Mechanical ventilation induces diaphragmatic mitochondrial dysfunction and increased oxidant production

Abstract

Mechanical ventilation (MV) is a life-saving intervention used in patients with acute respiratory failure. Unfortunately, prolonged MV results in diaphragmatic weakness, which is an important contributor to the failure to wean patients from MV. Our laboratory has previously shown that reactive oxygen species (ROS) play a critical role in mediating diaphragmatic weakness after MV. However, the pathways responsible for MV-induced diaphragmatic ROS production remain unknown. These experiments tested the hypothesis that prolonged MV results in an increase in mitochondrial ROS release, mitochondrial oxidative damage, and mitochondrial dysfunction. To test this hypothesis, adult (3-4 months of age) female Sprague-Dawley rats were assigned to either a control or a 12-h MV group. After treatment, diaphragms were removed and mitochondria were isolated for subsequent respiratory and biochemical measurements. Compared to control, prolonged MV resulted in a lower respiratory control ratio in diaphragmatic mitochondria. Furthermore, diaphragmatic mitochondria from MV animals released higher rates of ROS in both State 3 and State 4 respiration. Prolonged MV was also associated with diaphragmatic mitochondrial oxidative damage as indicated by increased lipid peroxidation and protein oxidation. Finally, our data also reveal that the activities of the electron transport chain complexes II, III, and IV are depressed in mitochondria isolated from diaphragms of MV animals. In conclusion, these results are consistent with the concept that diaphragmatic inactivity promotes an increase in mitochondrial ROS emission, mitochondrial oxidative damage, and mitochondrial respiratory dysfunction.

Fig. 1

Representative transmission electron microscopy image of mitochondria isolated from control diaphragms illustrating structural integrity.

Fig. 2

(A) State 3 respiration, (B) State 4 respiration, and (C) respiratory control ratio (RCR) in mitochondria isolated from diaphragms of control (n = 8) and mechanically ventilated (MV12) (n = 7) animals. The left illustrates data obtained using pyruvate/malate (PYR/MAL) as substrate, whereas the right shows data obtained using succinate as substrate. Data are presented as means ± standard error of the mean. *p < 0.05.

Fig. 3

(A) State 3 respiration, (B) State 4 respiration, and (C) RCR in mitochondria isolated from hind-limb muscles of control (n = 8) and mechanically ventilated (MV12) (n = 7) animals. The left illustrates data obtained using pyruvate/malate (PYR/MAL) as substrate, whereas the right shows data obtained using succinate as substrate. Data are presented as means ± standard error of the mean. *p < 0.05.

Fig. 4

Mitochondrial ROS production from (A) diaphragms of control (n = 7) and mechanically ventilated (MV12) (n = 7) animals and from (B) hind-limb muscles of control (n = 7) and mechanically ventilated (MV12) (n = 7) animals. Note that mitochondria isolated from diaphragms of MV12 animals released higher levels of ROS compared to controls in both State 3 and State 4. Data are presented as means ± standard error of the mean. *p < 0.05.

Fig. 5

(A) The levels of 4-hydroxynonenal in diaphragm mitochondria were analyzed as an indicator of lipid peroxidation via Western blotting. (B) Oxidized proteins in diaphragm mitochondria were quantified by Western blot using the oxy-blot technique. Representative Western blots for 4-hydroxynonenal and protein oxidation levels are shown above the graphs. Note that 12 h of mechanical ventilation (MV12) (n = 7) resulted in increased lipid peroxidation and protein oxidation in diaphragmatic mitochondria compared to control (n = 8). Data are presented as means ± standard error of the mean. *p < 0.05.

Fig. 6

(A) SOD1, (B) SOD2, (C) catalase, and (D) GPX1 protein levels in mitochondria isolated from diaphragms of control (n = 8) and mechanically ventilated (MV12) (n = 7) animals. Representative Western blots are shown above the graphs. Data are presented as means ± standard error of the mean. *p < 0.05.

Fig. 7

(A) Complex I, (B) complex II, (C) complex III, and (D) complex IV activity in mitochondria isolated from diaphragms of control (n = 8) and mechanically ventilated (MV12) (n = 7) animals was evaluated by spectrophotometric methods. Note that complex II, complex III, and complex IV activity was reduced in mitochondria isolated from mechanically ventilated animals compared to controls. Data are presented as means ± standard error of the mean. *p < 0.05.