Prevention of oxidative stress via antioxidants attenuates diaphragm myofiber atrophy associated with mechanical ventilation (MV). However, the specific redox-sensitive mechanisms responsible for this remain unknown. We tested the hypothesis that regulation of skeletal muscle proteolytic activity is a critical site of redox action during MV. Sprague-Dawley rats were assigned to five experimental groups: 1) control, 2) 6 h of MV, 3) 6 h of MV with infusion of the antioxidant Trolox, 4) 18 h of MV, and 5) 18 h of MV with Trolox. Trolox did not attenuate MV-induced increases in diaphragmatic levels of ubiquitin-protein conjugation, polyubiquitin mRNA, and gene expression of proteasomal subunits (20S proteasome α-subunit 7, 14-kDa E2, and proteasome-activating complex PA28). However, Trolox reduced both chymotrypsin-like and peptidylglutamyl peptide hydrolyzing (PGPH)-like 20S proteasome activities in the diaphragm after 18 h of MV. In addition, Trolox rescued diaphragm myofilament protein concentration (μg/mg muscle) and the percentage of easily releasable myofilament protein independent of alterations in ribosomal capacity for protein synthesis. In summary, these data are consistent with the notion that the protective effect of antioxidants on the diaphragm during MV is due, at least in part, to decreasing myofilament protein substrate availability to the proteasome.
- protein synthesis
- redox balance
mechanical ventilation (MV) is used clinically to maintain adequate alveolar ventilation in patients incapable of doing so on their own (19). Unfortunately, MV results in the rapid onset of diaphragmatic oxidative stress along with myofiber atrophy (10, 33, 34, 45, 55). Compared with other forms of disuse muscle atrophy, a unique characteristic of MV-induced diaphragmatic atrophy is the rapidity of the atrophic signaling that occurs (5, 33, 34, 45, 51, 52, 55). The molecular signaling events responsible for the swift progression of diaphragm atrophy during MV are not well understood but appear to be linked, at least in part, to oxidative stress (5, 34, 43).
Increased proteolysis is a well-established hallmark of disuse-induced skeletal muscle atrophy (13, 22, 25) and involves the coordinated interaction of numerous proteolytic systems. The ATP-dependent ubiquitin-proteasome pathway (UPP) is the major proteolytic system involved in the degradation of myofibrillar protein during disuse and includes ubiquitin-protein conjugate formation and subsequent degradation of proteins by the 26S proteasome (14). The activity of proteasome-mediated proteolysis in atrophying skeletal muscle is believed to be primarily transcriptionally regulated (8) and is influenced by levels of substrate (23), proteasome subunit protein (20), and proteasome regulatory complexes (i.e., PA700 and PA28; Ref. 20). In general, oxidative stress is known to stimulate activation of the ubiquitin-proteasome system in skeletal muscle (17, 18, 30). MV-induced oxidative stress activates the UPP in the diaphragm (8), and antioxidant administration during MV significantly retards protein breakdown and chymotrypsin-like activity of the 20S proteasome (5). These facts suggest that regulation of proteasome function may be a critical mechanism linking oxidative stress to MV-induced diaphragmatic atrophy. Nonetheless, it is unknown whether the protective effects of antioxidant administration on diaphragmatic atrophy and contractile function during MV are directly linked to the regulation of specific components governing proteasome activity.
To address these gaps in our knowledge, we hypothesized that the regulation of skeletal muscle proteolytic activity is a critical site of redox action altering protein balance during MV disuse. To test this postulate, we prevented MV-induced diaphragmatic oxidative stress via infusion of the antioxidant Trolox and examined key regulatory elements of proteasomal activity. Our results indicate that chymotrypsin-like and peptidylglutamyl peptide hydrolyzing (PGPH)-like 20S proteasome activities are attenuated by antioxidant administration independent of alterations in proteasome subunit gene expression or protein abundance. Because intact, sarcomeric myofilament protein cannot be recognized or degraded by the ubiquitin-proteasome system (43), we then determined whether antioxidant-induced alterations in proteasome activity are regulated, at least in part, by redox-mediated release of contractile protein substrates from the sarcomere. Our findings reveal that MV induces an increase in released myofilament protein within the diaphragm that is attenuated by antioxidant administration. Finally, depressed protein synthesis is a well-established hallmark of disuse-induced skeletal muscle atrophy (13, 22, 25) and occurs rapidly during mechanical ventilation (7, 50–52). In addition, recent research suggests that antioxidant administration increases the rate of protein synthesis in untreated rats (46). Therefore, we also determined whether antioxidant administration protected the diaphragm against MV-induced reductions in protein synthetic capacity. Our results demonstrate a singular effect of antioxidant administration on the activation of the translational initiation factor p70s6 kinase in the diaphragm and further support the concept that myofilament substrate release is a major source of antioxidant protection during MV.
