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1.  Preclinical evaluation of marketed sodium channel blockers in a rat model of myotonia discloses promising antimyotonic drugs 
Experimental Neurology  2014;255(100):96-102.
Although the sodium channel blocker mexiletine is considered the first-line drug in myotonia, some patients experiment adverse effects, while others do not gain any benefit. Other antimyotonic drugs are thus needed to offer mexiletine alternatives. In the present study, we used a previously-validated rat model of myotonia congenita to compare six marketed sodium channel blockers to mexiletine. Myotonia was induced in the rat by injection of anthracen-9-carboxylic acid, a muscle chloride channel blocker. The drugs were given orally and myotonia was evaluated by measuring the time of righting reflex. The drugs were also tested on sodium currents recorded in a cell line transfected with the human skeletal muscle sodium channel hNav1.4 using patch-clamp technique. In vivo, carbamazepine and propafenone showed antimyotonic activity at doses similar to mexiletine (ED50 close to 5 mg/kg); flecainide and orphenadrine showed greater potency (ED50 near 1 mg/kg); lubeluzole and riluzole were the more potent (ED50 near 0.1 mg/kg). The antimyotonic activity of drugs in vivo was linearly correlated with their potency in blocking hNav1.4 channels in vitro. Deviation was observed for propafenone and carbamazepine, likely due to pharmacokinetics and multiple targets. The comparison of the antimyotonic dose calculated in rats with the current clinical dose in humans strongly suggests that all the tested drugs may be used safely for the treatment of human myotonia. Considering the limits of mexiletine tolerability and the occurrence of non-responders, this study proposes an arsenal of alternative drugs, which may prove useful to increase the quality of life of individuals suffering from non-dystrophic myotonia. Further clinical trials are warranted to confirm these results.
Highlights
•Seven sodium channel blockers show antimyotonic activity in a rat model of myotonia.•The ED50 value ranges from 0.1 (riluzole, lubeluzole) to 5 mg/kg (mexiletine, carbamazepine).•The drugs use-dependently block hNav1.4 channels in cells in myotonic-like conditions.•The IC50 values in vitro were well linearly correlated with the ED50 values in vivo.•The study discloses promising new therapeutic options for myotonic patients.
doi:10.1016/j.expneurol.2014.02.023
PMCID: PMC4004800  PMID: 24613829
Non-dystrophic myotonia; Sodium channel blockers; Mexiletine; Rat model; Patch-clamp; Over-excitability
2.  In vivo evaluation of antimyotonic efficacy of β-adrenergic drugs in a rat model of myotonia 
Neuropharmacology  2013;65(C):21-27.
The sodium channel blocker mexiletine is considered the first-line drug in myotonic syndromes, a group of muscle disorders characterized by membrane over-excitability. We previously showed that the β-adrenoceptor modulators, clenbuterol and propranolol, block voltage-gated sodium channels in a manner reminiscent to mexiletine, whereas salbutamol and nadolol do not. We now developed a pharmacological rat model of myotonia congenita to perform in vivo preclinical test of antimyotonic drugs. Myotonia was induced by i.p. injection of 30 mg/kg of anthracene-9-carboxylic acid (9-AC), a muscle chloride channel blocker, and evaluated by measuring the time of righting reflex (TRR). The TRR was prolonged from <0.5 s in control conditions to a maximum of ∼4 s, thirty minutes after 9-AC injection, then gradually recovered in a few hours. Oral administration of mexiletine twenty minutes after 9-AC injection significantly hampered the TRR prolongation, with an half-maximum efficient dose (ED50) of 12 mg/kg. Both propranolol and clenbuterol produced a dose-dependent antimyotonic effect similar to mexiletine, with ED50 values close to 20 mg/kg. Antimyotonic effects of 40 mg/kg mexiletine and propranolol lasted for 2 h. We also demonstrated, using patch-clamp methods, that both propranolol enantiomers exerted a similar block of skeletal muscle hNav1.4 channels expressed in HEK293 cells. The two enantiomers (15 mg/kg) also showed a similar antimyotonic activity in vivo in the myotonic rat. Among the drugs tested, the R(+)-enantiomer of propranolol may merit further investigation in humans, because it exerts antimyotonic effect in the rat model, while lacking of significant activity on the β-adrenergic pathway. This study provides a new and useful in vivo preclinical model of myotonia congenita in order to individuate the most promising antimyotonic drugs to be tested in humans.
Highlights
► An in vivo pharmacological model of myotonia congenita was developed in the rat using 9-AC i.p. injection. ► A preclinical screening of antimyotonic drugs was performed. ► Propranolol and clenbuterol exert antimyotonic activity comparable to mexiletine. ► Both propranolol enantiomers block skeletal muscle hNav1.4 sodium channels in vitro. ► Both propranolol enantiomers exert similar antimyotonic effect in vivo.
doi:10.1016/j.neuropharm.2012.09.006
PMCID: PMC3546166  PMID: 23000075
Myotonia; Over-excitability; Propranolol; Mexiletine; In vivo rat model; hNav1.4; TRR, time of righting reflex; 9-AC, anthracene-9-carboxylic acid
3.  Correction of ClC-1 splicing eliminates chloride channelopathy and myotonia in mouse models of myotonic dystrophy 
The Journal of Clinical Investigation  2007;117(12):3952-3957.
