Blockade of Nitric Oxide Synthesis Reduces Myocardial Oxygen Consumption In Vivo
Background Although cardiac myocytes and coronary vascular endothelium are known to express a constitutive form of NO synthase, the in vivo effects of tonic endogenous production of NO on myocardial O2 consumption and contractile performance remain unclear.
Methods and Results The effects of blockade of NO synthase were determined in intact dogs. Myocardial O2 consumption decreased systematically over a wide range of hemodynamic demand after the systemic administration of Nω-nitro-l-arginine methyl ester (L-NAME) or Nω-nitro-l-arginine. Decreases after doses of 1 and 10 mg/kg L-NAME averaged 23±3.8% and 34±7.2% at a heart rate of 90 bpm in open-chest animals. Similar reductions occurred after the administration of L-NAME and Nω-nitro-l-arginine in chronically instrumented animals and were unaffected by β-adrenergic blockade. Intracoronary infusion of L-NAME in chronically instrumented animals reduced both myocardial O2 consumption and regional segment shortening, even at a dose that did not increase systemic arterial pressure.
Conclusions The blockade of NO synthesis reduces myocardial O2 consumption in vivo. The decrease in O2 consumption is accompanied by a decrease in segment shortening. It involves a direct myocardial action of NO, is unaffected by β-blockade, and is consistent with in vitro studies indicating that low levels of NO augment contractile performance by inhibition of a cGMP-dependent phosphodiesterase.
Although both cardiac myocytes and coronary vascular endothelial cells are now known to express a constitutive isoform of NO synthase, the effects of endogenous NO on myocardial contractile performance and O2 consumption remain unclear. Although some in vitro studies indicate that NO can exert a negative effect on myocardial contractile function,1 2 3 others have reported positive inotropic actions4 5 or no effect.6 In addition, the effects of NO on cardiac myocytes appear to be dose related, with contractile activity being augmented at low concentrations and reduced as NO levels are increased.4 5 In vivo responses of coronary blood flow to the blockade of NO synthesis have been variable under basal conditions, either showing no systematic change7 8 9 10 11 or a small decrease.12 13 14 15 16 17 The lack of increase or actual decrease in flow is surprising because systemic blood pressure usually increases after blockade of NO synthesis, often without a corresponding reduction in heart rate. An increase in afterload would be expected to increase myocardial O2 consumption unless counterbalanced by a reduction in some other determinant of O2 demand. Measurements of myocardial O2 consumption included in the in vivo studies of the effects of NO on other interventions10 14 15 16 18 19 have given varying results and are limited by potentially confounding effects of the other interventions.
The present study was undertaken to determine whether endogenous NO exerts a tonic effect on myocardial O2 consumption and contractile performance in vivo. To do so, measurements were made before and after administration of l-arginine analogues known to block NO synthase. Initially, the effects of L-NAME on O2 consumption were evaluated over a wide range of myocardial O2 demand in open-chest dogs.
Subsequently, L-NAME and L-NA, an additional arginine analogue without potential antimuscarinic activity,20 were studied in chronically instrumented dogs to avoid the potentially confounding effects of anesthesia, thoracotomy, and other acute instrumentation procedures. To establish that observed changes in O2 consumption represented a direct myocardial effect, the studies in chronically instrumented dogs included intracoronary administration of L-NAME, with measurement of both regional contractile activity and O2 consumption.
Studies were performed in mongrel dogs of either sex using procedures and protocols concordant with institutional guidelines for the care and use of experimental animals.
Eight dogs weighing 26 to 60 kg were studied after an overnight fast. After premedication with Innovar-Vet (1 mL IM of 0.4 mg/mL fentanyl and 20 mg/mL droperidol), anesthesia was induced with sodium pentobarbital (30 mg/kg IV). After intubation and institution of mechanical ventilation with a piston respirator, a left thoracotomy was performed, and the heart was suspended in a pericardial cradle. Catheters were placed for pressure measurement in the aorta and left atrium. A micromanometer (Konigsberg Instruments P 6.5) was inserted into the LV cavity through the ventricular apex. The proximal LCx was instrumented with a transit time ultrasonic flow probe (model 3RB, Transonic Systems Inc) and occluder. A sampling catheter was inserted into the coronary sinus through the right atrial appendage and advanced into the great cardiac vein. Atrioventricular block was produced by injection of formalin into the atrioventricular node, and the right ventricle was paced at a rate of 90 bpm with the use of epicardial electrodes and an external pacemaker (model 5525, Medtronic, Inc).
