Dystrophin expression in myotubes formed by the fusion of normal and dystrophic myoblasts

Abstract

Mdx mouse dystrophy is characterized by the absence in the muscle cytoplasmic membrane of a high molecular weight protein called dystrophin. A possible avenue for treatment of muscular dystrophies is to inject normal myoblasts in a dystrophic muscle to form hybrid muscle fibers. Hybrid myotubes were formed in vitro by the fusion of normal rat and dystrophic mouse (mdx) myoblasts. Staining with Hoechst dye 33258 permitted the clear distinction of mouse and rat nuclei. Immunostaining demonstrated that dystrophin was present over the entire membrane of all hybrid myotubes even when nuclei ratio normal/dystrophic was low.

Heterokaryon myotubes with normal mouse and Duchenne nuclei exhibit sarcolemmal dystrophin staining and efficient intracellular free calcium control.

“…These results showed that dystrophin was expressed throughout the myotube in all mouse-human heterokaryons as has been reported for heterokaryons between mdx mice and normal rat myoblasts (Huard et al, 1991). Heterokaryons with normal mouse nuclei and Duchenne nuclei expressed dystrophin over the areas occupied by the Duchenne nuclei.…”

“…A stock concentration of Hoechst 33342 (1 mg/ml) was diluted 1/1000 with rodent saline solution and incubated with myotubes for 10 min at 23°C. Mouse nuclei have punctate chromatin staining and human nuclei appeared smooth in fluorescence when excited at 385 nm (Blau et al, 1983;Huard et al, 1991).…”

“…Dystrophin, the missing gene product in Duchenne muscular dystrophy (Hoffman et al, 1987), is normally expressed on the cytoplasmic side of the muscle fiber sarcolemma Zubrzycka-Gaarn et al, 1988;Carpenter et al, 1990;Byers et al, 1991) and is also located at the plasma membrane in myotubes formed shortly after myoblast fusion Klamut et al, 1989;Huard et al, 1991). Dystrophin is a 125-nm rod-like protein (427 kDa, MW) with four domains and shares sequence homology with several cytoskeletal proteins (Koenig et al, 1988).…”

In Vivo Fusion of Circulating Fluorescent Cells with Dystrophin-Deficient Myofibers Results in Extensive Sarcoplasmic Fluorescence Expression but Limited Dystrophin Sarcolemmal Expression

“…22 Hybrid myotubes formed in vitro by the fusion of normal rat and dystrophic mdx mouse myoblasts showed that dystrophin was present over the entire membrane of all hybrid myotubes even when nuclei ratio normal/dystrophic was low (as low as 1 of 12). 22 In vivo transplantation of normal myoblasts into mdx mouse muscle 23 in which myogenic cells fuse with pre-existing fibers, showed that the dystrophin nuclear domain is ϳ300 to 400 m. 24,25 These results are very similar to those we observed after BM transplantation (116 m). In a previous study using direct myoblast injection in mdx muscle, the dystrophin domain was shown to be twofold to threefold smaller than that of soluble cytoplasmic ␤-galactosidase used as a reporter gene.…”

Contractile properties of skinned muscle fibres from young and adult normal and dystrophic (mdx) mice.

“…If the contractile properties of dystrophic muscle fibres are not compromised by the lack of dystrophin as we have shown, but rather non-specifically by the degeneration/regeneration processes, then procedures aimed at halting or reversing these deleterious cycles may represent sufficient treatment strategy. For example, the implantation of normal myoblasts into dystrophic muscle results in mosaic fibres possessing normal and dystrophic nuclei (Law, Goodwin & Wang, 1988;Karpati, Pouliot, Carpenter & Holland, 1989;Partridge, Morgan, Coulton, Hoffman & Kunkel, 1989;Huard, Labrecque, Dansareau, Robitaille & Tremblay, 1991), which can express dystrophin, and potentially reduce any susceptibility of the muscle fibre membrane to suffer stressinduced injury. If such treatments are carried out before significant deterioration in muscle function is detected then it is apparent from the results presented here, and in our related studies of muscle function in other dystrophies (Fink et al 1986(Fink et al , 1990Head et al 1990) that muscle function will be preserved at near-normal levels.…”