Nitric Oxide Inhibition Improved Myocardial Metabolism Independent of Tissue Perfusion During Ischemia But Not During Reperfusion

J Mol Cell Cardiol 32, 375–384 (2000) doi:10.1006/jmcc.1999.1082, available online at http://www.idealibrary.com on

Nitric Oxide Inhibition Improved Myocardial Metabolism Independent of Tissue Perfusion During Ischemia But Not During Reperfusion Makoto Araki1, Masaru Tanaka1, Koji Hasegawa∗1, Ryoji Yokota1, Takashi Maeda2, Makoto Ishikawa2, Youichi Yabuuchi2 and Shigetake Sasayama1 1

Department of Cardiovascular Medicine, Graduate School of Medicine, Kyoto University, Kyoto, Japan and 2Tokushima Research Institute, Otsuka Pharmaceutical Co. Ltd., Tokushima, Japan

(Received 8 April 1999, accepted in revised form 24 November 1999) M. A, M. T, K. H, R. Y, T. M, M. I, Y. Y  S. S. Nitric Oxide Inhibition Improved Myocardial Metabolism Independent of Tissue Perfusion During Ischemia But Not During Reperfusion. Journal of Molecular and Cellular Cardiology (2000) 32, 375–384. Nitric oxide (NO) is one of the important regulators of cardiac metabolism and function as well as of tissue perfusion. Myocardial NO formation is increased during ischemia and reperfusion. We investigated the roles of endogenous NO in myocardial metabolism during ischemia and reperfusion independent of tissue perfusion changes. In an open-chest pig model, a bolus infusion of 20 mg/kg of NG-nitro -arginine methyl ester (-NAME), a NO synthase inhibitor, did not alter the regional myocardial perfusion compared with a control saline injection, as measured by colored microsphares. Using 31P-nuclear magnetic resonance spectroscopy, we showed that the tissue levels of pH and adenosine triphosphate (ATP) but not those of creatine phosphate were significantly preserved in the -NAME group compared with the placebo group during the subsequent 15-min regional ischemia. Thus, -NAME reduced myocardial ATP utilization during ischemia, and the mechanism underlying these effects is independent of tissue perfusion changes. However, -NAME did not accelerate the recovery of ATP levels following reperfusion,  2000 Academic Press suggesting distinct roles of endogenous NO during reperfusion. K W: nitric oxide; myocardial ischemia; reperfusion; nuclear magnetic resonance.

Introduction It is becoming apparant that nitric oxide (NO) is one of the most important regulatory factors in many physiological and pathological processes of the cardiovascular system.1–3 NO is synthesized from -arginine by three types of isoforms of NO synthase (NOS); constitutive endothelial NOS, neuronal NOS and inducible NOS.4–8 A variety of cell types including neutrophils,9 vascular smooth muscle cells10 and heart muscle cells can produce NO.11 NO formation is increased in ischemic and ischemiareperfused myocardium.,13 NO not only functions

as a vasodilator but also prevents platelet aggregation to maintain coronary blood flow. On heart muscle cells, NO directly acts as a negative inotropic agent.14,15 These effects of NO are mediated through the stimulation of guanosine 3′, 5′-cyclic monophosphate (cGMP) production by guanylate cyclase.16 In addition, NO generates peroxinitrate (ONOO−) by rapidly interacting with superoxide (O2−) and inhibits mitochondrial enzymes.17,18 Because of these multiple actions, NO produced in the heart may play either a protective or a detrimental role in the pathogenesis of myocardial ischemia and reperfusion.

Please address all correspondence to: Koji Hasegawa, Department of Cardiovascular Medicine, Graduate School of Medicine, Kyoto University, 54 Kawara-cho, Shogoin, Sakyo-ku, Kyoto, 606-8507, Japan.

