Attenuation of cardiac mitochondrial dysfunction by melatonin in septic mice ´ ´ ´ ´ Germaine Escames1, Luis C Lopez1, Francisco Ortiz1, Ana Lopez1, Jose A Garcıa1, Eduardo Ros2 ´ ˜ and Darıo Acuna-Castroviejo1,3 ´ ´ Instituto de Biotecnologıa, Departamento de Fisiologıa, Universidad de Granada, Spain ´ ´ Servicio de Angiologıa y Cirugıa Vascular, Hospital Universitario San Cecilio, Granada, Spain ´lisis Clınicos, Hospital Universitario San Cecilio, Granada, Spain ´ Servicio de Ana Keywords ATP production; mitochondrial failure; mitochondrial nitric oxide synthase; oxidative stress; therapy Correspondence D Acuna-Castroviejo, Departamento de ˜ ´ Fisiologıa, Facultad de Medicina, Avenida de Madrid 11, E-18012, Spain Fax: +34 958246295 Tel: +34 958246631 E-mail: dacuna@ugr.es (Received December 2006, revised February 2007, accepted 23 February 2007) doi:10.1111/j.1742-4658.2007.05755.x The existence of an inducible mitochondrial nitric oxide synthase has been recently related to the nitrosative ⁄ oxidative damage and mitochondrial dysfunction that occurs during endotoxemia Melatonin inhibits both inducible nitric oxide synthase and inducible mitochondrial nitric oxide synthase activities, a finding related to the antiseptic properties of the indoleamine Hence, we examined the changes in inducible nitric oxide synthase ⁄ inducible mitochondrial nitric oxide synthase expression and activity, bioenergetics and oxidative stress in heart mitochondria following cecal ligation and puncture-induced sepsis in wild-type (iNOS+ ⁄ +) and inducible nitric oxide synthase-deficient (iNOS– ⁄ –) mice We also evaluated whether melatonin reduces the expression of inducible nitric oxide synthase ⁄ inducible mitochondrial nitric oxide synthase, and whether this inhibition improves mitochondrial function in this experimental paradigm The results show that cecal ligation and puncture induced an increase of inducible mitochondrial nitric oxide synthase in iNOS+ ⁄ + mice that was accompanied by oxidative stress, respiratory chain impairment, and reduced ATP production, although the ATPase activity remained unchanged Real-time PCR analysis showed that induction of inducible nitric oxide synthase during sepsis was related to the increase of inducible mitochondrial nitric oxide synthase activity, as both inducible nitric oxide synthase and inducible mitochondrial nitric oxide synthase were absent in iNOS– ⁄ – mice The induction of inducible mitochondrial nitric oxide synthase was associated with mitochondrial dysfunction, because heart mitochondria from iNOS– ⁄ – mice were unaffected during sepsis Melatonin treatment blunted sepsis-induced inducible nitric oxide synthase ⁄ inducible mitochondrial nitric oxide synthase isoforms, prevented the impairment of mitochondrial homeostasis under sepsis, and restored ATP production These properties of melatonin should be considered in clinical sepsis Sepsis-induced multiple organ failure is the major cause of mortality in critically ill patients, and its incidence is rising [1] The heart and cardiovascular sys- tems are seriously affected during sepsis [2] Although myocardial impairment in sepsis has been extensively studied, its etiology remains unclear [3] Some reports Abbreviations CLP, cecal ligation and puncture; ETC, electron transport chain; GPx, glutathione peroxidase; GRd, glutathione reductase; GSH, glutathione; GSSG, oxidized glutathione; iNOS, inducible nitric oxide synthase; i-mtNOS, inducible mitochondrial nitric oxide synthase; LPO, lipid peroxidation; mtNOS, constitutive mitochondrial nitric oxide synthase; nNOS, neuronal nitric oxide synthase FEBS Journal 274 (2007) 2135–2147 ª 2007 The Authors Journal compilation ª 2007 FEBS 2135 NOS and heart mitochondrial dysfunction in sepsis G Escames et al have shown that mitochondria are the primary targets injured in both vital and nonvital organs during inflammation [4–6] Besides other factors, mitochondrial dysfunction in sepsis is directly associated with an increase in reactive oxygen species and reactive nitrogen species [6–10] It has been shown that mitochondria from several organs, such as lungs, liver, diaphragm, and hind leg skeletal muscle, contain an inducible mitochondrial nitric oxide synthase (i-mtNOS) [9,11–13], which is encoded by the same gene as cytosolic inducible nitric oxide synthase (iNOS) [9,11,12,14,15] Moreover, mitochondria contain a constitutive NOS (mtNOS) derived from neuronal NOS (nNOS) [16,17] The expression and activity of i-mtNOS, but not those of mtNOS, increase during sepsis [9,11,12] Other studies have shown induction of mitochondrial NOS in the diaphragm and heart of septic rats, although these reports did not distinguish between constitutive and inducible forms [8,18] Increasing evidence suggests that the nitric oxide (NO) produced by i-mtNOS plays a role in mitochondrial dysfunction during sepsis [9,11,12] Because iNOS– ⁄ – mice not express i-mtNOS, and the mitochondria of these mice were unaffected by sepsis, it was suggested that the overproduction of NO by i-mtNOS is the main factor responsible for mitochondrial nitrosative ⁄ oxidative stress and impairment during endotoxemia [9,12] The induction of i-mtNOS after lipopolysaccharide administration leads to an increase in NO and other reactive species, such as superoxide anion (O2–), hydrogen peroxide (H2O2) and peroxynitrite (ONOO–), in heart and diaphragm mitochondria [8,18] Cecal ligation and puncture (CLP) also induces i-mtNOS in these tissues, increasing mitochondrial lipid peroxidation (LPO) and the oxidized glutathione ⁄ glutathione (GSSG ⁄ GSH) ratio, and reducing the activity of the electron transport chain (ETC) complexes [9,11,12] Although NO is a physiologic modulator of mitochondrial respiration [19,20], high levels of NO may inhibit the ETC, increasing the formation of O2– and H2O2 [21] NO reacts with O2– to yield ONOO–, which in turn impairs the ETC and ATP synthase [19] The parallel failure of the respiratory chain and oxidative phosphorylation leads to mitochondrial dysfunction, energy depletion, and cell death A reduction in the capacity of the mitochondria to produce ATP may be related to the organ failure in sepsis [4,5,22] There is evidence that antioxidants may be useful in protecting against mitochondrial damage induced by oxidative and ⁄ or nitrosative stress [23] Several reports have shown that melatonin (aMT) protects against 2136 mitochondrial oxidative stress, due to its antioxidant properties and its ability to enter mitochondria [24– 28] In muscular tissues such as skeletal muscle and diaphragm of septic mice, aMT administration inhibited the activity of i-mtNOS, restoring the mitochondrial GSH pool and the ETC activity in these animals [9,12,29] Mitochondrial dysfunction is an important pathophysiologic event related to heart failure during sepsis, and i-mtNOS may be directly related to it To address this question, we induced sepsis by CLP in iNOS+ ⁄ + and iNOS– ⁄ – mice, and explored in heart mitochondria: (a) the presence and source