INTRODUCTION

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NITRIC OXIDE (NO), released from endothelial cells or cardiomyocytes, affects not only vascular tone but also myocardial metabolism. Inhibition of NO synthases by N^G-nitro-L-arginine (L-NNA) decreased the myocardial utilization of free fatty acids (FFA) but increased carbohydrate utilization in conscious dogs. This shift was reversed by administration of NO donors (16), demonstrating its direct relation to the lack of NO. The details of the mechanisms by which NO acts on cardiac substrate metabolism are not clear. The following mechanisms have been discussed: 1) inhibition of glycolysis by inhibition of glyceraldehyde-3-phosphate dehydrogenase (14); 2) cGMP-dependent inhibition of glucose uptake and also augmentation of synthesis of malonyl-CoA, an inhibitor of long-chain FFA oxidation (5); and 3) increased activity of aldose reductase, which subsequently suppresses the rates of glycolysis and glucose oxidation, without affecting the rate of palmitate oxidation (9). In contrast, NO facilitates glucose utilization by increasing the action of insulin (1) or increases total substrate oxidation by activating cGMP-dependent protein kinases (21). Thus NO potentially interacts with myocardial FFA and carbohydrate metabolism; the reduction in myocardial FFA utilization with L-NNA could be due to stimulated glucose metabolism as well as to reduced FFA uptake and/or oxidation.

During acute myocardial ischemia, cardiac metabolism is shifted from FFA toward preferential glucose utilization (13, 15, 20). On the other hand, myocardial NO production is increased during ischemia (4, 7, 11). From these observations, the following question arises: Is NO still involved in the control of cardiac metabolism during ischemia, and, if so, to what extent? We therefore examined the effect of NO synthase inhibition during normoperfusion and moderate myocardial ischemia in pigs. The myocardial consumptions of the three main cardiac substrates, FFA, glucose, and lactate, were measured before and after intravenous L-NNA infusion.

	METHODS

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The experimental protocols used in this study were approved by the local authorities of the district of Düsseldorf and adhere to the guiding principles of the American Physiological Society.

Experimental model. The experimental model has been described in detail previously (8, 18, 19). Briefly, in 18 enflurane-anesthetized Göttinger minipigs, a left lateral thoracotomy was performed, and a micromanometer (model P7, Konigsberg Instruments, Pasadena, CA) was placed in the left ventricle (LV) through the apex. Ultrasonic dimension gauges were implanted in the LV myocardium to measure the thickness of the anterior wall (System 6, Triton Technologies, San Diego, CA). The proximal left anterior descending coronary artery (LAD) was cannulated and perfused from an extracorporeal circuit at constant flow. LAD perfusion pressure was measured from the sidearm of the extracorporeal circuit. The large epicardial vein parallel to the LAD was dissected and cannulated to sample coronary venous blood.

Regional myocardial blood flow and metabolism. Radiolabeled microspheres (NEN, DuPont, Boston, MA) were injected into the coronary perfusion circuit to determine regional myocardial blood flow (8, 18, 19). O₂ content and pH were measured (model ABL 510/615, Radiometer, Copenhagen, Denmark) using anaerobically sampled blood drawn simultaneously from the cannulated coronary vein and an artery, and O₂ consumption of the anterior myocardial wall (MO₂) was calculated by multiplying the arterial-coronary venous O₂ difference by the transmural blood flow at the crystal site. The blood concentrations of glucose and lactate were determined using an autoanalyzer (model ABL 510/615, Radiometer), and their consumptions were calculated accordingly. The plasma concentrations of FFA were measured using an enzymatic colorimetric test kit (NEFA C, Wako Chemicals, Neuss, Germany). Plasma concentrations of FFA were converted to blood concentrations from the respective hematocrit values, and FFA uptake was calculated again by multiplying the arterial-coronary venous difference by the transmural blood flow at the crystal site.

