Multi-analyte profiling and pathway analysis of plasma for proteins associated with cancer-related fatigue syndrome in disease-free breast cancer patients after primary treatment.

CONTEXT: A significant number of women treated for breast cancer develop long-term fatigue afterwards. Previous research has suggested that fatigue may be due to a prolonged inflammatory response. However, there are conflicting results and the exact nature of the disturbance remains unclear. OBJECTIVES: To identify inflammatory markers associated with fatigue. METHODS: We recruited women from a breast cancer follow-up clinic and categorised them on the basis of a diagnostic interview as to whether they met the criteria for cancer-related fatigue syndrome (cases) or not (controls). We took plasma samples from each participant to analyse subsequently using a panel of 88 biological markers. RESULTS: 90 samples were analysed in total (45 cases and 45 controls). A factorial analysis of variance (using age as a fixed factor) demonstrated a number of differences in inflammatory cytokines. There were 28 significantly different analytes in total. Granulocyte colony stimulating factor was the most significantly different analyte (p<0.001). Many of the significant analytes were chemokine ligands found to be linked through an inflammatory pathway promoting T-cell and granulocyte production and activation. CONCLUSIONS: Our results add further weight to the hypothesis that cancer-related fatigue syndrome is associated with an increased pro-inflammatory immune response. Our findings indicate that these cytokine changes could underpin the subjective symptoms, such as perceived muscle weakness and concentration difficulties, experienced by women who feel fatigued after treatment.

A comparative proteinomic analysis of nipple aspiration fluid from healthy women and women with breast cancer.

This pilot study examines the feasibility of nipple aspiration to distinguish women with breast cancer from healthy women using surface-enhanced laser desorption ionisation time-of-flight mass spectrometry (SELDI-TOF/MS). Nipple aspiration fluid (NAF) was collected from each breast in 21 women newly diagnosed with unilateral breast cancer and 44 healthy women. No differences were found when proteomic profiles of NAF from the cancer-bearing breast and the contralateral non-cancerous breast were compared. In contrast, 9 protein peaks were significantly different between the cancer-bearing breast compared with healthy women and 10 peaks were significantly different between the contralateral healthy breast and healthy women (P<0.05). These data suggest that invasive breast cancer may result in a field change across both breasts and that proteomic profiling of NAF may have more value in breast cancer risk assessment than as a diagnostic or screening tool.

Expression of Ankrd2 in fast and slow muscles and its response to stretch are consistent with a role in slow muscle function.

In striated muscle, the structural genes associated with muscle fiber phenotype determination as well as muscle mass accretion are regulated largely by mechanical stimuli. Passive stretch of skeletal muscle stimulates muscle growth/hypertrophy and an increased expression of slow muscle genes. We previously identified Ankyrin repeat-domain protein (Ankrd2) as a novel transcript expressed in fast tibialis anterior muscles after 7 days of passive stretch immobilization in vivo. Here, we test the hypothesis that the expression of Ankrd2 in stretched fast muscle is associated with the stretch-induced expression of slow muscle phenotype rather than the hypertrophic response. Our results show that, in 4- and 7-day stretched tibialis anterior muscle, the expression of Ankrd2 mRNA and protein was significantly upregulated (P > 0.001). However, in fast muscles of kyphoscoliotic mutant mice, which lack the hypertrophic response to overload but have a slower muscle phenotype than wild-type, Ankrd2 expression was significantly upregulated. The distribution pattern of Ankrd2 in fast and slow muscle is also in accord with their slow fiber composition. Furthermore, it was markedly downregulated in denervated rat soleus muscle, which produces a pronounced shift toward the fast muscle phenotype. Using a sensitive proteomics approach (Ciphergen Technology), we observed that Ankrd2 protein was undetectable in soleus after 4 wk of denervation. We suggest that Ankrd2, which is also a titin binding protein, is a stretch-response gene associated with slow muscle function and that it is part of a separate mechanotransduction system to the one that regulates muscle mass.

Identification of a novel stretch-responsive skeletal muscle gene (Smpx).

