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Am J Physiol Heart Circ Physiol 301: H1695–H1705, 2011. First published August 5, 2011; doi:10.1152/ajpheart.00276.2011.

Intermittent hypobaric hypoxia improves postischemic recovery of myocardial contractile function via redox signaling during early reperfusion Zhi-Hua Wang,1 Yi-Xiong Chen,1 Cai-Mei Zhang,1 Lan Wu,1 Zhuo Yu,1 Xiao-Long Cai,1 Yi Guan,1 Zhao-Nian Zhou,3 and Huang-Tian Yang1,2 1

Key Laboratory of Stem Cell Biology and Laboratory of Molecular Cardiology, Institute of Health Sciences, Shanghai Institutes for Biological Sciences (SIBS), Chinese Academy of Sciences (CAS), and Shanghai Jiao Tong University School of Medicine (SJTUSM), Shanghai; 2Shanghai Key Laboratory of Vascular Biology, Ruijin Hospital, SJTUSM, Shanghai; and 3Laboratory of Hypoxic Cardiovascular Physiology, SIBS, CAS, Shanghai, China Submitted 22 March 2011; accepted in final form 26 July 2011

Wang ZH, Chen YX, Zhang CM, Wu L, Yu Z, Cai XL, Guan Y, Zhou ZN, Yang H. Intermittent hypobaric hypoxia improves postischemic recovery of myocardial contractile function via redox signaling during early reperfusion. Am J Physiol Heart Circ Physiol 301: H1695–H1705, 2011. First published August 5, 2011; doi:10.1152/ajpheart.00276.2011.—Intermittent hypobaric hypoxia (IHH) protects hearts against ischemiareperfusion (I/R) injury, but the underlying mechanisms are far from clear. ROS are paradoxically regarded as a major cause of myocardial I/R injury and a trigger of cardioprotection. In the present study, we investigated whether the ROS generated during early reperfusion contribute to IHH-induced cardioprotection. Using isolated perfused rat hearts, we found that IHH significantly improved the postischemic recovery of left ventricular (LV) contractile function with a concurrent reduction of lactate dehydrogenase release and myocardial infarct size (20.5 ⫾ 5.3% in IHH vs. 42.1 ⫾ 3.8% in the normoxic control, P ⬍ 0.01) after I/R. Meanwhile, IHH enhanced the production of protein carbonyls and malondialdehyde, respective products of protein oxidation and lipid peroxidation, in the reperfused myocardium and ROS generation in reperfused cardiomyocytes. Such effects were blocked by the mitochondrial ATP-sensitive K⫹ channel inhibitor 5-hydroxydecanoate. Moreover, the IHH-improved postischemic LV performance, enhanced phosphorylation of PKB (Akt), PKC-ε, and glycogen synthase kinase-3␤, as well as translocation of PKC-ε were not affected by applying H2O2 (20 ␮mol/l) during early reperfusion but were abolished by the ROS scavengers N-(2-mercaptopropionyl)glycine (MPG) and manganese (III) tetrakis (1-methyl-4-pyridyl)porphyrin. Furthermore, IHH-reduced lactate dehydrogenase release and infarct size were reversed by MPG. Consistently, inhibition of Akt with wortmannin and PKC-ε with εV1-2 abrogated the IHH-improved postischemic LV performance. These findings suggest that IHHinduced cardioprotection depends on elevated ROS production during early reperfusion.

contractile dysfunction (3, 33), arrhythmias (31, 52), and cell death (8, 27). Recently, we (48) revealed a therapeutic effect of IHH on permanent coronary artery ligation-induced myocardial infarction by attenuating infarct size, myocardial fibrosis, and apoptosis and improving cardiac performance. Because IHH is a relatively simple intervention with a longer protection duration and fewer adverse effects and may offer profound benefit to patients with acute myocardial infarction (3, 37), elucidating the mechanistic insights underlying the cardioprotective effects of IHH is critical to potential clinic applications. Excessive generation of ROS during the early phase of reperfusion after myocardial ischemia has been proposed to contribute to reperfusion injury (2, 45, 55). Paradoxically, ROS generated at the same phase also act as signaling molecules, triggering the cardioprotection induced by ischemic or pharmacological conditioning (4, 16, 18, 36, 43). Mitochondrial permeability transition pore (MPTP) opening is a crucial event in lethal reperfusion injury and is regulated by glycogen synthase kinase (GSK)-3␤ activity (20, 21, 23). Activation of

reactive oxygen species; ischemia-reperfusion injury EARLY REPERFUSION during evolving myocardial infarction is essential for saving the myocardium, but lethal reperfusion injury can occur and limit the beneficial effects (49). A number of cardioprotective strategies have been developed to ameliorate or retard the irreversible injury. However, the clinical translation of these strategies has failed to achieve the anticipated results (13, 34). Intermittent hypobaric hypoxia (IHH) has been shown to protect the heart against ischemia-reperfusion (I/R) injury by improving the manifestations including

Address for reprint requests and other correspondence: H.-T. Yang, Institute of Health Sciences, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, and Shanghai Jiao Tong Univ. School of Medicine, 225 Chongqing Nan Rd., Bldg. 1, Shanghai 200025, China (e-mail: htyang@sibs. ac.cn). http://www.ajpheart.org

Fig. 1. Effects of intermittent hypobaric hypoxia (IHH) on body weight (A) and heart weight (B) of rats. Left ventricle (LV)-to-body weight and right ventricle (RV)-to-body weight ratios are shown. n ⫽ 46.

