Free Radical Biology & Medicine 40 (2006) 2223–2231www.elsevier.com/locate/freeradbiomed

Original Contribution

Induction of antioxidant gene expression in a mouse model of ischemiccardiomyopathy is dependent on reactive oxygen species

Saumya Sharma a, Oliver Dewald b,c, Julia Adrogue a, Rebecca L. Salazar a, Peter Razeghi a,James D. Crapo d, Russell P. Bowler d, Mark L. Entman b, Heinrich Taegtmeyer a,⁎

a Cardiology, The University of Texas Houston Medical School, Houston, TX, USAb Cardiovascular Sciences and DeBakey Heart Center, Baylor College of Medicine and The Methodist Hospital, Houston, TX, USA

c Department of Cardiac Surgery, University Clinical Center Bonn, Bonn, Germanyd Department of Medicine, National Jewish Medical and Research Center, Denver, CO, USA

Received 11 August 2005; revised 18 January 2006; accepted 28 February 2006Available online 31 March 2006

Abstract

Ischemia and reperfusion (I/R) are characterized by oxidative stress as well as changes in the antioxidant enzymes of the heart. However, littleis known about the transcriptional regulation of myocardial antioxidant enzymes in repetitive I/R and hibernating myocardium. In a mouse modelof ischemic cardiomyopathy induced by repetitive I/R, we postulated that induction of antioxidant gene expression was dependent on reactiveoxygen species (ROS). Repetitive closed-chest I/R (15 min) was performed daily in C57/BL6 mice and in mice overexpressing extracellularsuperoxide dismutase (EC-SOD). Antioxidant enzyme expression was measured at 3, 5, 7, and 28 days of repetitive I/R as well as 15 and 30 daysafter discontinuation of I/R. In order to determine whether ROS directly modulates antioxidant gene expression, transcript levels were measured incardiomyocytes exposed to hydrogen peroxide. Repetitive I/R caused an early and sustained increase in glutathione peroxidase (GPX) transcriptlevels, while heme oxygenase-1 (HO-1) expression increased only after 7 days of repetitive I/R. Overexpression of EC-SOD prevented theupregulation of GPX and HO-1 transcript levels by repetitive I/R, suggesting that both genes are regulated by ROS. However, while HO-1transcript levels increased in cardiomyocytes exposed to hydrogen peroxide, oxidative stress failed to induce the expression of GPX implying thatROS regulates GPX transcript levels only indirectly in repetitive I/R. In conclusion, repetitive I/R was associated with an early upregulation ofGPX expression as well as a delayed increase of HO-1 transcript levels in the heart. The induction of both antioxidant genes was dependent onROS, suggesting that alterations in redox balance mediate not only tissue injury but also components of “programmed cell survival” in hibernatingmyocardium.© 2006 Elsevier Inc. All rights reserved.

Keywords: Ischemic cardiomyopathy; Animal model; Reperfusion

Introduction

Repeated episodes of ischemia and reperfusion (I/R) canresult in reversible contractile dysfunction and are thought to

Abbreviations: IR, ischemia and reperfusion; ROS, reactive oxygen species;EC-SOD, extracellular superoxide dismutase; GPX, glutathione peroxidase;HO-1, heme oxygenase-1; NCS, neonatal calf serum; FBS, fetal bovine serum;CAT, catalase; PPARα, peroxisome proliferator-activated receptor α.⁎ Corresponding author. Department of Internal Medicine, Division of

Cardiology, University of Texas Houston-Medical School, 6431 Fannin, MSB1.246, Houston, TX 77030, USA. Fax: +1 713 500 0637.

E-mail address: Heinrich.Taegtmeyer@uth.tmc.edu (H. Taegtmeyer).

