NOVEL MECHANISMS UNDERLYING WARM-UP AND PERCUSSION MYOTONIA IN MYOTONIA CONGENITA

A Dissertation submitted in partial fulfillment of requirements for the degree of Doctor of Philosophy

By

Kevin Richard Novak B.S., University of Cincinnati, 2005 M.S., Wright State University, 2008

2017 Wright State University WRIGHT STATE UNIVERSITY GRADUATE SCHOOL

May 12, 2017

I HEREBY RECOMMEND THAT THE DISSERTATION PREPARED UNDER MY SUPERVISION BY Kevin Richard Novak ENTITLED Novel Mechanisms Underlying Warm-up and Percussion Myotonia in Myotonia Congenita BE ACCEPTED IN PARTIAL FULFUILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF Doctor of Philosophy.

Mark M. Rich, M.D. Ph.D. Dissertation Director

Mill W. Miller, Ph.D. Director, Biomedical Sciences Ph.D. Program

Robert E. W. Fyffe, Ph.D. Vice President for Research and Dean of the Graduate School

Committee on Final Examination

Mark M. Rich, M.D. Ph.D.

J. Ashot Kozak, Ph.D.

Andrew A. Voss, Ph.D.

Dan R. Halm, Ph.D.

Courtney Sulentic, Ph.D. Abstract Novak, Kevin Richard. Ph.D., Biomedical Sciences Ph.D. Program, Wright State University, 2017. Novel Mechanisms Underlying Warm-up and Percussion Myotonia in Myotonia Congenita.

Patients with myotonia congenita have muscle hyperexcitability due to loss-of- function mutations in the ClC-1 chloride channel in skeletal muscle, which causes spontaneous firing of muscle action potentials (myotonia), producing muscle stiffness.

Triggers for myotonia can occur voluntarily at the neuromuscular junction or involuntarily by striking the muscle with a reflex hammer (percussion myotonia). In patients, muscle stiffness lessens with exercise, a change known as the warm-up phenomenon. Our goal was to identify the mechanism underlying warm-up and percussion myotonia and to use this information to guide development of novel therapies.

To determine these underlying mechanisms, we used a drug to eliminate muscle contraction. This allowed for prolonged intracellular recording from individual muscle fibers during induction of warm-up and stretch-induced percussion myotonia in a mouse model of myotonia congenita. To investigate the warm-up phenomenon exercise was modelled in vitro by delivering 5000 action potentials at 20 Hz. Stretch-induced percussion myotonia was modelled in vitro by fabricating a blunt glass probe to stretch the muscle fiber mimicking the strike of the reflex hammer. Changes to action potential morphology following active exercise suggests slow inactivation of sodium channels as the mechanism contributing to warm-up. Reductions to stretch-induced depolarizations

iii and myotonia in response to mechanosensitive channel blockers strongly suggests the involvement of a stretch activated channel as the trigger for percussion myotonia. We propose that stretch seen during percussion myotonia is similar to stretch experienced by an antagonistic muscle while yielding to contraction. This stretch-induced myotonia may be contributing to patient stiffness. Drugs were investigated as potential therapeutics for accelerating warm-up or block of stretch activation. We found that enhancement of sodium channel slow inactivation using ranolazine reduces stimulation induced myotonic stiffness.

iv Table of Contents

Chapter I: Purpose and Specific Aims …..………………………………………………. 1

Chapter II: Significance / Background ………………………………………………….. 6

Chapter III: General Methods ……………………..…………………………………… 12

Chapter IV: Targeting sodium channel slow inactivation

as a treatment for stimulation induced myotonia ….……………..……… 17

Chapter V: Stretch-activated channels trigger stretch-induced myotonia …...…….….. 55

Chapter VI: General conclusions ...………...…………………………………………... 97

Chapter VII: References ……………………………...…..…………………………... 101

Appendix A: Commonly used abbreviations ……...…………………………………. 111

v List of Figures

Figure 1: In vitro model of warm-up ……………………………….………....……….. 30

Figure 2: Warm-up induced changes to single action potentials ……..………………... 39

Figure 3: Time and activity effect on resolution of warm-up phenomenon ….………... 41

Figure 4: Drug effects on myotonia…………………………………………….……..... 46

Figure 5: In vivo performance testing ...... 48

Figure 6: In vitro model of percussion myotonia.……..…………..………..…...... 61

Figure 7: Effects of stretch on ClCwt and ClCadr muscle fibers …………………….….. 69

Figure 8: Effects of impaled sharp recording electrode

manipulation on resting potential ……..……………………………………... 72

Figure 9: Degrees of depolarization in the presence of tetrodotoxin ….………….…… 75

Figure 10: Effect of HC-067047 on TRPV4+/+ and TRPV4-/- muscle ….………..……. 87

Figure 11: Depolarization in TRPV4+/+ and TRPV4-/- muscle

Exposed to HC-067047 ………………………….…………...…………….. 90

vi List of Tables

Table 1: Characteristics of Action Potentials in ClCadr Mice

and Unaffected Littermates …………..…………………………..……….... 32

Table 2: Characteristics of Action Potentials at Baseline and

After 5,000 APs ………………………………………………..…………… 36

Table 3: Summary of depolarization degrees in ClCwt and ClCadr ………………….... 77

Table 4: Effectiveness of HC-067047 for blocking stretch-induced myotonia .…...…. 81

Table 5: HC-067047 effect on excitability and

input resistance in ClCadr fibers ………………………………………..…… 84

Table 6: HC-067047 effect on excitability and

input resistance in ClCwt fibers …………………………………….....……. 85

Table 7: Summarized depolarization degrees in TRPV4+/+ and TRPV4-/- …………... 89

vii Chapter 1: Purpose & Specific Aims

Purpose

Myotonia congenita is a type of non-dystrophic skeletal muscle channelopathy characterized by a mutation to ClC-1 chloride channels. The lack of this channel causes muscles to experience a symptom known as myotonia. Patients describe myotonia as a feeling of delayed muscle relaxation and sustained muscle contraction. Humans as well as other mammals born with the nonfunctional ClC-1 channel experience the muscle stiffness and rigidity of myotonia upon contracting muscles forcefully after a period of muscle relaxation (Katzberg, Khan et al. 2010). This myotonic stiffness is thought to be triggered by stimulation of the muscles via the neuromuscular junction. If this were the case unwanted sustained contraction of the stimulated muscle would result in movement limited by that joints range of the motion. In order for stiffness to result the antagonistic muscle needs to oppose the sustained contraction generated by the stimulated muscle. We propose that during myotonic stiffness both the shortening stimulated muscle and the stretching antagonistic muscle are experiencing myotonia simultaneously. The purpose of this work is to identify possible targets to treat both the stimulation induced and stretch- induced components of myotonic stiffness.

This stiffness subsides with time, typically only lasting for a few minutes, and is referred to as the “warm-up phenomenon”. Once muscles are warmed-up they appear to function normally until they experience a period of relaxation (Lossin, Nam et al. 2012).

Neurological examination of patients with myotonia congenita reveals another

1 phenomenon called percussion myotonia. Percussion myotonia causes muscle stiffness or contraction from stretching the muscle belly by striking it with a reflex hammer (Rudel and Lehmann-Horn 1985). The purpose of this work is to identify possible mechanisms to explain warm-up and percussion myotonia, and possible pharmacological therapies to treat them.

Specific Aims

In order to address the specific aims in this dissertation, electrophysiological techniques were applied in vitro. These techniques will allow for experimentally induced muscle warm-up, determination of any changes that warm-up has on excitability, and identification of mechanism(s) responsible for those changes (Cannon and Corey 1993).

By targeting these mechanism(s) with FDA approved drugs, both in vitro as well as in vivo, we hope to activate the warm-up mechanism and inhibit the stimulation induced component of myotonic stiffness. Similarly, percussion or stretch-induced myotonia will be mimicked in vitro to identify an ion channel that maybe contributing to the stretch- induced component of myotonic stiffness.

2 Specific Aim I: To test the hypothesis that pharmacologically targeting sodium channel slow inactivation offers a beneficial alternative therapy to treat stimulation induced myotonia.

Published: Sodium Channel Slow Inactivation as a Therapeutic Target for Myotonia

Congenita. Annals of Neurology. 2015 February; Vol 77, Issue 2.

The sustained firing of muscle action potentials (myotonia) produces muscle stiffness following voluntary stimulation. With time and repeated muscle activation the stiffness subsides and the muscle function resembles that of a non-myotonic individual

(Birnberger, Rudel et al. 1975). Transitioning from this start of muscle stiffness to a state of normal function is known as the warm-up phenomenon. Mechanisms have been proposed to explain this warm-up (Birnberger and Klepzig 1979; Clausen 2003). The build-up of t-tubular potassium, increased sodium-potassium ATPase activity and slow inactivation of sodium channels all provide possible explanations. Warm-up occurs with continued muscle activity and muscle contraction, making it difficult to perform intracellular recordings. Contraction of the muscle results in movements that cause damaging tears to the sacrcolemea at the point of stationary electrode impalement. The application of N-benzyl-p-toluenesulfonamide (BTS) eliminates muscle contraction without affecting calcium handling or ion channel properties. BTS inhibition of muscle contraction will allow for long term stable recordings, permitting action potential recordings before and after induction of warm-up. Assessment of any changes to action potential morphology will indicate possible mechanism(s) contributing to warm-up. We

3 propose the altered state of muscle excitability following warm-up is the result of sodium channel slow inactivation. The use of drugs approved by the United States Food and

Drug Administration (FDA) could provide an alternative method for treating the stiffness associated with myotonia congentia. Currently no FDA-approved treatment for myotonia congenita exists (Trivedi, Cannon et al. 2014). A common method for managing symptoms of myotonia congenita is to avoid triggers that result in myotonia. Avoiding certain activities and cold temperatures have been suggested (Trivedi, Bundy et al. 2013).

Use-dependent blockers of the voltage gated sodium channels such as mexiletine

(Statland, Bundy et al. 2012) have been shown to decrease the degree of myotonia experienced (Desaphy, Carbonara et al. 2014; Trivedi, Cannon et al. 2014) and are the current standard of care. However, adverse side effects of mexiletine include epigastric discomfort, nausea, tremor, anxiety and headaches, and in patients with recessive myotonia congenita, there is a possibility of an increase in muscle weakness (Chrestian,

Puymirat et al. 2006). We propose that more effective therapy is available using an FDA approved drug that enhances sodium channel slow inactivation. Both lacosamide and ranolazine increase slow inactivation by inducing a hyperpolarized shift in the voltage dependence of slow inactivation (Errington, Stohr et al. 2008; El-Bizri, Kahlig et al.

2011; Niespodziany, Leclere et al. 2013; Peters, Sokolov et al. 2013; Kahlig, Hirakawa et al. 2014). We compared the in vitro and in vivo efficacy of lacosamide and ranolazine in treating myotonia to mexiletine.

4 Specific Aim II: To test the hypothesis that stretch-activated channels are the underlying trigger for percussion or stretch-induced myotonia.

Myotonia can be triggered through mechanical means and is known as percussion myotonia (Chrestian, Puymirat et al. 2006). During neurological assessment, percussion myotonia occurs when the belly of a muscle is struck with a reflex hammer (Rudel and

Lehmann-Horn 1985). Muscle contraction from the resulting myotonic run of action potentials forms a transient dimple in the muscle where it was struck. The myotonic action potentials formed are not the result of T-tubular potassium build up since there were no action potentials prior to the hammer strike, nor are they the result of muscle stimulation at the neuromuscular junction. The exact cause of the stretch-induced myotonic action potentials remains unknown. Stretch channels have been identified in many tissues including skeletal muscle (Kunert-Keil, Bisping et al. 2006; Gailly 2012).

Mechanical or stretch-activated channels exist on the skeletal muscle’s membrane, and are activated by membrane distortion causing fiber depolarization. Many of these channel types are known to depolarize cells in response to stretching by allowing the cations sodium and calcium to pass through them and enter the cell. Transient receptor potential

(TRP) channels, specifically TRPV4, have been found to respond to stretch in skeletal muscle (Ho, Horn et al. 2012). Through the use of a specific TRPV4 blocker and TRPV4 knockout mice we show the involvement of stretch activated TRPV4 in stretch-induced myotonia.

5 Chapter II: Significance/Background

Alterations to ion channels can result in channelopathies. These alterations can create changes in ion channel surface expression, ion channel gating, or functionality.

Channelopathies in skeletal muscle were the first to be studied and were found to result in a variety of disorders that influence muscle excitability (Cannon, Brown et al. 1993).

Since muscle excitability is coupled to muscle contraction, changes in the excitability can manifest as episodes of paralysis or weakness when excitability is lost or episodes of stiffness when excitability is increased. The majority of skeletal muscle channelopathies are inherited in an autosomal dominant fashion, while few have autosomal recessive inheritance. Regardless of their inheritance, skeletal muscle channelopathies are rare, and only affect 1 out every 100,000 people (Horga, Raja Rayan et al. 2013). It is common for these channelopathies to affect only skeletal muscle, however some may have additional effects on other excitable tissues like cardiac muscle, the central nervous system, or peripheral nerves. Numerous ion channels in skeletal muscle have been thoroughly studied (Catterall 2000) and the influence that they have on excitability is well known

(Jurkat-Rott, Fauler et al. 2006).

Skeletal muscle channelopathies have been shown to result from multiple ion channel types. At the neuromuscular junction mutations to both the presynaptic and postsynaptic parts of the neuromuscular junction result in congenital myasthenic syndrome (Engel, Ohno et al. 2003). Disruptions of excitation-contraction coupling and calcium homeostasis (Jurkat-Rott and Lehmann-Horn 2005) are seen in malignant hyperthermia and central core disease, resulting from mutations to the sarcoplasmic

6 reticulum ryanodine receptor (Ryr1) or the sarcolemma voltage gated calcium channel

(Cav1.1) (Monnier, Procaccio et al. 1997). Mutations to any of the various voltage-gated ion channels will have a profound influence on the muscle’s excitability. As previously mentioned alterations to excitability can result in episodes of reduced excitability leading to weakness or periodic paralysis, as well as episodes of heightened excitability leading to stiffness or myotonia (Cannon 2002; Jurkat-Rott and Lehmann-Horn 2005).

