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Departments of Medicine, Biochemistry, and Radiology and Center for Magnetic Resonance Research, University of Minnesota, Minneapolis, Minnesota
Submitted 3 June 2004 ; accepted in final form 9 September 2004
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ABSTRACT |
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O2) in open-chest dogs caused a decrease of myocardial PCr/ATP measured with 31P NMR from 2.04 ± 0.09 to 1.85 ± 0.08 (P < 0.05). Inhibition of NO synthesis with N
-nitro-L-arginine (L-NNA), while catecholamine infusion continued, caused PCr/ATP to increase to the control value. In a second group of animals, L-NNA administered before catecholamine stimulation (reverse intervention of the first group) increased PCr/ATP during basal conditions. In these animals L-NNA did not prevent a decrease of PCr/ATP at the high cardiac work state but, relative to MO2, PCr/ATP was significantly higher after L-NNA. In a third group of animals, pharmacological coronary vasodilation with carbochromen was used to prevent changes in coronary flow that might alter endothelial NO production. In these animals L-NNA again restored depressed myocardial PCr/ATP during catecholamine infusion. The finding that inhibition of NO production increased PCr/ATP suggests that during very high work states NO inhibition of mitochondrial respiration requires ADP to increase to drive oxidative phosphorylation.
nuclear magnetic resonance spectroscopy; high-energy phosphate; heart; phosphate; myocardial blood flow
O2, ATP production) with no change in cytosolic free ADP concentration. However, higher workloads produced by catecholamine administration consistently result in increased cytosolic free ADP levels (14, 33). Cytosolic ADP is increased during oxygen insufficiency, but oxygen availability is not limiting even during very high cardiac work states, because we found (4, 34) no evidence of myoglobin desaturation (detected with 1H NMR spectroscopy) during intense catecholamine stimulation. Several investigators have reported that endogenous NO production is increased during increases of cardiac work produced by exercise or catecholamine stimulation (1, 22, 28). In addition to influencing coronary blood flow and therefore oxygen delivery, NO can compete with O2 binding to cytochrome-c oxidase at the terminal complex of the mitochondrial respiratory chain. The functional effect of this interaction is to increase the apparent Km for O2 (2, 3, 31). Because NO competes with O2 at cytochrome oxidase, and because NO production is increased during high work states, it is possible that NO inhibition of respiration would require an increase of cytosolic free ADP (and consequently a decrease of myocardial PCr/ATP) to sustain adequate ATP production.
This study was designed to test the hypothesis that the decreased myocardial PCr-to-ATP ratio during very high cardiac workloads reflects increased NO-mediated inhibition of mitochondrial respiration. If this is the case, then blocking NO production should disinhibit mitochondrial respiration so that the high rate of myocardial ATP production required to support contractile function during catecholamine administration can be maintained at a lower ADP level. Because the increase of MO2 (and therefore coronary blood flow) would result in increased endothelial shear that might influence NO production, in a separate group of animals pharmacological coronary vasodilation was produced with carbochromen to eliminate possible interfering effects of flow.
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METHODS |
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TOPABSTRACTMETHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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Experimental Preparation
Animals were anesthetized with pentobarbital sodium (30 mg/kg iv bolus followed by iv infusion of 4 mg·kg–1·h–1), intubated, and ventilated with a respirator with supplemental oxygen to keep arterial blood gases within the physiological range. A heparin-filled polyvinyl chloride catheter was introduced into the right femoral artery and advanced into the ascending aorta. A left thoracotomy was performed, and a second catheter was introduced into the left ventricle (LV) through the apical dimple. A similar catheter was placed into the left atrium. A final catheter was introduced via the right atrium into the coronary sinus and advanced into the anterior interventricular vein. A double-tuned (31P and 1H) 28-mm-diameter NMR surface coil (4, 34) was sutured onto the anterior LV wall in the distribution of the left anterior descending coronary artery. The animals were then placed in a Lucite cradle and positioned in the magnet.