of i-mtNOS; (b) the relationship between i-mtNOS induction, ETC dysfunction, and oxidative phosphorylation activity; (c) the steady-state energy and ATP production; and (d) the protective effect of aMT against mitochondrial damage produced during sepsis Results Mitochondrial NOS activities Figure shows that heart mitochondria from iNOS+ ⁄ +mice contain two mitochondrial NOS isoforms: a constitutive, Ca2+-dependent form (mtNOS), and an inducible, Ca2+-independent form (i-mtNOS) In iNOS+ ⁄ + mice, sepsis induced a significant increase in i-mtNOS activity, whereas mtNOS activity remained unchanged (Fig 1A) Control iNOS– ⁄ – mice exhibited only the constitutive component of mitochondrial NOS that was partially inhibited during sepsis (Fig 1B) aMT administration counteracted sepsisinduced i-mtNOS activity in iNOS+ ⁄ + mice, without affecting mtNOS activity (Fig 1A) aMT also restored mtNOS activity that had been depressed by sepsis in mutant mice (Fig 1B) Some considerations should be borne in mind regarding the purity of the mitochondrial preparation used here Heart mitochondria were isolated by differential centrifugation, and purified by Percoll centrifugation [9,11,43] To remove contaminants, purified mitochondria were washed with high ionic strength solution (150 mm KCl) This protocol yields a very pure mitochondrial fraction without contaminating organelles and broken mitochondria, as reported elsewhere [11,12] The lack of mitochondrial contamination with cytosolic NOS was assessed by the absence of any detectable NOS activity and nitrite levels in the supernatant of the final centrifugation step (data not shown) These data confirm the purity of the mitochondria used in our experiments, and guarantee the mitochondrial origin of the NOS activity reported FEBS Journal 274 (2007) 2135–2147 ª 2007 The Authors Journal compilation ª 2007 FEBS G Escames et al NOS and heart mitochondrial dysfunction in sepsis Fig Total heart mitochondrial NOS activity comprises constitutive, Ca2+-dependent, and inducible, Ca2+-independent, components in iNOS+ ⁄ + mice (A) Deficient iNOS mice, however, show only the constitutive, Ca2+-dependent component (B) In both cases, mice were subjected to CLP to induce sepsis, and killed 24 h later Pure mitochondrial preparations were used to determine NOS activity with L-[3H]arginine as substrate Data represent the means ± SE of six experiments per group C, control; S, sepsis; S + aMT, sepsis + aMT *P < 0.05, **P < 0.01, ***P < 0.001 versus C; #P < 0.05, ###P < 0.001 versus S here Moreover, the method used for NOS measurement specifically detects mtNOS activity, and the addition of NG-monomethyl-l-arginine (l-NMMA) (300 lm) to the reaction mixture of mitochondrial samples from septic mice blocked the transformation of l-arginine to l-citrulline, due to mtNOS inhibition (14.67 ± 3.09 versus 1.09 ± 0.87 pmol citrullinmin)1Ỉmg)1 protein, CLP and CLP + l-NMMA, respectively) [9,11,12] iNOS+ ⁄ + mice exhibited a slight increase in nitrite level after sepsis, which was counteracted by aMT treatment (Table 1) Interestingly, iNOS– ⁄ – mice showed a significant decrease in nitrite level during sepsis, coinciding with the mtNOS activity inhibition, that was partially counteracted by aMT iNOS mRNA expression Figure shows the results obtained in quantitative RT-PCR experiments Because this is a semiquantita- tive technique, the data are expressed as the relative quantity of mRNA in experimental versus control samples, giving to the control samples a value ¼ after deducting basal and background values Sepsis resulted in a significant increase in the transcription of the mRNA encoding iNOS in heart of iNOS+ ⁄ + mice The expression of iNOS mRNA was not detected in iNOS– ⁄ – mice Treatment with aMT absolutely counteracted the transcription of iNOS mRNA induced by sepsis Mitochondrial oxidative stress Sepsis significantly increased LPO levels in heart mitochondria from iNOS+ ⁄ + mice, whereas aMT administration reduced LPO below the control values (Table 1) Sepsis, however, did not modify the levels of LPO in iNOS– ⁄ – mice, although aMT also reduced them below control values Table Effects of sepsis and aMT treatment on the mitochondrial nitrite and LPO levels, and on the activity of the mitochondrial ATPase in wild-type and iNOS knockout mice C, control; S, sepsis; S + aMT, sepsis + aMT Sepsis was induced by CLP, and the animals were killed 24 h later Nitrite, nmolỈmg protein)1; LPO, nmolỈmg protein)1; ATPase, nmol PiỈmin)1Ỉmg protein)1 Data are means ± SE, n ¼ iNOS+ ⁄ + iNOS– ⁄ – C Nitrite LPO ATPase S S + aMT C S S + aMT 3.07 ± 0.27 2.33 ± 0.1 2641 ± 169 3.23 ± 0.31 2.94 ± 0.1* 2753 ± 188 2.63 ± 0.29 2.05 ± 0.09*,  1430 ± 97 3.05 ± 0.24 2.53 ± 0.12 2280 ± 201 2.23 ± 0.34* 2.48 ± 0.9 2452 ± 134 2.48 ± 0.15 2.13 ± 0.05  2205 ± 142 *P < 0.05 versus C;  P < 0.05 versus S FEBS Journal 274 (2007) 2135–2147 ª 2007 The Authors Journal compilation ª 2007 FEBS 2137 NOS and heart mitochondrial dysfunction in sepsis G Escames et al aMT administration In these animals, mitochondrial glutathione reductase (GRd) activity decreased during sepsis, whereas aMT treatment increased it to above control values (Fig 3B) Heart mitochondria from iNOS– ⁄ – mice did not show changes in GPx and GRd activities with any treatment (Fig 3) Basal GPx activity was lower in iNOS– ⁄ – than in iNOS+ ⁄ + mice (Fig 3A) The mitochondrial level of GSH decreased and that of GSSG increased in hearts from iNOS+ ⁄ + mice after CLP (Fig 4A,B), raising the GSSG ⁄ GSH ratio (Fig 4C) Sepsis also reduced total glutathione levels in iNOS+ ⁄ + mice (Fig 4D) Treatment with aMT increased GSH levels and reduced GSSG levels in iNOS+ ⁄ + mice, normalizing the GSSG ⁄ GSH ratio (Fig 4A–C) aMT also increased the total glutathione pool in this mouse strain (Fig 4D) No changes in glutathione levels were found in heart mitochondria of iNOS– ⁄ – mice under any experimental conditions (Fig 4A–D) Fig Effects of aMT treatment on CLP-induced mRNA levels of iNOS Ten nanograms of RNA extracted from mouse heart was used, and quantification of iNOS mRNA was performed by real-time RT-PCR The relative level was calculated as the ratio of inflammatory mRNA expression to b-actin mRNA expression ***P < 0.001 versus C; ###P < 0.0001 versus S Each value represents the mean ± SE for three independent experiments Figure 3A shows that glutathione peroxidase (GPx) activity increased in heart mitochondria of iNOS+ ⁄ + mice after sepsis, and this increase was preserved after ETC complexes and ATPase activities Figure 5A–D shows that, after CLP, the activity of the four ECT complexes was significantly reduced in iNOS+ ⁄ + mice aMT administration increased the activity of these complexes above the control values (Fig 3A–D) The activity of the ETC complexes in heart mitochondria from iNOS– ⁄ – mice was unaffected by sepsis The basal activities of complex I and complex II were significantly lower, and those of complex III and IV were significantly higher, in iNOS– ⁄ – Fig Changes in heart mitochondrial GPx (A) and GRd (B) activities after sepsis and aMT treatment in iNOS+ ⁄ + and iNOS– ⁄ – mice Data represent the means ± SE of six experiments per group C, control; S, sepsis; S + aMT, sepsis + aMT *P < 0.05 and **P < 0.01 versus C; ##P < 0.005 versus S; + P < 0.05 versus iNOS+ ⁄ + mice 2138 FEBS Journal 274 (2007) 2135–2147 ª 2007 The Authors Journal compilation ª 2007 FEBS G Escames et al NOS and heart mitochondrial dysfunction in sepsis Fig GSH level (A), GSSG level (B), GSSG ⁄ GSH ratio (C) and GSH + GSSG level (D) in heart mitochondria of iNOS+ ⁄ + and iNOS– ⁄ – mice Data represent the mean ± SE of six experiments per group C, control; S, sepsis; S + aMT, sepsis + aMT **P < 0.01 versus C; #P < 0.05 versus S Fig Complex I (A), II (B), III (C) and IV (D) activities in heart mitochondria of iNOS+ ⁄ + and iNOS– ⁄ – mice Data represent the mean ± SE of six experiments per group C, control; S, sepsis; S + aMT, sepsis + aMT *P < 0.05 and **P < 0.01 versus C; #P < 0.05, #P < 0.05, ## P < 0.01 and ###P < 0.001 versus S; +P < 0.05, ++P < 0.01 and +++P < 0.001 versus iNOS+ ⁄ + mice than in iNOS+ ⁄ + mice (Fig 5A–D) The activity of the ETC complexes was also unchanged by aMT treatment in iNOS– ⁄ – mice No changes in ATPase activity were observed in any mouse strain under sepsis, and aMT treatment only slightly decreased ATPase activity in iNOS+ ⁄ + mice (Table 1) FEBS Journal 274 (2007) 2135–2147 ª 2007 The Authors Journal compilation ª 2007 FEBS 2139 NOS and heart mitochondrial dysfunction in sepsis G Escames et al Mitochondrial ATP production To assess whether sepsis modifies the bioenergetic status of heart mitochondria, ATP production was determined ATP production was significantly reduced in iNOS+ ⁄ + but not in iNOS– ⁄ – mice during sepsis, whereas aMT administration restored the ability of mitochondria to produce ATP in the former (Fig 6) After the ATP production assay, the amount of AMP in the samples was less that 3% of the total nucleotides, discounting extramitochondrial ATP production by adenylate kinase in our assays The experimental procedure used here allowed us to detect ATP inside (pellet, fraction p2) and outside (supernatant, fraction s1) the mitochondria The results indicated that 92–98% of the ATP produced was detected outside the mitochondria Animal survival To determine the mortality of CLP-induced sepsis in our experimental paradigm, and to assess whether the improvement in mitochondrial function after aMT treatment was followed by a reduction in mortality, mice survival was analyzed Figure shows the survi- Fig Survival curves obtained from untreated and aMT-treated septic iNOS+ ⁄ + and iNOS– ⁄ – mice The total number of animals used in this study was 20 in each group val curves obtained from untreated and aMT-treated septic mice The half-life of iNOS+ ⁄ + animals with sepsis was 26.5 h, increasing up to 35 h when they were treated with aMT Moreover, there was 100% mortality at 32.5 h in septic mice, whereas aMT treatment increased survival up to 50 h A significant improvement in survival was observed in iNOS– ⁄ – mice (Fig 7) Untreated animals with sepsis showed a half-life of 67.5 h, and aMT treatment increased it up to 129.5 h Also, there was 100% mortality at 90 h in septic mice, whereas aMT administration increased this time up to 150 h Discussion Fig Changes in mitochondrial ATP production in heart of iNOS+ ⁄ + and iNOS– ⁄ – mice, using succinate as substrate Data represent the mean ± SE of six experiments per group C, control; S, sepsis; S + aMT, sepsis + aMT *P < 0.05 and **P < 0.01 versus C; ##P < 0.01 versus S 2140 The results of this study demonstrate the presence of two NOS isoforms with constitutive and inducible kinetic properties in heart mitochondria During inflammation, i-mtNOS activity is increased, but not that of mtNOS The induction of i-mtNOS depends on iNOS expression, because mitochondria from iNOS– ⁄ – mice lack i-mtNOS These data, and the results obtained from iNOS mRNA expression, suggest that i-mtNOS found in heart mitochondria derives from the cytosolic iNOS and is encoded by the same gene Sepsis was also accompanied by increased oxidative stress and inhibition of the ETC complexes, leading to a reduction in ATP Because heart mitochondria from iNOS– ⁄ – FEBS Journal 274 (2007) 2135–2147 ª 2007 The Authors Journal compilation ª 2007 FEBS G Escames et al mice were unaffected by endotoxemia and they not express i-mtNOS, mitochondrial impairment during sepsis was probably related to i-mtNOS induction in iNOS+ ⁄ + mice aMT treatment counteracted sepsisinduced iNOS mRNA expression, a finding related to the reduction of i-mtNOS aMT also prevented mitochondrial dysfunction, increasing ATP production, and the survival of septic mice Since the discovery of NOS activity in the mitochondria [16,24,30], several reports have shown the presence of this enzyme in different tissues with properties of endothelial NOS, nNOS, and ⁄ or iNOS [14,31,32] In different models of sepsis and tissues, the existence of both mtNOS and i-mtNOS isoforms has been reported [9,11,12] The constitutive isoform was identified in liver mitochondria as an nNOS isoform that was post-translationally modified [11,17,33] Recent reports also support the existence of i-mtNOS in mitochondria from different tissues [9,11–13,15] Whereas the absence of mtNOS in nNOS knockout mice suggested that mtNOS is derived from cytosolic nNOS [16], the absence of i-mtNOS in iNOS knockout mice supported its relationship with cytosolic iNOS [9,12] NO is particularly important in the regulation of cardiac function [10] It is involved in vascular and nonvascular effects, including regulation of cardiomyocyte contractility, in which mitochondrial respiration and bioenergetics play an important role [15] Production of excessive quantities of NO leads to profound cellular disturbances and myocardial dysfunction [8,15,18,34] Mitochondrial dysfunction is a consequence of inflammation [6], and the induction of i-mtNOS in heart mitochondria may be responsible for mitochondrial failure during sepsis [9,11,12] The existence of an iNOS isoform was also recently confirmed in heart mitochondria from rats [13] Normally, the induction of i-mtNOS produces a significant increase in NO and nitrite [9,11,12] The lack of a significant increase in nitrite in iNOS+ ⁄ + mice after sepsis reported here could be explained by two main mechanisms In mitochondria, the major oxidative decay pathway of NO is its reaction with O2– to form ONOO– [20] In turn, ONOO– reacts with a variety of biomolecules [35] Moreover, ONOO– can react with H4-biopterin (BH), a cofactor necessary for NO synthesis by NOS, leading to formation of the BH3 radical [36], and causing NOS inactivation [37,38] An alternative explanation for the lack of changes in nitrite under sepsis is the presence of an NOS-independent NO source in mitochondria Alterations in the redox state of the ETC lead to the formation of reactive nitrogen species, including NO and ONOO–, and thus to nitrite [39] In turn, mitochondrial