In myotonic dystrophy (dystrophia myotonica [DM]), an increase in the excitability of skeletal muscle leads to repetitive action potentials, stiffness, and delayed relaxation. This constellation of features, collectively known as myotonia, is associated with abnormal alternative splicing of the muscle-specific chloride channel (ClC-1) and reduced conductance of chloride ions in the sarcolemma. However, the mechanistic basis of the chloride channelopathy and its relationship to the development of myotonia are uncertain. Here we show that a morpholino antisense oligonucleotide (AON) targeting the 3′ splice site of ClC-1 exon 7a reversed the defect of ClC-1 alternative splicing in 2 mouse models of DM. By repressing the inclusion of this exon, the AON restored the full-length reading frame in ClC-1 mRNA, upregulated the level of ClC-1 mRNA, increased the expression of ClC-1 protein in the surface membrane, normalized muscle ClC-1 current density and deactivation kinetics, and eliminated myotonic discharges. These observations indicate that the myotonia and chloride channelopathy observed in DM both result from abnormal alternative splicing of ClC-1 and that antisense-induced exon skipping offers a powerful method for correcting alternative splicing defects in DM.
doi:10.1172/JCI33355
PMCID: PMC2075481  PMID: 18008009
4.  Improvement of diaphragm and limb muscle isotonic contractile performance by K+ channel blockade 
The K+ channel blocking aminopyridines greatly improve skeletal muscle isometric contractile performance during low to intermediate stimulation frequencies, making them potentially useful as inotropic agents for functional neuromuscular stimulation applications. Most restorative applications involve muscle shortening; however, previous studies on the effects of aminopyridines have involved muscle being held at constant length. Isotonic contractions differ substantially from isometric contractions at a cellular level with regards to factors such as cross-bridge formation and energetic requirements. The present study tested effects of 3,4-diaminopyridine (DAP) on isotonic contractile performance of diaphragm, extensor digitorum longus (EDL) and soleus muscles from rats. During contractions elicited during 20 Hz stimulation, DAP improved work over a range of loads for all three muscles. In contrast, peak power was augmented for the diaphragm and EDL but not the soleus. Maintenance of increased work and peak power was tested during repetitive fatigue-inducing stimulation using a single load of 40% and a stimulation frequency of 20 Hz. Work and peak power of both diaphragm and EDL were augmented by DAP for considerable periods of time, whereas that of soleus muscle was not affected significantly. These results demonstrate that DAP greatly improves both work and peak power of the diaphragm and EDL muscle during isotonic contractions, which combined with previous data on isometric contractions indicates that this agent is suitable for enhancing muscle performance during a range of contractile modalities.
doi:10.1186/1743-0003-7-1
PMCID: PMC2821379  PMID: 20064261
5.  Cancer cachexia decreases specific force and accelerates fatigue in limb muscle 
Cancer cachexia is a complex metabolic syndrome that is characterized by the loss of skeletal muscle mass and weakness, which compromises physical function, reduces quality of life, and ultimately can lead to mortality. Experimental models of cancer cachexia have recapitulated this skeletal muscle atrophy and consequent decline in muscle force generating capacity. However, more recently, we provided evidence that during severe cancer cachexia muscle weakness in the diaphragm muscle cannot be entirely accounted for by the muscle atrophy. This indicates that muscle weakness is not just a consequence of muscle atrophy but that there is also significant contractile dysfunction. The current study aimed to determine whether contractile dysfunction is also present in limb muscles during severe Colon-26 (C26) carcinoma cachexia by studying the glycolytic extensor digitorum longus (EDL) muscle and the oxidative soleus muscle, which has an activity pattern that more closely resembles the diaphragm. Severe C-26 cancer cachexia caused significant muscle fiber atrophy and a reduction in maximum absolute force in both the EDL and soleus muscles. However, normalization to muscle cross sectional area further demonstrated a 13% decrease in maximum isometric specific force in the EDL and an even greater decrease (17%) in maximum isometric specific force in the soleus. Time to peak tension and half relaxation time were also significantly slowed in both the EDL and the solei from C-26 mice compared to controls. Since, in addition to postural control, the oxidative soleus is also important for normal locomotion, we further performed a fatigue trial in the soleus and found that the decrease in relative force was greater and more rapid in solei from C-26 mice compared to controls. These data demonstrate that severe cancer cachexia causes profound muscle weakness that is not entirely explained by the muscle atrophy. In addition, cancer cachexia decreases the fatigue resistance of the soleus muscle, a postural muscle typically resistant to fatigue. Thus, specifically targeting contractile dysfunction represents an additional means to counter muscle weakness in cancer cachexia, in addition to targeting the prevention of muscle atrophy.
doi:10.1016/j.bbrc.2013.05.018
PMCID: PMC3708303  PMID: 23673294
muscle weakness; muscle atrophy; C-26; contractile dysfunction
6.  Targeted mutation of mouse skeletal muscle sodium channel produces myotonia and potassium-sensitive weakness 
The Journal of Clinical Investigation  2008;118(4):1437-1449.
Hyperkalemic periodic paralysis (HyperKPP) produces myotonia and attacks of muscle weakness triggered by rest after exercise or by K+ ingestion. We introduced a missense substitution corresponding to a human familial HyperKPP mutation (Met1592Val) into the mouse gene encoding the skeletal muscle voltage-gated Na+ channel NaV1.4. Mice heterozygous for this mutation exhibited prominent myotonia at rest and muscle fiber-type switching to a more oxidative phenotype compared with controls. Isolated mutant extensor digitorum longus muscles were abnormally sensitive to the Na+/K+ pump inhibitor ouabain and exhibited age-dependent changes, including delayed relaxation and altered generation of tetanic force. Moreover, rapid and sustained weakness of isolated mutant muscles was induced when the extracellular K+ concentration was increased from 4 mM to 10 mM, a level observed in the muscle interstitium of humans during exercise. Mutant muscle recovered from stimulation-induced fatigue more slowly than did control muscle, and the extent of recovery was decreased in the presence of high extracellular K+ levels. These findings demonstrate that expression of the Met1592Val Na+ channel in mouse muscle is sufficient to produce important features of HyperKPP, including myotonia, K+-sensitive paralysis, and susceptibility to delayed weakness during recovery from fatigue.
doi:10.1172/JCI32638
PMCID: PMC2260907  PMID: 18317596
7.  Measuring quality of life impairment in skeletal muscle channelopathies 
European Journal of Neurology  2012;19(11):1470-1476.
