Our findings in the DMD human and the mdx mouse indicate that the absence of dystrophin in Duchenne-type muscular dystrophy results in elevated intracellular free calcium. Therefore, we propose to characterize the calcium leak (calcium background) channels in normal human and mouse skeletal myotubes in culture and compare them to the calcium leak channels in dystrophic DMD human and mdx mouse myotubes. In parallel experiments we will examine the degree of regulation of intracellular free calcium in response to levels of external calcium as the myotubes differentiate from populations of the respective myoblasts. In addition to the characterizations of channels and calcium regulation after myotube formation we will follow both channel properties and calcium regulation as the myoblasts undergo fusion, differentiate and develop as myotubes, over a period of 2-3 weeks in culture. By using heterocaryons formed by fusion of mdx mouse and normal rat myoblasts we will observe degree of rescue of normal channel properties and correlate degree of rescue with number of and proximity to fused normal nuclei. We will also study the restoration of normal regulation of channel properties by the expression of a full-length dystrophin cDNA in dystrophic mouse myotube cell lines. This would be a critical step in developing gene therapy for muscular dystrophy. We will extend these studies to cardiac myocytes since that tissue also expresses dystrophin. Unlike skeletal muscle, cardiac muscle preparations of RNA have been successfully used to express functional calcium channels in Xenopus oocytes, therefore we will use total RNA from cardiac tissue to develop an assay in Xenopus oocytes for the expression of calcium leak channels.
| Hopf, F W; Reddy, P; Hong, J et al. (1996) A capacitative calcium current in cultured skeletal muscle cells is mediated by the calcium-specific leak channel and inhibited by dihydropyridine compounds. J Biol Chem 271:22358-67 |
| Bi, G Q; Alderton, J M; Steinhardt, R A (1995) Calcium-regulated exocytosis is required for cell membrane resealing. J Cell Biol 131:1747-58 |
| Steinhardt, R A; Bi, G; Alderton, J M (1994) Cell membrane resealing by a vesicular mechanism similar to neurotransmitter release. Science 263:390-3 |
| Denetclaw Jr, W F; Bi, G; Pham, D V et al. (1993) Heterokaryon myotubes with normal mouse and Duchenne nuclei exhibit sarcolemmal dystrophin staining and efficient intracellular free calcium control. Mol Biol Cell 4:963-72 |
