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The defective gene responsible for Duchenne muscular dystrophy in humans and the dystrophic condition in mdx mice results in a lack of dystrophin at first thought to be localized to the triads, but more recently found on the cytoplasmic side of the sarcolemma of skeletal muscle fibres. Because the total calcium content of dystrophic fibres is significantly raised, we have compared the intracellular free calcium concentration [( Ca2+]i) in skeletal muscle in normal and mdx mice. We find that [Ca2+]i is markedly elevated in mdx muscle fibres compared with normal fibres, both at rest and during stimulation. By measuring protein degradation rates and manipulating [Ca2+]i, we have been able to demonstrate directly that the elevation of [Ca2+]i in mdx fibres results in an enhanced net degradation of muscle proteins.

Elevated free Ca2+ concentrations found in adult dystrophic muscle fibers result in enhanced protein degradation. Since the difference in concentrations may reflect differences in entry, Ca2+ leak channels in cultures of normal and Duchenne human myotubes, and normal and mdx murine myotubes, have been identified and characterized. The open probability of leak channels is markedly increased in dystrophic myotubes. Other channel properties, such as mean open times, single channel conductance, ion selectivity, and behavior in the presence of pharmacological agents, were similar among myotube types. Compared to the Ca2+ concentrations in normal human and normal mouse myotubes, intracellular resting free Ca2+ concentrations ([Ca2+]i) in myotubes of Duchenne and mdx origin were significantly higher at a time when dystrophin is first expressed in normal tissue. Taken together, these findings suggest that the increased open probability of Ca2+ leak channels contributes to the elevated free intracellular Ca2+ concentration in Duchenne human and mdx mouse myotubes.

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Abstract. We examined pathways which might result in the elevated resting free calcium ([Cal+] i) levels observed in dystrophic mouse (mdx) skeletal muscle fibers and myotubes and human Duchenne muscular dystrophy myotubes . We found that mdx fibers, loaded with the calcium indicator fura-2, were less able to regulate [Cal+]i levels in the region near the sarcolemma. Increased calcium influx or decreased efflux could lead to elevated [Cal+]i levels . Calcium transient decay times were identical in normal and mdx fibers if resting [Ca2+]; levels were similar, suggesting that calciumsequestering mechanisms are not altered in dystrophic muscle, but are slowed by the higher resting [Ca2 +] i . The defect appears to be specific for calcium since resting free sodium levels and sodium in-T HE gene product missing in Duchenne muscular dystrophy phy (DMD)' is dystrophin (Hoffman et al., 1987a,b), a 400-kD protein which subsequently has been localized to the sarcolemma of skeletal muscle (Sugita et al ., 1987; Zubryzycka-Gaarn et al ., 1988) . In the dystrophic mouse (mdx), muscle degeneration begins shortly after birth as a result of a mutation of the X chromosome (Bulfield et al., 1984) . As in the human, this mutation results in a lack ofdystrophin expression in muscle (Hoffman et al., 1987a,b) . Dystrophin has some sequence homology with cytoskeletal proteins and binds to several integral membrane glycoproteins (Campbell and Kahl, 1989 ;Ohlendieck et al ., 1991), but the function of dystrophin remains unknown .The location of dystrophin, and the finding that the total calcium content of dystrophic muscle is increased early in the disease (Bertorini et al., 1982), support a theory of altered calcium ion regulation in DMD, perhaps as the result of a defective sarcolemma (Duncan, 1978). Further support for this theory has come from studies showing that resting intracellular free calcium ([Caz+] i) levels are elevated in mdx muscle fibers (Turner et al., 1988;Williams et al ., 1990) and in cultured dystrophic human and mouse muscle flux rates in the absence of Na+/K±ATPase activity were identical in normal and dystrophic cells when measured with sodium-binding benzofuran isophthalate. Calcium leak channels, whose opening probabilities (Po) were voltage independent, could be the major calcium influx pathway at rest. We have shown previously that calcium leak channel Po is significantly higher in dystrophic myotubes . These leak channels were selective for calcium over sodium under physiological conditions. Agents that increased leak channel activity also increased [Ca 2+], in fibers and myotubes . These results suggest that increased calcium influx, as a result of increased leak channel activity, could result in the elevated [Ca2+] i in dystrophic muscle .