Histochemistry and Cell Biology

, Volume 131, Issue 5, pp 565–574 | Cite as

The ascorbic acid transporter SVCT2 is expressed in slow-twitch skeletal muscle fibres

  • Marcela Low
  • Daniel Sandoval
  • Evelyn Avilés
  • Fernando Pérez
  • Francisco Nualart
  • Juan Pablo Henríquez
Original Paper

Abstract

Ascorbic acid, the reduced form of vitamin C, functions as a potent antioxidant as well as in cell differentiation. Ascorbate is taken up by mammalian cells through the specific sodium/ascorbate co-transporters SVCT1 and SVCT2. Although skeletal muscle contains about 50% of the whole-body vitamin C, the expression of SVCT transporters has not been clearly addressed in this tissue. In this work, we analysed the expression pattern of SVCT2 during embryonic myogenesis using the chick as model system. We cloned the chick orthologue of SVCT2 (cSVCT2) that shares 93% identity with the mouse transporter. cSVCT2 mRNA and protein are expressed during chick embryonic muscle development. Immunohistochemical analyses showed that SVCT2 is preferentially expressed by type I slow-twitch muscle fibres throughout chick myogenesis as well as in post-natal skeletal muscles of several species, including human. Our results suggest that SVCT2-mediated uptake of ascorbate is relevant to the oxidative nature of type I muscle fibres.

Keywords

Slow muscle Ascorbate SVCT2 Chick Human 

Notes

Acknowledegments

The authors are indebted to Sylvain Marcellini, Nelson Osses, Maria de los Angeles García, Hugo Olguin and members of our laboratory for useful discussion and comments on the manuscript. This work was supported by grants Anillo PBCT-CONICYT ACT-02 and ACI-12, DIUC-UdeC 204.031.098-1.0 and Fundación Andes C-13960/28.

Supplementary material

418_2008_552_Fig8_ESM.jpg (2.7 mb)

Fig. 1 Cloning and comparison of cSVCT2 mRNA sequence with cloned mammalian orthologues. (a) Total RNA was obtained from stage HH42 chick brain (lane 2) and hindlimb skeletal muscles (lane 3) for RT-PCR analysis to detect cSVCT2 (2,126bp). Lane 1 corresponds to a 1Kb DNA ladder standard. (b) Partial alignment of cSVCT2 mRNA sequence with that of mouse (GenBank accession AY004874) and human (GenBank accession EF032501) mRNAs. Sequences of the primers used are highlighted by black shading. The coding sequence of cSVCT2 is underlined. (JPG 2.69 MB)

418_2008_552_Fig9_ESM.jpg (503 kb)

Fig. 2 A goat anti rat SVCT2 antibody specifically detects cSVCT2. Hindlimbs from chick embryos at stage HH40 were stained with a goat anti SVCT2 antibody (first panel). Control immunostaining experiments including those performed in the absence of any primary antibody (second panel), by using an irrelevant goat anti human MAP-1B antibody (third panel) and by coincubation of anti SVCT2 antibody along with its corresponding inhibitory peptide (IP, fourth panel), gave negative results. All sections were double stained with a mouse anti slow myosin heavy chain antibody (lower panels). Alexa488-conjugated anti goat and alexa633 conjugated anti mouse immunoglobulins were used as secondary antibodies. Bar, 20μm. (JPG 503 KB)

418_2008_552_Fig10_ESM.jpg (1.4 mb)

Fig. 3 SVCT2 is expressed in post-natal type I skeletal muscle fibres from different mammalian species. Skeletal muscle cryosections from post-natal guinea-pig (soleus), rabbit (tibialis anterior) and rat (soleus) were double stained with a goat anti SVCT2 antibody (upper panel) together with a mouse anti slow myosin heavy chain antibody (middle panel). Alexa488-conjugated anti goat and alexa546-conjugated anti mouse immunoglobulins were used as secondary antibodies. Merge images (lower panel) indicate the co-localisation of anti SVCT2 with anti slow myosin staining. Bar, 100μm. (JPG 1.39 MB)

