Identification of Disease Specific Pathways Using in Vivo SILAC Proteomics in Dystrophin Deficient mdx Mouse
2013; Elsevier BV; Volume: 12; Issue: 5 Linguagem: Inglês
10.1074/mcp.m112.023127
ISSN1535-9484
AutoresSree Rayavarapu, William Coley, Erdinç Çakır, Vanessa E. Jahnke, Shin’ichi Takeda, Yoshitsugu Aoki, Heather Grodish-Dressman, Jyoti K. Jaiswal, Eric P. Hoffman, Kristy J. Brown, Yetrib Hathout, Kanneboyina Nagaraju,
Tópico(s)Biotin and Related Studies
ResumoDuchenne muscular dystrophy (DMD) is an X-linked neuromuscular disorder caused by a mutation in the dystrophin gene. DMD is characterized by progressive weakness of skeletal, cardiac, and respiratory muscles. The molecular mechanisms underlying dystrophy-associated muscle weakness and damage are not well understood. Quantitative proteomics techniques could help to identify disease-specific pathways. Recent advances in the in vivo labeling strategies such as stable isotope labeling in mouse (SILAC mouse) with 13C6-lysine or stable isotope labeling in mammals (SILAM) with 15N have enabled accurate quantitative analysis of the proteomes of whole organs and tissues as a function of disease. Here we describe the use of the SILAC mouse strategy to define the underlying pathological mechanisms in dystrophin-deficient skeletal muscle. Differential SILAC proteome profiling was performed on the gastrocnemius muscles of 3-week-old (early stage) dystrophin-deficient mdx mice and wild-type (normal) mice. The generated data were further confirmed in an independent set of mdx and normal mice using a SILAC spike-in strategy. A total of 789 proteins were quantified; of these, 73 were found to be significantly altered between mdx and normal mice (p < 0.05). Bioinformatics analyses using Ingenuity Pathway software established that the integrin-linked kinase pathway, actin cytoskeleton signaling, mitochondrial energy metabolism, and calcium homeostasis are the pathways initially affected in dystrophin-deficient muscle at early stages of pathogenesis. The key proteins involved in these pathways were validated by means of immunoblotting and immunohistochemistry in independent sets of mdx mice and in human DMD muscle biopsies. The specific involvement of these molecular networks early in dystrophic pathology makes them potential therapeutic targets. In sum, our findings indicate that SILAC mouse strategy has uncovered previously unidentified pathological pathways in mouse models of human skeletal muscle disease. Duchenne muscular dystrophy (DMD) is an X-linked neuromuscular disorder caused by a mutation in the dystrophin gene. DMD is characterized by progressive weakness of skeletal, cardiac, and respiratory muscles. The molecular mechanisms underlying dystrophy-associated muscle weakness and damage are not well understood. Quantitative proteomics techniques could help to identify disease-specific pathways. Recent advances in the in vivo labeling strategies such as stable isotope labeling in mouse (SILAC mouse) with 13C6-lysine or stable isotope labeling in mammals (SILAM) with 15N have enabled accurate quantitative analysis of the proteomes of whole organs and tissues as a function of disease. Here we describe the use of the SILAC mouse strategy to define the underlying pathological mechanisms in dystrophin-deficient skeletal muscle. Differential SILAC proteome profiling was performed on the gastrocnemius muscles of 3-week-old (early stage) dystrophin-deficient mdx mice and wild-type (normal) mice. The generated data were further confirmed in an independent set of mdx and normal mice using a SILAC spike-in strategy. A total of 789 proteins were quantified; of these, 73 were found to be significantly altered between mdx and normal mice (p < 0.05). Bioinformatics analyses using Ingenuity Pathway software