Myotonia is a common symptom in a family of skeletal muscle channelopathies called non-dystrophic myotonia, and can result from mutations to either the Nav1.4 sodium channel (Cannon, Brown et al. 1993) or the chloride channel ClC-1. Myotonia has been described as sustained muscle contraction and delayed muscle relaxation after voluntary stimulation of the muscle (Heatwole, Statland et al. 2013). Myotonia congenita is a type of non-dystrophic myotonia that is caused by a reduction in skeletal muscle chloride conductance. A variety of different mutations to the ClC-1 gene have been identified as the culprit for the reduced chloride conductance (Lipicky, Bryant et al. 1971;

Koch, Steinmeyer et al. 1992). The mode of gene inheritance was first identified as autosomal dominant in the late 19th century by Thomsen. However approximately 100 years later in 1957 Becker discovered a recessive mode of inheritance which elicits a more severe myotonia. In both forms of myotonia bursts of muscle action potentials continue for seconds to minutes after the motor neuron has ceased firing, leading individuals to feel “stiff”. The duration of the unwanted muscle contracting stiffness is dependent on how long the muscle continues to fire action potentials after stimulation from the motor neuron has stopped. Duration and intensity of the post stimulus contraction will vary and is dependent on the degree of muscle activity prior to

7 stimulation (Trivedi, Bundy et al.). The most prominent stiffness being seen following forceful muscle contraction following prolonged relaxation. However with continuous movement and sustained contraction the intensity of myotonia (stiffness) begins to fade to the point that muscle excitability no longer seems impaired. This transition of muscle from a myotonic state to an asymptomatic state is referred to as the “warm-up phenomenon” (Birnberger, Rudel et al. 1975; Horlings, Drost et al. 2009).

When compared to other excitable cells like neurons or cardiomyocytes, skeletal muscle exhibits a high level of resting chloride conductance. 70% to 80% of skeletal muscle’s resting membrane conductance (Hodgkin and Horowicz 1959; Palade and

Barchi 1977) is attributed to chloride. This creates a buffer and stabilizes the muscle resting potential near the chloride equilibrium potential of -90mV (Dulhunty 1978). The majority of this chloride conductance is from the ClC-1 chloride channel found in abundance on the T-tubular network of skeletal muscles (Lamb, Murphy et al. 2011).

Over 130 different mutations to the ClC-1 gene have been identified and associated with myotonia congenita (Lossin and George 2008). These mutations result in low membrane expression of ClC-1 from the inability to translate functional channels

(Lorenz, Meyer-Kleine et al. 1994; Papponen, Nissinen et al. 2008) and a variety of changes that cause depolarizing shifts in the voltage dependence of activation (Fahlke,

Rudel et al. 1995; Pusch, Steinmeyer et al. 1995). These depolarizing shifts in activation result in reduced availability of ClC-1 at the negative 85mV resting potential. Regardless of the mutation type, myotonia congenita produces a non-function chloride channel.

The role of chloride conductance in myotonic muscle was first investigated by

Shirley Bryant when internal intercostal muscle from normal goats and goats with

8 myotonia congenita were compared, finding a significant loss of chloride conductance in the myotonia muscle fibers (Lipicky and Bryant 1966; Lipicky, Bryant et al. 1971).

- Normal muscle exposed to a solution containing impermeant sulfate (SO4 ) substituted for permeant chloride yields myotonic muscle similar to that found in the myotonic goats

(Adrian and Bryant 1974). This change in extracellular anion causes the electrochemical force for chloride to shift from inward to outward, and leaves potassium as the only component contributing to resting potential. No change in resting potential results from this change since the Nernst potential for potassium is very similar to that of chloride.

The large reduction in the resting membrane conductance leads to increased membrane resistance and a decrease in the current needed to elicit an action potential (rheobase).

Loss of chloride on the extracellular side of the cell leads to changes in the equilibrium

Others have provide insight through the use of aromatic carboxylic acids like 9-

Anthracene carboxylic acid (9-AC), an identified chloride channel blocker in rat muscle

(Furman and Barchi 1978; Skov, Riisager et al. 2013). Concentration related studies found that a reduction of at least 50% of the chloride conductance is needed to generate myotonia experimentally (Furman and Barchi 1978) which agreed with computer modeling (Adrian and Marshall 1976).

The most notable characteristic of myotonic skeletal muscle is that it continues to generate action potentials and contract after being stimulated. The lack of negatively charged chloride influx reduces the ability of the muscle to re-establish and hold its resting potential after being stimulated. The inability to re-establish a stable resting potential allows for bursting of post-stimulus action potentials that may continue for seconds to minutes and result in involuntary muscle stiffness.

9 While lack of chloride conductance would be expected to increase excitability, it does not fully explain the sustained firing following cessation of voluntary contraction. In

1974 Adrian and Bryant hypothesized that this sustained firing could be the result of potassium buildup in the extracellular space and that the transient increase in extracellular potassium was the origin of the post-stimulation depolarization (Adrian and Bryant

1974). Typically both chloride and potassium contribute to the repolarizing phase of the muscle action potential. However in myotonic muscles potassium is solely responsible for the repolarization. During repolarization potassium moves out of the myoplasm through channels on both sarcolemma and vast transverse tubule system. The T-tubular network holds the majority of the muscle’s membrane. Each T-tubule provides a very small space, measuring approximately 40nm in diameter and diving roughly 30m into the center of the myocyte (Peachey 1965). The microscopic nature of the T-tubular spaces make it very difficult for potassium ions to diffuse out of the T-tubular network into the surrounding extracellular space.

A local environment is created in the T-tubular network that can experience a

0.4mM elevation in potassium concentration with each action potential repolarization

(Adrian and Marshall 1976; Cannon, Brown et al. 1993; Wallinga, Meijer et al. 1999).

With each successive action potentials, the T-tubular potassium concentration increases, and depolarizes the potassium reversal potential. In normal muscle the abundance of ClC-

1 channels allows Cl- to play a role in repolarization, removing the high dependence on potassium for repolarization. The chloride conductance also prevents the depolarization induced by potassium build up in the T-tubular network. Potassium buildup in the T- tubular network has been shown to generate depolarizations that last for hundreds of

10 milliseconds in muscle fibers from both myotonic goats and those exposed to chloride- free extracellular solutions (Adrian and Bryant 1974).

There is a temporal effect to potassium buildup. With high frequency stimulation potassium buildup occurs more drastically and rapidly since there is not adequate time between successive action potentials for T-tubular potassium levels to diminish. After just 3 to 10 stimulated action potentials the T-tubular potassium concentrations become sufficient to generate myotonia and fuel sustained depolarizations for successive non- stimulus driven action potentials. This point was even further proven by detubulating the muscle fiber and checking for myotonia. Detubulation electrically disconnects the surface sarcolemma from the network of T-tubules. Detubulation has been accomplished by inducing temporary hyperosmolar shock, where the cell is exposed to solutions with different osmolarities to shrink and then swell the muscle fiber before returning to physiological buffer. This leaves the muscle fibers surface sarcolemma excitable and disconnected from the T-tubular network (Dulhunty and Gage 1973). Detubulation has been effective at eliminating the depolarization that occurs after high frequency action potential stimulation and blocks the resulting myotonia (Adrian and Bryant 1974; Cannon and Corey 1993).

11 Chapter III: General Methods

Using in vitro electrophysiology in combination with behavioral analysis, we investigate the mechanism(s) underlying warm-up phenomenon and percussion myotonia. To unexplained muscle phenomenon seen in non-dystrophic myotonia’s such as myotonia congenita. The general methods utilized are described in detail below.

Animal Use

Validity of the mouse model of myotonia congenita

A key aspect of any study using an animal model of a human condition is the validity of the animal model. We used the Clcn1adr-mto2J mouse model of myotonia congentia to study abnormal regulation of muscle excitability. ClCadr mice have a null mutation in the ClC-1 chloride channel and thus have a recessive form of myotonia congenita identical genetically to the recessive form of myotonia congentia in patients.

ClCadr mice faithfully recapitulate both the muscle stiffness due to myotonia and the warm-up phenomenon in which muscle performance improves with exercise (Mehrke,

Brinkmeier et al. 1988; Novak, Norman et al. 2015). We will do all experiments on both male and female mice and previously have found no difference between them.

All animal procedures used are approved by Wright State University Laboratory

Animal Care and Use Committee. Detailed electrophysiological analysis was performed on adult mice 8 to 12 weeks old (20-25g). Experiments were performed using a colony of

12 ClCn1adrmto2J (ClCadr) mice, which have a null mutation in the ClC-1 gene and came from the swr/j background. The mice were obtained from Jackson Laboratory (Bar Harbor,

ME) and a breeding colony was established. Myotonia was identified clinically in ClCadr mice via myotonic appearance of the animals as previously described (Hoppe, Lehmann-

Horn et al. 2013). Asymptomatic littermates were used as controls; two-thirds of asymptomatic mice were likely heterozygous for the ClC-1–null mutation. As unaffected littermates have previously been shown not to have myotonia or alteration in macroscopic chloride current, we did not make an effort to distinguish them from wild-type mice

(Mehrke, Brinkmeier et al. 1988; Reininghaus, Fuchtbauer et al. 1988). For this reason all unaffected littermates both heterozygous and wild type will be referred to as ClCwt while

ClCadr will refer to mice with Beckers myotonia congenita.

Intracellular Recordings and Stimulation Protocol:

Mice were euthanized by placing the animal in a chamber that was slowly filled with 100% carbon dioxide gas until the animal was unconscious, after which the flow rate was increased until the mouse was no longer breathing. After removing the mouse from the chamber cervical dislocation was performed as a secondary means of euthanasia. Immediately following euthanasia removal of the extensor digitorum longus

(EDL) muscle began. The EDL was chosen since all of its muscle fibers attach to bone via tendons, preventing muscle damage from cutting fibers. Extreme care was taken to avoid damage by over stretching or cutting muscle fibers. The proximal and distal tendons were cut leaving enough tendon to pin the muscle via its tendons to a sylgard

13 coated dish. Experiments were done on the first muscle while the second muscle was maintained in oxygenated physiological buffer. The second muscle was used within 3 hours of euthanizing the mouse. All muscles were maintained and recorded from at temperatures between 20 - 23°C. We have found that muscle electrical properties are stable for up to 6 hours if the muscle is maintained in adequately oxygenated solution.

Data from both muscles were pooled and considered one sample for statistical purposes.

The recording chamber was continuously perfused with 40ml of recirculating ringer solution containing (in millimolar) NaCl, 118; KCl, 3.5; CaCl2, 2; MgSO4, 0.7; NaHCO3,

26.2; NaH2PO4, 1.7; glucose, 5.5 (pH 7.3 – 7.4, 20 – 22°C), and equilibrated with 95%

O2 and 5% CO2.

To prevent contraction of muscle fibers, muscles were loaded with 50M N- benzyl-p-toluenesulfonamide (BTS) (Tokyo Chemical Industry, Tokyo, Japan)

(Macdonald, Pedersen et al. 2005) prior to recording. BTS was dissolved in dimethylsulfoxide (DMSO) and added to perfusate; maximal vehicle concentration of

DMSO was 0.1% which was tested against the control. The maximal DMSO concentration was less than 0.15%, which has been found to affect resting membrane properties of rat skeletal muscle (Pedersen, de Paoli et al. 2009). Muscle membranes were stained for 3 minutes with 10M 4-(4-diethylaminostyrl)-N-methylpyridinium iodide

(Molecular Probes, Eugene, OR) and imaged with an upright epifluorescence microscope

(Leica DMR, Bannockburn, IL) as previously described (Wang, Pinter et al.).

For intracellular recordings, muscle fibers were impaled with sharp microelectrodes filled with 3M KCl solution containing 1mM sulforhodamine to

14 visualize the electrodes with epifluorescence. Electrode resistances were between 15 and

30M, and capacitance compensation was optimized prior to recording.

Statistics and Analysis

Fibers with resting potentials more depolarized than -75mV were excluded from analysis. Input resistance was measured using a 60-millisecond, 10 nA hyperpolarizing current pulse. Input resistance was determined by dividing steady state voltage change by the current injected (V / I = R). Action potentials collected using positive current stimulation were analyzed for threshold, rate of rise, action potential peak as well as action potential half width, using Spike2 software. (Cambridge Electronic Design

Limited, Cambridge, England). To determine action potential threshold and rate of rise the derivative of voltage with respect to time was taken. Threshold was defined as the voltage when the dV/dt was 10mV/ms, and rate of rise of defined as the maximum dV/dt.

Action potential peak was recorded as the maximum voltage experienced during the action potential, while action potential half width was defined by the time difference at the half amplitude voltage (difference of resting potential and peak).

To determine statistical significance, paired T-tests were used when comparing two data sets collected from the same EDL muscle. In some cases 6 individual statistical comparisons were made from a family of data all collected from the same EDL muscle.

When making 5 or more comparisons from the same muscle the chances of a false positive result increase. In order to compensate for this potential error a Bonferroni correction is applied. Statically significant changes were defined by a p-value being less

15 than or equal to 0.05. When applying the Bonferroni correction this p value is divided by the number of individual tests performed from the family of data, in this case 6. The critical p value for significance with the applied Bonferroni correction is 0.008.

16 Chapter IV

Specific Aim I: To test the hypothesis that sodium channel slow inactivation is the mechanism underlying the warm-up phenomenon and that its pharmacological enhancement offers an alternative therapy to stimulation induced stiffness associated with myotonia congenita.

Introduction:

Warm-up Phenomenon

During myotonia, sustained firing of muscle action potentials presents as stiffness that mimics a muscle cramp without the pain or discomfort (Katzberg, Khan et al.). The sustained firing of myotonic action potentials and stiffness will eventually diminish and the muscle may appear almost asymptomatic if activity is continued (Lossin and George

2008). Reaching this asymptomatic state is known as the “warm-up phenomenon”. The ability of myotonic muscle to experience warm-up is dependent on time. The length of relaxation will influence the warm-up effect. With longer periods of relaxations there is a greater chance that the next stimulation will generate myotonia. The first 30 – 60 seconds of relaxation following full warm-up, muscles experience little to no myotonia when stimulated. However over the next one to five minutes of relaxation the degree of myotonia will continue to increase upon muscle stimulation (Birnberger and Klepzig

1979). Warm-up is spatially limited, affecting only the contracting muscle, muscle fibers

17 cannot generate warm-up for each other (Birnberger and Klepzig 1979). Contrary to the name temperature has not yet been identified as a factor in warm-up.

