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Cancer cachexia is common in many cancers and the loss of skeletal muscle mass compromises the response to therapies and quality of life. A contributing mechanism is oxidative stress and compounds able to attenuate it may be protective. Sulforaphane (SFN), a natural antioxidant in cruciferous vegetables, activates nuclear factor erythroid 2-related factor 2 (Nrf2) signaling to decrease oxidative stress. Although SFN has potential as a cancer therapeutic, whether it can attenuate muscle wasting in the absence or presence of chemotherapy is unknown. In healthy C2C12 myotubes, SFN administration for 48 h induced hypertrophy through increased myoblast fusion via Nrf2 and ERK signaling. To determine whether SFN could attenuate wasting induced by cancer cells, myotubes were cocultured with or without Colon-26 (C-26) cancer cells for 48 h and treated with 5-fluorouracil (5-FU, 5 µM) or vehicle (DMSO). SFN (10 µM) or DMSO was added for the final 24 h. Coculture with cancer cells in the absence and presence of 5-FU reduced myotube width by ∼30% ( P < 0.001) and ∼20% ( P < 0.01), respectively, which was attenuated by SFN ( P < 0.05). Exposure to C-26 conditioned media reduced myotube width by 15% ( P < 0.001), which was attenuated by SFN. Western immunoblotting and qRT-PCR confirmed activation of Nrf2 signaling and antioxidant genes. Coadministration of Nrf2 inhibitors (ML-385) or MEK inhibitors (PD184352) revealed that SFN’s attenuation of atrophy was blocked by ERK inhibition. These data support the chemoprotective and antioxidative function of SFN in myotubes, highlighting its therapeutic potential for cancer-related muscle wasting.

We investigated whether digoxin lowered muscle Na+,K+-ATPase (NKA), impaired muscle performance and exacerbated exercise K+ disturbances. Ten healthy adults ingested digoxin (0.25 mg; DIG) or placebo (CON) for 14 days and performed quadriceps strength and fatiguability, finger flexion (FF, 105%peak-workrate, 3 × 1 min, fourth bout to fatigue) and leg cycling (LC, 10 min at 33% VO2peak and 67% VO2peak, 90% VO2peak to fatigue) trials using a double-blind, crossover, randomised, counter-balanced design. Arterial (a) and antecubital venous (v) blood was sampled (FF, LC) and muscle biopsied (LC, rest, 67% , fatigue, 3 h after exercise). In DIG, in resting muscle, [3H]-ouabain binding site content (OB-Fab) was unchanged; however, bound-digoxin removal with Digibind revealed total ouabain binding (OB+Fab) increased (8.2%, P = 0.047), indicating 7.6% NKA–digoxin occupancy. Quadriceps muscle strength declined in DIG (−4.3%, P = 0.010) but fatiguability was unchanged. During LC, in DIG (main effects), time to fatigue and [K+]a were unchanged, whilst [K+]v was lower (P = 0.042) and [K+]a-v greater (P = 0.004) than in CON; with exercise (main effects), muscle OB-Fab was increased at 67% VO2peak (per wet-weight, P = 0.005; per protein P = 0.001) and at fatigue (per protein, P = 0.003), whilst [K+]a, [K+]v and [K+]a-v were each increased at fatigue (P = 0.001). During FF, in DIG (main effects), time to fatigue, [K+]a, [K+]v and [K+]a-v were unchanged; with exercise (main effects), plasma [K+]a, [K+]v, [K+]a-v and muscle K+ efflux were all increased at fatigue (P = 0.001). Thus, muscle strength declined, but functional muscle NKA content was preserved during DIG, despite elevated plasma digoxin and muscle NKA–digoxin occupancy, with K+ disturbances and fatiguability unchanged.

This file contains: • Supplemental Methods • Supplemental Figure S1. Muscle-specific overexpression of the AT1 but not the MasR regulates skeletal muscle fiber size in healthy mice. • Supplemental Figure S2. Representative images of differentiated C2C12 myotubes incubated for 48 h in normal horse serum and treated with vehicle control or the ACE/Ang II/AT1 axis activators Ang II or A779 • Supplemental Figure S3. Genetic MasR overexpression was induced by transducing C2C12 myoblasts with a Tet-regulated MasR lentivirus or empty lentivirus and treating differentiated myotubes with doxycylin • Supplemental Figure S4. Low dose AVE 0991 induces small but significant improvements in food and water intake and weight loss in severely cachectic C-26 tumor-bearing mice • Supplemental Figure S5. Low dose AVE 0991 (1 mg/kg) causes an oxidative-to-glycolytic shift in fiber types in TA muscles from severely cachectic C-26 tumor-bearing mice • Supplemental Figure S6. High dose AVE 0991 treatment enhanced cumulative water intake and movement as assessed by the number of beam breaks in severely cachectic C-26 tumor-bearing mice fed ad libitum but not in those pair-fed to vehicle treated controls • Supplemental Figure S7. Pharmacological Ang-(1-7)/MasR activation protects against Lewis Lung Carcinoma (LLC) cancer cell induced muscle fiber atrophy in vitro • Supplemental Figure S8. Specificity of our custom anti-AT1 peptide compared to a commercially available anti-AT1 antibody • Supplemental Table S1. Primers used to amplify AT1 and MasR genes • Supplemental Table S2. Human cancer cachexia study group characteristics • Supplemental Table S3. List of upregulated proteins in AVE 0991 treated serum starved myotubes • Supplemental Table S4. List of down regulated proteins in AVE 0991 treated serum starved myotubes

Cancer cachexia is a multifactorial syndrome characterized by a progressive loss of skeletal muscle mass associated with significant functional impairment. Cachexia robs patients of their strength and capacity to perform daily tasks and live independently. Effective treatments are needed urgently. Here, we investigated the therapeutic potential of activating the “alternative” axis of the renin-angiotensin system, involving ACE2, angiotensin-(1-7), and the mitochondrial assembly receptor (MasR), for treating cancer cachexia. Plasmid overexpression of the MasR or pharmacologic angiotensin-(1-7)/MasR activation did not affect healthy muscle fiber size in vitro or in vivo but attenuated atrophy induced by coculture with cancer cells in vitro. In mice with cancer cachexia, the MasR agonist AVE 0991 slowed tumor development, reduced weight loss, improved locomotor activity, and attenuated muscle wasting, with the majority of these effects dependent on the orexigenic and not antitumor properties of AVE 0991. Proteomic profiling and IHC revealed that mechanisms underlying AVE 0991 effects on skeletal muscle involved miR-23a–regulated preservation of the fast, glycolytic fibers. MasR activation is a novel regulator of muscle phenotype, and AVE 0991 has orexigenic, anticachectic, and antitumorigenic effects, identifying it as a promising adjunct therapy for cancer and other serious muscle wasting conditions.Significance:These findings demonstrate that MasR activation has multiple benefits of being orexigenic, anticachectic, and antitumorigenic, revealing it as a potential adjunct therapy for cancer.See related commentary by Rupert et al., p. 699

Twins Research Australia (TRA) is a community of twins and researchers working on health research to benefit everyone, including twins. TRA leads multidisciplinary research through the application of twin and family study designs, with the aim of sustaining long-term twin research that, both now and in the future, gives back to the community. This article summarizes TRA’s recent achievements and future directions, including new methodologies addressing causation, linkage to health, economic and educational administrative datasets and to geospatial data to provide insight into health and disease. We also explain how TRA’s knowledge translation and exchange activities are key to communicating the impact of twin studies to twins and the wider community. Building researcher capability, providing registry resources and partnering with all key stakeholders, particularly the participants, are important for how TRA is advancing twin research to improve health outcomes for society. TRA provides researchers with open access to its vibrant volunteer membership of twins, higher order multiples (multiples) and families who are willing to consider participation in research. Established four decades ago, this resource facilitates and supports research across multiple stages and a breadth of health domains.