To test the hypothesis that the regulation of skeletal muscle proteolytic activity is a critical site of redox action altering protein balance during MV disuse, adult (6 mo) female Sprague-Dawley rats were randomly assigned to 1) an acutely anesthetized control group (Con; n = 6), 2) a 6-h MV group (6-h MV; n = 6), 3) a 6-h MV group with exogenous administration of the antioxidant Trolox (6-h MVT; n = 6), 4) an 18-h MV group (n = 6), and 5) an 18-h MV group with exogenous administration of Trolox (18-h MVT; n = 6). Experiments were conducted in accordance with the policies contained in the Guide for the Care and Use of Laboratory Animals and were approved by the University of Florida Animal Care and Use Committee. Data for the systemic and biological response of animals to MV treatment have been published previously (34).
Acutely anesthetized controls.
Control animals were subjected to an acute plane of surgical anesthesia with an intraperitoneal injection of pentobarbital sodium (60 mg/kg body wt). Segments of the costal diaphragm were then removed, rapidly frozen in liquid nitrogen, and stored at −80°C for subsequent biochemical and molecular analyses.
All surgical procedures were performed with previously described aseptic techniques (7, 45, 52, 55). Briefly, animals randomly selected for MV were anesthetized with an intraperitoneal injection of pentobarbital sodium (60 mg/kg body wt). After reaching a surgical plane of anesthesia, the animals were tracheostomized and mechanically ventilated with a volume-driven small-animal ventilator (Harvard Apparatus, Cambridge, MA). The ventilator delivered all breaths; hence, this mode of ventilation (i.e., controlled MV) results in complete diaphragmatic inactivity (45). The tidal volume was established at ∼0.55 ml/100 g body wt, with a respiratory rate of 80 breaths/min. This respiratory rate was selected to mimic the breathing frequency of adult rats at rest. Additionally, a positive end-expiratory pressure of 1 cmH2O was used throughout the protocol. An arterial catheter was inserted into the carotid artery for constant measurement of blood pressure. Moreover, arterial blood samples (100 μl/sample) were removed during the first and last hour of MV and analyzed for arterial Pco2, Po2, and pH with a blood gas analyzer (model 1610, Instrumentation Laboratories, Lexington, MA). Anesthesia was maintained over the entire period of MV by continuous infusion of pentobarbital sodium (10 mg·kg body wt−1·h−1) via a venous catheter that was inserted into the jugular vein. Body temperature was maintained by use of a recirculating heating blanket. Additionally, heart rate and electrical activity of the heart were monitored via a lead II ECG using needle electrodes placed subcutaneously.
Body fluid homeostasis was maintained via the intravenous administration of 2.0 ml·kg−1·h−1 electrolyte solution. Continuing care during MV included expressing the bladder, removing airway mucus, lubricating the eyes, rotating the animal, and passive movement of the limbs. This care was maintained throughout the experimental period at hourly intervals. Finally, intramuscular injections of glycopyrrolate (0.04 mg/kg per 2 h) were used to reduce airway secretions during MV. On completion of MV, segments of the costal diaphragm were removed, rapidly frozen in liquid nitrogen, and stored at −80°C for subsequent biochemical and molecular analysis.
Exogenous administration of the antioxidant Trolox was performed as previously described (5). Briefly, a priming dose of Trolox (20 mg/kg) was infused over a 5-min period 20 min before the start of MV. During MV, Trolox was infused continuously at a rate of 4 mg·kg−1·h−1 for the entirety of the ventilation treatment. Verification of MV-induced increases in oxidative stress and diaphragmatic myofiber atrophy, as well as the efficacy of Trolox in both alleviating oxidative stress and myofiber atrophy, has previously been determined in these animals (34). In addition, we have previously demonstrated (34) that Trolox pretreatment of acutely anesthetized control animals does not alter diaphragm myofiber area or insulin/IGF-1 signaling to protein kinase B (PKB/Akt).
20S proteasome activity.