Background and purpose
Fatigue and pain have been previously shown to be important determinants for decreasing quality of life (QoL) in one report in patients with non-dystrophic myotonia. The aims of our study were to assess QoL in skeletal muscle channelopathies (SMC) using INQoL (individualized QoL) and SF-36 questionnaires.
Methods
We administered INQoL and SF-36 to 66 Italian patients with SMC (26: periodic paralysis, 36: myotonia congenita and 4: Andersen-Tawil) and compared the results in 422 patients with myotonic dystrophies (DM1: 382; and DM2: 40).
Results
(i) INQoL index in SMC is similar to that in DMs (P = 0.79). (ii) Patients with myotonia congenita have the worst perception of QoL. (iii) Myotonia has the most detrimental effect on patients with myotonia congenita, followed by patients with DM2 and then by patients with DM1 and hyperkalemic periodic paralysis. (iv) Pain is a significant complaint in patients with myotonia congenita, hypokalemic periodic paralysis and DM2 but not in DM1. (v) Fatigue has a similar detrimental effect on all patient groups except for patients with hyperkalemic periodic paralysis in whom muscle weakness and myotonia more than fatigue affect QoL perception. (vi) Muscle symptoms considered in INQoL correlate with physical symptoms assessed by SF-36 (R from −0.34 to −0.76).
Conclusions
QoL perception in patients with SMC is similar to that of patients with DMs, chronic multisystem disabling conditions. Our results provide information to target treatment and health care of these patients. The sensitivity of INQoL to changes in QoL in the SMC needs to be further explored in longitudinal studies.
doi:10.1111/j.1468-1331.2012.03751.x
PMCID: PMC3492909  PMID: 22607270
INQoL; myotonic dystrophy; non-dystrophic myotonias; quality of life; SF-36; skeletal muscle channelopathies
8.  Functional characterization of ClC-1 mutations from patients affected by recessive myotonia congenita presenting with different clinical phenotypes☆ 
Experimental Neurology  2013;248(100):530-540.
Myotonia congenita (MC) is caused by loss-of-function mutations of the muscle ClC-1 chloride channel. Clinical manifestations include the variable association of myotonia and transitory weakness. We recently described a cohort of recessive MC patients showing, at a low rate repetitive nerves stimulation protocol, different values of compound muscle action potential (CMAP) transitory depression, which is considered the neurophysiologic counterpart of transitory weakness. From among this cohort, we studied the chloride currents generated by G190S (associated with pronounced transitory depression), F167L (little or no transitory depression), and A531V (variable transitory depression) hClC-1 mutants in transfected HEK293 cells using patch-clamp. While F167L had no effect on chloride currents, G190S dramatically shifts the voltage dependence of channel activation and A531V reduces channel expression. Such variability in molecular mechanisms observed in the hClC-1 mutants may help to explain the different clinical and neurophysiologic manifestations of each ClCN1 mutation. In addition we examined five different mutations found in compound heterozygosis with F167L, including the novel P558S, and we identified additional molecular defects. Finally, the G190S mutation appeared to impair acetazolamide effects on chloride currents in vitro.
Highlights
•Myotonia congenita is a muscle disorder due to mutations in ClC-1 chloride channel.•Eight ClC-1 channel mutants were studied using patch-clamp technique.•Mutations induce a variety of molecular defects in ClC-1 channel function.•We discuss the relationship between genotype and clinical phenotype.
doi:10.1016/j.expneurol.2013.07.018
PMCID: PMC3781327  PMID: 23933576
Acetazolamide; Chloride channel mutation; ClC-1 chloride channel; Genotype–phenotype relationship; Myotonia congenita; Non-dystrophic myotonia; Patch-clamp; Transitory weakness
9.  Dominantly-Inherited Myotonia Congenita Resulting from a Mutation That Increases Open Probability of the Muscle Chloride Channel CLC-1 
Neuromolecular medicine  2012;14(4):328-337.
Myotonia congenita-inducing mutations in the muscle chloride channel CLC-1 normally result in reduced open probability (Po) of this channel. One well-accepted mechanism of the dominant inheritance of this disease involves a dominant-negative effect of the mutation on the function of the common gate of this homodimeric, double-barreled molecule. We report here a family with myotonia congenita characterized by muscle stiffness and clinical and electrophysiologic myotonic phenomena transmitted in an autosomal dominant pattern. DNA sequencing of DMPK and ZNF9 genes for myotonic muscular dystrophy types I and II was normal, whereas sequencing of CLC-1 encoding gene, CLCN1, identified a single heterozygous missense mutation, G233S. Patch-clamp analyses of this mutant CLC-1 channel in Xenopus oocytes revealed an increased Po of the channel’s fast gate, from ~ 0.4 in the wild type to > 0.9 in the mutant at −90 mV. In contrast, the mutant exhibits a minimal effect on the Po of the common gate. These results are consistent with the structural prediction that the mutation site is adjacent to the fast gate of the channel. Overall, the mutant could lead to a significantly reduced dynamic response of CLC-1 to membrane depolarization, from a 5-fold increase in chloride conductance in the wild type to a 2-fold increase in the mutant—this might result in slower membrane repolarization during an action potential. Since expression levels of the mutant and wild-type subunits in artificial model cell systems were unable to explain the disease symptoms, the mechanism leading to dominant inheritance in this family remains to be determined.
