Effect of saponin treatment on the sarcoplasmic reticulum of rat, cane toad and crustacean (yabby) skeletal muscle

1. Mechanically skinned fibres from skeletal muscles of the rat, toad and yabby were used to investigate the effect of saponin treatment on sarcoplasmic reticulum (SR) Ca2+ loading properties. The SR was loaded submaximally under control conditions before and after treatment with saponin and SR Ca2+ was released with caffeine. 2. Treatment with 10 mu g ml(-1) saponin greatly reduced the SR Ca2+ loading ability of skinned fibres from the extensor digitorum longus muscle of the rat with a rate constant of 0.24 min(-1). Saponin concentrations up to 150 mu g ml(-1) and increased exposure time up to 30 min did not further reduce the SR Ca2+ loading ability of the SR, which indicates that the inhibitory action of 10-150 mu g ml(-1) saponin is not dose dependent. The effect of saponin was also not dependent on the state of polarization of the transverse-tubular system. 3. Treatment with saponin at concentrations up to 100 mu g ml(-1) for 30 min did not affect the Ca2+ loading ability of SR in skinned skeletal muscle fibres from the twitch portion of the toad iliofibularis muscle but SR Ca2+ loading ability decreased markedly with a time constant of 0.22 min(-1) in the presence of 150 mu g ml(-1) saponin. 4. The saponin dependent increase in permeability could be reversed in both rat and toad fibres by short treatment with 6 mu M Ruthenium Red, a potent SR Ca2+ channel blocker, suggesting that saponin does affect the SR Ca2+ channel properties in mammalian and anuran skeletal muscle. 5. Treatment of skinned fibres of long sarcomere length (> 6 mu m) from the claw muscle of the yabby (a freshwater decapod crustacean) with 10 mu g ml(-1) saponin for 30 min abolished the ability of the SR to load Ca2+, indicating that saponin affects differently the SR from skeletal muscles of mammals, anurans and crustaceans. 6. is concluded that at relatively low concentrations, saponin causes inhibition of the skeletal SR Ca2+ loading ability in a species dependent manner, probably by increasing the Ca2+ loss through SR Ca2+ release channels.

MHC class I and II expression in juvenile dermatomyositis skeletal muscle

Objective To assess MHC I and II expressions in muscle fibres of juvenile dermatomyositis (JDM) and compare with the expression in polymyositis (PM), dermatomyositis (DM) and dystrophy. Patients and methods Forty-eight JDM patients and 17 controls (8 PM, 5 DM and 4 dystrophy) were studied. The mean age at disease onset was 7.1 +/- 3.0 years and the mean duration of weakness before biopsy was 9.4 +/- 12.9 months. Routine histochemistry and immunohistochemistry (StreptABComplex/HRP) for MHC I and II (Dakopatts) were performed on serial frozen muscle sections in all patients. Mann-Whitney, Kruskal Wallis, chi-square and Fisher`s exact statistical methods were used. Results MHC I expression was positive in 47 (97.9%) JDM cases. This expression was observed independent of time of disease corticotherapy previous to muscle biopsy and to the grading of inflammation observed in clinical, laboratorial and histological parameters. The expression of MHC I was similar on JDM, PM and DM, and lower in dystrophy. On the other hand, MHC II expression was positive in just 28.2% of JDM cases was correlated to histological features as inflammatory infiltrate, increased connective tissue and VAS for global degree of abnormality (p < 0.05). MCH II expression was similar in DM/PM and lower in JDM and dystrophy, and it was based on the frequency of positive staining rather than to the degree of the MCH II expression. Conclusions MHC I expression in muscle fibres is a premature and late marker of JDM patient independent to corticotherapy, and MHC II expression was lower in JDM than in PM and DM.

Changes in postnatal skeletal muscle development induced by alternative immobilization model in female rat

The objective of this study was to adapt a model of hind limb immobilization to newly weaned female rats and to determine the morphology of shortened soleus and plantaris muscles. Female Wistar rats were divided into three groups: control zero (n = 3) and control and free (n = 8), animals aged 21 and 31 days, respectively, submitted to no intervention, and immobilized (n = 25), animals aged 21 days submitted to immobilization for 10 days and sacrificed at 31 days of age. The device used for immobilization had advantages such as easy connection, good fit, and low cost. The immobilized rats showed a reduction in muscle fiber area and in connective tissue. The adaptation of this immobilization model originally used for adult rats was an excellent alternative for newly weaned rats and was also efficient in inducing significant hind limb disuse.

