Hypoxia induces severe right ventricular dilatation and infarction in heme oxygenase-1 null mice

Heme oxygenase (HO) catalyzes the oxidation of heme to generate carbon monoxide (CO) and bilirubin. CO increases cellular levels of cGMP, which regulates vascular tone and smooth muscle development. Bilirubin is a potent antioxidant. Hypoxia increases expression of the inducible HO isoform (HO-1) but not the constitutive isoform (HO-2). To determine whether HO-1 affects cellular adaptation to chronic hypoxia in vivo, we generated HO-1 null (HO-1^–/–) mice and subjected them to hypoxia (10% oxygen) for five to seven weeks. Hypoxia caused similar increases in right ventricular systolic pressure in wild-type and HO-1^–/– mice. Although ventricular weight increased in wild-type mice, the increase was greater in HO-1^–/– mice. Similarly, the right ventricles were more dilated in HO-1^–/– mice. After seven weeks of hypoxia, only HO-1^–/– mice developed right ventricular infarcts with organized mural thrombi. No left ventricular infarcts were observed. Lipid peroxidation and oxidative damage occurred in right ventricular cardiomyocytes in HO-1^–/–, but not wild-type, mice. We also detected apoptotic cardiomyocytes surrounding areas of infarcted myocardium by terminal deoxynucleotide transferase–mediated dUTP nick end-labeling (TUNEL) assays. Our data suggest that in the absence of HO-1, cardiomyocytes have a maladaptive response to hypoxia and subsequent pulmonary hypertension.

J.Clin. Invest.103:R23–R29 (1999).

Heme oxygenase (HO) catalyzes the oxidation of heme to carbon monoxide (CO) and biliverdin, which is reduced subsequently to bilirubin (1). CO, like nitric oxide, is a gas molecule that stimulates guanylyl cyclase and increases intracellular levels of cGMP (1, 2). cGMP is an important regulator of vascular tone and smooth muscle development (3, 4). By degrading the pro-oxidant heme and generating the antioxidant bilirubin (5, 6), HO may also protect cells against oxidative injury (1). Three HO isoforms have been identified. HO-1, also known as heat shock protein 32 (7, 8), is a 32-kDa protein induced by heat, heme, heavy metals, hypoxia, hyperoxia, shear stress, stretch, ultraviolet light, cytokines, and reactive oxygen species (1, 9). HO-2 is a constitutive, 36-kDa isoform (10, 11). HO-3, whose sequence is highly homologous to that of HO-2, has been cloned recently but has not been characterized fully (12).

Under normal physiological conditions, HO-1 and HO-2 are both expressed at low levels in vascular smooth muscle cells (VSMCs) and cardiomyocytes (13–15). Under pathophysiological conditions, however, HO-1, but not HO-2, is induced in the heart and blood vessels (13–17). For example, we and others have reported that bacterial endotoxin markedly increases HO-1 expression in VSMCs and cardiomyocytes in vivo (15, 17, 18). HO-1 is upregulated at both the mRNA and the protein levels in cultured VSMCs exposed to stretching and shear stress (19).

Hypoxia causes a remodeling of the pulmonary vasculature that is characterized by proliferation of VSMCs and deposition of matrix. Hypoxia also increases pulmonary vascular resistance (leading to pulmonary hypertension) and induces right ventricular hypertrophy in humans, rats, and mice (20–24). CO released by HO-1 has been implicated in the regulation of VSMC proliferation and may regulate vascular remodeling in response to hypoxia (13, 25). In addition, pressure overload of the right ventricle, induced experimentally by hypoxia or pulmonary artery banding, increases HO-1 expression in rat cardiomyocytes (26). Taken together, these data suggest an important role for HO-1 in cardiovascular adaptation to hypoxic stress.

To gain insight into the biologic functions of HO-1 in vivo, we generated HO-1 null (HO-1^–/–) mice by targeted mutation. We subjected the mice to chronic hypoxia, which induces pulmonary hypertension, to study the function of HO-1 in the cardiovascular system.

