Mitochondrial oxidative stress induces leaky ryanodine receptor during mechanical ventilation.

RATIONALE: Ventilator-induced diaphragm dysfunction (VIDD) increases morbidity and mortality in critical care patients. Although VIDD has been associated with mitochondrial oxidative stress and calcium homeostasis impairment, the underling mechanisms are still unknown. We hypothesized that diaphragmatic mitochondrial oxidative stress causes remodeling of the ryanodine receptor (RyR1)/calcium release channel, contributing to sarcoplasmic reticulum (SR) Caleak, proteolysis and VIDD. METHOD: In mice diaphragms mechanically ventilated for short (6 h) and long (12 h) period, we assessed mitochondrial ROS production, mitochondrial aconitase activity as a marker of mitochondrial oxidative stress, RyR1 remodeling and function, Cadependent proteolysis, TGFβ1 and STAT3 pathway, muscle fibers cross-sectional area, and diaphragm specific force production, with or without the mitochondrial targeted anti-oxidant peptide d-Arg-2', 6'-dimethyltyrosine-Lys-Phe-NH(SS31). MEASUREMENTS AND MAIN RESULTS: 6 h of mechanical ventilation (MV) resulted in increased mitochondrial ROS production, reduction of mitochondrial aconitase activity, increased oxidation, S-nitrosylation, S-glutathionylation and Ser-2844 phosphorylation of RyR1, depletion of stabilizing subunit calstabin1 from RyR1, increased SR Caleak. Preventing mROS production by SS31 treatment does not affect the TGFβ1 and STAT3 activation, which suggests that mitochondrial oxidative stress is a downstream pathway to TGFβ1 and STAT3, early involved in VIDD. This is further supported by the fact that SS-31 rescue all the other described cellular events and diaphragm contractile dysfunction induced by MV, while SS20, an analog of SS31 lacking antioxidant properties, failed to prevent these cellular events and the contractile dysfunction. Similar results were found in ventilated for 12 h. Moreover, SS31 treatment prevented calpain1 activity and diaphragm atrophy observed after 12 h of MV. This study emphasizes that mitochondrial oxidative stress during 6 h-MV contributes to SR Caleak via RyR1 remodeling, and diaphragm weakness, while longer periods of MV (12 h) were also associated with increased Ca-dependent proteolysis and diaphragm atrophy.