established that the integrin-linked kinase pathway, actin cytoskeleton signaling, mitochondrial energy metabolism, and calcium homeostasis are the pathways initially affected in dystrophin-deficient muscle at early stages of pathogenesis. The key proteins involved in these pathways were validated by means of immunoblotting and immunohistochemistry in independent sets of mdx mice and in human DMD muscle biopsies. The specific involvement of these molecular networks early in dystrophic pathology makes them potential therapeutic targets. In sum, our findings indicate that SILAC mouse strategy has uncovered previously unidentified pathological pathways in mouse models of human skeletal muscle disease. Dystrophin is an essential skeletal muscle protein that interacts with other glycoproteins such as the dystroglycans and sarcoglycans to form the dystrophin glycoprotein complex. This complex links the extracellular matrix and the cytoskeleton of the myofiber via F-actin, thereby protecting the skeletal muscle membrane against contraction-induced damage (1Petrof B.J. Shrager J.B. Stedman H.H. Kelly A.M. Sweeney H.L. Dystrophin protects the sarcolemma from stresses developed during muscle contraction.Proc. Natl. Acad. Sci. U.S.A. 1993; 90: 3710-3714Crossref PubMed Scopus (1176) Google Scholar). The absence of this complex due to the lack of expression of dystrophin makes the myofiber membrane susceptible to damage, which in turn activates various pathogenic processes and aberrant signaling cascades (2Oak S.A. Zhou Y.W. Jarrett H.W. Skeletal muscle signaling pathway through the dystrophin glycoprotein complex and Rac1.J. Biol. Chem. 2003; 278: 39287-39295Abstract Full Text Full Text PDF PubMed Scopus (76) Google Scholar, 3Spence H.J. Chen Y.J. Winder S.J. Muscular dystrophies, the cytoskeleton and cell adhesion.Bioessays. 2002; 24: 542-552Crossref PubMed Scopus (57) Google Scholar, 4Evans N.P. Misyak S.A. Robertson J.L. Bassaganya-Riera J. Grange R.W. Dysregulated intracellular signaling and inflammatory gene expression during initial disease onset in Duchenne muscular dystrophy.Am. J. Phys. Med. Rehabil. 2009; 88: 502-522Crossref PubMed Scopus (63) Google Scholar). Immune-mediated mechanisms are considered one of the key contributors to muscle degeneration in dystrophin-deficient subjects. However, the explicit role of specific pathogenic processes in this disease has not been thoroughly investigated. Dystrophin-deficient mdx mice are one of the most widely used animal models for studying disease pathophysiology and testing various therapeutic regimens (5Spurney C.F. Gordish-Dressman H. Guerron A.D. Sali A. Pandey G.S. Rawat R. Van Der Meulen J.H. Cha H.J. Pistilli E.E. Partridge T.A. Hoffman E.P. Nagaraju K. Preclinical drug trials in the mdx mouse: assessment of reliable and sensitive outcome measures.Muscle Nerve. 2009; 39: 591-602Crossref PubMed Scopus (124) Google Scholar). The mdx-23 mouse model on a C57BL/10 background is a spontaneous mutant with a point mutation in exon 23 of the dystrophin gene that eliminates the expression of dystrophin (6Sicinski P. Geng Y. Ryder-Cook A.S. Barnard E.A. Darlison M.G. Barnard P.J. The molecular basis of muscular dystrophy in the mdx mouse: a point mutation.Science. 1989; 244: 1578-1580Crossref PubMed Scopus (1000) Google Scholar). However, this mutation does not disrupt the expression of shorter isoforms that are also expressed from the dystrophin gene through differential promoter usage. Another mutant mdx mouse model, mdx-52 on a C57BL/6 background, has been generated by disrupting the dystrophin gene through gene targeting and the deletion of exon 52. The mdx-52 mice lack both dystrophin and the shorter dystrophin isoforms (Dp140 and Dp260). The skeletal muscles of mdx-52 mice exhibit a pathological profile similar to that of mdx-23 mice (7Araki E. Nakamura K. Nakao K. Kameya S. Kobayashi O. Nonaka I. Kobayashi T. Katsuki M. Targeted disruption of exon 52 in the mouse dystrophin gene induced muscle degeneration similar to that observed in Duchenne muscular dystrophy.Biochem. Biophys. Res. Commun. 