The Mechanism Underlying Transient Weakness in Myotonia Congenita

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bioRxiv preprint doi: https://doi.org/10.1101/2020.12.23.424129; this version posted December 24, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license. 1

1 The mechanism underlying transient weakness in myotonia congenita 2 3 Running head: Transient weakness in myotonia congenita 4 5 Jessica H Myers1, Kirsten Denman1, Chris DuPont1, Ahmed A Hawash2, Kevin R Novak3, 6 Andrew Koesters4, Manfred Grabner5, Anamika Dayal5, Andrew A Voss6, Mark M Rich1* 7 8 1Department of Neuroscience, Cell Biology and Physiology, Wright State University, Dayton, 9 OH, 45435. 2Department of Dermatology & Cutaneous Surgery, University of Miami, Miami, 10 FL 33136, 3Evokes LLC, Mason OH 45040, 4Naval Medical Research Unit, Wright Patterson 11 Air Force Base, Dayton, OH 45433; 5Department of Genetics and Pharmacology, Medical 12 University of Innsbruck, A-6020, Innsbruck, Austria; 6Department of Biology, Wright State 13 University, Dayton, OH, 45435. 14 15 *Please address all correspondence to: Mark Rich, email: [email protected] 16 17

18 Abstract: 19 In addition to the hallmark muscle stiffness, patients with recessive myotonia congenita (Becker 20 disease) experience debilitating bouts of transient weakness that remain poorly understood 21 despite years of study. We made intracellular recordings from muscle of both genetic and 22 pharmacologic mouse models of Becker disease to identify the mechanism underlying transient 23 weakness. Our recordings reveal transient depolarizations (plateau potentials) of the membrane 24 potential to -25 to -35 mV in the genetic and pharmacologic models of Becker disease. Both Na+ 25 and Ca2+ currents contribute to plateau potentials. Na+ persistent inward current (NaPIC) through 2+ 26 NaV1.4 channels is the key trigger of plateau potentials and current through Cav1.1 Ca channels 27 contributes to the duration of the plateau. Inhibiting NaPIC with ranolazine prevents the 28 development of plateau potentials and eliminates transient weakness in vivo. These data suggest 29 that targeting NaPIC may be an effective treatment to prevent transient weakness in myotonia 30 congenita. 31 32 Impact Statement: Transient weakness in myotonia congenita is caused by depolarization 33 secondary to activation of persistent Na+ current in skeletal muscle. 34

35 Introduction: 36 Myotonia congenita is one of the non-dystrophic muscle channelopathies. It is caused by 37 loss-of-function mutations affecting the muscle chloride channel (ClC-1) (Lipicky et al., 1971; 38 Steinmeyer et al., 1991; Koch et al., 1992). Patients with recessive myotonia congenita (Becker 39 disease) experience muscle stiffness due to hyperexcitability (Lehmann-Horn et al., 2008; 40 Trivedi et al., 2014; Cannon, 2015) as well as transient weakness due to unknown factors (Ricker 41 et al., 1978; Rudel et al., 1988; Zwarts and van Weerden, 1989; Deymeer et al., 1998; Trivedi et 42 al., 2013). Some patients with Becker disease report transient weakness in arm muscles as a 43 greater impediment than muscle stiffness (Rudel et al., 1988). This weakness can last up to 90 44 seconds and is brought on by exertion following rest (Ricker et al., 1978; Rudel et al., 1988; 45 Zwarts and van Weerden, 1989; Deymeer et al., 1998). 46 The mechanism underlying transient weakness in Becker disease has remained unknown 47 since its initial description close to 50 years ago (Ricker and Meinck, 1972). There appears to be 48 loss of muscle excitability, as weakness is accompanied by a drop in compound muscle action 49 potential amplitude (CMAP) during repetitive stimulation (Ricker and Meinck, 1972; Brown, 50 1974; Aminoff et al., 1977; Deymeer et al., 1998; Drost et al., 2001; Modoni et al., 2011). This 51 drop in CMAP is associated with reduction in muscle fiber conduction velocity, which has been 52 proposed to progress to depolarization block (Zwarts and van Weerden, 1989). What has 53 remained unclear, and perhaps counterintuitive, is why a loss-of-function mutation of the muscle 54 ClC-1 channels in myotonia congenita (Lipicky et al., 1971; Steinmeyer et al., 1991; Koch et al., 55 1992) leads to transient loss of excitability. The primary defect caused by loss of ClC-1 current is 56 hyperexcitability of muscle, which causes myotonia. 57 We established that a ClC-1 homozygous null (ClCadr) mouse model of Becker disease 58 has transient weakness in vivo, mimicking the condition in human patients. Intracellular 59 recording in both ClCadr muscle and a pharmacologic model of Becker disease (due to block of 60 ClC-1 with 9-AC) have elucidated a novel phenomenon: transient depolarizations to voltages 61 between -25 to -35 mV, lasting many seconds, which we termed “plateau potentials.” Blocking 62 Na+ persistent inward current (NaPIC) with ranolazine (a piperazine derivative) prevented both 63 development of plateau potentials and transient weakness. We conclude that NaPIC plays a 64 central role in the development of plateau potentials, which are the mechanism underlying 65 transient weakness in Becker disease.

66 Results: 67 Identification of transient weakness in mice with myotonia congenita 68 To study in situ isometric motor performance in the Clcn1adr-mto2J (ClCadr) mouse model of 69 recessive myotonia congenita (Becker disease), a muscle force preparation that we used 70 previously was employed (Dupont et al., 2019; Wang et al., 2020). Mice were anesthetized via 71 isoflurane inhalation and the distal tendon of the triceps surae (gastrocnemius, plantaris, and 72 soleus muscles) was dissected free and attached to a force transduction motor; then the sciatic 73 nerve was stimulated with 45 pulses delivered at 100 Hz. In unaffected littermates, there was no 74 myotonia following 45 pulses at 100 Hz, such that relaxation was immediate (Fig 1A). In ClCadr 75 mice, stimulation with 45 pulses caused full fusion of force, but relaxation was slowed, due to 76 the presence of myotonia (Fig 1C). 77 To determine whether transient weakness was present, the sciatic nerve was additionally 78 stimulated with 15 pulses at 100 Hz every 4 seconds for 1 minute. In unaffected littermates, this 79 caused stable force production with a mild, gradual reduction that was likely due to fatigue (Fig 80 1B). In myotonic mice, the same stimulation protocol revealed transient weakness, as force fell 81 over the first 10 to 15s and then recovered (Fig 1D). To avoid inclusion of fatigue in 82 measurement of transient weakness, we normalized to force at the end of the 1 minute of 83 intermittent stimulation. The plot of the mean normalized force revealed transient weakness, 84 which peaked in severity 15 to 20s after the initial stimulation and resolved within 1 minute (Fig 85 1E). These data indicate transient weakness is present in ClCadr mice. 86

