INTRODUCTION

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NITRIC OXIDE (NO) has been intensely investigated as a mediator of numerous physiological responses, and endothelial-derived NO has been shown to have a direct impact on the cardiovascular system. In this regard, NO has been shown to modulate vascular reactivity (2), leukocyte-endothelial cell interactions (12, 14), platelet function (4, 23), and smooth muscle cell proliferation (3). Many studies have shown exogenous and endogenous NO to be cardioprotective in animal models of myocardial ischemia-reperfusion (MI/R) injury. Previous studies have shown that NO donors decrease arrhythmias in association with MI/R (21, 25), improve postischemic blood flow and contractile function (19, 20), and reduce myocardial infarct size (15, 21). The NO precursor L-arginine has also been shown to protect against MI/R injury (18, 27). The mechanisms responsible for the cardioprotective actions of NO are thought to be related to inhibition of leukocyte-endothelial cell interactions and subsequent inflammation in the coronary microvasculature.

In contrast, some experimental studies (16, 28) suggest that NO is cytotoxic and may actually mediate myocardial cell death after MI/R. Pretreatment of animals with nonspecific NO synthase (NOS) inhibitors has been shown to reduce myocardial infarct size (22, 29). It has also been suggested that NO released in the coronary circulation contributes to cell signaling involved in the pathophysiology of MI/R injury (17).

The controversy of NO and postischemic myocardial injury has only become more complex with the development of endothelial NOS (eNOS) knockout mice. Two different strains of eNOS-deficient (eNOS^/) mice have been developed. Huang and colleagues (6) developed the first eNOS^/ mice at Massachusetts General Hospital (Har eNOS^/), and Shesely and colleagues (24) at the University of North Carolina (UNC eNOS^/) reported a second strain of eNOS^/ mouse. Our laboratory has previously reported that myocardial infarct size (8) and leukocyte-endothelial cell interactions (14) are both augmented in the Har eNOS^/ mouse. In contrast, it has recently been reported (10) that myocardial infarct size is significantly attenuated in the UNC eNOS^/ mouse, and other laboratories (30) investigating the UNC eNOS^/ mouse have demonstrated that the lack of eNOS has no effect on MI/R injury. In the present study, we sought to examine the response to MI/R of the two eNOS^/ strains of mice. Specifically, we examined systemic hemodynamics, myocardial cell injury after MI/R, myocardial inducible NO synthase (iNOS) mRNA levels, and myocardial iNOS and eNOS protein expression.

	MATERIALS AND METHODS

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Mice. Male, respective wild-type mice (Jackson Laboratory; Bar Harbor, ME) were used as control mice in the present study. C57/BL6 mice were used as a control for the UNC eNOS^/ mice and B6/129 mice were used as control for the Har eNOS^/ mice. eNOS-deficient mice were obtained from Dr. Paul Huang of Harvard University (6) (Har eNOS^/) and Jackson Laboratory (24) (UNC eNOS^/). The eNOS gene in the Har eNOS^/ mouse was replaced with a targeting vector with 5' and 3' flanking regions of homology, which replaced the HindIII to SalI fragment that contains exons encoding the reduced NADP ribose and adenine binding sites (amino acids 1010-1144). In the UNC eNOS^/ mouse, a premature translation stop codon was introduced via an eNOS target construct, pENOSX. This was prepared from the 4.7-kb XbaI fragment by the replacement of 129 bp in exon 12, with 1.2-kb sequences that include the neomycin-resistance gene (Neo) from pMC1Neo polA oriented oppositely to the eNOS gene. All animal experiments complied with the National Institutes of Health's Guide for the Care and Use of Laboratory Animals (NIH Publication No. 85-23, Revised 1985) and with state and federal regulations. All animal procedures and protocols were approved by the Louisiana State University Health Sciences Center Animal Care and Use Committee.

Mean arterial blood pressure and heart rate. Mice were anesthetized with pentobarbital sodium (50 mg/kg) and ketamine hydrochloride (60 mg/kg). A tracheotomy was performed, and the mouse was connected to a rodent ventilator. The right common carotid was cannulated with polyethylene (PE)-50 tubing tipped with PE-10. The cannula was attached to a transducer and monitor (World Precision Instruments; Sarasota, FL). The monitor was connected to a MacLab data collection system for analysis of mean arterial blood pressure (MABP) and heart rate (HR). The rate-pressure product was determined from the following equation: MABP × HR/1,000.

