Articles

Inhibition of Nitric Oxide Synthesis Causes Myocardial Ischemia in Endotoxemic Rats

J. A. M. Avontuur, H. A. Bruining, C. Ince
https://doi.org/10.1161/01.RES.76.3.418
Circulation Research. 1995;76:418-425
Originally published March 1, 1995

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Abstract

Abstract Inhibitors of nitric oxide (NO) synthesis have been used in the treatment of septic and endotoxic shock. However, several studies question the beneficial effect of inhibiting NO production in sepsis and endotoxemia. We have investigated the effect of inhibition of NO synthesis after endotoxemia in the isolated perfused rat heart. In hearts from endotoxin-treated animals, coronary flow was elevated 64% and oxygen consumption was elevated 20% compared with control hearts. NADH fluorescence imaging was used as an indicator of regional hypoperfusion. A homogeneous low-surface NADH fluorescence, indicative of adequate tissue perfusion, was observed in both control and endotoxin-treated hearts. The increase in coronary flow and oxygen consumption could only partially be prevented by pretreatment of the animals with dexamethasone. Addition of Nω-nitro-l-arginine (NNLA), an inhibitor of NO synthesis, to the perfusion medium eliminated differences in coronary flow and oxygen consumption between normal and endotoxin-treated hearts. However, NADH surface fluorescence images of endotoxin-treated hearts after NNLA revealed areas of high fluorescence, indicating local ischemia, whereas the control hearts remained without signs of ischemia. The ischemic areas were present at various perfusion pressures and disappeared after the infusion of l-arginine, the natural precursor of NO, or the exogenous NO donor sodium nitroprusside. Methylene blue (MB), an inhibitor of soluble guanylate cyclase, the effector enzyme of NO, also eliminated differences in coronary flow and produced similar areas of local myocardial ischemia in endotoxin-treated hearts but not in control hearts. Reducing coronary flow by direct vasoconstriction with vasopressin resulted in similar patterns of myocardial ischemia as with NNLA and MB. Our results suggest that the coronary vasodilation in the isolated rat heart after endotoxemia is caused by an increased release of NO. Coronary flow reduction with NNLA, MB, or direct vasoconstriction with vasopressin causes local areas of myocardial ischemia in endotoxin-treated hearts but not in untreated hearts. These data suggest that endotoxemia promotes myocardial ischemia in vulnerable areas of the heart after inhibition of the NO pathway or direct vasoconstriction.

Sepsis remains an important cause of mortality, especially when accompanied by shock or multiple organ failure, despite advances in antibiotic and vasopressant therapy.1 The initial cardiovascular changes observed during the hyperdynamic septic state are often characterized by profound vasodilation with normal to high cardiac output, low systemic vascular resistance, and severe hypotension.2 Persistent hypotension with low vascular resistance refractory to vasopressor therapy is present in ≈50% of the patients who die of septic shock.2 3 By promoting the release of cytokines and other mediators, bacterial lipopolysaccharide (LPS [endotoxin]) initiates many of the cardiovascular changes seen during sepsis in animals and in humans.4 5

Recent evidence suggests that a massive release of nitric oxide (NO), a very potent endogenous vasodilator, causes much of the vascular relaxation and hypotension of sepsis and endotoxemia.6 7 8 NO is formed from l-arginine by the constitutive NO synthase present in the vascular endothelium and stimulates the enzyme-soluble guanylate cyclase (GC) in the smooth muscle cell, which converts GTP to cGMP, resulting in vascular relaxation. Under physiological conditions, NO plays an important role in the regulation of blood pressure and tissue perfusion. In sepsis and endotoxemia, an inducible form of NO synthase, located in vascular smooth muscle, is formed; this process leads to a massive release of NO, resulting in profound vasodilation.6 The induction of this inducible type of NO synthase can be prevented by pretreatment with glucocorticoids, such as dexamethasone.7 8

In the coronary vessels, NO plays a role in the regulation of myocardial blood flow.9 10 11 12 13 In human septic shock, inappropriately high coronary flow with decreased oxygen extraction has been reported, closely mimicking the peripheral circulatory anomalies of vascular relaxation.14 15 This pattern of disordered coronary flow regulation may be due to the release of a putative vasodilator substance such as NO.8 16

