This Article Abstract Full Text (PDF) Data Supplement All Versions of this Article: 100/6/894    most recent 01.RES.0000261657.76299.ffv1 Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Permissions Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Doerries, C. Articles by Landmesser, U. Search for Related Content PubMed PubMed Citation Articles by Doerries, C. Articles by Landmesser, U. Related Collections Congestive Remodeling Heart failure - basic studies Oxidant stress

(Circulation Research. 2007;100:894.)
© 2007 American Heart Association, Inc.

Integrative Physiology

Critical Role of the NAD(P)H Oxidase Subunit p47^phox for Left Ventricular Remodeling/Dysfunction and Survival After Myocardial Infarction

Carola Doerries, Karsten Grote, Denise Hilfiker-Kleiner, Maren Luchtefeld, Arnd Schaefer, Steven M. Holland, Sajoscha Sorrentino, Costantina Manes, Bernhard Schieffer, Helmut Drexler, Ulf Landmesser

From the Abteilung Kardiologie und Angiologie (C.D., K.G., D.H.-K., M.L., A.S., S.S., C.M., B.S., H.D., U.L.), Medizinische Hochschule Hannover, Germany; and Laboratory of Clinical Infectious Diseases (S.H.), National Institutes of Health, Bethesda, Md.

Correspondence to Ulf Landmesser, MD, Medizinische Hochschule Hannover, Abteilung Kardiologie und Angiologie, Carl Neuberg Str. 1, 30625 Hannover, Germany. E-mail Landmesser.Ulf{at}mh-hannover.de

	Abstract

Top Abstract Introduction Materials and Methods Results Discussion References

 
Accumulating evidence suggests a critical role of increased reactive oxygen species production for left ventricular (LV) remodeling and dysfunction after myocardial infarction (MI). An increased myocardial activity of the NAD(P)H oxidase, a major oxidant enzyme system, has been observed in human heart failure; however, the role of the NAD(P)H oxidase for LV remodeling and dysfunction after MI remains to be determined. MI was induced in wild-type (WT) mice (n=46) and mice lacking the cytosolic NAD(P)H oxidase component p47^phox (p47^phox–/– mice) (n=32). Infarct size was similar among the groups. NAD(P)H oxidase activity was markedly increased in remote LV myocardium of WT mice after MI as compared with sham-operated mice (83±8 versus 16.7±3.5 nmol of O₂^{– ·}µg^–1·min^–1; P<0.01) but not in p47^phox–/– mice after MI (13.5±3.6 versus 15.5±3.5 nmol of O₂^{– ·}µg^–1·min^–1), as assessed by electron-spin resonance spectroscopy using the spin probe CP-H. Furthermore, increased myocardial xanthine oxidase activity was observed in WT, but not in p47^phox–/– mice after MI, suggesting NAD(P)H oxidase–dependent xanthine oxidase activation. Myocardial reactive oxygen species production was increased in WT mice, but not in p47^phox–/– mice, after MI. LV cavity dilatation and dysfunction 4 weeks after MI were markedly attenuated in p47^phox–/– mice as compared with WT mice, as assessed by echocardiography (LV end-diastolic diameter: 4.5±0.2 versus 6.3±0.3 mm, P<0.01; LV ejection fraction, 35.8±2.5 versus 22.6±4.4%, P<0.05). Furthermore, cardiomyocyte hypertrophy, apoptosis, and interstitial fibrosis were substantially reduced in p47^phox–/– mice as compared with WT mice. Importantly, the survival rate was markedly higher in p47^phox–/– mice as compared with WT mice after MI (72% versus 48%; P<0.05). These results suggest a pivotal role of NAD(P)H oxidase activation and its subunit p47^phox for LV remodeling/dysfunction and survival after MI. The NAD(P)H oxidase system represents therefore a potential novel therapeutic target to prevent cardiac failure after MI.

Key Words: myocardial infarction • remodeling • heart failure • NAD(P)H oxidase • superoxide anion

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion References

 
Left ventricular (LV) remodeling processes after myocardial infarction (MI) remain a major challenge and contribute to development and progression of heart failure.¹ Reactive oxygen species (ROS) have been implicated in LV remodeling and dysfunction.^2–4 Treatment with structurally different antioxidants prevented LV remodeling and dysfunction after MI.^2–4 However, the pathophysiologically important enzymatic sources leading to increased myocardial ROS production after MI remain to be further characterized. Notably, a markedly increased myocardial activity of the NAD(P)H oxidase, an important oxidant enzyme system, has recently been observed in human failing as compared with nonfailing hearts, raising the question of the relevance of this enzyme system for LV remodeling and dysfunction after MI.^5,6

Interestingly, NAD(P)H oxidase activity is significantly enhanced by several stimuli relevant to the pathophysiology of heart failure, such as mechanical stretch,^7,8 angiotensin II,^9–11 -adrenergic agonists,¹² endothelin-1,¹³ or tumor necrosis factor-.^14,15 We and others have recently shown that membrane translocation of the NAD(P)H oxidase subunit p47^phox is important for activation of the enzyme in response to angiotensin II.^10,16,17 Moreover, recent data suggest a potential interaction of the NAD(P)H oxidase with the xanthine oxidase (XO) system,¹⁸ an oxidant enzyme that has also been shown to be activated in experimental and human heart failure.^19–21

In the present study, we therefore examined the role of NAD(P)H oxidase activation for LV remodeling and dysfunction and survival after MI. Furthermore, we assessed the role of NAD(P)H oxidase for XO activation after MI.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion References

 
MI was induced by permanent ligation of the left anterior descending coronary artery, as described previously,^22,23 in male wild-type (WT) mice and mice lacking the cytosolic NAD(P)H oxidase component p47^phox (p47^phox–/– mice),^16,24 backcrossed more than 10 times to C57BL/6 background. Mice were 12 to 20 weeks of age. All groups of mice were housed under specific pathogen-reduced conditions receiving autoclaved chow and water ad libitum containing antibiotics (Cotrimoxazole; approximately 30 mg/kg). Electron-spin resonance (ESR) spectroscopy analysis, hemodynamic measurements, echocardiographic and histomorphometric analysis, and immunohistochemistry were performed 30 days after MI. Only mice with extensive MI (>30%) were included in the protocol.