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prosurvival signaling pathways [in particular, PKB (Akt) and PKC-ε] relies on the generation of ROS in the protected myocardium (14, 15, 41) and converge on the inhibition of GSK-3␤ activity by phosphorylation of Ser9 (23). It has been suggested that the concentration of ROS determines their different roles, i.e., ROS are cardioprotective at low levels but detrimental at high levels (6, 39). However, this theory is still clouded by the failure of a number of animal studies and most clinical studies with antioxidants to convey cardioprotection (24, 29, 42). An obvious increase of ROS in cardiomyocytes during early reperfusion after ischemia has been observed by several groups (24, 46, 55), but whether such an increase is already excess to cardiomyocytes remains uncertain. It also remains a debate whether the increase of ROS directly relates to cell injury (24, 42).

IHH increases myocardial oxidative stress during the intervals of hypoxia and reoxygenation. Treatment with the antioxidant N-acetylcysteine during exposure to IHH partially reduced the infarct size-limiting effect of IHH in the rat heart (26). However, it is unknown how IHH influences ROS production during myocardial I/R and whether ROS generated at this phase play a role in IHH-induced cardioprotection. To address these questions, we examined the effects of IHH on ROS generation during I/R in isolated perfused rat hearts and isolated cardiomyocytes and determined the roles of ROS as well as downstream signaling pathways in IHH-induced cardioprotection. Our results demonstrate that elevated ROS generation during early reperfusion is critical for triggering the cardioprotection induced by IHH. Our findings provide new insights into the

Fig. 2. Role of ROS generated during early reperfusion in the IHH-improved postischemic recovery of myocardial contractile function. A: representative traces of LV pressure (LVP) during ischemia-reperfusion (I/R) in isolated rat hearts from normoxic and IHH groups. H2O2 (20 ␮mol/l) and the ROS scavengers N-(2-mercaptopropionyl)glycine (MPG; 100 ␮mol/l) and manganese (III) tetrakis (1-methyl-4-pyridyl)porphyrin (MnTMPyP; 10 ␮mol/l) were added at the beginning of reperfusion for 5 min followed by washout, as indicated by the solid and open arrowheads, respectively. B–E: summarized effects of H2O2, MPG, and MnTMPyP on postischemic recovery of LV developed pressure (LVDP), LV end-diastolic pressure (LVEDP), and maximum rates of pressure development or decay over time (⫹dP/dtmax and ⫺dP/dtmax) in normoxic and IHH groups. Experimental numbers are indicated in each bar. *P ⬍ 0.05, **P ⬍ 0.01, and ***P ⬍ 0.001 vs. the corresponding normoxic group; #P ⬍ 0.05, ##P ⬍ 0.01, and ###P ⬍ 0.001 vs. the corresponding I/R control.

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explanation of the controversial roles of ROS in myocardial I/R injury and cardioprotection. MATERIALS AND METHODS

Animal care. The animals used in this study were maintained in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals (NIH Pub. No. 85-23), and all procedures were approved by the Institutional Review Board of the Institute of Health Sciences, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences and School of Medicine, Shanghai Jiao Tong University (Shanghai, China). IHH-adapted rat model. To establish animal models adapted to IHH, male Sprague-Dawley rats (Shanghai Slac Laboratory Animal, Shanghai, China) were intermittently exposed to hypobaric hypoxia (equivalent to an altitude of 5,000 m, barometric pressure: 404 mmHg, PO2: 84 mmHg) in a hypobaric chamber for one 4-h period each day for 4 wk as previously described (1, 51). During this period, their body weights rose from 100 –120 to 310 –360 g. Age-matched normoxic animals were maintained in a normoxic environment for a corresponding period. All animals had free access to water and standard laboratory diet. I/R injury model in Langendorff-perfused rat hearts. After rats has been anesthetized with pentobarbital sodium (45 mg/kg ip), hearts were rapidly excised and perfused with Krebs-Henseleit (K-H) solution at 37°C using a Langendorff apparatus at a constant pressure of 80 mmHg as previously described (54). A water-filled latex balloon connected to a pressure transducer (Gould P23Db, AD Instruments, Sydney, NSW, Australia) was inserted into the left ventricular (LV) cavity to achieve a stable LV end-diastolic pressure (LVEDP) of 5–10 mmHg during initial equilibration. After equilibration perfusion, the heart was subjected to 30 min of global noflow ischemia followed by 45 min of reperfusion. LV developed pressure (LVDP) and maximum rates of pressure development or decay over time (⫹dP/dtmax and ⫺dP/dtmax) were evaluated with the PowerLab system (AD Instruments). Experimental protocols. H2O2 (20 ␮mol/l) was perfused at the beginning of reperfusion for 5 min to examine the effects of exogenous ROS on the postischemic recovery of myocardial performance in normoxic and IHH groups. The ROS scavengers N-(2-mercaptopropionyl)glycine (MPG; 100 ␮mol/l, Sigma-Aldrich, St. Louis, MO) and manganese (III) tetrakis (1-methyl-4-pyridyl)porphyrin (MnTMPyP; 10 ␮mol/l, Merck, Darmstadt, Germany) were perfused at the beginning of reperfusion for 5 min. The phosphoinositide 3-kinase (PI3K) inhibitor wortmannin (300 nmol/l, Millipore) or the PKC-ε inhibitor εV1-2 (10 ␮mol/l, Anaspec) was perfused for 5 min with a 5-min washout before ischemia. The mitochondrial ATP-sensitive K⫹ (KATP) channel inhibitor 5-hydroxydecanoate (5-HD; 200 ␮mol/l, Sigma-Aldrich) was added during the last 5 min of equilibration perfusion and the first 5 min of reperfusion. At the end of 45 min of reperfusion, hearts were rapidly removed and frozen in liquid nitrogen for Western blot analysis and other tests. Equilibrationperfused hearts with a corresponding period were collected as balance controls. Lactate dehydrogenase activity. Coronary effluent was collected before ischemia and during reperfusion for the measurement of lactate dehydrogenase (LDH) release, an indicator of cell damage. The activity of LDH was measured based on the oxidation of lactate as previously described (24). Infarct size estimation. To determine whether IHH and MPG affect the irreversible cell injury after I/R, isolated rat hearts subjected to 30 min of ischemia followed by 2 h of reperfusion were frozen, and the LV was cut into 2-mm-thick slices. Sections were then incubated in 1% (wt/vol) triphenyltetrazolium chloride (phosphate buffer, pH 7.4) for 15 min for staining (54). Slices were fixed in 10% formaldehyde to enhance the contrast between stained viable and unstained necrotic tissue. Infarct size was calculated using Image Pro Plus software AJP-Heart Circ Physiol • VOL

