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Am J Physiol Heart Circ Physiol 280: H2030-H2037, 2001;
0363-6135/01 $5.00
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Vol. 280, Issue 5, H2030-H2037, May 2001

Oxygen supply and oxidative phosphorylation limitation in rat myocardium in situ

Ulrike Kreutzer, Yousry Mekhamer, Youngran Chung, and Thomas Jue

Department of Biological Chemistry, University of California, Davis, California 95616-8635.


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIAL AND METHODS
RESULTS
DISCUSSION
REFERENCES

The 1H-NMR signal of the proximal histidyl-Ndelta H of deoxymyoglobin is detectable in the in situ rat myocardium and can reflect the intracellular PO2. Under basal normoxic conditions, the cellular PO2 is sufficient to saturate myoglobin (Mb). No proximal histidyl signal of Mb is detectable. On ligation of the left anterior descending coronary artery, the Mb signal at 78 parts/million (ppm) appears, along with a peak shoulder assigned to the corresponding signal of Hb. During dopamine infusion up to 80 µg · kg-1 · min-1, both the heart rate-pressure product (RPP) and myocardial oxygen consumption (MVO2) increase by about a factor of 2. Coronary flow increases by 84%, and O2 extraction (arteriovenous O2 difference) rises by 31%. Despite the increased respiration and work, no deoxymyoglobin signal is detected, implying that the intracellular O2 level still saturates MbO2, well above the PO2 at 50% saturation of Mb. The phosphocreatine (PCr) level decreases, however, during dopamine stimulation, and the ratio of the change in Pi over PCr (Delta Pi/PCr) increases by 0.19. Infusion of either pyruvate, as the primary substrate, or dichloroacetate, a pyruvate dehydrogenase activator, abolishes the change in Delta Pi/PCr. Intracellular O2 supply does not limit MVO2, and the role of ADP in regulating respiration in rat myocardium in vivo remains an open question.

myoglobin; nuclear magnetic resonance; respiration; bioenergetics; heart


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIAL AND METHODS
RESULTS
DISCUSSION
REFERENCES

MYOCARDIAL OXYGEN CONSUMPTION (MVO2) can vary dramatically to meet changing energy demands of the myocardium. As MVO2 increases, the O2 delivery from air to mitochondria balances the supply and demand at either O2 transport, convection or diffusion, or metabolism step. Because the rate of O2 transport in the microcirculation correlates tightly with the maximal MVO2 (MVO2 max), some investigators have postulated the limiting step in O2 transport. Clearly, as blood flow increases, so also does O2 consumption (7). Increasing or decreasing acutely the O2 transport capacity alters MVO2 max and points to a limitation in diffusion rather than convection (26). However, others have postulated that the rate-limiting step is independent of the O2 supply and is controlled by the cytochrome a/a3 oxidation state, because the maximal mitochondrial O2 consumption varies across a wide range of muscle tissues (12, 31).

Measuring the intracellular O2 level in the myocardium during increased work load would certainly clarify the debate over the regulatory mechanisms. At least the intracellular oxygenation under different physiological states would signal whether the regulatory step is located in the vasculature or metabolism linked steps. Unfortunately, the measurement of intracellular O2 in physiological conditions has posed an experimental challenge (11, 28, 38).

With 1H NMR techniques, the detection of the proximal histidyl-Ndelta H F8 and the Val E11 signals of myoglobin in the perfused heart has established a methodology to quantify the relationship between cellular PO2 and bioenergetics and define the critical PO2 relationships in normal and postischemic perfused myocardium (5, 20, 22). Indeed, the experimental results suggest that O2 availability may not be the limiting factor. The buffer-perfused heart, however, is a simplified model and does not engender sufficient confidence about the function of the myocardium in situ. It neither responds fully to vascular regulation nor receives O2 from erythrocytes. Although in establishing an NMR technique, it is the appropriate model that avoids the potential signal interference between the Mb and Hb signals, it cannot establish definitively the relationship between O2 availability and consumption in the in vivo myocardium.

