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(Circulation. 2001;103:1695.)
© 2001 American Heart Association, Inc.

Basic Science Reports

H_mox-1 Constitutes an Adaptive Response to Effect Antioxidant Cardioprotection

A Study With Transgenic Mice Heterozygous for Targeted Disruption of the Heme Oxygenase-1 Gene

Tetsuya Yoshida, MD; Nilanjana Maulik, PhD; Ye-Shih Ho, PhD; Jawed Alam, PhD; Dipak K. Das, PhD

From the Cardiovascular Research Center, University of Connecticut School of Medicine, Farmington (T.Y., N.M., D.K.D.); the Institute of Chemical Toxicology, Wayne State University, Detroit, Mich (Y.-S.H.); and the Department of Molecular Genetics, Louisiana University Medical Center, New Orleans (J.A.).

Correspondence to Dipak K. Das, PhD, University of Connecticut, School of Medicine, Farmington, CT 06030-1110. E-mail ddas{at}neuron.uchc.edu

	Abstract

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Background—Heme oxygenase-1 (H_mox-1) has been implicated in protection of cells against ischemia/reperfusion injury.

Methods and Results—To examine the physiological role of H_mox-1, a line of heterozygous H_mox-1-knockout mice was developed by targeted disruption of the mouse H_mox-1 gene. Transgene integration was confirmed and characterized at the protein level. A 40% reduction of H_mox-1 protein occurred in the hearts of H_mox-1^+/- mice compared with those of wild-type mice. Isolated mouse hearts from H_mox-1^+/- mice and wild-type controls perfused via the Langendorff mode were subjected to 30 minutes of ischemia followed by 120 minutes of reperfusion. The H_mox-1^+/- hearts displayed reduced ventricular recovery, increased creatine kinase release, and increased infarct size compared with those of wild-type controls, indicating that these H_mox-1^+/- hearts were more susceptible to ischemia/reperfusion injury than wild-type controls. These results also suggest that H_mox-1^+/- hearts are subjected to increased amounts of oxidative stress. Treatment with 2 different antioxidants, Trolox or N-acetylcysteine, only partially rescued the H_mox-1^+/- hearts from ischemia/reperfusion injury. Preconditioning, which renders the heart tolerant to subsequent lethal ischemia/reperfusion, failed to adapt the hearts of the H_mox-1^+/- mice compared with wild-type hearts.

Conclusions—These results demonstrate that H_mox-1 plays a crucial role in ischemia/reperfusion injury not only by functioning as an intracellular antioxidant but also by inducing its own expression under stressful conditions such as preconditioning.

Key Words: heme oxygenase • genes • oxygen • stress • ischemia

	Introduction

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Heme oxygenase (HO) catalyzes the reaction for heme metabolism, yielding equimolar quantities of CO, iron, and biliverdin.¹ The HO system consists of 2 isoforms: oxidative stress–inducible HO-1 (H_mox-1), also known as heat-shock protein (HSP) 32, and the constitutive isozyme HO-2.² In addition to oxidants, a variety of other environmental stresses, including heat stress, hypoxia, metals, endotoxin, and certain hormones, can induce H_mox-1.^{3 4}

Recent evidence suggests that H_mox-1 induction plays a role in cellular protection against injury caused by the reactive oxygen species (ROS).⁵ Enhancement of HO activity by endotoxin attenuated renal failure, and this protection was lost when HO was inhibited with protoporphyrin.⁶ In another study, H_mox-1 mediated an adaptive response to oxidative stress in skin fibroblasts.⁷ ROS produced in the ischemic/reperfused myocardium has been shown to induce H_mox-1 gene expression.⁸

The physiological significance of H_mox-1 induction during myocardial ischemia remains unknown. It seems reasonable to speculate that H_mox-1 induction during ischemia/reperfusion is the heart’s own stress signal for survival against oxidative stress. Although a role of H_mox-1 in myocardial protection has been speculated upon, a definitive cardioprotective role of H_mox-1 is lacking. To fill this gap, we developed an H_mox-1^+/- mouse by targeted disruption of the mouse H_mox-1 gene. The isolated hearts with 1 functional copy of the H_mox-1 gene and those of matched wild-type mice were subjected to ischemia/reperfusion. To examine whether H_mox-1 plays a role in stress adaptation, the hearts were subjected to preconditioning by cyclic episodes of ischemia/reperfusion. The results demonstrated impaired ventricular recovery and increased infarct size for the H_mox-1^+/- hearts compared with those from wild-type controls, suggesting that H_mox-1^+/- hearts are vulnerable to ischemia/reperfusion injury. Improvement of cardiac function was less apparent for the H_mox-1^+/- mice after preconditioning.