Cancer cachexia is a multifactorial syndrome characterized by a progressive loss of skeletal muscle mass associated with significant functional impairment. Cachexia robs patients of their strength and capacity to perform daily tasks and live independently. Effective treatments are needed urgently. Here, we investigated the therapeutic potential of activating the “alternative” axis of the renin-angiotensin system, involving ACE2, angiotensin-(1-7), and the mitochondrial assembly receptor (MasR), for treating cancer cachexia. Plasmid overexpression of the MasR or pharmacologic angiotensin-(1-7)/MasR activation did not affect healthy muscle fiber size in vitro or in vivo but attenuated atrophy induced by coculture with cancer cells in vitro. In mice with cancer cachexia, the MasR agonist AVE 0991 slowed tumor development, reduced weight loss, improved locomotor activity, and attenuated muscle wasting, with the majority of these effects dependent on the orexigenic and not antitumor properties of AVE 0991. Proteomic profiling and IHC revealed that mechanisms underlying AVE 0991 effects on skeletal muscle involved miR-23a–regulated preservation of the fast, glycolytic fibers. MasR activation is a novel regulator of muscle phenotype, and AVE 0991 has orexigenic, anticachectic, and antitumorigenic effects, identifying it as a promising adjunct therapy for cancer and other serious muscle wasting conditions. Significance: These findings demonstrate that MasR activation has multiple benefits of being orexigenic, anticachectic, and antitumorigenic, revealing it as a potential adjunct therapy for cancer. See related commentary by Rupert et al., p. 699

Duchenne muscular dystrophy is a severe and progressive striated muscle wasting disorder that leads to premature death from respiratory and/or cardiac failure. We have previously shown that treatment of young dystrophic mdx and dystrophin/utrophin null ( dko ) mice with BGP-15, a coinducer of heat shock protein 72, ameliorated the dystrophic pathology. We therefore tested the hypothesis that later-stage BGP-15 treatment would similarly benefit older mdx and dko mice when the dystrophic pathology was already well established. Later stage treatment of mdx or dko mice with BGP-15 did not improve maximal force of tibialis anterior (TA) muscles ( in situ ) or diaphragm muscle strips ( in vitro ). However, collagen deposition (fibrosis) was reduced in TA muscles of BGP-15–treated dko mice but unchanged in TA muscles of treated mdx mice and diaphragm of treated mdx and dko mice. We also examined whether BGP-15 treatment could ameliorate aspects of the cardiac pathology, and in young dko mice it reduced collagen deposition and improved both membrane integrity and systolic function. These results confirm BGP-15's ability to improve aspects of the dystrophic pathology but with differing efficacies in heart and skeletal muscles at different stages of the disease progression. These findings support a role for BGP-15 among a suite of pharmacological therapies for Duchenne muscular dystrophy and related disorders.

The Brazilian Twin Registry (BTR) was established in 2013 and has impelled twin research in South America. The main aim of the initiative was to create a resource that would be accessible to the Brazilian scientific community as well as international researchers interested in the investigation of the contribution of genetic and environmental factors in the development of common diseases, phenotypes, and human behavior traits. The BTR is a joint effort between academic and governmental institutions from Brazil and Australia. The collaboration includes the Federal University of Minas Gerais (UFMG) in Brazil, the University of Sydney and University of Melbourne in Australia, the Australian Twin Registry, as well as the research foundations CNPq and CAPES in Brazil. The BTR is a member of the International Network of Twin Registries. Recruitment strategies used to register twins have been through participation in a longitudinal study investigating genetic and environmental factors for low back pain occurrence, and from a variety of sources including media campaigns and social networking. Currently, 291 twins are registered in the BTR, with data on demographics, zygosity, anthropometrics, and health history having been collected from 151 twins using a standardized self-reported questionnaire. Future BTR plans include the registration of thousands of Brazilian twins identified from different sources and collaborate nationally and internationally with other research groups interested on twin studies.

Numerous health problems including acute critical illness, cancer, diseases associated with chronic inflammation, and neurological disorders often result in skeletal muscle weakness and fatigue. Disease-related muscle atrophy and fatigue is an important clinical problem because acquired skeletal muscle weakness can increase the duration of hospitalization, result in exercise limitation, and contribute to a poor quality of life. Importantly, skeletal muscle atrophy is also associated with increased morbidity and mortality of patients. Therefore, improving our understanding of the mechanism(s) responsible for skeletal muscle weakness and fatigue in patients is a required first step to develop clinical protocols to prevent these skeletal muscle problems. This review will highlight the consequences and potential mechanisms responsible for skeletal muscle atrophy and fatigue in patients suffering from acute critical illness, cancer, chronic inflammatory diseases, and neurological disorders.

Cancer cachexia is a multifactorial syndrome characterized by a progressive loss of skeletal muscle mass associated with significant functional impairment. In addition to a loss of skeletal muscle mass and function, many patients with cancer cachexia also experience cardiac atrophy, remodeling, and dysfunction, which in the field of cancer cachexia is described as cardiac cachexia. The cardiac alterations may be due to underlying heart disease, the cancer itself, or problems initiated by the cancer treatment and, unfortunately, remains largely underappreciated by clinicians and basic scientists. Despite recent major advances in the treatment of cancer, little progress has been made in the treatment of cardiac cachexia in cancer, and much of this is due to lack of information regarding the mechanisms. This review focuses on the cardiac atrophy associated with cancer cachexia, describing some of the known mechanisms and discussing the current and future therapeutic strategies to treat this condition. Above all else, improved awareness of the condition and an increased focus on identification of mechanisms and therapeutic targets will facilitate the eventual development of an effective treatment for cardiac atrophy in cancer cachexia.

All same-sex dizygotic (DZ) twins and approximately one-third of monozygotic (MZ) twin pairs have separate placentas, making it impossible to use the number of placentas to determine zygosity. Zygosity determination is further complicated because incorrect assumptions are often made, such as that only DZ pairs have two placentas and that all MZ pairs are phenotypically identical. These assumptions, by twins, their families and health professionals, along with the lack of universal zygosity testing for same-sex twins, has led to confusion within the twin community, yet little research has been conducted with twins about their understanding and assumptions about zygosity. We aimed to explore and quantify understanding and assumptions about zygosity using twins attending an Australian twin festival. We recruited 91 twin pairs younger than 18 years of age and their parents, and 30 adult twin pairs who were all uncertain of their zygosity, to complete one pen and paper questionnaire and one online questionnaire about their assumed zygosity, reasons for their assumptions and the importance of accurate zygosity knowledge. Responses were compared with their true zygosity measured using a genetic test. We found a substantial proportion of parents and twins had been misinformed by their own parents or medical professionals, and that knowledge of their true zygosity status provided peace of mind and positive emotional responses. For these reasons we propose universal zygosity testing of same-sex twins as early in life as possible and an increase in education of medical professionals, twins and families of twins about zygosity issues.

