Muscle function and body composition profile in adolescents withrestrictive anorexia nervosa: Does resistance training help?

The aim of this study was to analyze the effects of short-term resistance training on the body composition profile and muscle function in a group of Anorexia Nervosa restricting type (AN-R) patients. The sample consisted of AN-R female adolescents (12.8 ± 0.6 years) allocated into the control and intervention groups (n¼18 each). Body composition and relative strength were assessed at baseline, after 8 weeks and 4 weeks following the intervention. Body mass index (BMI) increased throughout the study (p = 0.011). Significant skeletal muscle mass (SMM) gains were found in the intervention group (p = 0.045, d = 0.6) that correlated to the change in BMI (r = 0.51, p < 0.031). Meanwhile, fat mass (FM) gains were significant in the control group (p = 0.047, d = 0.6) and correlated (r > 0.60) with change in BMI in both the groups. Significant relative strength increases (p < 0.001) were found in the intervention group and were sustained over time.

Xanthine oxidase pathway and muscle damage: Insights from McArdle disease

The intent of this review is to summarize current body of knowledge on the potential implication of the xanthine oxidase pathway (XO) on skeletal muscle damage. The possible involvement of the XO pathway in muscle damage is exemplified by the role of XO inhibitors (e.g., allopurinol) in attenuating muscle damage. Reliance on this pathway (as well as on the purine nucleotide cycle) could be exacerbated in conditions of low muscle glycogen availability. Thus, we also summarize current hypotheses on the etiology of both baseline and exertional muscle damage in McArdle disease, a condition caused by inherited deficiency of myophosphorylase. Because myophosphorylase catalyzes the first step of muscle glycogen breakdown, patients are unable to obtain energy from their muscle glycogen stores. Finally, we provide preliminary data from our laboratory on the potential implication of the XO pathway in the muscle damage that is commonly experienced by these patients.

Muscle signaling in exercise intolerance: Insights from the McArdle mouse model

We recently generated a knock-in mouse model (PYGM p.R50X/p.R50X) of McArdle disease (myophosphorylase deficiency). One mechanistic approach to unveil the molecular alterations caused by myophosphorylase deficiency, which is arguably the paradigm of 'exercise intolerance', is to compare the skeletal-muscle tissue of McArdle, heterozygous, and healthy (wild type (wt)) mice. We analyzed in quadriceps muscle of p.R50X/p.R50X (n=4), p.R50X/wt (n=6) and wt/wt mice (n=5) (all male, 8 wk-old) molecular markers of energy-sensing pathways, oxidative phosphorylation (OXPHOS) and autophagy/proteasome systems, oxidative damage and sarcoplamic reticulum (SR) Ca handling. We found a significant group effect for total AMPK (tAMPK) and ratio of phosphorylated (pAMPK)/tAMPK (P=0.012 and 0.033), with higher mean values in p.R50X/p.R50X mice vs. the other two groups. The absence of massive accumulation of ubiquitinated proteins, autophagosomes or lysosomes in p.R50X/p.R50X mice suggested no major alterations in autophagy/proteasome systems. Citrate synthase activity was lower in p.R50X/p.R50X mice vs. the other two groups (P=0.036) but no statistical effect existed for respiratory chain complexes. We found higher levels of 4-hydroxy-2-nonenal-modified proteins in p.R50X/p.R50X and p.R50X/wt mice compared with the wt/wt group (P=0.011). Sarco(endo)plasmic reticulum ATPase 1 (SERCA1) levels detected at 110kDa tended to be higher in p.R50X/p.R50X and p.R50X/wt mice compared with wt/wt animals (P=0.076), but their enzyme activity was normal. We also found an accumulation of phosphorylated SERCA1 in p.R50X/p.R50X animals. Myophosphorylase deficiency causes alterations in sensory energetic pathways together with some evidence of oxidative damage and alterations in Ca handling but with no major alterations in OXPHOS capacity or autophagy/ubiquitination pathways, which suggests that the muscle tissue of patients is likely to adapt overall favorably to exercise training interventions.

