Treatment of myotonia congenita with retigabine in mice

Abstract

Patients with myotonia congenita suffer from muscle stiffness caused by muscle hyperexcitability. Although loss-of-function mutations in the ClC-1 muscle chloride channel have been known for 25 years to cause myotonia congenita, this discovery has led to little progress on development of therapy. Currently, treatment is primarily focused on reducing hyperexcitability by blocking Na⁺ current. However, other approaches such as increasing K⁺ currents might also be effective. For example, the K⁺ channel activator retigabine, which opens KCNQ channels, is effective in treating epilepsy because it causes hyperpolarization of the resting membrane potential in neurons. In this study, we found that retigabine greatly reduced the duration of myotonia in vitro. Detailed study of its mechanism of action revealed that retigabine had no effect on any of the traditional measures of muscle excitability such as resting potential, input resistance or the properties of single action potentials. Instead it appears to shorten myotonia by activating K⁺ current during trains of action potentials. Retigabine also greatly reduced the severity of myotonia in vivo, which was measured using a muscle force transducer. Despite its efficacy in vivo, retigabine did not improve motor performance of mice with myotonia congenita. There are a number of potential explanations for the lack of motor improvement in vivo including central nervous system side effects. Nonetheless, the striking effectiveness of retigabine on muscle itself suggests that activating potassium currents is an effective method to treat disorders of muscle hyperexcitability.

Keywords: Action potential; Excitability; Kv7; Muscle; Muscle contraction; Myotonia; Persistent inward current; Potassium channel; Retigabine; Sodium channel.

Fig 1.

Dose dependent reduction of the severity of myotonia by retigabine. A) Shown are three representative traces of myotonia in 9AC treated muscle following exposure to 0, 10, and 20 μM retigabine. B) Plot of the mean duration of myotonia following various doses of retigabine in 9AC treated muscle. ** = p < .01. n = 29 fibers from 5 mice for 0 μM retigabine, 20 fibers from 3 mice for 5 μM, 27 fibers from 3 mice for 10 μM and 22 fibers from 3 mice for 20 μM. C) Shown are traces from ClC^adr muscle fibers in the absence and presence of 20 μM retigabine. No myotonia is present following treatment with 20 μM retigabine.

Fig 2.

Examples of traces used to measure passive properties and single action potentials. A) Representative trace used to measure input resistance and time constant. B) Properties of action potentials that were measured.

Fig 3.

Retigabine lessens myotonia by reducing the afterdepolarization (AfD). Shown are two traces of myotonia from 9AC treated muscle. The top record is from a fiber in the absence of retigabine. The myotonia lasted for several seconds and continued beyond the time frame shown. The bottom record is from a fiber following treatment with 20 uM retigabine. The AfD peak fell below action potential threshold after only 3 myotonic action potentials. In the inset are shown the first two action potentials from myotonia in the absence and presence of 20 μM retigabine. The slope of the AfD is more than twice as steep in the absence of retigabine. AfD = after depolarization.

Fig 4.

Retigabine is effective in treating myotonia in vivo. A) Representative muscle force traces from a control and a ClC^adr mouse before and after treatment with retigabine. Top trace: the response of muscle from a wild type mouse to 2 s of 60 Hz stimulation of the sciatic nerve. Force is fully fused and there is immediate, complete relaxation following termination of nerve stimulation. Some fatigue is present during the 2s of stimulation. Middle trace: in a ClC^adr mouse, force generation during 60 Hz stimulation is normal, but relaxation following termination of stimulation is only partial and there is continued contraction secondary to myotonia for more than 5s. Bottom trace: force generation from the same myotonic gastrocnemius muscle shown in the middle trace 30 minutes after intraperitoneal injection of 30 mg/kg of retigabine. While myotonia is still present, it is reduced by close to 80%. B) A scatter plot of the integral of the post-stimulus muscle force*time relative to the normalized pre-treatment integral of a single twitch. Severity of myotonia was reduced by close to 75% following injection of retigabine (p < .01). The horizontal bar represents the mean. C) Scatter plot of peak muscle force in the 6 muscles recorded from before and 30 minutes after injection of retigabine. The horizontal bars represent the means before and after treatment with retigabine (p = 0.15).

Fig 5:

Lack of motor improvement following treatment with retigabine. Shown are scatter plots of the mean time of righting and mean time on the rotarod for 9 ClC^adr mice treated with either vehicle or retigabine. Each point represents the mean of 3 different treatment trials on different days. Error bars are not shown for clarity. The horizontal bars represent the means before and after treatment with retigabine. There was worsening of the time of righting following treatment with retigabine (p = 0.03). There was no difference in the time on the rotarod.

Fig 6:

The mechanism underlying efficacy of retigabine against myotonia. A) Firing of action potentials during voluntary movement causes build-up of K⁺ in t-tubules, which depolarizes the K⁺ equilibrium potential. This causes depolarization of the muscle membrane potential, which activates Na⁺ persistent inward current (NaPIC) and depolarizes the membrane potential to above action potential threshold. The result is involuntary repetitive firing (myotonia) that is responsible for muscle stiffness. B) Shown is a plot of the voltage dependence of activation of Kv7 channels in the presence and absence of retigabine (based on the work of (Tatulian, et al., 2001)). Retigabine causes a hyperpolarized shift in the voltage dependence of opening of Kv7 channels such that the channels activate in the subthreshold voltage range (light blue) between the K⁺ equilibrium potential and action potential threshold during runs of myotonia. With no retigabine, the voltage dependence of activation of Kv7 channels is relatively depolarized such that the channels are not significantly activated during myotonia. C) Following treatment with retigabine, Kv7 channels activate over the same voltage range as NaPIC and prevent NaPIC from depolarizing the membrane potential to action potential threshold. RMP = resting membrane potential, AP = action potential.