So as to avoid problems related to aging, the study used young adult male mice (age 2 months, body weight 21–23g) from the A/J strain, in whom emphysema was induced, along with control mice. We used a classic model of pulmonary emphysema through a single instillation (oropharyngeal aspiration) of elastase-high purity (EC134GI, Elastin Products Company), with a concentration of 0.15mg/100g of weight, or the equivalent of 20U of enzyme per 100g of weight. This is a methodology previously validated in other studies14,15 and in which the different stages of alveolar damage have been properly characterized. At the beginning of the study, the mice were randomly assigned to 2 groups: 1) mice with emphysema, slaughtered at 34 weeks into their disease (No.=6) and 2) control group, with only normal saline instilled into the oropharyngeal cavity and also slaughtered at 34 weeks following this single instillation (No.=7). Throughout the 34-week study period, the animals in both groups received daily nourishment and water ad libitum and remained under routine housing environment conditions. They also maintained a physical activity level typical for this type of animal throughout the course of the study.

This was a controlled study designed in accordance with our institution's current regulations on animal experimentation and with the Helsinki convention on the use and care of animals. The stipulations of current laws regulating the protection of the animals used for experimentation and other scientific purposes (Real Decreto [Royal Decree] 1201/2005 of 10 October) were also taken into account. All experiments were approved by the Experimentation Ethics Committee of the Centro para la Investigación Médica Aplicada (CIMA) [Centre for Applied Medical Research] in Pamplona (Navarra). It should be mentioned that, for reasons of an ethical nature, the mice used in this research had also been used in a previous study,15 the objective of which was to quantify the degree of emphysema using various methodologies.

Histological confirmation of the presence of emphysema in the lungs of the diseased mice, compared to the control animals, was quantitatively completed in a previous study.15 In the same study, lung distensibility was also evaluated in both groups of mice prior to slaughtering them, and the corresponding data were published.15

In our study, prior to their slaughter (week 34), computerized axial tomography (CAT scan) was also done on the 2 groups of mice for radiographic confirmation of the presence of emphysema in the diseased animals (Fig. 1A–B). All animals were weighed on day 0 and immediately prior to their slaughter (week 34). Given the animals’ age, they all had a tendency to gain weight over the course of the study. The percent gain in body weight for each animal, from day 0 (baseline weight) to week 34, was used as a study variable in all the mice.

Fig. 1.

(A) Computerized axial tomography of control mouse lung. (B) Computerized axial tomography of lung of mouse treated with elastase. (C) Haematoxylin and eosin stain of control mouse lung. (D) Haematoxylin and eosin stain of lung of mouse treated with elastase. The arrow indicates an area of lung parenchyma in which increased interalveolar space is appreciated.

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In the 2 groups of mice, the diaphragm, gastrocnemius muscle, and lungs were obtained. Prior to the slaughter, sodium pentobarbital 50mg/kg was administered intraperitoneally and, after confirming that the animal was completely anaesthetized and feeling no pain, these extractions were performed, beginning with the extremity muscle and ending with the diaphragm and lungs. Once the animal's muscles were extracted, it was immediately frozen with liquid nitrogen for subsequent preservation at −80°C. The extracted lungs were fixed in formalin at a constant pressure of 20cm H2O and then embedded in paraffin for making the appropriate histological sections and slices.15

The numerical variables (percent change in weight and optical densities for each marker) are expressed as mean and standard deviation for each group of mice. For each variable, the means for the 2 groups were compared using the t-test for independent samples. Statistical significance was set as a P value of <.05.

In our study, radiographic techniques (CAT scan) and haematoxylin and eosin staining (Fig. 1A–D, respectively) were used to qualitatively confirm the presence of emphysema in the mice treated with elastase, compared to the control animals. When the study period was over (week 34), total body weight gain for the emphysematous mice was significantly less than body weight gain for the control mice (Table 2). Likewise, the muscles examined in our study—diaphragm and gastrocnemius—also weighed significantly less in the emphysematous mice than in the control mice (Table 2).

No significant differences were observed in the diaphragm and gastrocnemius of the animals with emphysema, compared to the control animals, in terms of total levels of ubiquitinated protein, subunit C8 of the proteasome, ligase E2 atrogin-1, ubiquitin-conjugating enzyme E214k, or ligase E3 MuRF-1 (Table 3).

A significant reduction in diaphragm myosin levels (67%) was observed in the mice with emphysema, compared to the controls (Fig. 6), but not in the gastrocnemius (Table 3). Between the 2 groups of animals, no significant differences were shown in either respiratory or peripheral muscle levels of actin and creatine kinase protein (Table 3).