nitrite reductase can recycle NO NOS and heart mitochondrial dysfunction in sepsis from nitrite, masking the nitrite increase during sepsis [40] The existence of elevated nitrite levels in mitochondria from other tissues under conditions of sepsis [9,11,12] suggests that the presence of a nitrate reductase with higher activity in heart mitochondria than in the other tissues could explain the lack of changes in nitrite reported here However, differences in the relative activities of nitrate reductases in mitochondria from different tissues have not yet been found The reactive species produced as a consequence of i-mtNOS induction during sepsis are highly toxic, and they can impair the mitochondrial ETC and oxidative phosphorylation [19–21] Our results show a significant inhibition of the four complexes of the respiratory chain in septic iNOS+ ⁄ + mice Similar results were reported for other tissues [5,7–9,11,12] However, septic iNOS– ⁄ – mice did not show alterations in ECT activity during sepsis, suggesting that the oxidative ⁄ nitrosative stress derived from i-mtNOS induction is responsible for ETC dysfunction in inflammation Unlike the situation with ETC complexes, our results not show changes in ATPase activity in septic mice It was recently shown that the ETC complexes, but not ATPase, are damaged during the early and acute phases of Chagasic cardiomyopathy [41] In these phases, the innate inflammatory response corresponds with iNOS induction and a subsequent increase in NO These results suggest that ATPase is more resistant to oxidative ⁄ nitrosative stress than ETC complexes, probably because ETC complexes, unlike ATPase, have redox centers such as Fe–S that are very sensitive to NO [42] ETC coupled with oxidative phosphorylation is responsible for the production of 90–95% of the total ATP synthesized in the cell [26] Thus, ETC damage may alter the synthesis of ATP without any effect on ATPase Our results show a reduced ability of the mitochondria to produce ATP during sepsis, which may reduce cardiomyocyte contractility [43,44] Because the activity of the respiratory complexes was not affected in septic iNOS– ⁄ – mice, ATP production by heart mitochondria was not altered in this mouse strain Thus, the reduction in the production of ATP found in diaphragm and heart after endotoxin administration [5,22,45] probably reflects mitochondrial impairment due to i-mtNOS induction by the toxin Heart mitochondria from iNOS– ⁄ – mice show lower complex I and II activities and higher complex III and IV activities than iNOS+ ⁄ + mice, a finding also described in diaphragmatic and skeletal muscle mitochondria [9,12] Whereas the latter was attributed to the lack of the inhibitory effect of the NO derived from i-mtNOS, which is absent in iNOS– ⁄ – mice, the reasons for the former phenomenon remains unclear [9,12] In FEBS Journal 274 (2007) 2135–2147 ª 2007 The Authors Journal compilation ª 2007 FEBS 2141 NOS and heart mitochondrial dysfunction in sepsis G Escames et al any case, the data regarding ATP production suggest that the low activities of complex I and II in iNOS– ⁄ – mice are compensated by the higher activities of complex III and IV, allowing normal mitochondrial homeostasis Mitochondrial ETC impairment leads to electron leakage and formation of O2– through the partial reduction of oxygen by one electron Subsequent reduction by one or two electrons can yield H2O2, and HO, respectively [46] However, the main source of mitochondrial H2O2 is superoxide dismutase activity [47], whereas HO can be derived from H2O2 and ONOO– decomposition, although the latter is a minor process [35] The small effect on complex I compared with the strong inhibition of complex III produced during sepsis in iNOS+ ⁄ + mice suggests that the latter was the most important source of free radicals in our experimental model NO and O2– react to produce ONOO– in mitochondria [7,19,20], increasing ETC damage [19,20] and LPO activity [48] Besides causing direct oxidative damage, ONOO– can produce nitration, and to a lesser extent nitrosation, of mitochondrial components indirectly [35] The free radical pathways of ONOO– are mainly initiated secondary to the reaction of ONOO– with CO2, leading to the rapid formation of carbonate and nitrogen dioxide radicals The ONOO– ⁄ CO2 pathway becomes highly relevant in mitochondria, as these are the main organelles in which CO2 is produced, due to the decarboxylation reactions Although ONOO– can yield HO•, it is a rather minor pathway in mitochondria, as most ONOO– will react directly with either target or CO2 In any case, ONOO– promotes, to some degree, mitochondrial LPO activity; this could be initiated by nitrogen dioxide and HO• radicals Carbonate radicals, however, are poor direct initiators of LPO, due to their negative charge, which limits their diffusion to the hydrophobic domains of membrane phospholipids [35] These mechanisms explain the LPO increase found in heart mitochondria from septic iNOS+ ⁄ + mice, a finding related to the i-mtNOS induction, because iNOS– ⁄ – mice did not show changes in LPO levels Mitochondrial GPx activity increased in iNOS+ ⁄ + mice, reflecting a compensatory mechanism to reduce oxidative stress during sepsis However, the GSSG ⁄ GSH ratio remains elevated under these conditions, because the reduced activity of GRd, probably due to oxidative damage [53], prevents the recovery of GSH from GSSG Moreover, total glutathione levels in these mitochondria were also reduced, probably reflecting inhibition of GSH transport into the mitochondria [49] The lack of mitochondrial oxidative stress and the presence of a normal GSH pool in septic iNOS– ⁄ – mice further support the idea that i-mtNOS 2142 induction is the main event related to oxidative stress during sepsis in heart mitochondria Different types of antioxidants with beneficial effects against mitochondrial oxidative stress have been proposed [23] One of these molecules is aMT, an indoleamine with excellent antioxidant and antiinflammatory properties in the cell and mitochondria [24,27,28,50,51] First, aMT inhibits the expression and activity of both cytosolic iNOS and mitochondrial i-mtNOS in septic rats and mice [11,29,37], allowing the animals to recover from multiorgan failure Second, aMT directly scavenges reactive oxygen species and reactive nitrogen species [24,26–28,52], induces the expression of antioxidative enzymes [8], and restores mitochondrial GSH homeostasis [53] Third, aMT increases the activity of the ETC and ATP production in vitro and in vivo [54,55] Our results demonstrate these protective effects of aMT in heart mitochondria of septic iNOS+ ⁄ + mice aMT treatment counteracted iNOS expression, reducing the activity of i-mtNOS, increased the activity of the ETC complexes over the control values, and normalized the levels of LPO Reducing the levels of free radicals with aMT prevents them from causing oxidative damage to GRd [53], normalizing the GSSG ⁄ GSH ratio Low oxidative status also prevents the mitochondrial transition