doi:10.1007/s12017-012-8190-1
PMCID: PMC3508202  PMID: 22790975
myotonia congenita; muscle; chloride channel; CLCN1; dominant; gain of function
10.  The Effects of the KCNQ Openers Retigabine and Flupirtine on Myotonia in Mammalian Skeletal Muscle Induced by a Chloride Channel Blocker 
The purpose of this study was to investigate the effect of KCNQ (potassium channel, voltage-gated, KQT-like subfamily) openers in preventing myotonia caused by anthracene-9-carboxylic acid (9-AC, a chloride channel blocker). An animal model of myotonia can be elicited in murine skeletal muscle by 9-AC treatment. KCNQ openers, such as retigabine and flupirtine, can inhibit the increased twitch amplitude (0.1 Hz stimulation) and reduce the tetanic fade (20 Hz stimulations) observed in the presence of 9-AC. Furthermore, the prolonged twitch duration of skeletal muscle was also inhibited by retigabine or flupirtine. Lamotrigine (an anticonvulsant drug) has a lesser effect on the muscle twitch amplitude, tetanic fade, and prolonged twitch duration as compared with KCNQ openers. In experiments using intracellular recordings, retigabine and flupirtine clearly reduced the firing frequencies of repetitive action potentials induced by 9-AC. These data suggested that KCNQ openers prevent the myotonia induced by 9-AC, at least partly through enhancing potassium conductance in skeletal muscle. Taken together, these results indicate that KCNQ openers are potential alternative therapeutic agents for the treatment of myotonia.
doi:10.1155/2012/803082
PMCID: PMC3320144  PMID: 22536291
11.  Sarcolemmal-restricted localization of functional ClC-1 channels in mouse skeletal muscle 
The Journal of General Physiology  2010;136(6):597-613.
Skeletal muscle fibers exhibit a high resting chloride conductance primarily determined by ClC-1 chloride channels that stabilize the resting membrane potential during repetitive stimulation. Although the importance of ClC-1 channel activity in maintaining normal muscle excitability is well appreciated, the subcellular location of this conductance remains highly controversial. Using a three-pronged multidisciplinary approach, we determined the location of functional ClC-1 channels in adult mouse skeletal muscle. First, formamide-induced detubulation of single flexor digitorum brevis (FDB) muscle fibers from 15–16-day-old mice did not significantly alter macroscopic ClC-1 current magnitude (at −140 mV; −39.0 ± 4.5 and −42.3 ± 5.0 nA, respectively), deactivation kinetics, or voltage dependence of channel activation (V1/2 was −61.0 ± 1.7 and −64.5 ± 2.8 mV; k was 20.5 ± 0.8 and 22.8 ± 1.2 mV, respectively), despite a 33% reduction in cell capacitance (from 465 ± 36 to 312 ± 23 pF). In paired whole cell voltage clamp experiments, where ClC-1 activity was measured before and after detubulation in the same fiber, no reduction in ClC-1 activity was observed, despite an ∼40 and 60% reduction in membrane capacitance in FDB fibers from 15–16-day-old and adult mice, respectively. Second, using immunofluorescence and confocal microscopy, native ClC-1 channels in adult mouse FDB fibers were localized within the sarcolemma, 90° out of phase with double rows of dihydropyridine receptor immunostaining of the T-tubule system. Third, adenoviral-mediated expression of green fluorescent protein–tagged ClC-1 channels in adult skeletal muscle of a mouse model of myotonic dystrophy type 1 resulted in a significant reduction in myotonia and localization of channels to the sarcolemma. Collectively, these results demonstrate that the majority of functional ClC-1 channels localize to the sarcolemma and provide essential insight into the basis of myofiber excitability in normal and diseased skeletal muscle.
doi:10.1085/jgp.201010526
PMCID: PMC2995150  PMID: 21078869
12.  Incubating Isolated Mouse EDL Muscles with Creatine Improves Force Production and Twitch Kinetics in Fatigue Due to Reduction in Ionic Strength 
PLoS ONE  2011;6(8):e22742.
Background
Creatine supplementation can improve performance during high intensity exercise in humans and improve muscle strength in certain myopathies. In this present study, we investigated the direct effects of acute creatine incubation on isolated mouse fast-twitch EDL muscles, and examined how these effects change with fatigue.
Methods and Results
The extensor digitorum longus muscle from mice aged 12–14 weeks was isolated and stimulated with field electrodes to measure force characteristics in 3 different states: (i) before fatigue; (ii) immediately after a fatigue protocol; and (iii) after recovery. These served as the control measurements for the muscle. The muscle was then incubated in a creatine solution and washed. The measurement of force characteristics in the 3 different states was then repeated. In un-fatigued muscle, creatine incubation increased the maximal tetanic force. In fatigued muscle, creatine treatment increased the force produced at all frequencies of stimulation. Incubation also increased the rate of twitch relaxation and twitch contraction in fatigued muscle. During repetitive fatiguing stimulation, creatine-treated muscles took 55.1±9.5% longer than control muscles to lose half of their original force. Measurement of weight changes showed that creatine incubation increased EDL muscle mass by 7%.
Conclusion
Acute creatine application improves force production in isolated fast-twitch EDL muscle, and these improvements are particularly apparent when the muscle is fatigued. One likely mechanism for this improvement is an increase in Ca2+ sensitivity of contractile proteins as a result of ionic strength decreases following creatine incubation.
doi:10.1371/journal.pone.0022742
PMCID: PMC3151260  PMID: 21850234
13.  Mexiletine is an effective antimyotonia treatment in myotonic dystrophy type 1(LOE Classification) 
Neurology  2010;74(18):1441-1448.
Objective:
To determine if mexiletine is safe and effective in reducing myotonia in myotonic dystrophy type 1 (DM1).
