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Gene Transfer of Inducible Nitric Oxide Synthase AffordsCardioprotection by Upregulating Heme Oxygenase-1 Via a

Nuclear Factor-�B–Dependent PathwayQianhong Li, MD, PhD; Yiru Guo, MD; Qinghui Ou, MD; Chuanjue Cui, MD; Wen-Jian Wu, MSc;

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.)

Key Words: nitric oxide synthase � gene therapy � myocardial infarction � heme oxygenase-1 � NF-kappaB

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.,

G.D.R., S.W., R.B.); Department of Internal Medicine, Keio University School of Medicine, Tokyo, Japan (K.S.).The online-only Data Supplement is available with this article at http://circ.ahajournals.org/cgi/content/full/CIRCULATIONAHA.108.778688/DC1.Correspondence to Roberto Bolli, MD, Division of Cardiology, University of Louisville, Louisville, KY 40292. E-mail [email protected]© 2009 American Heart Association, Inc.

Circulation is available at http://circ.ahajournals.org DOI: 10.1161/CIRCULATIONAHA.108.778688

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Molecular Cardiology

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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.

Li et al iNOS Regulates HO-1 by Activating NF-�B 1223


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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

Occlusion Reperfusion

Groups Preocclusion 5 min 30 min 5 min 15 min

Temperature (°C)

WT�Av3/LacZ (n�8) 37.0�0.1 37.1�0.1 37.0�0.1 37.0�0.1 37.1�0.1

WT�Av3/iNOS (n�7) 36.8�0.0 37.1�0.1 37.1�0.1 36.8�0.0 36.8�0.1

HO-1�/��Av3/LacZ (n�8) 36.9�0.1 37.1�0.1 37.1�0.1 37.0�0.1 37.0�0.1

HO-1�/��Av3/iNOS (n�9) 36.9�0.0 37.1�0.0 37.0�0.1 37.0�0.1 36.9�0.1

Heart rate, bpm

WT�Av3/LacZ (n�8) 546�21 587�21 575�21 605�20 605�17

WT�Av3/iNOS (n�7) 564�14 581�19 593�8 587�12 574�13

HO-1�/��Av3/LacZ (n�8) 553�26 577�26 576�32 590�26 595�30

HO-1�/��Av3/iNOS (n�9) 585�19 613�14 607�11 597�18 587�27

Data are mean�SEM.

Figure 2. Effects of iNOS gene transfer on myocardial iNOS andHO-1 expression. Data are mean�SEM of experiments per-formed in duplicate.

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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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3. Bolli R. Preconditioning: a paradigm shift in the biology of myocardialischemia. Am J Physiol Heart Circ Physiol. 2007;292:H19–H27.

4. Li Q, Guo Y, Xuan YT, Lowenstein CJ, Stevenson SC, Prabhu SD, WuWJ, Zhu Y, Bolli R. Gene therapy with inducible nitric oxide synthaseprotects against myocardial infarction via a cyclooxygenase-2-dependentmechanism. Circ Res. 2003;92:741–748.

5. Li Q, Guo Y, Tan W, Stein AB, Dawn B, Wu WJ, Zhu X, Lu X, Xu X,Siddiqui T, Tiwari S, Bolli R. Gene therapy with iNOS provideslong-term protection against myocardial infarction without adverse func-tional consequences. Am J Physiol Heart Circ Physiol. 2006;290:H584–H589.

6. Jones SP, Bolli R. The ubiquitous role of nitric oxide in cardioprotection.J Mol Cell Cardiol. 2006;40:16–23.

7. Ryter SW, Alam J, Choi AM. Heme oxygenase-1/carbon monoxide: frombasic science to therapeutic applications. Physiol Rev. 2006;86:583–650.

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10. Immenschuh S, Hinke V, Ohlmann A, Gifhorn-Katz S, Katz N,Jungermann K, Kietzmann T. Transcriptional activation of the haemoxygenase-1 gene by cGMP via a cAMP response element/activatorprotein-1 element in primary cultures of rat hepatocytes. Biochem J.1998;334(part 1):141–146.

