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1.  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
2.  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.
•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.
PMCID: PMC3781327  PMID: 23933576
Acetazolamide; Chloride channel mutation; ClC-1 chloride channel; Genotype–phenotype relationship; Myotonia congenita; Non-dystrophic myotonia; Patch-clamp; Transitory weakness
3.  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.
PMCID: PMC3152724  PMID: 21886624
myotonia congenita; exercise; genetic; CLC-1 chloride channel; skeletal muscle
4.  Observations on the effects of low frequency electrical stimulation on fast muscles of dystrophic mice. 
The deterioration of tibialis anterior (TA) and extensor digitorum longus (EDL) muscles in dystrophic mice (C 57 BL dy/dy) was compared. The effects of chronic electrical stimulation on various characteristic properties of these muscles were also studied. The results indicate that EDL muscles are less affected by the disease than TA. This "selectivity" is difficult to explain since both muscles have similar fibre type composition. TA and EDL muscles that were stimulated for 10-28 days developed greater tetanic tensions than the contralateral muscles, but this effect was apparent only when the muscles were severely affected by the disease, that is the contralateral TA or EDL muscles developed less than 50% of the tension produced by muscles from normal animals. In all EDL muscles, stimulation increased the fatigue resistance. The time course of contraction and relaxation of dystrophic muscles is usually slower than that of normal muscles. The stimulation reduced this slowing effect, so that the stimulated muscles became similar to homologous muscles from normal littermates.
PMCID: PMC491203  PMID: 6977620
5.  Novel Mutations in the CLCN1 Gene of Myotonia Congenita: 2 Case Reports 
Introduction: Myotonia Congenita is an inherited myotonia that is due to a mutation in the skeletal muscle chloride channel CLCN1. These mutations lead to reduced sarcolemmal chloride conductance, causing delayed muscle relaxation that is evident as clinical and electrical myotonia.
Methods: We report the clinical presentations of two individuals with Myotonia Congenita (MC).
Results: Patient 1 has been diagnosed with the recessive form of MC, known as the Becker variant, and Patient 2 has been diagnosed with the dominant form of MC, known as the Thomsen variant. In both patients, the diagnosis was made based on the clinical presentation, EMG and CLCN1 gene sequencing. Patient 1 also had a muscle biopsy.
Conclusions: Genetic testing in both patients reveals previously unidentified mutations in the CLCN1 gene specific to Myotonia Congenita. We report the salient clinical features of each patient and discuss the effects and common types of CLCN1 mutations and review the literature.
PMCID: PMC3584487  PMID: 23483815
Myotonia Congenita; Becker variant; Thomsen variant; CLCN1 mutation
6.  Differences in Age-Related Alterations in Muscle Contraction Properties in Rat Tongue and Hindlimb 
Because of differences in muscle architecture and biomechanics, the purpose of this study was to determine whether muscle contractile properties of rat hindlimb and tongue were differentially affected by aging.
Deep peroneal and hypoglossal nerves were stimulated in 6 young and 7 old Fischer 344-Brown Norway rats to allow recording of muscle contractile properties of tongue and extensor digitorum longus (EDL) muscle in the hindlimb. In the same animals, the following measurements were made: (a) twitch contraction time (CT; in milliseconds), (b) half decay time (HDT; in milliseconds), (c) maximum twitch force (in grams), (d) tetanic force, and (e) fatigue index determined from repetitive stimulation of the muscles.
No significant differences were observed in young versus old groups in retrusive tongue forces, whereas a significant (p < .05) decrement in EDL tetanic forces was found in old rats. Slower CT in old rats was observed only in the tongue. Old and young groups were not significantly different in fatigue index or HDT for tongue or EDL.
Old animals generated equivalent maximum tongue forces with stimulation, but they were slower in achieving these forces than young animals. Limb and cranial muscles were not affected equally by aging. As such, information derived from limb muscle studies may not easily generalize to the cranial motor system.
PMCID: PMC2892886  PMID: 18658053
aging; tongue; extensor digitorum longus; muscle contraction
7.  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.
•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.
PMCID: PMC4004800  PMID: 24613829
Non-dystrophic myotonia; Sodium channel blockers; Mexiletine; Rat model; Patch-clamp; Over-excitability
8.  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.
