Inhibition of the ubiquitin-proteasome pathway does not protect against ventilator-induced accelerated proteolysis or atrophy in the diaphragm

Abstract

Background

Mechanical ventilation (MV) is a life-saving intervention in patients with acute respiratory failure. However, prolonged MV results in ventilator-induced diaphragm dysfunction (VIDD), a condition characterized by both diaphragm fiber atrophy and contractile dysfunction. Previous work has shown calpain, caspase-3 and the ubiquitin-proteasome pathway (UPP) are all activated in the diaphragm during prolonged MV. However, while it is established that both calpain and caspase-3 are important contributors to VIDD, the role that the UPP plays in VIDD remains unknown. These experiments tested the hypothesis that inhibition of the UPP will protect the diaphragm against VIDD.

Methods

We tested this prediction in an established animal model of MV using a highly specific UPP inhibitor, epoxomicin, to prevent MV-induced activation of the proteasome in the diaphragm (n = 8/group).

Results

Our results reveal that inhibition of the UPP did not prevent ventilator-induced diaphragm muscle fiber atrophy and contractile dysfunction during 12 hours of MV. Also, inhibition of the UPP does not impact MV-induced increases in calpain and caspase-3 activity in the diaphragm. Finally, administration of the proteasome inhibitor did not protect against the MV-induced increases in the expression of the E3 ligases, MuRF1 and atrogin-1/MaFbx.

Conclusions

Collectively, these results indicate that proteasome activation does not play a required role in VIDD during the first 12 hours of MV.

INTRODUCTION

Mechanical ventilation (MV) is used in critical care medicine to maintain adequate alveolar ventilation in patients. Common indications for MV include pulmonary disorders, heart failure, neuromuscular diseases, coma and surgery. Although MV can be a life-saving intervention, prolonged MV results in the rapid development of ventilator-induced diaphragm dysfunction (VIDD) which occurs due to diaphragm atrophy and contractile dysfunction ^1-3. From a clinical perspective, VIDD is important because respiratory muscle weakness is predicted to contribute to difficulties in weaning patients from the ventilator ⁴. However, there is currently no drug therapy or clinical standard of care to address this problem. Therefore, improving our understanding of the processes that promote VIDD is essential to develop a therapeutic strategy to prevent MV-induced diaphragm weakness. In this regard, we determined if targeting the ubiquitin-proteasome pathway via epoxomicin administration is sufficient to protect against VIDD. The clinical importance of VIDD and the rationale supporting our approach is further highlighted in the next section.

The development of VIDD occurs rapidly and has been reported to occur within 12-18 hours after the initiation of MV ^1,2. Reports indicate that ~33% of all adult patients admitted to an intensive care unit require MV and weaning procedures account for 40-50% of the total time spent in the critical care unit ⁵. Although numerous factors can contribute to weaning difficulties, the most frequent cause of weaning failure in patients is inspiratory (i.e. diaphragm) muscle weakness. Therefore, developing strategies to prevent VIDD is clinically important. Previous work reveals that MV-induced diaphragm atrophy occurs due to both decreased protein synthesis and increased proteolysis ^3,6,7. However, because of the rapid development of VIDD, MV-induced increases in diaphragm protein breakdown appear to play the dominant role 3. In regard to MV-induced proteolysis, all four primary proteolytic systems are activated in the diaphragm during MV, including calpain, caspase-3, autophagy and the ubiquitin-proteasome pathway (UPP) ^3,8,9. However, only calpain and caspase-3 have been shown to participate in the development of VIDD and the role that autophagy and the UPP play remains unknown ^9-11. The absence of studies investigating the role of the UPP in VIDD is surprising given the UPP is considered to be an important proteolytic system responsible for the breakdown of myofibrillar proteins and the UPP plays a required role in inactivity-induced atrophy in limb skeletal muscles ¹². In this regard, a recent study by Agten et al. delivered the protease inhibitor Bortezomib to animals during prolonged MV in an attempt to elucidate the role that the UPP plays in the development of VIDD ¹³. However, Bortezomib inhibited caspase-3 activation but did not inhibit the MV-induced increase in 20S proteasome activity in the diaphragm. Thus, additional work is needed to determine the contribution of the UPP to VIDD and thus forms the basis for the current experiments. Therefore, these experiments tested the hypothesis that pharmacological inhibition of the UPP will protect the diaphragm against VIDD.

