Differences in sodium voltage-gated channel properties according to myosin heavy chain isoform expression in single muscle fibres

J Physiol 587.21 (2009) pp 5249–5258 5249

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Differences in sodium voltage-gated channel propertiesaccording to myosin heavy chain isoform expressionin single muscle fibres

F. Rannou1,2,4, M. Droguet1,3,4, M. A. Giroux-Metges1,2,4, Y. Pennec3,4, M. Gioux1,2,4 and J. P. Pennec1,4

1Universite de Brest, Faculte de Medecine et des Sciences de la Sante, EA 4326, Laboratoire de Physiologie, Brest, F-29200 France2CHU Brest, Service d’Explorations Fonctionnelles Respiratoires, Brest, F-29200 France3Universite de Brest, Institut Universitaire et Technique, Brest, F-29200 France4Universite Europeenne de Bretagne, France

The myosin heavy chain (MHC) isoform determines the characteristics and shortening velocityof muscle fibres. The functional properties of the muscle fibre are also conditioned by itsmembrane excitability through the electrophysiological properties of sodium voltage-gatedchannels. Macropatch-clamp is used to study sodium channels in fibres from peroneus longus(PL) and soleus (Sol) muscles (Wistar rats, n = 8). After patch-clamp recordings, single fibresare identified by SDS-PAGE electrophoresis according to their myosin heavy chain isoform (slowtype I and the three fast types IIa, IIx, IIb). Characteristics of sodium currents are compared(Student’s t test) between fibres exhibiting only one MHC isoform. Four MHC isoforms areidentified in PL and only type I in Sol single fibres. In PL, maximal sodium current (I max),maximal sodium conductance (g Na,max) and time constants of activation and inactivation (τm

and τh) increase according to the scheme I→IIa→IIx→IIb (P < 0.05). τm values related tosodium channel type and/or function, are similar in Sol I and PL IIb fibres (P = 0.97) despitedifferent contractile properties. The voltage dependence of activation (V a,1/2) shows a shifttowards positive potentials from Sol type I to IIa, IIx and finally IIb fibres from PL (P < 0.05).These data are consistent with the earlier recruitment of slow fibres in a fast-mixed muscle likePL, while slow fibres of postural muscle such as soleus could be recruited in the same ways asIIb fibres in a fast muscle.

(Received 4 June 2009; accepted after revision 11 September 2009; first published online 14 September 2009)Corresponding author J. P. Pennec: Laboratoire de Physiologie, Faculte de Medecine, 22 avenue Camille Desmoulins,CS 93837, 29238 Brest cedex 3, France. Email: [email protected]

Abbreviations EDL, extensor digitorum longus; MHC, myosin heavy chain; PL, peroneus longus; Plan, plantaris;SDS-PAGE sodium dodecyl-sulfate polyacrylamide gel electrophoresis; Sol, soleus.

Introduction

Mammalian skeletal muscles express different functionalproperties, according to their mechanical role in theskeleton. Commonly, fast and slow twitch muscles areidentified according to their mechanical properties thatare related to their fibre type. Moreover it was shownthat skeletal muscles could have a large ability toevolve towards ‘slow’ or ‘fast’ phenotype, in physio-logical or pathological conditions (i.e. immobilization,Spector et al. 1982; or tendon transfer, Giroux-Metgeset al. 2003). The necessity of muscle characterizationpromoted the development of numerous techniques to

differentiate between fibres of slow and fast muscles,according to their histochemical properties (Brooke &Kaiser, 1970) or functional characteristics of motor units(Burke et al. 1973). More precisely, their myosin heavychain (MHC) isoform composition can differentiatebetween muscle fibres. Four myosin heavy chain (MHC)isoforms (i.e. slow type I, fast type IIa, fast type IIxand fast type IIb) were identified in whole muscles ofrat by sodium dodecyl-sulfate polyacrylamide gel electro-phoresis (SDS-PAGE) (Talmadge & Roy, 1993). Electro-phoresis analysis of MHC isoforms was also performedin single muscle fibres, currently allowing an exactcorrelation between physiological parameters with theidentified fibre type (Galler et al. 1994; Pette et al. 1999).

