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Published ahead of print on June 22, 2006, doi:10.1165/rcmb.2006-0103OC
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American Journal of Respiratory Cell and Molecular Biology. Vol. 35, pp. 689-696, 2006
© 2006 American Thoracic Society
DOI: 10.1165/rcmb.2006-0103OC

Muscle Wasting and Impaired Muscle Regeneration in a Murine Model of Chronic Pulmonary Inflammation

Ramon C. J. Langen, Annemie M. W. J. Schols, Marco C. J. M. Kelders, Jos L. J. van der Velden, Emiel F. M. Wouters and Yvonne M. W. Janssen-Heininger

Department of Respiratory Medicine, Maastricht University, Maastricht, The Netherlands; and Department of Pathology, University of Vermont, Burlington, Vermont

Correspondence and requests for reprints should be addressed to Ramon Langen, Maastricht University, Department of Respiratory Medicine, P.O. Box 5800, 6202 AZ Maastricht, The Netherlands. E-mail: r.langen{at}pul.unimaas.nl


   Abstract
Top
 Abstract
CLINICAL RELEVANCE
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 References
 
Muscle wasting and increased circulating levels of inflammatory cytokines, including TNF-{alpha}, are common features of chronic obstructive pulmonary disease. To investigate whether inflammation of the lung is responsible for systemic inflammation and muscle wasting, we adopted a mouse model of pulmonary inflammation resulting from directed overexpression of a TNF-{alpha} transgene controlled by the surfactant protein C (SP-C) promoter. Compared with wild-type mice, SP-C/TNF-{alpha} mice exhibited increased levels of TNF-{alpha} in the circulation and increased endogenous TNF-{alpha} expression in skeletal muscle, potentially reflecting an amplificatory response to circulating TNF-{alpha}. Decreased muscle and body weights observed in SP-C/TNF-{alpha} mice were indicative of muscle wasting. Further evaluation of the SP-C/TNF-{alpha} mouse musculature revealed a decreased muscle regenerative capacity, shown by attenuated myoblast proliferation and differentiation in response to reloading of disuse-atrophied muscle, which may contribute to skeletal muscle wasting. Importantly, incubation of cultured myoblasts with TNF-{alpha} also resulted in elevated TNF-{alpha} mRNA levels and inhibition of myoblast differentiation. Collectively, our results demonstrate that chronic pulmonary inflammation results in muscle wasting and impaired muscle regeneration in SP-C/TNF-{alpha} mice, possibly as a consequence of an amplificatory TNF-{alpha} expression circuit extending from the lung to skeletal muscle.

Key Words: muscle regeneration • pulmonary inflammation • skeletal muscle atrophy • TNF-{alpha} •



   CLINICAL RELEVANCE
Top
 Abstract
 CLINICAL RELEVANCE
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 References
 
This work adds to the understanding of how pulmonary inflammation results in systemic pathology and can direct further research aimed at prevention of the disabling consequences of skeletal muscle atrophy in disease conditions such as COPD.

 
Chronic obstructive pulmonary disease (COPD) is a slowly progressive condition characterized by irreversible airflow obstruction associated with an abnormal inflammatory response of the lung (1). Loss of body and muscle weight in COPD (pulmonary cachexia) significantly contributes to skeletal muscle weakness, impaired exercise capacity, decreased health status, and increased mortality independently of primary organ failure (2, 3). Besides inflammation of the diseased lung (4), pulmonary cachexia is accompanied by increased levels of circulating inflammatory cytokines (5–7), suggesting that systemic inflammation could trigger or contribute to muscle atrophy. In fact, studies in experimental models revealed that administration of the inflammatory cytokine TNF-{alpha} is sufficient to induce muscle atrophy, demonstrating a causal role for this cytokine in inflammation-associated muscle wasting (8, 9). However, whether inflammation-associated muscle atrophy also involves inflammatory signaling and gene expression in skeletal muscle is still unclear. Recently, muscle-specific activation of NF-{kappa}B, a transcription factor required to activate an array of TNF-{alpha}–dependent responses, was shown to be sufficient to induce muscle atrophy in mice, supporting the notion that inflammatory signaling in skeletal muscle results in atrophy (10). The loss of muscle mass in these mice was the result of a net increase in protein degradation, although in models of systemic inflammation, muscle atrophy has also been associated with decreased muscle protein synthesis (11). In addition to an inequity in muscle protein synthesis and degradation, an impaired ability to regenerate skeletal muscle may contribute to muscle atrophy. Muscle regeneration occurs constantly during normal muscle use (12), but is increased in response to muscle damage, increased muscle load, or resumed muscle use after inactivity (13). The recruitment of a reservoir of muscle precursor cells named satellite cells is essential for this process. Upon activation, satellite cells are stimulated to proliferate (and are called myoblasts at this stage), followed by exit from the cell cycle to engage in the myogenic differentiation program (13). Impaired satellite cell function as a result of interference with their ability to proliferate, differentiate, or fuse could ultimately result in loss of skeletal muscle tissue. We and others have demonstrated previously that myoblast differentiation and fusion are inhibited in response to inflammatory stimuli, including TNF-{alpha} (14–16), but the relevance of these findings for inflammation-associated muscle atrophy and impaired muscle plasticity remained to be addressed.