11. Guo Y, Stein AB, Wu WJ, Tan W, Zhu X, Li QH, Dawn B, Motterlini R,Bolli R. Administration of a CO-releasing molecule at the time of reper-fusion reduces infarct size in vivo. Am J Physiol Heart Circ Physiol.2004;286:H1649–H1653.

12. Ryter SW, Morse D, Choi AM. Carbon monoxide and bilirubin: potentialtherapies for pulmonary/vascular injury and disease. Am J Respir CellMol Biol. 2007;36:175–182.

13. Nakao A, Choi AM, Murase N. Protective effect of carbon monoxide intransplantation. J Cell Mol Med. 2006;10:650–671.

14. Jadhav A, Torlakovic E, Ndisang JF. Interaction among heme oxygenase,nuclear factor-{kappa}B, and transcription activating factors in cardiachypertrophy in hypertension. Hypertension. 2008;52:910–917.

15. Geraldes P, Yagi K, Ohshiro Y, He Z, Maeno Y, Yamamoto-Hiraoka J,Rask-Madsen C, Chung SW, Perrella MA, King GL. Selective regulationof heme oxygenase-1 expression and function by insulin through IRS1/PI3-kinase/AKT-2 pathway. J Biol Chem. 2008;283:34327–34336.

16. Hill-Kapturczak N, Voakes C, Garcia J, Visner G, Nick HS, Agarwal A.A cis-acting region regulates oxidized lipid-mediated induction of thehuman heme oxygenase-1 gene in endothelial cells. Arterioscler ThrombVasc Biol. 2003;23:1416–1422.

17. Maines MD, Gibbs PE. 30 some years of heme oxygenase: from a“molecular wrecking ball” to a “mesmerizing” trigger of cellular events.Biochem Biophys Res Commun. 2005;338:568–577.

18. Lavrovsky Y, Schwartzman ML, Levere RD, Kappas A, Abraham NG.Identification of binding sites for transcription factors NF-kappa B andAP-2 in the promoter region of the human heme oxygenase 1 gene. ProcNatl Acad Sci U S A. 1994;91:5987–5991.

19. Alam J, Cai J, Smith A. Isolation and characterization of the mouse hemeoxygenase-1 gene: distal 5� sequences are required for induction by hemeor heavy metals. J Biol Chem. 1994;269:1001–1009.

20. Xuan YT, Tang XL, Banerjee S, Takano H, Li RC, Han H, Qiu Y, Li JJ,Bolli R. Nuclear factor-kappaB plays an essential role in the late phase ofischemic preconditioning in conscious rabbits. Circ Res. 1999;84:1095–1109.

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28. Choi AM. Heme oxygenase-1 protects the heart. Circ Res. 2001;89:105–107.

29. Clark JE, Foresti R, Sarathchandra P, Kaur H, Green CJ, Motterlini R.Heme oxygenase-1-derived bilirubin ameliorates postischemic myo-cardial dysfunction. Am J Physiol Heart Circ Physiol. 2000;278:H643–H651.

30. L’Abbate A, Neglia D, Vecoli C, Novelli M, Ottaviano V, Baldi S,Barsacchi R, Paolicchi A, Masiello P, Drummond GS, McClung JA,Abraham NG. Beneficial effect of heme oxygenase-1 expression onmyocardial ischemia-reperfusion involves an increase in adiponectin inmildly diabetic rats. Am J Physiol Heart Circ Physiol. 2007;293:H3532–H3541.

31. Piantadosi CA, Carraway MS, Babiker A, Suliman HB. Heme oxygen-ase-1 regulates cardiac mitochondrial biogenesis via Nrf2-mediated tran-scriptional control of nuclear respiratory factor-1. Circ Res. 2008;103:1232–1240.

32. Bolli R, Li QH, Tang XL, Guo Y, Xuan YT, Rokosh G, Dawn B. The latephase of preconditioning and its natural clinical application— genetherapy. Heart Fail Rev. 2007;12:189–199.