MATERIALS AND METHODS

Experimental design

Young adult female Sprague-Dawley rats were used in these experiments. Animals were assigned to one of three experimental groups (n=8/group): 1) acutely anesthetized control animals (CON); 2) 12 hour mechanically ventilated animals, no treatment (MV); and 3) 12 hours of MV, treatment with the proteasome inhibitor epoxomicin (MV+epoxomicin). The Institutional Animal Care and Use Committee of the University of Florida (Gainesville, FL) approved these experiments. In an effort to comply with the U.S. Animal Welfare Act and Public Health Service Policy by using the absolute minimum number of animals required to achieve the science, measurements made using the control and mechanical ventilation groups were also utilized in a previously published manuscript ¹¹. Investigators were blinded during the analysis of the data when possible (diaphragm contractile force and cross-sectional area).

Acutely anesthetized controls

Control animals were acutely anesthetized with an intraperitoneal injection of sodium pentobarbital (100 mg/kg body weight). After reaching a surgical plane of anesthesia, the diaphragm was quickly removed and the costal diaphragm was divided into several segments. One strip of the medial costal diaphragm was stored for histological measurements, another strip was immediately used for specific force measurements and the remaining portion of the costal diaphragm was rapidly frozen in liquid nitrogen and stored at −80°C for subsequent biochemical analyses.

Mechanical ventilation

All surgical procedures were performed using aseptic techniques. Animals in the MV groups were anesthetized with an intraperitoneal injection of sodium pentobarbital (60 mg/kg body weight). Animals were then tracheostomized, and mechanically ventilated with a pressure-controlled ventilator (Servo Ventilator 300, Siemens AG; Munich, Germany) for 12 hours with the following settings: upper airway pressure limit: 20 cm H₂O; respiratory rate: 80 breathes per minute; positive end expiratory pressure: 1 cm H₂O. Previous work by our laboratory has demonstrated that the full support mode of MV utilized in this study results in the complete cessation of inspiratory electromyogram activity in the diaphragm ².

The carotid artery was cannulated to permit the continuous measurement of blood pressure and the collection of blood during the protocol. Arterial blood samples (150 μl per sample) were removed periodically and analyzed for arterial partial pressure of oxygen, carbon dioxide and pH using an electronic blood-gas analyzer (GEM Premier 3000; Instrumentation Laboratory; Lexington, MA). Ventilator adjustments were made if arterial partial pressure of oxygen exceeded 40-45 mm Hg. Moreover, arterial partial pressure of oxygen was maintained > 60 mm Hg throughout the experiment by increasing the F_IO₂ (22-27%). A venous catheter was inserted into the jugular vein for continuous infusion of sodium pentobarbital (10 mg/kg of body weight/hr).

Body temperature was maintained at 37°C by use of a recirculating water heating blanket and heart rate was monitored via a lead II electrocardiograph. Continuous care during the MV protocol included lubricating the eyes, expressing the bladder, removing airway mucus, rotating the animal, and passively moving the limbs. Animals also received an intramuscular injection of glycopyrrolate (0.04 mg/kg of body weight) every two hours during MV to reduce airway secretions. Upon completion of MV, the diaphragm was quickly removed and a section was stored for histochemical analyses, another strip was immediately used for contractile measurements, and the remaining portion was frozen in liquid nitrogen and stored at −80°C for subsequent biochemical analyses.

Proteasome inhibition

To prevent MV-induced proteasome activation in the diaphragm, we administered the highly selective proteasome inhibitor, epoxomicin (0.55 μg/kg body weight) (Boston Biochem, Boston, MA). This dose was chosen based on previous reports demonstrating the effectiveness of this dose 14 and the efficacy of this dose was confirmed in our preliminary experiments. The inhibitor was dissolved in 60% dimethyl sulfoxide (DMSO) and given intravenously as a bolus immediately prior to the initiation of MV. Importantly, preliminary work demonstrated that the use of DMSO as the vehicle had no additional effects on diaphragm contractile dysfunction following 12 hours of MV (Table 1).

Table 1.

