Human Muscle Biopsy Specimens Research Articles

Two-CyDye-Based 2D-DIGE Analysis of Aged Human Muscle Biopsy Specimens.

The gradual loss of skeletal muscle mass during aging and associated decline in contractile strength can result in reduced fitness, frailty, and loss of independence. In order to better understand the molecular and cellular mechanisms that underlie sarcopenia of old age and the frailty syndrome, as well as identify novel therapeutic targets to treat age-related fiber wasting, it is crucial to develop a comprehensive biomarker signature of muscle aging. Fluorescence two-dimensional gel electrophoresis (2D-DIGE) in combination with sensitive mass spectrometry presents an ideal bioanalytical tool for biomarker discovery in biogerontology. This chapter outlines the application of the 2D-DIGE method for the comparative analysis of human biopsy specimens from middle-aged versus senescent individuals using a two-CyDye-based method.

Targeting necroptosis in muscle fibers ameliorates inflammatory myopathies

Muscle cell death in polymyositis is induced by CD8+ cytotoxic T lymphocytes. We hypothesized that the injured muscle fibers release pro-inflammatory molecules, which would further accelerate CD8+ cytotoxic T lymphocytes-induced muscle injury, and inhibition of the cell death of muscle fibers could be a novel therapeutic strategy to suppress both muscle injury and inflammation in polymyositis. Here, we show that the pattern of cell death of muscle fibers in polymyositis is FAS ligand-dependent necroptosis, while that of satellite cells and myoblasts is perforin 1/granzyme B-dependent apoptosis, using human muscle biopsy specimens of polymyositis patients and models of polymyositis in vitro and in vivo. Inhibition of necroptosis suppresses not only CD8+ cytotoxic T lymphocytes-induced cell death of myotubes but also the release of inflammatory molecules including HMGB1. Treatment with a necroptosis inhibitor or anti-HMGB1 antibodies ameliorates myositis-induced muscle weakness as well as muscle cell death and inflammation in the muscles. Thus, targeting necroptosis in muscle cells is a promising strategy for treating polymyositis providing an alternative to current therapies directed at leukocytes.

Expression of the dermatomyositis autoantigen transcription intermediary factor 1γ in regenerating muscle.

Autoantibodies against transcription intermediary factor 1γ (TIF1γ) are found in many patients with dermatomyositis (DM). Although TIF1γ is known to play a role in the differentiation of other tissues, its functional role in muscle regeneration has not been elucidated. This study was undertaken to explore the regulation and functional role of this protein during muscle differentiation and regeneration. TIF1γ expression was analyzed in human muscle biopsy specimens using immunofluorescence microscopy. Immunofluorescence microscopy and immunoblotting analyses were used to study TIF1γ expression in a mouse model of muscle injury and repair. The effect of premature TIF1γ silencing on muscle differentiation was studied in cultured mouse myoblasts. In muscle biopsy specimens from DM patients, TIF1γ was expressed at low levels in the nuclei of histologically normal muscle cells but at high levels in the centralized nuclei of atrophic, perifascicular myofibers expressing markers of regeneration. TIF1γ levels were also increased in regenerating myonuclei following muscle injury in mice. Premature silencing of TIF1γ in vitro using small interfering RNA did not accelerate the expression of myogenin, a transcription factor that plays a central role in regulating relatively early stages of muscle differentiation. However, premature silencing of TIF1γ did accelerate myotube fusion and the expression of myosin heavy chain (MyHC), a later marker of muscle differentiation. The DM autoantigen TIF1γ is markedly up-regulated during muscle regeneration in human and mouse muscle cells. Premature silencing of this protein in cultured myoblasts accelerates MyHC expression and myoblast fusion. However, TIF1γ may function independently of, or downstream from, myogenin.

