Myostatin and its precursor protein are increased in the skeletal muscle of patients with Type-II muscle fibre atrophy

Preferential atrophy of Type-II muscle fibres occurs in several clinical situations, including cachexia, muscle disuse, chronic glucocorticoid treatment, remote neoplasia, and sometimes as an aspect of recent-denervation. For the patient, the Type-II atrophy itself might be unfavourable (as a glucocorticoid side-effect) or favourable (survivalistic via the muscle-alanine liver-gluconeogenesis pathway in starvation). The cellular mechanisms underlying Type-II fibre atrophy are unclear. Myostatin (Mstn) is physiologically a negative regulator of muscle mass and strength. In this study we evaluated a possible role of Mstn in Type-II fibre atrophy in human muscle. Mstn and Mstn precursor protein (MstnPP) were studied in 10-muscle biopsies containing Type-II fibre atrophy and in 17 disease and normal control muscle biopsies. When comparison was made with normal control fibres, we found the following: 1) by immunocytochemistry, diffusely increased Mstn/MstnPP in the atrophic Type-II muscle fibres; 2) by immunoblots, Mstn/MstnPP increased individually; 3) by RT-PCR, no increase in MstnPP mRNA. In conclusion, our results a) suggest that Mstn/ /MstnPP might play a role in the pathogenic cascade of Type-II muscle fibre atrophy; b) broaden our previously-described associations of Mstn in human muscle pathology, and c) could possibly lead to clinical prevention when Type-II muscle fibre atrophy is unfavourable, for instance in glucocorticoid therapy. (Folia Morphol 2008; 67: 1-7

Vacuolar myopathy in a dog resembling human sporadic inclusion body myositis

Sporadic inclusion body myositis (sIBM) is the most common myopathy in people over the age of 50 years. While immune-mediated inflammatory myopathies are well documented in dogs, sIBM has not been described. An 11-year-old dog with chronic and progressive neuromuscular dysfunction was evaluated for evidence of sIBM using current pathologic, immunohistochemical and electron microscopic diagnostic criteria. Vacuoles and congophilic intracellular inclusions were identified in cryostat sections of multiple muscle biopsies and immunostained with antibodies against amyloid-β peptide, amyloid-β precursor protein, and proteosome 20S of the ubiquitin–proteosome system. Cellular infiltration and increased expression of MHC Class I antigen were observed. Cytoplasmic filamentous inclusions, membranous structures, and myeloid bodies were identified ultrastructurally. These observations constitute the first evidence that both the inflammatory and degenerative features of human sIBM can occur in a non-human species

Interrelation of inflammation and APP in sIBM: IL-1β induces accumulation of β-amyloid in skeletal muscle

Distinct interrelationships between inflammation and β-amyloid-associated degeneration, the two major hallmarks of the skeletal muscle pathology in sporadic inclusion body myositis (sIBM), have remained elusive. Expression of markers relevant for these pathomechanisms were analysed in biopsies of sIBM, polymyositis (PM), dermatomyositis (DM), dystrophic and non-myopathic muscle as controls, and cultured human myotubes. By quantitative PCR, a higher upregulation was noted for the mRNA-expression of CXCL-9, CCL-3, CCL-4, IFN-γ, TNF-α and IL-1β in sIBM muscle compared to PM, DM and controls. All inflammatory myopathies displayed overexpression of degeneration-associated markers, yet only in sIBM, expression of the mRNA of amyloid precursor protein (APP) significantly and consistently correlated with inflammation in the muscle and mRNA-levels of chemokines and IFN-γ. Only in sIBM, immunohistochemical analysis revealed that inflammatory mediators including IL-1β co-localized to β-amyloid depositions within myofibres. In human myotubes, exposure to IL-1β caused upregulation of APP with subsequent intracellular aggregation of β-amyloid. Our data suggest that, in sIBM muscle, production of high amounts of pro-inflammatory mediators specifically induces β-amyloid-associated degeneration. The observations may help to design targeted treatment strategies for chronic inflammatory disorders of the skeletal muscle

Structure and behavior of rat primary and secondary Schwann cells in vitro

The structure and motility of isolated rat primary (I) Schwann cells (SC) have been compared to that of subcultured (II) SC during and after mitotic stimulation. I SC contain myelin components which persist for 2 weeks in serum-free medium while they rapidly disappear in medium containing serum and high glucose concentration. These components were never detected in II SC. Both I SC and II SC after their mitotic phase are spindle-shaped, contain many intermediate and actin filaments, have no basement membrane but show intense migratory and undulatory activities. Rare fibroblasts in I cultures are recognized by their extremely variable shape, the presence of Thy 1.1 antigen in their membrane and their intense edge ruffling alternating with abrupt translocation. In contrast, I SC movements consist of intracellular translocation of nuclei along SC processes, which retract and extend constantly, and in slow rhythmic undulation episodes (2.3 ± 0.2/min) alternating with migration at 135 ± 50 μ/h. The total number of these episodes per day in serum-free medium is rigorously identical for different cells (166.3 ± 0.2) and this uniformity of frequency suggests a genotypic basis. Cycles, consisting of an undulation episode followed by a resting interval, have mean durations of 8.6 ± 4.1 min and a sharp peak of occurrence at 6 min, with exponential distribution of the longer periods. Motility of II SC is considerably inhibited during mitotic stimulation by cholera toxin and a pituitary extract while SC phenotype has changed to a flat multipolar cell with prominent Golgi and ribosomes. Migration is reduced to 24 ± 2 μ/h and only 2% of the SC show pulsations of the same periodicity as the I SC undulations. A dramatic increase in pulsation frequency occurs 6–12 h after removal of mitogenic factors when 80% of II SC start pulsating twice as fast for 2–3 days. When mitoses cease, SC quickly recover their SC phenotype with rhythmic undulations while migration speed increased to 92 ± 20 μ/h. Thus, in spite of dramatic modification of shape, structure and behavior during mitotic stimulation, SC subsequently recover their unique motility pattern which might be essential for their myelinating functionPeer reviewe

