Muscle Proteomics Reveals Novel Insights into the Pathophysiological

Sep 11, 2014 - (6) In BM patients autophagic impairment and PTP pore opening are less severe or absent,(7, 8) leaving the question of pathophysiologic...
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Muscle Proteomics Reveals Novel Insights into the Pathophysiological Mechanisms of Collagen VI Myopathies Sara De Palma,†,‡,$ Daniele Capitanio,†,§,$ Michele Vasso,‡ Paola Braghetta,∥ Chiara Scotton,⊥ Paolo Bonaldo,∥ Hanns Lochmüller,¶ Francesco Muntoni,# Alessandra Ferlini,⊥ and Cecilia Gelfi*,†,‡,§ †

Department of Biomedical Sciences for Health, University of Milan, Segrate, Milan 20090, Italy Institute of Bioimaging and Molecular Physiology, National Research Council, Cefalù 90015 − Segrate 20090, Italy § IRCCS Policlinico San Donato, San Donato Milanese, Milan 20097, Italy ∥ Department of Molecular Medicine, University of Padova, Padova 35121, Italy ⊥ Department of Medical Sciences, University of Ferrara, Ferrara 44121, Italy ¶ Institute of Genetic Medicine, Newcastle University, Centre for Neuromuscular Diseases, Newcastle upon Tyne NE1 3BZ, United Kingdom # Dubowitz Neuromuscular Centre, University College London, Institute of Child Health, London WC1N 1EH, United Kingdom ‡

S Supporting Information *

ABSTRACT: Mutations in the collagen VI genes cause the Ullrich congenital muscular dystrophy (UCMD), with severe phenotype, and Bethlem myopathy (BM) with mild to moderate phenotype. Both, UCMD and BM patients show dystrophic features with degeneration/regeneration and replacement of muscle with fat and fibrous connective tissue. At molecular level, UCMD patients show autophagic impairment and increased PTP opening; these features are less severe in BM. To elucidate the biochemical mechanisms adopted by the muscle to adapt to collagen VI deficiency in BM and UCMD patients, a proteome analysis was carried out on human muscle biopsies. Qualitative and quantitative differences were assessed by 2D-DIGE coupled to MALDI-ToF/ ToF MS. Proteomics results, coupled with immunoblotting, indicate changes in UPR, hexosamine pathway, and amino acid and fatty acid metabolism, suggesting an association of ER stress, metabolic dysregulation, autophagic impairment, and alteration in mechanotransduction signaling. Overall, these results indicate that despite the common downregulation of hexosamine pathway in UCMD and BM, in BM the protein quality control system is sustained by a metabolic adaptation supporting energy requirements for the maintenance of autophagy, counteracting ER misfolded protein overload. In UCMD, this multilayered system may be disrupted and worsened by the metabolic rewiring, which leads to lipotoxicity. KEYWORDS: Collagen VI, Ullrich congenital muscular dystrophy, Bethlem myopathy, 2D-DIGE, endoplasmic reticulum, unfolded protein response, hexosamine, alpha-ketoglutarate

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INTRODUCTION Collagen VI is an ubiquitously expressed extracellular matrix (ECM) protein, composed of two chains of 140−150 kDa each, named alpha a1(VI) and alpha a2(VI), and one larger chain of 240−300 kDa named alpha a3(VI). In endoplasmic reticulum (ER), the three chains fold together into a triple-helical collagen VI molecule (monomer) that further assembles into dimers and tetramers. The tetramers are secreted into the extracellular space where they form collagen VI beaded microfibrils.1 In muscle, the microfibrillar network of collagen VI surrounds the basement membrane of fibers, binding components of the ECM and transferring mechanical and biochemical signals from ECM to muscle cell. Collagen VI is also present in the interstitial space of many other tissues © 2014 American Chemical Society

including tendon, skin, cartilage, and intervertebral discs. Disorders caused by collagen VI mutations affect muscle and connective tissue, leading to muscle weakness, joint laxity, contractures, and abnormal skin. Mutations in each of the COL6A1, COL6A2, and COL6A3 genes cause two major types of muscle disorders: Ullrich congenital muscular dystrophy (UCMD), characterized by severe phenotype, and Bethlem myopathy (BM) with mild to Special Issue: Proteomics of Human Diseases: Pathogenesis, Diagnosis, Prognosis, and Treatment Received: June 30, 2014 Published: September 11, 2014 5022

