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(Hypertension. 2005;45:860.)
© 2005 American Heart Association, Inc.

Original Articles

Role of NAD(P)H Oxidase- and Mitochondria-Derived Reactive Oxygen Species in Cardioprotection of Ischemic Reperfusion Injury by Angiotensin II

Shoji Kimura; Guo-Xing Zhang; Akira Nishiyama; Takatomi Shokoji; Li Yao; Yu-Yan Fan; Matlubur Rahman; Takeo Suzuki; Hajime Maeta; Youichi Abe

From the Department of Pharmacology (S.K., G-X.Z., A.N., T.S., L.Y., Y-Y.F., M.R., Y.A.) and the First Department of Surgery (T.S., H.M.), Kagawa University Medical School, Japan.

Correspondence to Shoji Kimura, MD, PhD, Department of Pharmacology, Kagawa University Medical School, 1750-1 Ikenobe, Miki, Kagawa 761-0793, Japan. E-mail kimura{at}kms.ac.jp

	Abstract

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Reactive oxygen species (ROS) participate in cardioprotection of ischemic reperfusion (I/R) injury via preconditioning mechanisms. Mitochondrial ROS have been shown to play a key role in this process. Angiotensin II (Ang II) exhibits pharmacological preconditioning; however, the involvement of NAD(P)H oxidase, known as an ROS-generating enzyme responsive to Ang II stimuli, in the preconditioning process remains unclear. We compared the effects of 5-hydroxydecanoate (5-HD; an inhibitor of mitochondrial ATP-sensitive potassium channels), apocynin (an NAD(P)H oxidase inhibitor), and 4-hydroxy-2,2,6,6-tetramethyl piperidinoxyl (tempol; a membrane permeable radical scavenger) on pharmacological preconditioning by Ang II in rat cardiac I/R injury in vivo. Treatment with a pressor dose of Ang II before a 30-minute coronary occlusion reduced infarct size as determined 24 hours after reperfusion. The protective effects of Ang II were eliminated by pretreatment with 5-HD or apocynin, similar to tempol. Both 5-HD and apocynin suppressed the enhanced cardiac lipid peroxidation and activation of the apoptosis signal-regulating kinase/p38, c-Jun NH₂-terminal kinase (JNK) pathways, but not the Raf/MEK/extracellular signal-regulated kinase pathway, elicited by acutely administered Ang II. Apocynin but not 5-HD suppressed Ang II–induced augmentations of the NAD(P)H oxidase complex formation (p47^phox, p22^phox, and Rac-1) and its activity in the heart. Finally, 5-HD suppressed superoxide production by isolated cardiac mitochondria without any effect on their respiration. These results suggest that the preconditioning effects of Ang II for cardiac I/R injury may be mediated by cardiac mitochondria-derived ROS enhanced through NAD(P)H oxidase via JNK and p38 mitogen-activated protein kinase activation.

Key Words: angiotensin • antioxidants • free radicals • heart

	Introduction

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The opening of cardiac mitochondrial ATP-sensitive potassium (mitoK_ATP) channels protects against subsequent ischemic reperfusion (I/R) tissue injury in the heart.^1,2 Receptor activation ligands, such as bradykinin, opioids, and acetylcholine, have been shown to trigger a preconditioning state³ similar to mitoK_ATP channel openers, which are inhibited by 5-hydroxydecanoate (5-HD), a specific mitoK_ATP channel inhibitor. In the process of mitoK_ATP channel–mediated preconditioning, the involvement of mitochondria-derived free radicals^4,5 and the posteffectors mitogen-activated protein (MAP) kinases, especially p38 MAP kinase^6–8 has been strongly suggested.^1,9