We report that the deoxymyoglobin (deoxy-Mb) proximal histidyl-Ndelta H signal is detectable in the rat myocardium in situ and is distinguishable from the corresponding Hb signals (4, 21, 34, 41). The visibility of the deoxy-Mb signal opens an opportunity to explore the cellular PO2 as a function of O2 consumption or work load. Indeed, under dopamine stimulation, the myocardial rate-pressure product (RPP) and MVO2 increase by about a factor of two. Yet no deoxy-Mb signal is observed. Stimulated respiration produces no shift in the MbO2 saturation, implying an unaltered intracellular PO2. Because the arterial PO2 remains constant, the observation suggests that the O2 gradient between the vasculature and the cell has not changed.

Despite a constant intracellular PO2, the ratio of the change in Pi over phosphocreatine (Delta Pi/PCr) increases, implying an elevated ADP level during stimulated respiration. Although isolated mitochondrial work supports an ADP-dependent control mechanism, a previous experiment with in situ canine myocardium observed insignificant changes in the Delta Pi/PCr ratio during enhanced respiration, which raises doubt about the role of ADP (18). Others, however, have observed a very slight shift in the Delta Pi/PCr ratio during dopamine stimulation. Nevertheless, the investigators have argued against an ADP-dependent mechanism, because the resting ADP level in canine myocardium is well above the Michaelis-Menten constant (Km) of the ATP synthetase. If ADP is not regulating oxidative phosphorylation, they have posited that intracellular O2 controls respiration (18).

In the present study with in situ rat myocardium, the intracellular oxygenation remains constant, but Delta Pi/PCr is significantly elevated during dopamine stimulation. Moreover, the infusion of dichloroacetate or pyruvate, well-known activators of pyruvate dehydrogenase, abolishes the changes in Delta Pi/PCr but does not affect respiration and work. Overall, the results indicate that the cellular O2 supply does not limit oxidative phosphorylation and that the issue of ADP or carbon substrate as a regulator of myocardial respiration remains moot.


    MATERIAL AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIAL AND METHODS
RESULTS
DISCUSSION
REFERENCES

Whole animal preparation. Male Sprague-Dawley rats (400-450 g) were anesthetized by intraperitoneal injection of 50 mg/kg pentobarbital. An optical sensor connected to a Nellcor pulse oximeter was taped over the region of the tail artery to monitor arterial O2 saturation and pulse rate. The left jugular vein and the right common carotid artery were cannulated with polyethylene tubing (PE-50, Becton-Dickinson). The carotid catheter was advanced through the aortic valve into the left ventricle and connected to a pressure transducer (Medex MX950) and an oscillographic recorder (Gould WindoGraf) to monitor left ventricular pressure and heart rate. Anesthesia was maintained over the experimental period by intravenous continuous-rate infusion of 15 mg · kg-1 · h-1 pentobarbital.

A tracheotomy was performed, and a polyethylene cannula (PE-240) connected to a rodent respirator (Harvard Instruments) was secured with a ligature. The animal was ventilated with room air supplemented with 30% O2 to yield a 44% O2 content in the inspired gas, which maintained the HbO2 saturation between 90 and 95% under all experimental conditions. Stroke volume was maintained between 2.5 and 3 ml, and ventilation rate was 60-65 strokes/min. The animal maintained an arterial O2 saturation >90%, a heart rate of ~338 ± 10 beats/min, and a left ventricular systolic pressure between 90 and 100 mmHg.

A medial sternotomy exposed the heart. The pericardium was then removed, and a suture (silk 6-0) was placed around the left anterior descending coronary artery (LAD) ~2 mm below its origin. The LAD was ligated during the last phase of the experiment. The animal was placed on a lucite holder, and a concentric 31P/1H surface coil was placed over the heart. Heart rate, left ventricular systolic pressure, and end-diastolic pressure were measured continuously. The animal's body temperature was maintained with a heating pad connected to a circulating water bath or with a stream of warm air circulating through the magnet bore.

After a control period of 30-45 min, dopamine was infused at a continuous rate of 80 µg · kg-1 · min-1 for a period of 30 min. The infusion was stopped, and the animal recovered for 30 min. The suture around the LAD was then tied. Animals were euthanized with 150 mg/kg of pentobarbital.

The dichloroacetate (DCA) experiments followed a similar protocol. Animals were monitored under control conditions for 30 min; DCA was then infused at a rate of 0.8 mg · kg-1 · min-1 for 30 min, followed by dopamine + DCA (80 µg and 0.8 mg · kg-1 · min-1, respectively) for 30 min. The pyruvate infusion followed the same protocol with the use of 12 mg · kg-1 · min-1 pyruvate instead of DCA.