Characterisation of the non-muscle α-actinins

Actinins are cytoskeleton proteins that cross-link actin filaments. Evolution of the actinin family resulted in the formation of Ca++-insensitive muscle isoforms (actinin-2 and- 3) and Ca++-sensitive non-muscle isoforms (actinin-1 and -4) with regard to their actin-binding function. Despite high sequence similarity, unique properties have been ascribed to actinin-4 compared with actinin-1. Actinin-4 is the predominant isoform reported to be associated with the cancer phenotype. Actinin-4, but not actinin-1, is essential for normal glomerular function in the kidney and and is able to translocate to the nucleus to regulate transcription. To understand the molecular basis for such isoform-specific functions I have comprehensively compared these proteins in terms of localisation, migration, alternative splicing, actin-binding properties, heterodimer formation and molecular interactions for the first time. This work characterises a number of commercially available actinin antibodies and in doing so, identifies actinin-1, -2 and -4 isoform-specific antibodies that enabled studies of actinin expression and localisation. This work identifies the actinin rod domain as the predominant domain that influences actinin localisation however localisation is likely to be effected by the entire actinin protein. si-RNA- mediated knockdown of actinin-1 and -4 did not affect migration in a number of cell lines highlighting that migration may only require a fraction of total non-muscle actinin levels. This work finds that the Ca++-insensitive variant of actinin-4 is expressed only in the nervous system and thus cannot be regarded as a smooth muscle isoform, as is the case for the Ca++-insensitive variant of actinin-1. This work also identifies a previously unreported exon 19a+19b expressing variant of actinin-4 in human skeletal muscle. This work finds that alternative splice variants of actinin-1 and -4 are co-expressed in a number of tissues, in particular the brain. In contrast to healthy brain, glioblastoma cells express Ca++-sensitive variants of both actinin-1 and -4. Actin-binding properties of actinin-1 and -4 are similar and are unlikely to explain isoform-specific functions. Surprisingly, this work reveals that actinin-1/-4 heterodimers, rather than homodimers, are the most abundant form of actinin in many cancer cell lines. Taken together this data suggests that actinin-1 and -4 cannot be viewed as distinct entities from each other but rather as proteins that can exist in both homodimeric and heterodimeric forms. Finally, this work employs yeast two-hybrid and proteomic approaches to identify actinin-interacting proteins. In doing so, this work identifies a number of putative actinin-4 specific interacting partners that may help to explain some of the unique functions attributed the actinin-4. The observation of alternative splice variants of actinin-1 and -4 combined with the observed potential of these proteins to form homodimers and heterodimers suggests that homodimers and heterodimers with novel actin-binding properties and interaction networks may exist. The ability to behave in this manner may have functional implications. This may be of importance considering that these proteins are central to such processes as cell migration and adhesion. This significantly alters our view of the non-muscle actinins.

Comparison of ELISA for tissue transglutaminase autoantibodies with antiendomysium antibodies in pediatric and adult patients with celiac disease.

BACKGROUND: Tissue transglutaminase (t-TG) is the main autoantigen recognized by the endomysium antibodies (EMA) observed in patients with celiac disease (CD). The aim of the study was to assess an ELISA method for t-TG antibodies (t-TGA) with respect to EMA IF assay in pediatric and adult patients. METHODS: t-TGA were analyzed by ELISA in 220 sera samples: 82 patients with biopsy-proven untreated CD (23 adults and 59 children), 14 CD children on gluten-free diet, 18 asymptomatic relatives of CD patients, and 106 age-matched control patients with gluten-unrelated gastrointestinal diseases (58 adults and 48 children). Serum IgA EMA were tested on umbilical cord sections in all patients. RESULTS: The great majority (92.7%) of untreated CD patients (both adults and children) were t-TGA positive (values ranging from 20.1 to > 300 AU). None of the child control patients and only two out of 58 (3.4%) of the adults with unrelated gastrointestinal diseases had serum t-TGA positivity; two out of 18 first-degree relatives with biopsy-proved silent CD were t-TGA (as well as EMA) positive. Finally, two out of 14 CD children, assuming a gluten-free diet, had serum t-TGA (as well as EMA). A highly significant correlation (P < 0.001) was observed between t-TGA concentrations and EMA. t-TGA showed a sensitivity of 87% and 95%, a specificity of 97% and 100% for adults and children, respectively. CONCLUSION: The method is highly sensitive and specific in the diagnosis of CD and is promising as a tool for routine diagnostic use and population screening, especially in children.

