Dihydropyridine receptors (DHPRs), ryanodine receptors (RyRs), and triadin are major components of triads of mature skeletal muscle and play crucial roles in Ca2+ release in excitation–contraction (E–C) coupling. We investigated the expression and localization of these proteins as well as intracellular Ca2+ transients during development of human muscle cells cultured aneurally and innervated with rat spinal cord. mRNAs encoding skeletal muscle isoforms of the DHPR α1 subunit (α1S-DHPR), the RyR, and triadin were scarce in myoblasts and increased remarkably after myotube formation. Immunocytochemically, α1S-DHPR was expressed after myoblast fusion and localized mainly within the cytoplasmic area of aneural myotubes whereas the cardiac isoform (α1C-DHPR) was abundant along the plasma membrane. RyRs and triadin were both detected after myotube formation and colocalized in the cytoplasm of aneural myotubes and innervated muscle fibers. Along the plasma membrane of aneural myotubes, colocalization of α1C-DHPR with the RyR was more frequently observed than that of α1S-DHPR. In innervated muscle fibers, α1S-DHPR and RyR were colocalized first along the plasma membrane and later in the cytoplasmic area and formed regular double rows of cross-striation. The α1C-DHPR diminished after innervation. In Ca2+ imaging, spontaneous irregular slow Ca2+ oscillations were observed in aneurally cultured myotubes whereas nerve-driven regular fast oscillations were observed in innervated muscle fibers. Both caffeine and depolarization induced Ca2+ transients in aneurally cultured myotubes and innervated muscle fibers. In aneurally cultured myotubes, depolarization-induced Ca2+ transients were highly dependent on extracellular Ca2+, suggesting immaturity of the Ca2+ release system. This dependence remarkably decreased after innervation. Our present results show that these proteins are expressed differently in aneurally cultured myotubes than in adult skeletal muscle, that Ca2+ release in aneurally cultured myotubes is different from in adult skeletal muscle, and that innervation induces formation of a mature skeletal muscle-like excitation–contraction coupling system in cultured human muscle cells.
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Adams BA, Tanabe T, Mikami A, Numa S and Beam KG (1990) Intramembrane charge movement restored in dysgenic skeletal muscle by injection of dihydropyridine receptor cDNAs. Nature 346: 569–572.
Airey JA, Beck CF, Murakami K, Tanksley SJ, Deerinck TJ, Ellisman MH and Sutko JL (1990) Identification and localization of two triad junctional foot protein isoforms in mature avian fast twitch skeletal muscle. J Biol Chem 265: 14187–14194.
Airey JA, Baring MD and Sutko JL (1991) Ryanodine receptor protein is expressed during differentiation in the muscle cell lines BC3H1 and C2C12. Dev Biol 148: 365–374.
Arai M, Otsu K, MacLennan DH and Periasamy M (1992) Regulation of sarcoplasmic reticulum gene expression during cardiac and skeletal muscle development. Am J Physiol 262: C614–620.
Armstrong CM, Bezanilla FM and Horowicz P (1972) Twitches in the presence of ethylene glycol bis (β-aminoethyl ether)-N,N′-tetraacetic acid. Biochim Biophys Acta 267: 605–608.
Askanas V and Engel WK (1975) A new program for investigating adult human skeletal muscle grown aneurally in tissue culture. Neurology 25: 58–67.
Askanas V, Kwan H, Alvarez RB, Engel WK, Kobayashi T, Martinuzzi A and Hawkins EF (1987) De novo neuromuscular junction formation on human muscle fibres cultured in monolayer and innervated by foetal rat spinal cord: ultrastructural and ultrastructural-cytochemical studies. J Neurocytol 16: 523–537.
Bennett DL, Cheek TR, Berridge MJ, De Smedt H, Parys JB, Missiaen L and Bootman MD (1996) Expression and function of ryanodine receptors in nonexcitable cells. J Biol Chem 271: 6356–6362.
Block BA, Imagawa T, Campbell KP and Franzini-Armstrong C (1988) Structural evidence for direct interaction between the molecular components of the transverse tubule/sarcoplasmic reticulum junction in skeletal muscle. J Cell Biol 107: 2587–2600.
Brandt NR, Caswell AH, Wen S-R and Talvenheimo JA (1990) Molecular interactions of the junctional foot protein and dihydro-pyridine receptor in skeletal muscle triads. J Membr Biol 113: 237–251.
Brandt NR, Caswell AH, Brunschwig J-P, Kang J-J, Antoniu B and Ikemoto N (1992) Effects of anti-triadin antibody on Ca2+ release from sarcoplasmic reticulum. FEBS Lett 299: 57–59.
Bulteau L, Cogné M, Cognard C and Raymond G (1997) Reversal of the relative expression of cardiac and skeletal alpha1 subunit isoforms of L-type calcium channel during in vitro myogenesis. Pflügers Arch Eur J Physiol 433: 376–378.
