AJP - Heart Calcium Transients and Cell-Sarcomere
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

Am J Physiol Heart Circ Physiol 275: H41-H49, 1998;
0363-6135/98 $5.00
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Suto, N.
Right arrow Articles by Okumura, K.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Suto, N.
Right arrow Articles by Okumura, K.
Vol. 275, Issue 1, H41-H49, July 1998

Nitric oxide modulates cardiac contractility and oxygen consumption without changing contractile efficiency

Naoyuki Suto1, Atsushi Mikuniya1, Tomoyuki Okubo1, Hiroyuki Hanada1, Nobuyo Shinozaki2, and Ken Okumura1

1 Second Department of Internal Medicine, Hirosaki University School of Medicine and 2 Department of Radiological Technology, School of Allied Medical Sciences, Hirosaki University, Hirosaki 036, Japan

    ABSTRACT
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

Nitric oxide (NO) affects myocardial contractility and myocardial oxygen consumption (MVO2) in vitro. In alpha -chloralose-anesthetized dogs instrumented for the measurements of left ventricular (LV) pressure, LV volume using a conductance catheter, coronary blood flow, and coronary venous oxygen saturation (ScvO2) using a fiber-optic catheter, LV end-systolic pressure-volume relationships (ESPVR) and the relationship between MVO2 and LV pressure-volume area (PVA) were analyzed before and after intravenous infusions of the NO synthase inhibitor NG-monomethyl-L-arginine acetate (L-NMMA; 5 mg/kg, 8 dogs) and the NO substrate L-arginine (600 mg/kg, 7 dogs). L-NMMA increased the slope of the ESPVR (Emax) (P < 0.05) without changing contractile efficiency indicated by the inverse of the slope of the MVO2-PVA line. L-NMMA also increased unloaded MVO2, indicated by the y-axis intercept of the MVO2-PVA line (P < 0.05). In contrast, L-arginine decreased Emax (P < 0.05) while decreasing MVO2 (P < 0.05), and without changing contractile efficiency. The basal oxygen metabolism was not affected by L-NMMA and L-arginine. These data imply that endogenous NO spares MVO2 by reducing oxygen use in excitation-contraction coupling and attenuates cardiac contractility without changing contractile efficiency.

myocardial oxygen consumption; pressure-volume area; excitation-contraction coupling

    INTRODUCTION
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

NITRIC OXIDE (NO) is produced in the vascular endothelium and shows a potent vasodilator effect (23, 26). Although NO is produced and released in the coronary artery system both in the basal condition and in response to diverse stimuli, its effect on cardiac contractility and myocardial oxygen consumption (MVO2) is not well characterized. Previous studies have shown that NO attenuates cardiac myocyte contraction (3, 4, 9), mediates vagal inhibition of the cardiac inotropic response to beta -adrenergic stimulation (12), and regulates oxygen consumption (10, 32, 33, 45). In patients with left ventricular (LV) dysfunction, NO was reported to attenuate the positive inotropic response to beta -adrenergic stimulation (13). Several isoforms of NO synthase (NOS) responsible for the conversion of L-arginine to L-citrulline plus NO have been identified (27, 29). Under physiological conditions, a constitutive form of NO synthase (cNOS) is associated with low levels of NO formation, which are continuously generated in the vascular endothelium. Another form of NOS, known as inducible NOS (iNOS), can be activated in endotoxin shock, heart failure, and ischemia (7, 8, 19, 21, 24, 30, 41). Most of the previous studies examined the role of NO produced by iNOS in cardiac contractility and MVO2 and only a few studies examined the role of NO produced by cNOS in a physiological condition (12).

The purpose of the present study was to investigate the effect of NO on cardiac contractility and MVO2 in hearts in situ. Using an in vivo canine heart, we evaluated 1) the slope (Emax) of the end-systolic pressure-volume relationship (ESPVR), 2) MVO2, and 3) the pressure-volume area (PVA). Emax allows estimation of cardiac contractility irrespectively of the changes in preload and afterload (38, 39). With the MVO2-PVA relationship, it is possible to partition total energy output into mechanical and nonmechanical components: 1) the excess MVO2 above the MVO2-axis intercept (unloaded MVO2) and 2) MVO2 at PVA = 0 (16, 34). In addition, the MVO2 for nonmechanical energy utilization can be divided into that for excitation-contraction coupling and that for basal metabolism. Thus cardiac contractile efficiency, an expression of the efficiency of chemomechanical energy transduction by the contractile proteins when both MVO2 and PVA are expressed in standard energy, can be evaluated from the inverse of the slope of the linear MVO2-PVA relation. We investigated the effects of the NOS inhibitor NG-monomethyl-L-arginine acetate (L-NMMA) and of L-arginine, a substrate of NO, on these parameters in order to clarify the role of NO in cardiac contractility and MVO2.

    MATERIALS AND METHODS
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

Surgical Preparation

Experiments were carried out in 31 mongrel dogs with a mean body weight of 23.0 ± 0.9 (mean ± SE) kg. All of the experiments were performed in accordance with the guidelines established for animal experiments in Hirosaki University. The dogs were premedicated with a subcutaneous injection of xylazine (4 mg/kg) and atropine sulfate (0.05 mg/kg) and were anesthetized with alpha -chloralose (100 mg/kg) and pancuronium bromide (0.7 mg/kg). A small supplemental dose of alpha -chloralose (20 mg · kg-1 · h-1) was continuously infused throughout the experiment. Under the anesthesia, dogs were intubated and ventilated by a fixed-volume positive-pressure respirator (model 613, Harvard Apparatus, South Natick, MA) with room air supplemented by oxygen. Arterial blood gases were measured every hour, and arterial PO2 (PaO2) and pH were corrected when necessary by adjustments of tidal volume, oxygen concentration, and administration of sodium bicarbonate. The arterial blood oxygen saturation especially was kept to be constant at a level >= 98% throughout the experiment. Blood volume loss resulting from the surgery was supplemented with 6% dextran and/or normal saline before the experimental protocol was initiated.