MR Spectroscopy: General Methods
Measurements were performed in a 40-cm-bore 4.7-T magnet interfaced with a computer console (SISCO, Fremont, CA). The LV pressure signal was used to gate magnetic resonance data acquisition to the cardiac cycle while respiratory gating was achieved by triggering the ventilator to the cardiac cycle between data acquisitions (4, 20). 31P and 1H NMR frequencies were 81 and 200.1 MHz, respectively. During each intervention, after steady-state conditions had been reached, myoglobin [1H magnetic resonance spectroscopy (MRS)] data and then high-energy phosphate (HEP; 31P MRS) data were acquired.
Spatially Localized 31P-NMR Spectroscopic Technique
Radio frequency transmission and signal detection were performed with the surface coil. A capillary containing 15 µl of 3 M phosphonoacetic acid was placed at the coil center to serve as a reference. The proton signal from water detected with the surface coil was used to homogenize the magnetic field and to adjust the position of the animal in the magnet so that the coil was at or near the magnet and gradient isocenters. 31P spectra were recorded in late diastole with a pulse repetition time (TR) of 6–7 s. This TR allowed full relaxation for ATP and Pi resonance and 
90% relaxation for PCr resonance. PCr resonance intensities were corrected for this minor saturation. Chemical shifts were measured relative to PCr, which was assigned a chemical shift of –2.55 ppm relative to 85% phosphoric acid at 0 ppm.
Spatial localization across the LV wall was performed with the rotating-frame experiment using adiabatic plane-rotation pluses for phase modulation-imaging-selected in vivo spectroscopy Fourier series window method (16). Detailed data with regard to voxel profiles and voxel volume and extensive documentation of the accuracy of the spatial localization obtained in phantom studies and in vivo have been published elsewhere (16, 20). Each set of spatially localized transmural spectra consisted of a total of 96 scans accumulated in 10-min blocks. Resonance intensities were quantified with integration routines provided by SISCO software. The numerical values for PCr and ATP in each voxel were expressed as ratios of PCr to ATP. Pi levels were measured as changes from baseline values (
Pi) with integrals obtained in the region covering the Pi resonance and reported as Pi/PCr.
1H NMR Spectroscopic Technique
1H NMR spectroscopy was used for detection of deoxymyoglobin (Mb-
) (4, 34, 35). In brief, radio frequency transmission and signal detection were performed with the dually tuned 28-mm-diameter surface coil. A single-pulse collection sequence with a frequency-selective, 1-ms Gaussian pulse was used to selectively excite the Mb- resonance. A short TR (25 ms) was used because of the short longitudinal relaxation time (T1) of Mb-. Each set of spectra was acquired in 5 min (10,000 free induction decays). Although the short T1 of Mb- and fast acquisition prevented gating to the cardiac cycle, signal loss due to motion was negligible because of the inherently broad line width of Mb- peak. To verify that Mb- could be detected when it was known to be present, a total coronary occlusion was induced at the end of the experiment and the Mb- resonance was observed.
Myocardial Blood Flow and MO2 Measurements
Myocardial blood flow was measured with radioactive microspheres, 15 µm in diameter, labeled with 141Ce, 51Cr, 95Nb, 85Sr, or 46Sc (NEN, Boston, MA) as previously described (33). MO2 was obtained as the coronary arteriovenous oxygen content difference multiplied by blood flow.
Tissue Preparation
At the end of the study, hearts were removed and fixed in 10% buffered formalin. The LV was sectioned into four transverse rings of approximately equal thickness parallel to the mitral valve ring. The region of myocardium beneath the surface coil was sectioned into three transmural layers (from epicardium to endocardium), weighed, and placed into vials for counting of radioactivity.
Experimental Protocol
Aortic and LV pressures were measured with fluid-filled pressure transducers positioned at midchest level. LV pressure was recorded at normal and high gain for measurement of end-diastolic pressure. Hemodynamic measurements and MRS spectra were first obtained under basal conditions. Midway through the 20-min data acquisition period, a microsphere injection was performed for determination of myocardial blood flow and blood samples were obtained for MO2 determination.