Background:
Myotonia is an early, prominent symptom in DM1 and contributes to decreased dexterity, gait instability, difficulty with speech/swallowing, and muscle pain. A few preliminary trials have suggested that the antiarrhythmic drug mexiletine is useful, symptomatic treatment for nondystrophic myotonic disorders and DM1.
Methods:
We performed 2 randomized, double-blind, placebo-controlled crossover trials, each involving 20 ambulatory DM1 participants with grip or percussion myotonia on examination. The initial trial compared 150 mg of mexiletine 3 times daily to placebo, and the second trial compared 200 mg of mexiletine 3 times daily to placebo. Treatment periods were 7 weeks in duration separated by a 4- to 8-week washout period. The primary measure of myotonia was time for isometric grip force to relax from 90% to 5% of peak force after a 3-second maximum grip contraction. EKG measurements and adverse events were monitored in both trials.
Results:
There was a significant reduction in grip relaxation time with both 150 and 200 mg dosages of mexiletine. Treatment with mexiletine at either dosage was not associated with any serious adverse events, or with prolongation of the PR or QTc intervals or of QRS duration. Mild adverse events were observed with both placebo and mexiletine treatment.
Conclusions:
Mexiletine at dosages of 150 and 200 mg 3 times daily is effective, safe, and well-tolerated over 7 weeks as an antimyotonia treatment in DM1.
Classification of Evidence:
This study provides Class I evidence that mexiletine at dosages of 150 and 200 mg 3 times daily over 7 weeks is well-tolerated and effective in reducing handgrip relaxation time in DM1.
GLOSSARY
= myotonic dystrophy type 1;
= maximal voluntary isometric contraction;
= peak force;
= relaxation time;
= 3 times daily.
doi:10.1212/WNL.0b013e3181dc1a3a
PMCID: PMC2871004  PMID: 20439846
14.  Impaired Wheel Running Exercise in CLC-1 Chloride Channel-Deficient Myotonic Mice 
Background: Genetic deficiency of the muscle CLC-1 chloride channel leads to myotonia, which is manifested most prominently by slowing of muscle relaxation. Humans experience this as muscle stiffness upon initiation of contraction, although this can be overcome with repeated efforts (the “warm-up” phenomenon). The extent to which CLC-1 deficiency impairs exercise activity is controversial. We hypothesized that skeletal muscle CLC-1 chloride channel deficiency leads to severe reductions in spontaneous exercise. Methodology/Principal Findings: To examine this quantitatively, myotonic CLC-1 deficient mice were provided access to running wheels, and their spontaneous running activity was quantified subsequently. Differences between myotonic and normal mice in running were not present soon after introduction to the running wheels, but were fully established during week 2. During the eighth week, myotonic mice were running significantly less than normal mice (322 ± 177 vs 5058 ± 1253 m/day, P = 0.025). Furthermore, there were considerable reductions in consecutive running times (18.8 ± 1.5 vs 59.0 ± 3.7 min, P < 0.001) and in the distance per consecutive running period (58 ± 38 vs 601 ± 174 m, P = 0.048) in myotonic compared with normal animals. Conclusion/Significance: These findings indicate that CLC-1 chloride deficient myotonia in mice markedly impairs spontaneous exercise activity, with reductions in both total distance and consecutive running times.
doi:10.3389/fphys.2011.00047
PMCID: PMC3152724  PMID: 21886624
myotonia congenita; exercise; genetic; CLC-1 chloride channel; skeletal muscle
15.  An analysis of myotonia in paramyotonia congenita1 
In two subjects with paramyotonia congenita myotonic delay in muscle relaxation, recorded electromyographically and with a displacement transducer, was found to increase with repeated forceful contractions. Myotonia was elicited readily in warm temperatures, was initially aggravated by cooling, but was invariably lost as muscle fatigue developed. The EMG evidence of myotonia usually subsided before complete muscle relaxation had occurred, suggesting that a defect of the contractile mechanism was present over and above any defect at membrane level.
PMCID: PMC494804  PMID: 4422263
16.  ClC1 chloride channel in myotonic dystrophy type 2 and ClC1 splicing in vitro 
Acta Myologica  2012;31(2):144-153.
Myotonic dystrophy type 2 (DM2) is caused by CCTG-repeat expansions. Occurrence of splicing and mutations in the muscle chloride channel gene CLCN1 have been reported to contribute to the phenotype. To examine the effect of CLCN1 in DM2 in Germany, we determined the frequency of a representative ClC1 mutation, R894X, and its effect on DM2 clinical features. Then, we examined CLCN1 mRNA splice variants in patient muscle functionally expressed the most abundant variant, and determined its subcellular localization. Finally, we established a cellular system for studying mouse clcn1 pre-mRNA splicing and tested effects of expression of (CCUG)18, (CUG)24 and (AAG)24 RNAs. The R894X mutation was present in 7.7% of DM2 families. DM2 R894X-carriers had more myotonia and myalgia than non-carriers. The most abundant CLCN1 splice variant in DM2 (80% of all transcripts) excluded exons 6-7 and lead to a truncated ClC1236X protein. Heterologous ClC1236X expression did not yield functional channels. Co-expression with ClC1 did not show a dominant negative effect, but a slightly suppressive effect. In C2C12 cells, the clc1 splice variants generated by (CCUG)18-RNA resembled those in DM2 muscle and differed from those generated by (CUG)24 and (AAG)24. We conclude that ClC1 mutations exert gene dose effects and enhance myotonia and pain in DM2 in Germany. Additionally, the ClC1236X splice variant may contribute to myotonia in DM2. Since splice variants depend on the types of repeats expressed in the cellular C2C12 model, similar cell models of other tissues may be useful for studying repeatdependent pathogenetic mechanisms more easily than in transgenic animals.