0022–2828/00/030375+10 $35.00/0

 2000 Academic Press

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A previous study demonstrated that NO inhibition by a NOS inhibitor, Nx-nitro -arginine methyl ester (-NAME) reduced the myocardial infarct size following ischemia and reperfusion.19 In that study, a bolus administration of -NAME before sustained ischemia by itself increased myocardial lactate levels, which may indicate tissue ischemia. These findings suggest that myocardial protection by -NAME is mediated, at least in part, through a preconditioning effect. Thus, direct effects of NAME independent of tissue perfusion changes have not been clarified. The purpose of the present study was to examine the direct effects of endogenous NO on myocardial metabolism during ischemia and reperfusion independent of tissue perfusion changes. We hypothesized that a relatively low concentration of NAME, which does not alter regional myocardial blood flow by itself, could evaluate this. To test this hypothesis, we serially measured regional myocardial blood flow (RMBF) by use of colored microsphere following the infusion of -NAME or saline as a control. To evaluate the effects of NAME on myocardial damage by ischemia and reperfusion, we serially measured myocardial adenosine triphosphate (ATP), creatine phosphate (CP), and inorganic phosphate (Pi) using 31P-nuclear magnetic resonance (NMR) spectroscopy20–26 in porcine hearts, which have no significant collateral circulation.

Methods Animal preparation This experiment conformed to the guiding principles of the American Physiological Society regarding the use of laboratory animals. Twenty-six farm pigs weighing 13–16 kg were sedated with ketamine (15 mg/kg), anesthetized with sodium pentobarbital (15 mg/kg), and randomly assigned to the saline group (n=14) or the -NAME group (n=12). After tracheotomy, they were mechanically ventilated at 18 ventilations per minute, and the tidal volume was adjusted from 13–17 ml/kg using a mixture of oxygen, nitrogen, and carbon dioxide (0.24:0.75:0.01) to keep the arterial blood gas within the physiological range. Anesthesia was maintained with fluorothane (0.5%–1.5%). An 8.5F introducer sheath was inserted into the ascending aorta via the left carotid artery to record the aortic pressure and heart rate on a polygraph system (Nihon Kohden, Tokyo,

Japan). Through the sheath, a 7F catheter-tipped micromanometer (Catheter Tip Pressure Sensor, Nihon Kohden) was advanced to the left ventricle (LV) for the measurement of LV pressure and LV dP/dt. The left external jugular vein was cannulated for fluid infusion. The left atrium was cannulated for the injection of colored microspheres, and the left femoral artery was cannulated to allow the withdrawal of reference blood samples during the measurement of the regional myocardial blood flow (RMBF). The heart was exposed through a midline thoracotomy and suspended in a pericardial cradle. A 3 mm segment of the center of the left anterior descending coronary artery was dissected free from the surrounding tissue and occluded by the air occluder. The area at risk was confirmed by the cyanotic color. The average area at risk was approximately 50×50 mm. A 17 mm diameter surface coil tuned to 32.7 MHz was positioned in the center of the area at risk. At the end of the experiment, we examined the position of the surface coil to determine whether it was at the center of the area at risk by reoccluding the artery.

Experimental protocol We set the time point of reperfusion at 0 min. After the resting period, initial baseline measurements of hemodynamics and NMR spectroscopy were performed. At −30 min, 20 mg/ml/kg of -NAME or 1 ml/kg of saline was administered through the cannula into the right atrium. At −5 min (15 min after the infusion of saline or -NAME), both groups of animals were subjected to 15 min of left anterior descending artery occlusion and 120 min of reperfusion.