Angiotensin II (Ang II) is well known as a powerful inducer of oxidative stress to cardiovascular tissues, and the reactive oxygen species (ROS) generated participate in Ang II–induced intracellular signaling pathways.¹⁰ We demonstrated that acutely administered Ang II stimulates redox-sensitive cardiac MAP kinase activation, which was eliminated by tempol, a superoxide dismutase mimetic, in vivo.¹¹ To date, NAD(P)H oxidase has been considered a source of ROS corresponding to Ang II effects in cardiovascular tissues.^12–14 In fact, NAD(P)H oxidase may play an important role in cardiac hypertrophy and tissue remodeling through chronic Ang II effects; for example, cardiac hypertrophy induced by a chronic subpressor dose of Ang II infusion was suppressed in mice genetically deficient in gp91^phox, a component of NAD(P)H oxidase.¹⁵ On the other hand, as the acute effects of Ang II, Liu et al demonstrated that pretreatment with Ang II exhibited preconditioning effects for cardioprotection against I/R injury in rabbits,¹⁶ which may lead to the opening of mitoK_ATP channels, as seen in common ligand-mediated preconditioning. However, it is not known whether ROS corresponding to the cardiac preconditioning effects of Ang II are derived predominantly from NAD(P)H oxidase or mitochondria, although Liu et al did not examine the involvement of mitoK_ATP channels or its redox sensitivity.¹⁶

These observations led us to evaluate the possible implications and interactions of NAD(P)H oxidase–derived and mitochondria-derived ROS in cardioprotection against I/R injury by Ang II. In this study, we compared the effects of 5-HD, apocynin, an NAD(P)H oxidase inhibitor, and tempol on Ang II–mediated preconditioning effects. Further, we examined the antioxidant properties and the site of action of 5-HD.

	Methods

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Pharmacological Preconditioning by Ang II for Cardiac I/R Injury
Pharmacological preconditioning by Ang II for cardiac I/R injury was evaluated essentially as described previously.¹⁷ The infarct size was determined 24 hours after reperfusion. Ang II was infused for 10 minutes at a dose of 200 ng · kg^–1 per minute, ending 10 minutes before occlusion of the coronary artery for 30 minutes. 5-HD (10 mg · kg^–1), 4-hydroxy-2,2,6,6-tetramethyl piperidinoxyl (tempol; a membrane-permeable radical scavenger; 30 mg · kg^–1), or 3-carboxy-2,2,5,5-tetramethyl-1-pyrrolidinyloxy (3-CP; an inactive compound of tempol; 30 mg · kg^–1) were given intravenously 10 minutes before or after Ang II infusion. Apocynin was given at 10 mg · kg^–1 over a period of 2 hours before Ang II. It is of note that the treatments of 5-HD and apocynin did not affect basal hemodynamics or the vasoconstrictor effects of Ang II. For details of the operative procedures, see the data supplement, available online at http://www.hypertensionaha.org.

Animal Preparation for Evaluations of 5-HD and Apocynin
To evaluate the site of action of 5-HD, we compared the effects of 5-HD and apocynin on cardiac MAP kinase, NAD(P)H oxidase, and cardiac thiobarbital reactive substances (TBARS) of Ang II–infused rats. Ang II, at a rate of 200 ng · kg^–1 per minute, was given intravenously for 30 minutes on conscious rats as described previously.¹¹ Before the start of Ang II or saline infusion, 5-HD was given at a dose of 10 mg · kg^–1 bolus, and apocynin at 10 mg · kg^–1 over a period of 2 hours. Arterial blood pressure was monitored continuously, and after 30 minutes of infusion, the heart was removed, quickly frozen in liquid nitrogen, and then stored at –80°C. All surgical and experimental procedures were performed according to the guidelines for the care and use of animals established by Kagawa University.

MAP Kinase Cascades
Measurements of the phosphorylated levels of MAP kinase elements (phospho–extracellular signal-regulated kinase [ERK]1/2, -MEK1/2, -Raf-1, -apoptosis signal-regulating kinase [ASK]-1, –c-Jun NH₂-terminal kinases [JNK], and -p38) in the cardiac left ventricle were conducted as described previously.¹¹ The antibodies used in this experiment were purchased from Cell Signaling.

Tissue Lipid Peroxidation
Lipid peroxidation levels in cardiac tissue were determined by measurements of tissue TBARS¹¹ and 8-iso-prostaglandin F₂ (8-iso-PGF₂).¹⁸ For details of the method of extraction of 8-iso-PGF₂, see the online data supplement.

Radical Scavenging Activity of 5-HD
Evaluations of the scavenging activities of 5-HD for superoxide and hydroxyl radicals were conducted according to the methods described by Wada et al.¹⁹ See the online data supplement for details.

