Mitochondria-targeted antioxidants protect against mechanical ventilation-induced diaphragm weakness

Abstract

Background: Mechanical ventilation is a life-saving intervention used to provide adequate pulmonary ventilation in patients suffering from respiratory failure. However, prolonged mechanical ventilation is associated with significant diaphragmatic weakness resulting from both myofiber atrophy and contractile dysfunction. Although several signaling pathways contribute to diaphragm weakness during mechanical ventilation, it is established that oxidative stress is required for diaphragmatic weakness to occur. Therefore, identifying the site(s) of mechanical ventilation- induced reactive oxygen species production in the diaphragm is important.

Objective: These experiments tested the hypothesis that elevated mitochondrial reactive oxygen species emission is required for mechanical ventilation-induced oxidative stress, atrophy, and contractile dysfunction in the diaphragm.

Design: Cause and effect was determined by preventing mechanical ventilation-induced mitochondrial reactive oxygen species emission in the diaphragm of rats using a novel mitochondria-targeted antioxidant (SS-31).

Interventions: None.

Measurements and main results: Compared to mechanically ventilated animals treated with saline, animals treated with SS-31 were protected against mechanical ventilation-induced mitochondrial dysfunction, oxidative stress, and protease activation in the diaphragm. Importantly, treatment of animals with the mitochondrial antioxidant also protected the diaphragm against mechanical ventilation-induced myofiber atrophy and contractile dysfunction.

Conclusions: These results reveal that prevention of mechanical ventilation-induced increases in diaphragmatic mitochondrial reactive oxygen species emission protects the diaphragm from mechanical ventilation-induced diaphragmatic weakness. This important new finding indicates that mitochondria are a primary source of reactive oxygen species production in the diaphragm during prolonged mechanical ventilation. These results could lead to the development of a therapeutic intervention to impede mechanical ventilation-induced diaphragmatic weakness.

Figure 1

Rates of hydrogen peroxide (H₂O₂) release from mitochondria isolated from diaphragms of control (CON), mechanically ventilated (MV), and mechanically ventilated rats treated with the mitochondrial-targeted antioxidant SS-31 (MVSS). Note that compared to CON, 12 hours of MV resulted in a significant increase in mitochondrial H₂O₂ emission during both state-3 and state-4 respiration. Importantly, treatment of animals with SS-31 significantly reduced the rates of H₂O₂ release from the mitochondria following prolonged MV. Values are mean ± SEM. * = CON different (p<0.05) from MV; (n =6/group).

Figure 2

Levels of oxidatively modified proteins in the diaphragm of control (CON), mechanically ventilated (MV), and mechanically ventilated rats treated with the mitochondrial-targeted antioxidant SS-31 (MVSS). A) Levels of 4-hydroxyl-nonenal-conjugated proteins in the diaphragm of the three experimental groups. The image above the histograph is a representative western blot of data from the three experimental groups. B) Levels of protein carbonyls in the diaphragm of the three experimental groups. The image above the histograph is a representative western blot of data from the three experimental groups. * = different (p<0.05) from both CON and MVSS (n = 6/group).

Figure 3

Effects of prolonged MV on the diaphragmatic force-frequency response (in vitro) in control and mechanically ventilated rats with/without mitochondrial targeted antioxidants. No significant differences in diaphragmatic force production existed between the CON and MVSS groups at any stimulation frequency. Values are means ± SEM. Note that some of the SEM bars are not visible because of the small size. * = MV different (p<0.05) from both CON and MVSS; ** = MVSS different (p<0.05) from CON (n = 6/group).

Figure 4

Fiber cross-sectional area (CSA) in diaphragm muscle myofibers from control (CON) and mechanically ventilated rats with (MVSS) and without mitochondrial targeted antioxidants (MV). Note that no significant differences in diaphragmatic fiber CSA existed between the CON and MVSS groups in any fiber type. Values are means ± SEM. * = different (p<0.05) from both CON and MVSS (n = 6/group).

Figure 5

Activity of the 20S proteasome (5A), mRNA and protein levels of both atrogin-1(5B) and MuRF-1 (5C) in the diaphragm from control (CON) and mechanically ventilated animals with (MVSS) and without mitochondrial-targeted antioxidants (MV). The images above the histograms in Figures 5B and 5C are representative western blots of data from the three experimental groups. Values are means ± SEM. * = different (p<0.05) from both CON and MVSS. ** = different (p<0.05) from both CON and MV (n = 6/group).

Figure 5

Activity of the 20S proteasome (5A), mRNA and protein levels of both atrogin-1(5B) and MuRF-1 (5C) in the diaphragm from control (CON) and mechanically ventilated animals with (MVSS) and without mitochondrial-targeted antioxidants (MV). The images above the histograms in Figures 5B and 5C are representative western blots of data from the three experimental groups. Values are means ± SEM. * = different (p<0.05) from both CON and MVSS. ** = different (p<0.05) from both CON and MV (n = 6/group).

Figure 5

Activity of the 20S proteasome (5A), mRNA and protein levels of both atrogin-1(5B) and MuRF-1 (5C) in the diaphragm from control (CON) and mechanically ventilated animals with (MVSS) and without mitochondrial-targeted antioxidants (MV). The images above the histograms in Figures 5B and 5C are representative western blots of data from the three experimental groups. Values are means ± SEM. * = different (p<0.05) from both CON and MVSS. ** = different (p<0.05) from both CON and MV (n = 6/group).

Figure 6

Calpain 1 and Caspase 3 activity in the diaphragm from control (CON) and mechanically ventilated animals with (MVSS) and without mitochondrial-targeted antioxidants (MVSS). A) The active form of calpain 1 in diaphragm muscle at the completion of 12 hours of MV. B) The cleaved and active band of caspase-3 in diaphragm muscle at the completion of 12 hours of MV. The images above the histograms in Figure 6A and 6B are representative western blots of data from the three experimental groups. Values are means ± SEM. §= different (p<0.05) from CON; * = different (p<0.05) from both CON and MVSS (n=6/group).

Figure 7

Calpain and caspase-3 activity in the diaphragm from control (CON) and mechanically ventilated animals with (MVSS) without mitochondrial-targeted antioxidants (MV). A) Levels of the 145 kDa α-II-spectrin break-down product (SBPD) in diaphragm muscle following 12 hours of MV. Note that the SBDP 145 kDa is an α-II-spectrin break-down product that is specific to calpain cleavage of intact α-II-spectrin and therefore, the cellular level of SBDP 145 kDa can be used as a biomarker of in vivo calpain activity. B) Levels of the 120 kDa α-II-spectrin break-down product (SBPD 120 kDa) in diaphragm muscle following 12 hours of MV. Note that the SBDP 120 kDa is a α-II-spectrin break-down product that is specific to caspase-3 cleavage of intact α-II-spectrin and therefore, the cellular levels of SBDP 120 kDa can be used as a biomarker of caspase-3 activity. The images above the histograms in Figure 7A and 7B are representative western blots of data from the three experimental groups. Values are means ± SEM. * = different (p<0.05) from both CON and MVSS. ‡= different (p<0.05) from MVSS (n=6/group).

Figure 8

Ratio of actin to total sarcomeric protein levels in the diaphragm from control (CON) and mechanically ventilated animals with (MVSS) without mitochondrial-targeted antioxidants (MV). Given that actin is preferentially degraded during disuse muscle atrophy, assessment of the ratio of actin to total sarcomeric protein levels provides a relative index of diaphragmatic proteolysis during prolonged MV. The image above the histogram is a representative western blot of data from the three experimental groups. Values are means ± SEM. * = different (p<0.05) from both CON and MVSS (n=6/group).