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(MediaCybernetics), and the infarct area was expressed as a percentage of the LV area at risk. ROS detection in isolated cardiomyocytes during simulated I/R. To directly monitor ROS production during I/R, a cellular model of simulated I/R in isolated cardiomyocytes was used as previously described (3, 5). Briefly, cardiomyocytes from the septal and LV free wall were isolated using a standard enzymatic method (3). Cardiomyocytes were then evenly plated on a 35-mm dish and equilibrated in modified K-H solution perfused by a peristaltic pump. Subsequently, the solution was switched to an ischemic solution containing (in mmol/l) 123.0 NaCl, 8.0 KCl, 1.8 CaCl2, 6.0 NaHCO3, 0.9 NaH2PO4, 0.5 MgSO4, and 20.0 Na-lactate and gassed with 95% N2-5% CO2 (pH 6.8) for 20 min followed by 30 min of reperfusion with modified K-H solution. The mitochondrial KATP channel inhibitor 5-HD (200 ␮mol/l) was perfused during the last 5 min of ischemia and the first 5 min of reperfusion. ROS production in isolated cardiomyocytes during simulated I/R was detected using 5(and 6)-carboxy-2=,7=-dichlorodihydrofluerescein diacetate (DCF; Invitrogen) as previously described (17, 46). Briefly, cardiomyocytes from normoxic or IHH groups were loaded with DCF (20 ␮mol/l) for 10 min before being plated on the dish. The acetate groups of the probe can be removed by intracellular esterases, which allow it to be retained by the cells. Because DCF is nonfluorescent until it is oxidized by ROS within the cell, the intracellular generation of ROS can be reflected by monitoring the increase in DCF fluores-

Fig. 3. Effects of IHH and MPG on cell injury during myocardial I/R. A: lactate dehydrogenase (LDH) activity was measured with coronary effluent collected at baseline, at 5 min of reperfusion (R5), and at 45 min of reperfusion (R45). n ⫽ 3. B: infarct size determined with triphenyltetrazolium chloride was expressed as a percentage of the LV area at risk from isolated hearts subjected to 30 min of ischemia followed by 2 h of reperfusion. n ⫽ 5. *P ⬍ 0.05 and **P ⬍ 0.01 vs. the corresponding normoxic group; #P ⬍ 0.05 and ##P ⬍ 0.01 vs. the corresponding IHH group.

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cence. Fluorescence was detected using an inverted microscope (Nikon, Tokyo, Japan) and recorded (excitation/emission: 495/525 nm) every 5 min during I/R with a fixed field of view including 30 –50 rod-shaped cardiomyocytes. The excitation light source was set at low power to avoid inducing oxidation and opened only when it was needed to get a photo. Fluorescence intensity was analyzed with Image Pro Plus software. Western blot analysis. Proteins were prepared as previously described (8). Briefly, freeze-clamped LV tissue (200 –300 mg) was homogenized in 10 volumes of lysis buffer containing 20 mmol/l Tris·HCl (pH 7.4), 150 mmol/l NaCl, 2.5 mmol/l EDTA, 50 mmol/l NaF, 0.1 mmol/l Na4P2O7, 1 mmol/l Na3VO4, 1 mmol/l PMSF, 1 mmol/l DTT, 0.2% (vol/vol) protease inhibitor cocktail (SigmaAldrich), 1% (vol/vol) Triton X-100, and 10% (vol/vol) glycerol. Homogenates were centrifuged twice at 20,000 g at 4°C for 15 min, and supernatants were saved as total proteins. To separate membrane and cytosolic fractions, LV tissue was homogenized in lysis buffer containing (in mmol/l) 20 Tris·HCl (pH 7.4), 250 sucrose, 1 EDTA, 1 EGTA, 1 NaF, 1 Na3VO4, 1 PMSF, and 1 DTT with 0.2% (vol/vol) protease inhibitor cocktail and centrifuged at 1,000 g for 10 min at 4°C. The supernatant was centrifuged at 10,000 g for 30 min at 4°C, and the pellet was washed once with lysis buffer by centrifugation, resuspended with 0.5% Triton X-100 in lysis buffer, sonicated on ice, and then centrifuged at 20,000 g for 10 min at 4°C. The resultant supernatant was defined as the membrane fraction. The supernatant after the membrane fraction had been pelleted was centrifuged at 100,000 g for 1 h at 4°C, and the resultant supernatant was defined as the cytosolic fraction. Triton X-100 was added at a final concentration of 0.5%. Protein concentrations were determined by the BCA method. Equal amounts of proteins were separated by SDS-PAGE and transferred to a polyvinylidene difluoride membrane (Bio-Rad). Western blot analysis was performed under standard conditions with specific antibodies including anti-phospho-Akt (Ser473), anti-Akt, antiphospho-PKC-ε (Ser729), anti-PKC-ε, anti-phospho-GSK-3␤ (Ser9), and anti-GSK-3␤. Antibodies were purchased from Cell Signaling. The immunoreaction was visualized using an enhanced chemiluminescent