In this study we investigated whether chronic pulmonary inflammation results in muscle atrophy and affects muscle regenerative capacity, and whether this is associated with increased circulating and muscle cytokine levels. Mice expressing a TNF-{alpha} transgene under the control of the surfactant protein-C (SP-C) promoter (SP-C/TNF-{alpha} mice) have marked chronic pulmonary inflammation (17, 18). We specifically evaluated whether the presence of inflammatory mediators extended beyond the pulmonary compartment to the circulation and skeletal muscle tissue, and determined whether SP-C/TNF-{alpha} mice displayed evidence of muscle atrophy and impaired muscle regeneration.


   MATERIALS AND METHODS
Top
 Abstract
 CLINICAL RELEVANCE
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 References
 
Animals
All protocols and procedures involving mice were approved by the University of Vermont Institutional Animal Care and Use Committee. SP-C/TNF-{alpha} transgenic mice (Tg-10 line), generated by Dr. Y. Miyazaki (17), were kindly provided by Dr. R. Mason (Department of Medicine, National Jewish Medical and Research Center, Denver, CO) and were crossed with C57BL/6 (Jackson Laboratories, Bar Harbor, ME). Offspring were genotyped via PCR analysis as previously described (17), with a slight adaptation in that a second primer pair was added to amplify a 183-bp fragment of the myosin heavy chain perinatal (MyHC-p) gene as an internal control of the PCR reaction to exclude false negative results (forward 5'-GAGGAGCGGGCTGACATC-3' and reverse 5'-ACCCAGAGAGGCAAGTGACC-3'). Animals were housed in filter-top cages in a temperature-controlled room on a 12:12 h light-dark cycle with food pellets and water provided ad libitum. For experiments, 18-wk-old male transgenic mice were compared with age-matched transgene-negative littermates.

To study potential differences in skeletal muscle plasticity between wild-type (WT) and SP-C/TNF-{alpha} mice, the animals were subjected to hind limb unloading (HU) as described previously (16). This model causes atrophy of the postural muscles, and subsequent reloading (RL) of the hind limbs evokes muscle regeneration, resulting in the restoration of muscle mass (19). Transgene-positive (Tg+) and WT mice were randomly assigned to one of the following groups: baseline (n = 12 of each genotype); HU for 14 d (n = 8); HU followed by RL for 3, 5, 7 (n = 4), or 14 d (n = 8). Following the experimental procedures, mice were killed by halothane overdose, body weight and length were determined, and blood was collected from the abdominal aorta. Soleus, gastrocnemius, and plantaris muscles were collected using standardized dissection methods, weighed, and frozen in liquid nitrogen. Tibia length was also measured to control for potential developmental or post-natal effects of the transgene on growth. In a subset of experiments, animals received intramuscular injections in the gastrocnemius/plantaris/soleus area of PBS or TNF-{alpha} (40 µg/kg, dissolved in PBS with a final volume of 50 µl administered using a 30-gauge needle) every 24 h for 5 d. The dose of TNF-{alpha} used here is in the concentration range known to affect skeletal muscle (16, 20). Muscles were excised 24 h after the last injection.

Brochoalveolar Lavage
After halothane overdose the chest cavity was opened and the heart, lungs, and trachea were dissected as a whole. This procedure also allowed confirmation of the genotype of each animal, as the lungs of transgenic animals have a distinct orange color (17, 18). Next, a tracheal tube was inserted, fixed, and the lungs were lavaged with 1 ml saline. The recovered bronchoalveolar lavage (BAL) fluid was centrifuged (5 min, 1,000 x g at 4°C), and the supernatant was stored at –80°C.