The in vitro trypsinlike, chymotrypsin-like, and PGPH activities of the 20S proteasome were measured fluorometrically in diaphragmatic extracts by following the release of free 7-amino-4-methylcoumarin (AMC) from synthetic substrates (BioMol International, Plymouth Meeting, PA). The proteasome substrates used were butyloxycarbonyl-Leu-Arg-Arg-AMC and benzyl oxycarbonyl-Leu-Leu-Glu-AMC for the measurement of trypsinlike and PGPH activities, respectively. Ten micrograms of protein (cytosolic fraction) were reacted with the respective synthetic peptide substrates in a reaction mixture containing 50 mM Tris·HCl and 1 mM dithiothreitol. One aliquot from each sample was incubated with 10 μM MG-132 (Sigma), an inhibitor of trypsinlike and PGPH proteasome activities (11), whereas the other aliquot was not incubated with the inhibitor. Release of AMC from the synthetic substrate in the presence of MG-132 indicates the action of nonproteasomal proteases. Samples were incubated for 30 min at 37°C before the addition of substrate. The change in fluorescence was measured at an excitation wavelength of 380 nm and an emission wavelength of 460 nm. The difference between the activities of the proteasome with and without inhibitor was used as the 20S proteasome activity.
Total and myofibrillar protein isolation and release.
Diaphragm total skeletal muscle protein concentration (μg protein/mg diaphragm muscle) was determined in whole muscle homogenates. Briefly, diaphragm strips of muscle (∼30 mg) were homogenized at 4°C with a glass-on-glass homogenizer in ice-cold cell lysis buffer (in mM: 10 NaCl, 1.5 MgCl2, and 20 HEPES, pH 7.4, and 1 DTT, with 20% glycerol, 0.1% Triton X-100). After a brief centrifugation (3 min) at 880 g and 4°C to pellet cellular debris, protein extracts were assayed by the Bradford method (Sigma, St. Louis, MO) and corrected for respective muscle weight. Myofibrillar protein isolation and easily releasable myofilament protein were assayed with a previously described protocol (54). Briefly, strips of diaphragm (∼50 mg) were homogenized at 4°C with a glass-on-glass homogenizer in ice-cold low-salt homogenizing buffer A (in mM: 10 Tris-maleate, 2 MgCl2, 2 EGTA, and 1 DTT, with 0.1 M KCl and 1% Triton X-100, pH 7.0). Homogenates were then centrifuged (10 min) at 1,500 g and 4°C. The resulting pellet was resuspended in an additional 9 ml of homogenizing buffer and filtered through two layers of gauze for 20 min at 4°C. The resulting supernatants were then centrifuged (10 min) at 1,500 g and 4°C, and the pellet was washed twice in buffer B (in mM: 10 Tris-maleate, 2 MgCl2, and 2 EGTA, with 0.1 M KCl, pH 7.0). Released myofilaments were then extracted from the myofibrillar protein fraction by repeated pipetting (10 passages through a Pasteur pipette) in 1.5 ml of buffer C (in mM: 10 Tris-maleate, 2 MgCl2, 2 EGTA, 5.0 ATP, and 1.0 DTT, with 0.1 M KCl, pH 7.0). Samples were then layered over 0.75 ml of buffer D (in mM: 10 Tris-maleate, 2 MgCl2, 2 EGTA, and 1.0 DTT, with 0.1 M KCl and 20% glycerol, pH 7.0) in a conical tube and subsequently centrifuged (10 min) at 1,500 g and 4°C. The final supernatant containing the released myofilaments was collected, and the pellet containing the residual myofibrillar protein fraction was resuspended in buffer D. Proteins from both fractions were then assayed by the Bradford method (Sigma). Myofibrillar protein (released myofibrillar protein + intact myofibrillar protein) was then corrected for muscle weight and expressed as myofibrillar protein concentration (μg myofibrillar protein/mg diaphragm muscle). Released myofilaments were expressed as a percentage of the combined amount of protein in the two fractions.
Fractionation of cellular homogenates.
Cytosolic protein fractions were obtained from costal segments of the diaphragm as previously described (53). Briefly, muscle was homogenized at 4°C with a glass-on-glass homogenizer in ice-cold cell lysis buffer (in mM: 10 NaCl, 1.5 MgCl2, 20 HEPES, pH 7.4, and 1 DTT, with 20% glycerol and 0.1% Triton X-100). After a brief centrifugation (3 min) at 880 g and 4°C to pellet nuclei and cellular debris, supernatants were subjected to three subsequent bouts of centrifugation (3,500 g at 4°C for 5 min each) to remove residual nuclei. The supernatant then received a protease inhibitor cocktail and was stored as the nuclei-free total cytosolic protein fraction. The purity of cytosolic fractions for the respective treatment groups was demonstrated previously (34).
