snRNA-seq analysis in multinucleated myogenic FSHD cells identifies heterogeneous FSHD transcriptome signatures associated with embryonic-like program activation and oxidative stress-induced apoptosis

Abstract

The sporadic nature of DUX4 expression in FSHD muscle challenges comparative transcriptome analyses between FSHD and control samples. A variety of DUX4 and FSHD-associated transcriptional changes have been identified, but bulk RNA-seq strategies prohibit comprehensive analysis of their spatiotemporal relation, interdependence and role in the disease process. In this study, we used single-nucleus RNA-sequencing of nuclei isolated from patient- and control-derived multinucleated primary myotubes to investigate the cellular heterogeneity in FSHD. Taking advantage of the increased resolution in snRNA-sequencing of fully differentiated myotubes, two distinct populations of DUX4-affected nuclei could be defined by their transcriptional profiles. Our data provides insights into the differences between these two populations and suggests heterogeneity in two well-known FSHD-associated transcriptional aberrations: increased oxidative stress and inhibition of myogenic differentiation. Additionally, we provide evidence that DUX4-affected nuclei share transcriptome features with early embryonic cells beyond the well-described cleavage stage, progressing into the 8-cell and blastocyst stages. Altogether, our data suggests that the FSHD transcriptional profile is defined by a mixture of individual and sometimes mutually exclusive DUX4-induced responses and cellular state-dependent downstream effects.

Single-cell spatial transcriptomics reveals a dystrophic trajectory following a developmental bifurcation of myoblast cell fates in facioscapulohumeral muscular dystrophy

“…The questions of how limited DUX4 expression impacts disease pathology and how dysregulation of DUX4 target genes contributes to the disease process are not well understood, although recent studies indicate the significant correlation between the clinical phenotype and DUX4 target expression ( Wang et al 2019 ; Wong et al 2024 ). Recent single-cell (sc) and single-nucleus (sn) RNA-seq studies have begun to address these questions ( van den Heuvel et al 2019 ; Jiang et al 2020 ; Zheng et al 2024 ). Our previous snRNA-seq analyses indicate heterogeneity of patient myotube nuclei and identify two discrete nuclei clusters, “FSHD-hi” and “FSHD-lo” ( Jiang et al 2020 ).…”