Microsphere measurement of coronary blood flow. The microsphere reference sample technique was utilized to obtain absolute coronary blood flow (13). Catheters were inserted into the femoral artery, right carotid artery, and left jugular vein. After the chest was opened, an additional catheter (PE-50) was inserted into the left atrium via the left auricle. Arterial pressure was recorded continuously. Approximately 20,000 fluorescent microspheres (NuFlo, Interactive Medical Technologies) were injected into the left atrium through the atrial catheter. A reference sample was withdrawn from the femoral catheter, starting 10 s before the microsphere injection, and continued for a total of 90 s with a constant withdrawal pump (model 22, Harvard Apparatus) at a rate of 0.8 ml/min. Blood was withdrawn into a preweighed, heparinized disposable syringe. The exact sampling rate was determined from the difference in weight of the syringe and connecting tubing before and after the withdrawal period. The blood was transferred into a test tube, and syringe and tubing were flushed with saline, which was then added to the sample. All blood withdrawn for the reference sample was replaced immediately with ringer. Twenty minutes after microsphere injection, the animal was euthanized with 150 mg/kg of pentobarbital. The heart and kidneys were excised, and microsphere counts in blood and organ samples were determined (Interactive Medical Technologies, Los Angeles, CA). Coronary blood flow (CBF) was obtained as follows
CBF<IT>=</IT>(sampling rate)<IT>·</IT>(total microspheres heart)<IT>/</IT>

(total microspheres reference sample)
where CBF and sampling rate are in milliliters per minute.

For relative blood flow measurements, parallel experiments performed outside the magnet instrumented and monitored the animals as described above. An additional catheter was placed in the right jugular vein. The length of the left jugular catheter was kept as short as possible. After a control period of 20 min after completion of surgery, the left jugular catheter was carefully advanced to the right atrium and into the left coronary vein. A coronary venous blood sample was withdrawn, and coronary flow was monitored by letting blood flow freely from the coronary catheter into a heparinized preweighed sample tube for 30 s. The catheter was then withdrawn from the coronary vein. Immediately afterwards, an arterial blood sample (~300 µl) was withdrawn through the arterial catheter. Coronary blood flow was estimated from the difference in sample tube weight before and after the collection of coronary venous blood. The collected blood (~0.4-0.8 ml) was reinfused via the right jugular vein. O2 content in blood samples was measured with an Oxycon O2 content analyzer (Cameron Instruments). Lactate and glucose in blood plasma were measured with a YSI-2700 lactate analyzer. The blood sampling procedure was performed during the control period as well as after 20 min of dopamine infusion and after 20 min of recovery.

Relative changes in O2 consumption were calculated from flow and O2 content (cO2) data as follows
%M<A><AC>V</AC><AC>˙</AC></A><SC>o</SC><SUB>2</SUB><IT>=</IT><FR><NU>flow<SUB>d</SUB><IT>·</IT>(arterial<IT>−</IT>coronary cO<SUB><IT>2</IT></SUB>)<SUB>d</SUB><IT>·100</IT></NU><DE>flow<SUB>b</SUB><IT>·</IT>(arterial<IT>−</IT>coronary cO<SUB><IT>2</IT></SUB>)<SUB>b</SUB></DE></FR>
with index b denoting values under baseline conditions and index d denoting values under dopamine infusion or after dopamine push.

NMR methods. In situ rat myocardium spectra were recorded with a GE Omega 7T 15-cm-diameter horizontal bore spectrometer system. A concentric 31P/1H coil system was utilized. The inner 1H coil was 2 cm in diameter; the outer 31P was 3 cm in diameter. The 1H and 31P 90° pulse at the coil were 25 and 35 µs, respectively.

A modified DANTE sequence was utilized to suppress the water signal and excite the Mb resonances (27). The sequence was tailored to give a maximum excitation at 76 ppm. A typical 1H spectrum consisted of 10,000 transients, requiring a total acquisition time of 7 min. The spectral width was 60 kHz; data block size was 4 k. All spectra were referenced to water at 4.76 ppm, at 25°C, which in turn was calibrated against 2,2-dimethyl-2-silapentane-5-sulfonate. Repetition time was 40 ms, and transmitter power was set to give a 90° pulse at the coil center. For the 31P spectra, the spectral width was 6 kHz; data block size was 1 K. Repetition time was 1 s. Total number of scans was 128.

