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1 Departments of Medicine, Biochemistry, and Radiology, and Center for Magnetic Resonance Research, University of Minnesota, Minneapolis 55455; and 2 Department of Veterans Affairs Medical Center, Minneapolis, Minnesota 55417
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ABSTRACT |
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 TOP
 ABSTRACT
 INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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This study
was performed to determine whether the fall in myocardial high-energy
phosphates (HEP) that occurs during high workstates can be ascribed to
either inadequate glycolytic pyruvate generation and conversion to
acyl-CoA or limitation of long-chain fatty acid transport into the
mitochondria. This was tested by using infusions of either pyruvate or
butyrate in anesthetized dogs. Pyruvate was used because it bypasses
the glycolytic sequence of reactions, activates pyruvate dehydrogenase,
and increases mitochondrial NADH concentration ([NADHm])
in isolated myocardium, whereas butyrate enters the mitochondria
without need for transport by the rate-limiting, palmitoyl-carnitine
transporter. Increasing blood pyruvate from 0.16 ± 0.016 mM to
>3 mM did not alter baseline HEP levels determined with
31P nuclear magnetic resonance, but caused an increase in
the rate-pressure product and a modest increase in myocardial oxygen
consumption (M
O2). Infusion of
dobutamine + dopamine (each 20 µg · kg
1 · min1 iv)
increased MO2 and caused decreases of
myocardial phosphocreatine (PCr)/ATP. Pyruvate partially reversed the
decrease of HEP levels produced by catecholamine stimulation, whereas
butyrate had no effect. Neither pyruvate nor butyrate caused an
increase of MO2 during catecholamine
infusion. Deoxymyoglobin was not detected by 1H magnetic
resonance spectroscopyy in any group. The data demonstrate that
carbon substrate availability to the mitochondria is not the only cause
of the reduction of PCr/ATP that occurs at high workstates.
Supplemental pyruvate (but not butyrate) attenuated the reduction of
PCr/ATP during the high workstates; this may have resulted from direct
effects on intermediary metabolism or from other effects such as the
free radical scavenging activity of pyruvate.
catecholamines; oxygen consumption; magnetic resonance spectroscopy
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INTRODUCTION |
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TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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IN THE IN VIVO
CANINE HEART high-energy phosphate (HEP) levels do not change
during moderate increases of cardiac workload produced by catecholamine
infusion, i.e., when rate-pressure products (RPP) are <35,000
mmHg · beats · min1 (1, 26,
36). However, catecholamine infusions that produce RPP >45,000
mmHg · beats · min1 do cause decreases of
myocardial phosphocreatine (PCr) and loss of ATP (36).
Furthermore, when a period of high cardiac workstate is terminated by
discontinuing catecholamine infusion, PCr levels quickly return to
control, whereas ATP levels remain depressed, indicating that a portion
of the adenine nucleotide pool has been lost during the high workstate
(36). Lastly, during catecholamine-induced high cardiac
workstates, pharmacological coronary vasodilation causes a significant
increase of myocardial oxygen consumption (MO2), but HEP levels do not recover
(36). These observations suggested that demand-induced
ischemia might be present during high workstates. However,
using 1H magnetic resonance spectroscopy to detect
myocardial deoxymyoglobin (Mb-
), we found that the reductions of HEP
at very high workstates were not associated with detectable myoglobin
desaturation (37). Hence, ischemia, if defined as
oxygen limitation of metabolism, was not present at the very high workstates.