C© 2009 The Authors. Journal compilation C© 2009 The Physiological Society DOI: 10.1113/jphysiol.2009.176446

5250 F. Rannou and others J Physiol 587.21

Fibre-type differentiation is also related to motor unitfiring patterns (Salmons & Sreter, 1976). The questionremains concerning a possible relation between fibre-typedifferentiation and their electrophysiological properties.Previous studies with sucrose gap (Duval & Leoty,1980a,b) aimed to point out the electrophysiologicalproperties of sodium channels in fast and slow musclesof rat hindlimb. Nowadays, the types of studied fibres arepresumed to correspond to the major type of a typicallyfast or slow muscle. For example, soleus (Sol) and extensordigitorum longus (EDL) have been extensively used asmodels of slow and fast muscles, respectively (McArdleet al. 1980; Goodman et al. 2003) because Sol expressesabout 90–95% type I and EDL 70% type IIb musclefibre types, respectively. However, when the propertiesof a single muscle fibre have to be considered accordingto its true type, this procedure appears approximatebecause mammalian muscle always exhibits a mixtureof fibre type. A first approach to identifying fibres byhistochemical ATPase staining was made by Ruff (1989)and Ruff & Whittlesey (1993) who correlated sodiumcurrents and IIa–IIb fibre types. Nevertheless, the relationbetween electrophysiological properties and a fibre typeis not simple because each fibre can express more thanone MHC isoform (Galler et al. 1994; Caiozzo et al.2003).

Therefore, we hypothesized that the electro-physiological properties of sodium channels could berelated to MHC isoform expression in single musclefibres. The purpose of our study is to show a relationshipbetween voltage-gated sodium channel characteristics,determined by the patch-clamp technique and MHCisoforms, identified by the SDS-PAGE electrophoreticapproach, in native single fibres. These fibres were iso-lated from a fast mixed-type muscle such as peroneuslongus (PL) and from Sol, which is a typical slow muscle(Armstrong & Phelps, 1984).

Methods

Muscle isolation and enzymatic dissociationof muscle fibres

All procedures were performed according to ourethical regional committee recommendations. The experi-ments were authorized by a departmental agreementno. A29-019-03 and performed according to therecommendations of the European Community directiveno. 86/609 (Drummond, 2009). Adult female Wistarrats (n = 8, body weight 250–300 g, age 2–3 months,Centre d’elevage Depre, Saint-Doulchard, France) wereanaesthetized by pentobarbital (40 mg kg−1 I.P.) thenkilled by cervical dislocation and exsanguinated.Plantaris (Plan), PL and Sol muscles were rapidly

excised from the hindlimb and then placed indenaturating lysis buffer (7 μl mg−1) for electrophoresis(see below) or in dissociation medium for patch-clampstudies. Patch-clamp studies were performed on singlefibres from PL and Sol after enzymatic dissociationin 4-(2-hydroxyethyl)-1-piperazine-ethanesulfonic acid(Hepes)-buffered saline supplemented with 3.0 mg ml−1

collagenase (type II, Gibco-BRL, Gaithersburg, MD, USA)for 2–3 h at 37◦C, depending on muscle type and size. Afterthis incubation period, dissociated fibres were sampledand rinsed several times with standard saline solutionbefore transfer to a 35 mm Petri dish for patch-clamprecordings. Only fibres showing a good appearance atvisual inspection (no break, no bleb, no constricted zoneor contracture, no granular aspect, good contrast, visibleradial striation) were used for patch clamp.

Patch clamp

Fast sodium currents were recorded in the cell-attachedconfiguration (Hamill et al. 1981) with macropatch-clamptechnique at room temperature (22 ± 2◦C). A GeneClamp500B amplifier equipped with a CV-5-1GU headstage,allowing clamping of currents up to 100 nA (AxonInstruments, Union City, CA, USA), was used. Micro-pipettes were pulled and polished from GC150TF-10borosilicate glass (Harvard Apparatus, Natick, MA, USA)using a horizontal pipette puller (Zeitz Instruments,Germany). Pipettes had resistances averaging 2 M� whenfilled with the standard saline solution (150 mM NaCl,5 mM KCl, 2 mM CaCl2, 1 mM MgCl2, 30 mM Hepes; pH:7.4). Voltage-clamp protocols and data acquisition wereperformed with WinWCP V3.8.5 (Whole Cell program,University of Strathclyde, Glasgow, UK) through a 12-bitanalog-to-digital/digital-to-analog interface (CED 1401,Cambridge Electronic Design, Cambridge, UK). Currentswere low-pass filtered at 5 kHz and digitized at48 kHz.