Group 1 (n = 9). After completion of baseline data acquisition, an infusion of dobutamine and dopamine (DbDp, each 20 µg·kg–1·min–1 iv) was started to produce a high cardiac work state. After allowing 10 min to achieve steady-state conditions, all measurements were repeated. The catecholamine infusion was then discontinued, and hemodynamics were allowed to return to baseline. Nitric oxide synthase (NOS) inhibition was then produced by infusion of L-NNA (20 mg/kg iv) over 20 min. Beginning 30 min after completion of the L-NNA infusion, the DbDp infusion was restarted, and beginning 10 min later all measurements were repeated.
Group 2 (n = 6). Because of concern that HEP changes occurring during the high workload produced by catecholamine infusion might alter the subsequent response to NOS inhibition, in a second group of animals the sequence of interventions was reversed, so that after baseline measurements were obtained, L-NNA (20 mg/kg iv) was administered over 20 min. All measurements were repeated 20 min after completion of the L-NNA infusion. The infusion of DbDp (each 20 µg·kg–1·min–1 iv) was then begun; after 10 min was allowed to achieve steady-state conditions, all measurements were repeated.
Group 3 (n = 8). Because changes in coronary blood flow could alter coronary shear forces or myocardial oxygen availability, in a third group of animals coronary blood flow was increased by infusion of the selective coronary vasodilator carbochromen throughout the experimental interventions. After completion of baseline measurements, carbochromen (6 mg·kg–1·min–1 iv) was given to cause coronary vasodilation. After 10 min was allowed to achieve steady-state conditions, DbDp infusion (each 20 µg·kg–1·min–1 iv) was started and all measurements were repeated. The catecholamine infusion was then discontinued. NOS inhibition was produced by infusion of L-NNA (20 mg/kg iv) over 20 min. Beginning 30 min after completion of the L-NNA infusion, the DbDp infusion was restarted, and 10 min later all measurements were repeated.
Data Analysis
Hemodynamic data were measured from the chart recordings. The integral numerical values for the ATP and Pi resonances were recorded as PCr-to-ATP and Pi-to-ATP ratios. 31P NMR spectra from the first, third, and fifth voxels were taken to represent subepicardium, midmyocardium, and subendocardium, respectively. Data obtained during different experimental conditions were compared by one-way analysis of variance with replications. A value of P < 0.05 was required for significance. When a significant result was found, individual comparisons were made with the method of Scheffé. Comparison between groups was made with Student's two-tailed independent t-test with the Bonferroni correction. All values are expressed as means ± SE.
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RESULTS |
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As shown in Table 1, DbDp infusion resulted in increases of heart rate, aortic pressure, and LV systolic pressure with a nearly threefold increase in the rate-pressure product. The addition of L-NNA during catecholamine infusion tended to further increase aortic and LV systolic pressures and to decrease heart rate, but these changes were not significant, so that the rate-pressure product was unchanged. Mean myocardial blood flow increased from a baseline value of 0.59 ± 0.11 to 1.44 ± 0.24 ml·min–1·g–1 during the DbDp infusion (P < 0.01; Table 2), with a further significant increase to 2.27 ± 0.40 ml·min–1·g–1 after L-NNA. MO2 increased from a baseline value of 7.2 ± 1.8 to 16.0 ± 3.5 ml O2·min–1·(100 g)–1 during DbDp (P < 0.01 vs. baseline; Table 3) and tended to further increase to 19.4 ± 3.7 ml O2·min–1·(100 g)–1 with the addition of L-NNA (P < 0.01 vs. baseline; P = 0.1 vs. DbDp).
