Proteomic screening analysis has detected myosin light chain (MLC) as a protein implied to be involved in chronic musculoskeletal neck and shoulder pain. Several analyses of MLC proteins have stated a difference in phosphorylation being the determining factor for protein activation hence altered contrability of the muscle in i.e. senescence. In continuation of a previous publication, this study is an attempt to analyze the different MLC isoforms by mass spectrometry and immune-analyses in myalgic and healthy trapezius muscle. In the present study no differences in phosphorylation level between the corresponding individual proteins were detected using LC-MSMS and immunoblotting; instead we assigned different isoforms of regulatory MLCs. To further elucidate the contrability: calcium (Ca²⁺) regulatory proteins, sarco(endo)plasmic reticulum Ca²⁺ ATPase 1 (SERCA-1) and calsequestrine (CSQ) were analyzed by western blot. The analysis revealed a significantly increased abundance of SERCA-1 protein in the myalgic muscle and a significantly increased abundance of CSQ in healthy muscle. Myalgic muscle contraction patterns have in previous studies shown to differ from healthy muscle which may be connected to the Ca²⁺ availability in the muscle. Here we present the proteomic characterization of differences in Ca²⁺ regulating proteins and particularly regulatory MLCs in trapezius muscle of women with chronic musculoskeletal neck and shoulder pain.

Chronic musculoskeletal pain exists either as localised to a single region or as widespread to multiple sites in several quadrants of the body. Prospective studies indicate that widespread pain could act as a far end of a continuum of musculoskeletal pain that started with chronic localised pain. The mechanism by which the transition from localised pain to widespread occurs is not clear, although many studies suggest it to be an altered metabolism. In this study, systemic metabolic differences between women with chronic localised neck-shoulder pain (NP), women with chronic widespread pain (CWP) and women who were healthy (CON) were assessed. Blood samples were analysed taking a metabolomics approach using gas chromatography mass spectrometry (GC-MS) and orthogonal partial least square discriminant analysis (OPLS-DA). The metabolomics analysis showed a clear systematic difference in the metabolic profiles between the subjects with NP and the CON but only a weak systematic difference between the subjects with CWP and the CON. This most likely reflects a difference in the portion of the metabolome influenced by the two pain conditions. In the NP group, the overall metabolic profile suggests that processes related to energy utilisation and lipid metabolism could be central aspects of mechanisms maintaining disorder.

Several hypotheses regarding the underlying mechanisms and the maintenance behind chronic work-related musculoskeletal disorders have been presented. Chronic low load work and psychosocial stress is believed to be the underlying causes to these pain conditions. The recent application of comprehensive screening methods: omnics methods; to this field of research could contribute to current knowledge regarding the pathophysiology of these disorders. The pathophysiological mechanisms behind chronic trapezius myalgia are discussed in the context of new findings obtained with proteomic and metabolomic methods. Proteins and metabolites which differ in abundance between healthy muscle and muscle suffering from chronic trapezius myalgia are presented. Primarily, the pathways and effects of the proteins and metabolites found in three recently published papers are discussed. Proteomics and metabolomics are efficient screening methods enabling the presentation of potential biomarkers and pathophysiological mechanisms explaining the pathophysiology of chronic work-related trapezius myalgia. The previous findings detecting systematic differences of proteins and metabolites when comparing chronic myalgic muscle to healthy muscle, indicating a higher glycogen metabolism, increased muscle turnover and increased neuronal signalling in the myalgic muscle, are discussed in this review.

PURPOSE:

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.

METHODS:

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.

RESULTS:

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.

CONCLUSION:

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

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.

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.

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.

The trapezius is a muscle of clinical interest due to its susceptibility to chronic work related musculoslteletal disorders. The mechanisms underlying these conditions are not fully understood. A comprehensive and comparative biochemical characterization of the trapezius muscle may reveal particular individual traits that make it more susceptible to chronic work related musculoskeletal disorders. We have examined the suitability and outcome of a proteomic method, two-dimensional difference gel electrophoresis (2D-DIGE), in the analysis of human skeletal muscle. This method allows a simultaneous comparison of large numbers of proteins within a limited pH and molecular weight range. A protocol using the 2D-DIGE method was adapted to human muscle and its repeatability was tested on human vastus lateralis muscle samples from one healthy male donor. The vastus lateralis is the most well studied muscle in the human body and was therefore used as the gold standard. The method was validated using western blot technique. Subsequently, a comparative 2D-DIGE analysis using the validated protocol was conducted on vastus lateralis and trapezius muscle biopsies from five healthy male subjects. Proteins were identified using matrix assisted laser desorption ionisation time of flight (MALDI-TOF) mass spectrometry and immunohistochemistry was performed on serial sections to confirm the findings. The 2D-DIGE method proved to be a good screening tool to further detect biochemical differences between muscles. The comparative study between the trapezius and vastus lateralis demonstrated significant differences in 25 important metabolic and structural proteins. In particular, it showed differences in proteins related to oxidative stress, which may be of specific importance for the susceptibility of the trapezius muscle to work related musculoskeletal disorders. These results provide a baseline for future studies on the trapezius muscle and puts further emphasis on the differences between different types of skeletal muscle.

Proteomic analyses are powerful methods and have the potential to yield vast amounts of data. The available proteomic methods have been hampered by large methodological errors in quantification, due to large gel-to-gel variations. The inclusion of an internal standard greatly reduces this variation, and the purpose of this investigation was 1) to develop a sample preparation protocol for human skeletal muscle for 2-dimensional differentiated gel electrophoresis (DIGE) and 2) to investigate the repeatability of one such system, the Ettan™ DIGE. To test repeatability, nine aliquots from the same homogenate were labeled with 3 different CyDye™ dyes (Cy2, Cy3, Cy5). Samples were run on 18 x 24 cm gels, scanned with a Typhoon™ 9410 laser scanner and analyzed in the DeCyder™ software. When selecting spots only appearing in triplicates (N = 1314) the mean error was 1.7 % (SD: 10.5%; 95% CI: 1.1-2.4%). By setting the significance level to 99%, no false positive changes in protein volume ratios were detected. The protocol presented here used only 0.5 mg tissue and allowed the separation of > 2500 distinct protein spots in the pH range 3-11 and MW 10-200 kDa. Changes in protein abundance of < 20% can 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