Because the decreased myocardial HEP content during high workstates
could not be ascribed to oxygen limitation, other explanations must be
considered including limitation of production of mitochondrial NADH
concentration ([NADHm]), which is primarily generated by the tricarboxylic acid cycle from acyl-CoA, which, in turn, is generated by carbohydrate or fatty acid metabolism. It possible that
the metabolic pathways leading to 1) pyruvate generation and/or its conversion to acyl-CoA and/or 2) transport of
long-chain fatty acids into the mitochondria and/or their rate of
conversion to acyl-CoA are sufficiently limited to cause a decrease of
steady-state acyl-CoA concentration and, in consequence,
[NADHm] during high workstates. On the basis of previous
studies of isolated and in vivo myocardium, we speculated that the
reductions of HEP might be prevented by the administration of
supplemental carbon substrate (5, 7, 14, 17, 23, 30). We
further speculated that if the postulated high workload associated
[NADHm] reduction was severe enough to limit the rate as
well as the kinetics of oxidative phosphorylation, then carbon
substrate supplementation during a high workstate might also increase
MO2.
Pyruvate and butyrate were chosen as the carbon substrates to be
administered for the following reasons: Pyruvate, which bypasses the
entire glycolytic sequence of reactions including glucose transport
into the cell, activates pyruvate dehydrogenase (PDH), and increases
[NADHm] in isolated mitochondria, has also been shown to
increase HEP in in vivo myocardium (5, 17, 30). Short-chain fatty acids enter mitochondria without need for transport by the palmitoyl-carnitine transporter by the palmitoyl-carnitine transporter and have been shown to increase myocyte acyl-CoA levels, to
prevent the reduction of acyl-CoA with increased workstates, and to
increase [NADHm] in in vitro myocardium. They also
increase HEP in in vivo myocardium (5, 14, 23, 30). The
present experiments sought to determine whether the administration of high concentrations of either pyruvate or butyrate could prevent or
reverse the reductions in PCr/ATP associated with high workstates and/or cause MO2 to increase during
high workstates.
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METHODS |
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TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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Studies were performed in 30 normal mongrel dogs weighing 18-27 kg. All experimental procedures were approved by the University of Minnesota Animal Care Committee and conformed to the Guide for the Care and Use of Laboratory Animals (National Institutes of Health, 1985).
Experimental Preparation
After sedation with ketamine (10 mg/kg im), animals were anesthetized with
-chloralose (100 mg/kg iv bolus followed by an infusion of 20 mg · kg1 · h1), intubated,
and ventilated with a respirator with supplemental oxygen to maintain
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) at the apical dimple. A similar catheter was placed into the left
atrium through the appendage. Another catheter was inserted via the
right atrial appendage and advanced into the coronary sinus and then
into the anterior coronary vein. The azygous vein was ligated proximal to its entrance into the coronary sinus to avoid contamination of
coronary venous blood with systemic venous blood. A double-tuned (31P and 1H) 28-mm diameter nuclear magnetic
resonance (NMR) surface coil (22, 37) was sutured onto the
anterior LV wall in the distribution of the left anterior descending
(LAD) coronary artery. An intracoronary microcatheter was placed in the
LAD (9) and a hydraulic occluder was placed around the
LAD. The surface coil leads were connected to a balanced-tuned circuit
external and perpendicular to the thoracotomy incision. The animals
were then placed in a lucite cradle and positioned within the magnet.
General Methods of NMR Spectroscopy
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 MR data acquisition to the cardiac cycle, whereas respiratory gating was achieved by triggering the ventilator to the cardiac cycle between data acquisitions (22, 37). 31P and 1H NMR frequencies were 81 and 200.1 MHz, respectively. During each intervention, after steady-state conditions had been reached, Mb- data and HEP data were
acquired by 1H magnetic resonance spectroscopy and
31P magnetic resonance spectroscopy, respectively.
