Late-Stage Skeletal Muscle Transcriptome in Duchenne Muscular Dystrophy Shows a BMP4-Induced Molecular Signature

Duchenne muscular dystrophy (DMD) is a fatal X-linked recessive disease due to loss-of-function variants in the DYSTROPHIN gene. DMD-related skeletal muscle wasting is typified by an aberrant immune response involving upregulation of the TGFβ family of cytokines, like TGFβ1 and BMP4. We previously demonstrated that bone morphogenetic protein 4 (BMP4) is increased in DMD and BMP4 stimulation induces a 20-fold upregulation of Smad8 transcription in muscle cells. However, the role of BMP4 in late-stage DMD skeletal muscle is unknown. We hypothesized that BMP4 signalling is a driver of aberrant gene expression in late-stage human DMD skeletal muscle detectable by a transcriptomic signature.

Transcriptomes from skeletal muscle biopsies of late-stage DMD versus non-DMD controls and C2C12 muscle cells with or without BMP4 stimulation were generated using RNA-Seq. We tested transcriptional differences at the single transcript level in skeletal muscle biopsy samples from three patients with DMD and compared them to three non-DMD. They were then analyzed by Ingenuity Pathway Analysis, weighted gene coexpression network analyses (WGCNA) and Gene Set Enrichment Analysis (GSEA). Key hub and high-fold change genes overlapping in the DMD and BMP4 muscle transcriptomes were validated in additional primary and bulk skeletal muscle samples.

A total of 3048 transcripts in the human muscle and 5291 transcripts in C2C12 muscle cells were differentially expressed. WGCNA identified an overlapping molecular signature of 1027 genes dysregulated in DMD muscle that were induced in BMP4-stimulated C2C12 muscle cells. SERPING1 and Aff3 were identified as the top hub genes. Highly upregulated DMD muscle transcripts that overlapped with BMP4-stimulated C2C12 muscle cells included ADAM12, SERPING1, SMAD8 and SFRP4. DMD skeletal muscle analysis showed aberrant upregulation of TGFβ signalling, extracellular matrix remodelling and collagen biosynthesis pathways, in contrast to inhibited mitochondrial and metabolic pathways.

In summary, the DMD transcriptome was characterized by dysregulation of immune function, ECM remodelling and muscle bioenergetic metabolism. We additionally define a late-stage DMD skeletal muscle transcriptome that overlaps with a BMP4-induced molecular signature in C2C12 muscle cells. This supports BMP4/Smad8 pathway as a disease-driving regulator of transcriptomic changes in late-stage DMD skeletal muscle. Further exploration of this cross-species transcriptomic signature may expand our understanding of the evolution of dystrophic signalling pathways and the associated gene networks, which could be evaluated for therapeutic development.