PMCID: PMC2075481  PMID: 18008009
9.  A Quantitative Measure of Handgrip Myotonia in Non-dystrophic Myotonia 
Muscle & nerve  2012;46(4):482-489.
Non-dystrophic Myotonia (NDM) is characterized by myotonia without muscle wasting. A standardized quantitative myotonia assessment (QMA) is important for clinical trials.
Myotonia was assessed in 91 individuals enrolled in a natural history study using a commercially available computerized handgrip myometer and automated software. Average peak force and 90% to 5% relaxation times were compared to historical normal controls studied with identical methods.
30 subjects had chloride channel mutations, 31 sodium channel mutations, 6 DM2, and 24 no identified mutation. Chloride channel mutations were associated with prolonged 1st handgrip relaxation times, and warm up on subsequent handgrips. Sodium channel mutations were associated with prolonged 1st handgrip relaxation times and paradoxical myotonia or warm-up, depending on underlying mutations. DM2 subjects had normal relaxation times but decreased peak force. Sample size estimates are provided for clinical trial planning.
QMA is an automated, non-invasive technique for evaluating myotonia in NDM.
PMCID: PMC3564214  PMID: 22987687
natural history; ion channel mutation; muscle disease; myotonia; non-dystrophic myotonia
10.  Myotonia and flaccid dysarthria in patients with adult onset myotonic dystrophy 
Objective: To specify and quantify possible defects in speech execution in patients with adult onset myotonic dystrophy.
Methods: Studies on speech production were done on 30 mildly affected patients with myotonic dystrophy. Special attention was paid to myotonia. Because muscle activity can result in a decrease of myotonia, speech characteristics were measured before and after warm up. The possibility that warming up causes increased weakness was also assessed.
Results: As with other motor skills, a warm up effect was found in speech production, resulting in an increase in repetition rate and a decrease in variability of repetition rate. Signs of fatigue did not occur.
Conclusions: Warming up is valuable for patients with myotonic dystrophy in reducing the influence of myotonia on speech production.
PMCID: PMC1738733  PMID: 15377703
11.  Myotonia Congenita Mutation Enhances the Degradation of Human CLC-1 Chloride Channels 
PLoS ONE  2013;8(2):e55930.
Myotonia congenita is a hereditary muscle disorder caused by mutations in the human voltage-gated chloride (Cl−) channel CLC-1. Myotonia congenita can be inherited in an autosomal recessive (Becker type) or dominant (Thomsen type) fashion. One hypothesis for myotonia congenita is that the inheritance pattern of the disease is determined by the functional consequence of the mutation on the gating of CLC-1 channels. Several disease-related mutations, however, have been shown to yield functional CLC-1 channels with no detectable gating defects. In this study, we have functionally and biochemically characterized a myotonia mutant: A531V. Despite a gating property similar to that of wild-type (WT) channels, the mutant CLC-1 channel displayed a diminished whole-cell current density and a reduction in the total protein expression level. Our biochemical analyses further demonstrated that the reduced expression of A531V can be largely attributed to an enhanced proteasomal degradation as well as a defect in protein trafficking to surface membranes. Moreover, the A531V mutant protein also appeared to be associated with excessive endosomal-lysosomal degradation. Neither the reduced protein expression nor the diminished current density was rescued by incubating A531V-expressing cells at 27°C. These results demonstrate that the molecular pathophysiology of A531V does not involve anomalous channel gating, but rather a disruption of the balance between the synthesis and degradation of the CLC-1 channel protein.
PMCID: PMC3570542  PMID: 23424641
12.  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.
PMCID: PMC3508202  PMID: 22790975
myotonia congenita; muscle; chloride channel; CLCN1; dominant; gain of function
13.  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.
