# Adaptive response to electrical pulse stimulation is impaired in FSHD myotubes by DUX4 gene network activation

## TL;DR

This study shows that FSHD muscle cells have impaired responses to electrical stimulation due to DUX4 gene activity, suggesting a possible mechanism for the disease's progression.

## Contribution

The study identifies a DUX4-related functional defect in FSHD muscle cells during electrical stimulation, linking it to impaired adaptive responses.

## Key findings

- EPS activates contractile genes and contractility in control cells but is impaired in FSHD patient cells.
- FSHD cells fail to activate musclin/OSTN, an exercise-responsive myokine, under EPS.
- DUX4 gene network activation is sufficient to recapitulate the FSHD cell phenotype.

## Abstract

Facioscapulohumeral dystrophy (FSHD) is one of the most common muscular dystrophies with no effective treatment. The disease is linked to abnormal derepression of DUX4 embryonic transcription factor in skeletal muscle, but at very low frequency. How this relates to the disease process and the exact nature of functional defects of FSHD muscle cells remain obscure. We used electrical pulse stimulation (EPS) substituting motor neuron activation to perform quantitative structural, functional, and gene expression analyses of in vitro differentiated control and FSHD skeletal myocytes. We found that EPS effectively stimulated muscle contractile gene expression and contractility activation in control cells, which are impaired in FSHD patient cells. This is accompanied by exacerbated organizational differences of sarcomeric striation. Importantly, this FSHD patient cell phenotype was confirmed in engineered mutant cells carrying D4Z4 repeat contraction and SMCHD1 mutation. Notably, FSHD patient/mutant cells fail to activate musclin/OSTN, an exercise-responsive muscle-protective myokine. Overexpression of LEUTX, a major downstream DUX4 target, was sufficient to recapitulate this phenotype, indicating that the phenotype is linked to DUX4 gene network activation. The results demonstrate DUX4-induced cell intrinsic functional defects of FSHD muscle cells in adaptive response to electrical stimulation, suggesting the possible activity-stimulated pathological mechanism of FSHD.

The online version contains supplementary material available at 10.1038/s41598-025-32385-0.