TRPV4 Antagonism Prevents Mechanically Induced Myotonia

Abstract

Objective: Myotonia is caused by involuntary firing of skeletal muscle action potentials and causes debilitating stiffness. Current treatments are insufficiently efficacious and associated with side effects. Myotonia can be triggered by voluntary movement (electrically induced myotonia) or percussion (mechanically induced myotonia). Whether distinct molecular mechanisms underlie these triggers is unknown. Our goal was to identify ion channels involved in mechanically induced myotonia and to evaluate block of the channels involved as a novel approach to therapy.

Methods: We developed a novel system to enable study of mechanically induced myotonia using both genetic and pharmacologic mouse models of myotonia congenita. We extended ex vivo studies of excitability to in vivo studies of muscle stiffness.

Results: As previous work suggests activation of transient receptor potential vanilloid 4 (TRPV4) channels by mechanical stimuli in muscle, we examined the role of this cation channel. Mechanically induced myotonia was markedly suppressed in TRPV4-null muscles and in muscles treated with TRPV4 small molecule antagonists. The suppression of mechanically induced myotonia occurred without altering intrinsic muscle excitability, such that myotonia triggered by firing of action potentials (electrically induced myotonia) was unaffected. When injected intraperitoneally, TRPV4 antagonists lessened the severity of myotonia in vivo by approximately 80%.

Interpretation: These data demonstrate that there are distinct molecular mechanisms triggering electrically induced and mechanically induced myotonia. Our data indicates that activation of TRPV4 during muscle contraction plays an important role in triggering myotonia in vivo. Elimination of mechanically induced myotonia by TRPV4 inhibition offers a new approach to treating myotonia. ANN NEUROL 2020;88:297-308.

Figure 1:

Generation and characterization of Trpv4^−/− mice. (A) Schematic representation of the targeting strategy. The structure of the endogenous mouse Trpv4 allele is shown at top, including the locations of the three genotyping primers, above the targeting vector in which Trpv4 exons 4 and 5 are flanked by loxP sites. Following excision of the Neo cassette, mice were outcrossed to a Sox2^Cre-expressing mouse strain enabling Cre recombinase (Cre)-mediated gene deletion. (B) Trpv4 mRNA levels in extensor digitorum longus muscle of adult WT and Trpv4^−/− mice, as assessed by RT-qPCR (n=3 for WT and Trpv4^−/−).

Figure 2:

Electrically- and mechanically-induced myotonia are present in both pharmacologic and genetic mouse models of myotonia congenita. A) Intracellular recording from muscle fibers during stimulation by injection of depolarizing current. In each of the three traces, a 200 ms injection of current (horizontal line under the trace) triggers repeated firing of action potentials during the current injection. In wild type muscle, there is no firing of action potentials following termination of current injection (0/63 fibers from 7 mice). In wild type muscle myofibers in which ClC-1 chloride channels are blocked with 100 μM 9AC and in muscle from ClC^adr mice, there is continued firing of action potentials (myotonia) following termination of current injection (58/58 fibers from 8 mice and 72/72 fibers from 9 mice, respectively). B) The arrangement of electrodes used to mechanically stimulate individual muscle fibers. Three muscles fibers are in focus in the image shown. A sharp electrode is impaled into fiber #1 on the right of the image. On the left, a blunt electrode is resting on the bottom of fiber #1, over 200 μm away from the sharp recording electrode. In the top image (No stimulation), the blunt electrode is gently resting on fiber #1. In the lower image the blunt electrode has been manually advanced to mechanically stimulate fiber #1. C) At the times indicated by vertical arrows, the blunt electrode was manually advanced. In wild type muscle, mechanical stimulation did not trigger myotonia (0/56 fibers from 8 mice). In both 9AC-treated and ClC^adr muscle, mechanical stimulation triggered myotonia in all fibers tested (42/42 fibers from 6 mice and 56/56 fibers from 8 mice, respectively). D) Examples of mechanically-induced depolarization in muscle when 1 μM TTX was added to the perfusate to block Na⁺ channels.

