Oxygen and glucose deprivation induces mitochondrial dysfunction and oxidative stress in neurones but not in astrocytes in primary culture

In order to investigate the potential neuroprotective role played by glucose metabolism during brain oxygen deprivation, the susceptibility of cultured neurones and astrocytes to 1 h of oxygen deprivation (hypoxia) or oxygen and glucose deprivation (OGD) was examined. OGD, but not hypoxia, promotes dihydrorhodamine 123 and glutathione oxidation in neurones but not in astrocytes reflecting free radical generation in the former cells. A specific loss of mitochondrial complex-I activity, mitochondrial membrane potential collapse, ATP depletion and necrosis occurred in the OGD neurones, but not in the OGD astrocytes. Furthermore, superoxide anion but not nitric oxide formation was responsible for these effects. OGD decreased neuronal but not astrocytic NADPH concentrations; this was not observed in hypoxia and was independent of superoxide or nitric oxide formation. These results suggest that glucose metabolism would supply NADPH, through the pentose-phosphate pathway, aimed at preventing oxidative stress, mitochondrial damage and neurotoxicity during oxygen deprivation to neural cells.     

Adrenomedullin protects neurons against oxygen glucose deprivation stress in an autocrine and paracrine manner

The understanding of mechanisms involved in ischaemic brain tolerance may provide new therapeutical targets for stroke. In vivo genomic studies revealed an up-regulation of adrenomedullin expression by hypoxic pre-conditioning. Furthermore, adrenomedullin reduced ischaemia-induced brain damage in rodents. However, whether adrenomedullin is involved in hypoxic pre-conditioning-induced tolerance and whether adrenomedullin protects directly neurons against ischaemia remain unknown. Using a neuronal model of hypoxic pre-conditioning and oxygen glucose deprivation (OGD), we showed that 0.1% or 0.5% of O₂ pre-conditioning reduced the OGD-induced neuronal death, whereas 1% or 2% of O₂ pre-treatment did not induce neuroprotection. Adrenomedullin expression increased following the hypoxic period, and following OGD only in pre-conditioned (0.1% or 0.5% of O₂) neurons. Adrenomedullin pre-treatment and post-treatment reduced the OGD-induced neuronal death, partly through PI3kinase-dependent pathway. However, adrenomedullin antagonism during hypoxic pre-conditioning failed to inhibit the neuroprotection whereas adrenomedullin antagonism following OGD abolished the hypoxic pre-conditioning-induced neuroprotection. Finally, we showed that adrenomedullin is involved in neuroprotection induced by endothelial cells and microglia. In contrast, neuroprotection induced by astrocytes occurred through adrenomedullin-independent mechanisms. Altogether, our results suggest that adrenomedullin is an effector of the hypoxic pre-conditioning-induced neuronal tolerance and a potent autocrine and paracrine neuroprotective factor during cerebral ischaemia.     

Glucose deprivation induces mitochondrial dysfunction and oxidative stress in PC12 cell line

Glucose metabolism plays a pivotal role in many physiological and pathological conditions. To investigate the effect of hypoglycemia (obtained by glucose deprivation) on PC12 cell line, we analyzed the cell viability, mitochondrial function (assessed by MTT reduction, cellular ATP level, mitochondrial transmembrane potential), and the level of reactive oxygen species (ROS) after glucose deprivation (GD). Upon exposure to GD, ROS level increased and MTT reduction decreased immediately, intracellular ATP level increased in the first 3 hours, followed by progressive decrease till the end of GD treatment, and the mitochondrial transmembrane potential (ΔΨ_m) dropped after 6 hours. Both necrosis and apoptosis occurred apparently after 24 hours which was determined by nuclei staining with propidium iodide(PI) and Hoechst 33342. These data suggested that cytotoxity of GD is mainly due to ROS accumulation and ATP depletion in PC12 cells.     

