Abstract-The aim of the present study was to determine whether cardiac nitric oxide (NO) production changes during the progression of pacing-induced heart failure and whether this occurs in association with alterations in myocardial metabolism. Dogs (nϭ8) were instrumented and the heart paced until left ventricular end-diastolic pressure reached 25 mm Hg and clinical signs of severe failure were evident. Every week, hemodynamic measurements were recorded and blood samples were withdrawn from the aorta and the coronary sinus for measurement of NO metabolites, O 2 content, free fatty acids (FFAs), and lactate and glucose concentrations. ne of the proposed mechanisms of cardiac dysfunction in heart failure attributes a major role to an excessive production of nitric oxide (NO) in the heart and, specifically, in myocytes.1,2 Circulating proinflammatory cytokines, found in high concentrations in plasma of patients with heart failure, 3 would stimulate the expression of inducible NO synthase (iNOS) with consequent overproduction of NO. NO has, among other functions, negative inotropic effects at high concentrations, as demonstrated in vitro.4-6 A similar mechanism of cardiac depression has been recognized previously in septic shock, although the levels of circulating cytokines and NO catabolites in this syndrome are much higher than those found during heart failure. 3,[7][8][9] However, to date, evidence of an increase in cardiac production of NO, sufficient to cause a negative inotropic action during heart failure, has not been provided. The finding that iNOS is expressed in tissue from failing hearts does not necessarily imply that cardiomyocytes are exposed to toxic concentrations of NO. Indeed, it is the amount of NO and not the enzyme isotype generating it that determines the degree to which cardiac function would be depressed. Numerous studies suggest that NO release is rather low in heart failure. In clinical and animal studies, 2,10 pharmacological blockade of NO synthases (NOS) did not alter baseline hemodynamics, indicating that, rather than being characterized by overproduction, systemic NO synthesis could already be minimal. Several investigations, including our own, in patients and in animal models of heart failure report an impairment of endothelial release of NO in large arteries and in coronary microvessels.11-14 The reduced vascular NO

This study determined the changes in NO production from the coronary circulation of the conscious dog during exercise. The role of endogenous NO as it relates to coronary flow, myocardial work, and metabolism was also studied. Mongrel dogs were chronically instrumented for measurements of coronary blood flow (CBF), ventricular and aortic pressure, and ventricular diameter, with catheters in the aorta and coronary sinus. Acute exercise (5 minutes at 3.6, 5.9, and 9.1 mph) was performed, and hemodynamic measurements and blood samples were taken at each exercise level. Nitro-L-arginine (NLA, 35 mg/kg IV) was given to block NO synthesis, and the exercise was repeated. Blood samples were analyzed for oxygen, plasma nitrate/nitrite (an index of NO), lactate, glucose, and free fatty acid (FFA) levels. Acute exercise caused significant elevations in NO production by the coronary circulation (46 +/- 23, 129 +/- 44, and 63 +/- 32 nmol/min at each speed respectively, P < .05). After NLA, there was no measurable NO production at rest or during exercise. Blockade of NO synthesis resulted in elevations in myocardial oxygen consumption and reductions in myocardial FFA consumption for comparable levels of CBF and cardiac work. The metabolic changes after NLA occurred in the absence of alterations in myocardial lactate or glucose consumptions. NO production by the coronary circulation is increased with exercise and blocked by NLA. The absence of NO in the coronary circulation during exercise does not affect levels of CBF, because it shifts the relationship between cardiac work and myocardial oxygen consumption, suggesting that endogenous NO modulates myocardial metabolism.

These studies indicate that nitrate is a reliable measure of NO metabolism in vivo but that because of the long half-life, nitrate will accumulate in plasma once it is produced. Because of the large volume of distribution (21% of body weight versus the 4% of body weight usually attributed to plasma volume, the compartment in which nitrate is measured), simple measures of plasma nitrate underestimate by a factor of 4 to 6 the actual production of nitrate or NO by the body. In disease states, such as heart failure, in which renal function and extracellular volume are altered, caution should be exercised when increases in nitrate in plasma as an index of NO formation are evaluated.