Generation of HO-1^–/– mice. To generate a null mutation at the HO-1 locus, we constructed a targeting vector in which HO-1 exon 3 had been replaced with the neomycin resistance gene (neo). The thymidine kinase gene (TK) was included as a negative selection marker to prevent nonhomologous recombination (Figure 1a). ES cells heterozygous for the targeted HO-1 allele were used to generate chimeric mice. The breeding of chimeras with wild types resulted in transmission of the mutation through the germline. Heterozygotes were intercrossed to generate HO-1^–/– homozygotes, as determined by Southern blot analysis (Figure 1b). To confirm the null mutation, we performed Northern blot analysis on RNA isolated at E18.5. HO-1 mRNA levels were lower in heterozygotes than in wild-type mice, and HO-1 mRNA was undetectable in HO-1^–/– mice (Figure 1c). HO-2 mRNA levels, in contrast, did not differ significantly among the three genotypes (Figure 1c). We confirmed the null mutation further by Western blot analysis for HO-1 protein. Consistent with the Northern blot analysis, HO-1 protein was not detectable in HO-1^–/– mice (Figure 1d). The level of HO-2 protein did not change in HO-1^–/– mice (Figure 1d).

Genotyping of three-week-old mice from HO-1 heterozygote matings revealed that the survival rate of HO-1^–/– mice was ∼7%, consistent with the observations of Poss and Tonegawa (33) about their line of HO-1^–/– mice, described while our work was in progress. Among the newborn mice from heterozygote breedings, ∼20% were homozygous mutants, indicating partial embryonic or neonatal lethality. We are currently investigating the mechanism of lethality. Surviving HO-1^–/– mice were grossly normal and were used for subsequent experiments.

Chronic hypoxia increases right ventricular systolic pressure and ventricular weight in wild-type and HO-1^–/– mice. To investigate the role of HO-1 in the response to hypoxia, we housed mice (five to six mice in each group) in a 10% oxygen environment for five to seven weeks. As expected, seven weeks of hypoxia caused hematocrit values to increase from 44.0 ± 1.4% to 73.0 ± 8.2% in wild-type mice. Similarly, hematocrit values increased from 40.0 ± 2.8% to 78.0 ± 5.9% in HO-1^–/– mice. In contrast to rats (20, 30, 34), mice subjected to chronic hypoxia sustained rather modest pulmonary vascular remodeling (defined as an increase in peripheral muscularized vessels) (24, 35). Moreover, in our study, there was no significant difference in hypoxia-induced remodeling between wild-type and HO-1^–/– mice at seven weeks (51% vs. 52.5% partially and completely muscularized peripheral vessels). We then determined the effect of hypoxia on right ventricular systolic pressure, which is an indicator of pulmonary arterial systolic pressure. Right ventricular systolic pressure in wild-type and HO-1^–/– mice did not differ under normoxic conditions (P = 0.80; Figure 2a, open bars). Although five weeks of hypoxia increased right ventricular systolic pressure, it did so to a similar degree in wild-type and HO-1^–/– mice (P = 0.43; Figure 2a, filled bars).

Figure 2

Chronic hypoxia increases right ventricular systolic pressure and ventricular weight in wild-type and HO-1^–/– mice. Wild-type (+/+) and HO-1 null (–/–) mice were exposed to normoxia or chronic hypoxia (10% oxygen). (a) Right ventricular systolic pressure was measured under normoxic conditions (open bars) or after five weeks of hypoxia (filled bars). Error bars indicate standard deviations. *P < 0.05 vs. animals exposed to normoxia within the same group (n = 5 in each group). (b) Ventricular weight in wild-type and HO-1^–/– mice was measured under normoxic conditions (n = 6 in each group) or after seven weeks of hypoxia (n = 5 in each group) and expressed as a ratio of ventricular weight (milligrams) to total body weight (grams). The value obtained for normoxic wild-type mice (4.46 ± 0.52) was set as 100%. The ventricular weight index for mice housed under normoxic (open bars) or hypoxic (filled bars) conditions is expressed relative to the value for normoxic wild-type mice. Error bars indicate standard deviations. *P < 0.05 vs. animals exposed to normoxia within the same group. ^#P < 0.05 vs. wild-type animals exposed to hypoxia.

