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Gene Transfer of Inducible Nitric Oxide Synthase AffordsCardioprotection by Upregulating Heme Oxygenase-1 Via a
Wei Tan, MD; Xiaoping Zhu, MD; Lilibeth B. Lanceta, BS; Santosh K. Sanganalmath, MD;Buddhadeb Dawn, MD; Ken Shinmura, MD, PhD; Gregg D. Rokosh, PhD;
Shuyan Wang, MD; Roberto Bolli, MD
Background—Although inducible nitric oxide synthase (iNOS) is known to impart powerful protection against myocardialinfarction, the mechanism for this salubrious action remains unclear.
Methods and Results—Adenovirus-mediated iNOS gene transfer in mice resulted 48 to 72 hours later in increasedexpression not only of iNOS protein but also of heme oxygenase (HO)-1 mRNA and protein; HO-2 protein expressiondid not change. iNOS gene transfer markedly reduced infarct size in wild-type mice, but this effect was completelyabrogated in HO-1�/� mice. At 48 hours after iNOS gene transfer, nuclear factor-�B was markedly activated. Intransgenic mice with cardiomyocyte-restricted expression of a dominant negative mutant of I�B� (I�B�S32A,S36A), bothbasal HO-1 levels and upregulation of HO-1 by iNOS gene transfer were suppressed. Chromatin immunoprecipitationanalysis of mouse hearts provided direct evidence that nuclear factor-�B subunits p50 and p65 were recruited to theHO-1 gene promoter (�468 to �459 bp) 48 hours after iNOS gene transfer.
Conclusions—This study demonstrates for the first time the existence of a close functional coupling between cardiac iNOSand cardiac HO-1: iNOS upregulates HO-1 by augmenting nuclear factor-�B binding to the region of the HO-1 genepromoter from �468 to �459 bp, and HO-1 then mediates the cardioprotective effects of iNOS. These results alsoreveal an important role of nuclear factor-�B in both basal and iNOS-induced expression of cardiac HO-1. Collectively,the present findings significantly expand our understanding of the regulation of cardiac HO-1 and of the mechanismwhereby iNOS exerts its cardioprotective actions. (Circulation. 2009;120:1222-1230.)
Extensive evidence indicates that the inducible isoform ofnitric oxide synthase (iNOS) is a major cardioprotective
protein.1,2 A large number of studies have shown that cardiacoverexpression of iNOS confers a protected phenotype andthat iNOS is an obligatory mediator of the infarct-sparingeffects of the late phase of preconditioning induced byischemia and several other stimuli, which indicates thatupregulation of this enzyme is a common pathway wherebythe heart adapts to stress.3 Consistent with these facts, iNOSgene transfer protects the myocardium from ischemia/reperfusion injury.4,5 Because iNOS has traditionally beenviewed as a deleterious enzyme,2 these results may appearpuzzling and raise the question of the mechanism ofiNOS-dependent cardioprotection.6 At present, the molec-ular basis for the salubrious effects of iNOS remainsincompletely understood and represents a major unre-solved issue in ischemic biology.
Clinical Perspective on p 1230
One possible explanation for the unexpected beneficial roleof iNOS in myocardial ischemia/reperfusion injury is that itmight involve the ubiquitously cytoprotective protein hemeoxygenase (HO)-1. HO is the rate-limiting enzyme in hemecatabolism; it catalyzes the breakdown of heme into equimo-lar amounts of carbon monoxide, biliverdin, and free iron.7
Three mammalian HO isoforms have been identified, one ofwhich, HO-1, is a stress-responsive protein induced by aremarkably vast panoply of stimuli.7–10 Of the metabolitesgenerated by HO-1 catalysis, biliverdin (and bilirubin) hasbeen shown to possess antioxidant activity, whereas carbonmonoxide has been found to exert many salutary effects invarious settings, including myocardial ischemia.7,11,12 Al-though induction of HO-1 is known to constitute a commonadaptive response that increases cellular resistance to oxida-
Received December 22, 2008; accepted July 21, 2009.From the Institute of Molecular Cardiology, University of Louisville, Louisville, Ky (Q.L., Y.G., Q.O., C.C., W.J.W., W.T., X.Z., L.B.L., S.K.S., B.D.,
tive injury and other types of injury,7,12,13 the role of HO-1 in thecardioprotection afforded by iNOS gene therapy remains unclear.