Spatially Localized 31P NMR Spectroscopic Technique
31P spectra were recorded in late diastole with a pulse repetition time of 6-7 s. This repetition time allowed full relaxation for ATP and inorganic phosphate (Pi) resonances, and ~90% relaxation for the PCr resonance. PCr resonance intensities were corrected for this minor saturation (22, 37). Radiofrequency transmission and signal detection were performed with the 28-mm diameter 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, to adjust the position of the animal in the magnet so that the coil was at or near the magnet and gradient isocenters, and to determine the spatial coordinates for spectroscopic localization (22, 37). 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
RAPP-ISIS/FSW method. Detailed data with regard to voxel profiles, voxel volume, and documentation of the accuracy of the spatial localization have been published elsewhere (11, 12, 27). Briefly, signal origin was restricted using B0 gradients
and adiabatic inversion pulses to a column coaxial with the surface
coil and perpendicular to the LV wall; column dimensions were 17 mm × 17 mm. Within this column, the signal was further localized
using the B1 gradient to five voxels centered about 45, 60, 90, 120, and 135° spin rotation. The position of the voxels relative
to the coil was set using the B1 magnitude at the coil
center that was experimentally determined in each case by measuring the
90° pulse length for the phosphonoacetic acid reference located in the coil center. Each set of spatially localized transmural spectra consisted of a total of 96 scans accumulated in a 10-min block. Resonance intensities were quantified using integration routines provided by SISCO software. The numerical values for PCr and ATP in
each voxel were expressed as ratios of PCr to ATP (PCr/ATP). Pi levels were measured as change from baseline values
(
Pi), using integrals obtained in the region covering
the Pi resonance and reported as
Pi/PCr.
1H NMR Spectroscopic Technique
1H NMR methods for measuring Mb- have been
previously described (6, 37). In brief, radiofrequency
transmission and signal detection were performed with the dual-tuned,
28-mm diameter surface coil. A single-pulse collection sequence with a
1-ms frequency selective Gaussian excitation pulse was used to
selectively excite the Mb- resonance. A short repetition time (25 ms) was used, due to the short T1 of Mb-. Each spectrum
was acquired in 5 min (10,000 free induction decays). Although
the short T1 of Mb- and fast acquisition prevent
gating to the cardiac cycle, signal loss due to motion was negligible
due to the inherently broad line width of the Mb- peak. Resonance
intensities were quantified using integration routines provided by the
SISCO software. To verify that Mb- could be detected when it was
known to be present, a total coronary occlusion was produced at the end
of the experiment and a large Mb- resonance was observed. For the
purpose of estimating intracellular PO2, a
myoglobin oxygen P50 value of 2.38 mmHg was assumed and
PO2 was calculated from the Hill equation
(37). It was assumed that at baseline when no Mb-
resonance could be detected, myoglobin was ~10% desaturated and
during coronary occlusion was ~95% desaturated (6).
Myocardial Blood Flow and MO2
Measurements
O2
were measured with radioactive microspheres, 15 µm in diameter,
labeled with 141Ce, 51Cr, 95Nb,
85Sr, or 46Sc (New England Nuclear, Boston, MA)
as previously described (37). MO2 was calculated using the
difference in oxygen content of aortic and anterior interventricular
vein blood multiplied by blood flow.
Tissue Preparation
At the end of the study, the heart was fixed in 10% buffered formalin. The atria, right ventricle, aorta, and large epicardial vessels were dissected from the LV. 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.Experimental Protocol
Aortic and LV pressures were measured with pressure transducers positioned at midchest level and recorded on an 8-channel direct writing recorder (Coulbourne Instruments, Lehigh Valley, PA). LV pressure was recorded at normal and high gain for measurement of end-diastolic pressure. Hemodynamic measurements and magnetic resonance spectroscopy 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 I.
After completing baseline measurements, we examined the response to
sodium pyruvate infusion (11 mg · kg1 · min1 iv)
(17). Once the data were obtained (after allowing 20 min to reach a steady state), dobutamine and dopamine were simultaneously infused (each at a dosage of 20 µg · kg1 · min1 iv) to
produce a high cardiac workstate while the pyruvate infusion was
continued. After we allowed 10 min to achieve steady-state conditions,
we repeated all measurements. Arterial blood samples were also obtained
for the determination of pyruvate.
Group II.
