Facioscapulohumeral muscular dystrophy (FSHD) is a progressive muscle disorder 
linked to a contraction of the D4Z4 repeat array in the 4q35 subtelomeric 
region. This deletion induces epigenetic modifications that affect the 
expression of several genes located in the vicinity. In each D4Z4 element, we 
identified the double homeobox 4 (DUX4) gene. DUX4 expresses a transcription 
factor that plays a major role in the development of FSHD through the initiation 
of a large gene dysregulation cascade that causes myogenic differentiation 
defects, atrophy and reduced response to oxidative stress. Because miRNAs 
variably affect mRNA expression, proteomic approaches are required to define the 
dysregulated pathways in FSHD. In this study, we optimized a differential 
isotope protein labeling (ICPL) method combined with shotgun proteomic analysis 
using a gel-free system (2DLC-MS/MS) to study FSHD myotubes. Primary CD56(+) 
FSHD myoblasts were found to fuse into myotubes presenting various proportions 
of an atrophic or a disorganized phenotype. To better understand the FSHD 
myogenic defect, our improved proteomic procedure was used to compare 
predominantly atrophic or disorganized myotubes to the same matching healthy 
control. FSHD atrophic myotubes presented decreased structural and contractile 
muscle components. This phenotype suggests the occurrence of atrophy-associated 
proteolysis that likely results from the DUX4-mediated gene dysregulation 
cascade. The skeletal muscle myosin isoforms were decreased while non-muscle 
myosin complexes were more abundant. In FSHD disorganized myotubes, myosin 
isoforms were not reduced, and increased proteins were mostly involved in 
microtubule network organization and myofibrillar remodeling. A common feature 
of both FSHD myotube phenotypes was the disturbance of several caveolar 
proteins, such as PTRF and MURC. Taken together, our data suggest changes in 
trafficking and in the membrane microdomains of FSHD myotubes. Finally, the 
adjustment of a nuclear fractionation compatible with mass spectrometry allowed 
us to highlight alterations of proteins involved in mRNA processing and 
stability.