Furthermore, to determine the relative contribution of NAD(P)H oxidase and XO to LV remodeling and dysfunction after MI, both WT and p47^phox–/– mice were randomized to treatment with the XO inhibitor allopurinol (20 mg·kg^–1·d^–1) or vehicle (0.5% methylcellulose) by gavage for 4 weeks starting on day 1 after MI (n=6 to 10). Of note, we have previously observed that this dose of allopurinol effectively inhibits myocardial XO activity in mice after MI.²³ All animal experiments were approved by the local committee on animal research.

An expanded Materials and Methods section is available in the online data supplement at http://circres.ahajournals.org.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
ESR Spectroscopic Analysis of Myocardial NAD(P)H Oxidase and XO Activities After MI
NAD(P)H oxidase activity, as determined by ESR spectroscopic analysis, was substantially increased in the remote LV myocardium of WT mice after MI as compared with sham-operated mice, but not in p47^phox–/– mice after MI (Figure 1A). Representative ESR scans of myocardial NAD(P)H oxidase activity in WT and p47^phox–/– mice after MI are shown in Figure 1B.

View larger version (40K): [in this window] [in a new window]   Figure 1. A, NAD(P)H oxidase activity of LV myocardium of sham- and MI-operated WT and p47phox–/– mice (n=5 to 6). B, Representative ESR spectra of NAD(P)H oxidase activity of LV from sham- and MI-operated mice (WT and p47phox–/–). C, Expression of the other NAD(P)H oxidase subunits: p22phox, Nox2 (gp91phox), p67phox, and Rac1. Representative Western blots of the subunits are shown at the bottom; GAPDH served as equal protein loading control. D, XO activity of LV myocardium of sham- and MI-operated WT and p47phox–/– mice (n=5 to 6). E, Measurement of superoxide production in LV myocardium of sham- and MI-operated WT and p47phox–/– mice (n=5 to 8) by lucigenin-enhanced chemiluminescence. F, Detection of LV superoxide production by dihydroethidium staining in sham- and MI-operated mice. Data are representative of 3 separate experiments. *P<0.05 vs sham WT, **P<0.01 vs sham WT, $P<0.05 vs sham p47phox–/–.

We and others have recently observed that myocardial XO activity is increased after MI.^23,25 Moreover, recent in vitro data have suggested that NAD(P)H oxidase activation may trigger XO activity.^18,26 We therefore determined myocardial XO activity in WT and p47^phox–/– mice after MI. These measurements revealed a markedly reduced myocardial XO activation in p47^phox–/– mice as compared with WT mice after MI (Figure 1D), suggesting that myocardial XO activation after MI is NAD(P)H oxidase dependent.

To further confirm specificity of NAD(P)H oxidase and XO activity measurements by ESR spectroscopy using the spin probe CP-H, superoxide dismutase (SOD) (100 U/mL), diphenyleneiodonium chloride (DPI) (25 µmol/L), or the XO inhibitor oxypurinol (10 mmol/L) were added to LV samples from WT mice after MI. Addition of SOD and DPI completely abolished NADPH-stimulated CP* formation in LV samples after MI (SOD versus DPI versus without inhibitor: 5±4 versus 6±1 versus 83±8 nmol of O₂^{– ·}µg^–1·min^–1; P<0.01). Importantly, NADPH-stimulated 3-carboxy-proxyl radical (CP*) formation was not changed after addition of oxypurinol (83±8 versus 90±8 nmol of O₂^{– ·}µg^–1·min^–1; P=NS). Xanthine-stimulated CP* formation was completely inhibited by both addition of SOD and oxypurinol to LV samples from mice after MI (SOD versus oxypurinol versus without inhibitor: 7±5 versus 4±2 versus 77±11 nmol of O₂^{– ·}µg^–1·min^–1; P<0.01).

Western Blot Analysis of NAD(P)H Oxidase Subunit Expression
Protein expression of the other NAD(P)H oxidase subunits after MI was analyzed by Western blotting. The myocardial expression of the membrane subunits p22^phox and gp91^phox and the cytosolic subunit p67^phox were significantly increased in both WT and p47^phox–/– mice after MI (Figure 1C). The expression of the GTP protein rac1 was elevated in WT mice after MI, but not in p47^phox–/– mice after MI (Figure 1C).

Myocardial Superoxide Production After MI
Myocardial superoxide production, as assessed by lucigenin-enhanced chemiluminescence and dihydroethidium fluorescence staining using confocal microscopy was markedly increased in remote LV myocardium of WT mice after MI, but not in p47^phox–/– mice after MI (Figure 1E and 1F).

Echocardiographic Analysis and Hemodynamic Measurements
LV cavity dilation after MI was substantially reduced in p47^phox–/– mice as compared with WT mice (LV end-diastolic diameter: p47^phox–/– versus WT 4.5±0.2 versus 6.3±0.3 mm; P<0.01; Figure 2A). Furthermore, LV systolic function as assessed by LV ejection fraction (Figure 2C) or LV fractional shortening (Table) was better preserved in p47^phox–/– mice as compared with WT mice after MI.

View larger version (23K): [in this window] [in a new window]   Figure 2. A, LV end-diastolic diameter of sham- and MI-operated WT and p47phox–/– mice (n=9 to 10). B, Representative M-mode echocardiograms obtained of sham- and MI-operated mice. C, LV ejection fraction of sham- and MI-operated WT mice and p47phox–/– mice (n=9 to 10). **P<0.01 vs sham WT, $$P<0.01 vs sham p47phox–/–.

View this table: [in this window] [in a new window]   Table 1. Echocardiographic and Hemodynamic Parameters and Histomorphometric Analysis in WT and p47phox–/– Mice After Sham or MI Operation

LV dP/dt_min was better preserved in p47^phox–/– mice as compared with WT mice after MI (Table). LV dP/dt_max was significantly reduced in WT and p47^phox–/– mice after MI. There was a trend for a better-preserved LV dP/dt_max in p47^phox–/– as compared with WT mice after MI that, however, did not reach statistical significance. Mean arterial pressure after MI as measured invasively by the micromanometer conductance catheter was not significantly different between WT and p47^phox–/– mice (Table).