After completing baseline data acquisition, we infused dobutamine and
dopamine as described above; then, after allowing 10 min to achieve
steady-state conditions, we repeated all measurements. Finally, while
the catecholamine infusion continued, pyruvate infusion (11 mg · kg1 · min1 iv) was
begun, and, after allowing 20 min to achieve a steady-state, we
repeated measurements. Arterial blood samples were obtained for the
determination of pyruvate.
Group III. After completion of baseline data acquisition, we infused dobutamine and dopamine as described above; then, after allowing 10 min to achieve steady-state conditions, we repeated all measurements. Finally, while the catecholamine infusion continued, intracoronary sodium butyrate (to achieve a coronary artery blood concentration of ~1.5 mM) was infused, and (after allowing 15 min to achieve a steady state) we repeated measurements.
Data Analysis
Hemodynamic data were measured directly from the chart recordings. Integral numerical values for ATP and Pi resonances during each experimental condition were expressed as PCr/ATP andPi/ATP. 31P NMR spectra from the first,
third, and fifth voxels were taken to represent subepicardium (EPI),
midwall myocardium (MID) and subendocardium (ENDO), respectively.
Data obtained during different experimental conditions within the same group were compared using one-way ANOVA with replications. A value of P < 0.05 was required for significance. When a significant result was found, individual comparisons were made using the Scheffé method. All values are expressed as means ± SE.
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RESULTS |
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TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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Tables 1,
2, and 3
summarize hemodynamic data, myocardial blood flow data, and the HEP
data, respectively. It should be noted that the
Pi resonance (and, therefore, the
Pi/PCr resonance) was not detectable in the
absence of catecholamine stimulation (Fig. 1). Table
4 summarizes the
MO2 data in the subsets of animals in
each group.
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Group I.
In the animals in group I, pyruvate infusion caused modest,
but significant, increases of LV systolic pressure and the heart rate
times systolic blood pressure (RPP), while the arterial pyruvate concentration increased from a baseline value of 160 ± 16 µM to 3.5 ± 0.4 mM before the onset of catecholamine infusion
(P < 0.01). Catecholamine
infusion markedly increased LV systolic pressure, heart rate,
mean aortic pressure, and RPP. Mean myocardial blood flow increased
from a baseline value of 0.55 ± 0.07 to 0.86 ± 0.12 (P < 0.05) and 2.62 ± 0.69 ml · min1 · g1
(P < 0.05) during pyruvate and pyruvate plus
catecholamine, respectively. MO2 (in
the subset of six dogs in which it was measured) increased modestly
from a baseline value of 6.4 ± 0.4 to 10.5 ± 0.8 ml · 100 g1 · min1 during
pyruvate infusion (P < 0.05) and then markedly
increased to 34.1 ± 4.3 ml · 100 g1 · min1 during catecholamine infusion.
Pi/PCr was undetectable at baseline and during
pyruvate alone but increased to 0.28 ± 0.04, 0.26 ± 0.05, and 0.21 ± 0.05 for EPI, MID, and ENDO, respectively, during catecholamine infusion.
Group II.
Catecholamine infusion resulted in significant increases of LV systolic
pressure, heart rate, mean aortic pressure, and RPP. Mean myocardial
blood flow increased from a baseline value of 0.64 ± 0.11 to
1.63 ± 0.23 ml · min1 · g1 during
catecholamine infusion (P < 0.05).
MO2 (in a subset of six dogs in which
it was measured) increased from 7.0 ± 1.0 to 13.4 ± 2.4 ml · 100 g1 · min1 during
catecholamine infusion (P < 0.05). Infusion of
pyruvate during continuing catecholamine infusion did not cause further significant increases in either of these variables, although arterial blood pyruvate levels were averaged ~4.6 mM at the time of the measurements.