Loss of skeletal muscle mass and function (cachexia) is severe in patients with colorectal liver metastases because of the large increase in resting energy expenditure but remains understudied because of a lack of suitable preclinical models. Our aim was to characterize a novel preclinical model of cachexia in colorectal liver metastases. We tested the hypothesis that mice with colorectal liver metastases would exhibit cachexia, as evidenced by a reduction in liver-free body mass, muscle mass, and physiological impairment. Twelve-week-old male CBA mice received an intrasplenic injection of Ringer solution (sham) or murine colorectal cancer cells (MoCR) to induce colorectal liver metastases. At end-point (20–29 days), the livers of MoCR mice were infiltrated completely with metastases, and MoCR mice had reduced liver-free body mass, muscle mass, and epididymal fat mass compared with sham controls ( P < 0.03). MoCR mice exhibited impaired rotarod performance and grip strength ( P < 0.03). Histochemical analyses of tibialis anterior muscles from MoCR mice revealed muscle fiber atrophy and reduced oxidative enzyme activity ( P < 0.001). Adipose tissue remodeling was evident in MoCR mice, with reduced adipocyte diameter and greater infiltration of nonadipocyte tissue ( P < 0.05). These findings reveal the MoCR mouse model exhibits significant cachexia and is a suitable preclinical model of cachexia in colorectal liver metastases. This model should be used for identifying effective treatments for cachexia to improve quality of life and reduce mortality in patients with colorectal liver metastases.

Cancer cachexia describes the progressive skeletal muscle wasting and weakness associated with many cancers. Cachexia reduces mobility and quality of life and accounts for 20-30% of all cancer-related deaths. Activation of the renin-angiotensin system causes skeletal muscle wasting and weakness. We tested the hypothesis that treatment with the angiotensin converting enzyme (ACE) inhibitor, perindopril, would enhance whole body and skeletal muscle function in cachectic mice bearing Colon-26 (C-26) tumors. CD2F1 mice received a subcutaneous injection of phosphate buffered saline or C-26 tumor cells inducing either a mild or severe cachexia. The following day, one cohort of C-26 mice began receiving perindopril in their drinking water (4 mg kg(-1) day(-1) ) for 21 days. In mild and severe cachexia, perindopril increased measures of whole body function (grip strength and rotarod) and reduced fatigue in isolated contracting diaphragm muscle strips (p

Patients with advanced cancer often succumb to complications arising from striated muscle wasting associated with cachexia. Excessive activation of the type IIB activin receptor (ActRIIB) is considered an important mechanism underlying this wasting, where circulating procachectic factors bind ActRIIB and ultimately lead to the phosphorylation of SMAD2/3. Therapeutics that antagonize the binding of ActRIIB ligands are in clinical development, but concerns exist about achieving efficacy without off-target effects. To protect striated muscle from harmful ActRIIB signaling, and to reduce the risk of off-target effects, we developed an intervention using recombinant adeno-associated viral vectors (rAAV vectors) that increase expression of Smad7 in skeletal and cardiac muscles. SMAD7 acts as an intracellular negative regulator that prevents SMAD2/3 activation and promotes degradation of ActRIIB complexes. In mouse models of cachexia, rAAV:Smad7 prevented wasting of skeletal muscles and the heart independent of tumor burden and serum levels of procachectic ligands. Mechanistically, rAAV:Smad7 administration abolished SMAD2/3 signaling downstream of ActRIIB and inhibited expression of the atrophy-related ubiquitin ligases MuRF1 and MAFbx. These findings identify muscle-directed Smad7 gene delivery as a potential approach for preventing muscle wasting under conditions where excessive ActRIIB signaling occurs, such as cancer cachexia.

Objective The development of skeletal muscle insulin resistance is an early physiological defect, yet the intracellular mechanisms accounting for this metabolic defect remained unresolved. Here, we have examined the role of glucose-6-phosphate dehydrogenase (G6PDH) activity in the pathogenesis of insulin resistance in skeletal muscle. Methods Multiple mouse disease states exhibiting insulin resistance and glucose intolerance, as well as obese humans defined as insulin-sensitive, insulin-resistant, or pre-diabetic, were examined. Results We identified increased glucose-6-phosphate dehydrogenase (G6PDH) activity as a common intracellular adaptation that occurs in parallel with the induction of insulin resistance in skeletal muscle and is present across animal and human disease states with an underlying pathology of insulin resistance and glucose intolerance. We observed an inverse association between G6PDH activity and nitric oxide synthase (NOS) activity and show that increasing NOS activity via the skeletal muscle specific neuronal (n)NOSμ partially suppresses G6PDH activity in skeletal muscle cells. Furthermore, attenuation of G6PDH activity in skeletal muscle cells via (a) increased nNOSμ/NOS activity, (b) pharmacological G6PDH inhibition, or (c) genetic G6PDH inhibition increases insulin-independent glucose uptake. Conclusions We have identified a novel, previously unrecognized role for G6PDH in the regulation of skeletal muscle glucose metabolism., Highlights • Defective skeletal muscle G6PDH activity in multiple insulin resistant animal models. • Demonstration of defective skeletal muscle G6PDH activity in pre-diabetic individuals. • Identification of nNOSμ as a regulator of G6PDH activity in skeletal muscle. • G6PDH activity modulates insulin-independent glucose uptake in skeletal muscle.

This study investigates the effects of infusion with the antioxidant N-acetylcysteine (NAC) on exercise-induced activation of signalling pathways and genes involved in exercise adaptation in human skeletal muscle.

Cancer cachexia describes the progressive skeletal muscle wasting and weakness in many cancer patients and accounts for >20% of cancer-related deaths. We tested the hypothesis that antibody-directed myostatin inhibition would attenuate the atrophy and loss of function in muscles of tumor-bearing mice. Twelve-week-old C57BL/6 mice received a subcutaneous injection of saline (control) or Lewis lung carcinoma (LLC) tumor cells. One week later, mice received either once weekly injections of saline (control, n = 12; LLC, n = 9) or a mouse chimera of anti-human myostatin antibody (PF-354, 10 mg·kg−1·wk−1, LLC+PF-354, n = 11) for 5 wk. Injection of LLC cells reduced muscle mass and maximum force of tibialis anterior (TA) muscles by 8–10% ( P < 0.05), but the muscle atrophy and weakness were prevented with PF-354 treatment ( P > 0.05). Maximum specific (normalized) force of diaphragm muscle strips was reduced with LLC injection ( P < 0.05) but was not improved with PF-354 treatment ( P > 0.05). PF-354 enhanced activity of oxidative enzymes in TA and diaphragm muscles of tumor-bearing mice by 118% and 89%, respectively ( P < 0.05). Compared with controls, apoptosis that was not of myofibrillar or satellite cell origin was 140% higher in TA muscle cross sections from saline-treated LLC tumor-bearing mice ( P < 0.05) but was not different in PF-354-treated tumor-bearing mice ( P > 0.05). Antibody-directed myostatin inhibition attenuated the skeletal muscle atrophy and loss of muscle force-producing capacity in a murine model of cancer cachexia, in part by reducing apoptosis. The improvements in limb muscle mass and function highlight the therapeutic potential of antibody-directed myostatin inhibition for cancer cachexia.

Chronic stimulation of β-adrenoceptors with β-adrenoceptor agonists (β-agonists) can induce substantial skeletal muscle hypertrophy, but the mechanisms mediating this muscle growth have yet to be elucidated. We investigated whether chronic β-adrenoceptor stimulation in mice with the β-agonist formoterol alters the muscle anabolic response following β-adrenoceptor stimulation. Twelve-week-old C57BL/6 mice were treated for up to 28 days with a once-daily injection of either saline (control, n= 9) or formoterol (100 μg kg−1; n= 9). Rates of muscle protein synthesis were assessed at either 1, 7 or 28 days of treatment, 6 h after injection. Protein synthesis rates were higher in formoterol-treated mice at day 7 (∼1.5-fold, P < 0.05), but not at day 1 or 28. The increased muscle protein synthesis was associated with increased phosphorylation of S6K1 (r= 0.49, P < 0.01). Formoterol treatment acutely reduced maximal calpain activity by ∼25% (P < 0.05) but did not affect atrogin-1 protein levels and proteasome-mediated proteolytic activity, despite significantly enhanced phosphorylation of Akt (P < 0.05). Formoterol increased CREB phosphorylation by ∼30% (P < 0.05) and PPARγ coactivator-1α (PGC-1α) by 11-fold (P < 0.05) on day 1 only. These observations identify that formoterol treatment induces muscle anabolism, by reducing calpain activity and by enhancing protein synthesis via increased PI-3 kinase/Akt signalling.

