the current criteria for diagnosis of amyotrophic lateral sclerosis ( als ) require neurophysiological evidences of ongoing denervation , defined by fibrillation potentials ( fibs ) or positive sharp waves , and of chronic partial reinnervation , involving enlarged , unstable motor units with a reduced interference pattern .
fibs are detected as long as denervation - reinnervation processes take place , and their assessment is considered a sensitive method to evaluate disease progression and a useful research tool ( see , for instance , ) . at later stages of disease they fade owing to replacement of muscle fibres with fibrous tissue . in mammalian denervated muscle fibres , fibrillations arise because spontaneous activation of voltage - gated na ( nav ) channels triggers action potentials and contractions .
disappearance of fibs has been reported in one als patient receiving fentanyl and propofol during surgery , two anaesthetics that block nav channels [ 7 , 8 ] .
riluzole is a well - established blocker of neuronal nav channels , but it also affects other channels in nonneuronal tissues , for instance , muscle acetylcholine receptor channels [ 912 ] .
riluzole also causes a mild block of recombinant and native muscular voltage - gated na current ( ina ) [ 10 , 13 ] , although at concentrations higher than those attained in patients ' plasma . in myotubes
given these premises , riluzole might affect fibs in als patients , but this point has received little attention to date , even if riluzole is the only approved drug for als treatment .
therefore , we undertook a study of riluzole action on fibs , aimed at filling this gap .
human muscle satellite cells can be cultured in vitro in the absence of nerve , and myotubes adequately represent denervated muscle fibres . moreover , satellite cells in vitro preserve traces of their in vivo environment   including denervation - induced alterations .
satellite cells derived from als patients display a reduced myogenic potential   but form multinucleated myotubes ( als myotubes ; ) .
as commonly observed for primary human myotubes [ 1921 ] , als myotubes do not contract spontaneously . in the context of nav channels , both denervated
muscle fibres and cultured myotubes express nav1.5 channels in addition to nav1.4 , the only form present in innervated muscle fibres [ 22 , 23 ] .
thus , als myotubes provide a valuable experimental model with functional properties conditioned by donor health status .
we therefore first evaluated the effect of riluzole on muscle ina and action potentials in vitro and then verified our conclusions in vivo , in a group of patients . in als
myotubes , riluzole affected the amplitude and voltage dependence of ina . as a consequence , spike amplitude and rate of rise were reduced , but action potential firing was not suppressed , suggesting that riluzole would not prevent muscle fibrillations .
indeed , fibs were routinely detected during riluzole administration in als patients and brief suspension of the treatment had no univocal effects on fibs frequency .
all our data suggest that riluzole , in spite of partially blocking muscle nav channels , does not interfere with muscle fibs .
human myotubes were grown from satellite cells derived from muscle biopsies performed at the als centre of policlinico umberto i , sapienza university .
all patients gave written informed consent to use part of the biopsy material for the research project entitled  study of nicotinic acetylcholine receptor in muscle tissue of als patients ,  approved by the human subjects ethical committee of policlinico umberto i. satellite cells were obtained immediately after biopsy as described [ 11 , 12 ] .
frozen aliquots were thawed for the present study and propagated as previously reported [ 11 , 12 ] .
differentiation was induced at 50% confluence by switching to low - serum differentiating medium ( dulbecco 's minimum essential medium plus 2% horse serum and penicillin / streptomycin ) .
whole - cell patch - clamp recordings were made at room temperature ( 2327c ) using an axopatch 200b amplifier ( molecular devices , union city , ca , usa ) , driven by pclamp 9 ( molecular devices ) .
cells were continuously superfused using a gravity - driven fast exchanger perfusion system ( rsc-200 , bio - logic , france ) . external solution contained ( mm ) 140 nacl , 2.8  kcl , 2 cacl2 , 2 mgcl2 , 10 hepes - naoh , and 10 glucose , ph 7.3 .
