Published online before print October 20, 2008, doi: 10.1161/CIRCULATIONAHA.108.791533
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(Circulation. 2008;118:1970-1978.)
© 2008 American Heart Association, Inc.
Molecular Cardiology |
Cardiac Myocyte–Specific Expression of Inducible Nitric Oxide Synthase Protects Against Ischemia/Reperfusion Injury by Preventing Mitochondrial Permeability Transition
From the Institute of Molecular Cardiology (M.B.W., G.R., Y.-T.X., B.G.H., R.J.K., Y.G., D.J.C., S.D.P., R.B., A.B.), Department of Biochemistry and Molecular Biology (M.B.W., B.G.H.), VA Medical Center (S.D.P.), and Department of Anesthesiology and Perioperative Medicine (D.O.), University of Louisville, Louisville, Ky; Department of Cardiovascular Physiology (J.S.) and Department of Anesthesiology (D.O.), University of Düsseldorf, Düsseldorf, Germany; and Center for Biomedical EPR Spectroscopy and Imaging, Davis Heart and Lung Research Institute, and Division of Cardiovascular Medicine, Department of Internal Medicine, Ohio State University College of Medicine, Columbus (M.V., J.L.Z.).
Correspondence to Aruni Bhatnagar, PhD, Division of Cardiology, Department of Medicine, Delia Baxter Bldg, 580 S Preston St, Room 421F, University of Louisville, Louisville, KY 40202. E-mail aruni{at}louisville.edu
Received October 10, 2007; accepted July 7, 2008.
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Abstract |
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Background— Inducible nitric oxide synthase (iNOS) is an obligatory mediator of the late phase of ischemic preconditioning, but the mechanisms of its cardioprotective actions are unknown. In addition, it remains unclear whether sustained elevation of iNOS in myocytes provides chronic protection against ischemia/reperfusion injury.
Methods and Results— Constitutive overexpression of iNOS in transgenic mice (
-myosin heavy chain promoter) did not induce contractile dysfunction and did not affect mitochondrial respiration or biogenesis, but it profoundly decreased infarct size in mice subjected to 30 minutes of coronary occlusion and 24 hours of reperfusion. In comparison with wild-type hearts, isolated iNOS-transgenic hearts subjected to ischemia for 30 minutes followed by 40 minutes of reperfusion displayed better contractile recovery, smaller infarct size, and less mitochondrial entrapment of 2-deoxy-[3H]-glucose. Reperfusion-induced loss of NAD+ and mitochondrial release of cytochrome c were attenuated in iNOS-transgenic hearts, indicating reduced mitochondrial permeability transition. The NO donor NOC-22 prevented permeability transition in isolated mitochondria, and mitochondrial permeability transition–induced NAD+ loss was decreased in wild-type but not iNOS-null mice treated with the NO donor diethylene triamine/NO 24 hours before ischemia and reperfusion ex vivo. iNOS-mediated cardioprotection was not abolished by atractyloside. Reperfusion-induced production of oxygen-derived free radicals (measured by electron paramagnetic resonance spectroscopy) was attenuated in iNOS-transgenic hearts and was increased in wild-type hearts treated with the mitochondrial permeability transition inhibitor cyclosporin A.
Conclusions— Cardiomyocyte-restricted expression of iNOS provides sustained cardioprotection. This cardioprotection is associated with a decrease in reperfusion-induced oxygen radicals and inhibition of mitochondrial swelling and permeability transition.
Key Words: infarction • ischemia • nitric oxide • nitric oxide synthase
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Introduction |
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Myocardial ischemic injury is attenuated in hearts subjected to brief bouts of ischemia before the onset of sustained ischemia (ischemic preconditioning). Both early and delayed phases of ischemic preconditioning have been described.1–3 The early phase occurs immediately after the preconditioning stimulus but disappears within 1 to 2 hours. The protective effects of early preconditioning have been attributed to posttranslational modification of proteins, particularly kinase-mediated protein phosphorylation events.3 Late preconditioning becomes manifest 12 to 24 hours after ischemia, lasts 3 to 4 days, and is associated with increased synthesis of cardioprotective proteins.1 Although the signaling pathways involved in triggering cardioprotection have been extensively characterized,1–3 the metabolic basis for the antiischemic phenotype of the preconditioned heart remains obscure.
