Extension and magnitude of denervation in skeletal muscle from ageing mice
1 Department of Physiology and Pharmacology
2 Department of Internal Medicine, Gerontology
3 Neuroscience Program, Wake Forest University School of Medicine, Winston-Salem, NC 27157, USA
Abstract
In this work we hypothesized that denervation in flexor digitorum brevis (FDB) muscle from ageing mice is more extensive than predicted by standard functional and structural assays used in the past. In addition, we asked whether denervation is a fully or partially developed process. Despite the reported alteration in skeletal muscle innervation, the quantification of the extension and magnitude of denervation in ageing rodents has remained elusive. To address these two questions we utilized a combination of electrophysiological and immunohistochemical assays directed to detecting the expression of tetrodotoxin (TTX)-resistant sodium channels (Nav1.5) in FDB muscles from young-adult and senescent mice. Sodium current density measured with the macropatch cell-attached technique did not show significant differences between FDB fibres from young and old mice. The TTX dose–response curve, using the whole cell voltage-clamp technique, showed three populations of fibres in senescent mice, one similar to fibres from young mice (TTX sensitive), another one similar to fibres from experimentally denervated muscle (TTX resistant), and a third group intermediate between these two. Partially and fully denervated fibres added up to approximately 50% of the total number of fibres tested, a number that concurs with the percentage of fibres positive for the Nav1.5 channel by specific immunostaining.
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Introduction
The underlying mechanism of loss in the intrinsic force generating capacity of muscle fibres occurring in ageing rodents and humans is only partially understood (Renganathan et al. 1998; Frontera et al. 2000; González et al. 2000). Muscle weakness in ageing mammals may result from primary neural or muscular aetiological factors, or a combination of both (Delbono, 2003). Experimental muscle denervation leads to loss in absolute and specific force (Finol et al. 1981; Dulhunty & Gage, 1985). Although denervation contributes to skeletal muscle functional impairment with ageing (Larsson & Ansved, 1995), its prevalence in human and animal models of ageing remains to be determined.
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In the present work we hypothesized that denervation in ageing skeletal muscle is more extensive than predicted by standard functional and structural assays used up to now (see below). In addition, we asked whether denervation is a fully or partially developed process. To address these two questions we utilized a combination of electrophysiological and immunohistochemical assays directed to detecting the expression of tetrodotoxin (TTX)-resistant sodium channels (Nav1.5) in flexor digitorum brevis (FDB) muscles from young-adult and senescent mice. The FDB muscle has been selected for this study due to the fast fibre type composition (70% type IIx, 13% IIa, and 17% type I) (González et al. 2003), and because of the shortness of the fibres that make them suitable for patch-clamp recordings (Wang et al. 1999).
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Two sodium channel isoforms are expressed in skeletal muscle, the TTX-sensitive Nav1.4 and the TTX-resistant Nav1.5. Both channels were originally isolated from rat skeletal muscle and were denominated SkM1 (Trimmer et al. 1989) and SkM2 (Kallen et al. 1990), respectively. To determine the status of denervation of individual fibres from adult and senescent mice, we took advantage of the following properties of the Nav1.5 channel: (1) its expression after denervation but absence in innervated adult muscle, (2) its early increase in expression, recorded at 24 h after denervation in hindlimb muscles (Yang et al. 1991), and (3) its relative insensitivity to TTX (Redfern et al. 1970; Pappone, 1980; Kallen et al. 1990; White et al. 1991).
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Here we propose that muscle denervation occurs in stages in ageing rodents, which is consistent with our findings that some fibres are either fully innervated or denervated and a third group expresses an intermediate level of TTX-resistant sodium channel, indicating partial denervation.
Methods
Flexor digitorum brevis muscle fibres
Single skeletal muscle fibres from the FDB muscle were obtained from 5- to 7-month-old (young-adult group) or 21- to 24-month-old (old) FVB mice raised at the Animal Research Program of Wake Forest University School of Medicine (WFUSM). Animal handling followed an approved protocol by the Animal Care and Use Committee of WFUSM. Mice were killed by cervical dislocation. FDB muscles were dissected and fibres enzymatically isolated as described (Wang et al. 1999, 2002).
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Sodium current recordings
For the analysis of the sodium current–voltage relationship and current sensitivity to TTX we used the cell-attached and whole-cell configurations of the patch-clamp technique, respectively (Desaphy et al. 1998b; Wang et al. 1999).
Sodium current recordings in macropatches. FDB fibres were voltage-clamped using an Axopatch-200B amplifier (Molecular Devices–Axon Instruments, Union City, CA, USA) in the cell-attached configuration of patch-clamp (Hamill et al. 1981). Patch pipettes were pulled from borosilicate glass (Boralex), using a Flaming Brown micropipette puller (P97; Sutter Instruments, Novato, CA, USA), and then fire-polished to obtain electrode resistance ranging from 1.5 to 2.0 M when filled with the recording pipette solution. The pipette solution contained (mM): 150 NaCl, 1 MgCl2, 1 CaCl2, 10 Hepes, with pH adjusted to 7.3 with NaCl. The bath solution contained (mM): 145 CsCl, 5 EGTA (ethylene glycol-bis(-aminoethyl ether)-N,N,N'N'-tetraacetic acid), 1 MgCl2, 10 Hepes, 5 glucose. The pH was adjusted to 7.3 with CsOH. Sodium currents were acquired and filtered at 5 kHz with pCLAMP 6.04 software (Axon Instruments). A Digidata 1200 interface (Axon Instruments) was used for A–D conversion. Currents were low-pass filtered at 10 kHz (–3dB) with the amplifier's four-pole Bessel filter and digitized at 20–100 kHz. Membrane current during a voltage pulse, P, was initially corrected by analog subtraction of the linear components. Any remaining linear components were digitally subtracted on-line using hyperpolarizing control pulses of one-quarter test pulse amplitude (–P/4 procedure) as described (Delbono et al. 1997). The sodium currents were recorded from the extrajunctional membrane (Ruff, 1992; Desaphy et al. 2001).
