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. 1994 Oct;5(10):1159–1167. doi: 10.1091/mbc.5.10.1159

Myotubes from transgenic mdx mice expressing full-length dystrophin show normal calcium regulation.

W F Denetclaw Jr 1, F W Hopf 1, G A Cox 1, J S Chamberlain 1, R A Steinhardt 1
PMCID: PMC301138  PMID: 7865881

Abstract

A lack of dystrophin results in muscle degeneration in Duchenne muscular dystrophy. Dystrophin-deficient human and mouse muscle cells have higher resting levels of intracellular free calcium ([Ca2+]i) and show a related increase in single-channel open probabilities of calcium leak channels. Elevated [Ca2+]i results in high levels of calcium-dependent proteolysis, which in turn increases calcium leak channel activity. This process could initiate muscle degeneration by further increasing [Ca2+]i and proteolysis in a positive feedback loop. Here, we tested the direct effect of restoration of dystrophin on [Ca2+]i and channel activity in primary myotubes from mdx mice made transgenic for full-length dystrophin. Transgenic mdx mice have been previously shown to have normal dystrophin localization and no muscle degeneration. Fura-2 calcium measurements and single-channel patch recordings showed that resting [Ca2+]i levels and open probabilities of calcium leak channels of transgenic mdx myotubes were similar to normal levels and significantly lower than mdx littermate controls (mdx) that lack dystrophin. Thus, restoration of normal calcium regulation in transgenic mdx mice may underlie the resulting absence of degeneration.

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Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Almers W., Neher E. The Ca signal from fura-2 loaded mast cells depends strongly on the method of dye-loading. FEBS Lett. 1985 Nov 11;192(1):13–18. doi: 10.1016/0014-5793(85)80033-8. [DOI] [PubMed] [Google Scholar]
  2. Bakker A. J., Head S. I., Williams D. A., Stephenson D. G. Ca2+ levels in myotubes grown from the skeletal muscle of dystrophic (mdx) and normal mice. J Physiol. 1993 Jan;460:1–13. doi: 10.1113/jphysiol.1993.sp019455. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Bertorini T. E., Bhattacharya S. K., Palmieri G. M., Chesney C. M., Pifer D., Baker B. Muscle calcium and magnesium content in Duchenne muscular dystrophy. Neurology. 1982 Oct;32(10):1088–1092. doi: 10.1212/wnl.32.10.1088. [DOI] [PubMed] [Google Scholar]
  4. Bulfield G., Siller W. G., Wight P. A., Moore K. J. X chromosome-linked muscular dystrophy (mdx) in the mouse. Proc Natl Acad Sci U S A. 1984 Feb;81(4):1189–1192. doi: 10.1073/pnas.81.4.1189. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Campbell K. P., Kahl S. D. Association of dystrophin and an integral membrane glycoprotein. Nature. 1989 Mar 16;338(6212):259–262. doi: 10.1038/338259a0. [DOI] [PubMed] [Google Scholar]
  6. Carnwath J. W., Shotton D. M. Muscular dystrophy in the mdx mouse: histopathology of the soleus and extensor digitorum longus muscles. J Neurol Sci. 1987 Aug;80(1):39–54. doi: 10.1016/0022-510x(87)90219-x. [DOI] [PubMed] [Google Scholar]
  7. Cox G. A., Cole N. M., Matsumura K., Phelps S. F., Hauschka S. D., Campbell K. P., Faulkner J. A., Chamberlain J. S. Overexpression of dystrophin in transgenic mdx mice eliminates dystrophic symptoms without toxicity. Nature. 1993 Aug 19;364(6439):725–729. doi: 10.1038/364725a0. [DOI] [PubMed] [Google Scholar]