Chronically Instrumented Animals
Twenty dogs weighing 27 to 44 kg were instrumented after an overnight fast and the usual period of on-site conditioning. After premedication with acepromazine (0.1 mg/kg IV) or Innovar-Vet, anesthesia was induced with sodium thiamylal (20 mg/kg IV) or methohexital (11 mg/kg). After intubation and institution of mechanical ventilation, a surgical plane of anesthesia was maintained with a gas mixture of nitrous oxide (≈60%), oxygen (≈40%), and halothane (1% to 2%). A left thoracotomy was performed under sterile conditions, and Tygon catheters were placed into the descending aorta and left atrium. A Konigsberg micromanometer was inserted into the LV through the LV apex. Bipolar pacing leads were sewn onto the left atrial appendage. In 17 animals, the LCx was instrumented with a Transonic Systems transit time ultrasonic flow probe (model 3RB) and hydraulic occluder, and a small plastic catheter was introduced directly into the coronary sinus. Three animals received an LAD flow probe (Transonic Systems model 2SB) and great cardiac vein sampling catheter. Also, in animals used for intracoronary studies, a 22-gauge angiocatheter connected to small-bore tubing was inserted into the artery (LCx or LAD) to be used for infusion. An ultrasonic Doppler flow probe was added onto the artery not used for infusion; ultrasonic crystal pairs were placed in the LCx and LAD distributions for measurement of subendocardial segment length. In five animals, a sampling catheter was placed in the great cardiac vein as well as the coronary sinus. All catheters and wires were exteriorized through the chest wall and placed in an external jacket. The chest was closed, and the pneumothorax was evacuated. Cephalothin (Keflin; 30 to 35 mg/kg IV or IM BID) was administered for 1 to 3 days after surgery, and narcotic analgesia (0.01 to 0.02 mg/kg buprenorphine hydrochloride [Buprenex] SC every 8 to 12 hours) was given as needed for postoperative discomfort. Catheters were flushed with saline and filled with heparin every 1 to 2 days (10 000 U/mL for the circumflex catheter and 1000 U/mL for other catheters). Aspirin (325 mg PO) was administered daily beginning on the third postoperative day. Animals were allowed to recover for 10 to 14 days before being studied in the unanesthetized state.
Catheters used for pressure measurement were connected to strain gauge transducers (Viggo-Spectramed P23XL), with the zero reference level taken at midchest. The strain gauges and the LV micromanometer were connected to transducer preamplifiers (model 1147, Gould, Inc). Ultrasonic flow probes were connected to a Transonic Systems model T-206 meter; the LCx zero flow level was established by momentary LCx occlusion. Ultrasonic segment shortening signals were processed using a Crystal Biotech VF-1 sonomicrometer.
Hemodynamic and sonomicrometric data were monitored continuously in analog (Vidco 516YT) and digital (DataFlow, Crystal Biotech) form. Average values for individual parameters were calculated from digitized data sampled for 30 to 40 seconds at a rate of 120 Hz. LV pressure was differentiated electronically. For calculations of systolic segment shortening, end diastole was taken as the onset of positive LV dP/dt, and end systole was taken as 20 ms before peak negative dP/dt. Double product was calculated as the product of peak aortic pressure and heart rate. Indexes of conductance in the LCx and LAD beds were calculated as the quotient of LCx or LAD flow and mean aortic pressure. Blood samples were analyzed for pH, Pco2, Po2, and O2 saturation using a standard blood gas analyzer (model ABL-30, Radiometer, Inc) and hemoximeter (model OSM3, Radiometer, Inc). Arterial and coronary venous O2 contents were determined from O2 saturation values, blood hemoglobin levels, and Po2 values, with the assumptions that hemoglobin has an O2 capacity of 1.34 mL/g and the solubility of O2 in blood is 0.0031 mL·mL−1·mm Hg−1. Myocardial O2 consumption was calculated as the product of LCx flow and the arteriovenous difference in O2 content. Animals were killed with an overdose of sodium pentobarbital and potassium chloride at the completion of the study.
Studies were initially conducted in open-chest animals. Arterial and coronary venous blood samples were collected at ventricular pacing rates of 90, 60, 120, and 150 bpm. After return of the pacing rate to 90 bpm, L-NAME (1 mg/kg) was infused systemically through the left atrial catheter. Arterial and coronary venous blood samples and hemodynamic data were collected 20 to 30 minutes later. Observations were made at pacing rates of 60 and 120 bpm as well as 90 bpm in six animals and at 150 bpm in four animals. An additional 9 mg/kg L-NAME was then infused through the left atrial catheter. Arterial and coronary venous blood samples were again collected 20 to 30 minutes later, at pacing rates of 90, 60, 120, and 150 bpm.
