Cardioprotection methods have been developed to protect the heart against deleterious effects of ischemia reperfusion (IR), among them, postconditioning (PC) was shown to decrease infarct size (IS). Thus, it was shown that deacetylation, a post-translational modification, was crucial for PC success. Our lab recently shown that modification on acetylation level of cyclophilin D (CypD) by sirtuin 3 (SIRT3), a mitochondrial deacetylase, modifies the efficiency of ischemic-postconditionning (IPC). Indeed, it was shown that in absence of SIRT3, CypD is hyperacetylated leading to mPTP opening and cell death and inefficacy of IPC. Moreover, numerous studies have linked the lack of SIRT3 with aging associated pathologies. The goal of this study was to decipher the impact of CypD deacetylation mediated by SIRT3 in PC in the aging context. We submitted young (2 months) and old (≤ 12 months) mice, WT and SIRT3 KO, to IR and IPC. IS were quantified, and mitochondrial protein acetylation was evaluated. In parallel, basal mitochondrial functions and protein acetylation were assessed, as well as deacetylase activity, to estimate the impact of aging. Our results enlightened the importance of SIRT3 for PC success in young mice (25% IS reduction), that is lost in the old mice. We reported no more modification of the acetylation profile neither after IR or IPC in old mice. Moreover, we quantified a significant decrease in deacetylase activity with age (25%), associated to a significant decrease in NAD+ content (60%) with no basal mitochondrial function modification in old mice that could be associated to the loss of IPC efficiency. In accordance with what we demonstrated in young mice, the failure of IPC in old mice could be explained by a lack of CypD deacetylation following IPC stimulus, linked with an alteration of the deacetylase mitochondrial activity associated to a significant decrease in NAD+ mitochondrial content.

Rationale. How ischemic postconditioning can inhibit opening of the mitochondrial permeability transition pore (PTP) and subsequent cardiac myocytes death at reperfusion remains unknown. Recent studies have suggested that de-acetylation of cyclophilin D (CyPD) by sirtuin 3 (SIRT3) can modulate its binding to the PTP.Objective. The aim of the present study was to examine whether ischemic postconditioning (PostC) might activate SIRT3 and consequently prevent lethal myocardial reperfusion injury through a deacetylation of CyPD.Methods and results. Using hypoxia–reoxygenation (H/R) in H9C2 cells, we showed that SIRT3 overexpression prevented CyPD acetylation, limited PTP opening and reduced cell death by 24%. In vitro modification of the CyPD acetylation status in MEFs by site-directed mutagenesis altered capacity of PTP opening by calcium. Calcium Retention Capacity (CRC) was significantly decreased with CyPD-KQ that mimics acetylated protein compared with CyPD WT (871±266 vs 1193±263nmolesCa2+/mg protein respectively). Cells expressing non-acetylable CyPD mutant (CyPD-KR) displayed 20% decrease in cell death compared to cells expressing CyPD WT after H/R. Correspondingly, in mice we showed that cardiac ischemic postconditioning could not reduce infarct size and CyPD acetylation in SIRT3 KO mice, and was unable to restore CRC in mitochondria as it is observed in WT mice.Conclusions. Our study suggests that the increased acetylation of CyPD following myocardial ischemia–reperfusion facilitates PTP opening and subsequent cell death. Therefore ischemic postconditioning might prevent lethal reperfusion injury through an increased SIRT3 activity and subsequent attenuation of CyPD acetylation at reperfusion.

