Resistance training inhibits the elevation of skeletal muscle derived-BDNF level concomitant with improvement of muscle strength in zucker diabetic rat

[Purpose]

In the present study, we investigated the effects of 8 weeks of progressive resistance training on the level of skeletal muscle derived BDNF as well as glucose intolerance in Zucker diabetic rats.

[Methods]

Six week-old male Zucker diabetic fatty (ZDF) and Zucker lean control (ZLC) rats were randomly divided into 3 groups: sedentary ZLC (ZLC-Con), sedentary ZDF (ZDF-Con), and exercised ZDF (ZDF-Ex). Progressive resistance training using a ladder and tail weights was performed for 8 weeks (3 days/week).

[Results]

After 8 weeks of resistance training, substantial reduction in body weight was observed in ZDF-Ex compared to ZDF-Con. Though the skeletal muscle volume did not change, grip strength grip strength was significantly higher in ZDF-Ex compared to ZDF-Con. In the soleus, the level of BDNF was increased in ZDF-Con, but was significantly decreased (p<0.05) in ZDF-Ex, showing a training effect. Moreover, we found that there was a negative correlation (r=-0.657; p=0.004) between grip strength and BDNF level whereas there was a positive correlation (r=0.612; p=0.008) between plasma glucose level and BDNF level in skeletal muscle.

[Conclusion]

Based upon our results, we demonstrated that resistance training inhibited the elevation of skeletal muscle derived-BDNF expression concomitant with the improvement of muscle strength in zucker diabetic rats. In addition, muscle-derived BDNF might be a potential mediator for the preventive effect of resistance training on the progress of type 2 diabetes.     

Systemic Myostatin Inhibition via Liver-Targeted Gene Transfer in Normal and Dystrophic Mice

Background

Myostatin inhibition is a promising therapeutic strategy to maintain muscle mass in a variety of disorders, including the muscular dystrophies, cachexia, and sarcopenia. Previously described approaches to blocking myostatin signaling include injection delivery of inhibitory propeptide domain or neutralizing antibodies.

Methodology/Principal Findings

Here we describe a unique method of myostatin inhibition utilizing recombinant adeno-associated virus to overexpress a secretable dominant negative myostatin exclusively in the liver of mice. Systemic myostatin inhibition led to increased skeletal muscle mass and strength in control C57 Bl/6 mice and in the dystrophin-deficient mdx model of Duchenne muscular dystrophy. The mdx soleus, a mouse muscle more representative of human fiber type composition, demonstrated the most profound improvement in force production and a shift toward faster myosin-heavy chain isoforms. Unexpectedly, the 11-month-old mdx diaphragm was not rescued by long-term myostatin inhibition. Further, mdx mice treated for 11 months exhibited cardiac hypertrophy and impaired function in an inhibitor dose–dependent manner.

Conclusions/Significance

Liver-targeted gene transfer of a myostatin inhibitor is a valuable tool for preclinical investigation of myostatin blockade and provides novel insights into the long-term effects and shortcomings of myostatin inhibition on striated muscle.     

Glucocorticoid-treated mice are an inappropriate positive control for long-term preclinical studies in the mdx mouse

Background

Dmd^mdx (mdx) mice are used as a genetic and biochemical model of dystrophin deficiency. The long-term consequences of glucocorticoid (GC) treatment on dystrophin-deficient skeletal and heart muscle are not yet known. Here we used systematic phenotyping to assess the long-term consequences of GC treatment in mdx mice. Our investigation addressed not only the effects of GC on the disease phenotype but also the question of whether GCs can be used as a positive control for preclinical drug evaluations.

Methods and Findings

We performed nine pre-clinical efficacy trials (treated N = 129, untreated N = 106) of different durations in 9-to-50-week-old dystrophic mdx mice over a 3-year time period using standardized methods. In all these trials, we used either 1 mg/kg body weight of prednisone or 5 mg/kg body weight of prednisolone as positive controls to compare the efficacy of various test drugs. Data from untreated controls and GC-treated mice in the various trials have been pooled and analyzed to assess the effects of GCs on dystrophin-deficient skeletal and cardiac muscles of mdx mice. Our results indicate that continuous GC treatment results in early (e.g., at 50 days) improvements in normalized parameters such as grip strength, motor coordination and maximal in vitro force contractions on isolated EDL muscle, but these initial benefits are followed by a progressive loss of muscle strength after 100 days. We also found a significant increase in heart fibrosis that is reflected in a significant deterioration in cardiac systolic function after 100 days of treatment.

