Inducible Nitric Oxide Synthase Deficiency Protects the Heart From Systolic Overload–Induced Ventricular Hypertrophy and Congestive Heart Failure
Inducible nitric oxide synthase (iNOS) protein is expressed in cardiac myocytes of patients and experimental animals with congestive heart failure (CHF). Here we show that iNOS expression plays a role in pressure overload–induced myocardial chamber dilation and hypertrophy. In wild-type mice, chronic transverse aortic constriction (TAC) resulted in myocardial iNOS expression, cardiac hypertrophy, ventricular dilation and dysfunction, and fibrosis, whereas iNOS-deficient mice displayed much less hypertrophy, dilation, fibrosis, and dysfunction. Consistent with these findings, TAC resulted in marked increases of myocardial atrial natriuretic peptide 4-hydroxy-2-nonenal (a marker of lipid peroxidation) and nitrotyrosine (a marker for peroxynitrite) in wild-type mice but not in iNOS-deficient mice. In response to TAC, myocardial endothelial NO synthase and iNOS was expressed as both monomer and dimer in wild-type mice, and this was associated with increased reactive oxygen species production, suggesting that iNOS monomer was a source for the increased oxidative stress. Moreover, systolic overload–induced Akt, mammalian target of rapamycin, and ribosomal protein S6 activation was significantly attenuated in iNOS-deficient mice. Furthermore, selective iNOS inhibition with 1400W (6 mg/kg per hour) significantly attenuated TAC induced myocardial hypertrophy and pulmonary congestion. These data implicate iNOS in the maladaptative response to systolic overload and suggest that selective iNOS inhibition or attenuation of iNOS monomer content might be effective for treatment of systolic overload-induced cardiac dysfunction.
Several investigators have demonstrated that inducible nitric oxide synthase (iNOS) protein is expressed in cardiac myocytes and endocardial endothelium of patients and animals with ventricular hypertrophy or congestive heart failure (CHF) regardless of cause.1–4 Thus, iNOS was coexpressed with tumor necrosis factor-α in cardiac myocytes from patients with dilated cardiomyopathy2 and increased in several animal models of ventricular hypertrophy or CHF.5 Although unregulated NO production by iNOS has been proposed to exert negative effects on cardiomyocyte function, the effect of iNOS expression on ventricular hypertrophy and CHF in the in vivo heart is controversial. Thus, Heger et al6 reported that overexpression of iNOS in cardiac myocytes increased myocardial NOS activity and NO production but had no effect on cardiac morphology or function. In contrast, Mungrue et al7 reported that cardiac-specific overexpression of iNOS resulted in inflammatory cell infiltrate, left ventricular (LV) hypertrophy, dilation, fibrosis, and contractile dysfunction. The level of iNOS expression in these transgenic mice would depend on the promoter activity, and the iNOS-related phenotypes might vary depending on the level of myocardial iNOS expression. Furthermore, the effects of stress-induced iNOS expression in normal hearts may be different from that in the transgenic mice. Therefore, the present study examined the role of iNOS in the ventricular hypertrophy and CHF that develops in response to sustained pressure overload produced by transverse aortic constriction (TAC) in mice with or without the iNOS gene. We provide the first evidence, to our knowledge, that iNOS deficiency (iNOS−/−) attenuates TAC-induced ventricular hypertrophy and CHF and that iNOS expressed in response to systolic overload serves as a source for myocardial reactive oxygen species (ROS) that contribute to LV dilatation and hypertrophy.
Materials and Methods
Mice and TAC
Body weight and age-matched (2 to 3 months old) male iNOS−/− (crossed back to C57BL/6J for 12 times) and wild-type controls (C57BL/6J) were purchased from The Jackson Laboratory. This study was approved by the Institutional Animal Care and Use Committee of University of Minnesota.