Cytosolic protein extracts were assayed by the Bradford method (Sigma). Proteins (100 μg for protein synthesis signaling or 50 μg for proteasome subcomponents) were then separated by polyacrylamide gel electrophoresis via 4–15% gradient and transferred to nitrocellulose membranes (100 V for 3 h at 4°C). The resulting membranes were then stained with Ponceau S and visually inspected for equal protein loading and transfer. Images of each Ponceau S-stained membrane were analyzed by computerized image analysis (Scion Image, Frederick, MD) to further verify equal loading and transfer between lanes (data not shown). Membranes were then washed and blocked in PBS-Tween buffer containing 5% skim milk and 0.05% Tween for 2 h. Membranes were incubated with antibodies against mammalian target of rapamycin (mTOR) (7C10; no. 2983), phospho-(Ser 2448)mTOR (no. 2971S), 4E-BP1 (no. 9452), phospho-(Thr 37/46)4E-BP1 (no. 9459S), p70s6 kinase (no. 9202), and phospho-(Thr 389)p70s6 kinase (no. 9234S), all purchased from Cell Signaling Technology (Carlsbad, CA). Primary antibodies were diluted 1:1,000 in blocking buffer and applied to the membranes with gentle rocking overnight at 4°C. Membranes were also incubated with antibodies (1:1,000 in blocking buffer) against polyubiquitin (pUb) (Santa Cruz Biotechnology, Santa Cruz, CA), 20S α-subunit 7 (C8), 14-kDa E2 (E214k), and proteasome-activating complex PA28 (Boston Biochem, Cambridge, MA). Membranes were then incubated with horseradish peroxidase-antibody conjugate (1:2,000) directed against the primary antibody for 2 h. Membranes were treated with chemiluminescent reagents (luminol and enhancer) and exposed to light-sensitive film. Film images were captured and subsequently analyzed by computerized image analysis (Scion Image). Values for 4E-BP1 and p70s6 kinase proteins are not corrected for Con values but are presented as the percentages of total phosphorylated protein abundance as an indicator of activity.
4-Hydroxynonenal (4-HNE, trans-4-hydroxy-2-nonenal, C9H16O2) is an α,β-unsaturated hydroxyalkenal that is produced by lipid peroxidation in cells. 4-HNE was analyzed as an indicator of oxidative stress via Western blotting. Proteins (100 μg) were separated by polyacrylamide gel electrophoresis via 4–15% gradient and transferred to nitrocellulose membranes (100 V for 3 h at 4°C). Membranes were then washed and blocked in PBS-Tween buffer containing 5% skim milk and 0.05% Tween for 2 h. Membranes were incubated with an antibody against 4-HNE (ab46545; Abcam, Cambridge, MA). Primary antibody was diluted 1:1,000 in blocking buffer and applied to the membranes with gentle rocking overnight at 4°C. Membranes were then incubated with horseradish peroxidase-antibody conjugate (1:2,000) directed against the primary antibody for 2 h. Membranes were treated with chemiluminescent reagents (luminol and enhancer) and exposed to light-sensitive film. Film images were captured and subsequently analyzed by computerized image analysis (Scion Image). Values for 4-HNE were corrected for acutely anesthetized (Con) values and presented as fold changes.
RNA isolation and cDNA synthesis.
Total RNA was isolated from muscle tissue with TRIzol Reagent (Life Technologies, Carlsbad, CA) according to the manufacturer's instructions. Total RNA and RNA content (μg/mg muscle) were evaluated by spectrophotometry, and the integrity and relative abundance of 18S and 28S were checked by agarose gel electrophoresis. Total RNA (5 μg) was then reverse transcribed with the Superscript III First-Strand Synthesis System for RT-PCR (Life Technologies), using oligo(dT)20 primers and the protocol outlined by the manufacturer.
Real-time polymerase chain reaction.
One microliter of cDNA was added to a 25-μl PCR reaction for real-time PCR using Taqman chemistry and the ABI Prism 7000 Sequence Detection System (ABI, Foster City, CA). Relative quantization of gene expression was performed by the comparative computed tomography method (ABI, User Bulletin no. 2). This method uses a single sample, the calibrator sample (β-glucuronidase) for comparison of every unknown sample's gene expression. ΔΔCT [ΔCT(calibrator) − ΔCT(sample), where CT is threshold cycle] was then calculated for each sample, and relative quantification was calculated as 2ΔΔCT. Fivefold dilution curves were assayed on selected samples to confirm the validity of this quantization method for each gene. Primers and probes for E214k (GenBank NM_M62388, NM_AF144083), C8 (GenBank NM_M58593), β-glucuronidase (GenBank NM_Y00717, NM_M13962), and 18S (GenBank NM_X03205.1) were obtained from Applied Biosystems (Assays on Demand). The sequences used by the manufacturer in the design of primers and probes from this service are proprietary and are therefore not reported. However, the context sequences (i.e., the nucleotide sequence surrounding the probe) consist of the following: E214k, 5′-ATCCAAATGTGTATGCTGACGGCAG-3′; C8, 5′-GTAGTTAAAGAAGTTGCAAAAATAA-3′; and β-glucuronidase, 5′-TACTTCAAGACGCTGATCGCCCACA-3′. The pUb and PA28α primers and probes were obtained from Applied Biosystems (Assays-by-Design). Primer and probe sequences for pUb (GenBank NM_D16554) are as follows: forward, 5′-ACCCTCTCTGATTACAACATCCA-3′; reverse, 5′-CGGTCAGGGTCTTCACGAA-3′; probe, 5′-CCTGCACCTGGTCCTC-3′. Primer and probe sequences for PA28α (GenBank NM_D45249) are as follows: forward, 5′-GCTTCCAAACGCAGATCTCTAAGTA-3′; reverse, 5′-TGCCGATAATCACCCACATGAG-3′; probe, 5′-CTTGGCTGCTTTGGCC-3′. β-Glucuronidase was chosen as the reference gene based on initial experiments and previous work showing unchanged expression with our experimental manipulations (9, 10).
