Oxidative injury induced by hypochlorous acid to Ca-ATPase from sarcoplasmic reticulum of skeletal muscle and protective effect of trolox

Hypochlorous acid (HOCl) concentration-dependently decreased ATPase activity and SH groups of pure Ca-ATPase from sarcoplasmic reticulum (SERCA) of rabbit skeletal muscle with IC(50) of 150 micromol/l and 6.6 micromol/l, respectively. This indicates that SH groups were not critical for impairment of Ca-ATPase activity. Pure Ca-ATPase activity was analysed individually with respect to both substrates, Ca(2+) and ATP. Concerning dependence of ATPase activity on HOCl (150 micromol/l) as a function of free Ca(2+) and ATP, V(max) of both dependences decreased significantly, while the affinities to individual substrates were not influenced, with the exception of the regulatory binding site of ATP. On increasing HOCl concentration, fluorescence of fluorescein-5-isothiocyanate (FITC) decreased, indicating binding of HOCl to nucleotide binding site of SERCA. A new fragment appeared at 75 kDa after HOCl oxidation of SR, indicating fragmentation of SERCA. Fragmentation may be associated with protein carbonyl formation. The density of protein carbonyl bands at 75 and 110 kDa increased concentration- and time-dependently. Trolox (250 micromol/l) recovered the Ca-ATPase activity decrease induced by HOCl, probably by changing conformational properties of the Ca-ATPase protein. Trolox inhibited FITC binding to SERCA.

Evaluation of the rabbit as a model for Chagas disease - II: histopathologic studies of the heart, digestive tract and skeletal muscle

In order to investigate the value of the rabbit as an experimental model for Chagas' disease, seventy one animals were inoculated with different Trypanosoma cruzi strains and routes. The rabbits were submitted to necropsy in acute (earlier than three months of infection), recent chronic (three to six months) and late chronic (later than six months) phases. Myocarditis, generally focal and endomysial, occurred in 94.1%, 66.7% and 70.8% of the infected rabbits respectively in the acute, recent chronic and late chronic phases. The myocardial inflammatory exudate was composed by mononuclear cells, and also polymorphonuclear cells in the acute phase. In most cases of the late chronic phase, the myocarditis was similar to that described in the indeterminate form of human chagasic patients. Initial fibrosis occurred in the three phases but was more severe and frequent in the early chronic. Advanced fibrosis occurred only in the late chronic phase. Tissue parasites occurred only in the acute phase. The digestive tract and skeletal muscles showed mild and occasional lesions. Our data indicate that experimentally infected chagasic rabbits repeat some lesions similar to that of humans chagasic patients, specially that of the indeterminate form. So, it may be a useful, however not an ideal, model.

Effects of Recession versus Tenotomy Surgery without Recession in Adult Rabbit Extraocular Muscle

PURPOSE. Surgical recession of an extraocular muscle (EOM) posterior to its original insertion is a common form of strabismus surgery, weakening the rotational force exerted by the muscle on the globe and improving eye alignment. The purpose of this study was to assess myosin heavy chain (MyHC) isoform expression and satellite cell activity as defined by Pax7 expression in recessed EOMs of adult rabbits compared with that in muscles tenotomized but not recessed and with that in normal control muscles. METHODS. The scleral insertion of the superior rectus muscle was detached and sutured either 7 mm posterior to its original insertion site (recession surgery) or at the same site (tenotomy). One day before euthanatization, the rabbits received bromodeoxyuridine (BrdU) injections. After 7 and 14 days, selected EOMs from both orbits were examined for changes in fast, slow, neonatal, and developmental MyHC isoform expression, Pax7 expression, and BrdU incorporation. RESULTS. Recession and tenotomy surgery resulted in similar changes in the surgical EOMs. These included a decreased proportion of fast MyHC myofibers, an increased proportion of slow MyHC myofibers, and increased BrdU-positive satellite cells. Similar changes were seen in the non-operated contralateral superior rectus muscles. The ipsilateral inferior rectus showed reciprocal changes to the surgical superior rectus muscles. CONCLUSIONS. The EOMs are extremely adaptive to changes induced by recession and tenotomy surgery, responding with modulations in fiber remodeling and myosin expression. These adaptive responses could be manipulated to improve surgical success rates. (Invest Ophthalmol Vis Sci. 2010;51:5646-5656) DOI:10.1167/iovs.10-5523