Ahnak1 abnormally localizes in muscular dystrophies and contributes to muscle vesicle release

Ahnak1 is a giant, ubiquitously expressed, plasma membrane support protein whose function in skeletal muscle is largely unknown. Therefore, we investigated whether ahnak would be influenced by alterations of the sarcolemma exemplified by dysferlin mutations known to render the sarcolemma vulnerable or by mutations in calpain3, a protease known to cleave ahnak. Human muscle biopsy specimens obtained from patients with limb girdle muscular dystrophy (LGMD) caused by mutations in dysferlin (LGMD2B) and calpain3 (LGMD2A) were investigated for ahnak expression and localization. We found that ahnak1 has lost its sarcolemmal localization in LGMD2B but not in LGMD2A. Instead ahnak1 appeared in muscle connective tissue surrounding the extracellular site of the muscle fiber in both muscular dystrophies. The entire giant ahnak1 molecule was present outside the muscle fiber and did only partially colocalize with CD45-positive immune cell infiltration and the extracelluar matrix proteins fibronectin and collagenVI. Further, vesicles shedded in response to Ca2+ by primary human myotubes were purified and their protein content was analysed. Ahnak1 was prominently present in these vesicles. Electron microscopy revealed a homogenous population of vesicles with a diameter of about 150 nm. This is the first study demonstrating vesicle release from human myotubes that may be one mechanism underlying abnormally localized ahnak1. Taken together, our results define ahnak1 in muscle connective tissue as a novel feature of two genetically distinct muscular dystrophies that might contribute to disease pathology.Electronic supplementary materialThe online version of this article (doi:10.1007/s10974-011-9271-8) contains supplementary material, which is available to authorized users.

Interferon‐stimulated gene 15 (ISG15) conjugates proteins in dermatomyositis muscle with perifascicular atrophy

We investigated interferon-stimulated gene 15 (ISG15), a poorly understood ubiquitin-like modifier, and its enzymatic pathway in dermatomyositis (DM), an autoimmune disease primarily involving muscle and skin. We generated microarray data measuring transcript abundance for approximately 18,000 genes in each of 113 human muscle biopsy specimens, and studied biopsy specimens and cultured skeletal muscle using immunohistochemistry, immunoblotting proteomics, real-time quantitative polymerase chain reaction, and laser-capture microdissection. Transcripts encoding ISG15-conjugation pathway proteins were markedly upregulated in DM with perifascicular atrophy (DM-PFA) muscle (ISG15 339-fold, HERC5 62-fold, and USP18 68-fold) compared with 99 non-DM samples. Combined analysis with publicly available microarray datasets showed that >50-fold ISG15 transcript elevation had 100% sensitivity and specificity for 28 biopsies from adult DM-PFA and juvenile DM patients compared with 199 muscle samples from other muscle diseases. Free ISG15 and ISG15-conjugated proteins were only found on immunoblots from DM-PFA muscle. Cultured human skeletal muscle exposed to type 1 interferons produced similar transcripts and ISG15 protein and conjugates. Laser-capture microdissection followed by proteomic analysis showed deficiency of titin in DM perifascicular atrophic myofibers. A large-scale microarray study of muscle samples demonstrated that among a diverse group of muscle diseases DM was uniquely associated with upregulation of the ISG15 conjugation pathway. Exposure of human skeletal muscle cell culture to type 1 interferons produced a molecular picture highly similar to that seen in human DM muscle. Perifascicular atrophic myofibers in DM were deficient in a number of skeletal muscle proteins including titin.

Expression of the dermatomyositis autoantigen Mi‐2 in regenerating muscle

Autoantibodies against the chromatin remodeler Mi-2 are found in a distinct subset of patients with dermatomyositis (DM). Previous quantitative immunoblotting experiments demonstrated that Mi-2 protein levels are up-regulated in DM muscle. This study was undertaken to define the population of cells expressing high levels of Mi-2 in DM muscle and to explore the regulation and functional role of Mi-2 during muscle regeneration. The expression of Mi-2 was analyzed by immunofluorescence microscopy in human muscle biopsy specimens. In an experimental mouse model, cardiotoxin was used to induce muscle injury and repair, and expression of Mi-2 during muscle regeneration was studied in this model by immunofluorescence and immunoblotting analyses. In addition, a cell culture system of muscle differentiation was utilized to artificially modulate Mi-2 levels during proliferation and differentiation of myoblasts. In human DM muscle tissue, increased Mi-2 expression was found preferentially in the myofibers within fascicles affected by perifascicular atrophy, particularly in the centralized nuclei of small perifascicular muscle fibers expressing markers of regeneration. In injured mouse muscle tissue, Mi-2 levels were dramatically and persistently up-regulated during muscle regeneration in vivo. Premature silencing of Mi-2 with RNA interference in vitro resulted in accelerated myoblast differentiation. Expression of Mi-2 is markedly up-regulated during muscle regeneration in a mouse model of muscle injury and repair. It is also up-regulated in human DM myofibers expressing markers of regeneration. Results of the in vitro studies indicate that this protein may play a role in modulating the kinetics of myoblast differentiation. Our findings thus suggest that high levels of Mi-2 expression in muscle biopsy tissue from patients with DM reflect the presence of incompletely differentiated muscle cells.