Because sodium channel density is five- to tenfoldhigher on the endplate border than away from the end-plate (Ruff, 1992) sodium currents were recorded awayfrom extra-junctional membrane at a site > 200 μm fromthe endplate. This could be visualized with phase contrastunder an inverted microscope (Olympus IX 70) and with aprogressive-scan digital camera (XC8500CE, DONPISHA,Sony, Japan). The fibres were placed in a bath recordingsolution containing Cs+ ions as the main cation (145 mM

CsCl, 5 mM EGTA, 1 mM MgCl2, 10 mM Hepes, pH 7.3) toinhibit potassium currents and to depolarize the surfacemembrane. Patch-clamp recordings were carried out after10 min incubation in a recording solution. An amplifiercompensation circuit cancelled capacitance currents. Inorder to eliminate the residual capacitance transient andthe leak current, we used the P/4 subtraction procedure

C© 2009 The Authors. Journal compilation C© 2009 The Physiological Society

J Physiol 587.21 Sodium channel properties related to myosin heavy chain isoforms 5251

(Almers et al. 1983). Briefly, four hyperpolarizing pulseswith amplitude one-quarter of the pulse test amplitudewere applied to the patch before the test pulse, thusallowing further determination and then subtraction ofthe residual leak current. The current was totally blockedby a high concentration of tetrodotoxin (3 × 10−6 M)showing that no contaminating current was recorded.

Current–voltage relationship, determinationof maximal conductance

The holding potential was set to −100 mV, a value closeto physiological values in intact skeletal muscle fibres andat which most of the channels are in a closed activatablestate. At this holding potential, direct transitions fromclosed to activated states can occur leading few sodiumchannels to be non-conductive (Bean et al. 1983). Thecurrent–voltage relationship was measured by applyingto the patch membrane a cycle of 20 ms test pulses fromthe holding potential of −100 mV to increasing potentials(from −60 to +130 mV in 10 mV increments). The inter-vals between each test pulse were long enough (3 s) toallow the complete recovery of the sodium channel frominactivation. This protocol was repeated three times foreach patch to ensure sodium current stability. The patcheswith no reliable peak current amplitude were discarded.Imax corresponds to the maximal sodium current relatedto membrane area under the patch pipette and is expressedas an absolute value (nA). In order to standardize currentvalues, the raw data were also normalized by takinginto account the surface of the pipette tip (7 μm2). Thecurrent–voltage relationship or I–V curve represents therelationship between sodium peak current and membranepotential. The maximal sodium conductance (gNa,max)is given by the slope of the quasi-linear part of thecurrent–voltage relationship.

Activation determination

The activation curve was obtained by plotting gNa/gNa,max

as a function of imposed membrane potential. gNa,max wascalculated as reported here above; gNa was calculated foreach imposed potential from −60 to +30 mV, according tothe following relation: gNa = INa/(V m − V Na) where INa isthe sodium current; V m the membrane potential and V Na

the equilibrium potential for sodium, determined on thecurrent–potential curve. Calculated values of gNa/gNa,max

were fitted with the following Boltzmann equation:

g Na

g Na, max= 1

(1 + e(V−Va,1/2)

)K a

V a,1/2, potential at which half of the channels are activated;K a, slope factor.

Time constants determination

The activation and inactivation constants of the sodiumcurrent (τm and τh, respectively) were calculated at−20 mV by fitting the current (I) with the followingHodgkin–Huxley relation:

Imax(t) = A(1 − e

(−t/τm

))p (hinf − (hinf − 1)e

(−t/τh

))

A, voltage-depending part of the current; p, exponent of m(around 3); hinf , equilibrium value reached by h accordingto the potential; t , time in milliseconds.

Fast inactivation determination

Steady-state fast inactivation was measured by applying50 ms conditioning prepulses to various holding potentialsfrom –120 to +10 mV, followed by a 20 ms test pulseup to −20 mV to activate Na+ current. To calculate theslope factor Kh and half-inactivation voltage Vh,1/2, thesteady-state fast inactivation relationships were fitted withthe Boltzmann equation:

INa

INa,max= 1

(1 + e(V−Va,1/2)

)K h

,

where INa is the sodium current and INa,max the maximalsodium current.