Fig. 6.

Myosin protein content in diaphragm muscle of mice with emphysema and control animals. OD, optical density; au: arbitrary units. Mean values and standard deviation. *P<.05.

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FoxO1 protein—a transcription factor involved in the IGF-1/AKT pathway and regulator of the expression of genes, such as atrogin-1 that are involved in protein degradation pathways and muscle atrophy18,19—was observed to be at increased levels in the diaphragm of emphysematous mice. In a previous study,20 transgenic mice characterized by overexpression of FoxO1 were bred; these, in comparison with control mice, lost weight and were noted to have reduced muscle mass in various muscles of the extremities.20 Diminished expression of genes related to structural proteins of type I fibre was also detected.20 From a histological standpoint, a reduction in the size of type I and II fibres was observed, as well as a decrease in the percentage of type I fibres and an increase in protein catabolism.20 It has been shown that there is increased expression of FoxO in the diaphragm of patients with COPD,12 as well as in the diaphragm of patients exposed to prolonged mechanical ventilation.13 However, peripheral muscle FoxO levels in the mice with emphysema were no different from those in the control animals. It could be concluded that, in experimental murine models and in patients with COPD, the FoxO transcription factor appears to play a major role in signalling increased protein degradation in skeletal muscle.

Four subfamilies of the MAPK pathway have been described: ERK1/2, JNK, ERK5, and p38. Among various cellular functions, this signalling pathway is involved in protein degradation.21 Compared to the control animals, ERK1/2 levels were significantly higher in the diaphragm of mice with emphysema, while JNK levels were higher in their gastrocnemius. In experimental models, both JNK and ERK1/2 have been involved in the signalling of cellular proteolytic systems.21 However, in recent research by our group (observations not published), the MAPK signalling pathway was shown to be unchanged in the quadriceps of patients with advanced COPD.

Increased levels of NF-kB subunit p50 protein were also observed in peripheral muscle, but this was not the case in the diaphragm. The NF-kB/IKKB pathway has been studied extensively; among the numerous other functions that have been described is its direct participation in processes and diseases involving muscle atrophy and a reduction in muscle mass.22 Thus, the loss of muscle mass seen in the gastrocnemius of the emphysematous mice could also be mediated by this signalling pathway. This data is consistent with other published data from a study in which increased signalling via the NF-kB pathway was demonstrated in the vastus lateralis of patients with severe COPD and loss of muscle mass.23 Another recent study24 has also shown increased NF-kB expression in the diaphragm of patients with severe COPD. In summary, this signalling pathway appears to play a key role in proteolytic processes in muscle in experimental models and in the respiratory and peripheral muscles of patients with advanced COPD.

In the diaphragm but not in the gastrocnemius of the mice with emphysema, in comparison with the control animals, levels of the muscle growth factor myogenin were reduced while levels of myostatin were increased. These results are clearly consistent with those obtained in a very recent study,24 which reported decreased myogenin levels and increased myostatin levels in the diaphragm of patients with severe and very severe COPD. Myostatin is a potent inhibitor of muscle mass growth, and it is thought that it may inhibit the proliferation and differentiation of muscle precursor cells that could, in turn, have the capacity to positively regulate protein degradation via the ubiquitin–proteasome system.24–26 On the other hand, myogenin is a growth factor that favours the proliferation and differentiation of muscle fibres. Recent data (observations not published) has revealed very similar findings in terms of myostatin and myogenin levels in the vastus lateralis muscle of patients with severe COPD and loss of muscle mass. In fact, it should be kept in mind that our study conforms to a model of less increase in the amount of body weight and muscle mass in the diseased mice rather than a model of weight loss. In this regard, increased levels of the muscle growth inhibitor myostatin in conjunction with reduced levels of the muscle growth potentiating factor myogenin appears to be consistent with our study's experimental model of less weight and muscle gain.

The significant reduction in the respiratory muscle—but not peripheral muscle—content of the contractile protein myosin in the mice with emphysema, compared to the controls, constitutes a major finding of our research. Moreover, in the mice with emphysema, compared to the controls, the percentage for no gain in diaphragm muscle mass was 34%, while for the gastrocnemius it was 21%. These differences between the 2 muscles in the same group of mice could also explain why reduced myogenin and myosin levels were seen only in the respiratory muscles in mice with emphysema, compared to the control animals, and were not seen in the peripheral muscle.

