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Altered sodium channel-protein associations in critical illness myopathy

Altered sodium channel-protein associations in critical illness myopathy Background: During the acute phase of critical illness myopathy (CIM) there is inexcitability of skeletal muscle. In a rat model of CIM, muscle inexcitability is due to inactivation of sodium channels. A major contributor to this sodium channel inactivation is a hyperpolarized shift in the voltage dependence of sodium channel inactivation. The goal of the current study was to find a biochemical correlate of the hyperpolarized shift in sodium channel inactivation. Methods: The rat model of CIM was generated by cutting the sciatic nerve and subsequent injections of dexamethasone for 7 days. Skeletal muscle membranes were prepared from gastrocnemius muscles, and purification and biochemical analyses carried out. Immunoprecipitations were performed with a pan-sodium channel antibody, and the resulting complexes probed in Western blots with various antibodies. Results: We carried out analyses of sodium channel glycosylation, phosphorylation, and association with other proteins. Although there was some loss of channel glycosylation in the disease, as assessed by size analysis of glycosylated and de-glycosylated protein in control and CIM samples, previous work by other investigators suggest that such loss would most likely shift channel inactivation gating in a depolarizing direction; thus such loss was viewed as compensatory rather than causative of the disease. A phosphorylation site at serine 487 was identified on the Na 1.4 sodium channel α subunit, but there was no clear evidence of altered phosphorylation in the disease. Co-immunoprecipitation experiments carried out with a pan-sodium channel antibody confirmed that the sodium channel was associated with proteins of the dystrophin associated protein complex (DAPC). This complex differed between control and CIM samples. Syntrophin, dystrophin, and plectin associated strongly with sodium channels in both control and disease conditions, while β-dystroglycan and neuronal nitric oxide synthase (nNOS) associated strongly with the sodium channel only in CIM. Recording of action potentials revealed that denervated muscle in mice lacking nNOS was more excitable than control denervated muscle. Conclusion: Taken together, these data suggest that the conformation/protein association of the sodium channel complex differs in control and critical illness myopathy muscle membranes; and suggest that nitric oxide signaling plays a role in development of muscle inexcitability. Keywords: Skeletal muscle, Na 1.4 sodium channel, Na 1.5 sodium channel, Nitric oxide (NO), Neuronal nitric V V oxide synthase (nNOS), Glycosylation, Phosphorylation, Action potential, Excitability, Denervation * Correspondence: mark.rich@wright.edu Department of Neuroscience, Cell Biology, and Physiology, Wright State University School of Medicine, 3640 Colonel Glenn Hwy, Dayton, OH 45435, USA Full list of author information is available at the end of the article © 2012 Kraner et al.; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Kraner et al. Skeletal Muscle 2012, 2:17 Page 2 of 13 http://www.skeletalmusclejournal.com/content/2/1/17 Background Preparation of muscle membranes and classical Critical illness myopathy (CIM) is the most common purification of sodium channels cause of severe weakness in patients in the intensive Membranes were prepared from the gastrocnemius mus- care unit [1,2]. One of the hallmarks of the acute phase cles as previously reported [10]. Samples were thawed of CIM is paralysis due to loss of muscle’s electrical on ice for 10 min and minced finely on a glass plate on excitability [3,4]. To determine the mechanism under- ice. Samples were transferred to tubes containing lying loss of excitability, we utilize a rat model of CIM in sucrose buffer with protease and phosphatase inhibitors, which corticosteroid treatment is combined with denerv- using 10 volumes of buffer per gram of tissue. The ation to mimic treatment of critically-ill patients with sucrose buffer contained 0.3 M sucrose, 75 mM NaCl, corticosteroids and neuromuscular blocking agents [5,6]. 10 mM EGTA, 10 mM EDTA, 10 mM Tris, pH 7.4, In the rat model, inactivation of sodium channels is the 1 mM phenylmethylsulfonyl fluoride (PMSF), 1 mM cause of inexcitability [7,8]. A major contributor to the iodoacetamide, 0.1 μg/mL pepstatin A, 10 μM leupeptin, increase in inactivation is a hyperpolarized shift in 2.5 μM ALLN, and 1 mM sodium fluoride. Samples the voltage dependence of inactivation of the adult were homogenized using a PowerGen 700 tissue homo- (Na 1.4) and embryonic (Na 1.5) skeletal muscle so- genizer at a setting of 6 for 30 s. The samples were cen- V V dium channel isoforms expressed in CIM muscle [9]. As trifuged in a SS34 rotor at 4,500 g for 15 min. The the rat model of CIM occurs in genetically normal rats, supernatants were removed and centrifuged again. The this hyperpolarized shift in sodium channel gating must final supernatants were centrifuged in a SW55 rotor at be due to post-translational modification of sodium 138,000 g for 1 h. The pellets from this last step were channels or an alteration in sodium channel association resuspended in fresh sucrose buffers with inhibitors and with other proteins that modify gating. used as the membrane fraction. The goal of this study was to identify biochemical For classical purification of sodium channels, mem- changes that could potentially underlie the hyperpolar- branes were solubilized using NP40 and the sodium chan- ized shift in the voltage dependence of sodium channel nel fraction sequentially purified on DEAE-Sepharose and inactivation in CIM. We found changes in several candi- wheat germ agglutinin-Sepharose columns as described dates, although at least one change appeared compensa- [11]. For all membrane preparation and channel purifica- tory rather than causative. We identified an increase in tion procedures, samples, equipment, and buffers were the amount of neuronal nitric oxide synthase (nNOS) kept at 0-4°C. associated with the sodium channel in CIM as a change that might underlie the hyperpolarized shift in the volt- SDS-PAGE, western blot analysis, and antibodies age dependence of inactivation. Membrane fractions were assayed for protein content using the Lowry protein assay, and equal amounts of protein were loaded for control and CIM samples for Methods each protein assessed (approximately 40 μg protein Preparation of control and critical illness myopathy per lane). For deglycosylation experiments and co- muscle samples immunoprecipitation experiments, the same amount of All animal protocols were approved by the Institutional membrane protein was used to initiate the experiment; Animal Care and Use Committee at Wright State Uni- and equal volumes of the final product were loaded for versity. Critical illness myopathy was induced by a com- each control and CIM sample analyzed. SDS-PAGE bination of denervation and dexamethasone treatment. was performed on Criterion 4% to 20% acrylamide gels Briefly, rat muscle was denervated by removing a 10-mm for most samples; but 5% gels were used for the degly- segment of the sciatic nerve in isoflurane-anesthetized cosylation experiments, and 10% to 14.5% gels were adult female Wistar rats (250 to 350 g body weight). used for analysis of syntrophin and β-dystroglycan co- Buprenorphine was administered subcutaneously for immunoprecipitations to better resolve the molecular -1 postoperative analgesia. Dexamethasone (4 mg kg ) and weight region being analyzed. Western blot transfers antibiotics (0.2 mL of 2.27% Baytril, Bayer, Shawnee were carried out in a Criterion transfer apparatus Mission, KS, USA) were administered intraperitoneally using nitrocellulose as the blotting medium. Following beginning on the day of denervation and continuing for transfer, blots were washed with PBS/0.1% Tween, 7 days. Rats were killed by carbon dioxide inhalation, blocked in 1% I-Block (Tropix) in PBS/0.1% Tween for and the tibialis anterior, soleus, and gastrocnemius 1 h; incubated with first antibody for 2 h; washed and muscles removed, weighed, and snap-frozen in liquid ni- incubated with mouse or rabbit secondary antibody trogen. For control muscles, non-treated rats from the conjugated to alkaline phosphatase (Tropix); and visua- same group were used and muscles harvested in the lized using CDP-Star (Tropix) and a Fujifilm LAS-3000 same way. All muscles were stored at −80°C until use. close-caption device camera. Kraner et al. Skeletal Muscle 2012, 2:17 Page 3 of 13 http://www.skeletalmusclejournal.com/content/2/1/17 A pan-sodium channel antibody (pan Na 1.x) was 37°C for 1 h. Samples were quenched by addition of gel raised against the highly conserved peptide, TEEQKKYY- sample buffer. For neuraminidase (New England Biolabs) NAMKKLGSKK, corresponding to SP19 [12] at Open treatments, 10 μg of control or CIM membranes were Biosystems, ThermoFisher Scientific. The affinity-purified adjusted to 100 μL with 10 μL of 500 mM sodium citrate antibody selectively reacted to sodium channel in skeletal (pH 6.0), 10 μL of 10% NP40, 5 μL neuraminidase muscle and other tissues and could be displaced by the (250 units), and water. Samples were heated at 37°C for peptide antigen in western blot analysis. The antibody 4 h. As a control, 10 μg of fetuin was treated in the same to FGF13 was provided by Dr Geoffrey Pitts (Duke way. Samples were quenched by addition of gel sample University). The Na 1.4-specific monoclonal antibody buffer. For all samples, mock treatments without enzyme was provided by Dr Robert L Barchi (Thomas Jefferson were also carried out. Samples were analyzed by western Medical School), and can be obtained commercially from blot with Na 1.4-specific antibody. Sigma (S9568). Other antibodies were obtained from the following commercial sources: FGF12 (Abgent AP6750b), Analysis of phosphorylation by tandem mass spec plectin (Epitomics 1399–1), dystrophin (Vector Labs VP- The mass spectrometric analysis was performed on an D508), Na 1.5 (Alomone ASC-005), nNOS (Invitrogen optimized proteomics platform as previously reported 37–2800), β-dystroglycan (Abcam ab 49515), and syntro- [13]. Briefly, protein gel bands were washed and sub- phin (Abcam ab11425). jected to standard in-gel trypsin digestion. Digested pep- Some gels were stained rather than processed for west- tides were analyzed by capillary reverse-phase liquid ern blots. For analysis of phosphoproteins, Peppermint chromatography coupled with tandem mass spectrom- Stick Phosphoprotein Molecular Weight Standards (Invi- etry (LC-MS/MS), in which the eluted peptides were trogen), which include both phosphoprotein and non- analyzed by an MS survey scan followed by 10 data- phosphoprotein standards, were included on the gels as dependent MS/MS scans on an LTQ-Orbitrap ion trap controls. After electrophoresis, gels were fixed in 50% mass spectrometer (Thermo Scientific). Acquired MS/ methanol and 10% acetic acid for 30 min, transferred to MS spectra were then searched against the rat reference the Pro-Q Diamond Phosphoprotein Stain (Invitrogen) database of the National Center for Biotechnology In- for 90 min, then transferred to Destain (Invitrogen) for formation with differential modifications on serine/ 30 min. The gel was de-stained twice more for 30 min; threonine/tyrosine (+79.9663 Da); then filtered by then washed twice in ultrapure water. The gel was matching scores and mass accuracy to reduce protein imaged on a flatbed fluorescent scanner (Fujifilm FLA- false discovery rate to less than 1% using a concatenated 5100). After the phosphoprotein stain, the gel was im- reversed database [14]. Furthermore, we manually vali- mediately processed for all-protein stain with SYPRO dated MS/MS spectra of matched phosphopeptides using Ruby Protein Stain (Invitrogen) overnight. The gel was the following criteria: (1) the presence of signature phos- de-stained with 10% MeOH and 7% acetic acid for phate neutral losses (−49 for doubly charged or −32.7 for 45 min, washed twice in ultrapure water, and imaged on triply charged) for serine/threonine phosphorylation; (2) the fluorescent scanner. The containers with the gels the strong intensity (usually the top peak) of the neutral were covered with foil to prevent photo-bleaching of loss peak; (3) possible ambiguity of modification site the stains. assignment; and (4) comparison of the MS/MS spectra of For proteins that were purified using gels, the gels modified peptides with the corresponding unmodified were not fixed prior to the SYPRO stain. After the water counterparts. wash, these gels were imaged on a UV light-box, their pictures taken with a digital camera, and the bands of Immunoprecipitation interest excised and sent to the proteomics facility. For each sample analyzed (control, CIM, control/ peptide, CIM/peptide), 200 μL of protein G dynabeads Deglycosylation (Invitrogen) was incubated with 40 μg of pan-Na 1.x For deglycosylation with PNGase F (New England Bio- antibody - and for the peptide controls, 40 μg of block- labs), 100 μg of control or CIM membranes was adjusted ing peptide - for 3 h to 4 h, rotating end-over-end. to 80 uL volume; then 20 uL of 5X denaturation mix During this incubation, 3.0 mg of membrane protein (5 mM PMSF, iodoacetamide, NaF, 12.5 μM ALLN, was prepared for each sample. In a total of 1.8 mL vol- 0.5 μg/mL pepstatin A, 2.5% SDS, and 0.2 M DTT) was ume, the sample was solubilized in a buffer containing added and the sample incubated at 65°C for 20 min. Fol- 20 mM KPO4 (pH 6.5), 180 mM KCl, 1 mM EGTA, lowing denaturation and cooling to room temperature, 0.5 mM MgCl2, 1 mM PMSF, 1 mM iodoacetamide, 20 uL of 10% NP40, 20 μL of 500 mM sodium phosphate 1 mM NaF, 2.5 μM ALLN, 10 μM leupeptin, 0.1 μg/ml (pH 7.5), 59 μL water, and 1 μL (500 units) of PNGase F pepstatin A, and 1% NP40. Samples were vortexed, incu- were added to each sample and the samples incubated at bated on ice for 1 h, centrifuged at 13,700 x g in a Kraner et al. Skeletal Muscle 2012, 2:17 Page 4 of 13 http://www.skeletalmusclejournal.com/content/2/1/17 refrigerated microfuge for 30 min, and the supernatants NaHCO , 26.2; NaH PO , 1.7; and glucose, 5.5 (pH 7.3- 3 2 4 recovered for the immunoprecipitation. The antibody- 7.4, 20-22°C) equilibrated with 95% O and 5% CO . 