Proteomic analysis of dystrophic muscle.
ABSTRACT Mass spectrometry-based proteomics had a major impact on the global characterization of skeletal muscles and has decisively enhanced the field of neuromuscular pathology. Proteomic profiling of x-linked muscular dystrophy has identified a large number of new signature molecules involved in fiber degeneration. Here, we describe the difference in-gel electrophoretic analysis of the dystrophic diaphragm muscle from the MDX mouse model of Duchenne muscular dystrophy. This chapter summarizes the various experimental steps involved in muscle proteomics, such as sample preparation, fluorescence labeling, isoelectric focusing, second-dimension slab gel electrophoresis, image analysis, in-gel digestion and electrospray ionization mass spectrometry.
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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 proteomic techniques would help to identify disease specific pathways. Recent advances in in vivo metabolic labeling of mammals by stable isotope-labeled amino acids [13C6-lysine (SILAC mouse) or 15N (SILAM)] have enabled accurate quantitative analysis of the proteomes of the whole organs and tissues as a function of disease. Here we describe the use of 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 versus wild type (normal) mice. Generated data was further confirmed in an independent set of mdx and normal mice using 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. Bioinformatics analyses using Ingenuity Pathway software established that the integrin-linked kinase pathway, actin cytoskeleton signaling, mitochondrial energy metabolism, calcium homeostasis are the initial pathways to be affected in dystrophin deficient muscle at early stage of the pathogenesis. Key proteins involved in these pathways were validated by immunoblotting and immunohistochemistry in independent set of mdx mice samples 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.Molecular & Cellular Proteomics 01/2013; · 7.25 Impact Factor
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ABSTRACT: In basic and applied myology, gel-based proteomics is routinely used for studying global changes in the protein constellation of contractile fibers during myogenesis, physiological adaptations, neuromuscular degeneration and the natural aging process. Since the main proteins of the actomyosin apparatus and its auxiliary sarcomeric components often negate weak signals from minor muscle proteins during proteomic investigations, we have here evaluated whether a simple pre-fractionation step can be employed to eliminate certain aspects of this analytical obstacle. In order to remove a large portion of highly abundant contractile proteins from skeletal muscle homogenates without the usage of major manipulative steps, differential centrifugation was used to decisively reduce the sample complexity of crude muscle tissue extracts. The resulting protein fraction was separated by two-dimensional gel electrophoresis and 2D-landmark proteins identified by mass spectrometry. To evaluate the suitability of the contractile protein-depleted fraction for comparative proteomics, normal versus dystrophic muscle preparations were examined. The mass spectrometric analysis of differentially expressed proteins, as determined by fluorescence difference in-gel electrophoresis, identified 10 protein species in dystrophic mdx hind limb muscles. Interesting new biomarker candidates included Hsp70, transferrin and ferritin, whereby their altered concentration levels in dystrophin-deficient muscle was confirmed by immunoblotting.Analytical Biochemistry 08/2013; · 2.58 Impact Factor
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ABSTRACT: Muscular dystrophy is a heterogeneous group of genetic disorders characterised by progressive muscle tissue degeneration. No effective treatment has been discovered for these diseases. Preclinical and clinical studies aimed at the development of new therapeutic approaches have been carried out, primarily in subjects affected with dystrophinopathies (Duchenne and Becker muscular dystrophy). In this review, we outline the current therapeutic approaches and past and ongoing clinical trials, highlighting both the advantages and limits of each one. The experimental designs of these trials were based on different rationales, including immunomodulation, readthrough strategies, exon skipping, gene therapy, and cell therapy. We also provide an overview of available outcome measures, focusing on their reliability in estimating meaningful clinical improvement in order to aid in the design of future trials. This perspective is extremely relevant to the field considering the recent development of novel therapeutic approaches that will result in an increasing number of clinical studies over the next few years.Cellular and Molecular Life Sciences CMLS 06/2013; · 5.62 Impact Factor