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moderate phenotype. Originally considered as separate diseases, the identification of COL6A1−COL6A3 mutations in both of them demonstrated that UCMD and BM are allelic disorders. Dominant and recessive mutations in the three collagen VI genes produce a deficient or dysfunctional microfibrillar network in the ECM of muscle, skin, and tendons. UCMD is characterized by severe muscle weakness, joint contractures, and distal joint hypermobility. Independent ambulation is only acquired in ∼50% of cases, but it is invariably lost even in the second decade of life. Conversely, BM shows a mild to moderate muscle weakness and multiple progressive joint contractures. It is a slowly progressive disorder, and about half of BM patients need aids for ambulation after age of 50.2 The mutational spectrum provides a clear molecular distinction between BM and UCMD. BM are characterized by either single amino acid substitutions disrupting the Gly-X-Y motif of the triple helical domain, splice site mutations shortening the alpha1(VI) chain and preventing further assembly into dimers and missense mutations interfering with protein folding. On the other hand, UCMD is characterized by premature termination codons and by splice mutations leading to in-frame exonic deletions interfering with the monomer assembly. De novo dominant mutations have been also reported. Genotype− phenotype correlations of collagen VI-related muscle disorders are emerging based on the analysis of the specific mutations associated with either a BM or a UCMD phenotype.3−5 It is known that both UCMD and BM patients show dystrophic features with degeneration/regeneration and replacement of muscle with fat and fibrous connective tissue, although to a different extent. Collagen VI immunolabeling in the endomysium and basal lamina ranges from absent to moderately or markedly reduced in UCMD, whereas in a variable proportion of BM cases, it is indistinguishable from controls. At a molecular level, UCMD patients show authophagic impairment and increased PTP opening, which can be ameliorated by cyclosporin A treatment.6 In BM patients autophagic impairment and PTP pore opening are less severe or absent,7,8 leaving the question of pathophysiological mechanisms of these diseases still open. Serum creatine kinase levels are frequently normal or only mildly elevated and cardiac involvement is always absent, suggesting that the mechanisms leading to muscle dystrophy in collagen VI deficiencies are different from those of other muscular dystrophies, such as Duchenne, making the discovery of molecules involved in disease progression challenging. Despite the findings discussed above, a comprehensive study linking ECM alterations and mitochondrial phenotype in BM and UCMD patients, particularly by proteomics, is lacking. In this work, we investigated muscle proteomic changes with the aim to throw some light into the biochemical mechanisms adopted by the muscle to adapt to collagen VI deficiency in BM and leading to muscle wasting and atrophy in UCMD. The data indicate, for the first time in human muscle diseases, alterations in the unfolded protein response (UPR), hexosamine pathway, amino acid and fatty acid metabolism, indicating the association of ER stress, metabolic dysregulation, autophagic impairment, and alteration in mechanotransduction signaling from ECM to muscle cell. Moreover, our findings allowed to identify some specific molecules that could be targeted for possible intervention aimed to improve the phenotype of UCMD patients.

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EXPERIMENTAL SECTION

Ethical Statement

The protocol was approved by the University College London, the University of Newcastle and the University of Ferrara research ethics committees and written informed consent was obtained. Patients

Skeletal muscle biopsies from 8 BM and 4 UCMD patients were collected after overnight fast by Bergstrom needle biopsy from vastus lateralis in the midthigh, frozen in prechilled isopentane, and stored in liquid nitrogen. MRI was adopted to minimize blood and fat contamination. Patients were diagnosed by genetic, biochemical, and immunohistochemical analysis (Supporting Information, Table S1). Biopsies from 12 young male healthy subjects were taken after an overnight fast and in absence of strenuous exercise, samples were frozen in liquid nitrogen. Proteomic Analysis