2 2 bound beads were washed twice with PBS/0.5% NP40 and incubated with the solubilized channel protein, ro- Statistical analysis tating end-over-end, overnight. The next morning, the Western blots were quantified using the software sup- beads were collected at the side of the tube using a mag- plied with the Fujifilm LAS-3000 close-caption device net, and the supernatants removed. The beads were camera. For western blots with multiple samples of con- washed five times with the same buffer used for trol and CIM samples (for Na 1.4, Na 1.5, pan-Na 1.x, V V V solubilization, except NP40/asolectin was used in the FGF12, and FGF13), the average of the control was used buffer instead of NP40. The samples were eluted from as the 100% standard. All individual control and CIM the beads with gel sample buffer. Equal volumes of sam- samples were calculated relative to this number, and ple were loaded for each sample (con IP, CIM IP, con IP/ errors shown are SEM. Statistical comparison between pep, CIM IP/pep), and starting membranes were used control and CIM were carried out using Student’s t-test. as a positive control (con memb, CIM memb). Six separ- For quantification of the co-immunoprecipitation, the CIM ate immunoprecipitations were carried out. Prior to elut- was calculated relative to the control (set at 100%) for ing with sample buffer, all procedures were maintained each individual protein in each co-immunoprecipitation. at 0°C to 4°C. The expression in CIM relative to control for all six co-immunoprecipitations was analyzed by Student’s Immunohistochemistry t-test, and errors shown are SEM. Muscle excitability Medial gastrocnemius muscles from control or CIM rats was compared by calculating the percent of inexcitable were removed, fixed in 4% paraformaldehyde for 1 h, fibers in each muscle. At least six fibers were studied cryoprotected in 15% sucrose solution overnight, and in each muscle. The percent for each muscle was frozen in liquid nitrogen. Ten-μm-thick cross-sections compared between control and nNOS-null mice using were cut. The same rabbit FGF12 and mouse mono- Student’s t-test with n as the number of muscles studied. clonal nNOS antibodies used in western analysis were used for staining. For dystrophin staining of the FGF12 Results samples, the same mouse monoclonal used in western CIM skeletal muscle expresses the Na 1.5 α subunit and analysis was used, but for analysis of the mouse nNOS- aNa 1.4 α subunit with altered glycosylation stained samples, a rabbit dystrophin was used. Labeling We utilize a rat model of critical illness myopathy (CIM) of mouse monoclonal antibodies was visualized using a in which corticosteroid treatment is combined with Dylight 488-conjugated donkey anti-mouse secondary denervation to mimic treatment of critically-ill patients antibody (Jackson ImmunoResearch Laboratories). with corticosteroids and neuromuscular blocking agents Labeling of rabbit antibodies was visualized using a [5,6]. Previous analysis of mRNA indicated that CIM rhodamine-conjugated donkey anti-rabbit antibody (Jackson skeletal muscle expresses the Na 1.4 and Na 1.5 V V ImmunoResearch Laboratories). Images were obtained isoforms of sodium channel [15]. To confirm this at the using a Fluoview FV 1000 confocal microscope and an level of the protein, we analyzed western blots of skeletal X60 oil objective (Olympus Optical). muscle membrane fractions with antibodies specific to the two channel isoforms (Na 1.4 and Na 1.5) and also V V Electrophysiology with a pan-sodium channel antibody (pan-Na 1.x) to Wild type and nNOS knockout adult mice (B6.129 S4- compare the amount of both of these isoforms simultan- tm1Plh Nos1 /J, Jackson Labs, 25 g to 30 g body weight) eously (Figure 1, A and B). Membranes from heart were were denervated by removing a 0.5-mm segment of the used both as a negative control for the Na 1.4-specific left sciatic nerve in the upper thigh under isoflurane antibody and as a positive control for the Na 1.5 anti- anesthesia (2% to 3% inhaled). Buprenorphine was body. The change in expression observed with each of administered subcutaneously for postoperative analgesia. these antibodies (Figure 1B) suggests that the increased Mice were sacrificed on days 3 or 7 by carbon dioxide expression with the pan-Na 1.x antibody is due to up- inhalation. The extensor digitorum longus (EDL) muscle regulation of the Na 1.5 and suggests that the Na 1.5 V V was dissected tendon to tendon; then muscle fibers represents approximately 28% of the total sodium chan- were labeled with 10 μM 4-Di-2-ASP, and imaged using nel protein in CIM. an upright epifluorescence microscope during recording While the amount of Na 1.4 α subunit remained of action potentials as previously described [8]. For constant, its migration was altered. One potential all experiments, the recording chamber was continu- explanation for altered migration of the Na 1.4 α sub- ously perfused with solution containing (in millimoles unit is an alteration in the level of glycosylation. The per liter) NaCl, 118; KCl, 3.5; CaCl , 1.5; MgSO , 0.7; Na 1.4 α subunit is known to be highly glycosylated 2 4 V Kraner et al. Skeletal Muscle 2012, 2:17 Page 5 of 13 http://www.skeletalmusclejournal.com/content/2/1/17 Figure 1 CIM muscle expresses the Na 1.5 α subunit and a Na 1.4 α subunit altered in its glycosylation. (A) Western blots were V V performed on membranes prepared from gastrocnemius muscles of individual control or CIM rats using antibodies specific to the Na 1.4 and 1.5 sodium channel isoforms or a pan-Na 1.x antibody. Heart was a positive control for Na 1.5. The mobility of the Na 1.4 changed in CIM V V V (see the upper arrow to normal channel versus lower arrowhead pointing to altered). It should be noted that 5% gels were used for the blots with the Na 1.4 and 1.5 antibodies, while a 4% to 20% gel was used for the Na 1.x antibody, thus reducing the apparent size difference V V between control and CIM for that antibody. (B) Quantification of blots with different sodium channel antibodies. The average expression of protein under control conditions was set as 100%, and expression in individual control and CIM was calculated relative to this average (n = 4). Error bars shown are SEM, and asterisks indicate P < 0.01. Since the total amount of Na 1.4 does not change in CIM, the increase observed with the pan-Na 1.x is attributed to the up-regulation of the Na 1.5. (C) Membranes from control and CIM muscles were treated with either PNGase V V F (to remove all N-linked glycosylation) or Neuraminidase (to remove only terminal sialic acid) and visualized by western blotting with a Na 1.4-specific Ab on 5% gels. Treatment with PNGase F eliminated the migration difference between control and CIM channel, while Neuraminidase did not. (D) Neuraminidase treatment of fetuin, a control protein that is highly sialyated, shifts the molecular weight, indicating that the enzyme was functional. [16,17]. Membranes from control or CIM tissue were moieties is insufficient to account for the change in treated with one of two enzymes, PNGase F, which migration of Na 1.4. removes N-linked glycosylation at the ASN-linkage [18] or a recombinant neuraminidase, which removes ter- Identification of a phosphorylation site in the Na 1.4 minal sialic acid moieties [19]. The samples were then α subunit probed in a western blot with the Na 1.4-specific anti- In the Na 1.2 brain sodium channel, a complex inter- V V body. As shown, removal of the entire carbohydrate play of phosphorylation of sites within the I-II loop and sugar tree at the N-linkage completely abolished migra- the III-IV loop carried out by protein kinase A and C tion differences between the control and CIM samples, reduce peak sodium currents [20]. Skeletal muscle while treatment with neuraminidase did not (Figure 1C). sodium channels are not as highly phosphorylated as As a positive control for neuraminidase function, we their brain and neuronal counterparts [21], but phos- treated fetuin (a protein with a large number of sialic phorylation could affect gating of sodium channels in acid moieties), under the same conditions and found a CIM. Na 1.4 is phosphorylated by protein kinase A shift in migration (Figure 1D). Taken together, these data in vitro at a single site [21]. In the Na 1.5 channel, indicate that a focused removal of terminal sialic acid phosphorylation of S1505 in the III-IV loop by protein Kraner et al. Skeletal Muscle 2012, 2:17 Page 6 of 13 http://www.skeletalmusclejournal.com/content/2/1/17 kinase C both reduces peak current and shifts inactiva- found to contain an in vitro cAMP-phosphorylation site tion gating in the hyperpolarizing direction [22]. [21]. However, its surrounding sequence [QALES*GEE] As an assessment of the degree of overall phospho- does not correspond to the conserved consensus rylation, classically-purified control and CIM sodium sequence of [RK] 2x [ST] for either cAMP or cGMP- channels [11] were comparatively stained with Pro-Q dependent protein kinase. The lack of a quantitative Diamond Phosphoprotein Stain (which stains only phos- difference between the control and CIM channels, based phoproteins) and SYPRO Ruby Protein Stain (which on the ratio of Pro Q: SYPRO (Figure 2B), suggests that stains all proteins) (Figure 2A). Quantitative comparison gross changes in levels of phosphorylation do not under- of the fluorescent signal intensities of the two dyes was lie the shift in voltage dependence of inactivation. made, and the ratio of Pro-Q to SYPRO was found to be constant in control versus CIM channel (Figure 2B). No difference in FGF sub-type associated with CIM To determine the site at which the phosphorylation sodium channel occurred, the sodium channel bands were excised from Fibroblast growth factors (FGFs) have emerged as a class control and CIM samples, trypsinized, and analyzed by of proteins that modulate sodium channel gating [23-25]. tandem mass spectrometry (Figure 3, control sample Different FGF isoforms/splice variants shift the inactiva- shown). The serine at position 487 was partially phos- tion gating curves of sodium channels to varying degrees, phorylated and lies within the general region previously such that a switch in expression of one sub-type over Figure 2 The Na 1.4 is phosphorylated at similar levels in control and CIM muscle. (A) To assess the degree of phosphorylation in CIM and control muscle, classically-purified sodium channel was stained with a dye that binds only phosphoproteins (Pro Q Diamond phosphoprotein dye) and the ratio of signal with this stain was compared to that of a dye that binds all proteins (SYPRO Ruby Red protein stain). As controls, a set of proteins that are non-phosphorylated (β-galactosidase, bovine serum albumin) or a known phosphoprotein (ovalbumin) were analyzed on the same gel (P/NP markers). Of these controls, only the ovalbumin stained on both gels, indicating that staining conditions were optimal for each dye. Control and CIM sodium channel, indicated by arrows, had similar phosphostaining. (B) Quantification of the ratio of Pro Q: SYPRO protein stains for each of the experimental and control proteins. The control and CIM proteins had very similar degrees of phosphorylation, while only the ovalbumin standard was phosphorylated. Kraner et al. Skeletal Muscle 2012, 2:17 Page 7 of 13 http://www.skeletalmusclejournal.com/content/2/1/17 2+ [MH-H PO ] 3 4 2+ [MH-H O] y 7 5 b 12 14 2+ [MH-H O] Figure 3 Identification of Ser as a phosphorylation site in Na 1.4 channel. Using previously reported methods [11], a purified fraction of sodium channels was prepared from control and CIM muscles and the excised band corresponding to the Na 1.4 α subunit was excised, trypsinized, and analyzed by tandem mass spectrometry. This analysis indicated that the serine at 487 was partially phosphorylated, as we detected both the phosphorylated and unmodified forms in the samples. The MS/MS spectra of both forms were shown. The main product ions (b and y ions) were indicated. It is clear that a number of b ions (b and b ) from the two peptides differ in a mass of 80 Da, the mass shift due 12 14 to phosphorylation. In addition, neutral loss of phosphorus group was also observed for the modified form. The spectra shown are from a control sample. Although both control and CIM spectra were partially phosphorylated at the Ser site, the exact percentage was difficult to determine due to the small amount of sodium channel analyzed. another could alter the voltage dependence of sodium associated protein complex (DAPC) [26,27]. To deter- channel inactivation in CIM [23-25]. Thus, relative expres- mine if the constituent members of this sodium channel sion of FGFs may be a mechanism through which cells complex are dynamically