Conclusion

Continuous administration of prednisone to mdx mice initially improves skeletal muscle strength, but further therapy result in deterioration of muscle strength and cardiac function associated with enhanced cardiac fibrosis. These results suggest that GCs may not serve as an appropriate positive control for long-term mdx mouse preclinical trials.     

The Proton Pump Inhibitor Lansoprazole Improves the Skeletal Phenotype in Dystrophin Deficient mdx Mice

Background

In Duchenne muscular dystrophy (DMD), loss of the membrane stabilizing protein dystrophin results in myofiber damage. Microinjury to dystrophic myofibers also causes secondary imbalances in sarcolemmic ion permeability and resting membrane potential, which modifies excitation-contraction coupling and increases proinflammatory/apoptotic signaling cascades. Although glucocorticoids remain the standard of care for the treatment of DMD, there is a need to investigate the efficacy of other pharmacological agents targeting the involvement of imbalances in ion flux on dystrophic pathology.

Methodology/Principal Findings

We designed a preclinical trial to investigate the effects of lansoprazole (LANZO) administration, a proton pump inhibitor, on the dystrophic muscle phenotype in dystrophin deficient (mdx) mice. Eight to ten week-old female mice were assigned to one of four treatment groups (n = 12 per group): (1) vehicle control; (2) 5 mg/kg/day LANZO; (3) 5 mg/kg/day prednisolone; and (4) combined treatment of 5 mg/kg/day prednisolone (PRED) and 5 mg/kg/day LANZO. Treatment was administered orally 5 d/wk for 3 months. At the end of the study, behavioral (Digiscan) and functional outcomes (grip strength and Rotarod) were assessed prior to sacrifice. After sacrifice, body, tissue and organ masses, muscle histology, in vitro muscle force, and creatine kinase levels were measured. Mice in the combined treatment groups displayed significant reductions in the number of degenerating muscle fibers and number of inflammatory foci per muscle field relative to vehicle control. Additionally, mice in the combined treatment group displayed less of a decline in normalized forelimb and hindlimb grip strength and declines in in vitro EDL force after repeated eccentric contractions.

Conclusions/Significance

Together our findings suggest that combined treatment of LANZO and prednisolone attenuates some components of dystrophic pathology in mdx mice. Our findings warrant future investigation of the clinical efficacy of LANZO and prednisolone combined treatment regimens in dystrophic pathology.     

Age- and Gender-Related Changes in Contractile Properties of Non-Atrophied EDL Muscle

Background

In humans, ageing causes skeletal muscles to become atrophied, weak, and easily fatigued. In rodent studies, ageing has been associated with significant muscle atrophy and changes in the contractile properties of the muscles. However, it is not entirely clear whether these changes in contractile properties can occur before there is significant atrophy, and whether males and females are affected differently.

Methods and Results

We investigated various contractile properties of whole isolated fast-twitch EDL muscles from adult (2–6 months-old) and aged (12–22 months-old) male and female mice. Atrophy was not present in the aged mice. Compared with adult mice, EDL muscles of aged mice had significantly lower specific force, longer tetanus relaxation times, and lower fatiguability. In the properties of absolute force and muscle relaxation times, females were affected by ageing to a greater extent than males. Additionally, EDL muscles from a separate group of male mice were subjected to eccentric contractions of 15% strain, and larger force deficits were found in aged than in adult mice.

Conclusion

Our findings provide further insight into the muscle atrophy, weakness and fatiguability experienced by the elderly. We have shown that even in the absence of muscle atrophy, there are definite alterations in the physiological properties of whole fast-twitch muscle from ageing mice, and for some of these properties the alterations are more pronounced in female mice than in male mice.     

