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Am J Physiol Heart Circ Physiol 292: H1541-H1550, 2007. First published November 17, 2006; doi:10.1152/ajpheart.00264.2006
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Endothelial nitric oxide synthase (NOS3) knockout decreases NOS2 induction, limiting hyperoxygenation and conferring protection in the postischemic heart

Xue Zhao,1,2 Yeong-Renn Chen,1 Guanglong He,1 Aiwen Zhang,1 Lawrence J. Druhan,1 Arthur R. Strauch,1 and Jay L. Zweier1

1Davis Heart and Lung Research Institute and the Division of Cardiovascular Medicine, Department of Internal Medicine, The Ohio State University College of Medicine, Columbus, Ohio; and 2Department of Cardiology, Changzheng Hospital, Second Military Medical University, Shanghai, China

Submitted 14 March 2006 ; accepted in final form 10 November 2006


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Although it has been shown that endothelial nitric oxide synthase (eNOS)-derived nitric oxide downregulates mitochondrial oxygen consumption during early reperfusion, its effects on inducible NOS (iNOS) induction and myocardial injury during late reperfusion are unknown. Wild-type (WT) and eNOS–/– mice were subjected to 30 min of coronary ligation followed by reperfusion. Expression of iNOS mRNA and protein levels and peroxynitrite production were lower in postischemic myocardium of eNOS–/– mice than levels in WT mice 48 h postreperfusion. Significantly improved hemodynamics (±dP/dt, left ventricular systolic pressure, mean arterial pressure), increased rate pressure product, and reduced myocardial infarct size (18 ± 2.5% vs. 31 ± 4.6%) were found 48 h after reperfusion in eNOS–/– mice compared with WT mice. Myocardial infarct size was also significantly decreased in WT mice treated with the specific iNOS inhibitor 1400W (20.5 ± 3.4%) compared with WT mice treated with PBS (33.9 ± 5.3%). A marked reperfusion-induced hyperoxygenation state was observed by electron paramagnetic resonance oximetry in postischemic myocardium, but PO2 values were significantly lower from 1 to 72 h in eNOS–/– than in WT mice. Cytochrome c-oxidase activity and NADH dehydrogenase activity were significantly decreased in postischemic myocardium in WT and eNOS–/– mice compared with baseline control, respectively, and NADH dehydrogenase activity was significantly higher in eNOS–/– than in WT mice. Thus deficiency of eNOS exerted a sustained beneficial effect on postischemic myocardium 48 h after reperfusion with preserved mitochondrial function, which appears to be due to decreased iNOS induction and decreased iNOS-derived peroxynitrite in postischemic myocardium.

oxygen; superoxide; peroxynitrite; mitochondria; electron paramagnetic resonance; ischemia-reperfusion

NITRIC OXIDE (NO) is an important modulator of cardiac performance and left ventricular (LV) remodeling after myocardial infarction. In the setting of ischemic preconditioning, there is evidence that endothelial nitric oxide synthase (eNOS)-derived NO is important for the acute window of protection as well as the subsequent induction of inducible NOS (iNOS), triggering the delayed window of protection (4). However, there has been considerable controversy regarding the effect of eNOS- and eNOS-derived NO on myocardial ischemia-reperfusion injury. In eNOS knockout mice (eNOS–/–), some studies have shown protection, whereas others have reported increased cardiac injury (6, 10, 14, 17, 24, 25). Sharp et al. (24) reported that different eNOS–/– mouse strains responded differently to myocardial reperfusion injury and that the protection seen in University of North Carolina (UNC) eNOS–/– mice was due to compensatory increases in constitutive iNOS expression; however, they only measured mRNA levels and not protein expression. A subsequent study did not find a compensatory increase of basal eNOS expression in iNOS–/– mice, and mice lacking iNOS had better preserved cardiac function and tended to have less severe chamber dilatation after myocardial infarction (18). Thus questions remain regarding the interrelationship of eNOS and iNOS expression and the role of eNOS in the process of iNOS induction in the normal and postischemic heart. Furthermore, the role of iNOS itself in the pathogenesis of postischemic injury remains unclear.

iNOS is thought to play the dominant role in NO production during the late phase of myocardial reperfusion. iNOS is prone to form superoxide and peroxynitrite and can induce cytotoxicity and cellular injury (32, 34). Maximum induction of iNOS activity has been noted 48–72 h after reperfusion (31). Myocardial performance has been shown to be improved and infarct size reduced by continuous iNOS inhibition during 48 h of reperfusion in rabbits (31). A significant increase in survival and improved cardiac function was found after myocardial infarction in iNOS–/– mice (5). These observations suggest that the expression of iNOS contributes to abnormal function and cellular injury in postischemic myocardium. However, using iNOS–/– mice, Zingarelli et al. (36) found an exacerbation of myocardial injury 60 min after reperfusion, and Jones et al. (15) reported no alteration of myocardial injury 24 h after reperfusion. Thus the consequences of iNOS induction in the postischemic heart remain controversial.