Swim training attenuates oxidative damage and promotes neuroprotection in cerebral cortical slices submitted to oxygen glucose deprivation

Although it is well known that regular exercise may promote neuroprotection, the mechanisms underlying this effect are still not fully understood. We investigated if swim training promotes neuroprotection by potentiating antioxidant pathways, thereby decreasing the effects of oxidative stress on glutamate and nitric oxide release. Male Wistar rats (n=36) were evenly randomized into a trained group (TRA) (5 days/week, 8 weeks, 30 min) and a sedentary group (SED). Forty‐eight hours after the last session of exercise, animals were killed and brain was collected for in vitro ischemia. Cortical slices were divided into two groups: a group in which oxidative stress was induced by oxygen and glucose deprivation (OGD), and a group of non‐deprived controls (nOGD). Interestingly, exercise by itself increased superoxide dismutase activity (nOGD, SED vs. TRA animals) with no effect on pro‐oxidative markers. In fact, TRA‐OGD slices showed lowered levels of lactate dehydrogenase when compared with SED‐OGD controls, reinforcing the idea that exercise affords a neuroprotective effect. We also demonstrated that exercise decreased glutamate and nitrite release as well as lipid membrane damage in the OGD cortical slices. Our data suggest that under conditions of metabolic stress, swim training prevents oxidative damage caused by glutamate and nitric oxide release.     

Nitro-oleic acid ameliorates oxygen and glucose deprivation/re-oxygenation triggered oxidative stress in renal tubular cells via activation of Nrf2 and suppression of NADPH oxidase

Nitroalkene derivative of oleic acid (OA-NO₂), due to its ability to mediate revisable Michael addition, has been demonstrated to have various biological properties and become a therapeutic agent in various diseases. Though its antioxidant properties have been reported in different models of acute kidney injury (AKI), the mechanism by which OA-NO₂ attenuates intracellular oxidative stress is not well investigated. Here, we elucidated the anti-oxidative mechanism of OA-NO₂ in an in vitro model of renal ischemia/reperfusion (I/R) injury. Human tubular epithelial cells were subjected to oxygen and glucose deprivation/re-oxygenation (OGD/R) injury. Pretreatment with OA-NO₂ (1.25?μM, 45?min) attenuated OGD/R triggered reactive oxygen species (ROS) generation and subsequent mitochondrial membrane potential disruption. This action was mediated via up-regulating endogenous antioxidant defense components including superoxide dismutase (SOD1), heme oxygenase 1 (HO-1), and γ-glutamyl cysteine ligase modulatory subunits (GCLM). Moreover, subcellular fractionation analyses demonstrated that OA-NO₂ promoted nuclear translocation of nuclear factor-E2- related factor-2 (Nrf2) and Nrf2 siRNA partially abrogated these protective effects. In addition, OA-NO₂ inhibited NADPH oxidase activation and NADPH oxidase 4 (NOX4), NADPH oxidase 2 (NOX2) and p22^phox up-regulation after OGD/R injury, which was not relevant to Nrf2. These results contribute to clarify that the mechanism of OA-NO₂ reno-protection involves both inhibition of NADPH oxidase activity and induction of SOD1, Nrf2-dependent HO-1, and GCLM.     

Glucose deprivation increases nuclear DNA repair protein Ku and resistance to radiation induced oxidative stress in human cancer cells

Recent studies have indicated that nutrient deprivation particularly glucose may play a major role in tumor cell tolerance to a generally oxidative stress environment in solid tumors. Here, we studied the impact of glucose deprivation on the response of human colon (HT29) and prostate (DU145) cancer cells to γ radiation. A significant decrease in intracellular glucose level was observed in glucose deprived cells as measured by bioreductive assay. The survival of HT29 and DU145 were increased by 30 and 100% respectively when these cells were exposed to γ radiation in the absence of glucose compared to that in the presence of glucose. In glucose depleted medium, glutathione (GSH), a free radical scavenger, content remained the same, and showed no correlation with the radiation resistance induced by glucose deprivation. Glucose regulated protein78 (GRP78), a stress response survival protein, was not significantly increased in cells deprived of glucose for 4 h compared to those cells in glucose. DNA repair protein Ku, which is known to play a major role in cellular resistance to radiation, was significantly increased in glucose deprived cancer cells that showed enhanced radiation resistance. These results have demonstrated, for the first time, that glucose deprivation mediated stress increased the expression of nuclear Ku and resistance to radiation induced oxidative stress in human cancer cells. The additional resistance caused by glucose deprivation in cancer cells has clinical significance since solid tumors are known to have low level of glucose due to diffusion limited blood supply and higher metabolic activity. Copyright © 2009 John Wiley & Sons, Ltd.     