Myocardial I/R protocol. The surgical protocol and infarct size determination were performed similar to methods described previously (5, 9). Mice were anesthetized with pentobarbital sodium (50 mg/kg) and ketamine hydrochloride (60 mg/kg) and orally intubated with PE-90 tubing and connected to a rodent ventilator (model 683, Harvard Apparatus). Core body temperature was maintained at 37°C with the use of a rectal thermometer and infrared heating lamp. A median sternotomy was performed, and the left anterior descending coronary artery (LAD) was visualized and completely ligated for 30 min with 7-0 silk suture. Ischemia was confirmed by the appearance of hypokinesis and pallor distal to the occlusion. Animals receiving the iNOS inhibitor (1400W) (5 mg · kg¹ · h¹) underwent intraperitoneal implantation of an osmotic minipump (Alzet) immediately after the initiation of ischemia. After 30 min of LAD occlusion, the ligature was removed and reperfusion was visually confirmed. The chest wall was closed, and the mice were allowed to recover in a temperature-controlled area with butorphanol tartrate (0.1 mg/kg) for analgesia. The next day (at the end of 24 h of reperfusion), mice were anesthetized with pentobarbital sodium (50 mg/kg) and ketamine hydrochloride (60 mg/kg). A tracheotomy was performed and the mouse was connected to the ventilator. The right common carotid artery was cannulated for Evans blue infusion. The LAD was religated and Evans blue (1.5 ml of 1.0% solution) was retrogradely infused through the carotid artery catheter to delineate the ischemic from the nonischemic zones. Ex vivo incubation for 5 min at 37°C in a 1% solution composed of 2,3,5-triphenyltetrazolium chloride allowed differentiation between the viable and necrotic areas of the myocardium previously rendered ischemic. Each of the five 1-mm-thick slices was weighed and the areas of infarction, risk, and left ventricle (LV) were assessed with the use of computer-assisted planimetry software (NIH Image version 1.57).

Determination of iNOS mRNA levels. Reverse transcriptase (RT)-polymerase chain reaction (PCR) was performed on whole heart homogenates from wild-type, UNC eNOS^/, and Har eNOS^/ mice as previously reported (11). Briefly, mice were anesthetized with pentobarbital sodium (50 mg/kg) and ketamine hydrochloride (60 mg/kg). The heart was excised and flushed with 37°C saline and snap-frozen in liquid nitrogen. RNA was extracted using TriZOL reagent (GIBCO-BRL). The RNA was precipitated and subjected to a harsh DNAse treatment, and RT-PCR analysis was carried out. cDNA was amplified for iNOS using the forward primer 5'-ATGTCCGAAGCAAACATCAC-3' and the reverse primer 5'-TAATGTCCAGGAAGTAGGTG-3'. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was amplified with the use of the forward primer 5'-CGGAGTCAACGGATTTGGTCGTAT-3' and the reverse primer 5'-AGCCTTCTCCATGGTGGTGAAGAC-3'. iNOS was semiquantitated with the use of Alpha Imager version 3 (Alpha Inotech) and reported as a percentage of GAPDH.

Western blot analysis. Myocardial protein was isolated as described by Transduction Laboratories. Briefly, mice were anesthetized as above, and the heart was rapidly excised and snap-frozen in liquid nitrogen. Hearts were then ground with the use of a mortar and pestle and subsequently placed into a tissue grinder containing lysis buffer. Protein concentrations were obtained with the use of DC Protein Assay II (Bio-Rad) and read at 690 nM. Protein (150 µg) was electrophoresed and transferred to nitrocellulose membrane. The membranes were incubated in primary antibodies specific for iNOS or eNOS and subsequently incubated in secondary antibody. The membranes were exposed to enhanced chemiluminescence (ECL+, Amersham) and imaging film for 3 min. Spot density was then obtained.

Statistical analysis. All data were subjected to analysis of variance with Scheffé's post hoc test or an unpaired t-test. All values are reported as means ± SE. Statistical significance was set at P < 0.05.

	RESULTS

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Hemodynamic measurements. MABP, HR, and rate-pressure product were measured in Har eNOS^/ and UNC eNOS^/ and in respective wild-type control mice (Table 1). MABP was significantly elevated in both the UNC eNOS^/ and the Har eNOS^/ mice (P < 0.05 vs. wild-type controls). The UNC eNOS^/ mice exhibited a significant increase in rate-pressure product compared with wild-type control; however, the Har eNOS^/ did not.