The exact cause of warm-up is still unknown, but many hypotheses have been proposed. It has been speculated that an increase in sodium potassium ATP-ase activity can provide an explanation. In normal muscle increased activity of the Na/K ATPase is thought to occur with increased muscle activity. This occurs in an attempt to restore the sodium and potassium concentrations across the membrane and prevent reduced muscle excitability. Within 10 seconds the sodium efflux can increase to 20 times the resting rate

(Nielsen and Clausen 1997). This timeframe for the increased ATPase activity coincides with the time frame for onset of warm-up (Clausen 2003). This provides an explanation for the warm-up phenomenon, as its increased activity would remove the depolarizing buildup of potassium in the T-tubules allowing for more stable resting potentials to obtained. However the use of the Na/K ATPase inhibitor ouabain was not able to prevent warming up of skeletal muscle in human (Van Beekvelt, Drost et al. 2006). Activity dependent decreases in intracellular pH have also been hypothesized to cause warm-up

(Birnberger and Klepzig 1979). In mouse muscle exposed to low chloride or to drugs that provoke myotonia, lowering pH reduced myotonia. At a pH of 7.0 the maximal amplitude of myotonic contraction was reduced 25%, and at a pH of 6.8 the contractions were decreased to an almost asymptomatic state. Lower pH has been shown to reduce maximal sodium conductance (Hille 1968; Drouin and Neumcke 1974).

Another proposed mechanism for warm-up is the buildup of potassium in the network of T-tubules during high rate of action potential firing. Potassium build up occurs in the T-tubules during repeated myotonic action potential production. Each action

18 potential can increase the local extracellular T-tubular potassium contraction by 0.4mM

(Cannon, Brown et al. 1993; Wallinga, Meijer et al. 1999). As the T-tubular potassium concentrations rise with repetitive firing they eventually reach a level where potassium will depolarize the muscle between action potentials. This would reduce the time spent at negative membrane potentials and slow the recovery of sodium channel inactivation.

With increased sodium channel inactivation the membrane becomes less excitable stopping the myotonic discharges (De Bellis, Carbonara et al. 2016).

Previous attempts to investigate warm-up have primarily measured muscle contraction and force to study myotonia, while others have used EMG

(electromyography).(Chrestian, Puymirat et al. 2006; Cannon 2015) Because of the lack of intracellular recordings during warm-up, the exact cause of warm-up still remains undetermined.

Slow Inactivation of Nav1.4

Changes in membrane potential, membrane conductance, and slow inactivation of sodium channels have all been proposed to underlie warm-up (Lossin and George 2008;

Lossin, Nam et al. 2012). Nav1.4 is the dominant sodium channel isoform found on skeletal muscle, and is responsible for controlling its excitability. Being the sole source of sodium current. Nav1.4 may offer some insight into explaining the warm-up phenomenon. Some parallels exist between slow inactivation of Nav1.4 and warm-up.

First, is the fact that both slow inactivation and warm-up occur following increased activity (Lossin, Nam et al. 2012). Secondly both warm-up and slow inactivation require

19 continuous activity to be maintained. Nav1.4 channels recover from slow inactivation hyperpolarized potentials similar to how muscle returns to its myotonic state following rest, with a comparable time course. Finally both slow inactivation and warm-up are limited spatially. Slow inactivation of Nav1.4 requires channel activity to develop similar to warm-up which also occurs with activity.

Unfortunately, it has been impossible to directly study the mechanism underlying warm-up because muscle contraction makes it impossible to perform intracellular recording from individual muscle fibers during the stimulation necessary to induce warm- up. Because of this technical challenge, the mechanism underlying warm-up has remained unknown since its original description almost 40 years ago.(Birnberger, Rudel et al. 1975) A better understanding of the mechanism underlying warm-up could help in the development of more effective therapy.

The drug BTS was found to eliminates muscle contraction with minimal effect on excitability (Cheung, Dantzig et al. 2002; Macdonald, Pedersen et al. 2005). BTS has made it possible to perform intracellular recordings in muscle fibers during thousands of action potentials (Pedersen, de Paoli et al. 2009; Pedersen, Macdonald et al. 2009). BTS blocks muscle contraction by inhibiting interaction between myosin and actin, while causing minimal alteration in calcium handling (Cheung, Dantzig et al. 2002), such that

Ca-dependent processes are unperturbed. Using BTS to eliminate muscle contraction in a mouse model of myotonia congenita, we were able to observe the electrophysiological correlate of stimulation induced myotonic stiffness at baseline, perform intracellular recordings, and induce warm-up in isolated muscle fibers in the absence of muscle contraction. This allowed us to examine mechanisms underlying warm-up.

20 There is currently no FDA-approved treatment for myotonia congenita (Trivedi,

Cannon et al. 2014). A common method for managing symptoms of myotonia congenita is to avoid triggers that result in myotonia. Avoiding certain activities and cold temperatures have been suggested (Trivedi, Bundy et al. 2013). Use-dependent blockers of the voltage gated sodium channels such as mexiletine (Statland, Bundy et al. 2012) have been shown to decrease the degree of myotonia experienced (Desaphy, Carbonara et al. 2014; Trivedi, Cannon et al. 2014). Possible adverse side effects of mexiletine include epigastric discomfort, nausea, tremor, anxiety and headaches, and in patients with recessive myotonia congenita, there is a possibility of an increase in muscle weakness

(Chrestian, Puymirat et al. 2006).

This provides the need for an improved or alternative therapy, one with better alleviation of stiffness or with fewer side effects. Muscles that are in the warmed up state do not exhibit stiffness. It would be ideal to find a way to get muscle into the warmed up state without first experiencing stiffness and prolonged activity. By looking at the mechanism of warm-up we hope to identify a pharmacological target to prevent stimulation induced myotonic stiffness.

Our inference that slow inactivation of sodium channels contributes to elimination of myotonic stiffness during warm-up suggested that enhancing slow inactivation of sodium channels might offer a more physiologic approach to reducing muscle sodium current. Ranolazine and lacosamide are FDA-approved drugs whose mechanism of action is an increase in sodium channel slow inactivation (Errington, Stohr et al. 2008; El-Bizri,

Kahlig et al. 2011; Niespodziany, Leclere et al. 2013; Peters, Sokolov et al. 2013; Kahlig,

Hirakawa et al. 2014). We tested these 2 potential novel therapies for myotonia congenita

21 both in vitro and in vivo in a mouse model of the disease. We compared the efficacy of ranolazine and lacosamide, both in vitro and in vivo, to mexiletine, the current standard pharmacological therapy for myotonia congenita (Statland, Bundy et al. 2012; Trivedi,

Cannon et al. 2014).

Methods

Model of Warm-up

To induce the warm-up phenomenon, stimulation at 20Hz was applied for differing lengths of time to trigger 1,000, 2,500, or 5,000 action potentials. Fiber excitability following the stimulation was classified based on excitability of normal muscle. Normal muscle is able to generate at least 3 action potentials during a 60- millisecond pulse, but does not fire action potentials following termination of current injection. If a fiber generated 3 action potentials and fired action potentials after cessation of a 60-millisecond current injection in ≥ 6 of 10 trials, it was classified as hyperexcitable. If a fiber was unable to generate 3 action potentials during a 60- millisecond current injection, it was classified as hypoexcitable. If a fiber generated 3 action potentials and fired action potentials after cessation of 60-millisecond current injection in ≤5 of 10 trials, it was classified as having normal excitability. In almost all cases, fibers had myotonia on either 10 of 10 trials or 0 of 10 trials.

22 Drugs Used:

Ranolazine and mexiletine were purchased from Sigma-Aldrich (St Louis, MO) and were directly dissolved in Ringer solution for in vitro experiments or phosphate – buffered saline for injection into mice. Premixed lacosamide (10mg/ml) was purchased from UCB (Smyrna, GA) and diluted in Ringer for in vitro experiments or phosphate buffered saline for intraperitoneal injections.

In Vitro Drug Study:

We compared the efficacy of lacosamide and ranolazine in treating myotonia to that of mexiletine, a sodium channel blocker that is the current standard of care (Statland,

Bundy et al. 2012). ClCadr muscle was prepared as described in the intracellular recording protocol above. Baseline recordings were made in a few fibers before drugs were added to the recirculating perfusate. Drug doses were titrated from a low dosage with modest effect on excitability, to a high dosage that caused hypoexcitability (mexiletine and ranolazine) or that greatly exceeded concentrations used clinically (lacosamide). The following doses were tested sequentially on a single EDL muscle (in micromoles per liter): mexiletine, 10, 25, 50, and 100; lacosamide, 50, 100, 300, and 600; ranolazine, 5,

25, 50, and 100. Each dose included collecting data from 10 muscle fibers before

23 stepping to the next dose and recording from 10 more muscle fibers. Each drug dose was tested on 3 different EDL muscles from 3 different mice. The data for each dose was pooled from these 30 muscle fibers. For each muscle fiber, we first qualitatively assessed the presence of spontaneous firing of action potentials at the time of impalement. Next, we quantitatively measured excitability by inducing myotonia via current injection.

Fibers were classified as hyperexcitable, normally excitable, or hypoexcitable as described in the intracellular recording protocol, and the number of each class for each dose was recorded.

In Vivo Dose Finding Study:

Mexiletine has been found to be effective in treating myotonia congenita in vivo in ClCadr mice at 5 and 10mg/kg, via subcutaneous injection (De Luca, Pierno et al.

2004). Neither ranolazine nor lacosamide have been studied as treatment for myotonia congenita. However, ranolazine has been given daily to mice at a dose of 50mg/kg via intraperitoneal injection looking for long term changes in excitability, (Nodera and

Rutkove 2012) and lacosamide has been given to mice at doses up to 100mg/kg via intraperitoneal injection looking for anticonvulsant effects during epilepsy (Stohr,

Kupferberg et al. 2007). Based on these studies and our in vitro data, we chose the following initial drug doses for our dose-finding study: mexiletine at 5mg/kg, ranolazine at 12.5mg/kg, and lacosamide at 12.5mg/kg. Drugs were delivered via intraperitoneal injection to 3 treatment groups of 4 mice each, with a fourth control group injected with vehicle. Dosages of each drug were doubled for each successive step in this study. Three

24 days were given before the next doubled dose was administered, to allow time for drug elimination. All 3 drugs have half-lives significantly less than 1 day in humans (Begg,

Chinwah et al. 1982; Chaitman 2006; Kelemen and Halasz 2010); thus, 3 days allowed for >5 half-lives of drug decay. Gross motor performance (walking and interacting with the environment) was monitored at each dosage, until dosage resulted in apparent toxicity. For lacosamide, clear sedation and ataxia were apparent at a dose of 50mg/kg.

For ranolazine, sedation and ataxia were apparent at 100mg/kg. As toxicity was obvious, higher doses of lacosamide and ranolazine were not tested. Mexiletine showed no beneficial effects at 5mg/kg, and possible mild ataxia was apparent at 40mg/kg. While monitoring motor performance during these trials, we also determined the time course of each drug’s maximal impact. All 3 drugs had positive effects on motor function within 5 minutes of injection and continued to have positive effects for at least 30 minutes.

In Vivo Study of Therapeutic Drug Effects:

Based on data from the dose-finding study, we decided to test 3 dosages of mexiletine (10, 20, and 40mg/kg), 2 dosages of lacosamide (12.5 and 25mg/kg), and 3 dosages of ranolazine (12.5, 25, and 50mg/kg). We treated 16 mice and measured motor performance at both 5 and 15 minutes after injection, with the examiner blind to treatment status. All mice were tested for baseline motor function before each treatment.

Motor function was analyzed by timing the righting reflex 3 times (De Luca, Pierno et al.

2004; Desaphy, Carbonara et al. 2014) and by scoring 2 performance trials on a modified rotarod test. In a traditional rotarod test, mice are placed on a stationary rod and the speed

25 of the rod is gradually increased until the mice can no longer stay on the rod. We found this method gave mice time to warm-up and was therefore insensitive to motor dysfunction. To better measure treatment effect on muscle stiffness prior to warm-up, we altered the rotarod test as follows. Mice were placed on a stationary rod; then the rod was turned on at a single, intermediate speed. Test length was 5 seconds, and motor performance was scored on a 0 to 3 scale as follows: 0 for falling off a stationary rod, 1 for falling off as soon as the rod was turned on, 2 for holding on and being rotated for >5 seconds without walking, and 3 for walking to stay upright for >5 seconds. After baseline was established, all 16 mice were given the same intraperitoneal treatment and dosage.

Motor function was again analyzed at 5 and 15 minutes after injection, which allowed mice sufficient time to recover from any warm-up induced by the baseline trials.

The averaged post-treatment righting times and the averaged post-treatment rotarod scores (with the 5- and 15-minute time points averaged together) were compared to the averaged baseline performance for each mouse. As the same 16 mice were used for all treatment and control groups, at least 3 days were given between studies to allow time for drug elimination, as described in the dose-finding study. As no mouse demonstrated significant variation in baseline motor performance from 1 pretreatment trial to another, there was no evidence that previous drug treatment had any lingering effect.

26 In Vivo Measure of Drug Impact on Myotonia:

The dose of each drug that gave the best improvement in motor performance in the previous trial was selected for this study (mexiletine, 20mg/kg; ranolazine, 50mg/kg; lacosamide, 25mg/kg), plus a saline control; thus, 12 total mice were used, 3 for each treatment group. Mice were anesthetized with inhaled isoflurane. Isoflurane appeared to have little effect on myotonia, as prolonged and frequent myotonia on electromyogram

(EMG) was present in all ClCadr mice at baseline. Muscles impaled in vivo included the left paraspinal and gastrocnemius muscles prior to drug injection, and the right paraspinal and gastrocnemius muscles after drug injection. Prior to treatment, the baseline degree of myotonia was assigned a subjective severity by 2 blinded EMG examiners using standard

EMG technique: 0 = no action potentials firing from needle insertion; 1 = firing upon insertion and subsiding within 2 seconds of needle movement; 2 = firing upon insertion and lasting >2 seconds, but subsiding within 10 seconds; 3 = continued spontaneous firing in the absence of needle movement. The severity of myotonia was reassessed 10 minutes after drug injection. Myotonia levels from both paraspinal and gastrocnemius were averaged together. The pretreatment and post-treatment myotonia averages were compared in each mouse to determine the degree of drug impact on myotonia. After recordings, mice were euthanized by carbon dioxide inhalation, followed by cervical dislocation.