HO-1 is regulated primarily at the transcriptional level.7,14,15
HO-1 gene expression is mediated through cis-regulatoryDNA sequences located in the promoter region,16 a processthat frequently involves transcriptional or structural activa-tion of transcription factors and their translocation to thenucleus.17 Among them, the presence of nuclear factor(NF)-�B–binding sequences in the HO-1 gene promoterregion implicates NF-�B in the regulation of the HO-1gene.18,19 Because we have previously found that cardiacNF-�B is activated by NO in vivo,20 it appears plausible thataugmented NO availability may lead to HO-1 gene expres-sion via NF-�B activity.
On the basis of these considerations, we postulated that thecardioprotection afforded by iNOS may be mediated byinduction of HO-1 via increased binding of NF-�B to theHO-1 gene promoter. To test this hypothesis, we combinedmolecular analyses with physiological studies in a well-characterized murine model of infarction.21 We examined 3issues: (1) Whether iNOS gene transfer induces HO-1 expres-sion in the myocardium; (2) if so, whether HO-1 is necessaryfor the protection afforded by iNOS gene transfer; and (3)whether iNOS regulates HO-1 expression by modulating theaccess of NF-�B to the HO-1 gene promoter. We used iNOSgene transfer to study the mechanism of iNOS-dependentprotection because this approach enabled us to achieveselective upregulation of iNOS without the numerous cellularperturbations associated with ischemic preconditioning orother interventions known to induce this protein.1 Further-more, we used genetically engineered mice rather thanpharmacological agents. That is, to conclusively establishwhether HO-1 plays an obligatory role in the cardioprotectionafforded by iNOS gene transfer, we studied mice withtargeted disruption of the HO-1 gene (HO-1�/�).22 To inves-tigate whether the upregulation of HO-1 induced by iNOSgene transfer is mediated by NF-�B activity, we used I�B�mutant transgenic mice with cardiac-specific abrogation ofNF-�B activation.23 Finally, to specifically identify NF-�Bbinding to the region of the HO-1 gene promoter, wedeveloped, for the first time, a technique that enabled us toperform chromatin immunoprecipitation (ChIP) analysis di-rectly on the mouse heart.
MethodsThis study was performed in accordance with the Guide for the Careand Use of Laboratory Animals (DHHS publication No. 85-23,revised 1996) and the guidelines of the Animal Care and UseCommittee of the University of Louisville, School of Medicine(Louisville, Ky). Detailed methods are available in the online-onlyData Supplement.
Genetically Engineered MiceThe HO-1�/� mice used in the present study were generated by Yetet al22; colonies were maintained by breeding HO-1�/� males withHO-1�/� females. Offspring were genotyped at the time of weaningby polymerase chain reaction to amplify the wild-type (WT) andmutant alleles of genomic DNA from tail DNA samples. WTlittermates were used as controls. Transgenic mice that express aphosphorylation-resistant mutant of I�B� (I�B�S32A,S36A) under the
control of a cardiac-specific promoter have been described previous-ly;23 in these mice, expression of the dominant negative mutant I�B�results in cardiomyocyte-restricted inhibition of NF-�B activation.23
I�B�S32A,S36A transgenic mice (I�B�S32A,S36ATg) were identified bypolymerase chain reaction–based DNA screening.23 Nontransgeniclittermates were used as controls. For all experiments, mice weremaintained in microisolator cages under specific pathogen-freeconditions in a room with a temperature of 24°C, 55% to 65%relative humidity, and a 12-hour light-dark cycle.