Aging-Associated Dysfunction of Akt/Protein Kinase B: S-Nitrosylation and Acetaminophen Intervention

Background

Aged skeletal muscle is characterized by an increased incidence of metabolic and functional disorders, which if allowed to proceed unchecked can lead to increased morbidity and mortality. The mechanism(s) underlying the development of these disorders in aging skeletal muscle are not well understood. Protein kinase B (Akt/PKB) is an important regulator of cellular metabolism and survival, but it is unclear if aged muscle exhibits alterations in Akt function. Here we report a novel dysfunction of Akt in aging muscle, which may relate to S-nitrosylation and can be prevented by acetaminophen intervention.

Principal Findings

Compared to 6- and 27-month rats, the phosphorylation of Akt (Ser473 and Thr308) was higher in soleus muscles of very aged rats (33-months). Paradoxically, these increases in Akt phosphorylation were associated with diminished mammalian target of rapamycin (mTOR) phosphorylation, along with decreased levels of insulin receptor beta (IR-β), phosphoinositide 3-kinase (PI3K), phosphatase and tensin homolog deleted on chromosome 10 (PTEN) and phosphorylation of phosphoinositide-dependent kinase-1 (PDK1) (Ser241). In vitro Akt kinase measurements and ex vivo muscle incubation experiments demonstrated age-related impairments of Akt kinase activity, which were associated with increases in Akt S-nitrosylation and inducible nitric oxide synthase (iNOS). Impairments in Akt function occurred parallel to increases in myocyte apoptosis and decreases in myocyte size and the expression of myosin and actin. These age-related disorders were attenuated by treating aged (27-month) animals with acetaminophen (30 mg/kg body weight/day) for 6-months.

Conclusions

These data demonstrate that Akt dysfunction and increased S-nitrosylation of Akt may contribute to age-associated disorders in skeletal muscle and that acetaminophen may be efficacious for the treatment of age-related muscle dysfunction.     

Functional and Molecular Effects of Arginine Butyrate and Prednisone on Muscle and Heart in the mdx Mouse Model of Duchenne Muscular Dystrophy

Background

The number of promising therapeutic interventions for Duchenne Muscular Dystrophy (DMD) is increasing rapidly. One of the proposed strategies is to use drugs that are known to act by multiple different mechanisms including inducing of homologous fetal form of adult genes, for example utrophin in place of dystrophin.

Methodology/Principal Findings

In this study, we have treated mdx mice with arginine butyrate, prednisone, or a combination of arginine butyrate and prednisone for 6 months, beginning at 3 months of age, and have comprehensively evaluated the functional, biochemical, histological, and molecular effects of the treatments in this DMD model. Arginine butyrate treatment improved grip strength and decreased fibrosis in the gastrocnemius muscle, but did not produce significant improvement in muscle and cardiac histology, heart function, behavioral measurements, or serum creatine kinase levels. In contrast, 6 months of chronic continuous prednisone treatment resulted in deterioration in functional, histological, and biochemical measures. Arginine butyrate-treated mice gene expression profiling experiments revealed that several genes that control cell proliferation, growth and differentiation are differentially expressed consistent with its histone deacetylase inhibitory activity when compared to control (saline-treated) mdx mice. Prednisone and combination treated groups showed alterations in the expression of genes that control fibrosis, inflammation, myogenesis and atrophy.

Conclusions/Significance

These data indicate that 6 months treatment with arginine butyrate can produce modest beneficial effects on dystrophic pathology in mdx mice by reducing fibrosis and promoting muscle function while chronic continuous treatment with prednisone showed deleterious effects to skeletal and cardiac muscle. Our results clearly indicate the usefulness of multiple assays systems to monitor both beneficial and toxic effects of drugs with broad range of in vivo activity.     