  8. Denetclaw W. F., Jr, Bi G., Pham D. V., Steinhardt R. A. Heterokaryon myotubes with normal mouse and Duchenne nuclei exhibit sarcolemmal dystrophin staining and efficient intracellular free calcium control. Mol Biol Cell. 1993 Sep;4(9):963–972. doi: 10.1091/mbc.4.9.963. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. DiMario J. X., Uzman A., Strohman R. C. Fiber regeneration is not persistent in dystrophic (MDX) mouse skeletal muscle. Dev Biol. 1991 Nov;148(1):314–321. doi: 10.1016/0012-1606(91)90340-9. [DOI] [PubMed] [Google Scholar]
  10. DiMario J., Strohman R. C. Satellite cells from dystrophic (mdx) mouse muscle are stimulated by fibroblast growth factor in vitro. Differentiation. 1988 Nov;39(1):42–49. doi: 10.1111/j.1432-0436.1988.tb00079.x. [DOI] [PubMed] [Google Scholar]
  11. Dunn J. F., Radda G. K. Total ion content of skeletal and cardiac muscle in the mdx mouse dystrophy: Ca2+ is elevated at all ages. J Neurol Sci. 1991 Jun;103(2):226–231. doi: 10.1016/0022-510x(91)90168-7. [DOI] [PubMed] [Google Scholar]
  12. Ervasti J. M., Campbell K. P. A role for the dystrophin-glycoprotein complex as a transmembrane linker between laminin and actin. J Cell Biol. 1993 Aug;122(4):809–823. doi: 10.1083/jcb.122.4.809. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Ervasti J. M., Ohlendieck K., Kahl S. D., Gaver M. G., Campbell K. P. Deficiency of a glycoprotein component of the dystrophin complex in dystrophic muscle. Nature. 1990 May 24;345(6273):315–319. doi: 10.1038/345315a0. [DOI] [PubMed] [Google Scholar]
  14. Fahlke C., Zachar E., Rüdel R. Single-channel recordings of chloride currents in cultured human skeletal muscle. Pflugers Arch. 1992 Jun;421(2-3):108–116. doi: 10.1007/BF00374816. [DOI] [PubMed] [Google Scholar]
  15. Fong P. Y., Turner P. R., Denetclaw W. F., Steinhardt R. A. Increased activity of calcium leak channels in myotubes of Duchenne human and mdx mouse origin. Science. 1990 Nov 2;250(4981):673–676. doi: 10.1126/science.2173137. [DOI] [PubMed] [Google Scholar]
  16. Franco A., Jr, Lansman J. B. Calcium entry through stretch-inactivated ion channels in mdx myotubes. Nature. 1990 Apr 12;344(6267):670–673. doi: 10.1038/344670a0. [DOI] [PubMed] [Google Scholar]
  17. Gailly P., Boland B., Himpens B., Casteels R., Gillis J. M. Critical evaluation of cytosolic calcium determination in resting muscle fibres from normal and dystrophic (mdx) mice. Cell Calcium. 1993 Jun;14(6):473–483. doi: 10.1016/0143-4160(93)90006-r. [DOI] [PubMed] [Google Scholar]
  18. Grynkiewicz G., Poenie M., Tsien R. Y. A new generation of Ca2+ indicators with greatly improved fluorescence properties. J Biol Chem. 1985 Mar 25;260(6):3440–3450. [PubMed] [Google Scholar]
  19. Hamill O. P., Marty A., Neher E., Sakmann B., Sigworth F. J. Improved patch-clamp techniques for high-resolution current recording from cells and cell-free membrane patches. Pflugers Arch. 1981 Aug;391(2):85–100. doi: 10.1007/BF00656997. [DOI] [PubMed] [Google Scholar]
  20. Head S. I. Membrane potential, resting calcium and calcium transients in isolated muscle fibres from normal and dystrophic mice. J Physiol. 1993 Sep;469:11–19. doi: 10.1113/jphysiol.1993.sp019801. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Hescheler J., Trautwein W. Modification of L-type calcium current by intracellularly applied trypsin in guinea-pig ventricular myocytes. J Physiol. 1988 Oct;404:259–274. doi: 10.1113/jphysiol.1988.sp017289. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Hoffman E. P., Brown R. H., Jr, Kunkel L. M. Dystrophin: the protein product of the Duchenne muscular dystrophy locus. Cell. 1987 Dec 24;51(6):919–928. doi: 10.1016/0092-8674(87)90579-4. [DOI] [PubMed] [Google Scholar]