It has been suggested that low doses of NO appear to be beneficial, but high doses are harmful to postischemic myocardium (16). There is evidence that expression of one NOS isoform may be altered after deletion of another NOS isoform (17, 18). To date, the role of eNOS in the induction of iNOS in postischemic myocardium and its subsequent role in postischemic injury are not well understood, especially in the late period of reperfusion. Our group (35) has recently shown that eNOS-derived NO markedly suppresses in vivo myocardial O2 consumption in the early period of reperfusion through modulation of mitochondrial respiration. In this study, we tested the hypothesis that eNOS deletion alters iNOS expression and exerts a critical influence on myocardial injury at 48 h reperfusion. Our results indicate that deletion of eNOS decreases the induction of iNOS, and this prevents chronic NO-mediated inhibition of mitochondrial respiration.


   MATERIALS AND METHODS
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 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Animals. Male wild-type (WT) C57BL/6 mice and eNOS knockout (eNOS–/–) mice were purchased from Jackson Laboratory (Bar Harbor, ME). eNOS–/– mice were developed by the group of Smithies at UNC. Mice were regenotyped by the PCR method using genomic DNA extracted from tails. All procedures were performed with the approval of the Institutional Animal Care and Use Committee at Ohio State University (Columbus, OH) and conform to the Guide for the Care and Use of Laboratory Animals (NIH publication 86-23, revised 1985).

In vivo ischemia-reperfusion mouse model. For the in vivo ischemia-reperfusion mouse model, we followed a technique similar to that described previously (35). Mice were anesthetized with ketamine (55 mg/kg) plus xylazine (15 mg/kg). Atropine (0.05 mg sc) was administered to reduce airway excretion. Animals were intubated and ventilated with room air (tidal volume = 250 µl, 100 breaths/min) with the use of a mouse respirator (Harvard Apparatus). A left intercostal thoracotomy was performed. The left anterior descending coronary artery was ligated with a 7-0 silk suture. After 30 min of ischemia, the occlusion was released and reperfusion was confirmed visually. Sham-operated mice underwent the same surgery minus the coronary artery ligation. The rectal temperatures of the mice were maintained at 37°C. Penicillin was used to prevent infection.

RT-PCR and real-time PCR analysis. PCR analysis was performed to determine the levels of iNOS in WT and eNOS–/– mouse nonischemic hearts, using GAPDH as the cDNA quality and loading control. The primer sequences (sense and antisense) designed to detect mouse iNOS mRNA, using Primer Express software (Applied Biosystems), were 5'-CTCTGACAGCCCAGAGTTCC-3' and 5'-GAAAGGGAGAGAGGGGAGG-3'. Triplicate heart specimens were frozen in liquid nitrogen and crushed. RNAs were extracted with TRIzol reagent (Invitrogen, Carlsbad, CA). Isolated RNA (10 µg) was reverse transcribed to first-strand cDNA with the Superscript II RT kit (Invitrogen). PCR amplification was performed with 1 µl of cDNA in 1x Promega buffer, 0.2 mM deoxynucleotide triphosphate, 15 mM MgCl2, 0.5 µM oligonucleotide primers, and 0.5 U Taq DNA polymerase in a 25-µl reaction. Temperature was cycled through 94°C for 4 min initially and then at 94°C for 30 s, 60°C for 45 s, and 72°C for 30 s for 35 cycles and finally at 72°C for 7 min. PCR products were electrophoresed on 2% agarose gels and stained with ethidium bromide. For real-time PCR analysis, 1 µl of cDNA was mixed with 10 µl of 1x SYBR green master mixture (Applied Biosystems, Foster City, CA) and amplified under standard conditions, 50°C for 2 min, 95°C for 10 min, and 40 cycles of a two-step PCR (95°C for 15 s, 60°C for 1 min) with a 7900 HT Fast real-time PCR system (Applied Biosystems). GAPDH was used as the endogenous control.