Results

27 ClC mice have the warm-up phenomenon in vivo:

ClCadr mice that are homozygous for the null mutation in the ClC-1 gene have severe muscle stiffness that manifests as impaired ability to run (van Lunteren, Spiegler et al. 2011), a stiff gait, and an inability to rapidly right after being placed in a supine position. When ClCadr mice were placed in a supine position, they initially took 3.5 ± 1.0 seconds (n = 10 mice) to right themselves. With repeated testing over 30 seconds, the mice were able to right themselves within an average of 1.7 ± 0.3 seconds (p<0.01). The motor improvement appears similar to the warm-up phenomenon experienced by patients with myotonia congenita. We used ClCadr mice to study the mechanism underlying the warm-up phenomenon. We were unable to flip unaffected littermates into the supine position, and they did not show any indication of stiffness. The inability to genotype for mutant alleles made it impossible to designate ClC+/+ from ClC+/-, unaffected littermate or

ClCwt is to describe these mice that did not display the phenotypical myotonic stiffness and could not be flipped supine.

Warm-up Can Be Induced In Vitro in ClCadr Muscle in the Absence of Muscle

Contraction:

When extensor digitorum longus muscle fibers from 8- to 12-week-old mice were impaled in vitro with two sharp electrodes, the electrophysiological correlate of the severe stiffness of ClCadr mice at baseline was easily observed. Impalement triggered

28 spontaneous runs of action potentials, something never seen in wild type muscle. After allowing the membrane potential to stabilize, ClCadr muscle fibers stopped firing spontaneously such that a 5-millisecond current injection of >10nA was necessary to trigger a single action potential. When a 60-millisecond pulse of 20nA or more was injected, action potentials continued to fire following termination of current injection

(Figure 1). In phenotypically normal, age-matched unaffected littermates, action potentials were never observed once stimulation was terminated (n = 6 mice, 30 fibers).

The increased excitability in ClCadr mice was accompanied by an increase in input resistance, but characteristics of action potentials were otherwise similar between ClCadr mice and unaffected littermates ClCwt mice (Table 1). These findings are similar to those reported previously for ClCadr muscle fibers (Mehrke, Brinkmeier et al. 1988).

29 Figure 1: In vitro model of warm-up

30 Figure 1: In vitro model of warm-up

Induction of reversible warm-up during intracellular recording: Shown on the left is the response of a normal skeletal muscle fiber to a 60 ms injection of depolarizing current.

The fiber is able to repeatedly fire action potentials during the current injection, but firing stops immediately after termination of current injection. The three traces on the right are from an individual ClCadr muscle fiber at baseline, after warm-up, and 5 minutes after warm-up. At baseline the ClCadr fiber is hyperexcitable and continues to fire action potentials after termination of the current injection. After 5000 action potentials have been triggered to induce warm-up, excitability of the fiber has normalized such that no action potentials are fired after termination of current injection. Following 5 minutes of rest, hyperexcitability has returned such that action potentials continue to be fired after termination of current injection. AP = action potential, min = minutes.

31 Table 1: Characteristics of Action Potentials in ClCadr Mice and Unaffected

Littermates

Resting Threshold Action Potential Action Input potential (mV) rate of rise Potential Resistance (mV) (mV/ms) Peak (mV) (M) ClCwt -82.2 ± 1.4 -61.6 ± 1.0 299.1 ± 16.0 12.9 ± 1.8 0.85 ± 0.05 (Unaffected littermates) ClCadr -82.1 ± 0.8 -61.7 ± 0.7 235.6 ± 19.1 11.9 ± 1.7 1.79 ± 0.11**

All values are shown ± SEM.

** = p < .01, n = 6 ClCwt unaffected littermates (30 fibers), n = 8 ClCadr mice (46 fibers)

32 Slow Inactivation of Sodium Channels Contributes to Warm-up of ClCadr Muscle in

Vitro:

To study the electrophysiological correlate of the reduction in excitability that underlies the warm-up phenomenon, it is necessary to be able to study warm-up in an isolated muscle in vitro. Warm-up has been shown to occur ex vivo in wild-type muscle made acutely myotonic by blockade of muscle chloride channels (van Lunteren, Spiegler et al. 2011). To study action potential and passive membrane properties associated with warm-up, it is necessary to record from individual muscle fibers before and after repeated activation of the fiber. After blocking muscle contraction with BTS, 5,000 action potentials were delivered at 20Hz in skeletal muscle from ClCadr mice using a protocol similar to one that has been used on wild-type skeletal muscle fibers (Pedersen, de Paoli et al. 2009; Pedersen, Macdonald et al. 2009). Skeletal muscle develops different degrees of tension in response to different durations and frequencies of action potentials.

Maximum firing rates can reach 100Hz generating maximum tension. At a frequency of

20Hz 5000 action potentials mimics four minutes of moderate exercise. We examined whether prolonged firing of action potentials led to resolution of myotonia. In normal muscle in vitro, no action potentials were fired after the depolarizing current terminated

(Adrian and Bryant 1974). In all muscle fibers from ClCadr mice, myotonia was present at baseline. Following a train of 5,000 action potentials, myotonia was eliminated in all 18 fibers studied from ClCadr mice (Figure 1). In 5 fibers, impalement was stable enough to allow for additional recording for 5 minutes following stimulation, and in all of these 5 fibers myotonia returned following inactivity. Elimination of myotonia following

33 prolonged firing and the return of myotonia following rest indicates that the warm-up phenomenon can be triggered in vitro in the absence of muscle contraction. We conclude that warm-up is triggered by action potentials rather than muscle contraction.

The ability to trigger warm-up while recording from an individual muscle fiber allowed for comparison of biophysical properties of individual fibers before and after warm-up, to determine potential mechanisms underlying warm-up. Action potential threshold was elevated in parallel with resolution of myotonia (Table 2). This demonstrated that resolution of myotonia with warm-up is paralleled by reduced excitability of fibers.

A number of mechanisms might contribute to reduced excitability following warm-up. One proposed mechanism for warm-up is accumulation of K+ in the t-tubules, which depolarizes the muscle and increases Na+ channel inactivation (Lossin and George

2008). Alternatively, K+ accumulation could stimulate the Na+-K+ pump, which because of its electrogenicity might hyperpolarize the membrane to eliminate myotonia (Van

Beekvelt, Drost et al. 2006). It is known that a brief period of muscle fiber depolarization occurs following 5 to 10 action potentials in myotonic muscle (Adrian and Bryant 1974).

We examined whether prolonged depolarization or hyperpolarization occurred after 5,000 action potentials. No change in resting potential occurred in parallel with warm-up

(Table 2). Suggests that the warm-up phenomenon does not involve a change in resting potential. Another potential mechanism underlying warm-up is an increase in resting membrane conductance (Pusch, Steinmeyer et al. 1995). In wild-type rat muscle, it was found that prolonged firing of action potentials triggered a marked increase in membrane conductance (Pedersen, de Paoli et al. 2009; Pedersen, Macdonald et al. 2009). Although

34 it appeared that most of the increase in conductance was mediated by ClC-1 chloride channels, there was also increased potassium conductance (Pedersen, de Paoli et al. 2009;

Pedersen, Macdonald et al. 2009). To measure changes in resting membrane conductance, input resistance was measured. Although there was a statistically significant decrease in input resistance following 5,000 action potentials (Table 2), the change was relatively small, seems unlikely to be a major contributor to the reduction of excitability that underlies warm-up.

35 Table 2: Characteristics of Action Potentials at Baseline and After 5,000 APs

Resting Threshold Action Action Action Input potential (mV) Potential Potential Potential Resistance (mV) rate of rise Peak (mV) Half-width (M) (mV/ms) (ms) ClCadr -80.5 ± -60.6 ± 0.5 256.4 ± 14.2 ± 1.3 1.16 ± 0.03 1.69 ± 0.07 Baseline 0.6 18.9 ClCadr -81.5 ± -56.3 ± 0.7* 149.4 ± 4.4 ± 1.8* 1.54 ± 1.45 ± Post- 1.0 11.0* 0.05* 0.06* 5000AP

The values shown are from 18 ClCadr muscle fibers at baseline and after warm-up induced by 5,000 action potentials. * = p < .01 using a paired t-test, n =18 fibers. All values are shown ± SEM.

36 Another mechanism that might underlie warm-up is slow inactivation of sodium channels (Lossin 2013). The relative density of sodium channels opening during the upstroke of the action potential can be estimated by measuring the maximal rate of action potential rise and action potential peak (Bean 2007). Thus, if slow inactivation of sodium channels contributes to warm-up, both action potential rate of rise and action potential peak will be reduced. When the action potential rate of rise and action potential peak were measured following warm-up, both were strongly reduced (Figure 2, Table 2).

These data are consistent with slow inactivation of sodium channels following warm-up.

Time Course of Warm-up Parallels Time Course of Sodium Channel Slow Inactivation:

In skeletal muscle there is a very slow form of sodium channel inactivation called slow inactivation. Slow inactivation has a time constant on the order of minutes (Ruff

1999; Todt, Dudley et al. 1999; Rich and Pinter 2003). If slow inactivation of sodium channels contributes to warm-up, the duration of warm-up should parallel the degree of sodium channel slow inactivation. We measured the reduction in rate of action potential rise to estimate the relative reduction in density of functional sodium channels to determine whether it correlated with duration of warm-up. Different durations of stimulation at 20Hz were used to induce warm-up. After 1,000 action potentials, rate of action potential rise was reduced by 15.1 ± 3.3% relative to baseline (n = 10 fibers); after

2,500 action potentials it was reduced by 18.2 ± 2.6% (n = 11 fibers), and after 5,000 action potentials it was reduced by 40.2 ± 3.7% (n = 16 fibers). All 3 durations of stimulation eliminated myotonia in 100% of fibers, but the duration of warm-up varied in

37 parallel with the reduction in rate of action potential rise (Figure 3). Following 1,000 action potentials, myotonia had returned in 100% of fibers 1 minute following termination of stimulation. After 2,500 action potentials it took 5 minutes, and after 5,000 action potentials it took 8 minutes. The parallel between the degree of reduction in rate of action potential rise and the duration of warm-up is consistent with the possibility that slow inactivation of sodium channels is an important contributor to warm-up.

38 Figure 2: Warm-up induced changes to single action potentials

39 Figure 2: Warm-up induced changes to single action potentials

Alteration of the action potential waveform induced by warm-up: Shown are three superimposed action potential traces from an individual muscle fiber before and after

5000 action potentials, and again after 5 minutes of inactivity. These action potentials are aligned by resting potential. Following 5000 action potentials, there is slight hyperpolarization of resting potential, slight elevation of threshold, reduction in both rate of rise and peak of the action potential, as well as slowing of repolarization. Following 5 minutes of inactivity, the action potential has recovered to closely resemble its initial waveform.

40 Figure 3: Time and activity effect on resolution of warm-up phenomenon

41 Figure 3: Time and activity effect on resolution of warm-up phenomenon

The duration of warm-up depends on the duration of stimulation. Shown is a plot of the percent of fibers in which myotonia returned at various times following termination of stimulation at 20 Hz. AP = action potential.

42 Increasing Slow Inactivation of Sodium Channels to Treat Myotonia of ClCadr Muscle In

Vitro:

If slow inactivation of sodium channels underlies warm-up, it might be possible to mimic warm-up with drugs that increase sodium channel slow inactivation. There are 2 drugs that are approved by the US Food and Drug Administration (FDA) that increase slow inactivation of sodium channels. One of the drugs (lacosamide) is used to treat epilepsy,(Abdelsayed and Sokolov 2013) whereas the other drug (ranolazine) is used to treat myocardial ischemia (Kloner 2013). Both lacosamide and ranolazine increase slow inactivation by inducing a hyperpolarized shift in the voltage dependence of slow inactivation (Errington, Stohr et al. 2008; El-Bizri, Kahlig et al. 2011; Niespodziany,

Leclere et al. 2013; Peters, Sokolov et al. 2013; Kahlig, Hirakawa et al. 2014). We compared the in vitro efficacy of lacosamide and ranolazine in treating myotonia to mexiletine, a sodium channel blocker that is the current standard of care (Statland, Bundy et al. 2012). Drug doses were titrated from low dosage with modest effect on excitability to high dosage, which caused hypoexcitability (mexiletine and ranolazine) or which greatly exceeded concentrations used clinically (lacosamide).

We measured the effect of drugs on hyperexcitability of ClCadr muscle fibers in 2 ways. The first was a qualitative evaluation of the presence of spontaneous firing of action potentials at the time of impalement (impalement myotonia). Normal muscle does not fire action potentials when it is impaled, but all ClCadr fibers have impalement myotonia. None of the 3 drugs eliminated impalement myotonia, except at high doses when muscle became hypoexcitable (see below). The second measure of excitability was

43 a quantitative evaluation of myotonia induced by current injection. Normal muscle fires repetitively during current injection, but firing stops as soon as current injection is terminated (see Figure 1). In ClCadr muscle, 100% of fibers continue to fire action potentials after current injection is terminated. Fibers were classified as hyperexcitable, normally excitable, or hypoexcitable as described in Methods and shown in Figure 4A.

There was no dose of mexiletine that normalized excitability in a majority of fibers (see

Figure 4). At 10 and 25M of mexiletine, most fibers remained hyperexcitable; at 50M, there was a wide range in excitability; and at 100M, almost all fibers were hypoexcitable. Lacosamide was effective in normalizing excitability, but only at a dose of

600M. Ranolazine normalized excitability in 100% of fibers at a dose of 50M and induced hypoexcitability at a dose of 100M.

Increasing Slow Inactivation of Sodium channels Improves Motor Performance of ClCadr

Mice:

We next studied the relative efficacy of mexiletine, lacosamide, and ranolazine in

ClCadr mice in vivo. We used previous studies (De Luca, Pierno et al. 2004; Stohr,

Kupferberg et al. 2007; Nodera and Rutkove 2012) as well as our in vitro data to guide initial choices of drug doses and performed a dose escalation study as described in

Methods. All 3 drugs improved motor function as measured by the decrease in time of the righting reflex (Figure 5). There was no statistically significant difference in efficacy between any of the highest doses of drugs on this measure of motor function. At their optimal doses, both mexiletine and ranolazine triggered greater improvement on the

44 rotarod than lacosamide (p<0.05). At 40mg/kg of mexiletine, some sedation and ataxia were present, and this appeared to account for the decline in rotarod performance.

