Regulation of cardiomyocyte apoptosis in ischemic reperfused mouse heart by glutathione peroxidase

Apoptosis, a genetically controlled programmed cell death, has been found to play a role in ischemic reperfusion injury in several animal species including rats and rabbits. To examine whether this is also true for other animals, an isolated perfused mouse heart was subjected to 30 min of ischemia followed by 2 h of reperfusion. Experiments were terminated before ischemia (baseline), after ischemia, and at 30, 60, 90 and 120 min of reperfusion. At the end of each experiment, hearts were processed for the evaluation of apoptosis and DNA laddering. The in situ end labeling (ISEL) technique was used to detect apoptotic cardiomyocyte nuclei while DNA laddering was evaluated by subjecting the DNA obtained from the cardiomyocytes to 1.8% agarose gel electrophoresis followed by photographing under UV illumination. The results of our study revealed that apoptotic cells appear only after 60 min of reperfusion as demonstrated by the intense fluorescence of the immunostained genomic DNA when observed under fluorescence microscopy. None of the ischemic hearts showed any evidence of apoptosis. These results were corroborated with the findings of DNA fragmentation showing increased ladders of DNA bands in the same reperfused hearts representing integer multiples of the internucleosomal DNA length (about 180 bp). Since our previous studies showed a role of glutathione peroxidase (GSHPx) in apoptotic cell death, we performed identical experiments using isolated hearts from GSHPx-l knockout mice and transgenic mice overexpressing GSHPx-l. GSHPx-l knockout mice showed evidence of apoptotic cell death even after 30 min of reperfusion. Significant number of apoptotic cells were found in the cardiomyocytes as compared to non-transgenic control animals. To the contrary, very few apoptotic cells were found in the hearts of the transgenic mice overexpressing GSHPx-l. Hearts of GSHPx-l knockout mice were more susceptible to ischemia/reperfusion injury while transgenic mice overexpressing GSHPx- 1 were less susceptible to ischemia reperfusion injury compared to non-transgenic control animals. The results of this study clearly demonstrate a role of GSHPx in ischemia/reperfusion-induced apoptosis in mouse heart.     

Absence of acute doxorubicin-induced dysfunction of heart mitochondrial oxidative phosphorylation and creatine kinase activities

Since reductions in cardiac high-energy phosphate content and dysfunction of mitochondrial activities have been demonstrated after doxorubicin exposure, one mechanism of doxorubicin cardiotoxicity has been thought to be an interference with mitochondrial energy metabolism. To determine whether mitochondrial dysfunction is induced by acute drug exposure, isolated rat hearts were perfused with 10(-5) M doxorubicin for 70 min followed by mitochondrial isolation. Rates of electron transport, creatine kinase activity, acceptor control, respiratory control, and ADP/O ratios were assayed and correlated to doxorubicin-induced abnormalities in left ventricular function. At doses of doxorubicin sufficient to cause a marked deterioration of left ventricular systolic pressure and a rise in end-diastolic pressure, no decreases were noted in the measured mitochondrial parameters with either glutamate plus malate or succinate as respiratory substrates. In fact, in some cases the rates of electron transport were higher in mitochondria isolated from the treated hearts. In addition, isolated heart mitochondria were directly incubated in doxorubicin at doses as high as 10(-4) M for up to 70 min at 0 and 20 degrees C and 1.5 min at 37 degrees C. Under these conditions functional impairment of mitochondrial respiration was also not detected. Therefore, it appears that acute doxorubicin cardiotoxicity cannot be related to primary mitochondrial defects in high-energy phosphate metabolism. These data lend further support to the notion that doxorubicin cardiotoxicity may be fundamentally related to changes in coronary vascular resistance and resultant damage induced by hypoperfusion.     

Control of doxorubicin-induced,reactive oxygen-related apoptosis by glutathione peroxidase 1 in cardiac fibroblasts