addition of 4-aminopyridine ( 2  mm ) to block voltage - gated k channels did not affect ina .
patch pipettes ( 25  m ) contained ( mm ) 130 cscl , 10 nacl , 5 bapta , 10 hepes - koh , 2 mg - atp , and 2 mgcl2 , ph 7.3 . to record action potentials ,
recordings were considered only if patch series resistance was less than 8  m and compensated by 9599% .
riluzole - induced shifts of activation and inactivation curves were unaltered if voltage drop over uncompensated access resistance ( ru ) was kept into account correcting the test potential ( vm ) as vcorr = vm  ruina and using vcorr in data analysis .
current - voltage curves were obtained applying membrane potential steps ( 5  ms at 1  s interval ) in 5  mv increments from 65  mv to + 60  mv , from a steady holding potential of 80  mv .
fast inactivation curve was obtained using a two - pulse protocol : prepulse potential ( 20  ms ) was changed from 140  mv to 15  mv in 5  mv increments , followed by a test pulse to 10  mv ( 5  ms at 1  s interval ) , as previously done in human muscle fibres .
current reversal potential ( ena ) , estimated from plot of peak ina versus vm , was 64  1  mv ( n = 30 ) , as predicted by nernst equation ( 66 mv ) .
conductance was calculated as g = ina/(vm  ena ) and normalized to the maximal value gmax. values of g / gmax were then fitted with boltzmann equation : 
 ( 1)ggmax=11+e(vva)/ka , 
 where va is the potential at which conductance is half of maximal value and ka is the slope factor .
fast inactivation relationships were constructed plotting current amplitude i , normalized to maximal value imax , versus prepulse potentials and fitting a boltzmann equation to data points : 
 ( 2)iimax=11+e(vvi)/ki , 
 where vi is the potential at which current is half of maximal value and ki is the slope factor .
action potentials were recorded under current clamp conditions using stimuli of increasing amplitude ( from 0.2 to 1  na ) at 1  s intervals .
phase plots , representing changes in membrane potential with time ( i.e. , the first derivative of membrane potential ) versus the value of membrane potential at the corresponding time , were built for each action potential .
two data sets were considered statistically different when p < 0.05 by anova or student 's paired t - test . eleven patients ( 6 females ) with probable or definite als according to el escorial criteria were recruited and analyzed at the als centre of policlinico umberto i , sapienza university ( table 1 ) .
inclusion criteria were as follows : male or female subjects older than 18 years ; disease duration lower than 60 months ; treatment with riluzole for at least 3 months before the study ; no history of alcohol or substance abuse ; no other neuromuscular disorders and diabetes mellitus ; no marked lower limbs spasticity ( > 2 on the modified ashworth scale ) ; absence of psychiatric disorders or cognitive impairment at first evaluation ; no concomitant therapy with psychoactive drugs .
muscle strength of lower limbs was assessed with the medical research council ( mrc ) score for muscle strength , an ordinal scale ranging from 0 ( absence of movement ) to 5 ( normal contraction against full resistance ) that quantifies muscle weakness in isolated muscles or muscle groups .
five muscle groups in lower limbs are tested : hip flexors , knee extensors , knee flexors , ankle plantar flexors , and ankle dorsiflexors . an electromyographic instrument ( system plus evolution 1.04.0104 ) employing a one - channel montage (
sensitivity 50  v / division ; low frequency filter 20  hz ; high frequency filter 5  khz ) was used to detect electrical activity . during the electrophysiological procedures ,
needle electromyographic signals were recorded from the right anterior tibialis muscle at a site approximating the demarcation between the proximal one - third and distal two - thirds of the muscle .
three consecutive needle insertions ( 10  s each ) with the same depth of insertion of needle ( 1.5  cm ) per session were performed in all patients .
a reference landmark by indelible pen allowed recording at the same muscle site in the second session .
right tibialis anterior compound muscle action potential ( cmap ) was evoked by peroneal nerve stimulation .