A set, composed of two 1H and 31P spectra, was acquired during the control period, during dopamine infusion after the hemodynamic values have stabilized, and during LAD ligation.

Calculations. ADP was calculated from the creatine kinase reaction with the use of the equilibrium constant of 1.66 × 10-9 · M-1, pH 7.1, and literature values for the baseline concentration of PCr (14 mM), ATP (7.7 mM), and Cr (5.1 mM) (2, 37). The pH remained constant at 7.1 in the control and dopamine-stimulated state. For the control state, the ADP concentration is then 21 µM. Statistical analysis was performed with the SigmaStat program (SPSS). Data are given as means ± SE. Statistical significance was determined by paired t-test: P < 0.05.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIAL AND METHODS
RESULTS
DISCUSSION
REFERENCES

Figure 1 shows the 1H spectra from the myocardium of an open-chest rat. During the control period, no signal is detected in the region between 100 and 60 ppm (Fig. 1A). With the infusion of dopamine at 80 µg · kg-1 · min-1, still no signal appears in the spectral region (Fig. 1B). Figure 1C also shows no detectable signal during the recovery period when dopamine infusion ceased. However, on ligation of the LAD, a signal corresponding to the deoxy-Mb proximal histidyl-Ndelta H emerges at 78 ppm (Fig. 1D). An upfield shoulder at 75 ppm is also visible, corresponding to the deoxy-Hb proximal histidyl-Ndelta H signal.


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  		Fig. 1.   1H spectra from in situ myocardium. A: during the control period, no 1H signal appears in the 100- to 60-parts/min (ppm) region. B: on infusion with 80 µg · kg-1 · min-1 of dopamine, the spectral region shows no signal of the proximal histidyl-Ndelta H of myoglobin (Mb). C: after dopamine infusion was discontinued. D: after left anterior descending coronary artery (LAD) ligation, the deoxy-Mb signal appears at 78 ppm. The upfield shoulder corresponds to the proximal histidyl-Ndelta H signal of Hb.

The corresponding 31P signals are shown in Fig. 2. During the control period, the signals of PCr, ATP, and Pi are clearly visible (Fig. 2A). However, during dopamine infusion, the PCr signal decreases significantly, whereas the Pi peak increases (Fig. 2B). PCr/ATP recovers during the postinfusion period (Fig. 2C). On LAD ligation, the PCr signal disappears, and the Pi signal increases (Fig. 2C).


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  		Fig. 2.   31P spectra from in situ myocardium. A: during control. B: during 80 µg · kg-1 · min-1 of dopamine. C: after discontinuation of dopamine infusion. D: after LAD ligation. The 31P signals correspond to Pi (5 ppm), phosphocreatine (PCr; 0 ppm), and ATP (-2.4, -7.5, and -16 ppm).

The physiological and high-energy phosphate data during the control, dopamine infusion, and recovery period are shown in Table 1. The RPP increases by a factor of two, originating from both a 60% increase in left ventricular systolic pressure and a 25% increase in heart rate. Within 10 min of recovery, the heart function returns to its control level.

                              
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  		Table 1.   Heart function and high-energy phosphates during dopamine infusion experiment

Figures 2 and 4 summarize the relative changes in PCr, Pi, and ATP during control, dopamine stimulation, and recovery period. During dopamine stimulation, PCr decreases by 18%, whereas the Delta Pi/PCr ratio increases from 0 to 0.19. ATP remains unperturbed. The ADP level rises from 21 to 31 µM.

Fluorescent microsphere experiments indicate a baseline coronary flow of 4.02 ± 0.49 ml · min-1 · g wet wt-1 (n = 4), which increases by 84 ± 23% during stimulation (Fig. 3). The mean arteriovenous O2 difference is 4.2 ± 0.4 and 5.5 ± 0.4 mM, respectively (Table 2). The data then lead to an estimate of the MVO2 of 17 and 40 µmol · min-1 · g-1. Dopamine stimulation enhances MVO2 by a factor of 2.4 (Fig. 3).