1997; 238: 492-497Crossref PubMed Scopus (98) Google Scholar). These mdx strains show a mild phenotype relative to the human disease, but nevertheless display substantial myofiber degeneration, muscle weakness, elevated serum creatine kinase, and extensive inflammatory infiltrates in the muscle tissue (5Spurney C.F. Gordish-Dressman H. Guerron A.D. Sali A. Pandey G.S. Rawat R. Van Der Meulen J.H. Cha H.J. Pistilli E.E. Partridge T.A. Hoffman E.P. Nagaraju K. Preclinical drug trials in the mdx mouse: assessment of reliable and sensitive outcome measures.Muscle Nerve. 2009; 39: 591-602Crossref PubMed Scopus (124) Google Scholar, 8Collins C.A. Morgan J.E. Duchenne's muscular dystrophy: animal models used to investigate pathogenesis and develop therapeutic strategies.Int. J. Exp. Pathol. 2003; 84: 165-172Crossref PubMed Scopus (149) Google Scholar). Initial disease onset in mdx mice occurs around 3 weeks of age, with recurring bouts of myofiber degeneration and regeneration. These bouts are limited by 12 to 16 weeks of age, but the tissue infiltration and muscle weakness continue for the remainder of the animal's life. Thus, this model continues to be important for studying the consequences of dystrophin deficiency and alteration in the molecular events that lead to muscle pathology. For our studies, we have used mdx-52 mice that are on the C57BL/6 background. The investigation of protein dynamics and their involvement in signaling pathways in the course of dystrophinopathy can provide valuable insight into its pathogenesis. Protein modulations can be monitored using mass-spectrometry-based quantitative strategies. In the past, two-dimensional gel electrophoresis and fluorescence difference in gel electrophoresis methods have been used to study protein changes in the muscle of dystrophic mdx mice. Traditional proteomic techniques (2-DE) suffer from a disadvantage in that they detect alterations predominantly in abundantly expressed proteins. Furthermore, prior studies using the mdx mouse model focused on established disease instead of early disease, in which limited pathways drive the pathology. Proteomic profiling of established disease has identified perturbations in Ca2+ handling and bioenergetic pathways but not specific mechanisms that are upstream in the pathogenesis (9Ge Y. Molloy M.P. Chamberlain J.S. Andrews P.C. Proteomic analysis of mdx skeletal muscle: great reduction of adenylate kinase 1 expression and enzymatic activity.Proteomics. 2003; 3: 1895-1903Crossref PubMed Scopus (77) Google Scholar, 10Doran P. Dowling P. Donoghue P. Buffini M. Ohlendieck K. Reduced expression of regucalcin in young and aged mdx diaphragm indicates abnormal cytosolic calcium handling in dystrophin-deficient muscle.Biochim. Biophys. Acta. 2006; 1764: 773-785Crossref PubMed Scopus (52) Google Scholar, 11Doran P. Dowling P. Lohan J. McDonnell K. Poetsch S. Ohlendieck K. Subproteomics analysis of Ca+-binding proteins demonstrates decreased calsequestrin expression in dystrophic mouse skeletal muscle.Eur. J. Biochem. 2004; 271: 3943-3952Crossref PubMed Scopus (79) Google Scholar, 12Doran P. Martin G. Dowling P. Jockusch H. Ohlendieck K. Proteome analysis of the dystrophin-deficient MDX diaphragm reveals a drastic increase in the heat shock protein cvHSP.Proteomics. 2006; 6: 4610-4621Crossref PubMed Scopus (113) Google Scholar). Therefore, we performed proteomic studies of early disease stages in the mdx mouse model. The stable isotope labeling by amino acids in cell culture (SILAC) 1The abbreviations used are:DMDDuchenne muscular dystrophyILKintegrin-linked kinaseIPAIngenuity Pathway AnalysisSILACstable isotope labeling with amino acids in cell culture. 