regulated in CIM, we immuno- regulate excitability. Membranes from control and CIM precipitated the complex from control versus CIM gastrocnemius muscles were analyzed for expression of muscle membranes solubilized in the non-ionic deter- FGF proteins (Figure 4). While we found that neither con- gent NP40. We used the pan-Na 1.x antibody, directed trol or CIM skeletal muscle expressed FGF 11 and 14 towards the highly conserved III-IV linker, to bring (data not shown), both control and CIM membranes down both the Na 1.4 and 1.5 isoforms. The recovery expressed the same ratio of FGF13 relative to the amount of sodium channel in the immunoprecipitates (IPs) of total sodium channel protein detected with pan-Na 1.x was specific since a blocking peptide prevented IP of antibody (Figure 4, A and B). In CIM membranes, there the sodium channel in both control and CIM samples was increased expression of FGF12B relative to the pan- (Figure 5). The starting membranes were used as a posi- Na 1.x antibody (Figure 4, A and B). However, further tive control. Co-immunoprecipitation (CoIP) of syntro- analysis of FGF12 localization by staining in muscle cross- phin and dystrophin, the proteins most closely linked to sections indicated the protein was expressed in nuclei, not sodium channel in the complex, was relatively high in the surface membrane, and thus was unlikely to interact both control and CIM samples (Figure 5B and D). Plec- with sodium channels to alter the voltage dependence of tin, which is associated with the sodium channel both inactivation (Figure 4C). Thus none of the FGFs appear to through its interaction with β-dystroglycan and dys- be candidates that could account for the hyperpolarized trophin [28], is present at lower levels in control than shift in the voltage dependence of inactivation in CIM. CIM samples. Other proteins (β-dystroglycan and nNOS) were present primarily in the CIM samples The sodium channel is part of a protein complex that is (Figure 5B and D). A number of CoIPs were carried out altered in CIM to assess the degree to which proteins were present and Work carried out by a number of investigators has absent in the control versus CIM complexes, such that shown that sodium channels are part of the dystrophin statistical significance was reached (Figure 5C). The Kraner et al. Skeletal Muscle 2012, 2:17 Page 8 of 13 http://www.skeletalmusclejournal.com/content/2/1/17 Figure 4 FGFs appear unlikely to contribute to altered sodium channel gating in CIM. (A) FGF12 and 13 levels in muscle membrane samples from control and CIM muscle. As positive controls, tissues or expression vectors known to produce the protein of interest were used: for the pan-Na 1.x Ab, skeletal muscle; for FGF13, a lysate of 293 cells transfected with an FGF13 expression vector; for FGF12, brain membranes. (B) FGF13 was expressed in both control and CIM muscle, such that the relative ratio of FGF13: sodium channel did not change in CIM. In contrast, FGF12B was expressed at low levels in control muscle, but increased in CIM muscle, such that the ratio of FGF12B: sodium channel significantly increased. Asterisk indicates P < 0.05. (C) Cross-section of control and CIM medial gastrocnemius muscle stained for FGF12, dystrophin, and overlay reveals that most FGF12 staining is in myonuclei and nuclei of cells in the interstitial space. cartoon summarizes the dynamic nature of the complex contributes to loss of excitability. To determine the role (Figure 5D), in that β-dystroglycan and nNOS, shown in of nNOS in regulation of muscle excitability following white, are primarily associated with CIM sodium channels. denervation, we recorded from muscle fibers in nNOS- In other models of muscle atrophy, nNOS was reported null and control mice. In innervated mouse muscle, to move from the surface membrane to an intracellular 100% of fibers were excitable (n = 4 muscles). We found pool [29]. To confirm that nNOS is co-localized with that denervation of mouse muscle in the absence of sodium channel in the surface membrane in the CIM treatment with corticosteroids was sufficient to induce muscle, cross-sections of CIM muscle were stained for inexcitability. In control muscle denervated for 3 days, nNOS using dystrophin as a marker for the surface mem- few muscle fibers were excitable (n = 4 muscles, Figure 7). brane (Figure 6). Both control and CIM surface mem- In contrast, in nNOS-null mice the majority of fibers brane were positive for nNOS (Figure 6). Taken together, remained excitable 3 days after denervation (n = 4 mus- the biochemical and immunostaining data indicate that cles, P < 0.05 vs. control). To determine whether absence nNOS is a dynamically regulated member of the sodium of nNOS lessened development of inexcitability at a channel-DAPC complex. longer time point, we measured excitability in a second set of mice in which muscle was denervated for 7 days. nNOS plays a role in loss of muscle excitability In control muscle, no fibers remained excitable 7 days following denervation following denervation (n = 4 muscles); whereas in nNOS- In the rat model of CIM, treatment of denervated null fibers, excitability was preserved in a fraction of muscle with corticosteroids in vivo results in inexcitabil- fibers (n = 4 muscles, P < 0.05 vs. control). These data are ity of muscle fibers [7,8,30]. The biochemical data pre- consistent with the possibility that increased association sented above are consistent with the possibility that of nNOS with sodium channels following denervation increased association of nNOS with sodium channels contributes to development of muscle inexcitability. Kraner et al. Skeletal Muscle 2012, 2:17 Page 9 of 13 http://www.skeletalmusclejournal.com/content/2/1/17 Figure 5 Na 1.4 co-precipitates with members of the dystrophin associated protein complex (DAPC). Using an antibody generated against a peptide corresponding to the highly conserved III-IV linker region (pan Na 1.x), sodium channels from NP40-solubilized control and CIM skeletal muscle membranes were immunoprecipitated. The immunoprecipitated (IP) control (Con) or CIM materials were resolved on SDS-PAGE gels and probed in western blots with the indicated antibodies. As a negative control, the IP was performed in the presence of blocking peptide (IP/pep), the same sequence used as the antigen; and as a positive control, starting membranes were used (Memb). (A) A full-panel western with the Na 1.4-specific monoclonal antibody LD3 is shown on the left in comparison to a protein gel stained with SYPRO Ruby protein stain on the right. The denatured antibody heavy chain (Ab HC) and light chain (Ab LC) are present in the immunoprecipitated samples, as best seen in the SYPRO stained gel. A large number of proteins co-precipitate with the sodium channel (Na 1.x), and many of these are shown to be specific since they are absent in the peptide control. (B) Using antibodies against plectin, dystrophin (dys), neuronal nitric oxide synthase (nNOS), syntrophin (syn), and β-dystroglycan (β-dys) confirms that many components of the DAPC are present in the control and CIM IPs. (C) Quantification of the data from panel B. For each antibody in each CoIP, the signal for the control was set as 100% and the signal for the CIM was determined relative to this. The average of the signals for the CIM: control for each antibody is shown, and error bars are SEM (n =6). For some antibodies, notably the sodium channel antibodies, approximately equal signals were seen in control and CIM CoIPs. For other antibodies, notably dystrophin and syntrophin, there were slightly elevated amounts of these proteins present in the CIM. Finally, for the nNOS and β-dystroglycan, there was considerably more protein in the CIM CoIPs. These results are summarized in cartoon form in (D) which shows ‘tightly’ associated proteins in grey, and ‘loosely’ associated proteins in white. The protein associations shown in the cartoon are based on work carried out by a number of investigators, which previously demonstrated that Na 1.x channels associate with proteins of the DAPC through their consensus S/TXV-COOH C-termini [26,27]. The dynamic regulation of the signaling protein nNOS in CIM suggests that it may play a role in the disease process, including affecting the inactivation gating of the adjacent sodium channels. Kraner et al. Skeletal Muscle 2012, 2:17 Page 10 of 13 http://www.skeletalmusclejournal.com/content/2/1/17 Figure 6 nNOS is expressed in the sarcolemma of control and CIM muscle. Cross-sections of medial gastrocnemius muscle were stained with the antibodies to nNOS as well as dystrophin to mark muscle surface membrane. In both control and CIM, nNOS is expressed in the surface membrane. The myofiber cross-sections are much smaller in CIM muscle, due to the previously reported muscle atrophy in the disease [10]. Discussion neuraminidase did not eliminate the size difference. We compared biochemical properties of control and Given that the Na 1.4 channel is known to have mul- CIM sodium channels to find candidates that might ac- tiple carbohydrate trees [16,17], the simplest explanation count for the hyperpolarized shift in inactivation gating for these observations is that some but not most of the seen in the acute phase of CIM. We identified several carbohydrate trees are removed in CIM, removing some biochemical changes in sodium channel in CIM, but the but not most of the sialic acids. Previous work shows most promising candidates appeared to be alterations in that removal of sialic acid from sodium channels shifts sodium channel-associated proteins in CIM. In particu- inactivation gating in a depolarizing direction [16,17,31]. lar, nNOS is a promising candidate that was more asso- This is opposite of the hyperpolarizing shift we observed ciated with sodium channels from CIM muscle. In mice in CIM [9]. Thus, removal of some carbohydrate trees lacking nNOS, the normal reduction in excitability fol- may be a compensatory mechanism that moves the volt- lowing denervation was greatly reduced. These data are age dependence of inactivation towards more depolar- consistent with the possibility that increased association ized potentials. of nNOS with sodium channels is involved in triggering One change we found in CIM muscle that could loss of muscle excitability in CIM. underlie the hyperpolarized shift in the voltage depend- We identified an increase in Na 1.5 in CIM muscle ence of inactivation was an alteration in the composition such that it is approximately 28% of the entire channel of the dystrophin protein associated complex (DAPC) as population. This is similar to our earlier estimate of 21% summarized in Figure 5D. This figure is based not only obtained by measuring current densities [9]. In our pre- on work in this paper, but also on work carried out by vious study, both the TTX-insensitive (Na 1.5) and other investigators that identified the components of the TTX-sensitive (Na 1.4) channels demonstrated similar DAPC (reviewed in [32]). In our hands, the DAPC hyperpolarizing shifts in inactivation gating in CIM [9], appeared to dissociate more easily in control samples so increased expression of Na 1.5 per se cannot be such that all components (except sodium channels) were responsible for the shift. present at higher levels in the CIM samples. This was es- A second change identified in membranes from CIM pecially true for β-dystroglycan and nNOS, which are muscle was reduced glycosylation of the Na 1.4 sodium present at much higher levels in CIM CoIPs. These channel. Removal of the entire carbohydrate ‘tree’ at observations suggested to us that the sodium channel- the asparagine-linkage eliminated the molecular weight DAPC complex is bound more tightly in CIM, perhaps difference between the control and CIM channel. How- indicating that the sodium channel and cytoskeleton are ever, selective removal of sialic acid moieties with in a different and more strongly ‘locked’ conformation in Kraner et al. Skeletal Muscle 2012, 2:17 Page 11 of 13 http://www.skeletalmusclejournal.com/content/2/1/17 The protein components that we identified in the DAPC are consistent with those identified by other investigators [26,27]. In skeletal muscle, the consensus C-termini (S/TXV-COOH) of Na 1.4 and 1.5 sodium channels bind the PDZ domain of syntrophin at a site overlapping and/or closely adjacent to the binding site for nNOS [26]. Through syntrophin, both sodium chan- nels and nNOS bind the C-terminus of dystrophin [26]. In cardiac muscle, the dynamic nature of this complex was shown by comparative analysis of control vs. syntro- phin point mutation that causes Long QT syndrome. The syntrophin point mutation altered the complex con- 2+ stituents, such that the plasma membrane Ca ATPase no longer bound syntrophin. This released inhibition of nNOS, allowed S-nitrosylation of the Na 1.5 sodium channel, and altered gating [27]. Dystrophin is part of the muscle cytoskeletal system. In mdx mice, which lack dystrophin, sodium channel in- activation gating is shifted 10 mV more positively than that of control mice [33]. This observation suggests that loss of cytoskeletal components shifts inactivation gating in a depolarizing direction, a finding consistent with our hypothesis that sodium channel inactivation gating is hyperpolarized in CIM because it is more tightly asso- ciated with cytoskeletal components. However, acute dis- ruption of cytoskeleton by pressure during formation of seals during patch clamp measurements has been found to trigger hyperpolarized shifts in the voltage dependent of Na 1.4 and Na 1.5 activation and fast