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  		Fig. 3.   Physiological response during dopamine infusion experiment. Rate-pressure product (RPP) increases by a factor of 2.0, and myocardial oxygen consumption (MVO2) increases by a factor of 2.4 during dopamine infusion and returns to baseline levels after discontinuation of dopamine infusion. The increase in O2 consumption is met by an 84 ± 23 and 31 ± 13% increase in coronary flow and O2 extraction (arteriovenous O2 difference; Delta avO2), respectively. Data are means ± SE; n = 6-8. * Significant difference from control based on paired t-test (P < 0.05).


                              
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  		Table 2.   Blood O2 content, glucose, and lactate during dopamine infusion experiment

Dopamine stimulation (Fig. 4) also leads to a twofold increase in arterial blood glucose level and a decrease in the lactate level (30). Despite the elevated concentration, the glucose and lactate extraction, however, remain the same.


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  		Fig. 4.   Changes in high-energy phosphates during dopamine infusion experiment. ATP and PCr levels are normalized to 100% and the ratio of change in Pi over PCr (Delta Pi/PCr) to 0% during control. Values are means ± SE; n = 8. * Significant difference from control based on paired t-test (P < 0.05). As RPP increases during dopamine infusion, PCr decreases significantly by 18%, and Delta Pi/PCr increases.

If the animals receive DCA (0.8 mg · kg-1 · min-1) or pyruvate infusion (12 mg · kg-1 · min-1) before dopamine stimulation, the 31P spectra no longer exhibit any significant change in the high-energy phosphate levels, despite the MVO2 enhancement (Tables 3 and 4, Fig. 5). With DCA or pyruvate infusion (Fig. 6), neither the PCr/ATP ratio nor the Delta Pi/PCr ratio shows any change during dopamine stimulation.

                              
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  		Table 3.   Heart function and high-energy phosphates during dopamine stimulation with DCA


                              
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  		Table 4.   Heart function and high-energy phosphates during dopamine stimulation with pyruvate



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  		Fig. 5.   Changes in high-energy phosphates during dichloroacetate (DCA) + dopamine infusion experiment. ATP and PCr levels are normalized to 100% and Delta Pi/PCr to 0% during DCA infusion. Solid bar in each group shows baseline before DCA infusion. Values are means ± SE; n = 6. * Significant difference from DCA infusion based on paired t-test (P < 0.05).



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  		Fig. 6.   Changes in high-energy phosphates during pyruvate (Pyr) + dopamine infusion experiment. ATP and PCr levels are normalized to 100% and Delta Pi/PCr to 0% during pyruvate infusion. Solid bar in each group shows baseline before pyruvate infusion. Values are means ± SE; n = 6. * Significant difference from pyruvate infusion based on paired t-test (P < 0.05).


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIAL AND METHODS
RESULTS
DISCUSSION
REFERENCES

Resting state PO2. The applicability of the 1H NMR technique to detect the Mb signal in a regionally ischemic in situ rat myocardium is clearly shown in Fig. 1. Unlike global ischemia in the perfused heart experiments, ligation of the LAD produces only a local ischemic region, which limits the deoxygenated tissue volume and therefore the signal-to-noise ratio of the deoxy-Mb peak. Nevertheless, the proximal histidyl-Ndelta H signal of Mb is clearly visible from the ischemic left ventricle, along with an upfield Hb signal at 75 ppm. These assignments are consistent with previously reported results from perfused myocardium and erythrocyte studies (19, 21, 34).

Although perfused heart and skeletal muscle studies indicate that resting-state cellular PO2 is sufficient to maintain Mb saturation, the myocardium in situ with blood perfusion and normal sinus rhythm may still be poised at the brink of O2 limitation, as some researchers have postulated (5, 7, 20, 39). Observation of a partially saturated Mb would confirm such a hypothesis and would also provide quintessential evidence for a significant Mb role in facilitating O2 diffusion. The result from Fig. 1A, however, shows no Mb desaturation under resting or even the dopamine-stimulated conditions. Despite the increased O2 demand during dopamine stimulation, the cellular O2 is sufficient to saturate Mb well above 90%; otherwise, the NMR would have detected a deoxy-Mb signal. As a result, the Mb-facilitated-diffusion hypothesis can no longer depend on a partially desaturated Mb in blood-perfused tissue as a precept.