Effects of dimethyl sulfoxide administration on diaphragm contractile function

	Stimulation frequency (Hz)
Treament	15	30	60	100	160
CON (N/cm2)	16.97 ± 2.65	22.25 ± 1.33	25.16 ± 0.95	25.93 ± 0.97	25.90 ± 1.05
MV (N/cm2)	12.26 ± 1.38	16.97 ± 1.05	19.58 ± 1.34	20.44 ± 1.06	20.39 ± 0.80
MV+DMSO (N/cm2)	12.19 ± 1.06	17.58 ± 0.62	20.49 ± 0.68	21.26 ± 0.47	21.27 ± 0.54

Preliminary experiments were conducted to determine the effects of dimethyl sulfoxide (DMSO) on the diaphragm (n = 6/group). Values are mean ± SD. Acutely anesthetized control animals (CON); 12 hour mechanically ventilated animals, no treatment (MV); and 12 hours of MV, treatment with 60% DMSO (MV+DMSO). CON vs MV (p<0.001); CON vs MV+DMSO (p<0.001); MV vs MV+DMSO (p=0.582)

Measurement of in vitro diaphragm contractile properties

Upon sacrifice or the completion of the MV period, the entire diaphragm was removed and placed in a dissecting chamber containing a Krebs-Hensleit solution equilibrated with 95% O₂-5% CO₂ gas. A muscle strip, including the tendinous attachments at the central tendon and rib cage was dissected from the midcostal region. The strip was suspended vertically between two lightweight Plexiglas clamps with one end connected to an isometric force transducer (model FT-03, Grass Instruments, Quincy, MA) within a jacketed tissue bath. The force output was recorded via a computerized data-acquisition system (Super Scope II, GW Instruments Somerville, MA; Apple Computer Cupertino, CA). Lo (i.e. the length at which peak twitch tension is achieved) was determined by systematically adjusting the length of the muscle using a micrometer while evoking single twitches. Thereafter, all contractile properties were measured isometrically at Lo. To measure maximal isometric twitch force each strip was stimulated supramaximally with 120-V pulses at 1 Hz and to measure the force frequency response each strip was stimulated supramaximally with 120-V pulses at 15–160 Hz. The duration of each train was 500 ms to achieve a force plateau. Contractions were separated by a 2-min recovery period. For comparative purposes, diaphragmatic (bundles of fibers) force production was normalized as specific Po. The total muscle cross-sectional area at right angles to the long axis was calculated by the following algorithm ¹⁵: Total muscle cross-sectional area (mm²) = [muscle mass/(fiber length × 1.056)], where 1.056 is the density of muscle (in g/cm³). Fiber length was expressed in centimeters measured at Lo ¹⁶.

Myofiber cross-sectional area

Sections from frozen diaphragm samples were cut at 10 μm using a cryotome (Shandon Inc., Pittsburgh, PA) and stained as described previously ⁹. Cross-sectional area was determined using Scion software (NIH, Bethesda, MD).

Myofibrillar protein isolation and release

Myofibrillar protein isolation and easily releasable myofilament protein was assayed with a previously described protocol ^17,18. The final supernatant containing the released myofilaments was collected, and the pellet containing the residual myofibrillar protein fraction was resuspended. Proteins from both fractions were then assayed by the Bradford method (Sigma-Aldrich; St. Louis, MO). 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.

Western blot analysis

Protein abundance was determined in diaphragm samples via Western Blot analysis. Briefly, diaphragm tissue samples were homogenized 1:10 (wt/vol) in 5 mM Tris (pH 7.5) and 5 mM EDTA (pH 8.0) with a protease inhibitor cocktail (Sigma-Aldrich) and centrifuged at 1500 g for 10 min at 4°C. After collection of the resulting supernatant, diaphragm protein content was assessed by the method of Bradford (Sigma-Aldrich). Proteins from the supernatant fraction were separated via polyacrylamide gel electrophoresis. Proteins were transferred to nitrocellulose membranes, blocked in 5% non-fat milk and incubated with primary antibodies directed against proteins of interest. 4-hydroxynonenal (4-HNE) (Abcam; Cambridge, MA), poly-ubiqutinated proteins (pUB) (Boston Biochem), active calpain (Cell Signaling; Beverly, MA), cleaved caspase-3 (Cell Signaling), spectrin (Santa Cruz; Santa Cruz, CA), MuRF-1 (ECM Biosciences; Versailles, KY) and Atrogin-1 (ECM Biosciences) were assessed. Finally, tubulin (Santa Cruz) protein content was measured to normalize for equal protein loading and transfer. Membranes were developed using autoradiographic film and images of the film were captured and analyzed using the 440CF Kodak Imaging System (Kodak; New Haven, CT).

RNA isolation and complementary DNA synthesis

Total RNA was isolated from muscle tissue with TRIzol Reagent (Life Technologies; Carlsbad, CA) according to the manufacturer’s instructions. RNA content (μg/mg muscle weight) was evaluated by spectrophotometry. RNA (5 μg) was then reverse transcribed with the Superscript III First-Strand Synthesis System for reverse transcription-polymerase chain reaction (Life Technologies), using oligo(dT)20 primers and the protocol outlined by the manufacturer.

