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Am J Physiol Heart Circ Physiol 287: H2115-H2121, 2004. First published July 29, 2004; doi:10.1152/ajpheart.00267.2004
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Control of myocardial oxygen consumption in transgenic mice overexpressing vascular eNOS

E. K. Walsh,1 H. Huang,1 Z. Wang,1 J. Williams,1 R. de Crom,3,4 R. van Haperen,3 C. I. Thompson,1 D. J. Lefer,2 and T. H. Hintze1

1Department of Physiology, New York Medical College, Valhalla, New York 10595; 2Department of Molecular and Cellular Physiology, Louisiana State University Health Sciences Center, Shreveport, Louisiana 71130-3932; and 3Department of Cell Biology and Genetics and 4Department of Vascular Surgery, Erasmus University Medical Center, Rotterdam 3000 DR, The Netherlands

Submitted 16 March 2004 ; accepted in final form 13 July 2004


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Our objective was to investigate the potential role of selective endothelial nitric oxide (NO) synthase (eNOS) overexpression in coronary blood vessels in the control of myocardial oxygen consumption (MVO2). Transgenic (Tg) eNOS-overexpressing mice (eNOS Tg) (n = 22) and wild-type (WT) mice (n = 24) were studied. Western blot analysis indicated greater than sixfold increase of eNOS in cardiac tissue. Echocardiography in awake mice indicated no difference in cardiac function between WT and eNOS Tg; however, systolic pressure in eNOS Tg mice decreased significantly (126 ± 2.3 to 109 ± 2.3 mmHg; P < 0.05), whereas heart rate (HR) was not different. Total peripheral resistance (TPR) was also decreased (9.8 ± 0.8 to 7.6 ± 0.4 4 mmHg·ml–1·min; P < 0.05) in eNOS Tg. Furthermore, female eNOS Tg mice showed even lower TPR (7.2 ± 0.4 mmHg·ml–1·min) compared with male eNOS mice (8.6 ± 0.5, mmHg·ml·min–1; P < 0.05). Left ventricular slices were isolated from WT and eNOS Tg mice. With the use of a Clark-type oxygen electrode in an airtight bath, MVO2 was determined as the percent decrease during increasing doses (10–10 to 10–4 mol/l) of bradykinin (BK), carbachol (CCh), forskolin (10–12 to 10–6 mol/l), or S-nitroso-N-acetyl penicillamine (SNAP; 10–7 to 10–4 mol/l). Baseline MVO2 was not different between WT (181 ± 13 nmol·g–1·min–1) and eNOS Tg (188 ± 14 nmol·g–1·min–1). BK decreased MVO2 (10–4 mol/l) in WT by 17% ± 1.1 and 33% ± 2.7 in eNOS Tg (P < 0.05). CCh also decreased MVO2, 10–4 mol/l, in WT by 20% ± 1.7 and 31% ± 2.0 in eNOS Tg (P < 0.05). Forskolin (10–6 mol/l) or SNAP (10–4 mol/l) also decreased MVO2 in WT by 24% ± 2.8 and 36% ± 1.8 versus eNOS 31% ± 1.8 and 37% ± 3.5, respectively. N-nitro-L-arginine methyl ester (10–3 mol/l) inhibited the MVO2 reduction to BK, CCh, and forskolin by a similar degree (P < 0.05), but not to SNAP. Thus selective overexpression of eNOS in cardiac blood vessels in mice enhances the control of MVO2 by eNOS-derived NO.

forskolin; nitric oxide

BEFORE THE DISCOVERY of nitric oxide (NO), Granger et al. (13) found that mitochondrial respiration was inhibited by activated macrophages by an unknown mechanism. This macrophage-induced cytotoxic effect on mitochondrial respiration was later found to be mediated by NO (15, 39). NO is a potent inhibitor of mitochondrial respiration through nitrosylation of aconitase in the tricarboxylic acid cycle, complexes I and II of the electron transport chain, and cytochrome c oxidase (4, 5, 12). In recent years, we (28, 38) and others (3) have demonstrated that endogenous NO plays a significant role in modulating myocardial O2 consumption (MVO2) during exercise, heart failure, and in response to various agonists. Furthermore, the control of MVO2 by NO was established in genetically altered mice in which the B2-kinin receptor as well as inducible NO synthase (iNOS) and endothelial NOS (eNOS) have been knocked out (27, 29).