33. Yet SF, Tian R, Layne MD, Wang ZY, Maemura K, Solovyeva M, Ith B,Melo LG, Zhang L, Ingwall JS, Dzau VJ, Lee ME, Perrella MA. Cardiac-specific expression of heme oxygenase-1 protects against ischemia andreperfusion injury in transgenic mice. Circ Res. 2001;89:168–173.

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35. Dawn B, Bolli R. HO-1 induction by HIF-1: a new mechanism fordelayed cardioprotection? Am J Physiol Heart Circ Physiol. 2005;289:H522–H524.

36. Datta PK, Lianos EA. Nitric oxide induces heme oxygenase-1 geneexpression in mesangial cells. Kidney Int. 1999;55:1734–1739.

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38. Kinobe RT, Vlahakis JZ, Vreman HJ, Stevenson DK, Brien JF, Szarek WA,Nakatsu K. Selectivity of imidazole-dioxolane compounds for in vitro inhi-bition of microsomal haem oxygenase isoforms. Br J Pharmacol. 2006;147:307–315.



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39. Wiesel P, Patel AP, Carvajal IM, Wang ZY, Pellacani A, Maemura K,DiFonzo N, Rennke HG, Layne MD, Yet SF, Lee ME, Perrella MA.Exacerbation of chronic renovascular hypertension and acute renalfailure in heme oxygenase-1-deficient mice. Circ Res. 2001;88:1088 –1094.

40. Alam J, Cook JL. How many transcription factors does it take to turn onthe heme oxygenase-1 gene? Am J Respir Cell Mol Biol. 2007;36:166–174.

41. Baeuerle PA, Baltimore D. I kappa B: a specific inhibitor of the NF-kappaB transcription factor. Science. 1988;242:540–546.

42. Hayden MS, Ghosh S. Shared principles in NF-kappaB signaling. Cell.2008;132:344–362.

43. Klement JF, Rice NR, Car BD, Abbondanzo SJ, Powers GD, Bhatt PH,Chen CH, Rosen CA, Stewart CL. IkappaBalpha deficiency results in asustained NF-kappaB response and severe widespread dermatitis in mice.Mol Cell Biol. 1996;16:2341–2349.

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.



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Rokosh, Shuyan Wang and Roberto BolliLilibeth B. Lanceta, Santosh K. Sanganalmath, Buddhadeb Dawn, Ken Shinmura, Gregg D. Qianhong Li, Yiru Guo, Qinghui Ou, Chuanjue Cui, Wen-Jian Wu, Wei Tan, Xiaoping Zhu,

B-Dependent PathwayκUpregulating Heme Oxygenase-1 Via a Nuclear Factor-Gene Transfer of Inducible Nitric Oxide Synthase Affords Cardioprotection by

Print ISSN: 0009-7322. Online ISSN: 1524-4539 Copyright © 2009 American Heart Association, Inc. All rights reserved.

is published by the American Heart Association, 7272 Greenville Avenue, Dallas, TX 75231Circulation doi: 10.1161/CIRCULATIONAHA.108.778688

2009;120:1222-1230; originally published online September 14, 2009;Circulation.

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CIRCULATIONAHA/2008/778688/R2-Suppl

1

SUPPLEMENTAL MATERIAL

SUPPLEMENTAL METHODS

Adenoviral vectors. Recombinant adenoviral vectors deleted in the E1, E2a, and E3 regions

and carrying either a nuclear targeted β-galactosidase reporter gene (Av3/LacZ) or the human

iNOS gene (Av3/iNOS) were constructed essentially as previously described by homologous

recombination between pAvS6/LacZ or pAvS6/iNOS and the large Cla I fragment constituting

the right side of a novel Ad5 mutant that contains deletions in the E2a and E3 regions 1-3.

Plaque-isolated viral clones were propagated at a high titer in an A549-derived cell line, AE1-2a,

which contains the Ad5 E1 and E2a region genes, and then purified over two CsCl gradients

and titered by plaque assay 1, 2.

