Proteomics identification of differentially expressed proteins in the muscle of dysferlin myopathy patients

The muscular dystrophies are a large and heterogeneous group of neuromuscular disorders that can be classified according to the mode of inheritance, the clinical phenotype and the molecular defect. To better understand the pathological mechanisms of dysferlin myopathy we compared the protein-expression pattern in the muscle biopsies of six patients with this disease with six patients with limb girdle muscular dystrophy 2A, five with facioscapulohumeral dystrophy and six normal control subjects. To investigate differences in the expression levels of skeletal muscle proteins we used 2-DE and MS. Western blot or immunohistochemistry confirmed relevant results. The study showed specific increase expression of proteins involved in fast-to-slow fiber type conversion (ankyrin repeat protein 2), type I predominance (phosphorylated forms of slow troponin T), sarcomere stabilization (actinin-associated LIM protein), protein ubiquitination (TRIM 72) and skeletal muscle differentiation (Rho-GDP-dissociation inhibitor ly-GDI) in dysferlin myopathy. As anticipated, we also found differential expression of proteins common to all the muscular dystrophies studied. This comparative proteomic analysis suggests that in dysferlin myopathy (i) the type I fiber predominance is an active process of fiber type conversion rather than a selective loss of type II fibers and (ii) the dysregulation of proteins involved in muscle differentiation further confirms the role of dysferlin in this process.     

Proteomic profile of differentially expressed plasma proteins from dystrophic mice and following suberoylanilide hydroxamic acid treatment

Purpose: Histone Deacetylase Inhibitors (DI) ameliorates dystrophic muscle regeneration restoring muscular strength in the mdx mouse model of Duchenne muscular dystrophy (DMD). The further development of these compounds as drugs for DMD treatment is currently hampered by the lack of knowledge about DIs effect in large dystrophic animal models and that of suitable biomarkers to monitor their efficacy. Experimental design: In this study we applied proteomic analysis to identify differentially expressed proteins present in plasma samples from mdx mice treated with the Suberoylanilide hydroxamic acid (SAHA) and relative normal controls (WT). Results: Several differentially expressed proteins were identified between untreated wild type and mdx mice. Among these, fibrinogen, epidermal growth factor 2 receptor, major urinary protein and glutathione peroxidase 3 (GPX3) were constitutively up‐regulated in mdx, while complement C3, complement C6, gelsolin, leukaemia inhibitory factor receptor (LIFr), and alpha 2 macroglobulin were down‐regulated compared to WT mice. SAHA determined the normalization of LIFr and GPX3 protein level while apoliprotein E was de novo up‐regulated in comparison to vehicle‐treated mdx mice. Conclusions and clinical relevance: Collectively, these data unravel potential serological disease biomarkers of mdx that could be useful to monitor muscular dystrophy response to DI treatment.     

Proteomic profiling of heat shock proteins: An emerging molecular approach with direct pathophysiological and clinical implications

The HSP family is one of the most ancient and evolutionarily conserved protective protein families found in nature. Originally discovered as inducible molecules capable of maintaining cellular homeostasis against abrupt temperature changes, HSPs were later determined to represent an adaptive physiological response that copes with a variety of different cellular proteotoxic stresses. These physiological molecular chaperones facilitate the synthesis, folding, assembly, trafficking and secretion of specific proteins in various cellular compartments. Most importantly, these proteins guard the whole cell proteome against misfolding and inappropriate aggregation. A series of diversified proteotoxic stresses, including heat, hypoxia/ischemia, free radicals, acidosis, ATP depletion and toxins are capable of inducing a typical cellular stress response characterised by rapid inhibition of overall protein synthesis, with a concomitant dramatic increase in HSP expression. From a pathophysiological point of view, HSP induction has been observed in a wide spectrum of inflammatory and degenerative diseases (from cancer to prion disease by passing to infective and autoimmune diseases) and, intriguingly, overexpression monitoring seems to have potential implications in terms of diagnosis, prognosis and, above all, therapy. Proteomics studies, identifying a series of modification of HSP expression patterns in different diseases, are confirming these promising clinical applications.     

