Progressive impairment of CaV1.1 function in the skeletal muscle of mice expressing a mutant type 1 Cu/Zn superoxide dismutase (G93A) linked to amyotrophic lateral sclerosis

Amyotrophic lateral sclerosis (ALS) is an adult-onset neurodegenerative disorder that is typically fatal within 3–5 years of diagnosis. While motoneuron death is the defining characteristic of ALS, the events that underlie its pathology are not restricted to the nervous system. In this regard, ALS muscle atrophies and weakens significantly before presentation of neurological symptoms. Since the skeletal muscle L-type Ca2+ channel (CaV1.1) is a key regulator of both mass and force, we investigated whether CaV1.1 function is impaired in the muscle of two distinct mouse models carrying an ALS-linked mutation. We recorded L-type currents, charge movements, and myoplasmic Ca2+ transients from dissociated flexor digitorum brevis (FDB) fibers to assess CaV1.1 function in two mouse models expressing a type 1 Cu/Zn superoxide dismutase mutant (SOD1G93A). In FDB fibers obtained from “symptomatic” global SOD1G93A mice, we observed a substantial reduction of SR Ca2+ release in response to depolarization relative to fibers harvested from age-matched control mice. L-type current and charge movement were both reduced by ~40 % in symptomatic SOD1G93A fibers when compared to control fibers. Ca2+ transients were not significantly reduced in similar experiments performed with FDB fibers obtained from “early-symptomatic” SOD1G93A mice, but L-type current and charge movement were decreased (~30 and ~20 %, respectively). Reductions in SR Ca2+ release (~35 %), L-type current (~20 %), and charge movement (~15 %) were also observed in fibers obtained from another model where SOD1G93A expression was restricted to skeletal muscle. We report reductions in EC coupling, L-type current density, and charge movement in FDB fibers obtained from symptomatic global SOD1G93A mice. Experiments performed with FDB fibers obtained from early-symptomatic SOD1G93A and skeletal muscle autonomous MLC/SOD1G93A mice support the idea that events occurring locally in the skeletal muscle contribute to the impairment of CaV1.1 function in ALS muscle independently of innervation status.


Background
Amyotrophic lateral sclerosis (ALS), known commonly as Lou Gehrig's disease in the USA or motoneurone disease in the UK, is an adult-onset neurodegenerative disorder that is usually lethal within 3 to 5 years of initial diagnosis [1,2]. As a consequence of disrupted nervemuscle communication, afflicted individuals progressively lose control of voluntary muscle function and die most frequently from respiratory failure. Nearly a quarter of familial ALS cases have been linked to point mutations in the type 1 Cu/Zn superoxide dismutase (SOD1) enzyme, which functions to mitigate cellular oxidative stress [3]. Since these mutations are autosomal dominant, transgenic mice overexpressing ALS-linked SOD1 mutations have proven to be useful models for investigation of ALS pathogenesis [4,5].
Two independent groups have found that skeletal muscle-targeted, transgenic overexpression of SOD1 G93A causes profound muscle atrophy [6,7] and initiates motoneuron death [7]. These congruent reports reinforce earlier challenges to the dogma that ALS is solely a neurological disorder and support the "dying-back" phenomenon by which motor unit loss and associated muscle function precede the death of motor neurons (reviewed in refs. [8][9][10][11]). Additional support for this view is provided by the observations that destruction of neuromuscular junctions has been linked to oxidative stress induced by tissue-specific breakdown of muscle mitochondria [12] and trophic factors secreted from the skeletal muscle promote motoneuron survival in an ALS model by stabilizing neuromuscular junctions (e.g., IGF-1, GDNF) [13][14][15].
The question of whether Ca V 1.1 function is disrupted during the progression of ALS was originally posed over 20 years ago [26][27][28], but the experimental approach used to investigate this premise left much ambiguity. In these earlier studies, serum of ALS patients was acutely applied to normal cut wild-type rat extensor digitorum longus (EDL) fibers and the Vaseline-gap voltage-clamp technique was used to record L-type currents and gating charge movements [26,27]; L-type currents and intramembrane gating charge movements were both found to be reduced. Similarly, acute IgG application also produced inhibitory effects on single Ca V 1.1 channels in planar lipid bilayers [28]. In the time that has passed since the experiments that employed acute application of ALS IgG were performed, it has become evident that that muscle dysfunction in ALS manifests early and slowly over time. Fortunately, a number of ALS mouse models have been developed since then, including SOD1 G93A transgenic lines [4,[29][30][31].