Reactive oxygen formation plays a mechanistic role in the cardiotoxicity of doxorubicin, a chemotherapeutic agent that remains an important component of treatment programs for breast cancer and hematopoietic malignancies. To examine the role of doxorubicin-induced reactive oxygen species (ROS) in drug-related cardiac apoptosis, murine embryonic fibroblast cell lines were derived from the hearts of glutathione peroxidase 1 (Gpx-1) knockout mice. Cells from homozygous Gpx-1 knockout mice and parental animals were propagated with (Se+) and without (Se-) 100 nM sodium selenite. Activity levels of the peroxide detoxifying selenoprotein glutathione peroxidase (GSHPx) were marginally detectable (<1.6 nmol/min/mg) in fibroblasts from homozygous knockout animals whether or not cells were supplemented with selenium. GSHPx activity in Se- cells from parental murine fibroblasts was also <1.6 nmol/min/mg, whereas GSHPx levels in Se+ parental murine fibroblasts were 12.9 ± 2.7 nmol/min/mg (mean ± SE; P < 0.05). Catalase, superoxide dismutase, glutathione reductase, glutathione S-transferase, glucose 6-phosphate dehydrogenase, and reduced glutathione activities did not differ amongst the four cell lines. Reactive oxygen production increased from 908 ± 122 (arbitrary units) for untreated control cells to 1668 ± 54 following exposure to 1 μM doxorubicin for 24 h in parental fibroblasts not supplemented with selenium (P < 0.03); reactive oxygen formation in doxorubicin-treated parental fibroblasts propagated in selenium was 996 ± 69 (P = not significant compared to untreated control cells). Reactive oxygen levels in homozygous Gpx-1 knockout fibroblasts, irrespective of selenium supplementation status, were increased and equivalent to that in selenium deficient wild type fibroblasts. When cardiac fibroblasts were exposed to doxorubicin (0.05 μM) for 96 h and examined for cell cycle alterations by flow cytometry, and apoptosis by TUNEL assay, marked G2 arrest and TUNEL positivity were observed in knockout fibroblasts in the presence or absence of supplemental selenium, and in parental fibroblasts propagated without selenium. Parental fibroblasts propagated with selenium and exposed to the same concentration of doxorubicin demonstrated modest TUNEL positivity and substantially diminished amounts of low molecular weight DNA. These results were replicated in cardiac fibroblasts exposed to doxorubicin (1–2 μM) for 2 h (to mimic clinical drug dosing schedules) and examined 96 h following initiation of drug exposure. Doxorubicin uptake in cardiac fibroblasts was similar irrespective of the mRNA expression level or activity of GSHPx. These experiments suggest that the intracellular levels of doxorubicin-induced reactive oxygen species (ROS) are modulated by GSHPx and play an important role in doxorubicin-related apoptosis and altered cell cycle progression in murine cardiac fibroblasts.     

Cellular glutathione peroxidase deficiency and endothelial dysfunction

Cellular glutathione peroxidase (GPx-1) is the most abundant intracellular isoform of the GPx antioxidant enzyme family. In this study, we hypothesized that GPx-1 deficiency directly induces an increase in vascular oxidant stress, with resulting endothelial dysfunction. We studied vascular function in a murine model of homozygous deficiency of GPx-1 (GPx-1(-/-)). Mesenteric arterioles of GPx-1(-/-) mice demonstrated paradoxical vasoconstriction to beta-methacholine and bradykinin, whereas wild-type (WT) mice showed dose-dependent vasodilation in response to both agonists. One week of treatment of GPx-1(-/-) mice with L-2-oxothiazolidine-4-carboxylic acid (OTC), which increases intracellular thiol pools, resulted in restoration of normal vascular reactivity in the mesenteric bed of GPx-1(-/-) mice. We observed an increase of the isoprostane iPF(2alpha)-III, a marker of oxidant stress, in the plasma and aortas of GPx-1(-/-) mice compared with WT mice, which returned toward normal after OTC treatment. Aortic sections from GPx-1(-/-) mice showed increased binding of an anti-3-nitrotyrosine antibody in the absence of frank vascular lesions. These findings demonstrate that homozygous deficiency of GPx-1 leads to impaired endothelium-dependent vasodilator function presumably due to a decrease in bioavailable nitric oxide and to increased vascular oxidant stress. These vascular abnormalities can be attenuated by increasing bioavailable intracellular thiol pools.     

Development of contractile dysfunction in rat heart failure: hierarchy of cellular events

The cellular mechanisms underlying the development of congestive heart failure (HF) are not well understood. Accordingly, we studied myocardial function in isolated right ventricular trabeculae from rats in which HF was induced by left ventricular myocardial infarction (MI). Both early-stage (12 wk post-MI; E-pMI) and late, end-stage HF (28 wk post-Mi; L-pMI) were studied. HF was associated with decreased sarcoplasmic reticulum Ca(2+) ATPase protein levels (28% E-pMI; 52% L-pMI). HF affected neither sodium/calcium exchange, ryanodine receptor, nor phospholamban protein levels. Twitch force at saturating extracellular [Ca(2+)] was depressed in HF (30% E-pMI; 38% L-pMI), concomitant with a marked increase in sensitivity of twitch force toward extracellular [Ca(2+)] (26% E-pMI; 68% L-pMI). Ca(2+)-saturated myofilament force development in skinned trabeculae was unchanged in E-pMI but significantly depressed in L-pMI (45%). Tension-dependent ATP hydrolysis rate was depressed in L-pMI (49%), but not in E-pMI. Our results suggest a hierarchy of cellular events during the development of HF, starting with altered calcium homeostasis during the early phase followed by myofilament dysfunction at end-stage HF.     