With the animal in a supine position, the right femoral artery was isolated and a 6-Fr pig-tail catheter (Cordis, Miami, FL) was inserted into the descending aorta to measure aortic pressure with a pressure transducer (model TP-400T, Nihon Kohden, Tokyo, Japan). A midline cervical incision was performed to expose the bilateral carotid arteries, and a 7-Fr catheter-tip micromanometer (model PC 780 N, Millar, Houston, TX) was advanced through the left carotid artery into the LV chamber for the measurement of LV pressure. A 6-Fr eight-electrode conductance catheter (model 2-RE-218-B, Leycom, The Netherlands) was also advanced through the right carotid artery into the LV chamber to measure LV volume. These catheters were positioned parallel to the long axis of the LV.

The chest was opened midsternally, and after pericardiectomy the pericardial cradle was created. The proximal portion of the left anterior descending coronary artery (LAD) was dissected free from the surrounding tissue before the first diagonal branch, and an ultrasound transit time flow probe (model 2SB, Transonic Systems, Ithaca, NY) was snugly applied around the artery for the measurement of coronary blood flow (CBF). For the continuous evaluation of rapid changes in coronary venous oxygen saturation (ScvO2 , %), a proximal portion of the coronary vein running along with the LAD was dissected and connected to the left femoral vein with a 7-Fr Teflon tube (Kawasumi, Tokyo, Japan) under the controlled perfusion using a pump (model AP-7000, Atto, Tokyo, Japan), and a 3-Fr fiber-optic catheter (Opticath, Abbott Lab, North Chicago, IL) was inserted into the bypass tube. A snare occluder was attached to the inferior vena cava (IVC) for a brief occlusion to measure Emax. To avoid undesirable reflexes, we isolated each stellate ganglion and ligated it tightly at its junction with the ansa subclavia. Each cervical vagus nerve was also crushed with a ligature.

Measurements

LV volume. The conductance catheter method was used for the measurement of LV volume (1, 2). Briefly, the method is based on the measurement of the time-varying electrical conductance of blood at five segments of the LV, estimating LV volume from the blood conductivity. An alternating current (0.07-mA root mean square at 20 kHz) was applied between the neighboring two electrodes of the conductance catheter. Five time-varying segmental conductances, Gi(t), were measured and converted to total LV volume, V(t), with the use of the formula
V(<IT>t</IT>) = (1/&agr;)(<IT>L</IT><SUP>2</SUP>/&sfgr;)[<IT>G</IT>(<IT>t</IT>) − <IT>G</IT><SUB>p</SUB>]
where sigma  represents blood conductivity, alpha  is the slope constant, L is the electrode distance, G(t) is the sum of the five segmental conductances, and Gp represented the parallel conductance formed by the tissue surrounding the LV cavity (myocardium, right ventricle contents, etc.). Gp was determined in each experiment by the injection of 12 ml of hypertonic saline (6 mol/l) through the pulmonary artery into the LV. Current generation, conductance measurement, and analog computations to obtain the desired volume variable G(t) were performed with the use of a model Sigma 5 signal-conditioner processor (Cardio-Dynamics, Rijnsburg, The Netherlands).

CBF. The LAD blood flow was continuously measured with an ultrasound transit time flowmeter (model T206, Transonic Systems) and was corrected with the weight of the LAD perfusion territory.

Perfusion territory. The perfusion territory of the LAD was defined at the conclusion of the experiment. A 7-Fr Teflon tube (Kawasumi, Tokyo, Japan) was inserted into the proximal segment of the LAD, and indocyanine green solution was injected through the tube keeping a similar perfusion pressure to that during the experiment with the use of a tonometer (Nihon Kohden). The weight of the area stained with indocyanine green was measured.

ScvO2. Using a fiber-optic catheter indwelled into the bypass tube between the coronary vein and femoral vein, we continuously measured ScvO2 with the oximeter (Oximetrix 3, Abbott Labs). This oximeter system displays successively an average of oxygen saturation measured during the latest 5-s period. Furthermore, ScvO2 was measured in the bypass tube and not in the intraluminal or pericardial coronary vein, so that there seemed to be a time lag between the changes in the coronary venous oxygen saturation and ScvO2 measured in this study. Therefore, in the estimation of ScvO2, we corrected its time course so as to make the nadir of ScvO2 during IVC occlusion coincide with that of the LV systolic pressure.

Plasma norepinephrine concentration. Coronary venous blood samples (5 ml) were collected in prechilled tubes containing 5 mg of Na2EDTA. The plasma was separated by centrifugation (4°C, 3,000 rpm, 15 min) and stored at -20°C until the assay. The plasma norepinephrine concentration was determined with an automatic fluorescence analyzer (model HLC-725CA, Toso, Tokyo, Japan).

Throughout the experiment, electrocardiographic (ECG) lead II, LV volume, aortic pressure, LV pressure, LAD flow, ScvO2, and LV pressure-volume loops were displayed on-line using a storage oscilloscope. Data were displayed on an eight-channel heat-stylus recorder (model WS-681G, Nihon Kohden) and also stored on a magnetic tape with the use of an analog tape recorder (model A614, Sony, Tokyo, Japan) for subsequent analysis.

Experimental Protocol

The following experiments were performed in a condition with stabilized hemodynamics lasting for >30 min.

Validation of IVC-occlusion method in estimating the MVO2-PVA relationship (n = 3). In the original reports (16, 35), MVO2-PVA relationship was analyzed in a steady-state loading condition using an isolated, cross-circulated canine heart. Thus both MVO2 and PVA were measured in several conditions consisting of different end-diastolic volumes and different systolic pressures. For the analysis of the effect of each drug on the MVO2-PVA relationship in an in vivo model with intact circulation, we measured beat-to-beat changes in MVO2 and PVA during IVC occlusion before and after the drug. To validate this IVC-occlusion method in estimating the MVO2-PVA relationship, we performed transient IVC occlusion and subsequent volume loading, and the ESPVR and MVO2-PVA relationship obtained from these two methods were compared. After Emax and the MVO2-PVA relationship were measured by transient IVC occlusion, the blood in the left atrium was removed to the reservoir through a catheter inserted to the left atrial appendage so as to obtain systolic pressures almost equal to that at the end of IVC occlusion. The removed blood supplemented with normal saline (500 ml) was then injected into the left atrium in a stepwise fashion to obtain systolic pressures of ~90, 110, and 130 mmHg. At each volume-loading state, at least 1 min was allowed for stabilization, and Emax and MVO2 were measured while both end-diastolic volume and systolic pressure were in steady states.