Methylated or nitro-substituted analogues of l-arginine competitively inhibit the formation of NO from l-arginine by NO synthase.17 18 Furthermore, at the end of the NO pathway, the effect of NO can be inhibited by methylene blue (MB), which inhibits the enzyme-soluble GC in the vascular smooth muscle cell.19 It has been suggested that such inhibitors have therapeutic value in the treatment of septic shock, since hypotension is corrected after the administration of these l-arginine analogues and MB.20 21 22 23 However, complete inhibition of NO formation has been shown to increase mortality in endotoxemic animals.24 25 26 Also, many investigators have reported reductions in cardiac output after the inhibition of NO production.21 22 23 24 25 27 Combined with the increased vascular resistance seen after the inhibition of NO formation, such reductions in cardiac output may compromise myocardial and tissue perfusion even further. Decreased coronary flow causing myocardial ischemia has been hypothesized to contribute to the fall in cardiac output seen after the administration of inhibitors of NO synthesis during sepsis and endotoxemia; however, this has never been shown directly.25 28

In the present study, we investigated the effect of Nω-nitro-l-arginine (NNLA)18 and MB19 on myocardial metabolism and coronary flow in hearts of normal and endotoxin-treated rats by use of a Langendorff heart preparation. To identify any inadequacies in oxygen supply causing myocardial ischemia in the perfused hearts, we used the fluorescence properties of reduced pyridine nucleotide (NADH), an indicator of mitochondrial respiration.29 30 We used this technique recently to visualize the existence of microcirculatory units vulnerable to ischemia in the isolated rat heart.29 In the present study, we hypothesize that such units might be susceptible for ischemia during endotoxemia when the NO pathway is inhibited.

Materials and Methods

Experimental Setup

The protocol was approved by the animal ethics board. Male Wistar rats (300 to 360 g) received either 0.7 mg/kg body wt Escherichia coli endotoxin (LPS, 0127:B8, Sigma Chemical Co) or saline by intraperitoneal injection 12 hours before the initiation of the experiments. Control and endotoxin-treated animals were used for in vivo measurement of blood pressure and heart rate. After anesthesia with pentobarbital (50 mg/kg body wt IP), the right carotid artery was cannulated, and blood pressure was measured by a pressure transducer (Hewlett Packard 8805B corner amplifier) in control and endotoxemic rats.

Animals to be used for isolated perfusion of the heart were anesthetized with ether, and rectal temperature was measured. These animals were heparinized (0.5 mL, 100 IU), and 1 mL of blood was withdrawn from the descending aorta. To assess the adequacy of endotoxin administration, collected blood samples were analyzed for lactate by using an enzymatic and colorimetric procedure.31 For indirect determination of global NO release, measurement of plasma nitrite and nitrate, the stable end products of NO oxidation, was performed by use of an automated procedure based on the Griess reaction.32 The hearts were removed, immediately cooled in ice-cold perfusion medium, and rapidly arranged for constant-pressure perfusion at 37°C according to the Langendorff technique. The perfusion medium consisted of (mmol/L) NaCl 128, KCl 4.7, MgCl2 1, NaH2PO4 0.4, NaHCO3 20.2, CaCl2 1.3, glucose 5.0, and pyruvic acid 2.0 and was oxygenated with 95% O2/5% CO2. All hearts were continuously paced at 5 Hz. To minimize metabolic demand, the left ventricle was drained by a cannula in the apex so that no external work was performed. Flow rates (expressed as milliliters per minute per gram of ventricular wet weight) were measured by an electromagnetic flow probe (Skalar-Medical) in the aortic perfusion line. For the determination of effluent oxygen pressure, the right pulmonary artery was cannulated, and a constant fraction of the coronary effluent was led along a Clark-type electrode (YSI biological oxygen monitor, model 5300, Yellow Springs Instruments). The coronary influent was similarly monitored for oxygen pressure via a side arm of the aortic cannula. Oxygen consumption (in micromoles per minute per gram of ventricular wet weight) was calculated from the product of the influent-effluent concentration difference and coronary flow by using the Bunsen solubility coefficient of oxygen in Krebs-Henseleit buffer (22.7 μL O2/mL per atmosphere at 37°C).