pore opening and uncoupling, a condition associated with ATP hydrolysis [27,56,57] In fact, aMT restored the ability of heart mitochondria to produce ATP In these circumstances, cardiomyocytes could have enough energy for muscle contraction, avoiding myocardial dysfunction, and probably heart failure, in sepsis The significant increase in survival of mice treated with aMT further supports this observation It is interesting that aMT had minor effects on heart mitochondria from iNOS– ⁄ – mice As reported in other pathophysiologic conditions, it seems that aMT can upregulate mitochondrial function when it is impaired [9,11,12,24,49,53,55], but the indoleamine had minor effects under normal conditions The half-life and maximum survival time of septic iNOS– ⁄ – mice were significantly higher than those of wild-type animals Moreover, aMT treatment significantly increased the half-life and maximum survival time of mutant mice Because iNOS– ⁄ – mice with sepsis did not show significant mitochondrial damage, they probably died by a mechanism different from iNOS-dependent dysfunction, such as cyclooxygenase-2 induction Because aMT treatment had only minor effects on mitochondria of iNOS– ⁄ – mice, the inhibitory effect of aMT on cyclooxygenase-2 expression may explain the significant improvement in survival of iNOS– ⁄ – mice This hypothesis, however, remains to be studied FEBS Journal 274 (2007) 2135–2147 ª 2007 The Authors Journal compilation ª 2007 FEBS G Escames et al In summary, this study demonstrates that, besides mtNOS, mouse heart mitochondria contain an i-mtNOS isoform that is induced during sepsis Among other consequences, mitochondrial dysfunction, oxidative stress and reduced ability to produce ATP follows an increase in i-mtNOS These alterations could contribute to the myocardial dysfunction that often occurs during sepsis The parallel increases in iNOS expression and i-mtNOS activity, the inhibition of both iNOS mRNA expression and i-mtNOS activity after aMT treatment, and the lack of i-mtNOS expression in mitochondria from iNOS– ⁄ – mice, suggest that the enzyme is encoded by the same gene that encodes cytosolic iNOS Moreover, the absence of any sign of mitochondrial dysfunction and the lack of i-mtNOS expression in iNOS– ⁄ – mice further support the role of i-mtNOS in mitochondrial impairment during sepsis Administration of aMT to septic iNOS+ ⁄ + mice normalized mitochondrial function, restoring their ability to produce ATP, and increasing mice survival These properties, together with the prevention of endotoxininduced circulatory failure in rats [58,59], and the mortality reduction in septic newborns after aMT therapy [60], suggest that the use of the indoleamine in septic patients should be seriously considered Experimental procedures Chemicals l-[2,3,4,5-3H]arginine monohydrochloride (58 CiỈmmol)1) was obtained from Amersham Biosciences Europe GmBH (Barcelona, Spain) Liquid scintillation cocktail (Ecolume) was purchased from ICN (Madrid, Spain) All other chemicals, of the purest available grade, were obtained from Sigma-Aldrich (Madrid, Spain) unless otherwise specified NOS and heart mitochondrial dysfunction in sepsis equithesin anesthesia (1 mLỈkg)1) Four doses of aMT (30 mgỈkg)1) were injected as follows: one dose 30 before surgery (intraperitoneal); the second dose just after surgery (subcutaneous); and the remaining doses h and h after surgery (subcutaneous) Animals were killed 24 h after CLP, except for the survival studies Preparation of cardiac mitochondria Pure mitochondria were isolated from 12–14-week-old wildtype and knockout mouse hearts by differential centrifugation and density gradient centrifugation with Percoll as follows [9,11] All procedures were carried out in the cold Briefly, cardiac muscle was excised, washed with saline, treated with proteinase K (1 mgỈmL)1) for 30 s, washed with buffer A (250 mm mannitol, 0.5 mm EGTA, mm Hepes, 0.1% BSA, pH 7.4, °C), and homogenized (1 : 10, w ⁄ v) in buffer A at 800 r.p.m at °C with a Teflon pestle The homogenate was aliquoted, and centrifuged at 600 g for at °C (twice) (rotor type F34-6-38 Eppendorf 5810R centrifuge), and the supernatants were centrifuged at 10 300 g for 10 at °C (rotor type F1255 Beckman TL-100 centrifuge) Then, the mitochondrial pellets were suspended in 0.5 mL of buffer A, and placed in ultracentrifuge tubes containing 1.4 mL of buffer B (225 mm mannitol, mm EGTA, 25 mm Hepes, 0.1% BSA, pH 7.4, °C) and 0.6 mL of Percoll The mixture was centrifuged at 105 000 g for 30 at °C (rotor type F1255 Beckman TL-100 centrifuge) The fraction corresponding to a pure mitochondrial fraction was collected, washed twice with buffer A at 10 300 g for 10 at °C (rotor type F1255 Beckman TL-100 centrifuge) to remove the Percoll, and washed again with a high ionic strength solution of KCl (150 mm) to yield a highly pure mitochondrial preparation without contaminating organelles and broken mitochondria [28,50] Aliquots of these pure mitochondrial fractions were frozen to ) 80 °C The purity of the mitochondrial preparations was assessed as described elsewhere [9,11] Experimental animals Mitochondrial NOS activity measurement All procedures involving animals were carried out under an approved protocol and in accordance with the Spanish Government Guide and the European Community Guide for animal care iNOS knockout B6.129P2-Nos2tm1Lau mice (iNOS– ⁄ –) and their respective wild-type control C57 ⁄ Bl ⁄ mice (iNOS+ ⁄ +) were obtained from Jackson’s Laboratory through Charles River Laboratories (Barcelona, Spain) The animals were housed in the university’s facility with a 12 h : 12 h light ⁄ dark cycle (lights on at 07:00 h) at 22 ± °C, and given regular chow and tap water Both iNOS+ ⁄ + and iNOS– ⁄ – mice 12–14 weeks of age were grouped (n ¼ 18 animals ⁄ group) as follows: (a) control group; (b) sepsis group; and (c) sepsis + aMT group Sepsis was induced by CLP [61] under intraperitoneal Measurement of constitutive, Ca2+-dependent, and inducible, Ca2+-independent, mitochondrial NOS activities was done as previously described [11,62] Briefly, an aliquot of frozen mitochondria was thawed and homogenized (0.1 gỈmL)1) in 25 mm Tris buffer (pH 7.6) containing 0.5 mm dithiothreitol, 10 lgỈmL)1 pepstatin, 10 lgỈmL)1 leupeptin, 10 lgỈmL)1 aprotinin and mm phenylmethanesulfonyl fluoride at °C (rotor type F34-6-38 Eppendorf 5810R centrifuge) The homogenate was centrifuged at 2500 g for at °C, and the supernatant was used immediately for determination of NOS activity; one aliquot was frozen at ) 80 °C for protein determination [63] Ten microliters of the supernatant (2 mgỈmL)1 protein) were added to the FEBS Journal 274 (2007) 2135–2147 ª 2007 The Authors Journal compilation ª 2007 FEBS 2143 NOS and heart mitochondrial dysfunction in sepsis G Escames et al incubation mixture (100 lL, final volume) prewarmed at 37 °C, and containing (final concentration) 25 mm Tris, mm dithiothreitol, 30 lm H4-biopterin, 10 lm FAD, 0.5 mm inosine, 0.5 mgỈmL)1 BSA, 0.1 mm CaCl2, 10 lm