detection kit (Amersham, London, UK), exposed to X-ray film, and quantified by densitometry with a video documentation system (Gel Doc 2000, Bio-Rad). Protein oxidation and lipid peroxidation. To evaluate the effect of IHH on oxidative stress during I/R, we measured protein carbonyls and malondialdehyde (MDA), respective products of protein oxidation and lipid peroxidation (22, 24), in LV tissue homogenated in protein lysis buffer without DTT. Protein carbonyls were measured using an immunoblot kit to detect the 2, 4-dinitrophenylhydrazine (DNPH) derivatization of protein carbonyls following the manufacturer’s instruction (Cell Biolabs). MDA content was determined by the thiobarbituric acid reaction as previously described (24). Statistical analysis. Data are expressed as means ⫾ SE. Significant differences between two mean values were estimated using Student’s t-test. For multiple comparisons, ANOVA or repeated ANOVA followed by a least-significant-difference post hoc test was used. All statistics were done using SPSS software (version 13.0, SPSS, Chicago, IL). P values of ⬍0.05 were considered statistically significant. RESULTS

IHH improves the postischemic recovery of myocardial contractile function and cell survival. IHH-adapted rats showed comparable body weights compared with normoxic controls (Fig. 1A). LV-to-body weight and right ventricle-tobody weight ratios were slightly increased after exposure to IHH for 4 wk but did not reach statistic significance (Fig. 1B). In Langendorff-perfused rat hearts, LV contractile function was markedly suppressed after 30 min of noflow ischemia followed by 45 min of reperfusion (Fig. 2A). IHH did not affect baseline LV contractile function but significantly improved the postischemic recovery of LVDP, LVEDP, ⫹dP/dtmax, and ⫺dP/dtmax compared with the normoxic group (Fig. 2, B–E). Consistently, IHH significantly inhibited the I/R-induced robust increase of LDH activity in coronary perfusate at 5 and 45

Fig. 4. Effects of IHH and 5-hydroxydecanoate (5-HD) on myocardial protein oxidation and lipid peroxidation during I/R. A: representative immunoblot of 2,4-dinitrophenylhydrazine (DNPH) derivatization of protein carbonyls, a product of protein oxidation, in the LV during I/R in normoxic and IHH groups. Parallel gels were stained with Coomassie brilliant blue R250 dye as a control for protein loading. B: averaged data of protein oxidation shown as the DNPH signal over Coomassie blue stain. C: lipid peroxidation as assessed by melondialdyhyde (MDA) content normalized against the protein concentration in the LV during I/R. n ⫽ 3. The mitochondrial ATP-sensitive K⫹ (KATP) channel inhibitor 5-HD (200 ␮mol/l) was added during the last 5 min of baseline perfusion and first 5 min of reperfusion. *P ⬍ 0.05 and **P ⬍ 0.01 vs. the corresponding normoxic group; #P ⬍ 0.05 and ##P ⬍ 0.01 vs. the corresponding IHH group; †P ⬍ 0.05, ††P ⬍ 0.01, and †††P ⬍ 0.001 vs. the corresponding baseline.

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min of reperfusion (Fig. 3A) and attenuated the I/R-induced myocardial infarct size after 2 h of reperfusion (20.5 ⫾ 5.3% in the IHH group vs. 42.1 ⫾ 3.8% in the normoxic group, P ⬍ 0.01; Fig. 3B), suggesting that IHH protects the heart against I/R injury. IHH increases ROS production during early reperfusion. To determine the effect of IHH on ROS, we examined myocardial protein oxidation and lipid peroxidation by measuring protein carbonyls and MDA contents during I/R. DNPH derivatization of myocardial protein carbonyls and MDA content were increased after reperfusion (Fig. 4). Compared with the normoxic group, IHH significantly increased the baseline level of protein carbonyls and further enhanced the I/R-induced increase of DNPH at the first 5 min of reperfusion (Fig. 4, A and B). MDA content was also significantly enhanced by IHH at the first 5 min of reperfusion (Fig. 4C). To directly confirm the influence of IHH on ROS, we then monitored ROS production in isolated cardiomyocytes subjected to simulated I/R (20/30 min) using DCF fluorescence. Fluorescence intensity was significantly enhanced during rep-