Editorial p 1915
Clinical Perspective p 1978
Mechanistic studies of late preconditioning have demonstrated that nitric oxide (NO) plays a central role in mediating cardioprotection. According to the NO hypothesis of late preconditioning, increased generation of NO from endothelial NO synthase (NOS) on day 1 triggers multiple signaling pathways.1,4 These lead to the upregulation of a number of proteins, including inducible NOS (iNOS), which in turn mediate the cardioprotective effects of late preconditioning 24 hours later (day 2).1,4 The postulated dual role of NO—as both a trigger of late preconditioning on day 1 and a mediator on day 2—is based on the observations that pretreatment with NO donors protects the heart from ischemia (24 hours later). NOS inhibitors given on day 1 abolish the development of delayed cardioprotection, and iNOS inhibitors given on day 2 abrogate the infarct-sparing effect of late preconditioning.1,4 The obligatory role of iNOS also is supported by the observation that targeted deletion of the iNOS gene abrogates late preconditioning induced by a variety of stimuli, including ischemia, adenosine A1 agonists, opioid 
1 agonists, endotoxin derivatives, and exercise, suggesting that iNOS is the final common effector of cardioprotection.1,4,5 Collectively, these data support a key role of iNOS-derived NO in mitigating ischemic injury. Nevertheless, it is unclear whether this protection is mediated by a myocyte-specific increase in iNOS and whether continuous expression of iNOS can confer chronic protection against ischemia/reperfusion (I/R) injury. These issues are important considerations in developing therapeutically viable antiischemic strategies and in understanding the mechanisms underlying the cardioprotective effects of iNOS.
Several mechanisms could account for the antiischemic actions of NO. These include regulation of mitochondrial respiration,6 antioxidant protection,7 activation of the mitochondrial KATP channels,8 and inhibition of cell death pathways.9,10 Because of the central role of mitochondria in governing cell death/survival decisions,11,12 it seems likely that the mechanism of NO protection may be related to mitochondrial injury. Indeed, mitochondrial swelling is the first sign of irreversible ischemic damage,13 and multiple cardioprotective signaling pathways converge on the mitochondria.14 Induction of mitochondrial permeability transition (MPT), in particular, has been suggested as the defining event in myocardial reperfusion injury15–17 and activation of cell death pathways.11,12 Therefore, we hypothesized that the beneficial actions of iNOS stem from mitochondrial protection. To test this hypothesis, we investigated whether chronic cardiomyocyte-specific expression of iNOS affects mitochondrial permeability and the generation of oxygen-derived free radicals in hearts subjected to I/R. The results demonstrate, for the first time, that cardiomyocyte-restricted expression of iNOS is sufficient to confer chronic cardioprotection and that transgenic upregulation of cardiac iNOS decreases free radical generation, which in turn prevents MPT and swelling and chronically protects the heart against I/R injury. Preliminary findings of this study have been reported.18
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Methods |
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Detailed methodology is provided in the online Data Supplement. Adult C57BL/6 mice were purchased from The Jackson Laboratory (Bar Harbor, Me). The iNOS-transgenic mice express the iNOS gene specifically in cardiomyocytes under the control of the
-myosin heavy chain promoter.19 These mice are healthy, and their cardiovascular function is normal.19 Baseline 2-dimensional echocardiography (Toshiba T380 Powervision) was performed as previously described.20
Ischemic Preconditioning and Acute Myocardial Infarction In Vivo
The murine model of ischemic preconditioning and infarction has been described previously.21 Briefly, mice were preconditioned with a sequence of six 4-minute occlusion/4-minute reperfusion cycles. Control mice were subjected to sham operation. Myocardial infarction was produced by subjecting mice to a 30-minute coronary occlusion followed by 24 hours of reperfusion.