  23. Hoffman E. P., Garcia C. A., Chamberlain J. S., Angelini C., Lupski J. R., Fenwick R. Is the carboxyl-terminus of dystrophin required for membrane association? A novel, severe case of Duchenne muscular dystrophy. Ann Neurol. 1991 Oct;30(4):605–610. doi: 10.1002/ana.410300414. [DOI] [PubMed] [Google Scholar]
  24. Hoffman E. P., Kunkel L. M. Dystrophin abnormalities in Duchenne/Becker muscular dystrophy. Neuron. 1989 Jan;2(1):1019–1029. doi: 10.1016/0896-6273(89)90226-2. [DOI] [PubMed] [Google Scholar]
  25. Hopf F. W., Steinhardt R. A. Regulation of intracellular free calcium in normal and dystrophic mouse cerebellar neurons. Brain Res. 1992 Apr 24;578(1-2):49–54. doi: 10.1016/0006-8993(92)90228-2. [DOI] [PubMed] [Google Scholar]
  26. Ibraghimov-Beskrovnaya O., Ervasti J. M., Leveille C. J., Slaughter C. A., Sernett S. W., Campbell K. P. Primary structure of dystrophin-associated glycoproteins linking dystrophin to the extracellular matrix. Nature. 1992 Feb 20;355(6362):696–702. doi: 10.1038/355696a0. [DOI] [PubMed] [Google Scholar]
  27. Karpati G., Ajdukovic D., Arnold D., Gledhill R. B., Guttmann R., Holland P., Koch P. A., Shoubridge E., Spence D., Vanasse M. Myoblast transfer in Duchenne muscular dystrophy. Ann Neurol. 1993 Jul;34(1):8–17. doi: 10.1002/ana.410340105. [DOI] [PubMed] [Google Scholar]
  28. Kurebayashi N., Ogawa Y. Discrimination of Ca(2+)-ATPase activity of the sarcoplasmic reticulum from actomyosin-type ATPase activity of myofibrils in skinned mammalian skeletal muscle fibres: distinct effects of cyclopiazonic acid on the two ATPase activities. J Muscle Res Cell Motil. 1991 Aug;12(4):355–365. doi: 10.1007/BF01738590. [DOI] [PubMed] [Google Scholar]
  29. López J. R., Briceño L. E., Sánchez V., Horvart D. Myoplasmic (Ca2+) in Duchenne muscular dystrophy patients. Acta Cient Venez. 1987;38(4):503–504. [PubMed] [Google Scholar]
  30. MacLennan P. A., McArdle A., Edwards R. H. Effects of calcium on protein turnover of incubated muscles from mdx mice. Am J Physiol. 1991 Apr;260(4 Pt 1):E594–E598. doi: 10.1152/ajpendo.1991.260.4.E594. [DOI] [PubMed] [Google Scholar]
  31. Malgaroli A., Milani D., Meldolesi J., Pozzan T. Fura-2 measurement of cytosolic free Ca2+ in monolayers and suspensions of various types of animal cells. J Cell Biol. 1987 Nov;105(5):2145–2155. doi: 10.1083/jcb.105.5.2145. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Mastrangeli A., Danel C., Rosenfeld M. A., Stratford-Perricaudet L., Perricaudet M., Pavirani A., Lecocq J. P., Crystal R. G. Diversity of airway epithelial cell targets for in vivo recombinant adenovirus-mediated gene transfer. J Clin Invest. 1993 Jan;91(1):225–234. doi: 10.1172/JCI116175. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Matsumura K., Lee C. C., Caskey C. T., Campbell K. P. Restoration of dystrophin-associated proteins in skeletal muscle of mdx mice transgenic for dystrophin gene. FEBS Lett. 1993 Apr 12;320(3):276–280. doi: 10.1016/0014-5793(93)80602-q. [DOI] [PubMed] [Google Scholar]