Differential regulation of redox proteins and chaperones in HbEβ-thalassemia erythrocyte proteome

Purpose: In (hemoglobin, Hb) HbEβ‐thalassemia, HbE (β‐26 Glu→Lys) interacts with β‐thalassemia to produce clinical manifestation of varying severity. This is the first proteomic effort to study changes in protein levels of erythrocytes isolated from HbEβ‐thalassemic patients compared to normal. Experimental design: We have used 2‐DE and MALDI‐MS/MS‐based techniques to investigate the differential proteome profiling of membrane and Hb‐depleted fraction of cytosolic proteins of erythrocytes isolated from the peripheral blood samples of HbEβ‐thalassemia patients and normal volunteers. Results: Our study showed that redox regulators such as peroxiredoxin 2, Cu‐Zn superoxide dismutase and thioredoxin and chaperones such as α‐hemoglobin stabilizing protein and HSP‐70 were upregulated in HbEβ‐thalassemia. We have also observed larger amounts of membrane associated globin chains and indications of disruption of spectrin‐based junctional complex in the membrane skeleton of HbEβ‐thalassemic erythrocytes upon detection of low molecular weight fragments of β‐spectrin and decrease in β‐actin and dematin content. Conclusion and clinical relevance: We have observed interesting changes in the proteomic levels of redox regulators and chaperons in the thalassemic hemolysates and have observed strong correlation or association of the extent of such proteomic changes with HbE levels. This could be important in understanding the role of HbE in disease progression and pathophysiology.     

Heat-shock protein-27, -70 and peroxiredoxin-II show molecular chaperone function in sickle red cells: Evidence from transgenic sickle cell mouse model

Sickle cell disease (SCD) is an autosomal recessive genetic red cell disorder characterized by the production of a defective form of hemoglobin, hemoglobin-S, that is worldwide-distributed. The acute clinical manifestations of SCD are related to hemoglobin cyclic-polymerization and to the generation of rigid, dense red blood cells (RBCs). We studied RBCs membrane proteome from human sickle RBCs, fractioned according to density compared to normal RBCs. 2-DE followed by MS analysis was carried out. We identified 65 proteins differently expressed, divided into five major clusters according to their functions: (i) membrane-cytoskeleton proteins; (ii) metabolic enzymes; (iii) ubiquitin-proteasome-system; (iv) flotillins; (v) chaperones. HSP27, HSP70 and peroxiredoxin-II (Prx-II) showed the most relevant changes. They were differently recruited to sickle RBCs membrane in response to in vitro hypoxia. Potential markers were then validated in a transgenic-mouse model for SCD, the SAD mice, exposed to hypoxia mimicking acute SCD vaso-occlusive-crisis (VOCs); we found that HSP70 and HSP27 bound to RBCs membrane respectively after 12?h and 48?h of hypoxia, while Prx-II membrane binding was modulated during hypoxia. Our data indicate that HSP27 and HSP70 play a novel role as RBCs membrane protein protectors and as possibly new markers of severity of RBCs membrane damage during acute VOCs.     

Discovery of putative oocyte quality markers by comparative ExacTag proteomics

Purpose: Identification of the biomarkers of oocyte quality, and developmental and reprogramming potential is of importance to assisted reproductive technology in humans and animals. Experimental design: PerkinElmer ExacTag? Kit was used to label differentially proteins in pig oocyte extracts (oocyte proteome) and pig oocyte‐conditioned in vitro maturation media (oocyte secretome) obtained with high‐ and low‐quality oocytes. Results: We identified 16 major proteins in the oocyte proteome that were expressed differentially in high‐ versus low‐quality oocytes. More abundant proteins in the high‐quality oocyte proteome included kelch‐like ECH‐associated protein 1 (an adaptor for ubiquitin‐ligase CUL3), nuclear export factor CRM1 and ataxia‐telangiectasia mutated protein kinase. Dystrophin (DMD) was more abundant in low‐quality oocytes. In the secretome, we identified 110 proteins, including DMD and cystic fibrosis transmembrane conductance regulator, two proteins implicated in muscular dystrophy and cystic fibrosis, respectively. Monoubiquitin was identified in the low‐quality‐oocyte secretome. Conclusions and clinical implications: A direct, quantitative proteomic analysis of small oocyte protein samples can identify potential markers of oocyte quality without the need for a large amount of total protein. This approach will be applied to discovery of non‐invasive biomarkers of oocyte quality in assisted human reproduction and in large animal embryo transfer programs.     