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Sodium current recordings in whole-cell configuration of patch-clamp. For tetrodotoxin (TTX) perfusion experiments, FDB muscle fibres were voltage-clamped in the whole-cell configuration of the patch-clamp technique (Hamill et al. 1981) according to procedures previously described (Wang et al. 1999; Payne et al. 2004). Potential voltage errors associated with whole-cell recording in large cells have been minimized by selecting small FDB fibres and by adequate compensation for whole cell capacitance transients. The pipette electrode was filled with the following solution (mM): 130 caesium aspartate; 2 MgCl2, 10 Cs2EGTA, 10 Hepes, 5 Na-ATP, 0.5 GTP, with pH adjusted to 7.3 with CsOH, and the resistance ranged from 450 to 650 k. The external solution containing (mM): 100 TEA-OH (tetraethylammonium hydroxide), 50 Na2SO4, 2 MgSO4, 2 CaSO4, 2 3,4 diaminopyridine (3–4DAP), 5 Na-Hepes. Solution pH was adjusted to 7.3 with CH4SO3. For muscle fibre perfusion we used a recording chamber of 130 μl, which allowed TTX solutions to be exchanged completely at least twice in less than 10 s. All experiments were recorded at room temperature (21–22°C).
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Muscle immunohistochemistry
For immunohistochemistry fresh FDB muscles were embedded in OCT medium, and rapidly frozen in 2-methylbutane cooled in liquid nitrogen. Muscles were kept at –80°C for subsequent use. Frozen samples were sectioned with a cryostat at –25°C. Cross-sections (8 μm) of the muscle were fixed with 4% paraformaldehyde in PBS for 30 min, rinsed in 0.1 M Tris-buffered saline (TBS), blocked in 5% goat serum for 30 min followed by avidin blocking solution (Vector laboratories, Burlingame, CA, USA), rinsed in TBS, blocked in biotin blocking solution (Vector), and again rinsed in TBS. The sections were incubated with the antibody against the TTX-R Nav1.5. This Nav1.5 antibody was generated against peptide SH1 (KTEPQAPGCGET-PEDS) as described (Maier et al. 2002) and was diluted 1 : 100 in TBS. This purified antibody was kindly provided by Dr William A. Catterall (Department of Pharmacology, University of Washington, Seattle). The immunostaining technique has been published (Maier et al. 2002). Muscle slices from young and old mice were paired, mounted on a slide, and thereby similarly stained. Heart muscle was used as a positive control. Images from young and old muscles were digitized using the same binning, camera gain, and exposure time. Muscle fibres were considered positive when they exhibited a clear fluorescent enhancement of the borders.
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Statistics
Values are given as means ± S.E.M. with the number of observations (n). Statistical analysis has been performed using Student's unpaired t test and the Mann-Whitney Rank Sum Test, when values were not normally distributed. P < 0.05 was considered significant.
Results
Sodium current recordings in macropatches of FDB muscle fibres from young and senescent mice
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To determine the properties of sodium currents in FDB fibres we analysed the current–voltage relationship in the cell-attached configuration of the patch-clamp technique in freshly dissociated mouse FDB fibres. Currents were elicited by 5 ms depolarizing pulses from the holding potential (–110 mV) to command potentials ranging from –100 to 80 mV with 10 mV intervals. Sodium currents from young and old mice were similarly detected between –80 and –60 mV. Currents reached a maximum between –20 and –10 mV, and inactivated completely at 80 mV. Figure 1 shows the sodium current–voltage relationship for FDB fibres from young (n = 15) and old (n = 13) mice. Normalized data points to the maximum current amplitude were fitted to the following equation:
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where Gmax is the maximum conductance, V is the membrane potential, Vr is the reversal potential, and V1/2 is the half-activation potential, z is the effective valence, F is the Faraday constant, R is the gas constant, and T is the absolute temperature. The V1/2 values for fibres from young and old mice were –23 ± 2.7 mV and –27.6 ± 3.1 mV, respectively (P > 0.05). Figure 1 illustrates typical sodium current recordings in FDB fibres from young (Fig. 1B) and old (Fig. 1C) mice from –40 to 10 mV in 10 mV intervals. To determine whether ageing is associated with changes in sodium channel expression, the peak sodium current amplitude and current density were measured. Peak sodium current amplitude was normalized to the square of the pipette conductance to calculate current density (Desaphy et al. 2001). Peak current values in fibres from young (n = 15) and old (n = 13) mice were 110 ± 15 pA and 107 ± 11 pA (P > 0.05), whereas the current density (in pA/arbitrary units) was 1115 ± 123 and 1056 ± 139, respectively (P > 0.05). In summary, these results suggest that neither the level of extra-junctional sodium channel expression nor the voltage dependence of sodium current is modified with ageing.