Loke et al. (29) found that in mice deficient in eNOS, the regulation of MVO2 was due to stimulation of NO synthesis and this is confirmed by the ability of L-arginine analogues to inhibit NOS. A transgenic (Tg) mouse overexpressing the constitutive eNOS gene in blood vessels has been engineered (23, 42). Studies in these transgenic mice have added to the notion that NO plays a key role in regulating the redox state of cardiac tissues. The overexpression of eNOS attenuates congestive heart failure by decreasing pulmonary and cardiac dysfunction, protects against ischemia-reperfusion injury, inhibits lesion formations in blood vessels, reverses diminished vasorelaxation during ischemic heart failure, and allows for increased oxygenation to skeletal muscle due to vasodilation (1, 2, 9, 19, 20, 32, 34). The goal of our study was to determine whether increased production of NO subsequent to vascular overexpression of eNOS in the mouse heart results in tighter control of MVO2. In part, the goal of this study was to substantiate the contention that blood vessel-derived NO can regulate MVO2.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Tg eNOS-overexpressing mice. A DNA fragment containing the human eNOS gene was isolated from a homemade human genomic cosmid library (18, 23) using eNOS cDNA (kindly donated by Dr. S. Janssens, Leuven, Belgium) as a probe. In addition, the DNA fragment contained ~6 kb of the 5'-natural flanking sequence, including the native eNOS promoter, and ~3 kb of the 3'-sequence to the gene. Vector sequences were removed by restriction endonucleases. A solution of 1–2 µg/ml DNA was used for microinjection into fertilized oocytes from FVB donor mice and transplanted into the oviducts of pseudopregnant B10 x CBA mice. Founder mice and offspring were genotyped by PCR using DNA isolated from tail biopsies. The primers used were sense, 5'-GTCCTGCAGACCGTGCAGC-3', and antisense, 5'-GGCTGTTGGTGTCTGAGCCG-3'. Mice were back-crossed to C57B/6 for at least five generations (>96%) C57B/6. All eNOS transgenic mice were hemizygous (18, 23, 42).

Hemodynamics. Male and female (n = 22) Tg eNOS and WT (C57B/6X129) (n = 24) mice (12–16 wk old) were used to measure blood pressure, cardiac function, and MVO2. All mice were housed in the same type of cage and supplied with standard mouse chow and had free access to water. All protocols were approved by the Institutional Animal Care and Use Committee of New York Medical College and conform to the National Institutes of Health Guide for the Care and Use of Laboratory Animals and American Physiological Society "Guidelines for the Use and Care of Laboratory Animals."

Western blot analysis. The right ventricle was harvested from anesthetized eNOS Tg mice and from C57B/6 WT controls. The tissue was pulverized in liquid nitrogen and resuspended in lysis buffer containing protease inhibitors (14). After sonification for 60 s, samples were centrifuged at 5,000 rpm for 10 min, and protein concentration was determined as previously described (14). Briefly, 100 µg of protein were separated on a 7.5% SDS-PAGE, followed by an electronic transfer to a polyvinylidene fluoride membrane (Amersham; Piscataway, NY). Human umbilical vein endothelial cell protein was loaded as a positive control for eNOS. The transferred proteins were incubated with a 1:500 dilution of a monoclonal anti-eNOS antibody (Affinity BioReagents; Golden, CO), a monoclonal 1:1,000 dilution anti-iNOS antibody, or a monoclonal 1:500 dilution anti-neuronal NOS (nNOS) antibody (BD Transduction Labs; San Diego, CA) at 4°C overnight. The bound primary antibody was detected by a peroxidase-coupled anti-mouse antibody (dilution 1:2,000; Amersham), followed by a chemiluminescent reaction using luminol (SuperSignal West Pico, Pierce; Rockford, IL). Afterward, the membrane was exposed to a film, and bands were analyzed by densitometry, as previously published (14, 26).

