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. 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. Myocardial preconditioning was initially described as an adaptive response of the heart to brief episodes of ischemia that decreased necrosis during subsequent prolonged ischemia (1Murry C.E. Jennings R.B. Reimer K.A. Circulation. 1986; 74: 1124-1136Crossref PubMed Scopus (7173) Google Scholar). Reactive oxygen species (ROS 1The abbreviations used are: ROS, reactive oxygen species; BSS, balanced salt solution; PI, propidium iodide; DCF, dichlorofluorescein; DHE, dihydroethidium; Eth, ethidium; SOD, superoxide dismutase; NOS, nitric-axide synthase; DCFH-DA, 2′,7′-dichlorofluorescin diacetate ; DCFH, 2′,7′-dichlorofluorescin; 2-MPG, 2-mercaptopriopionyl glycine; DPI, diphenyleneiodonium; DDC, diethyldithiocarbamic acid; DIDS, 4,4′-diisothiocyanato-stilbene-2,2′-disulfonate. ;e.g. superoxide, H2O2, hydroxyl radicals) generated from brief ischemia/reperfusion have been recognized as possible “triggers” in the initiation of preconditioning (2Baines C.P. Goto M. Downey J.M. J. Mol. Cell. Cardiol. 1997; 29: 207-216Abstract Full Text PDF PubMed Scopus (419) Google Scholar). Evidence for this role includes intact heart studies where exposure to superoxide or H2O2caused preconditioning-like protection (2Baines C.P. Goto M. Downey J.M. J. Mol. Cell. Cardiol. 1997; 29: 207-216Abstract Full Text PDF PubMed Scopus (419) Google Scholar, 3Tritto I. D'Andrea D. Eramo N. Scognamiglio A. De Simone C. Violante A. Esposito A. Chiariello M. Ambrosio G. Circ. Res. 1997; 80: 743-748Crossref PubMed Scopus (256) Google Scholar), and other studies demonstrating that antioxidants abolished the induction of preconditioning (4Tanaka M. Fujiwara H. Yamasaki K. Sasayama S. Cardiovasc. Res. 1994; 28: 980-986Crossref PubMed Scopus (130) Google Scholar, 5Osada M. Takeda S. Sato T. Komori S. Tamura K. Jpn. Circ. J. 1994; 58: 259-263Crossref PubMed Scopus (30) Google Scholar). Few studies have directly measured ROS generation during brief hypoxia or ischemia induction (6Zhou X. Zhai X. Ashraf M. Circulation. 1997; 93: 1177-1184Crossref Scopus (175) Google Scholar). Such direct measures are needed to clarify important questions that remain regarding the role of ROS as inducing agents, including their source, where they are metabolized, and the relative contributions of different oxidant species to the induction of preconditioning protection. Within the intact heart, possible sources of ROS include the cardiomyocytes, endothelial cells, neutrophils, or the auto-oxidation of catecholamines (7Ferrari R. Ceconi C. Curello S. Alfieri O. Visioli O. Eur. Heart. J. 1993; 14: 25-30Crossref PubMed Scopus (23) Google Scholar, 8Hess M.L. Manson N.H. J. Mol. Cell. Cardiol. 1984; 16: 969-985Abstract Full Text PDF PubMed Scopus (482) Google Scholar). Within cardiomyocytes, sources of ROS could include superoxide generation from NAD(P)H or other oxidases such as cytochrome P450 (9Thannickal V.J. Fanburg B.L. J. Biol. Chem. 1995; 270: 30334-30338Abstract Full Text Full Text PDF PubMed Scopus (386) Google Scholar, 10Griendling K.K. Minieri C.A. Ollerenshaw J.D. Alexander R.W. Circ. Res. 1994; 74: 1141-1148Crossref PubMed Scopus (2433) Google Scholar, 11Mohazzab-H K.M. Kaminski P.M. Wolin M.S. Circulation. 1997; 96: 614-620Crossref PubMed Scopus (138) Google Scholar), the mitochondrial electron transport chain (12Ambrosio G. Zweier J.L. Duilio C. Kuppusamy P. Santoro G. Elia P.P. Tritto I. Cirillo P. Condorelli M. Chiarello M. J. Biol. Chem. 1993; 268: 18532-18541Abstract Full Text PDF PubMed Google Scholar), or even nitric-oxide synthase under conditions where arginine is depleted (13Kitakaze M. Node K. Komamura K. Minamino T. Inoue M. Hori M. Kamada T. J. Mol. Cell. Cardiol. 