Here, we provide the first comprehensive voltage-clamp assessment of Ca V 1.1 function in the muscle of an engineered mammalian model of ALS. Specifically, we utilized both global SOD1 G93A mice and mice expressing SOD1 G93A only in skeletal muscle to systematically investigate whether Ca V 1.1 function is compromised during the progression of the disease. We found substantial reductions in myoplasmic Ca 2+ transient amplitude, peak L-type current density, and maximal intramembrane charge movement in flexor digitorum brevis (FDB) fibers obtained from "symptomatic" congenic SOD1 G93A mice. Consistent with a progressive muscle defect, we observed lesser reductions in L-type current amplitude and charge movement in FDB fibers obtained from "early-symptomatic" SOD1 G93A mice in which widespread denervation had yet to occur. To investigate the possibility that events occurring in muscle contribute, at least in part, to the impairment of Ca V 1.1 function, we performed voltage-clamp experiments with FDB fibers of mice expressing SOD1 G93A autonomously in skeletal muscle. In these experiments, we observed significant reductions in EC coupling, charge movement, and peak L-type current amplitude.

Ethical information/animals
All procedures involving mice were approved by the University of Colorado-Anschutz Medical Campus Institutional Animal Care and Use Committee (91813(05)1D). Male congenic SOD1 G93A mice (B6.Cg-Tg(SOD1*G93A)1Gur/J; 32 transgene copies) and age-matched male C57BL/6J mice (both Jackson Laboratories, Bar Harbor, ME) were used at two time points based on the criteria for disease progression described by Hatzipetros et al. [31]: (1) "symptomatic" and (2) "early-symptomatic." Nine symptomatic mice presented classical ALS symptoms (i.e., tremor, abnormal gait, comprised ability to stand on hindquarters, etc.) when they were sacrificed at 149 ± 1 days. Six mice from the early-symptomatic cohort were largely devoid of overt symptoms when they were sacrificed at 105 ± 1 days. Age-matched C57BL/6J mice were used as background wild-type controls for both symptomatic and early-symptomatic global SOD1 G93A mice (eight and seven mice, respectively). Seven MLC/SOD1 G93A mice were sacrificed at (111 ± 1 days) [6]. In this case, six age-matched FVB/NJ mice were used as the background wild-type control strain. The dissimilar backgrounds precluded direct comparisons between global SOD1 G93A and MLC/SOD1 G93A strains.

Assessment of SR Ca 2+ store content
Freshly dissociated FDB fibers were washed with Ca 2 + /Mg 2+ -free Ringer's solution (in mM: 146 NaCl, 5 KCl, 10 HEPES, and 11 glucose, pH 7.4, with NaOH) twice and loaded with 5 μM Fluo 3-AM (Invitrogen) dissolved in Rodent Ringer's solution for 20-30 min. Fibers were then washed three times in Rodent Ringer's solution with gentle agitation. Fluo 3-AM-loaded fibers bathed in Rodent Ringer's solution were then placed on the stage of an LSM META scanning laser confocal microscope (Carl Zeiss, Inc., Jena, Germany) and viewed under ×10 magnification. N-benzyl-p-toluenesulfonamide (BTS; 100 μM; Sigma-Aldrich) was always present in the bath solution (∼25°C) to prevent contractions. The Fluo-3 dye was excited with the 488 nm line of an argon laser (30 mW maximum output, operated at 50 % or 6.3 A, attenuated to 1-2 %). The emitted fluorescence was directed to a photomultiplier equipped with a 505-nm long-pass filter. Confocal fluorescence intensity data were digitized at 8 bits, with the photomultiplier gain and offset adjusted such that maximum pixel intensities were no more than ∼70 % saturated and cell-free areas had close to zero intensity. Application of 4-chloro-m-cresol (4-CmC; Pfaltz and Bauer, Waterbury, CT) via a manually operated, gravity-driven global perfusion system was used to deplete the SR of Ca 2+ . Fluorescence amplitude data are expressed as ΔF/F, where F represents the baseline fluorescence before the application of 4-CmC, and ΔF represents the change in fluorescence during the application of 4-CmC.