Protein extraction. Muscle biopsies were ground in a frozen mortar, suspended in lysis buffer (7 M urea, 2 M thiourea, 4% CHAPS, 30 mM Tris, 1 mM PMSF, 1% phosphatase inhibitor cocktail 1 and 2 (Sigma), pH 8.5), and solubilized by sonication on ice. Proteins were selectively precipitated using PlusOne 2DClean up kit (GE Healthcare) in order to remove nonprotein impurities, and resuspended in lysis buffer. Protein extracts were adjusted to pH 8.5 by addition of 1 M NaOH. Protein concentrations were determined by PlusOne 2D-Quant Kit (GE Healthcare). Two-dimensional difference in gel electrophoresis (2DDIGE). Protein labeling, 2D-separation, and analysis were performed exactly as previously described.9 Protein minimal labeling with cyanine dyes (Cy3 and Cy5) was performed, according to manufacturer’s recommendations, by mixing 50 μg of each sample extract with 400 pmol CyDye (GE Healthcare) and incubating, on ice, in the dark for 30 min. The labeling reaction was quenched with 1 mL L-lysine 10 mM on ice for 10 min in the dark. Sample proteins were labeled with Cy5 whereas the internal standard, generated by pooling individual samples (UCMD, BM, and control), was Cy3 labeled. The inclusion of an internal standard improved the matching of intra- and intergel images and allowed normalization across all gels. The combination of Cy5/Cy3, named “two dyes” protocol, was adopted, due to their labeling efficiency and reliability compared with other dye combinations. By using this protocol and manufacturer’s dye/protein ratio, no dye swap was needed because all samples undergoing the statistical analysis were labeled with the same dye (Cy5) and were normalized against the same internal standard. Samples from each subject (40 μg) were combined with an equal amount of internal standard. Each sample was run in triplicate on 24 cm, 3−10 nonlinear pH-gradient IPG strips, with a voltage gradient ranging from 200 to 8000 V, for a total of 75 000 Vh, using an IPGphor electrophoresis unit (GE Healthcare). The adopted pH gradient enabled separation of protein isoforms in the first dimension, providing a detailed pattern of the muscle proteome. After focusing, proteins were reduced and alkylated. The second dimension was carried out in 20 × 25 cm2, 12% T, 2.5% C constant concentration polyacrylamide gels at 20 °C and 15 mA per gel using the Ettan Dalt II system (GE Healthcare). CyDye-labeled gels were visualized and acquired using a Typhoon 9200 Imager (GE Healthcare). Image analysis 5023

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anti-PPARβ (1:1000), anti-PPARγ (1:1000), anti-GLUD1/2 (1:1000), anti-GLUL (1:1000), anti-ODC (1:1000), anti-HK (1:1000), anti-FBP1 (1:1000), anti-GFAT1 (1:1000), antiUAP1(1:1000), anti-OGT (1:1000), anti-OGA (1:1000), and anti-STT3B (1:1000). To confirm 2D-DIGE data, the following blots were performed: anti-ACO2, anti-ATP5B, anti-DES, antiHSPA5 (Santa Cruz Biotechnology), and anti-VIM (Monosan) (dilution: 1:1000) (Supporting Information, Figure S1). After washing, membranes were incubated with antirabbit (GE Healthcare) or antigoat (Santa Cruz Biotechnology) secondary antibodies conjugated with horseradish peroxidase. Signals were visualized by chemiluminescence using the ECL Prime detection kit and the Image Quant LAS 4000 (GE Healthcare) analysis system. Band quantification was performed using the Image Quant TL (Molecular Dynamics) software followed by statistical analysis (ANOVA + Tukey, n = 3, p < 0.05).