l-arginine, and 40 nm l-[3H]arginine (pH 7.6) The reaction was started by the addition of 10 lL of NADPH (0.75 mm final concentration), and continued for 30 at 37 °C To determinate the Ca2+-independent activity of NOS (i-mtNOS), 10 mm EDTA was added to the buffer before the reaction was started Control incubations were performed in the absence of NADPH The reaction was stopped by adding 400 lL of cold 0.1 m Hepes buffer containing 10 mm EGTA and mm l-citrulline (pH 5.5) The mixture was decanted onto a mL column packed with Dowex-50W ion exchange resin (Na+ form), and eluted with 1.2 mL of water l-[3H]Citrulline was quantified by liquid scintillation spectroscopy The retention of l-[3H]arginine by the column was greater than 98% Enzyme activity was determined as pmoL l-[3H]citrullinmin)1Ỉmg protein)1 Real-time quantitative RT-PCR assay of iNOS mRNA expression Quantification of the iNOS mRNA levels was done by SYBR green two-step real-time RT-PCR (Stratagene Mx 3005P; Stratagene, La Jolla, CA, USA) Total cellular RNA was isolated from the heart using the RNA isolation kit Real Total RNA Spin Plus (Durviz, S.L., Valencia, Spain) Ten nanograms of the total RNA extracted was used Gene-specific primers for iNOS (forward primer, 5¢AGACGGATAGGCAGAGATTGG-3¢, and reverse primer, 5¢-ACTGACACTTCGCACAAAGC-3¢) and b-actin (forward primer, 5¢-GCTGTCCCTGTATGCCTCTG-3¢, and reverse primer, 5¢-CGCTCGTTGCCAATAGTGA TG-3¢) were designed using the beacon designer software (Premier Biosoft Int., Palo Alto, CA, USA) and obtained from Thermo Electron GmbH (Ulm, Germany) Real-time PCR reactions were carried out in a final volume of 25 lL of reaction mixture containing 10 ng of RNA, 12.5 lL of 2X SYBR Green Master Mix (Stratagene), 75 nm each specific gene primer, and H2O The samples were run in triplicate in the amplification program, and the mean value was used as the final expression value A negative control without RNA template was run The PCR program was initiated by 10 at 95 °C before 40 thermal cycles, each of 30 s at 95 °C and at 55 °C Data were analyzed according to the relative standard curve method, constructed with triplicate serial dilutions (50, 5, 0.5 and 0.05 ng), and were normalized by b-actin expression Nitrite determination Mitochondrial fractions were thawed and suspended in icecold distilled water, and immediately sonicated to break the 2144 mitochondrial membranes Aliquots of these samples were used to calculate nitrite levels following the Griess reaction [64], and expressed in nmoL nitritmg protein)1 Determination of mitochondrial function The activities of the four respiratory complexes were determined as previously described [65,66], with slight modifications [9,12], and expressed as nmoLỈmin)1Ỉmg protein)1 Complex V (ATP synthase) activity was measured by following the rate of hydrolysis of ATP to ADP + Pi Ferrous sulfate ⁄ ammonium molybdate reagent was utilized to determinate Pi concentration [67] ATPase activity was expressed in nmoL PiỈmin)1Ỉmg protein)1 Determination of ATP production For the determination of ATP production, hearts were excised, washed with saline, treated with proteinase K (1 mgỈmL)1) for 30 s, washed with buffer A (220 mm mannitol, 70 mm sucrose, mm EGTA, 20 mm Hepes, 1% BSA, pH 7.2, °C), and homogenized (1 : 10, w ⁄ v) in buffer A at 800 r.p.m at °C with a Teflon pestle The homogenates were centrifuged at 1500 g for at °C (rotor type F1255 Beckman TL-100 centrifuge), and the supernatants were centrifuged again at 23 000 g for at °C (rotor type F1255 Beckman TL-100 centrifuge) Then, the mitochondrial pellets were suspended in mL of buffer A, and centrifuged at 10 300 g for at °C (rotor type F1255 Beckman TL-100 centrifuge) The resultant pellets (p1) were suspended in respiration buffer (225 mm mannitol, 75 mm sucrose, 10 mm KCl, 10 mm Tris ⁄ HCl, mm potassium phosphate, pH 7.2, saturated with O2, plus mm succinate, 30 °C), and ATP production was induced adding 125 nmol of ADP After 45 s, the sample was centrifuged at 13 000 g for at °C (rotor type F1255 Beckman TL-100 centrifuge) [68,69], and the ATP content in the pellet (p2) and supernatant (s1) was measured Ice-cold 0.5 m perchloric acid was rapidly added to the p1, p2 and s1 fractions, mixed for in a vortex mixer, and centrifuged at 25 000 g for 15 at °C (rotor type F1255 Beckman TL-100 centrifuge) to precipitate proteins The pellets were frozen to ) 80 °C for protein determination [63]; the supernatants were mixed with lL of m potassium carbonate to neutralize the pH, and centrifuged at 12 000 g for 10 at °C (rotor type F1255 Beckman TL-100 centrifuge) ATP was measured in the resultant supernatants by HPLC with a · 250 mm ProPac PA1 column (Dionex, Barcelona, Spain) [70] After stabilization of the column with the mobile phase, samples (20 lL) were injected onto the HPLC system The mobile phase consisted of water (phase A) and 0.3 m ammonium carbonate (pH 8.9) (phase B), and the following time schedule for the binary gradient (flow rate mLỈmin)1) was used: min, 50% A and 50% B; min, 50% to 100% B, and then 100% B for FEBS Journal 274 (2007) 2135–2147 ª 2007 The Authors Journal compilation ª 2007 FEBS G Escames et al 25 min; min, 100% to 50% B, and then another with 50% B [71] Water was used for calibration purposes A standard curve was constructed with 3.125 lgỈmL)1, 6.250 lgỈmL)1, 12.5 lgỈmL)1 and 25 lgỈmL)1 of ATP The absorbance of the samples was measured with a UV detector at 254 nm, and the concentration of each nucleotide in the samples was calculated according to the peak area [70] ATP production was expressed in lgỈmin)1Ỉmg protein)1 Determination of mitochondrial oxidative stress For LPO measurement (expressed in nmolỈmg protein)1), mitochondrial fractions were thawed and sonicated in icecold 20 mm Tris ⁄ HCl buffer (pH 7.6) to break the mitochondrial membranes Aliquots of these samples were either stored at ) 80 °C for total protein determination [63], or used for malondialdehyde plus 4-hydroxyalkenal determination as an index of LPO (Bioxytech LPO-568 assay kit; OxisResearch, Portland, OR, USA) [72] GSSG and GSH were measured by fluorescence [73], and expressed in nmoLỈmg protein)1 For GPx and GRd activity determination, aliquots of the mitochondrial fraction were suspended in 200 lL of 50 mm potassium phosphate buffer containing mm EDTA-K2 (pH 7.4), and the oxidation of NADPH was spectrophotometrically measured for at 340 nm [74] The activity of GPx and GRd was expressed in nmolỈmin)1Ỉmg protein)1 Statistical analysis Data are expressed as means ± SEM Significance was determined using two-way ANOVA followed by Dunnet’s post hoc test, when appropriate The level of statistical significance was taken as P < 0.05 Acknowledgements This study was partially supported by grants FIS01 ⁄ 1076, PI03 ⁄ 0817 and G03 ⁄ 137 from the Instituto de ´ Salud Carlos III, and Consejerı´ a de Educacion, Junta ´ de Andalucı´ a (CTS-101) L C Lopez is an FPI fellow ´ from the Ministerio de Educacion (Spain), and ´ F Ortiz and A Lopez are predoctoral fellows from