MPT Measurement
Induction of MPT was assessed by the mitochondrial uptake of 2-deoxy-[3H]-glucose (DOG),16,22 NAD+ measurement,15 and appearance of cytochrome c in the cytosol.12 Hearts were excised and perfused in the Langendorff mode at constant flow (3 mL/min) to ensure consistent perfusion. After 20 minutes of equilibration, the hearts were perfused in the recirculating mode with 50 mL modified Krebs-Henseleit buffer containing 0.5 mmol/L 2-[3H]-DOG (0.1 µCi/mL) for 20 minutes. Perfusion was then returned to normal for 10 minutes, and the hearts were subjected to 30 minutes of ischemia. After 15 minutes of reperfusion, mitochondria were isolated, and the radioactivity associated with the mitochondrial fraction was measured by scintillation counting. To ensure equal yield of mitochondria, citrate synthase activity was measured by colorimetric assay (Sigma-Aldrich, St Louis, Mo). Retention of 3H-DOG was calculated by dividing the radioactivity in the mitochondria by the total radioactivity recovered from the tissue.
Electron Paramagnetic Resonance Spin Trapping Measurement of Oxygen Radicals
After intraperitoneal induction of anesthesia (pentobarbital sodium, Abbott Laboratories, Abbott Park, Ill; 5 mg), the heart was excised, and the ascending aorta was cannulated and perfused at a constant flow (2 mL/min) with Krebs-Henseleit buffer. After ischemia, the spin trap, 5,5-dimethyl-1-pyrroline-N-oxide (DMPO; 1 mol/L; Dojindo Laboratories, Kumamoto, Japan) in buffer containing 100 µmol/L diethylenetriaminepentaacetic acid (Sigma Aldrich), was administered immediately on reperfusion with the trap infused at a rate of 100 µL/min through a side arm located close to the heart. During reperfusion, periodic collections of the effluent were made until 5 minutes of reperfusion at indicated intervals. On sample collection, each tube was immediately frozen in liquid nitrogen. Electron paramagnetic resonance spectra were recorded as described previously.23,24
Statistical Analysis
Data are reported as mean±SEM. Comparisons between 2 groups were performed with unpaired Student’s t tests. Comparisons among multiple groups or between 2 groups at multiple time points were performed by either 1-way or 2-way ANOVA, as appropriate, followed by paired or unpaired Student’s t tests with the Bonferroni correction. For comparing wild-type (WT) and iNOS-transgenic mice at multiple time points, a repeated-measures ANOVA was used.
The authors had full access to and take full responsibility for the integrity of the data. All authors have read and agree to the manuscript as written.
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Results |
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Constitutive Expression of iNOS Protects Against I/R Injury
Previous studies have shown that the late phase of ischemic preconditioning is associated with selective upregulation of iNOS and that targeted disruption of iNOS abrogates the infarct-sparing effect of late preconditioning.1,4,5 Nevertheless, it remains unclear whether continuous expression of iNOS in cardiac myocytes can establish a state of chronic cardioprotection and enhance ischemic tolerance. Accordingly, to test the duration and cellular dependence of iNOS-mediated cardioprotection, we used 12- to 20-week-old iNOS-transgenic mice (20 to 30 g) in which the iNOS gene is under the control of the cardiac myocyte–specific
-myosin heavy chain promoter. Despite an increase in cardiac iNOS, these mice are healthy and breed normally.19 Heger et al19 have also reported that cardiomyocyte-specific iNOS expression does not affect the ratio of left ventricular (LV) to body weight or the heart rate, and although cardiac output and mean arterial pressures were mildly depressed, there was no overt hypertrophy or heart failure. In agreement with these observations, we found no significant differences in echocardiographically assessed cardiac function between hearts of iNOS-transgenic and WT mice (the Table). The iNOS-transgenic hearts, however, displayed a slight concentric hypertrophy as evidenced by increased anterior and posterior wall thickness and elevated LV mass with no changes in LV diastolic or systolic dimension (the Table).