人体下肢肌力分布的仿真模型及参数影响研究

肌力的分布对于更好地理解单个肌肉作用和负载情况有着重要的指导意义。该文建立了三自由度9块肌肉的人体下肢肌力分布求解模型，并利用该模型仿真计算了跑步运动缓冲阶段和蹬伸阶段的肌力分布规律，比较了不同目标函数和肌肉生理横断面积的变化对肌力分布的影响作用。数值计算的结果显示以最小应力立方和为优化目标可获得较好的优化性能，而且肌肉生理横断面积的增加使得相应肌肉的肌力有所增加同时也影响其它肌肉的肌力变化。该方法可用于分析跑步运动过程中下肢肌肉相互协调的功能特性，并且为在体育训练中有针对性的进行肌肉力量训练以及改进技术动作提供了一定的参考数据。     

Prostate cancer serum biomarker discovery through proteomic analysis of alpha‐2 macroglobulin protein complexes

Alpha‐2 macroglobulin (A2M) functions as a universal protease inhibitor in serum and is capable of binding various cytokines and growth factors. In this study, we investigated if immunoaffinity enrichment and proteomic analysis of A2M protein complexes from human serum could improve detection of biologically relevant and novel candidate protein biomarkers in prostate cancer. Serum samples from six patients with androgen‐independent, metastatic prostate cancer and six control patients without malignancy were analyzed by immunoaffinity enrichment of A2M protein complexes and MS identification of associated proteins. Known A2M substrates were reproducibly identified from patient serum in both cohorts, as well as proteins previously undetected in human serum. One example is heat shock protein 90 alpha (HSP90α), which was identified only in the serum of cancer patients in this study. Using an ELISA, the presence of HSP90α in human serum was validated on expanded test cohorts and found to exist in higher median serum concentrations in prostate cancer (n = 18) relative to control (n = 13) patients (median concentrations 50.7 versus 27.6 ng/mL, respectively, p = 0.001). Our results demonstrate the technical feasibility of this approach and support the analysis of A2M protein complexes for proteomic‐based serum biomarker discovery.     

Upper trapezius muscle activity in healthy office workers: reliability and sensitivity of occupational exposure measures to differences in sex and hand dominance

Patterns of cervical muscle activity may contribute to overuse injuries in office workers. The purpose of this investigation was to characterise patterns of upper trapezius muscle activity in pain-free office workers using traditional occupational exposure measures and a modified Active Amplitude Probability Distribution Function (APDF), which considers only periods of active muscle contraction. Bilateral trapezius muscle activity was recorded in 77 pain-free office workers for 1–2 full days in their natural work environment. Mean amplitude, gap frequency, muscular rest and Traditional and Active APDF amplitudes were calculated. All measures demonstrated fair to substantial reliability. Dominant muscles demonstrated higher amplitudes of activity and less muscular rest compared to non-dominant, and women demonstrated less muscular rest with no significant difference in amplitude assessed by Active APDF compared to men. These findings provide normative data to identify atypical motor patterns that may contribute to persistence or recurrence of neck pain in office workers.

Practitioner Summary: Upper trapezius muscle activity was characterised in a large cohort of pain-free workers using electromyographic recordings from office environments. Dominant muscles demonstrated higher activity and less rest than non-dominant, and women demonstrated less rest than men. Results may be used to identify atypical trapezius muscle activity in office workers.     

A review of: “Applying Psychology: Understanding People” By R. C. Beck (New Jersey: Prentice Hall, 1982.) [Pp.484.] $15-70. ISBN 0-13-043463-9.