Eight additional studies of systemic blockade of NO synthesis were performed in chronically instrumented animals lightly sedated with Innovar-Vet and resting in a sling to which they had previously been acclimated. Because second-degree atrioventricular block sometimes develops after systemic administration of l-arginine analogues, VVI pacing was instituted through an ultrasonic crystal pair at ≈120 bpm. In five animals, myocardial O2 consumption was measured before and after left atrial infusion of L-NA (20 mg/kg in four studies and 10 mg/kg in the fifth). In the remaining three studies, we used L-NAME (10 mg/kg). To determine whether the effects of the l-arginine analogues were altered by β-adrenergic blockade, four of the eight studies were performed while the animals were receiving propranolol (0.15 mg·kg−1·h−1 IV) after a loading dose of 0.2 mg/kg. This dosage of propranolol blunted heart rate responses to isoproterenol (0.4 μg/kg IV) by 92% (range, 80% to 100%).
To separate direct myocardial effects from possible systemic-mediated effects, responses of myocardial O2 consumption to intracoronary administration of L-NAME were studied in 13 chronically instrumented animals. Atrial, rather than ventricular, pacing was used so that segmental function in the LCx and LAD beds could be evaluated simultaneously. Preliminary studies in other animals indicated that systemic arterial pressure could increase after an intracoronary dose of L-NAME as low as 0.1 mg/kg. Accordingly, intracoronary studies were conducted with doses of 0.03 and 1.0 mg/kg. In an animal that had been administered intracoronary doses of acetylcholine, the 0.03 and 1.0 mg/kg doses of L-NAME blunted peak acetylcholine-induced increases in flow by 68% and 81%, respectively.
Data are presented as mean±SEM. Differences between mean values in studies before and after a single dose of L-NAME or L-NA were assessed with two-tailed paired Student's t tests, with statistical significance taken as P<.05. Changes in hemodynamic parameters and myocardial O2 consumption in studies involving two dose levels of an l-arginine analogue were analyzed with an ANOVA for repeated measures; post hoc comparisons were performed with the Student-Newman-Keuls test with significance set at P<.05.
Effects of Systemic Administration of l-Arginine Analogues
Initial values of arterial pH, Pco2, and Po2 in open-chest animals averaged 7.39±0.01, 31±0.8 mm Hg, and 102±6.3 mm Hg. Hemodynamic, blood gas, and O2 consumption values at a ventricular rate of 90 are shown in Table 1⇓. LCx flow, LCx conductance index, and myocardial O2 consumption decreased systematically at all pacing rates after both 1 and 10 mg/kg L-NAME. At a rate of 90, decreases in O2 consumption averaged 23±3.8% and 34±7.2% (P<.05 in both cases). Fig 1⇓ illustrates values of myocardial O2 consumption in relation to the double product index of O2 consumption over the full range of pacing rates. The relation shifts downward after blockade of NO synthesis (ie, O2 consumption is reduced at any level of double product).
In chronically instrumented animals, initial values of arterial pH, Pco2, and Po2 averaged 7.38±0.01, 38±1.0 mm Hg, and 88±3.0 mm Hg. Systemic blockade of NO synthesis with L-NAME or L-NA increased mean arterial pressure by 24±3.9% (99±2.1 to 122±2.6 mm Hg; P<.001). Peak left ventricular dP/dt (1870±107 and 1797±151 mm/s) and left ventricular end-diastolic pressure (5.3±1.1 and 4.3±1.3 mm Hg) were unchanged. Myocardial O2 consumption was again reduced in relation to double product, in both the presence and absence of β-adrenergic blockade (Fig 2⇓). The control relation was restored after blockade of NO synthesis had abated, and the relation was unaffected by left atrial infusion of saline (without L-NAME or L-NA) (Fig 3⇓).
Effects of Intracoronary Administration of l-Arginine Analogues
Hemodynamic, blood gas, and O2 consumption data for animals studied with LCx infusion of L-NAME are presented in Table 2⇓. The initial values of arterial pH, Pco2, and Po2 averaged 7.37±0.01, 39±1.4 mm Hg, and 91±2.1 mm Hg. The 0.03 mg/kg dose of L-NAME was not associated with an increase in systemic arterial pressure. Myocardial O2 consumption in the LCx bed nevertheless decreased in each case, averaging 3.90±0.36 mL/min before and 3.39±0.34 mL/min after L-NAME (P<.05). LCx segment shortening also decreased in each case; reductions averaged 10±1.7% (P<.05). LAD segment shortening did not change systematically.