Figure 3:

Genetic deletion of TRPV4 channels eliminates mechanically-induced myotonia without affecting electrically-induced myotonia. A) Scatter plot of the relative Trpv4 transcript levels normalized to the reference gene Gusb for 6 wild type and 6 ClC^adr extensor digitorum longus muscles. The horizontal bars represent the mean of each group. B) In 9AC-treated muscle from Trpv4^+/+ mice, mechanically-induced myotonia was present in 21/28 fibers from 4 mice whereas in 9AC-treated muscle fibers from Trpv4^−/− mice, mechanically-induced myotonia was present in 0/28 fibers from 4 mice. At the times indicated by vertical arrows, a blunt electrode was manually advanced to mechanically stimulate the fiber being studied. C) Electrically-induced myotonia was present in 35/35 9AC-treated Trpv4^+/+ fibers from 4 mice and 22/22 Trpv4^−/− fibers from 3 mice.

Figure 4:

Treatment with small molecule inhibitors of TRPV4 greatly reduces mechanically-induced myotonia in ClC^adr muscle. A) Mechanically-induced myotonia was present in 28/28 vehicle-treated fibers from 4 mice, and 0/28 fibers treated with 1 μM HC-067047 from 4 mice. At the times indicated by vertical arrows, a blunt electrode was manually advanced to mechanically stimulate the fiber being studied. B) Electrically-induced myotonia was present in 33/33 vehicle-treated fibers from 5 mice and 48/48 HC-067047-treated fibers from 5 mice. C) Following application of vehicle 19/20 fibers from 3 mice had mechanically-induced myotonia. After application of GSK2193874, 3/20 fibers from 3 mice had mechanically-induced myotonia. D) Electrically-induced myotonia was present in 21/21 vehicle-treated fibers from 3 mice and 19/19 fibers treated with 1 μM GSK2193874 from 3 mice.

Figure 5:

Block of TRPV4 is effective in treating myotonia in vivo. A) The response of wild type muscle to 2 s of 60 Hz stimulation of the sciatic nerve. The horizontal bar under the trace indicates the period of 60 Hz stimulation of the sciatic nerve. Force is fully fused and there is immediate, complete relaxation following termination of stimulation. B) The response of muscle from ClC^adr mice to 60 Hz stimulation. The vertical dotted line in each trace represents the rapid relaxation of force that occurs in wild type muscle following termination of nerve stimulation. Pre-injection, force generation during 60 Hz stimulation is fused normally, but relaxation following termination of stimulation is only partial and there is continued contraction for many seconds secondary to myotonia. At 45 minutes post-injection of vehicle, myotonia is minimally changed, but is greatly decreased in mice that received either HC-067047 or GSK2193874. C) Scatter plot of the ratio of severity of myotonia post-injection to severity of myotonia pre-injection in each mouse. The horizontal bars represent the mean of each group. Following injection of vehicle there was no change in severity of myotonia. Following injection of either drug there was a marked reduction. ** indicates p < 0.01 versus vehicle.

Figure 6:

Distinct mechanisms underlying electrically- and mechanically-induced myotonia. Electrically-induced myotonia (top two rows): in wild type muscle Cl^- current plays a central role in controlling resting potential both at rest and during firing of action potentials during voluntary movement. Because of the stabilizing influence of Cl^- current, K⁺ build-up in t-tubules does not cause sufficient depolarization to trigger myotonia following voluntary activation of muscle. In myotonia congenita, muscle Cl^- current is reduced such that K⁺ build-up causes depolarization large enough to trigger myotonia. Mechanically-induced myotonia (bottom two rows): mechanical stimulation of wild type muscle activates TRPV4 channels, which depolarizes muscle. This depolarization is opposed by Cl^- current such that myotonia is not triggered. Activation of TRPV4 channels during mechanical stimulation of muscle with reduced Cl^- current causes depolarization sufficient to trigger myotonia.