TAC-induced LV hypertrophy. TAC was performed using the minimally invasive suprasternal approach described by Hu et al.8
Selective iNOS inhibition with 1400W. To study the effect of selective iNOS inhibition on TAC-induced ventricular hypertrophy and dysfunction, adult male C57BL/6J mice were randomly divided into 2 groups immediately after TAC; 1 group was treated with the selective iNOS inhibitor 1400W, whereas the other group was treated with saline vehicle. 1400W was delivered at a constant dose of 6 mg/kg per hour via an osmotic minipump (Alzet Model 2002). This dose of 1400W resulted in plasma 1400W concentrations that were 2.4- to 4.9-fold higher than the EC50 for tissue iNOS and decreased plasma nitric oxide metabolites generated by iNOS by 63% to 83%.9 This dose of 1400W had no effect on hemodynamic variables in normal animals, indicating lack of effect on constitutive NOS. We have found that male C57B6J mice develop ventricular dysfunction and pulmonary congestion in 4 weeks after moderate TAC (using a 26-gauge needle) and develop ventricular dysfunction and pulmonary congestion in 2 weeks after severe TAC (using a 27-gauge needle). To induce ventricular dysfunction in the mice within 2 weeks, because the capacity of the minipump to deliver the required dose of 1400W was 2 weeks, we produced severe TAC in these animals by ligating the aorta over a 27-gauge needle.
Echocardiography was performed when mice were anesthetized with 1.5% isoflurane by inhalation.
NOS Protein content was analyzed using Western blots as previously described.10 Primary antibodies against iNOS, eNOS, neuronal NOS (nNOS), atrial natriuretic peptide (ANP), protein arginine methyltransferase 1 (PRMT1), 4-hydroxy-2-nonenal (4-HNE), nitrotyrosine, total mammalian target of rapamycin (mTOR), phospho-mTOR, Akt, phospho-Akt, phosphor-S6, and total p70S6K were purchased from Transduction Laboratories, Santa Cruz Biotechnology, Sigma, Upstate, and Cell Signaling Technology, respectively. Dimethylarginine dimethylaminohydrolase 1 (DDAH1) antibody was a gift from Dr M. Kimoto (Okayama Prefectural University, Japan).10
Measurement of ROS
ROS production was determined by chemiluminescence of coelenterazine (4 μmol/L; Molecular Probes; for details, see the online data supplement at http://circres.ahajournals.org)11 and the red fluorescent dye dihydroethidium (DHE) (2 μmol/L; Invitrogen).
Matrix Metalloproteinase Activity
In vitro gelatin lysis by matrix metalloproteinase (MMP)-2 and MMP-9 was assessed by zymography.12
iNOS−/− Attenuates Moderate TAC-Induced Ventricular Hypertrophy and Dysfunction
Ventricular mass, lung weight, the ratio of ventricular weight to body weight, and the ratio of lung weight to body weight in iNOS−/− and wild-type mice in response to TAC are shown in Figure 1 and Table I in the online data supplement. Under control conditions, ventricular weight, lung weight, and the ratio of organ weight to body weight were not different between iNOS−/− and wild-type mice (Figure 1). Although TAC for 28 days resulted in ventricular hypertrophy in both wild-type and iNOS−/− mice, iNOS−/− significantly attenuated the TAC-induced increase of ventricular weight and the ratio of ventricular weight to body weight (Figure 1). As compared with wild-type mice, a significantly attenuated increase of ventricular weight and the ratio of ventricular weight to body weight was also observed in iNOS−/− mice 8 days after TAC (data not shown). In addition, lung weight and the ratio of lung weight to body weight were significantly greater in wild-type mice as compared with iNOS−/− mice 28 days after TAC, indicating increased pulmonary congestion in the wild-type mice (Figure 1). There was no difference in mortality rate between iNOS−/− and wild-type mice following TAC. To determine the degree of pressure overload produced by the TAC, systolic pressure proximal to the TAC site and the pressure gradient across the TAC site were determined in a group of wild-type mice and knockout mice immediately after the TAC procedure. The results showed that TAC produced a similar increase of LV systolic pressure and the pressure gradient across the aortic constriction (supplemental Figure III).