inactivation V V Figure 7 Muscle lacking nNOS is more excitable following [34-37]. Thus, while it is clear that changes in cytoskel- denervation. (A) Action potential traces from innervated and denervated wild-type and nNOS (NOS-1) null fibers. In innervated eton can have profound effects on the voltage depend- wild-type and nNOS null fibers, all or none action potentials are ence of sodium channel gating, we currently do not present. In the control 3 and 7 day denervated traces shown, no know which changes in cytoskeleton will translate into action potential is present and the only response is passive changes in sodium channel gating. depolarization of the membrane potential in response to current Our finding that nNOS is present at higher levels in the injection. In the 3-day denervated nNOS null fiber shown a nearly normal action potential is present. In the 7-day denervated nNOS sodium channel-DAPC complex in CIM raises the possi- null fiber shown an action potential is present, but it is smaller and bility that increased signaling through NO-dependent wider than action potentials from innervated muscle. (B) Plot of the pathways contributes to loss of muscle excitability in CIM percent of excitable fibers in innervated and denervated wild-type (see however, [38]). There are several cell signaling path- and nNOS null fibers. In both wild-type and nNOS null innervated ways that are regulated by NO, including protein phos- muscles 100% of fibers were excitable in all muscles studied (n =4 for wild-type, n = 3 for nNOS null). Three days following denervation phorylation through cGMP-protein kinase [39] and direct only 13% of fibers were excitable in wild type muscles (n = 4). In nitrosylation of cysteine or other amino acid side chains, nNOS null muscles 73% of fibers were excitable 3 days following as discussed above for the cardiac Na 1.5 [27]. We mea- denervation (P < 0.05 vs. wild-type, n = 4). Seven days following sured phosphorylation changes in CIM and found no denervation 0% of wild-type fibers were excitable (n = 4) while overall difference (Figure 2). 43% of fibers from nNOS null muscles were excitable (P < 0.05 vs. wild-type, n = 4). To determine whether increased association of nNOS with sodium channels could be involved in inducing inexcitability of muscle, we measured excitability follow- the disease. Alternatively, the constituent members of ing denervation in control and nNOS-null mice. In rats, the DAPC may be dynamically regulated. In either case, denervation alone induces inexcitability in only a minor- the presence of the important signaling protein nNOS in ity of fibers, so addition of corticosteroids is necessary the DAPC of CIM muscle suggests that NO signaling to induce inexcitability [8,30]. In control mice, denerv- through this protein could contribute to the altered ation alone was sufficient to induce inexcitability so it inactivation gating in CIM. was not necessary to co-administer corticosteroids. In Kraner et al. Skeletal Muscle 2012, 2:17 Page 12 of 13 http://www.skeletalmusclejournal.com/content/2/1/17 nNOS-null mice, a greater percentage of muscle fibers 2. Khan J, Harrison TB, Rich MM, Moss M: Early development of critical illness myopathy and neuropathy in patients with severe sepsis. Neurology 2006, remained excitable following denervation. There are 67:1421–1425. multiple mechanisms that could account for mainten- 3. Rich MM, Teener JW, Raps EC, Schotland DL, Bird SJ: Muscle is electrically ance of excitability following denervation in the absence inexcitable in acute quadriplegic myopathy [see comments]. Neurology 1996, 46:731–736. of nNOS. Further study will be necessary to determine 4. Rich MM, Bird SJ, Raps EC, McCluskey LF, Teener JW: Direct muscle if the contribution of nNOS to inexcitability is mediated stimulation in acute quadriplegic myopathy. Muscle Nerve 1997, by its association with sodium channels as part of 20:665–673. 5. Rouleau G, Karpati G, Carpenter S, Soza M, Prescott S, Holland P: the DAPC. Glucocorticoid excess induces preferential depletion of myosin in denervated skeletal muscle fibers. Muscle Nerve 1987, 10:428–438. 6. Mozaffar T, Haddad F, Zeng M, Zhang LY, Adams GR, Baldwin KM: Conclusion Molecular and cellular defects of skeletal muscle in an animal model of We surveyed sodium channels and their associated pro- acute quadriplegic myopathy. Muscle Nerve 2007, 35:55–65. teins in control versus CIM muscle using a variety of 7. Rich MM, Pinter MJ: Sodium channel inactivation in an animal model of acute quadriplegic myopathy. Ann Neurol 2001, 50:26–33. biochemical techniques to identify candidates that could 8. Rich MM, Pinter MJ: Crucial role of sodium channel fast inactivation in underlie the hyperpolarized shift in inactivation gating/ muscle fibre inexcitability in a rat model of critical illness myopathy. loss of electrical excitability that is characteristic of CIM. J Physiol 2003, 547:555–566. 9. Filatov GN, Rich MM: Hyperpolarized shifts in the voltage dependence of While we identified a number of changes in CIM, fast inactivation of Nav1.4 and Nav1.5 in a rat model of critical illness including increased expression of the Na 1.5 sodium myopathy. J Physiol 2004, 559:813–820. channel and partial de-glycosylation of the Na 1.4 10. Kraner SD, Wang Q, Novak KR, Cheng D, Cool DR, Peng J, Rich MM: sodium channel, it is the change in association of the Upregulation of the CaV 1.1-ryanodine receptor complex in a rat model of critical illness myopathy. Am J Physiol Regul Integr Comp Physiol 2011, sodium channels with members of the DAPC that seems 300:R1384–R1391. most promising as a potential explanation for the shift 11. Kraner S, Yang J, Barchi R: Structural inferences for the native skeletal in inactivation. muscle sodium channel as derived from patterns of endogenous proteolysis. J Biol Chem 1989, 264:13273–13280. 12. Gordon RD, Li Y, Fieles WE, Schotland DL, Barchi RL: Topological Abbreviations localization of a segment of the eel voltage-dependent sodium channel CIM: Critical illness myopathy; DAPC: Dystrophin associated protein complex; primary sequence (AA 927–938) that discriminates between models of DTT: Dithiothreitol; EDTA: Ethylenediaminetetraacetic acid; EGTA: Ethylene tertiary structure. J Neurosci 1988, 8:3742–3749. glycol-bis(2-aminoethylether)-N,N,N’,N’-tetraacetic acid; FGF: Fibroblast growth 13. Xu P, Duong DM, Seyfried NT, Cheng D, Xie Y, Robert J, Rush J, factor; FHF: Fibroblast growth factor homologous factors; Hochstrasser M, Finley D, Peng J: Quantitative proteomics reveals the IP: Immunoprecipitation; nNOS: Neuronal nitric oxide synthase; NO: Nitric function of unconventional ubiquitin chains in proteasomal oxide; NP40: Nonidet P-40; PBS: Phosphate-buffered saline; degradation. Cell 2009, 137:133–145. PMSF: Phenylmethylsulfonyl fluoride; PNGase F: Peptide: N-Glycosidase F; 14. Peng J, Elias JE, Thoreen CC, Licklider LJ, Gygi SP: Evaluation of SDS-PAGE: Sodium dodecyl sulfate polyacrylamide gel electrophoresis. multidimensional chromatography coupled with tandem mass spectrometry (LC/LC-MS/MS) for large-scale protein analysis: the yeast Competing interests proteome. J Proteome Res 2003, 2:43–50. None of the authors have competing interests. 15. Rich MM, Kraner SD, Barchi RL: Altered gene expression in steroid-treated denervated muscle. Neurobiol Dis 1999, 6:515–522. Authors’ contributions 16. Bennett E, Urcan MS, Tinkle SS, Koszowski AG, Levinson SR: Contribution SDK carried out all biochemical analyses on channel samples, prepared of sialic acid to the voltage dependence of sodium channel figures relating to that data, and co-wrote the manuscript. KRN carried out gating. A possible electrostatic mechanism. J Gen Physiol 1997, all surgical and drug treatments of animals as well as electrophysiologic 109:327–343. recordings. QW carried out immunostaining, analyzed data, and prepared 17. Bennett ES: Isoform-specific effects of sialic acid on voltage-dependent related figures. JP carried out and did all analysis and interpretation on Na + channel gating: functional sialic acids are localized to the S5–S6 tandem mass spectrometry. MMR supervised all experiments, co-wrote the loop of domain I. J Physiol 2002, 538:675–690. manuscript, and provided final interpretation of all data. All authors read and 18. Tarentino AL, Gomez CM, Plummer TH Jr: Deglycosylation of approved the final manuscript. asparagine-linked glycans by peptide:N-glycosidase F. Biochemistry 1985, 24:4665–4671. Acknowledgements 19. Roggentin P, Rothe B, Lottspeich F, Schauer R: Cloning and sequencing of This work was supported by NIH grant number NS040826 (MMR) and a Clostridium perfringens sialidase gene. FEBS Lett 1988, 238:31–34. quantification of westerns and stained gels were carried out in the Wright 20. Cantrell AR, Tibbs VC, Yu FH, Murphy BJ, Sharp EM, Qu Y, Catterall WA, State University Proteome Analysis Laboratory. Scheuer T: Molecular mechanism of convergent regulation of brain Na(+) channels by protein kinase C and protein kinase A anchored to AKAP- Author details 15. Mol Cell Neurosci 2002, 21:63–80. Department of Neuroscience, Cell Biology, and Physiology, Wright State 21. Yang J, Barchi R: Phosphorylation of the rat skeletal muscle sodium University School of Medicine, 3640 Colonel Glenn Hwy, Dayton, OH 45435, channel by cyclic AMP-dependent protein kinase. J Neurochem 1990, USA. Department of Structural Biology, St. Jude’s Children Research Hospital, 54:954–962. Memphis, TN 38105, USA. 22. Qu Y, Rogers JC, Tanada TN, Catterall WA, Scheuer T: Phosphorylation of S1505 in the cardiac Na + channel inactivation gate is required for Received: 16 May 2012 Accepted: 30 July 2012 modulation by protein kinase C. J Gen Physiol 1996, 108:375–379. Published: 30 August 2012 23. Liu CJ, Dib-Hajj SD, Renganathan M, Cummins TR, Waxman SG: Modulation of the cardiac sodium channel Nav1.5 by fibroblast growth factor homologous factor 1B. J Biol Chem 2003, 278:1029–1036. References 1. Lacomis D, Zochodne DW, Bird SJ: Critical illness myopathy. Muscle Nerve 24. Dover K, Solinas S, D’Angelo E, Goldfarb M: Long-term inactivation particle 2000, 23:1785–1788. for voltage-gated sodium channels. J Physiol 2010, 588:3695–3711. Kraner et al. Skeletal Muscle 2012, 2:17 Page 13 of 13 http://www.skeletalmusclejournal.com/content/2/1/17 25. Wang C, Hoch EG, Pitt GS: Identification of novel interaction sites that determine specificity between fibroblast growth factor homologous factors and voltage-gated sodium channels. J Biol Chem 2011, 286:24253–24263. 26. Gee SH, Madhavan R, Levinson SR, Caldwell JH, Sealock R, Froehner SC: Interaction of muscle and brain sodium channels with multiple members of the syntrophin family of dystrophin-associated proteins. J Neurosci 1998, 18:128–137. 27. Ueda K, Valdivia C, Medeiros-Domingo A, Tester DJ, Vatta M, Farrugia G, Ackerman MJ, Makielski JC: Syntrophin mutation associated with long QT syndrome through activation of the nNOS-SCN5A macromolecular complex. Proc Natl Acad Sci U S A 2008, 105:9355–9360. 28. Rezniczek GA, Konieczny P, Nikolic B, Reipert S, Schneller D, Abrahamsberg C, Davies KE, Winder SJ, Wiche G: Plectin 1f scaffolding at the sarcolemma of dystrophic (mdx) muscle fibers through multiple interactions with beta-dystroglycan. J Cell Biol 2007, 176:965–977. 29. Suzuki N, Motohashi N, Uezumi A, Fukada S, Yoshimura T, Itoyama Y, Aoki M, Miyagoe-Suzuki Y, Takeda S: NO production results in suspension-induced muscle atrophy through dislocation of neuronal NOS. J Clin Invest 2007, 117:2468–2476. 30. Rich MM, Pinter MJ, Kraner SD, Barchi RL: Loss of electrical excitability in an animal model of acute quadriplegic myopathy. Ann Neurol 1998, 43:171–179. 31. Montpetit ML, Stocker PJ, Schwetz TA, Harper JM, Norring SA, Schaffer L, North SJ, Jang-Lee J, Gilmartin T, Head SR, Haslam SM, Dell A, Marth JD, Bennett ES: Regulated and aberrant glycosylation modulate cardiac electrical signaling. Proc Natl Acad Sci U S A 2009, 106:16517–16522. 32. Burton EA, Davies KE: Muscular dystrophy–reason for optimism? Cell 2002, 108:5–8. 33. Hirn C, Shapovalov G, Petermann O, Roulet E, Ruegg UT: Nav1.4 deregulation in dystrophic skeletal muscle leads to Na + overload and enhanced cell death. J Gen Physiol 2008, 132:199–208. 34. Eickhorn R, Dragert C, Antoni H: Influence of cell isolation and recording technique on the voltage dependence of the fast cardiac sodium current of the rat. J Mol Cell Cardiol 1994, 26:1095–1108. 35. Tabarean IV, Juranka P, Morris CE: Membrane stretch affects gating modes of a skeletal muscle sodium channel. Biophys J 1999, 77:758–774. 36. Shcherbatko A, Ono F, Mandel G, Brehm P: Voltage-dependent sodium channel function is regulated through membrane mechanics. Biophys J 1999, 77:1945–1959. 37. Morris CE, Juranka PF: Nav channel mechanosensitivity: activation and inactivation accelerate reversibly with stretch. Biophys J 2007, 93:822–833. 38. Capasso M, Muzio AD, Pandolfi A, Pace M, Tomo PD, Ragno M, Uncini A: Possible role for nitric oxide dysregulation in critical illness myopathy. Muscle Nerve 2008, 137:196–202. 39. Madhusoodanan KS, Murad F: NO-cGMP signaling and regenerative medicine involving stem cells. Neurochem Res 2007, 32:681–694. doi:10.1186/2044-5040-2-17 Cite this article as: Kraner et al.: Altered sodium channel-protein associations in critical illness myopathy. Skeletal Muscle 2012 2:17. 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Altered sodium channel-protein associations in critical illness myopathy

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Copyright © 2012 by Kraner et al.; licensee BioMed Central Ltd.