View this table: [in this window] [in a new window]   Table 1.   Hemodynamic data for the UNC eNOS/, Har eNOS/, and respective wild-type controls

Myocardial protein levels. Western blot analyses for eNOS and iNOS protein levels were performed in wild-type, UNC eNOS^/, and Har eNOS^/ mice. Although eNOS protein was constitutively present in both wild-type strains, it was completely absent in both the UNC and Har eNOS^/ mice (Fig. 1). We also evaluated basal iNOS protein levels in myocardial tissue of the wild-type controls and both strains of eNOS^/ animals. Basal levels of iNOS were very low and similar in both strains of wild-type animals. In sharp contrast, levels were elevated by approximately fourfold in the UNC eNOS^/ mice compared with the Har eNOS^/ mice (P < 0.05).

View larger version (23K): [in this window] [in a new window]   Fig. 1.   A: representative gel of endothelial nitric oxide (NO) synthase (eNOS) protein in wild-type and University of North Carolina (UNC) eNOS/ mice. This shows that whereas the wild-type mice have eNOS protein, the UNC eNOS/ mice are completely deficient. B: representative gel of eNOS protein in wild-type mice and Harvard (Har) eNOS/ mice. This shows that the Har eNOS/ mice have an absence of eNOS protein, whereas the wild-type mice express the protein. C: bar graph of eNOS protein in wild-type, UNC eNOS/, and Har eNOS/ mice as a percentage of -actin. **P < 0.01 vs. wild-type control. The number of animals per group is indicated inside the bars.

Myocardial area-at-risk and infarct size. Summary data of area-at-risk (AAR) and infarct size (NEC) after 30 min of LAD occlusion and 24 h of reflow are shown in Fig. 2. All four groups experienced similar-sized AAR per LV (AAR/LV) (UNC eNOS^/ 50.2 ± 4.6%, wild-type 55.9 ± 3.4%, Har eNOS^/ 58.9 ± 3.3%, and wild-type 59.8 ± 2.1%). UNC eNOS^/ mice exhibited a significant (P < 0.05) reduction in NEC/AAR compared with wild-type controls (15.7 ± 1.4 vs. 36.7 ± 5.1%). However, the Har eNOS^/ experienced a significant (P < 0.01) increase in NEC/AAR compared with wild-type controls (62.5 ± 4.9 vs. 34.0 ± 5.7%).

View larger version (30K): [in this window] [in a new window]   Fig. 2.   Myocardial area-at-risk (AAR) per left ventricle (AAR/LV) and infarct size per AAR (NEC/AAR) for UNC eNOS/ mice, Har eNOS/ mice and their respective wild-type controls. The number of animals per group is indicated in the bars. The infarct size is significantly lower in the UNC eNOS/ mice compared with wild-type control. In contrast, the absence of eNOS in the Har strain exacerbates myocardial infarct size compared with wild-type mice.

Detection of iNOS mRNA. RT-PCR was performed on whole hearts from wild-type, UNC eNOS^/, and Har eNOS^/ (Fig. 3, A-C). Representative gels are shown in Fig. 3, A and B. Summary data of iNOS mRNA levels are presented in Fig. 3C. Constitutive levels of iNOS mRNA were significantly higher (P < 0.05) in UNC eNOS^/ (65.5 ± 11.4%) mice compared with wild-type controls (26.4 ± 10.5%). There was no significant difference between Har eNOS^/ mice and wild-type controls in iNOS mRNA expression (65.3 ± 4 vs. 62.5 ± 2.4%).

View larger version (37K): [in this window] [in a new window]   Fig. 3.   A: representative gel of constitutive inducible NOS (iNOS) mRNA in wild-type and UNC eNOS/ mice. This shows an increase in iNOS mRNA in UNC eNOS/ mice compared with wild-type. B: representative gel of constitutive iNOS mRNA in wild-type and Har eNOS/. This shows that there is no difference in iNOS mRNA in the Har eNOS/ and the wild-type control. C: bar graph of constitutive iNOS expression in UNC eNOS/, Har eNOS/, and wild-type mice as a percentage of glyceraldehyde-3-phosphate dehydrogenase (GAPDH). **P < 0.01 vs. wild-type control. Numbers per group are indicated inside the bars.

Inhibition of iNOS in UNC eNOS^/ mice. The iNOS-specific inhibitor 1400W was administered to UNC eNOS^/ mice at the onset of myocardial ischemia (Fig. 4). The AAR/LV was not significantly different among the wild-type mice, UNC eNOS^/, and UNC eNOS^/ mice treated with 1400W. Inhibition of iNOS increased the infarct size per AAR to 43 ± 5% compared with 17 ± 2% in the untreated UNC eNOS^/ group (P < 0.05).

