Skip to main content
PMC home page
Biochemical Journal logoLink to Biochemical Journal
. 2004 Apr 15;379(Pt 2):479–488. doi: 10.1042/BJ20031311

Drastic reduction of sarcalumenin in Dp427 (dystrophin of 427 kDa)-deficient fibres indicates that abnormal calcium handling plays a key role in muscular dystrophy.

Paul Dowling 1, Philip Doran 1, Kay Ohlendieck 1
PMCID: PMC1224066  PMID: 14678011

Abstract

Although the primary abnormality in dystrophin is the underlying cause for mdx (X-chromosome-linked muscular dystrophy), abnormal Ca2+ handling after sarcolemmal microrupturing appears to be the pathophysiological mechanism leading to muscle weakness. To develop novel pharmacological strategies for eliminating Ca2+-dependent proteolysis, it is crucial to determine the fate of Ca2+-handling proteins in dystrophin-deficient fibres. In the present study, we show that a key luminal Ca2+-binding protein SAR (sarcalumenin) is affected in mdx skeletal-muscle fibres. One- and two-dimensional immunoblot analyses revealed the relative expression of the 160 kDa SR (sarcoplasmic reticulum) protein to be approx. 70% lower in mdx fibres when compared with normal skeletal muscles. This drastic reduction in SAR was confirmed by immunofluorescence microscopy. Patchy internal labelling of SAR in dystrophic fibres suggests an abnormal formation of SAR domains. Differential co-immunoprecipitation experiments and chemical cross-linking demonstrated a tight linkage between SAR and the SERCA1 (sarcoplasmic/endoplasmic-reticulum Ca2+-ATPase 1) isoform of the SR Ca2+-ATPase. However, the relative expression of the fast Ca2+ pump was not decreased in dystrophic membrane preparations. This implies that the reduction in SAR and calsequestrin-like proteins plays a central role in the previously reported impairment of Ca2+ buffering in the dystrophic SR [Culligan, Banville, Dowling and Ohlendieck (2002) J. Appl. Physiol. 92, 435-445]. Impaired Ca2+ shuttling between the Ca2+-uptake SERCA units and calsequestrin clusters via SAR, as well as an overall decreased luminal ion-binding capacity, might indirectly amplify the Ca2+-leak-channel-induced increase in cytosolic Ca2+ levels. This confirms the idea that abnormal Ca2+ cycling is involved in Ca2+-induced myonecrosis. Hence, manipulating disturbed Ca2+ handling might represent new modes of abolishing proteolytic degradation in muscular dystrophy.

Full Text

The Full Text of this article is available as a PDF (443.4 KB).