31

P-NMR spectroscopy

31

P-NMR spectra were obtained using an in vivo spectrometer (BEM-250/80, Otsuka Electronics, Philadelphia, PA, USA) with a 1.9-T, 31 cm bore superconducting magnet. The spectrometer was interfaced with the surface coil. The homogeneity of the magnetic field in the region of the surface coil was optimized by shimming on the proton signal using a 12-channel shim supply to make the width smaller than 0.5 ppm. Respiration- and arterial pressure-gated spectra were obtained at end inspiration and peak systole, accumulating 90 free induction decays (FIDs) for each spectrum over 5 min. The pulse was 90 broad band (15 ls) with

Myocardial Metabolism by -NAME

a cycle time of 3.3±0.2 s and sweep width of 3 kHz. The FID was multiplied by an exponential to 10 Hz. The tissue levels of b-ATP, CP, and inorganic phosphate (Pi) were estimated by integrating the areas under the individual peaks using a computer software program (MEAS1, Graphtec Co, Tokyo, Japan) and a digitizer. The serial changes of these parameters during ischemia and reperfusion are expressed as percentages relative to the baseline values. We used a convolution difference algorithm for baseline correction. Intracellular pH was calculated from the chemical shift of the major Pi peak using the Flaherty equation.23 The CP level was used as a reference for the chemical shift of Pi. After the disappearance of CP, a small glass tube containing hexamethylphosphoric triamide set in the center of the surface coil was used as a reference; this was also used as a standard to correct the changes in spectrometer characteristics. The levels of CP and ATP are known to change during the cardiac cycle.27,28 In the present study, blood pressure gating eliminated such cardiac cyclic variation, and respiration gating kept the position of the heart constant in the magnetic field, which enhanced the accuracy of the metabolic information.20,21,24,26

Postmortem analysis After the pigs were sacrificed, the hearts were removed and both the left and right coronary arteries were cannulated. While the air occluder was kept inflated, 1% monastral blue dye (Sigma Chemicals, St. Louis, MO, USA) was injected into the coronary arteries at a pressure of 90 mmHg. The monastral blue staining allowed the visualization of the unstained ischemic area at risk that served as a guide for sampling tissues for regional blood flow. After fixation in 10% formalin, the hearts were cut into 5 mm serial slices in a plane parallel to the atrioventricular groove. The right ventricle, atria, and valvular structures were removed. The slices of the isolated left ventricle were weighed, and their apical surfaces were photographed. The area at risk was identified, traced from enlarged projections (×10) of the photographic slide of each ventricular slice, and quantified with a digitizer. The percent area at risk (area at risk divided by the slice area) was calculated for each slice and multiplied by the slice weight to obtain the weight of area at risk. The weights of each area at risk were summed and then divided by the LV weight to yield the percentage of the LV at risk.

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Measurement of RMBF Measurements of RMBF were made with 15 lm colored polystyrene microspheres (E-Z Trac, Los Angeles, CA, USA). Approximately 107 microspheres (red, yellow, blue, and black) were injected into the left atrium at –40 min (baseline, before the administration of -NAME or saline), –25 min (after the infusion of -NAME or saline, before occlusion), at −2 min (during occlusion) and at 10 min after reperfusion. Reference blood samples were withdrawn at a rate of 5 ml/min from the femoral artery starting 10 s before and continuing for 2 min after each injection. Tissue samples for RMBF measurements were taken transmurally from the central ischemic area and the remote non-ischemic area. Each sample was divided into endocardial and epicardial halves, each of which usually weighed 1.5–2.0 g. The extraction of microspheres from the blood and tissue samples was performed as described by Hale et al.29 RMBF was calculated from the formula RMBF= Cm×Qr/Cr, where Cm is the microsphere count per gram of tissue, Qr is the withdrawal rate of the reference blood sample (5 ml/min), and Cr is the microsphere count in the reference blood sample.

Statistical analysis For the comparison of hemodynamic parameters, each metabolite, intracellular pH and RMBF across time between the -NAME and saline groups, a two-factor ANOVA for repeated measures was used. When the ANOVA was significant, comparisons between the two groups were made by Student’s ttest. A value of P<0.05 was considered significant, and all results are expressed as mean ±...