1995; 27: 2149-2154Abstract Full Text PDF PubMed Scopus (73) Google Scholar, 14Kelly R.A. Balligand J.L. Smith T.W. Circ. Res. 1996; 79: 363-380Crossref PubMed Scopus (634) Google Scholar, 15Xia Y. Dawson V.L. Dawson T.M. Snyder S.H. Zweier J.L. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 6770-6774Crossref PubMed Scopus (657) Google Scholar). Although it is likely that superoxide is the initial oxidant generated from these systems, the relative importance of superoxide, or its reduced products H2O2 or hydroxyl radical, in the signal transduction system involved in preconditioning is not known. Some evidence suggests that either superoxide or hydrogen peroxide can initiate preconditioning (2Baines C.P. Goto M. Downey J.M. J. Mol. Cell. Cardiol. 1997; 29: 207-216Abstract Full Text PDF PubMed Scopus (419) Google Scholar, 16Gopalakrishna R. Anderson W.B. Proc. Natl. Acad. Sci. U. S. A. 1989; 86: 6758-6762Crossref PubMed Scopus (381) Google Scholar,17Downey J.M. Cohen M.V. Marber M.M. Yellon D.M. Ischaemia: Preconditioning and Adaptation. 1996: 21-34Google Scholar), so it is conceivable that H2O2 is the active signaling agent in this process. The purpose of our study was to investigate the role of mitochondrial ROS in the induction of hypoxic preconditioning, and to clarify which ROS are required for the preconditioning response. For this study, we used chick cardiomycytes, which have been shown to precondition with brief hypoxia (18Liang B.T. Am. J. Physiol. 1996; 271: H1769-H1777PubMed Google Scholar, 19Strickler J. Jacobson K.A. Liang B.T. J. Clin. Invest. 1996; 98: 1773-1779Crossref PubMed Scopus (117) Google Scholar). Embryonic ventricular cardiac myocytes were prepared as described previously (20Vanden Hoek T.L. Shao Z. Li C. Zak R. Schumacker P.T. Becker L.B. Am. J. Physiol. 1996; 270: H1334-H1341PubMed Google Scholar). Heart ventricles from 10-day-old chick embryos were dissected, minced, enzymatically dispersed with 0.025% trypsin (Life Technologies, Inc.), and centrifuged differentially to yield 5–6 × 105cells/embryo. Cells (0.7 × 106) were pipetted onto coverslips, incubated, and grown into contractile layers. Synchronous contractions were seen by the third day in culture. Cultures were checked for non-muscle cell contamination (greater than 95% of cells stain with anti-myosin heavy chain monoclonal antibodies, CCM-52). Experiments were performed with 3–5-day cardiac cell cultures, at which point viability exceeded 99%. Coverslips with synchronously contracting cells were placed inside a Sykes-Moore chamber (1.2-ml volume, Bellco Glass Inc., Vineland, NJ). The chamber and inflow tubing were maintained at 37 °C. Flow rate (0.25 ml/min), pH, and oxygen tension (PO2) of the perfusate were controlled. Hypoxic conditions were verified with an optical method of phosphorescence quenching (Oxyspot, Medical Systems Inc, Greenvale, NY) (21Lo L.W. Koch C.J. Wilson D.F. Anal. Biochem. 