Orphan GPCR GPRC5C Facilitates Angiotensin II-Induced Smooth Muscle Contraction.

BACKGROUND: GPCRs (G-protein-coupled receptors) play a central role in the regulation of smooth muscle cell (SMC) contractility, but the function of SMC-expressed orphan GPCR class C group 5 member C (GPRC5C) is unclear. The aim of this project is to define the role of GPRC5C in SMC in vitro and in vivo. METHODS: We studied the role of GPRC5C in the regulation of SMC contractility and differentiation in human and murine SMC in vitro, as well as in tamoxifen-inducible, SMC-specific GPRC5C knockout mice under basal conditions and in vascular disease in vivo. RESULTS: Mesenteric arteries from tamoxifen-inducible, SMC-specific GPRC5C knockout mice showed ex vivo significantly reduced angiotensin II (Ang II)-dependent calcium mobilization and contraction, whereas responses to other relaxant or contractile factors were normal. In vitro, the knockdown of GPRC5C in human aortic SMC resulted in diminished Ang II-dependent inositol phosphate production and lower myosin light chain phosphorylation. In line with this, tamoxifen-inducible, SMC-specific GPRC5C knockout mice showed reduced Ang II-induced arterial hypertension, and acute inactivation of GPRC5C was able to ameliorate established arterial hypertension. Mechanistically, we show that GPRC5C and the Ang II receptor AT1 dimerize, and knockdown of GPRC5C resulted in reduced binding of Ang II to AT1 receptors in HEK293 cells, human and murine SMC, and arteries from tamoxifen-inducible, SMC-specific GPRC5C knockout mice. CONCLUSIONS: Our data show that GPRC5C regulates Ang II-dependent vascular contraction by facilitating AT1 receptor-ligand binding and signaling.

Mitochondrial fusion and altered beta-oxidation drive muscle wasting in a Drosophila cachexia model.

Cancer cachexia is a tumour-induced wasting syndrome, characterised by extreme loss of skeletal muscle. Defective mitochondria can contribute to muscle wasting; however, the underlying mechanisms remain unclear. Using a Drosophila larval model of cancer cachexia, we observed enlarged and dysfunctional muscle mitochondria. Morphological changes were accompanied by upregulation of beta-oxidation proteins and depletion of muscle glycogen and lipid stores. Muscle lipid stores were also decreased in Colon-26 adenocarcinoma mouse muscle samples, and expression of the beta-oxidation gene CPT1A was negatively associated with muscle quality in cachectic patients. Mechanistically, mitochondrial defects result from reduced muscle insulin signalling, downstream of tumour-secreted insulin growth factor binding protein (IGFBP) homologue ImpL2. Strikingly, muscle-specific inhibition of Forkhead box O (FOXO), mitochondrial fusion, or beta-oxidation in tumour-bearing animals preserved muscle integrity. Finally, dietary supplementation with nicotinamide or lipids, improved muscle health in tumour-bearing animals. Overall, our work demonstrates that muscle FOXO, mitochondria dynamics/beta-oxidation and lipid utilisation are key regulators of muscle wasting in cancer cachexia.

Impaired skeletal muscle health in Parkinsonian syndromes: clinical implications, mechanisms and potential treatments.

There is increasing evidence that neurodegenerative disorders including the Parkinsonian syndromes are associated with impaired skeletal muscle health, manifesting as wasting and weakness. Many of the movement problems, lack of muscle strength and reduction in quality of life that are characteristic of these syndromes can be attributed to impairments in skeletal muscle health, but this concept has been grossly understudied and represents an important area of unmet clinical need. This review describes the changes in skeletal muscle health in idiopathic Parkinson's disease and in two atypical Parkinsonian syndromes, the most aggressive synucleinopathy multiple system atrophy, and the tauopathy progressive supranuclear palsy. The pathogenesis of the skeletal muscle changes is described, including the contribution of impairments to the central and peripheral nervous system and intrinsic alterations. Pharmacological interventions targeting the underlying molecular mechanisms with therapeutic potential to improve skeletal muscle health in affected patients are also discussed. Although little is known about the mechanisms underlying these conditions, current evidence implicates multiple pathways and processes, highlighting the likely need for combination therapies to protect muscle health and emphasizing the merit of personalized interventions for patients with different physical capacities at different stages of their disease. As muscle fatigue is often experienced by patients prior to diagnosis, the identification and measurement of this symptom and related biomarkers to identify early signs of disease require careful interrogation, especially for multiple system atrophy and progressive supranuclear palsy where diagnosis is often made several years after onset of symptoms and only confirmed post-mortem. We propose a multidisciplinary approach for early diagnosis and implementation of personalized interventions to preserve muscle health and improve quality of life for patients with typical and atypical Parkinsonian syndromes.

Sulforaphane attenuates cancer cell-induced atrophy of C2C12 myotubes.

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.

Oral digoxin effects on exercise performance, K+ regulation and skeletal muscle Na+ ,K+ -ATPase in healthy humans.