Echocardiographic imaging of the heart 28 days after TAC demonstrated significant increases of LV wall thickness, LV end systolic diameter and LV end diastolic diameter in both iNOS−/− and wild-type mice in comparison with mice of similar body weight without TAC (Figure 2 and supplemental Table I). However, TAC resulted in a significantly greater decrease of the LV systolic shortening fraction and ejection fraction in wild-type mice than in iNOS−/− mice (Figure 2 and supplemental Table I).
iNOS−/− Attenuates TAC-Induced Ventricular Fibrosis
Histological staining of LV tissue at 28 days after TAC demonstrated more interstitial fibrosis in wild-type mice as compared with iNOS−/− mice (Figure 3A and 3B). Under control conditions, the cross-sectional area of the cardiac myocytes was not different between wild-type mice (227±10 μmol/L2) and iNOS−/− mice (206±13 μmol/L2). Myocyte hypertrophy occurred in both groups of animals in response to TAC, but the increase in myocyte cross-sectional area was significantly less in iNOS−/− mice (395±20 μmol/L2) than in wild-type mice (wild type, 464±31 μmol/L2; P<0.05) (Figure 3C and 3D).
TAC-Induced Alterations of eNOS, iNOS, nNOS, and ANP
Western blots demonstrated that iNOS protein was expressed in wild-type mice at both 8 and 28 days after TAC (Figure 4). eNOS protein content was significantly increased 28 days after TAC in wild-type mice, and iNOS−/− attenuated the TAC-induced induction of eNOS. Myocardial nNOS was unchanged after TAC.
DDAH1 and protein arginine methyltransferase (PRMT1) regulate NO availability by either degrading or synthesizing the endogenous NOS inhibitor asymmetric dimethylarginine (ADMA). Therefore, the protein contents of DDAH1 and PRMT1 were determined. Interestingly, both DDAH1 and PRMT1 were increased in wild-type mice after TAC (Figure 4), whereas iNOS−/− attenuated the TAC-induced induction of DDAH1.
In addition, iNOS−/− attenuated TAC-induced increase of myocardial ANP (Figure 4), consistent with the finding of less ventricular hypertrophy and CHF in the iNOS−/− mice. Myocardial nitrotyrosine and 4-hydroxy-2-nonenal (4HNE) were increased in both wild-type and iNOS−/− mice after TAC (Figure 4), but the increases were significantly less in iNOS−/− mice than in wild-type mice, implying lower oxidative stress in the iNOS−/− mice.
Myocardial Fibrosis and MMP Activity
After TAC, iNOS−/− hearts developed less fibrosis as compared with wild-type mice (Figure 3). Furthermore, iNOS−/− mice had significantly lower myocardial MMP-2 activity as demonstrated by zymography (Figure 3). Although myocardial collagen-1 was increased in both wild-type and iNOS−/− mice 28 days after TAC, the TAC-induced increase of collagen-I expression was significantly less in the iNOS−/− mice (Figure 4).
iNOS−/− Attenuates TAC-Induced Akt-mTOR-S6 Activation
As increased oxidative stress can activate Akt, and activation of mTOR and ribosomal protein S6 (S6) regulates cell growth, total and activated Akt, mTOR, and S6 were determined. TAC caused significant increases of phospho-AktSer473, phospho-mTORSer2488, phospho-S6Ser235/236, and phospho-ErkThr202/204), whereas total Akt was unchanged (Figure 5). iNOS deletion attenuated the TAC-induced increases of AktSer473, mTORSer2488, S6Ser235/236, and ErkThr202/204 (Figure 5).
