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Applying proteomics and metabolomics for studying human skeletal muscle with a focus on chronic trapezius myalgia
Umeå University, Faculty of Medicine, Department of Integrative Medical Biology (IMB). (Anatomi)
2012 (English)Doctoral thesis, comprehensive summary (Other academic)Alternative title
Tillämpning av proteomiska och metabolomiska metoder på human skelettmuskel med inriktning mot kronisk trapezius myalgi (English)
Abstract [en]

Work related musculoskeletal disorders are the dominating causes of reported ill-health in industrialized countries. These chronic pain conditions are one of the most costly public health problems in Europe and North America. When work related musculoskeletal disorders are considered to be of muscular origin and the trapezius muscle is affected, the common appellation is trapezius myalgia. Since little is known about the genesis or how it is maintained, it is of great importance to better understand the pathophysiology of trapezius myalgia; doing so will better enable recommendations for prevention, treatment and rehabilitation. Several hypotheses have been presented based on biochemical alterations in the muscle, suggesting increased signaling of inflammatory substances and altered metabolism. Previous research has not been able to present the comprehensive picture of the muscle in pain. Thus there is a demand for more comprehensive research regarding the biochemical milleu of the chronic trapezius muscle.

Proteomic and metabolomic methods allow non-targeted simultaneous analyses of a large number of proteins and metabolites. The main emphasis in this thesis is on a proteomic method, two-dimensional differential gel electrophoresis (2D-DIGE). The method is validated to human skeletal muscle biopsy research with laboratory specific settings. In the baseline study, there were 14 metabolic, contractile, structural and regulatory proteins that differed significantly in abundance when trapezius and vastus lateralis muscles were compared. Using the validated 2D-DIGE method and the baseline study, a comparison between healthy and myalgic muscles was made. Biopsies from female cleaners with and without myalgia were compared to obtain results from women with the same type of work exposure. In the multivariate model, 28 identified unique proteins separated healthy and myalgic muscle and were grouped according to function: metabolic (n=10), contractile (n=9), regulatory (n=3), structural (n=4), and other (n=2). Finally, a second screening method, metabolomics, was introduced to analyze differences in metabolite content as a complement to and verification of the proteomic results. Gas chromatography-mass spectrometry (GC-MS) was performed on muscle interstitial fluid samples obtained with microdialysis, and differences in the abundance of extracellular metabolites were revealed.

 The 2D-DIGE method is a reliable method to analyze human skeletal muscle. The outcomes of the proteomic analyses were dependant on the statistical approach. Systematic differences in protein and metabolite content were detected using a multivariate approach. Univariate analyses were used to analyze individual proteins for their significance. The significant proteins in the baseline study were predominately related to muscle fiber type which correlated with the differences in fiber type content between trapezius and vastus lateralis. The proteomic and metabolomics studies where myalgic and healthy muscles were compared provide us with new clues and new aspects regarding the pathophysiology of the myalgic muscle.

Technically advanced methods employed in the thesis enabled an explorative screening of proteins of relevance for the pathophysiology of the myalgic muscle. The results of these analyses may contribute to the formulation of future hypothesis that need to be further evaluated.

Place, publisher, year, edition, pages
Umeå: Umeå Universitet , 2012. , 60 p.
Series
Umeå University medical dissertations, ISSN 0346-6612 ; 1533
Keyword [en]
Trapezius myalgia, proteomics, 2D-DIGE, metabolomics, microdialysate, biopsies
National Category
Other Basic Medicine
Research subject
Human Anatomy
Identifiers
URN: urn:nbn:se:umu:diva-61399ISBN: 978-91-7459-515-4OAI: oai:DiVA.org:umu-61399DiVA: diva2:567362
Public defence
2012-12-06, BiA 201, Biologihuset, Umeå Universitet, Umeå, 09:00 (English)
Opponent
Supervisors
Available from: 2012-11-15 Created: 2012-11-12 Last updated: 2012-11-15Bibliographically approved
List of papers
1. Evaluation of 2-D DIGE for skeletal muscle: Protocol and repeatability
Open this publication in new window or tab >>Evaluation of 2-D DIGE for skeletal muscle: Protocol and repeatability
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2008 (English)In: The Scandinavian Journal of Clinical & Laboratory Investigation, Vol. 68, no 8, 793-800 p.Article in journal (Refereed) Published
Abstract [en]