The ventricular weight index did not differ between wild-type and HO-1^–/– mice under normoxic conditions (Figure 2b, open bars). Exposure to hypoxia for seven weeks caused a 32% increase in the ventricular weight index in wild-type mice. In HO-1^–/– mice, however, the ventricular weight index after seven weeks of hypoxia increased 100% in comparison with that in normoxic wild-type mice (Figure 2b, filled bars). Studies in a subgroup of mice revealed that the changes in ventricular weight reflected mainly a right ventricular effect, as the RV/(LV + septum) increased in HO-1^–/– mice in comparison with wild-type mice exposed to hypoxia for seven weeks (data not shown).

Hypoxia induces right ventricular infarcts and mural thrombi in HO-1^–/– mice. Under normoxic conditions, there were no obvious histological differences between wild-type and HO-1^–/– mice (Figure 3, a and b). After five weeks of hypoxia, HO-1^–/– mice showed increased right ventricular dilatation (Figure 3d) in comparison with wild-type mice (Figure 3c). After seven weeks of hypoxia, the right ventricles in HO-1^–/– mice were even more dilated (Figure 3f) than those in similarly treated wild-type mice (Figure 3e). Note that the right ventricular free wall from the HO-1^–/– mouse (Figure 3f) does not appear thicker than the one from the wild-type mouse (Figure 3e). Remarkably, a large organized thrombus, attached to the infarcted right ventricular wall, is visible in the HO-1^–/– mouse (Figure 3f). Four of six HO-1^–/– hearts exposed to seven weeks of hypoxia showed evidence of right ventricular infarcts, and mural thrombi were present in all four infarcted right ventricles. Sections from one animal in each group (n = 5–6) are shown in Figure 3 because the histological analyses were similar under normoxic and hypoxic conditions within a particular group.

Figure 3

Right ventricular dilatation and thrombus formation in HO-1^–/– mice in response to hypoxia. Heart cross-sections (at the papillary muscle level) from wild-type (+/+) and HO-1 null (–/–) mice were stained with hematoxylin and eosin (n = 5–6 in each group). Mice were exposed to normoxia (a, +/+; b, –/–), five weeks of hypoxia (c, +/+; d, –/–), or seven weeks of hypoxia (e, +/+; f, –/–). RV, right ventricle; LV, left ventricle; T, thrombus. Original magnification: ×15.

Chronic hypoxia induces right ventricular infarction in HO-1^–/– mice. To search for additional evidence of infarcts in the right ventricle, we examined the tissue sections at higher magnifications. Cardiomyocytes were intact in ventricular sections from wild-type mice exposed to seven weeks of hypoxia (Figure 4, a and c). In contrast, ventricular sections from HO-1^–/– mice exposed to seven weeks of hypoxia (Figure 4, b and d) showed mononuclear inflammatory cell infiltration and extensive cardiomyocyte degeneration and death with focal calcification (indicated by an arrow in Figure 4d); these characteristics are consistent with infarcts one to two weeks old. Moreover, the right ventricular infarcts did not appear to result from vascular occlusion, because the coronary arteries supplying blood to the right ventricle were patent in HO-1^–/– mice (data not shown). To detect collagen accumulation indicative of fibrosis, we stained ventricular sections with Masson’s trichrome. After seven weeks of hypoxia, cells surrounding blood vessels stained positive for collagen in hearts from wild-type mice (Figure 4e). In hearts from HO-1^–/– mice exposed to seven weeks of hypoxia, however, early collagen deposition was present throughout the lesion (Figure 4f, beneath the thrombus). This degree of fibrosis is consistent with early scar formation and a lesion one to two weeks old.