Cloning of a differentially expressed tropomyosin isoform from cultured rabbit aortic smooth muscle cells

The four known tropomyosin genes have highly conserved DNA and amino acid sequences, and at least 18 isoforms are generated by alternative RNA splicing in muscle and non-muscle cells. No rabbit tropomyosin nucleotide sequences are known, although protein sequences for alpha- and beta-tropomyosin expressed by rabbit skeletal muscle have been described. Subtractive hybridisation was used to select for genes differentially expressed in rabbit aortic smooth muscle cells (SMC), during the change in cell phenotype in primary culture that is characterised by a loss of cytoskeletal filaments and contractile proteins. This led to the cloning of a tropomyosin gene predominantly expressed in rabbit SMC during this change. The full-length cDNA clone, designated rabbit TM-beta, contains an open reading frame of 284 amino acids, 5' untranslated region (UTR) of I 17 base pairs and 3' UTR of 79 base pairs. It is closely related to the beta-gene isoforms in other species, with the highest homology in DNA and protein sequences to the human fibroblast isoform TM-1 (91.7% identity in 1035 bp and 93.3% identity in the entire 284 amino acid sequence of the protein), It differs from rabbit skeletal muscle P-tropomyosin (81.7% homology at the protein level) mainly in two regions at amino acids 189-213 and 258-283 suggesting alternative splicing of exons 6a for 6b and 9d for 9a. Since this TM-P gene was the only gene strongly enough expressed in SMC changing phenotype to be observed by the subtractive hybridisation screen, it likely plays a significant role in this process. (C) 2002 Published by Elsevier Science Ltd.

Morphological aspects of rabbit masseter muscle after cervical sympathectomy

The objective of this paper was to study the effect of sympathetic innervation on morphological and histochemical aspects of skeletal muscle tissue. Rabbit masseter muscle was studied using histochemical and immunohistochemical methods for periods of up to 18 months post-sympathectomy. The morphological and enzymatic characteristics of control masseter muscles were similar on both the left and right sides. The main features were muscle fibres with a mosaic pattern and a predominance of type IIa fibres, followed by type I. Type IIb fibres showed very low frequency. Sympathectomized animals showed varying degrees of metabolic and morphological alterations, especially 18 months after sympathectomy. The first five groups showed a higher frequency of type I fibres, whilst the oldest group showed a higher frequency of type lib fibres. In the oldest group, a significant variation in fibre diameter was observed. Many fibres showed small diameter, atrophy, hypertrophy, splitting, and necrosis. Areas with fibrosis were observed. Thus cervical sympathectomy induced morphological alterations in the masseter muscles. These alterations were, in part, similar to both denervation and myopathy. These findings indicate that sympathetic innervation contributes to the maintenance of the morphological and metabolic features of masseter muscle fibres.

Morphological aspects of rabbit masseter muscle after cervical sympathectomy

The objective of this paper was to study the effect of sympathetic innervation on morphological and histochemical aspects of skeletal muscle tissue. Rabbit masseter muscle was studied using histochemical and immunohistochemical methods for periods of up to 18 months post-sympathectomy. The morphological and enzymatic characteristics of control masseter muscles were similar on both the left and right sides. The main features were muscle fibres with a mosaic pattern and a predominance of type IIa fibres, followed by type I. Type IIb fibres showed very low frequency. Sympathectomized animals showed varying degrees of metabolic and morphological alterations, especially 18 months after sympathectomy. The first five groups showed a higher frequency of type I fibres, whilst the oldest group showed a higher frequency of type IIb fibres. In the oldest group, a significant variation in fibre diameter was observed. Many fibres showed small diameter, atrophy, hypertrophy, splitting, and necrosis. Areas with fibrosis were observed. Thus cervical sympathectomy induced morphological alterations in the masseter muscles. These alterations were, in part, similar to both denervation and myopathy. These findings indicate that sympathetic innervation contributes to the maintenance of the morphological and metabolic features of masseter muscle fibres.