Results Mortality Twenty-six pigs were initially entered into this study. Five of the 14 pigs in the saline group and five of the 12 pigs in the -NAME group died during the sustained ischemia. Two of the pigs in the saline group and none of the pigs in the -NAME group died immediately after reperfusion following sustained ischemia. The cause of death was ventricular fibrillation in all cases. Thus, seven pigs in the saline group and seven pigs in the -NAME group were used for analysis. There was no significant

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difference in the mortality rate between the two groups. Hemodynamic changes The hemodynamic data are summarized in Table 1. The heart rate and systolic and diastolic aortic pressure are expressed as the mean values for each 5 min period. During the baseline measurements, the heart rate and systolic and diastolic aortic pressure values did not change significantly. At −25 min (following the administration of saline or -NAME), the systolic and diastolic aortic pressure became significantly higher in the -NAME group than in the saline group. In both groups, systolic and diastolic aortic pressure decreased at 0 min (after 15 min of ischemia) but recovered promptly at 15 min (after reperfusion) nearly to a level before occlusion. The elevation of systolic and diastolic aortic pressure in the -NAME group compared with the saline group continued throughout the reperfusion period. The heart rate was lower in the -NAME group than in the saline group, compatible with a previous report.30 As a result of the systolic aortic pressure elevation and heart rate decrease by -NAME, double product, calculated by multiplying heart rate by systolic blood pressure, did not differ betweeen the saline and -NAME groups throughout the experiments. There were no significant differences in LV dP/dt or LV end-diastolic pressure between the two groups throughout the experiment. Regional myocardial blood flow The RMBF values are summarized in Table 2. The area at risk was not stained by the monastral blue dye, indicating that there was no significant collateral circulation. Indeed, RMBF in the area at risk was below 0.03 ml/g/min at −2 min (after 13 min of ischemia, 2 min before reperfusion) in both groups. RMBF did not differ significantly between the two groups at −40 min (baseline), at −25 min (after the administration of -NAME or saline, before occlusion), at −2 min (during occlusion) or at 10 min (during reperfusion) in both the subendocardium and the subepicardium. 31

P-NMR spectroscopy

During the baseline measurements, CP, ATP and intracellular pH did not change significantly. Accordingly, the time course of changes in CP and

ATP are expressed as percentages relative to the baseline values. Representative 31P-NMR spectra are shown in Figure 1, and the time-course changes of ATP, pH and CP levels are shown in Figs 2, 3 and 4, respectively. As shown in Figure 2, the rate of ATP decrease between −20 min (before occlusion) and 0 min (after 15 min of ischemia) was significantly (P<0.01) lower in the -NAME group (1.5±0.2%/ min) than in the saline group (3.1±0.4%/min). The ATP level at 0 min (at 15 min of sustained ischemia) was 41.8±4.8% in the saline group and 81.8±6.7% in the -NAME group. Thus, during sustained ischemia, ATP decreased much more slowly in the -NAME group than in the control group. Following reperfusion, ATP showed prompt recovery in the saline group while the levels remained unchanged in the -NAME group. The ATP levels were 85.2±6.1% in the saline group and 86.9±6.4% in the -NAME group after 120 min of reperfusion. ATP values at 120 min after reperfusion are significantly (P<0.05) lower than the values at –20 min (before occlusion). These results are compatible with those of a previous report demonstrating that ATP recovered slightly during an early reperfusion period after 15 min of ischemia but remained depressed for at least 24 h in dog hearts.31 The ATP levels did not differ significantly between the -NAME and saline groups throughout the reperfusion period. In the saline group, the intracellular pH was 7.40±0.05 at −20 min (before ischemia) and fell to 6.88±0.03 at −10 min, 6.66±0.06 at −5 min, and 6.49±0.06 at 0 min (after 5, 10, and 15 min of sustained ischemia, respectively) (Fig. 3). In the -NAME group, intracellular pH was 7.33±0.04 at −20 min (before ischemia) and fell to 7.03±0.06 at −10 min, 6.83±0.05 at −5 min, and 6.73±0.06 at 0 min (after 5, 10, and 15 min of sustained ischemia, respectively). The intracellular pH was significantly higher at −10, −5, and 0 min in the -NAME group compared to the saline group (P<0.05). After reperfusion, the intracellular pH returned to baseline levels in both groups and did not significantly differ between the groups throughout the reperfusion period. The CP values decreased to 2.1±1.5% of baseline in the saline group and 9.8±5.3% of baseline in the -NAME group at 0 min (after 15 min of sustained ischemia) (Fig. 4). Following reperfusion, the CP returned to baseline in both groups. The CP levels did not differ significantly between the groups throughout the experiment. There was no significant difference in the area at risk between the saline group (44.5±2.4% of