Expression of eNOS and iNOS Monomer and Dimer
NOS monomer produces superoxide anion, whereas NOS dimer generates NO. Because evidence of increased myocardial oxidative stress was observed in the wild-type mice after TAC, and iNOS was robustly expressed in the wild-type mice early after TAC, relative myocardial iNOS and eNOS monomer and dimer were determined in wild-type mice 8 days after TAC by using nondenatured gel. As shown in Figure 6, iNOS was present as both monomer and dimer in the wild-type mice after TAC (18±2% monomer). Myocardial eNOS monomer was undetectable in sham mice but was present in the iNOS−/− mice (39±1% monomer) and wild-type mice (38±3% monomer) after TAC.
iNOS−/− Attenuates the TAC-Induced Increase of ROS
To determine whether the finding of NOS monomer was associated with increased ROS generation, superoxide anion content was determined with a chemiluminescence assay in myocardial tissue extracts. TAC resulted in increases of superoxide anion in both wild-type and iNOS-deficient mice (Figure 5D), but iNOS−/− partially attenuated TAC-induced myocardial superoxide anion production (Figure 5D). In separate samples obtained from wild-type mice after TAC, both the selective iNOS inhibitor 1400W (decreased to 75±10% after 1400W treatment) or the nonselective NOS inhibitor L-NAME (decreased to 62±13% after L-NAME treatment) attenuated myocardial superoxide production. These findings support the notion that NOS uncoupling contributed to myocardial superoxide production. Intracellular ROS generation was also estimated with red DHE (typically nuclear localization) staining in frozen sections; the results demonstrated that TAC caused an increase of ROS production and that iNOS−/− partially attenuated the TAC-induced superoxide production (supplemental Figure II).
Myocardial eNOS and iNOS Distribution
Under control conditions, eNOS was mainly distributed in capillaries and endothelial cells of larger blood vessels, with a similar expression pattern in wild-type and iNOS−/− mice (Figure 7). Interestingly, although eNOS was still highly expressed in endothelial cells of blood vessels after TAC, an apparent increase of eNOS expression was observed in areas of fibrosis and in cardiac myocytes around the fibrotic areas (Figure 7). This TAC-induced eNOS induction was attenuated iNOS−/− mice (Figure 7). Immunostaining demonstrated that iNOS was broadly expressed in cardiac myocytes and connective tissue in wild-type mice after TAC (supplemental Figure III). The TAC-induced myocardial iNOS expression pattern in wild-type mice was similar to the pattern of iNOS expression in mice following LPS stimulation.
1400W Attenuates TAC-Induced Ventricular Hypertrophy and Dysfunction
Because iNOS deficiency attenuated the TAC-induced ventricular hypertrophy and dysfunction, we examined whether selective iNOS inhibition with 1400W would have similar protective effects on TAC-induced ventricular hypertrophy in wild-type mice. 1400W significantly attenuated the TAC-induced ventricular hypertrophy (Figure 8B), pulmonary congestion (Figure 8B), and ventricular dysfunction (Figure 8E and 8F). 1400W also significantly attenuated the TAC-induced myocardial fibrosis (Figure 8J and 8K). 1400W did not affect heart rate or LV wall thickness (Figure 8) and had no effect on mean aortic pressure (86±3 mm Hg in mice treated with 1400W versus 83±5 mm Hg in mice without 1400W) or LV systolic pressure (98±3.7 in 1400W treated mice versus 102±3.6 mm Hg in untreated mice). 1400W had no effect on ventricular function and fibrosis in sham mice.