1996; 236: 153-160Crossref PubMed Scopus (228) Google Scholar). An extracellular Pd-porphine dye bound to albumin (1–10 μm) was added to the perfusate, and the PO2-dependent phosphorescence decay was recorded in response to pulsed excitation light. Perfusion with hypoxic media resulted in measured PO2 values of 3 torr within the chamber during steady state perfusion. Tubing supplying perfusate to the chamber was of low O2 permeability, constructed of PharMed (Cole-Parmer Instrument Co., Chicago, IL) or stainless steel to minimize O2 leaks. Standard perfusion media consisted of oxygenated balanced salt solution (BSS) with a PO2 of 100 torr, PCO2 of 40, pH of 7.4, K+ of 4.0 mEq/liter, and a glucose of 5.6 mm. Simulated ischemia consisted of BSS containing no glucose, with 2-deoxyglucose (20 mm) added to inhibit glycolysis and a K+ of 8.0 mEq/liter. This was bubbled with 80% N2 gas and 20% CO2 to produce a PO2 of less than 3 torr, a PCO2 of 144 torr, and a final pH of 6.8. Hypoxic media used for preconditioning consisted of BSS with no glucose, bubbled with 95% N2 gas and 5% CO2. Reperfusion was with standard media unless stated otherwise. Cells were imaged with an Olympus IMT-2 inverted phase/epifluorescent microscope equipped with Hoffman Modulation optics to accentuate surface topology of the cells. This facilitated detection of contractile movement in the confluent layer of cells. Phase-contrast images were recorded for contraction analysis with a CCD camera. Fluorescence was measured using a cooled with for of in of propidium to were using an excitation of with and used to oxidant generation was measured using excitation of with and An of oxidant which and bound as the was measured using the used to and oxidation studies were with or the other of these viability was using the stain an dye that to of This method is in to and been to the from to cell in J.M. Res. Chem. Google Scholar). is not to cells a of its addition to the perfusate the the of using PI, all in a of cells were by cells with of viability cell was relative to the seen exposure oxidant was by in from probe (1–10 the cell and can be by ROS including superoxide hydroxyl to yield to its J. Biol. 1994; PubMed Google Scholar). is but can be decreased with hydroxyl 1984; PubMed Scopus Google Scholar). increases in oxidation to increases in are of superoxide generation Hoek T.L. Li C. Shao Z. Schumacker P.T. Becker L.B. J. Mol. Cell. Cardiol. 1997; 29: Full Text PDF PubMed Scopus Google Scholar). The probe 2′,7′-dichlorofluorescin diacetate the cell and the is by the 2′,7′-dichlorofluorescin oxidation by ROS, hydrogen peroxide and hydroxyl radical, the J. Biol. 1994; PubMed Google Scholar). increases in oxidation to increases in are of H2O2 or hydroxyl generation Hoek T.L. Li C. Shao Z. Schumacker P.T. Becker L.B. J. Mol. Cell. Cardiol. 1997; 29: Full Text PDF PubMed Scopus Google Scholar). The of these for different ROS have been verified in and chick and have been described previously Hoek T.L. Li C. Shao Z. Schumacker P.T. Becker L.B. J. Mol. Cell. Cardiol. 1997; 29: Full Text PDF PubMed Scopus Google Scholar). contractions were observed as described previously Hoek T.L. Shao Z. Li C. Schumacker P.T. Becker L.B. J. Mol. Cell. Cardiol. 1997; 29: Full Text PDF PubMed Scopus Google Scholar). The for a of contraction was contractions were seen the of cells the of reperfusion. of cells was for contractions the ischemia/reperfusion were to 1 h of simulated ischemia hypoxia, and followed by 3 h of reperfusion. shown that this cell death during that to from oxidant (20Vanden Hoek T.L. Shao Z. Li C. Zak R. Schumacker P.T. Becker L.B. Am. J. Physiol. 1996; 270: H1334-H1341PubMed Google Scholar, Hoek T.L. Li C. Shao Z. Schumacker P.T. Becker L.B. J. Mol. Cell. Cardiol. 1997; 29: Full Text PDF PubMed Scopus Google Scholar). preconditioning, were to 10 min of hypoxia 3 glucose, followed by 10 min of in BSS to subsequent and oxidant generation were measured during preconditioning induction and during subsequent ischemia and reperfusion. were with non-preconditioned cells under were and were An was the of of a of cells a were performed are as or For of analysis of and were with to be As cell death in this of simulated ischemia/reperfusion during the cell death was seen during the ischemia (20Vanden Hoek T.L. Shao Z. Li C. Zak R. Schumacker P.T. Becker L.B. Am. J. Physiol. 