Antiendomysial and antihuman recombinant tissue transglutaminase antibodies in the diagnosis of coeliac disease: a biopsy-proven European multicentre study.

OBJECTIVE: To investigate the value of serum antitissue transglutaminase IgA antibodies (IgA-TTG) and IgA antiendomysial antibodies (IgA-EMA) in the diagnosis of coeliac disease in cohorts from different geographical areas in Europe. The setting allowed a further comparison between the antibody results and the conventional small-intestinal histology. METHODS: A total of 144 cases with coeliac disease [median age 19.5 years (range 0.9-81.4)], and 127 disease controls [median age 29.2 years (range 0.5-79.0)], were recruited, on the basis of biopsy, from 13 centres in nine countries. All biopsy specimens were re-evaluated and classified blindly a second time by two investigators. IgA-TTG were determined by ELISA with human recombinant antigen and IgA-EMA by an immunofluorescence test with human umbilical cord as antigen. RESULTS: The quality of the biopsy specimens was not acceptable in 29 (10.7%) of 271 cases and a reliable judgement could not be made, mainly due to poor orientation of the samples. The primary clinical diagnosis and the second classification of the biopsy specimens were divergent in nine cases, and one patient was initially enrolled in the wrong group. Thus, 126 coeliac patients and 106 controls, verified by biopsy, remained for final analysis. The sensitivity of IgA-TTG was 94% and IgA-EMA 89%, the specificity was 99% and 98%, respectively. CONCLUSIONS: Serum IgA-TTG measurement is effective and at least as good as IgA-EMA in the identification of coeliac disease. Due to a high percentage of poor histological specimens, the diagnosis of coeliac disease should not depend only on biopsy, but in addition the clinical picture and serology should be considered.

Effect of microRNA modulation on bioartificial muscle function.

Cellular therapies have recently employed the use of small RNA molecules, particularly microRNAs (miRNAs), to regulate various cellular processes that may be altered in disease states. In this study, we examined the effect of transient muscle-specific miRNA inhibition on the function of three-dimensional skeletal muscle cultures, or bioartificial muscles (BAMs). Skeletal myoblast differentiation in vitro is enhanced by inhibiting a proliferation-promoting miRNA (miR-133) expressed in muscle tissues. As assessed by functional force measurements in response to electrical stimulation at frequencies ranging from 0 to 20 Hz, peak forces exhibited by BAMs with miR-133 inhibition (anti-miR-133) were on average 20% higher than the corresponding negative control, although dynamic responses to electrical stimulation in miRNA-transfected BAMs and negative controls were similar to nontransfected controls. Immunostaining for alpha-actinin and myosin also showed more distinct striations and myofiber organization in anti-miR-133 BAMs, and fiber diameters were significantly larger in these BAMs over both the nontransfected and negative controls. Compared to the negative control, anti-miR-133 BAMs exhibited more intense nuclear staining for Mef2, a key myogenic differentiation marker. To our knowledge, this study is the first to demonstrate that miRNA mediation has functional effects on tissue-engineered constructs.

Fiber type-specific nitric oxide protects oxidative myofibers against cachectic stimuli.

