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Myotonia Congenita (MC) is a rare, inherited ion channelopathy caused by a loss-of-function mutation in the CLCN1 gene. The resulting downregulation of the skeletal muscle chloride channel (ClC-1) results in hyperexcitable skeletal muscle fibers that fire action potentials involuntarily. Patients with MC suffer from debilitating stiffness due to myotonia, described clinically as delayed muscle relaxation following voluntary contraction. Skeletal muscle is a unique system we use in this study to advance our understanding of the pathophysiology underlying MC, and other channelopathies characterized by hyperexcitable cells (i.e., forms of epilepsy and cardiac arrhythmia). Furthermore, re-assessing what makes anti-myotonic drugs such as mexiletine efficacious, yet imperfect, may help us improve the quality of life of affected patients through optimizing treatment outcomes.The current therapeutic approach to treating myotonia is to block voltage-gated Na+ channels in skeletal muscle (Nav1.4) to reduce the fast-transient Na+ current responsible for action potential generation (NaT) via use-dependent block (Statland et al., 2012),(Lo Monaco et al., 2015). Two assumptions are made by the field when taking this approach. First, open channel blockers, those that increase their block of Na channels during repetitive firing, are the preferred type of drug. Second, the block of NaT is the mechanism underlying efficacy. We tested both hypotheses utilizing both transgenic and pharmacologic models of MC in mice. Techniques used included current clamp of the extensor digitorum longus (EDL) muscle and voltage clamp of the flexor digitorum brevis (FDB) muscle. We compared the efficacy of two open-state NaCh blockers; mexiletine and ranolazine, to a closed-state blocker, µ-conotoxin GIIIA (uCTX). We quantitated efficacy against myotonia and determined the optimal concentration of each drug using intracellular recordings from EDL fibers. We were surprised to find that open-channel blockers were not more effective than the closed-channel blocker, uCTX. Using voltage clamp we quantified the reduction of both NaT and NaP caused by each drug in individual, dissociated muscle fibers from the flexor digitorum brevis (FDB). We found block of NaT does not correlate with the elimination of myotonia. In contrast, block of NaP (a non-inactivating, persistent Nav1.4 current) correlates well. In collaboration with Dr. Brent Foy, we performed a computer simulation of myotonia and found block of NaP, rather than NaT, appears to be the mechanism underlying efficacy. This work suggests a paradigm shift for the field in which selective targeting of NaP is the goal. A corollary is that block of NaT is an unwanted side effect of therapy. We studied whether findings in paralyzed single muscle fibers were predictive of the response of contracting whole EDL muscles to treatment with Na channel blockers. We were surprised to find that uCTX was significantly less effective compared to ranolazine and mexiletine in contracting muscles in the pharmacologic model of MC. The difference was significantly less in the genetic model of MC. These data are consistent with the possibility that both ranolazine and mexiletine block a second contributor to myotonia. That contributor appears to be downregulated in the genetic model of MC, in which myotonia is present chronically. We combined treatment with uCTX with a blocker of TRPV4, a stretch-activated channel, and found a modest improvement in efficacy. Efforts are ongoing to determine the additional channel type(s) blocked by mexiletine and ranolazine.Our data suggest 3 conclusions; (1) Block of NaP rather than NaT is the mechanism underlying efficacy of Na channel block in lessening myotonia, (2) Open channel block is not a superior approach to therapy. (3) A stretch-activated channel, which is blocked by ranolazine and mexiletine, is an important contributor to the generation of myotonia in contracting muscle.

Myotonia congenita is an inherited skeletal muscle disorder caused by loss-of-function mutation in the CLCN1 gene. This gene encodes the ClC-1 chloride channel, which is almost exclusively expressed in skeletal muscle where it acts to stabilize the resting membrane potential. Loss of this chloride channel leads to skeletal muscle hyperexcitability, resulting in involuntary muscle action potentials (myotonic discharges) seen clinically as muscle stiffness (myotonia). Stiffness affects the limb and facial muscles, though specific muscle involvement can vary between patients. Interestingly, respiratory distress is not part of this disease despite muscles of respiration such as the diaphragm muscle also carrying this mutation. In addition to stiffness, patients experience episodes of transient weakness that remained poorly understood despite years of study. Current treatment focuses on use-dependent block of sodium channels, with off-label use of the antiarrhythmic mexiletine and the antianginal ranolazine. We performed intracellular recordings from muscle of both genetic and pharmacologic mouse models of myotonia congenita to identify the mechanism underlying transient weakness. Our recordings revealed transient depolarizations (plateau potentials) of the membrane potential to –25 to –35 mV in our mouse models. Na+ persistent current (NaP) through Nav1.4 channels is the key trigger of plateau potentials. Inhibition of Nav1.4 with Ranolazine eliminates transient weakness in vivo and prevents the development of plateau potentials. These data suggest that targeting NaP may be an effectivevtreatment to prevent attacks of transient weakness in myotonia congenita. We also performed intracellular, and ex vivo force recordings on the diaphragm muscle of our myotonic mouse model and found that the diaphragm muscle is immune to myotonic discharges and relatively spared from myotonia. Through comparison with myotonia-affected EDL we propose that some of the resistance of diaphragm to myotonia is due to smaller fiber size. This suggests that muscles affected by myotonia may depend on fiber size and may explain lessening symptoms with age. Lastly, we compared use-dependent block of transient Na+ currents (NaT) via ranolazine and mexiletine with tonic block of NaP with μ-conotoxin GIIIA. Our data suggests the use-dependent block of NaT is not required for treatment and that tonic block of NaP may be superior because it eliminates myotonic discharges without drastically altering action potential characteristics.

Myotonia congenita is a rare skeletal muscle channelopathy caused by a reduced chloride channel (ClC-1) current, which results in debilitating muscle hyperexcitability, prolonged contractions, and transient episodes of weakness. The excitatory events that trigger myotonic action potentials in the absence of stabilizing ClC-1 current are not fully understood. My in vitro intracellular recordings from a mouse homozygous knockout of ClC-1 revealed a slow after-depolarization (AfD) that triggers myotonic action potentials. The AfD is well-explained by a tetrododoxin-sensitive and voltage-dependent Na+ persistent inward current (NaPIC). Notably, this NaPIC undergoes slow inactivation over seconds, thus providing the first mechanistic explanation for the end of myotonic runs. Highlighting the significance of this mechanism, we show that ranolazine and elevated serum divalent cations eliminate myotonia by inhibiting AfD and NaPIC. The electrophysiological events responsible for the transient weakness are not well understood either. My in vitro intracellular recordings revealed a novel behavior, in which the muscle is functionally inexcitable for seconds to minutes. This hanging behavior, as I refer to it, is likely to be responsible for periods of weakness described by patients and is explained by another persistent inward current. Partial pharmacological block of this other PIC decreases the hanging behavior in myotonic muscle.This work significantly changes our understanding of the mechanisms underlying myotonia and transient weakness seen in myotonia congenita and reveals a novel and highly effective therapeutic target.