Daily Supplementation of D-ribose Shows No Therapeutic Benefits in the MHC-I Transgenic Mouse Model of Inflammatory Myositis

Background

Current treatments for idiopathic inflammatory myopathies (collectively called myositis) focus on the suppression of an autoimmune inflammatory response within the skeletal muscle. However, it has been observed that there is a poor correlation between the successful suppression of muscle inflammation and an improvement in muscle function. Some evidence in the literature suggests that metabolic abnormalities in the skeletal muscle underlie the weakness that continues despite successful immunosuppression. We have previously shown that decreased expression of a purine nucleotide cycle enzyme, adenosine monophosphate deaminase (AMPD1), leads to muscle weakness in a mouse model of myositis and may provide a mechanistic basis for muscle weakness. One of the downstream metabolites of this pathway, D-ribose, has been reported to alleviate symptoms of myalgia in patients with a congenital loss of AMPD1. Therefore, we hypothesized that supplementing exogenous D-ribose would improve muscle function in the mouse model of myositis. We treated normal and myositis mice with daily doses of D-ribose (4 mg/kg) over a 6-week time period and assessed its effects using a battery of behavioral, functional, histological and molecular measures.

Results

Treatment with D-ribose was found to have no statistically significant effects on body weight, grip strength, open field behavioral activity, maximal and specific forces of EDL, soleus muscles, or histological features. Histological and gene expression analysis indicated that muscle tissues remained inflamed despite treatment. Gene expression analysis also suggested that low levels of the ribokinase enzyme in the skeletal muscle might prevent skeletal muscle tissue from effectively utilizing D-ribose.

Conclusions

Treatment with daily oral doses of D-ribose showed no significant effect on either disease progression or muscle function in the mouse model of myositis.     

Genetic Variations in the Androgen Receptor Are Associated with Steroid Concentrations and Anthropometrics but Not with Muscle Mass in Healthy Young Men

Objective

The relationship between serum testosterone (T) levels, muscle mass and muscle force in eugonadal men is incompletely understood. As polymorphisms in the androgen receptor (AR) gene cause differences in androgen sensitivity, no straightforward correlation can be observed between the interindividual variation in T levels and different phenotypes. Therefore, we aim to investigate the relationship between genetic variations in the AR, circulating androgens and muscle mass and function in young healthy male siblings.

Design

677 men (25–45 years) were recruited in a cross-sectional, population-based sibling pair study.

Methods

Relations between genetic variation in the AR gene (CAGn, GGNn, SNPs), sex steroid levels (by LC-MS/MS), body composition (by DXA), muscle cross-sectional area (CSA) (by pQCT), muscle force (isokinetic peak torque, grip strength) and anthropometrics were studied using linear mixed-effect modelling.

Results

Muscle mass and force were highly heritable and related to age, physical activity, body composition and anthropometrics. Total T (TT) and free T (FT) levels were positively related to muscle CSA, whereas estradiol (E₂) and free E₂ (FE₂) concentrations were negatively associated with muscle force. Subjects with longer CAG repeat length had higher circulating TT, FT, and higher E₂ and FE₂ concentrations. Weak associations with TT and FT were found for the rs5965433 and rs5919392 SNP in the AR, whereas no association between GGN repeat polymorphism and T concentrations were found. Arm span and 2D:4D finger length ratio were inversely associated, whereas muscle mass and force were not associated with the number of CAG repeats.

Conclusions

Age, physical activity, body composition, sex steroid levels and anthropometrics are determinants of muscle mass and function in young men. Although the number of CAG repeats of the AR are related to sex steroid levels and anthropometrics, we have no evidence that these variations in the AR gene also affect muscle mass or function.     

Estrogen Regulates Estrogen Receptors and Antioxidant Gene Expression in Mouse Skeletal Muscle

Background

Estrogens are associated with the loss of skeletal muscle strength in women with age. Ovarian hormone removal by ovariectomy in mice leads to a loss of muscle strength, which is reversed with 17β-estradiol replacement. Aging is also associated with an increase in antioxidant stress, and estrogens can improve antioxidant status via their interaction with estrogen receptors (ER) to regulate antioxidant gene expression. The purpose of this study was to determine if ER and antioxidant gene expression in skeletal muscle are responsive to changes in circulating estradiol, and if ERs regulate antioxidant gene expression in this tissue.