Electrophysiology and whole-cell recordings of myoplasmic Ca 2+ transients Borosilicate patch pipettes had resistances of ≤1.0 MΩ when filled with internal solution, which consisted of (mM): 140 Cs-aspartate, 10 Cs 2 -EGTA, 5 MgCl 2 , and 10 HEPES, pH 7.4 with CsOH. FDB fibers that were selected for voltage-clamp experiments were glossy, had rounded ends, and cross-striations. Fibers meeting our visual criteria were dialyzed in the whole-cell configuration for >20 min prior to recording. To record changes in intracellular Ca 2+ from FDB fibers, the pentapotassium salt form of Fluo 3 single wavelength Ca 2+ indicator dye (Invitrogen) was added to the standard internal solution (see above) for a final concentration of 200 μM. The external solution contained (mM): 145 TEAmethanesulfonic acid, 10 CaCl 2 , 10 HEPES, 2 MgCl 2 , 1 4-aminopyridine, 0.1 anthracene-9-carboxylic acid, 0.002 tetrodotoxin, pH 7.4 with TEA-OH. Ten micromolar BTS was added to the standard external solution for all whole-cell experiments. Following the establishment of the whole-cell configuration, the dye was allowed to diffuse into the cell interior for no less than 20 min. A 100 W mercury illuminator and a set of fluorescein filters were used to excite the dye present in the voltageclamped fiber. A computer-controlled shutter was used to block illumination in the intervals between test potentials. Fluorescence emission was measured by means of a fluorometer apparatus (Biomedical Instrumentation Group, University of Pennsylvania). Fluorescence data are expressed as ΔF/F, where ΔF represents the change in peak fluorescence from baseline during the test pulse and F is the fluorescence immediately prior to the test pulse minus the average background fluorescence. The peak value of the fluorescence change (ΔF/F) for each test potential (V) was fitted according to: where (ΔF/F) max is the maximal fluorescence change, V F is the potential causing half the maximal change in fluorescence, and k F is a slope parameter. All electrophysiological data were acquired with an Axon 200B patch-clamp amplifier and a CV 203BU headstage (both Molecular Devices, Sunnyvale, CA). L-type Ca 2+ currents were recorded with the same external solution used to record myoplasmic Ca 2+ transients described above. A solution containing (in mM): 145 TEA-methanesulfonic acid, 10 CaCl 2 , 10 HEPES, 2 MgSO 4 , 1 4-aminopyridine, 0.1 anthracene-9-carboxylic acid, 0.002 tetrodotoxin, 1 LaCl 3 , 0.5 CdCl 2 , pH 7.4 with TEA-OH were used to record intramembrane gating charge movements. Linear components of leak and capacitive current were corrected with −P/4 online subtraction protocols. Output filtering was at 2-5 kHz and digitization was either at 5 kHz (currents) or 10 kHz (charge movements). Cell capacitance (C m ) was determined by integration of a transient from −80 to −70 mV using Clampex 10.3 (Molecular Devices) and was used to normalize charge movement (nC/μF) and current amplitude (pA/pF). The average value of C m for all fibers used in the study was 2.4 ± 0.1 nF (n = 160 fibers; please see Additional file 1: Figure S1 for C m data for each experimental group). To minimize voltage error, the time constant for the decay of the whole-cell capacity transient was reduced as much as possible using the analog compensation circuit of the amplifier; the average values of τ m and R a were 1.0 ± 0.0 ms and 456 ± 21 kΩ, respectively. Current-voltage (I-V) curves were fitted according to: where I is the normalized current for the test potential V, V rev is the reversal potential, G max is the maximum channel conductance, V 1/2 is the half-maximal activation potential and k G is the slope factor. For charge movements, the initial non-linear outward transient "Q ON " was normalized to C m and plotted as a function of test potential (V) and the resultant Q ON − V relationships were fitted according to: where Q max is the maximal Q ON , V Q is the potential causing movement of half the maximal charge, and k Q is a slope parameter. All electrophysiological and Ca 2+ imaging experiments were performed at room temperature (~25°C).

Analysis
All data are presented as mean ± SEM. Statistical comparisons were made by two-tailed, unpaired t test with P < 0.05 considered significant. Figures were made using the software program SigmaPlot (version 11.0, SSPS Inc., San Jose, CA).