was performed using the DeCyder version 6.5 software (GE Healthcare). The proteomic profiles of vastus lateralis muscle of 8 BM and 4 UCMD patients were compared with 12 healthy control subjects. Statistically significant differences of 2D-DIGE data were computed by ANOVA and Tukey’s tests (p < 0.01). False discovery rate (FDR) was applied as a multiple test correction in order to keep the overall error rate as low as possible. Power analysis was conducted on statistically changed spots, and only spots that reached a sensitivity threshold >0.8 were considered as differentially expressed. Protein identification by MALDI-ToF/ToF mass spectrometry. For protein identification, semipreparative gels were loaded with unlabeled sample (400 μg per strip); electrophoretic conditions were the same as 2D-DIGE, and gels were stained with a total-protein fluorescent stain (Deep purple, GE Healthcare). Image acquisition was performed using a Typhoon 9200 laser scanner. Spots of interest were excised from gel using the Ettan spot picker robotic system (GE Healthcare), destained in 50% methanol/50 mM ammonium bicarbonate (AMBIC) and incubated with 30 μL of 6 ng/mL trypsin (Promega) dissolved in 10 mM AMBIC for 16 h at 37 °C. Released peptides were subjected to reverse phase chromatography (Zip-Tip C18 micro, Millipore), eluted with 50% acetonitrile/0,1% trifluoroacetic acid. Peptides mixture (1 μL) was diluted in an equal volume of 10 mg/mL a-cyano-4hydroxycinnamic acid matrix dissolved in 70% acetonitrile/30% citric acid and processed on a Ultraflex III MALDI-ToF/ToF (Bruker Daltonics) mass spectrometer. Mass spectrometry was performed at an accelerating voltage of 20 kV and spectra were externally calibrated using Peptide Mix calibration mixture (Bruker Daltonics); 1000 laser shots were taken per spectrum. Spectra were processed by FlexAnalysis software v. 3.0 (Bruker Daltonics) setting the signal-to-noise threshold value to 6 and search was carried out by correlation of uninterpreted spectra to Homo sapiens entries (178 655 sequences) in NCBInr 20100918 (11 833 178 sequences; 4 040 378 175 residues). The significance threshold was set at p value < 0.05. No mass and pI constraints were applied and trypsin was set as enzyme. One missed cleavage per peptide was allowed and carbamidomethylation was set as fixed modification while methionine oxidation as variable modification. Mass tolerance was set at 30 ppm for MS spectra. To confirm protein identification, a MS/ MS spectrum was collected by Ultraflex III MALDI-ToF/ToF (Bruker Daltonics) mass spectrometer, as acceptance criterium. Spectra were searched against the database using BioTools v. 3.2 (Bruker Daltonics) interfaced to the online MASCOT software, which utilizes a robust probabilistic scoring algorithm. The significance threshold was set at p value < 0.05. One missed cleavage per peptide was allowed and carbamidomethylation was set as fixed modification, whereas methionine oxidation as variable modification. Mass tolerance was set at 30 ppm and 0.5 Da for peptide and MS/MS fragment ion, respectively.

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RESULTS

Proteomic Analysis of BM and UCMD Patients vs Healthy Controls

Vastus lateralis muscle extracts were analyzed by 2D-DIGE to evaluate proteome changes in BM and UCMD patients compared to healthy controls. Overall, 60 spots over 1000 matched spots among all gels resulted as differentially expressed. Among them, 44 spots were identified by mass spectrometry. A representative proteomic map (Supporting Information, Figure S2) and the statistical analysis results are shown in Supporting Information together with MALDI-ToF/ ToF MS data (Table S2). The proteomic profile revealed common alterations in both BM and UCMD vs controls (Figure 1A). In particular, a shift toward a decrement of anaerobic metabolism (i.e., aldolase A, ALDOA; triosephosphate isomerase, TPI1; and phosphoglycerate mutase, PGAM2), oxidative metabolism (i.e., pyruvate dehydrogenase, PDHB; aconitase 2, ACO2; and malate dehydrogenase, MDH1), and in energy production (i.e., ATP synthase d subunit, ATP5H) was observed. Contractile and cytoskeletal regulatory proteins (fast skeletal myosin regulatory light chain, MYL1 and sarcosin, KBTBD10) were increased in patients vs controls, whereas structural proteins (alpha actin, ACTA1 and slow troponin T, TNNT1) were decreased. The cytosolic stress response proteins heat shock 70 kDa (HSPA8), and carbonic anhydrase 3 (CAIII) were increased, whereas the heat shock protein 27 kDa (HSPB1) located also in ER, decreased in patients compared to controls, together with the transport protein myoglobin (MB). However, a number of proteins were exclusively changed in one group of patients vs controls. BM patients were characterized by a decrement of glyceraldehyde-3-phosphate dehydrogenase (GAPDH), a dysregulation of two isoforms of beta-enolase (ENO3) and an increment of lactate dehydrogenase A (LDHA), together with the ATP synthase complex subunit beta (ATP5B), which was strongly increased. The structural/contractile proteins keratin type 1 (KRT10) and TNNT1 were increased, with the exception of desmin (DES) that was slightly decreased (Figure 1B). On the other hand, UCMD patients showed a decrement of additional isoforms of anaerobic/oxidative proteins (ALDOA, ACO2). The respiratory chain protein ubiquinol− cytochrome c reductase (UQCRC1) was decreased, as for the ATP5B subunit of the ATP synthase complex. The same protein was increased in BM, whereas UCMD are characterized