References

  1. Barnard RJ, Edgerton VR, Furukawa T, Peter JB (1971) Histochemical, biochemical, and contractile properties of red, white, and intermediate fibers. Am J Physiol 220:410–414PubMedGoogle Scholar
  2. Castro M, Caprile T, Astuya A, Millan C, Reinicke K, Vera JC, Vasquez O, Aguayo LG, Nualart F (2001) High-affinity sodium–vitamin C co-transporters (SVCT) expression in embryonic mouse neurons. J Neurochem 78:815–823PubMedCrossRefGoogle Scholar
  3. Castro T, Low M, Salazar K, Montecinos H, Cifuentes M, Yanez AJ, Slebe JC, Figueroa CD, Reinicke K, s Garcia M, Henriquez JP, Nualart F (2008) Differential distribution of the sodium–vitamin C cotransporter-1 along the proximal tubule of the mouse and human kidney. Kidney Int 74:1278–1286PubMedCrossRefGoogle Scholar
  4. Crow MT, Stockdale FE (1986) Myosin expression and specialization among the earliest muscle fibers of the developing avian limb. Dev Biol 113:238–254PubMedCrossRefGoogle Scholar
  5. Daruwala R, Song J, Koh WS, Rumsey SC, Levine M (1999) Cloning and functional characterization of the human sodium-dependent vitamin C transporters hSVCT1 and hSVCT2. FEBS Lett 460:480–484PubMedCrossRefGoogle Scholar
  6. DiMario JX, Stockdale FE (1997) Both myoblast lineage and innervation determine fiber type and are required for expression of the slow myosin heavy chain 2 gene. Dev Biol 188:167–180PubMedCrossRefGoogle Scholar
  7. Duarte TL, Lunec J (2005) Review: When is an antioxidant not an antioxidant? A review of novel actions and reactions of vitamin C. Free Radic Res 39:671–686PubMedCrossRefGoogle Scholar
  8. Englard S, Seifter S (1986) The biochemical functions of ascorbic acid. Annu Rev Nutr 6:365–406PubMedCrossRefGoogle Scholar
  9. Faaland CA, Race JE, Ricken G, Warner FJ, Williams WJ, Holtzman EJ (1998) Molecular characterization of two novel transporters from human and mouse kidney and from LLC-PK1 cells reveals a novel conserved family that is homologous to bacterial and Aspergillus nucleobase transporters. Biochim Biophys Acta 1442:353–360PubMedGoogle Scholar
  10. Fredette BJ, Landmesser LT (1991) Relationship of primary and secondary myogenesis to fiber type development in embryonic chick muscle. Dev Biol 143:1–18PubMedCrossRefGoogle Scholar
  11. Frei B, England L, Ames BN (1989) Ascorbate is an outstanding antioxidant in human blood plasma. Proc Natl Acad Sci USA 86:6377–6381PubMedCrossRefGoogle Scholar
  12. Garcia Mde L, Salazar K, Millan C, Rodriguez F, Montecinos H, Caprile T, Silva C, Cortes C, Reinicke K, Vera JC, Aguayo LG, Olate J, Molina B, Nualart F (2005) Sodium vitamin C cotransporter SVCT2 is expressed in hypothalamic glial cells. Glia 50:32–47PubMedCrossRefGoogle Scholar
  13. Godoy A, Ormazabal V, Moraga-Cid G, Zuniga FA, Sotomayor P, Barra V, Vasquez O, Montecinos V, Mardones L, Guzman C, Villagran M, Aguayo LG, Onate SA, Reyes AM, Carcamo JG, Rivas CI, Vera JC (2007) Mechanistic insights and functional determinants of the transport cycle of the ascorbic acid transporter SVCT2. Activation by sodium and absolute dependence on bivalent cations. J Biol Chem 282:615–624PubMedCrossRefGoogle Scholar