Adenoviral VectorsRecombinant adenoviral vectors (Av3/LacZ and Av3/iNOS) wereconstructed essentially as described previously.4,5
In Vivo Gene TransferAnesthetized mice received an intramyocardial injection in theanterior left ventricular wall of Av3/LacZ or Av3/iNOS. Two or 3days later, mice were euthanized for cardiac tissue collection orunderwent the infarction protocol summarized below (Figure 1).4,5
Assessment of Infarct SizeMyocardial infarction was produced as described previously(Figure 1).4,21,24
Western Immunoblotting Analysis,Immunohistochemistry, and Bilirubin AssayThe methods for these procedures are described in the online-onlyData Supplement.
Reverse-Transcription Polymerase ChainReaction StudyFor first-strand complementary DNA synthesis and polymerasechain reaction amplification with the One-Step Platinum Taq RT-PCR kit (Invitrogen, San Diego, Calif), 0.1 �g of total RNA wasused.25 A 360-bp fragment (for HO-1) or a 494-bp fragment (forGAPDH) was amplified with mouse HO-1– or GAPDH-specificprimers. Each sample was assayed in duplicate.
Figure 1. Experimental protocol. On day 1, mice were subjectedto intramyocardial injections of Av3/LacZ (LacZ group) or Av3/iNOS (iNOS group). On day 2 or 3, mice were euthanized forcardiac tissue collection or underwent a 30-minute coronaryocclusion followed by 4 hours of reperfusion for the infarctstudy. NTg indicates nontransgenic mice; I�B�S32A,S36ATg,I�B�S32A,S36A mutant transgenic mice.
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ChIP Analysis of Cardiac TissueChIP analysis was performed by use of a magnetic bead–based ChIPkit (Active Motif, Carlsbad, Calif) according to the manufacturer’sinstructions.26 Each sample was assayed in duplicate.
Statistical AnalysisData are reported as mean�SEM. Protein band density was normal-ized to the corresponding loading control and then to the mean of thecorresponding control group.4,24 All data are analyzed with a 1-wayor 2-way ANOVA, as appropriate, followed by Student t tests.Because of the small sample sizes, data were also analyzed withnonparametric tests (Kruskal-Wallace test and Mann–Whitney test);because the results were similar to those obtained with 1-way or2-way ANOVA and Student t tests, for the sake of simplicity andclarity, the latter (parametric) results are reported herein. A P value�0.05 was considered statistically significant. All statistical analyseswere performed with the SigmaStat software system (3.5V).
The authors had full access to and take full responsibility for theintegrity of the data. All authors have read and agree to themanuscript as written.
ResultsExclusionsA total of 105 mice were used for the present study: 57 forstudies of infarct size, 32 for studies of protein expression, 8for studies of HO-1 mRNA expression, and 8 for ChIPanalyses of NF-�B. Twenty-two mice died during or shortlyafter the surgical procedure, and 8 were excluded because oftechnical problems. Thus, a total of 75 mice were included inthe final analyses.
Fundamental Physiological ParametersHeart rate and body temperature, fundamental physiologicalparameters that may impact infarct size,21 were similar in all4 groups of mice used for studies of coronary occlusion(Table). By experimental design,21,27 rectal temperature re-mained within a narrow, physiological range (36.8°C to37.1°C) in all groups. Five minutes before the 30-minutecoronary occlusion, the average heart rate in the 4 groupsranged from 546�21 to 585�19 bpm (P�0.05). Heart ratedid not differ significantly among the 4 groups at any timeduring the 30-minute occlusion or the ensuing reperfusion(Table). The size of the region at risk, expressed as a
percentage of left ventricular weight, did not differ among the4 groups: WT�Av3/LacZ, 41�1%; WT�Av3/iNOS,41�2%; HO-1�/��Av3/LacZ, 37�3%; and HO-1�/��Av3/iNOS, 38�2% (P�0.05).