75.0±4.5 143.7±8.2∗∗ 104.9±6.4∗∗ 10803±933 5.3±1.1 1.45±0.19

6.1±0.6 1.53±0.21

0 min

74.3±3.0 137.9±7.5∗∗ 105.4±6.5∗∗ 10316±850 8.1±0.9 1.19±0.13

8.6±0.8 1.26±0.21

88.6±6.5 95.6±7.3 66.1±6.1 8704±1277

Occlusion

88.7±6.3 109.0±5.2 66.6±4.9 9792±1133

−15 min

70.9±3.8∗ 143.8±7.5∗∗ 113.4±7.0∗∗ 11023±867 8.9±1.1 1.19±0.12

7.6±1.0 1.28±0.18

90.3±6.6 105.3±6.8 71.6±6.1 9680±1244

15 min

75.1±2.4 149.6±6.7∗∗ 111.0±6.5∗∗ 10630±895 7.4±0.8 1.23±0.12

7.4±1.0 1.29±0.21

91.3±7.9 104.9±7.9 72.9±7.7 9816±1400

30 min

74.7±3.3 141.0±7.1∗ 109.7±6.1∗∗ 11023±870 7.6±0.6 1.16±0.10

7.0±1.0 1.20±0.22

91.9±11.2 101.6±9.4 72.6±8.3 9778±1840

Reperfusion 60 min

73.1±2.8 131.6±5.7∗ 105.3±6.2∗ 9705±726 7.0±1.6 1.09±0.09

6.9±0.8 1.09±0.24

84.7±11.9 100.7±11.6 68.0±9.2 9101±2074

90 min

71.3±2.7 130.1±6.4 104.0±6.5∗ 9336±695 8.3±1.3 1.01±0.10

7.8±1.5 1.26±0.27

87.6±15.1 105.0±10.9 72.0±12.1 9564±2267

120 min

Values are mean±.... HR, heart rate; bpm, beats per minute; SBP, systolic blood pressure; DBP, diastolic blood pressure; LVEDP, left ventricular end-diastolic pressure; LV dP/dt, rate of change of left ventricular pressure. ∗P<0.05 v saline. ∗∗P<0.01 v saline.

74.8±4.9 153.9±7.2∗∗ 105.8±5.9∗∗ 11512±887 5.3±1.0 1.46±0.15

6.3±0.8 1.55±0.2

6.4±1.0 1.56±0.19

-NAME (n=7) HR (bpm) 82.6±5.2 SBP (mmHg) 112.0±6.2 DBP (mmHg) 68.7±5.4 Double Product 9309±840 LVEDP (mmHg) 5.3±0.9 LV dP/dt (mmHg/ms) 1.47±0.10

87.9±5.6 114.5±5.6 73.5±5.6 10064±1075

After infusion −25 min

87.0±4.9 108.0±5.9 66.4±6.3 9225±1016

Baseline −40 min

Hemodynamic parameters and cardiac function in saline and -NAME administered pigs during coronary occlusion and reperfusion

Saline (n=7) HR (bpm) SBP (mmHg) DBP (mmHg) Double Product (mmHg×bpm) LVEDP (mmHg) LV dP/dt (mmHg/s)