0891-5849/$ - see front matter © 2006 Elsevier Inc. All rights reserved.doi:10.1016/j.freeradbiomed.2006.02.019

be an important mechanism in the pathogenesis of ischemiccardiomyopathy and myocardial hibernation. I/R involvesmyocardial damage due to ischemia itself and/or oxidativestress secondary to generation of reactive oxygen species(ROS). We have previously characterized a murine model ofischemic cardiomyopathy induced by repetitive I/R [1]. Inthis model, repetitive I/R leads to the development of areversible cardiomyopathy associated with interstitial fibrosisand ventricular dysfunction without myocardial infarction[1]—features resembling human myocardial hibernation.Furthermore, we demonstrated that overexpression ofextracellular superoxide dismutase (EC-SOD) attenuatedinflammation, fibrosis, and improved contractile function,

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indicating that cardiac dysfunction in this model isdependent on ROS [1]. However, ROS not only mediatemyocardial injury but also regulate a number of signalingpathways, suggesting a physiologic role for free radicalgeneration in the heart.

Ischemia and reperfusion are characterized by significantoxidative stress as well as changes in the endogenous myocar-dial antioxidant defense [2,3]. Although there are inconsis-tencies, many studies have shown decreased myocardialantioxidant enzyme levels in response to I/R [3]. Furthermore,overexpression of myocardial antioxidant enzymes in heartsexposed to I/R greatly attenuates cardiac dysfunction,suggesting the importance of this defense mechanism in I/R[1,4–8]. Until now, little is known about the expression ofendogenous myocardial antioxidant enzymes in repetitive I/R.We postulated that induction of endogenous antioxidantenzymes in a mouse model of ischemic cardiomyopathyinduced by repetitive I/R was dependent on ROS. We foundthat repetitive I/R induced an early upregulation of glutathioneperoxidase (GPX) expression as well as a delayed increase ofheme oxygenase-1 (HO-1) transcript levels in the heart. Theinduction of both antioxidant enzymes was dependent onROS.

Methods

Brief repetitive I/R

All animals received humane care in compliance with theGuide for the Care and Use of Laboratory Animals (NIHpublication 85-23, revised 1985). WT C57/BL6 mice wereobtained commercially (Harlan Sprague-Dawley) and EC-SOD mice were developed as previously described [1].Heterozygous EC-SOD (n = 7 at each time point) and WTmice (n = 5 at each time point) underwent initial surgery at 8–10 weeks of age by using a closed-chest model of I/R [1].Briefly, an 8-O Prolene (Ethicon, Sommerville, NJ) suture wasplaced around the left anterior descending artery; both ends ofit threaded through a piece of PE-10 plastic tube (BectonDickinson), exteriorized through the thorax wall, and storedsubcutaneously. After 7–9 days of recovery, the skin wasreopened and the ends of the suture were attached to heavymetal picks. The picks were pulled apart until ST elevationoccurred in electrocardiogram. The 15-min ischemia wasfollowed by reperfusion (confirmed by resolution of STelevation) until the next day. This brief repetitive I/R wasperformed daily for 3, 5, 7, and 28 days in WT and 3, 5, and7 days in the EC-SOD mice. For the recovery studies, miceunderwent a 7-day I/R protocol, and the animals stayed in thevivarium for 15 and 30 days. Sham animals underwent theinitial surgery only and waited for the same period of time asI/R groups.

Neonatal cardiomyocyte isolation

One- to 2-day-old neonatal Sprague-Dawley rats wereused. Each isolation was a single experimental replicate.