In vivo gene transfer. Mice (12-14 weeks old; body weight 26.4 ±1.1 g) were anesthetized

with sodium pentobarbital (50 mg/kg i.p.). After opening the chest through a midline

sternotomy, mice received an intramyocardial injection in the anterior left ventricular (LV) wall of

Av3/LacZ (1x107pfu [Av3/LacZ group]) or Av3/iNOS (1x107pfu [Av3/iNOS group]). Two or three

days later, mice underwent cardiac tissue collection or the infarction protocol described below

(Fig. 1). The intramyocardial injection was 10 μl in volume and was performed with a 50-μl

syringe using a 30-gauge needle; each mouse received one injection in the soon-to-be-ischemic

region of the LV 1, 2.

Coronary occlusion/reperfusion protocol. The murine model of myocardial ischemia and

reperfusion has been described in detail 4. Briefly, mice were anesthetized with sodium

pentobarbital (50 mg/kg i.p.) and ventilated using carefully selected parameters. After


2

administration of antibiotics, the chest was opened through a midline sternotomy, and a

nontraumatic balloon occluder was implanted around the mid-left anterior descending coronary

artery by using an 8-0 nylon suture. To prevent hypotension, blood from a donor mouse was

given at serial times during surgery 2, 4. Rectal temperature was carefully monitored and

maintained between 36.7oC and 37.3oC throughout the experiment. In all groups, myocardial

infarction was produced by a 30-min coronary occlusion followed by 4 h of reperfusion (Fig. 1).

The LacZ and iNOS groups received intramyocardial injections of Av3/LacZ or Av3/iNOS,

respectively, as described above, three days before the 30-min occlusion. Successful

performance of coronary occlusion and reperfusion was verified by visual inspection (i.e., by

noting the development of a pale color in the distal myocardium after inflation of the balloon and

the return of a bright red color due to hyperemia after deflation) and by observing S-T segment

elevation and widening of the QRS on the ECG during ischemia and their resolution after

reperfusion. After the coronary occlusion/reperfusion protocol was completed, the chest was

closed in layers, and a small catheter was left in the thorax for 10–20 min to evacuate air and

fluids. The mice were removed from the ventilator, kept warm with heat lamps, given fluids

(1.0–1.5 ml of 5% dextrose in water i.p.), and allowed 100% oxygen via nasal cone.

Postmortem tissue analysis. At the conclusion of the study, the occluded/reperfused vascular

bed and the infarct were identified by postmortem perfusion of the heart with phthalo blue dye

and triphenyltetrazolium chloride (TTC) 4, 5. The corresponding areas were measured by

computerized videoplanimetry (Adobe Photoshop version 7.0) and from these measurements

infarct size was calculated as a percentage of the region at risk 4, 5.

Western immunoblotting analysis. Protein samples were isolated from heart tissues as

previously described 2. Nuclear protein fractions were prepared from freshly isolated mouse


3

hearts using the Nuclear Extract Kit (Active Motif, Carlsbad, CA) according to the

manufacturer’s instructions 5, 6. The protein content in the cytosolic, membranous, and nuclear

fractions was determined by the Bradford technique (Bio-Rad). The expression of iNOS, HO-1,

HO-2, NF-κB subunits p50 and p65 was assessed by standard SDS/PAGE Western

immunoblotting techniques 2, 7. Briefly, 80 μg of protein was separated on an SDS-

polyacrylamide gel and transferred to a nitrocellulose membrane. Gel transfer efficiency was

recorded carefully by making photocopies of membranes dyed with reversible Ponceau staining;

gel retention was determined by Coomassie blue staining 1, 2, 7. Proteins were probed with

specific anti-iNOS (BD Transduction Laboratories, Lexington, KY), anti-HO-1 and anti-HO-2

(Stressgen, Ann Arbor, MI), anti-NF-κB subunits p50 and p65 (Santa Cruz Biotechnology, Santa

Cruz, CA) antibodies. Immunoreactive bands were visualized with horseradish peroxidase–

conjugated anti-rabbit IgG using an enhanced chemiluminescence detection kit (NEN),

quantified by densitometry, and normalized to the Ponceau stain density. In all samples, the

content of the protein of interest was expressed as a percentage of the corresponding mean

values in the Av3/LacZ group.

