Intermediate filaments in the human extraocular muscles.

PURPOSE: To investigate the distribution of the intermediate filament (IF) proteins desmin, vimentin, and nestin in human extraocular muscles (EOMs). METHODS: Healthy adult EOM samples were serially sectioned (5 and 1 µm) and processed for immunohistochemistry, with specific antibodies (Abs) against desmin, vimentin, and nestin and different myosin heavy chains (MyHCs), including the newly characterized Ab MYH7b against MyHC slow tonic. The distribution of desmin was also studied in EOMs at 16 to 18 weeks of gestation. RESULTS: Desmin was present in the vast majority of muscle fibers. Notably, muscle fibers that contained MyHC slow tonic were either unlabeled or very weakly labeled with three different Abs against desmin. These muscle fibers had normal cytoarchitecture and intact basement membrane. In fetal muscle, desmin was also absent or weak in myotubes containing MyHC slow tonic. Nestin was detected in a large proportion of muscle fibers in the orbital layer and to some extent also in the global layer, whereas no muscle fibers contained vimentin. Desmin and nestin were enriched at neuromuscular junctions, as in limb muscle. In contrast, some myotendinous junctions lacked desmin or nestin. CONCLUSIONS: The human EOMs differed significantly from the other muscles in the body with respect to their IF composition. Desmin, hitherto regarded as a ubiquitous muscle cytoskeletal protein, was absent or only present in trace amounts in a subset of normal muscle fibers in adult and fetal EOMs. Nestin, normally downregulated early in the postnatal period, was present in a high proportion of adult muscle fibers.

mRNA expression of fatty acid transporters in rainbow trout: in vivo and in vitro regulation by insulin, fasting and inflammation and infection mediators.

In the present study, we analyzed endocrine and nutritional regulation of fatty acid (FA) transporters mRNA expression fatty acid transport protein (FATP1) and fatty acid translocase (CD36) in rainbow trout in vivo and in adipocytes and myocytes in vitro. The expression of FATP1 increased with adipocyte and that of CD36 with myocyte in vitro differentiation suggesting a different role for each transporter during the two cell differentiation programs. Food deprivation (15, 25 and 35 days) increased FATP1 and CD36 mRNA expression in white muscle, red muscle and adipose tissue while insulin administration decreased the FATP1 expression in adipose tissue in vivo (21.6 pmol/g body mass) and in vitro (1 µM) in adipocytes. In trout myotubes insulin (1 µM) decreased FATP1 and increased CD36 mRNA expression. Thus, regulation of FA transporters expression by insulin is complex and directed to specific tissue needs. Although FATP1 and CD36 mRNA levels are controlled by insulin, it appears that FATP1 respond more clearly to situations of hyper and hypo-insulinemia in trout muscle and adipose tissue than CD36. FATP1 and CD36 transcription was also modulated by growth hormone in cultured myotubes and isolated adipocytes. Lipopolysaccharide administration (E. coli, serotype O26:B6, 6 µg/g body mass) decreased FATP1 mRNA expression in red muscle, adipose tissue and liver after 24h according to changes in lipid metabolism during infection. Tumor necrosis factor (TNFα) (100 ng/ml) reduced FATP1 expression in isolated adipocytes. Further, insulin (1µM) and IGF-I (100 nM) increased the FA uptake in rainbow trout myotubes through the PI3K/Akt signaling pathway. Overall, we demonstrated not only that feeding condition regulates FATP1 and CD36 mRNA expression in a tissue-specific manner, but also that insulin is an important regulator of these genes in vivo and in vitro and also it stimulates FA uptake in trout muscle cells.

Canine inflammatory myopathies: a clinicopathologic review of 200 cases.