Comparisons between groups for each dependent variable measured were made by one-way analysis of variance (ANOVA). When significant differences were observed, a Tukey honestly significant difference (HSD) test was implemented post hoc. Significance was established at P < 0.05. Additional two-way ANOVA analyses were also performed with only the 6- and 18-h treatment groups (ventilation duration × Trolox administration) for each dependent variable measured to determine any specific interactions (P < 0.05). Post hoc analysis of significant interactions was done with a Bonferroni test.
Oxidative stress and efficacy of Trolox as an antioxidant.
The development of MV-induced diaphragmatic oxidative stress and the efficacy of Trolox administration as an antioxidant were determined by measuring α,β-unsaturated hydroxyalkenal (4-HNE)-modified proteins. 4-HNE-modified proteins increased 31% (1.31 ± 0.12) and 55% (1.55 ± 0.13) from control values (1.00 ± 0.09) with 6 and 18 h of MV, respectively. Trolox administration attenuated the MV-induced increases in 4-HNE-modified proteins at both 6 (0.91 ± 0.02) and 18 (1.11 ± 0.05) h.
Ubiquitin-protein conjugates and polyubiquitin gene expression.
Diaphragm cytosolic proteins were analyzed via Western blotting to determine the effects of MV-induced oxidative stress and antioxidant administration on the level of ubiquitin protein conjugation during MV. Ubiquitin conjugation increased 28–33% with both 6 and 18 h of MV independent of Trolox administration (Fig. 1A). Ventilation-induced increase in ubiquitin protein expression has previously been shown to be, at least in part, transcriptionally regulated (8). pUb gene expression increased 175% and 135% with both 6 and 18 h of MV, respectively (Fig. 1B). Similar to protein expression, ventilation-induced elevations in pUb mRNA abundance were unaltered by Trolox administration (Fig. 1B).
Proteasome subunit protein abundance and gene expression.
Protein abundance of C8, E214k, and PA28 components of the proteasome were analyzed via Western blot. There were no effects of 6-h MV [1.16 ± 0.18 integrated optical density (IOD)], 6-h MVT (1.02 ± 0.15 IOD), 18-h MV (1.07 ± 0.10 IOD), or 18-h MVT (1.14 ± 0.05 IOD) on C8 protein abundance compared with Con (1.00 ± 0.09 IOD). E214k protein abundance was not altered from Con (1.0 ± 0.05 IOD) by 6-h MV (1.17 ± 0.10 IOD), 6-h MVT (1.03 ± 0.07 IOD), 18-h MV (1.06 ± 0.06 IOD), or 18-h MVT (0.98 ± 0.07 IOD) treatments. PA28 protein abundance was also not altered from Con (1.00 ± 0.08 IOD) by 6-h MV (1.11 ± 0.10 IOD), 6-h MVT (1.13 ± 0.04 IOD), 18-h MV (1.08 ± 0.06 IOD), or 18-h MVT (1.02 ± 0.05 IOD) treatments. Gene expression of the C8 proteasomal subunit increased with 6 (134%) and 18 (268%) h of MV, independent of Trolox administration at either time point (Fig. 2A). E214k gene expression increased 178% with 6 h of MV and remained elevated relative to control values with 18 h of MV (101%), although demonstrating a slight decrease from 6-h values (Fig. 2B). Trolox administration did not alter the pattern of E214k gene expression relative to MV alone. Finally, gene expression of the PA28 proteasomal subunit increased with 6 (160%) and 18 (175%) h of MV, independent of Trolox administration at either time point (Fig. 2C).
In vitro 20S proteasome activities.