the Instituto de Salud Carlos III (Spain) References Riedemann NC, Guo RF & Ward PA (2003) The enigma of sepsis J Clin Invest 112, 460–467 Court O, Kumar A, Parrillo JE & Kumar A (2002) Clinical review: myocardial depression in sepsis and septic shock Crit Care 6, 500–508 Levy RJ & Deutschman CS (2004) Evaluating myocardial depression in sepsis Shock 22, 1–10 NOS and heart mitochondrial dysfunction in sepsis Brealey D, Brand M, Hargreaves I, Heales S, Land J, Smolenski R, Davies NA, Cooper CE & Singer M (2002) Association between mitochondrial dysfunction and severity and outcome of septic shock Lancet 360, 219–223 Callahan LA & Supinski GS (2005) Sepsis induces diaphragm electron transport chain dysfunction and protein depletion Am J Respir Crit Care Med 172, 861–868 Crouser ED (2004) Mitochondrial dysfunction in septic shock and multiple organ dysfunction syndrome Mitochondrion 4, 729–741 Boczkowski J, Lisdero CL, Lanone S, Samb A, Carreras MC, Boveris A, Aubier M & Poderoso JJ (1999) Endogenous peroxynitrite mediates mitochondrial dysfunction in rat diaphragm during endotoxemia FASEB J 13, 1637–1646 ´ Boveris A, Alvarez S & Navarro A (2002) The role of mitochondrial nitric oxide synthase in inflammation and septic shock Free Radic Biol Med 33, 1186–1193 ´ Escames G, Lopez LC, Tapias V, Utrilla P, Reiter RJ, ´ Hitos AB, Leon J, Rodrı´ guez MI & Acuna-Castroviejo ˜ D (2006) Melatonin counteracts inducible mitochondrial nitric oxide synthase-dependent mitochondrial dysfunction in skeletal muscle of septic mice J Pineal Res 40, 71–78 10 Massion PB, Feron O, Dessy C & Balligand JJ (2003) Nitric oxide and cardiac function: ten years after, and continuing Circ Res 93, 388–398 ´ 11 Escames G, Leon J, Macı´ as M, Khaldy H & Acuna˜ Castroviejo D (2003) Melatonin counteracts lipopolysaccheride-induced expression and activity of mitochondrial nitric oxide synthase in rats FASEB J 17, 932–934 ´ ´ 12 Lopez LC, Escames G, Tapias V, Utrilla P, Leon J & Acuna-Castroviejo D (2006) Identification of an induci˜ ble nitric oxide synthase in diaphragm mitochondria from septic mice Its relation with mitochondrial dysfunction and prevention by melatonin Int J Biochem Cell Biol 38, 267–278 13 Zanella B, Giordano E, Muscari C, Zini M & Guarnieri C (2004) Nitric oxide synthase activity in rat cardiac mitochondria Basic Res Cardiol 99, 159–164 ´ 14 Acuna-Castroviejo D, Escames G, Lopez LC, Hitos AB ˜ ´ & Leon J (2005) Melatonin and nitric oxide: two required antagonists for mitochondrial homeostasis Endocrine 27, 159–168 15 Lisdero CL, Carreras MC, Meulemans A, Melani M, Aubier M, Boczkowski J & Poderoso JJ (2004) The mitochondrial interplay of ubiquinol and nitric oxide in endotoxemia Methods Enzymol 382, 67–81 16 Kanai AJ, Pearce LL, Clemens PR, Birder LA, VanBibber MM, Choi SY, de Groat WC & Peterson J (2001) Identification of a neuronal nitric oxide synthase in isolated cardiac mitochondria using FEBS Journal 274 (2007) 2135–2147 ª 2007 The Authors Journal compilation ª 2007 FEBS 2145 NOS and heart mitochondrial dysfunction in sepsis 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 G Escames et al electrochemical detection Proc Natl Acad Sci USA 98, 14126–11431 Tatoyan A & Giulivi C (1998) Purification and characterization of a nitric oxide synthase from rat liver mitochondria J Biol Chem 273, 11044–11048 ´ ´ Alvarez S & Boveris A (2004) Mitochondrial nitric oxide metabolism in rat muscle during endotoxemia Free Radic Biol Med 37, 1472–1478 Brown GC (2001) Regulation of mitochondrial respiration by nitric oxide inhibition of cytochrome c oxidase Biochim Biophys Acta 1504, 46–57 Cadenas E, Poderoso JJ, Antunes F & Boveris A (2000) Analysis of the pathways of nitric oxide utilization in mitochondria Free Radic Res 33, 747–756 Piantadosi CA, Tatro LG & Whorton AR (2002) Nitric oxide and differential effects of ATP on mitochondrial permeability transition Nitric Oxide 6, 45–60 ´ Poderoso JJ, Carreras MC, Lisdero C, Riobo N, Schoper F & Boveris A (1996) Nitric oxide inhibits ă electron transfer and increases superoxide radical production in rat heart mitochondria and submitochondrial particles Arch Biochem Biophys 308, 89–95 Albuszies G & Bruckner UB (2003) Antioxidant therapy in sepsis Intensive Care Med 29, 1632–1636 ´ Acuna-Castroviejo D, Escames G, Carazo A, Leon J, ˜ Khaldy H & Reiter RJ (2002) Melatonin, mitochondrial homeostasis and mitochondrial-related diseases Curr Top Med Chem 2, 133–151 ´ Acuna-Castroviejo D, Escames G, Leon J, Carazo A & ˜ Khaldy H (2003) Mitochondrial regulation by melatonin and its metabolites Adv Exp Med Biol 527, 549–557 Acuna-Castroviejo D, Martı´ n M, Macı´ as M, Escames ˜ ´ G, Leon J, Khaldy H & Reitrer RJ (2001) Melatonin, mitochondria and cellular bioenergetics J Pineal Res 30, 65–74 ´ Leon J, Acuna-Castroviejo D, Escames G, Tan DX & ˜ Reiter RJ (2005) Melatonin mitigates mitochondrial malfunction J Pineal Res 38, 1–9 ´ Leon J, Acuna-Castroviejo D, Sainz RM, Mayo JC, ˜ Tan DX & Reiter RJ (2004) Melatonin and mitochondrial function Life Sci 75, 765–790 Crespo E, Macias M, Pozo D, Escames G, Martin M, Vives F, Guerreo JM & Acuna-Castroviejo D (1999) ˜ Melatonin inhibits expression of the inducible NO synthase II in liver and lung and prevents endotoxemia in lipopolysaccharide-induced multiple organ dysfunction syndrome in rats FASEB J 13, 1537–1546 Bates TE, Loesch A, Burnstock G & Clark JB (1995) Immunocytochemical evidence for a mitochondrially located nitric oxide synthase in brain and liver Biochem Biophys Res Commun 213, 896–900 Brookes PS (2004) Mitochondrial nitric oxide synthase Mitochondrion 3, 187–204 Ghafourifar P & Cadenas E (2005) Mitochondrial nitric oxide synthase Trends Pharmacol Sci 26, 190–195 2146 33 Elfering SL, Sarkela TM & Giulivi C (2002) Biochemistry of mitochondrial nitric-oxide synthase J Biol Chem 277, 38079–38086 34 Barth E, Radermacher P, Thiemermann C, Weber S, Georgieff M & Albuszies G (2006) Role of inducible nitric oxide synthase in the reduced responsiveness of the myocardium to catecholamines in a hyperdynamic, murine model of septic shock Crit Care Med 34, 307–313 35 Radi R, Cassina A, Hodara R, Quijano C & Castro L (2002) Peroxynitrite reactions and formation in mitochondria Free Radic Biol Med 33, 1451–1464 36 Kuzkaya N, Weissmann N, Harrison DG & Dikalov S (2003) Interactions of peroxynitrite, tetrahydrobiopterin, ascorbic acid, and thiols: implications for uncoupling endothelial nitric-oxide synthase J Biol Chem 278, 22546–22554 37 Kobzik L, Stringer B, Balligand JL, Reid MB & Stamler JS (1995) Endothelial type nitric oxide synthase in skeletal muscle fibers: mitochondrial relationships Biochem Biophys Res Commun 211, 375–381 38 Kumagai Y, Kikushima M, Nakai Y, Shimojo N & Kunimoto M (2004) Neuronal nitric oxide synthase (nNOS) catalyzes one-electron reduction of 2,4,6-trinitrotoluene, resulting in decreased nitric oxide production and increased nNOS gene expression: implications for oxidative stress Free Radic Biol Med 37, 350–357 ´ 39 Lacza Z, Kozlov AV, Pankotai