SETDB1 modulates the TGFß response in Duchenne muscular dystrophy myotubes.

Overactivation of the transforming growth factor-ß (TGFß) signaling in Duchenne muscular dystrophy (DMD) is a major hallmark of disease progression, leading to fibrosis and muscle dysfunction. Here, we investigated the role of SETDB1 (SET domain, bifurcated 1), a histone lysine methyltransferase involved in muscle differentiation. Our data show that, following TGFß induction, SETDB1 accumulates in the nuclei of healthy myotubes while being already present in the nuclei of DMD myotubes where TGFß signaling is constitutively activated. Transcriptomics revealed that depletion of SETDB1 in DMD myotubes leads to down-regulation of TGFß target genes coding for secreted factors involved in extracellular matrix remodeling and inflammation. Consequently, SETDB1 silencing in DMD myotubes abrogates the deleterious effect of their secretome on myoblast differentiation by impairing myoblast pro-fibrotic response. Our findings indicate that SETDB1 potentiates the TGFß-driven fibrotic response in DMD muscles, providing an additional axis for therapeutic intervention.

Acute conversion of patient-derived Duchenne muscular dystrophy iPSC into myotubes reveals constitutive and inducible over-activation of TGFß-dependent pro-fibrotic signaling.

BACKGROUND: In Duchenne muscular dystrophy (DMD), DYSTROPHIN deficiency exposes myofibers to repeated cycles of contraction/degeneration, ultimately leading to muscle loss and replacement by fibrotic tissue. DMD pathology is typically exacerbated by excessive secretion of TGFß and consequent accumulation of pro-fibrotic components of the extra-cellular matrix (ECM), which in turn impairs compensatory regeneration and complicates the efficacy of therapeutic strategies. It is currently unclear whether DMD skeletal muscle fibers directly contribute to excessive activation of TGFß. Development of skeletal myofibers from DMD patient-derived induced pluripotent stem cells (iPSC), as an "in dish" model of disease, can be exploited to determine the myofiber contribution to pathogenic TGFß signaling in DMD and might provide a screening platform for the identification of anti-fibrotic interventions in DMD. METHODS: We describe a rapid and efficient method for the generation of contractile human skeletal muscle cells from DMD patient-derived hiPSC, based on the inducible expression of MyoD and BAF60C (encoded by SMARCD3 gene), using an enhanced version of piggyBac (epB) transposone vectors. DMD iPSC-derived myotubes were tested as an "in dish" disease model and exposed to environmental and mechanical cues that recapitulate salient pathological features of DMD. RESULTS: We show that DMD iPSC-derived myotubes exhibit a constitutive activation of TGFß-SMAD2/3 signaling. High-content screening (HCS)-based quantification of nuclear phosphorylated SMAD2/3 signal revealed that DMD iPSC-derived myotubes also exhibit increased activation of the TGFß-SMAD2/3 signaling following exposure to either recombinant TGFß or electrical pacing-induced contraction. CONCLUSIONS: Acute conversion of DMD patient-derived iPSC into skeletal muscles, by the ectopic expression of MyoD and BAF60C, provides a rapid and reliable protocol for an "in dish" DMD model that recapitulates key pathogenic features of disease pathology, such as the constitutive activation of the TGFß/SMAD signaling as well as the deregulated response to pathogenic stimuli, e.g., ECM-derived signals or mechanical cues. Thus, this model is suitable for the identification of new therapeutic targets in DMD patient-specific muscles.