45 Figure 4: Drug effects on myotonia

46 Figure 4: Drug effects on myotonia

Lacosamide and ranolazine are more effective than mexiletine in normalizing excitability of ClCadr muscle. A) Shown are the responses of 3 different ClCadr muscle fibers to a

60ms injection of depolarizing current following treatment with mexiletine. In the trace on the left, the fiber fired normally during current injection, but was hyperexcitable and fired an additional action potential following termination of current injection. In the trace in the middle, the fiber had normal excitability and was able to fire repetitively during current injection, but immediately stopped firing after termination of current injection. In the trace on the right, the fiber was hypoexcitable and unable to fire repetitively during the 60 ms current injection. B) Plotted for each drug is the percent of hyperexcitable

(black), normally excitable (dark grey) and hypoexcitable (light grey) fibers for each dose of drug tested. The bar graph for each drug dose is based on at least 22 fibers from 3 different animals.

47 Figure 5: In vivo performance testing

48 Figure 5: In vivo performance testing

Mexiletine and ranolazine cause greater improvement in motor function than lacosamide.

Shown on the left is a plot of the improvement in time of the righting reflex in mice 5 to

15 minutes following intraperitoneal injection of the doses indicated of mexiletine, lacosamide and ranolazine. The highest dose of all three drugs led to statistically significant improvement relative to saline injection (mexiletine p < .01, lacosamide p <

.05, ranolazine p < .01). On the right is a plot of the improvement on the Rotarod test of motor performance (see methods for details of scoring). Both mexiletine (20 mg/kg) and ranolazine (50 mg/kg) caused significant improvement in Rotarod function (p < .01) while lacosamide did not cause significant improvement relative to saline. N= 16 mice for all studies.

49 We measured the efficacy of each drug in treating myotonia in vivo. Mice were anesthetized with isoflurane, and the average duration of myotonia following needle movement in the paraspinal and gastrocnemius muscles was rated on a scale of 0 to 3 by

2 EMG examiners blinded to the drug given. The drug dose that gave the best improvement in motor performance in the previous study (mexiletine, 20mg/kg; lacosamide, 25mg/kg; ranolazine, 50mg/kg) was then administered; 10 minutes later, the degree of myotonia was again assessed by the same blinded reviewers. Both lacosamide and ranolazine appeared to be more effective in shortening the duration of myotonia than mexiletine. Lacosamide led to an improvement of 1.2 ± 0.2 on the scale of myotonia duration and ranolazine led to an improvement of 1.0 ± 0.1, whereas mexiletine only led to an improvement of 0.4 ± 0.2 and saline led to no improvement (n = 3 mice for each drug). Our in vivo studies suggest ranolazine is as effective in treating motor dysfunction and myotonia in ClCadr mice as either mexiletine or lacosamide.

Discussion

Mechanisms Contributing to Warm-up:

Ours is the first study to determine the changes in excitability that occur during warm-up. The first change identified was reduction of input resistance. Reduction in input resistance in wild-type muscle occurs following prolonged firing due to activation of both KATP and ClC-1 chloride channels (Pedersen, de Paoli et al. 2009; Pedersen,

Macdonald et al. 2009). As ClCadr muscle lacks functional ClC-1 channels, (Lipicky,

50 Bryant et al. 1971; Koch, Steinmeyer et al. 1992) the decrease in input resistance in

adr ClC muscle is likely due to activation of KATP channels. Although our simulation of action potentials (data not shown) suggests that activation of KATP channels has little effect on action potential waveform, activation of these channels may contribute to warm-up by increasing the amount of current required to reach the threshold for generation of action potentials. KATP channels are not contributing to resting potential as they are closed while intracellular ATP levels are normal. However, after prolonged stimulation ATP levels will deplete leading to opening of KATP channels. These channels will increase the potassium conductance allowing them to temporarily fulfill part of the dysfunctional ClC-1 channel role.

A depolarized shift in potassium’s equilibrium potential results from T-tubular potassium accumulation following repetitive firing and has been suggested as the source of depolarization leading to repetitive firing during myotonia (Adrian and Bryant 1974).

Although we found that warm-up was not accompanied by a change in resting potential, others have found a 5mV depolarized shift in the equilibrium potential for potassium when running computer simulation of potassium accumulation in the T-tubules during repetitive firing (Wallinga, Meijer et al. 1999; Fraser, Huang et al. 2011). This 5mV depolarizing shift in potassium equilibrium potential is necessary to computer model warmed up action potentials (Novak, Norman et al. 2015). A 5mV depolarizing shift in potassium equilibrium potential following warm-up would suggest a more depolarized resting potential. However, a change in resting potential was not found. In rat soleus muscle, increased Na+- K+ pump activity following stimulation provides enough current to cause a 10mV hyperpolarization of the resting potential (Hicks and McComas 1989).

51 We hypothesize that, during prolonged firing of ClCadr muscle, increased activity of the

Na+- K+ pump offsets a depolarized shift in the equilibrium potential for potassium, such that there is little net change in resting potential.

The significant increase in action potential half width following warm-up can be attributed to the reduced voltage activated potassium conductance. When modeling warmed up action potentials a reduction in voltage activated potassium conductance is necessary (Novak, Norman et al. 2015). There is a process of slow inactivation of potassium channels that has a time course of seconds, (Cheng, Fedida et al. 2013;

Ostmeyer, Chakrapani et al. 2013) and because we measured action potential waveform within seconds of warm-up, it is possible that slow inactivation underlies the reduction in voltage-activated potassium conductance. It seems unlikely that a reduction in voltage- activated potassium conductance plays an important role in warm-up, as its only effect is to cause a slight increase in action potential half-width. The time between successive myotonic action potentials is in the order of 20 milliseconds. The 0.38msec increase in action potential half width would be expected to have little effect on firing rate or triggering of myotonia.

The most dramatic changes seen post warm-up was reductions to maximum rate of action potential rise and peak. A possible explanation for the reductions to the rate of rise and peak amplitude is reduced sodium channel availability. Sodium channel availability can be reduced through a process known as slow inactivation which has a time constant on the order of minutes (Ruff 1999; Todt, Dudley et al. 1999; Rich and

Pinter 2003). This is similar to the time course of induction and recovery from warm-up.

Computer based modeling of action potentials following warm-up is only possible by

52 enhancing sodium channel slow inactivation (Novak, Norman et al. 2015). The reduction of sodium channel availability through slow inactivation causes action potential threshold to move toward more depolarized potentials such that excitability is reduced, giving the warm-up phenomenon.

Enhancing Sodium Channel Slow Inactivation to Treat Myotonia:

In vitro studies of muscle from ClCadr mice suggested that ranolazine and lacosamide were more effective than mexiletine in normalizing muscle excitability. In agreement with this finding, ranolazine and lacosamide appeared more effective than mexiletine in reducing the duration of myotonia in vivo. Reduction in the duration of myotonia following treatment with Lacosamide and ranolazine both function to increase slow inactivation leading to a reduction in duration of myotonia. However, there is no obvious reason that mexiletine should be any less effective in shortening the duration of myotonia. Its mechanism of action is a use-dependent reduction in sodium current that is mediated by a hyperpolarized shift in the voltage dependence of fast inactivation (De

Bellis, De Luca et al. 2006). However, despite mexiletine’s inferiority in lessening myotonia in vitro and in vivo, it was superior to lacosamide in improving motor function in vivo. This may be due to the propensity of lacosamide to cause ataxia and vertigo

(Zaccara, Perucca et al. 2013). These findings in mice raise the possibility that side effects of lacosamide may limit its efficacy in treating myotonia in patients.

In ClCadr mice, ranolazine appeared to cause less sedation and ataxia than lacosamide and was as good as or better than mexiletine on all in vitro and in vivo

53 measures of improvement in myotonia and motor function. It is not immediately obvious why reduction of sodium current due to a hyperpolarized shift in the voltage dependence of slow inactivation (ranolazine) should be more effective in improving motor function than reduction of sodium current due to a hyperpolarized shift in the voltage dependence of fast inactivation (mexiletine). We propose that ranolazine may be superior to mexiletine in treating myotonia congenita for 2 reasons. One advantage of using ranolazine may be that it is less likely to induce loss of muscle fiber excitability at high doses. Mexiletine and other sodium channel blockers can readily reduce sodium current to near zero, (Courtney 1981; De Bellis, De Luca et al. 2006; Desaphy, Carbonara et al.

2014) which induces loss of muscle excitability and weakness. Ranolazine reduces sodium current due to a hyperpolarized shift in the voltage dependence of slow inactivation of channels, but the voltage dependence of Nav1.4 slow inactivation is shallow, such that slow inactivation is almost never complete (Rich and Pinter 2003),(El-

Bizri, Kahlig et al. 2011); see however Ruff (Ruff 1999). Thus, ranolazine will not reduce sodium current to near zero unless there is prolonged, severe depolarization of muscle fibers. Because of this difference in mechanism, ranolazine may be less prone to induction of muscle weakness than mexiletine. The second advantage of using ranolazine is that mexiletine is not well tolerated by many patients due to gastrointestinal side effects and a possible increase in mortality (Trivedi, Cannon et al. 2014). We conclude that ranolazine has excellent therapeutic potential for treatment of stimulation induced myotonic stiffness in patients with myotonia congenita.

54 Chapter V

Specific Aim II: To test the hypothesis that stretch activated channels trigger percussion or stretch-induced myotonia.

Introduction

Percussion Myotonia:

Aims 1 focused on identification of the mechanism underlying the warm-up phenomenon, with that information we used ranolazine to enhance slow inactivation of sodium channels. In both in vitro and in vivo studies ranolazine reduced the stimulation induced component of myotonic stiffness. However, another component to myotonic stiffness is triggered through mechanical means and this is known as percussion or stretch-induced myotonia (Chrestian, Puymirat et al. 2006). During neurological assessment percussion myotonia occurs when the belly of a muscle is struck with a reflex hammer (Rudel and Lehmann-Horn 1985). Muscle contraction from a resulting myotonic run of action potentials forms a transient dimple in the muscle where it was struck. The myotonic action potentials are not the result of T-tubular potassium build up since there were no action potentials prior to the hammer strike, nor are they the result of muscle stimulation at the neuromuscular junction. The exact cause of the stretch-induced myotonic action potentials remains unknown.

Identification of the mechanism used to transduce the stretch into an excitable depolarization that triggers myotonic action potentials would provide a target for

55 alleviating stretch-induced myotonia. The mechanism for this depolarization has not been determined. In 1983 Brinberger showed that low doses of N-propyl-ajmalin had a small effect on reducing percussion myotonia (Birnberger, Rudel et al. 1975). This drug acts on sodium channels and is most frequently used as a cardiac antiarrhythmic drug. In 9 out of

10 patients the drug was effective and increased the speed of performing tasks but was unable to make patient movements resemble that of non-myotonic individuals. A common side effect among patients was dizziness, making it easy to discriminate between the drug and the placebo. Others have also noted a reduction in percussion myotonia with application of acetazolamide, a diuretic (Kwiecinski 1980).

Acetazolamide does not have a known mechanism of action for decreasing myotonia.

However the authors hypothesized that the metabolic acidosis typically induced by the drug may be altering chloride conductance. This conclusion was drawn from the observation that frog skeletal muscle has a pH sensitive chloride conductance. Another proposed mode of action for acetazolamide is through a change in potassium conductance. The altered potassium conductance is hypothesized to result from decreased serum potassium, as one side effect of the drug is increased potassium secretion in the urine. While these studies have looked for changes in the duration of myotonia following muscle percussion they did not look specifically at the depolarizing mechanism that is occurring. One hypothesis claims that this depolarization is the result of irritation at the neuromuscular junction in response to the mallet strike (Valenstein, Watson et al. 1978).

However it seems unlikely that every mallet strike hits the neuromuscular junction.

The clinical importance to studying stretch-induced myotonia is not in the percussion but rather in the resulting stretch. Typically as a muscle contracts the opposite

56 opposing muscle (antagonistic muscle) will relax and in doing so experiences a lengthening or stretching as it yields to the contraction. This stretching of the antagonistic muscle has no visible effect in non-myotonic muscle, however in muscle lacking chloride conductance this stretch may be able to induce a depolarization capable of triggering myotonia. If myotonic stiffness was the result of only voluntary muscle contraction, then contraction should continue to occur until the joint’s range of motion limits any further movement. However in myotonia congenita this type of muscle contraction is not observed, instead a state of stiffness is experienced. We propose this stiffness results from the antagonistic muscle experiencing stretch-induced myotonia, and the endogenously stimulated muscle experiencing myotonia simultaneously. We propose the source of depolarization to trigger myotonic action potentials in the antagonistic muscle is through stretch-activated channels. Treatment with inhibitors of stretch-activated channels may provide a therapeutic target to assist in treating stiffness experienced during myotonia congenita.

Stretch Channels:

Mechanical or stretch-activated channels exist in skeletal muscle membranes, and distortion of the membrane activates these channels and depolarizes the membrane.

Stretch channels have been identified in many tissues including skeletal muscle (Kunert-

Keil, Bisping et al. 2006; Gailly 2012). Many of these channel types depolarize cells in response to stretching by allowing cations to pass through them. Transient receptor potential (TRP) channels and piezo channels are two stretch channels that allow passage

57 of cations, such as calcium and sodium (Kunert-Keil, Bisping et al. 2006; Volkers,

Mechioukhi et al. 2015).

Piezo channels (piezo-1 and piezo-2) are the most recently discovered stretch- activated channels, and have been identified in sensory nerve ends of proprioceptors such as muscle spindles and Golgi tendon organs. Expression of these channels in skeletal muscle has yet to be determined, however they have been found in smooth muscle of the bladder (Michishita, Yano et al. 2016). Recessive mutations to piezo2 channels have been linked to arthrogryposis, which presents with contractures mainly involving the distal parts of the limbs. The hands have a characteristic position with medially overlapping fingers, clenched fists, ulnar deviation of fingers, and camptodactyly, and the feet have deformities. Contractures at other joints are variable; there are no associated visceral anomalies, and intelligence is normal (Haliloglu, Becker et al. 2017). The finding that mutation of piezo 2 channels causes arthrogryposis suggests piezo2 might be expressed in skeletal muscle.