Analysis of MHC isoforms

Immediately after the patch-clamp recordings the patchedfibre was removed under visual control from the dishby using specially sharpened tweezers (Dumont no. 5;Switzerland) or a 10 μl automatic pipette (the twomethods gave the same good results). The fibre wasthen placed in a 1.5 ml polypropylene microcentrifugetube that contained 20 μl of denaturating lysis buffer(0.3 M NaCl, 0.1 M NaH2PO4, 50 mM Na2HPO4, 0.01 M

Na4P2O7, 1 mM MgCl2.6H2O, 10 mM EDTA, 1.4 mM

β−mercapto-ethanol) for 24 h at 4◦C. Whole muscleextracts (Sol, Plan and PL) were placed in the denaturatinglysis buffer (7 μl mg−1 of whole muscle extract in a 1.5 mlpolypropylene microcentrifuge tube), for 24 h at 4◦C. Sol,Plan and PL samples were centrifuged (13 500 g for 15 minat 4◦C) and the supernatant was diluted 1/1 (v/v) withglycerol (20%).

Composition and preparation of gels

MHC isoforms of single fibre and whole muscles wereseparated on 8% polyacrylamide gels containing 30%glycerol with a protocol adapted from Talmadge &Roy (1993). Separating gel (8% acrylamide, 0.16%bis-acrylamide, 30% glycerol, 0.4% SDS, 0.1 M glycine,0.2 M Tris (pH 8.8)) and stacking gel (4% acrylamide,0.08% bis-acrylamide, 30% glycerol, 0.4% SDS, 70 mM



Tris (pH 6.8), 4 mM EDTA) were degassed for ∼10 min.The polymerization of the gels was accelerated byadding TEMED (N,N,N′,N′-Tetramethylethylenediamine;N,N,N′,N′-Di-(dimethylamino)ethane; N,N,N′,N′-Tetramethyl-1-,2-diaminomethane, 0.05%) and APS(ammomium persulfate) (0.1%) and terminated in theBiorad mini-Protean II dual slab cell system. Each sampleof single fibre or whole muscle was heated (100◦C for2 min), then loaded onto gel. Gels were run for 31 h at70 V, and during electrophoresis the temperature of thebuffer was maintained at 4◦C.

Gel staining and analysis

Following migration, the gels were silver-stained (SilverStain Plus kit, Biorad, Richmond, CA, USA). Isoformsof MHC were separated according to their electro-phoretic mobility (fast types IIa, IIx, IIb, and slowtype I in progressive order of migration). They wereidentified by comparison to whole muscles (Sol andPlan) exhibiting the known content of MHC iso-forms. Sol contained mainly slow type I (90–95%)with IIa (5–10%) (Talmadge & Roy, 1993), and Plancontained mainly IIx and IIb isoforms, in equal parts(Pette et al. 1999; Caiozzo et al. 2003). Silver-stainedgels were photographed and densitometric tracing wasobtained with densitometry software (Mesurim Pro,[email protected]). The content of thedifferent MHC isoforms in whole muscle and single fibrewas determined by the area under the peaks in absorbancecurves (Talmadge & Roy, 1993). Because single fibres cancontain different isoforms of MHC (Caiozzo et al. 2003),only fibres exhibiting a single peak representing more than95% of the total absorbance were analysed. In addition tothe single fibre of PL, patch-clamp and electrophoresiswere also conducted on Sol fibres to provide results for thetype I MHC isoform from a slow muscle.

Data analysis and statistics

After electrophoresis results, patch-clamp data for eachsingle muscle fibre were separated according to its MHCisoform content (I, IIa, IIx or IIb). Excel (Microsoft,Redmond, WA, USA) was used to analyse experimentaldata and to perform curve fittings. All values are given asmeans ± standard error of the mean (S.E.M.). Statisticaldifferences were determined by performing Student’st test, after checking the normality of distribution.Differences were considered significant for P < 0.05.