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Administration of L-NNA tended to increase LV systolic pressure and decrease heart rate, but neither of these changes was significant, so that the rate-pressure product was unchanged (Table 1). Both myocardial blood flow and MO2 tended to increase after L-NNA, but these changes were not significant (Tables 2 and 3). Infusion of DbDp in the presence of L-NNA caused an increase of heart rate similar to that observed in group 1; the increase of LV systolic pressure tended to be greater than in group 1, but this was not significant. Likewise, myocardial blood flow and MO2 during DbDp after L-NNA were not significantly different from those in group 1 (Tables 2 and 3). Administration of L-NNA during baseline conditions caused no change of PCr/ATP (Table 4). After L-NNA, the increased workload produced by DbDp caused a decrease of PCr/ATP relative to the value after L-NNA. However, PCr/ATP did not fall significantly below the baseline value during the DbDp infusion and remained significantly higher than PCr/ATP measured during DbDp in the absence of L-NNA in group 1 (P < 0.05). Calculated ADP did not decrease during DbDp after L-NNA and remained significantly higher than in group 1 when DbDp was administered in the absence of L-NNA (P < 0.05).
Group 3
During coronary vasodilation with carbochromen, infusion of DbDp caused a significantly greater increase of LV systolic pressure than observed in group 1 (Table 1); as expected, myocardial blood flow was substantially higher than in group 1 (P < 0.05; Table 2). Furthermore, MO2 was significantly higher during DbDp infusion in the animals treated with carbochromen than in the animals in group 1 (P < 0.05; Table 3). In group 3 animals, infusion of DbDp caused significant decreases of PCr/ATP in the LV subepicardial and midmyocardial layers and significant increases of Pi/PCr in all myocardial layers (Table 4). These changes were similar to those observed in group 1 animals. Administration of L-NNA in group 3 animals increased mean aortic pressure (Table 1); this was associated with a significant further increase of myocardial blood flow (Table 2) but no change of MO2 (Table 3). In the animals receiving carbochromen, the decrease of PCr/ATP (Table 4) and the increase of myocardial free ADP (Table 5) produced by DbDp were fully reversed after the administration of L-NNA. Furthermore, Pi/PCr was significantly decreased after administration of L-NNA (Table 4).
To better understand the effect of L-NNA, the relationship between MO2 and calculated ADP before and after treatment with L-NNA was plotted simultaneously for all three groups of animals (Fig. 1). During control conditions increases of MO2 produced by DbDp were associated with significant increases of ADP. NO synthesis blockade caused no change in the relationship between MO2 and PCr/ATP during basal work state conditions. However, L-NNA abolished the increase in ADP that normally occurred during the increases of cardiac work produced by catecholamine infusion. As a result, for a given value of MO2 during the high work state condition, ADP was lower after L-NNA than during control conditions. L-NNA produced a similar effect on the relationship between ADP and rate-pressure product (data not shown).
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No Mb- resonance was detected in any of the groups under basal conditions, during catecholamine infusion, or with the addition of L-NNA. During coronary artery occlusion (done as a positive control) a prominent Mb- resonance appeared at 71 ppm downfield of the water resonance with a high signal-to-noise ratio (data not shown).
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DISCUSSION |
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TOPABSTRACTMETHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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In the present study HEP content was monitored with 31P NMR spectroscopy, which allows direct detection of ATP and PCr, and calculation of myocardial free ADP content. Because the creatine kinase reaction is near equilibrium, increases in cytosolic free ADP are reflected by reciprocal changes in PCr/ATP, provided that total cellular creatine remains constant and there are no marked changes in ATP content (29). More than 90% of the ATP utilized by the heart is produced through OXPHOS in myocardial mitochondria. In the in vivo heart, the increased ATP generation required to support moderate increases of cardiac work occur with no change in cytosolic free ADP (14, 33). However, at high work states, as achieved during catecholamine infusion in the present study, ADP levels increase and are presumed to contribute to stimulation of the increased ATP production (8, 33). An increase in myocardial free ADP concentration ([ADP]) during catecholamine administration could be the result of insufficient oxygen availability during the high work state. However, that was excluded in the present study by the finding that Mb- was undetectable during the high workload, indicating that cytosolic PO2 remained far above the PO2 at which myoglobin is 50% saturated with O2 for cytochrome oxidase (35).