NADH Videofluorometry

NADH is a key intermediate in the transfer of reducing equivalents from metabolic substrates in the cytosol to oxygen in the mitochondria in the myocardial cells. During hypoxia, less NADH is oxidized to NAD+, leading to the accumulation of NADH. When excited with light at 365 nm, NADH, unlike NAD+, emits fluorescence light at wavelengths centered around 475 nm. Therefore, hypoxic regions will appear as areas of high fluorescence on the surface of the heart. NADH surface fluorescence provides a sensitive method for visualizing any regional inadequacies in oxygen supply and incipient myocardial ischemia. NADH videofluoroscopy allows on-line evaluation of spatial heterogeneity in hypoxia not detected by global parameters of flow, pressure, and metabolites. NADH surface fluorescence imaging was performed by excitation of ≈1 cm2 of the left ventricle of the Langendorff-perfused rat heart with light of 360 nm and by measurement of the emitted fluorescence (460 to 490 nm) by use of an image-intensified charge-coupled device (CCD) videocamera as described previously.30 Fluorescence images were recorded on a videorecorder and computer-analyzed off-line. A subtraction routine was used in all pictures to enhance contrast.

Experimental Protocol

Perfusion pressure is an important determinant of both oxygen delivery and oxygen consumption in the isolated Langendorff-perfused rat heart.33 Therefore, we investigated myocardial metabolism over a range of perfusion pressures in eight control and eight endotoxin-treated hearts. Hearts were perfused for a total of 130 minutes. After a period of 30 minutes, which allowed coronary flow to stabilize at 60 mm Hg, perfusion pressure was first lowered to 20 mm Hg and subsequently increased by stepwise changes in perfusion pressure of 10 mm Hg at 3-minute intervals to a maximum of 100 mm Hg. At the end of this series of pressure changes, perfusion pressure was set at the initial value of 60 mm Hg. When the flow was stable, the perfusion medium was switched to one containing NNLA in a concentration of 100 μmol/L. After a 30-minute interval, when a new steady state was achieved, the stepwise changes in perfusion pressure were repeated once again. In three hearts, the effects of either l-arginine (100 μmol/L), the precursor of NO production,6 or nitroprusside (10 μmol/L), a nitrovasodilator that acts as an exogenous NO donor,34 were studied after 30 minutes of NNLA. To study the effect of inhibition of soluble GC, the effector enzyme of NO, we used MB in four control and four endotoxin-treated hearts. MB was used at a concentration of 5 μmol/L since higher concentrations (>20 μmol/L) caused a paradoxical increase in coronary flow in control hearts, probably attributable to nonspecific toxic effects.35 To see whether the effect of direct vasoconstriction was different from the effect of inhibition of NO metabolism, in three separate hearts we switched to vasopressin, a direct smooth muscle vasoconstrictor, at a concentration of ≈1 nmol/L to reach approximately the same reduction in flow as did NNLA.

The LPS-stimulated induction of NO synthase can be prevented by pretreatment with dexamethasone without affecting NO release of the constitutive enzyme.7 8 To investigate the effect of dexamethasone pretreatment, five rats were injected with dexamethasone (4 mg/kg IV) 90 minutes before LPS infusion compared with three rats in the control group, which received only dexamethasone in combination with saline.

Histological examination by light microscopy was performed after fixation with 10% formaldehyde and hematoxylin/eosin staining to assess whether histopathological differences were present between control and endotoxin-treated hearts.

Chemicals

NNLA, MB, arginine vasopressin, and dexamethasone were obtained from Sigma. All the other chemicals were obtained from Merck.

Statistics

All values are expressed as mean±SD. Student’s t test for group comparisons or paired t test where appropriate was used to statistically compare values. To test whether drug effects were perfusion-pressure dependent, we used ANCOVA. A value of P<.05 was taken as statistically significant.

Results

Effects of Endotoxin Administration

Although most animals of the endotoxin-treated group showed signs of lethargy, all animals survived. As shown in the Table⇓, endotoxin-treated animals showed elevated rectal temperature, decreased mean arterial blood pressure, increased heart rate, and increased plasma levels of lactate. Values of serum nitrate plus nitrite (NO2−+NO3−) were elevated in endotoxin-treated animals, indicating increased systemic release of NO (Table⇓).