87 88 Fig 1: Transient weakness in the ClCadr mouse model of recessive myotonia. A) Shown is a 89 force trace from a triceps surae muscle group of an unaffected littermate in response to 90 stimulation of the sciatic nerve with 45 pulses at 100 Hz. B) The force trace generated by 91 following the initial stimulus with 15 pulses at 100 Hz delivered every 4 s. C) In a ClCadr mouse, 92 following the 45 pulses at 100 Hz (indicated by the dotted vertical line) there was continued 93 force generation secondary to myotonia. D) With stimulation every 4 s transient weakness was 94 revealed. E) Plot of the force normalized to force at 60 s in unaffected littermates and ClCadr 95 mice. Transient reduction in force was present in ClCadr mice (*, p = .00015 vs unaffected 96 littermates at 16s, t-test, 95% confidence interval 110 to 114 vs 75 to 93). n = 5 unaffected 97 littermates and n = 9 ClCadr mice. Error bars represent ± SD. 98 99 Characterization of plateau potentials in genetic and pharmacologic models of myotonia 100 congenita 101 In myotonic patients, transient weakness is paralleled by a drop in compound muscle 102 action potential amplitude (CMAP) (Ricker and Meinck, 1972; Modoni et al., 2011). This 103 finding suggests weakness is due to loss of excitability. To look for inexcitability of ClCadr 104 muscle, intracellular current clamp recordings were performed. In both unaffected littermates 105 and ClCadr mice, stimulation with a 200-ms injection of depolarizing current triggered repetitive 106 firing of action potentials during the stimulus. In muscle from unaffected littermates, the firing 107 ceased as soon as the stimulus was terminated (Fig 2A). In muscle from ClCadr mice, there was 108 myotonia (continued firing of action potentials following termination of the stimulus) in 100% of 109 fibers (Fig 2B). The myotonia often persisted for many seconds. While many runs of myotonia 110 ended with repolarization to the resting membrane potential (Fig 2B), in other instances, 111 myotonia terminated with development of depolarizations lasting 5 to >100s to a membrane 112 potential near -30 mV (Fig 2C, D). During the depolarizations there was gradual repolarization 113 of the membrane potential, which was followed by sudden repolarization back to the resting 114 potential. In some cases, the sudden repolarization was preceded by development of oscillations 115 in the membrane potential (Fig 2D). The depolarizations occurred in 62% of ClCadr muscle

116 fibers (n = 42 fibers from 10 mice). Depolarizations lasting less than 1s to a membrane potential 117 close to -60 mV have been described in a toxin-induced model of hyperkalemic periodic 118 paralysis and were termed plateau depolarizations (Cannon and Corey, 1993). While the 119 depolarizations we identified could be due to similar mechanisms as those described in 120 hyperkalemic periodic paralysis, they seemed more similar to transient depolarizations in spinal 121 motor neurons, which can last many seconds, and have been termed plateau potentials (Alaburda 122 et al., 2002; Heckman and Enoka, 2012; Hounsgaard, 2017). We thus chose the term plateau 123 potentials to describe them. 124 To determine whether plateau potentials cause inexcitability, ClCadr muscle fibers were 125 stimulated during the plateau phase. During, and immediately following plateau potentials, 126 action potential generation in response to current injection failed (Fig 2E). At later times 127 following repolarization, action potential generation was again possible. The inexcitability at 128 earlier times following repolarization is likely due to slow inactivation of Na channels following 129 the many-second depolarization (Ruff, 1996, 1999; Rich and Pinter, 2003). These data are 130 consistent with the possibility that plateau potentials and the resultant inexcitability of muscle are 131 the mechanism underlying transient weakness in myotonia congenita.

132 133 Fig 2- Plateau potentials in ClCadr muscle. For A through D, the insets show portions of the 134 traces on an expanded time base. A) The response of muscle from an unaffected littermate to 135 injection of 200ms of depolarizing current (horizontal bar below the voltage trace); note that 136 action potentials stop when current stops. B-D) traces of myotonia triggered by a 200-ms 137 injection of depolarizing current from 3 different ClCadr muscle fibers. The following membrane 138 potentials are identified in C and D: membrane potential prior to stimulation, initial membrane 139 potential during the plateau potential, membrane potential prior to the termination of the plateau 140 potential, and membrane potential following repolarization. E) Development of a plateau 141 potential during repetitive stimulation at 8 Hz (stimuli represented by vertical hash marks under 142 the recording).

143 144 In ClCadr muscle, ClC-1 chloride conductance has been absent throughout development 145 such that plateau potentials could be a compensatory response to muscle hyperexcitability. To 146 test this possibility, we acutely blocked ClC-1 chloride channels in muscle from unaffected 147 littermates with 100 µM 9-anthracene carboxylic acid (9-AC). This dose of 9-AC blocks more 148 than 95% of ClC-1 chloride channels in skeletal muscle (Palade and Barchi, 1977) and has been 149 used to model myotonia congenita both in vitro and in vivo (van Lunteren et al., 2011; Desaphy 150 et al., 2013; Desaphy et al., 2014; Skov et al., 2015). Acute blocking of ClC-1 triggered 151 myotonia and plateau potentials in 94% of fibers (Fig 3A, n= 53 fibers from 8 mice). These data 152 strongly suggest that the ion channels responsible for development of plateau potentials are 153 present in wild-type skeletal muscle. 154 In contrast to the lack of variability in voltage at onset and termination of plateau 155 potentials, the duration in both the ClCadr and 9-AC treated models of myotonia was highly 156 variable (Fig 3B, Table 1, variance = 3216s for ClCadr and 623s for 9-AC treated duration). The 157 reason for the high variance of duration was that the rate of repolarization during the plateau 158 potential varied by more than 100-fold (Fig 3B, Table 1). It was generally not possible to record 159 multiple plateau potentials in individual ClCadr fibers due to the long median duration. However, 160 it was possible to record multiple plateau potentials within individual fibers of 9-AC treated 161 muscle, as most lasted only a few seconds. Within individual fibers, the slope of repolarization 162 of plateau potentials was less variable such that duration was relatively constant with a mean 163 variance of 0.5s ± 0.7s (n = 17 9-AC treated fibers in which 4 or more plateau potentials were 164 recorded). 165 In both ClCadr muscle and 9-AC treated muscle, plateau potentials did not occur 166 following every run of myotonia (Fig 3C). To determine why some runs of myotonia ended in 167 plateau potentials while others did not, we compared the mean voltage at the end of runs of 168 myotonia that produced plateau potentials vs. the mean voltage at the end of runs that did not 169 produce plateau potentials, in 9-AC treated fibers. There was a strong correlation between the 170 mean membrane potential during the final 500-ms of runs of myotonia and development of 171 plateau potentials. In 22/22 fibers, the mean membrane potential was more depolarized in runs 172 of myotonia that produced plateau potentials (mean = -40.1 ± 3.1 mV vs -49.0 ± 4.5 mV, p = 5 x 173 10-11, paired t-test, Fig 3D). This suggested that a voltage-dependent current might be involved.