Biomechanical effects of immobilization and rehabilitation on the skeletal muscle of trained and sedentary rats

Because of the scarcity of information about the comparison of training to sedentarism beforehand immobilization and rehabilitation through muscle mechanical properties, the present work investigates this theme. Seventy rats were divided into 7 groups: 1-control (C); 2-trained (T); 3-sedentary (S); 4-trained and immobilized (TI); 5-sedentary and immobilized (SI); 6-trained, immobilized and rehabilitated (TIR); 7-sedentary, immobilized and rehabilitated (SIR). Interventions: Swimming training; Sedentarism (reduced size cages); Cast immobilization (pelvic limb) and water rehabilitation. Load at the limit of proportionality (LLP), maximum limit load (MLL) and stiffness (St) were the mechanical properties determined after a mechanical test of traction of the gastrocnemius. The training improved all mechanical properties when compared to sedentarism. After immobilization, LLP and MLL were reduced in TI and SI. However, there was no difference in St between C and TI. Additionally, TI showed improved MLL when compared to SI. The comparison of TI and TIR showed significant melioration in all properties after remobilization. SIR showed an improvement only in MLL when compared to SI. Significant melioration in LLP and St was observed in TIR compared to SIR. We demonstrated that the training before immobilization and rehabilitation had a positive effect on the muscle mechanical behavior compared to sedentarism. This analysis is of fundamental importance because it helps characterize the muscle tissue under different functional demands.

TIME COURSE OF SKELETAL MUSCLE LOSS AND OXIDATIVE STRESS IN RATS WITH WALKER 256 SOLID TUMOR

Reactive oxygen species oxidize proteins and modulate the proteasomal system in muscle-wasting cancer cachexia. On day 5 (D5), day 10 (D10), and day 14 (D14) after tumor implantation, skeletal muscle was evaluated. Carbonylated proteins and thiobarbituric acid reactive substances were measured. Chemiluminescence was employed for lipid hydroperoxide estimation. Glutathione, superoxide dismutase, and total radical antioxidant capacity were evaluated. The proteasomal system was assessed by mRNA atrogin-1 expression. Increased muscle wasting, lipid hydroperoxide, and superoxide dismutase, and decreased glutathione levels and total radical antioxidant capacity, were found on D5 in accordance with increased mRNA atrogin-1 expression. All parameters were significantly modified in animals treated with alpha-tocopherol. The elevation in aldehylde levels and carbonylated proteins observed on D10 were reversed by cc-tocopherol treatment. Oxidative stress may trigger signal transduction of the proteasomal system and cause protein oxidation. These pathways may be associated with the mechanism of muscle wasting that occurs in cancer cachexia. Muscle Nerve 42: 950-958, 2010

Mechanisms Involved in 3 `,5 `-Cyclic Adenosine Monophosphate-Mediated Inhibition of the Ubiquitin-Proteasome System in Skeletal Muscle