Synergistic depletion of astrocytic glutathione by glucose deprivation and peroxynitrite: correlation with mitochondrial dysfunction and subsequent cell death

Previously we reported that immunostimulated astrocytes were highly vulnerable to glucose deprivation. The augmented death was mimicked by the peroxynitrite (ONOO )-producing reagent 3-morpholinosydnonimine (SIN-1). Here we show that glucose deprivation and ONOO- synergistically deplete intracellular reduced glutathione (GSH) and augment the death of astrocytes via formation of cyclosporin A-sensitive mitochondrial permeability transition (MPT) pore. Astrocytic GSH levels were only slightly decreased by glucose deprivation or SIN-1 (200 microM) alone. In contrast, a rapid and large depletion of GSH was observed in glucose-deprived/ SIN-1-treated astrocytes. The depletion of GSH occurred before a significant release of lactate dehydrogenase (a marker of cell death). Superoxide dismutase and ONOO-scavengers completely blocked the augmented death, indicating that the reaction of nitric oxide with superoxide to form ONOO was implicated. Furthermore, nitrotyrosine immunoreactivity (a marker of ONOO-) was markedly enhanced in glucose-deprived/SIN-1 -treated astrocytes. Mitochondrial transmembrane potential (MTP) was synergistically decreased in glucose-deprived/SIN-1-treated astrocytes. The glutathione synthase inhibitor L-buthionine-(S,R)-sulfoximine markedly decreased the MTP and increased lactate dehydrogenase (LDH) releases in SIN-1-treated astrocytes. Cyclosporin A, an MPT pore blocker, completely prevented the MTP depolarization as well as the enhanced LDH releases in glucose-deprived/SIN-1-treated astrocytes.     

Atorvastatin prevents cell damage via modulation of oxidative stress,glutamate uptake and glutamine synthetase activity in hippocampal slices subjected to oxygen/glucose deprivation

Oxygen–glucose deprivation (OGD) in brain cells increases extracellular glutamate concentration leading to excitotoxicity. Glutamate uptake from the synaptic cleft is carried out by glutamate transporters, which are likely to be modulated by oxidative stress. Therefore, oxidative stress is associated with reduced activity of glutamate transporters and glutamine synthetase, thus increasing extracellular glutamate levels that may aggravate damage to brain cells. Atorvastatin, a cholesterol-lowering agent, has been shown to exert neuroprotective effects. The aim of this study was to investigate if in vivo atorvastatin treatment would have protective effects against hippocampal slices subjected to OGD, ex vivo. Atorvastatin pretreatment promoted increased cell viability after OGD and reoxygenation of hippocampal slices. Atorvastatin-induced neuroprotection may be related to diminished oxidative stress, since it prevented OGD-induced decrement of non-proteic thiols (NPSH) levels and increase in the production of reactive oxygen species (ROS). Atorvastatin pretreatment also prevented the OGD-induced decrease in glutamate uptake and glutamine synthetase activity, although it had no effect on OGD-induced excitatory aminoacids release. Addition of cholesterol before OGD and reoxygenation, abolished the protective effect of atorvastatin on cellular viability as well as on glutamate uptake and glutamine synthetase activity. Therefore, atorvastatin is capable of preventing OGD-induced cell death, an effect achieved due to modulation of glutamate uptake and glutamine synthetase activity, and associated with diminished oxidative stress. Additionally, atorvastatin effects were dependent on its action on cholesterol synthesis inhibition. Thus, atorvastatin might be a useful strategy in the prevention of glutamate exitotoxicity involved in brain injuries such as vascular disorders.     

Restoring mitochondrial DNA copy number preserves mitochondrial function and delays vascular aging in mice

Aging is the largest risk factor for cardiovascular disease, yet the molecular mechanisms underlying vascular aging remain unclear. Mitochondrial DNA (mtDNA) damage is linked to aging, but whether mtDNA damage or mitochondrial dysfunction is present and directly promotes vascular aging is unknown. Furthermore, mechanistic studies in mice are severely hampered by long study times and lack of sensitive, repeatable and reproducible parameters of arterial aging at standardized early time points. We examined the time course of multiple invasive and noninvasive arterial physiological parameters and structural changes of arterial aging in mice, how aging affects vessel mitochondrial function, and the effects of gain or loss of mitochondrial function on vascular aging. Vascular aging was first detected by 44 weeks (wk) of age, with reduced carotid compliance and distensibility, increased β‐stiffness index and increased aortic pulse wave velocity (PWV). Aortic collagen content and elastin breaks also increased at 44 wk. Arterial mtDNA copy number (mtCN) and the mtCN‐regulatory proteins TFAM, PGC1α and Twinkle were reduced by 44 wk, associated with reduced mitochondrial respiration. Overexpression of the mitochondrial helicase Twinkle (Tw⁺) increased mtCN and improved mitochondrial respiration in arteries, and delayed physiological and structural aging in all parameters studied. Conversely, mice with defective mitochondrial polymerase‐gamma (PolG) and reduced mtDNA integrity demonstrated accelerated vascular aging. Our study identifies multiple early and reproducible parameters for assessing vascular aging in mice. Arterial mitochondrial respiration reduces markedly with age, and reduced mtDNA integrity and mitochondrial function directly promote vascular aging.     

