Skip to main content

Advertisement

SpringerLink
Log in

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

  • Original Research
  • Published:
Cytotechnology Aims and scope Submit manuscript

Abstract

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.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5
Fig. 6
Fig. 7
Fig. 8
Fig. 9
Fig. 10

Similar content being viewed by others

References

  • Almers W, Stanfield PR, Stuhmer W (1983) Slow changes in currents through sodium channels in frog muscle membrane. J Physiol 339:253–271

    CAS  Google Scholar 

  • 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 9:305–330

    Article  CAS  Google Scholar 

  • Bers DM, Perez-Reyes E (1999) Ca channels in cardiac myocytes: structure and function in Ca influx and intracellular Ca release. Cardiovasc Res 42:339–360

    Article  CAS  Google Scholar 

  • 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 35:593–598

    Article  CAS  Google Scholar 

  • Cecil JT (1969) Mitoses in cell cultures from cardiac tissue of the surf clam Spisula solidissima. J Invertebr Pathol 14:407–410

    Article  CAS  Google Scholar 

  • Chardonnet Y, Pérès G (1963) Essai de culture de cellules provenant d’un mollusque: Mytilus gallovincialis. L C R Soc Biol Lyon 157:1593–1595

    CAS  Google Scholar 

  • Chen SN, Wen CM (1999) Establishment of cell lines derived from oyster, Crassostrea gigas Thunberg and hard clam, Meretrix lusoria Roding. Methods Cell Sci 21:183–192

    Article  CAS  Google Scholar 

  • 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 37:237–244

    CAS  Google Scholar 

  • Curtis TM, Depledge MH, Williamson R (1999) Voltage-activated currents in cardiac myocytes of the blue mussel, Mytilus edulis. Comp Biochem Physiol A 124:231–241

    Google Scholar 

  • 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 16:109–120

    Article  CAS  Google Scholar 

  • 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 14:245–251

    Article  CAS  Google Scholar 

  • Droguet M (2006) Etude des caractéristiques fonctionnelles des cardiomyocytes d’huître en culture. Thèse de doctorat de biologie. Université de Bretagne Occidentale, p 211

  • Ellington WR (1993) Studies of intracellular pH regulation in cardiac myocytes from the marine bivalve mollusk, Mercenaria campechiensis. Biol Bull 184:209–215

    Article  Google Scholar 

  • 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–H1626

    CAS  Google Scholar 

  • 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 230

  • Giles WR, Imaizumi Y (1988) Comparison of potassium currents in rabbit atrial and ventricular cells. J Physiol 405:123–145

    CAS  Google Scholar 

  • 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 395:233–253

    CAS  Google Scholar 

  • Kenyon JL, Sutko JL (1987) Calcium- and voltage-activated plateau currents of cardiac Purkinje fibers. J Gen Physiol 89:921–958

    Article  CAS  Google Scholar 

  • Le Deuff RM, Lipart C, Renault T (1994) Primary culture of pacific oyster, Crassostrea gigas, heart cells. J Tissue Cult Methods 16:67–72

    Article  Google Scholar 

  • 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)

  • Le Marrec-Croq F, Fritayre P, Chesn GuillouzoA, Dorange G (1998) Cryopreservation of Pecten maximus heart cells. Cryobiology 37:200–206

    Article  Google Scholar 

  • 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 35:289–295

    Article  CAS  Google Scholar 

  • Ödblom MP, Williamson R, Jones MB (2000) Ionic currents in cardiac myocytes of squid, Alloteuthis subulata. J Comp Physiol B 170:11–20

    Article  Google Scholar 

  • 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) 48:351–358

    CAS  Google Scholar 

  • 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 207:3935–3944

    Article  CAS  Google Scholar 

  • 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 59:121–134

    Article  CAS  Google Scholar 

  • Renault T, Flaujac G, Le Deuff RM (1995) Isolation and culture of heart cells from the European flat oyster, Ostrea edulis. Methods Cell Sci 17:199–205

    Article  Google Scholar 

  • Reynolds ES (1963) The use of lead citrate at high pH as an electron-opaque stain in electron microscopy. J Cell Biol 17:208–212

    Article  CAS  Google Scholar 

  • Rinkevich B (2005) Marine invertebrate cell cultures: new millennium trends. Mar biotechnol (NY) 7:429–439

    Article  CAS  Google Scholar 

  • Siegelbaum SA, Tsien RW (1980) Calcium-activated transient outward current in calf cardiac Purkinje fibres. J Physiol 299:485–506

    CAS  Google Scholar 

  • Souza MM, Stucchi-Zucchi A, Cassola AC, Scemes E (2002) Electrophysiology of cardiac myocytes of Aplysia brasiliana. Comp Biochem Physiol A Mol Integr Physiol 133:161–168

    Article  Google Scholar 

  • 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 90:153–168

    Article  CAS  Google Scholar 

  • Tseng GN, Robinson RB, Hoffman BF (1987) Passive properties and membrane currents of canine ventricular myocytes. J Gen Physiol 90:671–701

    Article  CAS  Google Scholar 

  • Varro A, Papp JG (1992) The impact of single cell voltage clamp on the understanding of the cardiac ventricular action potential. Cardioscience 3:131–144

    CAS  Google Scholar 

  • Wen CM, Kou GH, Chen SN (1993a) Cultivation of cells from the heart of the hard clam, Meretrix lusoria (RODING). J Tissue Cult Methods 15:123–130

    Article  Google Scholar 

  • Wen CM, Kou GH, Chen SN (1993b) Establishment of cell lines from the Pacific oyster. In Vitro Cell Dev Biol Anim 29A:901–903

    Article  CAS  Google Scholar 

  • 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 82:2415–2427

    CAS  Google Scholar 

  • Yeoman MS, Brezden BL, Benjamin PR (1999) LVA and HVA Ca(2+) currents in ventricular muscle cells of the Lymnaea heart. J Neurophysiol 82:2428–2440

    CAS  Google Scholar 

Download references

Author information

Authors and Affiliations

  1. Faculté de médecine, EA 4326, Université Européenne de Bretagne, Université de Bretagne Occidentale, 22 Avenue Camille Desmoulins, 29238, Brest Cedex 3, France

    H. Hanana, H. Talarmin, J. P. Pennec, M. Droguet & G. Dorange

  2. CHU Morvan, Service d’Anatomie Pathologique, 29609, Brest, France

    E. Gobin & P. Marcorelle

Authors
  1. H. Hanana
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. H. Talarmin
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. J. P. Pennec
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. M. Droguet
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. E. Gobin
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. P. Marcorelle
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. G. Dorange
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to H. Hanana.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Hanana, H., Talarmin, H., Pennec, J.P. et al. Establishment of functional primary cultures of heart cells from the clam Ruditapes decussatus . Cytotechnology 63, 295–305 (2011). https://doi.org/10.1007/s10616-011-9347-8

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s10616-011-9347-8

Keywords

Search

Navigation