Effects of iNOS Gene Transfer on HO-1 mRNA,HO-1 Protein, and BilirubinThree days after iNOS gene transfer, immunoblotting re-vealed a marked increase in myocardial iNOS protein expres-sion (�186% versus the LacZ group, n�6, P�0.009; Figure2). At the same time, the iNOS-transduced myocardial regionalso exhibited a significant increase in HO-1 protein expres-sion (�263% versus the LacZ group, n�6, P�0.041; Figure2). Consistent with the immunoblotting data, immunohisto-chemical analysis showed elevated expression of HO-1 (Fig-ure 3C) and iNOS (Figure 3F) in the transduced region of theanterior left ventricular wall 3 days after iNOS gene transfer.Figure 3C and 3F illustrate 2 adjacent sections from the sameheart and show that the spatial distribution of HO-1 immu-noreactivity coincided with that of iNOS immunoreactivity.At higher magnification (�300), intense HO-1 (Figure 3B)
Table. Rectal Temperature and Heart Rate on the Day of the 30-Minute Coronary Occlusion
and iNOS (Figure 3E) immunoreactivity can be appreciatedin cardiac myocytes but not in nonmyocytes. As illustrated inFigure 4, at 48 hours after iNOS gene transfer, there was amarked elevation in cardiac HO-1 mRNA levels (�212%,n�4, versus LacZ group, n�3; P�0.003). The content of
bilirubin, a byproduct of HO-1, was significantly increased inthe transduced myocardium 3 days after iNOS gene transfer(0.35�0.04 ng/�g protein [n�4] versus 0.16�0.01 ng/�gprotein [n�6] in the LacZ group; P�0.001).
Effect of iNOS Gene Transfer on Infarct Size inHO-1�/� MiceIn WT mice, 3 days after Av3 injection, infarct size wasreduced by an average of 50% in the Av3/iNOS-treatedversus the Av3/LacZ-treated group (Figure 5). In contrast,when HO-1�/� mice received Av3/iNOS, infarct size(47.5�3.4% of the risk region, n�9) did not differ signifi-cantly from that observed in HO-1�/� mice that receivedAv3/LacZ (51.9�3.3%, n�8; Figure 5), which demonstratesthat HO-1 plays an obligatory role in the cardioprotectionafforded by iNOS gene transfer. After LacZ gene transfer,infarct size was similar in HO-1�/� and WT mice (Figure 5),which implies that HO-1 does not modulate ischemia/reper-fusion injury under basal conditions.
Figure 3. Distribution of HO-1 and iNOS pro-tein expression 3 days after iNOS gene trans-fer. A–C illustrate HO-1 immunohistochemicalstaining, whereas D–F illustrate iNOS immuno-histochemistry. C and F show 2 adjacent sec-tions from the same heart; note that the spatialdistribution of HO-1 immunoreactivity coincideswith that of iNOS immunoreactivity. (A, B, D,and E: original magnification �300; C and F:original magnification �7.5; n�4/group.)
Figure 4. Representative reverse-transcription polymerase chainreaction analysis of myocardial HO-1 mRNA content 2 daysafter iNOS gene transfer. Cardiac HO-1 mRNA expression levelsincreased 2.1-fold, with no changes in cardiac GAPDH mRNAcontent, in the iNOS group. Data are mean�SEM of experi-ments performed in duplicate.
Figure 5. Effect of ablation of the HO-1 gene on the infarct-sparing effect of iNOS gene therapy. Myocardial infarct size isexpressed as a percentage of the region at risk. Open circlesrepresent individual mice, whereas solid circles representmean�SEM.
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Effect of iNOS Gene Transfer on NF-�BNuclear TranslocationNuclear proteins extracted from the transduced myocardiumwere assayed for the presence of the active p50 or p65 subunitof NF-�B in the nuclear fraction. Quantitative analysis ofWestern immunoblotting demonstrated increased nuclearcontent of NF-�B at 48 hours after injection of Av3/iNOSwith regard to both the p50 subunit (�58.1�1.3%, n�3,versus the LacZ group, n�3; P�0.001; Figure 6) and the p65subunit (�52.1�1.9%, n�3, versus the LacZ group, n�3;P�0.001; Figure 6).
