Age-Associated Changes in Antioxidants and Redox Proteins of Rat Heart.

Oxidative stress and decline in cellular redox regulation have been hypothesized to play a key role in cardiovascular aging; however, data on antioxidant and redox regulating systems in the aging heart are controversial. The aim of the present study was to examine the effect of aging on critical antioxidant enzymes and two major redox-regulatory systems glutathione (GSH) and thioredoxin (Trx) system in hearts from adult (6-month-old), old (15-month-old), and senescent (26-month-old) rats. Aging was associated with a non-uniform array of changes, including decline in contents of reduced GSH and total mercaptans in the senescent heart. The activities of Mn-superoxide dismutase (SOD2), glutathione peroxidase (GPx), glutathione reductase (GR), and thioredoxin reductase (TrxR) exhibited an age-related decline, whereas catalase was unchanged and Cu,Zn-superoxide dismutase (SOD1) displayed only slight decrease in old heart and was unchanged in the senescent heart. GR, Trx, and peroxiredoxin levels were significantly reduced in old and/or senescent hearts, indicating a diminished expression of these proteins. In contrast, SOD2 level was unchanged in the old heart and was slightly elevated in the senescent heart. Decline in GPx activity was accompanied by a loss of GPx level only in old rats, the level in senescent heart was unchanged. These results indicate age-related posttranslational protein modification of SOD2 and GPx. In summary, our data suggest that changes are more pronounced in senescent than in old rat hearts and support the view that aging is associated with disturbed redox balance that could alter cellular signaling and regulation.

Effect of maturation on the resistance of rat hearts against ischemia. Study of potential molecular mechanisms.

Reduced tolerance to ischemia/reperfusion (IR) injury has been shown in elder human and animal hearts, however, the onset of this unfavorable phenotype and cellular mechanisms behind remain unknown. Moreover, aging may interfere with the mechanisms of innate cardioprotection (preconditioning, PC) and cause defects in protective cell signaling. We studied the changes in myocardial function and response to ischemia, as well as selected proteins involved in "pro-survival" pathways in the hearts from juvenile (1.5 months), younger adult (3 months) and mature adult (6 months) male Wistar rats. In Langendorff-perfused hearts exposed to 30-min ischemia/2-h reperfusion with or without prior PC (one cycle of 5-min ischemia/5-min reperfusion), we measured occurrence of reperfusion-induced arrhythmias, recovery of contractile function (left ventricular developed pressure, LVDP, in % of pre-ischemic values), and size of infarction (IS, in % of area at risk size, TTC staining and computerized planimetry). In parallel groups, LV tissue was sampled for the detection of protein levels (WB) of Akt kinase (an effector of PI3-kinase), phosphorylated (activated) Akt (p-Akt), its target endothelial NO synthase (eNOS) and protein kinase Cepsilon (PKCepsilon) as components of "pro-survival" cascades. Maturation did not affect heart function, however, it impaired cardiac response to lethal IR injury (increased IS) and promoted arrhythmogenesis. PC reduced the occurrence of malignant arrhythmias, IS and improved LVDP recovery in the younger animals, while its efficacy was attenuated in the mature adults. Loss of PC protection was associated with age-dependent reduced Akt phosphorylation and levels of eNOS and PKCepsilon in the hearts of mature animals compared with the younger ones, as well as with a failure of PC to upregulate these proteins. Aging-related alterations in myocardial response to ischemia may be caused by dysfunction of proteins involved in protective cell signaling that may occur already during the process of maturation.

Hyperhomocysteinemia as a risk factor for the neuronal system disorders.

Elevated concentration of the homocysteine (Hcy) in human tissues, resulting either from mutations in genes enconding Hcy-metabolizing enzymes, or from deficiences of folic acid has recognized cytotoxic effect. Even a mild Hcy level increase is a risk factor for cardiovascular diseases and stroke in humans and also a risk factor for neurodegenerative disordes, such as dementia, or Alzheimer's disease. However, it is not yet clear whether homocysteine is a marker, or a causative agent. We present here an overview of recent data on the homocysteine metabolism and on the genetic and the metabolic causes of hyperhomocysteinemia-related pathologies in humans. In context of our results which detected an increased oxidative stress in hyperhomocysteinemic rats we discuss here the role of free radicals in this disorder. Imbalance between homocysteine auto-oxidation, production of reactive metabolites and cellular antioxidant defence induced by hyperhomocysteinemia results to cytotoxicity by oxidizing membrane lipids and proteins. Consequently, protein thiolation and homocysteinylation results in the structural and functional modifications of cells, including neuronal ones. It is our hope that identification of prophylacting factors effective in the prevention of toxic effect of Hcy would lead to improved therapeutics, especially the brain tissue.

p-ERK involvement in the neuroprotection exerted by ischemic preconditioning in rat hippocampus subjected to four vessel occlusion.