Results

Fibre-type distribution

Whole muscle extracts from Plan (Fig. 1A) and Sol wereused as markers of migration to differentiate betweensingle fibres from PL and Sol according to their MHC iso-

form content. The electrophoretic separation of PL, Planand Sol fibres allowed the distinction of four differentMHC isoforms, as previously described (Pette & Staron,1990). Whole PL exhibits the four MHC isoforms as shownin the electrophoregram (Fig. 1A), also illustrated withfour distinct peaks in densitometric analysis (Fig. 1D).This is consistent with previous results obtained by usingthe ATPase method in PL (Ranatunga & Thomas, 1990).

In addition, densitometric analysis allows thequantification of fibre content in terms of differentMHC isoforms by calculating the area under the curveof absorbance (Staron et al. 1999). Electrophoresis wasconducted on 35 single fibres from PL and 12 fibres fromSol. In PL, 23 fibres from PL were classified as follows:2 fibres were identified as type I, 5 fibres as type IIa, 12fibres as type IIx and 7 fibres as type IIb. The remaining 12fibres exhibiting more than 5% of a second MHC isoformwere excluded from the analysis. All fibres analysed in Solshowed only the type I MHC isoform.

Current–voltage relationship

Sodium currents in single fibres from PL and Sol wereelicited by test pulses between −60 and 130 mV, fromthe holding potential of –100 mV as shown with typicalrecords in Fig. 2A. Peak current amplitude (Imax) wasmeasured and plotted as a function of test pulse potential,illustrated in Fig. 2B with one Sol and one IIb PL fibre.The characteristics of inward sodium currents in singlefibres from Sol and PL are summarized in Table 1. Peakcurrent amplitude (Imax) increases from Sol I to PLIIb, with intermediate currents in PL IIa and IIx (meanvalues significantly differ from each other, P < 0.05). Themaximal sodium conductance gNa,max corresponding tothe linear part of the current–voltage curve (Fig. 2C)similarly increases from Sol type I to PL IIa, then IIx to IIb(see Table 1, values significantly differing from each other,P < 0.01). Notice that Imax and gNa,max values in Sol and PLtype I are in the same range. Time constants correspondingto activation (τm) and fast inactivation (τh) were calculatedfrom −20 mV (Table 1). In PL, τm increases from IIa toIIb with an intermediate value in IIx fibres (mean valuessignificantly differ from each other, P < 0.02). τh in Sol Iis higher than IIa, IIx and IIb fibres from PL (P = 0.007,P = 0.002 and P = 0.009, respectively). τh in IIa fibres issignificantly different from IIb fibres (P = 0.019), but notfrom IIx fibres (P = 0.09). τh in PL IIb fibres is not differentfrom PL IIx fibres (P = 0.12). Surprisingly, Sol type I fibresexhibit a similar τm value to PL IIb fibres (P = 0.97).

Activation and fast inactivation

Current values were normalized to maximal current(Fig. 3) to further compare curves of activation and fastinactivation in fibres corresponding to different MHC



isoforms because they were exhibiting various currentamplitudes. Values calculated by fitting experimentalpoints with the Boltzmann equation are shown in Table 2.The voltage dependence of activation is characterizedby V a,1/2 and shows a shift towards more positivepotentials from Sol type I to IIa, IIx and finally IIbfibres from PL (−27.7 ± 2.1, −25.6 ± 7.8, −10.1 ± 2.3

and −5.3 ± 4.1 mV, respectively, P < 0.05). The slopefactor of activation, K a, is significantly higher in IIb fibrescompared to other fibre types (P < 0.03) and correspondsto a larger current variation for the same depolarizingpulse.

For the different types of fibres, the voltage dependenceof fast inactivation of sodium current (Fig. 3B and

Figure 1. Electrophoretic separation(SDS-PAGE) of the four myosin heavychain (MHC) isoforms (fast types IIa, IIx,IIb, and slow type I, in progressive orderof migration) from rat hindlimb musclesA, silver-stained gel with plantaris, soleusand peroneus longus (whole muscles andsingle fibre). B, C, D and E, densitometrictracings with MHC percentage composition(relative area under the curve) of wholeplantaris (B), whole soleus (C), wholeperoneus longus (D), and single fibre fromperoneus longus identified as ‘IIa’ (E).