  14. Gordon T, Perry R, Srihari T, Vrbova G (1977) Differentiation of slow and fast muscles in chickens. Cell Tissue Res 180:211–222PubMedCrossRefGoogle Scholar
  15. Hamburger V, Hamilton HL (1992) A series of normal stages in the development of the chick embryo. 1951. Dev Dyn 195:231–272PubMedGoogle Scholar
  16. Hood DA (2001) Invited review: contractile activity-induced mitochondrial biogenesis in skeletal muscle. J Appl Physiol 90:1137–1157PubMedGoogle Scholar
  17. Jackson MJ, Pye D, Palomero J (2007) The production of reactive oxygen and nitrogen species by skeletal muscle. J Appl Physiol 102:1664–1670PubMedCrossRefGoogle Scholar
  18. Kaprielian Z, Fambrough DM (1987) Expression of fast and slow isoforms of the Ca2+-ATPase in developing chick skeletal muscle. Dev Biol 124:490–503PubMedCrossRefGoogle Scholar
  19. Kuo SM, MacLean ME, McCormick K, Wilson JX (2004) Gender and sodium-ascorbate transporter isoforms determine ascorbate concentrations in mice. J Nutr 134:2216–2221PubMedGoogle Scholar
  20. Lee JY, Chang MY, Park CH, Kim HY, Kim JH, Son H, Lee YS, Lee SH (2003) Ascorbate-induced differentiation of embryonic cortical precursors into neurons and astrocytes. J Neurosci Res 73:156–165PubMedCrossRefGoogle Scholar
  21. Li X, Huang J, May JM (2003) Ascorbic acid spares alpha-tocopherol and decreases lipid peroxidation in neuronal cells. Biochem Biophys Res Commun 305:656–661PubMedCrossRefGoogle Scholar
  22. Li Y, Schellhorn HE (2007) New developments and novel therapeutic perspectives for vitamin C. J Nutr 137:2171–2184PubMedGoogle Scholar
  23. Liang WJ, Johnson D, Jarvis SM (2001) Vitamin C transport systems of mammalian cells. Mol Membr Biol 18:87–95PubMedCrossRefGoogle Scholar
  24. Lutsenko EA, Carcamo JM, Golde DW (2004) A human sodium-dependent vitamin C transporter 2 isoform acts as a dominant-negative inhibitor of ascorbic acid transport. Mol Cell Biol 24:3150–3156PubMedCrossRefGoogle Scholar
  25. Miller JB, Stockdale FE (1986) Developmental regulation of the multiple myogenic cell lineages of the avian embryo. J Cell Biol 103:2197–2208PubMedCrossRefGoogle Scholar
  26. Nualart FJ, Rivas CI, Montecinos VP, Godoy AS, Guaiquil VH, Golde DW, Vera JC (2003) Recycling of vitamin C by a bystander effect. J Biol Chem 278:10128–10133PubMedCrossRefGoogle Scholar
  27. Pansarasa O, Bertorelli L, Vecchiet J, Felzani G, Marzatico F (1999) Age-dependent changes of antioxidant activities and markers of free radical damage in human skeletal muscle. Free Radic Biol Med 27:617–622PubMedCrossRefGoogle Scholar
  28. Peake JM (2003) Vitamin C: effects of exercise and requirements with training. Int J Sport Nutr Exerc Metab 13:125–151PubMedGoogle Scholar
  29. Pette D, Staron RS (2001) Transitions of muscle fiber phenotypic profiles. Histochem Cell Biol 115:359–372PubMedGoogle Scholar
  30. Rafuse VF, Milner LD, Landmesser LT (1996) Selective innervation of fast and slow muscle regions during early chick neuromuscular development. J Neurosci 16:6864–6877PubMedGoogle Scholar
  31. Rajan DP, Huang W, Dutta B, Devoe LD, Leibach FH, Ganapathy V, Prasad PD (1999) Human placental sodium-dependent vitamin C transporter (SVCT2): molecular cloning and transport function. Biochem Biophys Res Commun 262:762–768PubMedCrossRefGoogle Scholar