This study investigated how ergonomic design influences neck-and-shoulder muscle strain, through keyboard assessment. Muscular activity was measured electromyographically (EMG) from six muscles in the forearm and shoulders of eight experienced typists using each of five different types of keyboard: one mechanical, one electromechanical, and one electronic typewriter; one personal computer/word processor (PC-XT) keyboard; and one angled at 20° in the horizontal plane. The impact on muscular activity of using a palmrest was also studied. The mechanical typewriter induced a higher strain in the forearm and finger muscles than did the modern typewriters and keyboards. These induced no different strain on the neck-and-shoulder muscles, except for the right shoulder muscle, which was more active with the electronic typewriter than with the other machines. Using a palmrest did not decrease the strain on the muscles investigated. Use of the ‘angled’ PC-XT keyboard did not influence the measured muscular load on the forearm and finger muscles compared to typing on an ordinary PC-XT keyboard, but decreased the extensor muscular strain compared to the electronic typewriter.     

Proteomic profiling of animal models mimicking skeletal muscle disorders

Over the last few decades of biomedical research, animal models of neuromuscular diseases have been widely used for determining pathological mechanisms and for testing new therapeutic strategies. With the emergence of high-throughput proteomics technology, the identification of novel protein factors involved in disease processes has been decisively improved. This review outlines the usefulness of the proteomic profiling of animal disease models for the discovery of new reliable biomarkers, for the optimization of diagnostic procedures and the development of new treatment options for skeletal muscle disorders. Since inbred animal strains show genetically much less interindividual differences as compared to human patients, considerably lower experimental repeats are capable of producing meaningful proteomic data. Thus, animal model proteomics can be conveniently employed for both studying basic mechanisms of molecular pathogenesis and the effects of drugs, genetic modifications or cell-based therapies on disease progression. Based on the results from comparative animal proteomics, a more informed decision on the design of clinical proteomics studies could be reached. Since no one animal model represents a perfect pathobiochemical replica of all of the symptoms seen in complex human disorders, the proteomic screening of novel animal models can also be employed for swift and enhanced protein biochemical phenotyping.     

An electromyographic study of dental work

Musculoskeletal disorders are common among dentists, and have been ascribed to the demands of high precision work and sustained static loading in the neck-shoulder region, combined with a flexed and rotated cervical spine. In order to determine muscular load levels during dentistry, activity in neck, shoulder, and arm muscles was recorded using an electromyography technique (EMG). Normalized mean, median, 10th and 90th percentile EMG amplitude levels (% maximal reference contraction, %max-RVC) were calculated during ordinary dental work. Among the muscles investigated, the trapezius muscle on both sides had the highest mean (the right trapezius 9.0% and the left 7.6% of max-RVC) and 10th percentile amplitude levels (both about 2% of max-RVC). The trapezius muscles showed similar myoelectric activity on the right and left side, probably because of similar muscular static load on the both sides. The right extensor carpi radialis muscle had a significantly higher muscular load level than the left one, possibly due to stabilization demands on the dominant wrist during demanding precision work. The infraspinatus muscle had low activity level on both sides, reflecting that the dentists worked with a small degree of arm elevation and external rotation. The dentistry work thus seems to generate relatively high muscular load on both trapezius and dominant extensor-carpi-radialis, and relatively low load on the infraspinatus muscle.     

Characterization of cashier shoulder and low back muscle demands

Cashiers commonly report musculoskeletal discomfort in their shoulders, neck, and low back. This may result from excessive loading, awkward postures, insufficient rest, or task repetition. Recently, widespread introduction of reusable, consumer-supplied bags has introduced challenges in assessing exposures in this occupational group. Limited information exists on the physical demands associated with cashier work, particularly in the context of multiple bagging formats; this study was thus designed to generate a novel data set describing standard grocery packaging tasks. Twenty-five experienced cashiers completed 36 grocery packaging tasks consisting of twice performing all combinations of workload intensity (6, 20 items), workstation height (low, medium, high), and packaging type (plastic bags, reusable bags, bins). Surface electromyography (EMG) was measured bilaterally for 5 shoulder and 3 low back muscles and processed to generate integrated muscle demand for each combination evaluated. A mixed effect ANOVA was used to assess the influences of gender, intensity, package type, side, (muscles on the right or left side of the body) and workstation height on individual and total muscular demands. High workload intensity combined with several other factors to increase muscle demands, including using plastic or reusable bags for packaging and increasing workstation height. Gender and side also interacted with workload intensity to influence muscle activity. Encouraging rest breaks, the use of bins for packaging, and decreasing cashier workstation height may help reduce potentially injurious muscular effort for cashiers.     