The major new findings of this study are that chronic systolic overload resulted in expression of myocardial iNOS as both monomer and dimer in wild-type mice, and this was associated with greater ventricular hypertrophy, dilation, fibrosis, and dysfunction as compared with iNOS−/− mice. Consistent with these findings, selective iNOS inhibition with 1400W significantly attenuated the TAC-induced ventricular hypertrophy and dysfunction in wild-type mice. Expression of iNOS after TAC was associated with greater increases of myocardial nitrotyrosine, 4HNE, ANP, and phosphorylation of Akt, mTOR, S6 in wild-type mice as compared with iNOS−/− mice. The finding of myocardial iNOS monomer, a structural unit that generates superoxide anion, suggests that iNOS is a significant source of superoxide anion in the overloaded or failing heart. The finding that iNOS deletion protected against TAC-induced ventricular hypertrophy and CHF, in association with decreased myocardial nitrotyrosine and 4HNE, suggests that selective iNOS inhibition might potentially be an effective strategy for treatment of myocardial dysfunction and CHF in the chronically overloaded heart.
iNOS Effects on Ventricular Remodeling/Fibrosis and CHF
Although no previous reports have directly examined the effect of iNOS deletion on pressure overload-induced ventricular hypertrophy and CHF, several investigators have studied the effect of iNOS deficiency on ventricular remodeling after myocardial infarction.5,13–15 Studies from 3 different groups have demonstrated that iNOS deficiency caused mildly or moderately decreased infarct-induced mortality, improved ventricular function, reduced myocardial nitrotyrosine content, reduced plasma nitrate, and decreased programmed cell death during both the acute and chronic phases of myocardial infarction,5,14–15 suggesting a detrimental effect of iNOS expression. In addition, using conditional cardiac specific transgenic mice, Mungrue et al found that overexpression of human iNOS in cardiac myocytes resulted in increased myocardial peroxynitrite, myocardial fibrosis, ventricular hypertrophy, CHF and cardiac sudden death,7 indicating that high level overexpression of iNOS can induce ventricular hypertrophy and CHF. A recent study from our laboratory demonstrated that selective pharmacological iNOS inhibition significantly improved LV contractility and myocardial oxygen consumption in end-stage pacing-induced canine heart failure,3 indicating that iNOS inhibition can acutely improve ventricular function in failing hearts. It should be noted, however, that there are conflicting reports in which iNOS deficiency failed to reduce myocardial infarct-induced ventricular dysfunction or mortality.13 The differing results regarding the role of iNOS in ventricular remodeling may be related to variations in infarct size, the stage of heart failure and different genetic backgrounds of the mice.5,13–15 The differing results obtained from two cardiac specific transgenic lines of iNOS overexpression may be attributable to variations of promoter efficiency in these transgenic lines.6–7
The TAC-induced iNOS expression in the wild-type mice in the present study, in conjunction with less severe ventricular hypertrophy, dilation, fibrosis and dysfunction in the iNOS−/− mice in response to systolic overload, is consistent with previous reports that iNOS can exert detrimental effects on the heart. The finding that iNOS−/− only partially attenuated the TAC-induced myocardial ROS production and ventricular dysfunction is not unexpected, because previous reports have demonstrated that other factors such as eNOS uncoupling,16 and increases of NADPH oxidase17 and xanthine oxidase18 can contribute to increased oxidative stress in the failing heart. The finding that iNOS deficiency and selective iNOS inhibition with 1400W attenuated LV remodeling and dysfunction suggests that selective iNOS inhibition might be a useful approach for treating systolic overload-induced ventricular hypertrophy and CHF.