Callewaert G (1992) Excitation-contraction coupling in mammalian cardiac cells. Cardiovasc Res 26: 923–932.
Caswell AH, Brandt NR, Brunschwig J-P and Purkerson S (1991) Localization and partial characterization of the oligomeric disul-fide-linked molecular weight 95,000 protein (triadin) which binds the ryanodine and dihydropyridine receptors in skeletal muscle triadic vesicles. Biochemistry 30: 7507–7513.
Chaudhari N and Beam KG (1993) mRNA for cardiac calcium channel is expressed during development of skeletal muscle. Dev Biol 155: 507–515.
Cognard C, Rivet-Bastide M, Constantin B and Raymond G (1992) Progressive predominance of ‘skeletal’ versus ‘cardiac’ types of excitation-contraction coupling during in vitro skeletal myogenesis. Pflügers Arch Eur J Physiol 422: 207–209.
Constantin B, Cognard C and Raymond G (1996) Myoblast fusion requires cytosolic calcium elevation but not activation of voltage-dependent calcium channels. Cell Calcium 19: 365–374.
Endo M, Tanaka M and Ogawa Y (1970) Calcium induced release of calcium from the sarcoplasmic reticulum of skinned skeletal muscle fibres. Nature 228: 34–36.
Ercolani L, Florence B, Denaro M and Alexander M (1988) Isolation and complete sequence of a functional human glycer-aldehyde-3-phosphate dehydrogenase gene. J Biol Chem 263: 15335–15341.
Ferrari MB, Rohrbough J and Spitzer NC (1996) Spontaneous calcium transients regulate myofibrillogenesis in embryonic Xenopus myocytes. Dev Biol 178: 484–497.
Flucher BE, Terasaki M, Chin HM, Beeler TJ and Daniels MP (1991) Biogenesis of transverse tubules in skeletal muscle in vitro. Dev Biol 145: 77–90.
Flucher BE and Andrews SB (1993) Characterization of spontaneous and action potential-induced calcium transients in developing myotubes in vitro. Cell Motil Cytoskeleton 25: 143–157.
Flucher BE, Andrews SB, Fleischer S, Marks AR, Caswell A and Powell JA (1993) Triad formation: organization and function of the sarcoplasmic reticulum calcium release channel and triadin in normal and dysgenic muscle in vitro. J Cell Biol 123: 1161–1174.
Flucher BE, Andrews SB and Daniels MP (1994) Molecular organization of transverse tubule/sarcoplasmic reticulum junctions during development of excitation-contraction coupling in skeletal muscle. Mol Biol Cell 5: 1105–1118.
Franzini-Armstrong C and Jorgensen AO (1994) Structure and development of E-C coupling units in skeletal muscle. Annu Rev Physiol 56: 509–534.
Froemming GR, Murray BE and Ohlendieck K (1999) Self-aggregation of triadin in sarcoplasmic reticulum of rabbit skeletal muscle. Biochim Biophys Acta 1418: 197–205.
Furuya A, Kobayashi T, Kameda N and Tsukagoshi H (1991) Human myasthenia gravis thymic myoid cells: de novo immunohistochemical and intracellular electrophysiological studies. J Neurol Sci 101: 208–220.
Grouselle M, Koenig J, Lascombe M-L, Chapron J, Méléard P and Georgescauld D (1991) Fura-2 imaging of spontaneous and electrically induced oscillations of intracellular free Ca2+ in rat myotubes. Pflügers Arch Eur J Physiol 418: 40–50.
Guo W and Campbell KP (1995) Association of triadin with the ryanodine receptor and calsequestrin in the lumen of the sarco-plasmic reticulum. J Biol Chem 270: 9027–9030.
Hell JW, Westenbroek RE, Warner C, Ahlijanian MK, Prystay W, Gilbert MM, Snutch TP and Catterall WA (1993) Identification and differential subcellular localization of the neuronal class C and class D L-type calcium channel α1 subunits. J Cell Biol 123: 949–962.
Hogan K, Gregg RG and Powers PA (1996) The structure of the gene encoding the human skeletal muscle α1 subunit of the dihydropyridine-sensitive L-type calcium channel (CACNL1A3). Genomics 31: 392–394.
Kameda N, Kobayashi T, Park-Matsumoto YC, Tsukagoshi H and Shimizu T (1993) Developmental studies of the expression of myosin heavy chain isoforms in cultured human muscle aneurally and innervated with fetal rat spinal cord. J Neurol Sci 114: 85–98.
Kameda N, Ueda H, Ohno S, Shimokawa M, Usuki F, Ishiura S and Kobayashi T (1998) Developmental regulation of myotonic dystrophy protein kinase in human muscle cells in vitro. Neuroscience 85: 311–322.