Table 1. Characteristics of Na+ currents recorded with patch-clamp technique, in single fibres from soleusand peroneus longus identified according to their content in MHC isoforms

Soleus Peroneus longus

I (n = 12) I (n = 2) IIa (n = 5) IIx (n = 12) IIb (n = 7)

Imax (nA) 7.1 ± 0.7 7.0 ± 0.6 8.2 ± 1.6∗ 9.5 ± 1.0∗† 15.4 ± 4.4∗†‡Imax (mA cm−2) 282.4 ± 74.0 281.0 ± 25.6 326.3 ± 62.7∗ 377.9 ± 40.8∗† 617.5 ± 174.3∗†‡gNa,max (mS) 80.0 ± 4.2 76.9 ± 15.7 107.4 ± 8.3∗ 158.8 ± 6.4∗† 218.5 ± 10.4∗†‡τm (ms) 0.20 ± 0.01 0.06 ± 0.02 0.11 ± 0.01∗ 0.16 ± 0.01∗† 0.20 ± 0.02†‡τh (ms) 0.34 ± 0.02 0.15 ± 0.07 0.19 ± 0.03∗ 0.22 ± 0.01∗ 0.24 ± 0.02∗†

Single fibres were identified according to their content of MHC isoforms (slow type I or fast types IIa, IIx, IIb).Parameters are derived from the non-normalized current–voltage curve corresponding to maximal currentamplitude. Imax, maximal sodium current expressed in absolute values and relative to the membrane surfaceunder pipette of patch clamp; gNa,max, maximal sodium conductance; τm and τh are the activation andinactivation time constants of sodium channels determined at –20 mV. Values are means ± S.E.M. ∗Significantlydifferent from type I soleus fibres (P < 0.05). †Significantly different from type IIa peroneus longus fibres(P < 0.05). ‡Significantly different from type IIx peroneus longus fibres (P < 0.05) (Student’s t test).

Figure 2. Relationship between fastsodium currents and MHC content inisolated muscle fibre from peroneuslongus and soleusA, example of sodium current recorded incell-attached macropatch technique inCsCl-containing medium. Sodium currentswere elicited, from the holding potential of−100 mV, by depolarizing the membrane in10 mV steps for 20 ms, between −60 and+130 mV. B, representative current–voltagerelationship for sodium current in singlemuscle fibres from peroneus longus (type IIbfibre) and soleus (type I fibre). C, normalizedcurrent–voltage relationship in single fibresidentified according to their content in MHCisoforms (slow type I or fast types IIa, IIx andIIb). Peak amplitudes for each testedpotential were normalized to maximalamplitude of sodium current.



Figure 3. Activation and fast inactivation curves of voltage-gated sodium channels, in single fibresfrom peroneus longus and soleus, identified according to their MHC isoform content (slow type I orfast types IIa, IIx, IIb)Curves were obtained by fitting the data with the Boltzmann equation. A, gNa/gNa,max for each tested potentialbetween −60 and +30 mV from the holding potential of −100 mV. B, steady-state fast inactivation wasdetermined from the holding potential of −100 mV. Inactivation prepulses lasting 50 ms were applied to potentialsranging between −120 and +10 mV and peak sodium current amplitude was measured during the 20 ms testpulse, the amplitude of which was selected to give a response near to the maximum. Peak values of inward currentswere measured at the times indicated by arrows, plotted against prepulse voltage and normalized to maximalamplitude. Two currents are shown: A, record corresponding to the prepulse of −10 mV; B, record correspondingto the prepulse −80 mV.



Table 2. Activation and fast inactivation parameters of voltage-gated sodium channels from single fibres ofperoneus longus and soleus discriminated with respect to their content of MHC isoforms

Soleus Peroneus longus

I (n = 12) I (n = 2) IIa (n = 5) IIx (n = 12) IIb (n = 7)

ActivationVa,1/2 (mV) −27.7 ± 2.1 −25.6 ± 7.8 −18.0 ± 3.9∗ −10.1 ± 2.3∗† −5.3 ± 4.1∗†‡Ka (V−1) 116.4 ± 3.8 109.7 ± 15.6 99.5 ± 6.5∗ 105.9 ± 2.6∗ 130.1 ± 9.2∗†‡