A retrospective study was performed on 200 randomly selected cases of inflammatory myopathy in dogs from diagnostic muscle biopsies received at the Comparative Neuromuscular Laboratory, University of California, San Diego. The most common clinical signs in dogs diagnosed with an inflammatory myopathy were generalized weakness, stilted gait, dysphagia, masticatory or generalized muscle atrophy, inability to open the jaw, megaesophagus, and dysphonia. Myalgia was rarely described. Age of onset ranged from 0.25 to 14 years. Genders were equally represented. Breed distribution approximated the 2002 American Kennel Club registration statistics (r = .85) with the notable exception of Boxers and Newfoundlands. From the results of muscle biopsies, clinical signs, and presence or absence of antibodies against type 2M fibers, dogs were classified as a generalized inflammatory myopathy (gIM)--including immune-mediated polymyositis; infectious and preneoplastic myositis; and, rarely, dermatomyositislike or overlap syndromes or unclassified myositis-or a focal inflammatory myopathy (flM)--including masticatory muscle and extraocular myositis. Average creatine kinase (CK) and aspartate aminotransferase (AST) concentrations in gIMs were significantly higher than those with fIMs (P < .05). Neoplasia developed in 12 of 200 dogs within 12 months of diagnosis of polymyositis, with lymphoma diagnosed in 6 of 32 Boxers. Inflammatory myopathy was associated with antibody titers against infectious diseases in 38 dogs. Neospora caninum and Hepatozoon americanum cysts were found in tissues of 2 dogs not serologically tested. Antibodies against an unidentified sarcolemmal antigen were found in 9 of 19 Newfoundlands with polymyositis. The spectrum of canine inflammatory myopathies can be broad, with infectious etiologies relatively common, and can include preneoplastic and uncharacterized syndromes.

Complement activation alters myocellular sodium homeostasis during polymicrobial sepsis.

OBJECTIVE: To determine whether complement activation alters sodium homeostasis in fast-twitch skeletal muscles during sepsis, and if protein kinase-C is involved in this process. DESIGN: Prospective, randomized, controlled animal study. SETTING: Research laboratory. SUBJECTS: Male Sprague-Dawley rats weighing 60-75 g. INTERVENTIONS: Rats underwent cecal ligation and puncture (CLP) or sham-operation with or without soluble complement receptor-1 treatment. Soluble complement receptor-1 (20 mg/kg) was administered intraperitoneally 5 mins before operation. Twenty-four hours after operation, fast-twitch extensor digitorum longus muscles were isolated and incubated in normal Krebs-Henseleit buffer (pH 7.4). In addition, extensor digitorum longus muscles isolated from normal rats were incubated for 1 hr in the Krebs-Henseleit buffer media containing normal rat sera, zymosan-activated (4 or 10 mg/mL) rat sera, or heat-inactivated rat sera. Ten percent diluted rat sera were used as a complement source in all groups. Last, extensor digitorum longus muscles isolated from normal rats were incubated for 1 hr in the Krebs-Henseleit buffer media containing zymosan-activated or heat-inactivated rat sera in the presence of protein kinase-C inhibitors (i.e., 4 microM GF109203X or 5 microM rottlerin). Soluble C5b-9 complex concentrations in zymosan-activated human sera were determined by enzyme-linked immunosorbent assay to evaluate the degree of complement activation induced by zymosan. MEASUREMENTS AND MAIN RESULTS: Incubated extensor digitorum longus muscles from CLP, sham-operated, or normal rats were used to measure intracellular Na+ and K+ contents ([Na+]i or [K+]i). Polymicrobial sepsis, as produced by CLP, markedly increased [Na+]i and [Na+]i/[K+]i ratios in fast-twitch extensor digitorum longus muscles 24 hrs after CLP compared with sham operation. Administration of soluble recombinant complement receptor 1 before operation significantly decreased myocellular [Na+]i and [Na+]i/[K+]i ratios. Zymosan profoundly elevated soluble C5b-9 concentrations in human sera in vitro. Sublytic zymosan-activated rat sera significantly increased myocellular [Na+]i and [Na+]i/[K+]i ratios relative to heat-inactivated rat sera. No difference in myocellular [Na+]i and [Na+]i/[K+]i ratios was observed when we used 4 mg/mL compared with 10 mg/mL of zymosan for activation. Last, incubation of extensor digitorum longus muscles with GF109203X or rottlerin significantly attenuated increases in myocellular [Na+]i and [Na+]i/[K+]i ratios induced by sublytic zymosan-activated rat sera. CONCLUSIONS: Polymicrobial sepsis alters sodium homeostasis in fast-twitch skeletal muscles, which is significantly attenuated by administration of soluble complement receptor 1. Protein kinase-C inhibition completely blocks changes in myocellular [Na+]i and [Na+]i/[K+]i ratios induced by sublytic zymosan-activated rat sera. Collectively, these results suggest that an inappropriate activation of complement is, at least in part, responsible for changes in skeletal muscle sodium homeostasis during sepsis, and activation of PKC is one of the intracellular signaling pathways by which complement activation alters myocellular sodium homeostasis.