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Cardiac homogenates from iNOS-transgenic mice displayed an 
400-fold increase in iNOS protein expression and an 3-fold increase in NOx content compared with WT littermates. Although it has been reported that NO triggers mitochondrial biogenesis,25 we found no difference in the size distribution or the number of mitochondria in electron micrographs from WT and iNOS-transgenic hearts (Figure I of the online Data Supplement). The expression of cytochrome c and cytochrome c oxidase subunit IV also was similar between WT and iNOS-transgenic hearts (supplemental Figure II), and there was no difference in the ratio of the mitochondrial and nuclear DNA content between WT and iNOS-transgenic hearts (supplemental Figure II), indicating that mitochondrial synthesis is not stimulated by a chronic increase in iNOS abundance and activity. Rates of state 3 and 4 respiration and the ADP:O ratios measured in mitochondria isolated from iNOS-transgenic and WT mice were similar (supplemental Figure III), and no difference was observed in basal reactive oxygen species (ROS) production or ROS production after inhibition of complex I and III (supplemental Figure IV). Small increases in the abundance of complex II and IV, but not complex I, III, and V, were observed (supplemental Figure III). The reasons for the increase in complex II and IV are unclear, but because no changes in respiration and ROS generation were observed, it appears unlikely that these changes significantly affect mitochondrial function.
To examine the effects of chronic iNOS overexpression on ischemic tolerance, WT and iNOS-transgenic mice were subjected to 30 minutes of coronary occlusion followed by 24 hours of reperfusion. The iNOS-transgenic mice and their WT littermates were not different with respect to the size of the risk region (35±3 versus 41±2 mg); however, as shown in Figure 1, the infarct was significantly smaller in iNOS-transgenic hearts compared with WT hearts (25±3% versus 58±4% of the region at risk; P<0.05). These observations indicate that cardiomyocyte-specific iNOS expression produces a chronically protected cardiac phenotype.
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iNOS Decreases Ischemic Injury in Perfused Hearts
Ischemia and reperfusion cause extensive mitochondrial damage, and induction of MPT has been suggested to be the critical event in the evolution of I/R injury.15–17 To examine changes in MPT, we used the isolated perfused heart preparation, which, in contrast to in vivo models, allows direct measurements of MPT. Perfused heart preparations from either mouse strain were stable, with <10% loss of LV developed pressure per hour. During 30 minutes of ischemia, the hearts developed ischemic contracture, which was not significantly different between WT and iNOS-transgenic hearts (Figure 2A). After reperfusion, however, LV developed pressure recovered to a much greater extent in iNOS-transgenic than in WT hearts (Figure 2B). At 30 minutes of reperfusion, developed pressure was 75±11% of baseline in iNOS-transgenic hearts versus 17±3% in WT hearts (P<0.001). Similarly, LV dP/dt was strikingly greater in iNOS-transgenic than in WT hearts, averaging 2004±298 and 507±93 mm Hg/s, respectively, at the end of reperfusion (P<0.001; Figure 2C). In WT hearts, LV diastolic pressure rose markedly on reperfusion, averaging 56±8 mm Hg after 40 minutes; in contrast, in iNOS-transgenic hearts, diastolic pressure was much lower (19±8 mm Hg at 30 minutes; P<0.01; Figure 2A).
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) mice. *P<0.05 vs WT (n=7).The greater functional recovery of iNOS-transgenic hearts was associated with a reduction in tissue injury (Figure 3A through 3F). Total lactate dehydrogenase release during reperfusion was greater in WT than in iNOS-transgenic hearts (465±35 versus 296±63 arbitrary units, respectively; P<0.05; Figure 3A and 3C); creatine kinase release also was greater in WT (299±46 arbitrary units) than in iNOS-transgenic (165±27 arbitrary units; P<0.005; Figure 3B and 3D) hearts. The reduction in tissue injury was confirmed by tetrazolium-based measurements of infarct size, which was reduced from 68±9% of the LV in WT to 43±6% in iNOS-transgenic hearts (P<0.05; Figure 3E and 3F). Taken together, these data corroborate the in vivo results by demonstrating that constitutive overexpression of iNOS protects the heart against I/R injury in the same preparation that we used to assess MPT.