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Sodium current in fibres from young and old mice were recorded in the cell-attached mode. B and C illustrate typical sodium current recordings in fibres from young and old mice, respectively, in the range of voltages depicted on the right.
Sodium current sensitivity to TTX in FDB fibres from young and old mice
To investigate the status of denervation, we recorded the sodium current sensitivity to TTX in FDB fibres voltage-clamped in the whole cell configuration of the patch-clamp. Sodium currents were evoked by 20 ms depolarizing pulses from the holding potential (–80 mV) to command potentials ranging from –100 to 50 mV in 10 mV intervals. The effects of TTX on sodium current amplitude were tested on the peak sodium current. TTX dose–response curves were constructed by pulsing the fibre repeatedly in a series of 10 s intervals from the holding potential (–80 mV) to the command potential that elicited the maximum sodium current amplitude. After full current blockade, TTX was washed out completely. Only fibres that exhibited an 80% or better recovery of the initial peak current (before exposure to TTX) were included in the statistics. Figure 2A illustrates the stability of the peak sodium current over a period of 23 min in FDB fibres from old mice (n = 5). Arrows indicate the times at which the solution was changed. For these experiments we used the bath solution devoid of TTX. Figure 2B illustrates the time course of the sodium currents (the 10 s interpulse interval was omitted). The currents were recorded in fibres from an old mouse in response to the application of repetitive depolarizing command pulses to –10 mV. These experiments show that sodium currents, recorded in the most demanding preparation (old), maintain the amplitude within 80–90% of the initial value for a period that exceeds that needed to test a set of TTX concentrations (see below).
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Peak sodium current recorded in response to repetitive depolarizing command pulses to –10 mV in 5 FDB fibres. Arrows indicate the times at which the external solution was exchanged with the same bath solution. B illustrates the time course of the sodium currents, recorded in a fibre from an old mouse. The 10 s interpulse interval was omitted. The dashed line represents the baseline.
Figure 3 shows the dose–response relationship and analysis of the dissociation constant for TTX in young and old mice. Peak currents in TTX (ITTX) are expressed relatively to the average of the currents in toxin-free solution (Io,TTX). Data points for fibres from young control mice were fitted to the following equation:
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where K1 is the dissociation constant for TTX (Pappone, 1980).
Data points were fitted to either eqn (2) or eqn (3) (Methods). B illustrates the time course of the sodium currents for an old-TTX-I fibre in control (before TTX, time zero) and after the exposure to TTX (top bar). The dashed line represents the baseline.
Sodium currents recorded in fibres from young mice (n = 26) exhibited a quick drop in the peak sodium current amplitude and were fully blocked with 1 μM TTX. The K1 value for these fibres was 5.6 ± 0.7 nM. Fibres from old mice showed a marked variability in the TTX concentration needed to block sodium currents (n = 29). Approximately half of the fibres from old mice were well fitted to eqn (2) (old-TTX-S) giving a K1 value of 6.2 ± 0.8 nM (n = 15). This group of fibres was called old-TTX-S. The difference between fibres from old-TTX-S and young mice was not statistically significant (P > 0.05). Data points from another group of fibres from old mice (n = 4) were better fitted to the following equation:
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where K2, as K1, is a dissociation constant for TTX (Pappone, 1980). Data points for this group were 5.6 ± 0.6 nM and 990 ± 256 nM for K1 and K2, respectively. This group was called old-TTX-R. An intermediate group was detected in between the previous two (n = 10). The values for the intermediate group were 6.5 ± 0.8 nM and 257 ± 98 nM, respectively. This intermediate population of fibres was called old-TTX-I. Differences in K2 but not in K1 values between the old-TTX-R and old-TTX-I were statistically significant (P < 0.05). The interpretation of these results is consistent with the expression of a population of TTX-R sodium channels (SkM2) (Yang et al. 1991) in old-TTX-I and old-TTX-R fibres and the ratio between TTX-resistant and -sensitive sodium channel is higher in the latter group. Figure 3B illustrates the time course of the sodium currents for a fibre belonging to the old-TTX-I group in which eight TTX concentrations were tested. It appears that a quick drop in current amplitude in response to 1–5 nM TTX is followed by a less pronounced effect. In summary, these results show that FDB fibres from old mice exhibit three patterns in response to TTX, one group that is similar to innervated fibres from young mice (52%) and another group that has two subpopulations, those of partially (35%) and fully (13%) denervated fibres.