PMCID: PMC3476861  PMID: 23097607
PROMM; myotonic dystrophy; chloride channel
17.  Inactivation of excitation-contraction coupling in rat extensor digitorum longus and soleus muscles 
The Journal of General Physiology  1988;91(5):737-757.
K contractures and two-microelectrode voltage-clamp techniques were used to measure inactivation of excitation-contraction coupling in small bundles of fibers from rat extensor digitorum longus (e.d.l.) and soleus muscles at 21 degrees C. The rate of spontaneous relaxation was faster in e.d.l. fibers: the time for 120 mM K contractures to decay to 50% of maximum tension was 9.8 +/- 0.5 s (mean +/- SEM) in e.d.l. and 16.8 +/- 1.7 s in soleus. The rate of decay depended on membrane potential: in e.d.l., the 50% decay time was 14.3 +/- 0.7 s for contractures in 80 mM K (Vm = 25 mV) and 4.9 +/- 0.4 s in 160 mM K (Vm = -3 mV). In contrast to activation, which occurred with less depolarization in soleus fibers, steady state inactivation required more depolarization: after 3 min at -40 mV in 40 mM K, the 200 mM K contracture amplitude in e.d.l. fell to 28 +/- 10% (n = 5) of control, but remained at 85 +/- 2% (n = 6) of control in soleus. These different inactivation properties in e.d.l. and soleus fibers were not influenced by the fact that the 200 mM K solution used to test for steady state inactivation produced contractures that were maximal in soleus fibers but submaximal in e.d.l.: a relatively similar depression was recorded in maximal (200 mM K) and submaximal (60 and 80 mM K) contracture tension. A steady state "pedestal" of tension was observed with maintained depolarization after K contracture relaxation and was larger in soleus than in e.d.l. fibers. The pedestal tension was attributed to the overlap between the activation and inactivation curves for tension vs. membrane potential, which was greater in soleus than in e.d.l. fibers. The K contracture results were confirmed with the two- microelectrode voltage clamp: the contraction threshold increased to more positive potentials at holding potentials of -50 mV in e.d.l. or - 40 mV in soleus. At holding potentials of -30 mV in e.d.l. or 0 mV in soleus, contraction could not be evoked by 15-ms pulses to +20 mV. Both K contracture and voltage-clamp experiments revealed that activation in soleus fibers occurred with a smaller transient depolarization and was maintained with greater steady state depolarization than in e.d.l. fibers. The K contracture and voltage-clamp results are described by a model in which contraction depends on the formation of a threshold concentration of activator from a voltage-sensitive molecule that can exist in the precursor, activator, or inactive states.
PMCID: PMC2216151  PMID: 3418320
18.  Muscle channelopathies and electrophysiological approach 
Myotonic syndromes and periodic paralyses are rare disorders of skeletal muscle characterized mainly by muscle stiffness or episodic attacks of weakness. Familial forms are caused by mutation in genes coding for skeletal muscle voltage ionic channels. Familial periodic paralysis and nondystrophic myotonias are disorders of skeletal muscle excitability caused by mutations in genes coding for voltage-gated ion channels. These diseases are characterized by episodic failure of motor activity due to muscle weakness (paralysis) or stiffness (myotonia). Clinical studies have identified two forms of periodic paralyses: hypokalemic periodic paralysis (hypoKPP) and hyperkalemic periodic paralysis (hyperKPP), based on changes in serum potassium levels during the attacks, and three distinct forms of myotonias: paramyotonia congenita (PC), potassium-aggravated myotonia (PAM), and myotonia congenita (MC). PC and PAM have been linked to missense mutations in the SCN4A gene, which encodes α subunit of the voltage-gated sodium channel, whereas MC is caused by mutations in the chloride channel gene (CLCN1). Exercise is known to trigger, aggravate, or relieve symptoms. Therefore, exercise can be used as a functional test in electromyography to improve the diagnosis of these muscle disorders. Abnormal changes in the compound muscle action potential can be disclosed using different exercise tests. Five electromyographic (EMG) patterns (I-V) that may be used in clinical practice as guides for molecular diagnosis are discussed.
doi:10.4103/0972-2327.40221
PMCID: PMC2781140  PMID: 19966974
Channelopathy; electromyographic; ion channel; myotonia; periodic paralysis
19.  Enhanced Ca2+ transport and muscle relaxation in skeletal muscle from sarcolipin-null mice 
Sarcolipin (SLN) inhibits sarco(endo)plasmic reticulum Ca2+-ATPase (SERCA) pumps. To evaluate the physiological significance of SLN in skeletal muscle, we compared muscle contractility and SERCA activity between Sln-null and wild-type mice. SLN protein expression in wild-type mice was abundant in soleus and red gastrocnemius (RG), low in extensor digitorum longus (EDL), and absent from white gastrocnemius (WG). SERCA activity rates were increased in soleus and RG, but not in EDL or WG, from Sln-null muscles, compared with wild type. No differences were seen between wild-type and Sln-null EDL muscles in force-frequency curves or maximum rates of force development (+dF/dt). Maximum relaxation rates (−dF/dt) of EDL were higher in Sln-null than wild type across a range of submaximal stimulation frequencies, but not during a twitch or peak tetanic contraction. For soleus, no differences were seen between wild type and Sln-null in peak tetanic force or +dF/dt; however, force-frequency curves showed that peak force during a twitch and 10-Hz contraction was lower in Sln-null. Changes in the soleus force-frequency curve corresponded with faster rates of force relaxation at nearly all stimulation frequencies in Sln-null compared with wild type. Repeated tetanic stimulation of soleus caused increased (−dF/dt) in wild type, but not in Sln-null. No compensatory responses were detected in analysis of other Ca2+ regulatory proteins using Western blotting and immunohistochemistry or myosin heavy chain expression using immunofluorescence. These results show that 1) SLN regulates Ca2+-ATPase activity thereby regulating contractile kinetics in at least some skeletal muscles, 2) the functional significance of SLN is graded to the endogenous SLN expression level, and 3) SLN inhibitory effects on SERCA function are relieved in response to repeated contractions thus enhancing relaxation rates.