Methodology/Principal Findings

Adult C57BL/6 mice underwent ovariectomies or sham surgeries to remove circulating estrogens. These mice were implanted with placebo or 17β-estradiol pellets acutely or chronically. A separate experiment examined mice that received weekly injections of Faslodex to chronically block ERs. Skeletal muscles were analyzed for expression of ER genes and proteins and antioxidant genes. ERα was the most abundant, followed by Gper and ERβ in both soleus and EDL muscles. The loss of estrogens through ovariectomy induced ERα gene and protein expression in the soleus, EDL, and TA muscles at both the acute and chronic time points. Gpx3 mRNA was also induced both acutely and chronically in all 3 muscles in mice receiving 17β-estradiol. When ERs were blocked using Faslodex, Gpx3 mRNA was downregulated in the soleus muscle, but not the EDL and TA muscles.

Conclusions/Significance

These data suggest that Gpx3 and ERα gene expression are sensitive to circulating estrogens in skeletal muscle. ERs may regulate Gpx3 gene expression in the soleus muscle, but skeletal muscle regulation of Gpx3 via ERs is dependent upon muscle type. Further work is needed to determine the indirect effects of estrogen and ERα on Gpx3 expression in skeletal muscle, and their importance in the aging process.     

Alterations in Skeletal Muscle Cell Homeostasis in a Mouse Model of Cigarette Smoke Exposure

Background

Skeletal muscle dysfunction is common in chronic obstructive pulmonary disease (COPD), a disease mainly caused by chronic cigarette use. An important proportion of patients with COPD have decreased muscle mass, suggesting that chronic cigarette smoke exposure may interfere with skeletal muscle cellular equilibrium. Therefore, the main objective of this study was to investigate the kinetic of the effects that cigarette smoke exposure has on skeletal muscle cell signaling involved in protein homeostasis and to assess the reversibility of these effects.

Methods

A mouse model of cigarette smoke exposure was used to assess skeletal muscle changes. BALB/c mice were exposed to cigarette smoke or room air for 8 weeks, 24 weeks or 24 weeks followed by 60 days of cessation. The gastrocnemius and soleus muscles were collected and the activation state of key mediators involved in protein synthesis and degradation was assessed.

Results

Gastrocnemius and soleus were smaller in mice exposed to cigarette smoke for 8 and 24 weeks compared to room air exposed animals. Pro-degradation proteins were induced at the mRNA level after 8 and 24 weeks. Twenty-four weeks of cigarette smoke exposure induced pro-degradation proteins and reduced Akt phosphorylation and glycogen synthase kinase-3β quantity. A 60-day smoking cessation period reversed the cell signaling alterations induced by cigarette smoke exposure.

Conclusions

Repeated cigarette smoke exposure induces reversible muscle signaling alterations that are dependent on the duration of the cigarette smoke exposure. These results highlights a beneficial aspect associated with smoking cessation.     

Absence of Functional Leptin Receptor Isoforms in the POUND (Leprdb/lb) Mouse Is Associated with Muscle Atrophy and Altered Myoblast Proliferation and Differentiation

Objective

Leptin receptors are abundant in human skeletal muscle, but the role of leptin in muscle growth, development and aging is not well understood. Here we utilized a novel mouse model lacking all functional leptin receptor isoforms (POUND mouse, Lepr^db/lb) to determine the role of leptin in skeletal muscle.

Methods and Findings

Skeletal muscle mass and fiber diameters were examined in POUND mice, and primary myoblast cultures were used to determine the effects of altered leptin signaling on myoblast proliferation and differentiation. ELISA assays, integrated pathway analysis of mRNA microarrays, and reverse phase protein analysis were performed to identify signaling pathways impacted by leptin receptor deficiency. Results show that skeletal muscle mass and fiber diameter are reduced 30–40% in POUND mice relative to wild-type controls. Primary myoblast cultures demonstrate decreased proliferation and decreased expression of both MyoD and myogenin in POUND mice compared to normal mice. Leptin treatment increased proliferation in primary myoblasts from muscles of both adult (12 months) and aged (24 months) wild-type mice, and leptin increased expression of MyoD and myogenin in aged primary myoblasts. ELISA assays and protein arrays revealed altered expression of molecules associated with the IGF-1/Akt and MAPK/MEK signaling pathways in muscle from the hindlimbs of mice lacking functional leptin receptors.