Selected References

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

  1. Alderton J. M., Steinhardt R. A. Calcium influx through calcium leak channels is responsible for the elevated levels of calcium-dependent proteolysis in dystrophic myotubes. J Biol Chem. 2000 Mar 31;275(13):9452–9460. doi: 10.1074/jbc.275.13.9452. [DOI] [PubMed] [Google Scholar]
  2. Alderton J. M., Steinhardt R. A. How calcium influx through calcium leak channels is responsible for the elevated levels of calcium-dependent proteolysis in dystrophic myotubes. Trends Cardiovasc Med. 2000 Aug;10(6):268–272. doi: 10.1016/s1050-1738(00)00075-x. [DOI] [PubMed] [Google Scholar]
  3. Badalamente M. A., Stracher A. Delay of muscle degeneration and necrosis in mdx mice by calpain inhibition. Muscle Nerve. 2000 Jan;23(1):106–111. doi: 10.1002/(sici)1097-4598(200001)23:1<106::aid-mus14>3.0.co;2-d. [DOI] [PubMed] [Google Scholar]
  4. Berchtold M. W., Brinkmeier H., Müntener M. Calcium ion in skeletal muscle: its crucial role for muscle function, plasticity, and disease. Physiol Rev. 2000 Jul;80(3):1215–1265. doi: 10.1152/physrev.2000.80.3.1215. [DOI] [PubMed] [Google Scholar]
  5. Berridge Michael J., Bootman Martin D., Roderick H. Llewelyn. Calcium signalling: dynamics, homeostasis and remodelling. Nat Rev Mol Cell Biol. 2003 Jul;4(7):517–529. doi: 10.1038/nrm1155. [DOI] [PubMed] [Google Scholar]
  6. Blake Derek J., Weir Andrew, Newey Sarah E., Davies Kay E. Function and genetics of dystrophin and dystrophin-related proteins in muscle. Physiol Rev. 2002 Apr;82(2):291–329. doi: 10.1152/physrev.00028.2001. [DOI] [PubMed] [Google Scholar]
  7. Bradford M. M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem. 1976 May 7;72:248–254. doi: 10.1006/abio.1976.9999. [DOI] [PubMed] [Google Scholar]
  8. 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]
  9. Campbell K. P., MacLennan D. H., Jorgensen A. O. Staining of the Ca2+-binding proteins, calsequestrin, calmodulin, troponin C, and S-100, with the cationic carbocyanine dye "Stains-all". J Biol Chem. 1983 Sep 25;258(18):11267–11273. [PubMed] [Google Scholar]
  10. Campbell K. P. Three muscular dystrophies: loss of cytoskeleton-extracellular matrix linkage. Cell. 1995 Mar 10;80(5):675–679. doi: 10.1016/0092-8674(95)90344-5. [DOI] [PubMed] [Google Scholar]
  11. Clarke M. S., Khakee R., McNeil P. L. Loss of cytoplasmic basic fibroblast growth factor from physiologically wounded myofibers of normal and dystrophic muscle. J Cell Sci. 1993 Sep;106(Pt 1):121–133. doi: 10.1242/jcs.106.1.121. [DOI] [PubMed] [Google Scholar]
  12. Collet C., Csernoch L., Jacquemond V. Intramembrane charge movement and L-type calcium current in skeletal muscle fibers isolated from control and mdx mice. Biophys J. 2003 Jan;84(1):251–265. doi: 10.1016/S0006-3495(03)74846-2. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Culligan Kevin, Banville Niamh, Dowling Paul, Ohlendieck Kay. Drastic reduction of calsequestrin-like proteins and impaired calcium binding in dystrophic mdx muscle. J Appl Physiol (1985) 2002 Feb;92(2):435–445. doi: 10.1152/japplphysiol.00903.2001. [DOI] [PubMed] [Google Scholar]
  14. Dowling Paul, Culligan Kevin, Ohlendieck Kay. Distal mdx muscle groups exhibiting up-regulation of utrophin and rescue of dystrophin-associated glycoproteins exemplify a protected phenotype in muscular dystrophy. Naturwissenschaften. 2002 Feb;89(2):75–78. doi: 10.1007/s00114-001-0289-4. [DOI] [PubMed] [Google Scholar]
  15. Dowling Paul, Lohan James, Ohlendieck Kay. Comparative analysis of Dp427-deficient mdx tissues shows that the milder dystrophic phenotype of extraocular and toe muscle fibres is associated with a persistent expression of beta-dystroglycan. Eur J Cell Biol. 2003 May;82(5):222–230. doi: 10.1078/0171-9335-00315. [DOI] [PubMed] [Google Scholar]
  16. Dunn M. J., Bradd S. J. Separation and analysis of membrane proteins by SDS-polyacrylamide gel electrophoresis. Methods Mol Biol. 1993;19:203–210. doi: 10.1385/0-89603-236-1:203. [DOI] [PubMed] [Google Scholar]
  17. Emery Alan E. H. The muscular dystrophies. Lancet. 2002 Feb 23;359(9307):687–695. doi: 10.1016/S0140-6736(02)07815-7. [DOI] [PubMed] [Google Scholar]