LV Morphology and Morphometry Analysis
Myocardial hypertrophy after MI, as assessed by cardiomyocyte cross-sectional area, was largely prevented in p47^phox–/– mice as compared with WT mice (Figure 3A). Similarly, there was a significant increase of LV weight in WT mice after MI that was largely blunted in p47^phox–/– mice after MI (Figure 3C). Furthermore, mRNA expression of the cardiac hypertrophy marker skeletal muscle -actin was only increased in WT mice after MI, but not in p47^phox–/– mice after MI (Figure 3D and 3E).

View larger version (48K): [in this window] [in a new window]   Figure 3. A, Cardiomyocyte cross-sectional area (CSA) of sham- and MI-operated WT and p47phox–/– mice (n=5 to 8). B, Representative high-power photomicrographs of hematoxylin/eosin-stained and wheat germ agglutinin–stained (red) LV cross-sections, with nuclei stained with Hoechst (blue). C, LV weight of sham- and MI-operated WT and p47phox–/– mice. D, Northern blot analysis of the cardiac hypertrophy marker skeletal muscle -actin of sham- and MI-operated WT and p47phox–/– mice (n=3 to 5). E, Representative Northern blot analysis of the cardiac hypertrophy marker skeletal muscle (SkM) -actin in sham- and MI-operated WT and p47phox–/– mice. F, Low-power photomicrographs of hematoxylin/eosin-stained LV cross-sections obtained from WT and p47phox–/– mice after MI. G, Relative interstitial collagen fraction of sham- and MI-operated WT and p47phox–/– mice (n=10 to 12). H, Representative high-power photomicrographs of Sirius red–stained remote LV myocardium from MI-operated WT and p47phox–/– mice. *P<0.05 vs sham WT, **P<0.01 vs sham WT, $P<0.05 vs sham p47phox–/–, $$P<0.01 vs sham p47phox–/–.

The interstitial collagen volume fraction was increased to a greater extent in WT mice as compared with p47^phox–/– mice after MI (Table and Figure 3G and 3H). The infarct size was similar between both groups (Table).

Apoptosis Detection in LV Myocardium After MI
The number of apoptotic nuclei as detected by TUNEL staining was significantly reduced in the LV border zone of p47^phox–/– mice as compared with WT mice after MI (Figure 4A and 4B). Similarly, the analysis of cleaved caspase-3 revealed reduced levels of the large fragment (19 kDa) of activated caspase-3 in the LV border zone of p47^phox–/– mice as compared with WT mice after MI (Figure 4C and 4D). There was no significant change of uncleaved caspase-3 between WT and p47^phox-deficient mice after MI, suggesting that a NAD(P)H oxidase–dependent pathway is involved in caspase-3 activation after MI (Figure 4D).

View larger version (26K): [in this window] [in a new window]   Figure 4. Myocardial apoptosis after MI. A, Number of TUNEL-positive nuclei in sham- and MI-operated WT and p47phox–/– mice (n=4 to 7). B, Representative LV sections demonstrating TUNEL-positive nuclei (marked by arrows) in the infarct border zone of WT and p47phox–/– mice after MI. C, Western blot analysis of cleaved caspase-3 in sham- and MI-operated WT and p47phox–/– mice (n=4 to 5). D, Representative Western blot analysis of the large fragment (19 kDa) of cleaved caspase-3 and uncleaved caspase-3 (35 kDa) in the infarct border zone of WT and p47phox–/– mice. E, Gelatin zymography analysis of MMP-2 in sham- and MI-operated WT and p47phox–/– mice (n=6 to 7). F, Representative gelatin zymography analysis of myocardial MMP-2 in sham- and MI-operated WT and p47phox–/– mice. *P<0.05 vs sham WT, **P<0.01 vs sham WT, $P<0.05 vs sham p47phox–/–, $$P<0.01 vs sham p47phox–/–.

Matrix Metalloproteinase-2 Gelatinolytic Activity After MI
The echocardiographic analysis demonstrated a markedly reduced LV dilatation after MI in p47^phox–/– mice as compared with WT mice. Notably, we have previously observed an NAD(P)H oxidase–dependent activation of matrix metalloproteinase (MMP)-2 by mechanical stretch and angiotensin II in cultured vascular smooth muscle cells.^27,28 Furthermore, MMP-2–deficient mice have previously been shown to have a reduced LV dilatation after MI.²⁹ We therefore postulated that NAD(P)H oxidase is involved in MMP-2 activation after MI and determined MMP-2 gelatinolytic activity in p47^phox–/– and WT mice after MI. These studies revealed a significantly reduced gelatinolytic activity of MMP-2 in the remote LV myocardium of p47^phox–/– mice as compared with WT mice 5 days after MI (Figure 4E and 4F).

ESR Spectroscopic Analysis of NO Production After MI
NO production was similar in aortas of sham-operated WT and p47^phox–/– mice. NO production was reduced in WT mice and p47^phox–/– mice after MI. Importantly, however, NO production was markedly better preserved in p47^phox–/– as compared with WT mice after MI (Figure 5A). Representative ESR scans of vascular NO production of WT and p47^phox–/– mice are shown in Figure 5B.

View larger version (18K): [in this window] [in a new window]   Figure 5. A, NO production of whole aortas of sham- and MI-operated WT and p47phox–/– mice, as determined by ESR spectroscopy (n=5). B, Representative ESR NO spectra of aortas from sham- and MI-operated mice (WT and p47phox–/–). **P<0.01 vs sham WT; $$P<0.01 vs sham p47phox–/–.