Enzyme-Linked Immunosorbent Assay
The concentrations of TNF-{alpha}, sTNF-RI, and sTNF-RII were determined in BAL fluid and serum by enzyme-linked immunosorbent assay (ELISA). TNF-{alpha} was detected and quantified using the DuoSet ELISA kit for mouse TNF-{alpha} (R&D Systems, Minneapolis, MN) according to manufacturer's instructions. sTNF-R concentrations were assessed as described previously (21) using reagents kindly provided by Dr. Buurman (Dept. of Surgery, Maastricht University, Maastricht, The Netherlands).

Cell Culture
The murine skeletal muscle cell line C2C12 (#CRL1772; ATCC, Manassas, VA) was cultured in growth media (GM), composed of low-glucose Dulbecco's Modified Eagle's Medium (DMEM) containing antibiotics (50 U/ml penicillin and 50 µg/ml streptomycin) and 9% (vol/vol) FBS (all from Life Technologies, Rockville, MD), or differentiation media (DM), which contained DMEM with 1% heat-inactivated FBS and antibiotics (DM). Cells were grown on Matrigel (Becton Dickinson Labware, Bedford, MA) coated (1:50 in DMEM) dishes as described previously (22). Cells were plated at 104/cm2 and cultured in GM for 24 h, before induction of differentiation. When applicable, 10 ng/ml murine TNF-{alpha} (Calbiochem, La Jolla, CA) was added once to the culture dishes directly after induction of differentiation, or to fully (5 d) differentiated myotubes.

RNA Isolation and Assessment of mRNA Abundance
At the indicated times after TNF-{alpha} treatment, cells were washed twice with PBS and lysed using 1 ml solution-D (6.4 M guanidine thiocyanate, 40 mM Na-citrate, 0.8% sarcosyl, 100 mM beta-mercaptoethanol) per 60-mm dish. Alternatively, for muscle RNA isolation, tissue was homogenized (Polytron; Kinematica, Lucerne, Switzerland) in 0.75 ml solution-D. RNA was extracted by adding 0.1 vol 2 M sodium acetate pH 5.0, 1 vol of water saturated phenol, and 0.2 vol of chloroform:isoamyl alcohol (50:1). After 15 min incubation and centrifugation, RNA was precipitated with an equal volume of isopropanol. The pellet was resuspended in 0.5 ml solution D and precipitated again with isopropanol. After a wash step in 70% ethanol, the RNA was dissolved in nuclease-free water and stored at –80°C.

RNase Protection Assay (RPA) was performed to determine MyoD, myogenin, Troponin-I fast, muscle creatine kinase (MCK), myoglobin, or MyHC-p mRNA levels as previously described (16), using templates developed in our laboratory. Briefly, a radio-labeled multiprobe panel was prepared by transcription reaction using the MaxiScript Kit (Ambion, Houston, TX) followed by hybridization with 10 or 2 µg RNA (8 x 104 CPM per probe) per sample at 42°C for 18 h. The protection reaction was performed by RNase digestion (1:100 diluted RNase A/RNase T1 cocktail) using the RPA III kit (Ambion), and protected fragments were resolved on a 5% denaturing polyacrylamide gel. Gels were dried and exposed to film (Biomax MR-1; Kodak, Rochester, NY), or an imaging screen for quantification in a Personal Molecular Imager FX (Bio-Rad, Hercules, CA), and band intensity was analyzed using Quantity One software (Bio-Rad).

Semiquantitative RT-PCR was used to assess TNF{alpha} mRNA levels. Total RNA was isolated as indicated above and 1 µg was reverse transcribed to cDNA using Superscript II RT and oligo dT primers (Invitrogen, Carlsbad, CA) according to manufacturer's instructions. PCR reactions were performed using Platinum Supermix (Invitrogen). Total TNF-{alpha} (transgenic and endogenous) was amplified (38 cycli) using the following primers: forward 5'–ATGCACCACCATCAAGGACT-3', reverse 5'–GCACCTCAGGGAAGAGTCTG-3', yielding a 173-bp product. Selective detection of endogenous TNF-{alpha} mRNA was accomplished using a primer pair with a forward primer designed against the 5'UTR of TNF-{alpha} (absent in the transgene); forward 5'–GAGGACAG CAAGGGACTAGC-3', reverse 5'-GAGTGCCTCTTCTGCCAGTT-3', yielding a 200-bp product. TNF-{alpha} mRNA abundance was normalized to beta-actin within each individual reaction using QuantumRNA technology (Ambion) according to manufacturer's instructions.

