Mitochondrial oxidant stress triggers cell death in simulated ischemia–reperfusion

Flavonoids within Scutellaria baicalensis may be potent antioxidants on the basis of our studies of S. baicalensis extract. To further this work, we studied the antioxidative effects of baicalein, a flavonoid component of S. baicalensis, in a chick cardiomyocyte model of reactive oxygen species (ROS) generation during hypoxia, simulated ischemia-reperfusion, or mitochondrial complex III inhibition with antimycin A. Oxidant stress was measured by oxidation of the intracellular probes 2′,7′-dichlorofluorescin diacetate and dihydroethidium. Viability was assessed by propidium iodide uptake. Baicalein attenuated oxidant stress during all conditions studied and acted within minutes of treatment. For example, baicalein given only at reperfusion dose dependently attenuated the ROS burst at 5 min after 1 h of simulated ischemia. It also decreased subsequent cell death at 3 h of reperfusion from 52.3 ± 2.5% in untreated cells to 29.4 ± 3.0% (with return of contractions; P < 0.001). In vitro studies using electron paramagnetic resonance spectroscopy with the spin trap 5-methoxycarbonyl-5-methyl-1-pyrroline- N-oxide revealed that baicalein scavenges superoxide but does not mimic the effects of superoxide dismutase. We conclude that baicalein can scavenge ROS generation in cardiomyocytes and that it protects against cell death in an ischemia-reperfusion model when given only at reperfusion.

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Exogenous Nitric Oxide Protects Human Embryonic Stem Cell-Derived Cardiomyocytes against Ischemia/Reperfusion Injury

Oxidative Medicine and Cellular Longevity ◽

10.1155/2016/4298945 ◽

2016 ◽

Vol 2016 ◽

pp. 1-9 ◽

Cited By ~ 8

Author(s):

János Pálóczi ◽

Zoltán V. Varga ◽

Ágota Apáti ◽

Kornélia Szebényi ◽

Balázs Sarkadi ◽

...

Keyword(s):

Cell Death ◽

Stem Cell ◽

Embryonic Stem Cell ◽

Human Embryonic Stem Cell ◽

Ischemia Reperfusion Injury ◽

Ischemia Reperfusion ◽

Embryonic Stem ◽

No Donor ◽

Simulated Ischemia ◽

Screening Platform

Background and Aims. Human embryonic stem cell- (hESC-) derived cardiomyocytes are one of the useful screening platforms of potential cardiocytoprotective molecules. However, little is known about the behavior of these cardiomyocytes in simulated ischemia/reperfusion conditions. In this study, we have tested the cytoprotective effect of an NO donor and the brain type natriuretic peptide (BNP) in a screening platform based first on differentiated embryonic bodies (EBs, 6 + 4 days) and then on more differentiated cardiomyocytes (6 + 24 days), both derived from hESCs.Methods. Both types of hESC-derived cells were exposed to 150 min simulated ischemia, followed by 120 min reperfusion. Cell viability was assessed by propidium iodide staining. The following treatments were applied during simulated ischemia in differentiated EBs: the NO-donor S-nitroso-N-acetylpenicillamine (SNAP) (10−7, 10−6, and 10−5 M), BNP (10−9, 10−8, and 10−7 M), and the nonspecific NO synthase inhibitor Nω-nitro-L-arginine (L-NNA, 10−5 M).Results. SNAP (10−6, 10−5 M) significantly attenuated cell death in differentiated EBs. However, simulated ischemia/reperfusion-induced cell death was not affected by BNP or by L-NNA. In separate experiments, SNAP (10−6 M) also protected hESC-derived cardiomyocytes.Conclusions. We conclude that SNAP, but not BNP, protects differentiated EBs or cardiomyocytes derived from hESCs against simulated ischemia/reperfusion injury. The present screening platform is a useful tool for discovery of cardiocytoprotective molecules and their cellular mechanisms.