View larger version (18K): [in this window] [in a new window]   Fig. 4.   Myocardial infarct size in wild-type and UNC eNOS/ mice in the absence or presence of 1400W. Data are presented as AAR as a percentage of LV and as infarct size (NEC) as a percentage of AAR. Treatment with 1400W significantly increased myocardial infarct size in the UNC eNOS/ mouse. *P < 0.05 vs. wild-type control. Numbers per group are indicated inside the bars.

Inhibition of iNOS in Har eNOS^/ mice. As described above, 1400W was administered to Har eNOS^/ at the onset of myocardial ischemia (Fig. 5). The AAR/LV was not significantly different among the wild-type mice, Har eNOS^/, and Har eNOS^/ treated with 1400W. Inhibition of iNOS did not significantly increase the infarct size per AAR between untreated Har eNOS^/ (62 ± 4%) and treated Har eNOS^/ (45 ± 7%).

View larger version (18K): [in this window] [in a new window]   Fig. 5.   Myocardial infarct size in wild-type and Har eNOS/ mice in the absence or presence of the iNOS inhibitor 1400W. Data are presented as AAR as a percentage of the LV and as NEC as a percentage of AAR. Treatment with 1400W had no significant effect on infarct size in the Har eNOS/ mice. *P < 0.05 vs. wild-type control. Numbers per group are indicated inside the bars.

	DISCUSSION

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Endothelium-derived NO has been previously reported to protect against or to exacerbate myocardial reperfusion injury. The two strains of eNOS^/ mice used in the present study have revealed important findings. The most important finding is the disparity in infarct size between the two strains. Previous studies (8, 10) have shown opposite effects of these two strains in models of MI/R injury. The present study is the first demonstration of disparate effects of two different strains of eNOS^/ mice in the same model of myocardial reperfusion injury. Previously, studies (10, 30) of MI/R injury in the UNC eNOS^/ mouse have shown opposite effects. Yang et al. (30) used a 2.5-h in vivo model of MI/R and reported that in the UNC eNOS^/ mouse, MI/R injury was not different from control mice. In contrast, Kanno et al. (10) showed protection in the UNC eNOS^/ mouse using an ex vivo model of MI/R injury. We have previously shown (8) and in this study that with the Har eNOS^/ mouse, the absence of eNOS exacerbates myocardial necrosis in both an acute and more chronic in vivo models of MI/R injury.

In the present study, we demonstrate significant attenuation of MI/R injury in the UNC eNOS^/ mice. We hypothesized that this is due to increased constitutive levels of iNOS mRNA in the myocardium, which in turn might promote NO bioavailability during MI/R. The iNOS inhibitor 1400W ameliorated the cardioprotective effect observed in the UNC eNOS^/ mouse. As shown in previous studies, exogenous NO shows cardioprotective effects (7, 15, 20).

Har eNOS^/ mice have demonstrated that the absence of eNOS exacerbates the extent of necrosis in the murine myocardium during MI/R (8). This was also confirmed in the present study. As previously reported (6, 24), we have shown here that both strains of eNOS^/ mice exhibit elevated MABPs. Aortic vascular rings isolated from either the UNC eNOS^/ or Har eNOS^/ mice do not relax to the administration of acetylcholine, but exhibit high sensitivity to authentic NO and sodium nitroprusside (1, 13, 26).

Both strains of mice show that NO plays a very important role in the protection of the cardiovascular system and shed light on the controversy of NO. The Har eNOS^/ mice show an increase in area of necrosis during an acute MI/R. This is due to the lack of endothelium-derived NO, which here has been shown to be important and have protective effects in the cardiovascular system. The UNC eNOS^/ mice show protection, which may be a result of compensatory superinduction of iNOS (10). This superinduction of iNOS may cause NO to be released, giving protection to the murine myocardium. Although both lines of eNOS^/ mice demonstrate different responses to MI/R injury, the mechanism of these different responses ultimately may reflect the cardioprotective effects of NO.

The results of the present study suggest a unique compensatory response in the UNC eNOS^/ mouse, in which iNOS mRNA levels are costitutively increased in the myocardium. This interesting response to eNOS gene disruption may reveal a significant physiological signaling pathway, in which the level of eNOS gene expression regulates iNOS expression. Clearly, further studies are necessary to fully elucidate the intrinsic regulatory mechanisms responsible for iNOS and eNOS gene expression and NO physiology in the heart.

In conclusion, our results strongly suggest that iNOS induction in the heart may be responsible for the cardioprotection observed in the UNC eNOS^/ mice. However, it is possible that other compensatory mechanisms may be involved in this unique biological response. Clearly, there are hundreds of cardioprotective genes that have been previously described. Our study did not investigate other gene products; therefore, we cannot rule out any of the other protective moieties that may result in cardioprotection in the UNC eNOS^/ mice.