Neonatal rats were briefly cleaned with 70% ethanol. Theywere then sacrificed by decapitation with heads droppedimmediately into liquid nitrogen. Hearts were removed andminced in an enzyme solution containing collagenase type II(Worthington, Lakewood, NJ) and pancreatin (Sigma, St.Louis, MO). Minced tissue and solution were placed in atrypsinizing flask and shaken at 37.5°C for 5 min to allowdigestion. The supernatant was collected and discarded. Tenmilliliters of enzyme solution was added again to the flaskand shaken at 37.5°C for 20 min. The supernatant this timewas retained in 50-ml tubes and centrifuged at 660 rpm for5 min. The pellet was resuspended in media containing 5%neonatal calf serum (NCS) and placed in an incubator at37.0°C with 5% CO2. This digestion step was repeated 4more times for 25, 25, 15, and 15 min. Each time thesupernatant was retained, spun down, and resuspended inNCS. The isolates were then spun again at 660 rpm,resuspended in Ham’s F-10 media with 10% fetal bovineserum (FBS), and filtered into a new 50-ml tube. The cellswere then preplated in 60-mm dishes and kept in theincubator for 1 h. Then, using a hemocytometer and trypanblue dye, the cell harvest number was determined and thecells were plated in 60-mm dishes for 24 to 48 h prior toexperiments. After serum starvation for 12 h in F-10 + 0.1%FBS, cardiomyocytes were treated with F-10 media andhydrogen peroxide (0.05 and 0.1 mM) for 4 h. Thistreatment protocol has been shown to induce ROS-dependent gene expression without altering cell viability[9,10].

Carbonylated protein measurement

Tissue homogenate (20 μl) was mixed (10:1) with 10%streptomycin, kept at room temperature for 10 min, and thenmicrocentrifuged at 4000 rpm for 10 min. Forty microliters of10 mM 2,4-dinitrophenylhydrazine in 2 N HCl was added to10 μl of supernatant and then incubated for 1 h at roomtemperature in the dark. Proteins were precipitated by adding50 μl of 20% trichloroacetic acid and centrifuging at 5000 rpmfor 10 min. The pellet was washed 3 times with 20 μl ethylacetate/ethanol (1:1) and dissolved in 15 μl 6 M guanidine, andabsorbance was measured at 280 and 370 nm. Proteinconcentration (mg/ml) was calculating as Abs280 × 1.659–0.0431. Carbonyl content (nmol/ml) was calculating asAbs370 × 45.5.

Gene expression

The method for RNA extraction and for quantitative RT-PCR has been described previously [11]. The nucleotidesequences for primers and probes are listed in Table 1. Thetranscript for the constitutive gene cyclophilin was used ashousekeeping gene for data normalization for mouse studiesand 18S was used for cardiomyocyte studies. Cyclophilin and18S gene expression did not change with I/R. Internal standardswere prepared using the T7 RNA polymerase method (Ambion,Austin).

Table 1

Gene Primer/Probe Sequence

Cu/Zn-SOD Forward 5′-AGCATGGGTTCCACGTCC-3′Reverse 5′-CATGGTTCTTAGAGTGAGGATTAAAAT-3′Probe 5′-FAM-TCCTGCACTGGTACAGCCTTGTGTATTGT-TAMRA-3′

Mn-SOD Forward 5′-CACCATTTTCTGGACAAACCTG-3′Reverse 5′-TTAAACTTCTCAAAAGACCCAAAGTC-3′Probe 5′-FAM-GCGTTGATAGCCTCCAGCAACTCTCCTT-TAMRA-3′

EC-SOD Forward 5′-TCCGGTGTCGACTTAGCAGA-3′Reverse 5-CATCCAGATCTCCAGGTCTTTG-3′Probe 5′-FAM-TGTCGCCTATCTTCTCAACCAGGTCAAGC-TAMRA-3′

GPX Forward 5′-GACTGGTGGTGCTCGGTT-3′Reverse 5′-ACTTGAGGGAATTCAGAATCTCTTC-3′Probe 5′-FAM-AATCAGTTCGGACACCAGGAGAATGGC-TAMRA-3′

Catalase Forward 5′-AGGAGGCAGAAACTTTCCCAT-3′Reverse 5’TTTGCCAACTGGTATAAGAGGGTA-3′Probe 5′-FAM-CCTTGTGAGGCCAAACCTTGGTCAG-TAMRA-3′

HO-1 Forward 5′-GGAGATGACACCTGAGGTCAAG-3′Reverse 5′-GTCTTTGTGTTCCTCTGTCAGCA-3′Probe 5-FAM-CCTGCAGCTCCTCAAACAGCTCAATGTT-TAMRA-3′

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Statistical analysis

Data are expressed as means ± SE. Differences between thegroups were calculated by a Student t test. A value of P < 0.05was considered as significant.

