Increased Proteolysis, Myosin Depletion, and Atrophic AKT-FOXO Signaling in Human Diaphragm Disuse

Rationale: Patients on mechanical ventilation who exhibit diaphragm inactivity for a prolonged time (case subjects) develop decreases in diaphragm force-generating capacity accompanied by diaphragm myofiber atrophy. Objectives: Our objectives were to test the hypotheses that increased proteolysis by the ubiquitin-proteasome pathway, decreases in myosin heavy chain (MyHC) levels, and atrophic AKT-FOXO signaling play major roles in eliciting these pathological changes associated with diaphragm disuse. Methods: Biopsy specimens were obtained from the costal diaphragms of 18 case subjects before harvest (cases) and compared with intraoperative specimens from the diaphragms of 11 patients undergoing surgery for benign lesions or localized lung cancer (control subjects). Case subjects had diaphragm inactivity and underwent mechanical ventilation for 18 to 72 hours, whereas this state in controls was limited to 2 to 4 hours. Measurements and Main Results: With respect to proteolysis in cytoplasm fractions, case diaphragms exhibited greater levels of ubiquitinated-protein conjugates, increased activity of the 265 proteasome, and decreased levels of MyHCs and a-actin. With respect to atrophic signaling in nuclear fractions, case diaphragms exhibited decreases in phosphorylated AKT, phosphorylated FOXO1, increased binding to consensus DNA sequence for Atrogin-1 and MuRF-1, and increased supershift of DNA-FOXO1 complexes with specific antibodies against FOXO1, as well as increased Atrogin-1 and MuRF-1 transcripts in whole myofiber lysates. Conclusions: Our findings suggest that increased activity of the ubiquitin-proteasome pathway, marked decreases in MyHCs, and atrophic AKT-FOXO signaling play important roles in eliciting the myofiber atrophy and decreases in diaphragm force generation associated with prolonged human diaphragm disuse.