Fig. 5. Effects of IHH, MPG, and 5-HD on ROS production during simulated I/R in isolated cardiomyocytes. A and B: cardiomyocytes labeled with 5 (and 6)-carboxy-2=,7=- dichlorodihydrofluerescein diacetate (DCF; 20 ␮mol/l) were subjected to 20 min of ischemia followed by 30 min of reperfusion by switching the perfusate from simulated ischemic solution to Krebs-Henseleit buffer. Fluorescence was recorded (excitation/emission: 495/525 nm) every 5 min during I/R in a fixed field of view including 30 –50 rod-shaped cardiomyocytes. MPG (100 ␮mol/l) was added at the beginning of reperfusion for 5 min. 5-HD (200 ␮mol/l) was added during the last 5 min of ischemia and first 5 min of reperfusion. n ⫽ 3 each. *P ⬍ 0.05 vs. the corresponding normoxic group; #P ⬍ 0.05 and ##P ⬍ 0.01 vs. the corresponding IHH group; †††P ⬍ 0.001 vs. the corresponding baseline. AJP-Heart Circ Physiol • VOL

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erfusion in cardiomyocytes isolated from normoxic rat hearts (Fig. 5A). DCF fluorescence intensity during baseline perfusion and the ischemic phase was similar between IHH and normoxic groups, but IHH markedly augmented the fluorescence intensity during reperfusion, especially during the first 5 min (Fig. 5A). Such increases were abolished by the ROS scavenger MPG (100 ␮mol/l; Fig. 5A). These data suggest that IHH enhanced ROS production during early reperfusion in the ischemic heart. Mitochondrial KATP channels contribute to IHH-increased ROS production. To determine whether the increased ROS production during early reperfusion by IHH is derived from the mitochondria through the opening of mitochondrial KATP channels, we examined the effects of the mitochondrial KATP inhibitor 5-HD (200 ␮mol/l) on ROS production and IHHinduced cardioprotection. 5-HD did not alter I/R-increased protein carbonyls and MDA content in the normoxic group, but it attenuated IHH-increased protein carbonyls and MDA content at the first 5 min of reperfusion (Fig. 4). In isolated cardiomyocytes, 5-HD attenuated ROS production during reperfusion and blocked the IHH-enhanced increase of ROS production (Fig. 5B). These data support that the opening of the mitochondrial KATP channel contributes to IHH-increased ROS production during early reperfusion. IHH-induced cardioprotection depends on ROS generation during early reperfusion. Although it has been suggested that the ROS generated during early reperfusion participate in cardioprotection induced by ischemic or pharmacological conditioning (4, 16, 36, 43), little is known about the effect of introducing exogenous ROS into the heart during early reperfusion. To further determine the role of increased ROS during early reperfusion in IHH-induced cardioprotection, we treated hearts with H2O2 and ROS scavengers during the first 5 min of reperfusion and examined their effects on the postischemic recovery of contractile function. Postconditioning with H2O2 (20 ␮mol/l) significantly improved the postischemic recovery of LVDP, LVEDP, ⫹dP/dtmax, and ⫺dP/dtmax in the normoxic group but did not affect those functional parameters in the IHH group (Fig. 2). However, scavenging ROS with MPG (100 ␮mol/l) and MnTMPyP (10 ␮mol/l) completely blocked the IHH-improved postischemic recovery of LV contractile function without changes in the normoxic group (Fig. 2). Moreover, IHH-reduced LDH activity and myocardial infarct size were significantly reversed by MPG (Fig. 3). Consistently, inhibition of mitochondrial KATP channels with 5-HD also abolished IHH-improved LV performance after I/R (Fig. 6). These data reveal that the increased ROS during early reperfusion are critical for IHH-induced cardioprotection. ROS mediates the IHH-enhanced activation of protective signaling after I/R. To understand the underlying signaling pathways, we next examined the phosphorylation of Akt (Ser473), PKC-ε (Ser729), and GSK-3␤ (Ser9), the putative last step of the cytoplasmic cardioprotective signaling cascade (23). Compared with the normoxic group, IHH significantly increased the phosphorylation levels of Akt (Fig. 7A), PKC-ε (Fig. 7B), and GSK-3␤ (Fig. 7C) after I/R. Such effects were not additive to the enhancement induced by H2O2 (20 ␮mol/l) but were completely blocked by the ROS scavengers MPG and MnTMPyP (Fig. 7, A–C). To further confirm the activation of PKC-ε by IHH, we then measured the translocation of PKC-ε by Western blot analysis. IHH significantly promoted the

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Fig. 6. Mitochondrial KATP channels participate in the IHH-induced protection of myocardial contractile function during I/R. A: representative traces of LVP during I/R in isolated rat hearts with and without IHH treatment. The mitochondrial KATP channel inhibitor 5-HD (200 ␮mol/l) was added at the last 5 min of equilibration perfusion and washout at the first 5 min of reperfusion, as indicated by the solid and open arrowheads, respectively. B–E: summarized effects of 5-HD on the postischemic recovery of LVDP, LVEDP, ⫹dP/dtmax, and ⫺dP/dtmax in normoxic and IHH groups. n ⫽ 5. **P ⬍ 0.01 vs. the corresponding normoxic group; #P ⬍ 0.05 and ##P ⬍ 0.01 vs. the corresponding I/R control.