doi:10.1152/ajpcell.00409.2010
PMCID: PMC3654932  PMID: 21697544 CAMSID: cams2941
knockout mouse; muscle contractility; isolated skeletal muscle; Ca2+ pump
20.  Muscle Chloride Channel Dysfunction in Two Mouse Models of Myotonic Dystrophy 
Muscle degeneration and myotonia are clinical hallmarks of myotonic dystrophy type 1 (DM1), a multisystemic disorder caused by a CTG repeat expansion in the 3′ untranslated region of the myotonic dystrophy protein kinase (DMPK) gene. Transgenic mice engineered to express mRNA with expanded (CUG)250 repeats (HSALR mice) exhibit prominent myotonia and altered splicing of muscle chloride channel gene (Clcn1) transcripts. We used whole-cell patch clamp recordings and nonstationary noise analysis to compare and biophysically characterize the magnitude, kinetics, voltage dependence, and single channel properties of the skeletal muscle chloride channel (ClC-1) in individual flexor digitorum brevis (FDB) muscle fibers isolated from 1–3-wk-old wild-type and HSALR mice. The results indicate that peak ClC-1 current density at −140 mV is reduced >70% (−48.5 ± 3.6 and −14.0 ± 1.6 pA/pF, respectively) and the kinetics of channel deactivation increased in FDB fibers obtained from 18–20- d-old HSALR mice. Nonstationary noise analysis revealed that the reduction in ClC-1 current density in HSALR FDB fibers results from a large reduction in ClC-1 channel density (170 ± 21 and 58 ± 11 channels/pF in control and HSALR fibers, respectively) and a modest decrease in maximal channel open probability(0.91 ± 0.01 and 0.75 ± 0.03, respectively). Qualitatively similar results were observed for ClC-1 channel activity in knockout mice for muscleblind-like 1 (Mbnl1ΔE3/ΔE3), a second murine model of DM1 that exhibits prominent myotonia and altered Clcn1 splicing (Kanadia et al., 2003). These results support a molecular mechanism for myotonia in DM1 in which a reduction in both the number of functional sarcolemmal ClC-1 and maximal channel open probability, as well as an acceleration in the kinetics of channel deactivation, results from CUG repeat–containing mRNA molecules sequestering Mbnl1 proteins required for proper CLCN1 pre-mRNA splicing and chloride channel function.
doi:10.1085/jgp.200609635
PMCID: PMC2151606  PMID: 17158949
21.  Refined Exercise testing can aid DNA-based Diagnosis in Muscle Channelopathies 
Annals of neurology  2011;69(2):328-340.
Objective
To improve the accuracy of genotype prediction and guide genetic testing in patients with muscle channelopathies we applied and refined specialised electrophysiological exercise test parameters.
Methods
We studied 56 genetically confirmed patients and 65 controls using needle electromyography, the long exercise test, and short exercise tests at room temperature, after cooling, and rewarming.
Results
Concordant amplitude-and-area decrements were more reliable than amplitude-only measurements when interpreting patterns of change during the short exercise tests. Concordant amplitude-and-area pattern I and pattern II decrements of >20% were 100% specific for PMC and MC respectively. When decrements at room temperature and after cooling were <20%, a repeat short exercise test after rewarming was useful in patients with myotonia congenita. Area measurements and rewarming distinguished true temperature sensitivity from amplitude reduction due to cold-induced slowing of muscle fibre conduction. In patients with negative short exercise tests, symptomatic eye closure myotonia predicted sodium channel myotonia over myotonia congenita. Distinctive ‘tornado-shaped’ neuromyotonia-like discharges may be seen in patients with paramyotonia congenita. In the long exercise test, area decrements from pre-exercise baseline were more sensitive than amplitude decrements-from-maximum-CMAP in patients with Andersen-Tawil syndrome. Possible ethnic differences in the normative data of the long exercise test argue for the use of appropriate ethnically-matched controls.
Interpretation
Concordant CMAP amplitude-and-area decrements of >20% allow more reliable interpretation of the short exercise tests and aid accurate DNA-based diagnosis. In patients with negative exercise tests, specific clinical features are helpful in differentiating sodium from chloride channel myotonia. A modified algorithm is suggested..
doi:10.1002/ana.22238
PMCID: PMC3051421  PMID: 21387378
22.  Activation of the MEF2 transcription factor in skeletal muscles from myotonic mice 
The Journal of Clinical Investigation  2002;109(10):1327-1333.
Becker syndrome, a recessive nondystrophic myotonia caused by mutations in the chloride channel 1 gene (CLCN1), is characterized by delayed muscle relaxation after contraction. The ADR (arrested development of righting response) mouse is an animal model for Becker syndrome. Skeletal muscles from ADR myotonic animals show an increased number of oxidative fibers with a lack of glycolytic fibers as well as signs of muscle hypertrophy. Through breeding ADR myotonic mice with mice harboring a MEF2-dependent reporter gene, we found that the transcriptional activity of MEF2 was dramatically enhanced in myotonic muscles. Post-translational induction of MEF2 transcriptional activity correlated with the activation of p38 MAPK and did not affect MEF2 DNA-binding affinity. Expression of class II histone deacetylases (HDACs), which repress MEF2-dependent gene expression, was significantly reduced in skeletal muscles from myotonic mice. These findings suggest that the combined effects of class II HDAC deficiency and p38 MAPK activation lead to potent upregulation of MEF2 transcriptional activity, which contributes to the long-term changes in gene expression and fiber-type transformation observed in myotonic skeletal muscles. These findings provide new molecular targets for potential treatment of congenital myotonia.
doi:10.1172/JCI15417
PMCID: PMC150985  PMID: 12021248
23.  Multiple Causes of Fatigue during Shortening Contractions in Rat Slow Twitch Skeletal Muscle 
PLoS ONE  2013;8(8):e71700.