Statistical Analysis
Raw data were entered into SPSS (version 11.0; Chicago, IL) for statistical analysis. Values as expressed in the graphs were subjected to one-way ANOVA, and the various treatment groups were compared post hoc with an LSD test (P < 0.05).


   RESULTS
Top
 Abstract
 CLINICAL RELEVANCE
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 References
 
Increased TNF-{alpha} and sTNF-RII in Lungs and Circulation of SP-C/TNF-{alpha} Mice
SP-C/TNF-{alpha} mice exhibit chronic pulmonary inflammation resulting from local expression of a TNF-{alpha} transgene (17, 18). Assessment of TNF-{alpha} in the BAL fluid by ELISA confirmed the continuous production of TNF-{alpha} in the lungs of Tg+ mice (Figure 1A). The TNF-{alpha} concentration was increased 6-fold in the BAL fluid of Tg+ animals compared with WT littermates, whereas the sTNF-RI concentration in the BAL fluid did not differ between these groups (Tg+: 29.1 ± 3.5 versus WT: 29.4 ± 3.1 pg/ml, respectively). However, sTNF-RII levels were raised ~ 2-fold in the BAL fluid of Tg+ mice compared with WT littermates (Figure 1A).


Figure 1
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  		Figure 1. Increased TNF{alpha} and sTNF-RII in lungs and circulation of SP-C/TNF-{alpha} mice. TNF-{alpha} and TNF-RII protein levels were assessed by ELISA in (A) BAL fluid or (B) serum of WT or SP-C/TNF-{alpha} (Tg+) mice (n = 12/group; *P < 0.001, #P < 0.01, {dagger}P < 0.05 compared with control as determined by one-way ANOVA).

 
TNF-{alpha} and its soluble receptors were also assessed in serum. In line with observations in the BAL fluid, sTNF-RI concentration was not different in serum of Tg+ and WT mice (1.10 ± 0.18 versus 1.09 ± 0.11 ng/ml, respectively), while sTNF-RII levels were increased ~ 50% in the serum of Tg+ mice (Figure 1B). Importantly, no circulating TNF-{alpha} could be detected in any of the WT animals, whereas TNF-{alpha} was clearly detectable in the serum of most Tg+ animals (Figure 1B).

Increased TNF-{alpha} Expression in Skeletal Muscle of SP-C/TNF-{alpha} Mice
To assess whether the elevated circulating levels of TNF-{alpha} also affected skeletal muscle, endogenous TNF-{alpha} mRNA abundance was determined in muscle tissue by RT-PCR. In gastrocnemius (Figure 2A), plantaris, and soleus muscle (not shown) of Tg+ animals, endogenous TNF-{alpha} mRNA levels were increased 5- to 10-fold compared with WT mice.


Figure 2
Figure 2
Figure 2
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  		Figure 2. Increased TNF-{alpha} expression in skeletal muscle of SP-C/TNF-{alpha} mice. RNA extracted from (A) gastrocnemius muscle of WT or SP-C/TNF-{alpha} (Tg+) mice, (B) plantaris muscle of WT animals after intramuscular (IM) PBS or TNF-{alpha} injections, or (C) 5-d-old myotubes cultured in presence or absence of TNF-{alpha} (10 ng/ml), was subjected to RT-PCR analysis to determine endogenous TNF-{alpha} mRNA abundance. TNF-{alpha} mRNA signal intensity was normalized to beta-actin, and expressed as fold increase over control. *P < 0.001, {dagger}P < 0.05 compared with control as determined by one-way ANOVA.

 
To verify whether TNF-{alpha} is capable of inducing its own expression in skeletal muscle, mice were subjected to intramuscular (IM) injections with TNF-{alpha}. Compared with PBS, IM injections with TNF-{alpha} resulted in a 3-fold increase in TNF-{alpha} mRNA abundance (Figure 2B). Similarly, TNF-{alpha} administered to cultured C2C12 skeletal myotubes caused a rapid increase in TNF-{alpha} mRNA abundance, with a 3-fold increase still apparent after 24 h (Figure 2C). These data demonstrate that TNF-{alpha} is indeed capable of inducing its own expression in skeletal muscle.