Leptin receptors in human skeletal muscle

[EN] Human skeletal muscle expresses leptin receptor mRNA; however, it remains unknown whether leptin receptors (OB-R) are also expressed at the protein level. Fourteen healthy men (age = 33.1 +/- 2.0 yr, height = 175.9 +/- 1.7 cm, body mass = 81.2 +/- 3.8 kg, body fat = 22.5 +/- 1.9%; means +/- SE) participated in this investigation. The expression of OB-R protein was determined in skeletal muscle, subcutaneous adipose tissue, and hypothalamus using a polyclonal rabbit anti-human leptin receptor. Three bands with a molecular mass close to 170, 128, and 98 kDa were identified by Western blot with the anti-OB-R antibody. All three bands were identified in skeletal muscle: the 98-kDa and 170-kDa bands were detected in hypothalamus, and the 98-kDa and 128-kDa bands were detected in thigh subcutaneous adipose tissue. The 128-kDa isoform was not detected in four subjects, whereas in the rest its occurrence was fully explained by the presence of intermuscular adipose tissue, as demonstrated using an anti-perilipin A antibody. No relationship was observed between the basal concentration of leptin in serum and the 170-kDa band density. In conclusion, a long isoform of the leptin receptor with a molecular mass close to 170 kDa is expressed at the protein level in human skeletal muscle. The amount of 170-kDa protein appears to be independent of the basal concentration of leptin in serum.

Functional identity of receptors for proteolysis-inducing factor on human and murine skeletal muscle

Background: Cachexia in both mice and humans is associated with tumour production of a sulphated glycoprotein called proteolysis-inducing factor (PIF). In mice PIF binds with high affinity to a surface receptor in skeletal muscle, but little is known about the human receptor. This study compares the human PIF receptor with the murine. Methods: Human PIF was isolated from the G361 melanoma and murine PIF from the MAC16 colon adenocarcinoma. The human PIF receptor was isolated from human skeletal muscle myotubes. Protein synthesis and degradation induced by human and murine PIF was studied in human and murine skeletal muscle myotubes. Results: Both the human and murine PIF receptors showed the same immunoreactivity and Mr 40 000. Both murine and human PIF inhibited total protein synthesis and stimulated protein degradation in human and murine myotubes to about the same extent, and this was attenuated by a rabbit polyclonal antibody to the murine PIF receptor, but not by a non-specific rabbit antibody. Both murine and human PIF increased the activity of the ubiquitin-proteasome pathway in both human and murine myotubes, as evidenced by an increased 'chymotrypsin-like' enzyme activity, protein expression of the 20S and 19S proteasome subunits, and increased expression of the ubiquitin ligases MuRF1 and MAFbx, and this was also attenuated by the anti-mouse PIF receptor antibody. Conclusions: These results suggest that the murine and human PIF receptors are identical. © 2014 Cancer Research UK.

Morphological Aspects And Cox-2 Expression After Exposure To 780-nm Laser Therapy In Injured Skeletal Muscle: An In Vivo Study.