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Expression of Nav1.5 in ageing FDB skeletal muscle
To investigate the expression of Nav1.5 we performed immunocytochemistry assays on cross-sections of FDB muscles. Figure 4 illustrates areas of muscle cross-sections from young (Fig. 4A) and old (Fig. 4B) mice. The sections of these FDB fibres from old mice depict two distinct patterns, either (1) intense fluorescence at their borders (horizontal arrow in Fig. 4B) or (2) uniform fluorescence throughout (vertical arrow). The former fibres were considered positive for TTX-R sodium channel expression, while the latter fibres were similar to the homogeneous pattern recorded in muscles from young mice (Fig. 4A), and were counted as negative. All the fibres from each muscle section were counted and the number of positive or negative cells was normalized to the total number of cells per section. For this analysis, three mice, six FDB muscles and 115–140 fibres per muscle were analysed for each group, young (1.6 ± 0.13% positive fibres for Nav 1.5 antibody) and old (48 ± 4.6%) mice (P < 0.01). Fibres per muscle were counted in at least five cross-sections and values were averaged. The ample space between fibres corresponds to the abundant connective tissue and tendons separating bundles of the short piece of the FDB muscle. Figure 4C and D illustrates muscle sections from young and old mice, respectively, in which the primary antibody was omitted. Figure 4E shows that muscles from senescent, compared with young mice, exhibited a larger number of fibres positive for the TTX-R Nav1.5 channel.
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Digitized images of FDB muscles from young (A) and old (B) mice. The sections from old mice (B) depict positive (horizontal arrow) or negative fibres (vertical arrow). C and D correspond to muscle sections from young and old mice, respectively, in which the primary antibody was omitted. E shows the normalized number of Nav1.5 positive fibres analysed for young and old mice. The asterisk indicates a statistically significant difference (P < 0.05).
Discussion
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The main conclusion of the present study is that FDB muscles from senescent mice exhibit three groups of fibres concerning innervation status. In addition, the percentage of fibres depicting some degree of TTX resistance (50%) was similar to that detected by immunocytochemistry using a specific antibody against the Nav1.5 channel.
Sodium current in skeletal muscle
Sodium current amplitude and density in FDB fibres from young mice, reported here, are similar to those recorded in mouse FDB muscle fibres (Reddy et al. 2002). An increase in sodium current density in ageing rats (Desaphy et al. 1998a,b), but not in mouse (present work), has been reported. Experimentally denervated EDL muscles from young rat exhibited an increase in maximal conductance at 22°C, but not at 12°C, when recorded with the double Vaseline gap technique (Pappone, 1980). A potential explanation for this discrepancy is that denervation affects muscle fibre subtypes differently. Rat FDB and extensor digitorum longus muscles are predominantly composed of type IIa (Carlsen et al. 1985) and type IIb (Brown et al. 1992) fibres, respectively, whereas mouse FDB muscles are mainly type IIx fibres (González et al. 2003).
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The sodium current–voltage relationship and sodium channel conductance have been reported to not change with maturation (Reddy et al. 2002); however, two different conductances of 18 pS and 9 pS have been reported in FDB muscles from old rat. Cells exhibiting a conductance of 18 pS were called ‘young-phenotype’, while the remaining group, was called ‘old-phenotype’ (Desaphy et al. 1998a). The similarity in the values recorded in fibres from old rats and myoblasts (Weiss & Horn, 1986) suggests that the small conductance channel corresponds to SkM2, whose expression increases in response to muscle denervation (Yang et al. 1991). However, two fibres exhibiting an ‘old phenotype’ were sensitive to a fixed TTX concentration in the cell-attached mode (Desaphy et al. 1998a). In that study, the small number of fibres tested together with a selection of those fibres exhibiting a better appearance, most likely decreased the number of denervated fibres detected.
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A shift in activation and inactivation parameters of sodium currents by 10 mV to more negative potentials has been reported in homogeneously denervated fibres (Pappone, 1980). By contrast, the population of fibres in FDB muscle from ageing mice (present work) is heterogeneously denervated. Approximately 52% of the fibres do not show evidence of denervation, and only 13% are fully denervated. Therefore, the comparison between these two preparations is not straightforward.
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Denervation in ageing skeletal muscle
Several studies have reported the phenomenon of skeletal muscle denervation and reinervation, as well as motor unit remodelling in ageing rodents or humans (Larsson, 1995; Lexell, 1995; for a review see Payne & Delbono, 2004). Due to the heterogeneity of the fibres from old mice reported here, it is reasonable to postulate that the fibres that exhibited greater resistance to TTX correspond to the positive cells labelled for neural cell adhesion molecule (NCAM) by immunostaining in old rats (Urbancheck et al. 2001). Using this technique the authors reported that less than 10% of rat extensor digitorum longus muscle fibres are NCAM positive. Underestimation of the number of denervated fibres with this technique is possible, based on the time-dependent decline in NCAM protein expression after denervation (Daniloff et al. 1986). In the present study FDB muscle denervation detected with Nav1.5 antibody is more extensive than that reported with NCAM antibody in EDL muscle because our probe is probably more sensitive to detecting partially denervated fibres (old-TTX-I), which represent approximately 35% of the fibres in FDB muscle from senescent mice. In addition, these differences of magnitude and extension of denervation may vary between muscle types and species. Differences with previous publications may reside on the use of a specific Nav1.5 channel antibody and the whole-cell configuration of patch-clamp to assess functionally a larger number of fibres for TTX sensitivity than in previous publications. Results included here concur with reports in human in which between 25 and 50% of loss in spinal cord motor neurone and motor units have been reported (for a review see Lexell, 1997).
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In summary, the experimental approach used here allowed for the identification of three populations of fibres in senescent mice, based on their sensitivity to TTX. Partially and fully denervated fibres, taken together, represent 50% of the total fibres, a number that concurs with the percentage of fibres positive for Nav1.5 channel by specific immunostaining. The extension and magnitude of muscle denervation reported here prompts a reassessment of the mechanisms proposed for the loss in muscle structure and function with ageing.