Inhibition of the mitochondrial permeability transition pore (PTP) has proved to be an effective strategy for preventing oxidative stress-induced cell death, and the pore represents a viable cellular target for drugs. Here, we report that inhibition of complex I by rotenone is more effective at PTP inhibition than cyclosporin A in tissues that express low levels of the cyclosporin A mitochondrial target, cyclophilin D; and, conversely, that tissues in which rotenone does not affect the PTP are characterized by high levels of expression of cyclophilin D and sensitivity to cyclosporin A. Consistent with a regulatory role of complex I in the PTP-inhibiting effects of rotenone, the concentrations of the latter required for PTP inhibition precisely match those required to inhibit respiration; and a similar effect is seen with the antidiabetic drug metformin, which partially inhibits complex I. Remarkably (i) genetic ablation of cyclophilin D or its displacement with cyclosporin A restored PTP inhibition by rotenone in tissues that are otherwise resistant to its effects; and (ii) rotenone did not inhibit the PTP unless phosphate was present, in striking analogy with the phosphate requirement for the inhibitory effects of cyclosporin A [Basso et al. (2008) J. Biol. Chem. 283, 26307-26311]. These results indicate that inhibition of complex I by rotenone or metformin and displacement of cyclophilin D by cyclosporin A affect the PTP through a common mechanism; and that cells can modulate their PTP response to complex I inhibition by modifying the expression of cyclophilin D, a finding that has major implications for pore modulation in vivo.

Il est admis que le préconditionnement limite les lésions d'ischémiereperfusion cardiaques. Plusieurs résultats indiquent l'implication du pore de transition de perméabilité mitochondriale (mPTP) et des radicaux libres de l'oxygène (ROS) dans cette protection. Le but de ce travail est de comparer à un temps court de reperfusion les fonctions mitochondriales cardiaques des rats préconditionnés par rapport aux rats non protégés. Les rats (mâles Wistar, 350 g environ) sont soumis à une ischémie de 45 minutes par ligature de l'artère coronaire gauche, suivie d'une reperfusion de 2 heures (groupe Contrôle), ou sont préconditionnés par 3 cycles de 3 minutes d'ischémie-5 minutes de reperfusion avant l'ischémie prolongée (groupe Préconditionné). A la fin de la reperfusion, les cœurs sont prélevés pour quantifier la taille de l'infarctus par planimétrie (TTC). Pour les mesures biochimiques, la reperfusion ne dure que 15 minutes et un groupe sham est constitué. A la fin des 15 minutes de reperfusion, les mitochondries de la zone à risque sont isolées. La capacité de rétention calcique (CRC, index d'ouverture du mPTP), la production des ROS et la phosphorylation oxydative sont mesurées. Après 2 heures de reperfusion, l'infarctus représente 58,2 ± 3,3 % de la zone à risque chez le groupe contrôle, contre 44,1 ± 4,6 % pour le groupe préconditionné (p < 0,05). En présence de glutamate (20 mM), la respiration à l'état 3 est diminuée dans les groupes contrôle et préconditionné par rapport au groupe sham (respectivement 20,3 ± 7,8 ; 19,2 ± 4,9 et 61,8 ± 7,9 nmoles O2/min/mg prot). La CRC est également diminuée chez ces mêmes animaux (respectivement 157 ± 12 ; 152 ± 14 et 557 ± 31 nmoles Ca2+/mg prot), tout comme la production de ROS après stimulation du complexe II de la chaine respiratoire (respectivement 131 ± 24 ; 110 ± 25 et 180 ± 15 pmoles H2O2/min/mg prot). En conclusion, le préconditionnement ischémique diminue la taille d'infarctus chez le rat sans changement significatif des functions mitochondriales à un temps court de reperfusion.

La mort cellulaire au cours de l'ischémie-reperfusion cardiaque est associée à l'ouverture du pore de transition de perméabilité mitochondrial (mPTP). La cyclosporine A (CsA) limite l'étendue des dommages myocardiques en inhibant l'ouverture du mPTP. La production massive de radicaux libres de l'oxygène (ROS) pendant la reperfusion est soupçonnée être un facteur favorisant l'ouverture du mPTP. Nous avons réalisé sur des mitochondries isolées de cœur de rats sham des études doses-réponses de l'inhibiteur du complexe I (la roténone de 6,25 nM à 6,25 μm), ou de l'inhibiteur du complexe III (antimycine A de 2,5 nM à 2,5 μm) sur la capacité de rétention du calcium (CRC) et la production de ROS en présence ou absence de CsA (1 μm). La CRC et la production de ROS ont été mesurées par spectrofluorimétrie en présence de Calcium Green ou d'Amplex Red respectivement. La roténone n'a pas d'effet sur la CRC (853 ± 62 nmoles/mg de prot) et la CsA augmente le CRC à 1405 ± 89 nmoles/mg de prot. La combinaison des deux potentialise l'effet de la CsA de 154 ± 9 %. L'antimycine A diminue la CRC à partir de 25 nM, la CsA n'antagonise pas cet effet. La production de ROS par les mitochondries est de 65 ± 9 pmol H2O2/min/mg prot. En présence de CsA, cette production diminue à 49 ± 6 pmol/min/mg prot. Quelque soit la dose utilisée de roténone, elle diminue cette production en présence ou absence de CsA. L'antimycine A augmente la production de ROS à partir de la concentration de 25 nM en présence ou absence de CsA. En conclusion, les inhibiteurs de la chaine respiratoire modifient l'ouverture du mPTP et la production de ROS. Ces résultats sont en faveur du rôle délétère des ROS sur le mPTP et indiquent que le complexe I participe à la formation du mPTP.