Effect of iNOS Gene Transfer on HO-1 ProteinExpression in I�B�S32A,S36A Mutant Transgenic MiceConsistent with the results reported above, nontransgenicmice that received Av3/iNOS exhibited robust expression ofHO-1 in the transduced myocardium (Figure 7). On average,injection of Av3/iNOS in nontransgenic mice resulted in a2.2-fold increase in HO-1 protein content versus Av3/LacZ-treated nontransgenic mice. In contrast, myocardial HO-2protein expression did not change after iNOS gene transfer(n�3; Figure 7). Interestingly, HO-1 immunoreactivity wasweaker in the transduced myocardium of I�B�S32A,S36A mutanttransgenic mice treated with Av3/LacZ than in nontransgenicmice under the same conditions (�60% versus thenontransgenic�Av3/LacZ group, n�3; P�0.019; Figure 7)and in I�B�S32A,S36A mutant transgenic mice not subjected toiNOS gene transfer versus the corresponding nontransgenicmice (�38%, n�3; online-only Data Supplement Figure I),which suggests that NF-�B modulates cardiac HO-1 proteinexpression under basal conditions. Although in I�B�S32A,S36A
mutant transgenic mice subjected to iNOS gene transfer HO-1was still upregulated relative to I�B�S32A,S36A mutant trans-genic mice treated with Av3/LacZ, levels of HO-1 expressionwere much lower than in nontransgenic mice treated with
Av3/iNOS (�56% versus the nontransgenic�Av3/iNOSgroup, n�3; P�0.036; Figure 7), which indicates that NF-�Bplays an essential role not only in basal cardiac HO-1 proteinexpression but also in iNOS-dependent induction of HO-1.The increase in HO-1 in I�B�S32A,S36AdTg mice treated withAv3/iNOS (Figure 7) likely reflects the multifactorial natureof HO-1 regulation14,18 and the activation by NO of transcrip-tion factors other than NF-�B. In principle, it may also reflectsynthesis of HO-1 in noncardiac myocytes (because themutant I�B� is expressed selectively in cardiac myocytes23).However, as indicated above, immunohistochemical analysisconfirmed that induction of HO-1 by iNOS gene transferoccurred in cardiac myocytes (Figure 3).
Effect of iNOS Gene Transfer on NF-�B Bindingto the HO-1 Gene PromoterTo further investigate the mechanism whereby iNOS modu-lates HO-1, we used a ChIP method that enabled us to directlyassess the effect of iNOS gene transfer on the upstreamregulatory sequences of the HO-1 gene in cardiac tissue. Asshown in Figure 8, ChIP analysis demonstrated that 48 hoursafter iNOS gene transfer, NF-�B was recruited to the HO-1gene promoter (specifically, to the region from �468 to �459bp), as evidenced by the presence of both the p50 subunit(�230% versus the LacZ group, n�3; P�0.001) and the p65subunit (�179% versus the LacZ group, n�3; P�0.001), afinding consistent with the observation that iNOS enhancesHO-1 mRNA levels (Figure 4). These results suggest that anNF-�B binding element located in the �468- to �459-bpregion of the murine HO-1 gene promoter is involved in thetranscriptional activation of the HO-1 gene in response toiNOS gene transfer.
Figure 6. iNOS gene transfer produces translocation of NF-�Bsubunits p50/p65 to the nucleus. Forty-eight hours after iNOSgene transfer, there was significant elevation of p50 and p65 inthe nuclear fraction. Data are mean�SEM of experiments per-formed in duplicate.
Figure 7. Effect of disruption of NF-�B activation (by cardiac-specific expression of a mutant [I�B�S32A,S36A]) on basal levels ofcardiac HO-1 protein expression and upregulation of HO-1 byiNOS gene therapy. NTg indicates nontransgenic mice;I�B�S32A,S36ATg, I�B�S32A,S36A transgenic mice. Data aremean�SEM of experiments performed in duplicate.
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DiscussionAlthough studies of ischemic preconditioning1,27 and iNOSgene transfer4,5,24 have clearly shown that iNOS impartspowerful protection against myocardial infarction, the mech-anism of this salubrious action remains unclear. iNOS isupregulated by numerous and diverse stimuli and thus ap-pears to be a ubiquitous cardioprotective protein.2 Of similarpotential importance is HO-1, another cytoprotective enzymethat has been recognized to play a critical role in response tovarious forms of stress, including myocardial ischemia.28–31
At present, virtually nothing is known about the interactionbetween these 2 major protective systems in the myocardiumand the molecular mechanism whereby iNOS modulatescardiac HO-1. The present study provides considerable newinformation relevant to these issues.