View this table:
Table 1.

Effects of Endotoxin Administration

Histological examination of the hearts after perfusion revealed no signs of capillary thrombosis, leukocyte infiltration, intracellular edema, or tissue damage in control (n=3) or endotoxin-treated (n=3) hearts.

Effect of Endotoxin and Dexamethasone Pretreatment on Coronary Flow, Effluent Po2, and Oxygen Consumption

In steady state conditions at a perfusion pressure of 60 mm Hg, coronary flow was elevated 64% in endotoxin-treated hearts (n=8) (from 9.2±0.6 to 15.0±1.6 mL/min per gram wet weight, Fig 1a⇓), and oxygen consumption was elevated 20% (from 4.0±0.2 to 4.8±0.3 μmol/min per gram wet weight, Fig 1c⇓) compared with control hearts (n=8). Since the relative rise in coronary flow was greater than the rise in oxygen consumption, this was accompanied by a rise in coronary effluent Po2 (from 374±20 to 463±37 mm Hg, Fig 1b⇓). To see whether increased coronary flow could be prevented with glucocorticoids, we pretreated rats (n=5) with dexamethasone 90 minutes before the injection of endotoxin. Dexamethasone pretreatment could only partially prevent the increase in coronary flow and oxygen consumption in the endotoxin-treated hearts (11.3±1.9 mL/min per gram and 4.3±0.3 μmol/min per gram, respectively; Fig 1⇓). Dexamethasone pretreatment was without effect on coronary flow and oxygen consumption in control hearts (n=3) (8.9±0.7 mL/min per gram and 4.0±0.3 μmol/min per gram, respectively; P=NS).

Figure 1.
Figure 1.

Bar graphs showing the effect of endotoxin (lipopolysaccharide [LPS]) and dexamethasone pretreatment (LPS+DEXA) on coronary flow, effluent Po2, and oxygen consumption (MVO2) compared with untreated control hearts (C) at steady state perfusion pressure of 60 mm Hg. Each column represents the mean, and vertical bars indicate SD of C hearts (n=8), endotoxin-treated hearts (n=8), or LPS+DEXA–treated hearts (n=5). *Statistically significant difference for LPS vs C (P<.05). #Statistically significant difference for LPS+DEXA vs LPS (P<.05).

Effect of Perfusion Pressure on Coronary Flow, Effluent Po2, Oxygen Consumption, and Mean NADH Fluorescence

During perfusion at different pressures ranging from 20 to 100 mm Hg, coronary flow (Fig 2a⇓) and oxygen consumption (Fig 2c⇓) were significantly raised in hearts from endotoxic animals at perfusion pressures of >20 mm Hg. Decreased oxygen extraction resulted in a rise of coronary effluent Po2 (Fig 2b⇓). Mean NADH surface fluorescence was not significantly different between the control and endotoxin-treated hearts for all perfusion pressures. Signs of local ischemia, detected as small areas of high fluorescence and a rise of mean NADH surface fluorescence, were only present at a perfusion pressure of 20 mm Hg. At higher perfusion pressures, a homogeneous image with low NADH fluorescence intensity was seen in both groups (Fig 5a⇓ and 5c⇓).

Figure 2.
Figure 2.

Graphs showing the effect of perfusion pressures on coronary flow (a), effluent Po2 (b), and O2 consumption (c) in control (○) and endotoxin-treated (•) rat hearts. Control (□) and endotoxin-treated (▪) hearts are shown after inhibition of nitric oxide synthesis with 100 μmol/L Nω-nitro-l-arginine (NNLA). Each point is the mean±SD of eight hearts at a given perfusion pressure. *Statistically significant difference for lipopolysaccharide (LPS) vs control (C) (P<.05).