Real-time polymerase chain reaction

One μl of complementary DNA was added to a 25 μl polymerase chain reaction mixture for real-time polymerase chain reaction using Taqman chemistry and the ABI Prism 7000 Sequence Detection system (ABI; Foster City, CA). Relative quantification of gene expression was performed using the comparative computed tomography method (ABI, User Bulletin #2). β-Glucuronidase, a lysosomal glycoside hydrolase, was chosen as the reference gene based on previous work showing unchanged expression with our experimental interventions ^19,20. MuRF1 and atrogin-1/MaFbx microRNA (mRNA) transcripts were assayed using predesigned rat primer and probe sequences commercially available from Applied Biosystems (Assays-on-Demand).

20S proteasome activity assay

A section of the ventral costal diaphragm was homogenized 1:10 (vol: vol) (5 mM Tris-HCL, pH 7.5; 5 mM EDTA, pH 8.0) and centrifuged at 1500 g for 10 minutes at 4°C. The cytosolic fraction was centrifuged at 10,000 g for 10 minutes at 4°C, followed by an additional spin of the supernatant at 100,000 g for 1 hour at 4°C. The in vitro chymotrypsin-like activity of the 20S proteasome was measured fluorometrically using techniques described by Stein et al ²¹.

Statistical analysis

In vitro diaphragmatic specific force production was analyzed using a two-way mixed design Group × Frequency (3 × 5) ANOVA with repeated measures on frequency. Follow-up tests were conducted using the Bonferroni procedure. For the cross sectional area data, we compared the groups at each fiber type (i.e., I, IIa, and IIx/IIb) with oneway ANOVAs. Data for 4-HNE, easily releasable myofilaments, MuRF-1, Atrogin-1, pUB, 20S proteasome activity, calpain, spectrin breakdown product (SBDP) 145 kDa, caspase-3, and SBDP 120 kDa were compared between groups by one-way ANOVA. When the one-way ANOVAs revealed statistically significant differences, a conservative Bonferroni adjustment was performed to identify group differences. Physiological variables (i.e., heart rate, systolic blood pressure, arterial partial pressure of oxygen and carbon dioxide, and arterial pH) were compared using the independent t-test. Data points two standard deviations different from the average were removed from the analyses. All statistical comparisons were performed using two-tail tests. Overall significance was established at p<0.05 and adjusted (i.e., p=0.05/3=0.0167) with Bonferroni’s technique when examining the group main effect means. Data are presented as mean ± standard deviation (SD). All statistical analyses were performed using PASW Statistics 21 (SPSS, Inc., Chicago, IL).

RESULTS

Systemic and biologic response to MV

Prior to the initiation of MV, no significant differences existed in body weight between the experimental groups. Importantly, 12 hours of MV did not significantly alter body weight between groups (data not shown). In addition, heart rate and systolic blood pressure were maintained relatively constant during the 12 hours of MV and no significant differences existed between experimental groups in any of these measures at the completion of 12 hours of MV (Table 2). The arterial partial pressures of oxygen, carbon dioxide and pH were also maintained relatively constant during MV with no significant differences existing between groups (Table 2). In addition, the colonic (body) temperature was homeostatic and relatively constant (36°C-37°C) during the MV period. At the completion of the MV protocol, there were no visual abnormalities of the lungs or peritoneal cavity, no visible indication of lung injury, and no evidence of infection, indicating that our aseptic surgical technique was successful.

Table 2.

Systemic and biological response to MV.

Physiological variable	MV	MV+epoxomicin	p-value
Heart rate (beats/min)	373 ± 27	359 ± 17	0.235
Systolic blood pressure (mm/Hg)	106 ± 25	106 ± 22	0.954
Arterial partial pressure of oxygen (mm/Hg)	83 ± 25	85 ± 8	0.824
Arterial partial pressure of carbon dioxide (mm/Hg)	41 ± 11	42 ± 6	0.836
Arterial pH	7.40 ± 0.10	7.39 ± 0.02	0.772

Animal heart rate, systolic blood pressure, arterial blood gas tension, and arterial pH at the completion of 12 hours of mechanical ventilation. Values are mean ± SD. Note that no significant differences existed between the experimental groups in any of these physiological variables. 12 hour mechanically ventilated animals, no treatment (MV); and 12 hours of MV, treatment with the proteasome inhibitor epoxomicin (MV+epoxomicin).