Skeletal Muscle Atrophy in SP-C/TNF-{alpha} Mice
In vitro studies have demonstrated that proinflammatory signaling pathways activated by TNF-{alpha} can cause atrophy of myotubes (20, 23), or inhibit muscle formation (15, 24). Because the increased TNF-{alpha} mRNA expression observed in skeletal muscle of SP-C/TNF-{alpha} mice suggested that skeletal muscle of Tg+ animals responded to the elevated levels of circulating TNF-{alpha}, we investigated whether this was associated with a change in muscle weight. Evaluation of soleus, gastrocnemius, and plantaris muscles revealed a 9–14% reduction in the weight of these skeletal muscles of Tg+ compared with WT mice. Body weights of Tg+ animals were also reduced, whereas body and tibia length were not different between Tg+ and WT mice (Table 1).


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  		TABLE 1. BODY AND MUSCLE WEIGHT OF WT AND SP-C/TNF-{alpha} MICE

 
We next determined whether mRNA abundance of a number of muscle-specific genes encoding regulatory factors, structural proteins, or enzymes was affected using RNase Protection Assay (RPA) (Figure 3). No differences between the mRNA levels of MyoD, myogenin, Troponin-I fast, MCK, or myoglobin in skeletal muscle of WT and Tg+ mice could be detected (Table 2), suggesting that the decreases in muscle weight are not linked to changes in mRNA expression of these muscle proteins.


Figure 3
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  		Figure 3. Example of the muscle-specific mRNA analysis using RNase Protection Assay. RNA was extracted from gastrocnemius muscle and subjected to one of two multiprobe panels for RPA analysis to determine mRNA abundance of MyoD, myogenin, His 3.2, His 3.3, and GAPDH (lane 1) or troponin I-fast, MCK, myoglobin, and GAPDH (lane 2).

 

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  		TABLE 2. MUSCLE-SPECIFIC mRNA EXPRESSION IN WT AND SP-C/TNF-{alpha} MICE

 
Altered Skeletal Muscle Plasticity in SP-C/TNF-{alpha} Mice
Muscle plasticity refers to the ability of skeletal muscle to change composition or size according to functional demand. To test whether muscle atrophy observed in Tg+ mice was associated with impaired muscle plasticity, animals were subjected to a hind limb unloading (HU)/reloading (RL) protocol. Two weeks of unloading caused an ~ 40% and ~ 20% reduction in the weight of the soleus and gastrocnemius muscle, respectively, in WT animals (Figure 4A). Although initial muscle weight was decreased in Tg+ animals, unloading caused a relative smaller reduction (34% in soleus and 11% in gastrocnemius weight, respectively) in Tg+ animals when compared with WT mice (Figure 4A). In fact, no difference in absolute soleus or gastrocnemius weight between WT and Tg+ animals was detected after HU; even after extending the unloading period to 28 d, muscle weights were still equal (data not shown).


Figure 4
Figure 4
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  		Figure 4. Altered skeletal muscle plasticity and impaired muscle regeneration in SP-C/TNF-{alpha} mice. Soleus or gastrocnemius muscle weights were determined in WT or SP-C/TNF-{alpha} mice at baseline or after 14 d of hind-limb unloading (HU-14). (A) The cumulative weight (paired weight) of the left and right muscle is shown (n = 8/group, *P < 0.001 between baseline and HU-14 or between WT and Tg+ as determined by one-way ANOVA). (B) Alternatively, soleus or gastrocnemius muscle weights were determined in WT or SP-C/TNF-{alpha} mice after the indicated time of RL after HU, and expressed as a % of mean HU-weight to evaluate RL-induced weight gain (n = 4/group, {dagger}P < 0.05 between WT and Tg+ for the indicated time point as determined by one-way ANOVA).

