Chang Qing Li

Reactive oxygen species released from mitochondria during brief hypoxia induce preconditioning in cardiomyocytes.

Reactive oxygen species (ROS) have been proposed to participate in the induction of cardiac preconditioning. However, their source and mechanism of induction are unclear. We tested whether brief hypoxia induces preconditioning by augmenting mitochondrial generation of ROS in chick cardiomyocytes. Cells were preconditioned with 10 min of hypoxia, followed by 1 h of simulated ischemia and 3 h of reperfusion. Preconditioning decreased cell death from 47 ± 3% to 14 ± 2%. Return of contraction was observed in 3/3 preconditioned versus 0/6 non-preconditioned experiments. During induction, ROS oxidation of the probe dichlorofluorescin (sensitive to H2O2) increased ∼2.5-fold. As a substitute for hypoxia, the addition of H2O2 (15 μmol/liter) during normoxia also induced preconditioning-like protection. Conversely, the ROS signal during hypoxia was attenuated with the thiol reductant 2-mercaptopropionyl glycine, the cytosolic Cu,Zn-superoxide dismutase inhibitor diethyldithiocarbamic acid, and the anion channel inhibitor 4,4′-diisothiocyanato-stilbene-2,2′-disulfonate, all of which also abrogated protection. ROS generation during hypoxia was attenuated by myxothiazol, but not by diphenyleneiodonium or the nitric-oxide synthase inhibitor l-nitroarginine. We conclude that hypoxia increases mitochondrial superoxide generation which initiates preconditioning protection. Furthermore, mitochondrial anion channels and cytosolic dismutation to H2O2 may be important steps for oxidant induction of hypoxic preconditioning.

Generation of superoxide in cardiomyocytes during ischemia before reperfusion

Although a burst of oxidants has been well described with reperfusion, less is known about the oxidants generated by the highly reduced redox state and low O2 of ischemia. This study aimed to further identify the species and source of these oxidants. Cardiomyocytes were exposed to 1 h of simulated ischemia while oxidant generation was assessed by intracellular dihydroethidine (DHE) oxidation. Ischemia increased DHE oxidation significantly (0.7 ± 0.1 to 2.3 ± 0.3) after 1 h. Myxothiazol (mitochondrial site III inhibitor) attenuated oxidation to 1.3 ± 0.1, as did the site I inhibitors rotenone (1.0 ± 0.1), amytal (1.1 ± 0.1), and the flavoprotein oxidase inhibitor diphenyleneiodonium (0.9 ± 0.1). By contrast, the site IV inhibitor cyanide, as well as inhibitors of xanthine oxidase (allopurinol), nitric oxide synthase (nitro-l-arginine methyl ester), and NADPH oxidase (apocynin), had no effect. Finally, DHE oxidation increased with Cu- and Zn-containing superoxide dismutase (SOD) inhibition using diethyldithiocarbamate (2.7 ± 0.1) and decreased with exogenous SOD (1.1 ± 0.1). We conclude that significant superoxide generation occurs during ischemia before reperfusion from the ubisemiquinone site of the mitochondrial electron transport chain.Although a burst of oxidants has been well described with reperfusion, less is known about the oxidants generated by the highly reduced redox state and low O(2) of ischemia. This study aimed to further identify the species and source of these oxidants. Cardiomyocytes were exposed to 1 h of simulated ischemia while oxidant generation was assessed by intracellular dihydroethidine (DHE) oxidation. Ischemia increased DHE oxidation significantly (0.7 +/- 0.1 to 2.3 +/- 0.3) after 1 h. Myxothiazol (mitochondrial site III inhibitor) attenuated oxidation to 1.3 +/- 0.1, as did the site I inhibitors rotenone (1.0 +/- 0.1), amytal (1.1 +/- 0.1), and the flavoprotein oxidase inhibitor diphenyleneiodonium (0.9 +/- 0.1). By contrast, the site IV inhibitor cyanide, as well as inhibitors of xanthine oxidase (allopurinol), nitric oxide synthase (nitro-L-arginine methyl ester), and NADPH oxidase (apocynin), had no effect. Finally, DHE oxidation increased with Cu- and Zn-containing superoxide dismutase (SOD) inhibition using diethyldithiocarbamate (2.7 +/- 0.1) and decreased with exogenous SOD (1.1 +/- 0.1). We conclude that significant superoxide generation occurs during ischemia before reperfusion from the ubisemiquinone site of the mitochondrial electron transport chain.