Effect of Treatment With the XO Inhibitor Allopurinol on LV Remodeling and Dysfunction in WT and p47^phox-Deficient Mice After MI
To further assess the relative contribution of NAD(P)H oxidase and XO activation to LV remodeling and dysfunction after MI, both WT and p47^phox–/– mice were treated with allopurinol or vehicle (n=6 to 10). LV cavity dilation and LV dysfunction were significantly reduced in WT mice treated with allopurinol as compared with vehicle-treated mice (Table I in the online data supplement). In contrast, allopurinol treatment had no effect on LV dilation and dysfunction in p47^phox–/– mice after MI (supplemental Table I). Notably, LV cavity dilation and LV systolic function were better preserved in p47^phox–/– mice after MI as compared with allopurinol-treated WT mice, suggesting that prevention of NAD(P)H oxidase activation exerts both XO-dependent and XO-independent beneficial effects after MI (supplemental Table I).

LV weight, cardiomyocyte cross-sectional area, and interstitial collagen volume were reduced after allopurinol treatment in WT mice, but not in allopurinol treated p47^phox–/– mice after MI (supplemental Table I). Infarct size was similar among all groups with vehicle or allopurinol treatment (supplemental Table I).

Survival
Importantly, the survival rate was substantially higher in p47^phox–/– mice as compared with WT mice after MI (72% versus 48%; P<0.05; Figure 6).

View larger version (8K): [in this window] [in a new window]   Figure 6. Kaplan–Meier survival curve analysis for WT (n=46) and p47phox–/– (n=32) mice after MI.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

 
The present study provides direct evidence that a deficiency of the NAD(P)H oxidase and its subunit p47^phox protects from LV remodeling and dysfunction after MI. p47^phox deficiency reduced LV cavity dilatation and dysfunction as well as cardiomyocyte hypertrophy, apoptosis, and interstitial fibrosis after MI. All of these beneficial effects may have contributed to improved survival of p47^phox-deficient mice after MI. NAD(P)H oxidase activation was critical for increased myocardial ROS production after MI. Furthermore, our ESR spectroscopic analysis suggests that NAD(P)H oxidase triggers the increase of myocardial XO activity after MI that further augments myocardial oxidant stress.

Experimental and clinical studies have demonstrated increased ROS generation in failing hearts that was related to LV dysfunction and dilatation.^30,31 Accumulating evidence suggests that ROS may play a major role in the development and progression of LV remodeling and dysfunction. However, the pathophysiologically relevant source of increased myocardial ROS production after MI remains to be further characterized.

Of note, recent studies have demonstrated increased cardiac NAD(P)H oxidase activity in human heart failure.^5,6 Several studies have shown that NAD(P)H oxidase is expressed in vascular and myocardial cells in both rodents and humans.^5,6,32–34 Notably, the NAD(P)H oxidase system is activated by stimuli relevant to the pathophysiology of heart failure, ie, angiotensin II, -adrenergic agonists, tumor necrosis factor-, and increased mechanical stretch.^7–15,32 These studies have raised the question of the pathophysiological relevance of NAD(P)H oxidase activation for LV remodeling and dysfunction after MI. In the present study, mice deficient in the NAD(P)H oxidase subunit p47^phox had a markedly reduced myocardial oxidant stress and LV remodeling and dysfunction after MI. Of note, direct exposure of cardiomyocytes to ROS has been shown to promote hypertrophy and apoptosis,^35,36 so that it is conceivable that the reduction of myocardial oxidant stress in p47^phox–/– mice may have directly contributed to prevention of cardiomyocyte hypertrophy and apoptosis. Moreover, exposure of cardiomyocytes to superoxide anions has been shown to induce a contractile dysfunction, suggesting that, in particular, a prevention of the increase in myocardial superoxide production after MI may have preserved LV function in p47^phox–/– mice.^37–40

In addition to direct effects of ROS on LV remodeling and dysfunction, a reduced interaction of ROS with nitric oxide (NO), in particular, with endothelial NO synthase–derived NO, may contribute importantly to beneficial effects of NAD(P)H oxidase inhibition after MI.⁴¹ Notably, increased vascular inactivation of NO by superoxide has been observed in experimental heart failure after MI.⁴² Preserved NO availability, as observed in p47^phox–/– mice after MI in the present study, therefore likely represents an additional mechanism whereby reduced NADPH oxidase activity exerts beneficial effects after MI.^22,41

Of note, recent studies have demonstrated increased cardiac and vascular XO activity in experimental and human heart failure, another major source of ROS.^{19,20,23,25,43} The present study suggests that the increase in myocardial XO activity after MI is dependent on the NAD(P)H oxidase system. In this respect, recent in vitro data have suggested that XO may be activated in a redox-sensitive manner, dependent on the NAD(P)H oxidase.^18,26 This concept is strongly supported by our observations in vivo that p47^phox–/– mice had no increase of myocardial XO activity after MI, as suggested by ESR spectroscopic analysis.

Notably, in recent studies, we and others have observed beneficial effects of XO inhibition on LV dysfunction and remodeling.^23,25,43 To further assess the relative contribution of NAD(P)H oxidase and XO to LV remodeling and dysfunction after MI in the present study, both WT and p47^phox–/– mice were randomized to treatment with the XO inhibitor allopurinol or vehicle. Of note, we have previously observed that this dose of allopurinol results in an effective inhibition of myocardial XO activity in mice after MI.²³ The results of these studies suggest that NAD(P)H oxidase activation after MI promotes LV remodeling and dysfunction both by XO-dependent and XO-independent effects. In addition to XO activation, NAD(P)H oxidase–derived superoxide may directly contribute to LV remodeling and dysfunction or may trigger activation of other sources of oxygen radicals, ie, uncoupling of endothelial NO synthase⁴⁴ or increase mitochondrial ROS production. In this respect, it has recently been suggested that hydroxyl radicals, originated from superoxide anions, cause mitochondrial damage that may represent another mechanism whereby increased superoxide production may stimulate myocardial ROS production and augment myocardial damage.⁴⁵ This concept is further supported by a recent study demonstrating that overexpression of the mitochondrial antioxidant peroxiredoxin-3 (Prx-3) protected mice from mitochondrial dysfunction and LV failure after MI.⁴⁶

