Background Mechanical ventilation (MV) is a life-saving intervention in patients with

Background Mechanical ventilation (MV) is a life-saving intervention in patients with acute respiratory failure. animal model of MV using a highly specific CH5424802 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 Itgb5 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. CH5424802 Although numerous factors can contribute to weaning difficulties the most frequent cause of weaning failure in patients is inspiratory (i.e. diaphragm) muscle weakness. CH5424802 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 CH5424802 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.