L’arrêt cardio-circulatoire (ACC) réanimé est une modalité d’ischémie reperfusion (I/R) globale. L’ouverture du pore de transition de perméabilité mitochondrial (mPTP), inhibé par la ciclosporine A (CsA), est un événement majeur à l’origine des lésions cellulaires (nécrose et apoptose) et de la dysfonction cardio-circulatoire secondaires à l’I/R. Le but de l’étude a été de vérifier que l’inhibition pharmacologique du mPTP concomitante de la réanimation cardio pulmonaire (RCP) pouvait limiter in vivo les lésions cellulaires et leurs conséquences fonctionnelles. Des lapins NZW ont été soumis à 15 min d’ACC hypoxique suivi d’une RCP (massage cardiaque et adrénaline) et de 120 min de reperfusion. Trois groupes ont été étudiés (ACC sans autre intervention, CsA 5 mg/kg administrée au début de la RCP, sham) dans deux protocoles expérimentaux (n = 7 12/groupe) : (1) hémodynamique invasive (incluant sonomicrométrie, débit aortique et dP/dt) et mesure de la taille d’infarctus myocardique (triphényltétrazolium), (2) dosage de troponine Ic et mitochondries isolées avec mesure fluorimétrique de la capacité de rétention calcique (CRC) et oxygraphie. Le succès de la RCP et la survie étaient améliorés de façon significative dans le groupe CsA avec des doses d’adrénaline plus faibles que dans le groupe ACC (p < 0,05). L’ensemble des paramètres hémodynamiques altérés par l’ACC était significativement amélioré par la CsA (p < 0,05 vs ACC). L’aire myocardique nécrosée était moindre dans le groupe CsA vs ACC (p < 0,05). La CRC, significativement diminuée dans le groupe ACC (p < 0,01), était restaurée par la CsA (p = ns vs sham). Comparativement au groupe sham, la consommation maximale d’oxygène (état 3) était significativement diminuée dans le groupe ACC et restaurée par la CsA. L’augmentation significative de la troponine Ic observée dans le groupe ACC comparativement aux animaux sham était limitée par la CsA (p < 0,05 vs ACC). Ces résultats montrent que l’inhibition pharmacologique in vivo du mPTP par la CsA, administrée au début de la RCP de l’ACC, limite (1) les dysfonctions mitochondriales, (2) les lésions myocardiques irréversibles, (3) la défaillance cardiocirculatoire.

Inactivation of GSK3β via phosphorylation at serine-9 residue has been shown to play a role in ischemic preconditioning. Inhibition of the opening of the mitochondrial permeability transition pore (mPTP) is supposed to be important in pre- and postconditioning. Using GSK-S9A transgenic mice (whose cardiac GSK3β activity cannot be inactivated), we addressed whether postconditioning might require the inactivation of GSK-3β, upstream of the mPTP. Anesthetized wild-type (WT) and GSK-S9A mice underwent 60 min. of LAD occlusion followed by 24 h of reperfusion. WT and GSK-S9A mice were each randomized into 4 groups (n=8–9/g): control (C: no additional intervention), postconditioned (postC: 3 episodes of 1 min. of ischemia followed by 1 min. of reperfusion), CsA (IV bolus injection of 10 mg/kg of ciclosporin A, a mPTP inhibitor) or SB21 (IV bolus injection of SB216763, a GSK3β inhibitor) 1 min before reperfusion. Infarct size was assessed by TTC staining. Area at risk was comparable among groups. In WT mice, infarct size of PostC, SB21 and CsA groups was significantly reduced, averaging 39±3%, 37±4% and 35±5%, respectively, versus 56±5% in C (p<0.05 for all 3 groups). In GSK-S9A mice, infarct size averaged 66±7% in C (p=ns versus WT-C). Infarct size was significantly reduced in CsA, but not postC, groups, averaging 41±8% and 51±5%, respectively. These results suggest that S9-phosphorylation of GSK3β is required for cardioprotection by postconditioning and occurs upstream of the mPTP.