1The abbreviations used are:DMDDuchenne muscular dystrophyILKintegrin-linked kinaseIPAIngenuity Pathway AnalysisSILACstable isotope labeling with amino acids in cell culture.strategy introduced by Ong et al. has been successfully implemented in various cell culture systems and has been proven to be the most accurate method for proteome profiling (13Ong S.E. Blagoev B. Kratchmarova I. Kristensen D.B. Steen H. Pandey A. Mann M. Stable isotope labeling by amino acids in cell culture, SILAC, as a simple and accurate approach to expression proteomics.Mol. Cell. Proteomics. 2002; 1: 376-386Abstract Full Text Full Text PDF PubMed Scopus (4569) Google Scholar, 14Emadali A. Gallagher-Gambarelli M. [Quantitative proteomics by SILAC: practicalities and perspectives for an evolving approach].Med. Sci. (Paris). 2009; 25: 835-842Crossref PubMed Scopus (11) Google Scholar, 15Blagoev B. Kratchmarova I. Ong S.E. Nielsen M. Foster L.J. Mann M. A proteomics strategy to elucidate functional protein-protein interactions applied to EGF signaling.Nat. Biotechnol. 2003; 21: 315-318Crossref PubMed Scopus (607) Google Scholar, 16Kruger M. Kratchmarova I. Blagoev B. Tseng Y.H. Kahn C.R. Mann M. Dissection of the insulin signaling pathway via quantitative phosphoproteomics.Proc. Natl. Acad. Sci. U.S.A. 2008; 105: 2451-2456Crossref PubMed Scopus (207) Google Scholar, 17Amanchy R. Kalume D.E. Iwahori A. Zhong J. Pandey A. Phosphoproteome analysis of HeLa cells using stable isotope labeling with amino acids in cell culture (SILAC).J. Proteome Res. 2005; 4: 1661-1671Crossref PubMed Scopus (100) Google Scholar). More recently, this powerful technique has been extended to in vivo studies to develop heavy-stable-isotope-labeled mammals. This method has been evaluated in both mice (SILAC mouse) and rats (SILAM) (18Kruger M. Moser M. Ussar S. Thievessen I. Luber C.A. Forner F. Schmidt S. Zanivan S. Fassler R. Mann M. SILAC mouse for quantitative proteomics uncovers kindlin-3 as an essential factor for red blood cell function.Cell. 2008; 134: 353-364Abstract Full Text Full Text PDF PubMed Scopus (547) Google Scholar, 19McClatchy D.B. Liao L. Park S.K. Xu T. Lu B. Yates III, J.R. Differential proteomic analysis of mammalian tissues using SILAM.PLoS One. 2011; 6: e16039Crossref PubMed Scopus (28) Google Scholar, 20McClatchy D.B. Yates 3rd, J.R. Stable isotope labeling of mammals (SILAM).CSH Protoc. 2008; 2008 (pdb.prot4940)PubMed Google Scholar). In the SILAC mouse strategy, the mice feed consists of a balanced synthetic feed labeled with 6-Lys, whereas in SILAM, the feed consists of 15N-labeled algae. These techniques enabled accurate measurement of differentially expressed proteins in different organs and tissues of mice and rats under different conditions (18Kruger M. Moser M. Ussar S. Thievessen I. Luber C.A. Forner F. Schmidt S. Zanivan S. Fassler R. Mann M. SILAC mouse for quantitative proteomics uncovers kindlin-3 as an essential factor for red blood cell function.Cell. 2008; 134: 353-364Abstract Full Text Full Text PDF PubMed Scopus (547) Google Scholar, 19McClatchy D.B. Liao L. Park S.K. Xu T. Lu B. Yates III, J.R. Differential proteomic analysis of mammalian tissues using SILAM.PLoS One. 2011; 6: e16039Crossref PubMed Scopus (28) Google Scholar, 21Lu X.M. Tompkins R.G. Fischman A.J. SILAM for quantitative proteomics of liver Akt1/PKBalpha after burn injury.Int. J. Mol. Med. 2012; 29: 461-471PubMed Google Scholar, 22Huang T.C. Sahasrabuddhe N.A. Kim M.S. Getnet D. Yang Y. Peterson J.M. Ghosh B. Chaerkady R. Leach S.D. Marchionni L. Wong G.W. Pandey A. Regulation of lipid metabolism by dicer revealed