Northern blotting analysis. Total RNA was extracted from homogenized tissue samples with TRIzol reagent (Invitrogen) according to the manufacturer's instructions. Isolated RNA (15 µg) was subjected to gel electrophoresis with 1% agarose gel containing 6% formaldehyde and then transferred onto a nitrocellulose membrane. Blots were hybridized by a 32P-labeled iNOS cDNA probe (Alpha Diagnostic, San Antonio, TX). For quantitative purposes, the blots were exposed to a phosphor-imaging screen, and the exposed screens were analyzed in a phosphor imager (Molecular Dynamics, Sunnyvale, CA). Each signal was normalized to the 18S signal.

Western blotting analysis. Tissue samples were homogenized in 25 mM Tris buffer (pH 7.5) containing 0.5 mM EDTA-0.5 mM EGTA-1 mM PMSF-protease inhibitor cocktail and then centrifuged at 14,000 g for 15 min. The resulting supernatants were collected as cytosolic fractions. The protein content of the supernatant was determined by the DC protein assay (Bio-Rad, Hercules, CA). The protein (90 µg) was separated by SDS-PAGE with the use of 8% or 4–20% Tris-glycine gel (Invitrogen) and electrophoretically transferred onto nitrocellulose membranes (Amersham Biosciences). The membranes were incubated with rabbit anti-iNOS polyclonal antibody (Transduction Laboratories, San Jose, CA), anti-OxPhos complex IV subunit VIb (cytochrome-c oxidase VIb) monoclonal antibody, or anti-OxPhos complex I 39-kDa subunit [NADH dehydrogenase (NADH-DH)] monoclonal antibody, respectively. Blots were then washed and incubated with horseradish peroxidase-conjugated secondary antibody (Amersham Biosciences) and then visualized by enhanced chemiluminescence detection reagents (Amersham Biosciences). GAPDH was used as a loading control for Western blotting. The signal intensity of blotting was normalized to the Western signal of GAPDH (9).

Immunohistochemistry for iNOS and nitrotyrosine. For iNOS staining, paraformaldehyde-fixed, paraffin-embedded cardiac sections were incubated with purified rabbit anti-iNOS polyclonal antibody (1:200) overnight at 4°C and then reacted with biotin-conjugated secondary antibody and streptavidin-horseradish peroxidase-conjugate solution. Color was developed using diaminobenzidine (17). For nitrotyrosine staining, mouse heart infused with peroxynitrite (1 mM, 0.05 ml; Cayman Chemical) into the LV chamber was taken as the positive control, with the heart removed and fixed after 15 min. Immunohistochemistry was performed by the method published previously by our laboratory (29). Briefly, the formalin-fixed paraffin sections were incubated with rabbit polyclonal anti-nitrotyrosine antibody (1:100; Upstate, Charlottesville, VA) for 1 h, then with the biotinylated secondary antibody, and again with the tertiary, ExtrAvidin alkaline phosphatase (Vector Laboratories, Burlingame, CA). Color was developed with the use of fast red.

Myocardial infarct size determination. Infarct size was measured in eNOS–/– mice vs. WT mice and also in WT mice treated with N-3-aminomethyl-benzyl-acetamidine-dihydrochloride (1400W) (WT-1400W; Sigma) and WT mice treated with vehicle (PBS) (WT-PBS). 1400W, a specific iNOS inhibitor, was administered (2.0 mg/kg ip in sterile PBS) at 0.5 h before and 8, 20, 32, and 44 h after coronary ligation (13).

Forty-eight hours after reperfusion, the mouse was reintubated and ventilated as above, the left coronary artery was reoccluded, and 0.15 ml of 4.0% Evans blue (Sigma) was injected from the inferior vena cava to delineate the nonischemic myocardial tissue. The heart was then cut into four transverse slices. The slices were stained with 1.5% 2,3,5-triphenyltetrazolium chloride (Sigma) to determine the infarct area and photographed under a dissecting microscope. LV area, area at risk (AAR), and infarct area (IF) were determined by computerized planimetry. The area of myocardial tissue showing white color was determined as IF. Infarct size was expressed as percentage of the IF in AAR.

Measurement of hemodynamics. Forty-eight hours after reperfusion, the right carotid artery was cannulated with a Millar tip transducer catheter (model SPR-261, 1.4 Fr.) connected to a PowerLab system. After arterial blood pressure and heart rate were obtained, the catheter was advanced to the LV for measurement of LV systolic and end-diastolic pressures and the maximal rate of pressure development (+dP/dt) and rate of relaxation (–dP/dt) of LV. Rate pressure product was calculated by the following equation: rate pressure product (mmHg/min) = mean arterial pressure x heart rate.