Duchenne muscular dystrophy (DMD) is characterized by progressive skeletal muscle wasting and weakness, leading to premature death from respiratory and/or cardiac failure. A clinically relevant question is whether myostatin inhibition can improve function of the diaphragm, which exhibits a severe and progressive pathology comparable with that in DMD. We hypothesized that antibody-directed myostatin inhibition would improve the pathophysiology of diaphragm muscle strips from young mdx mice (when the pathology is mild) and adult mdx mice (when the pathology is quite marked). Five weeks treatment with a mouse chimera of anti-human myostatin antibody (PF-354, 10 mg/kg/week) increased muscle mass (P < 0.05) and increased diaphragm median fiber cross-sectional area (CSA, P < 0.05) in young C57BL/10 and mdx mice, compared with saline-treated controls. PF-354 had no effect on specific force (sPo, maximum force normalized to muscle CSA) of diaphragm muscle strips from young C57BL/10 mice, but increased sPo by 84% (P < 0.05) in young mdx mice. In contrast, 8 weeks of PF-354 treatment did not improve muscle mass, median fiber CSA, collagen infiltration, or sPo of diaphragm muscle strips from adult mdx mice. PF-354 antibody-directed myostatin inhibition completely restored the functional capacity of diaphragm strips to control levels when treatment was initiated early, but not in the later stages of disease progression, suggesting that such therapies may only have a limited window of efficacy for DMD and related conditions.

Cancer cachexia is a complex syndrome that describes the progressive muscle wasting and weakness in many cancer patients. Muscle wasting reduces the ability of affected patients to perform the tasks of daily living and causes severe fatigue, leading to a reduction in quality of life. Cancer cachexia reduces patient response to anti-neoplastic treatments, increases the risk of postoperative complications and accounts for20% of cancer-related deaths. The pathogenesis of cancer cachexia is multifactorial and includes anorexia, inflammation, metabolic disturbances and enhanced muscle proteolysis, and each of these presents as a potential therapeutic target for ameliorating cancer cachexia.This review provides an update on some of the emerging drugs for cancer cachexia.This is a review of the current status of emerging therapies for cancer cachexia.An increasing number of studies are focused on the development of novel therapies for cancer cachexia. Initial studies concentrated on the treatment of anorexia, but now aim to attenuate inflammation or muscle proteolysis, or to use different drugs in combination so as to treat several aspects of cachexia simultaneously.There are several existing and emerging drugs with the potential to ameliorate cancer cachexia, but the most efficacious treatment will probably come from combined drug therapies.

Exercise increases Na(+)-K(+) pump isoform gene expression and elevates muscle reactive oxygen species (ROS). We investigated whether enhanced ROS scavenging induced with the antioxidant N-acetylcysteine (NAC) blunted the increase in Na(+)-K(+) pump mRNA during repeated contractions in human and rat muscle. In experiment 1, well-trained subjects received saline or NAC intravenously prior to and during 45 min cycling. Vastus lateralis muscle biopsies were taken pre-infusion and following exercise. In experiment 2, isolated rat extensor digitorum longus muscles were pre-incubated without or with 10 mm NAC and then rested or stimulated electrically at 60 Hz for 90 s. After 3 h recovery, muscles were frozen. In both experiments, the muscles were analysed for Na(+)-K(+) pump alpha(1), alpha(2), alpha(3), beta(1), beta(2) and beta(3) mRNA. In experiment 1, exercise increased alpha(2) mRNA by 1.0-fold (P = 0.03), but alpha(2) mRNA was reduced by 0.40-fold with NAC (P = 0.03). Exercise increased alpha(3), beta(1) and beta(2) mRNA by 2.0- to 3.4-fold (P 0.32). Neither exercise nor NAC altered alpha(1) or beta(3) mRNA (P > 0.31). In experiment 2, electrical stimulation increased alpha(1), alpha(2) and alpha(3) mRNA by 2.3- to 17.4-fold (P 0.07). Electrical stimulation almost completely reduced beta(1) mRNA but only in the presence of NAC (P 0.09). In conclusion, NAC attenuated the increase in Na(+)-K(+) pump alpha(2) mRNA with exercise in human muscle and all alpha isoforms with electrical stimulation in rat muscle. This indicates a regulatory role for ROS in Na(+)-K(+) pump alpha isoform mRNA in mammalian muscle during repeated contractions.

We aimed to quantify the Na(+)-K(+) exchange occurring during exercise in rat skeletal muscle in vivo. Intracellular Na(+) and K(+) content, Na(+) permeability ((22)Na(+) influx), Na(+)-K(+) pump activity (ouabain-sensitive (86)Rb(+) uptake) and Na(+)-K(+) pump alpha(2) subunit content ([(3)H]ouabain binding) were measured. Six-week-old rats rested (control animals) or performed intermittent running for 10-60 min and were then killed or were killed at 15 or 90 min following 60 min exercise. In the soleus muscle, intracellular Na(+) was 80% higher than in control rats after 60 min exercise, was still elevated (38%) after 15 min rest and returned to control levels after 90 min rest. Intracellular K(+) showed corresponding decreases after 15-60 min exercise, returning to control levels 90 min postexercise. Exercise induced little change in Na(+) and K(+) in the extensor digitorum longus muscle (EDL). In soleus, the exercise-induced rise in Na(+) and reduction in K(+) were augmented by pretreatment with ouabain or by reducing the content of muscular Na(+)-K(+) pumps by prior K(+) depletion of the animals. Fifteen minutes after 60 min exercise, ouabain-sensitive (86)Rb(+) uptake in the soleus was increased by 30% but was unchanged in EDL, and there was no effect of exercise on [(3)H]ouabain binding measured in vitro or in vivo in either muscle. In conclusion, in the soleus, in vivo exercise induces a rise in intracellular Na(+), which reflects the excitation-induced increase in Na(+) influx and leads to augmented Na(+)-K(+) pump activity without apparent change in Na(+)-K(+) pump capacity.

SummaryThe cytokine TWEAK and its cognate receptor Fn14 are members of the TNF/TNFR superfamily and are upregulated in tumors. We found that Fn14, when expressed in tumors, causes cachexia and that antibodies against Fn14 dramatically extended lifespan by inhibiting tumor-induced weight loss although having only moderate inhibitory effects on tumor growth. Anti-Fn14 antibodies prevented tumor-induced inflammation and loss of fat and muscle mass. Fn14 signaling in the tumor, rather than host, is responsible for inducing this cachexia because tumors in Fn14- and TWEAK-deficient hosts developed cachexia that was comparable to that of wild-type mice. These results extend the role of Fn14 in wound repair and muscle development to involvement in the etiology of cachexia and indicate that Fn14 antibodies may be a promising approach to treat cachexia, thereby extending lifespan and improving quality of life for cancer patients.

