Dissertation/Thesis Abstract

Understanding the Pathogenesis of Muscle Diseases Using In vivo SILAC Proteomic Strategy
by Rayavarapu, Sree Veera VSS, Ph.D., The George Washington University, 2013, 156; 3588601
Abstract (Summary)

In recent years, large-scale 'omic' studies have helped to understand disease pathogenesis; however, these studies were done largely at the 'transcriptome' level. Understanding the biological processes at the protein level is also equally important. In general, mass spectrometry (MS)-based quantitative proteomic strategies are used to study protein alterations in different biological states. Among these strategies, stable isotope labeling by amino acids in cell culture (SILAC) is most widely used for comparative proteomics. In SILAC, proteins in cell populations are metabolically encoded with 'heavy' isotopes of lysine and arginine and are used as internal standards for relative quantification of differentially altered proteins. In this dissertation, the use of SILAC was extended to study in vivo proteomic modulations in mice. A stable isotope (13C-lysine)-labeled 'SILAC mouse' was generated in order to quantify and compare protein alterations between normal and pathological conditions.

The primary objective of this dissertation was to identify novel and disease specific pathogenic mechanisms implicated in duchenne muscular dystrophy (DMD: a genetic muscle disease) and myositis (an autoimmune muscle disease) in vivo considering the entire complexity of the tissue. More importantly, the goal is to study global protein alterations in an unbiased manner in the affected skeletal muscle tissue. However, it is impractical to study in-depth disease pathology at tissue level using human muscle biopsy samples due to heterogeneity, complexity, and limited availability of muscle tissues at different stages in the disease process. To overcome these issues, mouse models that closely mimic the human disease phenotype i.e. a dystrophin deficient 'mdx' for DMD and a conditional major histocompatibility complex (MHC) class-I transgenic mice for myositis, were used. It was hypothesized that identification of precise proteomic alterations in the affected muscle, using mass spectrometry based untargeted-stable isotope labeled-proteomic strategy, would help to discover disease specific pathogenic mechanisms.

Firstly, the untargeted-labeled-proteomics approach using in vivo SILAC mouse proved to be a robust technique to uncover previously unidentified pathological pathways in mouse models of human skeletal muscle diseases. With respect to DMD, SILAC mouse proteomic profiling identified 73 significantly altered proteins in the early stage of the disease in mdx muscle compared to healthy muscle. Bioinformatics analyses of the altered proteins identified that integrin-linked kinase (ILK), actin cytoskeleton signaling, and mitochondrial energy metabolic pathways are significantly altered very early in the disease process in dystrophin deficient muscle. Disease specific protein modulations were further validated using an independent set of samples, SILAC spike-in strategy and specific antibody based biochemical assays. Moreover, the potential candidates of ILK pathway such as vimentin, desmin and ILK were confirmed to be significantly up-regulated in dystrophin-deficient human DMD samples suggesting the importance of these findings in relation to human disease pathology.

A novel association between the reduced mitochondrial activity and impaired sarcolemmal healing was identified in dystrophin deficient muscle. Live imaging of isolated single muscle fibers determined that reduced mitochondrial translocation to the site of injury negates the membrane repair processes even in the presence of compensatory up-regulation of repair proteins (dysferlin and annexin). Thus, current studies provided the first comprehensive understanding of the dystrophic muscle pathologies at the proteomic level.

In auto-immune myositis, SILAC mouse proteomic profiling identified significant alteration in the levels of 179 proteins. These proteins belong to the endoplasmic reticulum (ER) stress response, ubiquitin proteasome pathway (UPP), ER associated degradation (ERAD), oxidative phosphorylation, glycolysis, cytoskeleton, and muscle contractile apparatus categories. A significant increase in the ubiquitination of muscle proteins as well as a specific increase in ubiquitin carboxyl-terminal hydrolase isozyme L1 (UCHL1) was observed in myositis, but not in normal or other dystrophic muscles. Furthermore, inhibition of UPP using a specific proteasome inhibitor, bortezomib, significantly improved muscle function and also significantly decreased TNF-α (pro-inflammatory cytokine) expression in the skeletal muscle of myositis mice. Treatment with bortezomib, decreased GRP-78 (ER stress sensor) levels and also enhanced muscle regeneration. Thus, it can be concluded that UPP and ERAD activation in myositis muscle contribute to muscle degeneration. UCHL-1 is a potential biomarker for myositis disease progression and inhibition of UPP offers a potential therapeutic strategy for myositis.

These studies not only provided information on the implicated pathways both in 'dystrophin-deficient' and 'myositis' muscle but also identified potential therapeutic targets. Nevertheless, future experiments will help to associate pathways identified using this proteomic strategy with gene expression profiling to comprehensively understand disease specific as well as general pathogenic pathways. These studies will pave the way to enhance development of improved therapies for these rare muscle diseases.

Indexing (document details)
Advisor: Nagaraju, Kanneboyina
Commitee: Colberg-Poley, Anamaris M., Hathout, Yetrib, Hoffman, Eric P., Jaiswal, Jyoti K.
School: The George Washington University
Department: Biomedical Sciences
School Location: United States -- District of Columbia
Source: DAI-B 74/11(E), Dissertation Abstracts International
Source Type: DISSERTATION
Subjects: Pharmacology, Pathology, Veterinary services
Keywords: Mass spectrometry, Muscular dystrophy, Myositis, Proteomics, Silac, Skeletal muscle
Publication Number: 3588601
ISBN: 9781303267925
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