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Abstract 14: Pim-1 Preserves Mitochondrial Morphology by Inhibiting Drp1 Translocation

Circulation Research ◽

10.1161/res.111.suppl_1.a14 ◽

2012 ◽

Vol 111 (suppl_1) ◽

Author(s):

Shabana Din ◽

Matt Mason ◽

Mirko Volkers ◽

Bevan Johnson ◽

Mathias Konstandin ◽

...

Keyword(s):

Cell Death ◽

Apoptotic Cell ◽

Morphological Changes ◽

Ischemia Reperfusion ◽

Ischemic Injury ◽

Dominant Negative ◽

Neonatal Rat ◽

Mitochondrial Morphology ◽

Mitochondrial Fragmentation ◽

Simulated Ischemia

Rationale: Mitochondrial morphological dynamics affect the outcome of ischemic heart damage. Mitochondrial fission protein Dynamin Related Protein 1 (Drp1) is a mediator of mitochondrial morphological changes and cell death during ischemic injury. Mitochondrial integrity is maintained by cardioprotective kinase Pim1, which enhances resistance to apoptotic challenge and ischemia reperfusion injury. In this study we examine the relationship between Pim1 activity and Drp1 regulation of mitochondrial morphology in cardiomyocytes challenged by ischemia. Objective: To demonstrate that Pim1 inhibits Drp1 translocation to the mitochondria in response to ischemic injury. Methods and Results: Simulated ischemia and simulated ischemia reperfusion (sI & sI/R) induced mitochondrial fragmentation and cell death in neonatal rat cardiac myocytes (NRCM), respectively. Mitochondrial fragmentation accompanied Drp1 translocation to the mitochondria in NRCM. Inhibition of Drp1 by mdivi1 preserved mitochondrial reticular morphology and inhibited apoptotic cell death. Mice subjected to sI/R injury displayed Drp1 mitochondrial localization, while exposure to mdivi1 led to reduced infarct size. Interestingly, transgenic hearts overexpressing Pim1 decreased total Drp1 levels, increased phosphorylation of Drp1 at serine 637, and inhibited Drp1 localization to mitochondria while preserving reticular morphology after sI. In contrast, Pim1 dominant negative (PDN) transgenic hearts and NRCM exhibit increased Drp1 translocation to mitochondria and fragmented mitochondria. PDN hearts exhibit decreased phosphorylation of serine 637 and upregulation of BH3 protein PUMA, inducing Drp1 accumulation at mitochondria and increased sensitivity to apoptotic stimuli. In PDN NRCMs, overexpression of Puma dominant negative (PumaDN) attenuated localization of Drp1 to mitochondria and inhibited cell death during sI. Conclusion: Pim1 activity prevents Drp1 compartmentalization to the mitochondria and preserves reticular mitochondrial morphology in response to simulated ischemia. Therefore, selective manipulation of Pim1 should be pursued as a therapeutic target to maintain mitochondrial morphology for cardioprotection.

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Abstract P133: Role of Akt and p38 in Oxidant Stress Injury and Hypothermic Protection in Murine Cardiomyocytes

Circulation ◽

10.1161/circ.118.suppl_18.s_1474 ◽

2008 ◽

Vol 118 (suppl_18) ◽

Author(s):

Kimberly R Wojcik ◽

Zuo-Hui Shao ◽

Chang-Qing Li ◽

Kimm J Hamann ◽

Terry L Vanden Hoek

Keyword(s):