Of note, in recent in vitro studies, we have demonstrated that MMP-2 activation in response to mechanical stretch or angiotensin II is prevented in vascular smooth muscle cells deficient in p47^phox.^27,28 In the present study, MMP-2 activation was reduced in p47^phox–/– mice as compared with WT mice in vivo, which may have contributed to prevention of LV dilatation after MI in these mice. This concept is supported by recent observations in MMP-2–deficient mice, which had a reduced LV dilatation after MI.²⁹ Theoretically, a reduction of the proteolytic activity of the gelatinase MMP-2 should lead to an accumulation of substrates, in particular, collagens. In the present study, however, there was a reduced interstitial fibrosis in p47^phox–/– mice after MI. The present study is, therefore, in line with previous studies⁴⁷ suggesting that MMP-2 is not only involved in degradation of collagens but also has additional functions in cardiac remodeling. For example, MMP-2 has been suggested to contribute to tissue invasion of inflammatory cells, which may promote interstitial fibrosis.⁴⁷

In contrast to the present study, a recent study by Frantz et al does not indicate a protective effect of a deficiency of the membranous NAD(P)H oxidase subunit gp91^phox on LV remodeling and dysfunction after MI.⁴⁸ Notably, however, these authors do not observe a reduced myocardial ROS production in gp91^phox-deficient mice after MI and demonstrate that Nox 1, a homolog of gp91^phox, is upregulated in these mice. A compensation for gp91^phox by gp91^phox homologs, therefore, represents 1 potential explanation for the different findings as compared with the present study using p47^phox-deficient mice. Similarly, in 2 recent studies analyzing mice with "pressure overload" hypertrophy, deficiency of gp91^phox did not inhibit cardiac hypertrophy.^49,50 However, gp91^phox–/– mice still had an increased myocardial NAD(P)H oxidase activity after "pressure overload," suggesting a compensation of gp91^phox by other homologs (Nox 1 and 4) of this NAD(P)H oxidase subunit.⁴⁹

Moreover, there are several cell types in the myocardium that express NAD(P)H oxidase and its subunit p47^phox, ie, cardiomyocytes, endothelial cells, infiltrating neutrophils, and fibroblasts. A conditional knockout approach or a cell-specific transgene restoration of NADPH oxidase subunits in NAD(P)H oxidase–deficient mice will be required to determine the relative contribution of NAD(P)H oxidase in these different cell types to LV remodeling and dysfunction after MI.

Furthermore, Grote et al have recently suggested that young p47^phox-deficient mice have an increased systolic blood pressure as measured by the tail cuff method.⁵¹ In the present study, we determined mean arterial pressure after MI that was similar in p47^phox-deficient and WT mice, as measured by the micromanometer conductance catheter. The different methods used (ie, invasive versus noninvasive measurement) may, at least in part, explain these different findings. A higher blood pressure, however, would also be unlikely to contribute to reduced LV remodeling and dysfunction in p47^phox-deficient mice after MI.

In summary, deficiency of the NAD(P)H oxidase subunit p47^phox prevented LV remodeling and dysfunction after MI and reduced cardiomyocyte hypertrophy, apoptosis, and interstitial fibrosis, which was associated with an improved survival. Importantly, p47^phox deficiency also prevented myocardial XO activation after MI, thereby providing evidence that this enzyme is activated in a redox-sensitive manner in vivo. Treatment strategies that reduce NAD(P)H oxidase activation may therefore represent a novel option to prevent cardiac failure after MI.

	Acknowledgments

 
We thank Silvia Gutzke and Silke Pretzer for excellent technical assistance.

Sources of Funding

This work was supported by the Deutsche Forschungsgemeinschaft (LA 1432/3-1).

Disclosures

None.

	Footnotes

 
Original received July 20, 2006; revision received January 4, 2007; accepted February 19, 2007.

	References

Top Abstract Introduction Materials and Methods Results Discussion References

 
1. Pfeffer MA, Braunwald E. Ventricular remodeling after myocardial infarction. Experimental observations and clinical implications. Circulation. 1990; 81: 1161–1172.[Abstract/Free Full Text]

2. Kinugawa S, Tsutsui H, Hayashidani S, Ide T, Suematsu N, Satoh S, Utsumi H, Takeshita A. Treatment with dimethylthiourea prevents left ventricular remodeling and failure after experimental myocardial infarction in mice: role of oxidative stress. Circ Res. 2000; 87: 392–398.[Abstract/Free Full Text]

3. Sia YT, Lapointe N, Parker TG, Tsoporis JN, Deschepper CF, Calderone A, Pourdjabbar A, Jasmin JF, Sarrazin JF, Liu P, Adam A, Butany J, Rouleau JL. Beneficial effects of long-term use of the antioxidant probucol in heart failure in the rat. Circulation. 2002; 105: 2549–2555.[Abstract/Free Full Text]

4. Sawyer DB, Siwik DA, Xiao L, Pimentel DR, Singh K, Colucci WS. Role of oxidative stress in myocardial hypertrophy and failure. J Mol Cell Cardiol. 2002; 34: 379–388.[CrossRef][Medline] [Order article via Infotrieve]

5. Heymes C, Bendall JK, Ratajczak P, Cave AC, Samuel JL, Hasenfuss G, Shah AM. Increased myocardial NADPH oxidase activity in human heart failure. J Am Coll Cardiol. 2003; 41: 2164–2171.[Abstract/Free Full Text]

6. Maack C, Kartes T, Kilter H, Schafers HJ, Nickenig G, Bohm M, Laufs U. Oxygen free radical release in human failing myocardium is associated with increased activity of rac1-GTPase and represents a target for statin treatment. Circulation. 2003; 108: 1567–1574.[Abstract/Free Full Text]

7. Hishikawa K, Luscher TF. Pulsatile stretch stimulates superoxide production in human aortic endothelial cells. Circulation. 1997; 96: 3610–3616.[Abstract/Free Full Text]

8. Matsushita H, Lee Kh K, Tsao PS. Cyclic strain induces reactive oxygen species production via an endothelial NAD(P)H oxidase. J Cell Biochem. 2001; 81: 99–106.[CrossRef]