During the high work state produced by DbDp, L-NNA caused an increase in PCr/ATP, indicating a decrease of [ADP] whereas Pi/PCr was decreased. NO is known to compete with O2 at cytochrome oxidase, the terminal component of the electron transport chain (2, 3, 27), and the production of NO is increased during high work states (1, 28). Haynes et al. (10) have suggested that NO can be produced endogenously in concentrations sufficient to exert a modulatory (inhibitory) effect on cytochrome-c oxidase. There is evidence that OXPHOS is kinetically regulated by the availability of its primary substrates (NADH, Pi, ADP, and oxygen) (8). In this context, a decrease of ADP could be the result of increased availability of one or more of the other primary substrates of OXPHOS. Therefore, we suggest that, although cytosolic oxygen levels were always high, the capacity of cytochrome oxidase to transfer electrons to O2 was modestly limited by NO reversibly bound to that enzyme. Consequently, a reasonable interpretation of the present findings is that during the high work state removal of NO by L-NNA resulted in disinhibition of mitochondrial respiration, thereby allowing increased utilization of ADP and Pi by ATP synthase, with a consequent increase of PCr/ATP. The competitive inhibition of NO with oxygen at cytochrome oxidase is particularly important at low ambient PO2 values, such as those that exist in the cardiac myocyte (3), and would be amplified at high work states, because perimitochondrial PO2 values are further reduced at high rates of oxygen flux (25). Although the decrease of ADP following blockade of NO synthesis in the present study was relatively modest (20%), it was consistent among animals. Alternate explanations for the mechanism by which endogenous NO might result in an increase of cytosolic free ADP include direct effects on components of the electron transport chain, the F1,F0-ATPase or the adenine nucleotide translocase. Pathological increases of NO produced by exposure of cardiomyocytes to interleukin-1
caused reduction in activities of mitochondrial iron-sulfur-containing enzymes, including NADH-CoQ reductase and succinate-CoQ reductase, although not the ATPase (26). These changes appeared to result from protein alterations that are not quickly reversible. The adenine nucleotide translocase can also undergo modifications by pathological elevations of NO (26); again, these changes are not quickly reversible and would not explain the decrease of ADP over the short time period observed in the present study.
There are several potential sources of NO that might act to modulate OXPHOS in the intact heart. Giulivi (9) reported that NO produced by mitochondrial NOS inhibited O2 consumption in isolated mitochondria from rat liver. However, French et al. (7) found that NOS protein associated with mitochondria is 10 times lower in myocardium than in liver and were unable to detect NO production in myocardial homogenates, presumably because the breakdown of NO exceeded the rate of NO production. They concluded that NO produced by mitochondria does not regulate myocardial OXPHOS, although the importance of mitochondrial NOS in the in vivo heart is uncertain. Loke et al. (17) reported that bradykinin, which stimulates endothelial NO production, significantly decreased O2 consumption in myocardial tissue slices from wild-type endothelial NOS [eNOS(+/+)] but not homozygous eNOS(–/–) mice, suggesting that NO derived from eNOS may have the potential to modulate MO2. Neuronal NOS (nNOS) is expressed in sarcoplasmic reticulum of cardiac myocytes (32); whether NO produced by nNOS might have an influence on respiration or energy metabolism in the heart is unknown. An additional possible source of NO is inducible NOS (iNOS), which can be expressed in cardiomyocytes in response to inflammation or cytokine activation (12, 13). This isoform is capable of producing large quantities of NO over sustained periods of time and has been reported to depress MO2 in failing hearts (5). However, iNOS is not found in the normal canine heart and would not be expected to contribute to the present findings.