Experimental protocol. After baseline measurements, the animals received L-NNA (20 mg/kg iv) starting 30 min before the onset of ischemia (group 1, n = 11). This dose of L-NNA has previously been shown in the same animal model to abolish the bradykinin-induced nitrite production and the decrease in mean coronary resistance and to inhibit myocardial NO synthases, even under ischemic conditions, insofar as a net myocardial nitrite uptake, rather than release, was measured before and during ischemia (7). In a control group (group 2, n = 7), after baseline measurements, the descending aorta was constricted with a tube to increase peak LV pressure (LVPP) by 20-25 mmHg, and LAD inflow was adjusted to increase mean coronary arterial pressure proportionately. After a further set of measurements, the protocol was identical to that of group 1. A subsequent period of 90 min of ischemia was followed by 120 min of reperfusion. During ischemia, coronary inflow was decreased to reduce coronary arterial pressure to ~50 mmHg. Sets of measurements were performed under control conditions, immediately before, and at 10 and 85 min of ischemia. These measurements included the simultaneous withdrawal of pairs of arterial and coronary venous blood samples. During the blood sampling, microspheres were injected into the LAD perfusion system for the measurement of regional myocardial blood flow, and systemic hemodynamic and regional dimension data were recorded.

Data analysis and statistics. Hemodynamic parameters reported are LV end-diastolic pressure, LVPP, the maximum of the first derivative of LV pressure, mean coronary blood flow (CBF), and mean coronary arterial pressure. Regional function of the anterior wall is reported as regional percent systolic wall thickening (AWT) and as a myocardial work index (AWI). AWI was calculated as the sum of the instantaneous LV pressure-wall thickness product over the time of the cardiac cycle (8). Regional myocardial blood flow is reported as subendocardial blood flow. Metabolic parameters include MO₂ and the consumptions of FFA (M_FFA; molarities of FFA are given as moles of stearic acid), glucose (M_glucose), and lactate (M_lactate). Positive values indicate myocardial uptake. ATP equivalents (ATP_eq) of total myocardial substrate consumption (M_FFA, M_glucose, and M_lactate) were calculated on the basis of the assumption of full efficiency of complete oxidative decomposition of the respective substrates.

	RESULTS

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Systemic hemodynamics, regional myocardial blood flow, and the increases in LVPP were identical with L-NNA infusion and aortic constriction. Coronary arterial pressure was increased with L-NNA (P < 0.05) but only tended to increase [P = not significant (NS)] during aortic constriction (Table 1). AWT and AWI remained unchanged after aortic constriction but decreased with L-NNA (P < 0.05). With the induction of ischemia, CBF fell to similar values in both groups (P < 0.05). AWT and AWI were decreased in both groups at 10 and 85 min of ischemia (P < 0.05), more markedly with L-NNA (P < 0.05).

Regional myocardial blood flow and metabolism. During normoperfusion, L-NNA did not change MO₂ and M_lactate. M_FFA decreased (P < 0.05) but, surprisingly, M_glucose tended to decrease (P = NS) as well. Consequently, ATP_eq were decreased (P < 0.05). In group 2, aortic constriction increased MO₂ (P < 0.05); M_lactate tended to increase (P = 0.06), whereas M_FFA, M_glucose, and ATP_eq remained unchanged (P = NS for all; Table 2).