translocation of PKC-ε from the cytosolic fraction to the membrane fraction in the LV after I/R. Such an effect was not additive to that of H2O2 but was blocked by MPG and MnTMPyP (Fig. 7D). These results suggest that IHH activates protective signaling pathways via enhancement of ROS generation during early reperfusion. Activation of Akt and PKC-␧ contributes to IHH-induced cardioprotection. To confirm the contribution of Akt and PKC-ε pathways in IHH-induced cardioprotection, we then examined the effects of wortmannin and εV1-2, respective inhibitors of PI3K (the upstream activator of Akt) and PKC-ε, on the postischemic recovery of LV contractile function. Treatment with wortmannin (300 nmol/l) or εV1-2 (10 ␮mol/l) did not affect the postischemic recovery of LVDP, LVEDP, ⫹dP/dtmax, and ⫺dP/dtmax in the normoxic group (Fig. 8, B–E). However, both wortmannin and εV1-2 abrogated the IHH-improved postischemic recovery of LV contractile function (Fig. 8). Consistently, both wortmannin and εV1-2 blocked IHH-increased GSK-3␤ phosphorylation during reperfusion but did not affect the control (Fig. 9C). AJP-Heart Circ Physiol • VOL

Interestingly, the IHH-increased Akt phosphorylation after I/R was inhibited by the PKC-ε inhibitor, and vice versa (Fig. 9, A and B). Consistently, the IHH-promoted translocation of PKC-ε to the membrane fraction was blocked by both εV1-2 and wortmannin (Fig. 9D). These data suggest that Akt and PKC-ε pathways may form a positive feedback loop and mediate IHH-induced ROS-dependent cardioprotection. DISCUSSION

In this study, we demonstrated, for the first time, that adaptation of rats to IHH confers protection of the heart against I/R injury through elevation of ROS production during early reperfusion. A moderate and sufficient increase of ROS during early reperfusion is required to efficiently activate the downstream protective signaling pathways, whereas the endogenous ROS generated during early reperfusion in I/R rat hearts appear to be insufficient to trigger efficient cardioprotection.

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Fig. 7. ROS mediate the IHH-increased activation of protective signaling pathways. A–C: Western blot analysis of total and phosphorylation levels of PKB (Akt), PKC-ε, and glycogen synthase kinase (GSK)-3␤ during I/R in LVs from normoxic and IHH groups. D: Western blot analysis of PKC-ε expression in membrane and cytosolic fractions of the LV from normoxic and IHH groups. PKC-ε translocation is presented as the ratio of the particulate fraction to the cytosolic fraction. H2O2 (20 ␮mol/l), MPG (100 ␮mol/l), and MnTMPyP (10 ␮mol/l) were added at the beginning of reperfusion for 5 min. n ⫽ 4 each. *P ⬍ 0.05, **P ⬍ 0.01 and ***P ⬍ 0.001 vs. the corresponding normoxic group; #P ⬍ 0.05, ##P ⬍ 0.01, and ###P ⬍ 0.001 vs. the corresponding I/R control; †P ⬍ 0.05, ††P ⬍ 0.01, and †††P ⬍ 0.001 vs. the corresponding balance (Bal) group.

Enhancement of ROS generation during early reperfusion is critical to IHH-induced cardioprotection. ROS have been shown to participate in cardioprotection induced by ischemic or pharmacological conditioning (4, 16, 18, 36, 43). However, how ROS perform the protective function at the reperfusion phase rather than inducing injury is controversial. Our data showed that IHH increases ROS generation during early reperfusion (Figs. 4 and 5), and this was accompanied with an antioxidant-sensitive improvement of myocardial contractile function (Fig. 2) and a reduction of cell damage (Fig. 3). The fact that postconditioning with H2O2 (20 ␮mol/l) during the first 5 min of reperfusion improved the postischemic recovery of myocardial contractile function (Fig. 2) confirms that the endogenous ROS generated during early reperfusion in I/R hearts are insufficient to trigger cardioprotection. Otherwise, an aggravated injury should be observed if ROS are already excessively produced during this phase. This is further supported by the observations of Ytrehus et al. (50) showing that treatment with H2O2 during the first 30 min of reperfusion reduces myocardial infarct size after I/R injury in a coronary artery occlusion model. Although a lower concentration (1 ␮mol/l) of H2O2 was used in their study, the much AJP-Heart Circ Physiol • VOL

longer treatment time (30 min) makes it explicable as the activation of protective signaling pathways by ROS accumulates with time (44). Guo et al. (12) found that IHH upregulated the expression of antioxidant enzymes after myocardial I/R injury in guinea pigs, whereas we did not observed a reduction of oxidative stress in rat hearts. Besides the difference in species, this may be due to a more significant generation of ROS in IHH-protected hearts. Ischemic preconditioning and postconditioning have been reported to attenuate oxidative stress during reperfusion (46, 53). However, the repetitive episodes in those procedures lead to cyclic release of ROS (46) that may alter the quantitative threshold for ROS to trigger cardioprotection. In the present model, we provided direct evidence showing that a moderate increase of ROS generation during early reperfusion in cardiomyocytes from IHH-adapted rats is sufficient to activate protective signaling and trigger cardioprotection. This is supported by observations showing that scavenging ROS generation during early reperfusion abolishes the cardioprotection induced by ischemic postconditioning (4, 36) as well as by ischemic preconditioning (16), indicating that the early phase of reperfusion is crucial for ROS to convey cardioprotection.