1996; 270: H1334-H1341PubMed Google Scholar). 1 h of cell death in the study was ± in cells, which was not different from ± cell 3 h of in cells ± versus ± in non-preconditioned cells the preconditioned contractile of 3 h with of in not with 10 min of hypoxia to simulated ischemia/reperfusion reduced cell death and the of We tested the role of ROS generation during hypoxic preconditioning. during preconditioning with hypoxia a and in ROS generation with ROS generation during preconditioning hypoxia, and decreased during to As seen in 3 the ROS generation during hypoxia was attenuated with the agent 2-mercaptopriopionyl μm) of nitric-oxide synthase a and superoxide source during μm) to inhibit and superoxide from J.L. P. A. Kuppusamy P. 1995; PubMed Scopus Google increased ROS generation during preconditioning hypoxia or oxidation during hypoxic preconditioning. ROS generation during 10 min of hypoxic preconditioning, by increased was attenuated by added during conditions for min and hypoxic preconditioning. However, the inhibitor μm) the oxidation and the of the NAD(P)H inhibitor diphenyleneiodonium 10 also been to inhibit superoxide from the of nitric-oxide synthase T. Biochem. Res. 1997; PubMed Scopus Google Scholar). to inhibit the ROS seen during hypoxic preconditioning. the mitochondrial electron transport inhibitor attenuated this ROS generation during hypoxia in a that were the source of ROS generation during hypoxic preconditioning. have been shown to superoxide electron to at the A. Biochem. PubMed Scopus Google Scholar). This superoxide may be to H2O2 by superoxide dismutase in the or in the whether hypoxia superoxide that is by Cu,Zn-superoxide dismutase in the we ROS generation using 10 and DCFH, μm) to superoxide and H2O2 The inhibitor diethyldithiocarbamic was used to inhibit the cytosolic of superoxide to H2O2 J. Biol. Chem. Full Text PDF PubMed Google Scholar). As seen in abolished the in seen during hypoxic preconditioning the of oxidation during hypoxic preconditioning that superoxide generated by during hypoxic preconditioning can the where it is to H2O2 by of with to an increased oxidation of the probe DHE, and a in oxidation of of ROS generation during hypoxic preconditioning. mm) was added min to and during 10 min hypoxic preconditioning. increases during hypoxic preconditioning were attenuated by was increased by that cytosolic is involved in superoxide generated by hypoxic preconditioning to study the importance of cytosolic for the induction of preconditioning, was during preconditioning hypoxia and the subsequent preconditioning protection was As shown this the of superoxide H2O2 superoxide was to preconditioning could this protection. H2O2 was the active signaling preconditioning protection. As seen in addition of during hypoxic preconditioning abolished preconditioning protection. in was at the of ischemia/reperfusion preconditioned cells during preconditioning and non-preconditioned cells. was no of contraction in of the preconditioned cells with by was not with directly this the of cell death was seen was to ischemia/reperfusion non-preconditioned and in cells to ischemia/reperfusion to ± cell death ischemia/reperfusion with to DDC, versus ± in non-preconditioned for h to no in and to contractions not The with that H2O2, than superoxide, was for the induction of preconditioning. We tested whether low of H2O2 to ischemia/reperfusion