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Constitutive Expression of iNOS Prevents MPT
Changes in MPT were determined by measuring the mitochondrial retention of DOG, the loss of NAD+, and the release of cytochrome c into the cytoplasm. Perfusion with aerobic buffer for 60 minutes led to basal retention of radioactivity in the mitochondrial fraction. This was significantly increased (P<0.05) in mitochondria isolated from WT hearts subjected to 30 minutes of ischemia followed by 15 minutes of reperfusion, indicating induction of MPT (Figure 4A). In contrast, mitochondria from iNOS-transgenic hearts subjected to I/R exhibited essentially no increase in radioactivity (Figure 4A). These observations indicate that MPT is inhibited in iNOS-transgenic hearts. In addition to MPT, loss of NAD+ is a measure of MPT.15 In WT hearts subjected to I/R, the NAD+ content was 36% of that in nonischemic hearts. No decrease in NAD+ was observed in iNOS-transgenic hearts (Figure 4B). Additionally, after I/R, the cytoplasmic cytochrome c level was significantly less in iNOS-transgenic than in WT hearts (Figure 4C). Collectively, all 3 indices indicate a decrease in MPT in iNOS-transgenic hearts subjected to I/R.
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To determine whether MPT also is prevented in hearts preconditioned in vivo, we used a pharmacological model of preconditioning.26 For this, WT mice received 4 intravenous injections of 0.1 mg/kg diethylenetriamine (DETA)/NO; 24 hours later, the hearts were excised and subjected to 30 minutes of ischemia followed by 30 minutes of reperfusion. Treatment with DETA/NO attenuated NAD+ depletion compared with vehicle-treated hearts (supplemental Figure V). Compared with vehicle-treated hearts, hearts preconditioned with DETA/NO also showed less oxidative stress as measured by the accumulation of protein adducts of the lipid peroxidation product 4-hydroxy-trans-2-nonenal (supplemental Figure V). DETA/NO pretreatment did not prevent NAD+ depletion in hearts from iNOS-null mice (supplemental Figure V), indicating that inhibition of MPT after pharmacological preconditioning is mediated by iNOS.
NO Prevents Permeability Transition in Isolated Mitochondria
Having observed that increased iNOS expression is associated with a decrease in MPT during I/R, we examined whether NO directly prevents MPT. For this, isolated mitochondria were treated in suspension with Ca2+. As shown in Figure 5, addition of Ca2+ led to an abrupt decrease in light absorbance (as a result of increased light scattering), indicating mitochondrial swelling. No change in absorbance was observed in the absence of Ca2+. The Ca2+-induced decrease in absorbance was inhibited by cyclosporin A, which prevents opening of the permeability pore, and cyanide (NaCN), which inhibits cytochrome c oxidase (Figure 5A and 5B). This decrease in absorbance was accompanied by depolarization (loss of 
). Mitochondrial membrane potential also was abolished by NaCN, and Ca2+ did not cause additional depolarization or loss of absorbance in NaCN-poisoned mitochondria. Taken together, these data demonstrate that the high negative potential generated in well-energized (metabolically active) mitochondria favors Ca2+ uptake and that excessive accumulation of Ca2+ triggers MPT.
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Preincubation with the NO donor spermine NONOate (NOC-22) prevented Ca2+-induced loss of absorbance in mitochondrial suspension (Figure 5). Similar to NaCN, NOC-22 induced depolarization, and the mitochondria depolarized with NOC-22 were insensitive to Ca2+. Using a t1/2 of 230 minutes, we estimate the flux of NO generated from NOC-22 under the experimental conditions to be 10 nmol · min–1 · mg–1 protein, which is comparable to the estimated steady-state concentration of NO in cardiac myocytes (between 0.1 and 0.2 µmol/L6). No differences in Ca2+-induced MPT were observed, however, in mitochondria isolated from WT and iNOS-transgenic hearts (data not shown). These observations suggest that overexpression of iNOS does not alter the intrinsic sensitivity of the mitochondria to undergo permeability transition; however, physiological levels of NO inhibit permeability transition, in part by dissipating the membrane potential required for Ca2+ uptake.
