Emphysema induces human skeletal muscle loss , reductions in locomotory skeletal muscle contractile function , lipid peroxidation, and alterations in the glutathione redox system in skeletal muscles of hamsters ; it also decreases skeletal muscle oxidative enzyme capacity in hamsters . In patients with COPD, there is a relationship between FEV1 (the most acceptable parameter on establishment of severity in humans) and the functional status. Accordingly, a relationship between peripheral muscle strength and the severity of airflow obstruction, if present, would support the idea that chronic inactivity and muscle deconditioning are important in explaining muscle weakness in patients with COPD .
Our results showed significantly impaired crossed alveolar intercepts in E20 animals, which was worse in the E40 group. The comparison between E20 and E40 was also significantly different, showing injury enhancement on E40. The damaged lungs are shown in tissue micrographs in Figure 1. The total body weight and gastrocnemius muscle weight were decreased in the E20 group and were statistically significant in the E40 as compared to the CS group. The cachexia index was high in E20 animals, but higher in the E40 group. A positive correlation between emphysema severity and both body and muscle weight indicated a weak relationship between lung damage and cachexia. Bernard et al.  suggested that, in patients with stable COPD, the muscle contractile apparatus is preserved due to the fact that loss of muscle mass is proportional to the reduction in strength. The preferential loss in lower limb muscle mass and strength in patients, and their relationship with FEV1 percentage of predicted also suggest that muscle deconditioning and the related disuse atrophy are important factors in explaining peripheral skeletal muscle dysfunction in COPD. Skeletal muscle loss and its relationship with severity are poorly discussed on literature. To our knowledge, these factors were not related with oxidative alterations and their consequences.
Mechanisms underlying muscle wasting observed in several diseases remain largely unknown. Since ROS are demonstrated to be involved on cellular adaptation, recent studies in our laboratory  and others [2, 15] have demonstrated the involvement of oxidative stress in skeletal muscle loss. In the present study, concomitant muscle wasting and lipid peroxidation indicated that lipid peroxidation might be an important factor in the mechanism of muscle protein hyper-catabolism. Tert-butyl hydroperoxide-initiated CL was originally used to analyze the integrity of non-enzymatic antioxidant defenses and the levels of lipid hydroperoxides in muscle homogenates of animals inoculated with tumor cells. Previous studies using this assay indicated that the increase in CL is closely related to the oxidative stress previously suffered by the tissue. Oxidative stress induces the consumption of antioxidants and augments the formation of lipid hydroperoxides, which results in increased photon emission [26, 31]. TRAP on E20 result is in accordance with CL. Increased values of CL area and diminished values of TRAP represent the classic concept of oxidative stress . While CL curves enhance on E20 the total antioxidant capacity diminishes. On the contrary, in E40, CL curves and total antioxidant capacity becomes higher. In respect with this data, Palace et al.  demonstrated that the supply of vitamin A to the myocardium by storage organs during increased oxidative stress subsequent to myocardial infarction was showed in hemodynamically assessed rats using compartment analysis of a radio-labeled vitamin A. In our study, it seems that the same phenomenon happens, with a transport of antioxidants stimulated by free radicals damages, which can not handle the deleterious action of radicals at all.
In addition, we observed a strong association between chymotrypsin-like activity and lipid peroxidation markers (CL, TBARS and carbonylated proteins) in this emphysema model. It appears that ROS contribute to skeletal muscle dysfunction in a several ways. For example, Brotto and Nosek  demonstrated a blunted Ca2+ release from the sarcoplasmic reticulum, and Andrade et al.  demonstrated reduced Ca2+ sensitivity in skeletal muscles exposed to H2O2. ROS have been implicated in enzymatic dysfunction within the glycolytic pathway, the citric acid cycle, and the electron transport system, suggesting that elevated ROS may impair cellular energetics within skeletal muscles . In addition, Mattson et al.  demonstrated increased lipid peroxidation (evaluated by MDA levels) in gastrocnemius muscles of hamsters with single-dose elastase-induced emphysema; these findings are in agreement with our results. Moreover, we further confirmed the association between oxidative stress and loss of muscle mass and chymotrypsin-like proteolytic activity by using a sensitive CL method [26, 31, 39], which estimates the chain reaction of lipid peroxidation earlier, i.e., it measures both membrane lipid hydroperoxide formation and antioxidant depletion [40, 41]. Our results showed a progressive increase in TBARS and CL, which were strongly correlated with chymotrypsin-like proteolytic activity and increased tissue damage. In addition, progressive protein carbonylation was observed, which was well correlated with chymotrypsin-like proteolytic activity and lung tissue damage. Although Mean, SE and N from each group were combined and used in order to establish the correlations between the variables, there is no difference when all data are put together and the comparison is made point-by-point (with each single animal data put in the analysis as a point), since the calculation of correlation takes in account the mentioned values even when row data is used on the calculus.
A previous study demonstrated that treatment of C2C12 myotube cells with FeSO4/H2O2 caused a significant rise in MDA levels, with a concomitant increase in the catabolism of myofibrillary proteins and expression of the major components of the ubiquitin-proteasome pathway . The authors suggested that mild oxidative stress increases protein degradation in skeletal muscles by causing upregulation of the ubiquitin-proteasome proteolytic pathway in this in vitro model. In line with these findings, in the present study, chymotrypsin-like proteolytic activity was well correlated with CL, TBARS, and carbonyl proteins, and a reduction of body and muscle weight and an increase in emphysema severity were observed. It is worth considering that increased MDA levels are associated with increased proteolysis, which is related to carbonyl proteins levels. It is likely that when MDA or low-molecular-weight adducts is present, the level of oxidized proteins increases, and proteolytic activity could be accelerated, leading to muscle atrophy. Some authors have demonstrated that mild oxidative stress induces protein oxidation, with increased intracellular proteolysis [20, 43, 44]. Additionally, it has been postulated that mammalian cells are able to selectively remove moderately aldehyde-modified proteins from their intracellular protein pools and that the proteasomal system is responsible for this activity . The 3 major proteolytic activities (chymotrypsin-like, trypsin-like, and post-glutamyl peptide hydrolytic or caspase-like activity), occurring within the 20S core of the 26S proteasome complex are responsible for most of the protein degradation, which includes degradation of damaged cellular proteins.
The bulk of oxidized proteins can be degraded by the proteasomal system [42, 45, 46], particularly those modified by aldehydes and peroxides [20, 43]. Of note, the assay employed in our study can be used to measure proteolysis related to 20S proteasome, and not only the proteasome connected to ubiquitin-marked proteins. The 20S proteasome is also important, as it can degrade oxidized proteins without ubiquitination . Only Debigaré et al.  demonstrated that ubiquitination and proteolysis occur in the limb and respiratory muscles of patients with COPD, although no links were established between these processes and the oxidative status.
For the first time, the present study demonstrated that emphysema promotes body weight and skeletal muscle loss in a severity-dependent manner and is related to oxidative stress and chymotrypsin-like proteolytic activity. Additionally, oxidative stress variables and muscle chymotrypsin-like proteolytic activity were well correlated. Thus, it is reasonable to assume that muscle atrophy observed in this model of emphysema is mediated by increased muscle proteolytic activity, with possible involvement of oxidative stress.