Although it is well known that catecholamines inhibit skeletal muscle protein degradation, the molecular underlying mechanism remains unclear. This study was undertaken to investigate the role of beta(2)-adrenoceptors (AR) and cAMP in regulating the ubiquitin-proteasome system (UPS) in skeletal muscle. We report that increased levels of cAMP in isolated muscles, promoted by the cAMP phosphodiesterase inhibitor isobutyl methylxanthine was accompanied by decreased activity of the UPS, levels of ubiquitin-protein conjugates, and expression of atrogin-1, a key ubiquitin-protein ligase involved in muscle atrophy. In cultured myotubes, atrogin-1 induction after dexamethasone treatment was completely prevented by isobutyl methylxanthine. Furthermore, administration of clenbuterol, a selective beta(2)-agonist, to mice increased muscle cAMP levels and suppressed the fasting-induced expression of atrogin-1 and MuRF-1, atrogin-1 mRNA being much more responsive to clenbuterol. Moreover, clenbuterol increased the phosphorylation of muscle Akt and Foxo3a in fasted rats. Similar responses were observed in muscles exposed to dibutyryl-cAMP. The stimulatory effect of clenbuterol on cAMP and Akt was abolished in muscles from beta(2)-AR knockout mice. The suppressive effect of beta(2)-agonist on atrogin-1 was not mediated by PGC-1 alpha (peroxisome proliferator-activated receptor-gamma coactivator 1 alpha known to be induced by beta(2)-agonists and previously shown to inhibit atrogin-1 expression), because food-deprived PGC-1 alpha knockout mice were still sensitive to clenbuterol. These findings suggest that the cAMP increase induced by stimulation of beta(2)-AR in skeletal muscles from fasted mice is possibly the mechanism by which catecholamines suppress atrogin-1 and the UPS, this effect being mediated via phosphorylation of Akt and thus inactivation of Foxo3. (Endocrinology 150: 5395-5404, 2009)

Effects of vitamin E and alpha-lipoic acid on skeletal muscle contractile properties

Initial experiments were conducted using an in situ rat tibialis anterior (TA) muscle preparation to assess the influence of dietary antioxidants on muscle contractile properties. Adult Sprague-Dawley rats were divided into two dietary groups: 1) control diet (Con) and 2) supplemented with vitamin E (VE) and alpha -lipoic acid (alpha -LA) (Antiox). Antiox rats were fed the Con rats' diet (AIN-93M) with an additional 10,000 IU VE/kg diet and 1.65 g/kg alpha -LA. After an 8-wk feeding period, no differences existed (P > 0.05) between the two dietary groups in maximum specific tension before or after a fatigue protocol or in force production during the fatigue protocol. However, in unfatigued muscle, maximal twitch tension and tetanic force production at stimulation frequencies less than or equal to 40 Hz were less (P < 0.05) in Antiox animals compared with Con. To investigate which antioxidant was responsible for the depressed force production, a second experiment was conducted using an in vitro rat diaphragm preparation. Varying concentrations of VE and dihydrolipoic acid, the reduced form of -LA, were added either individually or in combination to baths containing diaphragm muscle strips. The results from these experiments indicate that high levels of VE depress skeletal muscle force production at low stimulation frequencies.

Long-term metabolic and skeletal muscle adaptations to short-sprint training: Implications for sprint training and tapering

The adaptations of muscle to sprint training can be separated into metabolic and morphological changes. Enzyme adaptations represent a major metabolic adaptation to sprint training, with the enzymes of all three energy systems showing signs of adaptation to training and some evidence of a return to baseline levels with detraining. Myokinase and creatine phosphokinase have shown small increases as a result of short-sprint training in some studies and elite sprinters appear better able to rapidly breakdown phosphocreatine (PCr) than the sub-elite. No changes in these enzyme levels have been reported as a result of detraining. Similarly, glycolytic enzyme activity (notably lactate dehydrogenase, phosphofructokinase and glycogen phosphorylase) has been shown to increase after training consisting of either long (> 10-second) or short (< 10-second) sprints. Evidence suggests that these enzymes return to pre-training levels after somewhere between 7 weeks and 6 months of detraining. Mitochondrial enzyme activity also increases after sprint training, particularly when long sprints or short recovery between short sprints are used as the training stimulus. Morphological adaptations to sprint training include changes in muscle fibre type, sarcoplasmic reticulum, and fibre cross-sectional area. An appropriate sprint training programme could be expected to induce a shift toward type Ha muscle, increase muscle cross-sectional area and increase the sarcoplasmic reticulum volume to aid release of Ca2+. Training volume and/or frequency of sprint training in excess of what is optimal for an individual, however, will induce a shift toward slower muscle contractile characteristics. In contrast, detraining appears to shift the contractile characteristics towards type IIb, although muscle atrophy is also likely to occur. Muscle conduction velocity appears to be a potential non-invasive method of monitoring contractile changes in response to sprint training and detraining. In summary, adaptation to sprint training is clearly dependent on the duration of sprinting, recovery between repetitions, total volume and frequency of training bouts. These variables have profound effects on the metabolic, structural and performance adaptations from a sprint-training programme and these changes take a considerable period of time to return to baseline after a period of detraining. However, the complexity of the interaction between the aforementioned variables and training adaptation combined with individual differences is clearly disruptive to the transfer of knowledge and advice from laboratory to coach to athlete.

