The sodium background currents in cultured neonatal rat ventricular myocytes
In neonatal rat ventricular myocytes tetrodotoxin (TTX), a specific blocker of the Na current (I[subscript Na]) decreases the beating rate and the slope of the diastolic depolarization when these cells develop spontaneous activity in culture. The objective of this study was to measure and characterize the steady state component of I[subscript Na] in neonatal rat ventricular myocytes to see whether this current component can modulate the diastolic depolarization. Cardiocytes from one day old rats were isolated and studied 5-14 and 24-36 hours after explantation with the patch clamp method in the whole-cell configuration. Steady state and transient components of I[subscript Na] were recorded using depolarizing pulse protocols. In 27 of the total 43 cells an I-V relation of the steady state I[subscript Na], was found that contained peaks at -49.0±1.4mV (range -66 to -40mV, negative peak) and at -15.4±1.OmV (range -39 to +9mV, positive peak) for cells cultured <14 hours. The mean amplitude of the maximum steady state I[subscript Na], was4.0±0.3pA. In cells cultured > 24h, the potentials where the peaks occurred were shifted to -54.6±1.3mV and -21.4±1.3mV respectively. To test whether the 2 peaks represented 2 different I[subscript Na], TTX was used. A high concentration (200µM) abolished both peaks of the steady state current and the transient component of I[subscript Na]. A low concentration (10µM) inhibited the positive peak 51.3±7.8% in cells cultured < 14h whereas the negative peak did not change. In cells cultured > 24h the inhibition by 10µM TTX increased to91.5±7.5% for the positive peak but the negative peak remained unchanged. The transient component of I[subscript Na] was analyzed. During time in culture (a) the potential of half maximal steady state inactivation changed from -66.4 to -74.8mV and the slope factor decreased from 7.8 to 6.4mV; (b) the potential of the half maximal normalized conductance shifted from -29.3 to -41.8mV with a change in slope factor from 7.8 to 5.1mV; and (c) time in culture did not influence current density or maximum chord conductance. We concluded that in neonatal rat ventricular cells (a) two steady state I[subscript Na] are present that can be distinguished by their TTX sensitivity and the potential where the peak steady state current occurs; (b) the steady state current with the more negative peak is associated with the experimentally determined transient I[subscript Na]; (c) the shape of the I-V curves of both steady state currents is compatible with the I-V curve of a window current; (d) if the background current with the more positive peak is a window current, the conductance of its transient component must be low and comparable to that of the steady state component; (e) the 2 steady state components of I[subscript Na] appear to reflect the properties of 2 isoforms of the Na channel molecule whose relative importance changes with time so that at a later stage the more TTX sensitive isoform predominates; and (f) the current with negative peak can modulate diastolic depolarization whereas the current with positive peak can influence the plateau phase and duration of the action potential.