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2 Department of Internal Medicine, Gerontology
3 Neuroscience Program, Wake Forest University School of Medicine, Winston-Salem, NC 27157, USA
Abstract
In this work we hypothesized that denervation in flexor digitorum brevis (FDB) muscle from ageing mice is more extensive than predicted by standard functional and structural assays used in the past. In addition, we asked whether denervation is a fully or partially developed process. Despite the reported alteration in skeletal muscle innervation, the quantification of the extension and magnitude of denervation in ageing rodents has remained elusive. To address these two questions we utilized a combination of electrophysiological and immunohistochemical assays directed to detecting the expression of tetrodotoxin (TTX)-resistant sodium channels (Nav1.5) in FDB muscles from young-adult and senescent mice. Sodium current density measured with the macropatch cell-attached technique did not show significant differences between FDB fibres from young and old mice. The TTX dose–response curve, using the whole cell voltage-clamp technique, showed three populations of fibres in senescent mice, one similar to fibres from young mice (TTX sensitive), another one similar to fibres from experimentally denervated muscle (TTX resistant), and a third group intermediate between these two. Partially and fully denervated fibres added up to approximately 50% of the total number of fibres tested, a number that concurs with the percentage of fibres positive for the Nav1.5 channel by specific immunostaining.
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Introduction
The underlying mechanism of loss in the intrinsic force generating capacity of muscle fibres occurring in ageing rodents and humans is only partially understood (Renganathan et al. 1998; Frontera et al. 2000; González et al. 2000). Muscle weakness in ageing mammals may result from primary neural or muscular aetiological factors, or a combination of both (Delbono, 2003). Experimental muscle denervation leads to loss in absolute and specific force (Finol et al. 1981; Dulhunty & Gage, 1985). Although denervation contributes to skeletal muscle functional impairment with ageing (Larsson & Ansved, 1995), its prevalence in human and animal models of ageing remains to be determined.
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In the present work we hypothesized that denervation in ageing skeletal muscle is more extensive than predicted by standard functional and structural assays used up to now (see below). In addition, we asked whether denervation is a fully or partially developed process. To address these two questions we utilized a combination of electrophysiological and immunohistochemical assays directed to detecting the expression of tetrodotoxin (TTX)-resistant sodium channels (Nav1.5) in flexor digitorum brevis (FDB) muscles from young-adult and senescent mice. The FDB muscle has been selected for this study due to the fast fibre type composition (70% type IIx, 13% IIa, and 17% type I) (González et al. 2003), and because of the shortness of the fibres that make them suitable for patch-clamp recordings (Wang et al. 1999).
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Two sodium channel isoforms are expressed in skeletal muscle, the TTX-sensitive Nav1.4 and the TTX-resistant Nav1.5. Both channels were originally isolated from rat skeletal muscle and were denominated SkM1 (Trimmer et al. 1989) and SkM2 (Kallen et al. 1990), respectively. To determine the status of denervation of individual fibres from adult and senescent mice, we took advantage of the following properties of the Nav1.5 channel: (1) its expression after denervation but absence in innervated adult muscle, (2) its early increase in expression, recorded at 24 h after denervation in hindlimb muscles (Yang et al. 1991), and (3) its relative insensitivity to TTX (Redfern et al. 1970; Pappone, 1980; Kallen et al. 1990; White et al. 1991).
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Here we propose that muscle denervation occurs in stages in ageing rodents, which is consistent with our findings that some fibres are either fully innervated or denervated and a third group expresses an intermediate level of TTX-resistant sodium channel, indicating partial denervation.
Methods
Flexor digitorum brevis muscle fibres
Single skeletal muscle fibres from the FDB muscle were obtained from 5- to 7-month-old (young-adult group) or 21- to 24-month-old (old) FVB mice raised at the Animal Research Program of Wake Forest University School of Medicine (WFUSM). Animal handling followed an approved protocol by the Animal Care and Use Committee of WFUSM. Mice were killed by cervical dislocation. FDB muscles were dissected and fibres enzymatically isolated as described (Wang et al. 1999, 2002).
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Sodium current recordings
For the analysis of the sodium current–voltage relationship and current sensitivity to TTX we used the cell-attached and whole-cell configurations of the patch-clamp technique, respectively (Desaphy et al. 1998b; Wang et al. 1999).
Sodium current recordings in macropatches. FDB fibres were voltage-clamped using an Axopatch-200B amplifier (Molecular Devices–Axon Instruments, Union City, CA, USA) in the cell-attached configuration of patch-clamp (Hamill et al. 1981). Patch pipettes were pulled from borosilicate glass (Boralex), using a Flaming Brown micropipette puller (P97; Sutter Instruments, Novato, CA, USA), and then fire-polished to obtain electrode resistance ranging from 1.5 to 2.0 M when filled with the recording pipette solution. The pipette solution contained (mM): 150 NaCl, 1 MgCl2, 1 CaCl2, 10 Hepes, with pH adjusted to 7.3 with NaCl. The bath solution contained (mM): 145 CsCl, 5 EGTA (ethylene glycol-bis(-aminoethyl ether)-N,N,N'N'-tetraacetic acid), 1 MgCl2, 10 Hepes, 5 glucose. The pH was adjusted to 7.3 with CsOH. Sodium currents were acquired and filtered at 5 kHz with pCLAMP 6.04 software (Axon Instruments). A Digidata 1200 interface (Axon Instruments) was used for A–D conversion. Currents were low-pass filtered at 10 kHz (–3dB) with the amplifier's four-pole Bessel filter and digitized at 20–100 kHz. Membrane current during a voltage pulse, P, was initially corrected by analog subtraction of the linear components. Any remaining linear components were digitally subtracted on-line using hyperpolarizing control pulses of one-quarter test pulse amplitude (–P/4 procedure) as described (Delbono et al. 1997). The sodium currents were recorded from the extrajunctional membrane (Ruff, 1992; Desaphy et al. 2001).