Effect of L-NMMA (n = 8). After the measurements of ECG, LV volume, aortic pressure, LV pressure, LAD coronary blood flow, and ScvO2, IVC was occluded transiently for 10 s to determine the ESPVR (baseline 1). After the hemodynamic parameters recovered to the baseline values, IVC was occluded in the same way to observe the reproducibility of the ESPVR (baseline 2). L-NMMA (5 mg/kg; Wako, Osaka, Japan) diluted with 10 ml of normal saline was then administered intravenously. Fifteen minutes after injection of L-NMMA at which the hemodynamic parameters were stabilized, a transient IVC occlusion with the measurements of hemodynamics and oxygen saturation was repeated. Blood samples for the measurement of the plasma norepinephrine concentration were collected from the coronary vein before and after administration of L-NMMA. At the conclusion of the experiment, the dogs were euthanized with injection of potassium chloride (0.04 M) and the basal oxygen consumption was measured immediately to examine the effect of L-NMMA on it.

Effect of L-arginine (n = 7). As in the study of the effect of L-NMMA, the baseline measurements of hemodynamics, oxygen saturation, and ESPVR were carried out twice. L-Arginine (600 mg/kg; Sigma, St. Louis, MO) diluted with 10 ml of normal saline was then administered intravenously, and the same measurements were repeated. Blood samples for the measurement of plasma norepinephrine concentration were collected from the coronary vein before and after administration of L-arginine. At the conclusion of the experiment, the basal oxygen consumption was measured to examine the effect of L-arginine on it.

Effects of sequential administration of L-NMMA and L-arginine (n = 5). To examine whether the effect of L-NMMA, especially on cardiac contractility, was reversed by the administration of L-arginine, we performed the measurements of hemodynamics and ESPVR at baseline, after intravenous L-NMMA (5 mg/kg), and after intravenous L-arginine (600 mg/kg). The time interval between L-NMMA and L-arginine administration was 20 min. Venous blood was taken before and after L-NMMA and L-arginine, and the plasma NOx (nitrate and nitrite) level was examined with the use of a Griess method (Cayman Chemical, Ann Arbor, MI). In this protocol of experiments, to observe the influence of frequency of contraction, the measurements were done during sinus rhythm and atrial pacing at rates of 130 and 150 beats/min at each of the conditions at baseline, after L-NMMA, and after L-arginine.

Basal oxygen consumption in control dogs. In eight dogs with the experimental instruments attached in the same way as in the other experiments and without treatment of any L-NMMA and L-arginine, the basal oxygen consumption was measured immediately after euthanasia with potassium chloride. It was then compared with that obtained from dogs treated with L-NMMA and L-arginine.

Data Analysis

All data stored on a magnetic tape were digitized. The sample frequency for analog-to-digital conversion was 200 Hz at 12-bit accuracy. Data of systemic hemodynamics were analyzed by the acquisition-archive system (Po-Ne-Mah, Storrs, CT) and of LV pressure-volume loops by the pressure-volume analysis program (Cardio-Dynamics).

ESPVR and PVA. ESPVR was determined by linear regression of the individual end-systolic points in the combined LV pressure-volume loops. The slope of the ESPVR (Emax) during a transient IVC occlusion was then calculated. PVA was obtained as the specific area in the pressure-volume diagram circumscribed by the ESPVR line, the end-diastolic pressure volume curve, and the systolic segment of the pressure-volume trajectory.

MVO2-PVA relationships. With values of hemoglobin (Hb; g/dl), coronary arterial oxygen saturation (ScaO2, %) and ScvO2, LAD flow (ml · min-1 · 100 g-1), and heart rate (HR; beats/min), we calculated coronary arteriovenous oxygen content difference (a-vO2; ml O2/100 ml) and MVO2 per beat (ml O2 · beat-1 · 100 g-1) with the following formulas, respectively
a-vO<SUB>2</SUB> = (Sca<SUB>O<SUB>2</SUB></SUB> − Scv<SUB>O<SUB>2</SUB></SUB>) × 10<SUP>−2</SUP> × 1.34 × Hb(g/dl)
M<A><AC>V</AC><AC>˙</AC></A><SC>o</SC><SUB>2</SUB> per beat = (a-vO<SUB>2</SUB>) × 10<SUP>−2</SUP> × (LAD flow)/HR
The MVO2-PVA relationship was constructed by MVO2 per beat against PVA.

Contractile efficiency. It is known that 1 mmHg · ml of PVA is equal to 1.33 × 10-4 J on a physical basis and that 1 ml of oxygen consumed by myocardium is approximately equivalent to 20 J under normal aerobic conditions. Accordingly, MVO2 (in ml O2 · beat-1 · 100 g-1) and PVA (in mmHg · ml · beat-1 · 100 g-1) can each be converted to the same unit of energy (J · beat-1 · 100 g-1). We obtained contractile efficiency (%) from the ratio of PVA to MVO2 (with both in J · beat-1 · 100 g-1).

Statistics

All data are shown as means ± SE. The hemodynamic parameters before and after each of the drugs (L-NMMA and L-arginine) were compared by paired t-test. ESPVR, MVO2-PVA relationships, and plasma norepinephrine concentrations at baseline 1, at baseline 2, and after L-NMMA or L-arginine, and the plasma NOx levels before and after L-NMMA and L-arginine, were compared with a repeated-measures ANOVA. The effects of L-NMMA and L-arginine sequentially administered were also analyzed with ANOVA for repeated measures. The basal oxygen consumption in dogs with treatment of L-NMMA and L-arginine and without any treatment was compared by one-way ANOVA. The level of significance was P < 0.05.

