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NOS isoform activation is related to liver failure during sepsis, but the mechanisms driving mitochondrial impairment remain unclear. We induced sepsis by LPS administration to inducible nitric oxide synthase (iNOS -/- ) and neuronal nitric oxide synthase (nNOS -/- ) mice and their respective wild-type controls to examine the contribution of iNOS to mitochondrial failure in the absence of nNOS. To achieve this goal, the determination of messenger RNA (mRNA) expression and protein content of iNOS in cytosol and mitochondria, the mitochondrial respiratory complex content, and the levels of nitrosative and oxidative stress (by measuring 3-nitrotyrosine residues and carbonyl groups, respectively) were examined in the liver of control and septic mice. We detected strongly elevated iNOS mRNA expression and protein levels in liver cytosol and mitochondria of septic mice, which were related to enhanced oxidative and nitrosative stress, and with fewer changes in respiratory complexes. The absence of the iNOS, but not nNOS, gene absolutely prevented mitochondrial impairment during sepsis. Moreover, the nNOS gene did not modify the expression and the effects of iNOS here shown. Melatonin administration counteracted iNOS activation and mitochondrial damage and enhanced the expression of the respiratory complexes above the control values. These effects were unrelated to the presence or absence of nNOS. iNOS is a main target to prevent liver mitochondrial impairment during sepsis, and melatonin represents an efficient antagonist of these iNOS-dependent effects whereas it may boost mitochondrial respiration to enhance liver survival.

Analysis of mitochondrial function is crucial to understand their involvement in a given disease. High-resolution respirometry of permeabilized myocardial fibers in septic mice allows the evaluation of the bioenergetic system, maintaining mitochondrial ultrastructure and intracellular interactions, which are critical for an adequate functionality. OXPHOS and electron transport system (ETS) capacities were assessed using different substrate combinations. Our findings show a severe septic-dependent impairment in OXPHOS and ETS capacities with mitochondrial uncoupling at early and late phases of sepsis. Moreover, sepsis triggers complex III (CIII)-linked alterations in supercomplexes structure, and loss of mitochondrial density. In these conditions, melatonin administration to septic mice prevented sepsis-dependent mitochondrial injury in mitochondrial respiration. Likewise, melatonin improved cytochrome b content and ameliorated the assembly of CIII in supercomplexes. These results support the use of permeabilized fibers to identify properly the respiratory deficits and specific melatonin effects in sepsis., (Copyright © 2015 Elsevier B.V. and Mitochondria Research Society. All rights reserved.)

The connection between the innate immune system, clock genes, and mitochondrial bioenergetics was analyzed during aging and sepsis in mouse heart. Our results suggest that the sole NF-κB activation does not explain the inflammatory process underlying aging; the former also triggers the NLRP3 inflammasome that enhances caspase-1-dependent maturation of IL-1β. In this way, aged mice enter into a vicious cycle as IL-1β further activates the NF-κB/NLRP3 inflammasome link. The origin of NF-κB activation was related to the age-dependent Bmal1/Clock/RORα/Rev-Erbα loop disruption, which lowers NAD(+) levels, reducing the SIRT1 deacetylase ability to inactivate NF-κB. Consequently, NF-κB binding to DNA increases, raising the formation of proinflammatory mediators and inducing mitochondrial impairment. The cycle is then closed with the subsequent NLRP3 inflammasome activation. This paired contribution of the innate immune pathways serves as a catalyst to magnify the response to sepsis in aged compared with young mice. Melatonin administration blunted the septic response, reducing inflammation and oxidative stress, and enhancing mitochondrial function at the levels of nonseptic aged mice, but it did not counteract the age-related inflammation. Together, our results suggest that, although with different strengths, chronoinflammaging constitutes the biochemical substrate of aging and sepsis, and identifies the NLRP3 inflammasome as a new molecular target for melatonin, providing a rationale for its use in NLRP3-dependent diseases., (© 2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

We determined the NF-κB- and NOD-like receptor (NLR)P3-dependent molecular mechanisms involved in sepsis and evaluated the role of retinoid-related orphan receptor (ROR)-α in melatonin's anti-inflammatory actions. Western blot, RT-PCR, ELISA, and spectrophotometric analysis revealed that NF-κB and NLRP3 closely interact, leading to proinflammatory and pro-oxidant status in heart tissue of septic C57BL/6J mice. Moreover, mitochondrial oxygen consumption was reduced by 80% in septic mice. In vivo and in vitro analysis showed that melatonin administration blunts NF-κB transcriptional activity through a sirtuin1-dependent NF-κB deacetylation in septic mice. Melatonin also decreased NF-κB-dependent proinflammatory response and restored redox balance and mitochondrial homeostasis, thus inhibiting the NLRP3 inflammasome. In an important finding, the inhibition of NF-κB by melatonin, but not that of NLRP3, was blunted in RORα (sg/sg) mice, indicating that functional RORα transcription factor is necessary for the initiation of the innate immune response against inflammation. Our results are evidence of the NF-κB/NLRP3 connection during sepsis and identify NLRP3 as a novel molecular target for melatonin. The multiple molecular targets of melatonin in this study explain its potent anti-inflammatory efficacy against systemic innate immune activation and herald a promising therapeutic application for melatonin in the treatment of sepsis., (© FASEB.)

