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The highly progressive neuromuscular disorder dystrophinopathy is triggered by primary abnormalities in the Dmd gene, which causes cytoskeletal instability and loss of sarcolemmal integrity. Comparative organellar proteomics was employed to identify sarcolemma-associated proteins with an altered concentration in dystrophic muscle tissue from the mdx-4cv mouse model of dystrophinopathy. A lectin agglutination method was used to prepare a sarcolemma-enriched fraction and resulted in the identification of 190 significantly changed protein species. Proteomics established differential expression patterns for key components of the muscle plasma membrane, cytoskeletal network, extracellular matrix, metabolic pathways, cellular stress response, protein synthesis, immune response and neuromuscular junction. The deficiency in dystrophin and drastic reduction in dystrophin-associated proteins appears to trigger (i) enhanced membrane repair involving myoferlin, dysferlin and annexins, (ii) increased protein synthesis and the compensatory up-regulation of cytoskeletal proteins, (iii) the decrease in the scaffolding protein periaxin and myelin PO involved in myelination of motor neurons, (iv) complex changes in bioenergetic pathways, (v) elevated levels of molecular chaperones to prevent proteotoxic effects, (vi) increased collagen deposition causing reactive myofibrosis, (vii) disturbed ion homeostasis at the sarcolemma and associated membrane systems, and (viii) a robust inflammatory response by the innate immune system in response to chronic muscle damage. Significance: Duchenne muscular dystrophy is a devastating muscle wasting disease and represents the most frequently inherited neuromuscular disorder in humans. Genetic abnormalities in the Dmd gene cause a loss of sarcolemmal integrity and highly progressive muscle fibre degeneration. Changes in the neuromuscular system are associated with necrosis, fibrosis and inflammation. In order to evaluate secondary changes in the sa

Dystrophinopathies are multi-system disorders that affect the skeletal musculature, the cardio-respiratory system and the central nervous system. The systematic screening of suitable biofluids for released or altered proteins promises new insights into the highly complex pathophysiology of X-linked muscular dystrophy. However, standard detection approaches using antibody-based assays often fail to reproducibly detect low-abundance protein isoforms in dilute biological fluids. In contrast, mass spectrometric screening approaches enable the proteome-wide identification of minor protein changes in biofluids. This report describes the findings from the comparative proteomic analysis of whole saliva samples from wild type versus the established mdx-4cv mouse model of highly progressive muscular dystrophy, focusing on the kallikrein protein family. Kallikrein-1 (Klk1) and 13 Klk1-related peptidases were identified in saliva and serum from normal mice. Comparative proteomics revealed elevated saliva levels of the Klk1-related peptidases Klk1-b1, Klk1-b5 and Klk-b22, as well as an increased Klk-1 concentration, which agrees with higher Klk-1 levels in serum from mdx-4cv mice. This indicates altered cellular signaling, extracellular matrix remodeling and an altered immune response in the mdx-4cv mouse, and establishes liquid biopsy procedures as suitable bioanalytical tools for the systematic survey of complex pathobiochemical changes in animal models of muscular dystrophy.

The comparative proteomic data presented in this article provide supporting information to the related research article "Proteomic identification of elevated saliva kallikrein levels in the mdx-4cv mouse model of Duchenne muscular dystrophy " (Murphy et al., 2018). Here we provide additional datasets on the comparative proteomic analysis of saliva and serum proteins and the mass spectrometric identification of kallikrein isoform Klk-1 in wild type versus mdx-4cv saliva specimens. The data article presents the systematic identification of the assessable saliva proteome and the differential presence of proteins in saliva versus serum samples. Representative mass spectrometric scans of unique peptides that were employed to identify the kallikrein isoform Klk-1 in wild type versus mdx-4cv saliva specimens are provided. The dataset contains typical saliva-associated marker proteins, including alphaamylase and albumin, as well as distinct isoforms of cystatin, serpin, kallikrein, cathepsin, glutathione transferase, carbonic anhydrase, mucin, pyruvate kinase, and aldolase.

Duchenne muscular dystrophy is a highly progressive muscle wasting disease with a complex pathophysiology that is based on primary abnormalities in the dystrophin gene. In order to study potential changes in the oligomerization of high-molecular-mass protein complexes in dystrophic skeletal muscle, chemical crosslinking was combined with mass spectrometric analysis. The biochemical stabilization of protein interactions was carried out with the homo-bifunctional and amine-reactive agent bis[sulfosuccinimidyl]suberate, followed by protein shift analysis in one-dimensional gels. The proteomic approach identified 11 and 15 protein species in wild type versus dystrophic microsomal fractions, respectively, as well as eight common proteins, with an electrophoretic mobility shift to very high molecular mass following chemical crosslinking. In dystrophin-deficient preparations, several protein species with an increased tendency of oligomerisation were identified as components of the sarcolemma and its associated intra- and extracellular structures, as well as mitochondria. This included the sarcolemmal proteins myoferlin and caveolin, the cytoskeletal components vimentin and tubulin, extracellular collagen alpha-1(XII) and the mitochondrial trifunctional enzyme and oxoglutarate dehydrogenase. These changes are probably related to structural and metabolic adaptations, especially cellular repair processes, which agrees with the increased oligomerisation of myosin-3, myosin-9 and actin, and their role in cellular regeneration and structural adjustments in dystrophinopathy.

