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.
Highlights
From the ‡Research Center for Genetic Medicine, Children’s National Medical Center, 111 Michigan Ave NW, Washington, D.C.; §Institute of Biomedical Sciences, Department of Integrative Systems Biology, The George Washington University, 2300 Eye Street NW, Ross 605, Washington, D.C.; ¶Department of Molecular Therapy, National Institute of Neuroscience, National Center of Neurology and Psychiatry, 4-1-1 Ogawa-higashi, Kodaira, Tokyo
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 [6]
We found that the heavy-lysine diet did not affect the overall health of the C57BL/6 mice, including with regard to their body weight and fertility
Summary
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 [1]. 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. This model continues to be important for studying the consequences of dystrophin deficiency and alteration in the molecular events that lead to muscle pathology. We performed proteomic studies of early disease stages in the mdx mouse model
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