Results

Carbonylated protein

Because ROS are unstable and short lived, direct measure-ment of free radical generation is difficult. However, ROSinduces structural changes in proteins (e.g., protein carbonyl-ation) which can be used as indirect measures of oxidativestress. We found a significant increase in protein carbonylationin mice exposed to 3 and 5 days of brief repetitive I/R,

Fig. 1. Carbonylated protein content increases after 3 and 5 days of repetitiveI/R. However, after 7 day of repetitive I/R carbonylated protein levels returnto baseline. There is no change in carbonylated protein levels 15 days afterdiscontinuation of I/R. (n = 5 in each time point) *p < 0.05 compared tosham.

suggesting oxidative stress-induced injury (Fig. 1). Surpris-ingly, carbonylated protein levels were near normal after7 days of repetitive I/R and 15 days after discontinuation of I/R(Fig. 1).

SOD expression

SOD catalazes superoxide (O2U−) dismutation to hydrogen

peroxide (H2O2). There are three distinct superoxide dismutaseenzymes found in eukaryotes. Copper/zinc SOD (Cu/Zn-SOD)is localized in the cytoplasm, manganese SOD (Mn-SOD)which is found in the mitochondria, and extracellular SOD (EC-SOD) is found in the extracellular matrix and blood [12–14].Overexpression of all three enzymes individually protects theheart from ischemic injury [1,7,15]. Surprisingly, there was nochange in Cu/Zn-SOD, Mn-SOD, or EC-SOD transcript levelsin repetitive I/R (Figs. 2A, B, and C). However, there was amodest increase of Mn-SOD expression at 15 and 30 days afterdiscontinuation of I/R.

GPX expression

GPX not only catalyzes H2O2 (formed after SOD-catalyzeddismutation) into water, but also detoxifies the lipid hydro-peroxidases [2]. Overexpression of GPX prevents left ventric-ular remodeling and heart failure after myocardial infarction inmice [6]. We found an early and sustained increase in GPXtranscript levels starting 3 days of repetitive I/R (Fig. 3A).Furthermore, GPX expression returned to baseline afterdiscontinuation of I/R (Fig. 3A).

Catalase expression

Catalase (CAT) also converts H2O2 into water. AlthoughCAT activity in the myocardium is low, inhibition of catalaseincreases mitochondrial H2O2 production and worsens postis-chemic recovery in the isolated rat heart [2]. We found a

Fig. 2. (A) Cu/Zn-SOD gene expression does not change with repetitive I/R or after discontinuation of I/R. (B) Repetitive I/R did not alter Mn-SOD transcript levels,but discontinuation of I/R is associated with increased expression. (C) EC-SOD expression is not changed with repetitive I/R or discontinuation of I/R. (n = 5 in eachtime point) *p < 0.05 compared to sham.

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decrease in catalase expression during repetitive I/R thatnormalized after discontinuation of I/R (Fig. 3B).

HO-1 expression

HO-1 is a cytoprotective enzyme that degrades heme (apotent oxidant) to generate carbon monoxide (CO) which hasvasodilatory and anti-inflammatory properties, bilirubin

(which has antioxidant properties), and iron [16]. In modelsof I/R and myocardial infarction, HO-1 has been shown to beimportant for reduction of myocardial damage [5,17]. Inrepetitive I/R, HO-1 transcript levels were markedly upregu-lated at 7 days of repetitive I/R and remained elevated at28 days (Fig. 3C). Furthermore, there was completenormalization of HO-1 expression after discontinuation ofI/R (Fig. 3C).

