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Proteomic Profiling of Mdx-4Cv Serum Reveals Highly Elevated Levels of the Inflammation-Induced Plasma Marker Haptoglobin in Muscular Dystrophy

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Proteomic Profiling of Mdx-4Cv Serum Reveals Highly Elevated Levels of the Inflammation-Induced Plasma Marker Haptoglobin in Muscular Dystrophy INTERNATIONAL JOURNAL OF MOLECULAR MEDICINE 39: 1357-1370, 2017 Proteomic profiling ofmdx-4cv serum reveals highly elevated levels of the inflammation-induced plasma marker haptoglobin in muscular dystrophy SANDRA MURPHY1, PAUL DOWLING1, MARGIT ZWEYER2, MICHAEL HENRY3, PAULA MELEADY3, RUSTAM R. MUNDEGAR2, DIETER SWANDULLA2 and KAY OHLENDIECK1 1Department of Biology, Maynooth University, National University of Ireland, Maynooth, Co. Kildare, Ireland; 2Department of Physiology II, University of Bonn, D‑53115 Bonn, Germany; 3National Institute for Cellular Biotechnology, Dublin City University, Dublin 9, Ireland Received November 16, 2016; Accepted March 29, 2017 DOI: 10.3892/ijmm.2017.2952 Abstract. X-linked muscular dystrophy is caused by primary Introduction abnormalities in the Dmd gene and is characterized by the almost complete loss of the membrane cytoskeletal protein Duchenne muscular dystrophy is a devastating muscle dystrophin, which triggers sarcolemmal instability, abnormal wasting disease of early childhood (1) and the most frequently calcium homeostasis, increased proteolysis and impaired exci- inherited neuromuscular disorder (2). It has an X-linked tation-contraction coupling. In addition to progressive necrosis, mode of inheritance and is primarily caused by abnormalities crucial secondary pathologies are represented by myofibrosis in the Dmd gene, which trigger the almost complete and the invasion of immune cells in damaged muscle fibres. In absence of the membrane cytoskeletal protein dystrophin of order to determine whether these substantial changes within 427 kDa (3). The loss of dystrophin results in a significant the skeletal musculature are reflected by an altered rate of reduction of dystrophin-associated glycoproteins, which in protein release into the circulatory system or other plasma turn impairs the linkage between the extracellular matrix fluctuations, we used label‑free mass spectrometry to charac- component laminin and the actin membrane cytoskeleton (4). terize serum from the mdx‑4cv model of Duchenne muscular In addition to destabilizing the fibre periphery and thereby dystrophy. Comparative proteomics revealed a large number of making muscle cells more susceptible to micro-rupturing increased vs. decreased protein species in mdx‑4cv serum. A during excitation-contraction-relaxation cycles (5), a variety serum component with greatly elevated levels was identified as of secondary changes play a key role in the molecular the inflammation‑inducible plasma marker haptoglobin. This pathogenesis of X-linked muscular dystrophy (6). This includes acute phase response protein is usually secreted in relation reactive myofibrosis resulting in tissue scarring and loss of to tissue damage and sterile inflammation. Both immunoblot fibre elasticity, abnormal calcium regulation and increased analyses and enzyme‑linked immunosorbent assays confirmed proteolytic degradation, impaired cellular signalling and the increased concentration of haptoglobin in crude mdx‑4cv disturbed excitation-contraction coupling, as well as sterile serum. This suggests that haptoglobin, in conjunction with inflammation of dystrophin-deficient muscle tissue (7-10). other altered serum proteins, represents a novel diagnostic, Therefore, the immune system appears to play a critical role in prognostic and/or therapy-monitoring biomarker candidate X-linked muscular dystrophy (11-14). It is debatable whether to evaluate the inflammatory response in the mdx‑4cv animal the inflammatory pathology of dystrophic muscles is based on model of dystrophinopathy. a reactive and relatively non‑specific invasion of immune cells in damaged muscle fibres or represents a separate mechanism that promotes fibre degeneration independent of sarcolemmal destabilisation (15-17). Thus, although dystrophinopathies are triggered by a single gene defect, the pathophysiological Correspondence to: Professor Kay Ohlendieck, Muscle Biology consequences and adaptive responses of dystrophic muscles Laboratory, Department of Biology, Maynooth University, National are highly complex. University of Ireland, Maynooth, Co. Kildare, Ireland In the pre-proteomic era, a variety of muscle-derived E-mail: [email protected] protein markers have been established for general diagnostic purposes. Serum markers with an association to tissue damage Key words: acute phase plasma protein, biomarker, dystrophin, and muscular injury include creatine kinase, pyruvate kinase, dystrophinopathy, haptoglobin, sterile inflammation hemopexin, adenylate kinase, carbonic anhydrase and lactate dehydrogenase (18-22). However, the concentration of some of these serum markers vary considerably in healthy individuals, lack a high degree of specificity and/or exhibit inconsistencies 1358 MURPHY et al: INCREASED SERUM HAPTOGLOBIN IN MUSCULAR DYSTROPHY in their concentration changes during acute vs. chronic Ultrapure acrylamide stock solutions for gel electrophoresis disease stages. This potential lack of diagnostic reliability were purchased from National Diagnostics (Atlanta, GA, and limited robustness restricts the clinical usefulness of USA). Invitrogen (Carlsbad, CA, USA) supplied Whatman many general protein biomarkers of skeletal muscle damage nitrocellulose transfer membranes. The chemiluminescence for accurate sample screening, prognosis or clinical outcome substrate and protease inhibitors were obtained from Roche measures. More specific serum proteins that exhibit elevated Diagnostics GmbH (Mannheim, Germany). Superfrost Plus levels in Duchenne patients seem to be the tissue inhibitors positively-charged microscope slides were obtained from of metalloproteinase-1, transforming growth factor TGF-β1 Menzel Glaser (Braunschweig, Germany). Sequencing grade and the matrix metalloproteinase MMP-9 (23-26). In contrast modified trypsin and Lys‑C were purchased from Promega Corp. to traditional biochemical studies that usually focus on the (Madison, WI, USA). Pierce C18 Spin Columns were obtained characterization of a restricted number of specific candidate from Thermo Fisher Scientific (Dublin, Ireland). Primary molecules, large-scale biochemical approaches promise to antibodies were purchased from Abcam (Cambridge, UK) gain new global insights into the molecular and cellular (ab131236 to haptoglobin, ab14225-200 to albumin, ab52488 mechanisms of muscular dystrophy. Mass spectrometry-based to lactate dehydrogenase, ab9033-1 to transferrin, ab15277 proteomics is highly suitable to improve our general to dystrophin and ab11427 to parvalbumin) and Santa Cruz understanding of complex changes in dystrophinopathies (27). Biotechnology, Santa Cruz, CA, USA (sc‑25607 to myoglobin). Systems biological methods promise to determine potential Chemicon International, Inc. (Temecula, CA, USA) provided pathophysiological hierarchies and interconnectivities peroxidase-conjugated secondary antibodies. Enzyme-linked between differing secondary mechanisms within muscle immunosorbent assay assays (ELISA) were purchased from tissues that are downstream of the pathobiochemical Abcam (ab157714 to haptoglobin and ab157711 to complement collapse of the dystrophin-glycoprotein complex (28). Most C3) and R&D Systems (FABP‑1/L‑FABP). Normal goat serum importantly, the systematic proteomic identification of new and Cy3-conjugated antibodies were obtained from Jackson serum biomarkers is crucial for the improved diagnostic and ImmunoResearch Laboratories, Inc. (West Grove, PA, USA). prognostic evaluation of Duchenne muscular dystrophy and The embedding medium Fluoromount-G was obtained from animal models of dystrophin deficiency (29). Southern Biotech (Birmingham, AL, USA). A variety of other In the field of muscular dystrophy research, the systematic general chemicals, including bis-benzimide Hoechst-33342, screening of body fluids from Duchenne patients and were of analytical grade and obtained from Sigma Chemical established animal models of dystrophinopathies using Company (Dorset, UK). proteomic technology has recently established a variety of new biomarker candidates, including fibronectin, the coagulation Dystrophic mdx‑4cv animal model of Duchenne muscular factor XIIIa, titin, various myosin light chain isoforms, dystrophy. In order to investigate the serum proteome and myomesin-3, filamin-C, lysosomal-associated membrane identify potential biomarkers of dystrophinopathy, the mdx‑4cv protein LAMP1 and its accompanying vesicle proteins, aldolase, animal model was employed in the present study. This mouse phosphoglycerate mutase, enolase, glycogen phosphorylase, model has been generated using N-ethylnitrosourea to induce fatty acid-binding protein, myoglobin, cytochrome c, malate a premature stop codon in exon 53 of the Dmd gene (44). dehydrogenase, fibrinogen, parvalbumin, electron transfer The mdx‑4cv model has 10-fold fewer dystrophin-positive flavoprotein A, troponin, Ca2+/calmodulin-dependent protein revertant fibres (41) than the conventional mdx mouse (45) and kinase Camk2b, metalloproteinase Adamts5, troponin-1, thus its serum proteome may be more representative of the myoglobin and heat-shock protein HSPA1A (30-39). In human condition. Mice were bred in the Bioresource Unit of analogy to these studies and to establish new biomarker the University of Bonn under standard conditions (46) with candidates for the improved evaluation of animal models of the authorization by the District Veterinary Office in Bonn, dystrophinopathy, we analysed serum from the dystrophic Germany. Tissue and organ harvesting was regularly
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