Tail-cuff blood pressures. Blood pressures (systolic and diastolic) were measured by an automated tail-cuff method (model NIBP-8; Columbus Instruments) along with the heart rate in unanesthetized mice. Each mouse was trained in the morning for five sessions in holding chambers warmed to 37–39°C to maximize arterial dilation. Five consecutive stable readings were averaged in each mouse.

Transthoracic two-dimensional Doppler echocardiography studies for cardiac morphology and function. Transthoracic two-dimensional echocardiography was performed in awake mice trained for 3 days with the use of an electrocardiograph (model Sequoia 256, Acuson) equipped with a 15-MHz linear transducer (15L8) in a phased-array format. Aquasonic ultrasound transmission gel (model 100, Parker Labs; Fairfield, NJ) was centrifuged at 1,000 rpm for 15 min to release any resolution artifacts. The gel was then warmed for 30 min in a water bath to avoid inducing hypothermic bradycardia. Generally, the heart was first imaged by placing the probe parallel to the sternum to obtain a long-axis view. From this image, we traced the left ventricular (LV) area during diastole (LVDA) and systole (LVSA) and calculated the ejection fraction (EF) over three consecutive cardiac cycles. The probe was then placed to obtain the parasternal short-axis view at the level of the pulmonary valve to measure the velocity time index. The parasternal short-axis view also allowed for an additional calculation of EF using B-mode. In the short-axis view, the heart was imaged with the two-dimensionally guided M-mode at the level of the papillary muscles. End-diastolic (LVEDD) and systolic (LVESD) LV chamber dimensions as well as posterior wall thickness were measured using the American Society of Echocardiography leadingedge techniques (16, 17, 43).

The equations used for hemodynamic measurements are the following:




where SF is shortening fraction, IVST is interventricular septum thickness, and PWT is posterior systolic wall thickness, LVcavity refers to LV chamber dimension, and LVmass is LV weight.

Measurement of MVO2. A Clark-type platinum O2 electrode system was used (model 5331, Yellow Springs Instruments; Yellow Springs, OH) with isolated cardiac tissue slices to measure MVO2. The O2 electrode was inserted into a small glass reaction chamber (3–8 ml) maintained at a constant 37°C temperature bath (model 5301, Yellow Springs Instruments). A built-in magnetic stirrer (480 rpm at 60 Hz) located in the bath assembly was used to prevent clumping of the tissue in the incubation medium path of the Teflon electrode membrane.

Preparation of cardiac tissue slices. Mice were anesthetized with pentobarbital sodium (65 mg/kg), and 0.5 ml of plasma were immediately removed from the left ventricle for nitrate measurements (38). The heart was removed immediately. The atria, large coronary arteries, connective tissue, and fat were discarded. A piece of the right ventricle was dissected and frozen in liquid nitrogen for Western blot analysis. The left ventricle was then bisected such that each piece of muscle contained a portion of the septum, free wall, and apex. Myocardial tissues were incubated in Krebs bicarbonate solution containing (mmol/l) 118 NaCl, 4.7 KCl, 25 NaHCO3, 1.2 KH2PO4, 1.1 MgSO4, and 5.6 C6H12O6 at 37°C, bubbled with 21% O2-5% CO2-74% N2 (pH 7.4) to equilibrate for at least 2 h.