Cardiac NAD(P)H Oxidase
The complex formation and activity of cardiac NAD(P)H oxidase were measured according to the methods described in the online data supplement.

Rat Polymorphonuclear Leukocytes
Superoxide production and oxygen consumption of isolated rat polymorphonuclear leukocytes (RPNLs) were measured according to the method described in the online data supplement.

Mitochondrial Respiration and Superoxide Production
Mitochondrial oxygen consumption was measured using Clark-type electrodes (Hansatech). Superoxide production by cardiac mitochondria was measured by the lucigenin-chemiluminescence (LC) method, as reported by Li et al²⁰ (see the online data supplement).

Statistical Analysis
Results are presented as means±SEM. Data were evaluated by ANOVA, and Duncan’s multiple-range test was used to locate differences between the experimental groups using SAS 6.02 software. A value of P<0.05 was chosen as the indicating statistical significance.

	Results

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Pharmacological Preconditioning by Ang II Is Eliminated by 5-HD
The ratio of infarct area to risk area in I/R hearts is presented in Figure 1. In control sham rats, occlusion of the left descending coronary artery for 30 minutes followed by reperfusion resulted in an infarct size of 36.1±2.2% of the risk area determined at 24 hours after reperfusion. Pretreatment with Ang II reduced the infarct area (6.0±1.7%), indicating pharmacological preconditioning effects of Ang II. 5-HD abolished the reduction in infarct size afforded by the subsequent infusion of Ang II, whereas 5-HD given after Ang II treatment had no effect on the reduction in infarct size. Similarly, tempol given before but not after Ang II treatment suppressed the reduction of infarct size, whereas 3-CP had no effect. Apocynin completely reversed the Ang II–induced preconditioning effects.

View larger version (13K): [in this window] [in a new window]   Figure 1. Effects of Ang II, 5-HD, tempol, 3-CP, and apocynin on infarct size caused by occlusion and reperfusion of the left anterior descending coronary artery in anesthetized rat heart. 5-HD and tempol were given before and after Ang II infusion. 3-CP and apocynin were given only before Ang II infusion. Circles and columns represent individual experiments and the mean±SEM for each group, respectively. *P<0.05 vs control rats; P<0.05 vs Ang II–infused rats; #P<0.05 vs rats treated with 5-HD or tempol before Ang II infusion.

Effects of 5-HD and Apocynin on Activated Cardiac MAP Kinase Cascade by Ang II
As shown in Figure 2, augmentations of phosphorylated p38 and JNK MAP kinases by a 30-minute infusion of Ang II in the left ventricle were significantly suppressed by pretreatment with 5-HD or apocynin, whereas those of ERK1/2 were not affected. Ang II–induced augmentation of phosphorylated ASK-1, an upstream regulator of p38 and JNK, was also suppressed by 5-HD, whereas phosphorylations of Raf-1 and MEK1/2, key upstream regulators of ERK1/2, were not affected. 5-HD or apocynin alone had no effects on any basal cardiac MAP kinase activities.

View larger version (32K): [in this window] [in a new window]   Figure 2. Effects of 5-HD and apocynin on Ang II–induced activation of MAP kinase pathways in rat cardiac left ventricle. The phosphorylated forms of the MAP kinase elements were analyzed in tissue obtained 30 minutes after Ang II treatment. Top, Representative blots are shown. Bottom, Densitometric analysis of the phosphorylated forms of the MAP kinase elements. The mean value of each phosphorylated protein in the saline-infused control rats is represented as 1. Data are presented as the mean±SEM of 4 or 5 rats. *P<0.05 vs control rats; P<0.05 vs Ang II–infused rats.

Effects of 5-HD and Apocynin on Cardiac Lipid Peroxidation in Ang II–Infused Rats
We demonstrated previously that tempol eliminated the increase of cardiac TBARS, a maker of lipid peroxidation, induced in acutely Ang II–infused rats.¹¹ In this study, we compared the effects of pretreatment with 5-HD and apocynin on Ang II–induced increases of TBARS (Figure 3) as well as 8-iso-PGF₂ (supplemental Figure I, available online at http://www.hypertensionaha.org) contents in cardiac left ventricular tissue, resulting in significant suppression of the increases of these lipid peroxidation makers elicited by Ang II. 5-HD or apocynin alone had no effects on basal cardiac TBARS and 8-iso-PGF₂ levels.