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Fig. 8. Akt and PKC-ε pathways contribute to IHH-improved postischemic contractile function. A: representative traces of LVP during I/R in isolated rat hearts with and without IHH treatment. The phosphoinositide 3-kinase (PI3K; upstream activator of Akt) inhibitor wortmannin (300 nmol/l) and the PKC-ε inhibitor εV1-2 (10 ␮mol/l) were added before ischemia for 5 min followed by a 5-min washout, as indicated by the solid and open arrowheads, respectively. B–E: summarized effects of wortmannin and εV1-2 on the postischemic recovery of LVDP, LVEDP, ⫹dP/dtmax, and ⫺dP/dtmax in normoxic and IHH groups. Experimental numbers are indicated in each bar. **P ⬍ 0.01 and ***P ⬍ 0.001 vs. the corresponding normoxic group; #P ⬍ 0.05 and ##P ⬍ 0.01 vs. the corresponding I/R control.

Mitochondria as the main source of ROS in IHH-induced cardioprotection. Mitochondria represent a main source of ROS generation in cardiomyocytes (45). It has been shown that more ROS are generated in isolated mitochondria during reoxygenation after hypoxia than after anoxia, suggesting that the mitochondria-derived ROS rely to a certain extent on mitochondrial function (28). We (47, 54) have reported previously that IHH effectively protects mitochondrial structure and function after myocardial I/R injury. Moreover, the abolishment of the IHH-induced increase in ROS production and cardioprotection by the mitochondrial KATP channel inhibitor 5-HD (Figs. 4, 5B, and 6) further demonstrates that mitochondria-derived ROS contribute to IHH-induced cardioprotection. This is consistent with observations showing that the activation of mitochondrial KATP channels increases ROS generation, which subsequently activates prosurvival signaling pathways and inhibits MPTP opening during myocardial I/R (6, 18, 35, AJP-Heart Circ Physiol • VOL

38). However, mitochondrial KATP channels might not participate in the cardioprotection of right ventricular function induced by chronic hypoxia in rats (9). Other ROS-producing enzymes, e.g., NADPH oxidase and xanthine oxidase, have also been proposed to participate in cardioprotection (10, 25). Whether this mechanism contributes to IHH-induced cardioprotection needs to be investigated. ROS trigger the cardioprotection of IHH via the activation of Akt and PKC-␧ pathways. Of note, we found that the increased ROS generation during early reperfusion is essential for IHH-induced activation of Akt and PKC-ε pathways, suggesting a quantitative threshold of ROS in the efficient activation of these protective signaling pathways. Myocardial I/R has been shown to induced a noticeable activation of Akt and PKC-ε, which could be further enhanced in the protected myocardium (23, 30, 32). We also observed that adaptation of rats to IHH enhanced the I/R-increased phosphorylation of Akt

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Fig. 9. Effects of the PI3K inhibitor wortmannin and the PKC-ε inhibitor εV1-2 on IHH-activated protective signaling pathways. A–C: Western blot analysis of total and phosphorylation levels of Akt, PKC-ε, and GSK-3␤ during I/R in the LV from normoxic and IHH groups. D: PKC-ε expression in membrane and cytosolic fractions of the LV from normoxic and IHH groups. PKC-ε translocation is presented as the ratio of the particulate fraction to the cytosolic fraction. Wortmannin (300 nmol/l) and εV1-2 (10 ␮mol/l) were added before ischemia for 5 min followed by a 5-min washout. n ⫽ 4 each. *P ⬍ 0.05, **P ⬍ 0.01, and ***P ⬍ 0.001 vs. the corresponding normoxic group; #P ⬍ 0.05, ##P ⬍ 0.01, and ###P ⬍ 0.001 vs. the corresponding I/R control; †P ⬍ 0.05, ††P ⬍ 0.01, and †††P ⬍ 0.001 vs. the corresponding balance group.

and PKC-ε in LVs (Fig. 7, A and B). Considering their sensitivity to antioxidants, as confirmed by our data (Fig. 7A and 7B) and those of others (14, 15, 41), it is persuasive that ROS production during early reperfusion in I/R cardiomyocytes is lower than the threshold to effectively activate the signaling pathways responsible for cardioprotection. Moreover, as downstream signaling pathways of ROS, Akt and PKC-ε form a positive feedback loop in the IHH-protected myocardium (Fig. 9, A, B, and D). This may explain the quantitative threshold of ROS to confer cardioprotection. Three programs of protein kinases have been suggested to be involved in the cardioprotection induced by ischemic preconditioning and postconditioning by Heusch et al. (19). In the first program, nitrogen oxide is a central step with the upstream activation of PI3K/Akt and downstream activation of mitochondrial KATP channels and inhibition of MPTP. The second program involves the activation of PI3K/Akt and the ERK system with the downstream inhibition of GSK-3␤, although this pathway varies with species, e.g., it seems not to participate in ischemic preconditioning-induced protection of pig hearts (40). Activation of signal transducer and activator of transcription 3 (STAT3) constitutes the third program. A positive feedback loop between Akt and STAT3 has been shown AJP-Heart Circ Physiol • VOL

to mediate opioid-induced cardioprotection (11). Whether STAT3 contributes to IHH-induced cardioprotection needs to be determined. Potential clinical significance. Although a number of cardioprotective strategies have been discovered, their clinical translation is disappointing, probably because of the inadequacy of animal I/R models to simulate what happens at the bedside (13, 34). IHH appears to be a promising therapeutic strategy for coronary heart disease due to its easier manipulation, longer protection duration, and fewer adverse effects (3, 37, 48). Moreover, we (48) have shown that IHH has a significant therapeutic effect after 1 wk of coronary artery occlusion. IHH has also been demonstrated to improve myocardial perfusion in patients with severe coronary heart disease (7). As a relatively simple intervention, IHH may offer profound benefits to patients with acute myocardial infarction upon the elucidation of protective mechanisms (37). Considering the failure of clinical usage of antioxidants (29), understanding the precise regulation of ROS generation and subsequent signaling pathways in IHH is of clinical significance. Taken together, our data demonstrate that IHH confers cardioprotection against I/R injury by enhancing the production of ROS during early reperfusion, which subsequently