At the end of each incubation period, each piece of tissue was placed in a stirred bath with 3 ml of air-saturated Krebs bicarbonate solution containing 10 mmol/1 HEPES (pH 7.4). The bath was sealed using a Clark-type platinum O2 electrode. Succinate (5 x 10–4 mol/l), a substrate for complex II, and sodium cyanide (10–3 mol/l), an inhibitor of complex IV of the electron transport chain, were added in succession at the completion of the concentration-response curve to each agonist to confirm that the changes in O2 uptake were effects due to mitochondrial respiration. The 100% increase in O2 consumption in the presence of succinate suggest that O2 is not limiting in the bath, and the abolition of O2 consumption after the addition of sodium cyanide confirms that changes in MVO2 were of mitochondrial origin (24, 27–29, 37, 40, 48).

Effect of bradykinin, carbachol, and NG-nitro-L-arginine methyl ester on MVO2. Cumulative concentrations of the B2 kinin receptor agonist bradykinin (BK; 10–10-10–4 mol/l, Sigma) and the muscarinic receptor agonist carbachol (CCh; 10–10-10–4 mol/l, Sigma) were given alone or in the presence of the NOS inhibitor NG-nitro-L-arginine methyl ester (L-NAME) (10–3 mol/l, Sigma).

Effect of forskolin, SNAP, and L-NAME on MVO2. Forskolin (10–12-10–6 mol/l, Calbiochem-Novabiochem), an activator of adenylate cyclase, which phosphorylates eNOS, or an exogenous NO donor, S-nitroso-N-acetyl-penicillamine (SNAP) (10–7-10–4 mol/l, Sigma), were given alone or in the presence of L-NAME (10–7-10–4 mol/l).

Data and statistical analysis. Myocardial tissue respiration was calculated as the rate of decrease in PO2, assuming an initial O2 concentration of 224 nmol/l (41), and was expressed as nanomoles of O2 consumed per minute per gram of tissue. The effect of BK, CCh, forskolin, and SNAP on myocardial O2 uptake is expressed as the percent change from baseline O2 consumption. All data are expressed as means ± SE. Statistical analysis for echocardiographic and blood pressure data were carried out with a two-sample Student's t-test. Changes in MVO2 across groups were analyzed by means of a two-way ANOVA, followed by multiple comparison using Student-Newman-Keuls test. A value of P < 0.05 was considered statistically significant. Statistical software was used for analysis and production of data and graphics (NCSS 2001-PASS 2002, and SlideWrite version 6.0, Advanced Graphics Software).


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Western blot analysis. Western blot analysis of eNOS Tg (n = 6) mice showed an approximate sixfold increase in eNOS protein (Fig. 1). There was no significant difference in protein expression of iNOS (70 ± 9.8 vs. 74 ± 12) or nNOS (108 ± 19 vs. 106 ± 32) in WT and eNOS Tg mice, respectively.



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  		Fig. 1. Representative Western blot analysis showing a sixfold increase in endothelial nitric oxide synthase (eNOS) protein expression in eNOS transgenic (Tg) mice and wild-type (WT) mice (n = 3 pairs of mice).

 
Assessment of blood pressure by tail cuff. Table 1 summarizes hemodynamic measurements from five successive blood pressure measurements taken from each WT (n = 24) and eNOS Tg (n = 22) mouse. Systolic, diastolic, and mean arterial pressure (MAP) in eNOS Tg mice versus WT were significantly lower (126 ± 2.3 to 109 ± 2.3, 84 ± 1.3 to 70 ± 1.9, and 97 ± 1.3 to 82 ± 1.8 mmHg, P < 0.05, respectively). Total peripheral resistance (TPR), calculated as MAP divided by cardiac output (CO), normalized for body weight, was also significantly decreased in eNOS Tg mice (7.64 ± 0.35 vs. 9.81 ± 0.80 mmHg·ml–1·min; P < 0.05). Heart rate (HR) was not significantly different between the two groups.


