Mechanisms of atrial-selective block of Na⁺ channels by ranolazine: I. Experimental analysis of the use-dependent block

Abstract

Atrial-selective inhibition of cardiac Na(+) channel current (I(Na)) and I(Na)-dependent parameters has been shown to contribute to the safe and effective management of atrial fibrillation. The present study examined the basis for the atrial-selective actions of ranolazine. Whole cell I(Na) was recorded at 15°C in canine atrial and ventricular myocytes and in human embryonic kidney (HEK)-293 cells expressing SCN5A. Tonic block was negligible at holding potentials from -140 to -100 mV, suggesting minimal drug interactions with the closed state. Trains of 40 pulses were elicited over a range of holding potentials to determine use-dependent block. Guarded receptor formalism was used to analyze the development of block during pulse trains. Use-dependent block by ranolazine increased at more depolarized holding potentials, consistent with an interaction of the drug with either preopen or inactivated states, but was unaffected by longer pulse durations between 5 and 200 ms, suggesting a weak interaction with the inactivated state. Block was significantly increased at shorter diastolic intervals between 20 and 200 ms. Responses in atrial and ventricular myocytes and in HEK-293 cells displayed a similar pattern. Ranolazine is an open state blocker that unbinds from closed Na(+) channels unusually fast but is trapped in the inactivated state. Kinetic rates of ranolazine interactions with different states of atrial and ventricular Na(+) channels were similar. Our data suggest that the atrial selectivity of ranolazine is due to a more negative steady-state inactivation curve, less negative resting membrane potential, and shorter diastolic intervals in atrial cells compared with ventricular cells at rapid rates.

Fig. 1.

Use-dependent block of the Na⁺ channel by ranolazine (25 μM) is unaffected by test pulse duration. A train of 40 pulses was applied after a 15-s rest. The diastolic interval was kept constant at 150 ms, and pulse durations were 5, 20, or 200 ms. A: atrial myocytes (n = 6). B: ventricular myocytes (n = 5 cells) C: human embryonic kidney (HEK)-293 cells expressing SCN5A outward Na⁺ current (I_Na; n = 6). D: HEK-293 cells expressing SCN5A inward I_Na (n = 6). Insets in A–D show typical current traces during 200-ms test pulses. Data are means ± SD.

Fig. 2.

Abbreviation of the diastolic interval accentuates the development of use-dependent block of I_Na activated in atrial and ventricular cardiac myocytes by test pulses of constant duration in the presence of 10 μM (A and B) and 25 μM (C and D) ranolazine. A and C: inward I_Na recorded from ventricular myocytes during a train of pulses of 20-ms duration and diastolic intervals of 150, 70, 50, or 20 ms as a fraction of I_Na of the first pulse of the train (n = 6). B and D: I_Na recorded from atrial myocytes during a train of pulses of 20-ms duration and diastolic intervals of 150, 70, 50, or 20 ms as a fraction of I_Na of the first pulse of the train (n = 6). Data are means ± SD.

Fig. 3.

Rate of recovery of inward I_Na from use-dependent block by ranolazine. A and B: recovery of I_Na from block induced by a train of 40 pulses in ventricular (A) and atrial (B) myocytes in the presence of 40 μM ranolazine. Insets show the last two pulses of the train with four subsequent pulses overlaid as the interpulse interval was increased. n = 6 for each cell type. C: recovery in HEK-293 cells expressing SCN5A (n = 6) after a single 1,000-ms pulse to −20 mV in the control and in the presence of 30 μM ranolazine. Dashed and solid lines are the best double-exponential fits to averaged recovery data in the control and in the presence of ranolazine, respectively. The time scale is logarithmic.

Fig. 4.

Effects of holding potential (V_hold) on tonic and use-dependent block in cardiac myocytes by 10 μM ranolazine. Tonic block was defined as the magnitude of block during the 1st pulse, and use-dependent block was defined as the magnitude of block during the 40th pulse of a train relative to the 1st pulse. Trains were 40 pulses (50 ms) to −30 mV. A: percent tonic block as a function of V_hold in 10 ventricular cells and 5 atrial cells. Values are means ± SD. Tonic block in ventricular myocytes was not statistically significant, whereas tonic block in atrial cells was statistically different from zero (*P < 0.01). B: use-dependent block as a function of V_hold in 10 ventricular cells and 5 atrial cells. Values are means ± SD. The diastolic interval was 250 ms when holding at −100, −110, and −120 mV. The diastolic interval was 150 ms when holding at −140 mV. Block in atrial cells was significantly greater than ventricular cells at −100, −110, and −120 mV (‡P < 0.01). C and D: development of use-dependent block in ventricular (C) and atrial (D) myocytes during pulse trains delivered using different V_hold in the presence of 10 μM ranolazine at a diastolic interval of 250 ms. I_Na during each successive pulse was normalized to the current recorded during the first pulse to eliminate the contribution of tonic block. A small degree of accumulation of inactivation (<5%) was observed in the absence of drug (not shown). This effect was eliminated by dividing the relative current in the presence of ranolazine by the relative current in the control for each pulse in the train. These data were fit with monoexponential functions to obtain steady-state block and uptake rates as a function of V_hold in ventricular and atrial myocytes, which were analyzed as shown in E (steady-state block) and F (λ⁻¹). Arrows indicate the shift of values obtained in atrial cells by 8.6 mV (steady-state block) and by 10.7 mV (λ⁻¹), which was required to obtain the best fit (minimal sum of squared errors) of all six data points by the following single Boltzmann function: A/{1 + e^{[(V_1/2 − V)/s]}} (solid lines in E and F).