through SILAC mice.J. Proteome Res. 2012; 11: 2193-2205Crossref PubMed Scopus (23) Google Scholar, 23Zanivan S. Krueger M. Mann M. In vivo quantitative proteomics: the SILAC mouse.Methods Mol. Biol. 2012; 757: 435-450Crossref PubMed Scopus (71) Google Scholar). Here we have extended the use of the SILAC mouse strategy to study the underlying molecular alterations in gastrocnemius muscle in the early phase of dystrophic muscle disease. To our knowledge, this is the first proteomics study of dystrophic skeletal muscle using an in vivo labeling strategy. With this method, we have not only confirmed the previously identified pathways (mitochondria and energy metabolism) that are differentially modulated in Duchenne muscular dystrophy (DMD), but also uncovered novel pathways such as actin cytoskeletal and integrin-linked kinase (ILK) signaling pathways that are implicated in dystrophic pathology. Duchenne muscular dystrophy integrin-linked kinase Ingenuity Pathway Analysis stable isotope labeling with amino acids in cell culture. Duchenne muscular dystrophy integrin-linked kinase Ingenuity Pathway Analysis stable isotope labeling with amino acids in cell culture. C57BL/6 control mice and dystrophin-deficient mdx-52 mice weighing 20 to 25 g were used for breeding to generate SILAC-labeled and unlabeled mice using custom-made mouse feed as described below. The mdx-52 mice were on a C57BL/6 background (7Araki E. Nakamura K. Nakao K. Kameya S. Kobayashi O. Nonaka I. Kobayashi T. Katsuki M. Targeted disruption of exon 52 in the mouse dystrophin gene induced muscle degeneration similar to that observed in Duchenne muscular dystrophy.Biochem. Biophys. Res. Commun. 1997; 238: 492-497Crossref PubMed Scopus (98) Google Scholar). All animals were handled according to Institutional Animal Care and Use Committee guidelines at the Children's National Medical Center (Approved Protocol No. 199-07-01). We followed the method described by Kruger et al. to generate SILAC mice (18Kruger M. Moser M. Ussar S. Thievessen I. Luber C.A. Forner F. Schmidt S. Zanivan S. Fassler R. Mann M. SILAC mouse for quantitative proteomics uncovers kindlin-3 as an essential factor for red blood cell function.Cell. 2008; 134: 353-364Abstract Full Text Full Text PDF PubMed Scopus (547) Google Scholar). Mouse-Express feed containing "heavy" l-lysine (13C6, 99%) or "light" l-lysine (12C6, 99%) at the 1% level that adhered to standard laboratory mouse nutritional standards was purchased from Cambridge Isotope Laboratories (Andover, MA). In this study, we arbitrarily chose to label wild-type C57BL/6 mice. Breeding pairs were set up, and after the confirmation of pregnancy, dams were fed the custom 13C6-lysine diet and breeding was continued to obtain F2-generation litters. In parallel, dystrophin-deficient mdx-52 breeding pairs were maintained on unlabeled custom feed (12C6-lysine) and bred to obtain F2-generation mdx litters. For all validation experiments, an independent set of C57BL/6 and mdx-52 mice (n = 3/group) that had been maintained on non-custom (normal) feed was used. All animals were housed in an individually vented cage system under a controlled 12-hour light/dark cycle with free access to feed and water. SILAC mice and age-matched mdx mice were perfused with phosphate-buffered saline to remove excess blood from organs and tissues and were then euthanized using CO2. All organs including muscle tissues were harvested and flash-frozen in liquid nitrogen-chilled isopentane. The collected tissues were stored at −80 °C until use. Liver, gastrocnemius, and brain were collected from "labeled" C57BL/6 mice of each generation (F0, F1, and F2) in order to monitor the incorporation of 13C-lysine. For differential proteomic analysis between normal and dystrophic mdx mice, tissues were collected