174 However, another feature of myotonia that determines whether a plateau potential is triggered is 175 the firing rate, which might correlate with elevation of intracellular Ca2+. Thus we examined and 176 found a strong correlation between the firing rate of myotonia runs and subsequent development 177 of plateau potentials: In 21/22 fibers, the mean firing rate was higher for runs of myotonia ending 178 in plateau potentials (mean = 31.2 ± 5.7 Hz vs 23.4 ± 2.9, p = 7 x 10-6, paired t-test, Fig 3E). 179 Thus, while voltage-dependent channels might be involved, this indicated possible participation 180 of channels gated by other factors, such as elevation of intracellular Ca2+.

181 182 Fig 3: Characterization of plateau potentials. A) Three examples of plateau potentials of 183 different duration in 9-AC treated muscle. The 200-ms current injection is indicated by a 184 horizontal bar underneath the trace. Indicated on each trace is the membrane potential at the start 185 and end of the plateau potential. B) The duration of plateau potentials in the ClCadr and 9-AC 186 models of myotonia plotted against the rate of repolarization during the plateau potential. A 187 linear fit was performed on the log-log plot with an R2 value of 0.90 and a slope of -0.97. C) 188 Shown are two runs of myotonia from the same muscle fiber, one ending in a plateau potential 189 and one ending with repolarization. The mean membrane potential and firing rate in the final 190 500ms of myotonia (horizontal bar) are indicated by the arrows above each trace. D) Plot of the 191 average membrane potential during the final 500 ms of myotonia for 22 fibers in which there 192 were both a run of myotonia ending in a plateau potential and a run ending with repolarization 193 (No plateau potential). E) Plot of the average firing rate during the final 500 ms of myotonia for 194 the same 22 fibers.

195 Table 1: Plateau potential parameters in ClCadr and 9-AC treated unaffected muscle 196 Pre- Initial PP PP repol Final PP Max repol Post PP Vm Duration of PP myotonia Vm (mV) slope Vm (mV) rate (mV/s) (mV) (s) Vm (mV) (mV/s) ClCadr -79.3 ± 2.9 -29.7 ± 3.6 -1.3 ± 4.4 -43.0 ± 5.4 142 ± 41 -75.7 ± 4.3 57.6 ± 56.7 (-79.6) (-29.6) (-0.2) (-43.3) (138) (-76.0) (42.2) 9-AC -81.5 ± 2.6 -31.2 ± 3.1 -3.4 ± 1.8* -45.2 ± 2.3 197 ± 44** -80.8 ± 2.9 11.7 ± 25.0*** (-82.4) (-31.4) (-3.5) (-45.1) (192) (-81.6) (4.1) 197 198 Shown are the mean value ± the standard deviation for parameters of plateau potentials in ClCadr 199 fibers and 9-AC treated fibers. Below the mean value is the median value in parenthesis. n = 26 200 fibers with plateau potentials from 10 ClCadr mice and 50 fibers with plateau potentials from 8 201 unaffected muscles treated with 9-AC. Vm is membrane potential, repol is repolarization. Pre- 202 myotonia Vm is the resting membrane potential prior to stimulation. Initial PP Vm is the 203 membrane potential at the beginning of the plateau potential. Final PP Vm is the membrane 204 potential prior to the sudden repolarization terminating the plateau potential. Max repol rate is 205 the maximum rate of repolarization during the sudden repolarization. Post PP Vm is the 206 membrane potential following termination of the plateau potential. * indicates p = .017 of log 207 transformed data (t-test), ** p = 0.001 (t-test), *** p = 4.5 x 10-4 of log transformed data (t-test). 208

209 210 Ca2+ and Na+ currents contribute to generation of plateau potentials 211 In spinal motor neurons, L-type Ca2+ channels play a central role in generation of plateau 212 potentials (Alaburda et al., 2002; Heckman and Enoka, 2012; Hounsgaard, 2017). To determine 2+ 213 whether Ca current through skeletal muscle L-type channel (Cav1.1) triggers plateau potentials, 214 we performed recordings on skeletal muscle fibers from a mouse model (ncDHPR) in which the 2+ 215 pore region of Cav1.1 carries a point mutation leading to ablation of inward Ca current (Dayal 216 et al., 2017). We used voltage clamp of FDB/IO fibers to verify the absence of inward currents 217 in ncDHPR mice. In wild-type littermate controls, ramp depolarization following block of Na+, 218 K+, and Cl- channels triggered a large, inward Ca2+ current, which began to activate at -15.1 ± 219 6.9 mV (n= 4 fibers) with a mean amplitude of 165 ± 46 nA (Fig 4 A, B). Ramp depolarization 220 triggered no inward Ca2+ current in ncDHPR muscle fibers (mean = 0 ± 0 nA, n = 10 fibers (Fig 221 4C).