	Methods

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Targeted Disruption of the Mouse H_mox-1 Gene
As shown in Figure 1, the XhoI fragment of the mouse H_mox-1 gene⁹ spanning from part of exon 1 to intron 2 was cloned into the XhoI site of the targeting vector pPNT.¹⁰ The 3' XbaI-EcoRI fragment of the gene-containing sequence of intron 3 to 3' flanking region of the gene was subcloned into the corresponding XbaI and EcoRI sites in pPNT. The targeting vector was linearized with NotI digestion and then transfected into R1 embryonic stem (ES) cells¹¹ by electroporation. Five homologous recombinant ES clones were identified from 224 clones by DNA blot analysis using the 5' external probe encompassing sequences from nucleotide -133 to +73. Male chimeric mice, generated by microinjecting C57BL/6 blastocytes with 3 independent clones, were used in breeding with C57BL/6 female mice. Germ-line transmission of 129/Sv chromosomes was achieved by chimeric mice derived from each of the homologous recombinant ES clones. Line 183 H_mox-1^+/- mice were used in this study.

View larger version (17K): [in this window] [in a new window]   Figure 1. Targeted disruption of mouse Hmox-1 gene. a, Genomic structure and partial restriction map of mouse Hmox-1 locus (top), targeting vector (middle), and predicted structure of targeted locus (bottom). Solid and open boxes represent coding and noncoding regions of exons, respectively. Hatched box indicates DNA fragment used for hybridization probe. E indicates EcoRI; H, HindIII; N, NotI; P, PstI; X, XhoI; and Xb, XbaI. Expected sizes of PstI genomic fragments from wild-type and targeted alleles hybridized with probe are shown at top and bottom, respectively. b, DNA blot analysis of mouse offspring. Mouse tail DNA was digested with PstI and probed with 5' external probe shown in a. +/+ and +/- represent wild-type and heterozygous knockout mice, respectively. c, Protein blot analysis of Hmox-1 expression in hearts.

Protein Blot Analysis of H_mox-1 in Hearts
Hearts were homogenized in 50 mmol/L phosphate buffer, pH 7.8, containing 0.1% Triton X-100 with a Polytron homogenizer. The homogenates were centrifuged at 20 000g and stored at -70°C. Protein contents were determined with an assay kit (Pierce).

For protein blot analysis, total cellular protein was separated on a 12% SDS-polyacrylamide gel and electrophoretically transferred onto a nitrocellulose paper. The protein blot filter paper was incubated in 1x HBSS plus a 1:2000 dilution of a monoclonal anti-human H_mox-1 antibody (StressGen) for 2 hours. The filter paper was washed with HBSS containing 1% nonfat milk, incubated with a 1:3000 dilution of horseradish peroxidase–conjugated goat anti-mouse IgG (Bio-Rad, Hercules) in HBSS plus 5% nonfat milk for 1 hour, washed again with HBSS, and then subjected to autoradiography in the presence of an enhanced chemiluminescent substrate (Pierce). Western blot data were quantified by densitometric scanning.

Measurement of Contractile Function
Twenty-four mice (10 to 12 weeks old) were divided into 2 groups: H_mox-1^+/- (n=12) and wild-type (n=12). They were anesthetized with pentobarbital (200 mg/kg). The heart was excised, the aorta cannulated, and the heart perfused with Krebs-Henseleit buffer (KHB) by the Langendorff method.¹² The effluent was collected before ischemia and during reperfusion and stored at -20°C for subsequent measurement of creatine kinase (CK) and malondialdehyde (MDA). A 4-0 silk suture on a round-bodied needle was passed through the apex of the heart and attached to the apex, which in turn was attached to a force transducer. The heart rate, force developed by the heart (DF), and first derivative of developed force (dF/dt) were recorded.¹² The data on myocardial contractile function were recorded and analyzed in real time with the Cordat II data acquisition and presentation system (Triton Technologies). The hearts were subjected to 30 minutes of ischemia by clamping of the aortic cannula, followed by 2 hours of reperfusion.