In vivo electron paramagnetic resonance oximetry. In vivo electron paramagnetic resonance (EPR) oximetry was performed as reported previously by our laboratory (35). This technique has the unique capability of providing repeated noninvasive measurements of tissue PO2 over long periods of time, up to several months (35). The in vivo ischemia-reperfusion mouse model was produced as described above. After thoracotomy, ~10 µg of lithium octa-n-butoxy-naphthalocyanine crystals were implanted into the myocardium at the center of the risk region with a 25-gauge needle. The mouse was then placed into the EPR system with a surface coil resonator placed on the left sternum just above the heart, and myocardial oxygenation was measured continuously. At 24 and 72 h after reperfusion, the mice were again anesthetized and EPR measurements were performed. EPR measurements were performed with the use of a custom-made L-band spectrometer with a frequency of 1.1 GHz, microwave power of 16 mW, modulation field of 7 x 10–6 T, and scan width of 5 x 10–4 T (11, 12). The implanted crystals were confirmed by histology to be located at the middle layer of the LV myocardium.

Mitochondrial enzyme activity assay. Forty-eight hours after reperfusion, mouse hearts were excised, and the myocardium in the risk region was immediately frozen with liquid nitrogen. The hearts from intact animals were taken as the baseline control. The tissues were homogenized in ice-cold HEPES buffer (3 mM, pH 7.2) containing sucrose (0.25 M), EGTA (0.5 mM), and protease inhibitor cocktail (1:40; Roche). Cytochrome-c oxidase (CcO) activity was measured in the presence of phosphate buffer (50 mM, pH 7.4) and reduced cytochrome c (60 µM; Sigma) (3). NADH-DH activity was measured in the presence of Tris-Cl buffer (20 mM, pH 8.0), NADH (150 µM; Sigma), and coenzyme Q1 (100 µM; Sigma) (8). Enzyme activity per milligram of protein in supernatant was calculated based on the millimolar extinction coefficient, {epsilon}550 nm = 18.5 mM–1·cm–1 for cytochrome c and {epsilon}340 nm = 6.22 mM–1·cm–1 for NADH.

Statistical analysis. Two-way ANOVA was used for analysis of the data with time course followed by Newman-Keuls multiple-comparison test. The t-test was used for data comparison between WT and eNOS–/– mice. Data are presented as means ± SE. P < 0.05 was considered significant.


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
iNOS mRNA and protein expression in normal and postischemic myocardium. In normal control hearts not subjected to ischemia, iNOS mRNA expression measured by RT-PCR appeared to be a little bit lower in the eNOS–/– than in WT hearts (Fig. 1A). To further confirm the relative expression of iNOS mRNA in both strains, real-time PCR experiments that provide truly quantitative assessment of mRNA expression were also performed, and iNOS mRNA expression was not significantly different in normal control hearts between eNOS–/– and WT mice (Fig. 1B; P = 0.91). From the real-time PCR measurements, it was clear that the copy number was quite low, ~0.1% of the GAPDH copy number in the WT mice and eNOS–/– mice. Furthermore, there was no detectable iNOS protein on Western blotting in either strain of control nonischemic mice (Fig. 1D). In the risk region of hearts subjected to 30 min of coronary ligation followed by 48 h of reperfusion, iNOS mRNA expression was readily detected in both eNOS–/– and WT mice, but these levels were lower in eNOS–/– than in WT mice (Fig. 1C). Similarly, the levels of iNOS protein detected on immunoblotting were greatly increased in WT, but increased to a lesser extent in eNOS–/– heart tissue (Fig. 1D). On immunohistology, prominent iNOS staining was seen in postischemic myocardium, but the staining was weaker in eNOS–/– than in WT hearts (Fig. 2, D vs. C). No immunostaining was seen for iNOS in control nonischemic WT or eNOS–/– hearts (Fig. 2, A and B). Thus it is clear that both iNOS mRNA and protein expression are increased in postischemic myocardium.


Figure 1
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  		Fig. 1. Myocardial inducible nitric oxide synthase (iNOS) expression. A and B: RT-PCR banding and real-time PCR analysis of iNOS expression in normal control mouse hearts (control). C: Northern blotting of iNOS mRNA in postischemic hearts. D: representative Western blotting of iNOS and mean values of iNOS protein in normal control mouse hearts (control) and postischemic hearts. iNOS expression of mRNA and protein were significantly weaker in endothelial nitric oxide synthase knockout (eNOS–/–) than in wild-type (WT) mice postischemia. The signals were normalized to GAPDH or 18S as indicated.