Statin drugs can upregulate endothelial nitric oxide (NO) synthase (eNOS) in isolated endothelial cells independent of lipid-lowering effects. We investigated the effect of short-term simvastatin administration on coronary vascular eNOS and NO production in conscious dogs and canine tissues. Mongrel dogs were instrumented under general anesthesia to measure coronary blood flow (CBF). Simvastatin (20 mg. kg(-1). day(-1)) was administered orally for 2 wk; afterward, resting CBF was found to be higher compared with control (P < 0.05) and veratrine- (activator of reflex cholinergic NO-dependent coronary vasodilation) and ACh-mediated coronary vasodilation were enhanced (P < 0.05). Response to endothelium-independent vasodilators, adenosine and nitroglycerin, was not potentiated. After simvastatin administration, plasma nitrate and nitrite (NO(x)) levels increased from 5.22 +/- 1.2 to 7. 79 +/- 1.3 microM (P < 0.05); baseline and agonist-stimulated NO production in isolated coronary microvessels were augmented (P < 0.05); resting in vivo myocardial oxygen consumption (MVO(2)) decreased from 6.8 +/- 0.6 to 5.9 +/- 0.4 ml/min (P < 0.05); NO-dependent regulation of MVO(2) in response to NO agonists was augmented in isolated myocardial segments (P < 0.05); and eNOS protein increased 29% and eNOS mRNA decreased 50% in aortas and coronary vascular endothelium. Short-term administration of simvastatin in dogs increases coronary endothelial NO production to enhance NO-dependent coronary vasodilation and NO-mediated regulation of MVO(2).

Background-Our objective for this study was to investigate whether nitric oxide (NO) modulates tissue respiration in the failing human myocardium. Methods and Results-Left ventricular free wall and right ventricular tissue samples were taken from 14 failing explanted human hearts at the time of transplantation. Tissue oxygen consumption was measured with a Clark-type oxygen electrode in an airtight stirred bath containing Krebs solution buffered with HEPES at 37°C (pH 7.4). Rate of decrease in oxygen concentration was expressed as a percentage of the baseline, and results of the highest dose are indicated. Bradykinin (10 Ϫ4 mol/L, Ϫ21Ϯ5%), amlodipine (10 Ϫ5 mol/L, Ϫ14Ϯ5%), the ACE inhibitor ramiprilat (10 Ϫ4 mol/L, Ϫ21Ϯ2%), and the neutral endopeptidase inhibitor thiorphan (10 Ϫ4 mol/L, Ϫ16Ϯ5%) all caused concentrationdependent decreases in tissue oxygen consumption. Responses to bradykinin (Ϫ2Ϯ6%), amlodipine (Ϫ2Ϯ4%), ramiprilat (Ϫ5Ϯ6%), and thiorphan (Ϫ4Ϯ7%) were significantly attenuated after NO synthase blockade with N-nitro-L-arginine methyl ester (10 Ϫ4 mol/L; all PϽ0.05). Ϫ4 mol/L, Ϫ34Ϯ5%) and nitroglycerin (10 Ϫ4 mol/L, Ϫ21Ϯ5%), also decreased tissue oxygen consumption in a concentration-dependent manner. However, the reduction in tissue oxygen consumption in response to S-nitroso-Nacetyl-penicillamine (Ϫ35Ϯ7%) or nitroglycerin (Ϫ16Ϯ5%) was not significantly affected by N-nitro-L-arginine methyl ester. Conclusions-These results indicate that the modulation of oxygen consumption by both endogenous and exogenous NOis preserved in the failing human myocardium and that the inhibition of kinin degradation plays an important role in the regulation of mitochondrial respiration. (Circulation. 1999;100:1291-1297.)Key Words: nitric oxide Ⅲ oxygen Ⅲ heart failure T he vast amount of ATP produced by the cardiac mitochondria is used mainly for cardiac muscle contraction. Abnormalities of mitochondria in cardiomyocytes in heart failure have been well documented in both animals [1][2][3][4] and human studies, 5-7 which provided structural and metabolic evidence of mitochondrial dysfunction. Mitochondrial DNA damage with increased mitochondrial DNA deletion in patients with heart failure has also been reported, 8,9 a defect associated with the impairment of oxidative phosphorylation. 10 On the other hand, normal mitochondrial metabolism has also been documented in chronic heart failure. 11,12 However, the role of nitric oxide (NO) in the control of mitochondrial metabolism is not well established. Our laboratory and others have demonstrated that attenuation of NO production increases whole-body or organ oxygen consumption. [13][14][15][16] The initial observation of the interaction between NO and mitochondrial enzymes was reported in cell culture studies of macrophage-induced cytotoxicity of neoplastic cells. 17,18 The activated macrophage induced reduction of electron transfer by inactivating iron-sulfur-containing complexes I and II of the respiratory chain and aconitase in the Krebs cycle. This effect was shown to b...

