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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Acsadi, G., Dickson, G., Love, D.R., Jani, A., Walsh, F.S., Gurusinghe, A., Wolff, J.A., Davies, K.E. 1991. Human dystrophin expression in mdx mice after intramuscular injection of DNA constructs. Nature 352:815–818
Byers, T.J., Kunkel, L.M., Watkins, S.C. 1991. The subcellular distribution of dystrophin in mouse skeletal, cardiac, and smooth muscle. J. Cell Biol. 115:411–421
Bulfield, G., Siller, W.G., Wight, P.A.L., Moore, K.J. 1984. X chromosome-linked muscular dystrophy (mdx) in the mouse. Proc. Natl. Acad. Sci. USA 81(4): 1189–1192
Campbell, K.P., Kahl, S.D. 1989. Association of dystrophin and an integral membrane glycoprotein. Nature 338:259–262
Dimario, J., Strohman, R.C. 1988. Satellite cells from dystrophic (mdx) mouse muscles are stimulated by fibroblast growth factor in vitro. Differentiation 39:42–49
Duncan, C.J. 1978. Role of intracellular calcium in promoting muscle damage: a strategy for controlling the dystrophic condition. Experientia 34:1531–1535
Fong, P., Turner, P.R., Denetclaw, W.F., Steinhardt, R.A. 1990. Increased activity of calcium leak channels in myotubes of Duchenne human and mdx mouse origin. Science 250:673–676
Franco, A., Jr., Lansman, J.B. 1990a. Calcium entry through stretch-inactivated ion channels in mdx myotubes. Nature 344:670–673
Franco A., Jr., Lansman J.B. 1990b. Stretch-sensitive channels in developing muscle cells from a mouse cell line. J. Physiol. 427:361–380
Furono, K., Goldberg, A.L. 1986. The activation of protein degradation in muscle by Ca22+ or muscle injury does not involve a lysosomal mechanism. Biochem J. 237:859–864
Fulks, R.M., Li, J.B., Goldberg, A.L. 1975. Effects of insulin, glucose, and amino acids on protein turnover in the rat diaphram. J. Biol. Chem. 250:290–298
Grollman, A.P., Jarkosky, Z. 1974. Emetine and related alkaloids. In: Antibiotics: Mode of Action III. J.W. Corcoran, and F.E. Hahn, editors, pp. 424–435. Springer-Verlag, NewYork
Grynkiewicz, G., Poenie, M., Tsien, R.Y. 1985. A new generation of Ca22+ indicators with greatly improved fluorescence properties. J. Biol. Chem. 260 (6):3440–3450
Gussoni, E., Pavlath, G.K., Lanctot, A.M., Sharma, K.R., Miller, R.G., Steinman, L., Blau, H.M. 1992. Normal dystrophin transcripts detected in Duchenne muscular dystrophy patients after myoblast transplantation. Nature 356:435–438
Hamill, O.P., Marty, A., Neher, E., Sakmann, B., Sigworth, F.J. 1981. Improved patch clamp techniques for high-resolution current recording from cells and cell-free membrane patches. Pfluegers Arch. 391:85–100
Higgins, J., Lasslett, Y., Bardsley, R., Buttery, P. 1988. The relation between dietary restriction or clenbuterol treatment on muscle growth and calpain proteinase (EC 184.108.40.206) and calpastatin activities in lambs. Brit. J. Nutrition 60:645–652
Hoffman, E.P., Brown, R.H., Jr., Kunkel, L.M. 1987. Dystrophin: the protein product of the Duchenne muscular dystrophy locus. Cell 51:919–928
Kar, N.C., Pearson, C.M. 1976. A calcium-activated neutral protease in normal and dystrophic human muscle, Clin. Chim. Acta 73:293–297
Kazanietz, M.G., Enero, M.A. 1990. Desensitization of the beta-2 adrenoceptor-mediated vasodilation in rat aorta after prolonged treatment with the beta-2 adrenoceptor agonist clenbuterol. J. Pharm. Exp. Therap. 252:758–64
Lewis, S.A., Clausen, C. 1991. Urinary proteases degrade epithelial sodium channels. J. Membrane Biol. 122:77–88
Libby, P., Goldberg, A.L. 1978. Leupeptin, a protease inhibitor, decreases protein degradation in normal and diseased muscle. Science 199:534–536