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  		Table 1. Changes in hemodynamics in WT and eNOS Tg mice

 
Echocardiographic assessment of hemodynamics. Two successive echo measurements were taken from each individual mouse in the morning. There was no significant difference between WT and eNOS Tg mice (Table 1). There was an increase in plasma nitrate from 5.6 ± 0.4 to 8.1 ± 1.0 µM in eNOS Tg mice.

Gender differences in WT and eNOS mice. When each of the two groups, WT and eNOS Tg, were separated into male and female there were some significant differences in hemodynamics (Table 1). Most importantly, TPR was even further reduced in female eNOS Tg (7.17 ± 0.41 mmHg·ml–1·min) compared with male eNOS Tg (8.59 ± 0.53 mmHg·ml–1·min), whereas the WT females had a TPR of 8.83 ± 0.52 mmHg·ml–1·min and the male WT had a TPR of 11.54 ± 1.58 mmHg·ml–1·min; P < 0.05.

Baseline MVO2 in eNOS myocardial tissue. Baseline MVO2 was not significantly different between WT (181 ± 13 nmol·g–1·min–1) and eNOS Tg (188 ± 14 nmol·g–1·min–1) or WT (153 ± 12 nmol·g–1·min–1) and eNOS Tg (165 ± 11 nmol·g–1·min–1) and in the presence of L-NAME.

Effect of bradykinin on MVO2. BK (10–10-10–4 mol/l; Sigma, n = 6) caused a concentration-dependent decrease in MVO2 in WT (17 ± 1.1%), with enhanced inhibition of MVO2 in eNOS Tg mice (33 ± 2.7%) (Fig. 2). In the presence of L-NAME, the responses to BK were attenuated to 14 ± 1.3% and 15 ± 5.5% in both WT and in eNOS Tg mice, respectively.



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  		Fig. 2. Effect of cumulative doses of bradykinin (BK) on myocardial O2 consumption (MVO2) in eNOS Tg (n = 6) and WT (C57B/6) mice (n = 6). BK caused a dose-dependent decrease of MVO2 in WT mice, and this effect was significantly increased in eNOS Tg mice. *P < 0.05, two-way ANOVA, followed by multiple comparison using Student-Newman-Keuls test.

 
Effect of CCh on MVO2. Cumulative doses of CCh (10–10-10–4 mol/l, Sigma, n = 6) also caused a dose-dependent decrease in MVO2 in WT by 20 ± 1.7% and an even larger decrease in eNOS Tg by 31 ± 2.0% (Fig. 3). The addition of L-NAME attenuated the reduction in MVO2 (18 ± 2.7% and 23 ± 4.6%) in both WT and eNOS Tg, respectively.



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  		Fig. 3. Effect of cumulative doses of carbachol (CCh) on MVO2 in eNOS Tg (n = 6) and WT (C57B/6) mice (n = 6). CCh caused a dose-dependent decrease of MVO2 in WT mice, and this effect was significantly increased in eNOS Tg mice. *P < 0.05, two-way ANOVA, followed by multiple comparison using Student-Newman-Keuls test.

 
Effect of forskolin on MVO2. Forskolin (10–12-10–6 mol/l, n = 7), caused a dose-dependent decrease in MVO2 in WT by 24 ± 2.8% and in eNOS Tg by 31 ± 1.8% (Fig. 4). The addition of L-NAME attenuated the reduction of MVO2 in both WT (17 ± 2.0%) and eNOS Tg (24 ± 2.8%).



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  		Fig. 4. Effect of cumulative doses of forskolin on MVO2 in eNOS Tg (n = 7) and WT (C57B/6) mice (n = 6). Forskolin caused a dose-dependent decrease of MVO2 in WT mice, and this effect was significantly increased in eNOS Tg mice. *P < 0.05, two-way ANOVA, followed by multiple comparison using Student-Newman-Keuls test.

