Pathobiochemical Changes in Diabetic Skeletal Muscle as Revealed by Mass-Spectrometry-Based Proteomics

Insulin resistance in skeletal muscle tissues and diabetes-related muscle weakness are serious pathophysiological problems of increasing medical importance. In order to determine global changes in the protein complement of contractile tissues due to diabetes mellitus, mass-spectrometry-based proteomics has been applied to the investigation of diabetic muscle. This review summarizes the findings from recent proteomic surveys of muscle preparations from patients and established animal models of type 2 diabetes. The potential impact of novel biomarkers of diabetes, such as metabolic enzymes and molecular chaperones, is critically examined. Disease-specific signature molecules may be useful for increasing our understanding of the molecular and cellular mechanisms of insulin resistance and possibly identify new therapeutic options that counteract diabetic abnormalities in peripheral organ systems. Importantly, the biomedical establishment of biomarkers promises to accelerate the development of improved diagnostic procedures for characterizing individual stages of diabetic disease progression, including the early detection of prediabetic complications

Proteomic Profiling of Fast-To-Slow Muscle Transitions during Aging

Old age is associated with a large spectrum of physical ailments, including muscle wasting. Skeletal muscle degeneration drastically increases the risk of poor balance, frequent falling and impaired mobility in the elderly. In order to identify new therapeutic targets to halt or even reverse age-dependent muscle weakness and improve diagnostic methods to properly evaluate sarcopenia as a common geriatric syndrome, there is an urgent need to establish a reliable biomarker signature of muscle aging. In this respect, mass spectrometry-based proteomics has been successfully applied for studying crude extracts and subcellular fractions from aged animal and human muscle tissues to identify novel aging marker proteins. This review focuses on key physiological and metabolic aspects of sarcopenia, i.e., age-related muscle fiber transitions and metabolic shifts in aging muscle as revealed by proteomics. Over the last decade, proteomic profiling studies have clearly confirmed the idea that sarcopenia is based on a multi-factorial pathophysiology and that a glycolytic-to-oxidative shift occurs in slower-twitching senescent muscles. Both, newly identified protein factors and confirmed alterations in crucial metabolic and contractile elements can now be employed to establish a sarcopenia-specific biomarker signature

Skeletal muscle proteomics: current approaches, technical challenges and emerging techniques

Background: Skeletal muscle fibres represent one of the most abundant cell types in mammals. Their highly specialised contractile and metabolic functions depend on a large number of membrane-associated proteins with very high molecular masses, proteins with extensive posttranslational modifications and components that exist in highly complex supramolecular structures. This makes it extremely difficult to perform conventional biochemical studies of potential changes in protein clusters during physiological adaptations or pathological processes. Results: Skeletal muscle proteomics attempts to establish the global identification and biochemical characterisation of all members of the muscle-associated protein complement. A considerable number of proteomic studies have employed large-scale separation techniques, such as high-resolution two-dimensional gel electrophoresis or liquid chromatography, and combined them with mass spectrometry as the method of choice for high-throughput protein identification. Muscle proteomics has been applied to the comprehensive biochemical profiling of developing, maturing and aging muscle, as well as the analysis of contractile tissues undergoing physiological adaptations seen in disuse atrophy, physical exercise and chronic muscle transformation. Biomedical investigations into proteome-wide alterations in skeletal muscle tissues were also used to establish novel biomarker signatures of neuromuscular disorders. Importantly, mass spectrometric studies have confirmed the enormous complexity of posttranslational modifications in skeletal muscle proteins. Conclusions: This review critically examines the scientific impact of modern muscle proteomics and discusses its successful application for a better understanding of muscle biology, but also outlines its technical limitations and emerging techniques to establish new biomarker candidates

Proteomic profiling of naturally protected extraocular muscles from the dystrophin-deficient mdx mouse