Kim KC, Caswell AH, Talvenheimo JA and Brandt NR (1990) Isolation of a terminal cisterna protein which may link the dihydropyridine receptor to the junctional foot protein in skeletal muscle. Biochemistry 29: 9281–9289.
Knudson CM, Stang KK, Jorgensen AO and Campbell KP (1993) Biochemical characterization and ultrastructural localization of a major junctional sarcoplasmic reticulum glycoprotein (triadin). J Biol Chem 268: 12637–12645.
Kobayashi T, Askanas V and Engel WK (1987) Human muscle cultured in monolayer and cocultured with fetal rat spinal cord: importance of dorsal root ganglia for achieving successful functional innervation. J Neurosci 7: 3131–3141.
Kobayashi T, Michikawa M, Miyazaki H and Tsukagoshi H (1990) The effect of Ba ions on human muscle cultured in monolayer and innervated with fetal rat spinal cord. Neurosci Lett 111: 157–163.
Kobayashi T, Ohno S, Park-Matsumoto YC, Kameda N and Baba T (1995) Developmental studies of dystrophin and other cytoskeletal proteins in cultured muscle cells. Microsc Res Tech 30: 437–457.
Kyselovic J, Leddy JJ, Ray A, Wigle J and Tuana BS (1994) Temporal differences in the induction of dihydropyridine receptor subunits and ryanodine receptors during skeletal muscle development. J Biol Chem 269: 21770–21777.
Lai FA, Erickson HP, Rousseau E, Liu Q-Y and Meissner G (1988) Purification and reconstitution of the calcium release channel from skeletal muscle. Nature 331: 315–319.
Lazarides E (1982) Intermediate filaments: a chemically heterogeneous, developmentally regulated class of proteins. Annu Rev Biochem 51: 219–250.
Leung AT, Imagawa T and Campbell KP (1987) Structural characterization of the 1,4-dihydropyridine receptor of the voltage-dependent Ca2+ channel from rabbit skeletal muscle. Evidence for two distinct high molecular weight subunits. J Biol Chem 262: 7943–7946.
Li Z, Lilienbaum A, Butler-Browne G and Paulin D (1989) Human desmin-coding gene: complete nucleotide sequence, characteriza-tion and regulation of expression during myogenesis and development. Gene 78: 243–254.
Marks AR, Tempst P, Hwang KS, Taubman MB, Inui M, Chadwick C, Fleischer S and Nadal-Ginard B (1989) Molecular cloning and characterization of the ryanodine receptor/junctional channel complex cDNA from skeletal muscle sarcoplasmic reticulum. Proc Natl Acad Sci USA 86: 8683–8687.
Marks AR, Taubman MB, Saito A, Dai Y and Fleischer S (1991) The ryanodine receptor/junctional channel complex is regulated by growth factors in a myogenic cell line. J Cell Biol 114: 303–312.
Martinuzzi A, Askanas V, Kobayashi T, Engel WK and Di Mauro S (1986) Expression of muscle-gene-specific isozymes of phosphorylase and creatine kinase in innervated cultured human muscle. J Cell Biol 103: 1423–1429.
Martinuzzi A, Askanas V, Kobayashi T, Engel WK and Gorsky JE (1987) Developmental expression of the muscle-specific isozyme of phosphoglycerate mutase in human muscle cultured in monolayer and innervated by fetal rat spinal cord. Exp Neurol 96: 365–375.
Martinuzzi A, Askanas V, Kobayashi T and Engel WK (1988) Asynchronous regulation of muscle specific isozymes of creatine kinase, glycogen phosphorylase, lactic dehydrogenase and phosphoglycerate mutase in innervated and non-innervated cultured human muscle. Neurosci Lett 89: 216–222.
Michikawa M, Kobayashi T and Tsukagoshi H (1991) Early events of chemical transmission of newly formed neuromuscular junctions in monolayers of human muscle cells co-cultured with fetal rat spinal cord explants. Brain Res 538: 79–85.
Mori Y (1994) Molecular biology of voltage-dependent calcium channels. In: Peracchia C (ed) Handbook of Membrane Channels, (pp. 163–176) Academic Press, San Diego.
Narahashi T, Tsunoo A and Yoshii M (1987) Characterization of two types of calcium channels in mouse neuroblastoma cells. J Physiol (Lond) 383: 231–249.
Park-Matsumoto YC, Askanas V and Engel WK (1992) The influence of muscle contractile activity versus neural factors on morphologic properties of innervated cultured human muscle. J Neurocytol 21: 329–340.
Péréon Y, Navarro J, Sorrentino V, Louboutin J-P, Noireaud J and Palade P (1997a) Regulation of dihydropyridine receptor and ryanodine receptor gene expression in regenerating skeletal muscle. Pflügers Arch Eur J Physiol 433: 221–229.