could preconditioning-like protection. were with BSS containing H2O2 (15 μmol/liter) for 10 min followed by a to to H2O2 for 10 min during normoxia resulted in protection cell death during subsequent ischemia/reperfusion 3/3 a of contraction with 0/6 experiments. We to preconditioning using the thiol reductant at a shown previously to the ROS signal generated during hypoxia the cytosolic of reduced is to the of H2O2 of during the min of and 10 min of hypoxic preconditioning abolished preconditioning protection ± cell death in hypoxic preconditioned cells, ± in hypoxic preconditioned hypoxic preconditioned studies a of contraction with 3/3 experiments. the role of H2O2 in the induction of preconditioning in cardiomyocytes. that anion channels may be required for of superoxide cell M. K. Biochem. PubMed Scopus Google Scholar), and that this can be by A. T.M. Wolin M.S. J. Physiol. 1993; 74: PubMed Scopus Google Scholar). As in superoxide generated in the may the where it may be by to H2O2, which subsequent of preconditioning. mitochondrial anion channels are involved in superoxide movement into the of channels H2O2 generation in the and preconditioning protection. were with BSS containing μm) during 10 min of hypoxic preconditioning. As seen in during hypoxic preconditioning abolished ROS generation as measured by during hypoxic preconditioning also abolished preconditioning protection no as by an of increased h of under conditions not that 10 min of hypoxia in chick a in ROS This ROS signal was attenuated by the mitochondrial electron transport inhibitor myxothiazol, but not NAD(P)H or nitric-oxide synthase that the ROS generated during hypoxia from the subsequent ischemia and was by that attenuated this H2O2 that the agent 2-MPG, the cytosolic inhibitor DDC, and the anion channel inhibitor all abolished preconditioning protection. H2O2 during normoxia induced preconditioning-like protection. We conclude that ROS participate in the signal transduction involved in hypoxic preconditioning in this ROS to as superoxide from the mitochondrial electron transport which the anion dismutation by to be for the of subsequent steps involved in preconditioning protection. are with studies that have ROS as signaling that preconditioning. studies have been in intact and that antioxidants during preconditioning its ischemia/reperfusion (4Tanaka M. Fujiwara H. Yamasaki K. Sasayama S. Cardiovasc. Res. 1994; 28: 980-986Crossref PubMed Scopus (130) Google Scholar, C.E. V.J. Jennings R.B. Reimer K.A. Circulation. Scholar). However, our study by as the source of ROS for induction, and by that these ROS are generated during hypoxic preconditioning than at Some studies were not to preconditioning with antioxidants M. Takeda S. Sato T. Komori S. Tamura K. Jpn. Circ. J. 1994; 58: 259-263Crossref PubMed Scopus (30) Google Scholar, C. C. Cardiovasc. Res. 1993; 27: PubMed Scopus Google Scholar), the that the of its or the of not the oxidant signal for of ROS generation that the were as and directly a role for ROS in the induction of hypoxic preconditioning. that hypoxic preconditioning is with oxidation of (sensitive to and with oxidation (sensitive to is a ROS during hypoxic preconditioning to H2O2 generated from superoxide However, the of these for different reactive species is so the of ROS is not the role of H2O2 as the ROS for preconditioning is by the that H2O2 can preconditioning-like protection during normoxia and that which increases superoxide generation relative to the of preconditioning. are with other studies that superoxide or H2O2 can produce preconditioning-like protection in the intact heart (2Baines C.P. Goto M. Downey J.M. J. Mol. Cell. Cardiol. 