The effectiveness of low-level laser therapy in muscle regeneration is still not well known. To investigate the effects of laser irradiation during muscle healing. For this purpose, 63 rats were distributed to 3 groups: non-irradiated control group (CG); group irradiated at 10 J/cm(2) (G10); and group irradiated at 50 J/cm(2) (G50). Each group was divided into 3 different subgroups (n=7), and on days 7, 14 and 21 post-injury the rats were sacrificed. Seven days post-surgery, the CG showed destroyed zones and extensive myofibrillar degeneration. For both treated groups, the necrosis area was smaller compared to the CG. On day 14 post-injury, treated groups demonstrated better tissue organization, with newly formed muscle fibers compared to the CG. On the 21(st) day, the irradiated groups showed similar patterns of tissue repair, with improved muscle structure at the site of the injury, resembling uninjured muscle tissue organization. Regarding collagen deposition, the G10 showed an increase in collagen synthesis. In the last period evaluated, both treated groups showed statistically higher values in comparison with the CG. Furthermore, laser irradiation at 10 J/cm(2) produced a down-regulation of cyclooxygenase 2 (Cox-2) immunoexpression on day 7 post-injury. Moreover, Cox-2 immunoexpression was decreased in both treated groups on day 14. Laser therapy at both fluencies stimulated muscle repair through the formation of new muscle fiber, increase in collagen synthesis, and down-regulation of Cox-2 expression.

Chicken skeletal muscle-associated macroarray for gene discovery

Macro- and microarrays are well-established technologies to determine gene functions through repeated measurements of transcript abundance. We constructed a chicken skeletal muscle-associated array based on a muscle-specific EST database, which was used to generate a tissue expression dataset of similar to 4500 chicken genes across 5 adult tissues (skeletal muscle, heart, liver, brain, and skin). Only a small number of ESTs were sufficiently well characterized by BLAST searches to determine their probable cellular functions. Evidence of a particular tissue-characteristic expression can be considered an indication that the transcript is likely to be functionally significant. The skeletal muscle macroarray platform was first used to search for evidence of tissue-specific expression, focusing on the biological function of genes/transcripts, since gene expression profiles generated across tissues were found to be reliable and consistent. Hierarchical clustering analysis revealed consistent clustering among genes assigned to 'developmental growth', such as the ontology genes and germ layers. Accuracy of the expression data was supported by comparing information from known transcripts and tissue from which the transcript was derived with macroarray data. Hybridization assays resulted in consistent tissue expression profile, which will be useful to dissect tissue-regulatory networks and to predict functions of novel genes identified after extensive sequencing of the genomes of model organisms. Screening our skeletal-muscle platform using 5 chicken adult tissues allowed us identifying 43 'tissue-specific' transcripts, and 112 co-expressed uncharacterized transcripts with 62 putative motifs. This platform also represents an important tool for functional investigation of novel genes; to determine expression pattern according to developmental stages; to evaluate differences in muscular growth potential between chicken lines, and to identify tissue-specific genes.

MicroRNA transcriptome profiles during swine skeletal muscle development

Background: MicroRNA (miR) are a class of small RNAs that regulate gene expression by inhibiting translation of protein encoding transcripts. To evaluate the role of miR in skeletal muscle of swine, global microRNA abundance was measured at specific developmental stages including proliferating satellite cells, three stages of fetal growth, day-old neonate, and the adult. Results: Twelve potential novel miR were detected that did not match previously reported sequences. In addition, a number of miR previously reported to be expressed in mammalian muscle were detected, having a variety of abundance patterns through muscle development. Muscle-specific miR-206 was nearly absent in proliferating satellite cells in culture, but was the highest abundant miR at other time points evaluated. In addition, miR-1 was moderately abundant throughout developmental stages with highest abundance in the adult. In contrast, miR-133 was moderately abundant in adult muscle and either not detectable or lowly abundant throughout fetal and neonate development. Changes in abundance of ubiquitously expressed miR were also observed. MiR-432 abundance was highest at the earliest stage of fetal development tested (60 day-old fetus) and decreased throughout development to the adult. Conversely, miR-24 and miR-27 exhibited greatest abundance in proliferating satellite cells and the adult, while abundance of miR-368, miR-376, and miR-423-5p was greatest in the neonate. Conclusion: These data present a complete set of transcriptome profiles to evaluate miR abundance at specific stages of skeletal muscle growth in swine. Identification of these miR provides an initial group of miR that may play a vital role in muscle development and growth.