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Sodium current recordings in whole-cell configuration of patch-clamp. For tetrodotoxin (TTX) perfusion experiments, FDB muscle fibres were voltage-clamped in the whole-cell configuration of the patch-clamp technique (Hamill et al. 1981) according to procedures previously described (Wang et al. 1999; Payne et al. 2004). Potential voltage errors associated with whole-cell recording in large cells have been minimized by selecting small FDB fibres and by adequate compensation for whole cell capacitance transients. The pipette electrode was filled with the following solution (mM): 130 caesium aspartate; 2 MgCl2, 10 Cs2EGTA, 10 Hepes, 5 Na-ATP, 0.5 GTP, with pH adjusted to 7.3 with CsOH, and the resistance ranged from 450 to 650 k. The external solution containing (mM): 100 TEA-OH (tetraethylammonium hydroxide), 50 Na2SO4, 2 MgSO4, 2 CaSO4, 2 3,4 diaminopyridine (3–4DAP), 5 Na-Hepes. Solution pH was adjusted to 7.3 with CH4SO3. For muscle fibre perfusion we used a recording chamber of 130 μl, which allowed TTX solutions to be exchanged completely at least twice in less than 10 s. All experiments were recorded at room temperature (21–22°C).
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Muscle immunohistochemistry
For immunohistochemistry fresh FDB muscles were embedded in OCT medium, and rapidly frozen in 2-methylbutane cooled in liquid nitrogen. Muscles were kept at –80°C for subsequent use. Frozen samples were sectioned with a cryostat at –25°C. Cross-sections (8 μm) of the muscle were fixed with 4% paraformaldehyde in PBS for 30 min, rinsed in 0.1 M Tris-buffered saline (TBS), blocked in 5% goat serum for 30 min followed by avidin blocking solution (Vector laboratories, Burlingame, CA, USA), rinsed in TBS, blocked in biotin blocking solution (Vector), and again rinsed in TBS. The sections were incubated with the antibody against the TTX-R Nav1.5. This Nav1.5 antibody was generated against peptide SH1 (KTEPQAPGCGET-PEDS) as described (Maier et al. 2002) and was diluted 1 : 100 in TBS. This purified antibody was kindly provided by Dr William A. Catterall (Department of Pharmacology, University of Washington, Seattle). The immunostaining technique has been published (Maier et al. 2002). Muscle slices from young and old mice were paired, mounted on a slide, and thereby similarly stained. Heart muscle was used as a positive control. Images from young and old muscles were digitized using the same binning, camera gain, and exposure time. Muscle fibres were considered positive when they exhibited a clear fluorescent enhancement of the borders.
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Statistics
Values are given as means ± S.E.M. with the number of observations (n). Statistical analysis has been performed using Student's unpaired t test and the Mann-Whitney Rank Sum Test, when values were not normally distributed. P < 0.05 was considered significant.
Results
Sodium current recordings in macropatches of FDB muscle fibres from young and senescent mice
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To determine the properties of sodium currents in FDB fibres we analysed the current–voltage relationship in the cell-attached configuration of the patch-clamp technique in freshly dissociated mouse FDB fibres. Currents were elicited by 5 ms depolarizing pulses from the holding potential (–110 mV) to command potentials ranging from –100 to 80 mV with 10 mV intervals. Sodium currents from young and old mice were similarly detected between –80 and –60 mV. Currents reached a maximum between –20 and –10 mV, and inactivated completely at 80 mV. Figure 1 shows the sodium current–voltage relationship for FDB fibres from young (n = 15) and old (n = 13) mice. Normalized data points to the maximum current amplitude were fitted to the following equation:
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where Gmax is the maximum conductance, V is the membrane potential, Vr is the reversal potential, and V1/2 is the half-activation potential, z is the effective valence, F is the Faraday constant, R is the gas constant, and T is the absolute temperature. The V1/2 values for fibres from young and old mice were –23 ± 2.7 mV and –27.6 ± 3.1 mV, respectively (P > 0.05). Figure 1 illustrates typical sodium current recordings in FDB fibres from young (Fig. 1B) and old (Fig. 1C) mice from –40 to 10 mV in 10 mV intervals. To determine whether ageing is associated with changes in sodium channel expression, the peak sodium current amplitude and current density were measured. Peak sodium current amplitude was normalized to the square of the pipette conductance to calculate current density (Desaphy et al. 2001). Peak current values in fibres from young (n = 15) and old (n = 13) mice were 110 ± 15 pA and 107 ± 11 pA (P > 0.05), whereas the current density (in pA/arbitrary units) was 1115 ± 123 and 1056 ± 139, respectively (P > 0.05). In summary, these results suggest that neither the level of extra-junctional sodium channel expression nor the voltage dependence of sodium current is modified with ageing.