eNOS and iNOS expression in hypertrophied or failing hearts. Increased iNOS expression and activity have been documented in myocardial specimens from patients and animals with ventricular hypertrophy1 and CHF.1–4 In the present study, the finding of a faint iNOS band in the wild-type hearts 8 days after sham surgery was likely the result of tissue trauma from the sham surgery. The expression of myocardial iNOS protein in hearts of wild-type mice both 8 days and 28 days after TAC is consistent with previous reports of iNOS expression in hypertrophied and failing hearts,1–2,4 although the mechanism for upregulation of iNOS expression after TAC is not totally clear. The relatively higher expression of iNOS and eNOS protein early after TAC is consistent with a previous report in which iNOS and eNOS expression peaked 3 to 5 days after TAC.4 The increased expression of eNOS after TAC is consistent with previous reports that eNOS protein was increased in hearts with CHF4,10 or ventricular hypertrophy in response to pressure overload.4,19 The increased eNOS expression in cardiac myocytes near the area with fibrosis is consistent with a previous report in cardiomyopathy samples obtained from mice with mutations of γ-sarcoglycan or δ-sarcoglycan.20 In the context of a recent study demonstrating that eNOS uncoupling contributes to TAC-induced myocardial oxidative stress and ventricular remodeling,16 and the fact that myocardial eNOS monomer was present after TAC, the protective effect of iNOS−/− on TAC-induced ventricular remodeling may relate to an attenuation of eNOS induction. It should be noted that the effect of eNOS on TAC-induced cardiomyopathy is controversial; although 1 study demonstrated that eNOS deficiency profoundly attenuated TAC-induced ventricular hypertrophy and CHF, 2 other studies reported that eNOS deficiency exacerbated ventricular hypertrophy produced by mild TAC.19,21
DDAH1 is an enzyme that degrades the endogenous NOS inhibitor ADMA, whereas PRMT1 is an enzyme that regulates ADMA production. In a canine model of pacing-induced heart failure, we recently found that myocardial DDAH activity was decreased, whereas myocardial PRMT1 and DDAH1 protein contents were unchanged, after the development of CHF.10 Interestingly, in the present study TAC resulted in significant increases of both PRMT1 and DDAH1 in the wild-type mice, suggesting model- or strain-dependent responses of these proteins.
Contribution of iNOS to oxidative stress in hypertrophied and failing hearts. Myocardial hypertrophy and failure are associated with increased superoxide anion (O2−·) production,22 and accumulation of oxidized lipid and protein products such as nitrotyrosine and 4-HNE.5 We recently found that the SOD mimetic M40401 enhanced endothelium-dependent coronary vasodilation and increased LV dP/dtmax in dogs with pacing-induced CHF,10 implying that increased O2−· production is partially responsible for coronary endothelial dysfunction and ventricular dysfunction in the failing heart. Oxygen free radicals are linked to fibrosis and matrix turnover involving the activation of MMPs.23 Overexpressing glutathione peroxidase,24 or administering tetrahydrobiopterin (BH4) to decrease myocardial O2−· production16 decreased myocardial MMP abundance. In the present study, the decreased myocardial oxidative stress in the iNOS-deficient mice was associated with decreased MMP-2 activity, supporting the notion that oxidative stress affects myocardial matrix turnover.
NOS can produce NO, O2−·, or peroxynitrite. This unique property is a consequence of the dimeric nature of the enzyme, in which the 2 subunits are able to function independently.25 NOS optimally exists as a homodimer that generates NO and l-citrulline from l-arginine. However, when exposed to oxidant stress, or when deprived of its reducing cofactor BH4 or substrate l-arginine, NOS can uncouple to the monomeric form that generates O2−· rather than NO.16,26–28 BH4 is required for iNOS dimerization29 and also stabilizes nNOS and eNOS dimers. Thus, a decrease of BH4 or unregulated NO production by iNOS to decrease l-arginine availability can cause NOS uncoupling.25,30 Using purified iNOS protein, several studies have demonstrated that iNOS can produce both NO and O2−· and that deficiency of l-arginine or BH4 will induce iNOS to produce O2−·,27,30 indicating that iNOS can be uncoupled. In addition, in cultured macrophages, depletion of cytosolic l-arginine triggered O2−· production by iNOS that was blocked by a NOS inhibitor, suggesting that iNOS uncoupling was responsible for the O2−· production.26
In the cardiovascular system, most studies of NOS uncoupling have focused on eNOS. Thus, uncoupled eNOS produces O2−·31 in apoE-deficient mice, and BH4 improves the endothelial dysfunction associated with hypercholesterolemia, atherosclerosis, or hypertension.32–33 Takimoto et al recently demonstrated that TAC resulted in a decrease of plasma BH4, an increase of myocardial eNOS monomer content, and increased O2−· production.16 Moreover, administration of BH4 reduced the eNOS monomer content and decreased myocardial O2−· production and TAC-induced cardiomyopathy, indicating that eNOS uncoupling contributed to the TAC-induced ventricular dysfunction.16 In the present study, iNOS deletion reduced the evidence of TAC-induced myocardial oxidative stress, indicating that iNOS contributed to oxidative stress in the wild-type mice, either directly through iNOS uncoupling or by iNOS-dependent eNOS uncoupling. Based on the finding that myocardial iNOS was expressed as both monomer and dimer, and that iNOS deletion attenuated the evidence of oxidative stress in animals exposed to TAC, it can be concluded that iNOS uncoupling contributed to the increased oxidative stress in the wild-type mice. However, iNOS might also decrease intracellular BH4 and l-arginine availability to eNOS and thereby induce eNOS uncoupling. The relative contributions of iNOS uncoupling versus iNOS-dependent eNOS uncoupling in the TAC-induced increase of myocardial oxidative stress merit further investigation.