Group

Table 1

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Table 2 Regional myocardial blood flow Ischemic zone (ml/g/min) END EPI

Non-ischemic zone (ml/g/min) END EPI

−40 min (Baseline) Saline -NAME

0.74±0.08 0.75±0.11

0.73±0.07 0.79±0.07

0.88±0.14 0.82±0.07

0.76±0.14 0.73±0.10

−25 min (After infusion, before occlusion) Saline -NAME

0.79±0.07 0.72±0.09

0.85±0.05 0.74±0.11

0.84±0.08 0.90±0.12

0.70±0.07 0.73±0.11

−2 min (Occlusion) Saline -NAME

0.01±0.01 0.01±0.02

0.01±0.02 0.01±0.02

0.69±0.14 0.77±0.26

0.76±0.19 0.88±0.36

10min (Reperfusion) Saline -NAME

0.93±0.12 0.83±0.10

0.89±0.10 0.88±0.12

0.72±0.05 0.71±0.06

0.61±0.06 0.54±0.05

Values are mean±... of seven pigs per group. END, subendocardium; EPI, subepicardium.

Figure 1 Original 31P-NMR (nuclear magnetic resonance) spectra. ATP, adenosine triphosphate; CP, creatine phosphate; Pi, inorganic phosphate.

Myocardial Metabolism by -NAME

Figure 2 Time course changes of ATP (adenosine triphosphate). ∗∗P<0.01 different from saline group; Occ, occlusion.

Figure 3 Time course changes of pH. ∗P<0.05 different from saline group; Occ, occlusion.

the LV) and the -NAME group (53.8±5.0% of the LV).

Discussion The present study demonstrates that an administration of -NAME, a NOS inhibitor, markedly preserved the ATP level during myocardial ischemia without affecting the regional myocardial blood flow. However, this dosage of -NAME treatment did not show protective effects during the subsequent reperfusion. NO formation is reported to be increased in ischemic and ischemia-reperfused myocardium.12,13 In cardiac myocytes, NO may negatively regulate the L-type calcium current and contraction through the activation of cGMP-dependent protein kinase and cGMP-modulated phosphodiesterases.32–35

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Figure 4 Time course changes of CP (creatine phosphate). Occ, occlusion.

Thus, increased amounts of NO produced in the heart may be involved in the myocardial contractile depression. Compatible with this hypothesis, it was reported that during sustained ischemia in isolated rabbit hearts, NOS inhibition delayed the onset of ischemic contracture.36 The present experiments performed in the in vivo pig heart showed that NAME did not alter the left ventricular dP/dt despite the apparent protection of myocardial metabolism by this agent during ischemia. During ischemia as well, systolic and dialstolic blood pressure were significantly higher in the -NAME group than in the saline group. Although RMBF did not differ between the saline and -NAME groups, these indicate that coronary vascular resistance is higher in the -NAME group than in the saline group. Therefore, one possible reason for the finding that left ventricular contractility did not improve despite the improvement of myocardial metabolism might have been the increase of afterload. Another possibility is high coronary vascular resistance, which can increase oxygen consumption of the heart and depress the contractility. However, the mechanisms for this discrepancy are not clear at present. The precise evaluation of left ventricular contractility requires measurement of percentage segment shortening rather than global left ventricular function. Thus, further studies are needed for the role of endogenous NO in myocardial contraction and for the relationship between function and metabolism. The preservation of ATP and pH during ischemia indicates less damage to the myocardium. Interestingly, the protective effect of -NAME on myocardial metabolism observed during ischemia disappeared promptly following reperfusion. Although -NAME was administered by bolus injection before ischemia, the rise in blood pressure continued after reperfusion as well. Hence, the disappearance of the protective effect on myocardial

