Abstract :
[en] Facioscapulohumeral muscular dystrophy (FSHD) is linked to chromatin opening and DNA hypomethylation at the D4Z4 repeat array in the 4q35 subtelomeric region. The open chromatin facilitates the expression of the DUX4 gene located in the last D4Z4 unit. The DUX4 protein is a potent transcription factor that initiates a deregulation cascade affecting many genes and that causes the disease1.
We have previously compared the proteome of primary FSHD and control myotubes at day 4 of differentiation2. FSHD myotubes presented a disturbance of two major caveolar proteins: PTRF (cavin-1) and MURC (cavin-4). Caveolae are membrane nanodomains, considered as a subset of lipid rafts, enriched in cholesterol and sphingolipids. They play a major role in signal transduction for many biological processes. Caveolae contain the muscle specific caveolin (caveolin-3) and small clusters of GPI-anchor proteins, also increased in FSHD myotubes. The extracellular protein AHNAK, a member of the dysferlin protein complex, is also slightly increased in FSHD myotubes.
To evaluate the FSHD caveolar defect, we have investigated the expression of the four cavin proteins (PTRF, SDPR, SRBC, MURC) by immunodetection on western-blot and immunofluorescence on myotubes. We have validated the disturbance of PTRF and MURC expression on FSHD primary myoblasts and myotubes2 (kindly provided by Prof. D. Laoudj-Chenivesse, INSERM U1046, Montpellier). Because primary myoblasts are in limited supply we have switched to immortalized control and FSHD cell lines3 (kindly provided by Dr. Ch. Emerson, Wellstone Foundation, Boston) for further studies. We also use mouse myoblast lines with inducible DUX44 (iC2C12-DUX4, kindly provided by Prof. M. Kyba, Univ. Minnesota), to evaluate the correlation between DUX4 expression and caveolar protein disturbance. We have performed these immunodetections on western-blots with myoblast/tube total extracts and on isolated lipid raft extracts. We are also studying the putative relocations of these rafts during differentiation with a lipid raft tracer: FITC-conjugated cholera toxin B subunit.
If caveolar dysfunction was confirmed in FSHD, deregulation of MURC and PTRF that are essential for skeletal muscle membrane stability could lead to deregulation of many cellular processes. In addition, if a link with DUX4 can be demonstrated, these proteins could be used as additional FSHD biomarkers. Identification of such markers and their validation is essential to assess the therapeutic approaches developed by several groups to decrease DUX4 expression such as antisense tools used in our laboratory5 in collaboration with Prof. S. Wilton (Murdoch University, Australia).