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% V O 2 peak ${\rm{V}}_{{{\rm{O}}}_{\rm{2}}{\rm{peak}}}$ and 67% V O 2 peak ${\rm{V}}_{{{\rm{O}}}_{\rm{2}}{\rm{peak}}}$ , 90% V O 2 peak ${\rm{V}}_{{{\rm{O}}}_{\rm{2}}{\rm{peak}}}$ 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% V O 2 peak ${\rm{V}}_{{{\rm{O}}}_{\rm{2}}{\rm{peak}}}$ , fatigue, 3 h after exercise). In DIG, in resting muscle, [3 H]-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% V O 2 peak ${\rm{V}}_{{{\rm{O}}}_{\rm{2}}{\rm{peak}}}$ (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. KEY POINTS: The Na+ ,K+ -ATPase (NKA) is vital in regulating skeletal muscle extracellular potassium concentration ([K+ ]), excitability and plasma [K+ ] and thereby also in modulating fatigue during intense contractions. NKA is inhibited by digoxin, which in cardiac patients lowers muscle functional NKA content ([3 H]-ouabain binding) and exacerbates K+ disturbances during exercise. In healthy adults, we found that digoxin at clinical levels surprisingly did not reduce functional muscle NKA content, whilst digoxin removal by Digibind antibody revealed an ∼8% increased muscle total NKA content. Accordingly, digoxin did not exacerbate arterial plasma [K+ ] disturbances or worsen fatigue during intense exercise, although quadriceps muscle strength was reduced. Thus, digoxin treatment in healthy participants elevated serum digoxin, but muscle functional NKA content was preserved, whilst K+ disturbances and fatigue with intense exercise were unchanged. This resilience to digoxin NKA inhibition is consistent with the importance of NKA in preserving K+ regulation and muscle function.

Role for Plant-Derived Antioxidants in Attenuating Cancer Cachexia.

Cancer cachexia is the progressive muscle wasting and weakness experienced by many cancer patients. It can compromise the response to gold standard cancer therapies, impair functional capacity and reduce overall quality of life. Cancer cachexia accounts for nearly one-third of all cancer-related deaths and has no effective treatment. The pathogenesis of cancer cachexia and its progression is multifactorial and includes increased oxidative stress derived from both the tumor and the host immune response. Antioxidants have therapeutic potential to attenuate cancer-related muscle loss, with polyphenols, a group of plant-derived antioxidants, being the most widely investigated. This review describes the potential of these plant-derived antioxidants for treating cancer cachexia.

Phosphorylation of ERK and dystrophin S3059 protects against inflammation-associated C2C12 myotube atrophy.

The dystrophin-glycoprotein complex (DGC) is a multiprotein structure required to maintain muscle fiber membrane integrity, transmit force by linking the actin cytoskeleton with the extracellular matrix, and maintain muscle homeostasis. Membrane localization of dystrophin is perturbed in muscles wasting as a consequence of cancer cachexia, tenotomy, and advanced aging, which are all associated with low level, chronic inflammation. Strategies to preserve dystrophin expression at the sarcolemma might therefore combat muscle wasting. Phosphorylation of dystrophin serine 3059 (S3059) enhances the interaction between dystrophin and ß-dystroglycan. To test the contribution of amino acid phosphorylation to muscle fiber size changes, dystrophin constructs with phospho-null and phosphomimetic mutations were transfected into C2C12 muscle cells or AAV-293 cells in the presence or absence of kinase inhibitors/activators to assess effects on myotube diameter and protein function. Overexpression of a dystrophin construct with a phospho-null mutation at S3059 in vitro reduced myotube size in healthy C2C12 cells. Conversely overexpression of a phosphomimetic mutation at S3059 attenuated inflammation-induced myotube atrophy. Increased ERK activation by addition of phorbol myristate acetate (PMA) also reduced inflammation-associated myotube atrophy and increased the interaction between dystrophin and ß-dystroglycan. These findings demonstrate a link between increased ERK activation, dystrophin S3059 phosphorylation, stabilization of the DGC, and the regulation of muscle fiber size. Interventions that increase dystrophin S3059 phosphorylation to promote stronger binding of dystrophin to ß-dystroglycan may have therapeutic potential for attenuation of inflammation-associated muscle wasting.

Mas Receptor Activation Slows Tumor Growth and Attenuates Muscle Wasting in Cancer.

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.Graphical Abstract: http://cancerres.aacrjournals.org/content/canres/79/4/706/F1.large.jpg.See related commentary by Rupert et al., p. 699.

Glucose-6-phosphate dehydrogenase contributes to the regulation of glucose uptake in skeletal muscle.

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.

BGP-15 Improves Aspects of the Dystrophic Pathology in mdx and dko Mice with Differing Efficacies in Heart and Skeletal Muscle.

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.

Smad7 gene delivery prevents muscle wasting associated with cancer cachexia in mice.

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.

Disease-Induced Skeletal Muscle Atrophy and Fatigue.

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 experiencing acute critical illness, cancer, chronic inflammatory diseases, and neurological disorders.

The pathogenesis and treatment of cardiac atrophy in cancer cachexia.

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.

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.

Phosphorylation within the cysteine-rich region of dystrophin enhances its association with ß-dystroglycan and identifies a potential novel therapeutic target for skeletal muscle wasting.

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.

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-ß 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.

Glycine administration attenuates skeletal muscle wasting in a mouse model of cancer cachexia.

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.

Physiological characterization of a mouse model of cachexia in colorectal liver metastases.

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.

Inhibition of the renin-angiotensin system improves physiological outcomes in mice with mild or severe cancer cachexia.

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.

Disruption of muscle renin-angiotensin system in AT1a-/- mice enhances muscle function despite reducing muscle mass but compromises repair after injury.

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 (AT(1A)(-/-)) would exhibit enhanced whole body and skeletal muscle function and improved regeneration after severe injury. Despite 18- to 20-wk-old AT(1A)(-/-) 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 AT(1A)(-/-) 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 AT(1A)(-/-) mice (P < 0.05). Surprisingly, the recovery of muscle mass and fiber CSA following myotoxic injury was impaired in AT(1A)(-/-) 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.