  18. Froemming G. R., Murray B. E., Ohlendieck K. Self-aggregation of triadin in the sarcoplasmic reticulum of rabbit skeletal muscle. Biochim Biophys Acta. 1999 Apr 14;1418(1):197–205. doi: 10.1016/s0005-2736(99)00024-3. [DOI] [PubMed] [Google Scholar]
  19. Froemming G. R., Ohlendieck K. Native skeletal muscle dihydropyridine receptor exists as a supramolecular triad complex. Cell Mol Life Sci. 2001 Feb;58(2):312–320. doi: 10.1007/PL00013228. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Glover Louise, Quinn Sandra, Ryan Michelle, Pette Dirk, Ohlendieck Kay. Supramolecular calsequestrin complex. Eur J Biochem. 2002 Sep;269(18):4607–4616. doi: 10.1046/j.1432-1033.2002.03160.x. [DOI] [PubMed] [Google Scholar]
  21. Harmon S., Froemming G. R., Leisner E., Pette D., Ohlendieck K. Low-frequency stimulation of fast muscle affects the abundance of Ca(2+)-ATPase but not its oligomeric status. J Appl Physiol (1985) 2001 Jan;90(1):371–379. doi: 10.1152/jappl.2001.90.1.371. [DOI] [PubMed] [Google Scholar]
  22. Hoffman E. P. Muscular dystrophy: identification and use of genes for diagnostics and therapeutics. Arch Pathol Lab Med. 1999 Nov;123(11):1050–1052. doi: 10.5858/1999-123-1050-MD. [DOI] [PubMed] [Google Scholar]
  23. Isfort Robert J. Proteomic analysis of striated muscle. J Chromatogr B Analyt Technol Biomed Life Sci. 2002 May 5;771(1-2):155–165. doi: 10.1016/s1570-0232(02)00056-9. [DOI] [PubMed] [Google Scholar]
  24. Iwata Yuko, Katanosaka Yuki, Arai Yuji, Komamura Kazuo, Miyatake Kunio, Shigekawa Munekazu. A novel mechanism of myocyte degeneration involving the Ca2+-permeable growth factor-regulated channel. J Cell Biol. 2003 Jun 9;161(5):957–967. doi: 10.1083/jcb.200301101. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Kutchai H., Campbell K. P. Calcium transport by sarcoplasmic reticulum of skeletal muscle is inhibited by antibodies against the 53-kilodalton glycoprotein of the sarcoplasmic reticulum membrane. Biochemistry. 1989 May 30;28(11):4830–4839. doi: 10.1021/bi00437a047. [DOI] [PubMed] [Google Scholar]
  26. Leberer E., Charuk J. H., Green N. M., MacLennan D. H. Molecular cloning and expression of cDNA encoding a lumenal calcium binding glycoprotein from sarcoplasmic reticulum. Proc Natl Acad Sci U S A. 1989 Aug;86(16):6047–6051. doi: 10.1073/pnas.86.16.6047. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Leberer E., Timms B. G., Campbell K. P., MacLennan D. H. Purification, calcium binding properties, and ultrastructural localization of the 53,000- and 160,000 (sarcalumenin)-dalton glycoproteins of the sarcoplasmic reticulum. J Biol Chem. 1990 Jun 15;265(17):10118–10124. [PubMed] [Google Scholar]
  28. Mallouk N., Jacquemond V., Allard B. Elevated subsarcolemmal Ca2+ in mdx mouse skeletal muscle fibers detected with Ca2+-activated K+ channels. Proc Natl Acad Sci U S A. 2000 Apr 25;97(9):4950–4955. doi: 10.1073/pnas.97.9.4950. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Melzer W., Herrmann-Frank A., Lüttgau H. C. The role of Ca2+ ions in excitation-contraction coupling of skeletal muscle fibres. Biochim Biophys Acta. 1995 May 8;1241(1):59–116. doi: 10.1016/0304-4157(94)00014-5. [DOI] [PubMed] [Google Scholar]
  30. Monaco A. P., Neve R. L., Colletti-Feener C., Bertelson C. J., Kurnit D. M., Kunkel L. M. Isolation of candidate cDNAs for portions of the Duchenne muscular dystrophy gene. Nature. 1986 Oct 16;323(6089):646–650. doi: 10.1038/323646a0. [DOI] [PubMed] [Google Scholar]
  31. Mulvey Claire, Ohlendieck Kay. Use of continuous-elution gel electrophoresis as a preparative tool for blot overlay analysis. Anal Biochem. 2003 Aug 1;319(1):122–130. doi: 10.1016/s0003-2697(03)00321-x. [DOI] [PubMed] [Google Scholar]
  32. Murray B. E., Ohlendieck K. Cross-linking analysis of the ryanodine receptor and alpha1-dihydropyridine receptor in rabbit skeletal muscle triads. Biochem J. 1997 Jun 1;324(Pt 2):689–696. doi: 10.1042/bj3240689. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Ohkura M., Furukawa K., Fujimori H., Kuruma A., Kawano S., Hiraoka M., Kuniyasu A., Nakayama H., Ohizumi Y. Dual regulation of the skeletal muscle ryanodine receptor by triadin and calsequestrin. Biochemistry. 1998 Sep 15;37(37):12987–12993. doi: 10.1021/bi972803d. [DOI] [PubMed] [Google Scholar]
