Estudio bioenergético mitocondrial en la sepsis experimental y efecto protector de la melatonina

Resumen

Sepsis, a systemic inflammatory response of the organism against an infection, represents the major cause of death in the intensive care units of developed countries. Previous studies demonstrated that septic process courses with an oxidative and nitrosative stress able to damage cellular structures (such as proteins, including respiratory complexes, DNA and membrane lipids). A substantial body of evidence supports a mitochondrial dysfunction associated with sepsis. Mitochondria play an important role in the energetic metabolism, but also in other processes in physiological and pathological conditions. Therefore, the analysis of mitochondrial function is crucial to understand a specific pathology such as sepsis. Melatonin (N-acetyl-5-methoxytryptamine) is an amphipathic indoleamine, present in all cellular compartments including mitochondria. Melatonin acts as a potent antioxidant and anti-inflammatory molecule with other important actions. Several works have been demonstrated the beneficial effects of melatonin in sepsis. According with this background, the present work was focused in the study of mitochondrial function during sepsis. Moreover, was evaluated the properties of melatonin against mitochondrial failure during sepsis. The objectives of this research were: 1. To determine the mitochondrial bioenergetics in liver and heart of mice at early and late phases of sepsis. 2. To identify mitochondrial substrate-dependent behavior during septic process in these tissues. 3. To assess the protective effect of melatonin administration to mice on mitochondrial function of liver and heart of septic mice. 4. To evaluate the mechanistics effects of melatonin on mitochondria using different substrate combinations in liver and heart of septic mice. The first chapter of this thesis is focused in the mitochondrial respiration in isolated mouse liver mitochondria with different substrate combinations (glutamate/malate, glutamate/malate/succinate or succinate/rotenone) to identify mitochondrial defects and melatonin targets during sepsis. Our results report a severe mitochondrial dysfunction in liver from septic mice, which mainly depends on a CI impairment. For this reason, mitochondrial CI should be a central therapeutic approach in the treatment of sepsis. Our work demonstrates the beneficial effects of melatonin treatment against the mitochondrial dysfunction, acting fundamentally when the damage is present. In the second chapter of this thesis, the respirometric study was obtained with permeabilized myocardial fibers by HRR. Permeabilized fibers allow to identify specific defects in mitochondrial bioenergetics as a result of sepsis without disruption due to mitochondrial isolation process. In this case, OXPHOS and ETS capacities with substrate combinations for CI+I and CII were strongly decreased in both phases of sepsis. In parallel, we found a loss in the proportion of complex III (CIII) incorporated in mitochondrial respiratory SC. Therefore, melatonin treatment prevented mitochondrial failure, preserving OXPHOS and ETS capacities. In conclusion, our results evidence an early severe sepsis-related mitochondrial impairment either in liver and myocardial tissues, which is counteracted by melatonin treatment.