Immunoblotting

Protein extracts (50 μg) from pooled BM, UCMD and healthy control muscles were loaded in triplicate and resolved on 6%, 10%, and 12% polyacrylamide gels, according to protein molecular weight. Blots were incubated with rabbit or goat polyclonal primary antibodies (Cell Signaling Technology and Santa Cruz Biotechnology) as follows: anti-TN-C (1:500), antiFAK (1:500), anti-Sirt3 (1:1000), anti-IDH1 (1:500), antiACLY (1:1000), anti-FASN (1:500), anti-PPARα (1:1000), 5024

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Figure 1. continued TPI1, triosephosphate isomerase 1; PGAM2, phosphoglycerate mutase; PDHB, pyruvate dehydrogenase E1 component subunit Beta; ACO2, aconitase 2; MDH1, cytosolic malate dehydrogenase; ATP5H, ATP synthase, H+ transporting, mitochondrial F0 complex, subunit d isoform a; MYL1, fast skeletal myosin alkali light chain 1 isoform 1f; KBTBD10, sarcosin; ACTA1, alpha actin precursor; TNNT1, Troponin T, slow skeletal muscle; HSPA8, heat shock 70 kDa protein 8 isoform 1; HSPB1, heat shock protein 27; CAIII, carbonic anhydrase III; MB, myoglobin; GAPDH, glyceraldehyde-3phosphate dehydrogenase; ENO3, enolase 3; LDHA, lactate dehydrogenase A; ATP5B, mitochondrial ATP synthase, H+ transporting F1 complex beta subunit; KRT10, keratin, type I cytoskeletal 10; DES, desmin; UQCRC1, ubiquinol-cytochrome c reductase core protein I; ATP5A1, ATP synthase, H+ transporting, mitochondrial F1 complex, alpha subunit; GOT1, aspartate aminotransferase; CKM, creatine kinase; MYLPF, myosin light chain 2; KRT1, type II keratin subunit; TNNT3, troponin T3; VIM, vimentin; TF, transferrin; ALB, albumin; HSPA5, glucose regulated 78 kDa protein.)

by increment of alpha ATP synthase subunit (ATP5A1). Two isoforms of aspartate aminotransferase (GOT1) and three isoforms of muscle creatine kinase (CKM) were decreased in UCMD compared to controls. Structural/contractile proteins (myosin light chain 2, MYLPF, keratin type II, KRT1 and fast troponin T, TNNT3) were decreased, with the exception of vimentin (VIM). Transport proteins (transferrin, TF and albumin, ALB) were increased, whereas an additional isoform of MB was decreased. Glucose-regulated protein 78 kDa (HSPA5) was increased compared to control, suggesting the activation of the UPR signaling in the ER (Figure 1C). Validation of 2D-DIGE Results

The protein identification was validated performing a random analysis via immunoblotting of 16% of the identified proteins (see Supporting Information, Figure S1). Pathways Analysis by Immunoblotting