Reactive oxygen species (ROS) have been linked with both depressed Na(+),K(+)-pump activity and skeletal muscle fatigue. This study investigated N-acetylcysteine (NAC) effects on muscle Na(+),K(+)-pump activity and potassium (K(+)) regulation during prolonged, submaximal endurance exercise. Eight well-trained subjects participated in a double-blind, randomised, crossover design, receiving either NAC or saline (CON) intravenous infusion at 125 mg kg(-1) h(-1) for 15 min, then 25 mg kg(-1) h(-1) for 20 min prior to and throughout exercise. Subjects cycled for 45 min at 71% , then continued at 92% until fatigue. Vastus lateralis muscle biopsies were taken before exercise, at 45 min and fatigue and analysed for maximal in vitro Na(+),K(+)-pump activity (K(+)-stimulated 3-O-methyfluorescein phosphatase; 3-O-MFPase). Arterialized venous blood was sampled throughout exercise and analysed for plasma K(+) and other electrolytes. Time to fatigue at 92% was reproducible in preliminary trials (c.v. 5.6 +/- 0.6%) and was prolonged with NAC by 23.8 +/- 8.3% (NAC 6.3 +/- 0.5 versus CON 5.2 +/- 0.6 min, P < 0.05). Maximal 3-O-MFPase activity decreased from rest by 21.6 +/- 2.8% at 45 min and by 23.9 +/- 2.3% at fatigue (P < 0.05). NAC attenuated the percentage decline in maximal 3-O-MFPase activity (%Deltaactivity) at 45 min (P < 0.05) but not at fatigue. When expressed relative to work done, the %Deltaactivity-to-work ratio was attenuated by NAC at 45 min and fatigue (P < 0.005). The rise in plasma [K(+)] during exercise and the Delta[K(+)]-to-work ratio at fatigue were attenuated by NAC (P < 0.05). These results confirm that the antioxidant NAC attenuates muscle fatigue, in part via improved K(+) regulation, and point to a role for ROS in muscle fatigue.

The AMP-activated protein kinase (AMPK) cascade has been linked to many of the acute effects of exercise on skeletal muscle substrate metabolism, as well as to some of the chronic training-induced adaptations. We determined the effect of 3 wk of intensified training (HIT; 7 sessions of 8 × 5 min at 85% V̇o2 peak) in skeletal muscle from well-trained athletes on AMPK responsiveness to exercise. Rates of whole body substrate oxidation were determined during a 90-min steady-state ride (SS) pre- and post-HIT. Muscle metabolites and AMPK signaling were determined from biopsies taken at rest and immediately after exercise during the first and seventh HIT sessions, performed at the same (absolute) pre-HIT work rate. HIT decreased rates of whole body carbohydrate oxidation ( P < 0.05) and increased rates of fat oxidation ( P < 0.05) during SS. Resting muscle glycogen and its utilization during intense exercise were unaffected by HIT. However, HIT induced a twofold decrease in muscle [lactate] ( P < 0.05) and resulted in tighter metabolic regulation, i.e., attenuation of the decrease in the PCr/(PCr + Cr) ratio and of the increase in [AMPfree]/ATP. Resting activities of AMPKα1 and -α2 were similar post-HIT, with the magnitude of the rise in response to exercise similar pre- and post-HIT. AMPK phosphorylation at Thr172on both the α1 and α2 subunits increased in response to exercise, with the magnitude of this rise being similar post-HIT. Acetyl-coenzyme A carboxylase-β phosphorylation was similar at rest and, despite HIT-induced increases in whole body rates of fat oxidation, did not increase post-HIT. Our results indicate that, in well-trained individuals, short-term HIT improves metabolic control but does not blunt AMPK signaling in response to intense exercise.

Characterization of expression of, and consequently also the acute exercise effects on, Na(+),K(+)-ATPase isoforms in human skeletal muscle remains incomplete and was therefore investigated. Fifteen healthy subjects (eight males, seven females) performed fatiguing, knee extensor exercise at approximately 40% of their maximal work output per contraction. A vastus lateralis muscle biopsy was taken at rest, fatigue and 3 and 24 h postexercise, and analysed for Na(+),K(+)-ATPase alpha(1), alpha(2), alpha(3), beta(1), beta(2) and beta(3) mRNA and crude homogenate protein expression, using Real-Time RT-PCR and immunoblotting, respectively. Each individual expressed gene transcripts and protein bands for each Na(+),K(+)-ATPase isoform. Each isoform was also expressed in a primary human skeletal muscle cell culture. Intense exercise (352 +/- 69 s; mean +/-s.e.m.) immediately increased alpha(3) and beta(2) mRNA by 2.4- and 1.7-fold, respectively (P < 0.05), whilst alpha(1) and alpha(2) mRNA were increased by 2.5- and 3.5-fold at 24 h and 3 h postexercise, respectively (P < 0.05). No significant change occurred for beta(1) and beta(3) mRNA, reflecting variable time-dependent responses. When the average postexercise value was contrasted to rest, mRNA increased for alpha(1), alpha(2), alpha(3), beta(1), beta(2) and beta(3) isoforms, by 1.4-, 2.2-, 1.4-, 1.1-, 1.0- and 1.0-fold, respectively (P < 0.05). However, exercise did not alter the protein abundance of the alpha(1)-alpha(3) and beta(1)-beta(3) isoforms. Thus, human skeletal muscle expresses each of the Na(+),K(+)-ATPase alpha(1), alpha(2), alpha(3), beta(1), beta(2) and beta(3) isoforms, evidenced at both transcription and protein levels. Whilst brief exercise increased Na(+),K(+)-ATPase isoform mRNA expression, there was no effect on isoform protein expression, suggesting that the exercise challenge was insufficient for muscle Na(+),K(+)-ATPase up-regulation.

Mutations in dystrophin lead to Duchenne muscular dystrophy, which is among the most common human genetic disorders. Dystrophin nucleates assembly of the dystrophin-glycoprotein complex (DGC), and a defective DGC disrupts an essential link between the intracellular cytoskeleton and the basal lamina, leading to progressive muscle wasting. In vitro studies have suggested that dystrophin phosphorylation may affect interactions with actin or syntrophin, yet whether this occurs in vivo or affects protein function remains unknown. Utilizing nanoflow liquid chromatography mass spectrometry, we identified 18 phosphorylated residues within endogenous dystrophin. Mutagenesis revealed that phosphorylation at S3059 enhances the dystrophin-dystroglycan interaction and 3D modeling utilizing the Rosetta software program provided a structural model for how phosphorylation enhances this interaction. These findings demonstrate that phosphorylation is a key mechanism regulating the interaction between dystrophin and the DGC and reveal that posttranslational modification of a single amino acid directly modulates the function of dystrophin.

In models of cancer cachexia, inhibiting type IIB activin receptors (ActRIIBs) reverse muscle wasting and prolongs survival, even with continued tumor growth. ActRIIB mediates signaling of numerous TGF-β proteins; of these, we demonstrate that activins are the most potent negative regulators of muscle mass. To determine whether activin signaling in the absence of tumor-derived factors induces cachexia, we used recombinant serotype 6 adeno-associated virus (rAAV6) vectors to increase circulating activin A levels in C57BL/6 mice. While mice injected with control vector gained ~10% of their starting body mass (3.8±0.4 g) over 10 wk, mice injected with increasing doses of rAAV6:activin A exhibited weight loss in a dose-dependent manner, to a maximum of -12.4% (-4.2±1.1 g). These reductions in body mass in rAAV6:activin-injected mice correlated inversely with elevated serum activin A levels (7- to 24-fold). Mechanistically, we show that activin A reduces muscle mass and function by stimulating the ActRIIB pathway, leading to deleterious consequences, including increased transcription of atrophy-related ubiquitin ligases, decreased Akt/mTOR-mediated protein synthesis, and a profibrotic response. Critically, we demonstrate that the muscle wasting and fibrosis that ensues in response to excessive activin levels is fully reversible. These findings highlight the therapeutic potential of targeting activins in cachexia.