Twenty-seven different TRP channel genes exist in humans, all of pass cations, calcium among them. These 27 TRP channels can be divided into six subfamilies, with each subfamily divided up into subtypes, TRPC (6), TRPV (6), TRPM (8), TRPA (1),

TRPP (3), TRPML (3) (Venkatachalam and Montell 2007). These channels activate and in some cases inactivate in response to temperature changes, osmolarity changes, changes in intracellular calcium, specific ligands as well as to mechanical stimulation such as stretch (Alderton and Steinhardt 2000; Franco-Obregon and Lansman 2002). Real time

PCR, western blots, and immunohistochemistry mouse skeletal muscle demonstrate

58 expression of TRPC1, TRP3, TRPC4 and TRPC6, TRPV2, and TRPV4, TRPM4, and

TRPM7 (Gailly 2012), with TRPV4 and TRPM7 being expressed in the highest level.

The mechanosensitive nature of TRPV4 and its known presence in the mouse sarcolemma led us to investigate this channel as the possible depolarizing contribution to trigger percussion myotonia (Ho, Horn et al. 2012). We hypothesized that striking myotonic muscle with a reflex hammer distorts or stretches part of the membrane opening TRPV4 channels and allowing cations to enter the muscle. Since myotonic muscle lacks functional chloride channels the resulting influx of cations is sufficient to depolarize the muscle triggering myotonia. To test the possible involvement of TRPV4 channels we established an in vitro model of stretch-induced myotonia using a blunt glass pipette to mechanically induce membrane stretch while simultaneously recording voltage changes.

Methods

Single Fiber Model of Stretch-induced Myotonia:

To directly record stretch-induced myotonia the extensor digitorum longus (EDL) muscle was dissected from the mouse, using caution to avoid damage to the muscle during removal. Immediately following removal, the EDL muscle was carefully pinned to a sylgard coated dish and was bathed in the same physiological buffer mentioned in aim

1. The muscle was impaled with one sharp voltage sensing electrode filled with 3M KCl and a resistance between 15 and 25 M. Roughly 200m from the sharp electrode

59 impalement a heavily fire polished 20m diameter blunt glass pipette was placed against the muscle fiber. This blunt pipette was then manually advanced roughly 20m forward distorting the fiber, creating a slight bend in the normally straight muscle fiber (Figure

6). Any intracellular voltage change that occurred in response to the advancement of the blunt pipette was recorded through the impaled sharp electrode using Spike2 software.

Blunt glass probes were created by slowly advancing the tip of a low resistance sharp glass pipette towards the red hot electric filament of a pipette fire polisher. The advancement was stopped once the glass probe had a bulbous end of approximately

20m. Not every sharp pipette was capable of being formed into the spherical shape needed for the probe. The blunt glass probes were then filled with 3M KCl solution containing 1mM sulforhadamine for visualization. Unlike sharp pipettes used for intracellular recording, the blunt glass probes are reusable. The same glass probe was used to perform all experiments involving stretch induction.

60 Figure 6: In vitro model of percussion myotonia

A.

B.

61 Figure 6: In vitro model of percussion myotonia

A. Shows the muscle fiber, blunt glass probe (left side) and impaled sharp recording

electrode (right side). The blunt glass probe is positioned such that it was not

distorting the muscle fiber.

B. Shows the same fiber as in A with a 20m advancement of the blunt glass probe

causing distortion of the muscle fiber.

62 Mice:

There is currently no animal model of percussion myotonia. Lack of cooperation from mice makes it difficult to strike a relaxed non-warmed up muscle. For this reason no attempt was made to develop an in vivo model of percussion myotonia. Instead an in vitro model was developed using ClCadr mice. As previously mentioned these mice lack functional ClC-1 chloride channels and closely resemble the human condition of myotonia congenita. The lack of genotyping for the ClC-1 gene makes it difficult to identify potential breeders. This made it difficult to create a large population of these mice. When ClCadr mutants were not available unaffected littermates were again used

(ClCwt) and exposed to the chloride channel blocker 9-anthracenecarboxylic acid (9AC).

The use of 9AC to reduce chloride conductance created a myotonic state for otherwise non-myotonic muscle. TRPV4(-/-) knockout and TRPV4(+/+) wild type mice were a gift from Dr. Charlotte Sumner at Johns Hopkins University. The mice were housed in the

Lab Animal Recources center at Wright State University and were only accessible when a staff member granted access. Phenotypically the TRPV4 knockout and wild type mice appeared identical, an ear tag was used to identify each mouse with a number.

Pharmacology:

To see the depolarization created by membrane stretch, voltage gated sodium channels were blocked using 1 M tetrodotoxin (TTX) (Lee and Ruben 2008). The blockage of voltage gated sodium channels prevents action potential formation. The

63 absence of action potentials allows for the stretch-induced depolarization to be seen in isolation.

Previous published work (Ho, Horn et al. 2012) demonstrates that 200 M SKF-

96365 can be used to block TRPV4 channel activity in skeletal muscle. However others have shown SKF-96365 to block channels other than TRPV4. TRPC1, T-type calcium channels, and a small number of potassium channels are also affected by the inhibitor. A

200 M dose of SKF-96365 is a non-specific blocker of mechanically activated channels when used in these experiments (Singh, Hildebrand et al. 2010; Tanahashi, Wang et al.

2015). A specific blocker of TRPV4, HC-067047, has been shown to inhibit channel activity at 1M (Everaerts, Zhen et al. 2010).

Experimental Design:

All experiments in which TRP channel blockers were used were performed blinded. Two data sets were collected. The first data set collected from the EDL consisted of seven muscle fibers and was considered the baseline or control data. The second data set was collected from the same EDL as the first data set and consisted of 7 more muscle fibers, this was considered the treatment group. Control and treatment data sets were collected from the same EDL muscle, allowing for each muscle to serve as its own baseline. The treatment was with either vehicle (0.1% DMSO) or inhibitor treated

(200M SKF-96365 or 1M HC-067047). To ensure a muscle fiber was not sampled twice, fibers were sampled in order moving from the medial side of the muscle to the lateral side.

64 Since our in vitro model of stretch-induced myotonia relies on the manual manipulation of the blunt glass pipette, it was imperative that the investigator be blinded to prevent bias. To accomplish investigator blinding, each week inhibitor and vehicle were placed in unmarked identical micro-centrifuge tubes and were given to a second investigator who then randomized them and gave them back to the investigator performing the experiment. Details of the randomization are as follows: New inhibitor was made weekly. The tubes were filled with equivalent volumes of DMSO so that no difference could be determined between them. Both inhibitors (SKF-96365 or HC-

067047) came as a white powder and dissolved completely with no color change or opacity change to the solvent. Vehicle tubes were placed on the left hand side of a tube rack and tubes containing inhibitor dissolved in DMSO were placed on the right. The second investigator then randomly numbered the tubes starting with one. Following the random numbering the tubes were placed in numerical order in the center of the tube rack and placed in a -20oC freezer. The identity of each micro-centrifuge tubes contents was record by the second investigator and not revealed until after data analysis.

Experiments using TRPV4-/- and TRPV4+/+ mice were also blinded. TRPV4-/- and

TRPV4+/+ mice arrived with an ear tag containing a number. A second investigator would retrieve the mice from the housing facility. The experimenter would record the ear tag number after muscle harvest, but was not aware of the animal’s genotype. Following data analysis the genotypes corresponding to each ear tag were revealed. Each experiment involved using the EDL from either a TRPV4-/- or a TRPV4+/+ mouse. Three sets of data each consisting of seven muscle fibers were collected from a single EDL. The first set of data was collected while the muscle was exposed to 9AC, reducing the chloride

65 conductance to mimic that of myotonic muscle. While under these conditions the muscle was tested for its ability to experience stretch-induced myotonia. The second set of data collected from the same muscle was performed under the additional influence of 1M

TTX to block voltage gated sodium channels, allowing for measurement of stretch- induced depolarization. The final set of data collected from the EDL was done while the muscle was exposed to 1M HC-067047.

Stimulation Myotonia and Passive Properties:

Both ClCadr and ClCwt muscle fibers were stimulated using a second sharp electrode also filled with 3M KCl, and a resistance of 15 to 25 M. This was done to examine if the TRPV4 channel blocker HC-067047 had any effect on passive membrane properties or excitability. A 200ms negative current pulse was used to examine the passive membrane properties, allowing for detection of any HC-067047 or vehicle effect on muscle fiber input resistance. Positive 60ms current pulses were used to test for excitability and the ability of muscle fibers to develop stimulus induced myotonia.

Excitability was assessed by comparing values for resting potential, threshold, rate of rise

(dV/dt), and action potential peak both before and after either HC-067047 or vehicle treatment. For these experiments two data sets were collected from each EDL muscle, one before and one after drug / vehicle application. This allowed each muscle to act as its own baseline and be compared to itself.

66 Statistics and Data Analysis:

Data analysis of current and voltage traces were done using spike2 software.

Analysis and statistics were performed as previously mentioned in the general methods chapter.

Results

Development of the in vitro model of stretch-induced myotonia:

The first set of experiments were done to determine if ClCadr EDL muscle would generate myotonic action potentials in response to 20m manual advancements of a blunt glass probe. The result of this pushing triggered myotonic action potentials in 56 out of

56 muscle fibers taken from eight different ClCadr mice (Figure 7 bottom). To determine if myotonic action potentials were unique only to muscle lacking chloride conductance

ClCwt mice were subjected to the same stretch by the same blunt glass probe. In ClCwt

EDL muscle myotonic action potentials were triggered in 0 out of 56 muscle fiber taken from eight different mice, the only evidence of stretching was a small 3 or 4 mV depolarization (Figure 7 top).

Lack of myotonic action potentials in ClCwt was expected since the mice have normal chloride conductance. As described earlier, in skeletal muscle chloride conductance assists potassium with maintaining and establishing a resting potential. The lack of chloride conductance seen in ClCadr mice provides less opposition to

67 depolarization, allowing cation influx through stretch activated channels to trigger myotonic action potentials. We next wanted to determine whether ClCwt muscle was capable of producing stretch-induced myotonic action potentials. To do this 100M of the ClC-1 channel blocker 9AC was added to the perfusion solution. ClCwt muscle exposed to 9AC responded to stretching with myotonic action potentials in 42 out of 42 muscle fibers taken from six different mice (Figure 7 middle). This suggests that both the ClCadr and ClCwt muscle fibers shared the same stretch activated ion channels, and the effect of this channel can be better seen when chloride conductance is reduced.

68 Figure 7: Effects of stretch on ClCwt and ClCadr muscle fibers

69 Figure 7: Effects of stretch on ClCwt and ClCadr muscle fibers

Traces collected during 20m advancement of a blunt glass probe. Probe advancements are indicated by grey arrows.

- The top trace was taken from a ClCwt muscle fiber not treated with 9AC. A small

depolarization but no myotonic action potentials are produced in response to

stretching.

- The middle trace was collected from a ClCwt muscle fiber treated with 9AC.

Myotonic action potentials resulted from stretching with the blunt glass probe.

- The bottom trace was obtained from a ClCadr muscle fiber that lacks functional

ClC-1 chloride channels. Myotonic action potentials resulted from stretching with

the blunt glass probe.

70 Determining if Stretch-induced Depolarizations are Real or Artifact:

The manual advancement of the blunt glass probe could be stretching the membrane to the point of tearing at the impaled sharp electrode. While tearing of the membrane would allow for depolarization it would not permit the return to the original resting potential. For this reason only stretch-induced depolarizations that return to the original resting potential were accepted. Another possible explanation for the depolarization is movement of the impaled sharp recording electrode. The stretch- induced by the blunt glass probe could be causing the recording sharp electrode to slide out of the muscle fiber, giving false depictions of depolarization. To verify that this was not the case the impaled recording electrode was slightly and gently manipulated manually to mimic the possible movements that could result. Manipulation of the recording electrode resulted in a depolarized resting potential following any movement that triggered myotonia. In the presence of TTX the depolarization induced by sharp electrode moving also did not return to the original resting potential (Figure 8).

71 Figure 8: Effects of impaled sharp recording electrode manipulation on resting potential

72 Figure 8: Effects of impaled sharp recording electrode manipulation on resting potential

The results of moving the sharp recording electrode impaled into myotonic muscle fibers treated with TTX. The movements were performed manually and the corresponding movement is labeled under each trace. Movements were performed to resemble stretching induced from advancement of the blunt glass probe. Resting potential before (left) and after (right) each movement is shown.

73 Can stretch-induced depolarization be blocked:

All muscle fibers responded to stretching in some way and maintained resting potential in the process, suggesting the presence of a stretch-activated channel. To further study the source of this stretch-activated depolarization, 1M tetrodotoxin (TTX) was added to eliminate excitability by blocking the Nav1.4 voltage gated sodium channel.

200M of the non-specific blocker SKF-96365 was used as this has been shown to block multiple channel types including TRPV. 200M application of the SKF-96365 to 9AC treated ClCwt muscle fibers reduced the degree of depolarization from 8.5 +/- 0.4 mV to

4.9 +/- 0.2 mV (21 muscle fibers, n = 3 mice). Using paired sample T-tests this difference was found to be significant (p value of 0.003). A vehicle was also used to ensure the effect was not the result DSMO disruption and to allow blinding of the experimenter. The

0.1% DMSO did not significantly change the degree of stretch-induced depolarization in

9AC treated ClCwt muscle fibers. Prior to DSMO application the average depolarization was 8.6 +/- 0.5 mV following DMSO application the average depolarization was 8.4 +/-

0.5 mV (35 muscle fibers, n = 5 mice). Lack of a significant reduction in the presence of only the vehicle allowed confirmed that the SKF-96365 effect was not attributed to

DMSO (Figure 9, Table 3).

74 Figure 9: Stretch induced depolarization in the presence of tetrodotoxin A

B C

75 Figure 9: Stretch induced depolarization in the presence of tetrodotoxin A

Grey arrows indicate time points when the blunt glass probe was advanced to induce stretch. All muscle fibers were exposed to 1M TTX to block voltage gated Nav1.4 sodium channels to prevent action potential production. The resting potential of the muscle fiber is shown at the start of the trace and the conditions from which the fiber was recorded are detailed below each trace. All recordings are taken from different muscles.