Pi/PCr became detectable with
values of 0.27 ± 0.16, 0.22 ± 0.12, and 0.20 ± 0.10 for EPI, MID, and ENDO, respectively (Fig. 1). When the pyruvate
infusion was initiated while the catecholamine infusion continued,
PCr/ATP rose in all myocardial layers (1.90 ± 0.09, 1.85 ± 0.09, and 1.80 ± 0.06 in EPI, MID, and ENDO, respectively;
P < 0.05 for MID and ENDO). During pyruvate,
Pi/PCr trended downward, but this was not
significant. Of note, the magnitude in the reduction of PCr/ATP produced by catecholamine infusion in the group I hearts
that were pretreated with pyruvate was significantly less than in the group II hearts that had not been pretreated with pyruvate
(mean PCr/ATP values for the LV wall were 1.91 ± 0.06 vs.
1.68 ± 0.09, respectively; P < 0.01), and the
decrease in PCr/ATP for individual myocardial layers also tended to be
lower in the group I hearts (P = 0.08).
Group III.
In the animals in group III, catecholamine infusion resulted
in significant increases of LV systolic pressure, heart rate, mean
aortic pressure, and RPP. Mean myocardial blood flow increased from a
baseline value of 0.79 ± 0.13 to 1.93 ± 0.24 ml · min1 · g1 during
catecholamine infusion (P < 0.05).
MO2 (in a subset of five dogs in which
it was measured) increased from 7.6 ± 1.0 to 20.8 ± 2.9 ml · 100 g1 · min1 during
catecholamine infusion (P < 0.05). The infusion of
butyrate during continuing catecholamine infusion did not cause
significant changes in any hemodynamic variable. However, myocardial
blood flow increased to 3.05 ± 0.46 ml · min1 · g1
(P < 0.05 vs. both baseline and catecholamine) and
MO2 increased to 30.4 ± 5.7 ml · 100 g1 · min1,
although this was not significant.
Pi/PCr became detectable with
values of 0.23 ± 0.10, 0.22 ± 0.06, and 0.22 ± 0.06 for EPI, MID, and ENDO, respectively (Fig. 1). When butyrate infusion
was initiated with continuing catecholamine infusion, neither PCr/ATP nor Pi/PCr were significantly affected.
Myoglobin saturation.
No Mb- resonance was detected in any heart of any group under basal
conditions or during catecholamine and substrate infusions. 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/noise ratio (data not shown).
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DISCUSSION |
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TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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The main findings of this study are that 1)
supplemental pyruvate partially (but significantly) ameliorated the
reductions of PCr/ATP that occurred during the high cardiac workstates,
2) supplemental butyrate did not blunt the reductions of
PCr/ATP that occurred at the high workstate, and 3) neither
pyruvate or butyrate significantly increased
MO2 during the high workstate. The
lack of Mb- confirms our earlier finding that limited oxygen availability is not the cause of the high workstate-associated HEP
reductions (37). Taken together, the data imply that the availability of carbon substrate directly utilizable by mitochondria did not limit the rate of ATP synthesis at the high workstate. However, the modest but significant effect of pyruvate on PCr/ATP during the high workstate suggests that supraphysiological
concentrations of pyruvate may affect the kinetics of oxidative phosphorylation.
Implications of the PCr/ATP fall during catecholamine infusion. Creatine kinase is a near equilibrium enzyme and myocardial PCr levels are roughly inversely proportional to calculated cytosolic ADP concentration ([ADP]) levels if total cellular creatine remains constant and there are not marked changes in ATP levels (33). We have previously shown that these conditions are met in in vivo canine myocardium and that calculated free [ADP] is elevated during high workstates, whereas both PCr concentration ([PCr]) and [ATP] are reduced (36). Cytosolic [ADP] is primarily determined by mitochondrial ATP synthesis kinetics (7), and changes in cytosolic [ADP] are reflected by changes in cytosolic [PCr] and the PCr/ATP (33). Thus we can conclude that during moderately increased workstates (not associated with HEP changes), mechanisms other than ADP elevation mediate the increased rate of ATP synthesis (27), but at the higher workstates observed in the present study increases of [ADP] are also required to drive the ATP synthetic rate.
Cytosolic O2 during catecholamine stimulation.