Silibinin activates AMP-activated protein kinase to protect neuronal cells from oxygen and glucose deprivation-re-oxygenation

In this study, we explored the cytoprotective potential of silibinin against oxygen–glucose deprivation (OGD)-induced neuronal cell damages, and studied underling mechanisms. In vitro model of ischemic stroke was created by keeping neuronal cells (SH-SY5Y cells and primary mouse cortical neurons) in an OGD condition followed by re-oxygenation. Pre-treatment of silibinin significantly inhibited OGD/re-oxygenation-induced necrosis and apoptosis of neuronal cells. OGD/re-oxygenation-induced reactive oxygen species (ROS) production and mitochondrial membrane potential (MMP) reduction were also inhibited by silibinin. At the molecular level, silibinin treatment in SH-SY5Y cells and primary cortical neurons led to significant AMP-activated protein kinase (AMPK) signaling activation, detected by phosphorylations of AMPKα1, its upstream kinase liver kinase B1 (LKB1) and the downstream target acetyl-CoA Carboxylase (ACC). Pharmacological inhibition or genetic depletion of AMPK alleviated the neuroprotective ability of silibinin against OGD/re-oxygenation. Further, ROS scavenging ability by silibinin was abolished with AMPK inhibition or silencing. While A-769662, the AMPK activator, mimicked silibinin actions and suppressed ROS production and neuronal cell death following OGD/re-oxygenation. Together, these results show that silibinin-mediated neuroprotection requires activation of AMPK signaling.     

Effect of GRP75/mthsp70/PBP74/mortalin overexpression on intracellular ATP level, mitochondrial membrane potential and ROS accumulation following glucose deprivation in PC12 cells

Glucose regulated protein 75 (GRP75) is an important molecular chaperon belonged to the heat shock protein (HSP) family. To evaluate the effect of GRP75 overexpression on PC12 cells under glucose deprivation, cell viability and mitochondrial function of GRP75-overexpressing PC12 cells and the vector transfected control PC12 cells were monitored during glucose deprivation. Upon exposure to glucose deprivation, GRP75-overexpressing PC12 cells exhibited more moderate cell damage than control PC12 cells. Both of the two groups of cells showed a decreased ATP level following an early increase in the condition of glucose deprivation, and the mitochondrial potential were also reduced in the similar manner in the two groups of cells. Control PC12 cells showed an immediate and rapid increase in ROS accumulation after the onset of GD treatment, and this accumulation was slowed and reduced in GRP75-overexpressing PC12 cells. These findings suggested that GRP75 could inhibit the ROS accumulation, and it may be associated with the cytoprotective effect of GRP75 overexpression upon glucose deprivation. (Mol Cell Biochem 268: 45–51, 2005)     

Injury to primary cultures of rat heart endothelial cells by hypoxia and glucose deprivation

Summary Primary cultures of rat heart endothelial cells were subjected to simulated conditions of ischemia: hypoxia and glucose deprivation for 4 and 24 hr. Cellular injury was evaluated by measuring changes in viability, total protein, cellular morphology, and leakage of cytoplasmic enzymes from the cells into the culture medium. Deprivation of oxygen and glucose for 4 or 24 hr did not lethally injure the cells as noted by no change in cell viability, morphology, and total protein when compared to controls. However, reversible or nonlethal cellular injury was produced as reflected by a significant release of lactate dehydro-genase (LDH) from the cells into the medium after treatment with hypoxia and glucose deprivation for 4 or 24 hr. When the cultures were deprived of glucose, but were oxygenated, cellular injury was not evident after 24 hr. Deprivation of oxygen but not glucose resulted in significant loss of LDH after 4 or 24 hr. When the cultures were allowed to recover after oxygen and glucose deprivation in complete medium containing 1000 mg glucose per l and a normal atmosphere of 20% O₂, they had levels of LDH leakage comparable to those of control cultures. This study was supported by Research Grant HL 18647 from the National Heart, Lung, and Blood Institute and by a National Chicano Council on Higher Education Post-Doctoral Fellowship awarded to D. Acosta from the Ford Foundation. Additional support was provided to D. Acosta by a Faculty Research Assignment Award from the University of Texas Research Institute.     