An additional mechanism by which L-NNA might influence OXPHOS involves a change in substrate selection. Inhibition of NOS has been found to cause a reduction of fatty acid uptake by the heart with a concomitant increase in oxidation of glucose-derived pyruvate (19). However, an increase in glucose utilization would be expected to cause an increase (not a decrease) in ADP. Thus in perfused rat hearts ADP levels were highest when glucose was the primary substrate and lowest when pyruvate or fatty acids were available (8). To the best of our knowledge, ADP responses to a shift toward increased glucose utilization and reduced fatty acid utilization have not been examined in the in vivo left ventricle. Furthermore, a switch from predominantly fatty acid to glucose utilization would act to decrease oxygen consumption, rather than the tendency toward increase observed in the present study. Thus the present findings are not likely due to a switch in substrate utilization caused by inhibition of NO production.
The increase of [ADP] produced by L-NNA in the present study occurred only during the high workload produced by catecholamine infusion, whereas L-NNA did not alter ADP values during basal conditions. Increased endothelial shear forces during high coronary flow rates have the potential to activate eNOS and increase NO production (15) and might thereby have amplified the effects of L-NNA during catecholamine infusion (6). Furthermore, increases of coronary flow can cause flow-mediated increases of MO2 that appear to be independent of NO (Gregg phenomenon). To examine whether the increase in coronary flow could explain why L-NNA caused a decrease of PCr/ATP during the DbDp infusion (when coronary flow rates were increased) but not during basal conditions, we administered carbochromen to cause maximal coronary vasodilation and eliminate changes in blood flow during the catecholamine infusion. As previously observed, carbochromen produced intense coronary vasodilation that was associated with a significant increase of MO2 but no effect on myocardial HEP levels (33). Because there is no evidence that carbochromen causes mitochondrial uncoupling, the increased MO2 likely resulted from the increased LV systolic pressure that occurred after administration of the drug (30).
At the high rate of coronary flow produced by carbochromen, catecholamine infusion caused decreases of PCr/ATP and increases of Pi/PCr that were not different from those observed during control conditions. Furthermore, during catecholamine infusion in the presence of the high coronary flow rate produced by carbochromen, L-NNA caused an increase of PCr/ATP that was similar to that observed during control conditions. This indicates that the L-NNA-induced increase of PCr/ATP during DbDp was not the result of the increased flow during catecholamine infusion. Furthermore, the relatively low oxygen extraction during carbochromen, and the lack of change in oxygen extraction after L-NNA, support the conclusion that limited oxygen delivery was not responsible for the decreased PCr/ATP during the high work state.
Therefore, the inhibition of NOS with L-NNA caused an increase of myocardial PCr/ATP with a trend toward increased MO2 and coronary blood flow. These findings are compatible with the hypothesis that NO inhibition of respiration required cytosolic [ADP] to increase, with a consequent decrease of PCr/ATP. The findings support previous in vitro data suggesting that NO acts to increase cardiac efficiency (1), but this effect occurs at the expense of increased cytosolic free [ADP].
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FOOTNOTES |
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The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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REFERENCES |
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enhances cardiac myocyte NO production through MAP kinase-mediated NF-
B activation. Am J Physiol Heart Circ Physiol 277: H1641–H1646, 1999.This article has been cited by other articles:
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  F. Wu, E. Y. Zhang, J. Zhang, R. J. Bache, and D. A. Beard Phosphate metabolite concentrations and ATP hydrolysis potential in normal and ischaemic hearts J. Physiol., September 1, 2008; 586(17): 4193 - 4208. [Abstract] [Full Text] [PDF]
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 B. J. Behnke, M. D. Delp, D. C. Poole, and T. I. Musch Aging potentiates the effect of congestive heart failure on muscle microvascular oxygenation J Appl Physiol, November 1, 2007; 103(5): 1757 - 1763. [Abstract] [Full Text] [PDF]
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, Measurements obtained during control conditions; 
, measurements obtained after inhibition of NO production with L-NNA. During control conditions the increases of M