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ROS regulation of GPX and HO-1 expression

GPX and HO-1 gene expression was measured in miceoverexpressing EC-SOD exposed to 3, 5, and 7 days of brief

Fig. 3. (A) GPX transcript levels are increased at 3 days of repetitive I/R and remained(B) Catalase expression decreases with repetitive I/R reaching statistical significanctranscript levels which is sustained to 28 days. Discontinuation of I/R is associatedcompared to sham, # p < 0.05 compared to 7 days I/R.

repetitive closed chest I/R. The protective role of EC-SOD inreducing oxidative stress-related ischemic injury has beenestablished in transgenic and knockout animal models [1,18].Repetitive I/R failed to induce either GPX or HO-1 gene

elevated to 28 days. Discontinuation of I/R restored GPX expression to baseline.e at 28 days. (C) After 7 days of I/R there is a marked upregulation of HO-1with normalization of HO-1 expression. (n = 5 in each time point) *p < 0.05

Fig. 4. (A) Overexpression of EC-SOD prevents the increase in GPX transcriptlevels in repetitive I/R. (B) There is no induction of HO-1 in EC-SOD miceexposed to repetitive I/R. (n = 5 in each time point). Fig. 5. (A) In isolated cardiomyocytes, hydrogen peroxide exposure (0.05 or

0.1 mM) did not induce the expression of GPX. (B) There was a concentration-dependent induction of HO-1 transcript levels in cardiomyocytes exposed tohydrogen peroxide. (n = 5) p < 0.05 compared to control.

Fig. 6. Repetitive I/R increases ROS generation in the myocardium which notonly increases inflammation and apoptosis but also induces the expression ofGPX (indirectly) and HO-1 (directly). We suggest that this upregulation of bothGPX and HO-1 expression in repetitive I/R attempts to restore myocardialantioxidant enzyme levels in order to attenuate ROS-mediated tissue injury andis, therefore, a component of “programmed cell survival”.

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expression in mice overexpressing EC-SOD, suggesting thatROS regulates the expression of both genes (Figs. 4A and B).

In order to determine if ROS directly induces GPX and HO-1expression, we measured transcript levels in cardiomyocytesexposed to hydrogen peroxide. Although GPX expression didnot change in cardiomyocytes exposed to hydrogen peroxide(Fig. 5A), there was a concentration-dependent increase in HO-1 expression (Fig. 5B).

Discussion

We investigated ROS regulation of endogenous antioxidantgene expression in repetitive I/R. The two main findings are: (1)There is early induction of GPX gene expression in repetitive I/R, whereas HO-1 transcript levels increase only after 7 days ofI/R. (2) Both GPX and HO-1 gene expressions are regulated byROS in repetitive I/R.

ROS generation during repetitive I/R

Although free radical generation occurs under normalphysiological conditions, their generation is increased inpathological conditions (e.g., ischemia and reperfusion).Therefore, the increase in carbonylated protein (an indirect

marker of oxidative stress) after 3 and 5 days of repetitive I/Rwas not surprising. However, the reduction in carbonylatedprotein at 7 days was totally unexpected. This observationsuggests that an adaptive myocardial program which reducesoxidative stress-induced myocardial injury is activated at 7 days

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of repetitive I/R. Because repetitive I/R is associated with anincrease in GPX and HO-1 transcript levels, we speculate thatinduction of both of these genes is part of this adaptivemyocardial program (Fig. 6).