Results and discussion
EC coupling is impaired in symptomatic SOD1 G93A FDB fibers Like humans afflicted with ALS, mice engineered to carry ALS-linked mutations in SOD1 display substantial muscle atrophy and decreased specific force [30,32]. Declines in specific force that resemble that occurring in ALS [30] have previously been attributed to reduced membrane expression of Ca V 1.1 [33][34][35] and experimental knockdown of Ca V 1.1 expression causes marked atrophy [36]. Taken together, these observations raise the question of whether the primary function of Ca V 1.1-voltage-sensor for EC coupling-is impaired in skeletal muscle of a mutant SOD1 ALS model. To address this question, we dialyzed flexor digitorum brevis (FDB) fibers isolated from congenic SOD1 G93A mice and age-matched C57BL/6J wild-type control mice with cell-impermeant Fluo 3 Ca 2+ indicator dye and recorded depolarization-induced myoplasmic Ca 2+ transients in the whole-cell configuration (cf. ref. [37]). As shown in Fig. 1a, we observed robust SR Ca 2+ release in response to step depolarizations in Fluo 3-loaded wildtype control fibers (ΔF/F = 2.3 ± 0.4, n = 6). By contrast, depolarization-induced SR Ca 2+ release was substantially decreased in symptomatic SOD1 G93A fibers (ΔF/F = 1.0 ± 0.1, n = 9; P < 0.01; Fig. 1b, c; Table 1); no change in the voltage-dependence of transient activation was apparent (Table 1).
We also measured protein levels of Ca V 1.1 in the tibialis anterior, gastrocnemius, and FDB muscles via western blot. As shown in Fig. 4a-top, we observed a clear reduction in Ca V 1.1 levels in tibialis anterior preparations (band intensity relative to α-actin = 0.40 ± 0.03 for wild-type and 0.16 ± 0.02 for symptomatic; P < 0.001; both N = 4; Fig. 4a-top panel) that reflected our electrophysiological measurements (Fig. 3). For gastrocnemius, total Ca V 1.1 expression was only marginally insignificant (relative band intensity = 0.32 ± 0.04 and 0.20 ± 0.04 for wild-type and symptomatic, respectively; P = 0.074; N = 4:  Data are given as mean ± SEM, with the numbers in parentheses indicating the number of fibers tested. Data were fit by Eq. 1. The triple thin lines separate three distinct experimental groups: (1) symptomatic, (2) earlysymptomatic, and (3) MLC/SOD1 G93A . Significant differences within each group (i.e., between a group of SOD1 G93A -expressing fibers and the corresponding age-matched background control fibers) are indicated (* denotes P < 0.05; unpaired t test Fig. 4b-top panel). A reduction in Ca V 1.1 plasma membrane expression in FDB fibers was not detectable by western blot (relative band intensity = 0.54 ± 0.09 and 0.62 ± 0.12 for wild-type and symptomatic, respectively; Fig. 4c-top panel). It is important to note here that quantification of these biochemical data was extremely difficult because the expression level of all reference gene products used in our experiments also changed. α-actin was found to be the most stable marker, and we used it to calculate changes in Ca V 1.1 band intensity (Fig. 4a-c-middle panels). Still, α-actin is not an ideal reference as it does fluctuate somewhat between wild-type and symptomatic SOD1 G93A muscle. The levels of histone 3 were greatly and evenly increased in the symptomatic SOD1 G93A tibilias anterior and gastrocnemius muscles (Fig. 4a, b-bottom panels) and unchanged in FDB (Fig. 4c-bottom panel) preparations. These experiments further supported the idea that any observed downregulation of Ca V 1.1 was not a consequence of uneven gel loading. Likewise, Coomassie staining of the same samples used in western blots was similar between wild-type and SOD1 G93A muscle (Additional file 2: Figure S2A-C).