Inhibition of mitochondrial permeability transition pore (mPTP) opening by cyclosporin A or ischemic postconditioning attenuates lethal reperfusion injury. Its impact on major post-myocardial infarction events, including worsening of left ventricular (LV) function and death, remains unknown. We sought to determine whether pharmacological or postconditioning-induced inhibition of mPTP opening might improve functional recovery and survival following myocardial infarction in mice. Anesthetized mice underwent 25 min of ischemia and 24 h (protocol 1) or 30 days (protocol 2) of reperfusion. At reperfusion, they received no intervention (control), postconditioning (3 cycles of 1 min ischemia-1 min reperfusion), or intravenous injection of the mPTP inhibitor Debio-025 (10 mg/kg). At 24 h of reperfusion, mitochondria were isolated from the region at risk for assessment of the Ca(2+) retention capacity (CRC). Infarct size was measured by triphenyltetrazolium chloride staining. At 30 days of reperfusion, mortality and LV contractile function (echocardiography) were evaluated. Postconditioning and Debio-025 significantly improved Ca(2+) retention capacity (132 +/- 13 and 153 +/- 31 vs. 53 +/- 16 nmol Ca(2+)/mg protein in control) and reduced infarct size to 35 +/- 4 and 32 +/- 7% of area at risk vs. 61 +/- 6% in control (P < 0.05). At 30 days, ejection fraction averaged 74 +/- 6 and 77 +/- 6% in postconditioned and Debio-025 groups, respectively, vs. 62 +/- 12% in the control group (P < 0.05). At 30 days, survival was improved from 58% in the control group to 92 and 89% in postconditioned and Debio-025 groups, respectively. Inhibition of mitochondrial permeability transition at reperfusion improves functional recovery and mortality in mice.

Nitric oxide (NO) is a biological messenger synthesized by three main isoforms of NO synthase (NOS): neuronal (nNOS, constitutive calcium dependent), endothelial (eNOS, constitutive, calcium dependent) and inducible (iNOS, calcium independent). NOS is distributed in the brain either in circumscribed neuronal sets or in sparse interneurons. Within the laterodorsal tegmentum (LDT), pedunculopontine tegmentum and dorsal raphe nucleus, NOS-containing neurons overlap neurons grouped according to their contribution to steep mechanisms. The main target for NO is the soluble guanylate cyclase that triggers an overproduction of cyclic guanosine monophosphate. NO in neurons of the pontine tegmentum facilitates steep (particularly rapid-eye-movement steep), and NO contained within the LDT intervenes in modulating the discharge of the neurons through an auto-inhibitory process involving the co-synthesized neurotransmitters. Moreover, NO synthesized within cholinergic neurons of the basal forebrain, while under control of the LDT, may modulate the spectral components of the EEG instead of the amounts of different steep states. Finally, impairment of NO production (e.g. neurodegeneration, iNOS induction) has identifiable effects, including ageing, neuropathologies and parasitaemia. (c) 2004 Published by Elsevier Ltd.