Fatigue in muscles that shorten might have other causes than fatigue during isometric contractions, since both cross-bridge cycling and energy demand are different in the two exercise modes. While isometric contractions are extensively studied, the causes of fatigue in shortening contractions are poorly mapped. Here, we investigate fatigue mechanisms during shortening contractions in slow twitch skeletal muscle in near physiological conditions. Fatigue was induced in rat soleus muscles with maintained blood supply by in situ shortening contractions at 37°C. Muscles were stimulated repeatedly (1 s on/off at 30 Hz) for 15 min against a constant load, allowing the muscle to shorten and perform work. Fatigue and subsequent recovery was examined at 20 s, 100 s and 15 min exercise. The effects of prior exercise were investigated in a second exercise bout. Fatigue developed in three distinct phases. During the first 20 s the regulatory protein Myosin Light Chain-2 (slow isoform, MLC-2s) was rapidly dephosphorylated in parallel with reduced rate of force development and reduced shortening. In the second phase there was degradation of high-energy phosphates and accumulation of lactate, and these changes were related to slowing of muscle relengthening and relaxation, culminating at 100 s exercise. Slowing of relaxation was also associated with increased leak of calcium from the SR. During the third phase of exercise there was restoration of high-energy phosphates and elimination of lactate, and the slowing of relaxation disappeared, whereas dephosphorylation of MLC-2s and reduced shortening prevailed. Prior exercise improved relaxation parameters in a subsequent exercise bout, and we propose that this effect is a result of less accumulation of lactate due to more rapid onset of oxidative metabolism. The correlation between dephosphorylation of MLC-2s and reduced shortening was confirmed in various experimental settings, and we suggest MLC-2s as an important regulator of muscle shortening.
doi:10.1371/journal.pone.0071700
PMCID: PMC3745421  PMID: 23977116
24.  A Novel Mutation in CLCN1 Associated with Feline Myotonia Congenita 
PLoS ONE  2014;9(10):e109926.
Myotonia congenita (MC) is a skeletal muscle channelopathy characterized by inability of the muscle to relax following voluntary contraction. Worldwide population prevalence in humans is 1∶100,000. Studies in mice, dogs, humans and goats confirmed myotonia associated with functional defects in chloride channels and mutations in a skeletal muscle chloride channel (CLCN1). CLCN1 encodes for the most abundant chloride channel in the skeletal muscle cell membrane. Five random bred cats from Winnipeg, Canada with MC were examined. All cats had a protruding tongue, limited range of jaw motion and drooling with prominent neck and proximal limb musculature. All cats had blepharospasm upon palpebral reflex testing and a short-strided gait. Electromyograms demonstrated myotonic discharges at a mean frequency of 300 Hz resembling the sound of a ‘swarm of bees’. Muscle histopathology showed hypertrophy of all fiber types. Direct sequencing of CLCN1 revealed a mutation disrupting a donor splice site downstream of exon 16 in only the affected cats. In vitro translation of the mutated protein predicted a premature truncation and partial lack of the highly conserved CBS1 (cystathionine β-synthase) domain critical for ion transport activity and one dimerization domain pivotal in channel formation. Genetic screening of the Winnipeg random bred population of the cats' origin identified carriers of the mutation. A genetic test for population screening is now available and carrier cats from the feral population can be identified.
doi:10.1371/journal.pone.0109926
PMCID: PMC4214686  PMID: 25356766
25.  Effect of xanthine oxidase-generated extracellular superoxide on skeletal muscle force generation 
Skeletal muscle contractions increase superoxide anion in skeletal muscle extracellular space. We tested the hypotheses that 1) after an isometric contraction protocol, xanthine oxidase (XO) activity is a source of superoxide anion in the extracellular space of skeletal muscle and 2) the increase in XO-derived extracellular superoxide anion during contractions affects skeletal muscle contractile function. Superoxide anion was monitored in the extracellular space of mouse gastrocnemius muscles by following the reduction of cytochrome c in muscle microdialysates. A 15-min protocol of nondamaging isometric contractions increased the reduction of cytochrome c in microdialysates, indicating an increase in superoxide anion. Mice treated with the XO inhibitor oxypurinol showed a smaller increase in superoxide anions in muscle microdialysates following contractions than in microdialysates from muscles of vehicle-treated mice. Intact extensor digitorum longus (EDL) and soleus muscles from mice were also incubated in vitro with oxypurinol or polyethylene glycol-tagged Cu,Zn-SOD. Oxypurinol decreased the maximum tetanic force produced by EDL and soleus muscles, and polyethylene glycol-tagged Cu,Zn-SOD decreased the maximum force production by the EDL muscles. Neither agent influenced the rate of decline in force production when EDL or soleus muscles were repeatedly electrically stimulated using a 5-min fatiguing protocol (stimulation at 40 Hz for 0.1 s every 5 s). Thus these studies indicate that XO activity contributes to the increased superoxide anion detected within the extracellular space of skeletal muscles during nondamaging contractile activity and that XO-derived superoxide anion or derivatives of this radical have a positive effect on muscle force generation during isometric contractions of mouse skeletal muscles.
doi:10.1152/ajpregu.00142.2009
PMCID: PMC2806206  PMID: 19828843
contractile function; free radicals; exercise

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