We examined the effects of a high-fat diet (HFD-CHO) versus a habitual diet, prior to carbohydrate (CHO)-loading on fuel metabolism and cycling time-trial (TT) performance. Five endurance-trained cyclists participated in two 14-day randomized cross-over trials during which subjects consumed either a HFD (>65% MJ from fat) or their habitual diet (CTL) (30 ± 5% MJ from fat) for 10 day, before ingesting a high-CHO diet (CHO-loading, CHO > 70% MJ) for 3 days. Trials consisted of a 150-min cycle at 70% of peak oxygen uptake (V̇O2peak), followed immediately by a 20-km TT. One hour before each trial, cyclists ingested 400 ml of a 3.44% medium-chain triacylglycerol (MCT) solution, and during the trial, ingested 600 ml/hour of a 10% 14C-glucose + 3.44% MCT solution. The dietary treatments did not alter the subjects’ weight, body fat, or lipid profile. There were also no changes in circulating glucose, lactate, free fatty acid (FFA), and β-hydroxybutyrate concentrations during exercise. However, mean serum glycerol concentrations were significantly higher (p < .01) in the HFD-CHO trial. The HFD-CHO diet increased total fat oxidation and reduced total CHO oxidation but did not alter plasma glucose oxidation during exercise. By contrast, the estimated rates of muscle glycogen and lactate oxidation were lower after the HFD-CHO diet. The HFD-CHO treatment was also associated with improved TT times (29.5 ± 2.9 min vs. 30.9 ± 3.4 min for HFD-CHO and CTL-CHO, p < .05). High-fat feeding for 10 days prior to CHO-loading was associated with an increased reliance on fat, a decreased reliance on muscle glycogen, and improved time trial performance after prolonged exercise.

Summary Background and aims The non-essential amino acid, glycine, is often considered biologically neutral, but some studies indicate that it could be an effective anti-inflammatory agent. Since inflammation is central to the development of cancer cachexia, glycine supplementation represents a simple, safe and promising treatment. We tested the hypothesis that glycine supplementation reduces skeletal muscle inflammation and preserves muscle mass in tumor-bearing mice. Methods To induce cachexia, CD2F1 mice received a subcutaneous injection of PBS (control, n = 12) or C26 tumor cells ( n = 32) in accordance with the protocols developed by Murphy et al. [Murphy KT, Chee A, Trieu J, Naim T, Lynch GS. Importance of functional and metabolic impairments in the characterization of the C-26 murine model of cancer cachexia. Dis Models Mech 2012;5(4):533–545.]. Subcutaneous injections of glycine ( n = 16) or PBS ( n = 16) were administered daily for 21 days and at the conclusion of treatment, selected muscles, tumor and adipose tissue were collected and prepared for Real-Time RT-PCR or western blot analysis. Results Glycine attenuated the loss of fat and muscle mass, blunted increases in markers of inflammation (F4/80, P = 0.01 & IL-6 mRNA, P = 0.01) and atrophic signaling (MuRF, P = 0.047; atrogin-1, P = 0.04; LC3B, P = 0.06 and; BNIP3, P = 0.10) and tended to attenuate the loss of body mass ( P = 0.07), muscle function ( P = 0.06), and oxidative stress (GSSG/GSH, P = 0.06 and DHE, P = 0.07) seen in tumor-bearing mice. Preliminary studies that compared the effect of glycine administration with isonitrogenous doses of alanine or citrulline showed that the observed protective effect was specific to glycine. Conclusions Glycine protects skeletal muscle from cancer-induced wasting and loss of function, reduces the oxidative and inflammatory burden, and reduces the expression of genes associated with muscle protein breakdown in cancer cachexia. Importantly, these effects were glycine specific.

Cancer cachexia describes the progressive skeletal muscle wasting and weakness associated with many cancers. Cachexia reduces mobility and quality of life and accounts for 20-30% of all cancer-related deaths. Activation of the renin-angiotensin system causes skeletal muscle wasting and weakness. We tested the hypothesis that treatment with the angiotensin converting enzyme (ACE) inhibitor, perindopril, would enhance whole body and skeletal muscle function in cachectic mice bearing Colon-26 (C-26) tumors. CD2F1 mice received a subcutaneous injection of phosphate buffered saline or C-26 tumor cells inducing either a mild or severe cachexia. The following day, one cohort of C-26 mice began receiving perindopril in their drinking water (4 mg kg(-1) day(-1) ) for 21 days. In mild and severe cachexia, perindopril increased measures of whole body function (grip strength and rotarod) and reduced fatigue in isolated contracting diaphragm muscle strips (p 0.05). In severely cachectic mice, perindopril reduced tumor growth, improved locomotor activity and reduced fatigue of tibialis anterior muscles in situ (p 0.05), which was associated with increased oxidative enzyme capacity (succinate deyhydrogenase, p 0.05). Perindopril attenuated the increase in MuRF-1 and IL-6 mRNA expression and enhanced Akt phosphorylation in severely cachectic mice but neither body nor muscle mass was increased. These findings support the therapeutic potential of ACE inhibition for enhancing whole body function and reducing fatigue of respiratory muscles in early and late stage cancer cachexia and should be confirmed in future clinical trials. Since ACE inhibition alone did not enhance body or muscle mass, co-treatment with an anabolic agent may be required to address these aspects of cancer cachexia.

The role of the renin-angiotensin system (RAS) in vasoregulation is well established, but a localized RAS exists in multiple tissues and exerts diverse functions including autonomic control and thermogenesis. The role of the RAS in the maintenance and function of skeletal muscle is not well understood, especially the role of angiotensin peptides, which appear to contribute to muscle atrophy. We tested the hypothesis that mice lacking the angiotensin type 1A receptor (AT1A−/−) would exhibit enhanced whole body and skeletal muscle function and improved regeneration after severe injury. Despite 18- to 20-wk-old AT1A−/−mice exhibiting reduced muscle mass compared with controls ( P < 0.05), the tibialis anterior (TA) muscles produced a 25% higher maximum specific (normalized) force ( P < 0.05). Average fiber cross-sectional area (CSA) and fiber oxidative capacity was not different between groups, but TA muscles from AT1A−/−mice had a reduced number of muscle fibers as well as a higher proportion of type IIx/b fibers and a lower proportion of type IIa fibers ( P < 0.05). Measures of whole body function (grip strength, rotarod performance, locomotor activity) were all improved in AT1A−/−mice ( P < 0.05). Surprisingly, the recovery of muscle mass and fiber CSA following myotoxic injury was impaired in AT1A−/−mice, in part by impaired myoblast fusion, prolonged collagen infiltration and inflammation, and delayed expression of myogenic regulatory factors. The findings support the therapeutic potential of RAS inhibition for enhancing whole body and skeletal muscle function, but they also reveal the importance of RAS signaling in the maintenance of muscle mass and for normal fiber repair after injury.

