Published ahead of print on March 22, 2007, doi:10.1164/rccm.200609-1342OC
© 2007 American Thoracic Society doi: 10.1164/rccm.200609-1342OC
Leupeptin Inhibits Ventilator-induced Diaphragm Dysfunction in Rats1 Respiratory Muscle Research Unit, Laboratory of Pneumology, Katholieke Universiteit Leuven, Leuven, Belgium; and 2 Department of Applied Physiology and Kinesiology, University of Florida, Gainesville, Florida Correspondence and requests for reprints should be addressed to Ghislaine Gayan-Ramirez, Ph.D., Labo Ademspieren, O&N1 bus 706, Herestraat 49, B-3000 Leuven, Belgium. E-mail: ghislaine.gayan-ramirez{at}med.kuleuven.be
Rationale: Controlled mechanical ventilation (CMV) has been shown to result in elevated diaphragmatic proteolysis and atrophy together with diaphragmatic contractile dysfunction. Objectives: To test whether administration of leupeptin, an inhibitor of lysosomal proteases and calpain, concomitantly with 24 hours of CMV, would protect the diaphragm from the deleterious effects of mechanical ventilation. Methods: Rats were assigned to either a control group or 24 hours of CMV; animals in the ventilation group received either a single intramuscular injection of saline or 15 mg/kg of the protease inhibitor, leupeptin. Measurements and Main Results: Compared with control animals, mechanical ventilation resulted in a significant reduction of the in vitro diaphragm-specific force production at all stimulation frequencies. Leupeptin completely prevented this reduction in force generation. Atrophy of type IIx/b fibers was present after CMV, but not after treatment with leupeptin. Cathepsin B and calpain activities were significantly higher after CMV compared with the other groups; this was abolished by treatment with leupeptin. Significant inverse correlations were found between diaphragmatic force generation and cathepsin B and calpain activity, and illustrate the deleterious role of proteolysis in diminishing diaphragmatic force production after prolonged CMV. Conclusions: Administration of the protease inhibitor leupeptin concomitantly with mechanical ventilation completely prevented ventilation-induced diaphragmatic contractile dysfunction and atrophy.
Key Words: mechanical ventilation • protein degradation • respiratory muscles
Mechanical ventilation is commonly used in patients with respiratory failure. However, problems with weaning patients from the ventilator is a significant clinical issue, as about 20 to 30% of the patients receiving mechanical ventilation experience difficulties in weaning (1). Although weaning failure may be due to numerous factors, diaphragm dysfunction induced by mechanical ventilation probably plays an important role. Indeed, animal studies reveal that 18 hours of controlled mechanical ventilation (CMV) results in diaphragmatic contractile dysfunction and atrophy (2). Consistent with these findings, prolonged CMV results in a decrease in both insulin-like growth factor-I mRNA (3) and myogenic determination factor expression in the diaphragm (4) together with increased diaphragmatic proteolysis (5). Muscle proteolysis is a highly regulated process accomplished by at least three different proteolytic systems: the ubiquitin–proteosome pathway (UPP), the Ca2+-dependent system, and the lysosomal system. All three proteolytic systems have been shown to be implicated in the increased diaphragmatic proteolysis observed after CMV, as indicated by changes in the gene expression profile of several proteolytic enzymes (6). The UPP is a contributor to CMV-induced proteolysis, as muscle atrophy F-box (MAFbx) and muscle RING-finger protein-1 (MuRF1) mRNA levels, two skeletal muscle-specific ubiquitin ligases, are up-regulated after 12 hours of CMV (7). Moreover, an increase in diaphragmatic 20S proteasome activity occurs after 18 hours of CMV (5). The Ca2+-dependent protease calpain cleaves key cytoskeletal proteins to release myofilaments in skeletal muscle, and is also activated in the diaphragm after 18 hours of CMV (5). Moreover, because the UPP cannot degrade intact myofilaments (8), activation of calpain to release myofilaments for subsequent degradation by the UPP may represent a rate-limiting step in skeletal muscle proteolysis. Finally, although the exact role of the lysosomal system in muscle atrophy remains unclear, a coordinate stimulation of the lysosomal process with either the UPP and/or the Ca2+-dependent calpains has been reported in various models of muscle wasting, including that with prolonged CMV (6, 9, 10). Although it was demonstrated that CMV exerted several deleterious effects on the diaphragm, only few protective countermeasures have been developed to minimize CMV-induced diaphragm dysfunction and atrophy. Intermittent spontaneous breathing during the course of CMV has been shown to protect the diaphragm against the deleterious effects of CMV, but could not fully preserve diaphragm force and atrophy (11). Furthermore, administration of the antioxidant Trolox has been shown to prevent CMV-induced diaphragm contractile impairments and to retard proteolysis (12). Because proteases play an important role in CMV-induced diaphragm dysfunction, we investigated whether administration of the protease inhibitor leupeptin during CMV would protect the diaphragm from the deleterious effects of prolonged mechanical ventilation. We hypothesized that inhibition of both calpain and lysosomal proteases would provide diaphragmatic protection against both CMV-induced contractile dysfunction and fiber atrophy. Some of the results of this study have been previously reported in the form of an abstract (13).