Proteomic analysis has the potential to yield vast amounts of data. The available proteomic methods have been hampered by methodological errors in quantification due to large gel-to-gel variations. The inclusion of an internal standard greatly reduces this variation, and therefore the purpose of this investigation was: 1) to develop a sample preparation protocol for human skeletal muscle for two-dimensional differentiated gel electrophoresis (DIGE) and 2) to investigate the repeatability of one particular system, the Ettan™ DIGE. To test repeatability, nine aliquots from the same homogenate were labelled with three different CyDye™ dyes (Cy2, Cy3, Cy5). Samples were run on 1824 cm gels, scanned with a Typhoon™ 9410 laser scanner and analysed in the DeCyder™ software. When selecting spots appearing only in triplicate (n = 1314), the mean error was 1.7 % (SD: 10.5 %; 95 % CI: 1.1-2.4 %). When setting the significance level to 99 %, no false-positive changes in protein volume ratios were detected. In the protocol presented here, only 0.5 mg tissue was used and separation of >2500 distinct protein spots in the pH range 3-11 and MW 10-200 kDa. Changes in protein abundance of <20 % could be detected. The method is especially useful when comparing muscle proteins between different conditions; for example, healthy and diseased tissue, before and after treatment or different exercise protocols.

Place, publisher, year, edition, pages
Informa Healthcare, 2008
Keyword
DIGE, disease, exercise, human, mass spectrometry, skeletal muscle
National Category
Ophthalmology Cell and Molecular Biology
Research subject
Human Anatomy; Ophtalmology
Identifiers
urn:nbn:se:umu:diva-10396 (URN)10.1080/00365510802277464 (DOI)744 (Local ID)744 (Archive number)744 (OAI)
Available from: 2008-12-09 Created: 2008-12-09 Last updated: 2014-08-29Bibliographically approved
2. Protein differences between human trapezius and vastus lateralis muscles determined with a proteomic approach
Open this publication in new window or tab >>Protein differences between human trapezius and vastus lateralis muscles determined with a proteomic approach
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2011 (English)In: BMC Musculoskeletal Disorders, ISSN 1471-2474, Vol. 12, no 181, 11- p.Article in journal (Refereed) Published
Abstract [en]

Background: The trapezius muscle is a neck muscle that is susceptible to chronic pain conditions associated with repetitive tasks, commonly referred to as chronic work-related myalgia, hence making the trapezius a muscle of clinical interest. To provide a basis for further investigations of the proteomic traits of the trapezius muscle in disease, two-dimensional difference gel electrophoresis (2D-DIGE) was performed on the healthy trapezius using vastus lateralis as a reference. To obtain as much information as possible from the vast proteomic data set, both one-way ANOVA, with and without false discovery rate (FDR) correlation, and partial least square projection to latent structures with discriminant analysis (PLS-DA) were combined to compare the outcome of the analysis.

Results: The trapezius and vastus lateralis showed significant differences in metabolic, contractile and regulatory proteins, with different results depending on choice of statistical approach and pre-processing technique. Using the standard method, FDR correlated one-way ANOVA, 42 protein spots differed significantly in abundance between the two muscles. Complementary analysis using immunohistochemistry and western blot confirmed the results from the 2D-DIGE analysis.

Conclusions: The proteomic approach used in the present study combining 2D-DIGE and multivariate modelling provided a more comprehensive comparison of the protein profiles of the human trapezius and vastus lateralis muscle, than previously possible to obtain with immunohistochemistry or SDS-PAGE alone. Although 2D-DIGE has inherent limitations it is particularly useful to comprehensively screen for important structural and metabolic proteins, and appears to be a promising tool for future studies of patients suffering from chronic work related myalgia or other muscle diseases.

Place, publisher, year, edition, pages
BioMed Central, 2011
National Category
Basic Medicine
Identifiers
urn:nbn:se:umu:diva-46108 (URN)10.1186/1471-2474-12-181 (DOI)21831281 (PubMedID)
Available from: 2011-08-26 Created: 2011-08-26 Last updated: 2013-12-12Bibliographically approved
3. Multivariate Modeling of Proteins Related to Trapezius Myalgia, a Comparative Study of Female Cleaners with or without Pain
Open this publication in new window or tab >>Multivariate Modeling of Proteins Related to Trapezius Myalgia, a Comparative Study of Female Cleaners with or without Pain
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2013 (English)In: PLoS ONE, ISSN 1932-6203, Vol. 8, no 9, e73285- p.Article in journal (Refereed) Published
Abstract [en]