Oxidative skeletal muscles are more resistant than glycolytic muscles to cachexia caused by chronic heart failure and other chronic diseases. The molecular mechanism for the protection associated with oxidative phenotype remains elusive. We hypothesized that differences in reactive oxygen species (ROS) and nitric oxide (NO) determine the fiber type susceptibility. Here, we show that intraperitoneal injection of endotoxin (lipopolysaccharide, LPS) in mice resulted in higher level of ROS and greater expression of muscle-specific E3 ubiqitin ligases, muscle atrophy F-box (MAFbx)/atrogin-1 and muscle RING finger-1 (MuRF1), in glycolytic white vastus lateralis muscle than in oxidative soleus muscle. By contrast, NO production, inducible NO synthase (iNos) and antioxidant gene expression were greatly enhanced in oxidative, but not in glycolytic muscles, suggesting that NO mediates protection against muscle wasting. NO donors enhanced iNos and antioxidant gene expression and blocked cytokine/endotoxin-induced MAFbx/atrogin-1 expression in cultured myoblasts and in skeletal muscle in vivo. Our studies reveal a novel protective mechanism in oxidative myofibers mediated by enhanced iNos and antioxidant gene expression and suggest a significant value of enhanced NO signaling as a new therapeutic strategy for cachexia.

Biomimetic engineered muscle with capacity for vascular integration and functional maturation in vivo.

Tissue-engineered skeletal muscle can serve as a physiological model of natural muscle and a potential therapeutic vehicle for rapid repair of severe muscle loss and injury. Here, we describe a platform for engineering and testing highly functional biomimetic muscle tissues with a resident satellite cell niche and capacity for robust myogenesis and self-regeneration in vitro. Using a mouse dorsal window implantation model and transduction with fluorescent intracellular calcium indicator, GCaMP3, we nondestructively monitored, in real time, vascular integration and the functional state of engineered muscle in vivo. During a 2-wk period, implanted engineered muscle exhibited a steady ingrowth of blood-perfused microvasculature along with an increase in amplitude of calcium transients and force of contraction. We also demonstrated superior structural organization, vascularization, and contractile function of fully differentiated vs. undifferentiated engineered muscle implants. The described in vitro and in vivo models of biomimetic engineered muscle represent enabling technology for novel studies of skeletal muscle function and regeneration.

Glucocorticoid induced muscle atrophy is associated with upregulation of myostatin gene expression

The mechanisms by which excessive glucocorticoids cause muscular atrophy remain unclear. We previously demonstrated that dexamethasone increases the expression of myostatin, a negative regulator of skeletal muscle mass, in vitro. In the present study, we tested the hypothesis that dexamethasone-induced muscle loss is associated with increased myostatin expression in vivo. Daily administration (60, 600, 1,200 micro g/kg body wt) of dexamethasone for 5 days resulted in rapid, dose-dependent loss of body weight (-4.0, -13.4, -17.2%, respectively, P <0.05 for each comparison), and muscle atrophy (6.3, 15.0, 16.6% below controls, respectively). These changes were associated with dose-dependent, marked induction of intramuscular myostatin mRNA (66.3, 450, 527.6% increase above controls, P <0.05 for each comparison) and protein expression (0.0, 260.5, 318.4% increase above controls, P <0.05). We found that the effect of dexamethasone on body weight and muscle loss and upregulation of intramuscular myostatin expression was time dependent. When dexamethasone treatment (600 micro g. kg-1. day-1) was extended from 5 to 10 days, the rate of body weight loss was markedly reduced to approximately 2% within this extended period. The concentrations of intramuscular myosin heavy chain type II in dexamethasone-treated rats were significantly lower (-43% after 5-day treatment, -14% after 10-day treatment) than their respective corresponding controls. The intramuscular myostatin concentration in rats treated with dexamethasone for 10 days returned to basal level. Concurrent treatment with RU-486 blocked dexamethasone-induced myostatin expression and significantly attenuated body loss and muscle atrophy. We propose that dexamethasone-induced muscle loss is mediated, at least in part, by the upregulation of myostatin expression through a glucocorticoid receptor-mediated pathway.

