Sabah N. A. Hussain1,2, Mahroo Mofarrahi1,2, Ioanna Sigala3, Ho Cheol Kim4, Theodoros Vassilakopoulos3, Francois Maltais5, Ion Bellenis6, Rakesh Chaturvedi2, Stewart B. Gottfried1,2, Peter Metrakos7,8, Gawiyou Danialou1,9, Stefan Matecki10, Samir Jaber11, Basil J. Petrof1,2 and Peter Goldberg2.  American Journal of Respiratory and Critical Care Medicine Vol 182. pp. 1377-1386, (2010)
Rationale: Controlled mechanical ventilation (CMV) results in atrophy of the human diaphragm. The autophagy-lysosome pathway (ALP) contributes to skeletal muscle proteolysis, but its contribution to diaphragmatic protein degradation in mechanically ventilated patients is unknown.

Objectives: To evaluate the autophagy pathway responses to CMV in the diaphragm and limb muscles of humans and to identify the roles of FOXO transcription factors in these responses.

Methods: Muscle biopsies were obtained from nine control subjects and nine brain-dead organ donors. Subjects were mechanically ventilated for 2 to 4 hours and 15 to 276 hours, respectively. Activation of the ubiquitin-proteasome system was detected by measuring mRNA expressions of Atrogin-1, MURF1, and protein expressions of UBC2, UBC4, and the subunits of the 20S proteasome (MCP231). Activation of the ALP was detected by electron microscopy and by measuring the expressions of several autophagy-related genes. Total carbonyl content and HNE-protein adduct formation were measured to assess oxidative stress. Total AKT, phosphorylated and total FOXO1, and FOXO3A protein levels were also measured.

Measurements and Main Results: Prolonged CMV triggered activation of the ALP as measured by the appearance of autophagosomes in the diaphragm and increased expressions of autophagy-related genes, as compared with controls. Induction of autophagy was associated with increased protein oxidation and enhanced expression of the FOXO1 gene, but not the FOXO3A gene. CMV also triggered the inhibition of both AKT expression and FOXO1 phosphorylation.

Conclusions: We propose that prolonged CMV causes diaphragm disuse, which, in turn, leads to activation of the ALP through oxidative stress and the induction of the FOXO1 transcription factor.

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