Aerobic capacity and skeletal muscle function in children with asthma

Background Peripheral muscle strength and endurance are decreased in patients with chronic pulmonary diseases and seem to contribute to patients' exercise intolerance. However, the authors are not aware of any studies evaluating peripheral muscle function in children with asthma. It seems to be implied that children with asthma have lower aerobic fitness, but there are limited studies comparing the aerobic capacity of children with and without asthma. The present study aimed to evaluate muscle strength and endurance in children with persistent asthma and their association with aerobic capacity and inhaled corticosteroid consumption. Methods Forty children with mild persistent asthma (MPA) or severe persistent asthma (SPA) (N=20 each) and 20 children without asthma (control group) were evaluated. Upper (pectoralis and latissimus dorsi) and lower (quadriceps) muscle strength and endurance were assessed, and cardiopulmonary exercise testing was performed. Inhaled corticosteroid consumption during the last 6 and 24 months was also quantified. Results Children with SPA presented a reduction in peak oxygen consumption (VO(2)) (28.2 +/- 8.1 vs 34.7 +/- 6.9 ml/kg/min; p<0.01) and quadriceps endurance (43.1 +/- 6.7 vs 80.9 +/- 11.9 repetitions; p<0.05) compared with the control group, but not the MPA group (31.5 +/- 6.1 ml/kg/min and 56.7 +/- 47.7 repetitions respectively; p>0.05). Maximal upper and lower muscle strength was preserved in children with both mild and severe asthma (p>0.05). Finally, the authors observed that lower muscle endurance weakness was not associated with reductions in either peak VO(2) (r=0.22, p>0.05) or corticosteroid consumption (r=-0.31, p>0.05) in children with asthma. Conclusion The findings suggest that cardiopulmonary exercise and lower limb muscle endurance should be a priority during physical training programs for children with severe asthma.

Differential regulation of PGC-1 alpha expression in rat liver and skeletal muscle in response to voluntary running

Background: The beneficial actions of exercise training on lipid, glucose and energy metabolism and insulin sensitivity appear to be in part mediated by PGC-1 alpha. Previous studies have shown that spontaneously exercised rats show at rest enhanced responsiveness to exogenous insulin, lower plasma insulin levels and increased skeletal muscle insulin sensitivity. This study was initiated to examine the functional interaction between exercise-induced modulation of skeletal muscle and liver PGC-1 alpha protein expression, whole body insulin sensitivity, and circulating FFA levels as a measure of whole body fatty acid (lipid) metabolism. Methods: Two groups of male Wistar rats (2 Mo of age, 188.82 +/- 2.77 g BW) were used in this study. One group consisted of control rats placed in standard laboratory cages. Exercising rats were housed individually in cages equipped with running wheels and allowed to run at their own pace for 5 weeks. At the end of exercise training, insulin sensitivity was evaluated by comparing steady-state plasma glucose (SSPG) concentrations at constant plasma insulin levels attained during the continuous infusion of glucose and insulin to each experimental group. Subsequently, soleus and plantaris muscle and liver samples were collected and quantified for PGC-1 alpha protein expression by Western blotting. Collected blood samples were analyzed for glucose, insulin and FFA concentrations. Results: Rats housed in the exercise wheel cages demonstrated almost linear increases in running activity with advancing time reaching to maximum value around 4 weeks. On an average, the rats ran a mean (Mean +/- SE) of 4.102 +/- 0.747 km/day and consumed significantly more food as compared to sedentary controls (P < 0.001) in order to meet their increased caloric requirement. Mean plasma insulin (P < 0.001) and FFA (P < 0.006) concentrations were lower in the exercise-trained rats as compared to sedentary controls. Mean steady state plasma insulin (SSPI) and glucose (SSPG) concentrations were not significantly different in sedentary control rats as compared to exercise-trained animals. Plantaris PGC-1 alpha protein expression increased significantly from a 1.11 +/- 0.12 in the sedentary rats to 1.74 +/- 0.09 in exercising rats (P < 0.001). However, exercise had no effect on PGC-1 alpha protein content in either soleus muscle or liver tissue. These results indicate that exercise training selectively up regulates the PGC-1 alpha protein expression in high-oxidative fast skeletal muscle type such as plantaris muscle. Conclusion: These data suggest that PGC-1 alpha most likely plays a restricted role in exercise-mediated improvements in insulin resistance (sensitivity) and lowering of circulating FFA levels.