Skeletal muscle is able to respond to a range of stimuli, including stretch and increased load, by increasing in diameter and length in the absence of myofiber division. This type of cellular growth (hypertrophy) is a highly complex process involving division of muscle precursor cells (myoblasts) and their fusion to existing muscle fibers as well as increased protein synthesis and decreased protein degradation. Underlying the alterations in protein levels are increases in a range of specific mRNAs including those coding for structural proteins and proteins that regulate the hypertrophic process. Seven days of passive stretch in vivo of tibialis anterior (TA) muscle has been shown to elicit muscle hypertrophy. We have identified a cDNA corresponding to an mRNA that exhibits increased expression in response to 7 days of passive stretch imposed on TA muscles in vivo. This 944-bp novel murine transcript is expressed primarily in cardiac and skeletal muscle and to a lesser extent in brain. Translation of the transcript revealed an open reading frame of 85 amino acids encoding a nuclear localization signal and two overlapping casein kinase II phosphorylation sites. This gene has been called "small muscle protein (X chromosome)" (Smpx; HGMW-approved human gene symbol SMPX) and we hypothesize that it plays a role in skeletal muscle hypertrophy.

Identification of Serhl, a new member of the serine hydrolase family induced by passive stretch of skeletal muscle in vivo.

In response to extended periods of stretch, skeletal muscle typically exhibits cell hypertrophy associated with sustained increases in mRNA and protein synthesis. Several soluble hypertrophic agonists have been identified, yet relatively little is known as to how mechanical load is converted into intracellular signals regulating gene expression or how increased cell size is maintained. In skeletal muscle, hypertrophy is generally regarded as a beneficial adaptive response to increased workload. In some cases, however, hypertrophy can be detrimental as seen in long-term cardiac hypertrophy. Skeletal muscle wasting (atrophy) is a feature of both inherited and acquired muscle disease and normal aging. Elucidating the molecular regulation of cell size is a fundamental step toward comprehending the complex molecular systems underlying muscle hypertrophy and atrophy. Subtractive hybridization between passively stretched and control murine skeletal muscle tissue identified an mRNA that undergoes increased expression in response to passive stretch. Encoded within the mRNA is an open reading frame of 311 amino acids containing a highly conserved type 1 peroxisomal targeting signal and a serine lipase active center. The sequence shows identity to a family of serine hydrolases and thus is named serine hydrolase-like (Serhl). The predicted three-dimensional structure displays a core alpha/beta-hydrolase fold and catalytic triad characteristic of several hydrolytic enzymes. Endogenous Serhl protein immunolocalizes to perinuclear vesicles as does Serhl-FLAG fusion protein transiently expressed in muscle cells in vitro. Overexpression of Serhl-FLAG has no effect on muscle cell phenotype in vitro. Serhl's expression patterns and its response to passive stretch suggest that it may play a role in normal peroxisome function and skeletal muscle growth in response to mechanical stimuli.

Altered expression of Bag-1 in Coxsackievirus B3 infected mouse heart.

OBJECTIVE: The mechanisms by which Coxsackie B viruses cause myocarditis or dilated cardiomyopathy are not well understood. This study examined changes in the expression of cardiac genes resulting from Coxsackievirus B3 (CVB3) infection of mice. METHODS: Mice (five per group) were experimentally infected with CVB3 or mock-infected with diluent. Altered expression of genes was initially identified by cDNA array, and confirmed by semiquantitative RT-PCR, western blot and immunohistochemistry. RESULTS: Forty-two up-regulated or down-regulated genes were observed in cDNA arrays carrying 588 known mouse genes. Among these, one down-regulated gene, Bag-1, known to be involved in inhibition of apoptosis and modulation of chaperone activity, was investigated further. Semiquantitative RT-PCR showed that Bag-1 expression was down-regulated by up to 30% in virus-infected mouse heart on day 7 compared to the mock-infected. Cell fractionation and western blot analysis confirmed that Bag-1 isoform p32 was predominant in the cytoplasm of mouse myocardium and down-regulated at 4 days or 7 days after CVB3 infection. In contrast, Bag-1 isoform p50 appeared to increase in the nuclear fraction of mouse heart at 7 days after infection. Down regulated expression and distribution of Bag-1 protein or evidence of apoptosis in the infected mouse heart was demonstrated by immunostaining or histochemistry (TUNEL assay), respectively. CONCLUSION: CVB3 infection induced differential expression of Bag-1 in cytoplasmic and nuclear fractions of mouse heart and apoptosis. This may be important in the pathogenesis of enterovirus heart muscle disease.