 
Effect of SNAP on MVO2. SNAP (10–7-10–4, n = 4), also caused a dose-dependent decrease in MVO2. WT showed a 36 ± 1.7% decrease, whereas eNOS mice showed a 37 ± 3.6% decrease (Fig. 5). The addition of L-NAME did not alter the actions of SNAP in WT (39 ± 0.6%) or eNOS Tg (34 ± 1.1%).



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  		Fig. 5. Effect of cumulative doses of S-nitroso-N-acetyl penicillamine (SNAP) on MVO2 in eNOS Tg (n = 4) and WT (C57B/6) mice (n = 4). SNAP caused a dose-dependent decrease of MVO2 in WT mice, and this effect was not significantly increased in eNOS Tg mice. *P < 0.05, two-way ANOVA, followed by multiple comparison using Student-Newman-Keuls test.

 

   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
In this study, we have demonstrated that in heart from mice with coronary vascular overexpression of eNOS, NO plays an enhanced role in the regulation of MVO2. These findings support our previous studies indicating that vascular NO is coupled to the control of tissue respiration. Initial phenotypic and hemodynamic measurements of eNOS Tg mice showed approximately a sixfold increase in eNOS protein as seen by Western blot analysis, whereas systolic and diastolic pressures decreased, and there were no changes in ventricular contractile function by echocardiography. Furthermore, the overexpression of eNOS leads to an increased ability of drugs which stimulate NO production to regulate MVO2. BK and CCh reduced oxygen consumption to an even greater degree in heart from eNOS Tg compared with wild-type controls. Similarly, forskolin and SNAP administration reduced oxygen consumption in eNOS Tg mice. L-NAME reversed the inhibitory response of BK, CCh, and forskolin, but not that of SNAP. Vascular eNOS overexpression resulted in an enhanced inhibition in tissue respiration; hence, we conclude that vascular NO can control MVO2 and that introduction of an extra copy of the eNOS gene can further enhance the regulation of the redox state in myocardial tissue by NO.

Decreased blood pressures in eNOS Tg mice are consistent with data from several laboratories (22, 30, 32–34, 36). Other studies (2, 19), however, did not find significant hypotension; this could be due to either the depressive effects of anesthesia and/or differences in the transgenic model. We calculated TPR by normalizing MAP with our CO measurements from the echocardiograms. Overall, eNOS Tg mice had significantly lower resistance compared with WT. Interestingly, there were gender-specific decreases in TPR as well, for example, there was a further significant decrease in resistance in female mice (Table 1). Gender-specific differences in eNOS Tg mice are not surprising due to the fact that we have recently found a 50% increase in mortality of male eNOS–/– at 21 mo (25). In our calculations, the decrease in TPR was due to a significant decrease in MAP, and we assumed central venous pressure (CVP) was negligible. Even if one uses an estimate of CVP of a few mmHg, the marked reduction in MAP from 97–92 mmHg in WT mice to 81 mmHg in eNOS Tg mice, a difference of 11–16 mmHg, make it unlikely that a change in CVP would be great enough to alter our conclusions. In fact, we probably underestimated the change in TPR in eNOS Tg mice.