222 To determine whether current flow through Cav1.1 contributes to generation of plateau 223 potentials, current clamp recordings were performed. Application of 100 µM 9-AC led to 224 development of plateau potentials in 41/49 fibers from 4 wild-type mice and in 47/49 fibers from 225 3 ncDHPR mice (Fig 4D and E). Analysis of the characteristics of plateau potentials suggests 2+ 226 that Ca current flow through Cav1.1 channels may influence the duration of the plateau. While 227 there was no difference in the beginning or ending voltages of plateau potentials, the duration 228 was shorter in ncDHPR muscle (Fig 4D and E, Table 2). The cause of the shorter plateau 229 potential was an increase in the rate of repolarization (Table 2). These data suggest that Ca2+

230 influx through Cav1.1 does not play a role in initiation of plateau potentials, but is involved in 231 sustaining them. 232

233

234 Fig 4: Current flow through Cav1.1 does not initiate, but helps to sustain, plateau potentials in 9- 235 AC treated muscle. A) The voltage protocol applied to FDB/IO fibers consisted of a ramp 236 depolarization from -85 mV to +10 mV applied over 8 s. B) A large inward current is present in 237 wild-type muscle when Na+, K+, and Cl- currents were blocked. C) ncDHPR muscle fibers 238 confirm the absence of inward Ca2+ current as there is only the linear change in current due to the 239 changing command potential. D) A plateau potential in a 9-AC treated wild-type muscle fiber. 240 E) A plateau potential in a 9-AC treated ncDHPR fiber. 241 242 243 Table 2: Plateau potential parameters in 9-AC treated wild-type and ncDHPR muscle Pre-myotonia Initial plateau PP repol Final PP Vm Max repol Post PP Vm Duration of PP Vm (mV) Vm (mV) slope (mV/s) (mV) rate (mV/s) (mV) (s)

wild-type -79.3 ± 0.9 -36.0 ± 1.6 -3.3 ± 0.7 -48.1 ± 1.2 -125 ± 6 -79.5 ± 0.4 3.9 ± 0.7 (-79.5) (-36.0) (-3.1) (-48.5) (125) (-79.7) (3.8) ncDHPR -78.9 ± 0.6 -37.2 ± 0.8 -5.4 ± 0.6* -49.2 ± 1.1 -134 ± 10 -79 ± 0.8 2.4 ± 0.3* (-79.0) (-37.1) (-5.3) (-49.7) (138) (-79.3) (2.3) 244 Shown are the mean value and the standard deviation for parameters of plateau potentials in 9- 245 AC treated wild-type and ncDHPR fibers. Below the mean value in parentheses is the median 246 value. n = 41 fibers from 4 wild-type mice and n = 47 fibers from 3 ncDHPR mice. Vm is 247 membrane potential, repol is repolarization. Pre-myotonia Vm is the resting membrane potential 248 prior to stimulation. Initial PP Vm is the membrane potential at the beginning of the plateau 249 potential. Final PP Vm is the membrane potential prior to the sudden repolarization terminating 250 the plateau potential. Max repol rate is the maximum rate of repolarization during the sudden 251 repolarization. Post PP Vm is the membrane potential following termination of the plateau 252 potential. * indicates p = 0.01 for both significant differences (t-test).

253 + 254 The Nav1.4-mediated Na current responsible for generation of action potentials in 255 skeletal muscle inactivates within ms of depolarization, making it highly unlikely that it 256 contributes to development of plateau potentials. However, a Na+ current lacking fast 257 inactivation (Na+ persistent inward current, NaPIC) was found to be involved in the generation 258 of plateau depolarizations in muscle in a toxin model of hyperkalemic periodic paralysis 259 (Cannon and Corey, 1993). We recently determined that NaPIC contributes to repetitive firing 260 occurring during myotonia (Hawash et al., 2017; Metzger et al., 2020). NaPIC is present in

261 normal skeletal muscle and likely derives from a small subset of Nav1.4 channels that are in a 262 conformation that differs from fast-inactivating channels (Patlak and Ortiz, 1986; Gage et al., 263 1989). We term the Na+ channels responsible for action potentials “fast-inactivating Na+ 264 channels” and Na+ channels lacking fast inactivation “NaPIC”. 265 To determine whether NaPIC might play a role in generation of plateau potentials, we 266 applied ranolazine to 9-AC treated muscle. Ranolazine has been found to preferentially block 267 NaPIC in brain, heart, peripheral nerve, and skeletal muscle (El-Bizri et al., 2011; Kahlig et al., 268 2014). We previously found that ranolazine was effective in eliminating myotonia by blocking 269 NaPIC while sparing enough fast-inactivating Na+ channels to allow for repetitive firing of 270 action potentials triggered by current injection (Novak et al., 2015; Hawash et al., 2017). As 271 shown in Fig 5A, when myotonia was triggered by treatment of muscle with 9-AC, 94% of fibers 272 (n= 53 fibers from 8 mice) developed plateau potentials in response to a 200-ms injection of 273 depolarizing current. Following treatment with 40 µM ranolazine, 0/22 fibers from 3 mice 274 developed plateau potentials after 200-ms current injection (Fig 5A, p < .01 vs untreated). These 275 data were consistent with NaPIC playing a role in development of plateau potentials. 276 However, as myotonia was greatly reduced by ranolazine (Novak et al., 2015; Hawash et 277 al., 2017), it was possible that elimination of plateau potentials was secondary to a reduction in 278 the number of myotonic action potentials. We thus changed our stimulation protocol to a 2-s 279 train of 3-ms stimulus pulses delivered at 20 Hz. This firing rate and duration of firing mimics 280 the duration and rate of firing during runs of myotonia (Hawash et al., 2017). For trains of 281 stimuli, the amplitude of 3-ms pulses of current were first adjusted to find the lowest current 282 required to elicit an action potential. The current was then increased by 10 nA prior to delivering 283 a train of stimuli. 2s of 20 Hz stimulation triggered plateau potentials in 46/55 fibers (n = 5