Our salient findings can be summarized as follows: (1)iNOS gene transfer upregulates not only cardiac levels ofiNOS protein but also those of HO-1 mRNA and protein,which indicates that in the heart, HO-1 is coupled to iNOS;(2) targeted disruption of the HO-1 gene completely abro-gates the infarct-sparing effects of iNOS gene transfer, whichdemonstrates that HO-1 is a necessary mediator of iNOS-dependent cardioprotection; (3) iNOS gene transfer promotestranslocation of NF-�B to the nucleus and its binding to aspecific element in the promoter region of the HO-1 gene, asdemonstrated by both Western immunoblotting and ChIPanalysis of cardiac tissue, which suggests that the molecularmechanism whereby iNOS controls HO-1 expression in-volves transcriptional activation of HO-1 via an NF-�B–dependent pathway; and (4) cardiac-specific abrogation ofNF-�B activation via expression of a dominant negativemutant of I�B� (I�B�S32A,S36A) suppresses the HO-1 upregu-lation elicited by iNOS gene transfer and diminishes basallevels of HO-1 expression, which demonstrates that NF-�B isessential for cardiac HO-1 protein expression under basalconditions and for the induction of HO-1 by iNOS.
Previous investigations have shown that iNOS and HO-1exert a multitude of cytoprotective effects.1,2,27–33 To the bestof our knowledge, however, this is the first study to demon-strate the existence of a tight coupling between cardiac iNOSand cardiac HO-1, 2 inducible proteins that play a critical role
in the response of the heart to ischemia and other forms ofstress. This is also the first study to identify NF-�B activationas a key mechanism that mediates iNOS-dependent modula-tion of HO-1 in the heart.
Role of HO-1 in the Cardioprotection Affordedby iNOSMounting evidence indicates that HO-1 plays an importantcytoprotective role.28,33–35 This enzyme has been found tohave beneficial effects in a wide variety of pathologicalconditions, such as inflammation, atherosclerosis, and ische-mia/reperfusion injury.28,33,34 In noncardiac tissues, there isevidence that HO-1 is regulated by NO36,37 among otherfactors, and on this basis, we postulated that iNOS mayactivate HO-1 in the heart. Although iNOS can be induced bymany stimuli, including ischemic preconditioning,2 weelected to use iNOS gene transfer to study iNOS-dependentmodulation of HO-1 because this approach results in selectiveupregulation of iNOS (Figures 2 and 3) and thus in asustained increase in myocardial NO production without theconfounding effects of the multifarious cellular perturbationsand changes in gene expression that are associated withischemia/reperfusion or with pharmacological manipula-tions.1 As a consequence, the effect of NO on cardiac HO-1can be assessed independent of other cellular changes and inthe setting of a relatively steady-state NO generation. Indeed,we4 have previously demonstrated that concomitant with theelevation of iNOS protein expression, nitrate and nitrite levelsare increased significantly 3 days after iNOS gene transfer inthe transduced myocardium but not in the serum, whichindicates that the source of NO is cardiac. Our presentfindings that the increased myocyte expression of iNOS wasassociated with increased myocyte expression of HO-1 pro-tein (Figures 2 and 3) and mRNA (Figure 4) and withincreased content of bilirubin (a byproduct of HO-1), as wellas that iNOS and HO-1 were colocalized in the samemyocardial region that received iNOS gene transfer (Figure3), reveal a heretofore unrecognized coupling between theiNOS and HO-1 systems in the heart, even in the absence ofischemic stress or other pathological conditions.
Figure 8. ChIP analysis of NF-�B bindingto the HO-1 gene promoter 48 hours afteriNOS gene transfer. Data are mean�SEMof experiments performed in duplicate.