Vascular smooth muscle cell phenotypic modulation in culture is associated with reorganisation of contractile and cytoskeletal proteins

Smooth muscle cells (SMC) exhibit a functional plasticity, modulating from the mature phenotype in which the primary function is contraction, to a less differentiated state with increased capacities for motility, protein synthesis, and proliferation. The present study determined, using Western analysis, double-label immunofluorescence and confocal microscopy, whether changes in phenotypic expression of rabbit aortic SMC in culture could be correlated with alterations in expression and distribution of structural proteins. Contractile state SMC (days 1 and 3 of primary culture) showed distinct sorting of proteins into subcellular domains, consistent with the theory that the SMC structural machinery is compartmentalised within the cell. Proteins specialised for contraction (alpha -SM actin, SM-MHC, and calponin) were highly expressed in these cells and concentrated in the upper central region of the cell. Vimentin was confined to the body of the cell, providing support for the contractile apparatus but not co-localising with it. In line with its role in cell attachment and motility, beta -NM actin was localised to the cell periphery and basal cortex. The dense body protein alpha -actinin was concentrated at the cell periphery, possibly stabilising both contractile and motile apparatus. Vinculin-containing focal adhesions were well developed, indicating the cells' strong adhesion to substrate. In synthetic state SMC (passages 2-3 of culture), there was decreased expression of contractile and adhesion (vinculin) proteins with a concomitant increase in cytoskeletal proteins (beta -non-muscle [NM] actin and vimentin). These quantitative changes in structural proteins were associated with dramatic chan-es in their distribution. The distinct compartmentalisation of structural proteins observed in contractile state SMC was no longer obvious, with proteins more evenly distributed throughout die cytoplasm to accommodate altered cell function. Thus, SMC phenotypic modulation involves not only quantitative changes in contractile and cytoskeletal proteins, but also reorganisation of these proteins. Since the cytoskeleton acts as a spatial regulator of intracellular signalling, reorganisation of the cytoskeleton may lead to realignment of signalling molecules, which, in turn, may mediate the changes in function associated with SMC phenotypic modulation. (C) 2001 Wiley-Liss, Inc.

Skeletal muscle mitochondrial ATP production rate and walking performance in peripheral arterial disease

This study tested the hypotheses that skeletal muscle mitochondrial ATP production rate (MAPR) is impaired in patients with peripheral arterial disease (PAD) and that it relates positively to their walking performances. Seven untrained patients, eight exercise-trained patients and 11 healthy controls completed a maximal walking test and had muscle sampled from the gastrocnemius medialis muscle. Muscle was analysed for its MAPR in the presence of pyruvate, palmitoyl-L-carnitine or both, as well as citrate synthase (CS) activity. MAPRs were not different between untrained PAD and controls. In contrast, MAPRs (pyruvate) were significantly higher in trained PAD vs. controls. MAPR (pyruvate combinations) was also significantly higher in trained than untrained PAD muscle. MAPR and CS activity were highly correlated with walking performance in patients, but not in controls. These data do not support the hypothesis that isolated mitochondria are functionally impaired in PAD and demonstrate that the muscle mitochondrial capacity to oxidize carbohydrate is positively related to walking performance in these patients.

Diffusion characteristics of ethylene glycol in skeletal muscle

Part of the optical clearing study in biological tissues concerns the determination of the diffusion characteristics of water and optical clearing agents in the subject tissue. Such information is sufficient to characterize the time dependence of the optical clearing mechanisms—tissue dehydration and refractive index (RI) matching. We have used a simple method based on collimated optical transmittance measurements made from muscle samples under treatment with aqueous solutions containing different concentrations of ethylene glycol (EG), to determine the diffusion time values of water and EG in skeletal muscle. By representing the estimated mean diffusion time values from each treatment as a function of agent concentration in solution, we could identify the real diffusion times for water and agent. These values allowed for the calculation of the correspondent diffusion coefficients for those fluids. With these results, we have demonstrated that the dehydration mechanism is the one that dominates optical clearing in the first minute of treatment, while the RI matching takes over the optical clearing operations after that and remains for a longer time of treatment up to about 10 min, as we could see for EG and thin tissue samples of 0.5 mm.