Antioxidant Therapy
To examine whether antioxidants could protect the H_mox-1^+/- hearts from ischemia/reperfusion injury, 2 different protocols were used. (1) For in vivo studies, both wild-type and H_mox-1^+/- mice were injected once every 24 hours with either Trolox (10 mg/kg IV), an analogue of vitamin E, or N-acetylcysteine (NAC, 1.5 mg/kg IV), a precursor of glutathione synthesis and an antioxidant. Control hearts were injected with saline only. (2) In vitro experiments were performed by preperfusion of the isolated wild-type and H_mox-1^+/- hearts for 15 minutes with KHB buffer in the presence or absence of Trolox (10 mmol/L) or NAC (10 mmol/L). All hearts were subjected to 30 minutes of ischemia followed by 2 hours of reperfusion. The cardioprotective abilities of the antioxidants were examined by study of ventricular function, infarct size, and CK release.

Preconditioning Protocol
Another group of hearts from H_mox-1^+/- and wild-type mice were preconditioned by subjection to 3 cycles of 5 minutes of ischemia each, followed by another period of 5 minutes of reperfusion.^{13 14} This preconditioning protocol has been found to render the heart tolerant to the subsequent ischemic stress. All hearts were subjected to 30 minutes of ischemia followed by 2 hours of reperfusion.

Estimation of CK Release
CK release from the heart was estimated in the perfusate collected from the heart by use of a CK assay kit (Sigma). The results are expressed as IU/L of total CK release from the heart.

Estimation of MDA
MDA was assayed in the coronary perfusate as described previously¹⁵ to monitor the development of oxidative stress. The MDA was derivatized with DNPH.¹⁵ Aliquots of 25 µL of derivatized MDA in acetonitrile were injected onto a Beckman Ultrasphere C₁₈ (3-mm) column in a Waters high-performance liquid chromatograph (Waters Corp). The products were eluted isocratically and detected at 307, 325, and 356 nm. The amount of MDA was quantified by use of a Maxima software program (Waters).

Measurement of Infarct Size
At the end of reperfusion, a 10% (wt/vol) solution of triphenyl tetrazolium in phosphate buffer was infused into the aortic cannula.¹⁶ The hearts were excised and stored at -70°C. Sections (0.8 mm) of frozen heart were fixed in 2% paraformaldehyde, placed between 2 coverslips, and digitally imaged with a Microtek ScanMaker 600z. To quantify the areas of interest in pixels, an NIH Image 5.1 (a public-domain software package) was used. The infarct size (transmural) was quantified in pixels.

Statistical Analysis
For statistical analysis, a 2-way ANOVA followed by Scheffé’s test was first carried out to test for any differences between groups. If differences were established, the values were compared by Student’s t test for paired data. The values were expressed as mean±SEM. The results were considered significant at a level of P<0.05.

	Results

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Generation and Characterization of H_mox-1^+/- Mice
As shown in Figure 1a, the mouse H_mox-1 gene was inactivated by replacement of exon 2 with a neomycin resistance (neo) cassette. Insertion of the neo gene creates a new PstI restriction site in the H_mox-1 gene, resulting in a shorter PstI genomic fragment from the targeted allele than that from the wild-type allele (Figure 1b). Heterozygous H_mox-1^+/- mice were apparently healthy and showed no pathological or phenotypic differences from the wild-type littermates. No homozygous knockout mice could be detected in the progeny from heterozygote intercross, however, indicating that embryonic lethality occurs in the homozygous mice. A 40% reduction of H_mox-1 was evident in the heart of the heterozygous mouse compared with that of the wild-type mouse (Figure 1c). We reasoned that a decrease in expression of H_mox-1 protein due to the targeted mutation in H_mox-1^+/- mice might render the mice more susceptible to an increased oxidative stress than controls.

Myocardial Performance
All hearts recovered their beats spontaneously after 30 minutes of ischemia. The heart rates remained slightly but not significantly lowered, however, compared with baseline (results not shown). No significant difference was found in heart rate between groups throughout the experiment. Coronary flow progressively lowered during the reperfusion, but differences were not significant (Figure 2). The DF during reperfusion was lower for the H_mox-1^+/- mouse hearts than wild-type hearts (Figure 3, top). In control hearts, DF recovered to near baseline levels after 90 minutes of reperfusion. Conversely, DF did not recover beyond 87% of baseline for the H_mox-1^+/- hearts. At all points, DF showed significantly lower recovery for H_mox-1^+/- hearts than wild-type hearts after 30 minutes of reperfusion (P<0.05). A similar trend was seen in dF/dt_max (Figure 3, bottom). A significantly lower recovery of dF/dt_max occurred in H_mox-1^+/- hearts after 30 minutes of reperfusion (P<0.05). These results demonstrate that H_mox-1^+/- mouse hearts showed significantly lower contractile recovery after 30 minutes of ischemia.