Attenuation of cardiac mitochondrial dysfunction by melatonin in septic mice

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.     

Induction of cellular necrosis by the glutathione peroxidase mimetic ebselen

The selenium-based compound ebselen is a powerful antioxidant, a potent anti-inflammatory agent and a potential neuroprotective compound. Several studies have demonstrated that part of the biological effect of ebselen is the result of the inhibition of apoptosis. We show in this report that ebselen induced the necrotic cell death of Sp2/0-Ag14 hybridoma cells. This process was rapid, with over 90% of the cells being dead after a 2 h exposure to 50 microM ebselen. The toxic effect of ebselen could not be prevented by the caspase inhibitor Z-VAD-fmk but could be blocked with thiol-containing compounds. Interestingly, ebselen addition completely prevented caspase activation in cycloheximide-treated Sp2/O-Ag14 cells, indicating that this antioxidant interferes with the apoptotic machinery. Our results indicate that some cell types are acutely sensitive to the toxic effect of ebselen, and that ebselen-induced cell death interferes with apoptotic processes. These observations are of particular importance since ebselen is currently used in clinical trials for possible use as therapeutic agent for stroke.     

Attenuation of methylglyoxal-induced glycation and cellular dysfunction in wound healing by Centella cordifolia

Current pre-clinical evidences of Centella focus on its pharmacological effects on normal wound healing but there are limited studies on the bioactivity of Centella in cellular dysfunction associated with diabetic wounds. Hence we planned to examine the potential of Centella cordifolia in inhibiting methylglyoxal (MGO)-induced extracellular matrix (ECM) glycation and promoting the related cellular functions. A Cell-ECM adhesion assay examined the ECM glycation induced by MGO. Different cell types that contribute to the healing process (fibroblasts, keratinocytes and endothelial cells) were evaluated for their ability to adhere to the glycated ECM. Methanolic extract of Centella species was prepared and partitioned to yield different solvent fractions which were further analysed by high performance liquid chromatography equipped with photodiode array detector (HPLC-PDA) method. Based on the antioxidant [2,2-diphenyl-1-picrylhydrazyl (DPPH) assay] screening, anti-glycation activity and total phenolic content (TPC) of the different Centella species and fractions, the ethyl acetate fraction of C. cordifolia was selected for further investigating its ability to inhibit MGO-induced ECM glycation and promote cellular distribution and adhesion. Out of the three Centella species (C. asiatica, C. cordifolia and C. erecta), the methanolic extract of C. cordifolia showed maximum inhibition of Advanced glycation end products (AGE) fluorescence (20.20 ± 4.69 %, 25.00 ± 3.58 % and 16.18 ± 1.40 %, respectively). Its ethyl acetate fraction was enriched with phenolic compounds (3.91 ± 0.12 mg CAE/μg fraction) and showed strong antioxidant (59.95 ± 7.18 μM TE/μg fraction) and antiglycation activities. Improvement of cells spreading and adhesion of endothelial cells, fibroblasts and keratinocytes was observed for ethyl acetate treated MGO-glycated extracellular matrix. Significant reduction in attachment capacity of EA.hy926 cells seeded on MGO-glycated fibronectin (41.2%) and attachment reduction of NIH3t3 and HaCaT cells seeded on MGO-glycated collagen (33.7% and 24.1%, respectively) were observed. Our findings demonstrate that ethyl acetate fraction of C. cordifolia was effective in attenuating MGO-induced glycation and cellular dysfunction in the in-vitro wound healing models suggesting that C. cordifolia could be a potential candidate for diabetic wound healing. It could be subjected for further isolation of new phytoconstituents having potential diabetic wound healing properties.     

Purification and properties of rat liver mitochondrial glutathione peroxidase.