NO Prevents Reperfusion-Induced Oxygen Free Radical Generation
Reperfusion injury has been linked to the generation of oxygen-derived free radicals.2,27 Hence, to further elucidate the cardioprotective mechanisms elicited by NO, we studied free radical generation in WT and iNOS-transgenic hearts. Oxygen radicals generated on reperfusion were trapped with DMPO and quantified by electron paramagnetic resonance spectroscopy. As shown in Figure 6A, reperfusion was associated with a burst of free radical production. The levels of spin-trapped radicals returned to baseline after 120 seconds of reperfusion. The amount of radicals trapped from reperfused hearts was significantly attenuated in iNOS-transgenic hearts compared with WT hearts. In contrast, treatment with the MPT inhibitor cyclosporin A increased radical production (Figure 6B). Although cyclosporin A was found to promote functional recovery of WT hearts subjected to 30 minutes of ischemia and 40 minutes of reperfusion (supplemental Figure VI) and to decrease the release of cytochrome c in the cytosol, it did not affect mitochondrial retention of [3H]-DOG (supplemental Figure VII). Furthermore, iNOS-mediated cardioprotection was not abolished by the MPT opener atractyloside, and iNOS-transgenic hearts treated with atractyloside displayed better recovery of function and lower levels of lactate dehydrogenase and creatine kinase release than did the WT hearts (supplemental Figure VIII). Collectively, these observations indicate that iNOS decreases reperfusion-induced oxygen radical production. NO maintains the mitochondrial pore in the closed state, so its protection cannot be abolished by stabilizing the open state of the pore.
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Discussion |
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The major finding of this study is that a cardiomyocyte-specific increase in iNOS leads to sustained cardioprotection without causing overt myocyte injury or dysfunction. Transgenic overexpression of iNOS in cardiac myocytes reduced infarct size and decreased I/R-induced free radical production and MPT, indicating that overexpression of iNOS significantly enhances the ability of the heart to withstand oxidative and mitochondrial stress. Significantly, our observations demonstrate that manipulating a single myocyte gene can establish a chronically cardioprotected phenotype. This mode of cardioprotection differs in its time course from both early (which lasts 1 to 2 hours) and late (48 to 72 hours) preconditioning, both of which offer only transient protection. The permanent cardioprotection described here is due to the activity of iNOS, which is now widely recognized as an obligatory mediator of late preconditioning,1,4,28 and relates to favorable changes in 2 major determinants of I/R injury: free radical generation and MPT. These observations offer new insights into the mechanism of NO-mediated protection and have significant clinical implications for developing long-term prophylactic antiischemic interventions.
The results of this study show that a transgenic increase in iNOS is associated with a decrease in both I/R injury in isolated perfused hearts and infarct size after coronary occlusion in situ. These observations imply that the chronic cardioprotection afforded by iNOS is independent of the experimental model used to assess its efficacy. The concordance between in situ and ex vivo models also indicates that iNOS-mediated protection is not related to systemic changes in blood-borne responses, favorable neutrophil-endothelium interactions, or alterations in autonomic regulation but instead can be attributed to changes in the response of cardiac myocytes to ischemia.
NO has been shown to play an obligatory role not only in the delayed cardioprotection elicited by ischemic preconditioning but also in that provided by physical exercise, NO donors, adenosine A1 receptor agonists, opioid 1 receptor agonists, and endotoxin derivatives.1,4,28 Thus, generation of NO by iNOS appears to be a final common pathway that mediates the late phase of ischemic preconditioning, pharmacological preconditioning, and preconditioning induced by physical stimuli. Indeed, late preconditioning could be viewed as a state of increased NO availability. However, to the best of our knowledge, this is the first report that constitutive overexpression of iNOS in the heart is associated with sustained cardioprotection associated with a decrease in the production of oxygen-derived free radicals and alleviation of detrimental changes in the mitochondria. These observations reveal a new mechanism whereby iNOS imparts protection against I/R injury, ie, protection of the mitochondria (without increasing mitochondrial biogenesis or inducing permanent changes in the mitochondrial structure and function), and advance our understanding of the cardiovascular function of this protein.