the stimulating cathode was placed at the posterior - lateral area of the fibular head .
the point for the recording surface electrodes was placed 8  cm from the cathode over the tibialis anterior .
the latency of cmap was measured from the stimulus artefact to the onset point , and the amplitude was determined from baseline to the highest negative peak .
voltage - gated na current ( ina ) was recorded in all multinucleated myotubes ( 38 days differentiation ) examined .
current density was 0.27  0.03  na / pf ( n = 22 ) with the largest current obtained at test potentials of 20 to 0  mv ( figure 1(a ) ) .
current was reduced to 28  3% ( n = 10 ) of control value by ttx ( 1  m , not shown ) , as expected in myotubes which express a mixture of nav1.4 and nav1.5 channels , with half - inhibitory ttx concentration of 10  nm and 3  m , respectively , thus confirming that recorded signals were bona fide na currents . applying riluzole 1  m ,
a clinically relevant concentration , 30  s before stimulation , total ina amplitude was 79  2% ( n = 22 ) of untreated value ( figure 1(a ) ) .
in all cells tested , activation and steady - state fast inactivation curves were modified in the presence of riluzole .
the activation curve had a depolarizing shift ( figure 1(b ) ) , with a mean increase for va of 2.4  0.3  mv ( n = 22 , p < 0.0004 ) , and ka was significantly increased by 0.6  0.1  mv ( n = 22 , p = 0.0004 ) .
steady - state fast inactivation curves had a hyperpolarizing shift ( figures 1(c ) and 1(d ) ) , with vi decreasing by 8.7  1.5  mv ( n = 7 , p = 0.001 ) and ki by 3.5  0.5  mv
these riluzole - induced shifts in activation and fast inactivation of ina might reverberate on action potential generation , reducing spike amplitude ( because of enhanced fast inactivation ) and rate of rise ( due to reduced current amplitude ) .
we therefore recorded action potentials evoked by depolarizing currents of increasing amplitude , using current clamp mode .
in these myotubes , membrane resting potential ranged between 40 and 25  mv , so hyperpolarization ( to 89.0  1.4  mv , n = 5 ) was required to elicit action potentials .
depolarizing currents evoked overshooting spikes in all myotubes tested , but amplitude and rate of rise were reduced in the presence of riluzole ( figure 2(a ) ) . in the five myotubes tested , spike peak and rate of rise were 29.7  5.1  mv and 119  18  mvms under control conditions and 21.3  5.3  mv and 89  17  mvms , in the presence of riluzole ( p  0.002 by paired student 's t - test ) .
action potential peak was delayed by 1.8  0.7  ms ( n = 5 ; p = 0.03 ) .
furthermore , phase plots showed a small shift in threshold potential , compatible with the depolarizing shift of va measured in each myotube ( figure 2(b ) ) .
thus , riluzole reduces excitability of myogenic cells but does not prevent action potential generation in response to depolarizing stimuli , suggesting that it might be ineffective on muscle fibrillations .
all patients were treated with riluzole ( average treatment duration , 13.8 months ) and suspended treatment for one week for this study .
this time is adequate for riluzole washout , estimated to be complete within about 40 hours of withdrawal .
multiple fibs were detected in all patients ' anterior tibialis muscle ( figure 3 ) . across the 3 insertions performed , their number had small fluctuations in all but patients 5 and 7 , which suggests that variability due to needle position is limited . in the second recording session , one week after riluzole withdrawal , clinical status and cmap amplitude were unchanged for all patients ( table 1 ) , indicating that no relevant alteration in muscle innervation took place .
fibs continued to be present in all patients , and variability between insertions did not change . when compared to the first recording session , the total number of fibs changed by less than 25% in six patients ; in the other 5 , changes ranged between a 55% increase ( patient 2 ) and a 70% decrease ( patient 1 ) .
overall , there was no statistically significant variation in the number of fibs upon riluzole treatment suspension ( p = 0.69 ) ( table 1 ) .