 
Impaired Muscle Regeneration in SP-C/TNF-{alpha} Mice
Muscle plasticity also encompasses increases (growth) or restoration (regeneration) of muscle size, and muscle regeneration requires myoblast proliferation and differentiation (25). To evaluate whether muscle regeneration was affected, soleus and gastrocnemius muscle weights were determined in groups of mice that were subjected to 14 d of unloading atrophy followed by 3, 5, 7, or 14 d of hind limb reloading. Compared with the weight directly after HU, which was equal in WT and SP-C/TNF-{alpha} mice, soleus and gastrocnemius muscle weight gain during reloading was greater in WT compared with Tg+ animals (Figure 4B).

To assess myoblast proliferation during reloading, Histone 3.2 (H3.2) mRNA abundance was measured, as earlier reports have demonstrated that expression of H3.2 is restricted to dividing myoblasts (16, 26). While H3.2 mRNA levels were not different between WT and Tg+ skeletal muscle under baseline conditions (not shown) and did not change in response to HU, reloading induced a robust increase in H3.2 expression (Figure 5A). Interestingly, the proliferative response was stronger in WT muscle than in Tg+ muscle, and this difference was most apparent after 3 d of reloading, as H3.2 abundance in WT muscle was increased 6-fold over control value versus a 4-fold increase in Tg+ muscle.


Figure 5
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  		Figure 5. Impaired proliferative response and myogenic differentiation in regenerating muscle of SP-C/TNF-{alpha} mice. Soleus muscle was dissected at the indicated time (RL-3: 3 d of reloading, etc.), and RNA was extracted and subjected to RPA analysis to determine (A) mRNA abundance of the proliferation marker H3.2 or (B) the muscle differentiation marker MyHC-p. mRNA signal intensity was normalized to GAPDH, and expressed as a % of baseline levels (n = 4/group, *P < 0.001, #P < 0.01, {dagger}P < 0.05 between WT and Tg+, as determined by one-way ANOVA).

 
To evaluate myoblast differentiation during reloading, mRNA encoding perinatal myosin heavy chain (MyHC-p), a sensitive differentiation marker (27) was determined by RPA. MyHC-p mRNA abundance was not different between WT and Tg+ mice under baseline and HU conditions (data not shown). In soleus muscle of WT animals, MyHC-p mRNA was induced by reloading in a time-dependent fashion, with a maximal 4-fold induction after 5 d of reloading and levels returning toward baseline after 7 and 14 d (Figure 5B). In contrast, the reloading-induced increase in MyHC-p was not observed in the soleus muscles of Tg+ animals (Figure 5B). To investigate whether the absence of a robust increase in MyHC-p expression during RL in Tg+ animals may be attributed to TNF-{alpha} action, differentiating C2C12 myoblasts were incubated with this cytokine. The accumulation of MyHC-p mRNA transcripts observed during myoblast differentiation was strongly suppressed by TNF-{alpha} (Figure 6), in support of an inhibitory role of TNF-{alpha} during muscle regeneration.


Figure 6
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  		Figure 6. TNF-{alpha} inhibits MyHC-p expression in differentiating myoblasts. Myoblasts were induced to differentiate in the presence or absence of TNF-{alpha} (10 ng/ml) for, 24, 48, or 72 h (DM: differentiation medium). MyHC-p expression was evaluated by RPA analysis, and band intensity was normalized to GAPDH and expressed as fold increase over control (= GM; myoblasts cultured in growth medium). *P < 0.001 between sham and TNF-{alpha} as determined by one-way ANOVA.

 

   DISCUSSION
Top
 Abstract
 CLINICAL RELEVANCE
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
References
 
Besides primary organ failure and local inflammation, chronic wasting conditions such as COPD are associated with a systemic inflammatory response and skeletal muscle atrophy (5–7). In SP-C/TNF-{alpha} mice, which exhibit emphysematous changes (18, 28) and pulmonary inflammation (17, 18) analogous to COPD, we now demonstrate elevated levels of TNF-{alpha} and sTNF-RII in the circulation, and increased endogenous TNF-{alpha} mRNA expression in skeletal muscle, suggestive of a systemic inflammatory response. Importantly, SP-C/TNF-{alpha} mice exhibited decreased skeletal muscle and body weight, and impaired muscle regenerative capacity, which may contribute to skeletal muscle atrophy.