Preconditioning in Cardiomyocytes Protects by Attenuating Oxidant Stress at Reperfusion

Cardiomyocyte death after ischemia/reperfusion correlates with oxidant stress, and antioxidants confer protection in that model. Preconditioning (PC) with hypoxia or adenosine also confers protection, leading us to hypothesize that PC protects by attenuating oxidant generation during subsequent ischemia/reperfusion. Chick cardiomyocytes were preconditioned with 10 minutes of hypoxia or adenosine (100 micromol/L), followed by 1 hour of simulated ischemia and 3 hours of reperfusion. Adenosine PC decreased cell death from 50+/-3% to 18+/-4% and enhanced the return of contractions during reperfusion, as observed previously with hypoxic PC. A transient burst of dichlorofluorescein (sensitive to H2O2 oxidation that was significantly attenuated by PC initiated by hypoxia or adenosine was seen at reperfusion. The protein kinase C (PKC) inhibitor Go-6976 and the mitochondrial ATP-sensitive K(+) (K(ATP)) channel inhibitor 5-hydroxydecanoate each abolished protection and abrogated the PC-induced attenuation of reperfusion oxidant stress. By contrast, when given only at reperfusion, the K(+) channel opener pinacidil or the antioxidants 2-mercaptopropionylglycine and 1,10-phenanthroline decreased oxidant stress at reperfusion and improved survival and return of contractions. Thus, PC protection is associated with an attenuation of the oxidant burst at reperfusion, regardless of the method by which PC is triggered. Loss of PC protection associated with PKC inhibition or K(ATP) channel inhibitors is associated with a restoration of that oxidant stress. These results suggest a mechanism for PC protection and reveal a functional link between PKC activation and K(ATP) channel activation in that pathway.

Baicalein Protects Against Doxorubicin-Induced Cardiotoxicity by Attenuation of Mitochondrial Oxidant Injury and JNK Activation

The cardiotoxicity of doxorubicin limits its clinical use in the treatment of a variety of malignancies. Previous studies suggest that doxorubicin‐associated cardiotoxicity is mediated by reactive oxygen species (ROS)‐induced apoptosis. We therefore investigated if baicalein, a natural antioxidant component of Scutellaria baicalensis, could attenuate ROS generation and cell death induced by doxorubicin. Using an established chick cardiomyocyte model, doxorubicin (10 µM) increased cell death in a concentration‐ and time‐dependent manner. ROS generation was increased in a dose–response fashion and associated with loss of mitochondrial membrane potential. Doxorubicin also augmented DNA fragmentation and increased the phosphorylation of ROS‐sensitive pro‐apoptotic kinase c‐Jun N‐terminal kinase (JNK). Adjunct treatment of baicalein (25 µM) and doxorubicin for 24 h significantly reduced both ROS generation (587 ± 89 a.u. vs. 932 a.u. ± 121 a.u., P < 0.01) and cell death (30.6 ± 5.1% vs. 46.8 ± 8.3%, P < 0.01). The dissipated mitochondrial potential and increased DNA fragmentation were also ameliorated. Along with the reduction of ROS and apoptosis, baicalein attenuated phosphorylation of JNK induced by doxorubicin (1.7 ± 0.3 vs. 3.0 ± 0.4‐fold, P < 0.05). Co‐treatment of cardiomyocytes with doxorubicin and JNK inhibitor SP600125 (10 µM; 24 h) reduced JNK phosphorylation and enhanced cell survival, suggesting that the baicalein protection against doxorubicin cardiotoxicity was mediated by JNK activation. Importantly, concurrent baicalein treatment did not interfere with the anti‐proliferative effects of doxorubicin in human breast cancer MCF‐7 cells. In conclusion, baicalein adjunct treatment confers anti‐apoptotic protection against doxorubicin‐induced cardiotoxicity without compromising its anti‐cancer efficacy. J. Cell. Biochem. 112: 2873–2881, 2011.