Cell Death ◽

Cardiac Arrest ◽

Cell Injury ◽

Ischemia Reperfusion ◽

Oxidant Stress ◽

Ros Generation ◽

Akt Inhibitor ◽

Stress Injury ◽

P38 Inhibitor ◽

Sb 203580

Cardiac arrest is an ischemia/reperfusion (I/R) disease characterized by oxidant generation, inflammation, and cell death; and hypothermia (HT) has been shown to improve post-cardiac arrest reperfusion injury. We developed a neonatal mouse cardiomyocyte model of I/R (90 min I + 3 hr R) that demonstrates cell injury associated with increased reactive oxygen species (ROS) generation at reperfusion (as measured by DCFH). Mild HT (32°C) protects mouse cardiomyocytes from I/R injury, and we hypothesize that this protection may be related to the activation of the survival kinase Akt. The Akt inhibitor API-2 (10 ìM) reversed HT protection [32.4 ± 7.1% vs 65.7 ± 6.3% with API-2, p< .01(as measured by PI)] to cell death levels commensurate with normothermic I/R injury (60.7 ± 6.0%). Phospho-Akt (pAkt) levels declined during ischemia, and while both Ser473 and Thr308 were phosphorylated in normothermia and HT within 15 min reperfusion, HT showed an augmented level of pAkt at Thr308. Furthermore, this increase was sustained for the first 30 min of reperfusion. To further study this relationship, murine cardiomyocytes were exposed to exogenous H2O2 to mimic the oxidant stress associated with I/R. Mouse cardiomyocytes demonstrated a dose- and time-dependent activation of Akt to H2O2 that showed maximal activation of both the Ser473 and Thr308 sites within 30 min with 200 ìM H2O2. As in I/R-stimulated cells, the Thr308 site declined to near baseline levels within 1 hr while Ser473 remained elevated. Based on recent findings linking Akt and ROS with p38, we examined the effect of I/R and H2O2 on p38. Mouse cardiomyocytes demonstrated a rapid activation of p-p38 (Thr10/Tyr182) in the context of both stresses. Further, we studied the effect of the Akt inhibitor, API-2, as well as the p38 inhibitor, SB 203580, in H2O2-stimulated cells. As in I/R, API-2 blocked H2O2-induced pAkt, but this inhibitor did not have any effect on p-p38. However, when p38 activation was blocked using SB 203580, pAkt levels decreased by 2 hr. These data suggest that HT is, in part, mediated through Akt and that p38 lies upstream of Akt in the context of oxidant stress. These kinases may act as triggers for the initiation of survival pathways in cardiomyocytes to combat potential damage induced by ROS generation.

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Caspase-dependent cytochrome c release and cell death in chick cardiomyocytes after simulated ischemia-reperfusion

AJP Heart and Circulatory Physiology ◽

10.1152/ajpheart.01063.2003 ◽

2004 ◽

Vol 286 (6) ◽

pp. H2280-H2286 ◽

Cited By ~ 32

Author(s):

Yimin Qin ◽

Terry L. Vanden Hoek ◽

Kim Wojcik ◽

Travis Anderson ◽

Chang-Qing Li ◽

...

Keyword(s):

Cell Death ◽

Cytochrome C ◽

Ischemia Reperfusion ◽

Caspase 8 ◽

Caspase 9 ◽

Cytochrome C Release ◽

Simulated Ischemia ◽

Baseline Levels ◽

Caspase 2 ◽

Induced Apoptosis

We recently demonstrated that reperfusion rapidly induces the mitochondrial pathway of apoptosis in chick cardiomyocytes after 1 h of simulated ischemia. Here we tested whether ischemia-reperfusion (I/R)-induced apoptosis could be initiated by caspase-dependent cytochrome c release in this model of cardiomyocyte injury. Fluorometric assays of caspase activity showed little, if any, activation of caspases above baseline levels induced by 1 h of ischemia alone. However, these assays revealed rapid activation of caspase-2, yielding a 2.95 ± 0.52-fold increase (over ischemia only) within the 1st h of reperfusion, whereas activities of caspases-3, -8, and -9 increased only slightly from their baseline levels. The rapid and prominent activation of caspase-2 suggested that it could be an important initiator caspase in this model, and using specific caspase inhibitors given only at the point of reperfusion, we tested this hypothesis. The caspase-2 inhibitor benzyloxycarbonyl-Val-Asp(Ome)-Val-Ala-Asp(Ome)-CH2F was the only caspase inhibitor that significantly inhibited cytochrome c release from mitochondria. This inhibitor also completely blocked activation of caspases-3, -8, and -9. The caspase-3/7 inhibitor transiently and only partially blocked caspase-2 activity and was less effective in blocking the activities of caspases-8 and -9. The caspase-8 inhibitor failed to significantly block caspase-2 or -3, and the caspase-9 inhibitor blocked only caspase-9. Furthermore, the caspase-2 inhibitor protected against I/R-induced cell death, but the caspase-8 inhibitor failed to do so. These data suggest that active caspase-2 initiates cytochrome c release after reperfusion and that it is critical for the I/R-induced apoptosis in this model.