Our previous studies uncovered an inhibitory effect of nitric oxide (NO) on leg skeletal muscle respiration in dogs at rest. The role of NO in the modulation of O2 consumption and O2 extraction in hindlimb muscle during elevated metabolic states was investigated in chronically instrumented dogs while walking and at three exercise intensities which markedly increased hindlimb blood flow. Walking resulted in increased O2 consumption by 17 +/- 4 mL min-1 and O2 extraction from 24 +/- 1 to 37 +/- 8%, with no alteration in hindlimb blood flow (BFLeg) and vascular resistance (VRLeg). Running at the highest speed (9.1 mph) resulted in an increase in BFLeg from 0.67 +/- 0.05 to 2.2 +/- 0.1 L min-1, a reduction of VRLeg and elevation of hindlimb O2 consumption from 33 +/- 3 to 226 +/- 21 mL min-1 and O2 extraction from 29 +/- 2 to 61 +/- 5%, with a decrease in leg venous PO2 from 38 +/- 1 to 25 +/- 1 mmHg. After nitro-L-arginine (NLA) (35 mg kg-1, i.v.) to inhibit endogenous NO synthesis, walking caused greater increases in hindlimb O2 consumption (29 +/- 5 mL min-1) and O2 extraction (43 +/- 1 to 60 +/- 3%) (both P < 0.05), with no significant change in BFLeg. During running at the highest speed, BFLeg was 1.9 +/- 0.1 L min-1 (P < 0. 05) and VRLeg was higher, accompanied by increases in hindlimb O2 consumption from 49 +/- 7 to 318 +/- 24 mL min-1 and O2 extraction from 41 +/- 2 to 79 +/- 4% (both P < 0.05), with a greater decrease in leg venous PO2 from 33 +/- 1 to 20 +/- 1 mmHg (P < 0.05). Similar results were found for intermediate levels of exercise. Our results indicate that NO modulates hindlimb skeletal muscle O2 extraction and O2 usage whether blood flow increased or not during exercise.

Our previous studies have suggested that there is reduced nitric oxide (NO) production in canine coronary blood vessels after the development of pacing-induced heart failure. The goal of these studies was to determine whether flow-induced NO-mediated dilation is altered in coronary arterioles during the development of heart failure. Subepicardial coronary arterioles (basal diameter 80 microm) were isolated from normal canine hearts, from hearts with dysfunction but no heart failure, and from hearts with severe cardiac decompensation. Arterioles were perfused at increasing flow or administered agonists with no flow in vitro. In arterioles from normal hearts, flow increased arteriolar diameter, with one-half of the response being NO dependent and one-half prostaglandin dependent. Shear stress-induced dilation was eliminated by removing the endothelium. Arterioles from normal hearts and hearts with dysfunction but no failure responded to increasing shear stress with dilation that reached a maximum at a shear stress of 20 dyn/cm(2). In contrast, arterioles from failing hearts showed a reduced dilation, reaching only 55% of the dilation seen in vessels of normal hearts at a shear stress of 100 dyn/cm(2). This remaining dilation was eliminated by indomethacin, suggesting that the NO-dependent component was absent in coronary microvessels after the development of heart failure. Similarly, agonist-induced NO-dependent coronary arteriolar dilation was markedly attenuated after the development of heart failure. After the development of severe dilated cardiomyopathy and heart failure, the NO-dependent component of both shear stress- and agonist-induced arteriolar dilation is reduced or entirely absent.

Our results indicate that vagally mediated coronary vasodilation is selectively attenuated in conscious dogs after pacing-induced heart failure, whereas the vagally mediated bradycardia is preserved. Since muscarinic receptor-induced coronary vasodilation is mediated by NO, the disappearance of NO from blood vessels leads to a defect in the integrated neural regulation of coronary blood flow and myocardial function during heart failure.