Pulmonary inflammation is a well-documented feature of COPD, exemplified by local increases in levels of TNF-{alpha} and its receptors (4). However, the inflammatory response often extends beyond the diseased lung, as circulating levels of a number of cytokines, chemokines, and their receptors are elevated in COPD (6, 7, 29). In SP-C/TNF-{alpha} mice, the directed expression of the TNF-{alpha} transgene results in pulmonary inflammation (17, 18). Importantly, the presence of inflammatory mediators was not restricted to the lungs of these animals, as increased levels of circulating TNF-{alpha} and sTNF-RII were detected. This may indicate spill-over of inflammatory mediators from the pulmonary compartment into the circulation, but since TNF-{alpha} and sTNF-RII levels did not correlate in BAL fluid and serum (data not shown), tissues other than the lung may have contributed to the elevated TNF-{alpha} and sTNF-RII levels in the serum of SP-C/TNF-{alpha} mice.

Skeletal muscle is capable of expressing an array of cytokines and chemokines (30), which have mostly been studied in the context of muscle injury and repair. Analysis of various hind limb muscles of SP-C/TNF-{alpha} mice revealed increased TNF-{alpha} mRNA levels compared with control animals. This increase did not concern transgenic TNF-{alpha} due to a leaky SP-C promoter, because our detection method for TNF-{alpha} mRNA was set up to discriminate between endogenous and transgenic TNF-{alpha} mRNA. Although in vitro studies have demonstrated that skeletal muscle is capable of TNF-{alpha} secretion (30), it remains unclear whether the increased muscle TNF-{alpha} expression levels contribute to the raised TNF-{alpha} concentration in the circulation of SP-C/TNF-{alpha} mice. In fact, it is also conceivable that TNF-{alpha} expression in muscle is the consequence of the elevated circulating TNF-{alpha} levels, reflecting a positive feedback mechanism by which TNF-{alpha} binding to its receptor on the surface of myofibers stimulates its own expression in skeletal muscle. This notion is further supported by our data showing increased TNF-{alpha} mRNA expression in either mouse skeletal muscle after intramuscular TNF-{alpha} injections, or in cultured myotubes after treatment with TNF-{alpha}. Still, a contribution of other circulating cytokines such as IL-1 and -6, which are also associated with muscle wasting to increased muscular TNF-{alpha} expression in skeletal muscle, cannot be ruled out.

SP-C/TNF-{alpha} mice demonstrated a reduction in body weight which may be a direct or indirect effect of the observed systemic inflammatory response. An anorexic effect of chronic inflammation cannot entirely be ruled out, as food intake was not determined in this study. However, decreased food intake affects adipose tissue rather than skeletal muscle mass (31), whereas our data clearly demonstrate a reduction in muscle mass. In addition, body and tibia length were not decreased in transgenic animals, ruling out growth retardation, either through decreased food intake or other mechanisms, as a cause of the decreased body and muscle weights of SP-C/TNF-{alpha} mice. The balance between muscle protein synthesis and breakdown is a major determinant of whether skeletal muscle grows, atrophies, or remains at constant weight (32). Evaluation of the mRNA abundance of a number of genes encoding muscle structural or regulatory proteins did not reveal any alterations in muscle gene expression in SP-C/TNF-{alpha} mice. This suggests that decreased muscle gene expression is not responsible for inflammation-induced muscle atrophy, although the potential contribution of changes in mRNA translation efficiency cannot be ruled out.

Muscle atrophy observed in SP-C/TNF-{alpha} mice may likely be the consequence of increased protein degradation resulting in a net loss of muscle protein. Increased muscle proteolysis has been reported in a number of noninflammatory models of muscle atrophy and involves degradation of muscle proteins through the ubiquitin 26S-proteasome pathway (UPP) (33–35). Recently, an important role for NF-{kappa}B was postulated in the regulation of protein degradation through the UPP during muscle atrophy, as inhibition of NF-{kappa}B activation preserved muscle tissue in various experimental models of muscle atrophy (10, 36). Conversely, constitutive activation of NF-{kappa}B in skeletal muscle was sufficient to induce profound muscle atrophy via increased UPP-dependent proteolysis (10). Although we did not assess NF-{kappa}B activity directly, the elevated expression levels of endogenous TNF-{alpha} in skeletal muscle of SP-C/TNF-{alpha} mice could be considered as indirect evidence of NF-{kappa}B activation, since transcriptional regulation of TNF-{alpha} expression requires NF-{kappa}B activity (37).