Fast inactivationVh,1/2 (mV) −69.9 ± 3.3 −76.2 ± 4.0 −63.3 ± 4.8∗ −63.9 ± 3.4∗ −71.5 ± 5.7†‡Kh (V−1) −89.3 ± 4.5 −178.6 ± 19.8 −76.9 ± 3.9∗ −98.0 ± 5.4† −99.0 ± 8.1†

All parameters are derived from fits with the Boltzmann equation of activation and fast inactivationcurves. Va,1/2, potential at which half of the channels are activated; Ka, slope factor; Vh,1/2, midpointof inactivation curve, where half of sodium channels are inactivated; Kh, fast-inactivation slope factor.Statistics were conducted between fibres from peroneus longus exhibiting IIa, IIx and IIb fast types of MHCisoforms and soleus fibres. Only 2 peroneus fibres contained slow type I, preventing statistical analysis.Values are means ± S.E.M. ∗Significantly different from type I soleus fibres (P < 0.03). †Significantly differentfrom type IIa peroneus longus fibres (P < 0.02). ‡Significantly different from type IIx peroneus longus fibres(P < 0.02) (Student’s t test).

Table 2) does not follow the same order as observed inactivation curves. Vh,1/2 in Sol type I and PL type IIbare similar (−69.9 ± 3.3 vs. −71.5 ± 5.7 mV, respectively,P = 0.79) with higher values compared to IIa and IIxPL fibres (−63.3 ± 4.8 and −63.9 ± 3.4 mV, respectively).Nevertheless, Vh,1/2 values in PL IIa fibres are notdifferent from PL IIx fibres (P = 0.93). The meanvalue of Kh, the slope factor of fast inactivation, issignificantly lower in PL IIa fibres compared to IIx andIIb fibres from PL and Sol type I (−76.9 ± 3.9 V−1 vs.−98.0 ± 5.4 V−1, −99.0 ± 8.1 V−1 and −89.3 ± 4.5 V−1,respectively, P < 0.02).

Discussion

Whole muscle, even if selected according to its pre-dominant mechanical properties (‘slow’ or ‘fast’), exhibitsheterogeneous composition. Moreover, single musclefibres can co-express different MHC isoforms (‘hybridfibre’). These two sources of heterogeneity potentiallylessen discrimination between fibre characteristics. Thushighlighting the interest of a correlation between MHCisoforms and electrophysiological properties in isolatedfibres. Our main intention was to study sodium channelproperties (by patch clamp) in a single muscle fibre inrelation to its MHC isoform content (by SDS-PAGE).

When taking into account fibres expressing only oneMHC isoform, the distribution of single fibre types weobserved in PL and Sol muscles is in agreement withprevious ATPase studies (Armstrong & Phelps, 1984;Ranatunga & Thomas, 1990). However, in PL we found12/35 fibres showing more than 5% of a second MHC iso-form, thus giving a 33% index of polymorphism. Caiozzo

et al. (2003) reported index values ranging from 13% inSol to 58% in EDL, but gave no value for PL.

Previous studies using single fibres emphasized the linkbetween MHC content and mechanical properties, suchas the shortening velocity of a single fibre (Bottinelli et al.1991; Galler et al. 1994). Conversely, there is a lack ofstudies concerning the relation between fibre excitabilityand its MHC content. In addition, muscle mechanicalproperties are related to its nervous command andits electrophysiological properties. Sodium voltage-gatedchannels are involved in triggering and propagatingmuscle fibre action potential and thus fibre excitability.Desaphy et al. (2001) reported higher values for Imax infibres from flexor digitorum brevis (FDB) considered as‘fast’, compared to ‘slow’ fibres from soleus rat muscle. Ruff& Whittlesey (1993) noted larger current densities fromtype IIb compared with type IIa fibres in histochemicallyidentified fibres from human muscle biopsies. Our resultsprovide further precision, because we observed that INa,max

and gNa,max increase according to the recognizable schemeI→IIa→IIx→IIb. It should be emphasized that sodiumcurrent values in IIb fibres are approximately doublethose observed in the other types. This is certainlyrelated to contraction strength and velocity, throughexcitation–contraction coupling. Maximal current andconductance are linked to the number of open sodiumchannels and/or difference in the type or regulationof the sodium channel. Time constants are related tosodium channel types. We found that τm and τh exhibita progressive enhancement from I to IIb in PL indicatinga modification in type and/or regulation of the sodiumchannel. In spite of different mechanical properties, Soltype I and PL type IIb fibres exhibit similar activationconstants (τm). This is in keeping with previous studies