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metabolism during reperfusion cannot be explained by the disappearance of pharmacological efficacy. In contrast to the protective effect of the NOS inhibitor during ischemia, it is reported that arginine (the substrate of NOS) when added before reperfusion reduced the infarct size by limiting reperfusion injury.37 Thus, the NOS inhibitor protects against ischemic injury, whereas the NOS substrate protects against reperfusion injury, suggesting differential roles of endogenous NO in ischemia and reperfusion. ATP levels in myocardial cells are determined by ATP production and utilization. With zero-flow ischemia in pig hearts, ATP is produced by glycolysis and by the hydrolysis of any remaining CP. ATP hydrolysis after Pcr hydrolysis results in no net production of protons, while ATP hydrolysis after glycolysis accumulates protons. The present study showed that -NAME treatment preserved both the ATP and pH levels during ischemia without affecting the CP levels. Therefore, the protective effect of -NAME may be related mainly to reduced ATP utilization (hydrolysis). However, the double products did not differ between the saline and -NAME groups throughout the experiment, suggesting that the oxygen consumption did not differ greatly between the two groups. Thus, reduced ATP utilization may not be explained by changes of heart rate and blood pressure. Yellon et al. reported that -NAME administration caused transient myocardial ischemia and triggered a preconditioning effect, exerting protective effects on myocardial metabolism such as infarct size limitation.19 However, in the present experiment, NAME treatment did not alter the regional myocardial blood flow or the index of myocardial metabolism as assessed by NMR after the -NAME administration (before coronary occlusion). It is therefore unlikely that the -NAME administration induced ischemia leading to the preconditioning effect. The myocardial protection by the NOS inhibitor independent of tissue perfusion suggests that endogenous NO acts directly on the heart as a cytotoxic molecule of myocardial metabolism during ischemia. At present, we have no direct evidence that -NAME, at the dosage used in this study, did decrease NO formation in the ischemic pig heart in vivo. Further studies of NO formation in the heart are needed to identify the role of endogenous NO. Our results demonstrated that treatment with a relatively low concentration of -NAME, which does not alter regional myocardial blood flow by itself, improved the myocardial metabolism during ischemia. These suggest that endogenous NO acts directly on the heart as a cytotoxic molecule in

myocardial ischemia. In contrast, -NAME did not accelerate the recovery of myocardial metabolism following reperfusion, suggesting differing roles of endogenous NO in ischemia and reperfusion. There is evidence suggesting an injurious role for NO if it is present in substantial excess. NO-dependent toxicity results from the formation of NO-derived free radical species, such as the peroxynitrite anion. One possible mechanism for the better preservation of ATP by -NAME is the prevention of free radical formation from NO. Since free radicals have been shown to induce glycolytic inhibition, the prevention of free radical formation will lead to the increase of glycolysis, which results in the better preservation of ATP by the NOS inhibitor. It was recently suggested that NO is a mediator of myocardial cell apoptosis.38–40 The overexpression of human endothelial constitutive NOS gene in adult cardiac myocytes in vivo caused myocardial cell degradation, part of which was compatible with apoptosis.38 In addition, increased inducible NOS activity has a significant relationship with the induction of apoptosis in cardiac myocytes.39 Thus, another possible mechanism of the preferential effects on myocardial metabolism by -NAME is the prevention of NO-induced myocardial cell apoptosis. However, despite preferential effects of the NOS inhibitor during ischemia but not during reperfusion, free radical production and myocardial cell apoptosis mainly occur at the onset of reperfusion. The mechanisms of preservation of myocardial metabolism by -NAME thus remain to be clarified. Nevertheless, since the preservation of the myocardial ATP levels by -NAME was marked, it is of interest to explore how this preservation afforded by this agent can be applied to clinical situations.

Acknowledgement This work was supported in part by grants to K.H. from Yamanouchi Foundation for Research on Metabolic Disorders, Uehara Memorial Foundation, Tokyo Biochemical Research Foundation and the Ministry of Education, Science and Culture of Japan.

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