284 mice, Fig 5B). Out of the 46 fibers with plateau potentials triggered by 20 Hz trains of stimuli, 285 11 fibers entered plateau potentials with limited (3 or less myotonic APs) or no myotonia. When 286 40 µM ranolazine was applied, plateau potentials developed in 0/68 fibers (n = 5 mice, p < .01 vs 287 untreated, Fig 5B). These data suggest that ranolazine is not eliminating plateau potentials via a 288 secondary effect of prevention of repetitive firing. 289 As shown in Fig. 3C and D, the mean membrane potential at the end of a run of myotonia 290 correlated with whether myotonia terminated in a plateau potential or with repolarization. Thus, 291 we tested if ranolazine prevented plateau potentials by decreasing the mean membrane potential 292 prior to development of plateau potentials. Untreated fibers with additional action potentials and 293 plateau potentials occurring during the 2-s stimulation (myotonia) were excluded from analysis 294 to ensure that the presence of myotonia or plateau potentials did not account for the difference in 295 mean membrane potential. As all untreated fibers initially had plateau potentials or myotonia 296 during repetitive stimulation, the analysis was not possible. To address this issue, the warm-up 297 phenomenon was induced using 8 Hz 10s trains, which lessens myotonia due to slow inactivation 298 of Na channels (Novak et al., 2015). It must thus be noted that the untreated muscle was not at 299 baseline but had already undergone some Na channel inactivation. No induction of warm-up 300 was necessary following treatment with ranolazine, as no fibers had myotonia or plateau 301 potentials. In the absence of ranolazine, the mean membrane potential during the 500ms prior to 302 the plateau potential was -49.6 ± 2.5 mV (n = 5 muscles, 28 fibers). In the presence of 40 µM 303 ranolazine, the mean membrane potential was less depolarized (-56.2 ± 2.9 mV, p < .05 vs 304 untreated, n = 5 muscles, Fig 5D). These data suggest that ranolazine prevents plateau potentials 305 by lessening depolarization of the mean membrane potential during the 20 Hz stimulation. 306 There are two contributors to the mean membrane potential during repetitive stimulation: 307 1) the membrane potential during action potentials, and 2) the membrane potential during the 308 interspike interval. We analyzed the contribution of each of these to the hyperpolarization 309 caused by ranolazine. By the 30th action potential of the 20 Hz stimulation, action potential 310 duration had increased to close to 8ms. This widening of the spike-form was likely due to failure 311 of the membrane potential to fully repolarize between action potentials, given that depolarization 312 has previously been found to cause widening of action potentials (Renaud and Light, 1992; 313 Yensen et al., 2002; Miranda et al., 2017). We examined the mean membrane potential during 314 the 8ms encompassing the 30th action potential and found a trend (not statistically significant)

315 toward lessening of depolarization following treatment with ranolazine (Fig 5C, E: -28.2 ± 3.4 vs 316 -32.9 ± 3.8 mV, p = 0.07). With 20 Hz stimulation, there is an action potential every 50ms. 317 After taking the mean membrane potential for the 8ms encompassing the 30th action potential, 318 there remained 42ms in which there was no action potential. The membrane potential for this 319 42ms interspike interval before the 30th action potential was less depolarized following treatment 320 with ranolazine (Fig 5C, F: -56.0 ± 2.3 vs -61.7 ± 3.0 mV, p < .05). As the interspike interval 321 accounts for 84% (42/50ms) of the time between spikes during 20 Hz stimulation, 322 hyperpolarization of this interval is largely responsible for hyperpolarization of the mean 323 membrane potential. 324

325

326 327 Fig 5: Block of plateau potentials by ranolazine in 9-AC treated muscle is associated with 328 lessening of depolarization during repetitive firing. A) Examples of the response to a 200-ms 329 injection of depolarizing current at baseline and following treatment with 40 µM ranolazine. B) 330 Examples of the response to 2s of stimulation at 20 Hz. The vertical arrow points to the 30th 331 action potential in each trace, which was used for analysis of differences between ranolazine 332 untreated and ranolazine treated muscle (D-F). C) Plots of the membrane potential during the 8 333 ms encompassing the action potential and the 42ms encompassing the interspike interval for the 334 untreated and ranolazine traces shown in B. Each point represents the value for a single action 335 potential during the 2s of 20 Hz stimulation. action potential MP = the mean membrane 336 potential during the 8 ms encompassing the action potential, Inter-AP MP = the mean membrane 337 potential during the 42-ms interspike interval. D) Plot of the mean membrane potential during 338 the last 500 ms of stimulation. Each point represents an animal average derived from at least 4 339 fibers without (n = 5) and with treatment with ranolazine (n = 5). The horizontal line represents 340 the mean. E and F) Plots of the mean membrane potential during the 30th action potential and the 341 30th interspike interval. * indicates p = 0.005 (t-test), ** p= 0.01 (t-test). 342 343 Ranolazine prevents transient weakness in vivo 344 The finding that ranolazine eliminated plateau potentials allowed us to explore whether 345 plateau potentials are the mechanism underlying transient weakness in vivo. We recorded triceps 346 surae force in 5 ClCadr myotonic mice before and 45 minutes after intraperitoneal (i.p.) injection 347 of 50 mg/kg of ranolazine. As shown previously(Novak et al., 2015), treatment with ranolazine 348 decreased myotonia such that muscle was able to more rapidly relax following termination of 349 stimulation (Fig 6A). In addition to lessening myotonia, treatment with ranolazine eliminated 350 transient weakness in all 5 mice (Fig 6A and B, p < .01 vs untreated at 16s). The elimination of 351 both plateau potentials and transient weakness by ranolazine supports the hypothesis that plateau 352 potentials are the mechanism underlying transient weakness in vivo. 353

354 355 Fig 6: Ranolazine eliminates transient weakness in vivo. A) Shown are force traces from a 356 triceps surae muscle group before and 45 minutes after i.p. injection of 50 mg/kg ranolazine. B) 357 Shown is the mean normalized force in 5 ClCadr mice before and after injection of ranolazine. p

358 = .001 for the difference in force 16s (*) after the initial stimulation (paired t-test, 95% 359 confidence interval 70-86 vs 102-113). 360