Digital-analog hybrid control model for eukaryotic heat shock response illustrating the dynamics of heat shock protein 70 on exposure to thermal stress

We are introducing in this paper a digital-analog hybrid model approach for the study of a complete gene regulatory network; the heat shock response (HSR) network of eukaryotes. HSR is a crucial and widely studied cellular phenomenon occurring due to various stresses on the cell, and is characterised by the induction of heat shock genes resulting in the production of heat shock proteins (HSPs) which restores cellular homeostasis by maintaining protein integrity. We are proposing a model which incorporates simple digital and analog components which mimic the functioning of biological molecules involved in HSR and model their dynamics and behaviour. The simulation result of the circuit for the production of HSP70 has been found to be consistent with published experimental results. The qualitative behaviour of the HSR is expressed through a truth table. Through this novel approach, the authors have tried to develop a level of understanding of the interactions of the parts of the HSR system and of this system as a whole.     

Rapid Communication Effects of posture,weight and frequency on trunk muscular activity and fatigue during repetitive lifting tasks

Abstract

We examined the effects of posture, weight and frequency on trunk muscular activity and fatigue during repetitive dynamic lifting. Electromyographic (EMG) signals from eight primary trunk muscles were collected during 120 min for four different task conditions. The patterns of muscle recruitment and the levels of relative activation were analysed using the normalized EMG data. Median power frequency (MPF) shift patterns were analysed to examine muscular fatigue. The muscles in the dorsal part of the trunk were activated at the symmetric posture, while the muscles on the contralateral side to the workload were more strongly activated at the asymmetric posture. Decreasing trends of MPFs were found in some active muscles, and they were more pronounced for the asymmetric posture than for the symmetric posture. It was also seen that the muscles became fatigued faster for the light load-high frequency task than for the heavy load-low frequency task.     

Effects of heel cushioning elements in safety shoes on muscle–physiological parameters

Safety shoe designs are primarily based on safety requirements. But all-day comfort should not be luxury: Heel strike associated impact loads on joints need to be compensated by active muscular effort and safety shoes should support this protective function of muscle activation. In 10 healthy men, 12 trunk and leg muscles were analyzed with surface electromyography. Subjects walked on a walkway while wearing different safety shoes with the test shoes being equipped with exchangeable cushioning heel inserts according to individuals' body weight. While wearing the optimally cushioned shoes the cumulative muscle activity per distance travelled dropped clearly compared to the regular safety shoes, demonstrating reduced muscular effort. Also, the heel strike associated amplitude peak of back muscles occurred earlier within the stride while wearing the test shoes. Thus weight-balanced cushioning heel inserts in safety shoes proved able to reduce muscle strain, logically delaying muscular fatigue and extending muscular joint protection.Relevance to industryAdjustable heel inserts in safety shoes are suited to improve the health status of employees by reducing muscular effort so that active joint protection can be prolonged.     

THE STATIC MUSCLE LOAD IN DIFFERENT WORK POSITIONS: AN ELECTROMYOGRAPHIC STUDY∗

Abstract

In order to ascertain which muscles and muscle groups are engaged in various standing work positions an electromyographic study was performed.About twenty muscles or muscle groups were examined. Both coaxial needle electrodes and surface electrodes were employed.

