Proteomic reference map for sarcopenia research: mass spectrometric identification of key muscle proteins located in the sarcomere, cytoskeleton and the extracellular matrix.

Sarcopenia of old age is characterized by the progressive loss of skeletal muscle mass and concomitant decrease in contractile strength. Age-related skeletal muscle dysfunctions play a key pathophysiological role in the frailty syndrome and can result in a drastically diminished quality of life in the elderly. Here we have used mass spectrometric analysis of the mouse hindlimb musculature to establish the muscle protein constellation at advanced age of a widely used sarcopenic animal model.

Proteomic reference map for sarcopenia research: mass spectrometric identification of key muscle proteins of organelles, cellular signaling, bioenergetic metabolism and molecular chaperoning.

During the natural aging process, frailty is often associated with abnormal muscular performance. Although inter-individual differences exit, in most elderly the tissue mass and physiological functionality of voluntary muscles drastically decreases. In order to study age-related contractile decline, animal model research is of central importance in the field of biogerontology.

The potential of proteomics for in-depth bioanalytical investigations of satellite cell function in applied myology.

Introduction: Regenerative myogenesis plays a crucial role in mature myofibers to counteract muscular injury or dysfunction due to neuromuscular disorders. The activation of specialized myogenic stem cells, called satellite cells, is intrinsically involved in proliferation and differentiation, followed by myoblast fusion and the formation of multinucleated myofibers.

Areas Covered: This report provides an overview of the role of satellite cells in the neuromuscular system and the potential future impact of proteomic analyses for biomarker discovery, as well as the identification of novel therapeutic targets in muscle disease.

How Can Proteomics Help to Elucidate the Pathophysiological Crosstalk in Muscular Dystrophy and Associated Multi-System Dysfunction?

This perspective article is concerned with the question of how proteomics, which is a core technique of systems biology that is deeply embedded in the multi-omics field of modern bioresearch, can help us better understand the molecular pathogenesis of complex diseases. As an illustrative example of a monogenetic disorder that primarily affects the neuromuscular system but is characterized by a plethora of multi-system pathophysiological alterations, the muscle-wasting disease Duchenne muscular dystrophy was examined. Recent achievements in the field of dystrophinopathy research are described with special reference to the proteome-wide complexity of neuromuscular changes and body-wide alterations/adaptations.

Mass Spectrometry-Based Proteomic Technology and Its Application to Study Skeletal Muscle Cell Biology.

Voluntary striated muscles are characterized by a highly complex and dynamic proteome that efficiently adapts to changed physiological demands or alters considerably during pathophysiological dysfunction. The skeletal muscle proteome has been extensively studied in relation to myogenesis, fiber type specification, muscle transitions, the effects of physical exercise, disuse atrophy, neuromuscular disorders, muscle co-morbidities and sarcopenia of old age. Since muscle tissue accounts for approximately 40% of body mass in humans, alterations in the skeletal muscle proteome have considerable influence on whole-body physiology.

Cellular pathogenesis of Duchenne muscular dystrophy: progressive myofibre degeneration, chronic inflammation, reactive myofibrosis and satellite cell dysfunction.

Duchenne muscular dystrophy is a highly progressive muscle wasting disease of early childhood and characterized by complex pathophysiological and histopathological changes in the voluntary contractile system, including myonecrosis, chronic inflammation, fat substitution and reactive myofibrosis. The continued loss of functional myofibres and replacement with non-contractile cells, as well as extensive tissue scarring and decline in tissue elasticity, leads to severe skeletal muscle weakness. In addition, dystrophic muscles exhibit a greatly diminished regenerative capacity to counteract the ongoing process of fibre degeneration.

Biochemical and proteomic insights into sarcoplasmic reticulum Ca-ATPase complexes in skeletal muscles.

Introduction: Skeletal muscles contain large numbers of high-molecular-mass protein complexes in elaborate membrane systems. Integral membrane proteins are involved in diverse cellular functions including the regulation of ion handling, membrane homeostasis, energy metabolism and force transmission.

Areas Covered: The proteomic profiling of membrane proteins and large protein assemblies in skeletal muscles are outlined in this article.

Proteomic profiling of the brain from the mouse model of amyotrophic lateral sclerosis reveals elevated levels of the astrogliosis marker glial fibrillary acidic protein.

The wobbler mouse is a widely used model system of amyotrophic lateral sclerosis and exhibits progressive neurodegeneration and neuroinflammation in association with skeletal muscle wasting. This study has used wobbler brain preparations for the systematic and mass spectrometric determination of proteome-wide changes. The proteomic characterization of total protein extracts from wobbler specimens was carried out with the help of an Orbitrap mass spectrometer and revealed elevated levels of glia cell marker proteins, i.

Mass spectrometry-based proteomic characterization of the middle-aged mouse brain for animal model research of neuromuscular diseases.