Extensive evidences now suggest that an association between inducible nitric oxide synthase and oxidative stress takes place during aging. Since the part played by inducible nitric oxide synthase in the sleep impairments associated with aging still remains unexplored, we compared its involvement in old rats (20–24 months) versus adult ones (3–5 months) using polygraphic, biochemical, voltammetric and immunohistochemical techniques. The experiments were conducted either in basal condition or after a systemic injection of selected inducible nitric oxide synthase inhibitors. We found that 2-amino-5,6-dihydro-6-methyl-4H-1,3-thiazine (10mg/kg, i.p.) or aminoguanidine (400mg/kg, i.p.) was capable to suppress rapid-eye-movement sleep and induce a delayed enhancement in slow-wave sleep in old rats. These effects did not occur in adult animals. Within the frontal cortex, the laterodorsal tegmentum and dorsal raphe nuclei, the basal inducible nitric oxide synthase activity was 85–200% higher in old rats than in adult ones. In contrast, the neuronal nitric oxide synthase activity did not vary in both groups. 2-Amino-5,6-dihydro-6-methyl-4H-1,3-thiazine administration significantly reduced inducible nitric oxide synthase activity (70–80% according to the brain areas) independently of age, but significantly decreased the cortical nitric oxide release in old rats. Finally, in frontal cortex and dorsal raphe immunohistochemical analysis showed inducible nitric oxide synthase-positive cells again only in old animals. These data support the idea that nitric oxide produced by inducible nitric oxide synthase plays a role in the triggering and maintenance of rapid-eye-movement sleep during aging.

Evidence that nitric oxide (NO) is involved in the regulation of rapid-eye-movement sleep (REMS) is supported by recent studies. During aging, NO generation encounters marked changes mainly related to the activation of the inducible NO-synthase (iNOS). To investigate links existing between iNOS and REMS impairments related to aging, we examine the age-related variations occurring in: mRNA and activity of iNOS in brainstem and frontal cortex; sleep parameters under baseline and after treatment by a selective iNOS inhibitor (AMT) in Senescence Accelerated Mice (SAM). SAMR1 (control) mice are a model of aging while SAMP8 are adequate to study neurodegenerative processes. RT-PCR analysis does not reveal significant variation in iNOS mRNA expression in both strains. However, significant age-related increases in iNOS activity occur in SAMR1 but such variation is not observed in SAMP8. In baseline conditions, aging induces a slight increase in slow-wave sleep (SWS) amounts in both groups and deteriorates greatly REMS architecture in SAMP8 compared to SAMR1. AMT reduces REMS amounts for 4–6h after treatment in a dose and age-dependent manner in SAMR1. Almost no changes occur in SAMP8. Data reported suggest that NO derived from iNOS contributes to trigger and maintain REMS during aging.

The pollination of the DK 440 BTY (MON 810 genetic event) maize variety falls between the 74 and 88 day after sowing. The pollination of Hungarian maize varieties takes place during the course of July and August. In the case of the variety surveyed in our research – generating some 35 kg/hectares of pollen – the pollen coverage drops to 100 pollen grains / cm at a distance of 5 metres from the edge of the maize field. Caterpillars that hatch during the pollination period and feed on herbs in and near the maize field are most heavily affected by the toxin content of the pollen. The leaf surface/leaf mass ratio of stinging nettle (Urtica Dioica L., Urticaceae) is 2.85 times greater than that of common milkweed (Asclepias syriaca L. Asclepiadaceae). In the case of equal pollen coverage, this means an approximately three times higher Bt-toxin dose. There are 187 protected butterfly species in Hungary, 16% of which may develop on herbaceous weeds along maize fields as well. In the nettle growths in the drainage ditches of maize fields, the hatching of the larvae of two common butterfly species of the Nymphalidae family – peacock, Inachis io (L.) and red admiral (Vanessa atalanta) (L.) – coincides with pollination, as a consequence of which they are exposed to the Bt-contents of the maize pollen.

