the sarcoplasmic reticulum, particularly in the myofibers and these myofiber genes were higher in the VL. There was an enrichment of genes in "Collagen-Containing Extracellular Matrix" expressed by fibroblasts, endothelial, smooth muscle and pericytes, with most genes higher in the TA, as well as genes in "Regulation Of Apoptotic Process" expressed across all cell types. Previously reported genetic modifiers were also enriched within the differentially expressed genes. We also identify 6 genes with differential isoform usage between the VL and TA. Lastly, we integrate our findings with DMD RNA sequencing data from the TA, and identify "Collagen-Containing Extracellular Matrix" and "Negative Regulation Of Apoptotic Process" as differentially expressed between DMD compared to healthy. Collectively, these findings propose novel candidate mechanisms that may mediate differential muscle susceptibility in muscular dystrophies and provide new insight into potential therapeutic targets.

Keywords: DMD; gene expression; muscle; muscle susceptibility; single nuclei RNAseq; transcriptomics.

Grants and funding

This work was supported by the Center for Duchenne Muscular Dystrophy at UCLA. SN-R was supported by NIH T32HG002536 Genomic Analysis and Interpretation Training Grant and the CDMD Azrieli Graduate Award. KC was supported by NIH T32AR065972 Muscle Cell Biology Pathophysiology and Therapeutics Training Grant.