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 Lochmü ller, ¶ 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 impair- ment 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 differ- ences 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 ■ 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 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 Article pubs.acs.org/jpr © XXXX American Chemical Society A dx.doi.org/10.1021/pr500675e | J. Proteome Res. XXXX, XXX, XXX−XXX