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

Abstract

Duchenne and mdx muscle tissues lack dystrophin where it normally interacts with glycoproteins in the sarcolemma. Intracellular free calcium ([Ca2+]i) is elevated in Duchenne and mdx myotubes and is correlated with abnormally active calcium-specific leak channels in dystrophic myotubes. We fused Duchenne human and normal mouse myoblasts and identified heterokaryon myotubes by Hoechst 33342 staining to measure the degree to which dystrophin introduced by normal nuclei could incorporate throughout the myotube at the sarcolemma and restore normal calcium homeostasis. Dystrophin expression in myotubes was determined by immunofluorescence and confocal laser scanning microscopy. Dystrophin was expressed at the sarcolemma in normal mouse and heterokaryon myotubes, but not in Duchenne myotubes. In heterokaryons, extensive dystrophin localization occurred at the sarcolemma even where only Duchenne nuclei were present, indicating that dystrophin does not exhibit nuclear domains. Heterokaryon, normal mouse and Duchenne myotube [Ca2+]i was measured using fura-2 and fluorescence ratio imaging. Heterokaryon and normal mouse myotubes were found to maintain similar levels of [Ca2+]

Lipofection of a cDNA plasmid containing the dystrophin gene lowers intracellular free calcium and calcium leak channel activity in mdx myotubes

“…We thus of the same cultures that were not transfected (Figure 2). Restoration of dystrophin expression therefore improved extend our earlier results from myoblast fusion and transgenic mice, 24,25 and show that liposome-mediated transintracellular calcium regulation in originally dystrophic muscle cells.…”

“…As previously reported in studies using the extrapolated reversal potential were both similar to previously reported values. 12,16 fura-2, 12,13,16,17,24,25 the resting [Ca 2+ ] i here is significantly higher in mdx myotubes than that in age-matched normal…”

“…23 tiation were exposed to pRSVLacZ, a cDNA plasmid that contains the bacterial ␤-galactosidase gene driven by the In earlier reports we examined the effect on calcium regulation in dystrophic cells after the restoration of dysRous sarcoma virus promoter. 27 Lipofection with 10 g of DNA was performed on days 1, 3 and 6 after prolifertrophin expression by fusion of cultured mdx and normal rat myoblasts 24 and in mdx mice transgenic for dystroating cells were replated and induced to fuse by switching to low serum medium. Gene expression was assayed phin.…”

Myotubes from transgenic mdx mice expressing full-length dystrophin show normal calcium regulation.

“…Primary myotube cultures were prepared by isolating satellite cells from hind-limb muscles of 10-week-old adult mice and culturing as previously described (DiMario and Strohman, 1988;Denetclaw et al, 1993). Briefly, myoblasts were proliferated for 5-6 d in Ham's F10 medium with 20% FBS and 2% chick embryo extract.…”

“…In the absence of dystrophin, one observes greater total muscle calcium (Bertorini et al, 1982;Dunn and Radda, 1991), as well as a significant elevation in intracellular free calcium concentrations ([Ca21]i) in isolated myofibers (Lopez et al, 1987;Turner et al, 1988) and ©) 1994 by The American Society for Cell Biology cultured myotubes (Mongini et al, 1988;Sarabia and Klip, 1989;Fong et al, 1990;Turner et al, 1991Turner et al, , 1993Denetclaw et al, 1993;Bakker et al, 1993), although others have not observed differences in resting calcium (Gailly et al, 1993;Head, 1993;Pressmar et al, 1994). However, no difference is observed in calcium resequestration rates after stimulation (Turner et al, 1991), indicating that calcium efflux pathways are not impaired in dystrophic cells, and, instead, an influx pathway may be altered.…”