Akt, mTOR, and S6 Activation in Response to Oxidative Stress
Akt phosphorylation can regulate ventricular hypertrophy,34 and oxidative stress has been reported to activate Akt35 by regulating PTEN. Akt was reported to cause downstream activation of mTOR and p70S6K, and previous reports have demonstrated that inhibition of mTOR signaling with rapamycin attenuated TAC-induced ventricular hypertrophy36–37 and caused regression of cardiac hypertrophy produced by TAC.38 Interestingly, a recently study reported that activation of Akt-mTOR and nuclear factor κB participate in the development of ventricular hypertrophy, whereas the antioxidant pyrrolidine dithiocarbamate attenuated nuclear factor κB, Akt, and p70S6K activation and TAC-induced ventricular hypertrophy.37 Ribosomal protein S6 is a downstream target of p70S6K, and S6 phosphorylation increases the translation of a subset of mRNAs that promote protein synthesis. The finding of increased phospho-Akt and phospho-S6 in our study is consistent with the previous reports.16,37 Our finding that iNOS deletion attenuated TAC-induced myocardial oxidative stress, phospho-Akt, phospho-mTOR, phospho-S6, and ventricular hypertrophy supports the notion that oxidative stress exacerbates systolic overload-induced Akt-mTOR activation and ventricular hypertrophy.
The pressure gradient across the aortic constriction was not measured in the present study. However, care was taken to ensure that the identical TAC procedure was performed by the same surgeon who was blinded to the genotype of the mice. Because iNOS−/− attenuated the TAC-induced increase of eNOS expression, and eNOS monomer was present in wild-type mice exposed to TAC, we were unable to determine whether the protective effect of iNOS−/− was directly caused by the absence of iNOS or secondary to an attenuation of TAC-induced eNOS induction. We did not measure plasma BH4 levels in the present study. However, using an almost identical TAC model, a recent study reported that TAC resulted in a significant decrease of plasma BH4 in C57/B6J mice, and that administration of BH4 attenuated TAC-induced myocardial eNOS monomer formation and oxidative stress, suggesting that a decrease of BH4 after TAC may contribute to NOS monomer formation.16 An additional limitation is that the iNOS−/− and wild-type mice were not littermates. Finally, because of the relatively large amount of tissue required for assay of myocardial NOS activity, this determination was not performed in the present study.
We acknowledge the guidance, support, and encouragement of Dr Robert J. Bache, who contributed importantly to the successful completion of this study.
Sources of Funding
This study was supported by NIH/National Heart, Lung, and Blood Institute grants HL71790 and HL21872. P.Z. is supported by a Scientist Development Award from the American Heart Association.
↵*Both authors contributed equally to this work.
Original received February 15, 2006; first resubmission received October 3, 2006; second resubmission received February 7, 2007; accepted March 6, 2007.
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