Glutathione peroxidase (glutathione:hydrogen peroxide oxidoreductase, EC 1.11.1.9) was purified from rat liver mitochondria. The enzyme was shown to be pure by polyacrylamide-gel electrophoresis and to contain multiple forms that differed in charge. Selenium was specifically associated with the enzyme. The enzyme was inhibited by iodoacetic acid and iodoacetamide in an unusual pattern of reduction by sulfhydryl compounds and pH dependency. The mitochondrial and cytoplasmic forms of the enzyme were compared, and an explanation of the inhibition patterns is offered.     

Changes in mitochondrial redox state, membrane potential and calcium precede mitochondrial dysfunction in doxorubicin-induced cell death

Mitochondria play central roles in cell life as a source of energy and in cell death by inducing apoptosis. Many important functions of mitochondria change in cancer, and these organelles can be a target of chemotherapy. The widely used anticancer drug doxorubicin (DOX) causes cell death, inhibition of cell cycle/proliferation and mitochondrial impairment. However, the mechanism of such impairment is not completely understood. In our study we used confocal and two-photon fluorescence imaging together with enzymatic and respirometric analysis to study short- and long-term effects of doxorubicin on mitochondria in various human carcinoma cells. We show that short-term (< 30 min) effects include i) rapid changes in mitochondrial redox potentials towards a more oxidized state (flavoproteins and NADH), ii) mitochondrial depolarization, iii) elevated matrix calcium levels, and iv) mitochondrial ROS production, demonstrating a complex pattern of mitochondrial alterations. Significant inhibition of mitochondrial endogenous and uncoupled respiration, ATP depletion and changes in the activities of marker enzymes were observed after 48 h of DOX treatment (long-term effects) associated with cell cycle arrest and death.     

Acetaldehyde does not inhibit glutathione peroxidase and glutathione reductase from mouse liver in vitro

Acetaldehyde, the primary ethanol metabolite, has been implicated in the pathogenesis of alcoholic liver disease, but the mechanism involved is still under investigation. This study aims at the search for direct in vitro effects of different concentrations of acetaldehyde (30, 100 and 300microM) on the activities of glutathione reductase (GR), glutathione peroxidase (GPx) from liver supernatants, and the thiol-peroxidase activity of ebselen. They did not change after pre-incubation with acetaldehyde, which suggests that acetaldehyde does not have any direct effect. Nor were direct effects of acetaldehyde toward thiols, such as dithioerythritol and glutathione (GSH), observed either, even though GSH - measured as non-protein thiols from liver supernatants - were oxidized in the presence of acetaldehyde. In addition, acetaldehyde (up to 300microM) significantly oxidized GSH when incubated in the presence of commercially available gamma-glutamyltranspeptidase (GGT), but not in the presence of glutathione-S-transferase. The interaction between ebselen and GSH was also evaluated in an attempt to better understand the possible link between acetaldehyde and nucleophilic selenol groups. The formation and stability of ebselen intermediaries, produced in the chemical interaction between GSH and ebselen, were not affected by acetaldehyde either. Overall, the acetaldehyde oxidation of hepatic low-molecular thiols depends on mouse liver constituents and GGT is proposed as an important enzyme involved in this phenomenon. Thiol depletion, a phenomenon usually observed in the livers of alcoholic patients, can be related to GSH metabolism, and the involvement of GGT may reflect a molecular mechanism involved in thiol oxidation.     

Developmental expression of plasma glutathione peroxidase during mouse organogenesis

Plasma glutathione peroxidase (pGPx) is an extracellular antioxidative selenoenzyme which has been detected in various adult tissues, but little is known about the expression and distribution of pGPx during embryogenesis. To investigate the expression patterns of pGPx during embryogenesis, we performed quantitative real-time PCR, in situ hybridization, Western blot, and immunohistochemistry analyses in whole embryos or each developing organ of mice on embryonic days (E)7.5–18.5. In whole embryos of E7.5–8.5, pGPx mRNA was more typically expressed in extra-embryonic tissues including ectoplacental cone, trophectoderm, and decidual cells than in embryos. However, after E9.5, pGPx mRNA and protein levels were increased in the embryos with differentiation and growth, but trended to gradually decrease in the extra-embryonic tissues until E18.5. In sectioned embryonic tissues on E13.5–18.5, pGPx mRNA and protein were mainly expressed in the developing nervous tissues, the sensory organs, and the epithelia of lung, skin, and intestine, the heart and artery, and the kidney. In particular, pGPx immunoreactivity was very strong in the developing liver. These results indicate that pGPx is spatio-temporally expressed in various embryonic organs as well as extra-embryonic tissues, suggesting that pGPx may function to protect the embryos against endogenous and exogenous reactive oxygen species during organogenesis.     