Conclusion

These data support the hypothesis that the adipokine leptin is a key factor important for the regulation of skeletal muscle mass, and that leptin can act directly on its receptors in peripheral tissues to regulate cell proliferation and differentiation.     

Exercise Training Reverses Skeletal Muscle Atrophy in an Experimental Model of VCP Disease

Background

The therapeutic effects of exercise resistance and endurance training in the alleviation of muscle hypertrophy/atrophy should be considered in the management of patients with advanced neuromuscular diseases. Patients with progressive neuromuscular diseases often experience muscle weakness, which negatively impact independence and quality of life levels. Mutations in the valosin containing protein (VCP) gene lead to Inclusion body myopathy associated with Paget''s disease of bone and frontotemporal dementia (IBMPFD) and more recently affect 2% of amyotrophic lateral sclerosis (ALS)-diagnosed cases.

Methods/Principle Findings

The present investigation was undertaken to examine the effects of uphill and downhill exercise training on muscle histopathology and the autophagy cascade in an experimental VCP mouse model carrying the R155H mutation. Progressive uphill exercise in VCP^R155H/+ mice revealed significant improvement in muscle strength and performance by grip strength and Rotarod analyses when compared to the sedentary mice. In contrast, mice exercised to run downhill did not show any significant improvement. Histologically, the uphill exercised VCP^R155H/+ mice displayed an improvement in muscle atrophy, and decreased expression levels of ubiquitin, P62/SQSTM1, LC3I/II, and TDP-43 autophagy markers, suggesting an alleviation of disease-induced myopathy phenotypes. There was also an improvement in the Paget-like phenotype.

Conclusions

Collectively, our data highlights that uphill exercise training in VCP^R155H/+ mice did not have any detrimental value to the function of muscle, and may offer effective therapeutic options for patients with VCP-associated diseases.     

The effect of muscle power training with elastic band on blood glucose,cytokine, and physical function in elderly women with hyperglycemia

[Purpose]

The purpose of this study was to identify the effects of muscle power training with elastic band on body composition, glucose relation factor, and physical function in elderly women with hyperglycemia.

[Methods]

A total of 16 elderly women volunteered to participate in this study as subjects, and they were randomly assigned into one of the following two groups: muscle power training group (MPT: n = 8) and control group (CON: n = 8). The muscle power training group took exercise program using elastic band for 12 weeks, and the other group did not receive any exercise program during the same period. Before and after the experiment, both of the two groups received measurement in body composition (BMI, %Fat, skeletal muscle mass), glucose, cytokine (interleukin 6, adiponectin), and physical function (IPPB, grip strength). With these methods, the following conclusions were achieved.

[Results]

The results showed significant increases in adiponectin (p = 0.006), interleukin 6 (p = 0.018), SPPB (p = 0.024), and grip strength (P=.014). Blood glucose was significantly decreased in exercise group than contruo group.

[Conclusion]

It shows that the muscle power training with elastic band can give positive effects in elderly women with hyperglycemia.     

Effect of Testosterone on Insulin Stimulated IRS1 Ser Phosphorylation in Primary Rat Myotubes—A Potential Model for PCOS-Related Insulin Resistance

Background

Polycystic ovary syndrome (PCOS) is characterized by a hyperandrogenic state and frequently develops skeletal muscle insulin resistance. We determined whether testosterone adversely affects insulin action by increasing serine phosphorylation of IRS-1^636/639 in differentiated rat skeletal muscle myotubes. The phosphorylation of Akt, mTOR, and S6K, downstream targets of the PI3-kinase-IRS-1 complex were also studied.

Methods

Primary differentiated rat skeletal muscle myotubes were subjected to insulin for 30 min after 16-hour pre-exposure to either low (20 ng/ml) or high (200 ng/ml) doses of testosterone. Protein phosphorylation of IRS-1 Ser^636/639, Akt Ser⁴⁷³, mTOR-Ser²⁴⁴⁸, and S6K-Thr³⁸⁹ were measured by Western blot with signal intensity measured by immunofluorescence.