The 1.0 mg/kg dose of L-NAME produced systematic increases in mean arterial pressure averaging 11±1.6 mm Hg (P<.05). Myocardial O2 consumption (3.43±0.38 mL/min) remained significantly below the pre–L-NAME level despite the increases in arterial pressure and did not differ systematically from its level after 0.03 mg/kg L-NAME. LCx segment shortening fell 20±3.9% below its original level (P<.05 for both before L-NAME and 0.03 mg/kg L-NAME), whereas LAD segment shortening again did not change significantly. LCx conductance, which had decreased systematically after 0.03 mg/kg L-NAME, decreased further after the 1.0 mg/kg dose (P<.05). Conversely, LAD conductance, which was unchanged after the lower L-NAME dose, now increased in parallel with systemic arterial pressure (P<.05).
Findings in animals studied with LAD infusion of L-NAME were similar and are summarized in Table 3⇓. After 0.03 mg/kg, decreases in myocardial O2 consumption averaged 12% (range, 7% to 15%), and decreases in LAD segment shortening averaged 11% (range, 5% to 21%), without a change in aortic pressure. After 1.0 mg/kg, myocardial O2 consumption remained below its pre–L-NAME level despite a 20% increase in mean aortic pressure. LAD segment shortening decreased further, to 24% below its initial level.
The results of the present study indicate that blockade of NO synthesis with L-NAME or L-NA reduces myocardial O2 consumption and that the reduction involves an alteration in contractile performance. Decreased contractility is known to decrease myocardial O2 consumption under controlled hemodynamic conditions.21 The use of measurements of myocardial O2 consumption in chronically instrumented animals avoids the potentially confounding effects of anesthesia, thoracotomy, external perfusion circuits, and other acute instrumentation. The observations in chronically instrumented animals also indicate that the reductions are not dependent on β-adrenergic receptor stimulation or the use of barbiturates.22 The use of L-NA as well as L-NAME also indicates that the reductions are not related to an antimuscarinic action of alkyl esters of arginine.20 The physiological relevance of the findings is supported by their occurrence in an in vivo setting in which mechanisms that could potentially counteract NO-related changes remained available.
The studies involving intracoronary blockade of NO synthesis (Table⇑s 2 and 3) make several additional points. Our experience is that intracoronary doses of L-NAME as small as 0.1 mg/kg can produce measurable increases in systemic arterial pressure in chronically instrumented animals. The dose of 0.03 mg/kg avoided this effect but was nevertheless associated with a consistent decrease in regional segment shortening (ie, contractile performance was altered independent of a change in afterload). The reductions in O2 consumption and segment shortening with unchanged systemic hemodynamics verify that the findings involve a direct myocardial action of L-NAME. The dose of 1.0 mg/kg produced a further decrease in regional segment shortening. Because segment shortening in the noninfused arterial bed was not reduced concomitantly, the additional decrease is more likely related to the additional L-NAME than to the increase in systemic pressure. The directionally opposite responses of conductance in the infused and non-infused beds further indicate the predominantly regional effect of the 1.0 mg/kg dose. The intracoronary studies also indicate that the observed reductions in myocardial O2 consumption after systemic administration of l-arginine analogues cannot be attributed to systemic neural or hormonal factors without a local myocardial effect.
A number of the many documented biological actions of NO are of interest in relation to the present findings. Several of these actions involve the second messenger cGMP. Although NO is known to increase myocardial cGMP, effects of cGMP on myocardial performance may vary according to level and experimental preparation. As recently discussed by Paulus et al,23 elevations of cGMP can induce both negative and positive inotropic effects, probably depending on the relative activation of protein kinases, phosphodiesterases, and phosphatases. Not surprisingly, therefore, effects of NO on contractile performance have varied in reported studies.