View larger version (13K): [in this window] [in a new window]   Figure 3. Effects of 5-HD and apocynin on Ang II–induced increases in TBARS of rat cardiac left ventricle. Levels of TBARS were measured 30 minutes after Ang II treatment. Data are presented as the mean±SEM of 5 rats. *P<0.05 vs vehicle-infused control rats; P<0.05 vs Ang II–infused rats.

5-HD Is Not a Radical Scavenger
As shown in Figure 4, tempol strongly scavenged superoxide radicals generated by xanthine/xanthine oxidase reaction and also suppressed hydroxyl radicals formed by the deoxyribose method in a concentration-dependent manner, whereas 5-DH had no effects on these ROS parameters at concentrations up to 10 mmol/L, indicating that 5-HD might not reduce ROS levels through scavenging activity similar to that of tempol.

View larger version (20K): [in this window] [in a new window]   Figure 4. ROS scavenging activities of tempol and 5-HD for superoxide (left) and hydroxyl radicals (right). The concentration of 3-CP was 10 mmol/L. Data are presented as the mean±SEM of 3 different experiments. *P<0.05 vs control (C).

Effects of 5-HD and Apocynin on Cardiac NAD(P)H Oxidase Activity and Complex Formation Stimulated by Ang II
Complex formation of p47^phox, p22^phox, and Rac-1 of NAD(P)H oxidase in the cardiac left ventricle was determined by coimmunoprecipitation assays (Figure 5A; supplemental Figure II). The complex formation of these 3 components was promoted by a 30-minute infusion of Ang II. 5-HD or apocynin alone and pretreatment with 5-HD before Ang II infusion had no effects on basal or Ang II–induced increases in complex formation. However, pretreatment with apocynin before Ang II infusion completely suppressed any Ang II–induced increase in complex formation. As a control for the immunoprecipitations, the amounts of p47^phox, p22^phox, and Rac-1 immunoprecipitated in all the groups were essentially constant, as determined by direct immunoblotting (data not shown).

View larger version (20K): [in this window] [in a new window]   Figure 5. NAD(P)H oxidase in cardiac left ventricle of Ang II–infused rats. A, Cardiac NAD(P)H oxidase complex formation. The homogenates of cardiac left ventricle (LV) and PMA-stimulated RPNLs were immunoprecipitated with anti-p22phox antibody, and the precipitants obtained were immunoblotted by anti-p47phox or Rac-1 antibodies. The total amounts of p22phox immunoprecipitated in cardiac left ventricle of all the groups were essentially constant, as determined by direct immunoblotting (data not shown). Top, Representative blots are shown. Bottom, Densitometric analysis of the immunoreactivities. The mean value of each protein in the saline-infused control rats is represented as 1. B, Cardiac NAD(P)H oxidase activity. The enzyme activity was measured in cardiac membrane fraction obtained 30 minutes after treatment. Data are presented as the mean±SEM of 5 rats. *P<0.05 vs control rats; P<0.05 vs Ang II–infused rats.

NAD(P)H oxidase activity was analyzed in the membrane fraction obtained from Ang II–infused rat cardiac left ventricle. Reflecting the NAD(P)H oxidase complex formation, the activity was augmented by Ang II infusion, which was completely blocked by pretreatment with apocynin but not by 5-HD (Figure 5B).

Ang II Does Not Stimulate Phagocyte NAD(P)H Oxidase
As shown in Figure 6A, phorbol 12-myristate 13-acetate (PMA), a powerful activator of phagocyte NAD(P)H oxidase, stimulated the superoxide production of the RPNL suspension, whereas 1 µmol/L Ang II had no effect on the superoxide production from RPNLs. PMA-stimulated phagocyte NAD(P)H oxidase was significantly suppressed by pretreatment with diphenyleneiodonium but not by 5-HD. In addition, PMA caused a dramatic increase in the oxygen consumption of RPNLs, namely a respiratory burst (2.23±0.15 nmol/L O₂ · 10⁶ cells^–1 per 5 minutes),²¹ whereas Ang II did not affect the basal respiration of RPNLs (Figure 6B).