The kyphoscoliosis (ky) mouse is deficient in hypertrophic responses and is caused by a mutation in a novel muscle-specific protein.

The ky mouse mutant exhibits a primary degenerative myopathy preceding chronic thoraco-lumbar kyphoscoliosis. The histopathology of the ky mutant suggests that Ky protein activity is crucial for normal muscle growth and function as well as the maturation and stabilization of the neuromuscular junction. Muscle hypertrophy in response to increasing demand is deficient in the ky mutant, whereas adaptive fibre type shifts take place. The ky locus has previously been localized to a small region of mouse chromosome 9 and we have now identified the gene and the mutation underlying the kyphoscoliotic mouse. The ky transcript encodes a novel protein that is detected only in skeletal muscle and heart. The identification of the ky gene will allow detailed analysis of the impact of primary myopathy on idiopathic scoliosis in mice and man.

Identification of Ankrd2, a novel skeletal muscle gene coding for a stretch-responsive ankyrin-repeat protein.

Mechanically induced hypertrophy of skeletal muscles involves shifts in gene expression leading to increases in the synthesis of specific proteins. Full characterization of the regulation of muscle hypertrophy is a prerequisite for the development of novel therapies aimed at treating muscle wasting (atrophy) in human aging and disease. Using suppression subtractive hybridization, cDNAs corresponding to mRNAs that increase in relative abundance in response to mechanical stretch of mouse skeletal muscles in vivo were identified. A novel 1100-bp transcript was detected exclusively in skeletal muscle. This exhibited a fourfold increase in expression after 7 days of stretch. The transcript had an open reading frame of 328 amino acids encoding an ATP/GTP binding domain, a nuclear localization signal, two PEST protein-destabilization motifs, and a 132-amino-acid ankyrin-repeat region. We have named this gene ankyrin-repeat domain 2 (stretch-responsive muscle) (Ankrd2). We hypothesize that Ankrd2 plays an important role in skeletal muscle hypertrophy.

Differential effects of NT-3 on reinnervation of the fast extensor digitorum longus (EDL) and the slow soleus muscle of rat.

Previous studies of gastrocnemius muscle reinnervation showed specific normalization of the proportion and diameter of fast type 2b muscle fibres following NT-3 delivery to the proximal stump of the cut sciatic nerve. Here, we investigate if normalization was related to greater improvement of muscle reinnervation of fast (extensor digitorum longus; EDL) than slow (soleus) motor units. NT-3-impregnated (NT-3 group) or plain fibronectin (FN group) mats were inserted into a sciatic nerve gap. Neuromuscular junctions (NMJs) labelled with TRITC-alpha-bungarotoxin were colabelled with calcitonin gene-related peptide (CGRP) or 4E2 antisera and imaged using confocal microscopy. CGRP and 4E2 were used as markers for newly reinnervated and structurally mature NMJs, respectively. At 40 days postsurgery, denervated NMJs in EDL and soleus muscles of both groups presented a 50% decrease of surface area due to decreased width. At day 80 in EDL, more NMJs were reinnervated by CGRP-immunoreactive terminals in the NT-3 (7.1%) than in the FN group (4.2%); there was no difference between groups for soleus. At 120 days, 4E2-immunoreactive NMJs were more numerous in EDL of the NT-3 (40.0%) than in the FN group (7.3%), unlike in soleus (NT-3, 1. 6%; FN, 1.8%), and presented a partial size recovery. These results indicate that NT-3 preferentially improves reinnervation of fast muscles over slow muscle, although the mechanism of this improvement is still unclear.

A STS content physical and transcription map across the ky, kyphoscoliosis, nonrecombinant region.