Muscle regeneration after muscle damage or during recovery from atrophy requires the activation of local muscle precursor cells named satellite cells (25). Upon activation, these cells proliferate to form a pool of myoblasts, which differentiate and fuse to myofibers, and these processes were found to be essential for efficient muscle regeneration (19). Analysis of H3.2 mRNA abundance revealed a decreased proliferative response in soleus muscle of SP-C/TNF-{alpha} compared with WT mice during reloading. Although the contribution of cell types other than skeletal muscle to the H3.2 signal cannot completely be ruled out, proliferation signals in regenerating muscle have clearly been associated with dividing myoblasts in other studies (38, 39), suggesting a potential defect in myoblast proliferation in SP-C/TNF-{alpha} mice.

MyHC-p is a sensitive differentiation marker and its expression is positively regulated by the muscle specific transcription factor MyoD (27). Previously, we and others have demonstrated that myogenic differentiation is inhibited by TNF-{alpha} in an NF-{kappa}B–dependent fashion (14, 15). In line with these findings, TNF-{alpha} inhibited the expression of MyHC-p in differentiating myoblasts. Using the same in vitro model, we previously showed that inhibition of muscle gene expression by TNF-{alpha} was associated with decreased MyoD protein stability and abundance, with minor effects of TNF-{alpha} on MyoD mRNA levels (16). Accordingly, the reduced MyHC-p transcript accumulation (Figure 5B) despite a similar temporal induction of MyoD mRNA levels (data not shown) in reloaded muscle of SP-C/TNF-{alpha} compared with WT animals, may have resulted from MyoD protein destabilization and subsequent impaired transcriptional regulation of MyHC-p by TNF-{alpha}.

As these data demonstrate that inhibition of myoblast differentiation by TNF-{alpha} is characterized by decreased MyHC-p accumulation, increased levels of TNF-{alpha} and/or muscular NF-{kappa}B activation may be responsible for impaired myoblast differentiation and muscle regeneration in SP-C/TNF-{alpha} mice. Although it is currently unclear whether myoblast proliferation and differentiation are required for muscle maintenance, this study demonstrates that impaired muscle regeneration may contribute to skeletal muscle loss in disease conditions that are accompanied by chronic inflammation.

In conclusion, our results demonstrate that chronic pulmonary inflammation results in muscle atrophy and impaired muscle regeneration in SP-C/TNF-{alpha} mice, possibly as a consequence of an amplificatory TNF-{alpha} expression circuit extending from the lung to skeletal muscle.


   Acknowledgments
 
The authors thank Dr. B. Hunter and Dr. S. Kandarian, Department of Health Sciences, Boston University (Boston, MA) for sharing their expertise regarding the hind limb suspension protocol, and Dr. M. Toth, Department of Medicine, University of Vermont (Burlington, VT) for sharing his expertise regarding muscle dissection techniques used in this study and helpful discussion of the results. Dr. W. Buurman, Department of Surgery, Maastricht University (Maastricht University, Netherlands) is thanked for providing the sTNF-R ELISA reagents. SP-C/TNF-{alpha} mice were kindly provided by Dr. R. Mason, Department of Medicine, National Jewish Medical and Research Center (Denver, CO).


   Footnotes
 
Originally Published in Press as DOI: 10.1165/rcmb.2006-0103OC on June 22, 2006

Conflict of Interest Statement: R.C.J.L. does not have a financial relationship with a commercial entity that has an interest in the subject of this manuscript. A.M.W.J.S. received lecture fees from Boehringer Ingelheim. She received research grants between 2001-2004 from GlaxoSmithKline (GSK) and Numico Research. M.C.J.M.K. does not have a financial relationship with a commercial entity that has an interest in the subject of this manuscript. J.L.J.v.d.V. does not have a financial relationship with a commercial entity that has an interest in the subject of this manuscript. E.F.M.W. is a member of the scientific advisory boards for GSK, Boehringer Ingelheim, AstraZeneca, and Numico and received lecture fees from GSK, AstraZeneca, and Boehringer Ingelheim. He received research grants between 2002 and 2004 from GSK, AstraZeneca, Boehringer Ingelheim, Centocor, and Numico. Y.M.W.J.-H. does not have a financial relationship with a commercial entity that has an interest in the subject of this manuscript.

Received in original form March 9, 2006

Accepted in final form June 2, 2006


   References
Top
 Abstract
 CLINICAL RELEVANCE
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 References
 

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