Our laboratory has previously demonstrated (5, 8) increased 20S proteasome activity in the diaphragm after MV; however, the effects of antioxidant administration on proteasome activity remain unknown. Trypsinlike activity increases 20% with 18 h of MV and is returned to control values by Trolox administration (Fig. 3A). Because of variation within the results, however, Trolox administration with 18 h of MV does not decrease trypsinlike proteasome activity from 18-h MV-alone levels. Chymotrypsin-like proteasome activity increased 30% in the diaphragm with 6 h of MV, independent of Trolox administration (Fig. 3B). However, increased chymotrypsin-like activity with 18 h of MV (35%) was attenuated (40% decrease from 18-h MV) by Trolox administration (Fig. 3B). There were no alterations in diaphragm PGPH-like proteasome activity after 6 h of MV, 6 h of MV with Trolox, or 18 h of MV (Fig. 3C). Trolox administration to animals during 18 h of MV significantly reduced PGPH-like activity in the diaphragm by 23%.
Diaphragm total and myofibrillar protein concentrations.
Six hours of MV with or without antioxidant administration did not alter diaphragm total (Fig. 4A) or myofibrillar (Fig. 4B) protein concentrations. Decreased total diaphragm and myofibrillar protein concentrations with 18 h of MV were rescued by antioxidant administration. Because intact sarcomeric myofilament protein cannot be recognized or degraded by the proteasome (reviewed in Refs. 43, 44), the percentage of released myofilament protein can be utilized as an indicator of myofilament release for proteolysis. The percentage of easily released myofilament protein increased 54% with 18 h of MV, and this increase was attenuated with antioxidant administration (Fig. 4C).
Signaling to translational initiation of protein synthesis.
Signaling regulation of protein synthesis occurs through mediation of the activity of both the translational repressor protein 4E-BP1 and the translation initiation factor p70s6 kinase (4, 21, 40). Therefore, phosphorylation and expression of 4E-BP1 and p70s6 kinase proteins are commonly utilized indicators of protein synthesis initiation in skeletal muscle (Fig. 5A) (3, 4, 6, 21, 32, 35, 36, 40). Values for 4E-BP1 and p70s6 kinase proteins are not corrected for Con values but are presented as the percentages of total phosphorylated protein abundance as an indicator of activity. The percentage of total phosphorylated p70s6 kinase protein decreased 52% and 78% with 6 and 18 h of MV, respectively, and increased 146% with Trolox administration during 18 h of MV (Fig. 5B). The percentage of total phosphorylated 4E-BP1 decreased 27% with 18 h of MV independent of Trolox administration (Fig. 5C), indicating increased activation of this translational repressor.
MV-induced alterations in cellular capacity for protein synthesis.
Approximately 85% of total cellular RNA is ribosomal; therefore alterations in RNA content are reflective of changes in the cellular capacity for protein synthesis (40, 41). RNA concentration (μg RNA/mg diaphragm muscle) was not altered from control values with MV for 6 or 18 h (Fig. 6A). Trolox administration also did not alter RNA concentrations with 6 or 18 h of MV. In addition, the relative abundance of 18/28S rRNA (IOD) was not altered from Con with 6-h MV, 6-h MVT, 18-h MV, or 18-h MVT (Fig. 6B).
Alterations in cellular ribosomal RNA content are primarily due to alterations in the transcriptional activation of their respective genes (rDNA transcription) (38–41). Therefore, we also analyzed the mRNA abundance of 18S as an indication of rDNA transcriptional primary products. The relative abundance of 18S mRNA (corrected for β-glucuronidase and normalized to control values) was practically ablated (76–99% decrease) compared with Con with 6 or 18 h of MV, and Trolox administration failed to attenuate this response (Fig. 6C).
Overview of principal findings.
To determine the mechanisms responsible for the protective effect of antioxidants against diaphragmatic atrophy during MV, we tested the hypothesis that regulation of skeletal muscle proteolytic activity is a critical site of redox action that alters diaphragmatic protein balance during MV disuse. Our results confirmed that oxidative stress is a contributing mechanism to proteasome activation during MV-induced diaphragm disuse. Despite MV-induced decreases in proteasome subunit (C8, E214k, and PA28) gene expression, the protein abundances of proteasome subunits were unaltered by the attenuation of MV-induced oxidative stress. These observations led us to postulate that the redox-sensitive mechanism regulating diaphragmatic proteasome activity during MV was alteration in myofilament substrate release from the sarcomere. Additional experiments revealed that sarcomeric myofilament protein release occurs during MV and is attenuated by antioxidant administration. Finally, to verify whether the protective effects of antioxidants were primarily due to alterations in proteasome activity, we determined that diaphragm protein synthetic capacity was not altered by antioxidant administration during MV. A detailed discussion of these findings follows.
Oxidative stress and proteolysis.