Experimental Procedure and Study Design Adult male Wistar rats (12–14 wk old) were randomly assigned to one of three groups: (1) an acutely anesthetized control group (C; n = 9); (2) an anesthetized group submitted to 24 hours of CMV receiving a single intramuscular injection of saline (saline-CMV; n = 10); and (3) an anesthetized group submitted to 24 hours of CMV receiving a single intramuscular injection of 15 mg/kg leupeptin (Sigma, Bornem, Belgium) at the start of CMV (leupeptin-CMV; n = 8). The half-life of leupeptin in the circulation after an intramuscular injection was shown to be about 5 hours (Stracher and colleagues, unpublished data). The study was approved by the animal experiments committee of the Medical Faculty of the Katholieke Universiteit Leuven. For additional details on methods, see the online supplement. The mechanical ventilation protocol is the same as previously described (11). Briefly, animals were anesthetized with an intraperitoneal injection of sodium pentobarbital, tracheostomized, and mechanically ventilated (control mode; VT, about 0.55 ml/100 g; frequency of breathing, 60 breaths/min) with a volume-driven small-animal ventilator (model 665A; Harvard Apparatus, Holliston, MA) for 24 hours. Animals breathed humidified and oxygenated air maintained at 37°C and received a continuous dose of anesthesia (2 mg/100 g/h) and heparin (2.8 units/h) during the course of CMV. Body temperature was continuously maintained at 37°C. Arterial blood pressure was monitored during the protocol and blood gases were measured at 12 and 24 hours. After 24 hours, segments of the costal diaphragm were removed for measurement of in vitro contractile properties, as previously described (11). Diaphragm was stored for further histochemical and biochemical analysis. In addition, weights of the diaphragm and gastrocnemius were measured. Blood samples were collected for plasma analysis of liver enzymes, urea, and creatinine to check for potential liver or renal toxicity.
Histology and Histochemistry
Cathepsin B Activity Assay
Calpain Activity
Statistical Analysis
Systemic and Biologic Response to Mechanical Ventilation Values for arterial blood pressure and blood gases were maintained within the normal range during the course of 24-hour CMV and did not differ between the two ventilated groups, as shown in Table 1. Initial body weights and muscle weights were not different between the three groups (Table 1). The dose of anesthesia was similar in the two ventilated groups (pooled values, 1.13 ± 0.12 mg/100g/h).
Plasma levels of liver enzymes, urea, and creatinine (Table 1) did not differ between the two mechanical ventilation groups, underlining that the dose of 15 mg/kg leupeptin used in the present study was not toxic for either the liver or the kidneys.
Diaphragm Contractile Properties
Diaphragm Histology and Morphometry Morphologic analysis of the diaphragm sections stained with hematoxylin and eosin did not show any abnormalities. Diaphragm fiber proportions were similar in all groups. There were no changes in diaphragm fiber dimensions in the leupeptin-CMV group compared with control animals. Although diaphragm CSA of type-I fibers remained unchanged between the three groups, saline-CMV induced significant atrophy of type IIx/b fibers (–30%; p < 0.05 vs. control animals). This decrease was also significantly different from leupeptin-CMV (Figure 2). Although there was no significant difference in CSA of fiber type IIa, a clear trend toward a decrease in CSA of type IIa fibers was observed in the saline-CMV group compared with control animals and leupeptin-CMV (–20%; p = 0.06).
Diaphragm Cathepsin B Activity Saline-CMV significantly elevated in vitro cathepsin B activity in the diaphragm compared with control animals (+90%; p < 0.01) and leupeptin-CMV (+60% p < 0.05), while cathepsin B activity in the diaphragm of the leupeptin-CMV group was not significantly different from the control group (Figure 3, upper panel). A significant inverse correlation was found between diaphragm cathepsin B activity and diaphragm tetanic force (Figure 3, lower panel). Similarly significant negative correlations exist between diaphragm cathepsin B activity and the force produced by the diaphragm at all stimulation frequencies (–0.60 < r < –0.51; p < 0.01). Moreover, cathepsin B activity in the diaphragm was inversely correlated with the CSA of diaphragm type IIa and IIx/b fibers (r = –0.58 and –0.62, respectively; p < 0.05).
