In the mdx mouse model of Duchenne muscular dystrophy, the lack of dystrophin is associated with increased calcium levels and skeletal muscle myonecrosis. The intrinsic laryngeal muscles (ILM) are protected and do not undergo myonecrosis. We investigated whether this protection is related to an increased expression of calcium-binding proteins, which may protect against the elevated calcium levels seen in dystrophic fibers. The expression of sarcoplasmic-endoplasmic-reticulum Ca2+-ATPase and calsequestrin was examined in ILM and in nonspared limb muscles of control and mdx mice using immunofluorescence and immunoblotting. Dystrophic ILM presented a significant increase in the proteins studied when compared to controls. The increase of Ca2+-handling proteins in dystrophic ILM may permit better maintenance of calcium homeostasis, with the consequent absence of myonecrosis. The results further support the concept that abnormal Ca2+-handling is involved in dystrophinopathies. © 2009 Wiley Periodicals, Inc.395609615Alderton, J.M., Steinhardt, R.A., Calcium influx through calcium leak channels is responsible for the elevated levels of calcium-dependent proteolysis in dystrophic myotubes (2000) Journal of Biological Chemistry, 275 (13), pp. 9452-9460. , DOI 10.1074/jbc.275.13.9452Andrade, F.H., Porter, J.D., Kaminski, H.J., Eye muscle sparing by the muscular dystrophies: Lessons to be learned? (2000) Microsc Res Tech, 48, pp. 192-203Arai, M., Otsu, K., MacLennan, D.H., Periasamy, M., Regulation of sarcoplasmic reticulum gene expression during cardiac and skeletal muscle development (1992) Am J Physiol Cell Physiol, 262, pp. C614-C620Beard, N.A., Laver, D.R., Dulhunty, A.F., Calsequestrin and the calcium release channel of skeletal and cardiac muscle (2004) Progress in Biophysics and Molecular Biology, 85 (1), pp. 33-69. , DOI 10.1016/j.pbiomolbio.2003.07.001, PII S0079610703000774Blank, J.M., Schachat, F., Extraocular and laryngeal muscles exhibit differential amplification of protein involved in calcium homeostasis (1999) Mol Biol Cell, 10, pp. 246ABodensteiner, J.B., Engel, A.G., Intracellular calcium accumulation in Duchenne dystrophy and other myopathies: A study of 567,000 muscle fibers in 114 biopsies (1978) Neurology, 28 (5), pp. 439-446Brandl, C.J., De Leon, S., Martin, D.R., MacLennan, D.H., Adult forms of the Ca2+ ATPase of sarcoplasmic reticulum. Expression in developing skeletal muscle (1978) J Biol Chem, 262, pp. 3768-3774Brennan, T.J., Edmondson, D.G., Olson, E.N., Aberrant regulation of MyoD1 contributes to the partially defective myogenic phenotype of BC3H1 cells (1990) Journal of Cell Biology, 110 (4), pp. 929-937Bulfield, G., Siller, W.G., Wigth, P.A.L., Moore, K.J.X., Chromosome-like muscular dystrophy (mdx) in the mouse (1984) Proc Natl Acad Sci U S A, 81, pp. 1189-1192Carlsen, H., Gundersen, K., Helix-loop-helix transcription factors in electrically active and inactive skeletal muscles (2000) Muscle Nerve, 23, pp. 1374-1380Celio, M.R., Heizmann, C.W., Calcium-binding protein parvalbumin as a neuronal marker (1981) Nature, 293 (5830), pp. 300-302. , DOI 10.1038/293300a0Culligan, K., Banville, N., Dowling, P., Ohlendieck, K., Drastic reduction of calsequestrin-like proteins and impaired calcium binding in dystrophic mdx muscle (2002) Journal of Applied Physiology, 92 (2), pp. 435-445Divet, A., Huchet-Cadiou, C., Sarcoplasmic reticulum function in slow- and fast-twitch skeletal muscles from mdx mice (2002) Eur J Physiol, 444, pp. 