Previous work in our laboratory (5) revealed that MV-induced oxidative stress is associated with diaphragmatic proteolysis and an increase in chymotrypsin-like activity of the 20S proteasome. Although the assessment of the 20S proteasome activity is important, proteasome activity in combination with gene expression and protein abundance of α-subunits of the 20S proteasome core and PA28 regulatory complex may better reflect the activity of the proteasome in degrading skeletal muscle protein during disuse (8, 11, 12). Therefore, to determine the impact of oxidative stress on specific proteasome components, we analyzed the responses of ubiquitin-proteasome protein abundances, gene expression, and chymotrypsin-like, trypsinlike, and PGPH activities of the proteasome in diaphragm muscle during MV with and without antioxidant treatment. Interestingly, although Trolox administration attenuated chymotrypsin-like and PGPH proteasome activities, prevention of oxidative stress did not impact polyubiquitin or proteasome regulatory protein and/or gene expression during MV. Moreover, we previously reported (34) that prevention of MV-induced oxidative stress in the diaphragm does not prevent activation of muscle-specific ubiquitin ligases [muscle atrophy factor (MAFbx or atrogin) and muscle ring finger-1 (MuRF1)]. Collectively, these findings suggest that oxidative stress alone does not induce alterations in the substrate recognition component of the UPP. These findings do not preclude the possibility that posttranslational modification of proteasomal subunit proteins by oxidative stress and/or the alleviation of oxidative stress could provide an alternative explanation for the alterations in proteasomal activities in the present study. Further work is necessary to determine whether posttranslational modification of the substrate recognition component of the UPP is a critical site for activity regulation.
Intact, sarcomeric myofilament protein cannot be recognized or degraded by the ubiquitin-proteasome system (43), supporting the concept that myofilament release must precede myofiber degradation and muscle atrophy in disused skeletal muscle (34, 43, 44). Oxidatively modified proteins are selectively degraded by the 20S proteasome (47), and contractile proteins are specific targets for oxidative modification during MV (55), leading us to next hypothesize that decreased activity of the 20S proteasome due to Trolox may indicate a specific antioxidant-mediated alteration in contractile protein substrate release. Our findings support this hypothesis and demonstrate that antioxidant administration decreases myofilament contractile protein release before transcriptionally regulated alterations in proteasome subunit protein abundances. Together, these findings suggest that myofilament substrate availability may be the rate-limiting step in myofilament protein degradation during the rapid atrophy that occurs during prolonged MV.
The release of myofilament proteins for degradation is a complex process that has not yet been adequately described during disuse atrophy. Calpains, a class of Ca2+-sensitive cysteine proteases, are believed to mediate the disassembly of the sarcomere by cleaving titin and nebulin proteins at their point of attachment to the Z disk (16), allowing for the release of α-actinin from the myofibril (15). We have previously hypothesized (44) that redox control of calpain proteases could be the result of oxidative stress-induced Ca2+ disturbances or direct regulation of protease gene expression. Servais et al. (49) furthered this concept by demonstrating that vitamin E administration attenuates the postural muscle increase in μ-calpain gene expression that occurs with hindlimb suspension disuse. Although this finding does not establish a direct role for oxidants in the activation of calpains, it does demonstrate that vitamin E is sufficient to attenuate transcription of the μ-calpain gene during disuse. Additionally, scavenging of nitric oxide-induced reactive oxygen species with 3,4-dihydro-6-hydroxy-7-methoxy-2,2-dimethyl-1(2H)-benzopyran (CR-6) is sufficient to attenuate calpain and caspase activity in retinal photoreceptor cells (48). Collectively, these facts provide further evidence for a potential link between oxidative stress and protease activation. Although the mechanisms for the regulation of protease activities by oxidative stress remain a mystery, they could be related to intracellular Ca2+ disturbances, the abundance of the ubiquitous calpain inhibitor calpastatin, or protein phosphorylation.
Oxidative stress and protein synthesis.
Both mixed muscle and myosin heavy chain protein synthesis decrease in the diaphragm with as little as 6 h of MV, and this decrease in protein synthesis is exacerbated with more prolonged periods of ventilation (51). Decreased protein synthesis during skeletal muscle disuse is characterized by increases in translational repression (4E-BP1) and decreases in both translational initiation (p70s6 kinase) and cellular RNA content representing overall changes in the cellular capacity for protein synthesis (21, 24, 27, 28, 40, 41). The present study demonstrates that, similar to other models of disuse (37), decreased relative phosphorylation of p70s6 kinase is an initial event associated with MV-induced diaphragmatic inactivity. Interestingly, we also discovered that decreases in the relative phosphorylation of p70s6 kinase occur prior to alterations in the ribosomal capacity for protein synthesis, as indicated by a lack of alteration in diaphragm ribosomal RNA concentration or 18/28S ribosomal RNA abundance. These decreases occur at least as early as the onset of oxidative stress (6 h of MV) during MV and before either decreased total or myofilament protein loss or the appearance of diaphragmatic myofiber atrophy (33, 55). Downregulation of the initiation of protein synthesis via decreased p70s6 kinase activity and increased 4E-BP1 repressor activity are potential contributing mechanisms responsible for the MV-induced decreases in diaphragmatic protein synthesis. Furthermore, abundance of the 18S portion of the 45S rDNA gene decreases with 6 and 18 h of MV. Ribosomal RNA half-lives may range from 65 h to 12 days in skeletal muscle (31, 42), suggesting that a transcriptionally regulated decrease in ribosomal RNA and protein synthetic capacity would occur during longer periods of ventilation disuse.
