The lack of dystrophin in mdx mice and in Duchenne muscular dystrophy causes sarcolemmal breakdown and increased calcium influx followed by myonecrosis. We examined whether the calcium channel blockers diltiazem and verapamil protect dystrophic muscles from degeneration. Mdx mice received daily intraperitoneal injections of diltiazem or verapamil for 18 days, followed by removal of the sternomastoid, diaphragm, tibialis anterior, and cardiac muscles. Control mdx mice were injected with saline. Both drugs significantly decreased blood creatine kinase levels. Total calcium content was significantly higher in mdx muscles than in control C57Bl/10. Verapamil and diltiazem reduced total calcium content only in diaphragm and cardiac muscle. Histological analysis showed that diltiazem significantly attenuated myonecrosis in diaphragm. Immunoblots showed a significant increase of calsequestrin and β-dystroglycan levels in some diltiazem-and verapamil-treated muscles. Possible interactions of these drugs with the sarcoplasmic reticulum and sarcolemma may also contribute to the improvement of the dystrophic phenotype. © 2009 Wiley Periodicals, Inc.392167176Barresi, R., Campbell, K.P., Dystroglycan: From biosynthesis to pathogenesis of human disease (2006) J Cell Sci, 119, pp. 199-207Beard, N.A., Laver, D.R., Dulhunty, A.F., Calsequestrin and the calcium release channel of skeletal and cardiac muscle (2004) Prog Biophys Mol Biol, 85, pp. 33-69Beard, N.A., Sakowska, M.M., Dulhunty, A.F., Laver, D.R., Calsequestrin is an inhibitor of skeletal muscle ryanodine receptor calcium release channels (2002) Biophys J, 82, pp. 310-320Bertorini, T.E., Bhattacharya, S.K., Palmieri, G.M., Chesney, C.M., Pifer, D., Baker, B., Muscle calcium and magnesium content in Duchenne muscular dystrophy (1982) Neurology, 32, pp. 1088-1092Bertorini, T.E., Palmieri, G.M.A., Griffin, J.W., Igarashi, M., McGee, J., Brown, R., Effect of chronic treatment with the calcium antagonist diltiazem in Duchenne muscular dystrophy (1988) Neurology, 38, pp. 609-613Bulfield, G., Siller, W.G., Wight, P.A.L., Moore, K.J., X chromosome-linked muscular dystrophy (mdx) in the mouse (1984) Proc Natl Acad Sci U S A, 81, pp. 1189-1192Coulton, G.R., Curtin, N.A., Morgan, J.E., Partridge, T.A., The mdx mouse skeletal muscle myopathy: II. Contractile properties (1988) Neuropathol Appl Neurobiol, 14, pp. 299-314Courdier-Fruth, L., Barman, L., Wettstein, P., Meier, F., Detection of glucocorticoid-like activity in traditional Chinese medicine used for the treatment of Duchenne muscular dystrophy (2003) Neuromuscul Disord, 13, pp. 699-704Culligan, K., Banville, N., Dowling, P., Ohlendieck, K., Drastic reduction of calsequestrin-like proteins and impaired calcium binding in dystrophic mdx muscle (2002) J Appl Physiol, 92, pp. 435-445De Luca, A., Pierno, S., Liantonio, A., Cetrone, M., Camerino, C., Fraysse, B., Enhanced dystrophic progression in mdx mice by exercise and beneficial effects of taurine and insulin-like growth factor-1 (2003) J Pharmacol Exp Ther, 304, pp. 453-463Dehpour, A.R., Mousavizadeth, K., Gerayessh-Nejad, S., Calcium release by diltiazem from isolated sarcoplasmic reticulum of rabbit skeletal muscle (1998) Gen Pharmacol, 31, pp. 463-468Doran, 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 muscle (2006) Biochem Biophys Acta, 1764, pp. 773-785Dowling, 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) Eur J Cell Biol, 82, pp. 222-230Engel AG, Yamamoto M, Fischbeck