O2 and stimulated MVO2. On dopamine stimulation, the myocardial RPP and MVO2 rise by a factor of two. Many investigators have postulated that a rise in intracellular O2 should accompany the enhanced respiration. Indeed, optical studies have confirmed that MbO2 saturation does increase (9, 33). In that way, the cytochrome oxidase reaction rate, which is posited as the rate-limiting step in respiration, will also increase. Such a biochemical view coincides with a generally accepted tenet that Mb is only partially saturated in the in vivo myocardium and facilitates O2 transport in the cell, especially at low cellular PO2 (9, 16).

Neither postulate appears consistent with the experimental results. The 1H spectra show no sign of Mb desaturation in the resting state. Given the deoxy-Mb signal-to-noise ratio of ~20/1, which is assumed to reflect the fully deoxygenated state, a 10% change in Mb desaturation would certainly be detectable. It would imply that if MbO2 is 90% saturated, the NMR sensitivity would detect a 10% deoxygenated Mb proximal histidyl-Ndelta H signal. If the Mb p50 at 37°C is 2.37 Torr, then a 90% MbO2 saturation corresponds to a PO2 of 21.6 Torr (29). Above 90% MbO2 saturation, the cellular PO2 approaches the capillary PO2.

Quite clearly, given the undetected deoxy-Mb signal in the resting myocardium, Mb is not partially saturated in the myocardium in situ. To increase the O2 level to enhance the cytochrome oxidase reaction would require the cellular PO2 to rise above 21.6 Torr during dopamine stimulation, collapsing significantly any vascular-to-cellular O2 gradient and impairing the O2 transport into the cell.

The experimental observations imply that dopamine-stimulated respiration is independent of the O2 level. Despite the tight coupling between convective flow and respiration, the intracellular O2 concentration per se does not appear to limit respiration. A fully saturated MbO2 implies that the cellular O2 level has not decreased to any physiologically significant level, despite the factor of two increase in respiration. Given that the myocardial MVO2 max is still about two times higher in exercising animals than the observed MVO2 during dopamine stimulation, one might postulate that the MVO2 has not reached a maximum level and therefore has not taxed heavily the cellular O2 supply (26). However, skeletal muscle experiments militate against such a sharp shift in O2-dependent regulation of rate of O2 consumption (VO2), because cellular O2 decreases proportionately with increasing VO2 over a wide range (25). The deoxy-Mb signal would certainly appear in the 1H spectra at one-half MVO2 max.

Because the intracellular PO2 almost completely saturates MbO2 in the resting state and remains constant during enhanced respiration, some doubt arises about the role of Mb in facilitating O2 diffusion in the cell. The increased myocardial respiration does not elicit any measurable change in the cellular PO2. Moreover, at a resting-state PO2 sufficient to saturate MbO2, the contribution of Mb in facilitating O2 diffusion from the sarcoplasm to the mitochondria is small, consistent with the recent studies of Mb function in the myocardium (5a, 6, 39).

The 1H NMR results also suggest that the O2 gradient between the vasculature and the intracellular PO2 remains constant despite the increased MVO2. On the upfield shoulder at 75 ppm is a hint of a second signal from LAD ligated myocardium. It corresponds to the paramagnetic-shifted proximal histidyl-Ndelta H signal from the beta -heme of deoxy-Hb His F8 (the histidine at position 8 of helix F in myoglobin) Ndelta H (10, 19, 23, 32). The alpha -deoxy-Hb His F8 Ndelta H signal at 63 ppm is not visible under these signal-acquisition conditions, consistent with 1H NMR data from skeletal muscle (35). During dopamine stimulation, the blood flow increases without producing any observable deoxy-Hb signal. Meeting the enhanced MVO2 by increasing the blood flow and O2 extraction without perturbing the vascular-to-cellular O2 gradient implies an alteration in the capillary-to-cell distance or in the metabolic activity.

ADP-dependent regulation of MVO2. The in situ myocardium experiments cannot confidently investigate any changes in capillary-to-cell distance. However, they can focus on the role of metabolic control. If the O2 supply remains unperturbed during dopamine stimulation, then a rise in the ADP or NADH concentration can drive the increased respiration according to present theories of respiratory control. Indeed, on dopamine infusion, PCr decreases by 18% as RPP increases by a factor of two. The Delta Pi/PCr ratio increases to 0.19 (Table 1). On the basis of the creatine kinase reaction, the shift in PCr indicates that the ADP level has risen from 21 to 31 µM during enhanced respiration, consistent with the tight coupling between respiration and oxidative phosphorylation, as established from isolated mitochondria experiments (3).