The ky mouse mutant exhibits a degenerative muscle disease resulting in chronic deformation of the spinal column. Following a previous report describing the mapping of the ky locus to a small region of mouse chromosome 9 (Skynner et al., 1995, Genomics 25, 207-213), we have now undertaken a positional cloning approach to identify candidate genes for ky. A YAC/BAC contig encompassing the ky locus was constructed comprising 48 YAC clones and 48 newly generated STSs. The results from the combined physical and genetic analyses showed that only two overlapping BAC clones, which together do not exceed 260 kb, span the ky nonrecombinant region. A combination of gene hunting methods on the critical BACs has led to the identification of seven coding fragments, which have been tested for expression. The expression analysis and the position of the coding fragments on the contig suggest their grouping in at least four transcription units. One of these transcription units is expressed exclusively in skeletal muscle, making it a suitable candidate for this muscle defect in the ky mouse.

Neurotrophin-3-enhanced nerve regeneration selectively improves recovery of muscle fibers expressing myosin heavy chains 2b.

The purpose of this study was to evaluate the effect of neurotrophin 3 (NT-3) enhanced nerve regeneration on the reinnervation of a target muscle. Muscle fibers can be classified according to their mechanical properties and myosin heavy chain (MHC) isoform composition. MHC1 containing slow-type and MHC2a or 2b fast-type fibers are normally distributed in a mosaic pattern, their phenotype dictated by motor innervation. After denervation, all fibers switch to fast-type MHC2b expression and also undergo atrophy resulting in loss of muscle mass. After regeneration, discrimination between fast and slow fibers returns, but the distribution and fiber size change according to the level of reinnervation. In this study, rat gastrocnemius muscles (ipsilateral and contralateral to the side of nerve injury) were collected up to 8 mo after nerve repair, with or without local delivery of NT-3. The phenotype changes of MHC1, 2a, and 2b were analyzed by immunohistochemistry, and fiber type proportion, diameter, and grouping were assessed by computerized image analysis. At 8 mo, the local delivery of NT-3 resulted in significant improvement in gastrocnemius muscle weight compared with controls (NT-3 group 47%, controls 39% weight of contralateral normal muscle; P < 0.05). NT-3 delivery resulted in a significant increase in the proportion (NT-3 43.3%, controls 35.7%; P < 0.05) and diameter (NT-3 87.8 micron, controls 70.8 micron; P < 0.05) of fast type 2b fibers after reinnervation. This effect was specific to type 2b fibers; no normalization was seen in other fiber types. This study indicates that NT-3-enhanced axonal regeneration has a beneficial effect on the motor target organ. Also, NT-3 may be specifically affecting a subset of motoneurons that determine type 2b muscle fiber phenotype. As NT-3 was topically applied to cut nerves, our data suggest a discriminating effect of the neurotrophin on neuro-muscular interaction. These results would imply that muscle fibers may be differentially responsive to other neurotrophic factors and indicate the potential clinical role of NT-3 in the prevention of muscle atrophy after nerve injury.

Mechanical power and myosin composition of soleus and extensor digitorum longus muscles of ky mice.

Muscles of ky/ky homozygote mice exhibit neonatal muscle fiber necrosis and regeneration with subsequent motor nerve sprouting and development of a prominent kyphoscoliosis from approximately 100 days onward. Soleus and extensor digitorum longus (EDL) muscles from ky mice weighted < 50% of control muscles from age-matched NMRI mice. Maximal tetanic force was more reduced in soleus than in EDL. In EDL, the velocity constant of the force-velocity relation, maximal velocity, twitch time-to-peak, and isomyosin content were normal at all ages. The early mechanical changes seen in ky soleus muscles (47 day) were not accompanied by significant alterations in isomyosin or myosin heavy- and light-chain composition, since ky and NMRI expressed slow-twitch native myosin 2 (SM2, type I fibers) and intermediate-twitch native myosin (IM, type IIa fibers). Adult ky soleus (172 day) showed wholesale loss of IM and sole expression of SM2. This is sufficient to account for the markedly slowing of the force-velocity relation and the twitches observed in adult ky soleus. We propose that since shifts in muscle type only occurred in soleus, this reflects the persistent requirement to withstand the force of gravity.

Genetic mapping of the mouse neuromuscular mutation kyphoscoliosis.