PMCID: PMC2821379  PMID: 20064261
14.  Contractile Properties of EDL and Soleus Muscles of Myostatin-Deficient Mice 
Myostatin is a negative regulator of muscle mass. The impact of myostatin deficiency on the contractile properties of healthy muscles has not been determined. We hypothesized that myostatin deficiency would increase the maximum tetanic force (Po), but decrease the specific Po (sPo) of muscles and increase the susceptibility to contraction-induced injury. The in vitro contractile properties of EDL and soleus muscles from wild type (MSTN+/+), heterozygous-null (MSTN+/−) and homozygous-null (MSTN−/−) adult male mice were determined. For EDL muscles, the Po of both MSTN+/− and MSTN−/− mice were greater than the Po of MSTN+/+ mice. For soleus muscles, the Po of MSTN−/− mice was greater than that of MSTN+/+ mice. The sPo of EDL muscles of MSTN−/− mice was less than MSTN+/+ mice. For soleus muscles, however, no difference in sPo was observed. Following two lengthening contractions, EDL muscles from MSTN−/− mice had a greater force deficit than MSTN+/+ or MSTN+/− mice, whereas no differences were observed for the force deficits of soleus muscles. Myostatin deficient EDL muscles had less hydroxyproline, and myostatin directly increased type I collagen mRNA expression and protein content. The difference in the response of EDL and soleus muscles to myostatin may arise from differences in the levels of a myostatin receptor, ActRIIB. Compared with the soleus, the amount of ActRIIB was approximately two-fold greater in EDL muscles. The results support a significant role for myostatin not only in the mass of muscles, but also in the contractility and the composition of the ECM of muscles.
PMCID: PMC4088255  PMID: 16709649
GDF-8; muscle morphology; muscle injury
15.  Diagnostics and Therapy of Muscle Channelopathies – Guidelines of the Ulm Muscle Centre 
Acta Myologica  2008;27(3):98-113.
This article is dedicated to our teacher, Prof. Erich Kuhn, Heidelberg, on the occasion of his 88th birthday on 23th November 2008. In contrast to muscular dystrophies, the muscle channelopathies, a group of diseases characterised by impaired muscle excitation or excitation-contraction coupling, can fairly well be treated with a whole series of pharmacological drugs. However, for a proper treatment proper diagnostics are essential. This article lists state-of-the-art diagnostics and therapies for the two types of myotonic dystrophies, for recessive and dominant myotonia congenita, for the sodium channel myotonias, for the primary dyskalemic periodic paralyses, for central core disease and for malignant hyperthermia susceptibility in detail. In addition, for each disorder a short summary of aetiology, symptomatology, and pathogenesis is provided.
PMCID: PMC2858942  PMID: 19472919
Chloride and sodium channel myotonias; periodic paralyses; malignant hyperthermia and central core disease
16.  Differential Effect of Calsequestrin Ablation on Structure and Function of Fast and Slow Skeletal Muscle Fibers 
We compared structure and function of EDL and Soleus muscles in adult (4–6 m) mice lacking both Calsequestrin (CASQ) isoforms, the main SR Ca2+-binding proteins. Lack of CASQ induced ultrastructural alterations in ~30% of Soleus fibers, but not in EDL. Twitch time parameters were prolonged in both muscles, although tension was not reduced. However, when stimulated for 2 sec at 100 hz, Soleus was able to sustain contraction, while in EDL active tension declined by 70–80%. The results presented in this paper unmask a differential effect of CASQ1&2 ablation in fast versus slow fibers. CASQ is essential in EDL to provide large amount of Ca2+ released from the SR during tetanic stimulation. In contrast, Soleus deals much better with lack of CASQ because slow fibers require lower Ca2+ amounts and slower cycling to function properly. Nevertheless, Soleus suffers more severe structural damage, possibly because SR Ca2+ leak is more pronounced.
PMCID: PMC3173739  PMID: 21941434
17.  High-frequency fatigue of skeletal muscle: role of extracellular Ca2+ 
The present study evaluated whether Ca2+ entry operates during fatigue of skeletal muscle. The involvement of different skeletal muscle membrane calcium channels and of the Na+/Ca2+ exchanger (NCX) has been examined. The decline of force was analysed in vitro in mouse soleus and EDL muscles submitted to 60 and 110 Hz continuous stimulation, respectively. Stimulation with this high-frequency fatigue (HFF) protocol, in Ca2+-free conditions, caused in soleus muscle a dramatic increase of fatigue, while in the presence of high Ca2+ fatigue was reduced. In EDL muscle, HFF was not affected by external Ca2+ levels either way, suggesting that external Ca2+ plays a general protective role only in soleus. Calciseptine, a specific antagonist of the cardiac isoform (α1C) of the dihydropyridine receptor, gadolinium, a blocker of both stretch-activated and store-operated Ca2+ channels, as well as inhibitors of P2X receptors did not affect the development of HFF. Conversely, the Ca2+ ionophore A23187 increased the protective action of extracellular Ca2+. KB-R7943, a selective inhibitor of the reverse mode of NCX, produced an effect similar to that of Ca2+-free solution. These results indicate that a transmembrane Ca2+ influx, mainly through NCX, may play a protective role during HFF development in soleus muscle.