Effect of Inhibiting NO Synthesis With NNLA on Coronary Flow, Effluent Po2, and Oxygen Consumption

Fig 3⇓ shows two representative tracings of the response in time of coronary flow after inhibition of NO synthesis with 100 μmol/L NNLA in an endotoxin-treated and a control heart. In the endotoxin-treated heart, there is an initial steep reduction of 4.0 mL/min per gram in coronary flow the first 3 minutes, with a more sustained decrease in flow the following 20 minutes. In the control heart, a gradual decline in coronary flow of only 0.5 mL/min per gram is seen during the first 3 minutes after the administration of NNLA. As early as 5 minutes after starting the NNLA administration, the endotoxin-treated heart had reached approximately the same coronary flow as the control heart. Fig 4⇓ shows the steady state situation 30 minutes after inhibition of NO synthesis. NNLA decreased coronary flow by 8.5 mL/min per gram (57%) in the endotoxin group (P<.05) and by 3 mL/min per gram (34%) in the control group (P<.05). Thirty minutes after NNLA, coronary flow in endotoxin-treated hearts (6.5±0.6 mL/min per gram) was not significantly different from that in control hearts (6.1±0.6 mL/min per gram). Effluent Po2 was reduced 35% to 299±50 mm Hg in endotoxin-treated hearts and 26% to 276±27 mm Hg in control hearts. Oxygen consumption was significantly reduced in both endotoxin-treated (32% reduction to 3.3±0.3 μmol/min per gram) and control (18% reduction to 3.3±0.3 μmol/min per gram wet weight) hearts. In hearts of dexamethasone-pretreated endotoxemic animals, NNLA reduced coronary flow to 6.6±0.5 mL/min per gram and oxygen consumption to 3.4±0.4 μmol/min per gram wet weight (P<.05). No significant differences in coronary flow, effluent Po2, and oxygen consumption were present between the three groups receiving NNLA (Fig 4⇓).

Figure 3.
Figure 3.

Two representative tracings of coronary flow in a control (C) and an endotoxin-treated (lipopolysaccharide [LPS]) rat heart after inhibition of nitric oxide synthesis with Nω-nitro-l-arginine (NNLA, 100 μmol/L) at 0 minutes and a constant perfusion pressure of 60 mm Hg.

Figure 4.
Figure 4.

Bar graphs showing the effect of Nω-nitro-l-arginine (NNLA) on coronary flow, effluent Po2, and oxygen consumption (MVO2) in control (C), endotoxic (lipopolysaccharide [LPS]), and dexamethasone-pretreated (LPS+DEXA) rat hearts at 60 mm Hg of perfusion pressure. Each column represents the mean of control (n=8), endotoxic (n=8), or LPS+DEXA (n=5) hearts; vertical bars indicate SD. Differences between groups were not significant (P=NS).

Effect of Perfusion Pressure After NNLA

Fig 2⇑ shows the effect of different perfusion pressures on coronary flow (Fig 2a⇑), effluent Po2 (Fig 2b⇑), and oxygen consumption (Fig 2c⇑) after inhibition of NO synthesis with NNLA (100 μmol/L) in endotoxin-treated and control hearts. Relative changes in coronary flow induced by NNLA depended on perfusion pressure: they were larger at higher perfusion pressures in both control and endotoxin-treated hearts (ANCOVA, P<.05). After NNLA administration, no significant differences were present between endotoxic and control hearts at all perfusion pressures.

Effect of NNLA on Surface NADH Fluorescence of Rat Hearts

Switching to perfusate containing NNLA at a perfusion pressure of 60 mm Hg resulted in the development of small areas of high fluorescence in the endotoxin-treated hearts (Fig 5d⇓), which is indicative of local ischemia. The control hearts (Fig 5b⇓) remained without any sign of ischemia. The ischemic areas in the endotoxic hearts were present at all perfusion pressures. They disappeared after the addition of l-arginine, the natural precursor of NO, or sodium nitroprusside, an exogenous NO donor, indicating that the areas of ischemia are indeed caused by the action of NO. In normal hearts, areas of ischemia appeared only when the perfusion pressure was lowered to 20 and 30 mm Hg. The mean NADH fluorescence was not significantly different between groups and was raised only at a perfusion pressure of 20 mm Hg in both the endotoxin-treated and control groups. NNLA did not significantly affect mean NADH fluorescence in either group.

Figure 5.
Figure 5.