Monospecific antibodies against the three mammalian fast limb myosin heavy chains.

Skeletal muscle fibres in mammalian limb muscles are of four types: slow, 2A, 2X, and 2B, each characterized by a distinct myosin heavy chain (MyHC) isoform. Existing monoclonal antibodies (mabs) against fast MyHCs lack fibre-type specificity across species and could not positively identify 2X fibres. In this work, mabs were raised against each of the fast MyHCs. These mabs were shown to be monospecific by Western blots and immunohistochemistry in the rat. The advantages of using these mabs for identifying the three fast fibre types and hybrid fibres expressing multiple isoforms were illustrated using rat tibialis anterior muscle. Immunohistochemical analyses confirmed the monospecificity of these mabs in the following additional species: mouse, guinea pig, rabbit, cat, and baboon. 2B fibres were absent in limb muscles of the cat and baboon. These mabs constitute a set of powerful tools for studying muscle fibre types and their transformations.

Sublytic complement attack increases intracellular sodium in rat skeletal muscle.

BACKGROUND: Although excessive complement activation and deranged sodium homeostasis in skeletal muscle are characteristic in sepsis, their relationship has not been examined. This study was designed to determine if sublytic complement activation can directly mediate changes in myocellular sodium content. MATERIALS AND METHODS: Fast-twitch extensor digitorum longus muscles were freshly isolated from infant rats. Unsensitized muscles were incubated at 30 degrees C for 60 min in the media containing 10% human or rat serum under conditions of no complement activation, activation by zymosan, inactivation by heat, C7 or C9 deficiency, selective inhibition of complement pathway, and inhibition of Na(+)-K(+) ATPase by ouabain. Intracellular sodium ([Na(+)](i)) and potassium ([K(+)](i)) contents of the muscles, myocellular ATP, and LDH release from the muscles were then determined. RESULTS: Normal human serum significantly increased [Na(+)](i) and the [Na(+)](i)/[K(+)](i) ratio in the muscles as well as zymosan-activated serum. Heat inactivation, C7 deficiency, and inhibition of the alternative pathway completely abolished the cationic changes. Average LDH release was identical in all groups and less than 6%. Complement activation did not impair ouabain-sensitive Na(+)-K(+) ATPase activity in the muscles or alter myocellular ATP. Thus, the observed alterations are not likely due to dysfunction of Na(+)-K(+) pump or depletion of myocellular energy. Instead, alterations in [Na(+)](i) were dependent upon the amount of C9 added to C9-deficient serum, which suggests that the alterations are likely dependent on transmembrane pores created by membrane attack complexes (MAC). CONCLUSIONS: Sublytic amounts of MAC formed as a result of complement activation can directly alter [Na(+)](i) in ex vivo skeletal muscle.

Heterogeneity of myosin heavy-chain expression in fast-twitch fiber types of mature avian pectoralis muscle.

The aims of this study are to investigate the diversity of myosin heavy-chain (MyHC) expression among avian fast-twitch fibers, and to test the hypothesis that dissimilar MyHC isoforms are found in each of the principal avian fast-twitch fiber types. MyHCs within the muscle fibers of the pectoralis of 31 species of bird are characterized using immunocytochemical methods. A library of 11 monoclonal antibodies previously produced against chicken MyHCs is used. The specificity of these antibodies for MyHCs in each of the muscles studied is confirmed by Western blots. The results show that avian fast-twitch glycolytic fibers and fast-twitch oxidative-glycolytic fibers can contain different MyHCs. Among the species studied, there is also a conspicuous variety of MyHC isoforms expressed. In addition, the results suggest that two epitopes are restricted to chickens and closely allied gallinaceous birds. There are no apparent correlations between between MyHC epitope and presupposed contractile properties. However, the presence of different isoforms in different fast-twitch fiber types suggests a correlation between isoform and contractile function.