Myoglobin desaturation was not detected during catecholamine infusion,
indicating there was no evidence of inadequate convective or diffusive
oxygen transport at high workstates. As discussed in a previous report
(37), we assume that in the absence of a detectable Mb-
resonance ~10% desaturation is present; this level of desaturation
predicts an intracellular PO2 >20 mmHg (using the Hill equation and a P50 value of 2.38 mmHg)
(29). This PO2 value far exceeds
the Michaelis constant of cytochrome oxidase with regard to oxygen and
should not affect oxidative phosphorylation kinetics or limit ATP
synthesis. Thus the current data confirm our previous finding that
inadequate oxygen availability per se does not cause the high
workstate-associated reduction of PCr/ATP.
Effects of pyruvate infusion.
Pyruvate, when infused during basal workstates, caused
modest but significant increases of RPP and
MO2. However, pyruvate had no
effect on PCr/ATP. These observations are in contrast to findings in
the perfused heart utilizing only glucose; in that model the addition
of pyruvate markedly increased PCr/ATP and NADHm
(7). The modest nonsignificant response to pyruvate in the
basal state is not surprising because the in vivo heart has access to
abundant alternative carbohydrate and lipid substrates so that
[NADHm] is presumably high and nonlimiting. The current data differ somewhat from earlier in vivo canine data reported by
Laughlin et al. (17), who also reported a modest but
significant increase of PCr/ATP (but not of
MO2) when a comparable amount of
pyruvate was infused during basal workstate conditions. However, it
should be noted that the physiological changes in both studies were
small. The modest in vivo inotropic response to pyruvate under basal
conditions in the present study suggests that the much more pronounced
inotropic response to pyruvate in isolated myocardium and perfused
hearts may be due to the less physiological state of the experimental preparation.
O2. However, in these
hearts, pyruvate did cause an increase of PCr/ATP. Hence, pyruvate
modestly (but significantly) limited the decrease in PCr/ATP produced
by catecholamine infusion. However, in neither case was pyruvate
supplementation capable of maintaining PCr/ATP at normal levels during
the increased workstate.
Based on studies in perfused hearts, a high concentration of pyruvate
in the blood and myocytes should have increased [NADHm] and largely corrected the reduced PCr/ATP if the HEP reduction was
caused primarily by limited flux of acyl-CoA into the tricarboxylic acid cycle via PDH (5, 7, 16, 30). The inability of a high
concentration of pyruvate to prevent or fully reverse the PCr/ATP
reductions or to significantly increase
MO2 during the high workstate supports
the view that carbohydrate substrate availability and metabolism cannot
be the only cause of the PCr/ATP reductions and that carbohydrate
substrate availability does not limit the rate of ATP synthesis.
However, the modest but significant amelioration of the HEP changes in
response to supraphysiological concentrations of pyruvate suggests that
this intervention can affect the kinetics of oxidative phosphorylation.
There is evidence that oxidative phosphorylation is kinetically
regulated by the concentrations of its primary substrates, which
include O2, ADP, Pi, and NADH (7).
Over a broad range of workstates in the perfused heart (in which the
availability of Pi and O2 concentrations are
not limiting), the relationship between
MO2 and RPP is relatively independent
of the carbon substrate utilized (7). In contrast, the
relationship between ADP and MO2 is
highly sensitive to the specific substrate employed; thus ADP levels
are lowest with substrates that induce high NADHm levels
(supraphysiological concentrations of pyruvate or octanoate) and are
highest when glucose or glucose plus insulin are the substrate (7). In the latter cases, NADHlm levels are
relatively low (5, 16, 30). Hence, if in the current study
[NADHm] was within the kinetic regulatory range for
oxidative phosphorylation at high workstates, then the administration
of supraphysiological concentrations of pyruvate could have increased
acyl-CoA levels by means of PDH activation and/or a mass action effect
on PDH. In this regard, in an in vivo porcine model, flux through PDH was increased during dobutamine infusion, although the PDH activation state was not increased or decreased (unpublished data cited in Ref.