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Life Sciences; Cell Biology; Developmental Biology; Biochemistry, general; Systems Biology; Biotechnology
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22935229
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Abstract

Background: During the acute phase of critical illness myopathy (CIM) there is inexcitability of skeletal muscle. In a rat model of CIM, muscle inexcitability is due to inactivation of sodium channels. A major contributor to this sodium channel inactivation is a hyperpolarized shift in the voltage dependence of sodium channel inactivation. The goal of the current study was to find a biochemical correlate of the hyperpolarized shift in sodium channel inactivation. Methods: The rat model of CIM was generated by cutting the sciatic nerve and subsequent injections of dexamethasone for 7 days. Skeletal muscle membranes were prepared from gastrocnemius muscles, and purification and biochemical analyses carried out. Immunoprecipitations were performed with a pan-sodium channel antibody, and the resulting complexes probed in Western blots with various antibodies. Results: We carried out analyses of sodium channel glycosylation, phosphorylation, and association with other proteins. Although there was some loss of channel glycosylation in the disease, as assessed by size analysis of glycosylated and de-glycosylated protein in control and CIM samples, previous work by other investigators suggest that such loss would most likely shift channel inactivation gating in a depolarizing direction; thus such loss was viewed as compensatory rather than causative of the disease. A phosphorylation site at serine 487 was identified on the Na 1.4 sodium channel α subunit, but there was no clear evidence of altered phosphorylation in the disease. Co-immunoprecipitation experiments carried out with a pan-sodium channel antibody confirmed that the sodium channel was associated with proteins of the dystrophin associated protein complex (DAPC). This complex differed between control and CIM samples. Syntrophin, dystrophin, and plectin associated strongly with sodium channels in both control and disease conditions, while β-dystroglycan and neuronal nitric oxide synthase (nNOS) associated strongly with the sodium channel only in CIM. Recording of action potentials revealed that denervated muscle in mice lacking nNOS was more excitable than control denervated muscle. Conclusion: Taken together, these data suggest that the conformation/protein association of the sodium channel complex differs in control and critical illness myopathy muscle membranes; and suggest that nitric oxide signaling plays a role in development of muscle inexcitability. Keywords: Skeletal muscle, Na 1.4 sodium channel, Na 1.5 sodium channel, Nitric oxide (NO), Neuronal nitric V V oxide synthase (nNOS), Glycosylation, Phosphorylation, Action potential, Excitability, Denervation * Correspondence: mark.rich@wright.edu Department of Neuroscience, Cell Biology, and Physiology, Wright State University School of Medicine, 3640 Colonel Glenn Hwy, Dayton, OH 45435, USA Full list of author information is available at the end of the article © 2012 Kraner et al.; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Kraner et al. Skeletal Muscle 2012, 2:17 Page 2 of 13 http://www.skeletalmusclejournal.com/content/2/1/17 Background Preparation of muscle membranes and classical Critical illness myopathy (CIM) is the most common purification of sodium channels cause of severe weakness in patients in the intensive Membranes were prepared from the gastrocnemius mus- care unit [1,2]. One of the hallmarks of the acute phase cles as previously reported [10]. Samples were thawed of CIM is paralysis due to loss of muscle’s electrical on ice for 10 min and minced finely on a glass plate on excitability [3,4]. To determine the mechanism under- ice. Samples were transferred to tubes containing lying loss of excitability, we utilize a rat model of CIM in sucrose buffer with protease and phosphatase inhibitors, which corticosteroid treatment is combined with denerv- using 10 volumes of buffer per gram of tissue. The ation to mimic treatment of critically-ill patients with sucrose buffer contained 0.3 M sucrose, 75 mM NaCl, corticosteroids and neuromuscular blocking agents [5,6]. 10 mM EGTA, 10 mM EDTA, 10 mM Tris, pH 7.4, In the rat model, inactivation of sodium channels is the 1 mM phenylmethylsulfonyl fluoride (PMSF), 1 mM cause of inexcitability [7,8]. A major contributor to the iodoacetamide, 0.1 μg/mL pepstatin A, 10 μM leupeptin, increase in inactivation is a hyperpolarized shift in 2.5 μM ALLN, and 1 mM sodium fluoride. Samples the voltage dependence of inactivation of the adult were homogenized using a PowerGen 700 tissue homo- (Na 1.4) and embryonic (Na 1.5) skeletal muscle so- genizer at a setting of 6 for 30 s. The samples were cen- V V dium channel isoforms expressed in CIM muscle [9]. As trifuged in a SS34 rotor at 4,500 g for 15 min. The the rat model of CIM occurs in genetically normal rats, supernatants were removed and centrifuged again. The this hyperpolarized shift in sodium channel gating must final supernatants were centrifuged in a SW55 rotor at be due to post-translational modification of sodium 138,000 g for 1 h. The pellets from this last step were channels or an alteration in sodium channel association resuspended in fresh sucrose buffers with inhibitors and with other proteins that modify gating. used as the membrane fraction. The goal of this study was to identify biochemical For classical purification of sodium channels, mem- changes that could potentially underlie the hyperpolar- branes were solubilized using NP40 and the sodium chan- ized shift in the voltage dependence of sodium channel nel fraction sequentially purified on DEAE-Sepharose and inactivation in CIM. We found changes in several candi- wheat germ agglutinin-Sepharose columns as described dates, although at least one change appeared compensa- [11]. For all membrane preparation and channel purifica- tory rather than causative. We identified an increase in tion procedures, samples, equipment, and buffers were the amount of neuronal nitric oxide synthase (nNOS) kept at 0-4°C. associated with the sodium channel in CIM as a change that might underlie the hyperpolarized shift in the volt- SDS-PAGE, western blot analysis, and antibodies age dependence of inactivation. Membrane fractions were assayed for protein content using the Lowry protein assay, and equal amounts of protein were loaded for control and CIM samples for Methods each protein assessed (approximately 40 μg protein Preparation of control and critical illness myopathy per lane). For deglycosylation experiments and co- muscle samples immunoprecipitation experiments, the same amount of All animal protocols were approved by the Institutional membrane protein was used to initiate the experiment; Animal Care and Use Committee at Wright State Uni- and equal volumes of the final product were loaded for versity. Critical illness myopathy was induced by a com- each control and CIM sample analyzed. SDS-PAGE bination of denervation and dexamethasone treatment. was performed on Criterion 4% to 20% acrylamide gels Briefly, rat muscle was denervated by removing a 10-mm for most samples; but 5% gels were used for the degly- segment of the sciatic nerve in isoflurane-anesthetized cosylation experiments, and 10% to 14.5% gels were adult female Wistar rats (250 to 350 g body weight). used for analysis of syntrophin and β-dystroglycan co- Buprenorphine was administered subcutaneously for immunoprecipitations to better resolve the molecular -1 postoperative analgesia. Dexamethasone (4 mg kg ) and weight region being analyzed. Western blot transfers antibiotics (0.2 mL of 2.27% Baytril, Bayer, Shawnee were carried out in a Criterion transfer apparatus Mission, KS, USA) were administered intraperitoneally using nitrocellulose as the blotting medium. Following beginning on the day of denervation and continuing for transfer, blots were washed with PBS/0.1% Tween, 7 days. Rats were killed by carbon dioxide inhalation, blocked in 1% I-Block (Tropix) in PBS/0.1% Tween for and the tibialis anterior, soleus, and gastrocnemius 1 h; incubated with first antibody for 2 h; washed and muscles removed, weighed, and snap-frozen in liquid ni- incubated with mouse or rabbit secondary antibody trogen. For control muscles, non-treated rats from the conjugated to alkaline phosphatase (Tropix); and visua- same group were used and muscles harvested in the lized using CDP-Star (Tropix) and a Fujifilm LAS-3000 same way. All muscles were stored at −80°C until use. close-caption device camera. Kraner et al. Skeletal Muscle 2012, 2:17 Page 3 of 13 http://www.skeletalmusclejournal.com/content/2/1/17 A pan-sodium channel antibody (pan Na 1.x) was 37°C for 1 h. Samples were quenched by addition of gel raised against the highly conserved peptide, TEEQKKYY- sample buffer. For neuraminidase (New England Biolabs) NAMKKLGSKK, corresponding to SP19 [12] at Open treatments, 10 μg of control or CIM membranes were Biosystems, ThermoFisher Scientific. The affinity-purified adjusted to 100 μL with 10 μL of 500 mM sodium citrate antibody selectively reacted to sodium channel in skeletal (pH 6.0), 10 μL of 10% NP40, 5 μL neuraminidase muscle and other tissues and could be displaced by the (250 units), and water. Samples were heated at 37°C for peptide antigen in western blot analysis. The antibody 4 h. As a control, 10 μg of fetuin was treated in the same to FGF13 was provided by Dr Geoffrey Pitts (Duke way. Samples were quenched by addition of gel sample University). The Na 1.4-specific monoclonal antibody buffer. For all samples, mock treatments without enzyme was provided by Dr Robert L Barchi (Thomas Jefferson were also carried out. Samples were analyzed by western Medical School), and can be obtained commercially from blot with Na 1.4-specific antibody. Sigma (S9568). Other antibodies were obtained from the following commercial sources: FGF12 (Abgent AP6750b), Analysis of phosphorylation by tandem mass spec plectin (Epitomics 1399–1), dystrophin (Vector Labs VP- The mass spectrometric analysis was performed on an D508), Na 1.5 (Alomone ASC-005), nNOS (Invitrogen optimized proteomics platform as previously reported 37–2800), β-dystroglycan (Abcam ab 49515), and syntro- [13]. Briefly, protein gel bands were washed and sub- phin (Abcam ab11425). jected to standard in-gel trypsin digestion. Digested pep- Some gels were stained rather than processed for west- tides were analyzed by capillary reverse-phase liquid ern blots. For analysis of phosphoproteins, Peppermint chromatography coupled with tandem mass spectrom- Stick Phosphoprotein Molecular Weight Standards (Invi- etry (LC-MS/MS), in which the eluted peptides were trogen), which include both phosphoprotein and non- analyzed by an MS survey scan followed by 10 data- phosphoprotein standards, were included on the gels as dependent MS/MS scans on an LTQ-Orbitrap ion trap controls. After electrophoresis, gels were fixed in 50% mass spectrometer (Thermo Scientific). Acquired MS/ methanol and 10% acetic acid for 30 min, transferred to MS spectra were then searched against the rat reference the Pro-Q Diamond Phosphoprotein Stain (Invitrogen) database of the National Center for Biotechnology In- for 90 min, then transferred to Destain (Invitrogen) for formation with differential modifications on serine/ 30 min. The gel was de-stained twice more for 30 min; threonine/tyrosine (+79.9663 Da); then filtered by then washed twice in ultrapure water. The gel was matching scores and mass accuracy to reduce protein imaged on a flatbed fluorescent scanner (Fujifilm FLA- false discovery rate to less than 1% using a concatenated 5100). After the phosphoprotein stain, the gel was im- reversed database [14]. Furthermore, we manually vali- mediately processed for all-protein stain with SYPRO dated MS/MS spectra of matched phosphopeptides using Ruby Protein Stain (Invitrogen) overnight. The gel was the following criteria: (1) the presence of signature phos- de-stained with 10% MeOH and 7% acetic acid for phate neutral losses (−49 for doubly charged or −32.7 for 45 min, washed twice in ultrapure water, and imaged on triply charged) for serine/threonine phosphorylation; (2) the fluorescent scanner. The containers with the gels the strong intensity (usually the top peak) of the neutral were covered with foil to prevent photo-bleaching of loss peak; (3) possible ambiguity of modification site the stains. assignment; and (4) comparison of the MS/MS spectra of For proteins that were purified using gels, the gels modified peptides with the corresponding unmodified were not fixed prior to the SYPRO stain. After the water counterparts. wash, these gels were imaged on a UV light-box, their pictures taken with a digital camera, and the bands of Immunoprecipitation interest excised and sent to the proteomics facility. For each sample analyzed (control, CIM, control/ peptide, CIM/peptide), 200 μL of protein G dynabeads Deglycosylation (Invitrogen) was incubated with 40 μg of pan-Na 1.x For deglycosylation with PNGase F (New England Bio- antibody - and for the peptide controls, 40 μg of block- labs), 100 μg of control or CIM membranes was adjusted ing peptide - for 3 h to 4 h, rotating end-over-end. to 80 uL volume; then 20 uL of 5X denaturation mix During this incubation, 3.0 mg of membrane protein (5 mM PMSF, iodoacetamide, NaF, 12.5 μM ALLN, was prepared for each sample. In a total of 1.8 mL vol- 0.5 μg/mL pepstatin A, 2.5% SDS, and 0.2 M DTT) was ume, the sample was solubilized in a buffer containing added and the sample incubated at 65°C for 20 min. Fol- 20 mM KPO4 (pH 6.5), 180 mM KCl, 1 mM EGTA, lowing denaturation and cooling to room temperature, 0.5 mM MgCl2, 1 mM PMSF, 1 mM iodoacetamide, 20 uL of 10% NP40, 20 μL of 500 mM sodium phosphate 1 mM NaF, 2.5 μM ALLN, 10 μM leupeptin, 0.1 μg/ml (pH 7.5), 59 μL water, and 1 μL (500 units) of PNGase F pepstatin A, and 1% NP40. Samples were vortexed, incu- were added to each sample and the samples incubated at bated on ice for 1 h, centrifuged at 13,700 x g in a Kraner et al. Skeletal Muscle 2012, 2:17 Page 4 of 13 http://www.skeletalmusclejournal.com/content/2/1/17 refrigerated microfuge for 30 min, and the supernatants NaHCO , 26.2; NaH PO , 1.7; and glucose, 5.5 (pH 7.3- 3 2 4 recovered for the immunoprecipitation. The antibody- 7.4, 20-22°C) equilibrated with 95% O and 5% CO . 