X-linked muscular dystrophy is caused by primary abnormalities in the Dmd gene and is characterized by the almost complete loss of the membrane cytoskeletal protein dystrophin, which triggers sarcolemmal instability, abnormal calcium homeostasis, increased proteolysis and impaired excitation‑contraction coupling. In addition to progressive necrosis, crucial secondary pathologies are represented by myofibrosis and the invasion of immune cells in damaged muscle fibres. In order to determine whether these substantial changes within the skeletal musculature are reflected by an altered rate of protein release into the circulatory system or other plasma fluctuations, we used label‑free mass spectrometry to characterize serum from the mdx‑4cv model of Duchenne muscular dystrophy. Comparative proteomics revealed a large number of increased vs. decreased protein species in mdx‑4cv serum. A serum component with greatly elevated levels was identified as the inflammation‑inducible plasma marker haptoglobin. This acute phase response protein is usually secreted in relation to tissue damage and sterile inflammation. Both immunoblot analyses and enzyme‑linked immunosorbent assays confirmed the increased concentration of haptoglobin in crude mdx‑4cv serum. This suggests that haptoglobin, in conjunction with other altered serum proteins, represents a novel diagnostic, prognostic and/or therapy‑monitoring biomarker candidate to evaluate the inflammatory response in the mdx‑4cv animal model of dystrophinopathy.

In this article, we illustrate the application of difference in-gel electrophoresis for the proteomic analysis of dystrophic skeletal muscle. The mdx diaphragm was used as a tissue model of dystrophinopathy. Two-dimensional gel electrophoresis is a widely employed protein separation method in proteomic investigations. Although two-dimensional gels usually underestimate the cellular presence of very high molecular mass proteins, integral membrane proteins and low copy number proteins, this method is extremely powerful in the comprehensive analysis of contractile proteins, metabolic enzymes, structural proteins and molecular chaperones. This gives rise to two-dimensional gel electrophoretic separation as the method of choice for studying contractile tissues in health and disease. For comparative studies, fluorescence difference in-gel electrophoresis has been shown to provide an excellent biomarker discovery tool. Since aged diaphragm fibres from the mdx mouse model of Duchenne muscular dystrophy closely resemble the human pathology, we have carried out a mass spectrometry-based comparison of the naturally aged diaphragm versus the senescent dystrophic diaphragm. The proteomic comparison of wild type versus mdx diaphragm resulted in the identification of 84 altered protein species. Novel molecular insights into dystrophic changes suggest increased cellular stress, impaired calcium buffering, cytostructural alterations and disturbances of mitochondrial metabolism in dystrophin-deficient muscle tissue.*

The majority of patients afflicted with Duchenne muscular dystrophy develop cardiomyopathic complications, warranting large-scale proteomic studies of global cardiac changes for the identification of new protein markers of dystrophinopathy. The aged heart from the X-linked dystrophic mdx mouse has been shown to exhibit distinct pathological aspects of cardiomyopathy. In order to establish age-related alterations in the proteome of dystrophin-deficient hearts, cardiomyopathic tissue from young versus aged mdx mice was examined by label-free LC-MS/MS. Significant age-dependent alterations were established for 67 proteins, of which 28 proteins were shown to exhibit a lower abundance and 39 proteins were found to be increased in their expression levels. Drastic changes were demonstrated for 17 proteins, including increases in Ig chains and transferrin, and drastic decreases in laminin, nidogen and annexin. An immunblotting survey of young and old wild-type versus mdx hearts confirmed these proteomic findings and illustrated the effects of natural aging versus dystrophin deficiency. These proteome-wide alterations suggest a disintegration of the basal lamina structure and cytoskeletal network in dystrophin-deficient cardiac fibres, increased levels of antibodies in a potential autoimmune reaction of the degenerating heart, compensatory binding of excess iron and a general perturbation of metabolic pathways in dystrophy-associated cardiomyopathy.

The age-related loss of skeletal muscle mass and associated progressive decline in contractile strength is a serious pathophysiological issue in the elderly. In order to investigate global changes in the skeletal muscle proteome after the fifth decade of life, this study analysed total extracts from human vastus lateralis muscle by fluorescence difference in-gel electrophoresis. Tissue specimens were derived from middle-aged (47-62 years) vs. aged (76-82 years) individuals and potential changes in the protein expression profiles were compared between these two age groups by a comprehensive gel electrophoresis-based survey. Age-dependent alterations in the concentration of 19 protein spots were revealed and mass spectrometry identified these components as being involved in the excitation-contraction-relaxation cycle, muscle metabolism, ion handling and the cellular stress response. This indicates a generally perturbed protein expression pattern in senescent human muscle. Increased levels of mitochondrial enzymes and isoform switching of the key contractile protein, actin, support the idea of glycolytic-to-oxidative and fast-to-slow transition processes during muscle aging. Importantly, the carbonic anhydrase (CA)3 isoform displayed an increased abundance during muscle aging, which was independently verified by immunoblotting of differently aged human skeletal muscle samples. Since the CA3 isoform is relatively muscle-specific and exhibits a fibre type-specific expression pattern, this enzyme may represent an interesting new biomarker of sarcopenia. Increased levels of CA are indicative of an increased demand of CO2-removal in senescent muscle, and also suggest age-related fibre type shifting to slower-contracting muscles during human aging.