9. Griendling KK, Minieri CA, Ollerenshaw JD, Alexander RW. Angiotensin II stimulates NADH and NADPH oxidase activity in cultured vascular smooth muscle cells. Circ Res. 1994; 74: 1141–1148.[Abstract/Free Full Text]

10. Li JM, Shah AM. Mechanism of endothelial cell NADPH oxidase activation by angiotensin II. Role of the p47phox subunit. J Biol Chem. 2003; 278: 12094–12100.[Abstract/Free Full Text]

11. Touyz RM, Chen X, Tabet F, Yao G, He G, Quinn MT, Pagano PJ, Schiffrin EL. Expression of a functionally active gp91phox-containing neutrophil-type NAD(P)H oxidase in smooth muscle cells from human resistance arteries: regulation by angiotensin II. Circ Res. 2002; 90: 1205–1213.[Abstract/Free Full Text]

12. Xiao L, Pimentel DR, Wang J, Singh K, Colucci WS, Sawyer DB. Role of reactive oxygen species and NAD(P)H oxidase in alpha(1)-adrenoceptor signaling in adult rat cardiac myocytes. Am J Physiol Cell Physiol. 2002; 282: C926–C934.[Abstract/Free Full Text]

13. Duerrschmidt N, Wippich N, Goettsch W, Broemme HJ, Morawietz H. Endothelin-1 induces NAD(P)H oxidase in human endothelial cells. Biochem Biophys Res Commun. 2000; 269: 713–717.[CrossRef][Medline] [Order article via Infotrieve]

14. De Keulenaer GW, Alexander RW, Ushio-Fukai M, Ishizaka N, Griendling KK. Tumour necrosis factor alpha activates a p22phox-based NADH oxidase in vascular smooth muscle. Biochem J. 1998; 329 (pt 3): 653–657.[Medline] [Order article via Infotrieve]

15. Li JM, Mullen AM, Yun S, Wientjes F, Brouns GY, Thrasher AJ, Shah AM. Essential role of the NADPH oxidase subunit p47(phox) in endothelial cell superoxide production in response to phorbol ester and tumor necrosis factor-alpha. Circ Res. 2002; 90: 143–150.[Abstract/Free Full Text]

16. Landmesser U, Dikalov S, Price SR, McCann L, Fukai T, Holland SM, Mitch WE, Harrison DG. Oxidation of tetrahydrobiopterin leads to uncoupling of endothelial cell nitric oxide synthase in hypertension. J Clin Invest. 2003; 111: 1201–1209.[CrossRef][Medline] [Order article via Infotrieve]

17. Landmesser U, Cai H, Dikalov S, McCann L, Hwang J, Jo H, Holland SM, Harrison DG. Role of p47(phox) in vascular oxidative stress and hypertension caused by angiotensin II. Hypertension. 2002; 40: 511–515.[Abstract/Free Full Text]

18. McNally JS, Saxena A, Cai H, Dikalov S, Harrison DG. Regulation of xanthine oxidoreductase protein expression by hydrogen peroxide and calcium. Arterioscler Thromb Vasc Biol. 2005; 25: 1623–1628.[Abstract/Free Full Text]

19. Cappola TP, Kass DA, Nelson GS, Berger RD, Rosas GO, Kobeissi ZA, Marban E, Hare JM. Allopurinol improves myocardial efficiency in patients with idiopathic dilated cardiomyopathy. Circulation. 2001; 104: 2407–2411.[Abstract/Free Full Text]

20. Landmesser U, Spiekermann S, Dikalov S, Tatge H, Wilke R, Kohler C, Harrison DG, Hornig B, Drexler H. Vascular oxidative stress and endothelial dysfunction in patients with chronic heart failure: role of xanthine-oxidase and extracellular superoxide dismutase. Circulation. 2002; 106: 3073–3078.[Abstract/Free Full Text]

21. Naumova AV, Chacko VP, Ouwerkerk R, Stull L, Marban E, Weiss RG. Xanthine oxidase inhibitors improve energetics and function after infarction in failing mouse hearts. Am J Physiol Heart Circ Physiol. 2006; 290: H837–H843.[Abstract/Free Full Text]

22. Landmesser U, Engberding N, Bahlmann FH, Schaefer A, Wiencke A, Heineke A, Spiekermann S, Hilfiker-Kleiner D, Templin C, Kotlarz D, Mueller M, Fuchs M, Hornig B, Haller H, Drexler H. Statin-induced improvement of endothelial progenitor cell mobilization, myocardial neovascularization, left ventricular function, and survival after experimental myocardial infarction requires endothelial nitric oxide synthase. Circulation. 2004; 110: 1933–1939.[Abstract/Free Full Text]

23. Engberding N, Spiekermann S, Schaefer A, Heineke A, Wiencke A, Muller M, Fuchs M, Hilfiker-Kleiner D, Hornig B, Drexler H, Landmesser U. Allopurinol attenuates left ventricular remodeling and dysfunction after experimental myocardial infarction: a new action for an old drug? Circulation. 2004; 110: 2175–2179.[Abstract/Free Full Text]

24. Jackson SH, Gallin JI, Holland SM. The p47phox mouse knock-out model of chronic granulomatous disease. J Exp Med. 1995; 182: 751–758.[Abstract/Free Full Text]

25. Stull LB, Leppo MK, Szweda L, Gao WD, Marban E. Chronic treatment with allopurinol boosts survival and cardiac contractility in murine postischemic cardiomyopathy. Circ Res. 2004; 95: 1005–1011.[Abstract/Free Full Text]

26. McNally JS, Davis ME, Giddens DP, Saha A, Hwang J, Dikalov S, Jo H, Harrison DG. Role of xanthine oxidoreductase and NAD(P)H oxidase in endothelial superoxide production in response to oscillatory shear stress. Am J Physiol Heart Circ Physiol. 2003; 285: H2290–H2297.[Abstract/Free Full Text]