Our methods for echocardiographic assessment are those described by Yang et al. (46). We measured EF% from three different planes of the heart. Two measurements were taken parallel to the parasternal short axis of the left ventricle. With B-mode tracings, 5 were calculated from circumferential changes during systole and diastole, values ranged between ~55% and 65%. In an M-mode tracing, the values ranged from 80% to 90%. Even though our measurements for CO seem physiologically high, Janssen et al. (17) surveyed different methods of measuring CO in conscious mice with flow probes and echocardiography and determined an acceptable ranges of 12–27 ml/min. Baseline oxygen consumption was not different between WT and eNOS Tg hearts showing that although eNOS is upregulated, the production of NO during basal conditions remains small. In our buffer solution there are no agonists and no flow (shear) to stimulate eNOS until we add an agonist. Concurrently, in our in vitro preparation eNOS is not constitutively active, in contrast we have previously shown in tissues treated with lipopolysaccharide, where iNOS is constitutively active, there is a reduction in baseline MVO2, which is reversed by L-NAME (7). When stimulated with BK, however, the reduction in MVO2 is significantly greater in eNOS Tg than in WT controls. This shows that kinin/Ca2+-dependent mechanisms competitively inhibit mitochondrial respiration. Loke et al. (27), using B2-kinin receptor–/– mice, found that MVO2 was not decreased by BK. Later, Loke et al. (29) used eNOS(–/–) mice and stimulated the heart with BK. Not surprisingly, MVO2 was not altered in the eNOS knockouts showing that the coupling of kinin receptors to eNOS and the formation of NO was abolished. Muscarinic receptors are also expressed in coronary blood vessels. Shen et al. (37) and Xie et al. (44) demonstrated that CCh, a muscarinic agonist, stimulates eNOS to decrease MVO2 in the canine heart in vitro. Furthermore, in vivo we speculate the basal decrease in blood pressure is mostly due to Ca2+-independent shear stress mechanisms rather than Ca2+-dependent agonist activation of eNOS.

eNOS is thought to be activated by several Ca2+ and phosphorylation-dependent mechanisms, which allow for subsequent electron flux from the NH2 terminus reductase to the COOH terminal oxygenase, where L-arginine is converted to L-citrulline and NO. Various kinases drive activation of eNOS through phosphorylation, including; Akt, which phopshorylates Ser1177; AMPK, which phosphorylates Ser1177, and Thr495; PKA, which phosphorylates Ser1177 and Ser633; and PKG, which also phosphorylate Ser1177 and Ser633 (8). PKC and mitogen-activated protein kinases are also involved in phosphorylation of eNOS, but they negatively regulate eNOS activity (8). Zhang et al. (47) demonstrated that adenlyate cyclase is involved in phosphorylation of eNOS. They showed that stimulated cAMP signaling in canine coronary microvessels caused an increase in nitrite production during administration of an activator of adenylate cyclase, forskolin. To test whether the decrease in MVO2 is proportional to the increase in eNOS protein through phosphorylation, we stimulated the myocardium in eNOS Tg with forskolin. Our results indicated there was enhanced inhibition of MVO2 in eNOS Tg, showing that cAMP-dependent eNOS phosphorylation causes activation of the enzyme.

SNAP caused a dose-dependent decrease in MVO2 in eNOS Tg mice. L-NAME was given in the presence of BK, CCh, or forskolin, and there was inhibition of tissue respiration. L-NAME did not reverse the actions of SNAP.

