Abstract :
[en] The identification of FSHD biomarkers and their validation are essential to evaluate the therapeutic approach developed by our laboratory i.e. antisense tools preventing DUX4 expression1. These FSHD biomarkers which belong to the DUX4 deregulation cascade should return to healthy control values upon DUX4 suppression.
Fusion of FSHD primary myoblasts yields varying proportions of myotubes presenting either an atrophic or disorganized phenotype2. Our laboratory has compared the proteome of primary FSHD and control myotubes at day 4 of differentiation3. FSHD myotubes of both phenotypes presented a disturbance of several caveolar proteins such as PTRF (Cavin-1, Polymerase I and Transcript Release Factor), SDPR (Cavin-2, Serum Deprivation Response protein) and MURC (Cavin-4, MUscle Related Coiled-coil protein). Caveolae, considered as a subset of lipid rafts, are characterized by their own lipid and protein composition. Lipid rafts contain small clusters of GPI-anchor proteins, also increased in FSHD atrophic myotubes, such as Thy1 (Thy-1 membrane glycoprotein). Some proteins of the membrane repair complex were also deregulated such as AHNAK (Neuroblast differentiation-associated protein), MG53 (Mitsugumin 53) known to interact with PTRF, Dysferlin and CAV33. We have begun to validate these deregulations by western blot and immunofluorescence but this study would require primary myoblasts which are in limited supply. We decided in a first approach to use human immortalized myoblast clones derived from a mosaïc individual4. We received 3 clones (kindly provided by S. Van der Maarel and V. Mouly, Institute of Myology, Paris): a control line (54-6), and 2 lines expressing different amounts of DUX4 mRNA (54-A5 and 54-12). We have grown these cells and differentiated them for 4 days with either of two differentiation media. The first one contained a reduced amount of fetal bovine serum (2%) and the other one was complemented with insulin and apotransferrin. The cells were seeded at 2,5x105 cells/dish using 35mm matrigel-coated dish. At day 4 after induction of differentiation, cells were immunostained with an anti-troponinT (a differentiation marker) antibody and nuclei labelled with DAPI. The fusion index (MFI) was evaluated in ten fields per culture and the width of the myotubes was mesured at their largest place. Depending on the clone and the medium, we have observed distinct myotube phenotypes. The 54-12 myotubes present an atrophic phenotype and the 54-A5 myotubes presented many clusters of nuclei in the differentiating medium with insulin/apotransferrin as observed in disorganized primary myotubes.
Thus, we could reproduce the two phenotypes typical of FSHD primary myotubes and this cell model is being used for the validation of the deregulated proteins identified in primary myotubes.