Figure 4

Histological analysis of wild-type and HO-1^–/– mouse hearts after seven weeks of hypoxia. Six animals were examined in each group. Hematoxylin and eosin–stained right ventricular free wall from wild-type (+/+) mouse (a and c) and infarcted area of right ventricle (underneath thrombus) from HO-1 null (–/–) mouse (b and d). Original magnifications: ×200 (a and b) and ×400 (c and d). The dense areas of blue nuclei in b and d, not visible in a and c, represent mononuclear cell infiltrate. Wild-type (e) and HO-1^–/– (f) heart sections were stained with Masson’s trichrome to reveal collagen (blue). RV, right ventricle; T, thrombus. Original magnification: ×49. MAL-2–immunostained (pink) right ventricular free wall from wild-type mouse (g) and area of infarcted right ventricle (underneath thrombus) from HO-1^–/– mouse (h). Original magnification: ×200. TUNEL assay for apoptotic cardiomyocytes in right ventricular free wall from wild-type mouse (i) and free wall (surrounding infarcted region) from HO-1^–/– mouse (j). TUNEL-positive cardiomyocytes stained red. Original magnification: ×400.

Two of the six hearts examined in the HO-1^–/– mice exposed to seven weeks of hypoxia did not exhibit gross right ventricular infarcts; however, close examination of these hearts revealed focal areas of myocardial degeneration without evidence of extensive inflammatory cell infiltration. These hearts showed early evidence of myocardial damage. Hearts from wild-type and HO-1^–/– mice examined after five weeks of exposure to hypoxia displayed no cardiomyocyte degeneration or death and no extensive mononuclear inflammatory cell infiltration (data not shown). Cells surrounding blood vessels stained positive for collagen in hearts from wild-type and HO-1^–/– mice housed for five weeks under normoxic and hypoxic conditions (data not shown). However, in contrast with the right ventricular free walls from HO-1^–/– mice exposed to seven weeks of hypoxia, which showed deposition of collagen, no collagen deposition was evident in the right ventricular free walls from HO-1^–/– mice exposed to five weeks of hypoxia. Taken together, these data confirm that in the HO-1^–/– mice exposed to seven weeks of hypoxia, myocardial infarcts were less than two weeks old.

To assess oxidative damage in HO-1^–/– hearts with infarcts, we immunostained ventricular sections with polyclonal antibody MAL-2, which recognizes oxidation-specific lipid-protein adducts. In contrast to the minimal MAL-2 staining in hearts from wild-type mice (Figure 4g), MAL-2 staining was intense in cells (predominantly cardiomyocytes) beneath the infarcted area of the right ventricle in HO-1^–/– mice (Figure 4h). This difference suggests the presence of severe oxidative damage within and around the infarct site. We also performed TUNEL assays to detect the presence of DNA fragmentation and apoptosis in the hearts of HO-1^–/– mice exposed to hypoxia. No TUNEL-positive cardiomyocytes were detected in right ventricles from wild-type mice (Figure 4i). In right ventricles from HO-1^–/– mice, however, a significant number of TUNEL-positive cells surrounded the infarct site (Figure 4j).

We show by gene-deletion experiments in vivo that HO-1 plays an important, protective role in the adaptation of the cardiovascular system to hypoxia. Right ventricles from HO-1^–/– mice exposed to chronic hypoxia were severely dilated and contained right ventricular infarcts with mural thrombi (Figures 3 and 4).

The three characteristic responses of humans and animals to hypoxia are pulmonary vascular remodeling, pulmonary hypertension, and hypertrophy of the right ventricle (20–24). Hypoxia induces HO-1 expression in the lung (36), and CO generated by hypoxic VSMCs inhibits proliferation of these cells in culture (13, 25, 37). These links have suggested an important role for HO-1 in the regulation of hypoxia-induced pulmonary vascular remodeling. Our results indicate that chronic hypoxia–induced pulmonary vascular remodeling and pulmonary hypertension were similar in HO-1^–/– and wild-type mice (Figure 2). Thus, the loss of HO-1 appears to have had little effect on these two responses after prolonged hypoxia. Perhaps CO released by HO-2 is sufficient to inhibit excessive pulmonary vascular remodeling and hypertension in response to hypoxia. Alternatively, multiple vasoactive agents released within the pulmonary vasculature in the setting of chronic hypoxia may compensate for the lack of HO-1–derived CO.