Ethanol-induced oxidative stress in rat astrocytes: role of HSP70

Ethanol intake is associated with increase in lipid peroxidation and formation of reactive oxygen species in different cerebral areas, in neurons as well as in astrocytes. The latter's integrity is essential for the normal growth of neurons. In previous studies we observed, in different cerebral areas of both acutely and chronically ethanol-treated rats, correlation between ethanol-induced oxidative stress and the increased expression of HSP70 (70 kDa heat shock proteins), chaperonins having a protective and stabilizing effect on stress–induced cell injury. In this study we examined, in vitro, the role of HSP70 on chronically ethanol-treated rat astrocytes by transfection with an anti-HSP70 antisense oligonucleotide. The results show that treatment with ethanol, from 50 to 100 mmol/L, induces a dose-dependent increase in the production of reactive oxygen species and of HSP70 levels, together with an impairment of the respiratory chain activity and a decrease in cell viability. In addition, our data indicate a drastic reduction of cellular metabolism in HSP70-deprived astrocytes, particularly when these cells were also ethanol-treated. In fact, transfection with HSP70 antisense induced moderate oxidative damage in control astrocytes and, consequently, a drastic decrease in the viability of ethanol-treated cells, with the mitochondrial functionality being particularly affected. Our results confirm that heat shock proteins confer a survival advantage to the astrocytes, preventing oxidative damage and nuclear DNA damage as well, and suggest the development of new drugs exerting a cytoprotective role either in physiological, or pathological conditions. This revised version was published online in July 2006 with corrections to the Cover Date.     

Distinct H2O2 concentration promotes proliferation of tumour cells after transient oxygen/glucose deprivation

A solid tumour undergoes ischemia/reperfusion due to deficient vascularization and subsequent formation of new blood vessels. This study investigated the effect of transient oxygen and glucose deprivation (OGD) on proliferation of C6 glioma cells. The cells were subjected to 18 h of OGD followed by reoxygenation in the presence of glucose and different extra-cellular H₂O₂ concentrations since H₂O₂ affects cell proliferation. After reoxygenation, the cellular H₂O₂ concentration was increased returning to control levels within 24 h. Within this period, increase in cell number and MTT-reduction were impaired. Regeneration was completed on the third day of reoxygenation. MTT-reduction increased faster than cell number, indicating an OGD-dependent up-regulation of protein expression. It is concluded that ischemia/reperfusion stress promotes proliferation of tumour cells. An essential factor is a distinct H₂O₂ concentration. Massive elevation as well as significant reduction of H₂O₂ concentration impairs the proliferation process.     

Expression of glucose transporter GLUT3 by endotoxin in cultured rat astrocytes: the role of nitric oxide

The induction of nitric oxide (NO) synthase in astrocytes by endotoxin and/or cytokine treatment is associated with increased glucose consumption and glycolysis, but the mechanism whereby this phenomenon occurs remains obscure. In this work, we have addressed this issue and found that incubation of cultured rat astrocytes with lipopolysaccharide (LPS; 1 microg/mL) for 24 h increased the level of constitutively expressed GLUT1 glucose transporter mRNA, and triggered GLUT3 mRNA expression, which was absent in normal astrocytes. The occurrence of GLUT3 protein after LPS treatment was corroborated by western blotting and immunocytochemistry. A 4-h incubation of astrocytes in the absence of glucose, or under an oxygen-poor (3%) atmosphere also resulted in GLUT3 mRNA overexpression. Experiments performed with 2-deoxy-D-[U-14C]glucose (at 0.1 mM of D-glucose) confirmed that LPS (0.1-10 microg/mL) dose-dependently increased the rate of glucose uptake (by a factor of 1.6 at 1 microg/mL of LPS), which was paralleled with the increase in NO synthesis. Furthermore, blockade of NO synthase with 2-amino-5,6-dihydro-6-methyl-(4H)-1,3-thiazine (AMT; 50 microM) partially (by 45%) prevented the LPS-mediated increase in glucose uptake. Finally, incubation of astrocytes with the NO donor 1-[2-(2-aminoethyl)-N-(2-ammonioethyl)amino]diazen-1-ium-1,2-diolate (DETA; 100 microM) increased by a factor of 1.4 the rate of glucose uptake. We conclude that the increase in GLUT3-driven glucose uptake in astrocytes would have a neuroprotective role under conditions in which NO formation is combined with hypoglycaemia, such as in brain ischemia.     