2 2 bound beads were washed twice with PBS/0.5% NP40 and incubated with the solubilized channel protein, ro- Statistical analysis tating end-over-end, overnight. The next morning, the Western blots were quantified using the software sup- beads were collected at the side of the tube using a mag- plied with the Fujifilm LAS-3000 close-caption device net, and the supernatants removed. The beads were camera. For western blots with multiple samples of con- washed five times with the same buffer used for trol and CIM samples (for Na 1.4, Na 1.5, pan-Na 1.x, V V V solubilization, except NP40/asolectin was used in the FGF12, and FGF13), the average of the control was used buffer instead of NP40. The samples were eluted from as the 100% standard. All individual control and CIM the beads with gel sample buffer. Equal volumes of sam- samples were calculated relative to this number, and ple were loaded for each sample (con IP, CIM IP, con IP/ errors shown are SEM. Statistical comparison between pep, CIM IP/pep), and starting membranes were used control and CIM were carried out using Student’s t-test. as a positive control (con memb, CIM memb). Six separ- For quantification of the co-immunoprecipitation, the CIM ate immunoprecipitations were carried out. Prior to elut- was calculated relative to the control (set at 100%) for ing with sample buffer, all procedures were maintained each individual protein in each co-immunoprecipitation. at 0°C to 4°C. The expression in CIM relative to control for all six co-immunoprecipitations was analyzed by Student’s Immunohistochemistry t-test, and errors shown are SEM. Muscle excitability Medial gastrocnemius muscles from control or CIM rats was compared by calculating the percent of inexcitable were removed, fixed in 4% paraformaldehyde for 1 h, fibers in each muscle. At least six fibers were studied cryoprotected in 15% sucrose solution overnight, and in each muscle. The percent for each muscle was frozen in liquid nitrogen. Ten-μm-thick cross-sections compared between control and nNOS-null mice using were cut. The same rabbit FGF12 and mouse mono- Student’s t-test with n as the number of muscles studied. clonal nNOS antibodies used in western analysis were used for staining. For dystrophin staining of the FGF12 Results samples, the same mouse monoclonal used in western CIM skeletal muscle expresses the Na 1.5 α subunit and analysis was used, but for analysis of the mouse nNOS- aNa 1.4 α subunit with altered glycosylation stained samples, a rabbit dystrophin was used. Labeling We utilize a rat model of critical illness myopathy (CIM) of mouse monoclonal antibodies was visualized using a in which corticosteroid treatment is combined with Dylight 488-conjugated donkey anti-mouse secondary denervation to mimic treatment of critically-ill patients antibody (Jackson ImmunoResearch Laboratories). with corticosteroids and neuromuscular blocking agents Labeling of rabbit antibodies was visualized using a [5,6]. Previous analysis of mRNA indicated that CIM rhodamine-conjugated donkey anti-rabbit antibody (Jackson skeletal muscle expresses the Na 1.4 and Na 1.5 V V ImmunoResearch Laboratories). Images were obtained isoforms of sodium channel [15]. To confirm this at the using a Fluoview FV 1000 confocal microscope and an level of the protein, we analyzed western blots of skeletal X60 oil objective (Olympus Optical). muscle membrane fractions with antibodies specific to the two channel isoforms (Na 1.4 and Na 1.5) and also V V Electrophysiology with a pan-sodium channel antibody (pan-Na 1.x) to Wild type and nNOS knockout adult mice (B6.129 S4- compare the amount of both of these isoforms simultan- tm1Plh Nos1 /J, Jackson Labs, 25 g to 30 g body weight) eously (Figure 1, A and B). Membranes from heart were were denervated by removing a 0.5-mm segment of the used both as a negative control for the Na 1.4-specific left sciatic nerve in the upper thigh under isoflurane antibody and as a positive control for the Na 1.5 anti- anesthesia (2% to 3% inhaled). Buprenorphine was body. The change in expression observed with each of administered subcutaneously for postoperative analgesia. these antibodies (Figure 1B) suggests that the increased Mice were sacrificed on days 3 or 7 by carbon dioxide expression with the pan-Na 1.x antibody is due to up- inhalation. The extensor digitorum longus (EDL) muscle regulation of the Na 1.5 and suggests that the Na 1.5 V V was dissected tendon to tendon; then muscle fibers represents approximately 28% of the total sodium chan- were labeled with 10 μM 4-Di-2-ASP, and imaged using nel protein in CIM. an upright epifluorescence microscope during recording While the amount of Na 1.4 α subunit remained of action potentials as previously described [8]. For constant, its migration was altered. One potential all experiments, the recording chamber was continu- explanation for altered migration of the Na 1.4 α sub- ously perfused with solution containing (in millimoles unit is an alteration in the level of glycosylation. The per liter) NaCl, 118; KCl, 3.5; CaCl , 1.5; MgSO , 0.7; Na 1.4 α subunit is known to be highly glycosylated 2 4 V Kraner et al. Skeletal Muscle 2012, 2:17 Page 5 of 13 http://www.skeletalmusclejournal.com/content/2/1/17 Figure 1 CIM muscle expresses the Na 1.5 α subunit and a Na 1.4 α subunit altered in its glycosylation. (A) Western blots were V V performed on membranes prepared from gastrocnemius muscles of individual control or CIM rats using antibodies specific to the Na 1.4 and 1.5 sodium channel isoforms or a pan-Na 1.x antibody. Heart was a positive control for Na 1.5. The mobility of the Na 1.4 changed in CIM V V V (see the upper arrow to normal channel versus lower arrowhead pointing to altered). It should be noted that 5% gels were used for the blots with the Na 1.4 and 1.5 antibodies, while a 4% to 20% gel was used for the Na 1.x antibody, thus reducing the apparent size difference V V between control and CIM for that antibody. (B) Quantification of blots with different sodium channel antibodies. The average expression of protein under control conditions was set as 100%, and expression in individual control and CIM was calculated relative to this average (n = 4). Error bars shown are SEM, and asterisks indicate P < 0.01. Since the total amount of Na 1.4 does not change in CIM, the increase observed with the pan-Na 1.x is attributed to the up-regulation of the Na 1.5. (C) Membranes from control and CIM muscles were treated with either PNGase V V F (to remove all N-linked glycosylation) or Neuraminidase (to remove only terminal sialic acid) and visualized by western blotting with a Na 1.4-specific Ab on 5% gels. Treatment with PNGase F eliminated the migration difference between control and CIM channel, while Neuraminidase did not. (D) Neuraminidase treatment of fetuin, a control protein that is highly sialyated, shifts the molecular weight, indicating that the enzyme was functional. [16,17]. Membranes from control or CIM tissue were moieties is insufficient to account for the change in treated with one of two enzymes, PNGase F, which migration of Na 1.4. removes N-linked glycosylation at the ASN-linkage [18] or a recombinant neuraminidase, which removes ter- Identification of a phosphorylation site in the Na 1.4 minal sialic acid moieties [19]. The samples were then α subunit probed in a western blot with the Na 1.4-specific anti- In the Na 1.2 brain sodium channel, a complex inter- V V body. As shown, removal of the entire carbohydrate play of phosphorylation of sites within the I-II loop and sugar tree at the N-linkage completely abolished migra- the III-IV loop carried out by protein kinase A and C tion differences between the control and CIM samples, reduce peak sodium currents [20]. Skeletal muscle while treatment with neuraminidase did not (Figure 1C). sodium channels are not as highly phosphorylated as As a positive control for neuraminidase function, we their brain and neuronal counterparts [21], but phos- treated fetuin (a protein with a large number of sialic phorylation could affect gating of sodium channels in acid moieties), under the same conditions and found a CIM. Na 1.4 is phosphorylated by protein kinase A shift in migration (Figure 1D). Taken together, these data in vitro at a single site [21]. In the Na 1.5 channel, indicate that a focused removal of terminal sialic acid phosphorylation of S1505 in the III-IV loop by protein Kraner et al. Skeletal Muscle 2012, 2:17 Page 6 of 13 http://www.skeletalmusclejournal.com/content/2/1/17 kinase C both reduces peak current and shifts inactiva- found to contain an in vitro cAMP-phosphorylation site tion gating in the hyperpolarizing direction [22]. [21]. However, its surrounding sequence [QALES*GEE] As an assessment of the degree of overall phospho- does not correspond to the conserved consensus rylation, classically-purified control and CIM sodium sequence of [RK] 2x [ST] for either cAMP or cGMP- channels [11] were comparatively stained with Pro-Q dependent protein kinase. The lack of a quantitative Diamond Phosphoprotein Stain (which stains only phos- difference between the control and CIM channels, based phoproteins) and SYPRO Ruby Protein Stain (which on the ratio of Pro Q: SYPRO (Figure 2B), suggests that stains all proteins) (Figure 2A). Quantitative comparison gross changes in levels of phosphorylation do not under- of the fluorescent signal intensities of the two dyes was lie the shift in voltage dependence of inactivation. made, and the ratio of Pro-Q to SYPRO was found to be constant in control versus CIM channel (Figure 2B). No difference in FGF sub-type associated with CIM To determine the site at which the phosphorylation sodium channel occurred, the sodium channel bands were excised from Fibroblast growth factors (FGFs) have emerged as a class control and CIM samples, trypsinized, and analyzed by of proteins that modulate sodium channel gating [23-25]. tandem mass spectrometry (Figure 3, control sample Different FGF isoforms/splice variants shift the inactiva- shown). The serine at position 487 was partially phos- tion gating curves of sodium channels to varying degrees, phorylated and lies within the general region previously such that a switch in expression of one sub-type over Figure 2 The Na 1.4 is phosphorylated at similar levels in control and CIM muscle. (A) To assess the degree of phosphorylation in CIM and control muscle, classically-purified sodium channel was stained with a dye that binds only phosphoproteins (Pro Q Diamond phosphoprotein dye) and the ratio of signal with this stain was compared to that of a dye that binds all proteins (SYPRO Ruby Red protein stain). As controls, a set of proteins that are non-phosphorylated (β-galactosidase, bovine serum albumin) or a known phosphoprotein (ovalbumin) were analyzed