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"Goldin" : a triton-insoluble protein specifically upregulated in MDX skeletal muscle McCutcheon, Krista Maureen


Human Duchenne Muscular Dystrophy (DMD) is an X-linked recessive disorder with progressive muscle degeneration leading to death in the second decade of life. The pathogenesis of this disorder and its genetic correlate in the mutant mdx mouse, have been linked to the absence of dystrophin (Hoffman et al, 1987a; Sincinske, 1989), a cytoskeletal protein that makes up 5% of the membrane cytoskeleton of skeletal muscle (Ohlendieck and Campbell, 1991a). Dystrophin functions as part of a transmembrane complex (the dystrophin glycoprotein complex, or DGC) connecting the actin cytoskeleton to the extracellular matrix (reviewed by Matsumura and Campbell, 1994). The importance of dystrophin to the survival of skeletal muscle is emphasized by the necrosis of DMD and mdx muscle. However, whereas DMD muscle progressively degenerates, the mdx mouse experiences only transient muscle necrosis, lasting from 3-4 weeks of age (Torres and Duchen, 1987). The brief necrotic period in mdx skeletal muscle has led researchers to expect significant modifications in successfully regenerated mdx skeletal muscle. In particular, research interest has focused on changes that could compensate for the absence of dystrophin in the cytoskeletal matrix. However, the only modifications that have been directly correlated to the success of mdx regeneration are the upregulation of talin and vinculin in the subsarcolemmal cytoskeleton of the myotendinous junction (Law et al., 1994). The fact that dystrophin is localized at the sarcolemma, including all of its regional variations (myotendinous junctions, triads, neuromuscular junctions and acetylcholine receptor clusters) (Zhao etal, 1992; Hoffman et al, 1987b; Knudson et al, 1988; Dmytrenko et al, 1993) suggests that additional, perhaps more extensive cytoskeletal adaptations might occur in mdx myofibers To find these predicted alterations in mdx skeletal muscle, the total cytoskeletal protein composition of membrane vesicles normally enriched in dystrophin from normal and mdx adult soleus (SOL), extensor digitorum longus (EDL) and diaphragm (DIA) were compared by silver staining on 6.5% reducing SDS-PAGE. Total cytoskeletal protein was isolated based on the known insolubility of cytoskeletal matrices (including dystrophin) in the presence of relatively high concentrations of non-ionic detergents (Yu et al, 1973; Ohlendieck and Campbell, 1991a). It was of interest to include a comparison of the SOL, EDL and DIA in this study, because of the possibility that the reported differences in pathology between these skeletal muscles (Stedman et al, 1991; Dupont-Versteegden and McCarter, 1992; Louboutin etal, 1993) would be reflected by variations in their cytoskeletal adaptations. Using this biochemical approach to detect cytoskeletal changes in mdx skeletal muscle, novel alterations in two related and as yet unidentified bands (here collectively called "goldin") have been discovered in the Triton X-100 insoluble residue of a 142 000 x g muscle membrane fraction normally enriched in dystrophin. In particular, whereas normal skeletal muscles were seen to express ~245 kd and ~230 kd goldin molecules, the ~245 kd band became the predominant species in mdx at peak and post-regenerative ages. The three skeletal muscles SOL, EDL and DIA showed approximately equivalent changes in the expression of goldin. Appropriately, mdx tissues known to be unaffected by the absence of dystrophin (Torres and Duchen, 1987), such as cardiac muscle and liver and lung showed no change in the expression of goldin. The biochemical characterization of goldin's solubility, staining characteristics, protease resistance, tissue and species occurrence and variability has distinguished it from known proteins. Furthermore, it is possible that goldin is a universal cytoskeletal protein that can be tailored qualitatively and quantitatively to the specific and dynamic needs of cells. In the future, it will be important to determine the role of the ~245 kd goldin molecule in mdx skeletal muscle survival. To this end, the identity and cellular distribution of goldin should be determined and its expression correlated to other cytoskeletal proteins known to be affected in mdx and DMD skeletal muscle. Understanding the role of goldin in muscle regeneration may prove important to future avenues of DMD therapy.

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