Diaphragm Calpain Activity In the leupeptin-CMV group, the calpain activity, measured via Western blotting as the ratio of the 145/150-kD calpain-cleaved  II-spectrin to total II-spectrin, was not elevated in comparison with the control animals (Figure 4, upper panel). By contrast, diaphragm calpain activity was significantly higher in the diaphragm of the saline-CMV group compared with the other groups (+27% vs. control animals and +22% vs. leupeptin-CMV; p < 0.05). An inverse correlation was found between calpain activity and tetanic tension (Figure 4, lower panel). Moreover, the force produced by the diaphragm at all stimulation frequencies was negatively correlated with the calpain activity (–0.68 < r < –0.60; p < 0.01).
This study provides a new and important finding that administration of a protease inhibitor prevents diaphragmatic contractile dysfunction and atrophy caused by prolonged CMV. Indeed, our data reveal that administration of leupeptin, an inhibitor of lysosomal thiol proteases and calcium-activated protease, completely reversed diaphragm atrophy and contractile dysfunction induced by 24 hours of CMV. Moreover, leupeptin decreased the CMV-induced increase in both calpain and cathepsin B activity in the diaphragm.
Validation of the Model
Diaphragm Function and Atrophy A new and important finding is that a single injection of leupeptin completely abolished the CMV-induced diaphragmatic fiber atrophy and decrease in diaphragm force production. The mechanism responsible for this protection probably involves inhibition of proteolysis of diaphragm contractile proteins, as described subsequently here.
Diaphragm Proteolysis
Interestingly, all three proteolytic systems are likely to contribute to the elevated proteolysis observed in the diaphragm after CMV (5, 6). Identical to a previous report (5), our current data indicate that diaphragmatic calpain activity is significantly increased after 24 hours of CMV compared with control animals. Although cleavage of The physiologic role of the calpain system in the CMV-induced diaphragmatic contractile dysfunction is illustrated by the inverse correlation found between the calpain activity and the force produced by the diaphragm at all stimulation frequencies. Furthermore, the present study is the first showing that cathepsin B activity in the diaphragm is increased after 24 hours of CMV. Also, inverse correlations were found between the cathepsin B activity and diaphragmatic force production at all stimulation frequencies, as well as with the CSA of the type-IIx/b and -IIa fibers. This suggests that the CMV-induced diaphragm contractile dysfunction and diaphragm atrophy is likely the result of an activation of the calpain and the lysosomal system. Both may represent the earliest proteolytic events in the cascade of proteolysis, eventually leading to muscle atrophy. Indeed, several lines of evidence support this supposition. First, administration of leupeptin in a mouse model of muscular dystrophy has been shown to inhibit calpain activation and increase the diameter of diaphragmatic myofibers (14). Leupeptin is also known to reduce the elevated cathepsin B activity in skeletal muscles from septic rats, thereby limiting muscle atrophy and impairment of contractile force production (24). In the current study, administration of a single dose of leupeptin, concomitant with 24 hours of CMV, completely abolished the increase in diaphragmatic calpain and cathepsin B activity caused by CMV. Importantly, treatment with leupeptin also reversed the CMV-induced impairment in diaphragm contractile function and the diaphragmatic atrophy associated with prolonged CMV. We interpret these results as evidence that the deleterious effects of CMV on diaphragm function are, at least in part, mediated by the activation of the calpain and lysosomal protease systems.
Conclusions
The authors thank Mrs. Petra Weckx for cutting and staining the histologic sections.
Supported by Fonds voor Wetenschappelijk Onderzoek–Vlaanderen (FWO) (Scientific Research Foundation–Flanders) (G.0389.03), Katholieke Universiteit Leuven Research Foundation, and AstraZeneca.
* Dries Testelmans is a recipient of an FWO Aspirant fellowship. This article has an online supplement, which is accessible from this issue's table of contents at www.atsjournals.org Originally Published in Press as DOI: 10.1164/rccm.200609-1342OC on March 22, 2007 Conflict of Interest Statement: None of the authors has a financial relationship with a commercial entity that has an interest in the subject of this manuscript. Received in original form September 19, 2006; accepted in final form March 20, 2007
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