Epoxomicin partially protects against MV-induced diaphragm contractile dysfunction

To determine the role of the UPP in MV-induced diaphragm contractile dysfunction, we measured the in vitro specific force generation of diaphragm muscle strips. Similar to published reports, 12 hours of MV resulted in a significant decrease in specific force generation ^2,22,23. Animals treated with epoxomicin were partially protected against MV-induced diaphragm contractile dysfunction. However, diaphragmatic specific force production in the MV+epoxomicin group remained significantly lower than control animals (Figure 1).

Figure 1.

Diaphragmatic force-frequency response (in vitro). Values are mean ± SD (n=8/group). CON significantly different versus MV (p<0.001); CON significantly different versus MV+epoxomicin (p<0.001); MV significantly different versus MV+epoxomicin (p=0.007). Acutely anesthetized control animals (CON); 12 hour mechanically ventilated animals, no treatment (MV); 12 hours of MV, treatment with the proteasome inhibitor epoxomicin (MV+epoxomicin).

Epoxomicin administration does not prevent MV-induced diaphragm atrophy

To determine if the UPP is required for MV-induced diaphragm atrophy, we measured the cross-sectional area of diaphragm myofibers following 12 hours of MV. Our results demonstrate that compared to control, MV promotes significant atrophy in fiber type I in diaphragm muscle. Importantly, our data also reveal that inhibition of the 20S proteasome does not prevent this MV-induced diaphragm atrophy (Figure 2).

Figure 2.

Fiber cross-sectional area in diaphragm skeletal muscle myofibers expressing myosin heavy chain (MHC) I (Type I), MHC IIa (Type IIa), and MHC IIx/IIb (Type IIx/IIb) (CON n=7; MV n=8; MV+epoxomicin n=8). Values are mean ± SD. * Significantly different versus CON (p<0.0167). Exact p-values for all comparisons are listed below the graph. Acutely anesthetized control animals (CON); 12 hour mechanically ventilated animals, no treatment (MV); 12 hours of MV, treatment with the proteasome inhibitor epoxomicin (MV+epoxomicin).

Epoxomicin does not impact MV-induced oxidative damage in diaphragm

Oxidative stress has been linked to accelerated rates of proteolysis and diaphragm atrophy during prolonged MV ^7,24. 4-HNE is formed during the lipid peroxidation cascade and measurement of 4-HNE modified protein adducts is an excellent biomarker of oxidative damage in muscle. In this regard, MV resulted in a significant increase in the accumulation of 4-HNE modified proteins in the diaphragm. However, treatment of animals with epoxomicin failed to inhibit the increase in MV-induced oxidative damage in the diaphragm (Figure 3).

Figure 3.

The levels of 4 hydroxynonenal (4-HNE). A representative blot for 4-HNE protein conjugates is shown above the graph. Values are mean percentage change ± SD (n=8/group). * Significantly different versus CON (p<0.0167). Exact p-values for all comparisons are listed below the graph. Acutely anesthetized control animals (CON); 12 hour mechanically ventilated animals, no treatment (MV); 12 hours of MV, treatment with the proteasome inhibitor epoxomicin (MV+epoxomicin).

Epoxomicin fails to prevent diaphragm myofibrillar protein release

Easily releasable myofilaments were measured as an index of sarcomeric protein release in the diaphragm during MV. Our results reveal that MV produces a significant increase in the percentage of easily releasable myofibrillar proteins in the diaphragm and that inhibiting the 20S proteasome with epoxomicin does not attenuate this release of sarcomeric proteins from the myofibrillar lattice (Figure 4).

Figure 4.

Easily releasable myofilaments (CON n=7; MV n=8; MV+epoxomicin n=7). Values are mean percentage change ± SD. * Significantly different versus CON (p<0.0167). Exact p-values for all comparisons are listed below the graph. Acutely anesthetized control animals (CON); 12 hour mechanically ventilated animals, no treatment (MV); 12 hours of MV, treatment with the proteasome inhibitor epoxomicin (MV+epoxomicin).

Epoxomicin does not prevent MV-induced diaphragmatic expression of MuRF1 and atrogin-1/MaFbx

During periods of inactivity, the UPP relies on E3 ubiquitin ligases to tag damaged and misfolded muscle proteins, targeting them for degradation. To determine if inhibition of the 20S proteasome impacts muscle E3 ligase expression, we measured the mRNA expression and protein abundance of MuRF1 and atrogin-1/MaFbx. Compared to control, 12 hours of MV significantly increased the expression of both MuRF1 and atrogin-1/MaFbx in the diaphragm. However, proteasome inhibition did not prevent the MV-induced increases in the protein expression of either MuRF1 or atrogin-1/MaFbx. However, epoxomicin treatment did result in a significant reduction in MuRF-1 mRNA expression compared to MV (Figure 5A-D). In addition to MuRF1 and atrogin-1/MaFbx, the presence of ubiquitinated proteins can serve as a marker of proteasome activity and changes in protein turnover. Therefore, we measured the levels of ubiquitinated proteins in the diaphragm to determine if epoxomicin inhibited the formation of poly-ubiquitinated protein conjugates. Our results demonstrate that treatment of MV animals with epoxomicin results in a significant increase in ubiquitin protein concentration in the diaphragm (Figure 6A).