	DISCUSSION

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There is good evidence that NO is involved in the control of myocardial metabolism. In normoperfused hearts of conscious dogs, inhibition of NO synthesis shifted myocardial metabolism from FFA toward preferential carbohydrate utilization (16, 17). Our present study confirms the reduction in FFA utilization during inhibition of NO synthases in normoperfused hearts and supports the authors' suggestion that reduced M_FFA could not merely result from decreased arterial FFA concentrations with L-NNA. In our study, M_FFA decreased only with L-NNA, despite similar arterial FFA concentrations in both groups. MO₂ and M_lactate remained constant with L-NNA infusion, and, unexpectedly, M_glucose even tended to decrease. Thus less substrate was consumed at an unchanged MO₂, indicating impaired efficiency of oxidative metabolism during L-NNA infusion. The resulting decrease in ATP_eq was associated with decreased regional myocardial function. In contrast, during aortic constriction, the hearts were able to increase MO₂ and oxidative metabolism sufficiently to maintain regional myocardial function. ATP_eq were calculated under the assumption of complete myocardial oxidative substrate decomposition, which appears plausible for normoperfusion and is also likely for moderate ischemia; if there were no complete oxidation of substrate, this would apply to both groups. During normoperfusion and ischemia, AWI correlated to ATP_eq in both groups. The correlation tended to be shifted to lower AWI values in group 1, again supporting the idea of decreased efficiency of oxidative metabolism with L-NNA.

Reduced endogenous NO with L-NNA could affect oxidative myocardial metabolism by directly shifting myocardial substrate use or, alternatively, by acting on mitochondrial respiration or acting directly on the myofilaments. Direct action on myocardial substrate use could, e.g., result from inhibition of FFA uptake or oxidation. Such a decrease in myocardial FFA use would be expected to be compensated by an increased myocardial carbohydrate utilization to preserve energy production and myocardial contractile function, thereby affecting MO₂ through an increased cardiac respiratory quotient. If such compensation fails, myocardial contractile function and ATP_eq, as well as MO₂, are expected to be decreased, which was not the case in the present study with L-NNA. Alternatively to a shift in substrate utilization, L-NNA infusion could primarily act on the respiratory chain. In isolated mitochondria, NO modulates respiration by inhibiting complexes I, II, and IV of the electron transport chain (2, 3, 6). In pigs, at any given MO₂, myocardial contractile function was lower without than with endogenous NO (7). Thus reduced endogenous NO with L-NNA is expected to decrease the efficiency of myocardial O₂ utilization. A decreased efficiency of myocardial O₂ utilization could be compensated for by an increased MO₂ to maintain myocardial energy production and contractile function. At a given MO₂, the decreased efficiency of O₂ use would result in a decrease in myocardial substrate metabolism and contractile function. Finally, NO acts on the myofilaments itself, thereby increasing regional myocardial function by increasing the phosphorylation status of troponin I (10, 12). Reduced NO with L-NNA would decrease regional myocardial function, but the reduced energy requirement would result also in decreased substrate metabolism and MO₂.

The results of the present study are compatible with the hypothesis that inhibition of NO synthesis acts primarily on the mitochondrial respiratory chain, thereby reducing the efficiency of myocardial oxidative metabolism and, for a given MO₂, also reducing ATP_eq and regional myocardial function. Consequently, the reduction in M_FFA after L-NNA infusion could be viewed as a result of a decreased demand for substrates secondary to impaired O₂ utilization.

During ischemia, CBF and regional myocardial blood flow decreased to the same extent in both groups. The changes in carbohydrate metabolism were characteristic of moderate ischemia (20): M_glucose tended to increase, paralleled by net lactate production in early ischemia; net lactate production, however, partially recovered with prolongation of ischemia. These alterations of carbohydrate metabolism were identical in both groups. M_FFA decreased during ischemia, paralleled by a decrease in the arterial FFA concentration; again changes were identical in both groups. Thus L-NNA had no effect on carbohydrate or FFA consumption during ischemia. Potential explanations for the observed results are that NO was no longer effectively involved in the control of myocardial metabolism during ischemia or that ischemia-induced NO formation is independent of NO synthases (22) and, therefore, not attenuated by L-NNA. The latter explanation might become important during severe myocardial ischemia, such as in acute myocardial infarction. However, this latter explanation most likely does not play a major role in the present study with more moderate myocardial ischemia, because pH changes were small (Table 2) and insufficient to account for nonenzymatic NO production (22). The present results, however, indicate that NO synthases definitely did not contribute to the control of myocardial metabolism during ischemia.