The observed change in Delta Pi/PCr, however, brings into question the role of ADP in regulating respiration in the in situ myocardium. Some investigators have argued that in the in situ canine myocardium, an unaltered 31P NMR spectra during increased respiration reflects an ADP-independent mechanism of regulation (1, 40). Other in situ canine myocardial studies, however, have shown a modest change in the Delta Pi/PCr, suggesting an altered ADP level during dopamine stimulation (21).

However, researchers continue to posit that ADP does not play a key role as a regulator of myocardial respiration, because the resting ADP concentration is well above the Km for ATP synthetase reaction. In vitro biochemical assays have determined that the Km is ~20 µM (15). In the canine myocardium, the calculated ADP value is much higher than 20 µM and therefore cannot regulate oxidative phosphorylation, despite the observed Delta Pi/PCr change (18, 40). In the rat heart under normal conditions, however, the resting ADP concentration is uncertain. On the basis of the creatine kinase equilibrium, several studies have reported resting ADP concentration in the range of 20-50 µM (2, 8, 36). The uncertainty in resting ADP value in rat myocardium, where the respiration and heart rates are much higher than in canine myocardium, leaves unsettled the role of ADP in regulating respiration in the in situ rat myocardium (14).

Effect of pyruvate and DCA. One indirect way of probing the contribution of the ADP-dependent mechanism is to postulate that the rate-limiting steps involve the NADH production pathway. Stimulating pyruvate dehydrogenase activity should not significantly affect the observed Pi/PCr change with dopamine stimulation, if ADP and not NADH is the assumed limiting factor. However, with the infusion of DCA or pyruvate, well-established activators of pyruvate dehydrogenase (PDH) activity, the change in Delta Pi/PCr during dopamine stimulation disappears. These observations do not fully support the notion that ADP is the only regulator of respiration. Indeed the experimental data implicate a potential role for NADH, consistent with optical measurements (17). They are also consistent with a higher efficiency in O2 utilization on PDH activation with DCA in postischemic heart (24). Further studies are now underway to clarify the mechanism underlying the DCA or pyruvate effect.

In conclusion, in blood-perfused hearts, the physiological paradigm posits that the cellular PO2 is only sufficient to partially saturate MbO2. In that way, the increase in MVO2 would match the rise in MbO2 saturation. Moreover, a low cellular PO2 would accentuate the role of Mb in facilitating O2 diffusion. Our study provides no experimental evidence to support such a paradigm. Under normoxic conditions, the cellular PO2 is ample to saturate MbO2. Even during the dopamine-stimulated increase in MVO2, the cellular PO2 still remains high enough to continue saturating MbO2. Such an observation implies that O2 itself is not a key regulator during enhanced respiration.

Nevertheless, the dopamine stimulation increases MVO2 and concomitantly the Delta Pi/PCr. Although the data would suggest an ADP-dependent regulation of MVO2, comparative analysis with the expected cellular concentration of ADP and the in vitro Km of ADP in the oxidative phosphorylation reaction raises doubt about such an interpretation. The experiments with DCA, a PDH activator, and pyruvate are consistent with an ADP-independent regulatory mechanism and point to NADH as a regulator of respiration.


    ACKNOWLEDGEMENTS

We gratefully acknowledge scientific consultation with Drs. Tuan-Khanh Tran, Tim Musch, and Charles Stebbins and technical assistance from Tammi Myers.


    FOOTNOTES

This work was supported by National Institute of General Medical Sciences Grant GM-57355.

Address for reprint requests and other correspondence: T. Jue, Med: Biological Chemistry, Univ. of California Davis, Davis, CA 95616-8635 (E-mail: TJue{at}ucdavis.edu).

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.

Received 12 October 1999; accepted in final form 29 November 2000.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIAL AND METHODS
RESULTS
DISCUSSION
REFERENCES

1.   Balaban, RS, Kantor HL, Katz LA, and Briggs RW. Relation between work and phosphate metabolite in the in vivo paced mammalian heart. Science 232: 1121-1123, 1986[Abstract/Free Full Text].

2.   Bittl, JA, Balschi JA, and Ingwall JS. Effects of norepinephrine infusion on myocardial high-energy phosphate content and turnover in the living rat. J Clin Invest 79: 1852-1859, 1987.

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Am J Physiol Heart Circ Physiol 280(5):H2030-H2037
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