Effects of phorbol ester on immunoreactive protein kinase C,insulin binding,and glucose uptake in astrocytic glial and neuronal cells from the brain

Phorbol esters, potent stimulators of protein kinase C (PKC), stimulate [³H]2-deoxy-d-glucose (dGlc) uptake and [¹²⁵I] insulin binding in cultured glial cells but not neuronal cells from neonatal rat brains. Using an antibody to the and forms of PKC we have demonstrated that both neuronal and glial cells contain an immunoactive PKC of Mr 80 kD, although the PKC level in neurons is greater than 4-fold that in glia. The majority of immunoactive PKC (63%) is cytosolic in glial cells although the reverse is true in neuronal cells, in which 88% of the PKC is membrane-bound in the basal state. The most potent phorbol ester, 12-O-tetradecanoylphorbol-13-acetate (TPA), stimulates a redistribution of this enzyme in neuronal and glial cells. The TPA-stimulated translocation of PKC from cytosol to membrane precedes TPA's effecs of [³H]dGlc uptake and insulin binding in glial cells.     

Overexpression of GRP78 enhances survival of CHO cells in response to serum deprivation and oxidative stress

Chinese hamster ovary (CHO) cells are regarded as one of the most commonly used mammalian hosts, which decreases the productivity due to loss in culture viability. Overexpressing antiapoptosis genes in CHO cells was developed as a means of limiting cell death upon exposure to environmental insults. Glucose‐regulated protein 78 (GRP78) is traditionally regarded as a major ER chaperone that participates in protein folding and other cell processes. It is also a potent antiapoptotic protein and plays a critical role in cell survival, proliferation, and metastasis. In this study, the impact of GRP78 on CHO cells in response to environmental insults such as serum deprivation and oxidative stress was investigated. First, it was confirmed that CHO cells were very sensitive to environmental insults. Then, GRP78 overexpressing CHO cell line was established and exposed to serum deprivation and H₂O₂. Results showed that GRP78 engineering increased the viability and decreased the apoptosis of CHO cells. The survival advantage due to GRP78 engineering could be mediated by suppression of caspase‐3 involved in cell death pathways in stressed cells. Besides, GRP78 engineering also enhanced yields of antibody against transferrin receptor in CHO cells. GRP78 should be a potential application in the biopharmaceutical industries.     

A humanin analog decreases oxidative stress and preserves mitochondrial integrity in cardiac myoblasts

A potent analog (HNG) of the endogenous peptide humanin protects against myocardial ischemia–reperfusion (MI–R) injury in vivo, decreasing infarct size and improving cardiac function. Since oxidative stress contributes to the damage from MI–R we tested the hypotheses that: (1) HNG offers cardioprotection through activation of antioxidant defense mechanisms leading to preservation of mitochondrial structure and that, (2) the activity of either of a pair of non-receptor tyrosine kinases, c-Abl and Arg is required for this protection. Rat cardiac myoblasts (H₉C₂ cells) were exposed to nanomolar concentrations of HNG and to hydrogen peroxide (H₂O₂). Cells treated with HNG in the presence of H₂O₂ demonstrated reduced intracellular reactive oxygen species (ROS), preserved mitochondrial membrane potential, ATP levels and mitochondrial structure. HNG induced activation of catalase and glutathione peroxidase (GPx) within 5 min and decreased the ratio of oxidized to reduced glutathione within 30 min. siRNA knockdown of both Abl and Arg, but neither alone, abolished the HNG-mediated reduction of ROS in myoblasts exposed to H₂O₂. These findings demonstrate an HNG-mediated, Abl- and Arg-dependent, rapid and sustained activation of critical cellular defense systems and attenuation of oxidative stress, providing mechanistic insights into the observed HNG-mediated cardioprotection in vivo.