634-643Doran, P., Dowling, P., Donoghue, P., Buffini, M., Ohlendieck, K., Reduced expression of regucalcin in young and aged mdx diaphragm indicates abnormal cytosolic calcium handling in dystrophin-deficient msucle (2006) Biochem Biophys Acta, 1764, pp. 773-785Doran, P., Dowling, P., Lohan, J., McDonnell, K., Poetsch, S., Ohlendieck, K., Subproteomics analysis of Ca2+-binding proteins demonstrates decreased calsequestrin expression in dystrophic mouse skeletal muscle (2004) Eur J Biochem, 271, pp. 3943-3952Dowling, P., Doran, P., Ohlendieck, K., Drastic reduction of sarcalumenin in Dp427 (dystrophin of 427 kDa)-deficient fibres indicates that abnormal calcium handling plays a key role in muscular dystrophy (2004) Biochemical Journal, 379 (2), pp. 479-488. , DOI 10.1042/BJ20031311Dowling, P., Lohan, J., Ohlendieck, K., 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 (2003) European Journal of Cell Biology, 82 (5), pp. 222-230. , DOI 10.1078/0171-9335-00315Engel, A.G., Yamamoto, M., Fischbeck, K.H., Muscular dystrophies (1994) Myology, pp. 1133-1187. , Engel AG, Franzini-Armstrong C, editors. New York: McGraw-HillGailly, P., New aspects of calcium signaling in skeletal muscle cells: Implications in Duchenne muscular dystrophy (2002) Biochem Biophys Acta, 1600, pp. 38-44Goding Jr., G.S., Al-Sharif, K.I., McLoon, L.K., Myonuclear addition to uninjured laryngeal myofibers in adult rabbits (2005) Ann Otol Rhinol Laryngol., 114, pp. 552-557Han, R., Grounds, M.D., Bakker, A.J., Measurements of sub-membrane [Ca2+] in adult myofiber and cytosolic [Ca2+] in myotubes from normal and mdx mice using the Ca2+ indicator FFP-18 (2006) Cell Calcium, 40, pp. 299-307Haslett, J.N., Kang, P.B., Han, M., Kho, A., Sanoudou, D., Volinski, J.M., The influence of muscle type and dystrophin deficiency on murine expression profiles (2005) Mamm Genome, 16, pp. 739-748Hodgetts, S., Radley, H., Davies, M., Grounds, M.D., Reduced necrosis of dystrophic muscle by depletion of host neutrophils, or blocking TNF alpha function with Etanercept in mdx mice (2006) Neuromuscular Disorders, 16 (9-10), pp. 591-602. , DOI 10.1016/j.nmd.2006.06.011, PII S0960896606004457Hoffman, E.P., Brown Jr., R.H., Kunkel, L.M., Dystrophin: The protein product of the Duchenne muscular dystrophy locus (1987) Cell, 51 (6), pp. 919-928. , DOI 10.1016/0092-8674(87)90579-4Hoh, J.F.Y., Laryngeal muscle fibre types (2005) Acta Physiologica Scandinavica, 183 (2), pp. 133-149. , DOI 10.1111/j.1365-201X.2004.01402.xKarpati, G., Carpenter, S., Prescott, S., Small-caliber skeletal muscle fibers do not suffer necrosis in mdx mouse dystrophy (1988) Muscle and Nerve, 11 (8), pp. 795-803. , DOI 10.1002/mus.880110802Khurana, T.S., Prendergast, R.A., Alameddine, H.S., Tomé, F.M., Fardeau, M., Arahata, K., Absence of extraocular muscle pathology in Duchenne's muscular dystrophy: Role for calcium homeostasis in extraocular muscle sparing (1995) J Exp Med, 182, pp. 467-474Lucas, C.A., Rughani, A., Hoh, J.F.Y., Expression of extraocular myosin heavy chain in rabbit laryngeal muscle (1995) J Muscle Res Cell Motil, 16, pp. 368-378Lyons, P.R., Slater, C.R., Structure and function of the neuromuscular junction in young adult mdx mice (1991) J Neurocytol, 20, pp. 969-981Marques, M.J., Ferretti, R., Vomero, V.U., Minatel, E., Neto, H.S., Intrinsic laryngeal muscles are spared from myonecrosis in the mdx mouse model of Duchenne muscular dystrophy (2007) Muscle and Nerve, 35 (3), pp. 349-353. , DOI 10.1002/mus.20697Marques, M.J., Pertille, A., Carvalho, C.L.T., Santo Neto, H., Acetylcholine receptor