Figure 5.

Elucidation of Mechanism A as most likely cause for reduction in Ca²⁺ waves due to I_Na blockade. (A) Voltage clamp stimulation trains used to assess wave frequency with and without I_Na activity. Stimulation was induced by stepping from −80 to 0 mv (I_Na active) or −40 to 0 mV (I_Na inactive). Pulse duration was 100 ms and pulses were applied at 5 Hz. Waves were assessed in a subsequent 30 s interval during which membrane potential was held at −80 mV. (B) With I_Na inactive during the stimulation train (but available during the quiescent phase of the experiment), wave frequency was reduced (P = 0.002, n = 7 cells). (C) High-dose (50 µM) TTX was rapidly applied to cells to terminate stimulation following a period of external field stimulation at 5 Hz and compared with the control arm in which stimulation was terminated in the usual fashion at 30 s (see Supplementary material online, Figure S4 for further explanation). This produced the opposite situation to the previous experiment with I_Na active during the stimulation train but Na_v1.5 channels unavailable for stimulation during the quiescent phase. This produced no change in wave frequency (P = 0.99, n = 17 cells). (D) Similarly, there was no change in wave velocity (P = 0.66 by Student's t-test. Control: n = 88 waves; 50 µM TTX: n = 89 waves from 17 cells). (E) Voltage clamp experiments showing effects of flecainide on wave frequency with I_Na active vs. inactive. With I_Na active, flecainide reduces wave frequency (P = 0.001, n = 7 cells). (F) However, with I_Na inactive, no reduction in wave frequency was observed (P = 0.36, n = 7 cells).