FSHD Myotubes with Different Phenotypes Exhibit Distinct Proteomes

Abstract

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.

Antisense Oligonucleotides Used to Target the DUX4 mRNA as Therapeutic Approaches in FaciosScapuloHumeral Muscular Dystrophy (FSHD)

“…Each culture presents a mix of either myotube phenotype in different proportions [65,66]. We selected two FSHD primary myoblast lines that were previously shown to mostly differentiate into myotubes with either the atrophic (aFSHD) or disorganized (dFSHD) phenotype.…”

“…Primary human myoblasts were derived from muscle biopsies performed according to the current ethical and legislative rules of France, and written informed consent was obtained from all subjects, as directed by the ethical committee of the Centre Hospitalier Universitaire (CHU) Arnaud de Villeneuve (Montpellier, France) [65,66]. In addition, the use of this material was approved by the ethics committee of the University of Mons (ref # A901) and the ethics committee of ULB-Erasme (Brussels ref #B2011/003 and #P2015/516).…”

“…Primary human myoblasts from an unaffected control and a patient with FSHD were isolated from muscle biopsies, purified and established as described previously [65,66]. The myoblasts were grown in 35 mm collagen-coated dishes (Ywaki, Tokyo, Japan) in DMEM with 4.5 g/L glucose and l -glutamine (Lonza, Verviers, Belgium) as well as gentamycin (50 µg/mL, Sigma-Aldrich, Gillingham, UK), 10% fetal bovine serum (Invitrogen/Thermo-Fisher Scientific, Waltham, MA, USA ), and 1% Ultroser G (Pall BioSepra, Cergy-St-Christophe, France) at 37 °C under 5% CO 2 .…”

Ret function in muscle stem cells points to tyrosine kinase inhibitor therapy for facioscapulohumeral muscular dystrophy

“…However, Ret is expressed in the indirect flight muscles of Drosophila , and overexpression of constitutively active Ret (RET MEN2B ) in muscle via a MEF2-GAL4 driver causes increased muscle fibre size, irregular myofibrils and abnormal actin deposits (Klein et al, 2014). Interestingly, irregular myotube morphology is often reported in FSHD patient-derived cultures (Barro et al, 2010; Tassin et al, 2012). As we found that Ret was activated by DUX4 in murine satellite cells, it becomes a potential candidate for contributing to DUX4-induced myopathy.…”

“…Analysis of myoblasts from FSHD patients has revealed direct functional impairments, including increased susceptibility to oxidative stress (Winokur et al, 2003a, 2003b; Celegato et al, 2006; Macaione et al, 2007; Barro et al, 2010), up-regulation of apoptotic markers (Winokur et al, 2003a, 2003b; Sandri et al, 2001; Vanderplanck et al, 2011; Laoudj-Chenivesse et al, 2005) and repression of MYOD and MYOD-target genes (Winokur et al, 2003b; Celegato et al, 2006). Additionally, myogenic differentiation results in myotubes with either an atrophic, or hypertrophic and highly disorganised morphology (Barro et al, 2010; Vanderplanck et al, 2011; Tassin et al, 2012; Ansseau et al, 2009). Defects in myoblast function are likely to result in impaired muscle maintenance, directly contributing to clinical symptoms (Morgan and Zammit, 2010).…”