Metformin alleviates human cellular aging by upregulating the endoplasmic reticulum glutathione peroxidase 7

Metformin, an FDA‐approved antidiabetic drug, has been shown to elongate lifespan in animal models. Nevertheless, the effects of metformin on human cells remain unclear. Here, we show that low‐dose metformin treatment extends the lifespan of human diploid fibroblasts and mesenchymal stem cells. We report that a low dose of metformin upregulates the endoplasmic reticulum‐localized glutathione peroxidase 7 (GPx7). GP×7 expression levels are decreased in senescent human cells, and GPx7 depletion results in premature cellular senescence. We also indicate that metformin increases the nuclear accumulation of nuclear factor erythroid 2‐related factor 2 (Nrf2), which binds to the antioxidant response elements in the GPX7 gene promoter to induce its expression. Moreover, the metformin‐Nrf2‐GPx7 pathway delays aging in worms. Our study provides mechanistic insights into the beneficial effects of metformin on human cellular aging and highlights the importance of the Nrf2‐GPx7 pathway in pro‐longevity signaling.     

Transgenic mouse models for the vital selenoenzymes cytosolic thioredoxin reductase,mitochondrial thioredoxin reductase and glutathione peroxidase 4

Selenium, as an integral part of selenoproteins, is essential for mammals. Unequivocal evidence had been provided more than a decade ago when it was proven that mice incapable of producing any of the 24 selenoproteins failed to develop beyond the gastrulation stage (E6.5). Since then, more specific attempts have been made to unmask novel and essential functions of individual selenoproteins in mice. Genetic disruption of glutathione peroxidase 4 (GPx4; also referred to as phospholipid hydroperoxide glutathione peroxidase, PHGPx) in mice showed for the first time that a specific selenoenzyme is in fact required for early embryonic development. Later on, systemic ablation of cytosolic thioredoxin reductase (Txnrd1) or mitochondrial thioredoxin reductase (Txnrd2) yielded embryonic lethal phenotypes. Beside those three, no other selenoproteins have been found being indispensable for murine development so far. This review aims at summarizing mainly the in vivo findings on these important mammalian selenoenzymes, which have not only common attributes of being required for embryogenesis, but that they are also instrumental in the regulation of cellular redox metabolism.     

Doxorubicin induces mitochondrial permeability transition and contractile dysfunction in the human myocardium

In human atrial trabeculae, we examined the effects of doxorubicin on the isometric force of contraction, mitochondrial respiration, membrane potential and calcium retention capacity. Compared with untreated controls, doxorubicin induced contractile dysfunction and depression of mitochondrial respiration. Mitochondria isolated from doxorubicin-treated human atrial trabeculae displayed reduced transmembrane potential and calcium retention capacity. Cyclosporine A, a mitochondrial membrane transition pore opening blocker, prevented mitochondrial dysfunction and impaired contractile performance induced by doxorubicin. The study suggests that a mitochondrial membrane transition pore opening is involved in the development of doxorubicin cardiotoxicity in human hearts.     

Prevention of mitochondrial dysfunction in post-traumatic mouse brain by superoxide dismutase

Reactive oxygen species (ROS) are known to be involved in the pathogenesis of traumatic brain injury (TBI). Previous studies have shown that the susceptibility of mice to TBI-induced formation of cortical lesion is determined by the expression levels of copper-zinc and manganese superoxide dismutase (CuZnSOD and MnSOD, respectively). However, the underlying biochemical mechanisms are not understood. In this study, we measured the efficiency of mitochondrial respiration in mouse brains with altered expression of these two enzymes. While controlled cortical impact injury (CCII) with a deformation depth of 2 mm caused a drastic decrease in NAD-linked bioenergetic capacity in brain mitochondria of wild-type mice, the functional decrease was not observed in brains of littermate transgenic mice overexpressing CuZnSOD or MnSOD. In addition, a 1 mm CCII greatly compromised brain mitochondrial function in mice deficient in CuZnSOD or MnSOD, but not wild-type mice. Inclusion of the calcium-chelating agent, EGTA, in the assay solution could completely prevent dysfunction of oxidative phosphorylation in all mitochondrial samples, suggesting that the observed impairment of mitochondrial function was a result of calcium overloading. In conclusion, our results imply that mitochondrial dysfunction induced by superoxide anion radical contributes to lesion formation in mouse brain following physical trauma.