Abstract
BackgroundTissue ATP depletion and oxidative stress have been associated with the severe outcomes of septic shock. One of the compensatory mechanisms to alleviate the sepsis-induced mitochondrial dysfunction could be the increase in oxidative phosphorylation efficiency (ATP/O). We propose to study liver mitochondrial function and oxidative stress and the regulatory mechanism of mitochondrial oxidative phosphorylation efficiency in an animal model of sepsis.MethodsWe induced sepsis in rats by cecal ligation and perforation (CLP). Six, 24, or 36 h following CLP, we measured liver mitochondrial respiration, cytochrome c oxidase activity, and membrane permeability. We determine oxidative phosphorylation efficiency, by measuring ATP synthesis related to oxygen consumption at various exogenous ADP concentrations. Finally, we measured radical oxygen species (ROS) generation by liver mitochondria and mRNA concentrations of UCP2, biogenesis factors, and cytokines at the same end points.ResultsCLP rats presented hypotension, lactic acidosis, liver cytolysis, and upregulation of proinflammatory cytokines mRNA as compared to controls. Liver mitochondria showed a decrease in ATP synthesis and oxygen consumption at 24 h following CLP. A marked uncoupling of oxidative phosphorylation appeared 36 h following CLP and was associated with a decrease in cytochrome c oxidase activity and content and ATP synthase subunit β content (slip mechanism) and an increase in mitochondrial oligomycin-insensitive respiration, but no change in mitochondrial inner membrane permeability (no leak). Upregulation of UCP2 mRNA resulted in a decrease in mitochondrial ROS generation 24 h after the onset of CLP, whereas ROS over-generation associated with slip at cytochrome c oxidase observed at 36 h was concomitant with a decrease in UCP2 mRNA expression.ConclusionsDespite a compensatory increase in mitochondrial biogenesis factors, liver mitochondrial functions remain altered after CLP. This suggests that the functional compensatory mechanisms reported in the present study (slip at cytochrome c oxidase and biogenesis factors) were not strong enough to increase oxidative phosphorylation efficiency and failed to limit liver mitochondrial ROS over-generation. These data suggest that treatments based on cytochrome c infusion could have a role in mitochondrial dysfunction and/or ROS generation associated with sepsis.
Highlights
Tissue Adenosine triphosphate (ATP) depletion and oxidative stress have been associated with the severe outcomes of septic shock
The corresponding rates of ATP synthesis for the same amount of exogenous ADP added were lower at 24 h (144.51 ± 4.57, p = 0.0034; 82.02 ± 10.09, p = 0.0492; 47.47 ± 4.95 p = 0.0471; 12.84 ± 1.74, p = 0.0040) and 36 h (62.83 ± 12.71, p = 0.0446; 20.80 ± 3.43, p = 0.0020; 27.37 ± 3.32, p = 0.0274; 12.80 ± 7.50, p = 0.0080, n = 6) compared to matching sham-operated control rats at 24 h (265.31 ± 7.00, 121.06 ± 11.32, 93.33 ± 3.10, 38.83 ± 5.05, n = 6) and 36 h (144.16 ± 8.82, 117.83 ± 10.37, 81.16 ± 6.82, 32.16 ± 8.5, n = 6) but not at 6 h after the onset of cecal ligation and perforation (CLP). These results indicate that oxidative phosphorylation efficiency, i.e., the amount of ATP synthesized per amount of oxygen consumed (ATP/O ratios), was not altered early after the onset of CLP but decreased after 36 h of sepsis induction
The decrease in oxygen consumption was associated with a proportional decrease in ATP generation 24 h after the onset of CLP, whereas at 36 h the depletion in ATP synthesis was not associated with a proportional drop in oxygen consumption, indicating a reduction in the mitochondrial oxidative phosphorylation efficiency (ATP/O)
Summary
Tissue ATP depletion and oxidative stress have been associated with the severe outcomes of septic shock. To adjust mitochondrial activity to ATP demand in stress conditions such as septic shock, the activity and/or efficiency of oxidative phosphorylation have to be fine-tuned [16] This fine-tuning involves the control of (i) the inner mitochondrial membrane permeability (which regulates proton leakage), (ii) the activity and/or content of the mitochondrial respiratory chain proteins and ATP synthase, as well as (iii) the mechanisms preventing over-generation of mitochondrial reactive oxygen species (ROS). Among these steps, changes in the cytochrome c oxidase stoichiometry and/or activity [17, 18], the proton leak activity or the transcription of the UCP2 gene, and an increase in mitochondrial non-coupled respiration with phosphorylation are thought to play special roles [19, 20]
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