KH. Dystrophinopathies. In: Engel AG, Franzini-Armstrong C, editors. Myology. New York: McGraw-Hill1994. p 1133-1187Ervasti, J.M., Dystrophin, its interaction with other proteins, and implications for muscular dystrophy (2007) Biochem Biophys Acta, 1772, pp. 108-117Ervasti, J.M., Ohlendieck, K., Kahl, S.D., Gaver, M.G., Campbell, K., Deficiency of a glycoprotein component of the dystrophin complex in dystrophic muscle (1990) Nature, 345, pp. 315-319Fong, 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 (1990) Science, 250, pp. 673-676Franco-Obregon Jr, A., Lansman, J.B., Mechanosensitive ion channels in skeletal muscle from normal and dystrophic mice (1994) J Physiol, 481, pp. 299-309Gramolini, A.O., Barton, E.A., Tinsley, J.M., Ferris, M.J., Cartaud, A., Cartaud, J., Muscle and neural isoform of agrin increase utrophin expression in cultured myotubes via a transcriptional regulatory mechanism (1998) J Biol Chem, 273, pp. 736-743Grounds, M.D., Radley, H.G., Lynch, G.S., Nagaraju, K., De Luca, A., Towards developing standard operating procedures for preclinical testing in the mdx mouse model of Duchenne muscular dystrophy (2008) Neurobiol Dis, 31, pp. 1-19Grounds, M.D., Sorokin, L., White, J., Strength at the extracellular matrix-muscle interface (2005) Scand J Med Sci Sports, 15, pp. 381-391Grounds, M.D., Torrisi, J., Anti-TNFa (Remicade) therapy protects dystrophic skeletal muscle from necrosis (2004) FASEB J, 18, pp. 676-682Hamer, P.W., Mcgeachie, J.M., Davies, M.J., Grounds, M.D., Evans blue dye as an in vivo marker of myofibre damage: Optimising parameters for detecting initial myofibre membrane permeability (2002) J Anat, 200, pp. 69-79Hopf, F.W., Reddy, P., Hong, J., Steinhardt, R.A., A capacitative calcium current in cultured skeletal muscle cells is mediated by the calcium-specific leak channel and inhibited by dihydropyridine compounds (1996) J Biol Chem, 271, pp. 358-367Iwata, Y., Katanosaka, Y., Shijun, Z., Kobayashi, Y., Hanada, H., Shigekawa, M., Protective effects of Ca2+ handling drugs against abnormal Ca2+ homeostasis and cell damage in myopathic skeletal muscle cells (2005) Biochem Pharmacol, 70, pp. 740-751Janis, R.A., Silver, P.J., Triggle, D.J., Drug action and cellular calcium regulation (1987) Adv Drug Res, 16, pp. 309-591Johnson, P.L., Bhattacharya, S.K., Regulation of membrane-mediated chronic muscle degeneration in dystrophic hamsters by calcium-channel blockers - diltiazem, nifedipine and verapamil (1993) J Neurol Sci, 115, pp. 76-90Lefaucheur, J.P., Pastoret, C., Sebille, A., Phenotype of dystrophinopathy in old mdx mice (1995) Anat Rec, 242, pp. 70-76Lefkowitz, D.L., Lefkowitz, S.S., Fascioscapulohumeral muscular dystrophy: A progressive degenerative disease that responds to diltiazem (2005) Med Hypotheses, 65, pp. 716-721Mallouk, N., Jacquemond, V., Allard, B., Elevated subsarcolemmal Ca 2+ in mdx mouse skeletal muscle fibers detected with Ca 2+-activated K+ channels (2000) Proc Natl Acad Sci U S A, 97, pp. 4950-4955Marques, M.J., Luz, M.A.M., Minatel, E., Santo Neto, H., Muscle regeneration in dystrophic mdx mice is enhanced by isosorbide dinitrate (2005) Neurosci Lett, 382, pp. 342-345Marques, M.J., Machado, R.V., Minatel, E., Santo Neto, H., Disodium cromoglycate protects dystrophin-deficient muscle fibers from leakiness: Studies in mdx sternomastoid, diaphragm and tibialis anterior (2008) Muscle Nerve, 37, pp. 61-67Marques, M.J., Matsumura, C.Y., Santo Neto, H., Alterations in the permeability of dystrophic