Global brain ischemia-reperfusion causes delayed cell death in hippocampal CA1 (cornus ammonis 1) pyramidal neurons after reperfusion. Ischemic tolerance evoked by preconditioning (IPC) represents a phenomenon of CNS adaptation to any subsequent ischemia. This paper was designed to describe changes in the mitogen-activated protein kinases (MAPK) protein pathways of the hippocampal area following by IPC. Ischemia was induced by a 4-vessels occlusion (4VO) and the rats were preconditioned by a non-injurious ischemia. Apoptotic markers were used to follow the degeneration process. Western blot and immunohistochemistry identified p-ERK (phosphorylated extracellular signal-regulated protein kinase) and p38 proteins in injured hippocampal areas. P-ERK quantification increased after IPC and reached the highest level at 24 hours after ischemia. Interestingly, neuroprotection induced by IPC lead to the opposite effect on MAPK/p38, where the level was lowest at 24 hours after ischemia. Taken together, the present study clearly demonstrates that p-ERK takes part in complex cascades triggered by IPC in the selectively vulnerable hippocampal region. In addition, paper describes a crosstalk between p-ERK and p-p38 which occurs after preconditioning maneuver in 4VO model of global ischemia.

Why mitochondria are excellent targets for cancer therapy.

New insights into cancer cells - specific biological pathways are urgently needed to promote development of exactly targeted therapeutics. The role of oncoproteins and tumor suppressor proteins in proliferative signaling, cell cycle regulation and altered adhesion is well established. Chemicals, viruses and radiation are also generally accepted as agents that commonly induce mutations in genes encoding these cancer-inducing proteins, thereby giving rise to cancer. More recent evidence indicates the importance of two additional key factors imposed on proliferating cells - hypoxia and/or lack of glucose. These two additional triggers can initiate and promote the process of malignant transformation, when a low percentage of cells escape cellular senescence. Disregulated cell proliferation leads to formation of cellular masses that extend beyond the resting vasculature, resulting in oxygen and nutrient deprivation. Resulting hypoxia triggers a number of critical adaptations that enable cancer cell survival. The process of apoptosis is suppressed and glucose metabolism is altered. Recent investigations suggest that oxygen depletion stimulates mitochondria to compensate increased reactive oxygen species (ROS). It activates signaling pathways, such as hypoxia-inducible factor 1, that promote cancer cell survival and tumor growth. During the last decade, mitochondria have become key organelles involved in chemotherapy-induced apoptosis. Therefore, the relationship between mitochondria, ROS signaling and activation of survival pathways under hypoxic conditions has been the subject of increased study. Insights into mechanisms involved in ROS signaling may offer novel ways to facilitate discovery of cancer-specific therapies.

Effects of aging on activities of mitochondrial electron transport chain complexes and oxidative damage in rat heart.

Mitochondrial dysfunction and accumulation of oxidative damage have been implicated to be the major factors of aging. However, data on age-related changes in activities of mitochondrial electron transport chain (ETC) complexes remain controversial and molecular mechanisms responsible for ETC dysfunction are still largely unknown. In this study, we examined the effect of aging on activities of ETC complexes and oxidative damage to proteins and lipids in cardiac mitochondria from adult (6-month-old), old (15-month-old) and senescent (26-month-old) rats. ETC complexes I-IV displayed different extent of inhibition with age. The most significant decline occurred in complex IV activity, whereas complex II activity was unchanged in old rats and was only slightly reduced in senescent rats. Compared to adult, old and senescent rat hearts had significantly higher levels of malondialdehyde, 4-hydroxynonenal (HNE) and dityrosine, while thiol group content was reduced. Despite marked increase in HNE content with age (25 and 76 % for 15- and 26-month-old rats, respectively) Western blot analysis revealed only few HNE-protein adducts. The present study suggests that non-uniform decline in activities of ETC complexes is due, at least in part, to mitochondrial oxidative damage; however, lipid peroxidation products appear to have a limited impact on enzyme functions.

Molecular mechanisms leading to neuroprotection/ischemic tolerance: effect of preconditioning on the stress reaction of endoplasmic reticulum.