Figure 5.

(A) microRNA (mRNA) levels of muscle ring finger-1 (MuRF-1) (CON n=8; MV n=7; MV+epoxomicin n=7). (B) mRNA levels of atrogin-1/ Muscle Atrophy F-box (MaFbx) (CON n=8; MV n=8; MV+epoxomicin n=7). (C) Protein levels for MuRF-1 (CON n=8; MV n=7; MV+epoxomicin n=8). (D) Protein levels for atrogin-1/MaFbx (n=8/group). Representative blots for MuRF-1 and atrogin-1/MaFbx protein are shown above the graph. Values are mean percentage change ± SD. * Significantly different versus CON (p<0.0167). § Significantly different versus CON and MV (p<0.0167). Exact p-values for all comparisons are listed below the graph. Acutely anesthetized control animals (CON); 12 hour mechanically ventilated animals, no treatment (MV); 12 hours of MV, treatment with the proteasome inhibitor epoxomicin (MV+epoxomicin).

Figure 6.

(A) Protein levels for poly-ubiquitinated protein (pUB) (n=8/group). (B) Chymotrypsin-like activity of the 20S ubiquitin-proteasome system (n=7/group). Representative blots for pUB protein is shown above the graph. Values are mean percentage change ± SD. * Significantly different versus CON (p<0.0167). § Significantly different versus CON and MV+epoxomicin (p<0.0167). Exact p-values for all comparisons are listed below the graph. Acutely anesthetized control animals (CON); 12 hour mechanically ventilated animals, no treatment (MV); 12 hours of MV, treatment with the proteasome inhibitor epoxomicin (MV+epoxomicin).

Epoxomicin selectively inhibits proteasome activity in vivo

To verify that epoxomicin was effective in preventing proteasome activity, we measured the chymotrypsin-like activity of the 20S proteasome in the diaphragm. Importantly, our results verified that administration of epoxomicin prevents the MV-induced increase in 20S proteasome activity in the diaphragm (Figure 6B).

Epoxomicin does not prevent calpain or caspase-3 protease activation

Given that previous work has shown both calpain and caspase-3 are required for VIDD ^9-11, we determined whether diaphragmatic calpain and/or caspase-3 activity was inhibited by epoxomicin. Similar to previous results, we report that the protein abundance of active calpain-1 as well as the abundance of the 145kDa calpain-specific SBDP was significantly increased after 12 hours of MV in the diaphragm and treatment with epoxomicin did not attenuate this increase in calpain activity (Figure 7). In addition, we measured cleaved (active) caspase-3 and the 120kDa caspase-specific SBDP as markers of caspase-3 activity. MV resulted in a significant increase in caspase-3 activity in the diaphragm and this activity remained elevated in MV animals treated with epoxomicin (Figure 8).

Figure 7.

Calpain activation in the diaphragm was determined via western blot. (A) Active calpain 1 (n=8/group). (B) Calpain-specific spectrin breakdown product (SBDP) (n=8/group). Representative western blots are shown above the graphs. Values are mean percentage change ± SD. * Significantly different versus CON (p<0.0167). Exact p-values for all comparisons are listed below the graph. Acutely anesthetized control animals (CON); 12 hour mechanically ventilated animals, no treatment (MV); 12 hours of MV, treatment with the proteasome inhibitor epoxomicin (MV+epoxomicin).

Figure 8.

Caspase-3 activation in the diaphragm was determined via western blot. (A) Cleaved (active) caspase-3 (CON n=8; MV n=6; MV+epoxomicin n=6). (B) Caspase-3-specific spectrin breakdown product (SBDP) (n=8/group). Representative western blots are shown above the graphs. Values are mean percentage change ± SD. * Significantly different versus CON (p<0.0167). Exact p-values for all comparisons are listed below the graph. Acutely anesthetized control animals (CON); 12 hour mechanically ventilated animals, no treatment (MV); 12 hours of MV, treatment with the proteasome inhibitor epoxomicin (MV+epoxomicin).