It is important to note that there are different techniques to overexpress eNOS either constitutively or selectively; however, none of the previous studies addressed the role of NO in the control of cardiac oxygen consumption. Early studies [i.e., Ooboshi et al. (31)] involving eNOS overexpression with adenoviral transfections in rats, found that vasodilation was increased and that blood flow/oxygenation increased after hindlimb ischemia. Microinjection of eNOS into the rostral ventrolateral medulla and the nucleus tractus solitarius of the brain caused bradycardia and hypotension (21, 22, 36). Brunner et al. (2) found an attenuation of myocardial/reperfusion injury in myocyte-specific overexpression of eNOS using human cDNA amplified by PCR and then subcloned into vectors containing {alpha}-myosin heavy chain. The next step was to genetically modify mice to constitutively overexpress eNOS. One group used a bovine eNOS gene with a preproendothelin-1 promoter to produce a heterozygous strain with vascular-specific eNOS overexpression (20, 32–34). These mice exhibit hypotension, inhibition of lesion formation, attenuation of cardiac hypertrophy during isoproteronol perfusion, and consistent with Brunner et al. (2), protection against ischemia-reperfusion injury (20, 30, 32, 34). Finally, the transgenic mouse used in our experiments was created using a human cDNA eNOS promoter. Van Haperen et al. (42) performed initial phenotypic studies on two founder strains of eNOS Tg mice (eNOS Tg2 and eNOS Tg3). Immunohistochemical staining was performed to determine overexpression patterns in the aorta, heart, liver, kidney, adrenal, and testis. Sections from WT showed little or no staining in sections. Staining in eNOS Tg mice was clearly present in capillaries between myocytes, in sinusoids of the liver, and in peritubular capillaries and glomeruli of the kidney. In the adrenal gland, cortical capillaries and medullary capillary sinusoids were also stained as well as only blood vessels in the testis. All sections sampled did not exhibit staining in the parenchyma cells in eNOS Tg2 and eNOS Tg3 mice. This supports the conclusion that eNOS overexpression in these mice is specific to endothelial cells in blood vessels (42). With the use of those mice, Jones et al. (19) reported a decrease in the progression of congestive heart failure. Our studies have focused on the role of eNOS to control NO production and regulate mitochondrial respiration. To address the role of mitochondrial NOS selectively controlling MVO2, the most recent studies by Giulivi et al. (6, 10) suggest that a truncated nNOS protein is mitochondrial NOS (11). In our study, nNOS protein was not different in the WT or eNOS Tg mice, where eNOS protein was markedly elevated in eNOS Tg mice. Furthermore, BK and carbachol stimulate eNOS, not nNOS, providing evidence to suggest that upregulated eNOS is primarily responsible for the enhanced regulation of MVO2 in eNOS Tg mice.

In our previous studies (25), eNOS(–/–) male mice developed severe heart failure at 21 mo of age. Recent studies by Pacher et al. (35) also support age-related cardiac dysfunction eNOS(–/–) mice. In Pacher's study, they report 10-mo-old iNOS(–/–) mice showed no change in LV function, where eNOS(–/–) mice at 4 mo had increased TPR, increased contractility, and moderate diastolic dysfunction, and at 10 mo of age, eNOS(–/–) developed severe cardiac dysfunction characterized by decreased CO and EF (35). The use of adenoviral transfection or genetic modification suggests that if eNOS is restored, a disease process may be alleviated or reversed. There is, however, one important caveat to mention. If there is an abundance of eNOS and subsequent excessive NO generation then this may cause deleterious effects on lesion formation and cardiac function. In the presence of excess NO and low PO2, the formation of superoxide (O2–) and peroxynitrite (ONOO–) may effect tissue respiration. In our study, L-NAME acutely (in 30 min) inhibited the effects of BK, carbachol, or forskolin suggesting that the accumulation of nitrosylated proteins, with some long half-life, from ONOO– were likely not responsible for the significant reduction in MVO2. Our previous studies (40) also show that increased endogenous NO generation regulates tissue respiration independent of ONOO–. Xie et al. (45), showed that tiron, an intracellular scavenger of O2–, or uric acid, a scavenger of ONOO– did not modify the effects of SNAP on tissue oxygen consumption. Our hemodynamic and echocardiographic assessments also show that the relative vascular overexpression of eNOS does not cause deleterious effects subsequent to altered cardiac function or decreased blood pressures.

In summary, there is enhanced control of mitochondrial respiration to BK, carbachol, and forskolin in hearts from transgenic mice overexpressing vascular eNOS. The decreased blood pressures, TPR, and normal LV cardiac function in the eNOS Tg mouse indicate the lack of deleterious actions of eNOS overexpression in the coronary and peripheral circulations.


   GRANTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
This study was supported by National Heart, Lung, and Blood Institute Grants PO-1 HL-430231, ROI-HL-50142, and HL-61290 (to T. H. Hintze) and HL-60849 (to D. J. Lefer).


   FOOTNOTES
 

Address for reprint requests and other correspondence: T. H. Hintze, New York Medical College, Valhalla, NY 10595 (E-mail: Thomas_Hintze{at}nymc.edu)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


   REFERENCES
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 

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