Ischemic tolerance can be developed by prior ischemic non-injurious stimulus preconditioning. The molecular mechanisms underlying ischemic tolerance are not yet fully understood. The purpose of this study is to evaluate the effect of preconditioning/preischemia on ischemic brain injury. We examined the endoplasmic reticulum stress response (unfolded protein response (UPR)) by measuring the mRNA and protein levels of specific genes such as ATF6, GRP78, and XBP1 after 15 min 4-VO ischemia and different times of reperfusion (1, 3, and 24 h). The data from the group of naïve ischemic rats were compared with data from the group of preconditioned animals. The results of the experiments showed significant changes in the gene expression at the mRNA level in the all ischemic/reperfusion phases. The influence of preischemia on protein level of XBP was significant in later ischemic times and at 3 h, the reperfusion reached 230% of the controls. The protein levels of GRP78 in preischemic animals showed a significant increase in ischemic and reperfusion times. They exceeded to 50% levels of corresponding naïve ischemic/reperfusion groups. Preconditioning also induced remarkable changes in the levels of ATF6 protein in the ischemic phase (about 170%). The levels of ATF6 remained elevated in earlier reperfusion times (37 and 62%, respectively) and persisted significantly elevated after 24 h of reperfusion. This data suggest that preconditioning paradigm (preischemia) underlies its neuroprotective effect by the attenuation of ER stress response after acute ischemic/reperfusion insult.

Alterations induced by ischemic preconditioning on secretory pathways Ca2+-ATPase (SPCA) gene expression and oxidative damage after global cerebral ischemia/reperfusion in rats.

Ischemic preconditioning (IPC) represents the phenomenon of CNC adaptation, which results in increased tolerance of CNS to lethal ischemia. Brain ischemia/reperfusion (IRI) initiates a catastrophic cascade in which many subcellular organelles play an important role. The Golgi apparatus, which is a part of secretory pathways (SP), represents the Ca(2+) store and regulates secretion of proteins for growth/reorganization of neuronal circuit by secretory Ca(2+)ATPases (SPCA1). The purpose of this study is to evaluate the effect of IRI and preconditioning on SPCA1 gene expression and oxidative damage after 4-vessel occlusion for 15 min and after being exposed to different reperfusion periods. Rats were preconditioned by 5 min of sub-lethal ischemia and 2 days later, 15 min of lethal ischemia was induced. Our experiments conclusively showed IRI-induced depression of SPCA activity and lipo- and protein oxidation in rat hippocampal membranes. IRI also activates the induction of SPCA1 gene expression in later reperfusion periods. IPC partially suppresses lipo- and protein oxidation in hippocampal membranes and leads to partiall rovery of the ischemic-induced depression of SPCA activity. In addition, IPC initiates earlier cellular response to the injury by the significant elevation of mRNA expression to 142% comparing to 1 h of corresponding reperfusion and to 11% comparing to 24 h of corresponding reperfusion, respectively. Similar patterns were observed on the translational level by Western blot analysis. Our results indicate the specific SPCA1 expression pattern in ischemic hippocampus. It also shows that the SPCA expression and the post-translational changes induced by ischemia are modulated by the IPC. This might serve to understand the molecular mechanisms involved in the structural integrity and function of the SP after ischemic challenge. It also suggests that there is a correlation of SPCA function with the role of SP in the response to pre-ischemic challenge.

Effects of long-term oxygen treatment on alpha-ketoglutarate dehydrogenase activity and oxidative modifications in mitochondria of the guinea pig heart.

OBJECTIVE: Oxygen therapy is used for the treatment of various diseases, but prolonged exposure to high concentrations of O(2) is also associated with formation of free radicals and oxidative damage. METHODS: In the present study we compared alpha-ketoglutarate dehydrogenase (KGDH) activity and mitochondrial oxidative damage in the hearts of guinea pigs after long-term (17 and 60 h) oxygenation with 100% normobaric O(2) and with partially negatively (O(2 neg)) or positively (O(2 posit)) ionized oxygen. RESULTS: Inhalation of O(2) led to significant loss in KGDH activity and thiol group content and accumulation of bityrosines. Inhalation of O(2 neg) was accompanied by more pronounced KGDH inhibition, possibly due to additional formation of protein-lipid conjugates. In contrast, O(2 posit) prevented loss in KGDH activity and diminished mitochondrial oxidative damage. CONCLUSIONS: These findings suggest that oxygen treatment is associated with impairment of heart energy metabolism and support the view that inhalation of O(2 posit) optimizes the beneficial effects of oxygen therapy.

Molecular analysis of endoplasmic reticulum stress response after global forebrain ischemia/reperfusion in rats: effect of neuroprotectant simvastatin.