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Mechanisms of cell death in oxidant stress and ischemia‐reperfusion injury

The FASEB Journal ◽

10.1096/fasebj.21.5.a448-c ◽

2007 ◽

Vol 21 (5) ◽

Cited By ~ 1

Author(s):

Gabriel Loor ◽

Jyothi Kondapalli ◽

Bumihka Sharma ◽

Robert Guzy ◽

Paul Schumacker

Keyword(s):

Cell Death ◽

Reperfusion Injury ◽

Ischemia Reperfusion Injury ◽

Ischemia Reperfusion ◽

Oxidant Stress

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Targeted overexpression of α‐8‐oxoguanine DNA glycosylase in mitochondria (mt‐Ogg1) decreases cell death in response to oxidant stress or ischemia‐reperfusion injury.

The FASEB Journal ◽

10.1096/fasebj.21.6.a1377 ◽

2007 ◽

Vol 21 (6) ◽

Author(s):

Jyothisri Kondapalli ◽

Gabriel Loor ◽

Paul Schumacker

Keyword(s):

Cell Death ◽

Reperfusion Injury ◽

Ischemia Reperfusion Injury ◽

Ischemia Reperfusion ◽

Oxidant Stress ◽

Dna Glycosylase

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Signal Transduction of Opioid-induced Cardioprotection in Ischemia-Reperfusion

Anesthesiology ◽

10.1097/00000542-200106000-00024 ◽

2001 ◽

Vol 94 (6) ◽

pp. 1082-1088 ◽

Cited By ~ 34

Author(s):

Bradley C. McPherson ◽

Zhenhai Yao

Keyword(s):

Cell Death ◽

Opioid Receptor ◽

Oxygen Radicals ◽

Ischemia Reperfusion Injury ◽

Ischemia Reperfusion ◽

Oxidant Stress ◽

Katp Channels ◽

Molecular Probe

Background Morphine reduces myocardial ischemia-reperfusion injury in vivo and in vitro. The authors tried to determine the role of opioid delta1 receptors, oxygen radicals, and adenosine triphosphate-sensitive potassium (KATP) channels in mediating this effect. Methods Chick cardiomyocytes were studied in a flow-through chamber while pH, flow rate, oxygen, and carbon dioxide tension were controlled. Cell viability was quantified by nuclear stain propidium iodide, and oxygen radicals were quantified using molecular probe 2',7'-dichlorofluorescin diacetate. Results Morphine (1 microM) or the selective delta-opioid receptor agonist BW373U86 (10 pM) given for 10 min before 1 h of ischemia and 3 h of reoxygenation reduced cell death (31 +/- 5%, n = 6, and 28 +/- 5%, n = 6 [P < 0.05], respectively, 53 +/- 6%, n = 6, in controls) and generated oxygen radicals before ischemia (724 +/- 53, n = 8, and 742 +/- 75, n = 8 [P < 0.05], respectively, vs. 384 +/- 42, n = 6, in controls, arbitrary units). The protection of morphine was abolished by naloxone, or the selective delta1-opioid receptor antagonist 7-benzylidenenaltrexone. Reduction in cell death and increase in oxygen radicals with BW373U86 were blocked by the selective mitochondrial KATP channel antagonist 5-hydroxydecanoate or diethyldithiocarbamic acid (1,000 microM), which inhibited conversion of O2- to H2O2. The increase in oxygen radicals was abolished by the mitochondrial electron transport inhibitor myxothiazoL Reduction in cell death was associated with attenuated oxidant stress at reperfusion. Conclusion Stimulation of delta1-opioid receptors generates oxygen radicals via mitochondrial KATP channels. This signaling pathway attenuates oxidant stress and cell death in cardiomyocytes.

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