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Sodium current in fibres from young and old mice were recorded in the cell-attached mode. B and C illustrate typical sodium current recordings in fibres from young and old mice, respectively, in the range of voltages depicted on the right.
Sodium current sensitivity to TTX in FDB fibres from young and old mice
To investigate the status of denervation, we recorded the sodium current sensitivity to TTX in FDB fibres voltage-clamped in the whole cell configuration of the patch-clamp. Sodium currents were evoked by 20 ms depolarizing pulses from the holding potential (–80 mV) to command potentials ranging from –100 to 50 mV in 10 mV intervals. The effects of TTX on sodium current amplitude were tested on the peak sodium current. TTX dose–response curves were constructed by pulsing the fibre repeatedly in a series of 10 s intervals from the holding potential (–80 mV) to the command potential that elicited the maximum sodium current amplitude. After full current blockade, TTX was washed out completely. Only fibres that exhibited an 80% or better recovery of the initial peak current (before exposure to TTX) were included in the statistics. Figure 2A illustrates the stability of the peak sodium current over a period of 23 min in FDB fibres from old mice (n = 5). Arrows indicate the times at which the solution was changed. For these experiments we used the bath solution devoid of TTX. Figure 2B illustrates the time course of the sodium currents (the 10 s interpulse interval was omitted). The currents were recorded in fibres from an old mouse in response to the application of repetitive depolarizing command pulses to –10 mV. These experiments show that sodium currents, recorded in the most demanding preparation (old), maintain the amplitude within 80–90% of the initial value for a period that exceeds that needed to test a set of TTX concentrations (see below).
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Peak sodium current recorded in response to repetitive depolarizing command pulses to –10 mV in 5 FDB fibres. Arrows indicate the times at which the external solution was exchanged with the same bath solution. B illustrates the time course of the sodium currents, recorded in a fibre from an old mouse. The 10 s interpulse interval was omitted. The dashed line represents the baseline.
Figure 3 shows the dose–response relationship and analysis of the dissociation constant for TTX in young and old mice. Peak currents in TTX (ITTX) are expressed relatively to the average of the currents in toxin-free solution (Io,TTX). Data points for fibres from young control mice were fitted to the following equation:
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where K1 is the dissociation constant for TTX (Pappone, 1980).
Data points were fitted to either eqn (2) or eqn (3) (Methods). B illustrates the time course of the sodium currents for an old-TTX-I fibre in control (before TTX, time zero) and after the exposure to TTX (top bar). The dashed line represents the baseline.
Sodium currents recorded in fibres from young mice (n = 26) exhibited a quick drop in the peak sodium current amplitude and were fully blocked with 1 μM TTX. The K1 value for these fibres was 5.6 ± 0.7 nM. Fibres from old mice showed a marked variability in the TTX concentration needed to block sodium currents (n = 29). Approximately half of the fibres from old mice were well fitted to eqn (2) (old-TTX-S) giving a K1 value of 6.2 ± 0.8 nM (n = 15). This group of fibres was called old-TTX-S. The difference between fibres from old-TTX-S and young mice was not statistically significant (P > 0.05). Data points from another group of fibres from old mice (n = 4) were better fitted to the following equation:
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where K2, as K1, is a dissociation constant for TTX (Pappone, 1980). Data points for this group were 5.6 ± 0.6 nM and 990 ± 256 nM for K1 and K2, respectively. This group was called old-TTX-R. An intermediate group was detected in between the previous two (n = 10). The values for the intermediate group were 6.5 ± 0.8 nM and 257 ± 98 nM, respectively. This intermediate population of fibres was called old-TTX-I. Differences in K2 but not in K1 values between the old-TTX-R and old-TTX-I were statistically significant (P < 0.05). The interpretation of these results is consistent with the expression of a population of TTX-R sodium channels (SkM2) (Yang et al. 1991) in old-TTX-I and old-TTX-R fibres and the ratio between TTX-resistant and -sensitive sodium channel is higher in the latter group. Figure 3B illustrates the time course of the sodium currents for a fibre belonging to the old-TTX-I group in which eight TTX concentrations were tested. It appears that a quick drop in current amplitude in response to 1–5 nM TTX is followed by a less pronounced effect. In summary, these results show that FDB fibres from old mice exhibit three patterns in response to TTX, one group that is similar to innervated fibres from young mice (52%) and another group that has two subpopulations, those of partially (35%) and fully (13%) denervated fibres.
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Expression of Nav1.5 in ageing FDB skeletal muscle
To investigate the expression of Nav1.5 we performed immunocytochemistry assays on cross-sections of FDB muscles. Figure 4 illustrates areas of muscle cross-sections from young (Fig. 4A) and old (Fig. 4B) mice. The sections of these FDB fibres from old mice depict two distinct patterns, either (1) intense fluorescence at their borders (horizontal arrow in Fig. 4B) or (2) uniform fluorescence throughout (vertical arrow). The former fibres were considered positive for TTX-R sodium channel expression, while the latter fibres were similar to the homogeneous pattern recorded in muscles from young mice (Fig. 4A), and were counted as negative. All the fibres from each muscle section were counted and the number of positive or negative cells was normalized to the total number of cells per section. For this analysis, three mice, six FDB muscles and 115–140 fibres per muscle were analysed for each group, young (1.6 ± 0.13% positive fibres for Nav 1.5 antibody) and old (48 ± 4.6%) mice (P < 0.01). Fibres per muscle were counted in at least five cross-sections and values were averaged. The ample space between fibres corresponds to the abundant connective tissue and tendons separating bundles of the short piece of the FDB muscle. Figure 4C and D illustrates muscle sections from young and old mice, respectively, in which the primary antibody was omitted. Figure 4E shows that muscles from senescent, compared with young mice, exhibited a larger number of fibres positive for the TTX-R Nav1.5 channel.