on the same gel (P/NP markers). Of these controls, only the ovalbumin stained on both gels, indicating that staining conditions were optimal for each dye. Control and CIM sodium channel, indicated by arrows, had similar phosphostaining. (B) Quantification of the ratio of Pro Q: SYPRO protein stains for each of the experimental and control proteins. The control and CIM proteins had very similar degrees of phosphorylation, while only the ovalbumin standard was phosphorylated. Kraner et al. Skeletal Muscle 2012, 2:17 Page 7 of 13 http://www.skeletalmusclejournal.com/content/2/1/17 2+ [MH-H PO ] 3 4 2+ [MH-H O] y 7 5 b 12 14 2+ [MH-H O] Figure 3 Identification of Ser as a phosphorylation site in Na 1.4 channel. Using previously reported methods [11], a purified fraction of sodium channels was prepared from control and CIM muscles and the excised band corresponding to the Na 1.4 α subunit was excised, trypsinized, and analyzed by tandem mass spectrometry. This analysis indicated that the serine at 487 was partially phosphorylated, as we detected both the phosphorylated and unmodified forms in the samples. The MS/MS spectra of both forms were shown. The main product ions (b and y ions) were indicated. It is clear that a number of b ions (b and b ) from the two peptides differ in a mass of 80 Da, the mass shift due 12 14 to phosphorylation. In addition, neutral loss of phosphorus group was also observed for the modified form. The spectra shown are from a control sample. Although both control and CIM spectra were partially phosphorylated at the Ser site, the exact percentage was difficult to determine due to the small amount of sodium channel analyzed. another could alter the voltage dependence of sodium associated protein complex (DAPC) [26,27]. To deter- channel inactivation in CIM [23-25]. Thus, relative expres- mine if the constituent members of this sodium channel sion of FGFs may be a mechanism through which cells complex are dynamically regulated in CIM, we immuno- regulate excitability. Membranes from control and CIM precipitated the complex from control versus CIM gastrocnemius muscles were analyzed for expression of muscle membranes solubilized in the non-ionic deter- FGF proteins (Figure 4). While we found that neither con- gent NP40. We used the pan-Na 1.x antibody, directed trol or CIM skeletal muscle expressed FGF 11 and 14 towards the highly conserved III-IV linker, to bring (data not shown), both control and CIM membranes down both the Na 1.4 and 1.5 isoforms. The recovery expressed the same ratio of FGF13 relative to the amount of sodium channel in the immunoprecipitates (IPs) of total sodium channel protein detected with pan-Na 1.x was specific since a blocking peptide prevented IP of antibody (Figure 4, A and B). In CIM membranes, there the sodium channel in both control and CIM samples was increased expression of FGF12B relative to the pan- (Figure 5). The starting membranes were used as a posi- Na 1.x antibody (Figure 4, A and B). However, further tive control. Co-immunoprecipitation (CoIP) of syntro- analysis of FGF12 localization by staining in muscle cross- phin and dystrophin, the proteins most closely linked to sections indicated the protein was expressed in nuclei, not sodium channel in the complex, was relatively high in the surface membrane, and thus was unlikely to interact both control and CIM samples (Figure 5B and D). Plec- with sodium channels to alter the voltage dependence of tin, which is associated with the sodium channel both inactivation (Figure 4C). Thus none of the FGFs appear to through its interaction with β-dystroglycan and dys- be candidates that could account for the hyperpolarized trophin [28], is present at lower levels in control than shift in the voltage dependence of inactivation in CIM. CIM samples. Other proteins (β-dystroglycan and nNOS) were present primarily in the CIM samples The sodium channel is part of a protein complex that is (Figure 5B and D). A number of CoIPs were carried out altered in CIM to assess the degree to which proteins were present and Work carried out by a number of investigators has absent in the control versus CIM complexes, such that shown that sodium channels are part of the dystrophin statistical significance was reached (Figure 5C). The Kraner et al. Skeletal Muscle 2012, 2:17 Page 8 of 13 http://www.skeletalmusclejournal.com/content/2/1/17 Figure 4 FGFs appear unlikely to contribute to altered sodium channel gating in CIM. (A) FGF12 and 13 levels in muscle membrane samples from control and CIM muscle. As positive controls, tissues or expression vectors known to produce the protein of interest were used: for the pan-Na 1.x Ab, skeletal muscle; for FGF13, a lysate of 293 cells transfected with an FGF13 expression vector; for FGF12, brain membranes. (B) FGF13 was expressed in both control and CIM muscle, such that the relative ratio of FGF13: sodium channel did not change in CIM. In contrast, FGF12B was expressed at low levels in control muscle, but increased in CIM muscle, such that the ratio of FGF12B: sodium channel significantly increased. Asterisk indicates P < 0.05. (C) Cross-section of control and CIM medial gastrocnemius muscle stained for FGF12, dystrophin, and overlay reveals that most FGF12 staining is in myonuclei and nuclei of cells in the interstitial space. cartoon summarizes the dynamic nature of the complex contributes to loss of excitability. To determine the role (Figure 5D), in that β-dystroglycan and nNOS, shown in of nNOS in regulation of muscle excitability following white, are primarily associated with CIM sodium channels. denervation, we recorded from muscle fibers in nNOS- In other models of muscle atrophy, nNOS was reported null and control mice. In innervated mouse muscle, to move from the surface membrane to an intracellular 100% of fibers were excitable (n = 4 muscles). We found pool [29]. To confirm that nNOS is co-localized with that denervation of mouse muscle in the absence of sodium channel in the surface membrane in the CIM treatment with corticosteroids was sufficient to induce muscle, cross-sections of CIM muscle were stained for inexcitability. In control muscle denervated for 3 days, nNOS using dystrophin as a marker for the surface mem- few muscle fibers were excitable (n = 4 muscles, Figure 7). brane (Figure 6). Both control and CIM surface mem- In contrast, in nNOS-null mice the majority of fibers brane were positive for nNOS (Figure 6). Taken together, remained excitable 3 days after denervation (n = 4 mus- the biochemical and immunostaining data indicate that cles, P < 0.05 vs. control). To determine whether absence nNOS is a dynamically regulated member of the sodium of nNOS lessened development of inexcitability at a channel-DAPC complex. longer time point, we measured excitability in a second set of mice in which muscle was denervated for 7 days. nNOS plays a role in loss of muscle excitability In control muscle, no fibers remained excitable 7 days following denervation following denervation (n = 4 muscles); whereas in nNOS- In the rat model of CIM, treatment of denervated null fibers, excitability was preserved in a fraction of muscle with corticosteroids in vivo results in inexcitabil- fibers (n = 4 muscles, P < 0.05 vs. control). These data are ity of muscle fibers [7,8,30]. The biochemical data pre- consistent with the possibility that increased association sented above are consistent with the possibility that of nNOS with sodium channels following denervation increased association of nNOS with sodium channels contributes to development of muscle inexcitability. Kraner et al. Skeletal Muscle 2012, 2:17 Page 9 of 13 http://www.skeletalmusclejournal.com/content/2/1/17 Figure 5 Na 1.4 co-precipitates with members of the dystrophin associated protein complex (DAPC). Using an antibody generated against a peptide corresponding to the highly conserved III-IV linker region (pan Na 1.x), sodium channels from NP40-solubilized control and CIM skeletal muscle membranes were immunoprecipitated. The immunoprecipitated (IP) control (Con) or CIM materials were resolved on SDS-PAGE gels and probed in western blots with the indicated antibodies. As a negative control, the IP was performed in the presence of blocking peptide (IP/pep), the same sequence used as the antigen; and as a positive control, starting membranes were used (Memb). (A) A full-panel western with the Na 1.4-specific monoclonal antibody LD3 is shown on the left in comparison to a protein gel stained with SYPRO Ruby protein stain on the right. The denatured antibody heavy chain (Ab HC) and light chain (Ab LC) are present in the immunoprecipitated samples, as best seen in the SYPRO stained gel. A large number of proteins co-precipitate with the sodium channel (Na 1.x), and many of these are shown to be specific since they are absent in the peptide control. (B) Using antibodies against plectin, dystrophin (dys), neuronal nitric oxide synthase (nNOS), syntrophin (syn), and β-dystroglycan (β-dys) confirms that many components of the DAPC are present in the control and CIM IPs. (C) Quantification of the data from panel B. For each antibody in each CoIP, the signal for the control was set as 100% and the signal for the CIM was determined relative to this. The average of the signals for the CIM: control for each antibody is shown, and error bars are SEM (n =6). For some antibodies, notably the sodium channel antibodies, approximately equal signals were seen in control and CIM CoIPs. For other antibodies, notably dystrophin and syntrophin, there were slightly elevated amounts of these proteins present in the CIM. Finally, for the nNOS and β-dystroglycan, there was considerably more protein in the CIM CoIPs. These results are summarized in cartoon form in (D) which shows ‘tightly’ associated proteins in grey, and ‘loosely’ associated proteins in white. The protein associations shown in the cartoon are based on work carried out by a number of investigators, which previously demonstrated that Na 1.x channels associate with proteins of the DAPC through their consensus S/TXV-COOH C-termini [26,27]. The dynamic regulation of the signaling protein nNOS in CIM suggests that it may play a role in the disease process, including affecting the inactivation gating of the adjacent sodium channels. Kraner et al. Skeletal Muscle 2012, 2:17 Page 10 of 13 http://www.skeletalmusclejournal.com/content/2/1/17 Figure 6 nNOS is expressed in the sarcolemma of control and CIM muscle. Cross-sections of medial gastrocnemius muscle were stained with the antibodies to nNOS as well as dystrophin to mark muscle surface membrane. In both control and CIM, nNOS is expressed in the surface membrane. The myofiber cross-sections are much smaller in CIM muscle, due to the previously reported muscle atrophy in the disease [10]. Discussion neuraminidase did not eliminate the size difference. We compared biochemical properties of control and Given that the Na 1.4 channel is known to have mul- CIM sodium channels to find candidates that might ac- tiple carbohydrate trees [16,17], the simplest explanation count for the hyperpolarized shift in inactivation gating for these observations is that some but not most of the seen in the acute phase of CIM. We identified several carbohydrate trees are removed in CIM, removing some biochemical changes in sodium channel in CIM, but the but not most of the sialic acids. Previous work shows most promising candidates appeared to be alterations in that removal of sialic acid from sodium channels shifts sodium channel-associated proteins in CIM. In particu- inactivation gating in a depolarizing direction [16,17,31]. lar, nNOS is a promising candidate that was more asso- This is opposite of the hyperpolarizing shift we observed ciated with sodium channels from CIM muscle. In mice in CIM [9]. Thus, removal of some carbohydrate trees lacking nNOS, the normal reduction in excitability fol- may be a compensatory mechanism that moves the volt- lowing denervation was greatly reduced. These data are age dependence of inactivation towards more depolar- consistent with the possibility that increased association ized potentials. of nNOS with sodium channels is involved in triggering One change we found in CIM muscle that could loss of muscle excitability in CIM. underlie the hyperpolarized shift in the voltage depend- We identified an increase in Na 1.5 in CIM muscle ence of inactivation was an alteration in the composition such that it is approximately 28% of the entire channel of the dystrophin protein associated complex (DAPC) as population. This is similar to our earlier estimate of 21% summarized in Figure 5D. This figure is based not only