Muscle-specific mutations accumulate with aging in critical human mtDNA control sites for replication.

The recently discovered aging-dependent large accumulation of point mutations in the human fibroblast mtDNA control region raised the question of their occurrence in postmitotic tissues. In the present work, analysis of biopsied or autopsied human skeletal muscle revealed the absence or only minimal presence of those mutations. By contrast, surprisingly, most of 26 individuals 53 to 92 years old, without a known history of neuromuscular disease, exhibited at mtDNA replication control sites in muscle an accumulation of two new point mutations, i.e., A189G and T408A, which were absent or marginally present in 19 individuals younger than 34 years. These two mutations were not found in fibroblasts from 22 subjects 64 to 101 years of age (T408A), or were present only in three subjects in very low amounts (A189G). Furthermore, in several older individuals exhibiting an accumulation in muscle of one or both of these mutations, they were nearly absent in other tissues, whereas the most frequent fibroblast-specific mutation (T414G) was present in skin, but not in muscle. Among eight additional individuals exhibiting partial denervation of their biopsied muscle, four subjects >80 years old had accumulated the two muscle-specific point mutations, which were, conversely, present at only very low levels in four subjects <or =40 years old. The striking tissue specificity of the muscle mtDNA mutations detected here and their mapping at critical sites for mtDNA replication strongly point to the involvement of a specific mutagenic machinery and to the functional relevance of these mutations.

FMRFamide-like peptides (FLPs) enhance voltage-gated calcium currents to elicit muscle contraction in the human parasite Schistosoma mansoni.

Schistosomes are amongst the most important and neglected pathogens in the world, and schistosomiasis control relies almost exclusively on a single drug. The neuromuscular system of schistosomes is fertile ground for therapeutic intervention, yet the details of physiological events involved in neuromuscular function remain largely unknown. Short amidated neuropeptides, FMRFamide-like peptides (FLPs), are distributed abundantly throughout the nervous system of every flatworm examined and they produce potent myoexcitation. Our goal here was to determine the mechanism by which FLPs elicit contractions of schistosome muscle fibers. Contraction studies showed that the FLP Tyr-Ile-Arg-Phe-amide (YIRFamide) contracts the muscle fibers through a mechanism that requires Ca2+ influx through sarcolemmal voltage operated Ca2+ channels (VOCCs), as the contractions are inhibited by classical VOCC blockers nicardipine, verapamil and methoxyverapamil. Whole-cell patch-clamp experiments revealed that inward currents through VOCCs are significantly and reversibly enhanced by the application of 1 µM YIRFamide; the sustained inward currents were increased to 190% of controls and the peak currents were increased to 180%. In order to examine the biochemical link between the FLP receptor and the VOCCs, PKC inhibitors calphostin C, RO 31–8220 and chelerythrine were tested and all produced concentration dependent block of the contractions elicited by 1 µM YIRFamide. Taken together, the data show that FLPs elicit contractions by enhancing Ca2+ influx through VOCC currents using a PKC-dependent pathway.

Structure-activity relationships of FMRFamide-related peptides contracting Schistosoma mansoni muscle

