Inhibiting persistent inward sodium currents prevents myotonia

Abstract

Objective: Patients with myotonia congenita have muscle hyperexcitability due to loss-of-function mutations in the ClC-1 chloride channel in skeletal muscle, which causes involuntary firing of muscle action potentials (myotonia), producing muscle stiffness. The excitatory events that trigger myotonic action potentials in the absence of stabilizing ClC-1 current are not fully understood. Our goal was to identify currents that trigger spontaneous firing of muscle in the setting of reduced ClC-1 current.

Methods: In vitro intracellular current clamp and voltage clamp recordings were performed in muscle from a mouse model of myotonia congenita.

Results: Intracellular recordings revealed a slow afterdepolarization (AfD) that triggers myotonic action potentials. The AfD is well explained by a tetrodotoxin-sensitive and voltage-dependent Na⁺ persistent inward current (NaPIC). Notably, this NaPIC undergoes slow inactivation over seconds, suggesting this may contribute to the end of myotonic runs. Highlighting the significance of this mechanism, we found that ranolazine and elevated serum divalent cations eliminate myotonia by inhibiting AfD and NaPIC.

Interpretation: This work significantly changes our understanding of the mechanisms triggering myotonia. Our work suggests that the current focus of treating myotonia, blocking the transient Na⁺ current underlying action potentials, is an inefficient approach. We show that inhibiting NaPIC is paralleled by elimination of myotonia. We suggest the ideal myotonia therapy would selectively block NaPIC and spare the transient Na⁺ current. Ann Neurol 2017;82:385-395.

Figure 1

Myotonic firing in muscle from ClC mice due to both a steady depolarization and an afterdepolarization (AfD). Shown is the response of a ClC muscle fiber to a 200‐millisecond injection of stimulating current. The fiber fires multiple action potentials (APs) for the duration of the current injection, a normal behavior; it then also continues to fire APs after the cessation of current injection (myotonia). Contributors to myotonic firing of APs include a steady depolarization and an AfD occurring between APs. The AfD occurs over 20 to 50 milliseconds and brings the fiber to threshold. The inset shows the final 2 APs of the run of myotonia on an expanded timescale. Max Repol = the maximal repolarization following each AP; Threshold = AP threshold (dV/dt = 10mV/ms). An AfD is present following the last myotonic AP, but is too small to bring the fiber to threshold. Following the final AfD, there is a resolution of the steady depolarization as the fiber gradually returns to its resting membrane potential. Shown on the lower right is the mean value of the first and last AfD slopes (n = 51 fibers, 12 mice; p < 0.01, paired t test).

Figure 2

Characterization of a tetrodotoxin (TTX)‐sensitive persistent inward current in ClC muscle. (A) The voltage protocol used to identify persistent inward currents (PICs). From a holding potential of −85mV, fibers were depolarized to −10mV at a rate of 10mV/s. (B) The current trace generated by the ramp depolarization in normal K⁺ solution with 20μM nifedipine. A fit line (thin) is drawn using the first 0.5 seconds of the raw trace, representing the leak current. Deviations from the leak current/fit line in the negative direction are consistent with activation of a PIC. (C) Shown is a trace generated by subtracting the leak trace shown in B and plotting against voltage. (D) Plot of leak‐subtracted PIC in K⁺‐free solutions (−TTX). The PIC was blocked by the addition of 1μM TTX to the external solution (+TTX).

Figure 3

Current recorded during a voltage‐clamp ramp protocol with repeated slow depolarization. Current recorded during a voltage‐clamp ramp protocol in which the fiber was slowly depolarized from a holding potential of −85mV to −10mV, repolarized to −85mV, and again depolarized to −10mV is shown. The larger persistent inward current during the depolarizing ramps than the repolarizing ramp shows that the muscle Na⁺ PIC undergoes slow inactivation. The recording was performed in K⁺‐free solutions. The fit lines for linear leak currents are superimposed on all 3 ramps.

Figure 4

Changes in action potential (AP) parameters during a myotonic run. Shown at the top is a 10‐second run of myotonia triggered by a 200‐millisecond stimulation. On the lower left is a blowup of 2 APs from the middle of the myotonic run to demonstrate the AP parameters measured. On the lower right is a plot of the values of the various parameters, time‐matched to the run of myotonia shown above. Termination of myotonia correlates with a reduction in the slope of the afterdepolarization (AfD). Max dV/dt = the maximal rate of rise of the AP; Max Repol = the maximal repolarization reached between APs.

Figure 5

Elimination of myotonia and parallel reduction of Na⁺ persistent inward current (NaPIC) by ranolazine, and divalent cation‐treated ClC muscle. (A) In untreated ClC muscle (top), myotonia is triggered with a 200‐millisecond depolarizing current pulse, whereas 50μM ranolazine treatment (middle) and high extracellular divalent solutions (bottom) eliminate myotonia. (B) Representative NaPIC traces evoked by slow‐depolarizing voltage ramps (similar to those shown in Fig 2D) for untreated (top), ranolazine (middle), and elevated cation (bottom) groups. NaPIC current density is reduced by both ranolazine and elevated extracellular divalent concentration. (C) Top plot: ClC muscle (untreated) shows a maximal PIC current density of −1.25 ± 0.17nA/nF (n = 23 fibers, 4 mice) versus treatment with 50μM ranolazine (maximal PIC current density is reduced to −0.38 ± 0.11nA/nF; n = 20 fibers, 3 mice), and versus elevated cation solutions (maximal PIC current density is decreased to −0.61 ± 0.16nA/nF; n = 25 fibers, 3 mice). Lower plot: Mean threshold of the first AP of a myotonic run of APs in ClC muscle (control) compared to the peak afterdepolarization (AfD) following treatment with ranolazine and high divalent solution. Both treatments that eliminate myotonia cause the AfD to fail to reach the threshold at which myotonic firing is triggered in untreated, control muscle.

Figure 6

A new model of generation of myotonia. In the K⁺ buildup model on the left, myotonia is triggered solely by a steady depolarization caused by K⁺ buildup in t‐tubules, which is sufficient to depolarize the membrane to the action potential (AP) threshold and trigger myotonia. In the Na⁺ persistent inward current (NaPIC) and K⁺ buildup model on the right, K⁺ buildup is insufficient to trigger myotonia in isolation, but rather depolarizes the membrane sufficiently to activate NaPIC, which then contributes to the final depolarization that triggers myotonic APs. RMP = resting membrane potential.