Although the dysregulated glycolytic and TCA cycle enzymes operate at a fraction of their maximal enzymatic rate, their decrease could lead to a restriction of the entire pathway in patients. This observation opens the question of the use of an alternative energy source to sustain muscle function. As is well known, in the absence of glucose, glutamine is the major substrate available to the cells. In skeletal muscle, glutamine can act as a nitrogen donor for the synthesis of proteins and nucleosides10 and be converted to alphaketoglutarate (α-KG), thereby supporting anaplerotically the TCA cycle, or be reductively transformed by cytosolic isocitrate dehydrogenase (IDH1) to citrate.11−14 The latter enzyme was upregulated in the Col6a1 null animal model.15 The α-KG fate was investigated by immunoblotting. In particular, our attention was focused on the reductive/lipogenic pathway (isocitrate dehydrogensase 1, IDH1; citrate synthase, ACLY; fatty acid synthase, FASN) and related regulators (peroxisome proliferator-activated receptors, PPARs), on the conversion of α-KG to glutamate (glutamate dehydrogenase, GLUD1/2), on glutamine biosynthesis (glutamine syntethase, GLUL) and on the first step of polyamines biosynthetic pathway (ornithine decarboxylase, ODC). Although BM patients did not show significant changes compared to controls, UCMD showed increased levels of IDH1, ACLY, and FASN. The PPARβ and γ were increased as well. Conversely, GLUD1/2, GLUL, and

Figure 1. Histograms of differentially expressed proteins in BM (light gray bars) and UCMD patients (dark gray bars) vs healthy controls. Isoforms of proteins significantly altered (ANOVA and Tukey’s test, p < 0.01) are expressed as % of spot volume variation in BM or UCMD versus controls. Panel A: proteins differentially expressed in both BM and UCMD patients vs healthy controls. Panel B: proteins deregulated only in BM patients vs healthy controls. Panel C: proteins deregulated only in UCMD patients vs healthy controls. (ALDOA, aldolase A; 5025

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Figure 2. Representative histograms and immunoblot images of IDH1, ACLY, FASN, PPARα, β and γ, GLUD1/2, GLUL, and ODC (n = 3; mean ± S.D.; ANOVA and Tukey’s test, p < 0.05) in BM (gray bars) and UCMD (black bars) patients and healthy controls (white bars).

Hexosamine biosynthetic pathway (HBP) generates intermediates for N- and O-linked protein glycosylation, and it is a critical component of the protein homeostasis in close association with ER stress. The HBP relies on glucose and glutamine uptake.19 Fructose 6-phosphate, is converted through the enzymes of the HBP into the end product UDP-Nacetylglucosamine (UDP-GlcNAc), a substrate required for the biosynthesis of N- and O-glycans.20 It also serves as the substrate for UDP-N-acetylglucosamine/peptide N-acetylglucosaminyltransferase (OGT), an enzyme catalyzing the transfer of the GlcNAc moiety onto the free hydroxyl of specific serine and threonine residues of target proteins. This modification is removed by N-acetylglycosidase (OGA) that catalyzes cleavage of O-GlcNAc from proteins21 (Figure 4A). Results indicated, for both BM and UCMD, a decrement of glutamine/fructose-6phosphate aminotransferase (GFAT1) and UDP-N-acetylhexosamine pyrophosphorylase (UAP1), compared to healthy controls (Figure 4B). The O-glycosylating enzyme OGT was unchanged, whereas OGA decreased in both groups, suggesting a dysregulation in deglycosilation processes. The dolichyldiphosphooligosaccharide/protein glycosyltransferase subunit STT3B of the N-oligosaccharyl transferase complex was decreased in both BM and UCMD compared with controls (Figure 4B), indicating an impairment of N-linked protein glycosylation in the ER.

ODC were decreased in UCMD patients compared to controls (Figure 2). These results, combined with proteomic data, indicate in UCMD patients the presence of a metabolic rewiring at the level of α-KG (Figure 3) that leads to the shortage of glutamate-derived molecules with protective functions (glutamine, glutathione, polyamines), and alternatively, triggers lipogenesis, which can have detrimental effects causing lipotoxicity over time. The second question opened by proteomic results is the link between glucose depletion and ER stress. Because glucose is essential for protein glycosylation, if intracellular glucose levels decrease, the carbohydrate chain that is frequently used to glycosylate proteins cannot be assembled, leading to improper protein glycosylation, protein misfolding, activation of the UPR, and increased ER stress.16 The carbohydrate chain contains three glucose, nine mannose, and two N-acetylglucosamine residues.17 The insufficient supply of substrates (glucose and glucosamine), which is supported by the decrement of hexokinase (HK), glutamine syntethase (GLUL), and the gluconeogenetic enzyme fructose-1,6-bisphosphatase (FBP1) in both BM and UCMD can lead to impaired protein glycosylation contributing to ER stress and the activation of UPR, as suggested by HSPA8 increment18 in BM and UCMD patients. The phenotype is exacerbated in UCMD by HSPA5 increase and impaired autophagy.7 Overall these concepts suggests the analysis of pathways controlling glycosylation processes.