    RESULTS
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

Comparison of Emax and MVO2-PVA Relationships Obtained by Transient IVC-Occlusion and Volume-Loading Methods

Representative analog recordings of ECG, LV volume, aortic pressure, LAD flow, LV pressure, and ScvO2 during transient IVC occlusion and volume loading in one dog are shown in Fig. 1, left. Figure 1, middle, shows the pressure-volume loops obtained from these two methods, and Fig. 1, right, shows the MVO2-PVA relationships. It is noted that Emax values obtained from the two methods are almost equal and that MVO2 is linearly correlated with PVA in both methods. A similar observation was made in the other two dogs, and thus the MVO2 change during IVC occlusion was linearly related to the change in PVA.


View larger version (51K):
[in this window]
[in a new window]
  		Fig. 1.   Myocardial oxygen consumption (MVO2)-pressure-volume area (PVA) relationships measured by transient inferior vena caval (IVC)-occlusion method (A) and by steady-state, volume-loading method (B). A and B show representative analog recordings of electrocardiogram (ECG), left ventricular (LV) volume (LVV), aortic pressure (AoP), coronary blood flow (CBF), LV pressure (LVP), and coronary venous oxygen saturation (ScvO2) (left); end-systolic pressure-volume relationship (ESPVR) (middle); and MVO2-PVA relationship (right). See text for discussion.

Changes in Systemic and Coronary Hemodynamics After L-NMMA and L-Arginine

Table 1 summarizes changes in systemic and coronary hemodynamics after L-NMMA and L-arginine. There were no significant changes in HR, LV end-diastolic pressure (LVEDP), LV end-diastolic volume (LVEDV), and LV end-systolic volume (LVESV) after L-NMMA. However, L-NMMA increased LV end-systolic pressure (LVESP) (P < 0.05) and decreased LAD flow (P < 0.05). There were no significant changes in HR, LVEDP, and LVESV after L-arginine. There were trends toward a decrease in LVESP (P = 0.08) and increases in LVEDV (P = 0.08) and LAD flow (P = 0.14) after L-arginine.

                              
View this table:
[in this window]
[in a new window]
  		Table 1.   Systemic and coronary hemodynamics before and after L-NMMA and L-arginine

Effects of L-NMMA on Cardiac Contractility and MVO2

As shown in Table 2 and Fig. 2, neither ESPVR (Emax) nor the MVO2-PVA relationship differs between the measurements at baseline 1 and baseline 2, indicating the reproducibility of the measurements with the IVC-occlusion method. After L-NMMA administration, Emax was significantly increased (P < 0.05), and the y-axis intercept of the MVO2-PVA relationship (unloaded MVO2) was also significantly increased (P < 0.05). The slope of the MVO2-PVA relationship (the inverse of the contractile efficiency) remained unchanged after L-NMMA. Contractile efficiency (%), calculated by the ratio of PVA to MVO2, was 48.4 ± 1.5% at baseline and 47.3 ± 1.7% after L-NMMA [P = not significant (NS)]. An example of the change in MVO2-PVA relationship after L-NMMA is shown in Fig. 3A. L-NMMA shifts the line upward, with an increase of the y-axis intercept and without changing the slope, indicating that L-NMMA increased unloaded MVO2 without changing contractile efficiency.

                              
View this table:
[in this window]
[in a new window]
  		Table 2.   ESPVR, MVO2-PVA relationship and plasma norepinephrine concentration before and after L-NMMA and L-arginine


View larger version (13K):
[in this window]
[in a new window]
  		Fig. 2.   Slope (Emax) of ESPVR at baseline 1, at baseline 2, and after NG-monomethyl-L-arginine acetate (L-NMMA) (A; n = 8) or L-arginine (B; n = 7).


View larger version (13K):
[in this window]
[in a new window]
  		Fig. 3.   Example of effects of L-NMMA (A) and L-arginine (B) on MVO2-PVA relationship. Each baseline indicated before an administration of drug. A: y = 1.38 × 10-5x + 2.43 × 10-2 at baseline, and y = 1.42 × 10-5x + 3.09 × 10-2 after L-NMMA. B: y = 1.49 × 10-5x + 2.44 × 10-2 at baseline, and y = 1.47 × 10-5x + 1.47 × 10-2 after L-arginine.

Effects of L-Arginine on Cardiac Contractility and MVO2

As shown in Table 2 and Fig. 2, Emax was significantly decreased after L-arginine (P < 0.05). The y-axis intercept of the MVO2-PVA relationship was significantly decreased after L-arginine (P < 0.05). The slope of the MVO2-PVA relationship remained unchanged after L-arginine. Contractile efficiency was 44.8 ± 1.1% at baseline and 45.5 ± 0.9% after L-arginine (P = NS). As shown in Fig. 3B, L-arginine shifts the line downward, with a decrease of the y-axis intercept and without changing the slope, indicating that L-arginine decreased unloaded MVO2 without changing contractile efficiency.

Effects of Sequential Administration of L-NMMA and L-Arginine on Emax and Plasma NOx Level

Figure 4 shows Emax measured at baseline, after L-NMMA, and after L-arginine that was administered following L-NMMA. The measurement was done during sinus rhythm (110 ± 5 beats/min at baseline, 106 ± 4 beats/min after L-NMMA, and 110 ± 5 beats/min after L-arginine; P = NS among the 3 conditions) and during atrial pacing at rates of 130 and 150 beats/min. At all HRs, Emax was significantly increased after L-NMMA (all P < 0.05) and then decreased after L-arginine (all P < 0.05). When the changes in Emax after L-NMMA and L-arginine from baseline were compared among experiments during sinus rhythm and atrial pacing, no significant difference was noted.


View larger version (33K):
[in this window]
[in a new window]
  		Fig. 4.   Emax measured at baseline, after L-NMMA, and after L-arginine that was administered following L-NMMA. Measurements were done during sinus rhythm and atrial pacing at rates of 130 and 150 beats/min. See text for discussion.

The plasma NOx levels at baseline and after L-NMMA and after L-arginine were 2.3 ± 0.7, 2.8 ± 0.6, and 2.7 ± 0.6 µM, respectively. There was no statistical difference among them.