Results

Cells exposed to 100 nM of insulin had increased IRS-1 Ser^636/639 and Akt Ser⁴⁷³ phosphorylation. Cells pre-exposed to low-dose testosterone had significantly increased insulin-induced mTOR-Ser²⁴⁴⁸ and S6K-Thr³⁸⁹ phosphorylation (p<0.05), and further increased insulin-induced IRS-1 Ser^636/639 phosphorylation (p = 0.042) compared to control cells. High-dose testosterone pre-exposure attenuated the insulin-induced mTOR-Ser²⁴⁴⁸ and S6K-Thr³⁸⁹ phosphorylation.

Conclusions

The data demonstrated an interaction between testosterone and insulin on phosphorylation of intracellular signaling proteins, and suggests a link between a hyperandrogenic, hyperinsulinemic environment and the development of insulin resistance involving serine phosphorylation of IRS-1 Ser^636/639. These results may guide further investigations of potential mechanisms of PCOS-related insulin resistance.     

Direct Regulation of Striated Muscle Myosins by Nitric Oxide and Endogenous Nitrosothiols

Background

Nitric oxide (NO) has long been recognized to affect muscle contraction [1], both through activation of guanylyl cyclase and through modification of cysteines in proteins to yield S-nitrosothiols. While NO affects the contractile apparatus directly, the identities of the target myofibrillar proteins remain unknown. Here we report that nitrogen oxides directly regulate striated muscle myosins.

Principal Findings

Exposure of skeletal and cardiac myosins to physiological concentrations of nitrogen oxides, including the endogenous nitrosothiol S-nitroso-L-cysteine, reduced the velocity of actin filaments over myosin in a dose-dependent and oxygen-dependent manner, caused a doubling of force as measured in a laser trap transducer, and caused S-nitrosylation of cysteines in the myosin heavy chain. These biomechanical effects were not observed in response to S-nitroso-D-cysteine, demonstrating specificity for the naturally occurring isomer. Both myosin heavy chain isoforms in rats and cardiac myosin heavy chain from human were S-nitrosylated in vivo.

Significance

These data show that nitrosylation signaling acts as a molecular “gear shift” for myosin—an altogether novel mechanism by which striated muscle and cellular biomechanics may be regulated.     

Evaluation of functional erythropoietin receptor status in skeletal muscle in vivo: acute and prolonged studies in healthy human subjects

Background

Erythropoietin receptors have been identified in human skeletal muscle tissue, but downstream signal transduction has not been investigated. We therefore studied in vivo effects of systemic erythropoietin exposure in human skeletal muscle.

Methodology/Principal Findings

The protocols involved 1) acute effects of a single bolus injection of erythropoietin followed by consecutive muscle biopsies for 1–10 hours, and 2) a separate study with prolonged administration for 16 days with biopsies obtained before and after. The presence of erythropoietin receptors in muscle tissue as well as activation of Epo signalling pathways (STAT5, MAPK, Akt, IKK) were analysed by western blotting. Changes in muscle protein profiles after prolonged erythropoietin treatment were evaluated by 2D gel-electrophoresis and mass spectrometry. The presence of the erythropoietin receptor in skeletal muscle was confirmed, by the M20 but not the C20 antibody. However, no significant changes in phosphorylation of the Epo-R, STAT5, MAPK, Akt, Lyn, IKK, and p70S6K after erythropoietin administration were detected. The level of 8 protein spots were significantly altered after 16 days of rHuEpo treatment; one isoform of myosin light chain 3 and one of desmin/actin were decreased, while three isoforms of creatine kinase and two of glyceraldehyd-3-phosphate dehydrogenase were increased.

Conclusions/Significance

Acute exposure to recombinant human erythropoietin is not associated by detectable activation of the Epo-R or downstream signalling targets in human skeletal muscle in the resting situation, whereas more prolonged exposure induces significant changes in the skeletal muscle proteome. The absence of functional Epo receptor activity in human skeletal muscle indicates that the long-term effects are indirect and probably related to an increased oxidative capacity in this tissue.