Unusual organization of desmin intermediate filaments in muscular dysgenesis and TTX-treated myotubes

Cytoskeletal intermediate filaments were studied in muscular dysgenesis (mdg) and tetrodotoxin-treated inactive mouse embryo muscle cultures during myofibrillogenesis. Both muscular dysgenesis and tetrodotoxin-treated muscles are characterized in vitro by a total lack of contractile activity and an abnormal development of myofibrils. We studied the organization of the microtubule and intermediate filament networks with immunofluorescence, using anti-tubulin, anti-vimentin, and anti-desmin antibodies during normal and mdg/mdg myogenesis in vitro. Mdg/mdg myotubes present a heterogeneous microtubule network with scattered areas of decreased microtubule density. At the myoblast stage, cells expressed both vimentin and desmin. After fusion only desmin expression is revealed. In mutant myotubes the desmin network remains in a diffuse position and does not reorganize itself transversely, as it does during normal myogenesis. The absence of a mature organization of the desmin network in mdg/mdg myotubes is accompanied by a lack of organization of myofibrils. The role of muscle activity in the organization of myofibrils and desmin filaments was tested in two ways: (i) mdg/mdg myotubes were rendered active by coculturing with normal spinal cord cells, and (ii) normal myotubes were treated with tetrodotoxin (TTX) to suppress contractions. Mdg/mdg innervated myotubes showed cross-striated myofibrils, whereas desmin filaments remained diffuse. TTX-treated myotubes possessed disorganized myofibrils and a very unusual pattern of distribution of desmin: intensively stained desmin aggregates were superimposed upon the diffuse network. We conclude, on the basis of these results, that myofibrillar organization does not directly involve intermediate filaments but does need contractile activity.