The ky mouse mutant, kyphoscoliosis, exhibits a degenerative muscle disease resulting in chronic deformation of the spinal column. Using an interspecific backcross segregating the ky mutation, we have mapped the ky locus to a small region of mouse chromosome 9. ky is nonrecombinant with the microsatellites D9Mit24 and D9Mit169 and lies in a conserved linkage group that encompasses human chromosome 3. s-Laminin (LAMS) and the gene for dystrophin-associated glycoprotein 1 (DAG1), which map to human chromosome 3, are both recombinant with ky, ruling them out as candidates.

In situ localisation of single-stranded DNA breaks in nuclei of a subpopulation of cells within regenerating skeletal muscle of the dystrophic mdx mouse.

Degeneration of muscle fibres during the early stages of Duchenne Muscular Dystrophy (DMD) is accompanied by muscle fibre regeneration where cell division and myoblast fusion to form multinucleate myotubes within the lesions appear to recapitulate the events of normal muscle development. The mechanisms that govern the expression of genes regulating differentiation of myoblasts in regenerating skeletal muscle are of great interest for the development of future therapies designed to stimulate muscle regeneration. We show here that single-stranded breaks in DNA are localised in nuclei, using an exogenously applied medium containing labelled deoxynucleotides and the Klenow fragment of DNA polymerase I. The nuclei of a sub-population of cells lying in the inflammatory infiltrate of lesions in the skeletal muscle of the muscular dystrophic mouse (mdx), a genetic homologue of DMD, were labelled in this fashion. By contrast, labelled cells were completely absent from the muscles of normal non-myopathic animals (C57BL/10) and non-lesioned areas of mdx muscles. Cells expressing the muscle-specific regulatory gene, myogenin, were also found within mononucleate cells and myotubes within similar mdx muscle lesions. While we cannot yet say that the cells labelled by the DNA polymerase reaction are in fact differentiating, they were found only in significant numbers within mdx muscle lesions where new muscle fibres appear, providing strong circumstantial evidence that they are intimately associated with the regenerative process. Using a range of nucleases and different DNA polymerases, we show that the DNA polymerase-labelling reaction observed was DNA-dependent and most probably due to infilling of naturally occurring single-stranded gaps in DNA. Since the regenerative process in human Duchenne Muscular Dystrophy is apparently less effective than that seen in mdx mice, continued study of single-stranded DNA breaks may help to elucidate further the mechanisms controlling the expression of genes that characterise the myogenic process during skeletal muscle regeneration. Such findings might be applied in the development of future therapies designed to stimulate muscle regeneration in human dystrophies.

The neuromuscular basis of hereditary kyphoscoliosis in the mouse.

We describe a new neuromuscular disorder in the kyphoscoliotic mouse mutant (ky). Mice were killed at ages from birth to 210 days, and tissues were taken for standard light microscopy, histochemistry, nerve ending studies, and electron microscopy. At birth a few myofibers showed phagocytosis ultrastructurally. Between 6 and 25 days there was prominent necrosis and regeneration in soleus, gracilis, paraspinal, and back muscles. At 47 days, these muscles were atrophic and necrosis and regeneration were rare. At 136 days, all muscle groups, including head muscles, showed some degree of myofiber atrophy and gracilis was fibrotic. Prominent intramuscular axonal sprouting was present from 31 days. Peripheral nerves and anterior horn cells were normal. The findings indicate a neuromuscular basis of hereditary kyphoscoliosis in the mouse. The animal may be useful as a model of human muscle disease and scoliosis.

Localization of donor nuclei in skeletal muscle grafts by in situ hybridization to a cDNA probe.

The development of therapies, based upon implantation of normal muscle cell precursors, for the treatment of skeletal muscle diseases such as Duchenne Muscular Dystrophy is in its infancy. Detailed analysis of the genetic and phenotypic contribution made by donor myoblasts to the regenerated muscle is critical. Using non-radioactive in situ hybridization of a Y chromosome-specific DNA probe to sections of muscle, we have localized the position of male donor nuclei within female host muscles after myoblast implantation. These results were compared with the distribution of immunocytochemically-localized dystrophin and the expression of donor-specific glucose phosphate isomerase by isoelectric-focussing. We found consistent male-specific nuclear hybridization and a close spatial relationship between the distribution of male donor nuclei and dystrophin-positive muscle fibres within female, dystrophin-negative host muscles. This approach will be useful in the further analysis of myoblast implantation experiments.