from F2-generation labeled C57BL/6 mice and age-matched unlabeled mdx mice. Tissues were collected at 3, 6, and 12 weeks of age from labeled C57BL/6 and mdx mice (n = 2/age group) for this study, as well as for other projects. In the current study, we analyzed 3-week-old mdx gastrocnemius to identify early changes in the dystrophic skeletal muscle proteome. Protein lysates were prepared from tissue samples harvested from the labeled C57BL/6 at F0, F1, and F2 generations. Aliquots (50 μg) of protein lysate from each extract were separated via SDS-PAGE and stained with Bio-Safe Coomassie (Bio-Rad, Hercules, CA). Individual bands were excised and digested with trypsin, and the resulting peptides were analyzed via LC-MS/MS as described below. Raw spectra were analyzed using Integrated Proteomics Pipeline (IP2) software (version 1.01), developed by Integrated Proteomics Applications, Inc (San Diego, CA). Labeling efficiency was determined from unlabeled to labeled peptide ratios obtained for all identified proteins in each tissue (liver, brain, and muscle). These ratios were converted into percentages and then averaged to obtain the overall labeling efficiency for each respective tissue at each generation. Gastrocnemius muscle was collected from 3-week-old F2-generation SILAC-labeled C57BL/6 mice and age-matched unlabeled dystrophic mdx mice. Gastrocnemius muscle was also collected from age-matched unlabeled C57BL/6 mice. Total proteins were extracted from each muscle with RIPA buffer (50 mm Tris-HCl, pH 8.0, with 150 mm sodium chloride, 1.0% Igepal CA-630 (Nonidet P-40), 0.5% sodium deoxycholate, and 0.1% sodium dodecyl sulfate) with protease inhibitors (Halt protease inhibitor mixture 100X). Aliquots of the protein extracts from the muscles of unlabeled C57BL/6 and unlabeled mdx mice were each mixed 1:1 (50 μg) with protein extract from the muscle of a SILAC-labeled mouse. The protein concentration was estimated via BCA protein assay (Pierce). Labeled and unlabeled protein mixtures were further resolved via SDS-PAGE. The gel was stained with Bio-Safe Coomassie (Bio-Rad, Hercules, CA), and each lane was cut into 30 to 35 serial slices. Proteins in each gel slice were in-gel digested with trypsin. The resulting peptides from each band were injected via an autosampler (6 μl) and loaded onto a Symmetry C18 trap column (5 μm, 300 μm inner diameter × 23 mm, Waters Milford, MA) for 10 min at a flow rate of 10 μl/min with 0.1% formic acid. The sample was subsequently separated on a C18 reversed-phase column (3.5 μm, 75 μm × 15 cm, LC Packings Sunnyvale, CA) at a flow rate of 250 nl/min using a Nano-HPLC system from Eksigent (Dublin, CA). The mobile phases consisted of water with 0.1% formic acid (A) and 90% acetonitrile (B). A 65-min linear gradient from 5% to 40% B was employed. Eluted peptides were introduced into the mass spectrometer via a 10-μm silica tip (New Objective Inc., Ringoes, NJ) adapted to a nano-electrospray source (ThermoFisher Scientific). The spray voltage was set at 1.2 kV, and the heated capillary at 200 °C. The LTQ-Orbitrap-XL (ThermoFisher Scientific) was operated in data-dependent mode with dynamic exclusion, in which one cycle of experiments consisted of a full MS survey scan in the Orbitrap (300–2000 m/z, resolution = 30,000) and five subsequent MS/MS scans in the LTQ of the most intense peaks, using collision-induced dissociation with the collision gas (helium) and normalized collision energy value set at 35%. Protein identification and quantification were performed using IP2 software (version 1.01). Mass spectral data were uploaded into the IP2 software. Files from each lane were searched against the forward and reverse Uniprot mouse database (UniProt release 15.15, March 2010, 16,333 forward entries) for partially tryptic