It is clear that NO produces negative inotropic effects when its levels are increased markedly by cytokines.24 25 26 However, in vitro studies intended to address the effects of endogenous NO production have given conflicting results.1 2 3 5 6 16 27 28 Complexities in the interpretation of these reports have been discussed by Kaye et al.3 Also, some of the variation in NO effects observed in nonstimulated preparations may be dose related. Kodja et al5 reported a biphasic effect of NO-induced increases in cGMP in adult rat ventricular myocytes. Low concentrations of NO donors (1 μmol/L) produced small increases in cGMP, cAMP, and contractile activity, whereas high concentrations (100 μmol/L) produced marked increases in cGMP but reduced contractile activity. The results were interpreted as compatible with inhibition of a cGMP-regulated cAMP phosphodiesterase at low NO concentrations and stimulation of a cGMP-dependent protein kinase by high NO levels. Similarly, Mohan et al4 demonstrated a concentration-dependent biphasic contractile response to 8-bromo-cGMP as well as NO donors in cat papillary muscle. They too attribute their increased inotropic response to inhibition of cGMP-regulated cAMP phosphodiesterase. In addition, in an earlier study of 8-bromo-cGMP in adult rat myocytes, Shah et al27 noted a transient positive inotropic effect in ≈50% of cells before negative inotropic effects predominated. Studies indicating that cGMP inhibition of cardiac phosphodiesterase may be clinically relevant have been summarized by Beavo.29
NO-related modulation of myocardial responses to β-adrenergic stimulation has also received considerable attention. Blockade of NO synthase accentuates the contractile response to isoproterenol in cultured rat myocytes28 30 but produces a negative inotropic effect in isoproterenol-stimulated rat hearts.31 This reduction in contractile performance in the setting of augmented β-adrenergic stimulation has been attributed to increased phosphodiesterase activity caused by reduced NO and cGMP levels.31 Similarly, cGMP-induced inhibition of a cAMP phosphodiesterase has been suggested as the basis by which low levels of cGMP (1 to 10 μmol/L) potentiate β-adrenergic effects on Ca2+ current in guinea pig ventricular cells32 and by which NO stimulates cardiac Ca2+ channel current in human atrial myocytes.33 In addition, Mohan et al4 demonstrated that effects of NO and cGMP can be modulated by endothelial products and cholinergic as well as adrenergic stimulation in cat papillary muscle. The effects of blockade of NO synthesis on myocardial responses to β-adrenergic stimulation in intact animals have also varied. Hare et al34 found that vagal inhibition of the inotropic response to dobutamine is attenuated, whereas Bernstein et al35 reported reductions in isoproterenol-induced increases in myocardial O2 consumption. In the study of Kaneko et al,17 the effects of L-NAME on responses of regional wall thickening and LV dP/dt to β-adrenergic stimulation varied with the dose of L-NAME. The complexity of possible actions of NO on contractile behavior is further illustrated by the recent suggestion that NO can play a role in the regulation of cellular energetics independent of its effects on signal transduction.22 36
Measurements of myocardial O2 consumption have sometimes been included in the in vivo studies of the effects of l-arginine analogues on other interventions and have also given variable results. In studies of hypercapnic acidosis and isoflurane in open-chest animals in which a cannulated coronary artery was perfused through an external circuit, Crystal et al18 and Gurevicius et al19 found O2 consumption to be unchanged after the administration of intracoronary L-NAME and L-NMMA. Conversely, while studying the response to acute hypoxemic stress in fetal lambs, Reller et al16 noted a 47% decrease in myocardial O2 consumption after L-NA. While studying functional hyperemia in open-chest dogs, Maekawa et al15 reported 14% and 12% decreases in regional O2 consumption at heart rates of 168 and 214 after intracoronary L-NA, respectively. A study of regional inhibition of NO synthesis by L-NMMA and L-NA by Kirkeboen et al14 included measurements of myocardial O2 consumption in 13 of 28 open-chest pigs. Although O2 consumption did not change systematically, hemodynamic indexes of myocardial O2 demand were presented only for the total group of animals studied. In a study of canine coronary flow during exercise, Altman et al10 administered intracoronary L-NA 1 hour after an initial exercise period and obtained values of O2 consumption that were higher than before the initial exercise. Because the present study was directed solely at the effects of endogenous NO production, potentially confounding effects (secondary as well as primary) of interventions other than blockade of NO synthesis were avoided.
In summary, blockade of endogenous NO synthesis has been found to reduce myocardial O2 consumption in vivo in intact animals. The reductions involve a direct myocardial effect, include a reduction in regional contractile activity, and are not affected by β-adrenergic blockade. Although the mechanisms underlying these findings require additional clarification, they appear to be compatible with suggested inhibitory effects of low NO concentrations on cGMP-dependent phosphodiesterase.
Selected Abbreviations and Acronyms
|L-NAME||=||Nω-nitro-l-arginine methyl ester|
|LAD||=||left anterior descending coronary artery|
|LCx||=||left circumflex artery|
|LV||=||left ventricular; left ventricle|
Dr Sherman is a Research Fellow of the Thoracic Surgery Foundation for Research and Education.
- Received August 14, 1996.
- Revision received October 3, 1996.
- Accepted October 14, 1996.
- Copyright © 1997 by American Heart Association
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