Mdx muscle grafts retain the mdx phenotype in normal hosts.

Whole muscle grafts were made between mdx and normal mice to investigate whether the mdx myopathic lesion is intrinsic to mdx muscle or is a property of its environment. Grafts were examined between 20 and 101 days. Unequivocal necrotic muscle fibers and/or newly formed basophilic myotubes were noted in 8 of 16 grafts of mdx muscle made in normal hosts but in none of 16 grafts of normal muscle made in mdx hosts. In older grafts, the proportion of centrally nucleated fibers and variability of fiber diameter were both higher in mdx muscle grafted into normal hosts than in normal muscle grafted into either mdx or normal hosts. Analysis of the glucose-6-phosphate isomerase (GPI) isoenzyme content of the grafts indicated that the muscle formed was predominantly of donor origin. These findings provide evidence that the mdx lesion is a primary myopathy rather than secondary to an extramuscular primary lesion.

Conversion of mdx myofibres from dystrophin-negative to -positive by injection of normal myoblasts.

An important corollary to the recent advances in our understanding of the primary cause of Duchenne muscular dystrophy, is the validation of genuine genetic homologues as animal models of the disease in which potential therapies can be tested. The persistent skeletal muscle necrosis that characterizes human Duchenne muscular dystrophy is also seen in the mdx mouse and is, in both, a consequence of a deficiency of dystrophin, probably within the muscle fibres themselves. As injected muscle precursor cells of one genotype can fuse with host muscle fibres of a different genotype and express the donor genes, we decided to test grafts of normal muscle precursor cells to see if they could induce synthesis of dystrophin in innately dystrophin-deficient mdx muscle fibres. We show that injected normal muscle precursor cells can fuse with pre-existing or regenerating mdx muscle fibres to render many of these fibres dystrophin-positive and so to partially or wholly rescue them from their biochemical defect.

Effects of starvation, feeding, and time of day on the activity of proton transport adenosine triphosphatase in the parietal cells of the mouse gastric glands.

The effects of starvation, feeding, and time of day on mouse gastric glands were studied by means of an enzyme histochemical method for K+-dependent p-nitrophenyl phosphatase (K+-NPPase), a partial reaction of the proton pump ATPase which drives gastric acid secretion. The stomachs of mice starved for 24 h showed very low levels of parietal cell K+-NPPase histochemical reaction. However, a brief meal following such a period of starvation produced an abrupt increase in K+-NPPase reaction within most of the parietal cell-containing glands though not all parietal cells were equally susceptible to stimulation. The number of glands containing K+-NPPase-reactive parietal cells fell slowly in the hours following a feeding stimulus. These changes were shown to be caused by feeding rather than by general arousal and to follow the feeding cycle in ad libitum fed animals. The reasons that parietal cells in the basal parts of mouse gastric glands cannot be induced to show K+-NPPase reactivity by a feeding stimulus are not understood.

The mdx mouse skeletal muscle myopathy: II. Contractile properties.

The contractile properties of soleus muscles from mdx and control mice aged between 26 and 350 days were compared with those of muscles from similarly aged control mice. Mdx mice were in general heavier (their individual soleus muscles were also heavier), of greater cross-sectional area and greater standard length than age-matched controls. Isometric forces produced by soleus muscles from young mdx mice (less than or equal to 100 days) were similar to controls, but were weaker when force was normalized for cross-sectional area. Conversely, although the absolute isometric forces produced by older (greater than 100 days) mdx muscles were greater than age-matched controls, when normalized for cross-sectional area they were similar. No differences were found between mdx and control muscles in terms of length-force or force-velocity relationships. Thus, young mdx control muscles produce similar absolute isometric force but mdx mouse muscles are larger. When muscle size is accounted for, in terms of cross-sectional area, younger mdx muscles are, therefore, weaker than controls. Inefficient contraction of young mdx muscles may result from lack of contractile fibres, physiological inefficiency of contractile fibres, or loss of tendon-fibre continuity during muscle fibre necrosis and regeneration. The striking supernormal size and strength of older mdx muscles reflects their considerable regenerative capacity; whether this is due to an increase in muscle fibre number rather than fibre hypertrophy remains unclear.