Effect of inhibiting nitric oxide synthesis with Nω-nitro-l-arginine (NNLA) on NADH fluorescence images in control and endotoxin-treated rat hearts. Before administration of NNLA, both the control heart (a) and the endotoxic heart (c) show a homogeneous low fluorescence. After NNLA (100 μmol/L), the control heart (b) shows no changes in NADH fluorescence, whereas in the endotoxic heart (d) local areas of high fluorescence are visible, indicative of myocardial ischemia.

Effect of MB on Coronary Flow and Surface NADH Fluorescence

To study the effect of inhibiting soluble GC, the effector enzyme of NO, we used MB (5 μmol/L) in control hearts (n=4) and endotoxin-treated hearts (n=4). MB reduced coronary flow from 8.7±0.6 to 7.6±0.9 mL/min per gram wet weight (P=NS) in control hearts and from 16.0±1.3 to 8.9±1.4 mL/min per gram wet weight (P<.05) in endotoxin-treated hearts at a perfusion pressure of 60 mm Hg. There was no significant difference in coronary flow between control and endotoxin-treated hearts after the administration of MB. However, MB produced areas of local ischemia in endotoxin-treated hearts (Fig 6d⇓) but not in the control hearts (Fig 6b⇓). The areas of ischemia remained present in endotoxin-treated hearts at all perfusion pressures, whereas control hearts showed signs of ischemia only at 20 and 30 mm Hg of perfusion pressure.

Figure 6.
Figure 6.

Effect of methylene blue (MB), an inhibitor of soluble guanylate cyclase, on NADH fluorescence images in control and endotoxin-treated rat hearts. Before administration of MB, both the control heart (a) and the endotoxic heart (c) show a homogeneous low fluorescence. After MB (5 μmol/L), the control heart (b) shows no change in NADH fluorescence, whereas in the endotoxic heart (d) local areas of high fluorescence are visible, indicative of myocardial ischemia.

MB in a dose of 10 μmol/L did not further reduce flow in control or endotoxin-treated hearts. Higher doses of MB (20 μmol/L) caused a paradoxical vasodilatation with a sustained increase in coronary flow, probably attributable to nonspecific toxic effects on cardiomyocytes.35

Effect of Direct Smooth Muscle Vasoconstriction With Vasopressin on Surface NADH Fluorescence

It has been suggested that prevention of excess vasodilatation might less likely cause ischemia and poor tissue perfusion than the use of powerful vasoconstrictors.21 We investigated whether the effect of reduction of coronary flow by a vasoconstrictor on local tissue oxygenation was different from the reduction of flow by inhibition of the NO pathway with NNLA or MB in control and endotoxin-treated hearts. We used vasopressin, a direct smooth muscle vasoconstrictor, in a concentration (≈1 nmol/L) that achieved a reduction in coronary flow similar to that of 100 μmol/L NNLA at 60 mm Hg of perfusion pressure. At 60 mm Hg of perfusion pressure, vasopressin produced areas of local ischemia in endotoxin-treated hearts (Fig 7d⇓) but not in the control group (Fig 7b⇓). With vasopressin, the areas of ischemia remained present in endotoxin-treated hearts at all perfusion pressures, whereas control hearts showed signs of ischemia only at 20 and 30 mm Hg of perfusion pressure.

Figure 7.
Figure 7.

Effect of direct smooth muscle vasoconstriction with vasopressin on NADH fluorescence images in a control and endotoxin-treated rat heart. Before the administration of vasopressin, both the control heart (a) and the endotoxic heart (c) show a homogeneous low fluorescence. After smooth muscle vasoconstriction with vasopressin (≈1 nmol/L), the control heart (b) shows no changes in NADH fluorescence, whereas in the endotoxic heart (d) local areas of high fluorescence exist, indicative of myocardial ischemia.

Discussion

Myocardial circulation during septic shock is often characterized by an inappropriately high coronary flow.14 15 NO plays an important role in the regulation of myocardial flow.9 10 11 12 13 Therefore, we investigated the role of NO in the hyperdynamic changes of coronary flow in hearts of endotoxin-treated rats. The data in the present study indicate that these vascular changes are predominantly due to the vasodilatory action of NO. However, inhibition of the NO pathway can result in focal areas of ischemia in endotoxin-treated hearts, suggesting an imbalance of local oxygen supply to demand.

