, Volume 63, Issue 3, pp 295–305 | Cite as

Establishment of functional primary cultures of heart cells from the clam Ruditapes decussatus

  • H. HananaEmail author
  • H. Talarmin
  • J. P. Pennec
  • M. Droguet
  • E. Gobin
  • P. Marcorelle
  • G. Dorange
Original Research


Heart cells from the clam Ruditapes decussatus were routinely cultured with a high level of reproducibility in sea water based medium. Three cell types attached to the plastic after 2 days and could be maintained in vitro for at least 1 month: epithelial-like cells, round cells and fibroblastic cells. Fibroblastic cells were identified as functional cardiomyocytes due to their spontaneous beating, their ultrastructural characteristics and their reactivity with antibodies against sarcomeric α-actinin, sarcomeric tropomyosin, myosin and troponin T-C. Patch clamp measurements allowed the identification of ionic currents characteristic of cardiomyocytes: a delayed potassium current (I K slow) strongly suppressed (95%) by tetraethylammonium (1 mM), a fast inactivating potassium current (I K fast) inhibited (50%) by 4 amino-pyridine at 1 mM and, at a lower level (34%) by TEA, a calcium dependent potassium current (I KCa) activated by strong depolarization. Three inward voltage activated currents were also characterized in some cardiomyocytes: L-type calcium current (I Ca) inhibited by verapamil at 5 × 10−4 M, T-type Ca2+ current, rapidly activated and inactivated, and sodium current (I Na) observed in only a few cells after strong hyperpolarization. These two currents did not seem to be physiologically essential in the initiation of the beatings of cardiomyocytes. Potassium currents were partially inhibited by tributyltin (TBT) (1 μM) but not by okadaic acid (two marine pollutants). DNA synthesis was also demonstrated in few cultured cells using BrdU (bromo-2′-deoxyuridine). Observed effects of okadaic acid and TBT demonstrated that cultured heart cells from clam Ruditapes decussatus can be used as an experimental model in marine toxicology.