View larger version (13K): [in this window] [in a new window]   Figure 6. Effects of PMA and Ang II on RPNL superoxide production and oxygen consumption. A, Superoxide production by RPNLs was estimated by the LC method, and chemiluminescence from 2 to 7 minutes after the addition of 2 µg/mL PMA or 1 µmol/L Ang II were averaged. Concentrations of 5-DH and DPI were 100 and 1 µmol/L, respectively. Data are presented as the mean±SEM of 4 different experiments. *P<0.05 compared with the control; P<0.05 compared with PMA-treated RPNLs. B, Oxygen consumption of RPNLs was measured using a Clark-type oxygen electrode. Typical changes of [O2] in chamber (black line) and calculated rate of O2 consumption (red dot) of RPNLs stimulated by PMA (2 µg/mL; left) and Ang II (1 µmol/L; right) are shown.

5-HD Suppresses Mitochondrial Superoxide Production
As shown in Figure 7A, superoxide production from isolated cardiac mitochondria gradually increased over 30 minutes, as also demonstrated by Li et al,²⁰ whereas 5-HD at a dose of 100 µmol/L completely abolished the increase in mitochondrial superoxide production. With respect to mitochondrial respiration, the same dose of 5-HD had no significant effect on state 3 respiration (control 362±32 nmol/L; 5-HD 355±28 nmol/L; O₂ · mg^–1 per minute for 5 separate experiments), respiratory control index (control 3.6±0.4; 5-HD 3.8±0.6), or ADP/O ratio (control 1.45±0.1; 5-HD 1.32±0.1) of isolated cardiac mitochondria (Figure 7B).

View larger version (29K): [in this window] [in a new window]   Figure 7. Effects of 5-HD on the superoxide production and respiration of isolated cardiac mitochondria. A, Superoxide production by mitochondria isolated from rat hearts was determined by the LC method. (right; representative charts are shown). Data are presented the mean±SEM of 4 different experiments. *P<0.05 compared with control (C). B, Mitochondrial respiration was monitored using a Clark-type oxygen electrode. Typical changes of [O2] in chamber (black line) and calculated rate of O2 consumption (red dot) of mitochondrial suspension in the absence (left) or presence (right) of 100 µmol/L 5-HD are shown. Mito indicates mitochondrial; Suc, 3 mmol/L succinate; ADP, 250 µmol/L adenosine diphosphate.

	Discussion

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In this study, we demonstrated that the cardioprotective effects of Ang II in preconditioning for I/R injury are mediated via an ROS-sensitive mechanism. Our study of acute Ang II administration further showed that ROS derived from cardiac NAD(P)H oxidase as well as mitochondria participate in the phosphorylation of ASK-1/p38, the JNK pathway of the heart, indicating that these stress-activated MAP kinases may contribute to the cardiac pharmacological preconditioning effects of Ang II.

Although the contribution of mitoK_ATP channels to ischemic preconditioning is controversial,²² many chemical and biological compounds exhibit pharmacological preconditioning through the opening of mitoK_ATP channels,^3,23–25 and in fact, pretreatment with mitoK_ATP channel openers have been shown to exhibit cardioprotective effects against subsequent I/R injury.^2,4,5 In this study, we demonstrated that 5-HD effectively reduced the Ang II–induced enhancement of cardiac lipid peroxidation levels by means other than that of radical scavenging activity, a phenomenon that may well be reflected in the result that 5-HD suppressed superoxide production from isolated cardiac mitochondria without any effects on the respiratory parameters. We also examined the effect of Ang II on RPNLs, which could be considered a probable candidate as a source of ROS in the preconditioning effect.²⁶ The results clearly indicated that RPNLs are not involved in the Ang II–mediating pharmacological preconditioning effects and that the inhibitory action by 5-HD does not depend on suppression of RPNL activity, if any. Therefore, ROS in heart tissue generated from mitochondria through the opening of the mitoK_ATP channel might be key mediators for Ang II–induced pharmacological preconditioning, similar to other receptor-activating ligands.