Basal Oxygen Metabolism

Immediately after the animal was euthanized, the basal oxygen consumption was measured. It was 1.1 ± 0.1 ml O2 · min-1 · 100 g-1 in dogs treated with L-NMMA, 1.2 ± 0.1 ml O2 · min-1 · 100 g-1 in dogs treated with L-arginine, and 1.2 ± 0.1 ml O2 · min-1 · 100 g-1 in dogs without L-NMMA or L-arginine treatment (P = NS among three groups).

Norepinephrine Concentrations Before and After L-NMMA and L-Arginine

As shown in Table 2, the plasma norepinephrine concentration in the coronary vein did not change after L-NMMA. Also, it did not change after L-arginine.

    DISCUSSION
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

The present study showed that blockade of NOS with L-NMMA resulted in the increases in Emax (the slope of ESPVR), MVO2, and unloaded MVO2 (the y-axis intercept of the MVO2-PVA line) without changing contractile efficiency (the inverse of the slope of the MVO2-PVA line). In contrast, L-arginine, a substrate of NO, decreased Emax and unloaded MVO2 without changing contractile efficiency. Furthermore, the increasing effect of L-NMMA on Emax was reversed by the additional administration of L-arginine. These indicate that endogenous NO reduces cardiac contractility and MVO2 without affecting contractile efficiency.

Previous studies in isolated myocytes have shown that NO reduces cardiac contractility and MVO2 (3, 4, 9, 45). Most of the experiments were done in the settings of endotoxic shock, heart failure, and ischemia (7, 9, 19, 21, 24, 30, 41). In these pathological conditions, NO was most likely to be produced via an iNOS pathway and not a cNOS one. On the other hand, a positive inotropic effect (18, 22) or no detectable effect of NO (44) was also reported. Only a few studies have examined the role of NO in cardiac contractility and MVO2 in vivo (12). Thus it remains to be elucidated whether endogenous NO is involved in the regulation of cardiac contractility and MVO2

In the present study, we used in vivo canine hearts with intact circulation and evaluated Emax as an index of cardiac contractility and the MVO2-PVA relationship from the point of view of cardiac energetics. Suga et al. (36) reported that PVA, an expression of the total mechanical energy output of ventricular contraction on the basis of the time-varying elastance model (35), is linearly related to MVO2 per beat (37). In their original reports, the PVA and MVO2 relationship was determined in a steady-state, isovolumic contraction model. Thus the relationship was determined in several states of ventricular contraction from different end-diastolic volumes and against different systolic pressures using excised, cross-circulated canine hearts. In order to examine the effects of NOS inhibition and NO substrate supplement in vivo, we evaluated Emax and the MVO2-PVA relationship with a transient IVC-occlusion method, and not with a steady-state, volume-loading method, because the latter method was considered not to be appropriate for the evaluation of the effects of drugs in an in vivo model. Previous studies with in vivo experiments (20, 25, 28) showed that MVO2 and PVA were linearly related during steady-state isovolumic contraction with alternation in loading conditions, whereas they were not during a beat-to-beat alternating condition such as that during transient IVC occlusion. In these previous studies, however, a-vO2 was not measured on a beat-by-beat basis. We measured the oxygen saturation in the coronary sinus continuously using a fiber-optic catheter, keeping the oxygen saturation in the arterial blood constant at a level >= 98%, and analyzed the MVO2-PVA relationship on a beat-to-beat basis. The present IVC-occlusion method in estimating the MVO2-PVA relationship was validated because Emax obtained from the IVC-occlusion and steady-state volume-loading methods are almost equal and, furthermore, MVO2 was linearly correlated with PVA in both methods.

L-NMMA and L-arginine were administered intravenously in this study. L-NMMA significantly increased systolic blood pressure and decreased CBF, which was consistent with previous observations (6, 14). L-Arginine showed a tendency to decrease systolic blood pressure. The use of Emax and the MVO2-PVA relationship for the estimation of the effects of L-NMMA and L-arginine on cardiac contractility and MVO2 has a distinct advantage because these parameters are not affected by the hemodynamic changes induced by the drugs. L-NMMA increased Emax and unloaded MVO2, whereas L-arginine decreased Emax and unloaded MVO2.

The effect of L-arginine on MVO2 shown was greater than that reported. Previous studies showed that L-arginine at doses up to 10 mM had no effect on rat papillary muscle contractility and a very high concentration of L-arginine at 50 mM had a significant negative inotropic effect unrelated to NOS (44). A previous in vitro study reported that the intracellular concentration of L-arginine was estimated to be ~100 µM (11), so that NOS might be expected to be saturated by endogenous L-arginine. However, other experiments have shown that the endothelial production of nitrite, an indicator of NO formation, is not saturated at 2.5 mM of L-arginine in vitro (17) and that L-arginine infusion significantly decreased blood pressure with increments of L-citrulline, the by-product of NOS from L-arginine, and cGMP, the second messenger for NO in human models (15). These indicate that NOS may not be saturated by endogenous L-arginine, and thus an increased amount of NO production via the supplement of a large dose of L-arginine in the present study (~45 mM) may explain the present effects of L-arginine at least in part. Further studies on the mechanism for the effects of L-arginine are required.

The effect of L-NMMA on Emax was reversed by L-arginine administered after L-NMMA. L-NMMA shifted the MVO2-PVA relationship upward, whereas L-arginine shifted it downward. Unloaded MVO2 is considered to represent the MVO2 for nonmechanical energy utilization, consisting of the oxygen consumption for excitation-contraction coupling and basal metabolism. Neither L-NMMA nor L-arginine affected myocardial basal metabolism. Thus the changes in unloaded MVO2 induced by L-NMMA and L-arginine were due to the changes in the oxygen consumption for excitation-contraction coupling and not to those in the basal metabolism.