B

Bar graph displaying the degree of depolarization seen in ClCwt muscle exposed to 9AC and various conditions. The average data represented by the left black bar was collected from the same EDL muscle as the average data found in the grey bar to its right. Each of the three comparisons was made using a paired T test to determine statistical significance.

C

Bar graph displaying the degree of depolarization seen in ClCadr muscle. The black bar represents average data without the application of HC-067047 while the grey bar is average data with HC-067047. Comparisons were made using a paired T test.

76 Table 3: Summary of depolarization in response to stretch in 98AC treated ClCwt and ClCadr muscle

Condition Amount of depolarization (mV) P value

ClCwt + 9AC 8.6 +/- 0.5 (35 fibers n = 5) 0.34 ClCwt + 9AC + DMSO 8.4 +/- 0.5 (35 fibers n = 5)

ClCwt + 9AC 8.5 +/- 0.4 (21 fibers n = 3) 0.003 ClCwt + 9AC + SKF-96365 4.9 +/- 0.2 (21 fibers n = 3)

ClCwt + 9AC 8.5 +/- 0.3 (21 fibers n = 3) 0.007 ClCwt + 9AC + HC-067047 4.6 +/- 0.6 (21 fibers n = 3)

ClCadr 9.5 +/- 0.6 (21 fibers n = 3) 0.008 ClCadr + HC-067047 5.5 +/- 0.6 (21 fibers n = 3)

Table 3 shows the average depolarization experienced by muscle fibers exposed to vehicle or drug treatement. Represent the same data that is found in Figure 9B and 9C.

The total number of muscle fibers sampled as well as the number of mice sampled (n) is shown. The p values are the result of a paired T test. All values are shown +/- SEM

77 Could TRPV4 be the Source of Depolarization:

The reduced depolarization seen in the presences of SKF-96365 indicated the possible involvement of either TRPC or TRPV channel types (Schwarz, Droogmans et al.

1994; Singh, Hildebrand et al. 2010), as they are two known stretch activated channels found in skeletal muscle. Previous work in mouse skeletal muscle used SKF-96365 to block single channel TRPV4 activity in response to suction induced stretching with a patch pipette (Ho, Horn et al. 2012). TRPV4-/- knockout mice experienced no change in stretch-induced response when exposed to SKF-96365, leading to the conclusion that

TRPV4 is the mechanosensitive channel found in skeletal muscle. To further our investigation of TRPV4 as the source of stretch-induced depolarization, a more specific mechanosensitive channel blocker was used. HC-067047 blocks TRPV4 at a concentration of 1M (Ho, Horn et al. 2012). 9AC treatment of ClCwt muscle fibers was used for exploration to preserve the ClCadr mice for confirmation should an effect be seen. The experiments were conducted in the same fashion as the SKF-96365 experiments. Before the treatment with HC-067047 ClCwt muscle fibers treated with 9AC responded with an average stretch-induced depolarization of 8.5 +/- 0.3 mV and post treatment the depolarization fell to 4.6 +/- 0.6 mV (21 muscle fibers n = 3 mice). This decrease was confirmed as statistically significant using a paired sample T-test giving a p value of 0.007.

Both HC-067047 and SKF-96365 had an effect on the stretch-induced depolarization in 9AC treated ClCwt muscle fibers (Table 3). ClCadr muscle fibers were used to determine if these findings could be replicated in the mouse model of myotonia

78 congenita. We did not test the effect of SKF-96365 in ClCadr mice since HC-067047 was capable of reducing the response to a similar degree and was more specific. The EDL from ClCadr mice were exposed to either 1M HC-067047 or to 0.1% DMSO vehicle.

ClCadr muscle fibers experienced a stretch-induced depolarization of 9.5 +/- 0.6 mV when distorted with the blunt glass probe. Following treatment with 1M HC-067047 the average stretch-induced depolarization fell to 5.5 +/- 0.6 mV. This reduction in depolarization was again proven to be statistically significant using a paired T-test yielding a p value of 0.008.

Can Stretch-induced Myotonic Action Potentials be Blocked:

HC-067047 has been shown to reduce the stretch-induced depolarization in ClCadr muscle fibers as well as in 9AC treated ClCwt muscle fibers. In previous experiments the degree of depolarization was assessed in the presence of 1M TTX. With the application of 1M TTX the skeletal muscle is not capable of generating action potentials. This prevented us from seeing if 1M HC-067047 is capable of blocking depolarization to the degree of preventing stretch-induced myotonic action potentials. For these experiments

HC-067047 was applied to both ClCadr and 9AC treated ClCwt muscle fibers. The investigator was blinded to whether HC-067047 or vehicle was being used. ClCadr muscle fibers experienced stretch-induced myotonia in 56 out of 56 muscle fibers (n = 8 mice) when distorted with the blunt glass probe. After application of the vehicle 28 out of 28 muscle fibers (n = 4 mice) still experienced stretch-induced myotonia. However after

79 application of HC-067047, stretch-induced myotonia was triggered in 0 out of 28 (n = 4 mice) ClCadr muscle fibers.

ClCwt muscle fibers treated with 100M 9AC were shown to experience stretch- induced myotonia in 42 out of 42 muscle fibers (n = 6 mice). After application of DMSO

21 out of 21 muscle fibers (n = 3 mice) still experienced stretch-induced myotonia in response to the blunt glass probe. When in the presence of 1M HC-067047 (n = 3 mice)

0 out of 21 muscle fibers generated stretch-induced myotonia.

80 Table 4: Effectiveness of HC-067047 for blocking stretch-induced myotonia

Condition % fibers with myotonia Number of fibers / mice(n)

ClCadr 100 56 fibers / n = 8

ClCadr + HC-067047 0 28 fibers / n = 4

ClCadr + DMSO 100 28 fibers / n = 4

ClCwt + 9AC 100 42 fibers / n = 6

ClCwt + 9AC + HC-067047 0 21 fibers / n = 3

ClCwt + 9AC + DMSO 100 21 fibers / n = 3

Table 4 summaries the results from blinded studies of the effectiveness of HC-067047 to block stretch-induced myotonia. The percentage of fibers as well as the sampled number of muscle fibers and number of mice (n) are listed. Only muscles with reduced chloride conductance were studied since they were capable of producing myotonic action potentials in response to stretching

81 Is HC-067047 blocking myotonia by blocking the underlying stretch channel or by reducing excitability in a non-specific manner:

The absence of stretch-induced myotonia in the presence of 1M HC-067047 strongly suggests that TRPV4 is the channel contributing to stretch-induced myotonia.

However, other channel types may have also been affected. Muscle excitability and passive membrane properties were examined as follows. Current was injected through an impaled second sharp electrode rather than distorting with the blunt glass probe. The investigator was blinded to the identity of HC-067047 or DSMO until after experimental analysis. All 112 out of 112 (n = 8 mice) muscle fibers studied were able to generate action potentials in response to stimulus injection. An assessment of sodium channel availability was done through analysis of threshold, maximum rate of rise, and action potential peak. Table 5 shows the results taken from ClCadr muscle fibers stimulated with intracellular current injection. Also shown in Table 5 is the muscle fiber input resistance and resting potential. No statistically significant difference was seen between any of the five values tested when 28 ClCadr muscle fibers were compared against 28 ClCadr DSMO treated muscle fibers taken from the same n of 4 mice. In a different set of mice 28 ClCadr muscle fibers were tested against 28 ClCadr muscle fibers treated with 1M HC-

067047.These 56 fibers were taken from the same n of 4 animals, and no statistical significance was found between any of the tested values.

In Table 6 the results are listed for the same action potential characteristics and input resistance but taken from ClCwt muscle fibers treated with 9AC and again stimulated with intracellular current injection. No statistical significance was seen

82 between any of these five tests when 21 ClCwt muscle fibers were compared against 21

ClCwt DSMO treated muscle fibers taken from the same n of 3 mice. In a different set of mice 21 ClCwt muscle fibers were tested against 21 ClCwt muscle fibers treated with 1M

HC-067047. These 42 fibers were taken from the same n of 4 animals, and no statistical significance was found between any of the tested values.

83 Table 5: HC-067047 effects on excitability and input resistance in ClCadr fibers

4-mice Resting Threshold Rate of Rise Action Input 28-fibers potential (mV) (mV) (mV/ms) potential resistance Peak (mV) (M) ClCadr -79.3 ± 0.9 -54.7 ± 0.6 226.8 ± 30.8 29.6 ± 4.9 0.72 ± 0.06 ClCadr + DSMO -80.6 ± 0.6 -56.7 ± 0.8 230.2 ± 32.7 28.1 ± 5.4 0.75 ± 0.04 P-value 0.06 0.03 0.68 0.4 0.08 ClCadr -80.9 ± 0.5 -56.7 ± 0.5 234.1 ± 30.4 30.1 ± 4.7 0.85 ± 0.03 ClCadr + HC-067047 -80.9 ± 0.5 -56.9 ± 0.6 236.6 ± 30.6 29.9 ± 3.9 0.79 ± 0.05 P-value 0.95 0.76 0.6 0.95 0.29

Table 5 Summarized results taken from ClCadr muscle fibers that experienced stimulation myotonia. Action potential properties relating to excitably as well as input resistance were analyzed before (top row) and after (row directly underneath) application of DMSO or HC-067047. Each of the four conditions is an average of 28 muscle fibers taken from four different mice. Statistical significance were determined using a paired T test and a

Bonferroni correction was applied generating a critical p value of 0.01. All values are shown +/- SEM.

84 Table 6: HC-067047 effect on excitability and input resistance in ClCwt fibers treated with 9AC

3-mice Resting Pot. Threshold Rate of Rise Action pot. Input 21-fibers (mV) (mV) (mV/ms) Peak (mV) resistance (M) ClCwt -79.3 ± 0.5 -58.7 ± 0.7 267.9 ± 24.2 30.4 ± 2.5 0.54 ± 0.05 ClCwt + DSMO -80.1 ± 0.5 -59.7 ± 0.5 269.6 ± 17.9 26.3 ± 3.6 0.57 ± 0.06 P-value 0.45 0.3 0.83 0.06 0.49 ClCwt -80.5 ± 0.2 -59.1 ± 0.7 276.4 ± 34.6 34.2 ± 4.0 0.52 ± 0.04 ClCwt + HC-067047 -79.5 ± 0.4 -58.9 ± 0.2 270.6 ± 37.8 32.6 ± 3.9 0.54 ± 0.003 P-value 0.09 0.75 0.21 0.34 0.61

Table 6 Summarized results taken from ClCwt muscle fibers that experienced stimulation myotonia. Action potential properties relating to excitably as well as input resistance were analyzed before (top row) and after (row directly underneath) application of DMSO or HC-067047. Each of the four conditions is an average of 21 muscle fibers taken from three different mice. Statistical significance was determined using a paired T test and a

Bonferroni correction was applied generating a critical p value of 0.01. All values are shown +/- SEM.

85 Does HC-067047 have any Effect on TRPV4-/- Muscle fibers? :

Both the TRPV4-/- knockout and TRPV4+/+ muscle fibers were exposed to 100M

9AC and distorted with the blunt glass probe to determine. TRPV4+/+ muscle fibers triggered myotonic action potentials in 21 out of 28 muscle fibers (n = 4 muscles). In the

TRPV4-/- muscle fibers 0 out of 28 (n = 4) muscle fibers experienced stretch-induced myotonia. The absence of stretch-induced myotonia in the TRPV4-/- supports the previous findings that HC-067047 was blocking TRPV4 channels.

Muscle from both the TRPV4-/- and TRPV4+/+ mice were next exposed to 1M

TTX in addition to the 100M 9AC. The degree of depolarization was then measured and compared to the degree of depolarization seen after exposing to 1M HC-067047. In the

TRPV4+/+ an average depolarization of 5.6 +/- 0.5 mV (28 fibers, n = 4) was seen prior to

HC-067047 application while an average depolarization of 3.3 +/- 0.6 mV (28 fibers, n =

4) was seen after it was applied (Figure 10, Table 7). This change was statistically significant with a p value of 0.002. This result indicated that TRPV4 channels are present on TRPV4+/+ muscle fibers and that HC-067047 is blocking them. In the TRPV4-/- an average stretch-induced depolarization from 28 muscle fibers was found to be 2.8 +/- 0.5

(n = 4) when exposed to 9AC and TTX (Figure 11). In the presence of HC-067047 the average stretch-induced depolarization from 28 TRPV4-/- muscle fibers was 2.7 +/- 0.4 mV (n = 4). This change was found to not be statistically significant. The decreased stretch-induced depolarization and the absence of a response to HC-067047 in the

TRPV4-/- muscle fibers support TRPV4 as the most likely candidate for triggering stretch-induced myotonia.

86 Figure 10: Effect of HC-067047 on TRPV4+/+ and TRPV4-/-

87 Figure 10: Effect of HC-067047 on TRPV4+/+ and TRPV4-/- muscle Labels for each condition are listed below each trace. The resting potential is listed on the right of each trace, and grey arrows indicate points in time when advancement of the blunt glass probe occurred.

88 Table 7: Summary of depolarization in response to stretch in TRPV4+/+ and

TRPV4-/- muscle

Condition Amount of depolarization P value 100M 9AC + 1M TTX TRPV4+/+ 5.6 +/- 0.5 mV (28 fibers, n = 4) 0.002

TRPV4+/+ + HC-067047 3.3 +/- 0.6 mV (28 fibers, n = 4)

TRPV4-/- 2.8 +/- 0.5 mV (28 fibers, n = 4) 0.39

TRPV4-/- + HC-067047 2.7 +/- 0.4 mV (28 fibers, n = 4)

Table 7 Degree of depolarization experienced by TRPV4+/+ and TRPV4-/- muscle when exposed to HC-067047. All experiments performed in the presence of 100M 9AC and

1M TTX to eliminate chloride currents, as well as voltage gated sodium currents. All values are shown +/- SEM.