View larger version (22K): [in this window] [in a new window]   Figure 2. Coronary flow in Hmox-1+/- and wild-type mice during ischemia/reperfusion. Sequential changes of coronary flow during reperfusion in wild-type (), wild-type preconditioned (•), Hmox-1+/- (), and Hmox-1+/- preconditioned () hearts. Results are expressed as mean±SEM for n=12 per group.

View larger version (28K): [in this window] [in a new window]   Figure 3. Sequential changes of DF (top) and dF/dtmax (bottom) during reperfusion in wild-type (), wild-type preconditioned (•), Hmox-1+/- (), and Hmox-1+/- preconditioned () hearts. Results are expressed as mean±SEM for n=12 for each group. *P<0.05 vs wild-type control.

Preconditioning rendered the hearts of the wild-type mice tolerant to ischemia/reperfusion injury, as evidenced by improved DF and dF/dt compared with wild-type hearts not subjected to preconditioning. In contrast, the preconditioned hearts of the H_mox-1 mice did not exhibit similar improvement in functional recovery, and there were no significant differences of DF and dF/dt between preconditioned and nonpreconditioned hearts.

Both in vivo and in vitro, Trolox and NAC treatments partially improved the contractile function. For example, postischemic DF and dF/dt were improved significantly but only partially compared with those of H_mox-1^+/- hearts (Table). Interestingly, both antioxidants (in vivo or in vitro) improved DF and dF/dt to the same extent. Baseline values of the antioxidant-treated (in vivo or in vitro) and untreated groups were identical.

View this table: [in this window] [in a new window]   Table 1. Effects of Dimethylthiourea and NAC on Ventricular Function and Myocardial Infarct Size During Ischemia-Reperfusion of Wild-Type and Hmox-1+/- Mouse Hearts

View this table: [in this window] [in a new window]   Table 2.

CK Release From Heart
Total CK release from the heart (Figure 4) was negligible for all the groups before ischemia. After ischemia, CK release increased in both groups, but the amount of release was much higher for H_mox-1^+/- hearts. At 30 minutes of reperfusion, CK release was 70±9.7 IU/mL for H_mox-1^+/- mice versus 50±11.5 IU/mL (P<0.05) for nontransgenic controls. At 60 minutes of reperfusion, CK release was 120±8.7 IU/mL for H_mox-1^+/- hearts versus 82±9.4 IU/mL (P<0.05) for nontransgenic controls. Similarly, after 120 minutes of reperfusion, CK release from the knockout mouse hearts amounted to 239±8.1 IU/mL compared with 135±7.4 IU/mL for controls.

View larger version (22K): [in this window] [in a new window]   Figure 4. CK release from hearts of wild-type (), wild-type preconditioned (•), Hmox-1+/- (), and Hmox-1+/- preconditioned () hearts. CK was assayed in coronary effluent as described in Methods. Results are expressed as mean±SEM for n=12 per group. *P<0.05 vs wild-type control. P<0.05 vs Hmox-1+/-.

Preconditioning significantly reduced the amount of CK in wild-type hearts compared with the nonpreconditioned group. The amount of CK release was also less for the preconditioned H_mox-1^+/- hearts than for nonpreconditioned hearts, but the differences did not reach significance except at 90 and 120 minutes of reperfusion.

In vivo and in vitro, Trolox and NAC treatments partially but significantly reduced the CK release compared with that from the H_mox-1^+/- hearts (Table). Again, both antioxidants reduced the CK release to the same extent. Baseline values of the antioxidant-treated and untreated groups were identical.

Myocardial Infarction
The portion of the heart not stained by tetrazolium was measured and calculated as the infarct area. Mean value of infarct size in the knockout group was significantly higher than that in the wild-type group (48.5±3.2% versus 28.5±2.4% for wild-type, P<0.05) (Figure 5). Our results indicated that H_mox-1^+/- hearts had significantly higher myocardial necrosis.

