title: Identification of disease specific pathways using in vivo SILAC proteomics in dystrophin deficient mdx mouse.
abstract: 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 (13)C6-lysine or stable isotope labeling in mammals (SILAM) with (15)N 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.
authors.affiliation: Research Center for Genetic Medicine, Children's National Medical Center, 111 Michigan Ave NW, Washington, DC, USA
authors.forename: Sree
authors.initials: S
authors.lastname: Rayavarapu
authors.forename: William
authors.initials: W
authors.lastname: Coley
authors.forename: Erdinc
authors.initials: E
authors.lastname: Cakir
authors.forename: Vanessa
authors.initials: V
authors.lastname: Jahnke
authors.forename: Shin'ichi
authors.initials: S
authors.lastname: Takeda
authors.forename: Yoshitsugu
authors.initials: Y
authors.lastname: Aoki
authors.forename: Heather
authors.initials: H
authors.lastname: Grodish-Dressman
authors.forename: Jyoti K
authors.initials: JK
authors.lastname: Jaiswal
authors.forename: Eric P
authors.initials: EP
authors.lastname: Hoffman
authors.forename: Kristy J
authors.initials: KJ
authors.lastname: Brown
authors.forename: Yetrib
authors.initials: Y
authors.lastname: Hathout
authors.forename: Kanneboyina
authors.initials: K
authors.lastname: Nagaraju
mesh_terms: D000818:Animals; D001921:Brain / Q000378:metabolism; D016189:Dystrophin / Q000172:deficiency; D006801:Humans; D008099:Liver / Q000378:metabolism; D053858:Metabolic Networks and Pathways; D051379:Mice; D008810:Mice, Inbred C57BL; D018101:Mice, Inbred mdx; D024101:Mitochondrial Proteins / Q000378:metabolism; D018482:Muscle, Skeletal / Q000378:metabolism; D020388:Muscular Dystrophy, Duchenne / Q000378:metabolism*; D009928:Organ Specificity; D020543:Proteome / Q000378:metabolism*; D040901:Proteomics
chemical_list: D016189:Dystrophin; D024101:Mitochondrial Proteins; D020543:Proteome