PMCID: PMC2969177  PMID: 18560877
Dihydropyridine receptors; Stretch-activated Ca2+ channels; Store-operated Ca2+ channels; P2X receptors; Na+/Ca2+ exchanger
18.  Physiology and Pathophysiology of CLC-1: Mechanisms of a Chloride Channel Disease, Myotonia 
The CLC-1 chloride channel, a member of the CLC-channel/transporter family, plays important roles for the physiological functions of skeletal muscles. The opening of this chloride channel is voltage dependent and is also regulated by protons and chloride ions. Mutations of the gene encoding CLC-1 result in a genetic disease, myotonia congenita, which can be inherited as an autosmal dominant (Thomsen type) or an autosomal recessive (Becker type) pattern. These mutations are scattered throughout the entire protein sequence, and no clear relationship exists between the inheritance pattern of the mutation and the location of the mutation in the channel protein. The inheritance pattern of some but not all myotonia mutants can be explained by a working hypothesis that these mutations may exert a “dominant negative” effect on the gating function of the channel. However, other mutations may be due to different pathophysiological mechanisms, such as the defect of protein trafficking to membranes. Thus, the underlying mechanisms of myotonia are likely to be quite diverse, and elucidating the pathophysiology of myotonia mutations will require the understanding of multiple molecular/cellular mechanisms of CLC-1 channels in skeletal muscles, including molecular operation, protein synthesis, and membrane trafficking mechanisms.
PMCID: PMC3237021  PMID: 22187529
19.  CLCN1 Mutations in Czech Patients with Myotonia Congenita, In Silico Analysis of Novel and Known Mutations in the Human Dimeric Skeletal Muscle Chloride Channel 
PLoS ONE  2013;8(12):e82549.
Myotonia congenita (MC) is a genetic disease caused by mutations in the skeletal muscle chloride channel gene (CLCN1) encoding the skeletal muscle chloride channel (ClC-1). Mutations of CLCN1 result in either autosomal dominant MC (Thomsen disease) or autosomal recessive MC (Becker disease). The ClC-1 protein is a homodimer with a separate ion pore within each monomer. Mutations causing recessive myotonia most likely affect properties of only the mutant monomer in the heterodimer, leaving the wild type monomer unaffected, while mutations causing dominant myotonia affect properties of both subunits in the heterodimer. Our study addresses two points: 1) molecular genetic diagnostics of MC by analysis of the CLCN1 gene and 2) structural analysis of mutations in the homology model of the human dimeric ClC-1 protein. In the first part, 34 different types of CLCN1 mutations were identified in 51 MC probands (14 mutations were new). In the second part, on the basis of the homology model we identified the amino acids which forming the dimer interface and those which form the Cl- ion pathway. In the literature, we searched for mutations of these amino acids for which functional analyses were performed to assess the correlation between localisation of a mutation and occurrence of a dominant-negative effect (corresponding to dominant MC). This revealed that both types of mutations, with and without a dominant-negative effect, are localised at the dimer interface while solely mutations without a dominant-negative effect occur inside the chloride channel. This work is complemented by structural analysis of the homology model which provides elucidation of the effects of mutations, including a description of impacts of newly detected missense mutations.
PMCID: PMC3859631  PMID: 24349310
20.  Evaluation of diaphragmatic fatigue in obstructive sleep apnoeas during non-REM sleep 
Thorax  1997;52(8):731-735.