NO could protect the heart from ischemic injury by several mechanisms. Previous studies have shown that NO stimulates the opening of the mitochondrial KATP channel8 and inhibits apoptosis by nitrosylating caspases.10 In addition, an increase in iNOS can enhance the expression of antioxidant proteins.4 Our results suggest that the cardioprotection afforded by iNOS is not due to permanent changes in mitochondria because no difference in MPT was observed in mitochondria isolated from WT and iNOS-transgenic hearts. This observation is in agreement with results of other studies showing that mitochondria isolated from naïve or preconditioned hearts show no difference in their ability to undergo MPT.29–31 Thus, inhibition of MPT in iNOS-transgenic hearts, similar to that in hearts preconditioned by ischemia, does not appear to be due to a change in the intrinsic sensitivity of the mitochondria to undergo permeability transition per se. This view is consistent with our previous observation that NO must be generated during I/R; ie, NO is not only a trigger but also a mediator of preconditioning.1,28 On the basis of these considerations, we propose that the cardioprotective networks stimulated by iNOS converge on the mitochondria, strengthening their defense against ischemic changes and preventing them from triggering cell death pathways. The protective effects of iNOS on the mitochondria may be facilitated by its localization. In iNOS-transgenic hearts, significant levels of iNOS were associated with the mitochondria (supplemental Figure IX). Mitochondrial association of iNOS also was observed in WT hearts preconditioned by ischemia (supplemental Figure X). No change in the distribution of endothelial NOS or neuronal NOS was observed. Although further experiments are required to understand the mechanism by which iNOS associates with the mitochondria and to assess the significance of these findings fully, these data suggest that mitochondrial protection by iNOS may be facilitated by the generation of NO near the mitochondria.
Although MPT induction has been proposed as the key event that triggers myocyte cell death during reperfusion,32,33 the mechanisms that trigger MPT during I/R are poorly understood. Increased generation of free radicals could be 1 trigger, which could increase mitochondrial calcium overload and trigger pore opening. Hence, our observation that cardiomyocyte-specific expression of iNOS prevents radical generation supports the notion that the protective effect of iNOS relates primarily to a decrease in free radical production, which in turn could prevent MPT and decrease I/R injury. The relationship between MPT and free radical generation is further clarified by the data obtained with cyclosporin A. Although cyclosporin A prevented cytochrome c release and decreased I/R injury (this study and others15,34–36) in WT mice, it increased free radical generation. This observation is consistent with in vitro data showing that MPT decreases ROS generation by inhibiting complex I activity.37 Hence, we propose that because cyclosporin A prevents MPT, it preserves complex I activity and and maintains the electron transport in a highly charged state, conditions that favor free radical generation. It follows then that the protective effects of iNOS (which are associated with a decrease in free radical generation) cannot be ascribed to cyclosporin A–like inhibition of MPT but could be attributed instead to a decrease in the triggers of MPT, presumably oxygen-derived free radicals. That NO acts upstream of MPT also is supported by our observation, and NO-mediated cardioprotection was not abolished by atractyloside, indicating that stabilization of the open state of the pore does not abrogate NO-mediated cardioprotection. Such a finding implies that NO-dependent cardioprotection relates to suppression of upstream triggers of pore opening rather than to the intrinsic ability of the pore to open. This also is consistent with the observation that mitochondria isolated from WT and iNOS-transgenic hearts were equally susceptible to MPT. It follows from these considerations that clinical interventions to directly inhibit MPT are likely to be less successful than those that increase iNOS in the heart (eg, exercise, pharmacological preconditioning, or iNOS gene therapy) because NO, in addition to inhibiting MPT, prevents free radical generation. Therefore, the dual protection provided by iNOS, which could be chronically induced in the heart, may be a therapeutically accessible pathway for establishing sustained resistance to myocardial I/R injury.
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Acknowledgments |
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Sources of Funding
This work was supported in part by National Institutes of Health grants HL55477 and HL59378 (to Dr Bhatnagar); HL78825, HL55757, HL68088, HL76794, and HL70897 (to Dr Bolli); HL65660 (to Dr Xuan); HL89380 (to Dr Conklin); and HL63744 and HL65608 (to Dr Zweier); a VA merit award (to Dr Prabhu); and American Heart Association predoctoral fellowships (to Drs West and Hill).
Disclosures
None.
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*The first 2 authors contributed equally to this work.
The online Data Supplement is available with this article at http://circ.ahajournals.org/cgi/content/full/CIRCULATIONAHA.108.791533/DC1.
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Circulation 2008 118: 1911-1912.
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