  34. Ohlendieck K., Briggs F. N., Lee K. F., Wechsler A. W., Campbell K. P. Analysis of excitation-contraction-coupling components in chronically stimulated canine skeletal muscle. Eur J Biochem. 1991 Dec 18;202(3):739–747. doi: 10.1111/j.1432-1033.1991.tb16428.x. [DOI] [PubMed] [Google Scholar]
  35. Ohlendieck K., Campbell K. P. Dystrophin constitutes 5% of membrane cytoskeleton in skeletal muscle. FEBS Lett. 1991 Jun 3;283(2):230–234. doi: 10.1016/0014-5793(91)80595-t. [DOI] [PubMed] [Google Scholar]
  36. Ohlendieck K., Frömming G. R., Murray B. E., Maguire P. B., Leisner E., Traub I., Pette D. Effects of chronic low-frequency stimulation on Ca2+-regulatory membrane proteins in rabbit fast muscle. Pflugers Arch. 1999 Oct;438(5):700–708. doi: 10.1007/s004249900115. [DOI] [PubMed] [Google Scholar]
  37. Ohlendieck K. Towards an understanding of the dystrophin-glycoprotein complex: linkage between the extracellular matrix and the membrane cytoskeleton in muscle fibers. Eur J Cell Biol. 1996 Jan;69(1):1–10. [PubMed] [Google Scholar]
  38. Pandey A., Mann M. Proteomics to study genes and genomes. Nature. 2000 Jun 15;405(6788):837–846. doi: 10.1038/35015709. [DOI] [PubMed] [Google Scholar]
  39. Robert V., Massimino M. L., Tosello V., Marsault R., Cantini M., Sorrentino V., Pozzan T. Alteration in calcium handling at the subcellular level in mdx myotubes. J Biol Chem. 2000 Oct 11;276(7):4647–4651. doi: 10.1074/jbc.M006337200. [DOI] [PubMed] [Google Scholar]
  40. Sanchez J. C., Chiappe D., Converset V., Hoogland C., Binz P. A., Paesano S., Appel R. D., Wang S., Sennitt M., Nolan A. The mouse SWISS-2D PAGE database: a tool for proteomics study of diabetes and obesity. Proteomics. 2001 Jan;1(1):136–163. doi: 10.1002/1615-9861(200101)1:1<136::AID-PROT136>3.0.CO;2-1. [DOI] [PubMed] [Google Scholar]
  41. Shoshan-Barmatz V., Ashley R. H. The structure, function, and cellular regulation of ryanodine-sensitive Ca2+ release channels. Int Rev Cytol. 1998;183:185–270. doi: 10.1016/s0074-7696(08)60145-x. [DOI] [PubMed] [Google Scholar]
  42. Shoshan-Barmatz V., Orr I., Weil S., Meyer H., Varsanyi M., Heilmeyer L. M. The identification of the phosphorylated 150/160-kDa proteins of sarcoplasmic reticulum, their kinase and their association with the ryanodine receptor. Biochim Biophys Acta. 1996 Aug 14;1283(1):89–100. doi: 10.1016/0005-2736(96)00079-x. [DOI] [PubMed] [Google Scholar]
  43. 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]
  44. Spencer Melissa J., Mellgren Ronald L. Overexpression of a calpastatin transgene in mdx muscle reduces dystrophic pathology. Hum Mol Genet. 2002 Oct 1;11(21):2645–2655. doi: 10.1093/hmg/11.21.2645. [DOI] [PubMed] [Google Scholar]
  45. Squire S., Raymackers J. M., Vandebrouck C., Potter A., Tinsley J., Fisher R., Gillis J. M., Davies K. E. Prevention of pathology in mdx mice by expression of utrophin: analysis using an inducible transgenic expression system. Hum Mol Genet. 2002 Dec 15;11(26):3333–3344. doi: 10.1093/hmg/11.26.3333. [DOI] [PubMed] [Google Scholar]
  46. Suk J. Y., Kim Y. S., Park W. J. HRC (histidine-rich Ca2+ binding protein) resides in the lumen of sarcoplasmic reticulum as a multimer. Biochem Biophys Res Commun. 1999 Oct 5;263(3):667–671. doi: 10.1006/bbrc.1999.1432. [DOI] [PubMed] [Google Scholar]
  47. 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]
  48. Vandebrouck Clarisse, Martin Dominique, Colson-Van Schoor Monique, Debaix Huguette, Gailly Philippe. Involvement of TRPC in the abnormal calcium influx observed in dystrophic (mdx) mouse skeletal muscle fibers. J Cell Biol. 2002 Sep 16;158(6):1089–1096. doi: 10.1083/jcb.200203091. [DOI] [PMC free article] [PubMed] [Google Scholar]
  49. Watchko Jon F., O'Day Terrence L., Hoffman Eric P. Functional characteristics of dystrophic skeletal muscle: insights from animal models. J Appl Physiol (1985) 2002 Aug;93(2):407–417. doi: 10.1152/japplphysiol.01242.2001. [DOI] [PubMed] [Google Scholar]

Articles from Biochemical Journal are provided here courtesy of The Biochemical Society

RESOURCES