This study reports the potent myoactivity of flatworm FMRFamide-related peptides (FaRPs) on isolated muscle fibers of the human blood fluke, Schistosoma mansoni. The turbellarian peptides YIRFamide (EC50 4 eta M), GYIRFamide (EC50 1 eta M). and RYIRFamide (EC50 7 eta M), all induced muscle contraction more potently than the cestode FaRP GNFFRFamide (EC50 500 eta M). Using a series of synthetic analogs of the flatworm peptides YIRFamide, GYIRFamide and RYIRFamide, the structure-activity relationships of the muscle FaRP receptor were examined. With a few exceptions, each residue in YIRFamide is important in the maintenance of its myoactivity. Alanine scans resulted in peptides that were inactive (Ala(1), Ala(2), Ala(3) and Ala(4) YIRFamide; Ala(4) and Ala(5) RYIRFamide) or had much reduced potencies (Ala(1), Ala(2) and Ala(3) RYIRFamide). Substitution of the N-terminal (Tyr(1)) residue of YIRFamide with the non-aromatic residues Thr or Arg produced analogs with greatly reduced potency. Replacement of the N-terminal Tyr with aromatic amino acids resulted in myoactive peptides (FIRFamide, EC50 100 eta M; WIRFamide, EC50 0.5 eta M). The activity of YIRFamide analogs which possessed a Leu(2), Phe(2) or Met(2) residue (EC50's 10, 1 and 3 eta M, respectively) instead of Ile(2) was not significantly altered, whereas, YVRFamide had a greatly reduced (EC50 200 eta M) activity. Replacement of the Phe(4) with a Tyr(4) (YIRYamide) also greatly lowered potency. Truncated analogs were either inactive (FRFamide, YRFamide, HRFamide, RFamide, Famide) or had very low potency (IRFamide and MRFamide), with the exception of nLRFamide (EC50 20 eta M). YIRF free acid was inactive. In summary, these data show the general structural requirements of this schistosome muscle FaRP receptor to be similar, but not identical, to those of previously characterized molluscan FaRP receptors. (C) 1997 Elsevier Science Inc.

Cloning and analysis of a Trichinella pseudospiralis muscle larva secreted serine protease gene

Nematode parasites of the genus Trichinella are intracellular and distinct life cycle stages invade intestinal epithelial and skeletal muscle cells. Within the genus, Trichinella spiralis and Trichinella pseudospiralis exhibit species-specific differences with respect to host-parasite complex formation and host immune modulation. Parasite excretory-secretory (ES) proteins play important roles at the host-parasite interface and are thought to underpin these differences in biology. Serine proteases are among the most abundant group of T. spiralis ES proteins and multiple isoforms of the muscle larvae-specific TspSP-1 serine protease have been identified. Recently, a similar protein (TppSP-1) in T. pseudospiralis muscle larvae was identified. Here we report the cloning and characterisation of the full-length transcript of TppSP-1 and present comparative data between TspSP-1 and TppSP-1.

Comparative analysis of the excretory-secretory proteome of the muscle larva of Trichinella pseudospiralis and Trichinella spiralis

The nematodes Trichinella spiralis and Trichinella pseudospiralis are both intracellular parasites of skeletal muscle cells and induce profound alterations in the host cell resulting in a re-alignment of muscle-specific gene expression. While T. spiralis induces the production of a collagen capsule surrounding the host-parasite complex, T. pseudospiralis exists in a non-encapsulated form and is also characterised by suppression of the host inflammatory response in the muscle. These observed differences between the two species are thought to be due to variation in the proteins excreted or secreted (ES proteins) by the muscle larva. In this study, we use a global proteomics approach to compare the ES protein profiles from both species and to identify individual T. pseudospiralis proteins that complement earlier studies with T. spiralis. Following two-dimensional gel electrophoresis, tandem mass spectrometry was used to identify the peptide spots. In many cases identification was aided by the determination of partial peptide sequence from selected mass ions. The T. pseudospiralis spots identified included the major secreted glycoproteins and the secreted 5'-nucleotidase. Furthermore, two major groups of T. spiralis-specific proteins and several T. pseudospiralis-specific proteins were identified. Our results demonstrate the value of proteomics as a tool for the identification of ES proteins that are differentially expressed between Trichinella species and as an aid to identifying key parasite proteins that are involved in the host-parasite interaction. The value of this approach will be further enhanced by data arising out the current T. spiralis genome sequencing project.