Investigations on the FSHD disorganized myotube phenotype

Primary CD56+ FSHD myoblasts fuse into myotubes presenting various proportions of an atrophic or a disorganized phenotype. To better define those phenotypes, we optimized a differential isotope protein labeling (ICPL) combined with 2DLC-MS/MS to study their proteome as compared to healthy control myotubes. FSHD atrophic myotubes presented decreased relative abundance of structural and contractile muscle components. This phenotype suggests atrophy-associated proteolysis that likely results from the DUX4-mediated dysregulation cascade. The skeletal muscle myosin isoforms and non-muscle myosin complexes presented a lower or higher relative abundance, respectively, that likely reflects a different defect. In FSHD disorganized myotubes, myosin isoforms were unchanged, while proteins with increased relative abundance were mostly involved in microtubule network organization and myofibrillar remodeling.

Because the predominantly atrophic or disorganized FSHD cultures that we have analyzed are derived from comparable patients in terms of the number of D4Z4 units, sex and age, we assume that other factors could intervene to explain the emergence of a non-atrophic phenotype, despite the expression of DUX4. The open chromatin at the D4Z4 repeat array in FSHD facilitates transcription of several genes in the vicinity, among which DUX4 within the D4Z4 unit itself and DUX4c located 42 kb centromeric. The DUX4c protein, expressed in myoblasts from healthy control individual and induced in FSHD, could bind to DUX4-target promoters through its identical double homeodomain but has a shorter carboxyl terminal domain and does not share DUX4 toxicity. However, its overexpression in myoblasts led to an increased proliferation rate. We have now evaluated the impact of DUX4c overexpression on myotubes phenotypes. Control myoblasts overexpressing DUX4c fuse to form disorganized myotubes with clusters of nuclei, associated with the induction of ß-catenin and an abnormal troponin T distribution and sarcomere structure. We developed siRNAs targeting the DUX4c mRNA. The addition of these inhibitors to primary disorganized FSHD myoblasts cultures suppressed DUX4c expression and restored a normal myotube phenotype.

In conclusion, these results suggest that besides DUX4, DUX4c could be an additional target for the development of a therapeutic approach against FSHD.