  34. Mongini T., Ghigo D., Doriguzzi C., Bussolino F., Pescarmona G., Pollo B., Schiffer D., Bosia A. Free cytoplasmic Ca++ at rest and after cholinergic stimulus is increased in cultured muscle cells from Duchenne muscular dystrophy patients. Neurology. 1988 Mar;38(3):476–480. doi: 10.1212/wnl.38.3.476. [DOI] [PubMed] [Google Scholar]
  35. Negulescu P. A., Machen T. E. Intracellular ion activities and membrane transport in parietal cells measured with fluorescent dyes. Methods Enzymol. 1990;192:38–81. doi: 10.1016/0076-6879(90)92062-i. [DOI] [PubMed] [Google Scholar]
  36. Ohlendieck K., Campbell K. P. Dystrophin-associated proteins are greatly reduced in skeletal muscle from mdx mice. J Cell Biol. 1991 Dec;115(6):1685–1694. doi: 10.1083/jcb.115.6.1685. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Poenie M., Alderton J., Steinhardt R., Tsien R. Calcium rises abruptly and briefly throughout the cell at the onset of anaphase. Science. 1986 Aug 22;233(4766):886–889. doi: 10.1126/science.3755550. [DOI] [PubMed] [Google Scholar]
  38. Pressmar J., Brinkmeier H., Seewald M. J., Naumann T., Rüdel R. Intracellular Ca2+ concentrations are not elevated in resting cultured muscle from Duchenne (DMD) patients and in MDX mouse muscle fibres. Pflugers Arch. 1994 Apr;426(6):499–505. doi: 10.1007/BF00378527. [DOI] [PubMed] [Google Scholar]
  39. Sarabia V., Klip A. Regulation of cytosolic Ca2+ in clonal human muscle cell cultures. Biochem Biophys Res Commun. 1989 Dec 29;165(3):1130–1137. doi: 10.1016/0006-291x(89)92720-4. [DOI] [PubMed] [Google Scholar]
  40. Sicinski P., Geng Y., Ryder-Cook A. S., Barnard E. A., Darlison M. G., Barnard P. J. The molecular basis of muscular dystrophy in the mdx mouse: a point mutation. Science. 1989 Jun 30;244(4912):1578–1580. doi: 10.1126/science.2662404. [DOI] [PubMed] [Google Scholar]
  41. Tsien R. Y., Rink T. J., Poenie M. Measurement of cytosolic free Ca2+ in individual small cells using fluorescence microscopy with dual excitation wavelengths. Cell Calcium. 1985 Apr;6(1-2):145–157. doi: 10.1016/0143-4160(85)90041-7. [DOI] [PubMed] [Google Scholar]
  42. Turner P. R., Fong P. Y., Denetclaw W. F., Steinhardt R. A. Increased calcium influx in dystrophic muscle. J Cell Biol. 1991 Dec;115(6):1701–1712. doi: 10.1083/jcb.115.6.1701. [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. Turner P. R., Schultz R., Ganguly B., Steinhardt R. A. Proteolysis results in altered leak channel kinetics and elevated free calcium in mdx muscle. J Membr Biol. 1993 May;133(3):243–251. doi: 10.1007/BF00232023. [DOI] [PubMed] [Google Scholar]
  44. Turner P. R., Westwood T., Regen C. M., Steinhardt R. A. Increased protein degradation results from elevated free calcium levels found in muscle from mdx mice. Nature. 1988 Oct 20;335(6192):735–738. doi: 10.1038/335735a0. [DOI] [PubMed] [Google Scholar]
  45. Vainzof M., Takata R. I., Passos-Bueno M. R., Pavanello R. C., Zatz M. Is the maintainance of the C-terminus domain of dystrophin enough to ensure a milder Becker muscular dystrophy phenotype? Hum Mol Genet. 1993 Jan;2(1):39–42. doi: 10.1093/hmg/2.1.39. [DOI] [PubMed] [Google Scholar]
  46. Vaughan P. C., French A. S. Non-ligand-activated chloride channels of skeletal muscle and epithelia. Prog Biophys Mol Biol. 1989;54(1):59–79. doi: 10.1016/0079-6107(89)90009-6. [DOI] [PubMed] [Google Scholar]

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