Antioxidant transcriptional profile in repetitive I/R

The partial reduction of oxygen produces activated oxygenspecies such as singlet oxygen, superoxide, hydrogen peroxide,and hydroxyl radical which are unstable, extremely reactive,and, therefore, highly toxic to tissues [19]. Oxidative stressplays an important role in the pathogenesis of ischemic heartdisease, and an immense repertoire of antioxidant defensesystems exists to eliminate or reduce oxidative stress [3].Although results are often contradictory, many studies havedemonstrated a depletion of antioxidant enzyme levels in I/R[3]. Furthermore, overexpression or gene transfer of anynumber of antioxidant enzymes attenuates ventricular remodel-ing after myocardial infarction and improves cardiac function inresponse to I/R, indicating the importance of endogenousantioxidants in reducing oxidative stress-induced cardiacdysfunction in I/R [1,4–8]. However, most of these studiesutilized a single ischemia-reperfusion episode or nonreperfusedmyocardial infarction models. In this study, the antioxidantenzyme transcriptional profile of mice exposed to repetitive I/Rwithout myocardial infarction was examined. We foundupregulation of two antioxidant genes (GPX and HO-1) andthe downregulation of CAT.

The first line of defense mechanism against ROS-mediatedmyocardial injury comprises several antioxidant enzymesincluding SOD, CAT, and GPX [2,19]. In response to oxidativestress there is a decline in SOD and glutathione levels [2]. Wefound that among these three endogenous antioxidants onlyGPX transcript levels were increased in repetitive I/R. CATgene expression actually decreased with repetitive I/R. BecauseCATactivity is low in the myocardium [1], we suspect that GPXplays a more important role in repetitive I/R. Furthermore, invitro studies have demonstrated that GPX confers greaterprotection against oxidative damage than either SOD or catalase[20]. The early and sustained induction of GPX expression inrepetitive I/R may be an important mechanism that attempts toreplenish reduced glutathione levels and to decrease freeradical-mediated injury.

We observed a delayed upregulation of HO-1 transcriptlevels in response to repetitive I/R. Overexpression of HO-1 oradenoviral gene therapy with HO-1 attenuates myocardialinjury in a model of myocardial infarction [5,16]. Furthermore,HO-1 has been implicated as part of the delayed protectiveadaptation to I/R stress—termed “late preconditioning” [21].Therefore, we speculate that HO-1 is part of the secondarydefense mechanism that is upregulated in severe oxidativestress. Haramaki et al. demonstrated that antioxidants areutilized in a certain order, therefore supporting the concept thatthey are hierarchically organized in a systematic manner in I/R[22]. According to this concept, glutathione and ascorbateserve as the first line of defense against free radical attack.Only when oxidative stress overwhelms the first line of

antioxidant defense do distal antioxidant enzymes get utilized.Our findings also suggest hierarchy in antioxidant geneexpression in repetitive I/R. Induction of GPX expressionmay be part of an early defense mechanism, whereas HO-1transcript upregulation may be part of a delayed strategy forreducing ROS-mediated injury.

Role of ROS in antioxidant gene expression

The concept of redox balance currently implies that ROS notonly mediate tissue injury but also regulate metabolism, signaltransduction, and, ultimately, tissue function [3]. For example,ROS can activate the transcription factor NF-κB which isknown to be activated in I/R [23,24]. Similarly, activation ofMAP kinases (e.g., p38, JNK) in I/R is influenced by ROS [25].Furthermore, oxidative stress is also considered a signal for theinduction of cardiac hypertrophy [26]. We have shown thatdownregulation of peroxisome proliferator-activated receptor α(PPARα) a transcription factor regulating fatty acid metabolismin the heart, in repetitive I/R is dependent on ROS [27].

Here, we demonstrated that repetitive I/R failed to inducethe upregulation of GPX or HO-1 transcript levels in miceoverexpressing the antioxidant enzyme EC-SOD. Becauseoverexpression of EC-SOD has been shown to reduce oxidativestress-induced ischemic injury in I/R [1,4], our findingsindicate that both GPX and HO-1 expressions are regulatedby ROS. Therefore, ROS not only contributes to inflammationand fibrosis, as we have previously shown [1], but alsoregulates changes in antioxidant defense mechanisms that mayprotect the heart from further oxidative stress (Fig. 5). Thedelayed upregulation of HO-1 transcript levels and the gradualincrease in GPX transcript levels may represent a cumulativeeffect of ROS in repetitive I/R. Nonetheless, ROS-mediatedtranscriptional changes are likely to be complex and requirefurther investigation.