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The mere fact that iNOS expression is associated withHO-1 upregulation, however, does not necessarily imply afunctional role of HO-1 in iNOS-dependent effects, becauseHO-1 upregulation may simply be an epiphenomenon.Clearly, elucidation of the role of HO-1 in iNOS-dependentprotection requires inhibition of HO-1 activity. This could beachieved pharmacologically, but the utility of HO-1 inhibitorsis limited by their relative lack of specificity.38,39 Conse-quently, we used a molecular-genetic approach by studyingmice with targeted disruption of the HO-1 gene. The similar-ity in infarct size between WT and HO-1�/� mice afterAv3/LacZ administration (Figure 5) implies that HO-1 doesnot play a significant cardioprotective role under basalconditions, possibly because of its low level of expression innormal myocardium (Figure 7). However, the fact that HO-1gene knockout ablated the infarct-sparing effects of iNOSgene transfer (Figure 5) provides conclusive evidence thatupregulation of HO-1 is necessary for the acquisition ofischemic tolerance afforded by iNOS and that HO-1 is anobligatory mediator of iNOS-dependent protection. Thus, anincrease in iNOS in itself (in the absence of ischemia or otherstimuli) is sufficient to induce myocardial HO-1 expression invivo, which reveals a new aspect of the regulation of cardiacHO-1. On the basis of these observations, we propose that aclose functional coupling exists between cardiac iNOS andcardiac HO-1 and that induction of HO-1 is a criticalmechanism responsible for the cardioprotective effects ofiNOS. Inasmuch as iNOS appears to be a common mediatorof the protection induced by various types of precondition-ing,1 this concept implies that HO-1 plays a major role inthese adaptations as well.
Role of NF-�B in iNOS-Dependent Upregulationof HO-1The mechanism whereby iNOS induces expression of HO-1in the heart is unknown. One of the major transcriptionfactors known to govern HO-1 expression is NF-�B,40 whichhas also been implicated in the cardioprotection afforded byiNOS gene therapy.24 At present, however, nothing is knownabout whether NF-�B is involved in iNOS-dependent mod-ulation of cardiac HO-1.
NF-�B is most commonly a heterodimer of p50 and p65and is maintained in an inactive state in the cytoplasm byI�B�.41 In response to various stresses, phosphorylation ofthe serine residues at positions 32 and 36 results in degrada-tion of I�B�, which allows NF-�B to translocate to thenucleus and activate NF-�B-dependent genes.23,41 To over-come the limitations inherent in pharmacological manipula-tions of NF-�B, we have created a transgenic mouse thatexpresses cardiac-specifically a dominant negative mutantI�B� protein in which both serine residues 32 and 36 arereplaced by alanine (I�B�S32A,S36A).23 These I�B�S32A,S36A mu-tant transgenic mice exhibit normal cardiac development,morphology, and histology.23,24 The effectiveness of NF-�Bsuppression is demonstrated by the fact that tumor necrosisfactor-� and lipopolysaccharide, 2 of the most potent stimuliknown to activate NF-�B,42 fail to increase its nuclear levelsin I�B�S32A,S36A mutant transgenic mice.23,24
To gain insights into the role of NF-�B in iNOS-dependentmodulation of HO-1, we first examined the nuclear content ofNF-�B at 48 hours after iNOS gene transfer. Our finding ofincreased nuclear content of p50 and p65 (Figure 6) isconsistent with our previous observation that iNOS genetransfer results in increased phosphorylation of I�B� at serineresidues 32 and 36 and increased NF-�B DNA binding activityin the nuclear fraction.24 The finding that cardiomyocyte-restricted abrogation of NF-�B activation in I�B�S32A,S36A mutanttransgenic mice given Av3/LacZ significantly reduced basallevels of cardiac HO-1 protein expression compared withnontransgenic mice given Av3/LacZ (Figure 7) implies thatNF-�B modulates cardiac HO-1 protein expression underbasal conditions. The observation that upregulation of HO-1protein expression by iNOS gene transfer in nontransgenicmice was blocked in I�B�S32A,S36ATg mice (Figure 7) demon-strates that NF-�B is obligatorily involved in this process.Thus, taken together, these results reveal that NF-�B plays anessential role not only in the basal cardiac expression of HO-1but also in the upregulation of HO-1 by iNOS. Our previousfinding that the reduction in infarct size afforded by iNOSgene therapy is abolished in I�B�S32A,S36A mutant transgenicmice24 indicates that NF-�B is also essential for iNOS-dependent protection and that abrogation of the expression ofNF-�B–dependent genes (among which is HO-1) renders theheart more susceptible to lethal ischemic/reperfusion injury.On the other hand, the fact that HO-2 protein levels weresimilar in nontransgenic and I�B�S32A,S36A mutant transgenicmice regardless of iNOS gene transfer (Figure 7) indicatesthat the increased iNOS expression does not affect cardiacHO-2 protein and that NF-�B is not involved in the regulationof HO-2.