Immunohistochemical analysis. Immunohistochemical analysis for HO-1 and iNOS protein

expression 3 days after gene transfer was performed by using the ABC kit (Vector

Laboratories). Frozen 10-µm sections were fixed in 10% formalin for 2 min at room temperature,

washed in PBS, and blocked with Mouse-On-Mouse (MOM) IgG blocking reagent. Antigenic

epitopes were equilibrated in MOM diluent and then incubated at 37 °C with a monoclonal anti-

HO-1 antibody (Stressgen) (Fig 3. A to C) or with a monoclonal anti-iNOS antibody (BD

Transduction Laboratories) (Fig 3. D to F). Alternate sections were incubated in the absence of

the primary antibodies (negative control) as previously reported 2.


4

Bilirubin assay. Total bilirubin levels in myocardium were determined by using a bilirubin

assay kit (Wako Chemicals USA, Inc., Richmond, VA) 8. The sample absorbance at 450nm was

normalized to a concomitantly performed standard curve constructed from commercially

available bilirubin standard solution (Wako Chemicals USA, Inc., Richmond, VA) to determine

total bilirubin levels.

Reverse-Transcription PCR study. Total RNA was isolated from the LV using the TRI reagent

(Sigma). After total RNA extraction, all samples were tested for RNA integrity by electrophoresis

5. For reverse-transcription PCR detection of HO-1 and GAPDH transcripts, 100 ng of total RNA

was used for first-strand cDNA synthesis and PCR amplification with the One-Step Platinum

Taq reverse-transcription - PCR kit (Invitrogen, San Diego, CA ) according to the manufacturer’s

instructions 9. A 360-bp fragment for HO-1 or a 494-bp fragment for GAPDH was amplified for

20 or 28 cycles, respectively, with the following mouse HO-1 specific primers: forward, 5’-

CAGGTCCTGAAGAAGATTGC-3’, reverse, 5’-GGATGAGCTAGTGCTGATCT-3’ (Genbank

Accession No. NM010442), or with the following mouse GAPDH specific primers: forward, 5'-

GGCGCCTGGTCACCAGGGCTG-3', reverse, 5'-ATGGACTGTGGTCATGAGCCC-3' (Genbank

Accession No. NG005915). PCR products were then visualized on a 2% agarose gel and

quantitated by ImageJ 1.41o software. Each sample was assayed in duplicate.

ChIP analysis of cardiac tissue. ChIP analysis was performed with a magnetic bead-based

ChIP kit (Active Motif, Carlsbad, CA) according to the manufacturer’s instructions 10. Briefly, the

fresh heart was perfused with 1% formaldehyde for 10 min and then with glycine stop-fix

solution for 5 min for in situ cross-linking of transcription factor proteins with genomic DNA. The

myocardial tissue in the gene-transduced area of LV was isolated, minced in the PMSF-PBS,


5

and then centrifuged. The resulting pellet was used to isolate chromatin in ice-cold lysis buffer

provided in the kit by a dounce homogenizer. After centrifuge, the resulting pellet (chromatin)

was sheared with an enzymatic shearing mix at 37°C, immunoprecipitation was performed

overnight at 4°C with 2 µg of specific anti-NF-κB p50 or p65 antibodies (Santa Cruz

Biotechnology); a negative control was performed with an anti-mouse IgG antibody (Santa Cruz

Biotechnology). The immunoprecipitates were recovered with magnetic protein G-coated beads.