BACKGROUND: Resistive load applied to the airways may induce diaphragmatic fatigue, and hypoxaemia has been shown to predispose to the development of fatigue. Inspiratory muscle fatigue may occur in patients with obstructive sleep apnoea syndrome (OSAS), as these patients repetitively develop both inspiratory loading and hypoxaemia. The results of previous studies on this topic are inconclusive, probably because of the methodological approaches used. METHODS: Six obese patients with OSAS underwent a polysomnographic study. The diaphragmatic pressure time index (PTI) was evaluated as an indicator of diaphragmatic contraction, and the mean frequency of the diaphragmatic electromyogram power spectrum (Fm) and the maximum relaxation rate of transdiaphragmatic pressure (MRR) as indices of a fatiguing diaphragm. A total of 119 randomly selected apnoeas (each including 5-13 occluded efforts) were analysed throughout the night in non-REM sleep to assess possible muscle fatigue due to the high pressure generation in each apnoea. A breath-by-breath within-apnoea analysis was performed on the first three pre-apnoeic breaths, on all the occluded efforts, and on the first three unoccluded breaths following the apnoea interruption. Possible fatigue development due to the cumulative effect of apnoeas over the night was also evaluated. RESULTS: A progressive increase of Fm and MRR was found during the obstructive phase in all the subjects in the within-apnoea analysis. The overnight analysis did not show a reduction in either PTI, Fm, or MRR secondary to recurrent upper airway obstruction during the night. CONCLUSIONS: No evidence of diaphragmatic fatigue or impaired diaphragmatic contraction was found either within each apnoea or throughout the whole night, despite the generation of high PTI values during the apnoeic occluded phases. It is concluded that diaphragmatic fatigue does not occur in OSAS during non-REM sleep. 

PMCID: PMC1758615  PMID: 9337834
21.  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.
► 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.
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
22.  New immunohistochemical method for improved myotonia and chloride channel mutation diagnostics 
Neurology  2012;79(22):2194-2200.
The objective of this study was to validate the immunohistochemical assay for the diagnosis of nondystrophic myotonia and to provide full clarification of clinical disease to patients in whom basic genetic testing has failed to do so.
An immunohistochemical assay of sarcolemmal chloride channel abundance using 2 different ClC1-specific antibodies.
This method led to the identification of new mutations, to the reclassification of W118G in CLCN1 as a moderately pathogenic mutation, and to confirmation of recessive (Becker) myotonia congenita in cases when only one recessive CLCN1 mutation had been identified by genetic testing.
We have developed a robust immunohistochemical assay that can detect loss of sarcolemmal ClC-1 protein on muscle sections. This in combination with gene sequencing is a powerful approach to achieving a final diagnosis of nondystrophic myotonia.
PMCID: PMC3570820  PMID: 23152584
23.  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.
PMCID: PMC3320144  PMID: 22536291
24.  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.
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).
(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).
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.
PMCID: PMC3492909  PMID: 22607270
INQoL; myotonic dystrophy; non-dystrophic myotonias; quality of life; SF-36; skeletal muscle channelopathies
25.  Linkage of aerobic glycolysis to sodium-potassium transport in rat skeletal muscle. Implications for increased muscle lactate production in sepsis. 
Journal of Clinical Investigation  1996;98(10):2388-2397.
Although a linkage between aerobic glycolysis and sodium-potassium transport has been demonstrated in diaphragm, vascular smooth muscle, and other cells, it is not known whether this linkage occurs in skeletal muscle generally. Metabolism of intact hind-leg muscles from young rats was studied in vitro under aerobic incubation conditions. When sodium influx into rat extensor digitorum longus (EDL) and soleus muscles was facilitated by the sodium ionophore monensin, muscle weight gain and production of lactate and alanine were markedly stimulated in a dose-dependent manner. Although lactate production rose in both muscles, it was more pronounced in EDL than in soleus. Monensin-induced lactate production was inhibited by ouabain or by incubation in sodium-free medium. Preincubation in potassium-free medium followed by potassium re-addition also stimulated ouabain-inhibitable lactate release. Replacement of glucose in the incubation medium with pyruvate abolished monensin-induced lactate production but exacerbated monensin-induced weight gain. Muscles from septic or endotoxin-treated rats exhibited an increased rate of lactate production in vitro that was partially inhibited by ouabain. Increases muscle lactate production in sepsis may reflect linked increases in activity of the Na+, K+-ATPase, consumption of ATP and stimulation of aerobic glycolysis.
PMCID: PMC507691  PMID: 8941658

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