Regarding the lack of differences in content for other structural proteins, besides myosin, that were analysed in our study, the findings were similar to those reported in the group's previous studies of the diaphragm7 and vastus lateralis (observations not published) in patients with severe COPD. In fact, using electron microscopy, it has recently been confirmed that there is a myopathy in the respiratory and peripheral muscles of critically ill patients that affects only the thick filaments of the sarcomeric structure—that is, it affects the myosin filaments but not the actin filaments. This condition is commonly known as thick filament myopathy.27 Along the same line of reasoning, it is suggested that the reduced myosin content detected in the diaphragm of mice with emphysema in our model is analogous to the myopathy affecting only thick filaments of the sarcomere in critically ill patients. Even though, in our work, there was no access to the study of muscle structure due to the limitations explained below (see “Limitations of the Study”), it is conceivable that, if structural changes occurred in the diaphragm of the mice with emphysema, it is very likely that these changes would also affect the myosin filaments.

We must also comment that, in our study, no differences were seen between the 2 groups of mice in terms of the proteolytic pathways analysed (ubiquitinated proteins). The fact that differences were observed in the signalling pathways but not in the markers of proteolysis analysed in our study suggests that other mechanisms of protein degradation, such as calcium-activated proteases, lysosomes, and autophagy, could be involved in the muscles of the mice with emphysema. For reasons related to the size of the muscle specimens, these proteolytic systems were not analysed in our research; however, they could be the object of study in future research.

Our study also shows increased total levels of muscle protein oxidation (carbonylated proteins and MDA–protein adducts) in the diaphragm but not in the gastrocnemius of the diseased mice, compared to the controls. These results are consistent with those obtained in previous studies by our group5,7 that showed increased protein oxidation levels in the diaphragm of patients with COPD,5 these proteins being creatine kinase, carbonic anhydrase III, actin, and myosin.7 Given that carbonylated proteins and MDA–protein adducts are potent markers of oxidative stress, it could be concluded that the diaphragm of emphysematous mice in our study showed a clear increase in protein oxidation levels. That may be a response to the sustained and intense muscle contractions to which this muscle was subjected in the mice with emphysema throughout the entire course of the study. On the other hand, the relatively limited activity of the peripheral muscle, as opposed to the diaphragm—especially in the animals with emphysema—could explain why there were no changes in oxidative stress levels in the extremity muscle in these animals.

The content of the mitochondrial antioxidant Mn–SOD was reduced in the diaphragm of the diseased mice, leading to the conclusion that mitochondrial antioxidant systems were impaired in the respiratory muscle. That could contribute to increased oxidant production in the mitochondrial compartment because impaired antioxidant systems would not have sufficient capacity to neutralize the abundant production of oxygenated radicals in the diaphragm of the mice with emphysema, chronically subjected to elevated respiratory loads. Therefore, this would result in an equilibrium in favour of the oxidants. It is important to highlight that Mn–SOD and catalase levels were unchanged in the diaphragm of patients with severe COPD, compared to healthy controls.5 In the case of cytoplasmic antioxidant systems, such as CuZn–SOD and peroxiredoxin-2, increased protein levels of these were observed in the diaphragm of the diseased mice. This could be due to the attempt of cellular antioxidant systems to neutralize the excess oxidants but without fully achieving it, given that, in the end, protein oxidation levels were increased.

Then again, it is important to point out that the moderately reduced content of some cytosolic antioxidant systems in the gastrocnemius of the diseased animals ultimately had no effect on the redox equilibrium in the peripheral muscles. These findings are contrary to those reported for the quadriceps of patients with COPD, where an increased content of antioxidant enzymes Mn–SOD and CuZn–SOD has been shown.3,8 Differences between the murine model and patients with COPD in terms of cellular metabolism and muscle activity could explain the variations in the content of various antioxidant enzymes as well as in the redox equilibrium observed, in the end, in each muscle.

The following are the most notable limitations of our study: First, owing to the lack of histological preparations of the 2 muscles—because of the size of mouse muscle specimens—it was impossible to analyse potential structural changes, such as the fibrillar composition of each muscle, as well as their sizes. Second, it was also impossible to evaluate muscle function in the animals—again, for experiment logistics reasons related to the animals’ size. In our study, given the specimen size limitations, absolute priority was given, first of all, to an in-depth determination of the molecular mechanisms that may be involved in the loss of muscle mass in this experimental model of pulmonary emphysema. Once the principal molecular mechanisms have been identified and the existence of molecular changes has been confirmed in the muscles of mice with an experimental emphysema, it will be possible, in future studies, to earmark a large portion of the specimens for describing structural changes that may be present in the respiratory and peripheral muscles in mice with emphysema.