fibers during neuromuscular junction development (2007) Acta Biol Hung, 58, pp. 1-9Muntoni, F., Mateddu, A., Marchei, F., Clerk, A., Serra, G., Muscular weakness in the mdx mouse (1993) J Neurol Sci, 120, pp. 71-77Pernice, W., Beckmann, R., Ketelsen, U.P., Frey, M., Schmidt-Redemann, B., Haap, K.P., A double-blind placebo controlled trial of diltiazem in Duchenne dystrophy (1988) Klin Wochenschr, 66, pp. 565-570Petrof, 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) Proc Natl Acad Sci U S A, 90, pp. 3710-3714Porter, J.D., Merrian, A.P., Leahy, P., Gong, B., Khann, S., Dissection of temporal gene expression signatures of affected and spared muscle groups in dystrophin-deficient (mdx) mice (2003) Hum Mol Genet, 12, pp. 1813-1821Pozzan, T., Rizzuto, R., Volpe, P., Meldolesi, J., Molecular and cellular physiology of intracellular calcium stores (1994) Physiol Rev, 74, pp. 595-636Robert, V., Massimino, M.L., Tosselo, V., Marsault, R., Cantini, M., Sorrentino, V., Alterations in calcium handling at the subsarcolemmal level in mdx myotubes (2001) J Biol Chem, 276, pp. 4647-4651Rossi, A.E., Dirksen, R.T., Sarcoplasmic reticulum: The dynamic calcium governor of muscle (2006) Muscle Nerve, 33, pp. 715-731Shavlakadze, T., White, J., Hoh, J.F.Y., Rosenthal, N., Grounds, M.D., Targeted expression of insulin-like growth factor-I reduces early myofiber necrosis in dystrophic mdx mice (2004) Mol Ther, 10, pp. 829-843Stedman, H.H., Sweeny, 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. 536-538Straub, V., Rafael, J.A., Chamberlain, J.S., Campbell, K.P., Animal models for muscular dystrophy show different patterns of sarcolemma disruption (1997) J Cell Biol, 139, pp. 375-385Tanabe, Y., Esaki, K., Nomura, T., Skeletal muscle pathology in X chromosome-linked muscular dystrophy (mdx) mouse (1986) Acta Neuropathol, 69, pp. 91-95Tidball, J.G., Wehling-Henriks, M., Evolving therapeutics strategies for Duchenne muscular dystrophy: Targeting downstream events (2004) Pediatr Res, 56, pp. 831-841Turner, P.R., Fong, P., Denetclaw, W., Steinhardt, R.A., Increased calcium influx in dystrophic muscle (1991) J Cell Biol, 115, pp. 1701-1712Vandebrouck, A., Martin, D., Colson-Van Schoor, M., Debaix, H., Gailly, P., Involvement of TRPC in the abnormal calcium influx observed in dystrophic (mdx) mouse skeletal muscle fibers (2002) J Cell Biol, 158, pp. 1089-1096Vandebrouck, A., Sabourin, J., Rivet, J., Balghi, H., Sebille, S., Kitzis, A., Regulation of capacitative calcium entries by α1-syntrophin: Association of TRPC1 with dystrophin complex and the PDZ domain of α1-syntrophin (2007) FASEB J, 21, pp. 608-617Voisin, V., Sébrié, C., Matecki, S., Yu, H., Gillet, B., Ramonatxo, M., L-arginine improves dystrophic phenotype in mdx mice (2005) Neurobiol Dis, 20, pp. 123-130Wang, T., Tsai, L.I., Schwartz, A., Effects of verapamil, diltiazem, nisoldipine and felodipine on sarcoplasmic-reticulum (1984) Eur J Clin Pharmacol, 100, pp. 253-261Wells, D.J., Therapeutic restoration of dystrophin expression in Duchenne muscular dystrophy (2006) J Muscle Res Cell Motil, 27, pp. 387-398Whitehead, N.P., Yeung, E.W., Allen, D.G., Muscle damage in mdx (dystrophic) mice: Role of calcium and reactive oxygen species (2006) Clin Exp Pharmacol Physiol, 33, pp. 657-662Yoshida, M., Yonetani, A., Shirasaki, T., Wada, K., Dietary NaCl supplementation prevents muscle necrosis in a mouse model of Duchenne muscular dystrophy (2006) Am J Physiol Regul Integr Comp Physiol, 290, pp. R449-R455