 

Figure 2
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  		Fig. 2. Immunostaining of myocardial iNOS in mouse hearts postischemia using a rabbit anti-iNOS polyclonal antibody. A: negative control (no primary antibody) in WT heart. B: negative control (no primary antibody) in eNOS–/– heart. C: immunostaining for iNOS in the postischemic heart of WT. D: immunostaining for iNOS in the postischemic heart of eNOS–/– (x200).

 
Nitrotryosine staining as a marker of peroxynitrite production. The positive control shown in Fig. 3A was created by in vivo injection of a bolus of peroxynitrite into the LV chamber, which results in a portion of it being infused into the coronary arteries. Because of this, the nitrotyrosine levels are higher in the capillaries than in the myocytes, and one sees lines or patches of strong positive red staining in capillaries that were exposed to this peroxynitrite and formed high levels of nitrotyrosine. On the basis of the negative charge of peroxynitrite, only low levels would reach the myocytes so that the staining in myocytes is much weaker than that seen in the endothelium. In the myocardium of reperfused hearts (Fig. 3C), staining for nitrotyrosine is positive within myocytes and also in inflammatory cells. In postischemic myocardium of eNOS–/– hearts, the myocardium appears blue with little or very weak red staining; however, some positive red staining is seen in inflammatory cells. There was no staining in negative control (Fig. 3B).


Figure 3
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  		Fig. 3. Immunostaining of nitrotyrosine in mouse hearts postischemia using a rabbit polyclonal anti-nitrotyrosine antibody. A: positive control mouse heart infused with peroxynitrite (1 mM, 0.05 ml) into the left ventricular chamber. Marked positive (red) staining is seen in capillaries. B: negative control (no primary antibody). No positive (red) staining is seen. C and D: immunostaining of nitrotyrosine in the risk region of WT and eNOS–/– hearts (x200). In C (WT), diffuse positive red staining is seen in myocytes and inflammatory cells, whereas in D (eNOS–/–) weaker patchy staining is present mostly in inflammatory cells. Arrows show examples of areas of positive staining.

 
Myocardial infarct size. With 30 min of coronary ligation followed by 48 h of reperfusion, infarct size in eNOS–/– mice (IF/AAR = 18 ± 2.5%) was significantly less than that in WT mice (IF/AAR = 31 ± 4.6%) (Fig. 4A; P < 0.05, n = 8). In WT-1400W mice (IF/AAR = 20.5 ± 3.4%), a 39.6% reduction of infarct size was noted compared with WT-PBS mice (IF/AAR = 33.9 ± 5.3%) (Fig. 4B; P < 0.05, n = 10). No significant difference was observed in AAR/LV between WT and eNOS–/– mice and between WT-PBS and WT-1400W mice.


Figure 4
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  		Fig. 4. Measurement of myocardial infarct size. Mice were subjected to 30 min of coronary ligation followed by 48 h of reperfusion. A: infarct size in eNOS–/– mice (18 ± 2.5%) was significantly less than that in WT mice (31 ± 4.6%) (*P < 0.05; n = 8). B: infarct size in WT mice treated with N-3-aminomethyl-benzyl-acetamidine-dihydrochloride (1400W; 20.5 ± 3.4%) was significantly less than that in WT mice treated with PBS vehicle (33.9 ± 5.3%) (*P < 0.05; n = 10). AAR, area at risk; LV, left ventricle; IF, infarct area.

 
Hemodynamics. In vivo hemodynamics was measured in mice 48 h after reperfusion by LV catheterization. eNOS–/– mice exhibited significantly higher mean arterial pressure, LV systolic pressure, +dP/dt, –dP/dt, and rate pressure product compared with those in WT mice (Table 1).


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  		Table 1. Hemodynamics in mice after myocardial ischemia-reperfusion

 
In vivo myocardial tissue PO2. In vivo myocardial tissue PO2 was monitored acutely and for 72 h by EPR oximetry. Basal levels of myocardial PO2 were measured as ~8–10 Torr, consistent with earlier studies (35). After 30 min of coronary ligation, PO2 values declined to <2 Torr in both groups (Fig. 5). A marked reperfusion-induced hyperoxygenation state was observed in postischemic myocardium of WT mice with a maximum value of 61.7 ± 2.7 Torr at 24 h reperfusion. In eNOS–/– mice, the hyperoxygenation was less with a maximum value of 32.0 ± 2.6 Torr seen at 72 h reperfusion. The PO2 values were significantly lower from 1 to 72 h postischemia in eNOS–/– mice than in WT mice (P < 0.01, n = 7) (Fig. 5).