Impaired Ca V 1.1 function in early-symptomatic SOD1 G93A
In Fig. 7, we summarize our results with symptomatic and early-symptomatic SOD1 G93A FDB fibers. There, EC coupling (ΔF/F), L-type channel current amplitude (peak I L ) and intramembrane charge movement (Q max ) are normalized by the corresponding age-matched wild-type control value for each parameter. For symptomatic SOD1 G93A fibers, the reductions in EC coupling, channel function, and charge movement are quite evident (Fig. 7-black bars). Moreover, these impairments of Ca V 1.1 function were already developing in early-symptomatic Fig. 2 SR Ca 2+ store content is similar in wild-type and symptomatic SOD1 G93A FDB fibers. Representative records of 4-CmC (1 mM)-induced SR Ca 2+ release in dissociated FDB fibers harvested from a wild-type mouse (a) and from a symptomatic SOD1 G93A mouse (b). Insets show images of Fluo 3-loaded fibers before 4-CmC application (left) and at the peak of fluorescence (right). c Data summary SOD1 G93A mice before substantial denervation had occurred ( Fig. 7-white bars), suggesting that muscle-specific events are contributing to SOD1 G93A -dependent alterations in Ca V 1.1 function.
EC coupling, L-type currents, and charge movement are reduced in FDB fibers expressing SOD1 G93A autonomously in the skeletal muscle Our experiments with early-symptomatic SOD1 G93A mice raise the possibility that the reduction of Ca V 1.1 expression precedes overt neurological symptoms of ALS. Nonetheless, a degree of uncertainty exists concerning the contribution of denervation. On a more stringent level, the innervation profiles of the individual fibers used in our patch-clamp experiments were virtually impossible to assess accurately. To determine nerve-independent effects, we utilized a mouse model where transgenic expression of SOD1 G93A is limited to skeletal muscle (MLC/SOD1 G93A ) [6]. This mouse line enabled us to examine whether events occurring locally in skeletal muscle can cause impaired Ca V 1.1 function because substantial neuromuscular junction defects in such models do not develop until at least 8 months of age ( [6,7]; A.M., unpublished results).

Discussion
In this study, we found that FDB fibers harvested from symptomatic global SOD1 G93A mice had substantially reduced EC coupling relative to fibers of age-matched wild-type mice (Fig. 1). The impairment of EC coupling was not a consequence of a depleted SR Ca 2+ store (Fig. 2), but appeared to be the result of reduced L-type Ca 2+ channel membrane expression (though we cannot discount the presence of electrically silent channels). In particular, L-type Ca 2+ current (Fig. 3a-c) and intramembrane charge movement (Fig. 3d-f) were both decreased Fig. 4 Western blot analysis of Ca V 1.1 protein levels in symptomatic SOD1 G93A skeletal muscle. a western blots of lysates obtained from four different tibialis anterior (b) gastrocnemius muscles of age-matched wild-type and symptomatic SOD1 G93A mice are shown (N = 4 for both groups). In each case, the blots were probed with a monoclonal antibody directed to Ca V 1.1 (top panels), then stripped and reprobed sequentially for α-actin (middle panels) and then histone H3 (bottom panels). A protein ladder standard resides in lane 1 of each blot; molecular weights are indicated in kDa. c A western blot of lysates obtained from FDB muscles of age-matched wild-type and symptomatic SOD1 G93A mice are shown (N = 2 for both groups). The blot was first probed with a monoclonal antibody directed to Ca V 1.1 (top panel) and then stripped and reprobed sequentially with antibodies directed to α-actin (middle panel) and then histone H3 (bottom panel). For each blot, changes in expression were determined by comparing the intensity of each Ca V 1.1 band in the top panel to the corresponding actin band the middle panel. For each panel, similar results were observed in at least three experiments  Table 2 by~40 % in symptomatic SOD1 G93A FDB fibers. The observed reductions in Ca V 1.1 function in symptomatic SOD1 G93A fibers resembled the effect of application of ALS patient serum to normal rat extensor digitorum longus muscle [26,27], but our results imply that these impairments develop more slowly over time.