Contractile efficiency is indicated by the inverse of the slope of the MVO2-PVA relationship. The results of the present study showed that L-NMMA and L-arginine did not affect the contractile efficiency. These data imply that endogenous NO plays an important role in the regulation of cardiac performance by attenuating cardiac contractility and sparing MVO2 without affecting contractile efficiency. A previous in vitro study showed that the inotropic effect of NOS inhibition was dependent on the frequency of contraction (9). We examined the influence of the increase in HR on the effects of L-NMMA and L-arginine on cardiac contractility and found that the changes in Emax after L-NMMA and after L-arginine were similar among the experiments during sinus rhythm and atrial pacing. Although the changes in the rate induced in this study were small, endogenous NO was indicated to attenuate cardiac contractility at least in the physiological range of HR.

The autonomic nervous system is a major determinant for cardiac contractility and MVO2. Norepinephrine augments cardiac contractility and increases MVO2. A previous study showed that an NOS inhibitor was reported to enhance the evoked norepinephrine release in isolated rat hearts (31), whereas others showed that norepinephrine release was unaffected in several preparations (5, 34, 40, 42). Thus the relationship between endogenous NO and norepinephrine release remains unclear. We measured plasma norepinephrine concentration before and after L-NMMA and L-arginine and failed to demonstrate any changes after the drugs. Thus the effects of L-NMMA and L-arginine shown in this study were independent of norepinephrine release, and NO per se modulates cardiac performance.

Study Limitations

For the analysis of LV MVO2, the measurement of total left coronary artery blood flow may be required. In this study, we only measured the LAD flow and not the circumflex artery flow. Because both L-NMMA and L-arginine were administered intravenously, the changes in circumflex artery flow after these drugs were likely to be similar to those in LAD flow. Thus the coronary flow change per unit mass was likely to be uniform in the LV, and the total left coronary artery blood flow was possibly calculated from the LAD flow.

We measured the plasma NOx level but could not find any significant change after administration of L-NMMA and L-arginine. The present study, however, clearly showed that L-NMMA increased cardiac contractility and MVO2, which were associated with the increase in systolic blood pressure and the decrease in LAD flow. L-Arginine showed effects opposite to those of L-NMMA. Therefore, the changes demonstrated after L-NMMA and L-arginine were most likely to be caused by the inhibition and augmentation of endogenous NO, respectively. The plasma NOx level seems not to be affected by the acute administration of L-NMMA and L-arginine (43).

    FOOTNOTES

Address for reprint requests: K. Okumura, The Second Dept. of Internal Medicine, Hirosaki Univ. School of Medicine, Zaifu-cho 5, Hirosaki 036, Japan.

Received 22 July 1997; accepted in final form 11 March 1998.

    REFERENCES
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

1.   Baan, J., T. T. Jong, P. L. Kerkhof, R. J. Moene, A. D. van Dijk, E. T. van der Velde, and J. Koops. Continuous stroke volume and cardiac output from intra-ventricular dimensions obtained with impedance catheter. Cardiovasc. Res. 15: 328-334, 1981[Medline].

2.   Baan, J., E. T. van-der-Velde, H. G. de-Bruin, G. J. Smeenk, J. Koops, A. D. van-Dijk, D. Temmerman, J. Senden, and B. Buis. Continuous measurement of left ventricular volume in animals and humans by conductance catheter. Circulation 70: 812-823, 1984[Abstract/Free Full Text].

3.   Balligand, J. L., R. A. Kelly, P. A. Marsden, T. W. Smith, and T. Michel. Control of cardiac muscle cell function by an endogenous nitric oxide signaling system. Proc. Natl. Acad. Sci. USA 90: 347-351, 1993[Abstract/Free Full Text].

4.   Brady, A. J., J. B. Warren, P. A. Poole-Wilson, T. J. Williams, and S. E. Harding. Nitric oxide attenuates cardiac myocyte contraction. Am. J. Physiol. 265 (Heart Circ. Physiol. 34): H176-H182, 1993[Abstract/Free Full Text].

5.   Bucher, B., S. Ouedraogo, M. Tschopl, D. Paya, and J. C. Stoclet. Role of the L-arginine-NO pathway and of cyclic GMP in electrical field-induced noradrenaline release and vasoconstriction in the rat tail artery. Br. J. Pharmacol. 107: 976-982, 1992[Medline].

6.   Chu, A., D. E. Chambers, C. C. Lin, W. D. Kuehl, R. M. Palmer, S. Moncada, and F. R. Cobb. Effects of inhibition of nitric oxide formation on basal vasomotion and endothelium-dependent responses of the coronary arteries in awake dogs. J. Clin. Invest. 87: 1964-1968, 1991.

7.   De-Belder, A. J., M. W. Radomski, H. J. Why, P. J. Richardson, C. A. Bucknall, E. Salas, J. F. Martin, and S. Moncada. Nitric oxide synthase activities in human myocardium. Lancet 341: 84-85, 1993[Medline].

8.   Finkel, M. S., C. V. Oddis, T. D. Jacob, S. C. Watkins, B. G. Hattler, and R. L. Simmons. Negative inotropic effects of cytokines on the heart mediated by nitric oxide. Science 257: 387-389, 1992[Abstract/Free Full Text].

9.   Finkel, M. S., C. V. Oddis, O. H. Mayer, B. G. Hattler, and R. L. Simmons. Nitric oxide synthase inhibitor alters papillary muscle force-frequency relationship. J. Pharmacol. Exp. Ther. 272: 945-952, 1995[Abstract/Free Full Text].

10.   Geng, Y., G. K. Hansson, and E. Holme. Interferon-gamma and tumor necrosis factor synergize to induce nitric oxide production and inhibit mitochondrial respiration in vascular smooth muscle cells. Circ. Res. 71: 1268-1276, 1992[Abstract/Free Full Text].

11.   Gold, M. E., P. A. Bush, and L. J. Ignarro. Depletion of arterial L-arginine causes reversible tolerance to endothelium-dependent relaxation. Biochem. Biophys. Res. Commun. 164: 714-721, 1989[Medline].

12.   Hare, J. M., J. F. Keaney, Jr., J. L. Balligand, J. Loscalzo, T. W. Smith, and W. S. Colucci. Role of nitric oxide in parasympathetic modulation of beta -adrenergic myocardial contractility in normal dogs. J. Clin. Invest. 95: 360-366, 1995.