obtained by measuring current densities [9]. In our pre- on work in this paper, but also on work carried out by vious study, both the TTX-insensitive (Na 1.5) and other investigators that identified the components of the TTX-sensitive (Na 1.4) channels demonstrated similar DAPC (reviewed in [32]). In our hands, the DAPC hyperpolarizing shifts in inactivation gating in CIM [9], appeared to dissociate more easily in control samples so increased expression of Na 1.5 per se cannot be such that all components (except sodium channels) were responsible for the shift. present at higher levels in the CIM samples. This was es- A second change identified in membranes from CIM pecially true for β-dystroglycan and nNOS, which are muscle was reduced glycosylation of the Na 1.4 sodium present at much higher levels in CIM CoIPs. These channel. Removal of the entire carbohydrate ‘tree’ at observations suggested to us that the sodium channel- the asparagine-linkage eliminated the molecular weight DAPC complex is bound more tightly in CIM, perhaps difference between the control and CIM channel. How- indicating that the sodium channel and cytoskeleton are ever, selective removal of sialic acid moieties with in a different and more strongly ‘locked’ conformation in Kraner et al. Skeletal Muscle 2012, 2:17 Page 11 of 13 http://www.skeletalmusclejournal.com/content/2/1/17 The protein components that we identified in the DAPC are consistent with those identified by other investigators [26,27]. In skeletal muscle, the consensus C-termini (S/TXV-COOH) of Na 1.4 and 1.5 sodium channels bind the PDZ domain of syntrophin at a site overlapping and/or closely adjacent to the binding site for nNOS [26]. Through syntrophin, both sodium chan- nels and nNOS bind the C-terminus of dystrophin [26]. In cardiac muscle, the dynamic nature of this complex was shown by comparative analysis of control vs. syntro- phin point mutation that causes Long QT syndrome. The syntrophin point mutation altered the complex con- 2+ stituents, such that the plasma membrane Ca ATPase no longer bound syntrophin. This released inhibition of nNOS, allowed S-nitrosylation of the Na 1.5 sodium channel, and altered gating [27]. Dystrophin is part of the muscle cytoskeletal system. In mdx mice, which lack dystrophin, sodium channel in- activation gating is shifted 10 mV more positively than that of control mice [33]. This observation suggests that loss of cytoskeletal components shifts inactivation gating in a depolarizing direction, a finding consistent with our hypothesis that sodium channel inactivation gating is hyperpolarized in CIM because it is more tightly asso- ciated with cytoskeletal components. However, acute dis- ruption of cytoskeleton by pressure during formation of seals during patch clamp measurements has been found to trigger hyperpolarized shifts in the voltage dependent of Na 1.4 and Na 1.5 activation and fast inactivation V V Figure 7 Muscle lacking nNOS is more excitable following [34-37]. Thus, while it is clear that changes in cytoskel- denervation. (A) Action potential traces from innervated and denervated wild-type and nNOS (NOS-1) null fibers. In innervated eton can have profound effects on the voltage depend- wild-type and nNOS null fibers, all or none action potentials are ence of sodium channel gating, we currently do not present. In the control 3 and 7 day denervated traces shown, no know which changes in cytoskeleton will translate into action potential is present and the only response is passive changes in sodium channel gating. depolarization of the membrane potential in response to current Our finding that nNOS is present at higher levels in the injection. In the 3-day denervated nNOS null fiber shown a nearly normal action potential is present. In the 7-day denervated nNOS sodium channel-DAPC complex in CIM raises the possi- null fiber shown an action potential is present, but it is smaller and bility that increased signaling through NO-dependent wider than action potentials from innervated muscle. (B) Plot of the pathways contributes to loss of muscle excitability in CIM percent of excitable fibers in innervated and denervated wild-type (see however, [38]). There are several cell signaling path- and nNOS null fibers. In both wild-type and nNOS null innervated ways that are regulated by NO, including protein phos- muscles 100% of fibers were excitable in all muscles studied (n =4 for wild-type, n = 3 for nNOS null). Three days following denervation phorylation through cGMP-protein kinase [39] and direct only 13% of fibers were excitable in wild type muscles (n = 4). In nitrosylation of cysteine or other amino acid side chains, nNOS null muscles 73% of fibers were excitable 3 days following as discussed above for the cardiac Na 1.5 [27]. We mea- denervation (P < 0.05 vs. wild-type, n = 4). Seven days following sured phosphorylation changes in CIM and found no denervation 0% of wild-type fibers were excitable (n = 4) while overall difference (Figure 2). 43% of fibers from nNOS null muscles were excitable (P < 0.05 vs. wild-type, n = 4). To determine whether increased association of nNOS with sodium channels could be involved in inducing inexcitability of muscle, we measured excitability follow- the disease. Alternatively, the constituent members of ing denervation in control and nNOS-null mice. In rats, the DAPC may be dynamically regulated. In either case, denervation alone induces inexcitability in only a minor- the presence of the important signaling protein nNOS in ity of fibers, so addition of corticosteroids is necessary the DAPC of CIM muscle suggests that NO signaling to induce inexcitability [8,30]. In control mice, denerv- through this protein could contribute to the altered ation alone was sufficient to induce inexcitability so it inactivation gating in CIM. was not necessary to co-administer corticosteroids. In Kraner et al. Skeletal Muscle 2012, 2:17 Page 12 of 13 http://www.skeletalmusclejournal.com/content/2/1/17 nNOS-null mice, a greater percentage of muscle fibers 2. Khan J, Harrison TB, Rich MM, Moss M: Early development of critical illness myopathy and neuropathy in patients with severe sepsis. Neurology 2006, remained excitable following denervation. There are 67:1421–1425. multiple mechanisms that could account for mainten- 3. Rich MM, Teener JW, Raps EC, Schotland DL, Bird SJ: Muscle is electrically ance of excitability following denervation in the absence inexcitable in acute quadriplegic myopathy [see comments]. Neurology 1996, 46:731–736. of nNOS. Further study will be necessary to determine 4. Rich MM, Bird SJ, Raps EC, McCluskey LF, Teener JW: Direct muscle if the contribution of nNOS to inexcitability is mediated stimulation in acute quadriplegic myopathy. Muscle Nerve 1997, by its association with sodium channels as part of 20:665–673. 5. Rouleau G, Karpati G, Carpenter S, Soza M, Prescott S, Holland P: the DAPC. Glucocorticoid excess induces preferential depletion of myosin in denervated skeletal muscle fibers. Muscle Nerve 1987, 10:428–438. 6. Mozaffar T, Haddad F, Zeng M, Zhang LY, Adams GR, Baldwin KM: Conclusion Molecular and cellular defects of skeletal muscle in an animal model of We surveyed sodium channels and their associated pro- acute quadriplegic myopathy. Muscle Nerve 2007, 35:55–65. teins in control versus CIM muscle using a variety of 7. Rich MM, Pinter MJ: Sodium channel inactivation in an animal model of acute quadriplegic myopathy. Ann Neurol 2001, 50:26–33. biochemical techniques to identify candidates that could 8. Rich MM, Pinter MJ: Crucial role of sodium channel fast inactivation in underlie the hyperpolarized shift in inactivation gating/ muscle fibre inexcitability in a rat model of critical illness myopathy. loss of electrical excitability that is characteristic of CIM. J Physiol 2003, 547:555–566. 9. Filatov GN, Rich MM: Hyperpolarized shifts in the voltage dependence of While we identified a number of changes in CIM, fast inactivation of Nav1.4 and Nav1.5 in a rat model of critical illness including increased expression of the Na 1.5 sodium myopathy. J Physiol 2004, 559:813–820. channel and partial de-glycosylation of the Na 1.4 10. Kraner SD, Wang Q, Novak KR, Cheng D, Cool DR, Peng J, Rich MM: sodium channel, it is the change in association of the Upregulation of the CaV 1.1-ryanodine receptor complex in a rat model of critical illness myopathy. Am J Physiol Regul Integr Comp Physiol 2011, sodium channels with members of the DAPC that seems 300:R1384–R1391. most promising as a potential explanation for the shift 11. Kraner S, Yang J, Barchi R: Structural inferences for the native skeletal in inactivation. muscle sodium channel as derived from patterns of endogenous proteolysis. J Biol Chem 1989, 264:13273–13280. 12. Gordon RD, Li Y, Fieles WE, Schotland DL, Barchi RL: Topological Abbreviations localization of a segment of the eel voltage-dependent sodium channel CIM: Critical illness myopathy; DAPC: Dystrophin associated protein complex; primary sequence (AA 927–938) that discriminates between models of DTT: Dithiothreitol; EDTA: Ethylenediaminetetraacetic acid; EGTA: Ethylene tertiary structure. J Neurosci 1988, 8:3742–3749. glycol-bis(2-aminoethylether)-N,N,N’,N’-tetraacetic acid; FGF: Fibroblast growth 13. Xu P, Duong DM, Seyfried NT, Cheng D, Xie Y, Robert J, Rush J, factor; FHF: Fibroblast growth factor homologous factors; Hochstrasser M, Finley D, Peng J: Quantitative proteomics reveals the IP: Immunoprecipitation; nNOS: Neuronal nitric oxide synthase; NO: Nitric function of unconventional ubiquitin chains in proteasomal oxide; NP40: Nonidet P-40; PBS: Phosphate-buffered saline; degradation. Cell 2009, 137:133–145. PMSF: Phenylmethylsulfonyl fluoride; PNGase F: Peptide: N-Glycosidase F; 14. Peng J, Elias JE, Thoreen CC, Licklider LJ, Gygi SP: Evaluation of SDS-PAGE: Sodium dodecyl sulfate polyacrylamide gel electrophoresis. multidimensional chromatography coupled with tandem mass spectrometry (LC/LC-MS/MS) for large-scale protein analysis: the yeast Competing interests proteome. J Proteome Res 2003, 2:43–50. None of the authors have competing interests. 15. Rich MM, Kraner SD, Barchi RL: Altered gene expression in steroid-treated denervated muscle. Neurobiol Dis 1999, 6:515–522. Authors’ contributions 16. Bennett E, Urcan MS, Tinkle SS, Koszowski AG, Levinson SR: Contribution SDK carried out all biochemical analyses on channel samples, prepared of sialic acid to the voltage dependence of sodium channel figures relating to that data, and co-wrote the manuscript. KRN carried out gating. A possible electrostatic mechanism. J Gen Physiol 1997, all surgical and drug treatments of animals as well as electrophysiologic 109:327–343. recordings. QW carried out immunostaining, analyzed data, and prepared 17. Bennett ES: Isoform-specific effects of sialic acid on voltage-dependent related figures. JP carried out and did all analysis and interpretation on Na + channel gating: functional sialic acids are localized to the S5–S6 tandem mass spectrometry. MMR supervised all experiments, co-wrote the loop of domain I. J Physiol 2002, 538:675–690. manuscript, and provided final interpretation of all data. All authors read and 18. Tarentino AL, Gomez CM, Plummer TH Jr: Deglycosylation of approved the final manuscript. asparagine-linked glycans by peptide:N-glycosidase F. Biochemistry 1985, 24:4665–4671. Acknowledgements 19. Roggentin P, Rothe B, Lottspeich F, Schauer R: Cloning and sequencing of This work was supported by NIH grant number NS040826 (MMR) and a Clostridium perfringens sialidase gene. FEBS Lett 1988, 238:31–34. quantification of westerns and stained gels were carried out in the Wright 20. 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Shcherbatko A, Ono F, Mandel G, Brehm P: Voltage-dependent sodium channel function is regulated through membrane mechanics. Biophys J 1999, 77:1945–1959. 37. Morris CE, Juranka PF: Nav channel mechanosensitivity: activation and inactivation accelerate reversibly with stretch. Biophys J 2007, 93:822–833. 38. Capasso M, Muzio AD, Pandolfi A, Pace M, Tomo PD, Ragno M, Uncini A: Possible role for nitric oxide dysregulation in critical illness myopathy. Muscle Nerve 2008, 137:196–202. 39. Madhusoodanan KS, Murad F: NO-cGMP signaling and regenerative medicine involving stem cells. Neurochem Res 2007, 32:681–694. doi:10.1186/2044-5040-2-17 Cite this article as: Kraner et al.: Altered sodium channel-protein associations in critical illness myopathy. Skeletal Muscle 2012 2:17. 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Skeletal MuscleSpringer Journals

Published: Aug 30, 2012

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