Duchenne muscular dystrophy is the most frequent neuromuscular disorder of childhood. Although this x-linked muscle disease is extremely progressive, not all subtypes of skeletal muscles are affected in the same way. While extremities and trunk muscles are drastically weakened, extraocular muscles are usually spared in Duchenne patients. In order to determine the global protein expression pattern in these naturally protected muscles we have performed a comparative proteomic study of the established mdx mouse model of x-linked muscular dystrophy. Fluorescence difference in-gel electrophoretic analysis of 9-week-old dystrophin-deficient versus age-matched normal extraocular muscle, using a pH 4-7 gel range, identified out of 1088 recognized protein spots a moderate expression change in only seven protein species. Desmin, apolipoprotein A-I binding protein and perilipin-3 were found to be increased and gelsolin, gephyrin, transaldolase, and acyl-CoA dehydrogenase were shown to be decreased in mdx extraocular muscles. Immunoblotting revealed a drastic up-regulation of utrophin, comparable levels of beta-dystroglycan and key Ca(2+)-regulatory elements, and an elevated concentration of small stress proteins in mdx extraocular muscles. This suggests that despite the lack of dystrophin only a limited number of cellular systems are perturbed in mdx extraocular muscles, probably due to the substitution of dystrophin by its autosomal homolog. Utrophin appears to prevent the loss of dystrophin-associated proteins and Ca(2+)-handling elements in extraocular muscle tissue. Interestingly, the adaptive mechanisms that cause the sparing of extraocular fibers seem to be closely linked to an enhanced cellular stress response

Diversity of the Brain Dystrophin-Glycoprotein Complex

Duchenne muscular dystrophy (DMD), the most common inherited neuromuscular disorder, is characterized by progressive muscle wasting and weakness. One third of Duchenne patients suffer a moderate to severe, nonprogressive form of mental retardation. Mutations in the DMD gene are thought to be responsible, with the shorter isoforms of dystrophin implicated in its molecular brain pathogenesis. It is becoming clear that region-specific variations in dystrophin isoforms delegate the composition of the dystrophin-glycoprotein complex in brain, and hence, the function of the specific membrane assembly. Here we summarize the recent advances in the understanding of brain dystrophin, dystrophin-related proteins and dystrophin-associated proteins

Proteomic analysis of the mitochondria-enriched fraction from diabetic rat skeletal muscle

Mitochondrial dysfunction in muscle has been implicated to play a causative role or being an indirect consequence of insulin resistance in type-2 diabetes. In order to investigate potential diabetes-related alterations in the mitochondrial proteome of muscle, we have carried out a mass spectrometry-based proteomic analysis of the gastrocnemius muscle from normal versus diabetic Goto-Kakizaki rats. A generally perturbed protein expression pattern was observed in the mitochondria-enriched fraction from diabetic muscle. Various mitochondrial markers, including NADH dehydrogenase, cytochrome b-c1 complex and isocitrate dehydrogenase were reduced in diabetic preparations. Isoforms of pyruvate dehydrogenase and ATP synthase exhibited differential changes in their abundance. The altered protein expression levels of these key metabolic enzymes might trigger a diabetes-dependent decrease in mitochondrial oxidative phosphorylation levels. The proteomic findings presented here support the idea that mitochondrial abnormalities are involved in the molecular pathogenesis of type-2 diabetes and may be crucial for the development of insulin resistance

Proteomic profiling of naturally protected extraocular muscles from the dystrophin-deficient mdx mouse

Duchenne muscular dystrophy is the most frequent neuromuscular disorder of childhood. Although this x-linked muscle disease is extremely progressive, not all subtypes of skeletal muscles are affected in the same way. While extremities and trunk muscles are drastically weakened, extraocular muscles are usually spared in Duchenne patients. In order to determine the global protein expression pattern in these naturally protected muscles we have performed a comparative proteomic study of the established mdx mouse model of x-linked muscular dystrophy. Fluorescence difference in-gel electrophoretic analysis of 9-week-old dystrophin-deficient versus age-matched normal extraocular muscle, using a pH 4-7 gel range, identified out of 1088 recognized protein spots a moderate expression change in only seven protein species. Desmin, apolipoprotein A-I binding protein and perilipin-3 were found to be increased and gelsolin, gephyrin, transaldolase, and acyl-CoA dehydrogenase were shown to be decreased in mdx extraocular muscles. Immunoblotting revealed a drastic up-regulation of utrophin, comparable levels of beta-dystroglycan and key Ca(2+)-regulatory elements, and an elevated concentration of small stress proteins in mdx extraocular muscles. This suggests that despite the lack of dystrophin only a limited number of cellular systems are perturbed in mdx extraocular muscles, probably due to the substitution of dystrophin by its autosomal homolog. Utrophin appears to prevent the loss of dystrophin-associated proteins and Ca(2+)-handling elements in extraocular muscle tissue. Interestingly, the adaptive mechanisms that cause the sparing of extraocular fibers seem to be closely linked to an enhanced cellular stress response