Influence of Laser Photobiomodulation on Collagen IV During Skeletal Muscle Tissue Remodeling After Injury in Rats

Objective: The aim of the present study was to determine the effect of GaAlAs low-level laser therapy (LLLT) on collagen IV remodeling of the tibialis anterior (TA) muscle in rats after cryolesion. Background: Considerable interest exists in skeletal muscle regeneration in situations such as repair after exercise-induced muscle injury, after muscle transplantation, in muscular dystrophy, exercise-induced muscle injury, and the recovery of strength after atrophy due to disuse. A number of studies have demonstrated the potential of LLLT in facilitating the muscle-healing process; however, no consensus is found in the literature regarding the best laser-irradiation parameters. Methods: Adult male Wistar rats (n = 45) were used and randomly divided into three groups: control (n = 5); nontreated cryolesioned group (n = 20), and LLLT-cryolesioned group (n = 20). The cryolesioned groups were analyzed at 1, 7, 14, and 21 days after the injury procedure. Laser irradiation was performed 3 times per week on the injured region by using the GaAlAs laser (660 nm; beam spot of 0.04 cm(2), output power of 20 mW, power density of 500 mW/cm(2), and energy density of 5 J/cm(2), for 10 sec). The muscles were removed, frozen, cryosectioned, and then stained with hematoxylin-eosin for the visualization of general morphology or used for immunohistochemical analysis of collagen IV. Results: It was demonstrated that LLLT promotes an increase in collagen IV immunolabeling in skeletal muscle in the first 7 days after acute trauma caused by cryoinjury, but does not modify the duration of the tissue-repair process. Even with LLLT, the injured muscle tissue needs similar to 21 days to achieve the same state of organization as that in the noninjured muscle. Conclusion: The collagen IV content is modulated in regenerating skeletal muscle under LLLT, which might be associated with better tissue outcome, although the histologic analysis did not detect tissue improvement in the LLLT group.

Effect of 655-nm Low-Level Laser Therapy on Exercise-Induced Skeletal Muscle Fatigue in Humans

Objective: To investigate if development of skeletal muscle fatigue during repeated voluntary biceps contractions could be attenuated by low-level laser therapy (LLLT). Background Data: Previous animal studies have indicated that LLLT can reduce oxidative stress and delay the onset of skeletal muscle fatigue. Materials and Methods: Twelve male professional volleyball players were entered into a randomized double-blind placebo-controlled trial, for two sessions (on day 1 and day 8) at a 1-wk interval, with both groups performing as many voluntary biceps contractions as possible, with a load of 75% of the maximal voluntary contraction force (MVC). At the second session on day 8, the groups were either given LLLT (655 nm) of 5 J at an energy density of 500 J/cm(2) administered at each of four points along the middle of the biceps muscle belly, or placebo LLLT in the same manner immediately before the exercise session. The number of muscle contractions with 75% of MVC was counted by a blinded observer and blood lactate concentration was measured. Results: Compared to the first session (on day 1), the mean number of repetitions increased significantly by 8.5 repetitions (+/- 1.9) in the active LLLT group at the second session (on day 8), while in the placebo LLLT group the increase was only 2.7 repetitions (+/- 2.9) (p = 0.0001). At the second session, blood lactate levels increased from a pre-exercise mean of 2.4 mmol/L (+/- 0.5 mmol/L), to 3.6 mmol/L (+/- 0.5 mmol/L) in the placebo group, and to 3.8 mmol/L (+/- 0.4 mmol/L) in the active LLLT group after exercise, but this difference between groups was not statistically significant. Conclusion: We conclude that LLLT appears to delay the onset of muscle fatigue and exhaustion by a local mechanism in spite of increased blood lactate levels.