Kate T. Murphy

N‐acetylcysteine attenuates the decline in muscle Na+,K+‐pump activity and delays fatigue during prolonged exercise in humans

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 (%Δactivity) at 45 min (P < 0.05) but not at fatigue. When expressed relative to work done, the %Δactivity‐to‐work ratio was attenuated by NAC at 45 min and fatigue (P < 0.005). The rise in plasma [K+] during exercise and the Δ[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.

Antibody-directed myostatin inhibition in 21-mo-old mice reveals novel roles for myostatin signaling in skeletal muscle structure and function

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<0.05). PF‐354 increased fiber cross‐sectional area by 12% and enhanced maximum in situ force of tibialis anterior (TA) muscles by 35% (P<0.05). PF‐354 increased the proportion of type IIa fibers by 114% (P<0.01) and enhanced activity of oxidative enzymes (SDH) by 39% (P<0.01). PF‐354 reduced markers of apoptosis in TA muscle cross‐sections by 56% (P<0.03) and reduced caspase3 mRNA by 65% (P<0.04). Antibody‐directed myostatin inhibition attenuated the decline in mass and function of muscles of aging mice, in part, by reducing apoptosis. These observations identify novel roles for myostatin in regulation of muscle mass and highlight the therapeutic potential of antibody‐directed myostatin inhibition for sarcopenia.—Murphy, K. T., Koopman, R., Naim, T., Léger, B., Trieu, J., Ibebunjo, C. Lynch, G. S. Antibody‐directed myostatin inhibition in 21‐mo‐old mice reveals novel roles for myostatin signaling in skeletal muscle structure and function. FASEB J. 24, 4433–4442 (2010). www.fasebj.org

Elevated expression of activins promotes muscle wasting and cachexia

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.—Chen, J. L., Walton, K. L., Winbanks, C. E., Murphy, K. T., Thomson, R. E., Makanji, Y., Qian, H., Lynch, G. S., Harrison, C. A., Gregorevic, P. Elevated expression of activins promotes muscle wasting and cachexia. FASEB J. 28, 28–1711 (1723). www.fasebj.org

Infusion with the antioxidant N-acetylcysteine attenuates early adaptive responses to exercise in human skeletal muscle

Aim:  Production of reactive oxygen species (ROS) in skeletal muscle is markedly increased during exercise and may be essential for exercise adaptation. We, therefore, investigated 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.

Antibody-directed myostatin inhibition enhances muscle mass and function in tumor-bearing mice

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.

Intense exercise up-regulates Na+,K+-ATPase isoform mRNA, but not protein expression in human skeletal muscle.

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 ∼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 α1, α2, α3, β1, β2 and β3 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 α3 and β2 mRNA by 2.4‐ and 1.7‐fold, respectively (P < 0.05), whilst α1 and α2 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 β1 and β3 mRNA, reflecting variable time‐dependent responses. When the average postexercise value was contrasted to rest, mRNA increased for α1, α2, α3, β1, β2 and β3 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 α1–α3 and β1–β3 isoforms. Thus, human skeletal muscle expresses each of the Na+,K+‐ATPase α1, α2, α3, β1, β2 and β3 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.

Cellular mechanisms underlying temporal changes in skeletal muscle protein synthesis and breakdown during chronic β‐adrenoceptor stimulation in mice

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.

Antibody-Directed Myostatin Inhibition Improves Diaphragm Pathology in Young but not Adult Dystrophic mdx Mice

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.

Importance of functional and metabolic impairments in the characterization of the C-26 murine model of cancer cachexia

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.

Targeting of Fn14 Prevents Cancer-Induced Cachexia and Prolongs Survival

The 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.