10). It is of interest that the substantial
increase of fatty acid uptake induced by dobutamine might have been
expected to cause inhibition of PDH (10). The authors
suggested that this increase of flux through PDH was likely mediated by
increased pyruvate availability resulting from increased lactate and
glucose uptake. These findings support the view that increased pyruvate levels can increase the rate of conversion of pyruvate to acyl-CoA during dobutamine infusion, which could, in turn, cause
[NADHm] to increase. An increase of [NADHm]
would be expected to result in lower [ADP] and higher [PCr]. As
indicated above, if at high workstates [NADHm] was not
sufficiently low to limit the rate of oxidative phosphorylation, then
the postulated increase in [NADHm] produced by pyruvate
would not be expected to increase MO2
despite the fact that it could reduce [ADP] (5, 7, 16,
30).
Effects of butyrate infusion.
Butyrate infused during the high workstate had no significant effect on
hemodynamics or PCr/ATP. However, during butyrate infusion, myocardial
blood flow rose significantly and MO2
tended to increase, although the latter did not reach statistical
significance (perhaps because of the relatively small number of
animals). Moreover, these changes occurred in the absence of an
increase of RPP. This observation is not surprising because predominant
fatty acid utilization has been shown to increase
MO2 (as compared with predominant glucose utilization) in the catecholamine-stimulated canine heart without affecting hemodynamics (20, 21). The mechanisms of this MO2 increase are complex and
involve an obligatory component due to the lesser efficiency of ATP
production through 
-lipid oxidation compared with the aerobic
glycolytic production of ATP as well as energy wasting resulting from
fatty acid cycling within the myocyte (20, 21). The trend
toward increased MO2 is of importance
because it demonstrates that, although the high workstate in this model
was associated with reduced PCr/ATP and ATP loss, this alteration does
not appear to compromise the capacity to synthesize ATP. The latter
statement assumes that butyrate did not cause mitochondrial uncoupling.
Our earlier work in the perfused heart (15) and the
failure of PCr/ATP to fall during butyrate infusion argue against
uncoupling at the concentration of butyrate employed. Furthermore, even
if some uncoupling were present, the current data would still indicate
that electron transport chain capacity did not limit the rate of ATP
synthesis at high workstates because PCr/ATP did not fall further when
butyrate was infused. The lack of a response of PCr/ATP during butyrate infusion is puzzling. Short-chain fatty acids bypass the
palmitoyl-carnitine transporter and increase mitochondrial acyl-CoA
(and [NADHm]) in perfused hearts (5, 7, 16, 23,
30). Furthermore, -hydroxybutyrate has been shown to increase
PCr/ATP in basal-state canine myocardium (14). Hence, if
both pyruvate and butyrate can raise [NADHm], why did
only pyruvate cause PCr/ATP to increase during the high workstate?
Because butyrate increased myocardial blood flow and tended to increase
MO2, it is difficult to argue that
butyrate did not enter the mitochondria; however, this does not prove
that butyrate affected [NADHm] as well. However, if butyrate did increase [NADHm], it is possible that the
pyruvate effect on PCr/ATP may not have been mediated by elevation of
[NADHm].
O2 and fatty acid uptake (31,
32). However, when fatty acid uptake was blocked with
oxfenicine, catecholamine infusion caused a fall in PCr/ATP, although
the catecholamine-induced increase of
MO2 was not affected. Presumably, the
MO2 response to catecholamine was
maintained because compensatory increases of glucose and lactate uptake
were sufficient to support the increased rate of ATP synthesis, albeit
with the requirement for an elevation of cytosolic ADP (31). Importantly, these data indicate that at least in
the in vivo right ventricle, alterations of substrate mix can alter the
kinetics of oxidative phosphorylation dramatically without reducing the
rate of ATP synthesis.
