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

摘要

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 (IK?slow) strongly suppressed (95%) by tetraethylammonium (1?mM), a fast inactivating potassium current (IK?fast) inhibited (50%) by 4 amino-pyridine at 1?mM and, at a lower level (34%) by TEA, a calcium dependent potassium current (IKCa) activated by strong depolarization. Three inward voltage activated currents were also characterized in some cardiomyocytes: L-type calcium current (ICa) inhibited by verapamil at 5?×?10?4?M, T-type Ca2+ current, rapidly activated and inactivated, and sodium current (INa) 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.