Expression of platelet-derived growth factor and its receptor at human and mouse neuromuscular junctions

Objective To investigate the immunoreactivity of platelet-derived growth factor-A (PDGF-A) and its receptor at human and mouse neuromuscular junctions and to explore their regulatory effects on neuromuscular junctions. Methods Specific polyclonal antibodies were used to detect PDGF-A and PDGF a-receptor expression. Double immunohistochemistry for platelet-derived growth factor and the acetylcholine receptor was performed on normal human muscle biopsy specimens. Double fluorescence labeling was applied to detect immunoreactivity for PDGF-A, its receptor and acetylcholine receptors. Results PDGF-A and its receptor was closely co-localized with acetylcholine receptors at human and mouse neuromuscular junctions. Conclusion PDGF-A and its receptor concentrated at human and mouse neuromuscular junctions. PDGF might be involved in the interaction between the presynaptic and postsynaptic components, PDGF-A and its receptor might play regulatory role in signaling at neuromuscular junctions in normal muscle. Key words: Platelet-derived growth factor; Receptor; Neuromuscular junction

Ultrastructural findings at the satellite cell-myofiber border in normal and diseased human muscle biopsy specimens.

Satellite cells (SC) are mononuclear myoblasts located between the plasma membrane and the basement membrane of a myofiber. In normal adult muscle, SC are quiescent in the G0 phase of the cell cycle. The contact of the SC with the myofiber plasma membrane imposes a mitotic inhibition on the SC. Sarcolemmal molecules which might explain this membrane-imparted mitotic inhibition have not yet been identified. In this study we examined the border of the SC and the adjacent myofiber electron microscopically, assessing the number of SC showing encroachments of basement membrane (BM) material, secretion of cellular degradation products into the intercellular space, and caveolae. We studied normal and diseased muscle including Duchenne muscular dystrophy, Becker muscular dystrophy, idiopathic inflammatory myopathies, and neurogenic atrophies. Caveolae were present in SC from normal muscle, but they were more abundant in SC from diseased muscle, and they significantly prevailed at the outer surface of SC in all of the diseased muscle groups. Encroachments of BM material was only present in SC from diseased muscle, and mostly so in neurogenic atrophies. Secretion of cellular degradation products into the intercellular cleft occurred in normal and diseased muscle. We conclude that degradation products in the intercellular cleft do not disturb SC adhesion and that there is a neural influence on SC adhesion. The significance of the abundance of caveolae at the outer surface of the SC when compared with the inner surface in diseased muscle remains at present unknown.

One-step immunoaffinity purification of complex I subunits from beef heart mitochondria

Polypeptides of beef heart mitochondrial complex I were isolated from 15 mg of solubilized beef heart mitochondria using antibodies immobilized on an agarose chromatography column. The preparation was examined by SDS electrophoresis and Western blotting using affinity-purified antibodies to complex I and compared to beef heart complex I purified according to the conventional method of Hatefi and Rieske. There was a high degree of homology between the two preparations as judged by SDS-polyacrylamide electrophoresis and by immunoblotting with seven affinity-purified antibodies to various complex I subunits. This method could be applied to the preparation of complex I subunits from small samples such as human muscle biopsy specimens.

Dystrophin: A sensitive and reliable immunochemical assay in tissue and cell culture homogenates

A modified polyacrylamide gel electrophoresis system is described which provides excellent resolution of very high molecular weight proteins. This system has been successfully applied to the immunochemical detection of dystrophin in mouse and rat skeletal muscle, mouse myotubes in cell culture, and in human muscle-biopsy specimens. The mass of total homogenate protein (3–12 μg) and the relative quantity of dystrophin detected immunologically were found to be strongly correlated (r = 0.970 – 0.995). The method described here requires minute quantities of tissue or cells to accurately evaluate the relative amount of dystrophin present. The entire procedure for the detection of dystrophin is simple, rapid and cost efficient compared to other available techniques.