89 Figure 11: Stretch induced depolarization in 9AC and TTX treated TRPV4+/+ and

TRPV4-/- muscle exposed to HC-067047

90 Figure 11: Depolarization in TTX treated TRPV4+/+ and TRPV4-/- muscle exposed to

HC-067047

Bar graph displaying the average stretch-induced depolarization experienced by

TRPV4+/+ and TRPV4-/- muscle in the presence of 100M 9AC and 1M TTX, the black bar. The grey bar to the immediate right of each black bar represents the average stretch- induced depolarization under the same conditions plus HC-067047.

91 Discussion

We demonstrated that the mechanosensitive channel TRPV4 contributes to generation of percussion myotonia. The first evidence for our conclusion comes from the finding that in the presence of both SKF-96365 and HC-067047 stretch of muscle fibers does not generate myotonia nor does it depolarize to the same extent. Specificity of HC-

067047 for TRPV4 has been demonstrated in rat and mouse urothelial cells. They explored the selectivity of HC-067047 for a variety of ion channels including representative channels of the TRPV, TRPC, TRPA, and TRPM families, voltage gated sodium and potassium channels, hERG and endogenous TRPM7-like and TRPV2-like channels from isolated mouse urothelial cells (Everaerts, Zhen et al. 2010). They found that apart from TRPV4 the only other channel type affected at sub-micromolar ranges were the cold sensing TRPM7 channel and repolarizing hERG channels. Neither of these channel types responds to stretch. The specificity of HC-067047 for TRPV4 supports their involvement in stretch-induced myotonia.

The lack of stretch-induced myotonia experienced by TRPV4-/- muscle fibers in the presences of 9AC confirms the involvement of TRPV4. The exposure of these same fibers to HC-067047 showed no change in the degree of depolarization with TTX application, indicating specificity of HC-067047 for TRPV4.

The TRPV4+/+ muscle only experienced stretch-induced myotonia in 75% of fibers, 21 out of 28 fibers. The 7 muscle fibers that did not experience stretch-induced myotonia were all from the same mouse. This was considered an isolated occurrence attributed to the preparation of 9AC. In the presence of TTX the muscle still experienced

92 a depolarization similar to other TRPV4+/+ muscles, and experienced a similar reduction in the depolarization with HC-067047 application.

The lack of response seen in the TRPV4-/- muscle was surprising as it is common for knockout muscle to compensate by changing expression of other channel types.

Upregulation of TRPV2 channels has been shown to occur with the knockout of TRPV4

(O'Neil and Heller 2005). However, we did not see any stretch-induced myotonia in the

TRPV4-/- muscle fibers exposed to 9AC. If the mechanosensitive TRPV2 channel expression was upregulated it was not done to an extent that myotonic action potentials could be triggered.

TRPV4 channels are known to open in response to changes in temperature. In

Xenopus Oocytes TRPV4 channels respond with inward currents when activated at 34 degrees Celsius, with maximum currents being obtained at 45 degrees Celsius (Guler,

Lee et al. 2002). Surprisingly temperature responses of TRPV4 currents where inhibited when the cell was made hypertonic inducing membrane stretch through cell swelling

(Guler, Lee et al. 2002). TRPV1 channels in contrast continue to have robust responses to temperature change when exposed to hyperosmotic membrane stretch. We did not notice any temperature dependences for stretch-induced myotonia. There was no change in the stretch-induced depolarization or in the generation of myotonic action potentials when a temperature ramp was given from 22 to 45 degree Celsius (data not shown). An increased or prolonged stretch-induced depolarizations at 45°C would have indicated possible

TRPV1 involvement. The lack of change to the stretch-induced responses with temperature supported the role of TRPV4 as the mechanosensitive channel triggering

93 stretch-induced myotonia. Others have also found stretch-induced activation of TRPV4 to not be temperature dependent in skeletal muscle (Ho, Horn et al. 2012).

To rule out non-specific effects that might account for the efficacy of HC-

067047HC in treating myotonia we performed experiments looking at passive properties of muscle fibers. HC-067047 did not have a statistically significant effect on the input resistance of ClCadr or ClCwt fibers treated with 9AC. HC-067047 also had no effect on stimulation induced myotonia. We found that in the presence of HC-067047 that stretch- induced myotonia was inhibited but stimulation-induced myotonia was still present.

Indicating that the inhibitor had no effect on the muscle fibers excitability or sodium channel availability. Thus it appears likely that the elimination of stretch-induced myotonia was due to an effect specifically on stretch-activated channels.

The depolarization seen in the presence of TTX was never reduced to zero. In the presence of HC-067047 an approximate 43% reduction was seen in the depolarization in all mice tested. This indicates that TRPV4 is not the only mechanosensitive channel found on skeletal muscle that responses to stress. The remaining 57% of the depolarization is the result of other stretch-activated channels that are not affected by

HC-067047. Piezo channels, especially piezo-2 channels have been linked with arthrogryposis where distal skeletal muscles experience contractures. Peizo-2 channels may also provide a contribution to the stretch-induced depolarization. Other TRP channels besides TRPV4 can be activated by stretch and have been found on skeletal muscle (Gailly 2012). TRPV2, TRPM4, TRPM7, TRPC6 and TRPC1 have all been previously identified to respond to stretch as well as be found in skeletal muscle (Gailly

94 2012). The possible involvement of these channels types may also be contributing to the depolarization seen.

The degree of depolarization experienced by all muscle fibers exposed to TTX was not sufficient to depolarize the muscle fiber to threshold. Threshold values were between -53 and -60 mV with an approximately -80mV resting potential. This indicates that an approximate 20 to 27 mV depolarization would be necessary to reach threshold and trigger an action potential. However the maximum depolarization recorded over the

200micron separation in any of the muscle fibers tested with stretching was 9.5 mV. The cable properties of the muscle fiber allow for the loss of current across the membrane through channels open at rest. This reduction in current results in the gradual return of the membrane potential back to resting potential with distance. The distance between the point of stretch induction and the impaled recording electrode could allow for reductions in the depolarization (Bryant 1969). No changes to the resting leak of the muscle fibers were noticed with inhibitor application as input resistance did not change.

The clinical manifestation of percussion myotonia is muscle contraction following stretch-induced by the reflex hammer. The reflex hammer provides a sudden stretch of the sarcolemma which leads to activation of mechanosensitive channels. The stretch- induced depolarization triggers myotonia resulting in stiffness to a muscle that was in the relaxed state. Muscle experiences rapid stretch even when it is not being struck with a reflex hammer. During every concentric isotonic contraction the antagonistic muscle is relaxed and stretched as it yields to the contracting muscle. The stretch in the antagonistic muscle activates TRPV4 channels leading to a depolarization. In normal muscle this cation entry through TRPV4 has little to no effect since the high chloride conductance

95 assists in holding the resting potential. However, in muscle lacking chloride conductance the stretch-induced depolarization becomes sufficient to trigger myotonic action potentials resulting in muscle stiffness. The use of TRPV4 inhibitors such as HC-067047 could provide relieve from this stretch-activated stiffness seen in myotonic muscle.

96 Chapter VI

General Discussion and Conclusions

We propose that in skeletal muscle with reduced chloride conductance that myotonic stiffness is the result of two different triggers. We sought to develop treatment for this myotonic stiffness by studying the warm-up phenomenon and percussion myotonia.

Stimulation-induced myotonia results from intracellular current injection mimicking voluntary activation of the neuromuscular junction. Over time and with continuous activity the muscle will transition from the state of stiffness to a state that resembles non-myotonic muscle. This transition is known as the warm up phenomenon.

Through study of warm-up we identified sodium channel slow inactivation as a target to treat stimulus-induced myotonia. We identified the FDA approved drug ranolazine which acts in a dose dependent fashion on Nav1.4 to pharmacologically increase sodium channel slow inactivation (Wang, Calderon et al. 2008) and hence warm-up. Through the use of both a single muscle fiber model and a whole animal behavior study, ranolazine was shown to be just as effective if not better than the current standard of care for myotonia congenita.

In trying to identify why increasing slow inactivation treats myotonia, Ahmed

Hawash (another student in the Rich lab) identified a persistant inward sodium current as the cause of repetitive firing in stimulus induced myotonia. This persistent inward sodium current (NaPIC) was reduced by 80% in the presence of 50M ranolazine, the dose I found eliminates myotonia. Other published work has shown ranolazine to inhibit

97 late sodium currents in cardiac muscle (Belardinelli, Shryock et al. 2006; Sokolov, Peters et al. 2013). It thus appears that ranolazine decreases NaPIC in both skeletal and cardiac muscle. Our current hypothesis is thus that ranolazine is acting to eliminate stimulus- induced myotonia by enhancing slow inactivation of the opened NaPIC that triggers stimulus induced myotonia.

The effectiveness of ranolazine on both the in vitro and in vivo models of warm- up, along with the fact that no current FDA approved drug currently exists to treat myotonia congenita led to a collaboration with Dr. Kissel MD at The Ohio State

University Wexner Medical Center Department of Neurology. We designed a pilot clinical study to investigate ranolazine in patients with myotonia congenita. This trial found that ranolazine was able to alleviate a large degree of patient stiffness but not completely. (Neurology as a “Smaller Scope Study” and is titled “Open Label Trial of

Ranolazine for the treatment of Myotonia Congenita.” Arnold et al in press).

Three men and ten women with a mean age of 47.5 +/- 16.2 years old (ages 18 –

67) were used in the study. All had clinically evident stiffness and EMG myotonia, as well as genetically proven myotonia congenita or confirmed in a first degree relative. No participants discontinued the study due to side effects or intolerance of ranolazine. After four weeks of ranolazine treatment, statistically significant improvements to muscle stiffness, weakness, and pain were noted by self-reporting. Also following the four week treatment period, participates showed statistically significant reductions times for grip myotonia as well as in timed-up-and-go testing. Myotonic EMG activity of participants was significantly reduced but not eliminated after four weeks of ranolazine treatment.

This study did not compare mexiletine to ranolazine, but did conclude that ranolazine

98 would serve as a good alternative. Mexiletine is thus still the current treatment of choice, but about 25% of patients do not respond to it (Statland, Bundy et al. 2012; Trivedi,

Cannon et al. 2014). The fact that mexiletine is sometimes not well tolerated due to gastrointestinal side effects (Trivedi, Cannon et al. 2014) also gives strong support for having an alternative therapy available. This trial is moving on to its next phase as a large, prospective, placebo controlled trial, with the hope of providing a novel therapy targeting stimulus-induced myotonic stiffness.

The second component of myotonic stiffness is the stretch-induced or percussion myotonia. This stretch-induced myotonic stiffness is triggered by lengthening of antagonistic muscles during concentric contraction of the agonist. Developing a treatment for stretch-induced myotonic stiffness would be the first of its kind. We have shown that stretch-induced myotonia is triggered in muscle with reduced chloride conductance through stretch activation of TRPV4 channels. This stretch activated channel provides adequate depolarization to trigger stretch-induced myotonia. The TRPV4 specific blocker

HC-067047 has been demonstrated by others as well as ourselves to effectively block

TRPV4 at 1M (Everaerts, Zhen et al. 2010). Development of HC-067047 by Hydra

Biosciences Inc in collaboration with Catholic University Leuven was done with hopes of becoming a treatment for overactive bladder. The success of HC-067047 in its first in vivo tests with mice and rats (Everaerts, Zhen et al. 2010) give strong indication for the use of the drug at treating the stretch-induced component of myotonic stiffness in animals.

Unpublished data collected from stretching whole rat EDL muscle in the presence of 100M 9AC indicates that muscle stretching similar to that experienced by an

99 antagonistic muscle can trigger myotonic contraction. Every movement involves simultaneous shortening/contracting of a muscle being stimulated and lengthening/stretching of the antagonistic. If these two muscles experience myotonia simultaneously, stiffness results and the intended movement cannot be carried out. Even with an effective treatment for the stimulation induced component of myotonic stiffness, muscle may not truly behave as non-myotonic muscle does without effective treatment for the stretch-induced component as well. The use of HC-067047 in blinded animal behavior and clinical trials could provide further insight to development of a more comprehensive treatment for myotonia congenita. One that might involve the combine efforts of both ranolazine and HC-067047 to target the two triggers for stiffness.

To summarize, my work has led to the identification of two different novel approaches to treatment of myotonia in the muscle disease myotonia congenita. The first treatment has already been translated to a preliminary clinical trial. Work is underway to expand the study to a national placebo controlled trial. To move studies of the second treatment forward, we plan to perform in vivo mouse studies. Unfortunately, as HC-

067047 is not yet FDA approved, translating this finding to patients will take more time.

Our hope is that by combining these treatments in patients we will be able to greatly reduce, or even eliminate, myotonia such that patients can lead more normal lives.

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110 Appendix A

Common terms and abbreviations:

1. TTX (Tetrodotoxin) – blocks voltage gated sodium channels, reduces excitability and ability to generate action potentials 2. 9AC (9-anthracenecarboxylic acid) – blocks ClC-1 chloride channels, generating hyperexcitable muscle fibers that function similar to those in myotonic patients 3. BTS (N-benzyl-p-toluene sulphonamide) – inhibitor of the actin myosin cross bridge formation, preventing contraction of muscle while leaving excitation and ion channel function unaffected. 4. HC-067047 – specific blocker of TRPV4, has been shown to block channel activation from multiple stimuli; temperature, osmotic swelling, membrane stretch and ligand activation. 5. SKF-96365 – general blocker of mechanosensitive channels including TRPV4 and TRPC1. Has also been shown to block other channel types that are not mechanosensitive 6. NaPIC (persistent inward sodium current) – small subset of sodium channels that do not inactivate as typical voltage gated sodium channels do. Thought to be responsible for repetitive action potentials during myotonia 7. Nav1.4 – sodium channel isoform found predominantly in skeletal muscle. 8. EDL (Extensor digitorum longus) – muscle found in the leg with tendinous attachements for all muscle fibers 9. Ranolazine – slow inactivator of sodium channels including Nav1.4, more effective on the channel when it is in the open state 10. Mexiletine – sodium channel blocker 11. Lacosamide – slow inactivator of sodium channels including Nav1.4 12. DMSO (dimethylsulfoxide) – vehicle used to deliver lipophilic drugs 13. ClCadr / ClCwt – mice that lack functional ClC-1 chloride channels due to a recessive mutation to the gene. Wild type functions similar with 9AC applied 14. TRPV4-/- / TRPV4+/+ - mice that lack functional TRPV4 channels.

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