E, Csordas A, Wolf G, Redl H, Kollai M, Szabo C, Busija DW & Horn TF (2006) Mitochondria produce reactive nitrogen species via an arginine-independent pathway Free Radic Res 40, 369–378 40 Nohl H, Staniek K & Kozlov AV (2005) The existence and significance of a mitochondrial nitrite reductase Redox Rep 10, 281–286 41 Vyatkina G, Bhatia V, Gerstner A, Papaconstantinou J & Garg N (2004) Impaired mitochondrial respiratory chain and bioenergetics during chagasic cardiomyopathy development Biochim Biophys Acta 1689, 162–173 42 Pearce LL, Epperly MW, Greenberger JS, Pitt BR & Peterson J (2001) Identification of respiratory complexes I and III as mitochondrial sites of damage following exposure to ionizing radiation and nitric oxide Nitric Oxide 5, 128–136 43 Ataullakhanov FI & Vitvitsky VM (2002) What determines the intracellular ATP concentration Biosci Rep 22, 501–511 44 Saks V, Dzeja P, Schlattner U, Vendelin M, Terzic A & Wallimann T (2006) Cardiac system bioenergetics: metabolic basis of the Frank–Starling law J Physiol 571, 253–273 45 Supinski GS & Callahan LA (2006) Polyethylene glycolsuperoxide dismutase prevents endotoxin induced cardiac dysfunction Am J Respir Crit Care Med 173, 1240–1247 FEBS Journal 274 (2007) 2135–2147 ª 2007 The Authors Journal compilation ª 2007 FEBS G Escames et al 46 Naqui A, Chance B & Cadenas E (1986) Reactive oxygen intermediates in biochemistry Annu Rev Biochem 55, 137–166 47 Fridovich I (1995) Superoxide radical and superoxide dismutases Annu Rev Biochem 64, 97–112 ´ 48 Szabo C (2003) Multiple pathways of peroxynitrite cytotoxicity Toxicol Lett 140–141, 105–112 49 Anderson MF & Sims NR (2002) The effects of focal ischemia and reperfusion on the glutathione content of mitochondria from rat brain subregions J Neurochem 81, 541–549 50 Antolı´ n I, Rodrı´ guez C, Sainz RM, Mayo JC, Urı´ a H, Kotler ML, Rodrı´ guez-Colunga MJ, Tolivia D & ´ ´ Menendez-Pelaez A (1996) Neurohormone melatonin prevents cell damage: effect on gene expression for antioxidant enzymes FASEB J 10, 882–890 ´ 51 Mayo JC, Sainz RM, Tan DX, Hardeland R, Leon J, Rodrı´ guez C & Reiter RJ (2005) Anti-inflammatory actions of melatonin and its metabolites, N1-acetylN2-formyl-5-methoxykynuramine (aFMK) and (N1acetyl-5-methxykynuramine (AMK), in macrophages J Neuroimmunol 165, 139–149 52 Tan DX, Manchester LC, Reiter RJ, Qi WB, Karbownik M & Calvo JR (2000) Significance of melatonin in antioxidative defense system: reactions and products Biol Signals Recep 9, 137–159 ´ 53 Martı´ n M, Macı´ as M, Escames G, Leon J & Acuna˜ Castroviejo D (2000) Melatonin but not vitamin C and E maintains glutathione homeostasis in t-butyl hydroperoxide-induced mitochondrial oxidative stress FASEB J 14, 1677–1679 54 Castillo C, Salazar V, Ariznavarreta C, Vara E & Tresguerres JA (2005) Effect of melatonin administration on parameters related to oxidative damage in hepatocytes isolated from old Wistar rats J Pineal Res 38, 140–146 ´ 55 Martı´ n M, Macias M, Leon J, Escames G, Khaldy H & Acuna-Castroviejo D (2001) Melatonin increases the ˜ activity of the oxidative phosphorylation enzymes and the production of ATP in rat brain and liver mitochondria Int J Biochem Cell Biol 1212, 1–10 56 He L & Lemasters JJ (2002) Regulated and unregulated mitochondrial permeability transition pores: a new paradigm of pore structure and function? FEBS Lett 512, 1–7 57 Kowaltowski AJ, Castilho RF & Vercesi AE (2001) Mitochondrial permeability transition and oxidative stress FEBS Lett 495, 12–15 58 Wu CC, Chiao CW, Hsiao G, Chen A & Yen MH (2001) Melatonin prevents endotoxin-induced circulatory failure in rats J Pineal Res 30, 147–156 ´ 59 Escames G, Acuna-Castroviejo D, Lopez LC, Tan DX, ˜ ´ ´ Maldonado MD, Sanchez-Hidalgo M, Leon J & Reiter RJ (2006) The pharmacological utility of melatonin in the treatment of septic shock J Pharm Pharmacol 58, 1153–1165 NOS and heart mitochondrial dysfunction in sepsis 60 Gitto E, Karbownik M, Reiter RJ, Tan DT, Cuzzocrea S, Chiurazzi P, Cordaro S, Corona G, Troimarchi G & Barberi I (2001) Effect of melatonin treatment in septic newborns Pediatr Res 50, 756–760 61 Wichterman KA, Baue AE & Chaudry IH (1980) Sepsis and septic shock ) a review of laboratory models and a proposal J Surg Res 29, 189–201 62 Bredt DS & Snyder SH (1989) NO mediates glutamatelinked enhancement of cGMP levels in the cerebellum Proc Natl Acad Sci USA 86, 9030–9033 63 Lowry OH, Rosenbrough NJ, Farr AL & Randall RJ (1951) Protein measurement with Folin phenol reagent J Biol Chem 193, 265–275 64 Green LC, Ruiz de Luzuriaga K & Wagner DA (1981) Nitrate biosynthesis in man Proc Natl Acad Sci USA 78, 7764–7768 65 Barrientos A (2002) In vivo and in organello assessment of OXPHOS activities Methods 26, 307–316 66 Brusque AM, Borba Rosa R, Schuck PF, Dalcin KB, Ribeiro CA, Silva CG, Wannmacher CM, Dutra-Filho CS, Wyse AT, Briones P et al (2002) Inhibition of the mitochondrial respiratory chain complex activities in rat cerebral cortex by methylmalonic acid Neurochem Int 40, 593–601 67 Taussky HH & Shorr E (1953) A microcolorimetric method for the determination of inorganic phosphorus J Biol Chem 202, 675–685 68 Manfredi G, Yang L, Gajewski CD & Mattiazzi M (2002) Measurements of ATP in mammalian cells Methods 26, 317–326 69 Monteiro P, Duarte AI, Moreno A, Goncalves LM & ¸ Providencia LA (2003) Carvedilol improves energy production during acute global myocardial ischemia Eur J Pharmacol 482, 245–253 70 Pissarek M, Reinhardt R, Reichelt C, Manaenko A, Krauss G & Illes P (1999) Rapid assay for one-run determination of purine and pyrimidine mucleotide contents in neocortical slices and cell cultures Brain Res Protoc 4, 314–321 71 Krauss GJ, Pissarek M & Blasig I (1997) HPLC of nucleic acid components with volatile mobile phases: Part Separations on polymeric supports J High Resolut Chromatogr 20, 693–696 72 Esterbauer H & Cheeseman KH (1990) Determination of aldehydic lipid peroxidation products: malonaldehyde and 4-hydroxynonenal Methods Enzymol 186, 407–421 73 Hissin PJ & Hilf R (1976) A fluorometric method for determination of oxidized and reduced glutathione in tissues Anal Biochem 74, 214–216 74 Jaskot RH, Charlet EG, Grose EC, Grady MA & Roycroft JH (1983) An automatic analysis of glutathione peroxidase, glutathione-S-transferase, and reductase activity in animal tissue J Anal Toxicol 7, 86–88 FEBS Journal 274 (2007) 2135–2147 ª 2007 The Authors Journal compilation ª 2007 FEBS 2147 ... ¼ after deducting basal and background values Sepsis resulted in a significant increase in the transcription of the mRNA encoding iNOS in heart of iNOS+ ⁄ + mice The expression of iNOS mRNA was... cytosolic iNOS Moreover, the absence of any sign of mitochondrial dysfunction and the lack of i-mtNOS expression in iNOS– ⁄ – mice further support the role of i-mtNOS in mitochondrial impairment during... versus iNOS+ ⁄ + mice than in iNOS+ ⁄ + mice (Fig 5A–D) The activity of the ETC complexes was also unchanged by aMT treatment in iNOS– ⁄ – mice No changes in ATPase activity were observed in any