Summary Cancer cachexia describes the progressive skeletal muscle wasting and weakness that is associated with many cancers. It impairs quality of life and accounts for >20% of all cancer-related deaths. The main outcome that affects quality of life and mortality is loss of skeletal muscle function and so preclinical models should exhibit similar functional impairments in order to maximize translational outcomes. Mice bearing colon-26 (C-26) tumors are commonly used in cancer cachexia studies but few studies have provided comprehensive assessments of physiological and metabolic impairment, especially those factors that impact quality of life. Our aim was to characterize functional impairments in mildly and severely affected cachectic mice, and determine the suitability of these mice as a preclinical model. Metabolic abnormalities are also evident in cachectic patients and we investigated whether C-26-tumor-bearing mice had similar metabolic aberrations. Twelve-week-old CD2F1 mice received a subcutaneous injection of PBS (control) or C-26 tumor cells. After 18-20 days, assessments were made of grip strength, rotarod performance, locomotor activity, whole body metabolism, and contractile properties of tibialis anterior (TA) muscles (in situ) and diaphragm muscle strips (in vitro). Injection of C-26 cells reduced body and muscle mass, and epididymal fat mass. C-26-tumor-bearing mice exhibited lower grip strength and rotarod performance. Locomotor activity was impaired following C-26 injection, with reductions in movement distance, duration and speed compared with controls. TA muscles from C-26-tumor-bearing mice had lower maximum force (-27%) and were more susceptible to fatigue. Maximum specific (normalized) force of diaphragm muscle strips was reduced (-10%) with C-26 injection, and force during fatiguing stimulation was also lower. C-26-tumor-bearing mice had reduced carbohydrate oxidation and increased fat oxidation compared with controls. The range and consistency of functional and metabolic impairments in C-26-tumor-bearing mice confirm their suitability as a preclinical model for cancer cachexia. We recommend the use of these comprehensive functional assessments to maximize the translation of findings to more accurately identify effective treatments for cancer cachexia.

Sarcopenia is the progressive age-related loss of skeletal muscle mass associated with functional impairments that reduce mobility and quality of life. Overt muscle wasting with sarcopenia is usually preceded by a slowing of the rate of relaxation and a reduction in maximum force production. Parvalbumin (PV) is a cytosolic Ca2+ buffer thought to facilitate relaxation in muscle. We tested the hypothesis that restoration of PV levels in muscles of old mice would increase the magnitude and hasten relaxation of submaximal and maximal force responses. The tibialis anterior (TA) muscles of young (6 month), adult (13 month), and old (26 month) C57BL/6 mice received electroporation-assisted gene transfer of plasmid encoding PV or empty plasmid (pcDNA3.1). Contractile properties of TA muscles were assessed in situ 14 days after transfer. In old mice, muscles with increased PV expression had a 40% slower rate of tetanic force development (p

We examined whether abnormal skeletal muscle Na(+),K(+)-pumps underlie impaired exercise performance in haemodialysis patients (HDP) and whether these are improved in renal transplant recipients (RTx).Peak oxygen consumption ( O(2peak)) and plasma [K(+)] were measured during incremental exercise in 9RTx, 10 HDP and 10 healthy controls (CON). Quadriceps peak torque (PT), fatigability (decline in strength during thirty contractions), thigh muscle cross-sectional area (TMCSA) and vastus lateralis Na(+),K(+)-pump maximal activity, content and isoform (α(1)-α(3), β(1)-β(3)) abundance were measured.O(2peak) was 32 and 35% lower in RTx and HDP than CON, respectively (P0.05). PT was less in RTx and HDP than CON (P0.05) but did not differ when expressed relative to TMCSA. Fatigability was ∼1.6-fold higher in RTx (24 ± 11%) and HDP (25 ± 4%) than CON (15 ± 5%, P0.05). Na(+),K(+)-pump activity was 28 and 31% lower in RTx and HDP, respectively than CON (P0.02), whereas content and isoform abundance did not differ. Pooled (n = 28) O(2peak) correlated with Na(+),K(+)-pump activity (r = 0.45, P = 0.02).O(2peak) and muscle Na(+),K(+)-pump activity were depressed and muscle fatigability increased in HDP, with no difference observed in RTx. These findings are consistent with the possibility that impaired exercise performance in HDP and RTx may be partially due to depressed muscle Na(+),K(+)-pump activity and relative TMCSA.

Chronic stimulation of β-adrenoceptors with β-adrenoceptor agonists (β-agonists) can induce substantial skeletal muscle hypertrophy, but the mechanisms mediating this muscle growth have yet to be elucidated. We investigated whether chronic β-adrenoceptor stimulation in mice with the β-agonist formoterol alters the muscle anabolic response following β-adrenoceptor stimulation. Twelve-week-old C57BL/6 mice were treated for up to 28 days with a once-daily injection of either saline (control, n= 9) or formoterol (100 μg kg−1; n= 9). Rates of muscle protein synthesis were assessed at either 1, 7 or 28 days of treatment, 6 h after injection. Protein synthesis rates were higher in formoterol-treated mice at day 7 (∼1.5-fold, P < 0.05), but not at day 1 or 28. The increased muscle protein synthesis was associated with increased phosphorylation of S6K1 (r= 0.49, P < 0.01). Formoterol treatment acutely reduced maximal calpain activity by ∼25% (P < 0.05) but did not affect atrogin-1 protein levels and proteasome-mediated proteolytic activity, despite significantly enhanced phosphorylation of Akt (P < 0.05). Formoterol increased CREB phosphorylation by ∼30% (P < 0.05) and PPARγ coactivator-1α (PGC-1α) by 11-fold (P < 0.05) on day 1 only. These observations identify that formoterol treatment induces muscle anabolism, by reducing calpain activity and by enhancing protein synthesis via increased PI-3 kinase/Akt signalling.

Sarcopenia is the progressive loss of skeletal muscle mass and function with advancing age, leading to reduced mobility and quality of life. We tested the hypothesis that antibody-directed myostatin inhibition would attenuate the decline in mass and function of muscles of aged mice and that apoptosis would be reduced. Eighteen-month-old C57BL/6 mice were treated for 14 wk with a once-weekly injection of saline (control, n=9) or a mouse chimera of anti-human myostatin antibody (PF-354, 10 mg/kg; n=12). PF-354 prevented the age-related reduction in body mass and increased soleus, gastrocnemius, and quadriceps muscle mass (P

Physical function is limited in patients with kidney disease, although previous studies have been confounded by anemia. What is not clear is how physical performance changes over time as renal function deteriorates. A cohort of 12 patients (10 male, two female; mean +/- SD age 49 +/- 11 yr) who had stages 3 to 4 chronic kidney disease without previous anemia were examined, and nine were followed for a 2-yr period. Assessments were made of peak oxygen consumption (VO2peak) by cycle ergometry, leg extension strength, and fatigue on an isokinetic dynamometer and thigh muscle cross-sectional area (TMCSA) by computed tomography. At baseline, creatinine clearance was 31 +/- 13 ml/min and hemoglobin concentration ([Hb]) was 129 +/- 9 g/L. VO2peak was low (1.88 L/min, 82% of predicted), and maximal isometric voluntary contraction was 188 +/- 42 Nm, with a TMCSA of 144 +/- 27 cm2. VO2peak correlated with creatinine clearance corrected for body surface area (r = 0.613, P = 0.034) but not to [Hb]. VO2peak adjusted for patient weight correlated with leg fatigue (r = -0.693, P = 0.012). For those with follow-up tests, there were falls in renal function by 28% (P = 0.007) and VO2peak by 9% (P = 0.03), whereas [Hb] did not change. Leg strength fell across a range of isokinetic speeds (P = 0.04), whereas no change in TMCSA was observed. In conclusion, exercise performance as measured by aerobic (VO2peak) and leg strength tests were reduced in patients with stages 3 to 4 chronic kidney disease. As renal function declined over time, there was a corresponding decline in exercise performance even when [Hb] was maintained.