However, a rather important question has been raised regarding the ROS source corresponding to Ang II stimulation in heart tissue because numerous independent lines of evidence have supported and proven the critical role of NAD(P)H oxidase in the actions of Ang II.²⁷ Accordingly, we demonstrated in this study that pretreatment with apocynin suppressed augmentation of cardiac lipid peroxidation and the preconditioning effects of Ang II, pointing to the crucial role of NAD(P)H oxidase in cardiac Ang II signaling. However, activation of NAD(P)H oxidase does not directly link to the preconditioning effects because 5-HD reversed the Ang II–induced preconditioning effects similarly to apocynin without affecting NAD(P)H oxidase complex formation and its activity. Thus, it would be reasonable to assume that activation of NAD(P)H oxidase is required for the actions of Ang II, but the direct contribution of ROS derived from NAD(P)H oxidase for the preconditioning effects in response to acute Ang II stimuli is limited, and NAD(P)H oxidase activation is upstream of mitochondria in Ang II–induced ROS signaling.

There is no direct evidence available to show how ROS derived from membrane NAD(P)H oxidase interact with mitochondria. Interestingly, Zhang et al reconstituted mitoK_ATP channels of bovine heart using planar lipid bilayers and clearly demonstrated that superoxide stimulates the opening of reconstituted mitoK_ATP channels via a direct action on the sulfhydryl groups of this channel.²⁸ Therefore, it could be speculated that Ang II first stimulates NAD(P)H oxidase in the heart via protein kinase C–mediated mechanism,¹⁶ but the ROS generated in this step are insufficient to exhibit preconditioning effects. However, it could serve as a trigger to a subsequent activation of mitoK_ATP channels on the mitochondrial inner membrane, enhancing ROS production to increase tissue lipid peroxidation and contribute to Ang II–mediated preconditioning effects. Zorov et al further demonstrated ROS-induced ROS release, the mechanisms of which are primarily based on the mitochondrial permeability transition in cardiomyocytes.²⁹ Collectively, the results of this study could be explained by intracellular ROS accelerating mechanisms from NAD(P)H oxidase to mitochondria.

Ang II signaling via Ang II type-1 receptors proceeds to activate stress-activated MAP kinase (ie, JNK and p38) as well as ERK1/2, and the signaling has also been implicated in cardiac hypertrophy^30–32 and heart failure.³³ We demonstrated previously that Ang II–induced activation of these MAP kinases in the heart was tempol quenchable.¹¹ Interestingly, the Ang II–induced activation of stress-inducible MAP kinases was eliminated by 5-HD as well as apocynin, whereas those of the Raf/MEK/ERK pathway were not affected by 5-HD or apocynin. Among the MAP kinase family, p38 MAP kinase is thought to be responsible for ischemic preconditioning in isolated heart^5,6 and cultured cardiac cells.^7,8 Although NO-mediated cardioprotection for I/R injury via ERK activation through mitochondrial ROS has also been proposed,³⁴ the results of the present study support that stress-activated ASK pathway activation rather than that of the Raf/MEK/ERK pathway may play an important role in Ang II–induced cardioprotection against I/R injury.

Perspective
We demonstrated that acutely administered Ang II promotes cardiac lipid peroxidation and that this effect is eliminated by pretreatment with 5-HD and apocynin. We also found that among the cardiac MAP kinase cascades activated by Ang II, the ASK-1/p38, JNK pathways are suppressed by 5-HD and apocynin. Considering the different sites of action of each compound, we conclude that cardiac mitochondria are responsible for the ROS that contribute to the activation of redox-sensitive MAP kinase cascades and the preconditioning effects in response to acute Ang II treatment and that activation of NAD(P)H oxidase is also essential because it leads to mitochondrial ROS production by Ang II stimulation. Further studies regarding the relationship between NAD(P)H oxidase and mitochondria will be needed to clarify the physiological and pathophysiological roles of intracellular ROS signaling.

	Acknowledgments

 
This work was supported in part by a grant-in-aid for scientific research from the Ministry of Education, Science, and Culture of Japan.

Received October 8, 2004; first decision October 31, 2004; accepted February 22, 2005.

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