Oxidants Regulated Diaphragm Proteolysis during Mechanical Ventilation in Rats

Background: Diaphragm dysfunction and atrophy develop during prolonged controlled mechanical ventilation. Fiber atrophy has been attributed to activation of the proteasome and autophagy proteolytic pathways. Oxidative stress activates the proteasome during controlled mechanical ventilation, but it is unclear whether it also activates autophagy. This study investigated whether pretreatment with the antioxidant N-acetylcysteine affects controlled mechanical ventilation-induced diaphragm contractile dysfunction, fiber atrophy, and proteasomal and autophagic pathway activation. The study also explored whether proteolytic pathway activity during controlled mechanical ventilation is mediated by microRNAs that negatively regulate ubiquitin E3 ligases and autophagy-related genes. Methods: Three groups of adult male rats were studied (n = 10 per group). The animals in the first group were anesthetized and allowed to spontaneously breathe. Animals in the second group were pretreated with saline before undergoing controlled mechanical ventilation for 24 h. The animals in the third group were pretreated with N-acetylcysteine (150 mg/kg) before undergoing controlled mechanical ventilation for 24 h. Diaphragm contractility and activation of the proteasome and autophagy pathways were measured. Expressions of microRNAs that negatively regulate ubiquitin E3 ligases and autophagy-related genes were measured with quantitative polymerase chain reaction. Results: Controlled mechanical ventilation decreased diaphragm twitch force from 428 +/- 104 g/cm(2) (mean +/- SD) to 313 +/- 50 g/cm(2) and tetanic force from 2,491 +/- 411 g/cm(2) to 1,618 +/- 177 g/cm(2). Controlled mechanical ventilation also decreased diaphragm fiber size, increased expression of several autophagy genes, and augmented Atrogin-1, MuRF1, and Nedd4 expressions by 36-, 41-, and 8-fold, respectively. Controlled mechanical ventilation decreased the expressions of six microRNAs (miR-20a, miR-106b, miR-376, miR-101a, miR-204, and miR-93) that regulate autophagy genes. Pretreatment with N-acetylcysteine prevented diaphragm contractile dysfunction, attenuated protein ubiquitination, and downregulated E3 ligase and autophagy gene expression. It also reversed controlled mechanical ventilation-induced microRNA expression decreases. N-Acetylcysteine pretreatment had no affect on fiber atrophy. Conclusions: Prolonged controlled mechanical ventilation activates the proteasome and autophagy pathways in the diaphragm through oxidative stress. Pathway activation is accomplished, in part, through inhibition of microRNAs that negatively regulate autophagy-related genes.