27. Grote K, Flach I, Luchtefeld M, Akin E, Holland SM, Drexler H, Schieffer B. Mechanical stretch enhances mRNA expression and proenzyme release of matrix metalloproteinase-2 (MMP-2) via NAD(P)H oxidase-derived reactive oxygen species. Circ Res. 2003; 92: e80–e86.[CrossRef][Medline] [Order article via Infotrieve]

28. Luchtefeld M, Grote K, Grothusen C, Bley S, Bandlow N, Selle T, Struber M, Haverich A, Bavendiek U, Drexler H, Schieffer B. Angiotensin II induces MMP-2 in a p47phox-dependent manner. Biochem Biophys Res Commun. 2005; 328: 183–188.[CrossRef][Medline] [Order article via Infotrieve]

29. Hayashidani S, Tsutsui H, Ikeuchi M, Shiomi T, Matsusaka H, Kubota T, Imanaka-Yoshida K, Itoh T, Takeshita A. Targeted deletion of MMP-2 attenuates early LV rupture and late remodeling after experimental myocardial infarction. Am J Physiol Heart Circ Physiol. 2003; 285: H1229–H1235.[Abstract/Free Full Text]

30. Ide T, Tsutsui H, Kinugawa S, Suematsu N, Hayashidani S, Ichikawa K, Utsumi H, Machida Y, Egashira K, Takeshita A. Direct evidence for increased hydroxyl radicals originating from superoxide in the failing myocardium. Circ Res. 2000; 86: 152–157.[Abstract/Free Full Text]

31. Mallat Z, Philip I, Lebret M, Chatel D, Maclouf J, Tedgui A. Elevated levels of 8-iso-prostaglandin F2alpha in pericardial fluid of patients with heart failure: a potential role for in vivo oxidant stress in ventricular dilatation and progression to heart failure. Circulation. 1998; 97: 1536–1539.[Abstract/Free Full Text]

32. Griendling KK, Sorescu D, Ushio-Fukai M. NAD(P)H oxidase: role in cardiovascular biology and disease. Circ Res. 2000; 86: 494–501.[Abstract/Free Full Text]

33. Gao L, Wang W, Li YL, Schultz HD, Liu D, Cornish KG, Zucker IH. Superoxide mediates sympathoexcitation in heart failure: roles of angiotensin II and NAD(P)H oxidase. Circ Res. 2004; 95: 937–944.[Abstract/Free Full Text]

34. Li JM, Gall NP, Grieve DJ, Chen M, Shah AM. Activation of NADPH oxidase during progression of cardiac hypertrophy to failure. Hypertension. 2002; 40: 477–484.[Abstract/Free Full Text]

35. Siwik DA, Tzortzis JD, Pimental DR, Chang DL, Pagano PJ, Singh K, Sawyer DB, Colucci WS. Inhibition of copper-zinc superoxide dismutase induces cell growth, hypertrophic phenotype, and apoptosis in neonatal rat cardiac myocytes in vitro. Circ Res. 1999; 85: 147–153.[Abstract/Free Full Text]

36. Pimentel DR, Amin JK, Xiao L, Miller T, Viereck J, Oliver-Krasinski J, Baliga R, Wang J, Siwik DA, Singh K, Pagano P, Colucci WS, Sawyer DB. Reactive oxygen species mediate amplitude-dependent hypertrophic and apoptotic responses to mechanical stretch in cardiac myocytes. Circ Res. 2001; 89: 453–460.[Abstract/Free Full Text]

37. Flesch M, Maack C, Cremers B, Baumer AT, Sudkamp M, Bohm M. Effect of beta-blockers on free radical-induced cardiac contractile dysfunction. Circulation. 1999; 100: 346–353.[Abstract/Free Full Text]

38. Gao WD, Liu Y, Marban E. Selective effects of oxygen free radicals on excitation-contraction coupling in ventricular muscle. Implications for the mechanism of stunned myocardium. Circulation. 1996; 94: 2597–2604.[Abstract/Free Full Text]

39. MacFarlane NG, Miller DJ. Depression of peak force without altering calcium sensitivity by the superoxide anion in chemically skinned cardiac muscle of rat. Circ Res. 1992; 70: 1217–1224.[Abstract/Free Full Text]

40. Zeitz O, Maass AE, Van Nguyen P, Hensmann G, Kogler H, Moller K, Hasenfuss G, Janssen PM. Hydroxyl radical-induced acute diastolic dysfunction is due to calcium overload via reverse-mode Na(+)-Ca(2+) exchange. Circ Res. 2002; 90: 988–995.[Abstract/Free Full Text]

41. Hare JM, Stamler JS. NO/redox disequilibrium in the failing heart and cardiovascular system. J Clin Invest. 2005; 115: 509–517.[CrossRef][Medline] [Order article via Infotrieve]

42. Bauersachs J, Bouloumie A, Fraccarollo D, Hu K, Busse R, Ertl G. Endothelial dysfunction in chronic myocardial infarction despite increased vascular endothelial nitric oxide synthase and soluble guanylate cyclase expression: role of enhanced vascular superoxide production. Circulation. 1999; 100: 292–298.[Abstract/Free Full Text]

43. Minhas KM, Saraiva RM, Schuleri KH, Lehrke S, Zheng M, Saliaris AP, Berry CE, Vandegaer KM, Li D, Hare JM. Xanthine oxidoreductase inhibition causes reverse remodeling in rats with dilated cardiomyopathy. Circ Res. 2006; 98: 271–279.[Abstract/Free Full Text]

44. Takimoto E, Champion HC, Li M, Ren S, Rodriguez ER, Tavazzi B, Lazzarino G, Paolocci N, Gabrielson KL, Wang Y, Kass DA. Oxidant stress from nitric oxide synthase-3 uncoupling stimulates cardiac pathologic remodeling from chronic pressure load. J Clin Invest. 2005; 115: 1221–1231.[CrossRef][Medline] [Order article via Infotrieve]

45. Ide T, Tsutsui H, Hayashidani S, Kang D, Suematsu N, Nakamura K, Utsumi H, Hamasaki N, Takeshita A. Mitochondrial DNA damage and dysfunction associated with oxidative stress in failing hearts after myocardial infarction. Circ Res. 2001; 88: 529–535.[Abstract/Free Full Text]