Our data indicating that EC coupling and L-type current were compromised in symptomatic SOD1 G93A muscle fibers contrast with those of another group who found that depolarization-dependent Ca 2+ transients were slightly augmented in SOD1 G93A mice (B6SJL-Tg(SOD1*G93A)1Gur/J) as a consequence of insufficient local mitochondrial Ca 2+ buffering near the neuromuscular junction [40,41]. Our findings also differ with those of another study which also examined Ca 2+ handling in FDB fibers obtained from symptomatic SOD1 G93A mice (B6.Cg-Tg(SOD1*G93A)1Gur/J). In this latter study [42], the authors observed virtually no difference in the amplitude of Ca 2+ transients evoked by maximal field stimulation frequencies (100 Hz) using ratiometric Ca 2+ -sensitive dyes. It was concluded that the ability of Ca V 1.1 to engage EC coupling was normal in symptomatic SOD1 G93A superficial gastrocnemius muscle because western blots showed no change in Ca V 1.1 expression [42]. By contrast, our western blot analysis of whole-cell lysates obtained from symptomatic SOD1 G93A tibialis anterior muscles indicated a clear reduction in total Ca V 1.1 expression (Fig. 4a). We did not observe significant differences for either gastrocnemius or FDB (Fig. 4b, c), though the band intensity difference in gastrocnemius was marginal (P = 0.074). The ambiguous nature of our biochemical results underscores the critical importance of our voltage-clamp approach to determine the relative number of functional Ca V 1.1 channels in the plasma membrane.
One reasonable explanation for the observed decreases in Ca 2+ transient amplitude, peak L-type current density, and maximal charge movement is that SOD1 G93A -expressing muscle undergoes fast-twitch (type IIb or IIx) to slow or intermediate (type I or type Ia, respectively) fiber-type switching [6,30]. In an earlier study, slow-twitch rat soleus fibers were found to support voltage-dependent SR Ca 2+ release only about half as well as fast-twitch fibers dissected from EDL of the same animals [43,44]. Likewise, radioactive 1,4-dihydropyridine binding of the soleus was reduced~50 % compared to that of EDL in the same study. In view of this difference in Ca V 1.1 expression between general fiber types, it is possible that we recorded from a population more heavily weighted in type I or type IIa fibers and that our data reflect fiber-type switching. Nevertheless, the dramatic alteration in the expression profile of Table 2 Conductance and charge movement fit parameters Data are given as mean ± SEM, with the numbers in parentheses indicating the number of FDB fibers tested. Conductance and charge movement data were fit by Eqs. 2 and 3, respectively. As in Table 1, the triple thin lines separate three distinct experimental groups: (1) symptomatic, (2) early-symptomatic, and (3) MLC/ SOD1 G93A . Again, significant differences within each group are indicated (* denotes P < 0.05; *** denotes P < 0.001; unpaired t test) Ca V 1.1 that accompanies fiber-type switching appears to be a downstream consequence of mutant SOD1 expression. Future experiments with more complex models that enable the distinction amongst fiber types in live cells are required to investigate this idea further.
Importantly, we also found that the impairment of Ca V 1.1 function in SOD1 G93A mice began to develop prior the presentation of classical ALS symptoms.
Though the reduction in depolarization-induced Ca 2+ transients were not significantly reduced in earlysymptomatic SOD1 G93A muscle fibers (Fig. 5), decreased L-type current amplitude and intramembrane charge movement were clearly evident (Fig. 6). A number of explanations could account for this apparently discordant result. For example, the early-symptomatic SOD1 G93A mice may have had a dissimilar RyR (type 1 or type 3) complement than the symptomatic mice or there have been a compensatory reversion to a juvenile RyR1 splice variant (ASI+) that supports greater SR Ca 2+ release in response to depolarization [45,46]. In any event, the results of our experiments with early-symptomatic SOD1 G93A fibers suggested that the impairment of Ca V 1.1 function manifested in symptomatic SOD1 G93A fibers arises, at least in part, from signals that originate in muscle (Figs. 5, 6, and 7). To test this idea, we assessed Ca V 1.1 function in fibers obtained from 4-month-old MLC/SOD1 G93A mice in which expression of the mutant SOD1 protein is restricted to skeletal muscle. In these experiments, we observed~35,~20, and~15 % reductions in Ca 2+ transient amplitude, peak L-type current density, and maximal charge movement, respectively (Figs. 8 and 9). The reductions in L-type current amplitude and charge movement resembled the reductions in the parameters observed in early-symptomatic global SOD1 G93A fibers. Interestingly, the impact of muscle-specific expression of SOD1 G93A on EC coupling was greater than that observed in early-symptomatic global fibers. Previous ultrastructural analysis of the MLC/SOD1 G93A strain suggests that concurrent uncoupling of triad junctions with transverse tubules may amplify the EC coupling impairment [6]. Together, the ultrastructural and electrophysiological changes induced by muscle-specific expression of SOD1 G93A can reasonably account for marked reductions in specific force observed in both fast-and slow-twitch hindlimb muscles of MLC/SOD1 G93A mice [6]. Thus, the  Fig. 7 Summary of results obtained with global SOD1 G93A fibers. EC coupling (ΔF/F), L-type Ca 2+ current (peak I L ) , and maximal charge movement (Q max ) for early-symptomatic (105 ± 1 d; N = 6; white bars) and symptomatic global SOD1 G93A (149 ± 1 d; N = 9; black bars) fibers are represented as a percentage of corresponding values for the respective age-matched wild-type fiber groups results of our experiments with MLC/SOD1 G93A fibers support the idea that muscle-specific events also make a significant contribution to ALS pathology, similar to what has been observed previously with myotonic dystrophy [47] and Huntington's disease [48].