Neuromuscular diseases with primary muscle wasting symptoms may also display multi-systemic changes in the body and exhibit secondary pathophysiological alterations in various non-muscle tissues. In some cases, this includes proteome-wide alterations and/or adaptations in the central nervous system. Thus, in order to provide an improved bioanalytical basis for the comprehensive evaluation of animal models that are routinely used in muscle research, this report describes the mass spectrometry-based proteomic characterization of the mouse brain.

Extracellular Matrix Proteomics: The Mouse Diaphragm as a Surrogate for Studying Myofibrosis in Dystrophinopathy.

The progressive degeneration of the skeletal musculature in Duchenne muscular dystrophy is accompanied by reactive myofibrosis, fat substitution, and chronic inflammation. Fibrotic changes and reduced tissue elasticity correlate with the loss in motor function in this X-chromosomal disorder. Thus, although dystrophinopathies are due to primary abnormalities in the gene causing the almost-complete absence of the cytoskeletal Dp427-M isoform of dystrophin in voluntary muscles, the excessive accumulation of extracellular matrix proteins presents a key histopathological hallmark of muscular dystrophy.

Fiber-Type Shifting in Sarcopenia of Old Age: Proteomic Profiling of the Contractile Apparatus of Skeletal Muscles.

The progressive loss of skeletal muscle mass and concomitant reduction in contractile strength plays a central role in frailty syndrome. Age-related neuronal impairments are closely associated with sarcopenia in the elderly, which is characterized by severe muscular atrophy that can considerably lessen the overall quality of life at old age. Mass-spectrometry-based proteomic surveys of senescent human skeletal muscles, as well as animal models of sarcopenia, have decisively improved our understanding of the molecular and cellular consequences of muscular atrophy and associated fiber-type shifting during aging.

Histological and Histochemical Microscopy Used to Verify 2D-DIGE Pathoproteomics.

Comparative gel electrophoretic analyses of normal versus pathological specimens can swiftly identify proteome-wide changes in the concentration of specific protein isoforms. The application of fluorescence two-dimensional difference gel electrophoresis (2D-DIGE) can be employed for the characterization of complex protein populations in health and disease. In order to verify pathoproteomic findings and correlate them to histopathological alterations, standardized histological and histochemical methodology can be applied for the cell biological analysis of normal versus pathological tissue samples.

Verification of Protein Changes Determined by 2D-DIGE Based Proteomics Using Immunofluorescence Microscopy.

Fluorescence two-dimensional difference gel electrophoresis (2D-DIGE) is a key biochemical method for the comparative analysis of complex protein mixtures. The technique focuses on the identification and characterization of individual protein species following gel electrophoretic separation making it an important analytical tool of top-down proteomics. In order to verify changes in the expression levels of a particular protein, as determined by 2D-DIGE analysis, and evaluate the subcellular localization of the proteoform of interest, immunofluorescence microscopy is very well suited.

Bioinformatic Analysis of the Subproteomic Profile of Cardiomyopathic Tissue.

Following large-scale protein separation by two-dimensional gel electrophoresis or liquid chromatography, mass spectrometry-based proteomics can be used for the swift identification and characterization of cardiac proteins and their various proteoforms. Comparative cardiac proteomics has been widely applied for the systematic analysis of heart disease and the establishment of novel diagnostic protein biomarkers. The X-linked neuromuscular disorder Duchenne muscular dystrophy is a multisystemic disease that is characterized by late-onset cardiomyopathy.

Identification of Subproteomic Markers for Skeletal Muscle Profiling.

The biochemical and cell biological profiling of contractile fiber types and subcellular structures plays a central role in basic and applied myology. Mass spectrometry-based proteomics presents an ideal approach for the systematic identification of proteomic and subproteomic markers. These representative components of fast versus slow muscle fibers and their subcellular fractions are highly useful for in-depth surveys of skeletal muscle adaptations to physiological challenges, as well as the improvement of diagnostic, prognostic, and therapy-monitoring methodologies in muscle pathology.

Proteomic Identification of Saliva Proteins as Noninvasive Diagnostic Biomarkers.

Many biomedically relevant biomarkers are proteins with characteristic biochemical properties and a relatively restricted subcellular distribution. The comparative and mass spectrometry-based proteomic analysis of body fluids can be particularly instrumental for the targeted identification of novel protein biomarkers with pathological relevance. In this respect, new research efforts in biomarker discovery focus on the systematic mapping of the human saliva proteome, as well as the pathobiochemical identification of disease-related modifications or concentration changes in specific saliva proteins.

Proteomic Identification of Markers of Membrane Repair, Regeneration and Fibrosis in the Aged and Dystrophic Diaphragm.

Deficiency in the membrane cytoskeletal protein dystrophin is the underlying cause of the progressive muscle wasting disease named Duchenne muscular dystrophy. In order to detect novel disease marker candidates and confirm the complexity of the pathobiochemical signature of dystrophinopathy, mass spectrometric screening approaches represent ideal tools for comprehensive biomarker discovery studies. In this report, we describe the comparative proteomic analysis of young versus aged diaphragm muscles from wild type versus the dystrophic mouse model of X-linked muscular dystrophy.