As it does in rats (26), hypoxia increases HO-1 protein abundance (by approximately threefold in hearts of wild-type mice after seven weeks; data not shown). Our data show that the absence of HO-1 results in a maladaptive response in cardiomyocytes exposed to hypoxia-induced pulmonary hypertension. Although the myocardium in HO-1^–/– mice appears to be normal under normoxic conditions, under hypoxic conditions, severe right ventricular dilatation and infarcts with mural thrombi develop in HO-1^–/–, but not wild-type, mice — despite exposure to similar hypoxia and pulmonary hypertension. These results are supported by reports indicating that HO-1 is induced in the heart after exposure to hypoxia and increased pulmonary arterial pressure and that HO-1 may have a protective effect on cardiomyocytes subjected to stress (1, 16, 17, 26). Mural thrombi can be detected in 28–40% of patients with acute myocardial infarction (38, 39). We observed mural thrombi in every mouse that had a right ventricular infarct. This high percentage of mural thrombi may be related to a combination of marked dilatation of the right ventricle and high hematocrit and blood viscosity values (even though the hematocrit value was similar in wild-type and HO-1^–/– mice exposed to hypoxia). The absence of HO-1 may also increase platelet aggregation and predispose to thrombus formation, because CO has been shown to inhibit platelet aggregation (19, 40).

Our results demonstrate that cardiomyocytes in HO-1^–/– mice die in response to hypoxia/pulmonary hypertension. Although some cardiomyocyte death appears to be due to necrosis, we did detect TUNEL-positive cells in the zone around the infarct (Figure 4j), which suggests apoptosis as one of the mechanisms leading to cardiomyocyte death. Hypoxia and elevated pulmonary arterial pressure increase cardiac production of reactive oxygen species (41, 42), which play a significant role in myocardial cell death during ischemia/reperfusion (43, 44). Also, overexpression of HO-1 protects against damage caused by oxidative stress in several cell types in vitro (45, 46). Thus, it is likely that the absence of HO-1 in cardiomyocytes leads to an accumulation of reactive oxygen species that causes cardiomyocyte death. This conclusion is supported by our finding of extensive lipid peroxidation in the zone of right ventricular infarction (Figure 4h) and by the findings of Poss and Tonegawa (47), who showed that HO-1^–/– embryonic fibroblasts are more sensitive to cytotoxicity caused by hydrogen peroxide. Although myocardial infarction has been shown to increase oxidative stress (48, 49), we have found a two- to threefold increase in the nitration of protein tyrosine residues, which indicates the presence of the potent oxidant peroxynitrite, in noninfarcted HO-1^–/– hearts exposed to seven weeks of hypoxia (data not shown). Thus, an increase in oxidative stress may precede gross myocardial infarction.

In summary, our results show that HO-1 is essential for cardiomyocytes to adapt to stresses such as hypoxia and subsequent pulmonary hypertension. The protective effect of HO-1 may be related to its antioxidant activity. The absence of coronary occlusion in HO-1^–/– mice in combination with myocardial infarction and severe ventricular dilatation limited to the right side of the heart suggests a maladaptive response of cardiomyocytes to increased pulmonary arterial pressure. On the basis of these findings, we postulate that HO-1 may play a central role in cardiac physiology by protecting cardiomyocytes from pressure-induced injury and secondary oxidative damage. Our findings may lead to novel approaches for preventing cardiomyocyte damage due to hemodynamic stress or ischemia/reperfusion.

We are grateful to R. Rosenberg (Massachusetts Institute of Technology, Cambridge, Massachusetts, USA) for providing us with D3 ES cells, and H. Rayburn (Massachusetts Institute of Technology) for advice on ES cell culture. We thank T. Minamino (The Children’s Hospital) for his assistance with the hypoxic chamber setup, and J. Witztum for providing the MAL-2 antibody. We also thank S. Folta, P. Marria, D. Zhang, D. Tenenholz, A. Patel, C. Sim, and B. Ith for technical assistance, and T. McVarish for editorial assistance.

This work was supported in part by an American Heart Association Grant-in-Aid (to M.A. Perrella); National Institutes of Health grants HL-60788 (to M.A. Perella), HL-53249 (to M.-E. Lee), HL-10113 (to M.D. Layne), and HL-55454 and HL-56398 (to S. Kourembanas); a grant from Novartis and the SICPA Foundation (to P. Wiesel); and a grant from the CHRC New Project Development Fund of the National Institute of Child Health and Human Development (to H. Christou).

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