Influence of skeletal muscle mass on ventilatory and hemodynamic variables during exercise in patients with chronic heart failure

OBJECTIVE: To assess the influence of skeletal muscle mass on ventilatory and hemodynamic variables during exercise in patients with chronic heart failure (CHF). METHODS: Twenty-five male patients underwent maximum cardiopulmonary exercise testing on a treadmill with a ramp protocol and measurement of the skeletal muscle mass of their thighs by using magnetic resonance imaging. The clinically stable, noncachectic patients were assessed and compared with 14 healthy individuals (S) paired by age and body mass index, who underwent the same examinations. RESULTS: Similar values of skeletal muscle mass were found in both groups (CHF group: 3863 ± 874 g; S group: 3743 ± 540 g; p = 0.32). Significant correlations of oxygen consumption in the anaerobic threshold (CHF: r = 0.39; P= 0.02 and S: r = 0.14; P = 0.31) and of oxygen pulse also in the anaerobic threshold (CHF: r = 0.49; P = 0.01 and S: r =0.12; P = 0.36) were found only in the group of patients with chronic heart failure. CONCLUSION: The results obtained indicate that skeletal muscle mass may influence the capacity of patients with CHF to withstand submaximal effort, due to limitations in their physical condition, even maintaining a value similar to that of healthy individuals. This suggests qualitative changes in the musculature.

Treatment of Dyslipidemia with Statins and Physical Exercises: Recent Findings of Skeletal Muscle Responses

Statin treatment in association with physical exercise practice can substantially reduce cardiovascular mortality risk of dyslipidemic individuals, but this practice is associated with myopathic event exacerbation. This study aimed to present the most recent results of specific literature about the effects of statins and its association with physical exercise on skeletal musculature. Thus, a literature review was performed using PubMed and SciELO databases, through the combination of the keywords “statin” AND “exercise” AND “muscle”, restricting the selection to original studies published between January 1990 and November 2013. Sixteen studies evaluating the effects of statins in association with acute or chronic exercises on skeletal muscle were analyzed. Study results indicate that athletes using statins can experience deleterious effects on skeletal muscle, as the exacerbation of skeletal muscle injuries are more frequent with intense training or acute eccentric and strenuous exercises. Moderate physical training, in turn, when associated to statins does not increase creatine kinase levels or pain reports, but improves muscle and metabolic functions as a consequence of training. Therefore, it is suggested that dyslipidemic patients undergoing statin treatment should be exposed to moderate aerobic training in combination to resistance exercises three times a week, and the provision of physical training prior to drug administration is desirable, whenever possible.

Effect of drugs on Trypanosoma cruzi and on its interaction with heart muscle cell in vitro

Megazol, nifurtimox, benznidazol and allopurinol were investigated, by light and electron µscopy, for their action on T. cruzi. Both the direct effect upon amastigote and trypomastigote forms and the effect upon the interaction of heart muscle cells (HMC) with bloodstream trypomastigotes were studied. The proliferation of amastigotes in Warren medium was inhibited in a dose-dependent manner by megazol, nifurtimox and benznidazol. Treatment of amastigotes (25-50 µM/24 h) and trypomastigotes (25 µM/24h) led to several ultrastructural alterations in the parasites. These three drugs also had a potent effect on the treatment of infected heart muscle cells when added at the beginning of the interaction or after one or three days of infection. The interiorized parasites showed a similar pattern of ultrastructural alterations as observed by the direct effect on the amastigotes. The primary heart muscle cell culture proved to be a suitable model for the study of drugs on intracellular parasites. Likewise, the amastigote proliferation in axenic medium was shown to be an adequate assay for an initial trial of drugs. These parameters seem very reliable to us for a systematic investigation of the mechanism of action of new drugs.