38 heart [14] and released to the circulation) and binds to cell-surface receptors causing muscle loss. The myostatin protein circulates in the blood in a latent form as a full-length precursor that is cleaved into an amino-terminal pro-peptide and a carboxy-terminal mature region, which is the active form of the molecule. Once activated, myostatin has high affinity for the activin IIB receptor (Acvr2b, also known as ActRIIB) and weak affinity for Acvr2a (also known as ActRII and ActRIIA), both of which, like other receptors for TGF-β family members, bind multiple ligands [15, 16]. Liu et al. used a myostatin anti-sense RNA and found that the oligonucleotides could suppress myostatin expression in vivo resulting in an increase in muscle growth both in healthy and cachectic mice [17]. Interestingly, the effect of the RNA oligonucleotides was a significant upregulation of MyoD expression, therefore reinforcing the role of this transcription factor in muscle wasting [18, 19]. Although the use of the deacetylase inhibitors to increase the levels of follistatin (an antagonist of myostatin) has not lead to any positive results in the treatment of cachexia in experimental animals [20, 21], the use of the activin receptor extracellular domain/Fc fusion protein (ACVR2B-Fc) has been shown to be effective in the treatment of muscle wasting in tumor-bearing animals [21]. Finally, and very recently, Zhou et al. have shown that the administration of a high-affinity activin type II receptor leads to prolonged survival in tumor-bearing mice [22]. This receptor regulates the expression of target genes through to a TGFβ pathway. Myostatin signaling acts through this receptor in skeletal muscle by setting in motion an intracellular cascade of events involving SMAD proteins, p38 MAPK, ERK1/2, PI3-K/AKT, and Wnt pathways [23–26]. Bearing all this in mind, the objective of the present investigation was to analyze in animals bearing the Lewis lung carcinoma the effects of ActRIIB antagonism on both muscle weights and force. 2 Material and methods 2.1 Animals C57Bl/6 male mice (Criffa, Barcelona, Spain), of about 20 g were used in the different experiments. The animals were maintained at 22 ± 2°C with a regular light–dark cycle (light on from 08:00 a.m. to 08:00 p.m.) and had free access to food and water. The diet (Panlab, Barcelona, Spain) consists of 54% carbohydrate, 17% protein, and 5% fat (the residue was nondigestible material). The food intake was measured daily. All animal manipulations were made in accordance with the European Community guidelines for the use of laboratory animals. J Cachexia Sarcopenia Muscle (2012) 3:37–43 2.2 Tumor inoculation and treatment Mice received an intramuscular (hind leg) inoculum of 5 × 105 Lewis lung carcinoma cells obtained from exponential tumors. The Lewis lung carcinoma is a highly cachectic rapidly growing mouse tumor containing poorly differentiated cells, with a relatively short doubling time [27]. The animals were divided into three groups: control (C), tumor-bearing mice (TB), and tumor-bearing mice treated with the soluble receptor antagonist of myostatin (sActRIIB) [22] at the dose of 10 mg/kg s.c. twice a week (Fig. 1). sActRIIB sequesters Activin A and Myostatin in vivo [22]. At day 14, after tumor transplantation, the animals were weighed and anesthesized with a ketamine/xylacine mixture (i.p.) (Imalgene® and Rompun® respectively). The tumor was harvested from the hind leg, its volume and mass evaluated, and number of lung metastasis evaluated under the microscope. The metastases weight was evaluated according to the methodology used by Donati et al. [28]. Tissues were rapidly excised, weighed, and frozen in liquid nitrogen. 2.3 Real-time polymerase chain reaction Total RNA from tibialis muscle was extracted by TriPureTM kit (Roche, Barcelona, Spain), a commercial modification of the acid guanidinium isothiocyanate/ phenol/chloroform method [29]. First-strand cDNA was synthesized from total RNA with oligo dT15 primers and random primers pdN6 by using a cDNA synthesis kit (Transcriptor Reverse Transcriptase, Roche, Barcelona, Spain). Analysis of mRNA levels for the genes from the different proteolytic systems was performed with primers designed to detect the following gene products: ubiquitin (FORWARD 5′ GAT CCA GGA CAA GGA GGG C 3′, REVERSE 5′ CAT CTT CCA GCT GCT TGC CT3′); E2 (FORWARD: 5′ AGG CGA AGA TGG CGG T 3′; REVERSE 5′ TCA TGC CTG TCC ACC TTG TA 3′); C8 proteasome subunit (FORWARD 5′ AGA CCC CAA CAT GAA ACT GC 3′; REVERSE 5′ AGG TTT GTT GGC AGA TGC TC 3′); MuRF-1 (FORWARD 5′ TGT CTG GAG GTC GTT TCC G 3′; REVERSE 5′ ATG CCG GTC CAT GAT CAC TT 3′); Atrogin-1(FORWARD 5′ Fig. 1 Experimental protocol