Clam Ruditapes decussatus Cardiomyocyte Patch clamp Ionic currents In vitro 


  1. Almers W, Stanfield PR, Stuhmer W (1983) Slow changes in currents through sodium channels in frog muscle membrane. J Physiol 339:253–271Google Scholar
  2. Bebianno MJ, Geret F, Hoarau P, Serafim MA, Coelho MR, Gnassia-Barelli M, Romeo M (2004) Biomarkers in Ruditapes decussatus: a potential bioindicator species. Biomarkers 9:305–330CrossRefGoogle Scholar
  3. Bers DM, Perez-Reyes E (1999) Ca channels in cardiac myocytes: structure and function in Ca influx and intracellular Ca release. Cardiovasc Res 42:339–360CrossRefGoogle Scholar
  4. Buchanan JT, La Peyre JF, Cooper RK, Tiersch TR (1999) Improved attachment and spreading in primary cell cultures of the eastern oyster, Crassostrea virginica. In Vitro Cell Dev Biol Anim 35:593–598CrossRefGoogle Scholar
  5. Cecil JT (1969) Mitoses in cell cultures from cardiac tissue of the surf clam Spisula solidissima. J Invertebr Pathol 14:407–410CrossRefGoogle Scholar
  6. Chardonnet Y, Pérès G (1963) Essai de culture de cellules provenant d’un mollusque: Mytilus gallovincialis. L C R Soc Biol Lyon 157:1593–1595Google Scholar
  7. Chen SN, Wen CM (1999) Establishment of cell lines derived from oyster, Crassostrea gigas Thunberg and hard clam, Meretrix lusoria Roding. Methods Cell Sci 21:183–192CrossRefGoogle Scholar
  8. Cheng TC, La Peyre JF, Buchanan JT, Tiersch TR, Cooper RK (2001) Cryopreservation of heart cells from the eastern oyster. In Vitro Cell Dev Biol Anim 37:237–244Google Scholar
  9. Curtis TM, Depledge MH, Williamson R (1999) Voltage-activated currents in cardiac myocytes of the blue mussel, Mytilus edulis. Comp Biochem Physiol A 124:231–241Google Scholar
  10. Domart-Coulon I, Doumenc D, Auzoux-Bordenave S, Le Fichant Y (1994) Identification of media supplements that improve the viability of primarily cell cultures of Crassostrea gigas oysters. Cytotechnology 16:109–120CrossRefGoogle Scholar
  11. Domart-Coulon I, Auzoux-Bordenave S, Doumenc D, Khalanski M (2000) Cytotoxicity assessment of antibiofouling compounds and by-products in marine bivalve cell cultures. Toxicol In Vitro 14:245–251CrossRefGoogle Scholar
  12. Droguet M (2006) Etude des caractéristiques fonctionnelles des cardiomyocytes d’huître en culture. Thèse de doctorat de biologie. Université de Bretagne Occidentale, p 211Google Scholar
  13. Ellington WR (1993) Studies of intracellular pH regulation in cardiac myocytes from the marine bivalve mollusk, Mercenaria campechiensis. Biol Bull 184:209–215CrossRefGoogle Scholar
  14. Ferrier GR, Redondo IM, Mason CA, Mapplebeck C, Howlett SE (2000) Regulation of contraction and relaxation by membrane potential in cardiac ventricular myocytes. Am J Physiol Heart Circ Physiol 278:H1618–H1626Google Scholar
  15. Fritayre P (2004) Culture des cellules atriales de coquille Saint-Jacques, Pecten maximus: valeur et limites du modèle. Applications en toxicologie. Thèse en Océanologie Biologique et Environnement marin. Université de Bretagne Occidentale, p 230Google Scholar
  16. Giles WR, Imaizumi Y (1988) Comparison of potassium currents in rabbit atrial and ventricular cells. J Physiol 405:123–145Google Scholar
  17. Hagiwara N, Irisawa H, Kameyama M (1988) Contribution of two types of calcium currents to the pacemaker potentials of rabbit sino-atrial node cells. J Physiol 395:233–253Google Scholar
  18. Kenyon JL, Sutko JL (1987) Calcium- and voltage-activated plateau currents of cardiac Purkinje fibers. J Gen Physiol 89:921–958CrossRefGoogle Scholar
  19. Le Deuff RM, Lipart C, Renault T (1994) Primary culture of pacific oyster, Crassostrea gigas, heart cells. J Tissue Cult Methods 16:67–72CrossRefGoogle Scholar
  20. Le Marrec-Croq F, Dorange G, Chesné C (1997) Procédé de culture de cellules d’invertébrés marins et cultures obtenues, French Patent no 9506921; no of publication: 2735146 (1996)Google Scholar
  21. Le Marrec-Croq F, Fritayre P, Chesn GuillouzoA, Dorange G (1998) Cryopreservation of Pecten maximus heart cells. Cryobiology 37:200–206CrossRefGoogle Scholar
  22. Le Marrec-Croq F, Glaise D, Guguen-Guillouzo C, Chesne C, Guillouzo A, Boulo V, Dorange G (1999) Primary cultures of heart cells from the scallop Pecten maximus (mollusca-bivalvia). In Vitro Cell Dev Biol Anim 35:289–295CrossRefGoogle Scholar
  23. Ödblom MP, Williamson R, Jones MB (2000) Ionic currents in cardiac myocytes of squid, Alloteuthis subulata. J Comp Physiol B 170:11–20CrossRefGoogle Scholar
  24. Pennec JP, Gallet M, Gioux M, Dorange G (2002) Cell culture of bivalves: tool for the study of the effects of environmental stressors. Cell Mol Biol (Noisy-le-grand) 48:351–358Google Scholar
  25. Pennec JP, Talarmin H, Droguet M, Giroux-Metges MA, Gioux M, Dorange G (2004) Characterization of the voltage-activated currents in cultured atrial myocytes isolated from the heart of the common oyster Crassostrea gigas. J Exp Biol 207:3935–3944CrossRefGoogle Scholar
  26. Quinn B, Costello MJ, Dorange G, Wilson JG, Mothersill C (2009) Development of an in vitro culture method for cells and tissues from the zebra mussel (Dreissena polymorpha). Cytotechnology 59:121–134CrossRefGoogle Scholar
  27. Renault T, Flaujac G, Le Deuff RM (1995) Isolation and culture of heart cells from the European flat oyster, Ostrea edulis. Methods Cell Sci 17:199–205CrossRefGoogle Scholar
  28. Reynolds ES (1963) The use of lead citrate at high pH as an electron-opaque stain in electron microscopy. J Cell Biol 17:208–212CrossRefGoogle Scholar
  29. Rinkevich B (2005) Marine invertebrate cell cultures: new millennium trends. Mar biotechnol (NY) 7:429–439CrossRefGoogle Scholar
  30. Siegelbaum SA, Tsien RW (1980) Calcium-activated transient outward current in calf cardiac Purkinje fibres. J Physiol 299:485–506Google Scholar
  31. Souza MM, Stucchi-Zucchi A, Cassola AC, Scemes E (2002) Electrophysiology of cardiac myocytes of Aplysia brasiliana. Comp Biochem Physiol A Mol Integr Physiol 133:161–168CrossRefGoogle Scholar
  32. Talarmin H, Droguet M, Pennec JP, Schröder HC, Muller WEG, Gioux M, Dorange G (2008) Effects of a phycotoxin, okadaic acid, on oyster heart cell survival. Toxicol Environ Chem 90:153–168CrossRefGoogle Scholar
  33. Tseng GN, Robinson RB, Hoffman BF (1987) Passive properties and membrane currents of canine ventricular myocytes. J Gen Physiol 90:671–701CrossRefGoogle Scholar
  34. Varro A, Papp JG (1992) The impact of single cell voltage clamp on the understanding of the cardiac ventricular action potential. Cardioscience 3:131–144Google Scholar
  35. Wen CM, Kou GH, Chen SN (1993a) Cultivation of cells from the heart of the hard clam, Meretrix lusoria (RODING). J Tissue Cult Methods 15:123–130CrossRefGoogle Scholar
  36. Wen CM, Kou GH, Chen SN (1993b) Establishment of cell lines from the Pacific oyster. In Vitro Cell Dev Biol Anim 29A:901–903CrossRefGoogle Scholar
  37. Yeoman MS, Benjamin PR (1999) Two types of voltage-gated K(+) currents in dissociated heart ventricular muscle cells of the snail Lymnaea stagnalis. J Neurophysiol 82:2415–2427Google Scholar
  38. Yeoman MS, Brezden BL, Benjamin PR (1999) LVA and HVA Ca(2+) currents in ventricular muscle cells of the Lymnaea heart. J Neurophysiol 82:2428–2440Google Scholar

Copyright information

© Springer Science+Business Media B.V. 2011

Authors and Affiliations

  • H. Hanana
    • 1
    Email author
  • H. Talarmin
    • 1
  • J. P. Pennec
    • 1
  • M. Droguet
    • 1
  • E. Gobin
    • 2
  • P. Marcorelle
    • 2
  • G. Dorange
    • 1
  1. 1.Faculté de médecine, EA 4326Université Européenne de Bretagne, Université de Bretagne OccidentaleBrest Cedex 3France
  2. 2.CHU Morvan, Service d’Anatomie PathologiqueBrestFrance

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