Efficacy of sodium channel blockers in treating myotonia is independent of action potential sodium current block

Figures

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  Figure 1. Concentration dependence of Na+ channel blocker efficacy against myotonia A, a representative trace of a normally excitable fibre with one AP per stimulus throughout a 2‐s train at 20 Hz. B, a representative trace of a hyperexcitable fibre where extra, unstimulated APs (arrows) appear after the third stimulus. C, a representative trace of a hyperexcitable fibre with a plateau potential: a transient depolarization to –30 to –45 mV. D, a representative trace of a hypoexcitable fibre: the last eight stimuli fail to trigger APs exceeding –10 mV. E, box‐and‐whisker plots showing the percentage of hyperexcitable fibres after 9AC treatment and varying concentrations of Na+ channel blockers. F, box‐and‐whisker plots showing the percentage of hypoexcitable fibres under the same conditions. In E and F, boxes represent the interquartile range, horizontal lines the median and whiskers values below Q1 – 1.5×IQR or above Q3 + 1.5×IQR. Each point represents the percentage of fibres from one mouse.

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  Figure 2. Ranolazine and mexiletine block NaT in a use‐dependent manner A, voltage protocol: 2 s of 5‐ms steps to –30 mV at 20 Hz, with resulting NaT currents from a control fibre. B, the mean 1st and 40th NaT currents superimposed. The solid line represents the average 1st current, and the dashed line represents the average 40th current. C, mean normalized NaT amplitude for each of the 40 steps in each treatment group. Error bars indicate SEM. D, box‐and‐whisker plots of animal means for the ratio of the 40th to the 1st current. Mexiletine and ranolazine both significantly reduced normalized current compared to control. Control (n = 8 mice), mexiletine (n = 5), ranolazine (n = 5), µCTX (n = 5). Boxes represent the interquartile range, horizontal lines the median and whiskers values below Q1 – 1.5×IQR or above Q3 + 1.5×IQR.

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  Figure 3. A 59% reduction in AP upstroke velocity precedes the termination of myotonic discharges A, voltage response of a 9AC‐treated EDL fibre during the first 11 stimuli of a 20‐Hz train. Arrows indicate the first stimulated, first myotonic and last myotonic APs. The period of myotonic firing is marked by a horizontal red bar. B, APs and corresponding dV/dts for the first stimulated, first myotonic and last myotonic APs from the trace in A. C, scatter plot of upstroke velocity for the first and last myotonic APs across 20 fibres from 11 mice. Mean values decreased from 261 ± 27 to 112 ± 33 mV/ms (P < 0.001, paired t test, SD).

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  Figure 4. Na+ channel blockers prevent myotonia without significantly reducing AP upstroke velocity at the onset of myotonic firing A, representative traces of the third stimulated APs and corresponding dV/dt from 9AC‐treated fibres under control conditions and after 20 µM mexiletine, 40 µM ranolazine or 80 nM µCTX. B, box‐and‐whisker plots of the third AP upstroke velocity for each treatment group. Control (n = 11 mice), mexiletine (n = 5), ranolazine (n = 7), µCTX (n = 6). Horizontal lines indicate medians, boxes the 25th–75th percentiles and whiskers the 5th–95th percentiles. P‐values versus control: mexiletine = 1.000, ranolazine = 1.000, µCTX = 0.069.

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  Figure 5. Na+ channel blockers reduce depolarization of the interspike membrane potential A, first 150 ms of representative voltage traces during 20 Hz stimulation of 9AC‐treated EDL fibres recorded with and without 20 µM mexiletine, 40 µM ranolazine or 80 nM µCTX. Without drug the membrane potential depolarized to AP threshold after the third stimulus. AP peaks are truncated at –20 mV to emphasize subthreshold potentials. B, box‐and‐whisker plot of depolarization measured 15–50 ms after the second stimulus. Groups: control (n = 11 mice), mexiletine (n = 5), ranolazine (n = 7), µCTX (n = 6). Horizontal line = median; box = 25th–75th percentiles; whiskers = 5th–95th percentiles. AfD, after depolarization.

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  Figure 6. Effect of Na+ channel blockers on NaP at therapeutically effective concentrations A, average NaP traces during a ramp depolarization from –85 to –25 mV under control conditions and after treatment with each of the three Na+ channel blockers. B, box‐and‐whisker plot of animal averages of NaP current at –40 mV. Horizontal line = median; box = 25th–75th percentiles; whiskers = 5th–95th percentiles.

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  Figure 7. Reduction of NaP, not NaT, normalizes excitability A, C, intracellular recordings from EDL fibres. B, D, simulations of intracellular recordings under matching conditions. In all cases fibres were stimulated for 2 s at 20 Hz. Left: full trace. Right: responses to the first six stimuli. A, wild‐type fibre, firing one AP per stimulus throughout the train. B, simulation of wild‐type firing, reproducing one AP per stimulus. C, recording from a 9AC‐treated fibre showing myotonic APs after the 3rd–12th triggered APs. D–H, simulations of myotonia with a 90% reduction in Cl− conductance, producing myotonic APs. D, normal NaT and NaP with myotonia resembling experimental records. E, use‐dependent block of NaT reduced to 50% by the end of the train; myotonic firing persists. F, stronger use‐dependent block of NaT to 15% remaining; myotonia still present. G, tonic reduction of NaT to 30% remaining reduces AP peak amplitude to ∼0 mV, yet myotonic firing remains after the 6th–12th APs. H, reduction of NaP to 60% remaining eliminates myotonia while preserving AP peak amplitude. Horizontal red bars denote periods of myotonic firing.

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  Figure 8. Block of NaP is the primary mechanism underlying the efficacy of Na+ channel blockers against myotonic hyperexcitability Plot of excitability versus simulated combinations of NaT and NaP reduction after the third stimulated AP. Hyperexcitability was defined as APs occurring without stimulation (myotonic discharges). Normal excitability was defined as the absence of myotonia with APs peaking above –10 mV throughout the 20 Hz train. Hypoexcitability was defined as the absence of myotonia but APs falling below –10 mV by the end of the train. Superimposed are estimates of NaT and NaP remaining after the third AP (relative to baseline current) for control fibres and for each drug. The third AP was chosen because it coincides with the onset of myotonia.