To determine whether oxidative stress-induced diaphragmatic protein loss during MV occurs solely via an increase in proteolysis or because of a combination of proteolysis along with a decrease in protein accretion in the diaphragm, we investigated MV-induced alterations in p70s6 kinase in the diaphragm. We selected p70s6 kinase for study because this protein functions specifically in the translational initiation of protein synthesis for critical ribosomal proteins, elongation factors, and poly(A) binding proteins necessary for increasing protein synthetic capacity in the cell (26). Our finding revealed that relative phosphorylation of p70s6 kinase protein in the diaphragm increased with antioxidant administration. However, this increase was insufficient to alter the concentration of total or myofibrillar protein in the diaphragm during MV. Therefore, alterations in signaling pathways for translational initiation or ribosomal synthetic capacity do not appear to be significant components of the protective effect of Trolox administration in the diaphragm during MV.
Trolox as an antioxidant.
We previously demonstrated the effectiveness of Trolox in the attenuation of the progressive contractile dysfunction (5) and myofiber atrophy (34) that occur during ventilation disuse in the diaphragm. During our studies we have demonstrated that Trolox consistently fails to alleviate MV-induced decreases in glutathione content but is effective in reducing the levels of protein carbonyl formation, and now 4-HNE-modified proteins (5, 34). These findings do not definitively establish that the only function of Trolox is as an antioxidant. Other studies have suggested that antioxidants, particularly vitamin E, function in non-antioxidant-related signaling processes (1, 2, 49, 56). Vitamin E has been linked to cellular signaling regulating inflammation, cysteine protease abundance, heat shock protein abundance, ubiquitin ligases, and apoptosis (49, 56). Taken together with the results from the present study, it is important to interpret effects of Trolox and other antioxidants in the context that the protection they confer may be linked to both their direct oxidant scavenging properties and their involvement in cellular signaling and gene regulation.
Skeletal muscle is subjected to oxidant stress during disuse atrophy, and this redox disturbance is linked to several signaling processes that lead to muscle wasting (24, 29, 44). It is noteworthy that Servais et al. (49) recently suggested that that the protective effects of vitamin E (α-tocopherol) against unloading-induced locomotor skeletal muscle (i.e., soleus) atrophy could be due to signaling mechanisms involved in the modulation of proteolytic gene expression. However, alleviation of diaphragmatic oxidative stress with the vitamin E analog Trolox during MV does not suppress signaling pathways or gene expression known to be critically involved in postural muscle disuse atrophy. Specifically, atrophic signaling through insulin-like growth factor (IGF)-I-PKB/Akt to the forkhead box O (FoxO) class of transcription factors (including FoxO1, FoxO3, and FoxO4) and subsequent transcriptional regulation of the muscle-specific ubiquitin ligases [e.g., MAFbx (atrogin) and MuRF1] are unaltered by the attenuation of diaphragmatic oxidative stress with Trolox during MV (34). In conclusion, our collective findings reveal that independent of alterations in protein synthetic signaling or ribosomal synthetic capacity, administration of the antioxidant Trolox protects against muscle wasting in the rat diaphragm during MV by attenuating myofilament protein substrate release and retarding chymotrypsin-like and PGPH-like proteasome activity during prolonged MV. Given the important clinical ramifications of muscle wasting in both postural and respiratory skeletal muscle, it is critical to develop therapeutic countermeasures to circumvent morbidity and mortality outcomes in patient populations experiencing muscle wasting (e.g., prolonged bed rest, cancer, and MV). In this regard, the present basic investigation provides insight into the mechanisms responsible for antioxidant (Trolox)-mediated protection of diaphragmatic wasting during MV and provides the basis for future translation studies to develop therapeutic countermeasures to retard inactivity-induced skeletal muscle atrophy.
This work was supported by National Heart, Lung, and Blood Institute Grant R01-HL-072789 awarded to S. K. Powers.
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