The analysis of the muscle activity for different postures was based on action potentials recorded in a normal symmetric standing rest position. Among the different postures the rest position appears to require the least muscular effort for counterbalancing the effect of gravity on the various parts of the body. This state of equilibrium seems to be maintained by certain muscle groups, referred to hero a.s the “ prime postural muscles ”. Only a part of the potential power of the postural muscles is utilized in maintaining the standing symmetric rest position. Some persons engage other muscles besides the prime postural muscles, possibly because they stand in such a way that the load distribution is not optimal, or thoy havo weak prime postural muscles or because of poor muscular co-ordination as a consequence of “ non-physiologic” postural habits.

When changing from the rest position to most of the other standing positions the load on the prime postural muscles is increased, and in many cases other muscle groups arc activated as well. The sacrospiualis appears to be particularly susceptible to change in the load distribution. The muscles of the lower leg, which control the foot articulation, are also very susceptible to changes in body posture. However, a change in the position of the- trunk need not result in an appreciable increase in the load on the muscles of the lower legs if the displacement of the centre of gravity of the trunk is compensated by a postural adjustment at the ankle, such that the point of intersection of tho line of gravity of the body and the supporting area will be the same as in the symmetric standing rest position.

A change in posture of the hip- and knee-joints does not result in such a wide variation in activity of the muscles rogulating the position of these joints as a postural change in the foot articulation and the vertebrae.     

Arm positioning in manual tasks An electromyographic study of localized muscle fatigue

Myoelectric activity in four different shoulder muscles was collected and interpreted as indirect indicators of the load in eight different arm positions. The power spectrum changes associated with localized muscle fatigue were measured using a new dedicated instrument. Localized muscle fatigue was present in all muscles, in overhead and shoulder level work, and in some cases in waist level work ? as well. For instance, in overhead work, the supraspinatus load was significantly affected by elbow positioning. The infraspinatus muscle held the highest fatigue effects of all muscles investigated. The results indicate the possibility of finding positions entailing reduction of the total muscular load in different working situations.     

The effect of vibration on the skeleton, joints and muscles. A review of the literature

Owing to the elasticity and plasticity of the skeleton, joints and muscles, the musculoskeletal system is capable of absorbing and damping mechanical vibration without damage as long as the vibration level is within tolerable limits. However, technical developments have led to the exposure of many people to intolerable variation levels with destructive changes as a result.

These injuries to the musculoskeletal system continue to be the subject of research interest. Initially, the joints and joint complaints attracted the greatest attention. Vibration damping takes place mainly in the joints. The incidence of destructive joint changes has been examined in comprehensive clinical, epidemiological and radiographic studies, mainly concentrating on the joints of the hand and arm. The response of muscles to vibration is often expressed in the form of a tonic vibration reflex (TVR) which arises as a result of stimulation of the muscle spindles and therefore resembles the classic tonic stretch reflex. There is increased muscular activation for stabilisation of the joint positions, especially during whole-body vibration. Studies have also disclosed how vibration affects body equilibrium and equilibrium control and how vibration can elicit muscle pain, cramps and reduced muscular strength.     

Vascular proteomics

The characterization of patients with acute coronary syndromes (ACS) at the molecular and cellular levels provides a novel vision for understanding the pathological and clinical expression of the disease. Recent advances in proteomic technologies permit the evaluation of systematic changes in protein expression in many biological systems and have been extensively applied to cardiovascular diseases (CVD). The cardiovascular system is in permanent intimate contact with blood, making blood-based biomarker discovery a particularly worthwhile approach. Thus, proteomics can potentially yield novel biomarkers reflecting CVD, establish earlier detection strategies, and monitor response to therapy. Here we review the different proteomic strategies used in the study of atherosclerosis and the novel proteins differentially expressed and secreted by atherosclerotic lesions which constitute novel potential biomarkers (HSP-27, Cathepsin D). Special attention is paid to MS-Imaging of atheroma plaque and the generation, for the first time, of 2-D images of lipids, showing the distribution of these molecules in the different areas of the atherosclerotic lesions. In addition new potential biomarkers have been identified in plasma (amyloid A1α, transtherytin), circulating cells (protein profile in monocytes from ACS patients) and individual cells constituents of atheroma plaques (endothelial, VSMC, macrophages) which provide novel insights into vascular pathophysiology.