We did not observe a decrease in total membrane capacitance in SOD1 G93A -expressing fibers, indicating that there was not substantial atrophy and/or loss of tubular surface area in the fibers that we examined electrophysiologically (Additional file 1: Figure S1). Based on the indisputable fact that atrophy is characteristic of both global and muscle-targeted SOD1 G93A muscles, we think that our data are representative of a subpopulation of fibers healthy enough to survive the dissociation process and to meet the set visual criteria for voltage-clamp experiments (see Methods). If this is indeed the case, the reductions in voltage-dependent SR Ca 2+ release, charge movement, and L-type current in the entire population of SOD1 G93A fibers are likely more severe (and earlier onset) than we report.
Until recently, the involvement of Ca V 1.1 in the maintenance of muscle mass and composition was impossible to investigate because mice null for Ca V 1.1 (dysgenic) die immediately following parturition [49]. To circumvent the perinatal lethality of Ca V 1.1 deletion, Piétri-Rouxel and colleagues delivered anti-Ca V 1.1 siRNA to the mouse hindlimb muscle with a serotype 1 Adeno-Associated Virus (AAV1) vector [36]. Using this approach, they demonstrated that targeted, long-term suppression of L-type channel expression via induced exon-skipping produced prominent atrophy and extensive fibrosis. This atrophic effect of experimental downregulation of Ca V 1.1 expression suggested that pathological decreases in L-type channel activity, such as those we have observed in the two SOD1 G93A models utilized in this study, may also lead to muscle degeneration and, possibly, contribute to the destabilization of neuromuscular junctions early in the progression of ALS. Our current work provides initial support for this idea, but further work is needed to reveal the  Table 2 precise mechanism. The first step will be to determine whether the downregulation of the channel and/or its auxiliary subunits occurs at the message level or posttranslationally. For the former, quantification of Ca V 1.1 subunit message levels by qRT-PCR should be revealing. In regard to the latter, the chronic upregulation of expression the RGK family small GTP binding protein Rad (Ras-like Associated with Diabetes)a potent constitutively active inhibitor of Ca V 1.1 [50,51]-has been demonstrated in muscle of sporadic ALS patients of unknown aetiology and two established familial ALS mouse models (SOD1 G93A and SOD1 G86R ) [52,53]. The increases in muscle Rad expression were attributed to elevated cellular oxidative stress levels and coincided with the onset of spinal motoneuron death [53]. Without being overspeculative, these observations raise the possibility that such chronic enhancement of muscle Rad expression may contribute to both atrophy and the dissolution of neuromuscular junctions in human ALS [53].

Conclusions
We report that myoplasmic Ca 2+ transient amplitude, Ltype current density, and intramembrane charge movement are progressively downregulated in flexor digitorum brevis (FDB) fibers obtained from SOD1 G93A mice, the most extensively utilized mouse model of familial ALS. We also observed impairments of EC coupling, L-type current density, and charge movement in fibers of mice expressing SOD1 G93A only in the skeletal muscle, signaling a musclespecific contribution to SOD1 G93A -induced downregulation of Ca V 1.1 expression and/or function. Since Ca V 1.1 is a positive regulator of muscle mass, our results suggest that altered Ca V 1.1 activity is a contributor to the muscle remodeling that occurs in ALS patients prior to the presentation of overt neurological symptoms.