1997; 29: 207-216Abstract Full Text PDF PubMed Scopus (419) Google Scholar, 3Tritto I. D'Andrea D. Eramo N. Scognamiglio A. De Simone C. Violante A. Esposito A. Chiariello M. Ambrosio G. Circ. Res. 1997; 80: 743-748Crossref PubMed Scopus (256) Google Scholar, M.L. Circulation. 1995; Scholar). superoxide and H2O2 have been shown to of preconditioning such as and R. Anderson W.B. Proc. Natl. Acad. Sci. U. S. A. 1989; 86: 6758-6762Crossref PubMed Scopus (381) Google Scholar, R. P. Res. 1989; Google Scholar, M.M. Z. J. Biol. Chem. 1993; 268: Full Text PDF PubMed Google Scholar). As with our study, it is possible that superoxide could have induced preconditioning in studies increased However, studies superoxide not an so it is to whether superoxide or H2O2 was for preconditioning protection. to be a likely signaling it can than superoxide, and been shown to directly the of in its R. Anderson W.B. Proc. Natl. Acad. Sci. U. S. A. 1989; 86: 6758-6762Crossref PubMed Scopus (381) Google Scholar). of ROS in the intact heart M.L. Manson N.H. J. Mol. Cell. Cardiol. 1984; 16: 969-985Abstract Full Text PDF PubMed Scopus (482) Google Scholar, V.J. Fanburg B.L. J. Biol. Chem. 1995; 270: 30334-30338Abstract Full Text Full Text PDF PubMed Scopus (386) Google Scholar, 10Griendling K.K. Minieri C.A. Ollerenshaw J.D. Alexander R.W. Circ. Res. 1994; 74: 1141-1148Crossref PubMed Scopus (2433) Google Scholar, 11Mohazzab-H K.M. Kaminski P.M. Wolin M.S. Circulation. 1997; 96: 614-620Crossref PubMed Scopus (138) Google Scholar, G. Zweier J.L. Duilio C. Kuppusamy P. Santoro G. Elia P.P. Tritto I. Cirillo P. Condorelli M. Chiarello M. J. Biol. Chem. 1993; 268: 18532-18541Abstract Full Text PDF PubMed Google Scholar, 15Xia Y. Dawson V.L. Dawson T.M. Snyder S.H. Zweier J.L. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 6770-6774Crossref PubMed Scopus (657) Google Scholar, R. Ceconi C. Curello S. A. De A. Am. J. Clin. PubMed Scopus Google Scholar). and other in the may ROS in intact heart (7Ferrari R. Ceconi C. Curello S. Alfieri O. Visioli O. Eur. Heart. J. 1993; 14: 25-30Crossref PubMed Scopus (23) Google Scholar). 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PubMed Scopus Google Scholar). abolished the ROS generation during hypoxia, and to inhibit the no it is likely that is the source of The also that the ROS generation and protection with preconditioning is abolished with the anion channel inhibitor superoxide to the mitochondrial anion channels are to transport superoxide A. T.M. Wolin M.S. J. Physiol. 1993; 74: PubMed Scopus Google Scholar), and have been described the and mitochondrial J. Biol. Chem. 1997; Full Text Full Text PDF PubMed Scopus Google Scholar, H. J. 1996; 28: PubMed Scopus Google Scholar). studies to have the importance of such channels to the induction of preconditioning, but studies are needed to clarify their Although studies have the role of ROS generated during prolonged ischemia/reperfusion (12Ambrosio G. Zweier J.L. Duilio C. Kuppusamy P. Santoro G. Elia P.P. Tritto I. Cirillo P. Condorelli M. Chiarello M. J. Biol. Chem. 1993; 268: 18532-18541Abstract Full Text PDF PubMed Google Scholar), a of suggests that signaling of ROS generated by may signaling involved in this studies have shown that mitochondrial ROS generated during hypoxia to participate in the of and contraction in J. A. Shao Z. Schumacker P.T. J. Biol. Chem. Full Text Full Text PDF Scopus Google Scholar). The study by that mitochondrial ROS generated during brief can also signaling involved in from subsequent ischemia/reperfusion