Fig. 10

Proposed model on the impact of DUX4 mis- or DUX4c over-expression in FSHD muscles. (Upper panels) Punctate laminin-α2 disruptions or partial loss around myocytes/fibers are observed in all the FSHD muscles we analyzed including those with low clinical severity and histological score (Table S4). These laminin-α2 alterations might reflect basal membrane defects (as reported in FSHD, Figs. 10) that could either induce satellite cell (SC) activation or result from SC activation [100] (Figs. 10). We also observed cells containing intracellular laminin-α2 co-detected with either intense desmin (Fig. 3B, C), dMyHC (Fig. 4) or cytoplasmic MYOD staining (Fig. 5). These cells therefore correspond to activated SCs that are found next to specific myofiber(s) either hypotrophic, or with central nuclei or unusual shape, or several of these features. The fact we observed such cells could be that FSHD muscle cell fusion failed at some stage in the process as proposed for their classification as satellite cell-opathies [99]. Two features of FSHD muscles could affect the contribution of SCs to regeneration: DUX4 expression is known to inhibit myogenesis [30, 39, 101] and the extracellular matrix (ECM) thickening (Table S4, [31]) might affect the SC niche. (Bottom panels) Because DUX4c favors cell proliferation [2, 3], its overexpression would increase the myoblast proliferation rate. If DUX4 was expressed in proliferating SCs (myoblasts) or early myotubes, it would induce their death [4, 66, 67]. Myoblasts expressing DUX4 might have perturbed migration or increase the one of mesenchymal stem cells [67, 102]. Both DUX4- or DUX4c-overexpression negatively impact myoblast fusion [14]. Altogether, this might result in the formation of CD56- or MYOD-positive cell clusters corresponding to “frozen” satellite cells (blocked in differentiation) between myofibers (Figs. S9D, S11). DUX4 misexpression might perturb protein synthesis at the mRNA level ([97, 103, 104]) via its interaction with specific RBPs (major regulators of mRNA transport, translation and decay) and result in hypotrophic myotubes [3, 12] (Figs. 6 and 7, Fig. S11) in which DUX4 could diffuse among nuclei and thus expand its transcriptional deregulation cascade [9]. Moreover, DUX4 might compete with DUX4c for C1qBP binding since this interaction occurs via their identical homeodomains [12] and at similar intracellular locations next to clusters of large and close nuclei at the (ghost) myofiber periphery (Figs. 7 and 8, Fig. S13). DUX4c-overexpression in myotubes leads to the formation of disorganized myotubes presenting non-aligned nuclei (in clusters), that might favor DUX4 diffusion since they are closer to each other, as previously proposed [3]. Moreover, DUX4c-overexpression induced troponin T and α-tubulin delocalization, as well as β-catenin accumulation [3]. The latter is known to impact myogenesis [105] and to be a central coordinator of FSHD signaling pathways [106]. In mature fibers, Lassche et al. [107] reported sarcomeric dysfunction that might be associated to myofibril anomalies. Altogether, this would lead to non-functional myofibers and therefore SC activation. The higher proportion of M2 macrophages in FSHD muscle (Fig. S17B) is in favor of an active regeneration, as proposed by Banerji et al. [32]. However, early fibrosis ([31], Table S4) might interfere with a proper muscle regeneration. Moreover, Ki67 expression is not concomitant with DUX4c expression as is the case in DMD muscles (Fig. S10). In addition, other factors involved in muscle regeneration are deregulated by DUX4 misexpression (reviewed in [9]) and could further affect the myogenesis process. Finally, DUX4C (DUX4L9) might be a DUX4-target gene. Indeed, it was listed in the 228 most robust DUX4 targets (about a sixfold induction) [63]