Characterization of a monoclonal antibody to myosin specific for mammalian and human type II muscle fibers

We have characterized a monoclonal antibody (McAb), ALD-19, generated against slow myosin from chicken anterior latissimus dorsi (ALD) muscle for use in studies of human and animal muscle fiber types. This McAb bound selectively to the 200 kDa myosin heavy chain band in immunoblots against chicken, rat and human myosins and showed selective staining of A bands in the myofibrils. The reactivity of ALD-19 with various myosin types was quantitated by radioimmunoassays. Fiber type analysis revealed unexpected specificity of McAb ALD-19 for type II mammalian muscle fibers. This antibody should, therefore be useful for identification and quantification of normal type II fibers in human muscle biopsy specimens.

Levels of adenylate deaminase, adenylate kinase, and creatine kinase in frozen human muscle biopsy specimens relative to type 1/type 2 fiber distribution: evidence for a carrier state of myoadenylate deaminase deficiency.

Myoadenylate deaminase deficiency is believed to reflect a genetic deficiency of skeletal muscle, but its pattern of inheritance has not been established. We examined, histochemically and by quantitative biochemical assay, muscle biopsy specimens from 3 putative carriers of this disorder. Adenylate kinase and creatine kinase were assayed in parallel with adenylate deaminase in order to establish enzyme activity ratios and the variation of each enzyme with fiber-type distribution. Control tissue consisted of 34 biopsy specimens without notable abnormalities from 30 patients, and included 4 specimen pairs with disparate fiber-type contributions. By linear regression analysis, adenylate deaminase level averaged 2.8-fold higher, and adenylate kinase 4.5-fold higher, in type 2 than in type 1 fibers, whereas creatine kinase level did not differ. The slopes of the regression lines resulting from analysis of the four specimen pairs from individual patients agreed well with the overall regression line in each plot. The 3 putative carriers had adenylate deaminase levels 2.5 to 5.7 times lower than the mean control value for their fiber-type distribution, but at least 20 times higher than their enzyme-deficient kinfolk. This finding indicates that a carrier state does exist, and that the deficiency state reflects an autosomal recessive inheritance pattern. Three additional biopsy specimens were excluded from evaluation when preliminary analysis showed elevated adenylate kinase/adenylate deaminase ratios that were outliers at the 1% level. This result suggests a carrier incidence of 10% in the muscle biopsy specimen population, which would markedly bias population estimates if undetected.

A fixative for use in muscle histochemistry.

A fixative solution that preserves the activity of some relevant enzymes in muscle histochemistry is described. Portions of human muscle biopsy specimens and selected murine muscles were fresh frozen or placed in the fixative at room temperature for up to 1 month before freezing. Cryostat sections of fresh frozen and fixed frozen tissue were assayed for nicotinamide adenine dinucleotide phosphate (NADH)-tetrazolium reductase (NADH), several adenosine triphosphatases (ATPases), myoadenylate deaminase (MD), and phosphorylase. NADH, ATPase, and MD activity were preserved following fixation but phosphorylase was not preserved. Murine spleen and kidney were similarly tested for acid phosphatase (acid phos), alkaline phosphatase (alk phos), and nonspecific esterase (NSE). Alk phos activity was preserved but acid phos and NSE activity were significantly reduced following fixation. This fixative is useful in some circumstances for processing or shipping human muscle biopsy specimens and experimental tissues.

Incomplete Palmitate Oxidation

A study of palmitate oxidation in 200 consecutive human muscle biopsy specimens showed 14 patients in whom there was abnormal, incomplete palmitate oxidation when the rate of oxidation of palmitate 14C (ul) was compared with that of palmitate 14C (at carbon 1). Five patients had denervation as a primary diagnosis, and the remaining nine had a primary muscle disease. Of this latter group, six had clinical similarities, including proximal weakness, necrotic fibers on muscle biopsy, and extreme elevations of serum creatine kinase. With one exception, lipid storage was not part of the syndrome. The possibility of incomplete palmitate oxidation due to a defect in beta-oxidation producing a human myopathy is discussed.

Effect of exercise on AMP deaminase and adenylosuccinase in rat skeletal muscle.

Effect of exercise on AMP deaminase and adenylosuccinase in rat skeletal muscle.