The classic NF-�B isoform (a p50 and p65 heterodimer) iscapable of binding to the �B DNA binding site, a 10- or 11-bpsequence.43 The present ChIP analysis identified, in the intactmouse, a specific DNA element (GGGTTTGCCC) locatedupstream of the transcription initiation site of the mousecardiac HO-1 gene (from �468 to �459 bp) that binds boththe p50 and p65 subunits (Figure 8), which provides amolecular substrate for the upregulation of the HO-1 gene byiNOS. To the best of our knowledge, this is the first study touse ChIP analysis in the mouse heart, a powerful approach foridentifying transcription factors associated with specific re-gions of the target gene promoter.26
ConclusionsWe have used ChIP analysis of intact cardiac tissue combinedwith a genetically molecular approach in a well-establishedand physiologically relevant murine model of infarction.With this approach, we have demonstrated for the first timethat iNOS modulates the expression of HO-1 in the heart byaugmenting NF-�B nuclear localization and binding to theHO-1 gene promoter, resulting in increased transcription ofthe HO-1 gene. The present data indicate that NF-�B activa-tion is important both for basal levels of cardiac HO-1 proteinexpression and for iNOS-dependent upregulation of HO-1.We have also shown that HO-1 plays an obligatory role in thecardioprotection afforded by iNOS gene therapy. The presentfindings significantly expand our understanding of the regu-
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lation of cardiac HO-1 and of the mechanism whereby iNOSexerts its cardioprotective effects. The data reveal the exis-tence of an iNOS–HO-1 cardioprotective module in whichthese proteins effectively function together to limit myocar-dial ischemia/reperfusion injury.
Sources of FundingThis study was supported in part by National Institutes of Healthgrants R01 HL55757, HL-70897, HL-76794, P01HL78825, and P20RR024489.
DisclosuresNone.
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CLINICAL PERSPECTIVEAlthough nitric oxide donors such as nitroglycerin have been used as antianginal and preload-reducing agents for more thana century, their cardiovascular effects are still not entirely understood. In 1997, we proposed the nitric oxide hypothesisof ischemic preconditioning, which postulates that the cardioprotection afforded by the late phase of preconditioning isunderlain by the upregulation of the inducible isoform of nitric oxide synthase (iNOS) and the attendant increase in theproduction of nitric oxide. This hypothesis subsequently has been validated and extended to other organs, including kidneyand intestine, which implies that upregulation of iNOS is a central axis whereby tissues protect themselves from ischemia.However, the molecular basis responsible for the protective effects of iNOS in the heart remains largely unknown. In thepresent investigation, we have discovered the existence of a close functional coupling between cardiac iNOS and cardiacheme oxygenase-1 (HO-1), another major cytoprotective protein. We found that upregulation of cardiac iNOS via genetransfer leads to increased expression of HO-1 and that this occurs via nuclear translocation of the transcription factornuclear factor-�B and its binding to the promoter of the HO-1 gene. The identification of a coupling between iNOS andHO-1 has significant implications for the fields of gene therapy and ischemia/reperfusion injury, both from a conceptualand a therapeutic standpoint. For example, activation of the iNOS–HO-1 module via pharmacological means or genetherapy may prove beneficial in patients with ischemic heart disease.
1230 Circulation September 29, 2009
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