Aims: Previous data showed that melatonin maintains liver mitochondrial homeostasis during sepsis, but neither the mechanisms underlying mitochondrial dysfunction nor the target of melatonin are known., Main Methods: Here, we analyzed mitochondrial respiration in isolated mouse liver mitochondria with different substrate combinations (glutamate/malate, glutamate/malate/sucinate or succinate/rotenone) to identify mitochondrial defects and melatonin targets during sepsis. Other bioenergetic parameters including a + a3, b, and c + c1 content, mitochondrial mass, and mitochondrial supercomplexes formation were analyzed. Mitochondrial function was assessed during experimental sepsis induced by cecal ligation and puncture (CLP) in livers of 3 mo. C57BL/6 mice at early and late phases of sepsis, i.e., at 8 and 24 h after sepsis induction., Key Findings: Septic mice showed mitochondrial injury with a decrease in state 3, respiratory control rate, mitochondrial mass, and cytochrome b and c + c1 content, which was prevented by melatonin treatment. Mitochondrial dysfunction in sepsis was mainly linked to complex I damage, because complex II was far less impaired. These mitochondria preserved the respiratory supramolecular organization, maintaining their electron transport system capacity., Significance: This work strengthens the use of substrate combinations to identify specific respiratory defects and selective melatonin actions in septic mitochondria. Targeting mitochondrial complex I should be a main therapeutical approach in the treatment of sepsis, whereas the use of melatonin should be considered in the therapy of clinical sepsis.

While it is accepted that the high production of nitric oxide (NO˙) by the inducible nitric oxide synthase (iNOS) impairs cardiac mitochondrial function during sepsis, the role of neuronal nitric oxide synthase (nNOS) may be protective. During sepsis, there is a significantly increase in the expression and activity of mitochondrial iNOS (i-mtNOS), which parallels the changes in cytosolic iNOS. The existence of a constitutive NOS form (c-mtNOS) in heart mitochondria has been also described, but its role in the heart failure during sepsis remains unclear. Herein, we analyzed the changes in mitochondrial oxidative stress and bioenergetics in wild-type and nNOS-deficient mice during sepsis, and the role of melatonin, a known antioxidant, in these changes. Sepsis was induced by cecal ligation and puncture, and heart mitochondria were analyzed for NOS expression and activity, nitrites, lipid peroxidation, glutathione and glutathione redox enzymes, oxidized proteins, and respiratory chain activity in vehicle- and melatonin-treated mice. Our data show that sepsis produced a similar induction of iNOS/i-mtNOS and comparable inhibition of the respiratory chain activity in wild-type and in nNOS-deficient mice. Sepsis also increased mitochondrial oxidative/nitrosative stress to a similar extent in both mice strains. Melatonin administration inhibited iNOS/i-mtNOS induction, restored mitochondrial homeostasis in septic mice, and preserved the activity of nNOS/c-mtNOS. The effects of melatonin were unrelated to the presence or the absence of nNOS. Our observations show a lack of effect of nNOS on heart bioenergetic impairment during sepsis and further support the beneficial actions of melatonin in sepsis., (© 2013 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

While it is accepted that the high production of nitric oxide ( NO˙) by the inducible nitric oxide synthase ( iNOS) impairs cardiac mitochondrial function during sepsis, the role of neuronal nitric oxide synthase ( nNOS) may be protective. During sepsis, there is a significantly increase in the expression and activity of mitochondrial iNOS (i-mt NOS), which parallels the changes in cytosolic iNOS. The existence of a constitutive NOS form (c-mt NOS) in heart mitochondria has been also described, but its role in the heart failure during sepsis remains unclear. Herein, we analyzed the changes in mitochondrial oxidative stress and bioenergetics in wild-type and nNOS-deficient mice during sepsis, and the role of melatonin, a known antioxidant, in these changes. Sepsis was induced by cecal ligation and puncture, and heart mitochondria were analyzed for NOS expression and activity, nitrites, lipid peroxidation, glutathione and glutathione redox enzymes, oxidized proteins, and respiratory chain activity in vehicle- and melatonin-treated mice. Our data show that sepsis produced a similar induction of iNOS/i-mt NOS and comparable inhibition of the respiratory chain activity in wild-type and in nNOS-deficient mice. Sepsis also increased mitochondrial oxidative/nitrosative stress to a similar extent in both mice strains. Melatonin administration inhibited iNOS/i-mt NOS induction, restored mitochondrial homeostasis in septic mice, and preserved the activity of nNOS/c-mt NOS. The effects of melatonin were unrelated to the presence or the absence of nNOS. Our observations show a lack of effect of nNOS on heart bioenergetic impairment during sepsis and further support the beneficial actions of melatonin in sepsis. [ABSTRACT FROM AUTHOR]