In repetitive I/R, ROS can not only directly regulate geneexpression by activating redox-sensitive transcription factorssuch as NF-κB [24] but can also indirectly modulate transcriptlevels via ROS-mediated inflammation or contractile dysfunc-tion. For example, we have previously shown that ROS-mediated interstial fibrosis is mediated indirectly via thechemokine—monocyte chemotactic protein 1 (MCP-1) [28].Because reduction of oxidative stress by EC-SOD overexpres-sion in repetitive I/R will attenuate both the direct and theindirect effects of ROS, we exposed cardiomyocytes tohydrogen peroxide in order to determine whether GPX andHO-1 expression was directly regulated by ROS. Surprisingly,we found no change in GPX expression in cardiomyocytesexposed to hydrogen peroxide, indicating that ROS does notdirectly modulate GPX transcript levels. This observationsuggests that the induction of GPX transcript levels in repetitiveI/R are indirectly dependent on ROS (e.g., ROS-mediatedinflammation). However, it is also possible that cells in the heartother than cardiomyocytes (e.g., fibroblasts, endothelial cells)are responsible for the induction of GPX gene expression inresponse to oxidative stress. Nonetheless, the mechanism ofROS-mediated GPX expression is likely to be complex and

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requires further investigation. In contrast, HO-1 transcript levelswere induced in cardiomyocytes exposed to hydrogen peroxidein a concentration-dependent manner, indicating a direct effectof ROS on HO-1 gene expression.

We have previously suggested that the heart, when stressed(e.g., ischemia), activates a transcriptional profile that protect itfrom further injury—a phenomenon termed “programmed cellsurvival.” [28]. For example, the heart in response to pressureoverload, hypoxia, or ischemia downregulates PPARα, whichinduces a switch in substrate preference from fatty acids tocarbohydrate utilization, thereby resulting in greater efficiencyin energy conversion [27,29,30]. Furthermore, several othergenes which mediate cytoprotection (e.g., HSP70, HIF-1α, IAP)were also upregulated in experimental and human hibernatingmyocardium [31]. We suggest that the upregulation of GPX andHO-1 expression in response to repetitive I/R is part ofprogrammed cell survival representing an adaptive mechanismin hibernating myocardium which reduces oxidative stress-induced injury and ultimately preserves cardiomyocytes.Because the expression of both genes is dependent on ROS,our findings imply that programmed cell survival may bemediated, at least in part, by ROS, thus reinforcing theimportance of redox balance as a fundamental regulator ofcardiac function and dysfunction.

Limitations

Because this study was designed to analyze the regulation ofantioxidant gene expression by oxidative stress, we did notmeasure antioxidant activity in the heart in response torepetitive I/R. Therefore, the functional significance of thechanges in gene expression described in this study isspeculative. Determination of the functional significance ofthe initial observations described in this study is important andis part of ongoing investigations in our laboratory.

Conclusion

In a mouse model of reversible ischemic cardiomyopathyinduced by repetitive I/R, there was an early upregulation ofGPX expression followed by a delayed induction of HO-1transcript levels, suggesting a coordinated hierarchical antiox-idant defense mechanism. Furthermore, we found that bothGPX and HO-1 expressions in repetitive I/R are dependent onROS, indicating that alterations in redox balance mediate notonly tissue injury but also components of programmed cellsurvival in myocardial hibernation.

Acknowledgments

This study was supported by grants from the NHLBI (T32-HL 07591 and RO1-HL073162-01 to H.T.) and (HL-42550 toM.L.E). O. Dewald was supported by the Deutsche For-schungsgemeinschaft (DE801/1-1). We thank Thuy Pham,Christine Peigney, and Helge Doerr for their expert technicalassistance. We also thank Martin E. Young for his assistancewith the design of the Taqman primer/probe assays.

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