HETEROKARYON MYOTUBES WITH NORMAL MOUSE AND DUCHENNE NUCLEI EXHIBIT SARCOLEMMAL DYSTROPHIN STAINING AND EFFICIENT INTRACELLULAR FREE CALCIUM CONTROL

Duchenne and mdx muscle tissues lack dystrophin where it normally inte racts 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 my otubes. We fused Duchenne human and normal mouse myoblasts and identif ied heterokaryon myotubes by Hoechst 33342 staining to measure the deg ree to which dystrophin introduced by normal nuclei could incorporate throughout the myotube at the sarcolemma and restore normal calcium ho meostasis. Dystrophin expression in myotubes was determined by immunof luorescence and confocal laser scanning microscopy. Dystrophin was exp ressed at the sarcolemma in normal mouse and heterokaryon myotubes, bu t not in Duchenne myotubes. In heterokaryons, extensive dystrophin loc alization occurred at the sarcolemma even where only Duchenne nuclei w ere present, indicating that dystrophin does not exhibit nuclear domai ns. Heterokaryon, normal mouse and Duchenne myotube [Ca2+]i was measur ed using fura-2 and fluorescence ratio imaging. Heterokaryon and norma l mouse myotubes were found to maintain similar levels of [Ca2+]i. In contrast, Duchenne myotubes had significantly higher [Ca2+]i (p < 0.00 1). Furthermore, the ability of heterokaryons to maintain normal [Ca2]i did not depend on greater numbers of normal nuclei than Duchenne be ing present in the myotube. These results support the view that dystro phin expression in heterokaryons allows for efficient control of [Ca2]i.