peptides, allowing two missed cleavages and the possible modification of oxidized methionine (15.99492 Da) and heavy Lys (6.020 Da). IP2 uses the Sequest 2010 (06 10 13 1836) search engine. The mass tolerance was set at ±30 ppm for MS and ±1.5 Da for MS/MS. Data were filtered by setting the protein false discovery rate at less than 1%. Only proteins that were identified by at least two unique peptides were retained for further quantitative analysis. Census software (version 1.77), built into the IP2 platform, was used to determine the ratios of unlabeled to labeled peptide using an extracted chromatogram approach. Quantitative data were filtered based on a determinant value of 0.5 and an outlier p value of 0.1. To increase the robustness and to statistically validate the data obtained in the initial differential SILAC experiments using mdx and wild-type mice, we employed a spike-in SILAC strategy that was previously used in cell culture systems (24Monetti M. Nagaraj N. Sharma K. Mann M. Large-scale phosphosite quantification in tissues by a spike-in SILAC method.Nat. Methods. 2011; 8: 655-658Crossref PubMed Scopus (127) Google Scholar, 25Geiger T. Wisniewski J.R. Cox J. Zanivan S. Kruger M. Ishihama Y. Mann M. Use of stable isotope labeling by amino acids in cell culture as a spike-in standard in quantitative proteomics.Nat. Protoc. 2011; 6: 147-157Crossref PubMed Scopus (225) Google Scholar). In brief, gastrocnemius muscle lysates were obtained from both unlabeled C57BL/6 (n = 3) and unlabeled mdx (n = 3) mice. These lysates were spiked with equal amounts of lysate from labeled C57BL6 mice that was used as reference. Downstream sample preparation and MS analysis were performed as described above. To identify significant protein alterations between the mdx and wild-type groups, the mean relative ratio (unlabeled/labeled values) was compared for each protein between mdx (n = 3) and control (n = 3) mice using a non-parametric Wilcoxon rank sum test. Significance was set at p < 0.05, and no adjustments for multiple testing were performed. A bioinformatics approach was used to elucidate the global implications of differentially expressed proteins in dystrophic muscle. Ingenuity computational pathway analysis (IPA) (Ingenuity Systems, Redwood City, CA) software was applied to identify potentially perturbed molecular pathways in dystrophic muscle. The IPA program uses a knowledge database derived from the literature to relate the proteins to each other based on their interaction and function. The knowledge base consists of a high-quality expert-curated database containing 1.5 million biological findings consisting of more than 42,000 mammalian genes and pathway interactions extracted from the literature. In brief, proteins that were confidently identified in at least two samples (of both C57BL/6 and mdx comparisons) were considered for IPA analysis. All proteins that fell into the specified criteria were shortlisted, SILAC ratios were converted to fold changes and uploaded into the IPA software. Ingenuity then used these proteins and their identifiers to navigate the curated literature database and extract the overlapping network(s) among the candidate proteins. Associated networks were generated, along with a score representing the log probability of a particular network being found by random chance. Top canonical pathways associated with the uploaded data were presented, along with a p value. The p values were calculated using right-tailed Fisher's exact tests. Validation Using Biochemical Assays Gastrocnemius protein lysates (25 μg of protein) from dystrophic (n = 3) and control (n = 3) muscles were mixed with 4x NuPage LDS buffer (Invitrogen) supplemented with 50 mm DTT, heated for 5 min at 85 °C, loaded on Novex® 4%–12% Tris acetate mini gels (Invitrogen), and electrophoresed at 150 V in MOPS running buffer (20X) for 90 min at room temperature. Separated proteins were transferred at 300 mA for 90 min at room temperature onto a nitrocellulose membrane (Millipore Billerica, MA). Membranes were blocked in TBS-T (20 mm Tris, 500 mm NaCl, pH 7.5, with 0.1% Tween 20) supplemented with 5% nonfat dry milk (Bio-Rad) for 1 h at room temperature. The membranes were then incubated overnight at 4 °C with primary antibodies against desmin (1:2000; Santa Cruz), annexin-2 (1:1000; Santa Cruz), ILK (1:1000; Santa Cruz), vimentin, cofilin, and profilin (1:1000; Epitomics Burlingame, CA). All antibodies were diluted in TBS-T-5% milk. Membranes were washed three times (for 10 min each time) in TBS-T and incubated with goat anti-rabbit or rabbit anti-mouse secondary antibodies (Dako, Carpinteria, CA) conjugated to horseradish peroxidase (1:3000 in TBS-T-5% milk) for 1 h at room temperature. The protein bands were revealed with ECL chemi-luminescence substrate (Amersham Biosciences). Membranes were then stripped and exposed to β-actin antibody as a loading control. For quantification, the x-ray films were scanned, and densitometry analysis was carried out using a BioRad GS-800 calibrated densitometer running Quantity One software (Bio-Rad). Ratios of the optical density of each specific protein to the corresponding β-actin were compared between mdx and C57BL/6 samples in order determine significant differences. Lactate dehydrogenase activity was monitored in mdx (n = 3) and C57BL/6 (n = 3) muscle lysates as described earlier (26Jahnke V.E. Sabido O. Defour A. Castells J. Lefai E. Roussel D. Freyssenet D. Evidence for mitochondrial respiratory deficiency in rat rhabdomyosarcoma cells.PLoS One. 2010; 5: e8637Crossref PubMed Scopus (22) Google Scholar). In brief, 2.5 μl of protein extract (1:2 dilution) and 225 μl of assay buffer (2.5 ml of 1 m Tris (pH 7.6), 500 μl of 200 mm EDTA, 500 μl of 5 mm NADH,H+, and 48 ml water) were used to measure enzyme activity. The oxidation of NADH,H+ was recorded after pyruvate addition (10 μl, 100 mm). NADH fluorescence was detected using a luminescence/fluorescence analyzer (Mithras LB 940, Berthold Technologies Bad Wildbad, Germany). Lactate dehydrogenase activity was normalized to the protein concentration and expressed as the mean ± S.E. Citrate synthase activity (EC 4.1.3.7) was measured in mdx (n = 3) and C57BL/6 (n = 3) muscle lysates as described earlier (26Jahnke V.E. Sabido O. Defour A. Castells J. Lefai E. Roussel D. Freyssenet D. Evidence for mitochondrial respiratory deficiency in rat rhabdomyosarcoma cells.PLoS One. 2010; 5: e8637Crossref PubMed Scopus (22) Google Scholar). In brief, 2.5 μl of protein extract (1:30 dilution, v/v) was added to 225 μl of assay buffer (100 mm Tris, pH 8.0, 2 mm EDTA, 1.25 mm l-malate, 0.25 mm NAD), 0.01% Triton X-100 (v/v), and 6 U/ml malate dehydrogenase (Sigma) to monitor the enzyme activity. The production of NADH,H+ was recorded after the addition of 5 μl acetyl-CoA (50 μm). Enzyme activities were fluorometrically measured (excitation, λ 340 nm; emission, λ 450 nm) and represented as the mean ± S.E. Frozen human muscle biopsies of mutation-defined dystrophin-deficient (DMD) (n = 3) and control (normal) (n = 3) tissues were obtained without any identifiers (IRB Protocol No. 2405). Muscle tissues were sectioned and immunostained using rabbit anti-vimentin (Epitomics) and mouse anti-ILK antibodies (Santa Cruz) and HRP-conjugated anti-rabbit or anti-mouse (Dako, Carpinteria, CA) as the primary and secondary antibodies, respectively. As a specificity control, other serial sections were stained with secondary antibody alone. For all validation assays, the statistical significance betw
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