The transcription factor-DNA cross-links in the immunoprecipitates were then reversed, the

proteins were removed by treatment with Proteinase K, and the ChIP DNA samples were

recovered. The same amount of the sheared chromatin from each sample was used to purify

the Input DNA as controls in PCR analysis. To identify ChIP DNA bound to NF-κB, ChIP DNA

fragments associated with NF-κB p50 or p65 subunits in the range from 200 to 1500 bp were

analyzed by PCR using a pair of the specific primers (forward: 5'-

CCAAGCAGAACTGTGAACAT-3'; reverse: 5'-TGACAGGAAGACGGGAGTAG-3') that spanned

the putative NF-κB p50/p65 response element in the HO-1 gene promoter from -468 bp to -459

bp. A 2-µl aliquot of a 20-µl ChIP DNA extraction sample was amplified for 33 cycles of 94°C for

45 s, 55°C for 30 s, and 72°C for 1 min 30 s. PCR products were analyzed on a 2% agarose

gel, quantitated by ImageJ 1.41o software, and normalized to the Input DNA band density. Each

sample was assayed in duplicate.


6

HO

PR

OTE

IN (

% o

f N

Tg)

0

50

100

150

200

IκBαS32A,S36ATgNTg

HO-1HO-2

NTg IκBαS32A,S36ATg IκBαS32A,S36ATgNTg

HO-2HO-1

n=3 / group

Supplemental Figure 1


7


8

LEGENDS

Online Figure 1. Basal levels of cardiac HO-1 protein expression in mice without gene transfer.

NTg, nontransgenic mice; IκBα S32A,S36A Tg, IκBα S32A,S36A transgenic mice. Data are means +

SEM of experiments performed in duplicate.

Online Figure 2. Ponceau staining (PS) records corresponding to each Western blot in the

Results. The PS was used to correct for possible errors introduced during the gel loading and

gel transfer processes. The Ponceau stain density of total proteins in each lane on the

nitrocellulose membrane after gel transfer in Western blot (WB) was used to normalize the

protein measurements of iNOS, HO-1, HO-2, NF-κB subunits p50 and p65, respectively. A: PS

for iNOS (130 KDa) WB in Fig. 2; B: PS for HO-1 (32 KDa) WB in Fig. 2; C: PS for NF-κB

subunits p50 (50 KDa) and p65 (65 KDa) in Fig. 6. NF-κB subunit p50 or p65 protein was

assessed on the same WB membrane by reprobing with the specific anti-NF-κB subunit p50 or

p65 antibody, respectively. D: PS for HO WB in Fig. 7 (HO-1: 32 KDa, HO-2; 34 KDa); E: PS for

HO WB in Online Fig. 1.


9

REFERENCES

1. Li Q, Guo Y, Tan W, Stein AB, Dawn B, Wu WJ, Zhu X, Lu X, Xu X, Siddiqui T, Tiwari S,

Bolli R. Gene therapy with iNOS provides long-term protection against myocardial

infarction without adverse functional consequences. Am J Physiol Heart Circ Physiol.

2006;290:H584-589.

2. Li Q, Guo Y, Xuan YT, Lowenstein CJ, Stevenson SC, Prabhu SD, Wu WJ, Zhu Y, Bolli

R. Gene therapy with inducible nitric oxide synthase protects against myocardial

infarction via a cyclooxygenase-2-dependent mechanism. Circ Res. 2003;92:741-748.

3. Gorziglia MI, Kadan MJ, Yei S, Lim J, Lee GM, Luthra R, Trapnell BC. Elimination of

both E1 and E2 from adenovirus vectors further improves prospects for in vivo human

gene therapy. J Virol. 1996;70:4173-4178.

4. Guo Y, Wu WJ, Qiu Y, Tang XL, Yang Z, Bolli R. Demonstration of an early and a late

phase of ischemic preconditioning in mice. Am J Physiol. 1998;275:H1375-1387.

5. Li Q, Guo Y, Tan W, Ou Q, Wu WJ, Sturza D, Dawn B, Hunt G, Cui C, Bolli R.

Cardioprotection afforded by inducible nitric oxide synthase gene therapy is mediated by

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