The Core Protein to Myogenesis NFAT5 Forms Aggresomes in Normal and Duchenne Muscular Dystrophy Myotubes Exposed to Cell Stressors

source: American Academy of Neurology

year: 2016

authors: Sandrine Herbelet, Elly De Vlieghere, Gert Van Peer, Boel De Paepe, Eline Nys, Laurens Weynants, Jo Vandesompele, Olivier De Wever, Jens Schmidt, Jan De Bleecker

Objective:
The impact of chronic inflammation on muscle cell regeneration in idiopathic inflammatory myopathies (IIM) and Duchenne muscular dystrophy (DMD) was assessed by studying nuclear factor of activated T-cells 5 (NFAT5), an essential factor for myogenesis.

Background:
In virtually all cells, NFAT5 is responsive to stress stimuli, such as cytokines or osmotic stress, leading to its nuclear translocation and production of organic osmolytes (e.g. taurine) and cytokines TNF-α and LTβ. In DMD, lack of dystrophin induces enhanced Ca2+ and Na+ entry in the myocyte, exposing the cell to cellular osmotic stress.

Design/Methods:
This study was performed using immunofluorescence microscopy (IF), Western-blotting (WB), qPCR, confocal microscopy (CM) and cell cultures. Statistical analysis was performed using IBM SPPS 22.0.

Results:
By IF, in DMD patients’ muscle biopsies, NFAT5 was absent on the muscle fiber surface and more expressed in the myonuclei and perinuclear region than in normal controls. In PM and DM, increased myonuclear and perinuclear staining was observed, very discrete muscle fiber staining could be observed in some patients. In IBM, NFAT5 myonuclear staining and variable muscle fiber surface staining was present without perinuclear staining. These observations were validated by means of WB and CM. In human myotubes from non-myopathic and DMD patients, exposure to pro-inflammatory cytokines IL-1β, TNF-α and IFN-γ or hyperosmolar NaCl induced NFAT5 upregulation as reflected by IF, WB, qPCR and upregulation of mRNA of taurine and TNF. Under these stress conditions, surprisingly, we observed the formation of NFAT5 aggresomes which we could also retrieve in DMD, PM and DM biopsies by IF and CM. In IBM, NFAT5 aggregates were located inside the nuclear compartment.

Conclusions:
Our data suggest that NFAT5 becomes upregulated and misfolded in skeletal muscle cells exposed to pro-inflammatory cell stress conditions.