  32. Rando TA, Disatnik MH, Yu Y, Franco A (1998) Muscle cells from mdx mice have an increased susceptibility to oxidative stress. Neuromuscul Disord 8:14–21PubMedCrossRefGoogle Scholar
  33. Russell PJ, Williams A, Abbott A, DeRosales B, Vargas R (2006) Characteristics of rabbit muscle adenylate kinase inhibition by ascorbate. J Enzyme Inhib Med Chem 21:61–67PubMedCrossRefGoogle Scholar
  34. Savini I, Catani MV, Arnone R, Rossi A, Frega G, Del Principe D, Avigliano L (2007a) Translational control of the ascorbic acid transporter SVCT2 in human platelets. Free Radic Biol Med 42:608–616PubMedCrossRefGoogle Scholar
  35. Savini I, Catani MV, Duranti G, Ceci R, Sabatini S, Avigliano L (2005) Vitamin C homeostasis in skeletal muscle cells. Free Radic Biol Med 38:898–907PubMedCrossRefGoogle Scholar
  36. Savini I, Rossi A, Catani MV, Ceci R, Avigliano L (2007b) Redox regulation of vitamin C transporter SVCT2 in C2C12 myotubes. Biochem Biophys Res Commun 361:385–390PubMedCrossRefGoogle Scholar
  37. Savini I, Rossi A, Pierro C, Avigliano L, Catani MV (2008) SVCT1 and SVCT2: key proteins for vitamin C uptake. Amino Acids 34:347–355PubMedCrossRefGoogle Scholar
  38. Thoma WJ, Henderson LM (1984) Effect of vitamin C deficiency on hydroxylation of trimethylaminobutyrate to carnitine in the guinea pig. Biochim Biophys Acta 797:136–139PubMedGoogle Scholar
  39. Toutain PL, Bechu D, Hidiroglou M (1997) Ascorbic acid disposition kinetics in the plasma and tissues of calves. Am J Physiol 273:R1585–R1597PubMedGoogle Scholar
  40. Tsukaguchi H, Tokui T, Mackenzie B, Berger UV, Chen XZ, Wang Y, Brubaker RF, Hediger MA (1999) A family of mammalian Na+-dependent l-ascorbic acid transporters. Nature 399:70–75PubMedCrossRefGoogle Scholar
  41. Urso ML, Clarkson PM (2003) Oxidative stress, exercise, and antioxidant supplementation. Toxicology 189:41–54PubMedCrossRefGoogle Scholar
  42. Wang Y, Mackenzie B, Tsukaguchi H, Weremowicz S, Morton CC, Hediger MA (2000) Human vitamin C (L-ascorbic acid) transporter SVCT1. Biochem Biophys Res Commun 267:488–494PubMedCrossRefGoogle Scholar
  43. Webster C, Silberstein L, Hays AP, Blau HM (1988) Fast muscle fibers are preferentially affected in Duchenne muscular dystrophy. Cell 52:503–513PubMedCrossRefGoogle Scholar
  44. Wilson JX (1990) Regulation of ascorbic acid concentration in embryonic chick brain. Dev Biol 139:292–298PubMedCrossRefGoogle Scholar
  45. Wu X, Itoh N, Taniguchi T, Hirano J, Nakanishi T, Tanaka K (2004) Stimulation of differentiation in sodium-dependent vitamin C transporter 2 overexpressing MC3T3–E1 osteoblasts. Biochem Biophys Res Commun 317:1159–1164PubMedCrossRefGoogle Scholar

Copyright information

© Springer-Verlag 2009

Authors and Affiliations

  • Marcela Low
    • 1
  • Daniel Sandoval
    • 1
  • Evelyn Avilés
    • 1
  • Fernando Pérez
    • 2
  • Francisco Nualart
    • 1
  • Juan Pablo Henríquez
    • 1
  1. 1.Research Ring for the Study of the Nervous System (PBCT-CONICYT), Laboratory of Developmental Neurobiology, Department of Cell Biology, Faculty of Biological SciencesUniversidad de ConcepciónConcepciónChile
  2. 2.Hospital Guillermo Grant BenaventeConcepciónChile

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