Oxidative stress, Nrf2 and keratin up‐regulation associate with Mallory‐Denk body formation in mouse erythropoietic protoporphyria

Mallory‐Denk bodies (MDBs) are hepatocyte inclusions commonly seen in steatohepatitis. They are induced in mice by feeding 3,5‐diethoxycarbonyl‐1,4‐dihydrocollidine (DDC) for 12 weeks, which also causes porphyrin accumulation. Erythropoietic protoporphyria (EPP) is caused by mutations in ferrochelatase (fch), and a fraction of EPP patients develop liver disease that is phenocopied in Fech m1Pas mutant (fch/fch) mice, which have an inactivating fch mutation. fch/fch mice develop spontaneous MDBs, but the molecular factors involved in their formation and whether they relate to DDC‐induced MDBs are unknown. We tested the hypothesis that fch mutation creates a molecular milieu that mimics experimental drug‐induced MDBs. In 13‐ and 20‐week‐old fch/fch mice, serum alkaline phosphatase, alanine aminotransferase, and bile acids were increased. The 13‐week‐old fch/fch mice did not develop histologically evident MDBs but manifested biochemical alterations required for MDB formation, including increased transglutaminase‐2 and keratin overexpression, with a greater keratin 8 (K8)‐to‐keratin 18 (K18) ratio, which are critical for drug‐induced MDB formation. In 20‐week‐old fch/fch mice, spontaneous MDBs were readily detected histologically and biochemically. Short‐term (3‐week) DDC feeding markedly induced MDB formation in 20‐week‐old fch/fch mice. Under basal conditions, old fch/fch mice had significant alterations in mitochondrial oxidative‐stress markers, including increased protein oxidation, decreased proteasomal activity, reduced adenosine triphosphate content, and Nrf2 (redox sensitive transcription factor) up‐regulation. Nrf2 knockdown in HepG2 cells down‐regulated K8, but not K18. Conclusion : Fch/fch mice develop age‐associated spontaneous MDBs, with a marked propensity for rapid MDB formation upon exposure to DDC, and therefore provide a genetic model for MDB formation. Inclusion formation in the fch/fch mice involves oxidative stress which, together with Nrf2‐mediated increase in K8, promotes MDB formation. (H epatology 2012;56:322–331)Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/92074/1/25664_ftp.pd

Inclusion Body Myositis: Laser Microdissection Reveals Differential Up-Regulation of IFN-γ Signaling Cascade in Attacked versus Nonattacked Myofibers

Sporadic inclusion body myositis (IBM) is a muscle disease with two separate pathogenic components, degeneration and inflammation. Typically, nonnecrotic myofibers are focally surrounded and invaded by CD8+ T cells and macrophages. Both attacked and nonattacked myofibers express high levels of human leukocyte antigen class I (HLA-I) molecules, a prerequisite for antigen presentation to CD8+ T cells. However, only a subgroup of HLA-I+ myofibers is attacked by immune cells. By using IHC, we classified myofibers from five patients with sporadic IBM as attacked (AIBM) or nonattacked (NIBM) and isolated the intracellular contents of myofibers separately by laser microdissection. For comparison, we isolated myofibers from control persons (HCTRL). The samples were analyzed by microarray hybridization and quantitative PCR. HLA-I up-regulation was observed in AIBM and NIBM, whereas HCTRL were negative for HLA-I. In contrast, the inducible chain of the interferon (IFN) γ receptor (IFNGR2) and several IFN-γ–induced genes were up-regulated in AIBM compared with NIBM and HCTRL fibers. Confocal microscopy confirmed segmental IFNGR2 up-regulation on the membranes of AIBM, which positively correlated with the number of adjacent CD8+ T cells. Thus, the differential up-regulation of the IFN-γ signaling cascade observed in the attacked fibers is related to local inflammation, whereas the ubiquitous HLA-I expression on IBM muscle fibers does not require IFNGR expression

Selective culture of mitotically active human Schwann cells from adult sural nerves

We devised a simple method to isolate mitotically active human Schwann cells from sural nerve biopsy specimens and expand the population in culture. Nerve fascicles were treated with cholera toxin for 7 days in culture before dissociation, which increased the cell yield at least twenty-five–fold over immediated tissue dissociation. Digesting the tissue completely with enzymes in serum-containing medium resulted in the highest cell viability, and released 2 to 6 × 10 4 cells/mg of tissue. Seeding the cells on a poly- L -lysine substrate in a small volume of serum-free medium optimized the plating efficiency. Although Schwann cells comprised 90% of the initial culture population, their numbers declined over time due to a faster mitotic rate of the fibroblasts in the presence of cholera toxin alone. However, treating the cultures with a combination of cholera toxin and forskolin, which act synergistically to elevate cyclic AMP levels, inhibited fibroblast growth without causing Schwann cell toxicity. Adding glial growth factor to the adenyl cyclase activators maximized Schwann cell proliferation, and the population rapidly and selectively expanded. Therefore, it should be possible to generate large numbers of Schwann cells from diseased nerves to study defects in cell function or from normal nerves to study the effects of Schwann cell grafts on neuronal regeneration.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/50351/1/410310603_ftp.pd