Grape seed proanthocyanidins protect cardiomyocytes from ischemia and reperfusion injury via Akt‐NOS signaling

Ischemia/reperfusion (I/R) injury in cardiomyocytes is related to excess reactive oxygen species (ROS) generation and can be modulated by nitric oxide (NO). We have previously shown that grape seed proanthocyanidin extract (GSPE), a naturally occurring antioxidant, decreased ROS and may potentially stimulate NO production. In this study, we investigated whether GSPE administration at reperfusion was associated with cardioprotection and enhanced NO production in a cardiomyocyte I/R model. GSPE attenuated I/R‐induced cell death [18.0 ± 1.8% (GSPE, 50 µg/ml) vs. 42.3 ± 3.0% (I/R control), P < 0.001], restored contractility (6/6 vs. 0/6, respectively), and increased NO release. The NO synthase (NOS) inhibitor Nω‐nitro‐L‐arginine methyl ester (L‐NAME, 200 µM) significantly reduced GSPE‐induced NO release and its associated cardioprotection [32.7 ± 2.7% (GSPE + L‐NAME) vs. 18.0 ± 1.8% (GSPE alone), P < 0.01]. To determine whether GSPE induced NO production was mediated by the Akt‐eNOS pathway, we utilized the Akt inhibitor API‐2. API‐2 (10 µM) abrogated GSPE‐induced protection [44.3% ± 2.2% (GSPE + API‐2) vs. 27.0% ± 4.3% (GSPE alone), P < 0.01], attenuated the enhanced phosphorylation of Akt at Ser473 in GSPE‐treated cells and attenuated GSPE‐induced NO increases. Simultaneously blocking NOS activation (L‐NAME) and Akt (API‐2) resulted in decreased NO levels similar to using each inhibitor independently. These data suggest that in the context of GSPE stimulation, Akt may help activate eNOS, leading to protective levels of NO. GSPE offers an alternative approach to therapeutic cardioprotection against I/R injury and may offer unique opportunities to improve cardiovascular health by enhancing NO production and increasing Akt‐eNOS signaling. J. Cell. Biochem. 107: 697–705, 2009.

Synergistic effect of Scutellaria baicalensis and grape seed proanthocyanidins on scavenging reactive oxygen species in vitro.

Scutellaria baicalensis (SbE) is a commonly used Chinese herb medicine and grape seed proanthocyanidins is a popular herbal supplement in the United States. Both herbs have been shown to possess potent antioxidant effects. Using an in vitro model to produce the reactive oxygen species (ROS) generation (H2O2/FeSO4 for hydroxyl radicals, xanthine/xanthine oxidase for suproxide), we observed that Scutellaria baicalensis and grape seed proanthocyanidins acted synergistically to scavenge ROS. Our data suggest that a combination of these two herbs can potentially enhance their antioxidant efficacy, allowing lower dosages of each drug to be used. This has the advantage of avoiding possible side effects that may arise when higher doses of a single herb are used in an attempt to achieve a maximum degree of antioxidant activity.

Cytotoxicity induced by grape seed proanthocyanidins: role of nitric oxide.

Grape seed proanthocyanidin extract (GPSE) at high doses has been shown to exhibit cytotoxicity that is associated with increased apoptotic cell death. Nitric oxide (NO), being a regulator of apoptosis, can be increased in production by the administration of GSPE. In a chick cardiomyocyte study, we demonstrated that high-dose (500 μg/ml) GSPE produces a significantly high level of NO that contributes to increased apoptotic cell death detected by propidium iodide and terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) staining. It is also associated with the depletion of intracellular glutathione (GSH), probably due to increased consumption by NO with the formation of S-nitrosoglutathione. Co-treatment with L-NAME, a NO synthase inhibitor, results in reduction of NO and apoptotic cell death. The decline in reduced GSH/oxidized GSH (GSSG) ratio is also reversed. N-Acetylcysteine, a thiol compound that reacts directly with NO, can reduce the increased NO generation and reverse the decreased GSH/GSSG ratio, thereby attenuating the cytotoxicity induced by high-dose GSPE. Taken together, these results suggest that endogenous NO synthase (NOS) activation and excessive NO production play a key role in the pathogenesis of high-dose GSPE-induced cytotoxicity.

The effects of ginsenoside Rb1 on JNK in oxidative injury in cardiomyocytes.