The prevalence of chronic trapezius myalgia is high in women with high exposure to awkward working positions, repetitive movements and movements with high precision demands. The mechanisms behind chronic trapezius myalgia are not fully understood. The purpose of this study was to explore the differences in protein content between healthy and myalgic trapezius muscle using proteomics. Muscle biopsies from 12 female cleaners with work-related trapezius myalgia and 12 pain free female cleaners were obtained from the descending part of the trapezius. Proteins were separated with two-dimensional differential gel electrophoresis (2D-DIGE) and selected proteins were identified with mass spectrometry. In order to discriminate the two groups, quantified proteins were fitted to a multivariate analysis: partial least square discriminate analysis. The model separated 28 unique proteins which were related to glycolysis, the tricaboxylic acid cycle, to the contractile apparatus, the cytoskeleton and to acute response proteins. The results suggest altered metabolism, a higher abundance of proteins related to inflammation in myalgic cleaners compared to healthy, and a possible alteration of the contractile apparatus. This explorative proteomic screening of proteins related to chronic pain in the trapezius muscle provides new important aspects of the pathophysiology behind chronic trapezius myalgia.

Place, publisher, year, edition, pages
Public Library of Science, 2013
Keyword
human, muscle, proteomics, pain, 2D-DIGE, trapezius, myalgia
National Category
Physiology
Research subject
Biochemistry
Identifiers
urn:nbn:se:umu:diva-60613 (URN)10.1371/journal.pone.0073285 (DOI)000324515600091 ()
Funder
Swedish Research Council, K2011-69X-21874-01-6Forte, Swedish Research Council for Health, Working Life and Welfare, 2009-1761, 2010-0913
Available from: 2012-10-19 Created: 2012-10-19 Last updated: 2014-02-17Bibliographically approved
4. Comparative metabolomics of muscle interstitium fluid in human trapezius myalgia: an in vivo microdialysis study
Open this publication in new window or tab >>Comparative metabolomics of muscle interstitium fluid in human trapezius myalgia: an in vivo microdialysis study
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2013 (English)In: European Journal of Applied Physiology, ISSN 1439-6319, E-ISSN 1439-6327, Vol. 113, no 12, 2977-2989 p.Article in journal (Other academic) Published
Abstract [en]

The mechanisms behind trapezius myalgia are unclear. Many hypotheses have been presented suggesting an altered metabolism in the muscle. Here, muscle microdialysate from healthy and myalgic muscle is analysed using metabolomics. Metabolomics analyse a vast number of metabolites, enabling a comprehensive explorative screening of the cellular processes in the muscle.

Microdialysate samples were obtained from the shoulder muscle of healthy and myalgic subjects that performed a work and stress test. Samples from the baseline period and from the recovery period were analysed using gas chromatography-mass spectrometry (GC-MS) together with multivariate analysis to detect differences in extracellular content of metabolites between groups. Systematic differences in metabolites between groups were identified using multivariate analysis and orthogonal partial least square discriminate analysis (OPLS-DA). A complementary Mann-Whitney U test of group difference in individual metabolites was also performed.

A large number of metabolites were detected and identified in this screening study. At baseline, no systematic differences between groups were observed according to the OPLS-DA. However, two metabolites, l-leucine and pyroglutamic acid, were significantly more abundant in the myalgic muscle compared to the healthy muscle. In the recovery period, systematic difference in metabolites between the groups was observed according to the OPLS-DA. The groups differed in amino acids, fatty acids and carbohydrates. Myristic acid and putrescine were significantly more abundant and beta-d-glucopyranose was significantly less abundant in the myalgic muscle.

This study provides important information regarding the metabolite content, thereby presenting new clues regarding the pathophysiology of the myalgic muscle.

Place, publisher, year, edition, pages
Springer, 2013
Keyword
Metabolomics, trapezius myalgia, microdialysis, repetitive work, GC-MS, metabolites
National Category
Other Basic Medicine
Research subject
Human Anatomy
Identifiers
urn:nbn:se:umu:diva-61396 (URN)10.1007/s00421-013-2716-6 (DOI)
Funder
Swedish Research Council, K2011-69X-21874-01-6Forte, Swedish Research Council for Health, Working Life and Welfare, 2009-1761, 2010-0913
Note

Originally published in thesis in manuscript form.

Available from: 2012-11-12 Created: 2012-11-12 Last updated: 2013-12-18Bibliographically approved

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