13.   Hare, J. M., E. Loh, M. A. Creager, and W. S. Colucci. Nitric oxide inhibits the positive inotropic response to beta -adrenergic stimulation in humans with left ventricular dysfunction. Circulation 92: 2198-2203, 1995[Abstract/Free Full Text].

14.   Haynes, W. G., J. P. Noon, B. R. Walker, and D. J. Webb. L- NMMA increases blood pressure in man. Lancet 342: 931-932, 1993[Medline].

15.   Hishikawa, K., T. Nakaki, H. Suzuki, T. Saruta, and R. Kato. L-Arginine-induced hypotension. Lancet 337: 683-684, 1991[Medline].

16.   Khalafbeigui, F., H. Suga, and K. Sagawa. Left ventricular systolic pressure-volume area correlates with oxygen consumption. Am. J. Physiol. 237 (Heart Circ. Physiol. 6): H566-H569, 1979.

17.   Kilbourn, R. G., and P. Belloni. Endothelial cell production of nitrogen oxides in response to interferon gamma in combination with tumor necrosis factor, interleukin-1, or endotoxin. J. Natl. Cancer Inst. 82: 772-776, 1990[Abstract/Free Full Text].

18.   Kojda, G., K. Kottenberg, P. Nix, K. D. Schluter, H. M. Piper, and E. Noack. Low increase in cGMP induced by organic nitrates and nitrovasodilators improves contractile response of rat ventricular myocytes. Circ. Res. 78: 91-101, 1996[Abstract/Free Full Text].

19.   Levine, B., J. Kalman, L. Mayer, H. M. Fillit, and M. Packer. Elevated circulating levels of tumor necrosis factor in severe chronic heart failure. N. Engl. J. Med. 323: 236-241, 1990[Abstract].

20.  Lilly, R., S. Gall, J. Davis, V. Keller, J. Rankin, and D. Glower. Evaluation of contractile energetics models using indirect calorimetry in the consious dogs (Abstract). Circulation 84, Suppl. II: II-95, 1991.

21.   Matsumori, A., T. Yamada, H. Suzuki, Y. Matoba, and S. Sasayama. Increased circulating cytokines in patients with myocarditis and cardiomyopathy. Br. Heart J. 72: 561-566, 1994[Abstract/Free Full Text].

22.   Mohan, P., D. L. Brutsaert, W. J. Paulus, and S. U. Sys. Myocardial contractile response to nitric oxide and cGMP. Circulation 93: 1223-1229, 1996[Abstract/Free Full Text].

23.   Moncada, S., R. M. Palmer, and E. A. Higgs. Nitric oxide: physiology, pathophysiology, and pharmacology. Pharmacol. Rev. 43: 109-142, 1991[Medline].

24.   Node, K., M. Kitakaze, H. Kosaka, K. Komamura, T. Minamino, M. Inoue, M. Tada, M. Hori, and T. Kamada. Increased release of NO during ischemia reduces myocardial contractility and improves metabolic dysfunction. Circulation 93: 356-364, 1996[Abstract/Free Full Text].

25.   Nozawa, T., C. P. Cheng, T. Noda, and W. C. Little. Relation between left ventricular oxygen consumption and pressure-volume area in conscious dogs. Circulation 89: 810-817, 1994[Abstract/Free Full Text].

26.   Palmer, R. M., A. G. Ferrige, and S. Moncada. Nitric oxide release accounts for the biological activity of endothelium-derived relaxing factor. Nature 327: 524-526, 1987[Medline].

27.   Palmer, R. M., D. D. Rees, D. S. Ashton, and S. Moncada. L-Arginine is the physiological precursor for the formation of nitric oxide in endothelium-dependent relaxation. Biochem. Biophys. Res. Commun. 153: 1251-1256, 1988[Medline].

28.  Prabhu, S., L. Mulligan, R. O'Rourke, and G. Freeman. Effect of dobutamine on ventricular energetics in closed-chest dogs (Abstract). Circulation 86, Suppl. I: I-231, 1992.

29.   Schmidt, H. H., H. Nau, W. Wittfoht, J. Gerlach, K. E. Prescher, M. M. Klein, F. Niroomand, and E. Bohme. Arginine is a physiological precursor of endothelium-derived nitric oxide. Eur. J. Pharmacol. 154: 213-216, 1988[Medline].

30.   Schulz, R., E. Nava, and S. Moncada. Induction and potential biological relevance of a Ca2+-independent nitric oxide synthase in the myocardium. Br. J. Pharmacol. 105: 575-580, 1992[Medline].

31.   Schwarz, P., R. Diem, N. J. Dun, and U. Forstermann. Endogenous and exogenous nitric oxide inhibits norepinephrine release from rat heart sympathetic nerves. Circ. Res. 77: 841-848, 1995[Abstract/Free Full Text].

32.   Shen, W., T. H. Hintze, and M. S. Wolin. Nitric oxide. An important signaling mechanism between vascular endothelium and parenchymal cells in the regulation of oxygen consumption. Circulation 92: 3505-3512, 1995[Abstract/Free Full Text].

33.   Shen, W., X. Xu, M. Ochoa, G. Zhao, M. S. Wolin, and T. H. Hintze. Role of nitric oxide in the regulation of oxygen consumption in conscious dogs. Circ. Res. 75: 1086-1095, 1994[Abstract/Free Full Text].

34.   Shinozuka, K., Y. Kobayashi, K. Shimoura, and K. Hattori. Role of nitric oxide from the endothelium on the neurogenic contractile responses of rabbit pulmonary artery. Eur. J. Pharmacol. 222: 113-120, 1992[Medline].

35.   Suga, H. Total mechanical energy of a ventricle model and cardiac oxygen consumption. Am. J. Physiol. 236 (Heart Circ. Physiol. 5): H498-H505, 1979[Abstract/Free Full Text].

36.   Suga, H., T. Hayashi, and M. Shirahata. Ventricular systolic pressure-volume area as predictor of cardiac oxygen consumption. Am. J. Physiol. 240 (Heart Circ. Physiol. 9): H39-H44, 1981[Abstract/Free Full Text].

