Rabbit left ventricular cardiomyocytes were isolated enzymatically as prescribed previously.21 (link) Only cardiomyocytes with smooth and glossy edges and surfaces, clear transverse striations, and that could be stably voltage-clamped for 8 min without significant contraction were used. Seal resistance was above 1GΩ with less than 10 % variability. Currents were obtained with a patch-clamp amplifier (EPC-10, Heka Electronic, Lambrecht, Pfalz, Germany), filtered at 1 kHz and digitized at 10 kHz. During recording of late INa, the bath solution contained (in mmol/L):135 NaCl, 5.4 CsCl2, 1.8 CaCl2, 1 MgCl2, 0.3 BaCl2, 0.33 NaH2PO4, 10 glucose, 10 HEPES, 0.001 nicardipine, pH 7.3. The patch pipette solution contained (in mmol/L):120 CsCl2, 1.0 CaCl2, 5 MgCl2, 5 Na2ATP, 10 TEACl, 11 EGTA, 10 HEPES (pH 7.3, adjusted with CsOH). All experiments were carried out at a temperature of 23±1 °C.
To elicit a late INa in a single cell, twenty 300-ms depolarizing pulses to -20 mV from a holding potential of -90 mV were applied at CLs of 500, 667, 1000, 2000 and 4000 ms using a serial repeated-measurement protocol, with 3 min between each change of stimulation CL, during which membrane potential was held at -90 mV. In cardiomyocytes isolated from 8 rabbit hearts, the amplitude of late INa was measured at 200 ms after initiation of the depolarization step before (control, no drug) and 3 min after exposure of a myocyte to 1 and 4 μmol/L TTX, respectively.
To elicit a late INa in a single cell, twenty 300-ms depolarizing pulses to -20 mV from a holding potential of -90 mV were applied at CLs of 500, 667, 1000, 2000 and 4000 ms using a serial repeated-measurement protocol, with 3 min between each change of stimulation CL, during which membrane potential was held at -90 mV. In cardiomyocytes isolated from 8 rabbit hearts, the amplitude of late INa was measured at 200 ms after initiation of the depolarization step before (control, no drug) and 3 min after exposure of a myocyte to 1 and 4 μmol/L TTX, respectively.