the effect of riluzole withdrawal showed no correlation with patients ' age , sex , disease duration , or fibs frequency at baseline ( data not shown ) .
in this paper we studied the effect of riluzole on voltage - gated sodium channels in human myotubes and on fibs in als patients . in vitro , we show that riluzole at a therapeutically used concentration reduces ina and shifts the voltage dependence of gating .
as a consequence , action potentials evoked by depolarizing currents have lower amplitude and reduced rate of rise . these results clearly indicate that riluzole acts on channels present on muscle fibres .
the therapeutic implications of this nonneuronal effect , if any , are presently unknown . for instance , the documented partial block of muscle nav channels may result in dampened spontaneous activations of these channels and limit fibs occurrence in patients ' denervated muscle fibres . here
we report that fibs were consistently recorded during riluzole treatment in als patients , a clear indication that riluzole does not suppress muscle fibrillations .
we were unable to detect univocal effects on fibs upon riluzole withdrawal in the group of patients considered .
the in vitro findings here reported indicate that riluzole has a small effect on action potential firing .
data obtained in patients do not disclose even a trend in the change of fibs frequency upon riluzole withdrawal .
thus , extension of the in vivo study to a larger number of patients appears of little significance , also considering the discomfort of the procedure and the burden of repeated hospital access for heavily disabled patients .
none of the previous studies showing that nav channel blockers can suppress fibs in vivo [ 5 , 6 ] assessed the plasma concentration of the drugs , so it is not possible to correlate disappearance of fibs with the extent of nav channels blockade . in organ cultures of rat muscle , ttx prevented fibs when used at 1  m , which we show here to block a larger fraction of ina than riluzole at the concentration used .
thus , it is likely that riluzole - induced block of muscle ina is not sufficient to prevent fibs .
the aforementioned studies [ 46 ] report effects observed within minutes of drug application , whereas our data were collected in patients routinely taking riluzole or after a washout lasting one week , so that it can not be ruled out that adaptations to long - term presence / absence of riluzole contribute to its inability to prevent fibs in the examined patients . a reduced rate of rise of evoked action potentials under riluzole , here reported for cultured myotubes , was also observed in guinea pig cardiac purkinje fibres   but not in rat ventricular cardiomyocytes .
this study shows that riluzole at a typical clinical concentration partially blocks muscle nav channels and dampens  but does not abolish  the action potentials evoked by depolarizing stimuli in als myotubes . as suggested by in vitro inability to fully suppress spikes
, riluzole does not prevent muscle fibs in als patients , apparently having very limited impact on this symptom .
therefore , the pattern of fibs disappearance during disease progression is unlikely to be directly influenced by riluzole - induced block of muscle nav channels .
 denervated muscles undergo fibrillations due to spontaneous activation of voltage - gated sodium ( na+ ) channels generating action potentials 
 . fibrillations also occur in patients with amyotrophic lateral sclerosis ( als ) . 
 riluzole , the only approved drug for als treatment , blocks voltage - gated na+ channels , but its effects on muscle na+ channels and fibrillations are yet poorly characterized . using patch - clamp technique , we studied riluzole effect on na+ channels in cultured myotubes from als patients . 
 needle electromyography was used to study fibrillation potentials ( fibs ) in als patients during riluzole treatment and after one week of suspension . 
 patients were clinically characterized in all recording sessions . in myotubes , 
 riluzole ( 1  m , a therapeutic concentration ) reduced na+ current by 20% . the rate of rise and amplitude of spikes evoked by depolarizing stimuli were also reduced . 
 fibs were detected in all patients tested during riluzole treatment and riluzole washout had no univocal effect . 
 our study indicates that , in human myotubes , riluzole partially blocks na+ currents and affects action potentials but does not prevent firing . in line with this in vitro finding , muscle fibs in als patients appear to be largely unaffected by riluzole . 
1. Introduction
2. Materials and Methods
3. Results
4. Discussion
5. Conclusions