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activate downstream protective signaling pathways, Akt and PKC-ε. These results also suggest that the endogenous ROS generated during early reperfusion in I/R hearts seem not reach the threshold to trigger efficient cardioprotection. Our findings provide a new angle to interpret the controversial roles of ROS. ACKNOWLEDGMENTS The authors thank Dr. Yi Zhu for technical assistance in animal experiments and Lai-Wen Fu for assistance with microscopy. GRANTS This work was funded by Major State Basic Research Development Program of China Grants 2006CB504106, 2007CB512100, and 2012CB518203. DISCLOSURES No conflicts of interest, financial or otherwise, are declared by the author(s). REFERENCES 1. Asemu G, Papousek F, Ostadal B, Kolar F. Adaptation to high altitude hypoxia protects the rat heart against ischemia-induced arrhythmias. Involvement of mitochondrial KATP channel. J Mol Cell Cardiol 31: 1821–1831, 1999. 2. Becker LB. New concepts in reactive oxygen species and cardiovascular reperfusion physiology. Cardiovasc Res 61: 461–470, 2004. 3. Chen L, Lu XY, Li J, Fu JD, Zhou ZN, Yang HT. Intermittent hypoxia protects cardiomyocytes against ischemia-reperfusion injury-induced alterations in Ca2⫹ homeostasis and contraction via the sarcoplasmic reticulum and Na⫹/Ca2⫹ exchange mechanisms. Am J Physiol Cell Physiol 290: C1221–C1229, 2006. 4. Cohen MV, Yang XM, Downey JM. Acidosis, oxygen, and interference with mitochondrial permeability transition pore formation in the early minutes of reperfusion are critical to postconditioning’s success. Basic Res Cardiol 103: 464 –471, 2008. 5. Cordeiro JM, Howlett SE, Ferrier GR. Simulated ischaemia and reperfusion in isolated guinea pig ventricular myocytes. Cardiovasc Res 28: 1794 –1802, 1994. 6. Costa AD, Jakob R, Costa CL, Andrukhiv K, West IC, Garlid KD. The mechanism by which the mitochondrial ATP-sensitive K⫹ channel opening and H2O2 inhibit the mitochondrial permeability transition. J Biol Chem 281: 20801–20808, 2006. 7. del Pilar V, Garcia-Godos F, Woolcott OO, Marticorena JM, Rodriguez V, Gutierrez I, Fernandez-Davila L, Contreras A, Valdivia L, Robles J, Marticorena EA. Improvement of myocardial perfusion in coronary patients after intermittent hypobaric hypoxia. J Nucl Cardiol 13: 69 –74, 2006. 8. Dong JW, Zhu HF, Zhu WZ, Ding HL, Ma TM, Zhou ZN. Intermittent hypoxia attenuates ischemia/reperfusion induced apoptosis in cardiac myocytes via regulating Bcl-2/Bax expression. Cell Res 13: 385–391, 2003. 9. Forkel J, Chen X, Wandinger S, Keser F, Duschin A, Schwanke U, Frede S, Massoudy P, Schulz R, Jakob H, Heusch G. Responses of chronically hypoxic rat hearts to ischemia: KATP channel blockade does not abolish increased RV tolerance to ischemia. Am J Physiol Heart Circ Physiol 286: H545–H551, 2004. 10. Gelpi RJ, Morales C, Cohen MV, Downey JM. Xanthine oxidase contributes to preconditioning’s preservation of left ventricular developed pressure in isolated rat heart: developed pressure may not be an appropriate end-point for studies of preconditioning. Basic Res Cardiol 97: 40 –46, 2002. 11. Gross ER, Hsu AK, Gross GJ. The JAK/STAT pathway is essential for opioid-induced cardioprotection: JAK2 as a mediator of STAT3, Akt, and GSK-3␤. Am J Physiol Heart Circ Physiol 291: H827–H834, 2006. 12. Guo HC, Zhang Z, Zhang LN, Xiong C, Feng C, Liu Q, Liu X, Shi XL, Wang YL. Chronic intermittent hypobaric hypoxia protects the heart against ischemia/reperfusion injury through upregulation of antioxidant enzymes in adult guinea pigs. Acta Pharmacol Sin 30: 947–955, 2009. 13. Hausenloy DJ, Baxter G, Bell R, Botker HE, Davidson SM, Downey J, Heusch G, Kitakaze M, Lecour S, Mentzer R, Mocanu MM, Ovize M, Schulz R, Shannon R, Walker M, Walkinshaw G, Yellon DM. Translating novel strategies for cardioprotection: the Hatter Workshop Recommendations. Basic Res Cardiol 105: 677–686, 2010. AJP-Heart Circ Physiol • VOL

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