Péréon Y, Sorrentino V, Dettbarn C, Noireaud J and Palade P (1997b) Dihydropyridine receptor and ryanodine receptor gene expression in long-term denervated rat muscles. Biochem Biophys Res Commun 240: 612–617.
Phillips MS, Fujii J, Khanna VK, DeLeon S, Yokobata K, de Jong PJ and MacLennan DH (1996) The structural organization of the human skeletal muscle ryanodine receptor (RYR1) gene. Genomics 34: 24–41.
Protasi F, Franzini-Armstrong C and Flucher BE (1997) Coordinated incorporation of skeletal muscle dihydropyridine receptors and ryanodine receptors in peripheral couplings of BC3H1 cells. J Cell Biol 137: 859–870.
Protasi F, Franzini-Armstrong C and Allen PD (1998) Role of ryanodine receptors in the assembly of calcium release units in skeletal muscle. J Cell Biol 140: 831–842.
Rios E and Brum G (1987) Involvement of dihydropyridine receptors in excitation-contraction coupling in skeletal muscle. Nature 325: 717–720.
Saito K, Kobayashi T, Askanas V, Engel WK and Ishikawa K (1990) Electrical properties of human muscle cultured in monolayer aneurally and co-cultured with fetal rat spinal cord. Biomedical Research 11: 19–28.
Seigneurin-Venin S, Parrish E, Marty I, Rieger F, Romey G, Villaz M and Garcia L (1996) Involvement of the dihydropyridine receptor and internal Ca2+ stores in myoblast fusion. Exp Cell Res 223: 301–307.
Soldatov NM (1992) Molecular diversity of L-type Ca2+ channel transcripts in human fibroblasts. Proc Natl Acad Sci USA 89: 4628–4632.
Soldatov NM (1994) Genomic structure of human L-type Ca2+ channel. Genomics 22: 77–87.
Spitzer NC (1994) Spontaneous Ca2+ spikes and waves in embryonic neurons: signaling systems for differentiation. Trends Neurosci 17: 115–118.
Strube C, Beurg M, Georgescauld D, Bournaud R and Shimahara T (1994) Extracellular Ca2+-dependent and independent calcium transient in fetal myotubes. Pflügers Arch Eur J Physiol 427: 517–523.
Takeshima H, Nishimura S, Matsumoto T, Ishida H, Kangawa K, Minamino N, Matsuo H, Ueda M, Hanaoka M, Hirose T and Numa S (1989) Primary structure and expression from complementary DNA of skeletal muscle ryanodine receptor. Nature 339: 439–445.
Tanabe T, Takeshima H, Mikami A, Flockerzi V, Takahashi H, Kangawa K, Kojima M, Matsuo H, Hirose T and Numa S (1987) Primary structure of the receptor for calcium channel blockers from skeletal muscle. Nature 328: 313–318.
Tanabe T, Beam KG, Powell JA and Numa S (1988) Restoration of excitation-contraction coupling and slow calcium current in dysgenic muscle by dihydropyridine receptor complementary DNA. Nature 336: 134–139.
Tanabe T, Beam KG, Adams BA, Niidome T and Numa S (1990a) Regions of the skeletal muscle dihydropyridine receptor critical for excitation-contraction coupling. Nature 346: 567–569.
Tanabe T, Mikami A, Numa S and Beam KG (1990b) Cardiac-type excitation-contraction coupling in dysgenic skeletal muscle injected with cardiac dihydropyridine receptor cDNA. Nature 344: 451–453.
Tanabe T, Adams BA, Numa S and Beam KG (1991) Repeat I of the dihydropyridine receptor is critical in determining calcium channel activation kinetics. Nature 352: 800–803.
Taske NL, Eyre HJ, O'Brien RO, Sutherland GR, Denborough MA and Foster PS (1995) Molecular cloning of the cDNA encoding human skeletal muscle triadin and its localisation to chromosome 6q22–6q23. Eur J Biochem 233: 258–265.
Varadi G, Orlowski J and Schwartz A (1989) Developmental regula-tion of expression of the α1 and α2 subunits mRNAs of the voltage-dependent calcium channel in a differentiating myogenic cell line. FEBS Lett 250: 515–518.
Verkhratsky A and Shmigol A (1996) Calcium-induced calcium release in neurones. Cell Calcium 19: 1–14.
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Tanaka, H., Furuya, T., Kameda, N. et al. Triad proteins and intracellular Ca2+ transients during development of human skeletal muscle cells in aneural and innervated cultures. J Muscle Res Cell Motil 21, 507–526 (2000). https://doi.org/10.1023/A:1026561120566
- Human Skeletal Muscle
- Myoblast Fusion