A rat model of endotoxemia using a sublethal low dose of endotoxin was selected; this model produced a hyperdynamic coronary circulation36 that is also seen in human sepsis.14 15 Comparable to human sepsis, the model resulted in raised temperature,36 reduced blood pressure, and increased levels of plasma lactate37 and nitrate plus nitrite. The 12 hours needed to establish this model provides sufficient time for the inducible type of NO synthase to form, estimated to take a lag phase of ≈4 to 6 hours.38 Pretreatment with dexamethasone, which inhibits only the inducible type of NO synthase without affecting the constitutive enzyme,7 8 inhibited the increase in coronary flow in this model of endotoxemia. This suggests that the inducible type of NO synthase is to a large extent responsible for the increase in coronary flow. Dexamethasone, however, could not totally prevent the increase in coronary flow, suggesting that the effect of dexamethasone is temporary or that part of increased NO release is produced by the increased activity of the constitutive NO synthase.39 40 Since dexamethasone inhibits the LPS-induced synthesis and release of interleukin-1 and tumor necrosis factor41 (both cytokines stimulate the production of inducible NO synthase38 ), the inhibitory effect could also be the result of diminished release of these mediators in addition to direct inhibition of expression of the enzyme.42

Not only the coronary vessels but also the myocardium itself has the capacity to express constitutive and inducible NO synthase.38 43 44 The constitutive form of NO synthase has been shown to be present in normal rat myocardium, and the inducible NO synthase is expressed after endotoxemia.38 Isolated myocytes of the rat express the inducible NO synthase only after stimulation with cytokines; this expression could be inhibited with dexamethasone.38 Increased release of NO by the inducible NO synthase within the cardiac myocytes themselves has been suggested to contribute to the reduced contractility of isolated guinea pig cardiac ventricular myocytes after endotoxemia.43 Human ventricular tissue of patients with dilated cardiomyopathy shows a significant activity of the inducible enzyme accompanied by a low activity of the constitutive NO synthase.44 This suggests that the inducible enzyme plays a pathological role in the myocardium, whereas the constitutive enzyme plays a more physiological role. To what extent the direct expression of NO synthase within cardiac myocytes itself contributed to the vascular relaxation in our model of endotoxemia remains to be established.

Our data indicate that reduction of excess coronary flow in endotoxemia, due to inhibition of the NO pathway with NNLA or MB, can cause local myocardial ischemia. In a recent study by Duncker and Bache,45 who used radioactive microspheres, NNLA was shown to exacerbate myocardial hypoperfusion during exercise in the presence of coronary stenosis in awake dogs. Administration of NG-monomethyl-l-arginine, another inhibitor of NO synthesis, has been reported to exacerbate global myocardial ischemia in vivo in a rabbit model of endotoxemia, as indicated by changes on the electrocardiogram.24 In the present study, foci of ischemia are indicated by areas of enhanced NADH fluorescence. This inhomogeneity of NADH fluorescence may be explained by a heterogeneously distributed blood flow throughout the heart,46 47 leading to an uneven distribution of ischemic areas in the myocardium during low coronary perfusion pressure or coronary flow restriction.48 The present study shows that heterogeneously distributed ischemia also exists after flow restriction by inhibition of the NO pathway in endotoxin-treated rat hearts but not in normal rat hearts. Since histological examination revealed no differences between groups and since overall parameters of coronary flow, effluent Po2, and oxygen consumption in endotoxin-treated hearts were not significantly different from those in control hearts after NNLA, the mismatch in local oxygen balance may reflect changes in local myocardial blood flow. The size of the ischemic areas, as revealed by NADH videofluorometry, are approximately the same size as those seen during near hypoxia in normal hearts. Using NADH videofluorometry, we have recently shown that such areas represent microcirculatory units at a capillary level that are consistently vulnerable to ischemia in the isolated rat heart.30 Our results suggest that endotoxemia promotes ischemia in these vulnerable areas.