organization at the dystrophic extraocular muscle neuromuscular junction (2007) Anat Rec, 290, pp. 846-854McLoon, L.K., Thorstenson, K.M., Solomon, A., Lewis, M.P., Myogenic precursor cells in craniofacial muscles (2007) Oral Diseases, 13 (2), pp. 134-140. , DOI 10.1111/j.1601-0825.2006.01353.xPan, Y., Zvaritch, E., Tupling, R., Rice, W.J., De Leon, S., Rudnicki, M., Targeted disruption of the ATP2A1 gene encoding the sarco(endo)plasmic reticulum Ca2+ ATPase isoform 1 (SERCA1) impairs diaphragm function and is lethal in neonatal mice (2003) J Biol Chem, 278, pp. 13367-13375Pastoret, C., Sebille, A., mdx mice show progressive weakness and muscle deterioration with age (1995) J Neurol Sci, 129, pp. 97-105Petrof, B.J., Shrager, J.B., Stedman, H.H., Kelly, A.M., Sweeney, H.L., Dystrophin protects the sarcolemma from stresses developed during muscle contraction (1993) Proceedings of the National Academy of Sciences of the United States of America, 90 (8), pp. 3710-3714Porter, J.D., Baker, R.S., Muscles of a different 'color': The unusual properties of the extraocular muscles may predispose or protect them in neurogenic and myogenic disease (1996) Neurology, 46 (1), pp. 30-37Porter Jr., G.A., Makuck, R.F., Rivkees, A.A., Reduction in intracellular calcium levels inhibits myoblast differentiation (2002) J Biol Chem, 277, pp. 28942-28947Porter, J.D., Rafael, J.A., Ragusa, R.J., Brueckner, J.K., Trickett, J.I., Davies, K.E., The sparing of extraocular muscle is lost in mice lacking utrophin and dystrophin (1998) J Cell Sci, 111, pp. 1801-1811Rossi, A.E., Dirksen, R.T., Sarcoplasmic reticulum: The dynamic calcium governor of muscle (2006) Muscle and Nerve, 33 (6), pp. 715-731. , DOI 10.1002/mus.20512Sicinski, 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 (1989) Science, 244 (4912), pp. 1578-1580Stedman, H.H., Sweeney, H.L., Shrager, J.B., Maguire, H.C., Panettieri, R.A., Petrof, B., The mdx mouse diaphragm reproduces the degenerative changes of Duchenne muscular dystrophy (1991) Nature, 352, pp. 536-539Tidball, J.G., Spencer, M.J., Calpains and muscular dystrophies (2000) International Journal of Biochemistry and Cell Biology, 32 (1), pp. 1-5. , DOI 10.1016/S1357-2725(99)00095-3, PII S1357272599000953Torres, L.F., Duchen, L.W., The mutant mdx: Inherited myopathy in the mouse. Morphological studies of nerves, muscle and end-plates (1987) Brain, 110, pp. 269-299Turner, P.R., Fong, P., Denetclaw, W.F., Steinhardt, R.A., Increased calcium influx in dystrophic muscle (1991) Journal of Cell Biology, 115 (6), pp. 1701-1712Vandebrouck, A., Sabourin, J., Rivet, J., Balghi, H., Sebille, S., Kitzis, A., Regulation of capacitative calcium entries by alpha1-syntrophin: Association of TRPC1 with dystrophin complex and the PDZ domain of alpha1-syntrophin (2007) FASEB J, 21, pp. 608-617Whitehead, N.P., Yeung, E.W., Allen, D.G., Muscle damage in mdx (dystrophic) mice: Role of calcium and reactive oxygen species (2006) Clinical and Experimental Pharmacology and Physiology, 33 (7), pp. 657-662. , DOI 10.1111/j.1440-1681.2006.04394.xYoshida, M., Minamisawa, S., Shimura, M., Komazaki, S., Kume, H., Zhang, M., Impaired Ca2+ store functions in skeletal and cardiac muscle cells from sarcalumenin-deficient mice (2005) J Biol Chem, 280, pp. 3500-3506Zador, E., Vangheluwe, P., Wuytack, F., The expression of the neonatal sarcoplasmic reticulum Ca2+ pump (SERCA1b) hints to a role in muscle growth and development (2007) Cell Calcium, 41 (4), pp. 379-388. , DOI 10.1016/j.ceca.2006.08.001, PII S0143416006001631