Simvastatin is a cholesterol-lowering agent whose functional significance and neuroprotective mechanism in ischemic brain injury is not yet solved. The purpose of this study is to evaluate the effect of simvastatin on ischemic brain injury. We examined the endoplasmic reticulum stress response (UPR/unfolded protein response), by measuring the mRNA and protein levels of specific genes such as ATF6, GRP78, and XBP1 after 15 min 4-VO ischemia and different times of reperfusion (1, 3, and 24 h). The results from the group of naïve ischemic rats were compared with results from the group of pre-treated animals with simvastatin. The results of the experiments showed significant increase in all genes at the mRNA level in ischemic phase (about 43% for XBP1, 58% for GRP78, and 39% for ATF6 more than control). The protein level of XBP1 was decreased in pre-treated animals at ischemic phase and first hour of reperfusion (about 15% less), and did not reach control levels. The protein levels of GRP78 were maximal at third hour of reperfusion in statin group with a small decrease at 24 h of reperfusion in both groups. The levels of ATF6 mRNA in statin-treated animals was higher in comparison to non-statin animals at the ischemic phase and the third hour of reperfusion (about 35% higher), which was also translated into the higher protein level. This could indicate that one of the main proteins targeted to enhance neuroprotective effect to ER during the first two hours of reperfusion was ATF6 protein, the levels of which were 60% higher than in non-treated animals. These data suggest that simvastatin, in addition to the proposed neuroprotective effect, exerts a neuroprotective role in the attenuation of ER stress response after acute ischemic/reperfusion insult.

Ischemia-reperfusion induces inhibition of mitochondrial protein synthesis and cytochrome c oxidase activity in rat hippocampus.

Dysfunction of mitochondria induced by ischemia is considered to be a key event triggering neuronal cell death after brain ischemia. Here we report the effect of ischemia-reperfusion on mitochondrial protein synthesis and activity of cytochrome c oxidase (EC 1.9.3.1, COX). By performing 4-vessel occlusion model of global brain ischemia, we have observed that 15 min of global ischemia led to the inhibition of COX subunit I (COXI) synthesis to 56 % of control. After 1, 3 and 24 h of reperfusion, COXI synthesis was inhibited to 46, 50 and 72 % of control, respectively. Depressed synthesis of COXI was not a result of either diminished transcription of COXI gene or increased proteolytic degradation of COXI, since both Northern hybridization and Western blotting did not show significant changes in COXI mRNA and protein level. Thus, ischemia-reperfusion affects directly mitochondrial translation machinery. In addition, ischemia in duration of 15 min and consequent 1, 3 and 24 h of reperfusion led to the inhibition of COX activity to 90.3, 80.3, 81.9 and 83.5 % of control, respectively. Based on our data, we suggest that inhibition of COX activity is rather caused by ischemia-induced modification of COX polypeptides than by inhibition of mitochondrial translation.

Aminophylline treatment in meconium-induced acute lung injury in a rabbit model.

Administration of methylxanthines may diminish meconium-induced acute lung injury. Meconium-instilled rabbits intravenously received aminophylline (2.0 mg/kg) at two doses 0.5 h and 2.5 h after meconium instillation or were left without treatment, and were oxygen-ventilated for additional 5 h. At the end of experiment, lungs and trachea were excised. Within 5 h after the first dose of treatment, aminophylline significantly improved gas exchange and decreased right-to-left pulmonary shunts, central venous pressure, and ventilatory pressures. Moreover, aminophylline reduced meconium-induced lung edema formation, airway hyperreactivity to histamine, count of neutrophils in bronchoalveolar lavage fluid associated with higher total white blood cells and neutrophils in the blood, and diminished oxidative modifications of proteins and lipids in lung tissue compared with the non-treated meconium-instilled group. In a rabbit model of the meconium aspiration syndrome, aminophylline treatment enhanced pulmonary functions and alleviated oxidative injury and changes in airway reactivity related to lung inflammation.

Relationship between antioxidant potential and oxidative damage to lipids, proteins and DNA in aged rats.

Oxidative stress has been implicated to play a major role in aging and age-related diseases. In the present study, we investigated the effects of aging on the total antioxidant capacity, uric acid, lipid peroxidation, total sulfhydryl group content and damage to DNA in adult (6 months), old (15 months) and senescent (26 months) male Wistar rats. The antioxidant capacity, determined by phycoerythrin-based TRAP method (total peroxyl radical-trapping potential) was significantly decreased in the plasma and myocardium of old and senescent rats, whereas plasma level of uric acid was elevated in 26-month-old rats. Age-related decline in plasma and heart antioxidant capacity was accompanied by a significant loss in total sulfhydryl group content, increased lipid peroxidation and higher DNA damage in lymphocytes. Correlations between TRAP and oxidative damage to lipids, proteins and DNA suggest that the decline in antioxidant status may play an important role in age-related accumulation of cell damage caused by reactive oxygen species.