MacLennan, P.A., McArdle, A., Edwards, R.H.T. 1991. Effects of calcium on protein turnover of incubated muscles from mdx mice. Am. J. Physiol. 260 (4 Pt 1):E594-E598
Menke, A., Jockusch, H. 1991. Decreased osmotic stability of dystrophin-less muscle cells from the mdx mouse. Nature 349:69–71
McElligott, M.A., Barreto, A., Jr., Chaung, L.Y. 1989. Effect of continuous and intermittent clenbuterol feeding on rat growth rate and muscle. Comp. Biochem. Physiol. C: Comp. Pharmacol. 92:135–138
Mongini, T., Ghigo, D., Doriguzzi, C., Bussolino, F., Pescarmona, G., Polio, B., Schiffer, D., Bosia, A. 1988. Free cytoplasmic Ca2+2+ at rest and after cholinergic stimulus is increased in cultured muscle cells from Duchenne muscular dystrophy patients. Neurology 38(3):476–480
Neerunjun, J.S., Dubowitz, V. 1979. Increased calcium-activated neutral protease activity in muscles of dystrophic hamsters and mice. J. Neurol. Sci. 40:105–111
Ohlendieck, K., Ervasti, J.M., Snook, J.B., Campbell, K.P. 1991. Dystrophin-glycoprotein complex is highly enriched in isolated skeletal muscle sarcolemma. J. Cell Biol. 112(1):135–148
Poenie, M., Alderton, J., Steinhardt, R., Tsien, R. 1986. Calcium rises abruptly and briefly throughout the cell at the onset of anaphase. Science 233:886–889
Porter, G.A., Dmytrenko, G.M., Winkelmann, J.C., Bloch, R.J. 1992. Dystrophin colocalizes with β-spectrin in distinct subsarcolemmal domains in mammalian skeletal muscle. J. Cell Biol. 117 (5):997–1005
Rojas, C.V., Hoffman, E.P. 1991. Recent advances in dystrophin research. Curr. Op. Neurobiol. 1:420–429
Rothwell, N.J., Stock, M.J. 1985. Modification of body composition by clenbuterol in normal and dystrophic (mdx) mice. Biosci. Rep. 5:755–761
Sanchez, V., Lopez, J.R., Briceno, L.E. 1988. Dysfunction of [Ca22+], in Duchenne muscular dystrophy. Biophys. J. 53:438a (Abstr.)
Sugita, H., Arahata, K., Ishiguro, T., Suhara, Y., Tsukahara, T., Ishiura, S., Eguchi, C., Nonaka, I., Ozawa, E. 1987. Negative immunostaining of Duchenne muscular dystrophy and muscle surface membrane with antibody against synthetic peptide fragment predicted from DMD cDNA. Proc. Jap. Acad.[B] 64:37–39
Tischler, M., Desautels, M., Goldberg, A.L. 1982. Does leucine, leucyl-tRNA, or some metabolite of leucine regulate protein synthesis and degradation in skeletal and cardiac muscle. J. Biol. Chem. 257:1613–1621
Turner, P.R., Westwood, T., Regan, C.M., Steinhardt, R.A.. 1988. Increased protein degradation results from elevated free calcium levels found in muscle from mdx mice. Nature 335:735–738
Turner, P.R., Fong, P., Denetclaw, W., Steinhardt, R.A. 1991. Calcium influx in dystrophic muscle. J. Cell Biol. 115(6):1701–1712
Wang, S.Y., Beermann, D.H. 1988. Reduced calcium-dependent proteinase activity in cimaterol-induced muscle hypertrophy in lambs. J. Animal Sci. 66:2545–50
Williams, D.A., Head, S.I., Bakker, A.J., Stephenson, D.G. 1990. Resting calcium concentrations in isolated skeletal muscle fibers of dystrophic mice. J. Physiol. 428:243–256
Zeman, R.J., Kameyama, T., Matsumoto, K., Bernstein, P., Etlinger, J.D. 1985. Regulation of protein degradation in muscle by calcium. Evidence for enhanced nonlysosomal proteolysis associated with elevated cytosolic calcium. J. Biol. Chem. 260:13619–13624
Zubryzycka-Gaarn, E.E., Bulman, D.E., Karpati, G., Burghes, A.H.M., Beifall, B., Klamut, H.J., Talbot, J., Hodges, R.S., Ray, P.N., Worton, R.G. 1988. The Duchenne muscular dystrophy gene product is localized in sarcolemma of human skeletal muscle. Nature 333:466–469
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
- calcium leak channels
- Duchenne muscular dystrophy