using tetrodotoxin showing similar effects on the sodiumchannel in fibres from Sol (type I) and EDL (type IIx,IIb). These authors concluded that the type of channelwas not different between these two muscles (McArdleet al. 1980; Desaphy et al. 1998), although they donot characterize the single fibre types. Furthermore, weobserved an analogous voltage-dependence relationshipof fast inactivation between Sol I fibres and PL IIb fibres(Fig. 3B). Likewise Filatov & Rich (2004) showed that theinactivation process depends on the channel type.

Conversely, we found that the voltage dependence ofactivation in the sodium voltage-gated channel is differentaccording to the MHC content of single fibres. This isillustrated by the normalized current–voltage relationshipand confirmed by normalized conductance for each testedpotential. In our experimental conditions, activationcurves shift towards more positive potentials, from type Ito IIb fibres (see Fig. 3A and V a,1/2 in Table 2). This 22 mVshift suggests that fast fibres need more depolarizingcurrents to trigger an action potential, especially as theystart from a more negative resting potential (Duval &Leoty, 1978). This progressive increase in the actionpotential threshold from slow to the fastest fibre types leadsto an excitability decrease related to channel properties. Inan experimental model of septic rats, we observed botha shift in gNa/gNa,max towards more positive potentials(Rossignol et al. 2007) and a delayed trigger of the muscleaction potential in isolated motor units (Rannou et al.2007). Such a shift also contributes to the increase in thecapacity of repetitive firing in fast phenotypes (McArdleet al. 1980). Desaphy et al. (2001), in their Fig. 3, observeda trend in the current–voltage relationship in Sol whichshifted towards more negative potentials, compared withFDB, in spite of no statistical difference. Although notsignificant, these results are consistent with our findings.The whole muscle approach and polymorphism in selectedfibres could explain this lack of significance.

Inside a mixed muscle like PL, such differencesin excitability among the different fibres should beconsidered with regard to contractile implications. Lowervalues in τm and τh for the slower fibre types (I and IIain PL) increase excitability through faster depolarizationand a rapid return to resting state for the sodium channel.Both these factors appear in accordance with a progressiverecruitment of fibre types. This recalls the ‘Henneman’ssize principle’, postulating that slow motor units, andtherefore, slow fibres in a fast muscle like PL, are firstactivated before fast motor units. There are few slowfibres in ‘fast’ muscles and they develop little tetanicforce. Thus, our results trigger an absorbing questionconcerning the exact mechanical role of slow fibres in ‘fast’muscles. We hypothesize that the PL slow fibres have tobe activated early in order to optimize the mechanicalperformance of the predominant fast fibres. Such anobservation inside a single muscle can be compared to

the postural adjustment preceding voluntary movements(Bouisset & Zattara, 1981). Also, the mechanical forcesdeveloped by the PL fast motor units are closer to theSol slow motor units (Emonet-Denand et al. 1988). Thiscould explain why the time-dependent parameters of theirsodium channels are in the same range.

In conclusion, the properties of voltage-gated sodiumchannels could be different according to both the myosinexpression of a fibre type and to the muscle to which theybelong. Conductance and current parameters increaseprogressively from I to IIb fibres and this evolution isparallel to the mechanical performances of these fibresincluding power and shortening velocity. On the otherhand type I fibres have very different properties in a fastmuscle like PL and a homogeneous postural muscle likesoleus. This could be in relation to the neural drive ofthese fibres inside each muscle, leading to a paradoxicalsimilarity in the time constant activation of soleus type Ifibres and PL IIb fibres.

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Author contributions

All authors contributed to the different steps of analysis andinterpretation of data, drafting and discussion of the manuscript.All experiments were performed at the Laboratory of Physiology,School of Medicine, 22 Avenue Camille Desmoulins, 29238 Brest,France.

Acknowledgements

The authors thank Mrs Pauline Desjoyeaux for helpful assistanceconcerning electrophoresis. Public funding from the FrenchMinistry of Research supported this work.


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Differences in sodium voltage-gated channel properties according to myosin heavy chain isoform expression in single muscle fibres