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Proteolysis results in altered leak channel kinetics and elevated free calcium in mdx muscle

Summary

Resting free calcium levels ([Ca2−] i ) are elevated in Duchenne human myotubes 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 [Ca22+] i . The [Ca22+] i rise may 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 by leupeptin, a thiol protease inhibitor. In muscle homogenates, leupeptin also abolished calcium-induced increases in protein degradation. When mouse myotubes were cultured in the continuous presence of leupeptin (50 μm), the elevation in mdx resting [Ca22+] i was prevented. Leak channel P o increased with age in mdx myotubes, whereas leupeptin-treated mdx leak channel opening probabilities were always lower or equal to the P o for untreated normal myotubes.

These results indicate that increased leak channel activity in dystrophic muscle results in elevated [Ca22+] i levels, but also suggest that dystrophin does not directly regulate channel activity. Instead the results suggest that proteolysis may be responsible for the altered gating of calcium leak channels. The resultant increased channel P o in turn elevates [Ca22+] i , which further increases proteolytic activity in a positive feedback loop, leading to the eventual necrosis of the muscle fibers.

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Additional information

We acknowledge gratefully support by the NIH (to R.S.), the Muscular Dystrophy Association of America (to R.S.), and the Association Française contre les Myopathies (to P.T.) and private donations. We thank Dr. John Forte for assistance with the tyrosine assays, Janet Alderton and Dr. Wilfred Denetclaw for assistance with the cultures and Dr. Peying Fong for comments on the manuscript.

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Turner, P.R., Schultz, R., Ganguly, B. et al. Proteolysis results in altered leak channel kinetics and elevated free calcium in mdx muscle. J. Membarin Biol. 133, 243–251 (1993). https://doi.org/10.1007/BF00232023

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KeyWords

  • calcium leak channels
  • muscle
  • Duchenne muscular dystrophy
  • mdx
  • myotubes
  • protease