Figure 5
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  		Fig. 5. Myocardial tissue PO2 was monitored by electron paramagnetic resonance (EPR) oximetry in mice postischemia (n = 7). EPR microcrystal O2 probes were implanted into the myocardium at the center of the risk region before introduction of ischemia. **P < 0.01 vs. WT.

 
Enzymatic activities of mitochondrial electron transport chain in myocardium. With a consideration of the dependence of cardiac function on bioenergetic metabolism and the regulation of iNOS-derived NO on mitochondrial respiration, the enzymatic activities of CcO and NADH-DH were assayed in postischemic myocardium. In WT and eNOS–/– mice, both myocardial CcO activity and NADH-DH activity were significantly decreased at 48 h after reperfusion in WT (P < 0.01) and eNOS–/– mice (P < 0.05–0.01) compared with that in baseline control. No significant difference was found for CcO activity between WT and eNOS–/– mice (Fig. 6A). However, NADH-DH activity was significantly higher 48 h after reperfusion in eNOS–/– mice than in WT mice (P < 0.05) (Fig. 6B).


Figure 6
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  		Fig. 6. Mitochondrial enzyme activity in myocardium of mice postischemia (n = 5). A: cytochrome-c oxidase (CcO) activity. B: NADH dehydrogenase (NADH-DH) activity. *P < 0.05 and **P < 0.01 vs. baseline control. +P < 0.05 vs. WT.

 
Protein expression of mitochondrial electron transport chain in myocardium. To explore the regulatory mechanisms of mitochondrial respiration at the level of translation, protein expressions of CcO and NADH-DH were probed by Western blotting. Protein expression of the CcO was mildly decreased in postischemic myocardium in WT and eNOS–/– mice compared with baseline control, and there was no significant difference between the two groups (Fig. 7A). However, protein expression of the NADH-DH in postischemic myocardium was mildly but significantly lower in eNOS–/– than in WT mice (n = 3, P < 0.05) (Fig. 7B).


Figure 7
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  		Fig. 7. Protein expression of mitochondrial enzyme by Western blotting in postischemic myocardium of mice (n = 3 per group). A: Western blotting of CcO and mean values of CcO expression in WT and eNOS–/– hearts. B: Western blotting of NADH-DH and mean values of NADH-DH expression in WT and eNOS–/– hearts.

 

   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
In this study, we have demonstrated that a deficiency of eNOS exerted a beneficial effect on postischemic myocardium 48 h after reperfusion with decreased infarct size, improved cardiac function, and preserved mitochondrial function. This protection appears to be due to a decrease of iNOS induction and iNOS-derived NO and peroxynitrite in postischemic myocardium.

The induction of myocardial iNOS in the early postischemic phase in eNOS–/– mice has been reported in isolated hearts but remains controversial (17, 20). Mild iNOS induction in eNOS–/– mice has been suggested to contribute to the protective effect at 60 min (17) or 24 h reperfusion (24). It has been noted in failing hearts that because of the high NO output of iNOS, even modest expression of this enzyme might account for significant NO production (27). However, it is also known that iNOS is prone to produce superoxide, with this occurring from the reductase site as well as the oxygenase site (32, 34). Thus iNOS can produce the potent oxidant peroxynitrite, and this is favored by tetrahydrobiopterin consumption or L-arginine depletion (32–34).

In this study, we have demonstrated that myocardial iNOS expression at both mRNA and protein levels was significantly lower in eNOS–/– than in WT mice 48 h after reperfusion. Moreover, we detected weaker nitrotyrosine staining, a marker of peroxynitrite formation, in the risk region of eNOS–/– hearts. The mechanism for downregulation of iNOS expression in eNOS–/– mice was not clear. Expression of iNOS can be induced in response to inflammation or cytokine activation in postischemic myocardium (2). The burst of oxidant and oxygen radical formation that occurs during the early period of reperfusion can trigger postischemic inflammation (23, 37–39) and may also oxidize the critical NOS cofactor tetrahydrobiopterin, resulting in a shift of eNOS from the production of NO to superoxide (33). This study demonstrated less lethal injury in postischemic myocardium at 48 h in eNOS–/– hearts than in WT hearts.