View larger version (30K): [in this window] [in a new window]   Figure 5. Infarct size expressed as % left ventricle in wild-type (solid bar), wild-type-preconditioned (stippled bar), Hmox-1+/- (hatched bar), and Hmox-1+/- preconditioned (open bar) hearts. Results are expressed as mean±SEM for n=12 per group. *P<0.05 vs wild-type control.

A reduced amount of infarct size was apparent in the hearts subjected to preconditioning. Significantly less infarct was noticed, however, for the preconditioned wild-type hearts than nonpreconditioned hearts.

Both in vivo and in vitro, Trolox and NAC treatments partially but significantly reduced the infarct size compared with the H_mox-1^+/- hearts (Table). Similar to the previous results, both antioxidants reduced the infarct size to the same extent.

MDA Formation
MDA, measured as MDA-DNPH, increased progressively during reperfusion for the nonpreconditioned hearts (Figure 6). The amount of MDA production, however, was significantly higher at all points in the H_mox-1^+/- hearts than in control hearts, demonstrating that H_mox-1^+/- hearts were subjected to increased amounts of oxidative stress. Preconditioning reduced the amount of MDA formation in both groups of hearts. A significantly higher amount of MDA was found in the H_mox-1 hearts than in the wild-type control hearts.

View larger version (25K): [in this window] [in a new window]   Figure 6. MDA formation during reperfusion of ischemic myocardium. Coronary effluents were taken from wild-type (), wild-type preconditioned (•), Hmox-1+/- (), and Hmox-1+/- preconditioned () mouse hearts to estimate MDA. Results are expressed as mean±SEM for n=12 per group. *P<0.05 vs wild-type control. P<0.05 vs Hmox-1+/-.

	Discussion

Top Abstract Introduction Methods Results Discussion References

 
As mentioned earlier, H_mox-1^+/- mouse hearts showed 40% reduction of H_mox-1 protein compared with H_mox-1^+/+ hearts. The activities of other key antioxidant enzymes, including manganese superoxide dismutase (Mn-SOD), catalase, glutathione peroxidase, glutathione reductase, and glucose-6-phosphate-dehydrogenase, remained unaltered in the H_mox-1^+/- hearts compared with wild-type controls (data not shown). The results thus truly reflect the effects of H_mox-1^+/- in myocardial ischemia/reperfusion injury.

H_mox-1^+/- hearts were more susceptible to ischemia/reperfusion injury, as evidenced by greater infarct size, increased CK release, and reduced ventricular recovery compared with wild-type controls. In addition, these H_mox-1^+/- hearts could not be preconditioned like the hearts of the wild-type controls, suggesting that H_mox-1 plays a role in the adaptation to stress. This is further supported by the results with MDA, because MDA formation, a presumptive marker for oxidative stress, was higher for the preconditioned H_mox-1^+/- mouse hearts than the wild-type hearts.

During the course of our studies, Poss and Tonegawa^{17 18} have reported generation of H_mox-1^+/- mice in which exons 3, 4, and part of 5 were replaced by the neo cassette. Whereas they were able to obtain homozygous knockout mice from heterozygous breeding at a lower-than-expected frequency, an indication of embryonic lethality due to H_mox-1 deficiency, we could not detect any homozygous knockout mice in our breeding colony. The discrepancy in our breeding results is not understood. It may result from the fact that different ES cells were being used in each laboratory. The studies by Poss and Tonegawa using homozygous knockout mice have clearly established the antioxidant role of H_mox-1 against the toxicity resulting from treatment with endotoxin, heme, and hydrogen peroxide. We rationalized that a modest decrease in H_mox-1 gene expression in H_mox-1^+/- hearts might also cripple the cellular antioxidant defense mechanism, which is protective in certain disease models whose pathogenesis is known to be associated with an overproduction of ROS. Similar to what was observed by Poss and Tonegawa,¹⁸ the H_mox-1^+/- mice generated in our laboratory showed no increased susceptibility to endotoxin toxicity compared with that of wild-type mice (unpublished data). This report documents that heterozygous H_mox-1^+/- mice with an inactivated H_mox-1 allele are more vulnerable to myocardial ischemia/reperfusion injury.

Heme oxygenase, which catalyzes the initial rate-limiting step of heme catabolism, is known to be induced by a wide variety of oxidative stresses, including H₂O₂, UV radiation, and reduced glutathione depletion.^{3 4} A study from our laboratory documented that ischemia/reperfusion could induce the expression of H_mox-1 mRNA in the heart.⁸ Induction of H_mox-1 gene expression increased as a function of duration of reperfusion, which could be inhibited with ROS scavengers and SOD, suggesting that oxidative stress developed during ischemia/reperfusion induces the HO-1 gene. Our results are consistent with these previous reports, indicating that H_mox-1 is induced by oxidative stress.