DISCUSSION

Overview of Principal Findings

Our results reveal that inhibition of 20S proteasome activity is not sufficient to protect the diaphragm against MV-induced diaphragm fiber atrophy but inhibition of 20S activity does provide partial protection against MV-induced diaphragmatic contractile dysfunction. A detailed discussion of these findings follows.

UPP activation is not a major contributor to VIDD during 12 hours of MV

The ubiquitin-proteasome system is a key proteolytic system in muscle fibers that plays an important role during muscle wasting 25-27. The total proteasome complex (26S) is comprised of a core proteasome subunit (20S) coupled with two regulatory complexes (19S) ^28,29. The primary function of this system is to degrade proteins that have been poly-ubiquitinated. In this regard, previous work from our laboratory suggests that proteolytic processing by the UPP is increased in the diaphragm during prolonged MV ³⁰. However, the contribution of this pathway to VIDD remains unknown. Historically, the UPP has been characterized as the dominant proteolytic pathway in disuse muscle atrophy ^26,31. Therefore, we investigated the role that the UPP plays in MV-induced diaphragm atrophy using the 20S proteasome inhibitor epoxomicin. Our results reveal that treatment of mechanically ventilated animals with epoxomicin partially attenuates the MV-induced decrease in diaphragm contractile force production, but does not protect against MV-induced diaphragmatic fiber atrophy. Our interpretation of these findings is that while the UPP is active during MV, this proteolytic system does not play a dominant role in the development of VIDD during the first 12 hours of MV.

Proteasome inhibition does not impact MV-induced oxidative damage in the diaphragm

Protein oxidation can promote protein recognition and degradation by many proteolytic systems (i.e. calpain, caspase-3, UPP). Indeed, previous work has demonstrated that protein oxidation can enhance proteins susceptibility to degradation by altering the proper folding of proteins resulting in increased recognition and proteolysis of damaged proteins ^29,32,33. Regulatory proteins that influence force production include actin, myosin, tropomyosin and the troponin complex (reviewed in ³⁴). Each of these proteins appears to be sensitive to oxidation and therefore, to modifications that diminish force production rendering them potential targets for proteolytic degradation ^29,32,34. In addition, to oxidative modification of myofibrillar proteins, reports indicate that oxidative stress promotes the increased expression of proteins necessary for UPP function. Specifically, exposure of myotubes to hydrogen peroxide is sufficient to increase the expression of specific ubiquitin ligases ^35-37. Therefore, increased reactive oxygen species production appears to promote muscle protein degradation via the UPP. In this regard, we measured the abundance of 4-HNE modified proteins to determine whether inhibition of 20S proteasome activity results in a reduction of MV-induced oxidative damage in the diaphragm. In agreement with numerous reports, our results confirm that prolonged MV increases diaphragmatic oxidative stress ^7,22,38 and treatment of animals with epoxomicin does not attenuate the MV-induced accumulation of 4-HNE in the diaphragm.

MV-induced activation of diaphragm proteases

MV-induced diaphragm atrophy results from an imbalance in the rate of muscle protein synthesis and degradation, with increased protein degradation playing the dominant role ^39,40. In this regard, myofibrillar proteins constitute 50-60% of muscle protein and are degraded at a faster rate than other classes of muscle proteins during disuse muscle atrophy ⁴¹. In addition, myofibrillar proteins have also been shown to be the principle protein target of the UPP ⁴². The degradation of myofibrillar proteins is a process that requires the coordination of several proteolytic systems, including the calpain, caspase-3 and the UPP. Specifically, it is predicted that the UPP is unable to cleave intact sarcomeric proteins and that release of these myofilaments from the sarcomeric lattice is required for UPP degradation of actin and myosin ^40,43.

As discussed previously, the breakdown of muscle proteins by the UPP requires ubiquitination of the targeted proteins. This poly-ubiquitination is a three-step process in which activation of specific protein ligases leads to the activation and conjugation of ubiquitin to protein substrates followed by the degradation of poly-ubiquitin-tagged proteins by the proteasome. Specifically, ubiquitin monomers are activated by E1 or ubiquitin-activating proteins and transferred to E2 ubiquitin-conjugating proteins. Finally, in coordination with the E3 ligases, ubiquitin is attached to the protein substrate ⁴⁴. In reference to substrate recognition and degradation, expression of E2 and E3 ligases determines the specificity of the UPP and transcriptional levels of these proteins strongly correlates with muscle catabolism ^40,43,44. Further, expression of the muscle specific E3 ligases MuRF1 and atrogin-1/MaFbx has been reported to be essential for skeletal muscle atrophy ^25-27. Similarly, Bodine et al. demonstrated that these ligases are critical regulators of muscle atrophy and the deletion of either ligase during denervation is sufficient to protect muscle size and/or mass ²⁶.