361 Discussion: 362 Motor dysfunction in recessive myotonia (Becker disease) involves both muscle stiffness 363 and transient weakness. Using intracellular recordings from a mouse model of myotonia 364 congenita (ClCadr), we discovered that, while some runs of myotonia resolved with 365 repolarization, others terminated with a plateau potential; that is, depolarization to a membrane 366 potential between -30 and -45 mV, lasting up to 100s. There was gradual repolarization during 367 plateau potentials until the membrane potential reached -45 mV, at which point a sudden 368 repolarization to the resting membrane potential occurred. During plateau potentials, muscle 369 fibers were inexcitable. Studies of genetic and pharmacologic mouse models suggest both 2+ 370 current through voltage-activated CaV1.1 Ca channels and sodium persistent inward current 371 (NaPIC) may contribute to plateau potentials. Na+ persistent inward current (NaPIC) through 2+ 372 NaV1.4 channels is the key trigger of plateau potentials and current through Cav1.1 Ca channels 373 contributes to sustaining plateau potentials. Blocking NaPIC with ranolazine eliminated both 374 plateau potentials in vitro and transient weakness in vivo. Our results suggest plateau potentials 375 are the mechanism underlying transient weakness in Becker disease. 376 377 Rapid transition between hyperexcitability and inexcitability in Becker Disease 378 Our data suggest that muscle from a mouse model of Becker disease undergoes a rapid 379 transition between the states of hyperexcitability (myotonia) and inexcitability (due to plateau 380 potentials), shown in Fig 7. Repeated firing of action potentials during voluntary contraction 381 triggers myotonia, which often transitions to a depolarization that forms a plateau potential. 382 During plateau potentials, fibers cannot generate action potentials in response to stimulation, 383 providing an explanation for the drop in CMAP amplitude reported in patients (Ricker and 384 Meinck, 1972; Brown, 1974; Aminoff et al., 1977; Deymeer et al., 1998; Drost et al., 2001; 385 Modoni et al., 2011). The reason for the rapid transition between myotonia (hyperexcitability) 386 and plateau potentials (inexcitability) is that both states are caused by depolarization secondary 387 to loss of muscle Cl- current. The difference is one of degree: when depolarization is mild, Na+ 388 channels are not inactivated, such that repetitive firing of action potentials is triggered. When 389 depolarization worsens, Na+ channels inactivate, such that inexcitability and paralysis ensue. 390 This proposal is similar to the current understanding of hyperkalemic periodic paralysis, in which 391 there is often myotonia at the beginning of attacks (during the initial, mild depolarization) and

392 weakness at the height of an attack (when depolarization is maximal) (Cannon, 2015; Statland et 393 al., 2018). 394

395 396 397 Fig 7: Voluntary contraction in myotonia congenita triggers a sequential progression through 398 states of hyperexcitability and inexcitability. Shown on the left is a motor unit consisting of a 399 motor neuron and the muscle fibers it innervates. At rest, both the motor neuron and muscle 400 fibers are hyperpolarized and muscle is relaxed. To initiate voluntary contraction, the motor 401 neuron fires repeated action potentials, which activate the neuromuscular junction to trigger 402 repeated firing of action potentials in muscle (indicated by a yellow outline around the fiber). 403 With the rapid firing of muscle action potentials, there is sustained contraction and force 404 production (blue line). At the end of voluntary contraction, the motor neuron stops firing and the 405 neuromuscular junction (NMJ) repolarizes, but in myotonic muscle there is continued 406 involuntary firing of action potentials, which slows relaxation of muscle. When myotonia is 407 terminated by transition into a plateau potential, muscle remains depolarized but cannot fire 408 action potentials (black outline of the fiber), such that muscle is paralyzed. Finally, there is 409 sudden repolarization and return of muscle to the resting state. ACh = acetylcholine. 410

411 412 Ion channels contributing to generation and maintenance of plateau potentials 413 Involvement of voltage-gated channels in the generation of plateau potentials is 414 suggested by the strong correlation between membrane potential and the initiation and 415 termination of plateau potentials. In runs of myotonia that terminated in a plateau potential, the 416 mean membrane potentials averaged to include both action potentials and interspike intervals 417 prior to development of a plateau potential was -40 mV; whereas, within the same fibers, runs of 418 myotonia terminating with repolarization had a mean membrane potential prior to repolarization 419 of -49 mV. The midpoint of the difference between these values is close to -44 mV. In ClCadr 420 muscle, the mean membrane potential prior to termination of plateau potentials was -43 mV, and 421 in 9-AC treated muscle it was -45 mV. Thus, a mean membrane potential of -44 mV appears to 422 predict both entry into, as well as termination of, plateau potentials.

423 Both Cav1.1 and Nav1.4 are voltage-gated channels that could depolarize muscle to

424 potentials achieved during plateau potentials. In spinal motor neurons, CaV1.3 channels play a 425 central role in generation of plateau potentials that last many seconds (Alaburda et al., 2002; 426 Heckman and Enoka, 2012; Hounsgaard, 2017). Here, we show that the development of plateau 2+ 427 potentials was not affected in ncDHPR muscle that lack Ca current through Cav1.1. However, 428 there was a reduction in duration of plateau potentials in ncDHPR muscle. These data suggest 2+ 429 that Ca flux through Cav1.1 channels contributes to sustaining plateau potentials. This was a 430 surprise, as the membrane potential during plateau potentials is more negative than voltages at

431 which there is significant current through Cav1.1 channels (Garcia and Beam, 1994; Bannister 432 and Beam, 2013). One explanation is that in intact, mature fibers, prolonged depolarization

433 during plateau potentials allows for activation of Cav1.1 at more negative potentials than during 434 the shorter step depolarizations typically used for voltage clamp studies. Our findings suggest 2+ 435 that despite Ca current through Cav1.1 having no essential role in healthy muscle (Dayal et al., 436 2017), it may contribute to pathologic depolarization in muscle channelopathies.

437 Partial block of Nav1.4 channels with ranolazine eliminated plateau potentials, suggesting

438 involvement of Nav1.4. However, the majority of Nav1.4 channels inactivate within ms (fast- 439 inactivating Nav1.4 channels), such that they cannot contribute to prolonged depolarization 440 during plateau potentials. There is a Na+ persistent inward current (NaPIC) in skeletal muscle

441 that appears to arise from a subset of Nav1.4 channels that lack fast inactivation (Patlak and

