Identification of mitochondrial deficits and melatonin targets in liver of septic mice by high-resolution respirometry
Introduction
Sepsis, a systemic inflammatory response of the organism against infection, represents the major cause of death in the intensive care units of developed countries [50], [51]. Liver dysfunction during sepsis is closely associated with high mortality in septic patients [54]. Moreover, a substantial body of evidence supports a relationship between mitochondrial dysfunction and sepsis [14], [15]. Thus, exhaustive study of the mitochondrial function may reveal new insights related to the pathophysiology of sepsis.
There are several pathways involved in mitochondrial dysfunction and ATP deficit during sepsis. At the early-onset of sepsis, the inflammatory response triggers mitochondrial morphological and structural alterations [8], [22], [66]. Then, poly (A) polymerase 1 (PARP-1) activation leads to a massive consumption of NAD+, decreasing its mitochondrial availability and reducing ATP production [38]. Later, proinflammatory cytokines generated during sepsis participate in the inhibition of pyruvate dehydrogenase (PDH), which reduces the substrates of OXPHOS, favoring an additional drop in ATP production [62], [73]. In parallel, the induction of inducible nitric oxide synthase (iNOS) during sepsis results in a significant elevation of nitric oxide (NO•) levels. In the mitochondria, NO• reversibly inhibits the complex IV of the ETC and, in a concentration-dependent manner, progressively inhibits other respiratory complexes [16], [19], [20], [72]. Moreover, NO• reacts with O2•− generating the highly toxic peroxynitrite (ONOO−) anions which irreversibly inhibit mitochondrial respiration [16]. Therefore, sepsis courses with an oxidative–nitrosative stress able to damage proteins, including respiratory complexes, DNA and membrane lipids, yielding severe mitochondrial dysfunction and bioenergetic failure [6], [23], [45]. Moreover, mitochondrial respiratory complexes can be assembled in supramolecular structures called supercomplexes (SC). This association provides greater efficiency facilitating the electron transfer through enzymatic respiratory complexes. SC formation is associated with an increase in electron flow efficiency and, therefore, in the ATP synthesis, minimizing the ROS generation [48]. The participation of each respiratory complex and/or SC in mitochondrial dysfunction during sepsis remains, however, unclear.
Melatonin (N-acetyl-5-methoxytryptamine) is a highly conserved and amphipathic indoleamine, present in all cellular compartments including mitochondria [4], [68]. There is consistent experimental background showing that mitochondria are the main intracellular target of melatonin [2], [3], [5]. Melatonin prevents septic shock and multiple organ failure reducing, iNOS expression and ROS generation, restoring ETC activity and ATP production [18], [24], [25], [44], [45], [47]. Therefore, an exhaustive study of the mitochondrial function with different substrate combinations and with the aid of high-resolution respirometry may identify new insights related to the pathophysiology of sepsis and the targets of melatonin.
Section snippets
Animals
Adult C57/BL6 male mice (3 months old) were provided by Harland (Barcelona, Spain). The animals were maintained in the University of Granada's facility under a 12 h light/dark cycle with lights on at 08:00 h, and a constant room temperature (22 °C ± 1 °C). Mice were housed in groups of five individuals per clear plastic cage with food and water ad libitum. All experiments were conducted in accordance with the University of Granada's Ethical Committee; the Spanish Protection Guide for Animal
Measurement of leak state
Leak state represents the oxygen consumption mainly used to compensate for the proton leak across the inner mitochondrial membrane in a non-phosphorylating respiration. To avoid an overestimation of leak state due to any contamination of ATPase activity (where ATP recycling can happen), we determined the leak state without adenylates (for CI and CI + II) or with oligomycin (for CII) [12]. Septic mice exhibited an upshift of leak during the early phase of sepsis (S8) with substrates for CI and for
Discussion
Mitochondrial impairment and bioenergetic failure have been related to elevated mortality in sepsis [13], [23], [73], although the mechanisms underlying mitochondrial dysfunction are unclear. The excess of NO• and ROS produced during the inflammatory response may inhibit the mitochondrial ETC complexes, reducing the electron flow through the ETC [6], [9], [23], [46], [62]. The subsequent electron leak promotes the formation of ROS and RNS, including the highly toxic ONOO−, which irreversibly
Conclusion
Here, using HRR, the analysis of mitochondrial respiration allowed us to identify the different changes in CII and CI function with sepsis, which could not be identify when respiration is analyzed through CI + CII together. Thus, we confirmed that liver mitochondrial dysfunction in sepsis mainly depends on CI impairment, and identified the targets involved in the beneficial effects of melatonin treatment. The results support that mitochondrial CI should be a main therapeutical approach in the
Conflict of interest statement
The authors declare that there are no conflicts of interest.
Acknowledgments
This study was partially supported by grants from the Instituto de Salud Carlos III (RD12/0043/0005, PI08-1664), and from the Consejería de Innovación, Ciencia y Empresa, Junta de Andalucía (P07-CTS-03135 and CTS-101), Spain; CD and JAG are supported by the Instituto de Salud Carlos III, Spain; HV is a PhD student supported by a FPU fellowship (Ministerio de Educación, Spain); MED-C and ML-S are PhD students from the Consejería de Innovación, Ciencia y Empresa, Junta de Andalucía, Spain, and
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