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DISCUSSION UCMD and BM are allelic conditions due to deficiency of collagen VI, a major muscle ECM protein that has both 5026

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Figure 3. Alpha-ketoglutarate rewiring. Schematic representation of metabolic enzymes deregulated in BM and UCMD muscle patients compared to healthy control subjects. Arrows indicate the protein trend in BM and UCMD muscle patients vs controls. Dark gray rectangles indicate results obtained by immunoblotting, whereas light gray rectangles indicate 2D-DIGE results.

structural and signaling roles. The consequences of collagen VI deficiency are becoming increasingly clear, and in this study, we concentrated on proteomics analysis of muscle biopsies from patients with molecularly confirmed UCMD and BM. Our study suggests that the muscle of UCMD patients is metabolically characterized by a decrement of glycolytic flux, TCA cycle, and respiratory chain. This profile is associated with increased IDH1 levels, which generate lipogenic citrate. Lipotoxicity and the resulting cell death are further sustained by an increment of PPAR γ and β, triggering detrimental lipid signaling, and inhibiting glycolytic genes. Recently, a proteomic

study of muscles of Col6a1 null mice revealed a metabolic dysfunction characterized by rewiring at the α-ketoglutarate level, generating lipotoxicity associated with changes in mechanotransduction signaling from ECM to muscle cell.15 In UCMD patients, lipid accumulation, sparked off by FASN upregulation, resembles the profile previously observed in the diaphragm, which is the most affected muscle of Col6a1 null mice.22 This condition is further sustained in patients by a decrease of polyamine synthesis.23 In keeping with results from diaphragm of Col6a1 null mice,15 tenascin-C increased in UCMD muscle, suggesting changes in the ECM−integrin− 5027

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Figure 4. Panel A: schematic representation of hexosamine biosynthetic pathway. Gray rectangles indicate results obtained by immunoblotting. Panel B: immunoblot image of HK, FBP1, GLUL, GFAT1, UAP1, OGT, OGA, STT3B (n = 3; mean ± S.D.; ANOVA and Tukey’s test, p < 0.05) in BM (gray bars) and UCMD (black bars) patients and healthy controls (white bars). 5028

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costameric system.24,25 The latter transduces altered signals to muscle fibers, resulting in sarco/endoplasmic reticulum stress,26−28 Ca2+ deregulation,4,25 and activation of signaling pathways including FAK4,26,29 (see Supporting Information, Figure S3). Our result in muscle from BM patients identified a glycolytic flux decrement similar to UCMD patients, whereas in BM, we observed an increase of LDHA and ATP5B, suggesting a possible compensatory mechanism. The increment of LDHA may result in a conversion of pyruvate to lactate, which generates NAD+ from NADH, allowing glycolysis to persist. The lactate is exported from cells generating glucose through the Cori cycle. For this reason, only a fraction of pyruvate is converted to acetyl-CoA by pyruvate dehydrogenase (PDH). In BM, pyruvate enters the TCA cycle, where it generates citrate to sustain, even partially, the cycle. Conversely in UCMD, the fraction of pyruvate that does not flux into the TCA is used for the synthesis of fatty acids to generate lipids leading to lipotoxicity. In BM, the increment of ATP5B subunit of the ATP synthase complex, which is characterized by 10 potential lysine acetylation sites, associated with unchanged levels of Sirt3, suggests that this protein can possibly be stabilized through acetylation.30 The dysregulation of the latter was observed also in tibialis and gastrocnemius muscles of Col6a1 null mice. To clarify the role of ATP5B subunit overexpression, further investigations, focused on post-translational modifications, are in progress in animal models. Noteworthy, in BM the autophagic impairment is less severe,7 FASN is not significantly increased, enzymes regulating polyamine synthesis are at physiological level. However, due to the decrement of HK, GLUL, FBP1, GFAT1 (the first limiting enzyme of the hexosamine pathway), UAP1, and STT3B, a blunt of enzymes regulating N- and OGlcNAcylation processes is expected. As it is well known, the proper ER protein folding requires a covalent attach of Nlinked glycan oligosaccharides, suggesting that a decrement of N-glycan precursors, induced by a decrease of enzymes regulating the hexosamine pathway, can affect ER protein homeostasis. Furthermore, O-GlcNAcylation can alter protein function directly or, in some cases, by competing with phosphorylation sites regulating cellular signaling and transcription regulatory pathways in response to altered nutrients and stress.31 The results of this proteomic study demonstrate that a decrement of enzymes regulating N- and OGlcNAcylation occurs in both BM and UCMD patients. This observation can be directly correlated to mutation in collagen VI genes, with a decrease in glycosylation processes, which can result in ER stress and in the activation of UPR. This signaling cascade could result in attenuated translation and induction of ER-resident chaperones, as suggested by the increase in HSPA5 observed in UCMD patients.32 In addition, the decrease in autophagy, previously observed in UCMD, may affect the protein quality control and contributes to disruption of the ER system, affecting proteostasis.33 In fact, it is well known that an impairment in autophagy blunts the ER-associated protein degradation (ERAD) machinery in combination with the ubiquitin-proteasome system, both of them committed to remove ER luminal unfolded proteins for cytosolic degradation.34 Nevertheless, BM patients can cope with this dysregulation conceivably by keeping NAD+/NADH imbalance under control, sustaining anaerobic metabolism and