Maldistribution of heterogeneous coronary blood flow has been demonstrated during endotoxic shock in vivo and may play a role in the development of depressed cardiac function.49 50 This idea is supported by the results of the present study. There could be several explanations for the appearance of ischemic foci during inhibition of the NO pathway. Regional maldistribution of coronary flow leading to underperfusion of myocardial tissue could have caused a local decrease in oxygen supply in endotoxin-treated hearts. No signs of ischemia, either local or global, were present in the isolated endotoxin-treated rat hearts when the NO pathway was not inhibited. This might indicate that maldistribution of coronary blood flow did not exist before flow limitation or that maldistribution of blood flow was compensated by increased overall coronary flow. Reducing coronary flow by inhibiting NO metabolism or direct smooth muscle vasoconstriction could have unmasked relatively underperfused (low-flow) areas in the endotoxin-treated hearts by causing myocardial ischemia. These ischemic areas probably represent the vulnerable units in the capillary network of the rat myocardium, which are the first to be compromised when oxygen supply becomes limited.30 An alternative explanation may be that oxygen demand is higher while distribution of flow remains the same. However, our results showed equal oxygen consumption for both groups after the inhibition of NO synthesis. Increasing coronary flow by restoring NO synthesis with l-arginine or providing exogenous NO with the NO donor nitroprusside restored perfusion in the ischemic areas in both cases. These findings support the idea that NO plays a role in maintaining organ perfusion.24 26

In general, it is thought that the constitutive NO synthase plays a role in maintaining organ perfusion, whereas the expression of the inducible form (as in sepsis and endotoxemia) leads to the production of excessive amounts of NO, resulting in vascular relaxation and tissue damage.24 The therapeutic use of NO inhibitors during sepsis should, therefore, be directed to inhibition of the inducible NO synthase. In the present study, we used inhibitors that affect constitutive and inducible NO synthase. The inhibition of only the inducible NO synthase might have prevented myocardial ischemia in endotoxin-treated hearts. However, to our knowledge, no selective inhibitors of only the inducible NO synthase are currently available. To prevent tissue hypoperfusion, total inhibition of NO synthesis combined with an exogenous NO donor could be helpful.24 In the present study, the exogenous NO donor sodium nitroprusside restored myocardial hypoperfusion when endogenous NO synthesis was totally inhibited with NNLA. Development of selective inhibitors of inducible NO synthase must be awaited; such selective inhibitors may be of special value in the future management of septic patients. For now, we suggest that the therapeutic use of nonselective NO inhibitors during sepsis must be done with caution and under careful cardiac monitoring.

The present study supports the notion that the massive coronary vasodilation seen in the isolated endotoxin-treated rat heart is caused by an increased release of NO. Inhibition of NO metabolism with NNLA or MB after endotoxemia reduced the coronary flow and caused areas of myocardial ischemia. Reducing coronary flow by direct vasoconstriction resulted in similar areas of myocardial ischemia, as did inhibition of the NO pathway. This finding suggests that the local balance between oxygen supply and oxygen demand in endotoxin-treated hearts is disturbed after flow restriction. Inhibition of NO synthesis during sepsis and endotoxemia has been reported to be accompanied by reductions in cardiac output21 22 23 24 25 27 and increased mortality.24 25 26 It could be that localized myocardial ischemia reported in the present study may have contributed to these deleterious effects. Conversely, it could be that the induction of NO synthase in the myocardium during endotoxemia may be a protective mechanism that prevents the development of local ischemia of malperfused cardiac tissue and that the inhibition of NO synthesis could be counterproductive.

Acknowledgments

The authors wish to thank Dr J. van Amsterdam of the National Institute for Public Health and Environment for determination of nitrate plus nitrite concentration and Dr J.P. van der Sluijs for critical suggestions. The authors are also thankful for support by friends of the Rotterdam Blood Bank.

Footnotes

  • Previously published as preliminary results in abstract form at the 22nd annual meeting of the International Society of Oxygen Transport to Tissue, 1994.

  • Received June 9, 1994.
  • Accepted November 21, 1994.

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  • Inhibition of Nitric Oxide Synthesis Causes Myocardial Ischemia in Endotoxemic Rats
    J. A. M. Avontuur, H. A. Bruining and C. Ince
    Circulation Research. 1995;76:418-425, originally published March 1, 1995
    https://doi.org/10.1161/01.RES.76.3.418
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