The role of HO-1 in oxidative stress adaptation was further supported by the observation that the hearts of the H_mox-1^+/- mice could not be preconditioned to make them resistant to subsequent oxidative stress compared with wild-type hearts. Preconditioning by repeated brief episodes of ischemia/reperfusion is known to develop oxidative stress.¹⁹ The development of oxidative stress presumably leads to adaptive modification of the hearts, which is achieved through a chain of reactions including generation of intracellular mediators and modification of signal transduction processes and gene expression.²⁰ Oxidative stress has been shown to function as a second messenger in this process.²¹ The results of our study suggest that induction of HO-1 by oxidative stress plays a crucial role in preconditioning-mediated adaptive modification of the heart.

HO, an oxidative stress–inducible protein, is also an HSP, HSP 32. HO-1 is evolutionarily conserved in the primary amino acid and nucleotide sequences, and its extent of homology between mouse, rat, and human is >80%. H_mox-1 is readily induced not only by oxidative stress but also with many other pathological and environmental stresses, such as hypoxia, hyperoxia, cellular transformations, and heat shock.²² This enzyme catalyzes the conversion of heme into biliverdin and bilirubin, which function as in vivo antioxidants.¹ In addition, a messenger molecule, carbon monoxide (CO), is generated during the production of biliverdin. Previous studies demonstrated that production of cGMP from CO signaling is beneficial for the heart.²³

To examine whether antioxidants could rescue the H_mox-1^+/- hearts from increased ischemia/reperfusion injury, the H_mox-1^+/- mice were treated with 2 different antioxidants, Trolox and NAC. Both of these antioxidants are cell-permeable. Trolox, an analogue of vitamin E, is a hydrophilic antioxidant that has been found to be cardioprotective. NAC is a precursor of glutathione and functions as an antioxidant. NAC also prevents intracellular oxidation and maintains glutathione levels by reducing cystine to cysteine. Both Trolox and NAC provided similar degrees of cardioprotection when used in vitro or in vivo. Neither of these antioxidants, however, could completely rescue the hearts from the ischemia/reperfusion injury. Nevertheless, they significantly reduced the amount of ischemia/reperfusion injury compared with control H_mox-1^+/- hearts. Interestingly, the amount of cardioprotection afforded by these antioxidants was comparable to that obtained by preconditioning the H_mox-1^+/- hearts.

Numerous reports in the literature indicate cardioprotective effects of preconditioning.^{24 25} Preconditioning potentiates a signal transduction mechanism coupled with both G-protein and tyrosine kinase receptors, leading to the activation of protein kinase C and mitogen-activated protein kinases and redox-sensitive transcription factors such as nuclear factor-B and activating protein-1.²⁶ Preconditioning-mediated cardioprotection is believed to be achieved through its ability to induce several cardioprotective genes and proteins, including HSP 27, HSP 70, HSP 89, SOD, and reduced glutathione peroxidase-1.²⁷ These genes/proteins have been shown to constitute the defense system of the myocardium. Our results demonstrate that HO-1 is also an important member of the antioxidant defense system of the heart.

In summary, our study documents the crucial role of H_mox-1 in cardioprotection. A decrease in HO-1 in the heart makes it vulnerable to ischemia/reperfusion injury. In vivo or in vitro antioxidant therapy only partially rescued the H_mox-1^+/- hearts from the ischemia/reperfusion injury, suggesting that HO-1 plays an important role in cardioprotection. Preconditioning, which provokes oxidative stress and induces a large variety of oxidative stress–inducible genes, was unable to rescue the hearts of heterozygous H_mox-1^+/- mice from cellular injury, presumably because of the reduced level of H_mox-1. This paves the way for the potential of H_mox-1 gene therapy for cardioprotection in pathological conditions in which oxidative stress is implicated.

	Acknowledgments

 
This study was supported by NIH grants HL-22559, HL-33889, and HL-34360 (Dr Das), a Grant-in-Aid from the American Heart Association and HL-56803 (Dr Maulik), HL-56421 and P30-ES-06639 (Dr Ho), and DK-43135 (Dr Alam).

Received August 25, 2000; revision received October 9, 2000; accepted October 10, 2000.

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