PROTEOLYSIS RESULTS IN ALTERED LEAK CHANNEL KINETICS AND ELEVATED FREE CALCIUM IN MDX MUSCLE

Resting free calcium levels ([Ca2+]i) are elevated in Duchenne human m yotubes and mdx mouse muscle and myotubes which lack the gene product dystrophin at the sarcolemma. Increased net muscle protein degradation has been directly related to this elevated [Ca2+]i. The [Ca+]i rise m ay result from increased calcium influx via leak channels, which have increased opening probabilities (P(o)) in dystrophic cells. Dystrophin , therefore, might directly regulate leak channel activity. In intact mdx soleus muscles, protein degradation was reduced to normal levels b y leupeptin, a thiol protease inhibitor. In muscle homogenates, leupep tin also abolished calcium-induced increases in protein degradation. W hen mouse myotubes were cultured in the continuous presence of leupept in (50 muM), the elevation in mdx resting [Ca2+]i was prevented. Leak channel P(o) increased with age in mdx myotubes, whereas leupeptin-tre ated mdx leak channel opening probabilities were always lower or equal to the P(o) for untreated normal myotubes. These results indicate tha t increased leak channel activity in dystrophic muscle results in elev ated [Ca2+]i levels, but also suggest that dystrophin does not directl y regulate channel activity. Instead the results suggest that proteoly sis may be responsible for the altered gating of calcium leak channels . The resultant increased channel P(o) in turn elevates [Ca2+]i, which further increases proteolytic activity in a positive feedback loop, l eading to the eventual necrosis of the muscle fibers.