Impact of Ginkgo Biloba Extract EGb 761 on ischemia/reperfusion - induced oxidative stress products formation in rat forebrain.

Dysbalance in reactive oxygen/nitrogen species is involved in the pathogenesis of cerebral ischemia/reperfusion injury (IRI). Ginkgo biloba extract (Egb 761) pre-treatment was used to observe potential antioxidant/neuroprotective effect after global ischemia/reperfusion. Egb 761 significantly decreased the level of lipoperoxidation (LPO) in rat forebrain total membrane fraction (homogenate) induced by in vitro oxidative stress (Fe(2+)+H(2)O(2)). In animals subjected to four-vessel global ischemia for 15 min and 2-24 h reperfusion the EGb pretreatment slightly decreased LPO in forebrain homogenate. However, as detected in EGb treated group, the LPO-induced lysine conjugates are attenuated in comparison to non-treated IRI animals. EGb significantly improved parameters which indicate forebrain protein oxidative damage after IRI. The intensity of tryptophane fluorescence was increased by the 18.2% comparing to non-treated IRI group and bityrosine fluorescence was significantly decreased in ischemic (21%) and 24 h reperfused (15.9%) group in comparison non-treated IRI group. In addition, the level of total free SH- groups in pre-treated animals was significantly higher comparing to non-treated animals. Our results indicate that extract of EGb 761 has potent antioxidant activity and could play a role to attenuate the IRI-induced oxidative protein modification and lipoperoxidation in the neuroprotective process.

Effect of ischemia and reperfusion on protein oxidation in isolated rabbit hearts.

Reactive oxygen species and other oxidants are involved in the mechanism of postischemic contractile dysfunction, known as myocardial stunning. The present study investigated the oxidative modification of cardiac proteins in isolated Langendorff-perfused rabbit hearts subjected to 15 min normothermic ischemia followed by 10 min reperfusion. Reperfusion under these conditions resulted in only 61.8+/-2.7 % recovery of developed pressure relative to preischemic values and this mechanical dysfunction was accompanied by oxidative damage to cardiac proteins. The total sulfhydryl group content was significantly reduced in both ventricle homogenates and mitochondria isolated from stunned hearts. Fluorescence measurements revealed enhanced formation of bityrosines and conjugates of lipid peroxidation-end products with proteins in cardiac homogenates, whereas these parameters were unchanged in the mitochondrial fraction. Reperfusion did not alter protein surface hydrophobicity, as detected by a fluorescent probe 1-anilino-8-naphthalenesulfonate. Our results indicate that oxidation of proteins in mitochondria and possibly in other intracellular structures occurs during cardiac reperfusion and might contribute to ischemia-reperfusion injury.

Time course of ischemia/reperfusion-induced oxidative modification of neural proteins in rat forebrain.

Time course of oxidative modification of forebrain neural proteins was investigated in the rat model of global and partial cerebral ischemia/reperfusion. Animals were subjected to 4-vessel occlusion for 15 min (global ischemia). After the end of ischemia and at different reperfusion times (2, 24 and 48 h), lipoperoxidation-dependent and direct oxidative modification neural protein markers were measured in the forebrain total membrane fraction (tissue homogenate). Ischemia itself causes significant changes only in levels of tryptophan and bityrosine fluorescence when compared to controls. All tested parameters of protein modification altered significantly and were maximal at later reperfusion stage. Content of carbonyl group in re-flow period steadily increased and culminated at 48 h of reperfusion. The highest increase in the fluorescence of bityrosines was detected after 24 h of reperfusion and was statistically significant to both sham operated and ischemic groups. The changes in fluorescence intensity of tryptophan decreased during a reperfusion time dependent manner. Formation of lysine conjugates with lipoperoxidation end-products significantly increased only at later stages of reperfusion. Total forebrain membranes from animals subjected to 3-vessel occlusion model to 15 min (partial ischemia) show no altered content of oxidatively modified proteins compared to controls. Restoration of blood flow for 24 h significantly decreased only fluorescence of aromatic tryptophan. Partial forebrain ischemia/reperfusion resulted in no detectable significant changes in oxidative products formation in extracerebral tissues (liver and kidney) homogenates. Our results suggest that global ischemia/reperfusion initiates both the lipoperoxidation-dependent and direct oxidative modifications of neural proteins. The findings support the view that spatial and temporal injury at later stages of ischemic insult at least partially involves oxidative stress-induced amino acid modification. The results might have important implications for the prospective post-ischemic antioxidant therapy.