While the importance of both MuRF1 and atrogin-1/MaFbx has been demonstrated in other models of disuse muscle atrophy, the role that these ligases play in VIDD remains unknown. In regard to E3 ligase expression, the current results demonstrate that 12 hours of MV is sufficient to increase the transcription of both MuRF1 and atrogin-1/MaFbx in the diaphragm ^22,23. However, treatment of mechanically ventilated animals with epoxomicin did not reduce the expression of these E3 ligases in the diaphragm. Our data also demonstrates that 12 hours of MV increases protein ubiquitination in the diaphragm and the levels of ubiquitinated proteins in the diaphragm tended to be higher in the MV animals treated with epoxomicin. This increased protein ubiquitination in MV animals is likely due to an accumulation of poly-ubiquitinated proteins resulting from increased MuRF1 and Atrogin-1/MaFbx in the diaphragm. Further, the increase in ubiquitinated proteins in the diaphragms of MV animals treated with epoxomicin is expected because these tagged proteins cannot be degraded by the UPP because of epoxomicin-induced inhibition of 20S proteasome activity.

Finally, while it has been proposed that the UPP is the primary proteolytic system essential for the degradation of myofibrillar proteins, it has also been demonstrated that the activity of other proteases play an obligatory role in diaphragm protein breakdown during prolonged MV ^7,9-11. Specifically, calpain and caspase-3 activation both appear to play a required role in MV-induced diaphragm muscle turnover ^9-11. In this regard, it has been hypothesized that actomyosin complexes and other myofilaments must first be released from the sarcomere, unfolded and ubiquitinated for UPP degradation ^45-49. This suggests that the rate-limiting step in muscle contractile protein degradation is the processing required for UPP proteolysis. Further, evidence indicates that both calpain and caspase-3 can promote actomyosin disassociation ^45,49,50. Therefore, it has been speculated that activation of calpain and caspase-3 is required for UPP degradation of myofibrillar proteins ^32,50. A long-standing rationale for the prediction that the UPP is essential for disuse muscle atrophy is that the two major contractile proteins actin and myosin are reported to be weak substrates for calpain and caspase-3 45. However, recent evidence indicates that oxidized myofibrillar proteins can be directly degraded by both calpain and caspase-3 which demonstrates that the UPP may not be required for the degradation of myofibrillar proteins in skeletal muscles exposed to oxidative stress ³². Indeed, work from our laboratory demonstrates that prolonged MV results in oxidation of both actin and myosin in the diaphragm and that oxidation greatly increases the susceptibility of actin and myosin along with other skeletal muscle myofibrillar proteins to degradation by calpain and caspase-3 ^32,51. Therefore, during prolonged MV where myofibrillar proteins in the diaphragm are exposed to oxidizing conditions, activation of the UPP system may not be required for the rapid atrophy that occurs within the first 12 hours of prolonged MV.

Conclusions

This study provides the first evidence that the UPP plays a limited role in the development of VIDD during the first 12 hours of MV. Indeed, although the UPP is activated in the diaphragm during MV, inhibition of 20S proteasome activity is not sufficient to protect the diaphragm against MV-induced contractile dysfunction and atrophy during the first 12 hours of MV. Therefore, these results along with previous reports suggest that activation of calpain and caspase-3 play a key role in the development of VIDD ^32,45,50. Hence, while the proteasome has been considered to be the primary protease responsible for myofibrillar protein degradation during locomotor muscle atrophy, its contribution to VIDD during the first 12 hours of ventilator support appears to be limited. In regard to the impact of epoxomicin on diaphragm contractile function at high stimulation frequencies, it is feasible that the autophagy/lysosomal system can degrade poly-ubiquitinated proteins. Therefore, it is possible that activation of the autophagy/lysosomal system is upregulated in when UPP activity is inhibited. This is a testable hypothesis that is worthy of future experimentation.

Final Boxed Summary Statement.

What We Already Know about This Topic

* Mechanical ventilation is associated with rapid development of atrophy of the diaphragm, which contributes to respiratory failure

What This Article Tells Us That Is New

* Pharmacologic inhibition of the ubiquitin-proteasome pathway, using epoxomicin, did not protect the diaphragm against oxidative stress or atrophy in anesthetized, mechanically-ventilated rats