This study investigated the effects of endurance training status and sex differences on skeletal muscle Na+,K+-pump mRNA expression, content and activity. Methods: Forty-five endurance-trained males (ETM), 11 recreationally active males (RAM), and nine recreationally active females (RAF) underwent a vastus lateralis muscle biopsy. Muscle was analysed for Na+,K+-pump α1, α2, α3, β1, β2 and β3 isoform mRNA expression (real-time reverse transcription-polymerase chain reaction), content ([3H]-ouabain-binding site) and maximal activity (3- O-methylfluorescein phosphatase, 3- O-MFPase). Results: ETM demonstrated lower α1, α3, β2 and β3 mRNA expression by 74%, 62%, 70% and 82%, respectively, than RAM ( P < 0.04). In contrast, [3H]-ouabain binding and 3- O-MFPase activity were each higher in ETM than in RAM, by 16% ( P < 0.03). RAM demonstrated a 230% and 364% higher α3 and β3 mRNA expression than RAF, respectively ( P < 0.05), but no significant sex differences were found for α1, α2, β1 or β2 mRNA, [3H]-ouabain binding or 3- O-MFPase activity. No significant correlation was found between years of endurance training and either [3H]-ouabain binding or 3- O-MFPase activity. Significant but weak correlations were found between the number of training hours per week and 3- O-MFPase activity ( r = 0.31, P < 0.02) and between incremental exercise O2(peak) and both [3H]-ouabain binding ( r = 0.33, P < 0.01) and 3- O-MFPase activity ( r = 0.28, P < 0.03). Conclusions: Isoform-specific differences in Na+,K+-pump mRNA expression were found with both training status and sex differences, but only training status influenced Na+,K+-pump content and maximal activity in human skeletal muscle. Corrected by Corrigendum: Effects of endurance training status and sex differences on Na+, K+-pump mRNA expression, content and maximal activity in human skeletal muscle, Vol. 190, Issue 3, 261, 2007

This study investigated the effects of electrical stimulation on Na+-K+-ATPase isoform mRNA, with the aim to identify factors modulating Na+-K+-ATPase mRNA in isolated rat extensor digitorum longus (EDL) muscle. Interventions designed to mimic exercise-induced increases in intracellular Na+and Ca2+contents and membrane depolarization were examined. Muscles were mounted on force transducers and stimulated with 60-Hz 10-s pulse trains producing tetanic contractions three times at 10-min intervals. Ouabain (1.0 mM, 120 min), veratridine (0.1 mM, 30 min), and monensin (0.1 mM, 30 min) were used to increase intracellular Na+content. High extracellular K+(13 mM, 60 min) and the Ca2+ionophore A-23187 (0.02 mM, 30 min) were used to induce membrane depolarization and elevated intracellular Ca2+content, respectively. Muscles were analyzed for Na+-K+-ATPase α1–α3and β1–β3mRNA (real-time RT-PCR). Electrical stimulation had no immediate effect on Na+-K+-ATPase mRNA; however at 3 h after stimulation, it increased α1, α2, and α3mRNA by 223, 621, and 892%, respectively ( P = 0.010), without changing β mRNA. Ouabain, veratridine, and monensin increased intracellular Na+content by 769, 724, and 598%, respectively ( P = 0.001) but did not increase mRNA of any isoform. High intracellular K+concentration elevated α1mRNA by 160% ( P = 0.021), whereas A-23187 elevated α3mRNA by 123% ( P = 0.035) but reduced β1mRNA by 76% ( P = 0.001). In conclusion, electrical stimulation induced subunit-specific increases in Na+-K+-ATPase mRNA in isolated rat EDL muscle. Furthermore, Na+-K+-ATPase mRNA appears to be regulated by different stimuli, including cellular changes associated with membrane depolarization and increased intracellular Ca2+content but not increased intracellular Na+content.

We investigated whether depressed muscle Na+-K+-ATPase activity with exercise reflected a loss of Na+-K+-ATPase units, the time course of its recovery postexercise, and whether this depressed activity was related to increased Na+-K+-ATPase isoform gene expression. Fifteen subjects performed fatiguing, knee extensor exercise at ∼40% maximal work output per contraction. A vastus lateralis muscle biopsy was taken at rest, fatigue, 3 h, and 24 h postexercise and analyzed for maximal Na+-K+-ATPase activity via 3- O-methylfluorescein phosphatase (3- O-MFPase) activity, Na+-K+-ATPase content via [3H]ouabain binding sites, and Na+-K+-ATPase α1-, α2-, α3-, β1-, β2- and β3-isoform mRNA expression by real-time RT-PCR. Exercise [352 (SD 267) s] did not affect [3H]ouabain binding sites but decreased 3- O-MFPase activity by 10.7 (SD 8)% ( P < 0.05), which had recovered by 3 h postexercise, without further change at 24 h. Exercise elevated α1-isoform mRNA by 1.5-fold at fatigue ( P < 0.05). This increase was inversely correlated with the percent change in 3- O-MFPase activity from rest to fatigue (%Δ3- O-MFPaserest-fatigue) ( r = −0.60, P < 0.05). The average postexercise (fatigue, 3 h, 24 h) α1-isoform mRNA was increased 1.4-fold ( P < 0.05) and approached a significant inverse correlation with %Δ3- O-MFPaserest-fatigue( r = −0.56, P = 0.08). Exercise elevated α2-isoform mRNA at fatigue 2.5-fold ( P < 0.05), which was inversely correlated with %Δ3- O-MFPaserest-fatigue( r = −0.60, P = 0.05). The average postexercise α2-isoform mRNA was increased 2.2-fold ( P < 0.05) and was inversely correlated with the %Δ3- O-MFPaserest-fatigue( r = −0.68, P < 0.05). Nonsignificant correlations were found between %Δ3- O-MFPaserest-fatigueand other isoforms. Thus acute exercise transiently decreased Na+-K+-ATPase activity, which was correlated with increased Na+-K+-ATPase gene expression. This suggests a possible signal-transduction role for depressed muscle Na+-K+-ATPase activity with exercise.

The production of reactive oxygen species in skeletal muscle is linked with muscle fatigue. This study investigated the effects of the antioxidant compound N-acetylcysteine (NAC) on muscle cysteine, cystine, and glutathione and on time to fatigue during prolonged, submaximal exercise in endurance athletes. Eight men completed a double-blind, crossover study, receiving NAC or placebo before and during cycling for 45 min at 71% peak oxygen consumption (V̇o2 peak) and then to fatigue at 92% V̇o2 peak. NAC was intravenously infused at 125 mg·kg−1·h−1 for 15 min and then at 25 mg·kg−1·h−1 for 20 min before and throughout exercise. Arterialized venous blood was analyzed for NAC, glutathione status, and cysteine concentration. A vastus lateralis biopsy was taken preinfusion, at 45 min of exercise, and at fatigue and was analyzed for NAC, total glutathione (TGSH), reduced glutathione (GSH), cysteine, and cystine. Time to fatigue at 92% V̇o2 peak was reproducible in preliminary trials (coefficient of variation 5.6 ± 0.6%) and with NAC was enhanced by 26.3 ± 9.1% (NAC 6.4 ± 0.6 min vs. Con 5.3 ± 0.7 min; P < 0.05). NAC increased muscle total and reduced NAC at both 45 min and fatigue ( P < 0.005). Muscle cysteine and cystine were unchanged during Con, but were elevated above preinfusion levels with NAC ( P < 0.001). Muscle TGSH ( P < 0.05) declined and muscle GSH tended to decline ( P = 0.06) during exercise. Both were greater with NAC ( P < 0.05). Neither exercise nor NAC affected whole blood TGSH. Whereas blood GSH was decreased and calculated oxidized glutathione increased with exercise ( P < 0.05), both were unaffected by NAC. In conclusion, NAC improved performance in well-trained individuals, with enhanced muscle cysteine and GSH availability a likely mechanism.