utilizing polyamines to provide anaplerotic substrates for maintenance of autophagy. Our results suggest that the signaling provided by mutated collagen VI affects glycosylation processes both in BM and UCMD, even though the outcome of this signal is at variance: (a) in BM the protein quality control system is probably still active and can be sustained by a metabolic adaptation that can support the energy requirements for the autophagic process maintenance, counteracting ER misfolded protein overload and (b) in UCMD this multilayered system may be disrupted and worsened by the metabolic rewiring which leads to lipotoxicity. These results provide useful information about potential targets for therapy, highlighting specific pathways (i.e., glucose uptake, lactate production by modulating PDH activity and polyamines synthesis) that can be targeted with commercially available drugs (e.g., metformin, phenformin, the PDH inhibitor AZD754, polyamines supplementation) in order to modify the onset and progression of the disease. A major strength of this study lies in the combined analysis of the human muscle proteome with some specific signaling molecules underlying metabolic rewiring and their impact on protein glycosylation and ER stress. One of the limitations, beside the inability of 2D-DIGE to identify species under 125 picomoles, is represented by the limited availability of human muscle biopsies (less than 10 mg). The latter hampers an extensive validation on all samples, more specific analysis such as enzymes activity assays, fiber type characterization, a deeper analysis on target molecules involved in hexosamine pathway, and UPR and functional assays targeting ER stress−autophagy system. These studies are in progress in collagen VI null mouse where, even with the well-known limitations of animal models, additional information may provide further valuable clues on targets for therapy in collagen VI myopathies.

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ASSOCIATED CONTENT

S Supporting Information *

2D-DIGE data confirmation by immunoblotting is shown in Figure S1. A representative vastus lateralis muscle 2D-map, showing identified spot localization, is presented in Figure S2. Immunoblottings of tenascin C, focal adhesion kinase and sirtuin 3 are supplied in Figure S3. Patients characterization is shown in Table S1. MS identification data are listed in Table S2. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*C. Gelfi. E-mail: cecilia.gelfi@unimi.it. Tel.: +39(02)5033 0475. Fax: +39(02)2171 7558. Address: Department of Biomedical Sciences for Health, University of Milan,Via F.lli Cervi 93, 20090 Segrate, Milan, Italy. Author Contributions $

These authors contributed equally. All authors have given approval to the final version of the manuscript. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work has been supported by EC, FP7 BIO-NMD project (project number: 241665), and the Telethon Foundation (project GGP08017D and GGP110827). 5029

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dx.doi.org/10.1021/pr500675e | J. Proteome Res. 2014, 13, 5022−5030