It has been reported in the literature, based on RT-PCR measurements of mRNA, that constitutive iNOS expression has a compensatory increase in UNC eNOS–/– mice (24); however, RT-PCR is not quantitative and measurements of the levels of mRNA alone cannot determine the level of protein expression. In contrast to this study, other investigators reported that basal eNOS expression was not found to have a compensatory increase in iNOS–/– mice (18). Therefore, from prior studies, it has not been clear whether knockout of one isoform induces a compensatory change in the other. In our studies, we see that the level of iNOS mRNA in nonischemic hearts is not significantly different in UNC eNOS–/– mice from that in WT. We performed both RT-PCR and quantitative real-time PCR, which provides a reliable quantitative measure. Mice were also regenotyped at the time of assay to ensure no errors in strain assignment. Our results appear opposite to those of Sharp et al. (24). Nevertheless, it was clear that the mRNA copy number in nonischemic hearts is quite low and that iNOS protein was not detectable by Western blotting from either nonischemic WT or eNOS–/– control hearts. In contrast to this, both iNOS mRNA and protein are much higher and clearly detected in the risk region of postischemic myocardium after 48 h of reperfusion. When we compare the difference between the control and ischemic hearts, after 48 h reperfusion, iNOS expression increased >100-fold in WT and 40-fold in eNOS–/– hearts. It is unclear why the RT-PCR results of Sharp et al. showed an increase in iNOS mRNA in eNOS–/– hearts; however, without any measure of protein expression and only nonquantitative RT-PCR data reported, it is not valid to conclude that expression of iNOS is increased.

Although ischemia-reperfusion-induced iNOS expression was detected in both WT and eNOS–/– hearts, its induction was more than twofold lower with eNOS–/– and the pathophysiological response was markedly different. We hypothesize that this is due to the higher amount of NO and NO-derived peroxynitrite produced from iNOS in postischemic myocardium. With this higher level of iNOS induction, higher levels of the peroxynitrite product nitrotyrosine were seen in the postischemic myocardium of the WT mice. Interestingly, although nitrotyrosine staining was more prominent in WT, lower levels were still detectable in the eNOS–/– hearts. Because it has been shown that NOS-independent NO formation from nitrite can occur in ischemic myocardium, this lower level nitrotyrosine staining may arise in part from these NOS-independent pathways (41).

Lower amounts of NO production have been reported to be beneficial, but high amounts of NO have been shown to be harmful (16). Increased NO formation during ischemia or the early period of reperfusion has also been shown to occur in hearts subjected to global ischemia by our laboratory and others (40, 41). In general, it has been shown that during the acute process of myocardial ischemia and reperfusion that myocardial levels of NO and nitrosyl-heme proteins are greatly increased (26, 29). The protection observed in this study appears to be partially induced by the abolishment of the acute increase in eNOS-derived NO. The balance between NO and oxygen free radicals is also crucial in modulating the outcome after an ischemic insult (29). If NO is formed in the presence of superoxide, as occurs during the early period of reperfusion, it rapidly reacts to form the potent oxidant peroxynitrite, which oxidizes and nitrates proteins, inducing cellular injury (1, 6, 30). Higher NO concentrations (in the µM range) can also directly reduce cardiomyocyte function, mediate inflammatory processes following ischemia-reperfusion, impair mitochondrial respiration, and even induce cardiomyocyte death (21, 22).

In the present study, infarct size was found to be decreased in eNOS–/– mice compared with WT mice at 48 h reperfusion. In accordance with this, improved cardiac function was observed 48 h postischemia in eNOS–/– mice. Previous in vitro studies have reported a significant improvement of the postischemic myocardial functional recovery in eNOS–/– mice (6). Sharp et al. (24) have also noticed a reduction of infarct size 24 h after reperfusion in UNC eNOS–/– mice. Furthermore, in the present study, treatment with the specific iNOS inhibitor 1400W was shown to significantly decrease infarct size in WT mice. The decrease of infarct size in WT mice treated with 1400W appears to be related to decreased NO production from iNOS. Sharp et al. reported an increase of infarct size in UNC eNOS–/– mice treated with high doses of 1400W, 122.5 mg/kg over 24 h (24). One can speculate that the apparently opposite results of Sharp et al. reporting an increase of infarct size in eNOS–/– mice treated with 1400W could be due to a near total absence of NO production from both eNOS and iNOS in their study. Whereas lower physiological levels of NO may be beneficial, pathologically increased NO could induce injury (21, 22). Another concern is that the total dose of 1400W used in their study is high, and high doses of the drug can cause adverse hemodynamic effects (7). Overall, our studies clearly suggest that the increased iNOS expression in the postischemic heart is involved in exacerbation of myocardial injury and that less iNOS induction results in less injury.