Reactive oxygen species (ROS) can induce oxidative injury via iron interactions (i.e. Fenton chemistry and hydroxyl radical formation). Our prior work suggested that American ginseng berry extract and ginsenoside Re were highly cardioprotective against oxidant stress. To extend this study, we evaluated the protective effect of protopanaxadiol-type ginsenoside Rb1 (gRb1) on H2O2-induced oxidative injury in cardiomyocytes and explored the ROS-mediated intracellular signaling mechanism. Cultured embryonic chick cardiomyocytes (4–5 day) were used. Cell death was assessed by propidium iodide and lactate dehydrogenase release. Pretreatment with gRb1 (0.01, 0.1, or 1 μM) for 2 h and concurrent treatment with H2O2 (0.5 mM) for 2 h resulted in a dose-dependent reduction of cell death, 36.6 ± 2.9% (n = 12, p < 0.05), 30.5 ± 5.1% (n = 12, p < 0.05) and 28.6 ± 3.1% (n = 12, p < 0.01) respectively, compared to H2O2-exposed cells (48.2 ± 3.3%, n = 12). This cardioprotective effect of gRb1 was associated with attenuated intracellular ROS generation as measured by 6-carboxy-2′, 7′-dichlorodihydrofluorescein diacetate, preserved the mitochondrial membrane potential as determined using JC-1. In the ESR study, gRb1 exhibited the scavenging DPPH and hydroxyl radical activities. Furthermore, our data showed the increased JNK phosphorylation (p-JNK) in H2O2-exposed cells was suppressed by the pretreatment with gRb 1 (1 μM) (p < 0.01). Co-treatment of gRb1 with a specific inhibitor of JNK SP600125 (10 μM) further reduced the p-JNK and enhanced the cell survival after H2O2 exposure. Collectively, our results suggest that gRb1 conferred cardioprotection that was mediated via attenuating ROS and suppressing ROS-induced JNK activation.

Grape seed proanthocyanidins induce pro-oxidant toxicity in cardiomyocytes.

Grape seed proanthocyanidin extract (GSPE), a polyphenolic compound with antioxidant properties, may protect against cardiac ischemia and reperfusion injury. However, its potential toxicity at higher doses is unknown. The authors tested the effects of GSPE on reactive oxygen species (ROS) generation, cell survival, lactate dehydrogenase (LDH) release, and caspase-3 activity using chick cardiomyocytes incubated with GSPE at 5, 10, 50, 100, or 500 μg/mL in medium for 8 h. Exposure to increasing concentrations of GSPE (100 or 500 μg/mL) resulted in an increase in ROS generation and cell death as measured by propidium iodide uptake and LDH release. Caspase-3 activity was significantly increased fourfold in cells exposed to GSPE 500 μg/mL compared to controls; this was abolished by the selective caspase-3 inhibitor AC-Asp-Gln-Thr-Asp-H(50 μM), which aslso significantly reduced the cell death resulting from GSPE (500 μg/mL). The antioxidant N-acetylcysteine (NAC, 100 μM) reduced cell death induced by GSPE (500 μg/mL) but failed to attenuate caspase-3 activation. Collectively, the authors conclude that higher doses of GSPE could cause apoptotic cell injury via effector caspase-3 activation and subsequent induction of ROS generation. Consumers may take higher doses of dietary supplements in the belief that natural herbs have no major side effects. This study demonstrates that dosages of GSPE should be optimized to avoid potential harmful pro-oxidant effects.

Akt activates NOS3 and separately restores barrier integrity in H2O2-stressed human cardiac microvascular endothelium.

The integrity of microvascular endothelium is an important regulator of myocardial contractility. Microvascular barrier integrity could be altered by increased reactive oxygen species (ROS) stress seen within minutes after cardiac arrest resuscitation. Akt and its downstream target nitric oxide (NO) synthase (NOS)3 can protect barrier integrity during ROS stress, but little work has studied these oxidant stress responses in human cardiac microvascular endothelial cells (HCMVEC). We, therefore, studied how ROS affects barrier function and NO generation via Akt and its downstream target NOS3 in HCMVEC. HCMVEC exposed to 500 microM H2O2 had increased Akt phosphorylation within 10 min at both Ser-473 and Thr-308 sites, an effect blocked by the phosphatidylinositol 3-kinase inhibitor LY-294002. H2O2 also induced NO generation that was associated with NOS3 Ser-1177 site phosphorylation and Thr-495 dephosphorylation, with Ser-1177 effects attenuated by LY-294002 and an Akt inhibitor, Akt/PKB signaling inhibitor-2 (API-2). H2O2 induced significant barrier disruption in HCMVEC within minutes, but recovery started within 30 min and normalized over hours. The NOS inhibitor Nomega-nitro-L-arginine methyl ester (200 microM) blocked NO generation but had no effect on H2O2-induced barrier permeability or the recovery of barrier integrity. By contrast, the Akt inhibitor API-2 abrogated HCMVEC barrier restoration. These results suggest that oxidant stress in HCMVEC activates NOS3 via Akt. NOS3/NO are not involved in the regulation of H2O2-affected barrier function in HCMVEC. Independent of NOS3 regulation, Akt proves to be critical for the restoration of barrier integrity in HCMVEC.