Proteomic profiling of impaired excitation-contraction coupling and abnormal calcium handling in muscular dystrophy.

The X-linked inherited neuromuscular disorder Duchenne muscular dystrophy is characterised by primary abnormalities in the membrane cytoskeletal component dystrophin. The almost complete absence of the Dp427-M isoform of dystrophin in skeletal muscles renders contractile fibres more susceptible to progressive degeneration and a leaky sarcolemma membrane. This in turn results in abnormal calcium homeostasis, enhanced proteolysis and impaired excitation-contraction coupling.

Proteomic profiling of carbonic anhydrase CA3 in skeletal muscle.

Introduction: Carbonic anhydrase (CA) is a key enzyme that mediates the reversible hydration of carbon dioxide. Skeletal muscles contain high levels of the cytosolic isoform CA3. This enzyme has antioxidative function and plays a crucial role in the maintenance of intracellular pH homeostasis.

Histopathology of Duchenne muscular dystrophy in correlation with changes in proteomic biomarkers.

Duchenne muscular dystrophy is an inherited disorder of early childhood that affects multiple systems in the body. Besides late-onset cardio-respiratory syndrome and various body-wide pathophysiological changes, X-linked muscular dystrophy is primarily classified as a disorder of the skeletal musculature. This is reflected by severe histopathological alterations in voluntary contractile tissues, including progressive fibre degeneration, fat substitution, reactive myofibrosis and chronic inflammation.

Complexity of skeletal muscle degeneration: multi-systems pathophysiology and organ crosstalk in dystrophinopathy.

Duchenne muscular dystrophy is a highly progressive muscle wasting disorder due to primary abnormalities in one of the largest genes in the human genome, the DMD gene, which encodes various tissue-specific isoforms of the protein dystrophin. Although dystrophinopathies are classified as primary neuromuscular disorders, the body-wide abnormalities that are associated with this disorder and the occurrence of organ crosstalk suggest that a multi-systems pathophysiological view should be taken for a better overall understanding of the complex aetiology of X-linked muscular dystrophy. This article reviews the molecular and cellular effects of deficiency in dystrophin isoforms in relation to voluntary striated muscles, the cardio-respiratory system, the kidney, the liver, the gastrointestinal tract, the nervous system and the immune system.

Mass Spectrometric Profiling of Extraocular Muscle and Proteomic Adaptations in the Model of Duchenne Muscular Dystrophy.

Extraocular muscles (EOMs) represent a specialized type of contractile tissue with unique cellular, physiological, and biochemical properties. In Duchenne muscular dystrophy, EOMs stay functionally unaffected in the course of disease progression. Therefore, it was of interest to determine their proteomic profile in dystrophinopathy.

Proteomic profiling of the interface between the stomach wall and the pancreas in dystrophinopathy.

The neuromuscular disorder Duchenne muscular dystrophy is a multi-systemic disease that is caused by a primary abnormality in the X-chromosomal Dmd gene. Although progressive skeletal muscle wasting and cardio-respiratory complications are the most serious symptoms that are directly linked to the almost complete loss of the membrane cytoskeletal protein dystrophin, dystrophic patients also suffer from gastrointestinal dysfunction. In order to determine whether proteome-wide changes potentially occur in the gastrointestinal system due to dystrophin deficiency, total tissue extracts from the interface between the stomach wall and the pancreas of the mdx-4cv model of dystrophinopathy were analysed by mass spectrometry.

The Dystrophin Node as Integrator of Cytoskeletal Organization, Lateral Force Transmission, Fiber Stability and Cellular Signaling in Skeletal Muscle.

The systematic bioanalytical characterization of the protein product of the gene, which is defective in the pediatric disorder Duchenne muscular dystrophy, led to the discovery of the membrane cytoskeletal protein dystrophin. Its full-length muscle isoform Dp427-M is tightly linked to a sarcolemma-associated complex consisting of dystroglycans, sarcoglyans, sarcospan, dystrobrevins and syntrophins. Besides these core members of the dystrophin-glycoprotein complex, the wider dystrophin-associated network includes key proteins belonging to the intracellular cytoskeleton and microtubular assembly, the basal lamina and extracellular matrix, various plasma membrane proteins and cytosolic components.

Protocol for the Bottom-Up Proteomic Analysis of Mouse Spleen.

This protocol describes the comparative proteomic profiling of the spleen of wild type versus mouse, a model of dystrophinopathy. We detail sample preparation for bottom-up proteomic mass spectrometry experiments, including homogenization of tissue, protein concentration measurements, protein digestion, and removal of interfering chemicals. We then describe the steps for mass spectrometric analysis and bioinformatic evaluation.