Contribution of the late sodium current to intracellular sodium and calcium overload in rabbit ventricular myocytes treated by anemone toxin

Abstract

Pathological enhancement of late Na+ current ( INa) can potentially modify intracellular ion homeostasis and contribute to cardiac dysfunction. We tested the hypothesis that modulation of late INa can be a source of intracellular Na+ ([Na+]i) overload. Late INa was enhanced by exposing rabbit ventricular myocytes to Anemonia sulcata toxin II (ATX-II) and measured using whole cell patch-clamp technique. [Na+]i was determined with fluorescent dye Asante NaTRIUM Green-2 AM. Pacing-induced changes in the dye fluorescence measured at 37°C were more pronounced in ATX-II-treated cells than in control (dye washout prevented calibration). At 22–24°C, resting [Na+]i was 6.6 ± 0.8 mM. Treatment with 5 nM ATX-II increased late INa 8.7-fold. [Na+]i measured after 2 min of electrical stimulation (1 Hz) was 10.8 ± 1.5 mM and 22.1 ± 1.6 mM ( P < 0.001) in the absence and presence of 5 nM ATX-II, respectively. Inhibition of late INa with GS-967 (1 μM) prevented Na+i accumulation. A strong positive correlation was observed between the late INa and the pacing-induced increase of [Na+]i ( R2 = 0.88) and between the rise in [Na+]i and the increases in cytosolic Ca2+ ( R2 = 0.96). ATX-II, tetrodotoxin, or GS-967 did not affect [Na+]i in quiescent myocytes suggesting that late INa was solely responsible for triggering the ATX-II effect on [Na+]i. Experiments with pinacidil and E4031 indicate that prolongation of the action potential contributes to as much as 50% of the [Na+]i overload associated with the increase in late INa caused by ATX-II. Enhancement of late INa can cause intracellular Na+ overload in ventricular myocytes.