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Digitized images of FDB muscles from young (A) and old (B) mice. The sections from old mice (B) depict positive (horizontal arrow) or negative fibres (vertical arrow). C and D correspond to muscle sections from young and old mice, respectively, in which the primary antibody was omitted. E shows the normalized number of Nav1.5 positive fibres analysed for young and old mice. The asterisk indicates a statistically significant difference (P < 0.05).
Discussion
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The main conclusion of the present study is that FDB muscles from senescent mice exhibit three groups of fibres concerning innervation status. In addition, the percentage of fibres depicting some degree of TTX resistance (50%) was similar to that detected by immunocytochemistry using a specific antibody against the Nav1.5 channel.
Sodium current in skeletal muscle
Sodium current amplitude and density in FDB fibres from young mice, reported here, are similar to those recorded in mouse FDB muscle fibres (Reddy et al. 2002). An increase in sodium current density in ageing rats (Desaphy et al. 1998a,b), but not in mouse (present work), has been reported. Experimentally denervated EDL muscles from young rat exhibited an increase in maximal conductance at 22°C, but not at 12°C, when recorded with the double Vaseline gap technique (Pappone, 1980). A potential explanation for this discrepancy is that denervation affects muscle fibre subtypes differently. Rat FDB and extensor digitorum longus muscles are predominantly composed of type IIa (Carlsen et al. 1985) and type IIb (Brown et al. 1992) fibres, respectively, whereas mouse FDB muscles are mainly type IIx fibres (González et al. 2003).
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The sodium current–voltage relationship and sodium channel conductance have been reported to not change with maturation (Reddy et al. 2002); however, two different conductances of 18 pS and 9 pS have been reported in FDB muscles from old rat. Cells exhibiting a conductance of 18 pS were called ‘young-phenotype’, while the remaining group, was called ‘old-phenotype’ (Desaphy et al. 1998a). The similarity in the values recorded in fibres from old rats and myoblasts (Weiss & Horn, 1986) suggests that the small conductance channel corresponds to SkM2, whose expression increases in response to muscle denervation (Yang et al. 1991). However, two fibres exhibiting an ‘old phenotype’ were sensitive to a fixed TTX concentration in the cell-attached mode (Desaphy et al. 1998a). In that study, the small number of fibres tested together with a selection of those fibres exhibiting a better appearance, most likely decreased the number of denervated fibres detected.
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A shift in activation and inactivation parameters of sodium currents by 10 mV to more negative potentials has been reported in homogeneously denervated fibres (Pappone, 1980). By contrast, the population of fibres in FDB muscle from ageing mice (present work) is heterogeneously denervated. Approximately 52% of the fibres do not show evidence of denervation, and only 13% are fully denervated. Therefore, the comparison between these two preparations is not straightforward.
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Denervation in ageing skeletal muscle
Several studies have reported the phenomenon of skeletal muscle denervation and reinervation, as well as motor unit remodelling in ageing rodents or humans (Larsson, 1995; Lexell, 1995; for a review see Payne & Delbono, 2004). Due to the heterogeneity of the fibres from old mice reported here, it is reasonable to postulate that the fibres that exhibited greater resistance to TTX correspond to the positive cells labelled for neural cell adhesion molecule (NCAM) by immunostaining in old rats (Urbancheck et al. 2001). Using this technique the authors reported that less than 10% of rat extensor digitorum longus muscle fibres are NCAM positive. Underestimation of the number of denervated fibres with this technique is possible, based on the time-dependent decline in NCAM protein expression after denervation (Daniloff et al. 1986). In the present study FDB muscle denervation detected with Nav1.5 antibody is more extensive than that reported with NCAM antibody in EDL muscle because our probe is probably more sensitive to detecting partially denervated fibres (old-TTX-I), which represent approximately 35% of the fibres in FDB muscle from senescent mice. In addition, these differences of magnitude and extension of denervation may vary between muscle types and species. Differences with previous publications may reside on the use of a specific Nav1.5 channel antibody and the whole-cell configuration of patch-clamp to assess functionally a larger number of fibres for TTX sensitivity than in previous publications. Results included here concur with reports in human in which between 25 and 50% of loss in spinal cord motor neurone and motor units have been reported (for a review see Lexell, 1997).
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In summary, the experimental approach used here allowed for the identification of three populations of fibres in senescent mice, based on their sensitivity to TTX. Partially and fully denervated fibres, taken together, represent 50% of the total fibres, a number that concurs with the percentage of fibres positive for Nav1.5 channel by specific immunostaining. The extension and magnitude of muscle denervation reported here prompts a reassessment of the mechanisms proposed for the loss in muscle structure and function with ageing.
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