Oligomerization of β-dystroglycan in rabbit diaphragm and brain as revealed by chemical crosslinking

AbstractThe surface component β-dystroglycan is a member of the dystrophin–glycoprotein complex providing a trans-sarcolemmal linkage between the actin membrane cytoskeleton and the extracellular matrix component laminin-α2. Although abnormalities in this complex are involved in the pathophysiology of various neuromuscular disorders, little is known about the organization of dystrophin-associated glycoproteins in diaphragm and brain. We therefore investigated the oligomerization of β-dystroglycan and its connection with the most abundant dystrophin homologues in these two tissues. Employing detergent solubilization and alkaline extraction procedures of native membranes, it was confirmed that β-dystroglycan behaves like an integral surface molecule as predicted by its cDNA sequence. Immunoblot analysis following chemical crosslinking of native membranes showed that β-dystroglycan has a tendency to form high-molecular-mass complexes. Within these crosslinkable complexes, immuno-reactive overlaps were observed between β-dystroglycan, α-dystroglycan, laminin and 427 kDa dystrophin in diaphragm and skeletal muscle. In synaptosomes, the major brain dystrophin isoform Dp116 also exhibited an immuno-reactive overlap with members of the dystroglycan complex. These findings demonstrate that β-dystroglycan does not exist as a monomer in native membranes and imply that certain dystrophin isoforms and dystrophin-associated components interact with this surface protein in diaphragm and brain as has been previously shown for skeletal and heart muscle

Proteomic DIGE analysis of the mitochondria-enriched fraction from aged rat skeletal muscle

Skeletal muscle aging is associated with a loss in tissue mass and contractile strength, as well as fiber type shifting and bioenergetic adaptation processes. Since mitochondria represent the primary site for energy generation via oxidative phosphorylation, we investigated potential changes in the expression pattern of the mitochondrial proteome using the highly sensitive DIGE approach. The comparative analysis of the mitochondria-enriched fraction from young adult versus aged muscle revealed an age-related change in abundance for 39 protein species. MS technology identified the majority of altered proteins as constituents of muscle mitochondria. An age-dependent increase was observed for NADH dehydrogenase, the mitochondrial inner membrane protein mitofilin, peroxiredoxin isoform PRX-III, ATPase synthase, succinate dehydrogenase, mitochondrial fission protein Fis1, succinate-coenzyme A ligase, acyl-coenzyme A dehydrogenase, porin isoform VDAC2, ubiquinol-cytochrome c reductase core I protein and prohibitin. Immunoblotting, enzyme testing and confocal microscopy were used to validate proteomic findings. The DIGE-identified increase in key mitochondrial elements during aging agrees with the concept that sarcopenia is associated with a shift to a slower contractile phenotype and more pronounced aerobic-oxidative metabolism. This suggests that mitochondrial markers are reliable candidates that should be included in the future establishment of a biomarker signature of skeletal muscle aging

Illustration of Skeletal Muscle Calsequestrin Complex Formation by Cell Overlay and Two-Dimensional Blot Overlay Methodology

Based on the recent finding that a peroxidase-conjugated calsequestrin probe retains its biological binding properties, we have used it in two-dimensional blot and cell overlay procedures. Labeling of nitrocellulose replicas of electrophoretically separated microsomal proteins from predominantly fast versus slow fibres revealed self-aggregation of fast calsequestrin with molecular species of differing isolectric points. Incubation of transverse cryosections showed restricted calsequestrin interactions with elements in the fibre interior, mostly representing calsequestrin itself. This confirms that fast calsequestrin exists in large aggregates under native conditions and demonstrates the usefulness of blot and cell overlay techniques in identifying and locating supramolecular protein assemblies