J Cachexia Sarcopenia Muscle (2012) 3:37–43 Table 5 Effects of sActRIIB treatment on muscle force in mice bearing the Lewis lung carcinoma Grip force (day 0) Grip force (day 14) C 5.1±0.21a,c 5.0±0.23a,c T 4.7 ±0.20a,c 2.8 ±0.16b,d T+A 5.2±0.24a,c 4.4±0.3a,c Grip force is expressed as grams per gram IBW. Results are mean±S. E.M. Statistical significance of the results by one-way analysis of variance (ANOVA). Statistically significant difference by post hoc Duncan test. Different letters in superscript indicate significant differences between groups (a and b: differences between C, T, and T+A groups the same day of measurement; and c and d: differences between days 0 and 14 in the same group) C control mice (n=4), T tumor-bearing mice (n=8), T+A tumor-bearing mice treated with sActRIIB (n=8) (81%), and total traveled distance (80%). Resting time was increased and the time involved in different types of movements was decreased. Similar results from our laboratory have been obtained in another tumor model [36]. As a result of the sActRIIB treatment, there was a decrease in the resting time percentage and an increase in the period of time related with slow movements (Table 4). These data suggest that sActRIIB treatment caused a slight improvement in physical performance. In order to see if the increase of muscle weight and physical performance was related with increased muscle performance, grip force was determined using a specialized device dynamometer. The results presented in Table 5 41 clearly show an increase in muscle force as a result of sActRIIB treatment. Finally, and since muscle wasting during cancer has been related with the activity of different proteolytic systems involved in enhanced muscle protein breakdown during catabolic conditions [5, 34, 37, 38], the mRNA expression levels of different genes related to proteolysis was measured following sActRIIB treatment. Indeed, tumor burden resulted in generalized increases in the majority of the components of the ubiquitin-dependent proteolytic system analyzed. An increase in m-Calpain (calcium- dependent system) and Cathepsin B (lysosomal system) were also observed in the tibialis muscle of the tumor- bearing animals (Table 6). Surprisingly, sActRIIB treatment did not influence the mRNA expression levels of the different components of the ubiquitin-dependent proteolytic system. However, sActRIIB treatment significantly decreases both calcium-dependent and lysosomal markers (Table 6). PCR real-time analysis of tibialis muscle revealed that tumor-bearing mice myostatin expression increased 1.5-fold over non-tumor bearing mice and that tumor- bearing mice treated with sActRIIB expressed similar levels of myostatin as found in non-treated tumor-bearing mice (Table 6). It has to be pointed out that the calpain system could well play an important role in muscle proteolysis during cancer. From this point of view, Costelli et al. have reported an important role of calcium-dependent proteolysis both in animals [39] and humans [40]. In addition, Hasselgren et al. Table 6 Effects of sActRIIB treatment on tibialis muscle mRNA content of the different proteolytic systems and myostatin in mice bearing the Lewis lung carcinoma Proteolytic system Ubiquitin-dependent Ubiquitin C8 proteasome subunit MuRF-1 Atrogin-1 E2 Calcium-dependent m-Calpain Lysosomal Cathepsin B Myostatin C 100±5 100±5 100±24 100±24 100±5 100±7 100±6 100±5 T 159±9 159±8 207±15 266±21 115±5 146±6 126±7 159±9 T+A 152±9 144±8 206±49 250±36 108±6 107±5 90±8 152±9 ANOVA A 0.0001 0.0000 0.0116 0.0001 ns 0.0001 0.0430 0.0158 B ns ns ns ns ns 0.0004 0.0040 ns Results are mean±S.E.M. for four to eight animals. The results are expressed as a percentage of controls. Statistical significance of the results by two-way analysis of variance (ANOVA) C control animals, T tumor-bearing mice, T+A tumor-bearing mice treated with sActRIIB, ns nonsignificant differences, A (tumor effect), B (treatment effect)