Sleep–wake homeostasis is crucial for behavioral performances and memory both in the general population and in patients with learning disability, among whom were Down syndrome (DS) patients. We investigated, in mouse models of DS, cortical EEG and sleep–wake architecture under baseline conditions and after a 4-h sleep deprivation (SD). Young hemizygous mice (hSODwt/+) transgenic for the human CuZn superoxide dismutase (hSOD1) or for the human amyloid precursor protein (HuAPP695; hAPPwt/+) were obtained on the same FVB/N inbred background. Baseline records for slow wave sleep (SWS) and wake (W) parameters were unchanged, whereas paradoxical sleep (PS) episode numbers were decreased and PS latency increased after lights off in hSODwt/+ mice versus controls. hSODwt/+ mice did not experience SWS or PS rebounds after SD but EEG activity in the delta-SWS activity (SWA) was enhanced. hAPPwt/+ mice exhibited no change in PS but an increase in W and a decrease in SWS before light transition as well as an increase in theta-power in PS and W. After SD, hAPPwt/+ mice exhibited SWS and PS rebounds as well as enhancement of SWA. We investigated also the nitrite/nitrate levels in all mice and found an increase in the brainstem of hSODwt/+ mice only versus control ones. These preliminary data provide useful results to investigate other genetically manipulated mice and to better understand the biochemical basis of sleep disorders in DS patients.

Variations occurring in cortical nitric oxide (NO) release were analysed with a voltametric method in rats (i) placed in control conditions, (ii) while being paradoxical sleep deprived (PSD), or (iii) recovering from a PSD. Activities of neuronal (nNOS) and inducible (iNOS) NO-synthases as well as nNOS expression were also determined in several brain regions. In baseline conditions, circadian variations in nNOS expression and activity were maximal during the dark period and minimal during the light one for all the structures analysed (frontal cortex, pons and medulla). In the same way, cortical NO release occurred through a circadian rhythm exhibiting maxima and minima during dark and light periods, respectively. In the same experimental conditions, iNOS activity did not exhibit time-dependent changes. The correlative changes observed in baseline conditions between NO release, nNOS expression and activity within the frontal cortex were disrupted during PSD and subsequent recovery. Still again, iNOS activity remained unchanged. Results obtained point out that the tight coupling existing in control conditions between nNOS expression-activity and NO release is disrupted by a PSD and remains affected during the subsequent 24 h recovery. Their significance is discussed.

Changes in sleep-wake states and nitric oxide release were examined in aged rats versus young-adult ones. Sleep-wake recordings and nitric oxide measurements were taken from animals chronically equipped with polygraphic and voltametric electrodes. Animals were examined in baseline conditions and in response to a 24-hour paradoxical sleep deprivation. In aged rats, basal amount of paradoxical sleep is decreased during the light phase versus young-adult animals. After paradoxical sleep deprivation, a paradoxical sleep rebound occurs with an amount and intensity that are less marked in aged animals than in young-adult rats. The amplitude of the circadian distribution for wakefulness, slow-wave sleep and paradoxical sleep amounts is reduced with age. Finally, delta-slow-wave sleep and theta-paradoxical sleep power spectra are attenuated either in baseline conditions or after paradoxical sleep deprivation in aged animals. It is also reported that cortical nitric oxide release exhibits a circadian rhythm with higher amplitude in aged rats than in young-adult ones. However, after paradoxical sleep deprivation, a limited overproduction of nitric oxide is obtained compared with young-adult ones. These results, evidencing the dynamics of the nitric oxide changes occurring in relation to the sleep-wake cycle, point out the homeostatic paradoxical sleep regulation as an age-dependent process in which the nitric oxide molecule is possibly involved.

The effects of a lethal gamma irradiation were investigated on cerebral NO-ergic system by using a voltammetric method in freely moving rats. It is reported that the cortical NO concentration increases right from the end of the radiation exposure (15 Gy) and reaches a maximal magnitude (+120%) 24 h later. A dose-effect relationship from 2 to 15 Gy for gamma-ray exposure has also been observed. The effects, obtained with either an NO synthase inhibitor nonselective for the different NO synthase isoforms or an NO synthase inhibitor selective for the constitutive isoform, suggest that the radiation-induced increase in NO is likely to be dependent on the inducible NO synthase isoform. Moreover, experiments performed under ex vivo conditions showed that the cortical mRNA level for Ca++-independent NO synthase, the brain NOS activity, and urinary nitrites/nitrates increased significantly 24 h after gamma-ray exposure. These results demonstrate that a supralethal wholebody irradiation alters the NO-ergic pathways. The increase in NO obtained under such conditions might constitute a good index of central nervous system radiosensitivity during the acute phase of the radiation syndrome. (C) 2003 by Radiation Research Society.