Alternative mechanisms for pyruvate effects on PCr/ATP. In addition to its metabolic properties, pyruvate has been shown to act as an antioxidant (2, 34, 35). Therefore, it is possible that the effect of pyruvate on PCr/ATP may depend on antioxidant or other properties rather than effects on [NADHm]. For example, the function of the electron transport chain is facilitated by increases of mitochondrial matrix volume (19), and the terminal component cytochrome oxidase is regulated allosterically by ATP/ADP (13) and also by nitric oxide (NO), which competitively inhibits the binding of O2 to cytochrome oxidase (4, 8). It has recently been reported that the rate of free radical generation by mitochondria is increased by NO and that exercise, which is mimicked by dobutamine infusion, increases both NO and free radical production by the heart (3, 28). Inhibition of the electron transport chain or of its terminal component cytochrome oxidase by NO or by increased levels of other free radical species might reduce the mitochondrial proton motive force sufficiently to require an increase of cytosolic [ADP] to maintain a given rate of ATP synthesis. These effects might be limited by free radical scavenging by pyruvate (2, 34, 35).
Limitations. A critical question is whether the pyruvate infusion increased intracellular [pyruvate] sufficiently to induce activation of pyruvate dehydrogenase. It has recently been reported that a 1 mM coronary artery blood concentration of pyruvate [pyruvate] failed to increase intracellular [pyruvate] in an in vivo porcine model (25). However, earlier work in perfused hearts demonstrated that a [pyruvate] perfusate of 2 mM or greater resulted in a substantial increase in the active fraction of PDH (24). In the current study, arterial plasma [pyruvate] was 3.5-4.6 mM at the time HEP measurements were obtained. Laughlin et al. (17) demonstrated that plasma pyruvate levels of 2.86 ± 0.27 mM increased myocardial pyruvate extraction and markedly decreased glucose and lactate extraction in open-chest dogs; the effects on glucose and lactate uptake likely resulted from the well-known intracellular inhibitory effect of pyruvate on glycolysis and the effects of increased extracellular pyruvate on trans-sarcolemmal lactate transport (17). Furthermore, blood [pyruvate] of ~3 mM was able to increase glycogen accumulation rates sixfold in the canine heart when glucose uptake was maintained by glucose and insulin infusion; again, this could only result from inhibition of glycolysis by pyruvate entry into the myocyte (17). Although we were unable to measure the pyruvate uptake rate or intracellular [pyruvate] in the present study, our observations, together with the data cited above, indicate that it is probable that the coronary blood [pyruvate] achieved in the present study resulted in an increased rate of pyruvate uptake and a significant elevation of the cytosolic pyruvate level. An additional limitation of the present study is that we were not able to determine myocardial [NADHm]. Therefore, our discussion of the effects of pyruvate on [NADHm] are speculative, although based on reasonable inferences from studies of isolated myocardium and perfused hearts.
The present findings demonstrate that the reductions of PCr/ATP that occur at high workstates in the canine heart do not indicate limitation of the rate of ATP synthesis as the result of carbon substrate inadequacy or limitation of O2 availability. Hence, other explanations must be sought. High workstate-associated functional limitations at the level of the electron transport chain and especially at its terminal component, cytochrome oxidase, or possibly of ATP synthase could profoundly affect the kinetics of oxidative phosphorylation and are candidates for future study.| |
ACKNOWLEDGEMENTS |
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This work was supported by National Heart, Lung, and Blood Institute Grants HL-33600, HL-50470, HL-61353, HL-21872, and HL-58840, Department of Veterans Affairs Medical Research Funds, and an Established Investigator Award from the American Heart Association (to J. Zhang).
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FOOTNOTES |
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Address for reprint requests and other correspondence: J. Zhang, Univ. of Minnesota Health Science Center, 420 Delaware St. SE, Mayo Mail Code 508, Minneapolis, MN 55455 (E-mail: zhang047{at}maroon.tc.umn.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 25 October 2000; accepted in final form 29 June 2001.
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METHODS
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