Single-myofiber RNA-sequencing reveals myofiber-level transcriptomic heterogeneity and DUX4 activation in FSHD muscle biopsies

Although expression of the transcription factor DUX4 in skeletal muscle is widely regarded as the primary driver of facioscapulohumeral muscular dystrophy (FSHD), its assumed sporadic and transient nature complicates full characterization of its transcriptional impact. Cellular heterogeneity within FSHD muscles remains only partially understood. Here, we applied single-myofiber RNA-sequencing to FSHD muscle biopsies to address this heterogeneity. Our data revealed myofiber heterogeneity, with DUX4-signature expression detected in only a small subset of myofibers. We identified 119 genes differentially expressed in DUX4-positive myofibers, of which most were downregulated. These genes showed similar but attenuated expression patterns in bulk muscle RNA-seq data, highlighting the benefit of single-myofiber RNA-sequencing over the masking-affected bulk approaches, especially for downregulated genes. DUX4 signature-positive myofibers showed a reduction in factors involved in RNA processing and surveillance and several factors of the mediator complex, perhaps reflecting an overall response to the cellular stress these myofibers endure. Our study demonstrates the potential value of single-myofiber transcriptomics for studying transcriptional heterogeneity in FSHD, but may also provide insights into challenges in capturing disease-affected myofibers during quality control procedures.