Characterization of the voltage-activated currents in cultured atrial myocytes isolated from the heart of the common oysterCrassostrea gigas

Abstract

SUMMARY Using the macro-patch clamp technique, we show that cardiac myocytes isolated from the heart of the oyster Crassostrea gigas possess several types of voltage-activated ionic currents. (1) A classical non-inactivating potassium current of the IK type that is inhibited by tetraethyl ammonium and shows an outward rectification and a slow activation. (2) A potassium current of the IA type that shows rapid activation and inactivation, and is blocked by 4-amino pyridine or preliminary depolarisation. (3) A potassium calcium-dependent current that is inhibited by charybdotoxin, activated by strong depolarisations and shows a large conductance. (4) A calcium inward current of the L-type that is inhibited by verapamil, cobalt and high concentrations of cadmium. This current is identified in most cells, but a T-type calcium current and classical fast sodium current are only identified in few cells, and only after a strong hyperpolarizing pulse. This suggests that these channels are normally inactivated in cultured cells and are not involved in the spontaneous activity of these cells. When they exist, the fast sodium channel is blocked by tetrodotoxin. The L-type calcium conductance is increased by serotonin. The identification in cultured oyster atrial cells of classical ionic currents,which are observed in most vertebrate species but only in a few species of molluscs, demonstrates that these cells are an interesting model. Moreover the viability and the electrophysiological properties of these cells are not significantly modified by freezing and thawing, thus increasing their usefulness in various bioassays.

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

“…Our experiments showed that cells isolated from R. decussatus using Fritayre 2004;Pennec et al 2002Pennec et al , 2004Droguet 2006). To our knowledge it is the first time that pronase has been used to dissociate clam heart.…”

“…In the literature; data obtained using patch clamp and/ or immunostaining, ultrastructural studies concerned cultured cardiomyocytes from scallop, oyster and mussel (Wen et al 1993a;Le Deuff et al 1994;Curtis et al 1999;Le Marrec-Croq et al 1999;Pennec et al 2002Pennec et al , 2004Droguet 2006). Chen and Wen (1999) for oyster and Ellington (1993) for clam concluded that cultured fibroblastic cells were cardiomyocytes only by distinctive rhythmic contraction in vitro.…”

“…Electrophysiology A macro-patch clamp technique was used to characterize the main ionic currents according to the method previously described (Pennec et al 2004). The diameter of opening of the used pipettes (3 ± 0.02 lm) was checked by electron microscopy.…”

“…Le Deuff et al 1994;Renault et al 1995;Buchanan et al 1999;Chen and Wen 1999;DomartCoulon et al 2000;Pennec et al 2002Pennec et al , 2004Droguet 2006;Talarmin et al 2008). The aim of such attempts was to establish experimental models for applications in pathology and toxicology.…”

“…Cardiomyocytes e.g. from oysters and scallops can be now routinely maintained functional in culture up to 1 month (Domart-Coulon et al 1994;Le MarrecCroq et al 1997, 1998Pennec et al 2002Pennec et al , 2004Fritayre 2004;Droguet 2006;Talarmin et al 2008). Primary cultures can be obtained from freshly isolated cardiomyocytes and from cryopreserved cells (Le Marrec-Croq et al 1998;Cheng et al 2001).…”