Although a hyperoxygenation state was demonstrated persisting more than 72 h in the postischemic myocardium of both WT and eNOS–/– mice, the values of PO2 in eNOS–/– mice were significantly lower than in WT and the time of the peak in PO2 was delayed. Our group (35) has previously observed in the in vivo mouse model of regional ischemia that myocardial oxygen consumption in the risk region during the first 60 min of reperfusion is decreased because of NO-mediated inhibition of mitochondrial function. Thus it is likely that the NO-mediated impairment of mitochondrial function persists and is further exacerbated by the induction of iNOS that occurs with prolonged periods of reperfusion. In view of prior studies showing similar perfusion in the risk region of eNOS–/– and WT mice, the lower levels of tissue oxygenation in eNOS–/– than in WT likely suggest that mitochondrial O2 consumption is decreased to a greater extent in the risk region of WT hearts (35). As previously suggested, this decrease in O2 consumption could arise because of inhibition of critical sites in the electron transport chain, including CcO and NADH-DH.

At low levels of tissue PO2, NO reversibly inhibits the respiratory chain because NO competes with O2 in binding to CcO. The sensitivity of cellular respiration to NO depends on the O2 concentration, substrate type, and respiration rate in rat heart mitochondria. The effect of NO is more effective at low O2 concentrations (19). The NO-derived oxidant, peroxynitrite, can induce irreversible inhibition on many mitochondrial components, especially NADH-DH (28). NO and its derivatives may contribute to LV dysfunction by inhibition of CcO and NADH-DH in mitochondrial respiration and thus reduction of ATP synthesis (30). Our previous study showed that CcO activity was not decreased or influenced by eNOS-derived NO in postischemic myocardium at early times of reperfusion of 60 min (35). The present study, however, demonstrated that the activity and protein expression of myocardial CcO were decreased at later times of reperfusion of 48 h but to a similar extent in WT and eNOS–/– mice.

NO-derived peroxynitrite can induce irreversible inhibition of the mitochondrial components with iron sulfur clusters, such as complex I (NADH-DH) and complex II (1). Our previous study showed that, at early reperfusion times, eNOS-derived NO inhibited NADH-DH activity without change of NADH-DH expression (35). In the present study, decreased NADH-DH protein expression and increased NADH-DH activity were demonstrated in eNOS–/– mice, indicating that the activity of NADH-DH was preserved in the postischemic myocardium of eNOS–/– hearts compared with WT. This is likely due to less formation of eNOS and iNOS-derived peroxynitrite. In WT mice, NADH-DH activity was markedly decreased and protein expression was modestly upregulated in what may constitute a partial compensation for the loss of its enzymatic function.

Overall, in mice with deficiency of eNOS, a beneficial effect on postischemic recovery was seen, with smaller infarct size at 48 h after reperfusion and improved cardiac function. With deficiency of eNOS, less induction of iNOS and less evidence of peroxynitrite formation were seen. Marked myocardial hyperoxygenation was present within the risk region after 48 h of reperfusion but decreased in eNOS–/– mice compared with WT, primarily because of preservation of mitochondrial function and mitochondrial NADH-DH activity. Thus, in eNOS–/– mice, less myocardial iNOS expression occurs, with less injury to postischemic myocardium and preserved mitochondrial function.


   GRANTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
This work was supported by National Heart, Lung, and Blood Institute Grants HL-63744, HL-65608, and HL-38324 (J. L. Zweier), National Heart, Lung, and Blood Institute Grant HL-081630 and American Heart Association Grant GRT962974 (G. He), National Heart, Lung, and Blood Institute Grant HL-70294 (A. R. Strauch).


   ACKNOWLEDGMENTS
 
We thank Dr. Susie Jones for immunohistology support, as well as Dr. Yong Xia for valuable technical advice. We also acknowledge and thank Dr. Periannan Kuppusamy and Dr. Ramasamy Pandian for generously providing the EPR oximetry probes.


   FOOTNOTES
 

Address for reprint requests and other correspondence: J. L. Zweier, 473 West 12th Ave., Suite 110, Dept. of Cardiology Columbus, OH 43210 (e-mail: Jay.Zweier{at}osumc.edu); and X. Zhao, Changzheng Hospital, 415 Fengyang Rd., Shanghai 200003, China (e-mail: xue.zhao{at}osumc.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.


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 DISCUSSION
 GRANTS
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