46. Matsushima S, Ide T, Yamato M, Matsusaka H, Hattori F, Ikeuchi M, Kubota T, Sunagawa K, Hasegawa Y, Kurihara T, Oikawa S, Kinugawa S, Tsutsui H. Overexpression of mitochondrial peroxiredoxin-3 prevents left ventricular remodeling and failure after myocardial infarction in mice. Circulation. 2006; 113: 1779–1786.[Abstract/Free Full Text]

47. Panek AN, Bader M. Matrix reloaded: the matrix metalloproteinase paradox. Hypertension. 2006; 47: 640–641.[Free Full Text]

48. Frantz S, Brandes RP, Hu K, Rammelt K, Wolf J, Scheuermann H, Ertl G, Bauersachs J. Left ventricular remodeling after myocardial infarction in mice with targeted deletion of the NADPH oxidase subunit gp91PHOX. Basic Res Cardiol. 2006; 101: 127–132.[CrossRef][Medline] [Order article via Infotrieve]

49. Byrne JA, Grieve DJ, Bendall JK, Li JM, Gove C, Lambeth JD, Cave AC, Shah AM. Contrasting roles of NADPH oxidase isoforms in pressure-overload versus angiotensin II-induced cardiac hypertrophy. Circ Res. 2003; 93: 802–805.[Abstract/Free Full Text]

50. Maytin M, Siwik DA, Ito M, Xiao L, Sawyer DB, Liao R, Colucci WS. Pressure overload-induced myocardial hypertrophy in mice does not require gp91phox. Circulation. 2004; 109: 1168–1171.[Abstract/Free Full Text]

51. Grote K, Ortmann M, Salguero G, Doerries C, Landmesser U, Luchtefeld M, Brandes RP, Gwinner W, Tschernig T, Brabant EG, Klos A, Schaefer A, Drexler H, Schieffer B. Critical role for p47phox in renin-angiotensin system activation and blood pressure regulation. Cardiovasc Res. 2006; 71: 596–605.[CrossRef][Medline] [Order article via Infotrieve]

This article has been cited by other articles:

				Z. Kassiri, J. Zhong, D. Guo, R. Basu, X. Wang, P. P. Liu, J. W. Scholey, J. M. Penninger, and G. Y. Oudit Loss of Angiotensin-Converting Enzyme 2 Accelerates Maladaptive Left Ventricular Remodeling in Response to Myocardial Infarction Circ Heart Fail, September 1, 2009; 2(5): 446 - 455. [Abstract] [Full Text] [PDF]

				S. Matsushima, S. Kinugawa, T. Yokota, N. Inoue, Y. Ohta, S. Hamaguchi, and H. Tsutsui Increased myocardial NAD(P)H oxidase-derived superoxide causes the exacerbation of postinfarct heart failure in type 2 diabetes Am J Physiol Heart Circ Physiol, July 1, 2009; 297(1): H409 - H416. [Abstract] [Full Text] [PDF]

				H. Tsutsui, S. Kinugawa, and S. Matsushima Mitochondrial oxidative stress and dysfunction in myocardial remodelling Cardiovasc Res, February 15, 2009; 81(3): 449 - 456. [Abstract] [Full Text] [PDF]

				U. Landmesser, K. C. Wollert, and H. Drexler Potential novel pharmacological therapies for myocardial remodelling Cardiovasc Res, February 15, 2009; 81(3): 519 - 527. [Abstract] [Full Text] [PDF]

				X. W. Cheng, T. Murohara, M. Kuzuya, H. Izawa, T. Sasaki, K. Obata, K. Nagata, T. Nishizawa, M. Kobayashi, T. Yamada, et al. Superoxide-Dependent Cathepsin Activation Is Associated with Hypertensive Myocardial Remodeling and Represents a Target for Angiotensin II Type 1 Receptor Blocker Treatment Am. J. Pathol., August 1, 2008; 173(2): 358 - 369. [Abstract] [Full Text] [PDF]

				J. M. Hare, B. Mangal, J. Brown, C. Fisher Jr, R. Freudenberger, W. S. Colucci, D. L. Mann, P. Liu, M. M. Givertz, R. P. Schwarz, et al. Impact of oxypurinol in patients with symptomatic heart failure. Results of the OPT-CHF study. J. Am. Coll. Cardiol., June 17, 2008; 51(24): 2301 - 2309. [Abstract] [Full Text] [PDF]

				P. Pignatelli, R. Cangemi, A. Celestini, R. Carnevale, L. Polimeni, A. Martini, D. Ferro, L. Loffredo, and F. Violi Tumour necrosis factor {alpha} upregulates platelet CD40L in patients with heart failure Cardiovasc Res, June 1, 2008; 78(3): 515 - 522. [Abstract] [Full Text] [PDF]

				S. Rohrbach, A. Martin, B. Niemann, and A. Cherubini Enhanced myocardial vitamin C accumulation in left ventricular hypertrophy in rats is not attenuated with transition to heart failure Eur J Heart Fail, March 1, 2008; 10(3): 226 - 232. [Abstract] [Full Text] [PDF]

				P. Chitano, L. Wang, S. N. Mason, R. L. Auten, E. N. Potts, W. M. Foster, A. Sturrock, T. P. Kennedy, J. R. Hoidal, and T. M. Murphy Airway smooth muscle relaxation is impaired in mice lacking the p47phox subunit of NAD(P)H oxidase Am J Physiol Lung Cell Mol Physiol, January 1, 2008; 294(1): L139 - L148. [Abstract] [Full Text] [PDF]

This Article Abstract Full Text (PDF) Data Supplement All Versions of this Article: 100/6/894    most recent 01.RES.0000261657.76299.ffv1 Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Permissions Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Doerries, C. Articles by Landmesser, U. Search for Related Content PubMed PubMed Citation Articles by Doerries, C. Articles by Landmesser, U. Related Collections Congestive Remodeling Heart failure - basic studies Oxidant stress