442 Ortiz, 1986; Gage et al., 1989; Hawash et al., 2017). This subset of Nav1.4 channels could play a 443 role in the development of plateau potentials. 444 Ranolazine has been found to preferentially block NaPIC (El-Bizri et al., 2011; Kahlig et 445 al., 2014). When myotonic muscle was treated with ranolazine to block NaPIC, depolarization 446 of the mean membrane potential during repetitive firing was decreased and plateau potentials 447 were prevented. An important contributor was lessening of depolarization of the membrane 448 potential during the interspike interval. Previously, the only identified contributor to 449 depolarization of the interspike membrane potential was K+ build-up in the transverse (t)-tubules 450 (invaginations of the sarcolemma), which depolarizes the K+ equilibrium potential (Adrian and 451 Bryant, 1974; Adrian and Marshall, 1976; Wallinga et al., 1999; Fraser et al., 2011). The finding 452 that block of NaPIC lessens depolarization of the interspike membrane potential suggests that 453 NaPIC is activated during this interval. 454 We hypothesize that activation of NaPIC combines with K+ build-up in t-tubules to 455 depolarize muscle to a mean membrane potential of -44 mV during myotonia, such that a plateau 456 potential is triggered. An additional contributor to this depolarization may be the lessening of 457 inward rectifier potassium channel (Kir) conductance with depolarization (Standen and Stanfield, 458 1980; Struyk and Cannon, 2008). It is unclear whether NaPIC, K+ build-up, and decreased Kir 459 conductance can fully account for depolarization during plateau potentials. Based on studies of 460 sustained depolarizations (sometimes termed plateau potentials) in neurons, a family of ion 461 channels that might contribute is the transient receptor potential (TRP) ion channel family (Yan 462 et al., 2009; Phelan et al., 2012). Members of the TRP ion channel family are expressed in 463 skeletal muscle (Brinkmeier, 2011; Gailly, 2012), and we recently found that activation of 464 TRPV4 plays a role in triggering percussion myotonia (Dupont et al., 2020). 465 It is likely that voltage-gated ion channels promoting repolarization are involved in the 466 sudden termination of plateau potentials. Oscillations in membrane potential often occurred at 467 the beginning or end of plateau potentials. In motor neurons, oscillations in membrane potential 468 are caused by a balance between 2 voltage-gated ion channels: one promoting depolarization and 469 one promoting hyperpolarization (Iglesias et al., 2011; Sciamanna and Wilson, 2011; Nardelli et

470 al., 2017). Kv channels could participate in oscillations occurring during plateau potentials given

471 that Kv conductance in muscle is large and the channels begin to activate at voltages reached 472 during plateau potentials (Beam and Donaldson, 1983; DiFranco et al., 2012). Thus, it may be

473 possible to prevent plateau potentials by either blocking channels promoting depolarization such

474 as NaPIC, or by opening channels promoting repolarization such as Kv channels. 475 476 Blocking NaPIC as therapy for transient weakness 477 In a mouse model of myotonia congenita, ranolazine prevented both development of 478 plateau potentials in vitro and transient weakness in vivo. In open label trials of ranolazine in 479 both myotonia congenita and paramyotonia congenita, there were statistically significant 480 reductions in the degree of self-reported weakness (Arnold et al., 2017; Lorusso et al., 2019). 481 Taken together, these data suggest that the clinical benefit of blocking Na+ channels in some 482 diseases with myotonia may result, in part, from prevention of transient weakness secondary to 483 development of plateau potentials.

484 Mutations of Nav1.4 responsible for hyperkalemic periodic paralysis increase NaPIC 485 (Cannon et al., 1991; Cannon and Strittmatter, 1993), which appears to play a central role in 486 triggering the depolarization that underlies attacks of transient weakness (Lehmann-Horn et al., 487 1987; Jurkat-Rott et al., 2010; Cannon, 2015). This suggests that blocking NaPIC should be 488 effective in treating hyperkalemic periodic paralysis. However, treating patients with Na channel 489 blockers that are effective in treating myotonia, such as mexiletine, has previously been found to 490 be ineffective (Ricker et al., 1983; Ricker et al., 1986). The finding that blocking NaPIC with 491 ranolazine lessens depolarization of the interspike membrane potential suggests it might be worth 492 considering a trial of this FDA-approved drug’s efficacy in hyperkalemic periodic paralysis. 493 494 Conclusion 495 We identified currents contributing to plateau potentials that are responsible for transient 496 weakness in recessive myotonia congenita. We also determined that blocking a current 497 contributing to plateau potentials provided effective amelioration of transient weakness. 498 Currents contributing to plateau potentials are present in wild-type muscle; thus, they might 499 contribute to depolarization in other muscle channelopathies with transient weakness, such as 500 hyper- and hypokalemic periodic paralysis. Identification of channels involved in generation of 501 plateau potentials in skeletal muscle may thus advance understanding of regulation of excitability 502 in both healthy and diseased muscle. 503

504 Materials and Methods: 505 Mice 506 All animal procedures were performed in accordance with the policies of the Animal 507 Care and Use Committee of Wright State University and were conducted in accordance with the 508 United States Public Health Service's Policy on Humane Care and Use of Laboratory Animals. 509 The genetic mouse model of myotonia congenita used was Clcn1adr-mto/J (ClCadr) mice, 510 which have a homozygous null mutation in the Clcn1 gene (Jackson Laboratory Stock #000939). 511 The pharmacologic model of Becker disease involved treatment of muscle with 100 µM 9- 2+ 512 anthracenecarboxylic acid (9-AC). The mouse model of non-Ca -conducting CaV1.1 used was 513 ncDHPR, carrying point mutation N617D in pore loop II in the Cacna1S gene (Dayal et al., 514 2017). 515 Genotyping of ClCadr mice was performed as previously described to select heterozygous 516 mice for breeding (Dupont et al., 2019). Otherwise, homozygous myotonic mice were identified 517 by appearance and behavior as previously described (Novak et al., 2015). Unaffected littermates 518 were used as controls. Genotyping for selection of homozygous ncDHPR was performed as 519 previously described (Dayal et al., 2017). Both male and female mice were used from 2 months 520 to 6 months of age. As mice with myotonia have difficulty climbing to reach food, symptomatic 521 mice were supplied with moistened chow paste (Irradiated Rodent Diet; Harlan Teklad 2918) on 522 the floor of the cage. 523 524 Electrophysiology 525 Current and voltage clamp recordings were performed at 20–22°C. 526 Current clamp:

527 Mice were sacrificed using CO2 inhalation followed by cervical dislocation, and both 528 extensor digitorum longus (EDL) muscles were dissected out tendon-to-tendon. Muscles were 529 maintained and recorded at 22oC within 6 hours of sacrifice. The recording chamber was

530 continuously perfused with Ringer solution containing (in mM) NaCl, 118; KCl, 3.5; CaCl2, 1.5; o 531 MgSO4, 0.7; NaHCO3, 26.2; NaH2PO4, 1.7; glucose, 5.5 (pH 7.3-7.4 at 20-22 C), and

532 equilibrated with 95% O2 and 5% CO2. 533 Intracellular recordings were performed as previously described (Novak et al., 2015; 534 Hawash et al., 2017; Dupont et al., 2019). Briefly, muscles were loaded with 50 μM N-benzyl-p-