37.   Suga, H., T. Hayashi, S. Suehiro, R. Hisano, M. Shirahata, and I. Ninomiya. Equal oxygen consumption rates of isovolumic and ejecting contractions with equal systolic pressure-volume areas in canine left ventricle. Circ. Res. 49: 1082-1091, 1981[Abstract/Free Full Text].

38.   Suga, H., and K. Sagawa. Instantaneous pressure-volume relationships and their ratio in the excised, supported canine left ventricle. Circ. Res. 35: 117-126, 1974[Abstract/Free Full Text].

39.   Suga, H., K. Sagawa, and A. A. Shoukas. Load independence of the instantaneous pressure-volume ratio of the canine left ventricle and effects of epinephrine and heart rate on the ratio. Circ. Res. 32: 314-322, 1973[Abstract/Free Full Text].

40.   Toda, N., K. Yoshida, and T. Okamura. Analysis of the potentiating action of NG-nitro-L-arginine on the contraction of the dog temporal artery elicited by transmural stimulation of noradrenergic nerves. Naunyn Schmiedebergs Arch. Pharmacol. 343: 221-224, 1991[Medline].

41.   Tsujino, M., Y. Hirata, T. Imai, K. Kanno, S. Eguchi, H. Ito, and F. Marumo. Induction of nitric oxide synthase gene by interleukin-1beta in cultured rat cardiocytes. Circulation 90: 375-383, 1994[Abstract/Free Full Text].

42.   Vo, P. A., J. J. Reid, and M. J. Rand. Attenuation of vasoconstriction by endogenous nitric oxide in rat caudal artery. Br. J. Pharmacol. 107: 1121-1128, 1992[Medline].

43.   Wennmalm, A., A. Edlund, E. F. Granstrom, and O. Wiklund. Acute supplementation with the nitric oxide precursor L-arginine does not improve cardiovascular performance in patients with hypercholesterolemia. Atherosclerosis 118: 223-231, 1995[Medline].

44.   Weyrich, A. S., X. L. Ma, M. Buerke, T. Murohara, V. E. Armstead, A. M. Lefer, J. M. Nicolas, A. P. Thomas, D. J. Lefer, and J. Vinten-Johansen. Physiological concentrations of nitric oxide do not elicit an acute negative inotropic effect in unstimulated cardiac muscle. Circ. Res. 75: 692-700, 1994[Abstract/Free Full Text].

45.   Xie, Y. W., W. Shen, G. Zhao, X. Xu, M. S. Wolin, and T. H. Hintze. Role of endothelium-derived nitric oxide in the modulation of canine myocardial mitochondrial respiration in vitro. Implications for the development of heart failure. Circ. Res. 79: 381-387, 1996[Abstract/Free Full Text].

Am J Physiol Heart Circ Physiol 275(1):H41-H49
0002-9513/98 $5.00 Copyright © 1998 the American Physiological Society


This article has been cited by other articles:


Home page
 Am. J. Physiol. Heart Circ. Physiol.Home page
U. Hofmann, E. Domeier, S. Frantz, M. Laser, B. Weckler, P. Kuhlencordt, S. Heuer, B. Keweloh, G. Ertl, and A. W. Bonz
Increased myocardial oxygen consumption by TNF-alpha is mediated by a sphingosine signaling pathway
Am J Physiol Heart Circ Physiol, June 1, 2003; 284(6): H2100 - H2105.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Physiol. Endocrinol. Metab.Home page
F. A. Recchia, J. C. Osorio, M. P. Chandler, X. Xu, A. R. Panchal, G. D. Lopaschuk, T. H. Hintze, and W. C. Stanley
Reduced synthesis of NO causes marked alterations in myocardial substrate metabolism in conscious dogs
Am J Physiol Endocrinol Metab, January 1, 2002; 282(1): E197 - E206.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Physiol. Heart Circ. Physiol.Home page
S. Setty, X. Bian, J. D. Tune, and H. F. Downey
Endogenous nitric oxide modulates myocardial oxygen consumption in canine right ventricle
Am J Physiol Heart Circ Physiol, August 1, 2001; 281(2): H831 - H837.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Physiol. Heart Circ. Physiol.Home page
T. Stumpe, U. K. M. Decking, and J. Schrader
Nitric oxide reduces energy supply by direct action on the respiratory chain in isolated cardiomyocytes
Am J Physiol Heart Circ Physiol, May 1, 2001; 280(5): H2350 - H2356.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Physiol. Regul. Integr. Comp. Physiol.Home page
M. Iemitsu, T. Miyauchi, S. Maeda, K. Yuki, T. Kobayashi, Y. Kumagai, N. Shimojo, I. Yamaguchi, and M. Matsuda
Intense exercise causes decrease in expression of both endothelial NO synthase and tissue NOx level in hearts
Am J Physiol Regulatory Integrative Comp Physiol, September 1, 2000; 279(3): R951 - R959.
[Abstract] [Full Text] [PDF]

Home page
 Circ. Res.Home page
J. M. Canty Jr.
Nitric Oxide and Short-Term Hibernation : Friend or Foe?
Circ. Res., July 21, 2000; 87(2): 85 - 87.
[Full Text] [PDF]

Home page
 Am. J. Physiol. Heart Circ. Physiol.Home page
C. Korvald, O. P. Elvenes, and T. Myrmel
Myocardial substrate metabolism influences left ventricular energetics in vivo
Am J Physiol Heart Circ Physiol, April 1, 2000; 278(4): H1345 - H1351.
[Abstract] [Full Text] [PDF]

Home page
 HypertensionHome page
X.-P. Yang, Y.-H. Liu, E. G. Shesely, M. Bulagannawar, F. Liu, and O. A. Carretero
Endothelial Nitric Oxide Gene Knockout Mice : Cardiac Phenotypes and the Effect of Angiotensin-Converting Enzyme Inhibitor on Myocardial Ischemia/Reperfusion Injury
Hypertension, July 1, 1999; 34(1): 24 - 30.
[Abstract] [Full Text] [PDF]

This Article