535 toluenesulfonamide (BTS, Tokyo Chemical Industry) for at least 30 minutes prior to recording to 536 prevent contraction. BTS was dissolved in DMSO and added to the perfusate prior to bubbling

537 with 95% O2 and 5% CO2, as we have found that BTS is more soluble at a basic pH. Prior to 538 recording, muscles were stained for 3 minutes with 10µM 4-(4-diethylaminostyrl)-N- 539 methylpyridinium iodide (4-Di-2ASP, Molecular Probes) to allow imaging muscle with an 540 upright epifluorescence microscope (Leica DMR, Bannockburn, IL). 541 Micro-electrodes were filled with 3M KCl solution containing 1 mM sulforhodamine to 542 visualize the electrodes with epifluorescence. Resistances were between 15 and 30 MΩ, and 543 capacitance compensation was optimized prior to recording. Fibers with resting potentials more 544 depolarized than –74 mV were excluded from analysis. 545 In cases where there was failure of action potentials during trains of stimulation, we did 546 not determine whether the failure was due to the presence of an absolute or relative refractory 547 period by altering current injection during the train of stimuli. 548 549 Voltage clamp: 550 Flexor digitorum brevis (FDB) and interosseous (IO) muscle fibers were isolated as 551 previously described (Waters et al., 2013; Hawash et al., 2017). Briefly, muscles were surgically 552 removed and enzymatically dissociated at 37 °C under mild agitation for ∼1 h using 1,000 U/mL 553 of collagenase type IV (Worthington Biochemical). Mechanical dissociation was completed 554 using mild trituration in buffer with no collagenase. The fibers were allowed to recover at 20-22 555 °C for 1 hour before being used for electrical measurements. 556 Both the current-passing and voltage-sensing electrodes were filled with internal solution 557 (see below) and had resistances of ~15 MΩ. After impalement, 10 minutes of hyperpolarizing 558 current injection was allowed for equilibration of the electrode solution. Data were acquired at 559 20 kHz and low-pass filtered with the internal Axoclamp 900A filters at 1 kHz. The voltage 560 clamp command signal was low-pass filtered with an external Warner LFP-8 at 1 kHz.

561 Internal solution (in mM) was as follows: 75 aspartate, 30 EGTA, 15 Ca(OH)2, 5 MgCl2, 562 5 ATP di-Na, 5 phosphocreatine di-Na, 5 glutathione, 20 MOPS, and pH 7.2 with CsOH.

563 Extracellular solution (in mM) was as follows: 144 NaCl, 4 CsCl, 1.2 CaCl2, 0.6 MgCl2, 5

564 glucose, 1 NaH2PO4, 10 MOPS, 0.05 BaCl2, 0.1 9-AC, 0.001 TTX, 0.01 Ouabain, 0.1 3,4- 565 diaminopyridine (3,4-DAP), and pH 7.4 with NaOH.

566 567 Drugs and doses (when used, in mM): 568 0.05 BTS/N-benzyl-p-toluenesulfonamide (TCI America, Portand OR) 569 0.1 9-AC/9-anthracenecarboxylic acid (Tocris Bioscience, Minneapolis, MN) 570 0.05 Ranolazine (Sigma-Aldrich, St. Louis, MO) 571 0.001 TTX/Tetrodotoxin (Alomone Labs, Jerusalem, Israel) 572 0.1 3,4-DAP/3,4-Diaminopyridine (Sigma-Aldrich, St. Louis, MO) 573 0.01 Ouabain (Sigma-Aldrich, St. Louis, MO) 574 575 In situ muscle force recording: 576 In situ muscle force recordings were performed as previously described (Dupont et al., 577 2019; Wang et al., 2020). Briefly, mice were anesthetized via isoflurane inhalation; then the 578 distal tendon of the triceps surae muscles was attached to a force transduction motor and the 579 sciatic nerve was stimulated while isometric muscle force generation was measured. The sciatic 580 nerve was stimulated with constant current injection. The amplitude of the current pulse was 581 adjusted to 150% of the current required to trigger a single action potential. To induce myotonia, 582 45 pulses of 1ms duration were delivered at 100 Hz. To follow development of transient 583 weakness, 15 pulses delivered at 100 Hz were delivered every 4s. Muscle temperature was 584 monitored with a laser probe and maintained between 29oC and 31oC with a heat lamp. The 585 muscle was kept moist by applying mineral oil. Ranolazine was administered via intraperitoneal 586 (i.p.) injection at a dose of 50 mg/kg dissolved in water. The typical volume of water injected 587 was 100 µl. 588 589 Statistics 590 Sample size was determined by past practice, where we have found an n of 5 muscles 591 from 5 different mice, studied on different days, yields statistically significant differences in 592 muscle action potential properties. At least 5 muscle fibers were recorded from in each muscle. 593 However, for studies of plateau potentials in ClCadr mice we obtained recordings with adequate 594 preservation of resting membrane potential in only a subset of fibers so fewer fibers per muscle 595 were included in the final analysis. This was due to the prolonged duration of plateau potentials, 596 which required maintaining prolonged impalement of individual fibers with two electrodes. No

597 outlier data points were excluded. Intracellular recording data from different mice were analyzed 598 using nested analysis of variance with n as the number of mice, with data presented as mean ± 599 SD. p < 0.05 was considered to be significant. The numbers of animals and fibers used are 600 described in the corresponding figure legends and text. For parameters that were not normally 601 distributed, such as duration of the plateau potential and slope of the repolarization, differences 602 between two data sets were analyzed after applying a natural log transformation, which yielded 603 normally distributed data. 604 For comparisons of data within individual muscle fibers (mean membrane potential and 605 mean firing rate for runs of myotonia, without and with plateau potentials), the paired student’s t- 606 test was used with n as the number of fibers. For force recording comparisons before and after 607 ranolazine treatment, the paired student’s t-test was used with n as the number of mice. For 608 comparisons of force recordings between myotonic mice and unaffected littermates, a 2-sample 609 student’s t-test was used. 610 611 612 Acknowledgements: This work was supported by NIH grants AR074985 (M.M.R.), and MDA 613 grant 602459 (M.M.R.). 614 615 Author contributions: JM, KD, CD, AH, KN and AK all acquired, analyzed data, and made 616 figures. MG, AD, AV and MR drafted and revised the manuscript. 617 618 Competing interests: Nothing to report. 619

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