© 1997 American Heart Association, Inc.
Articles |
Altered Metabolite Exchange Between Subcellular Compartments in Intact Postischemic Rabbit Hearts
From the NMR Center (E.D.L., X.Y., L.T.W., J.M.O.), Department of Radiology, Massachusetts General Hospital and Harvard Medical School, Boston, Mass, and the Department of Physiology (K.F.L., C.D.), Hershey Medical Center, Pennsylvania State University, Hershey.
Correspondence to E. Douglas Lewandowski, PhD, NMR Center, Massachusetts General Hospital, Building 149, 13th St, Charlestown, MA 02129. E-mail doug{at}nmr.mgh.harvard.edu
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Abstract To examine metabolic regulation in postischemic hearts, we examined oxidative recycling of 13C within the glutamate pool (GLU) of intact rabbit hearts. Isolated hearts oxidized 2.5 mmol/L [2-13C]acetate during normal conditions (n=6) or during reperfusion after 10 minutes of ischemia (n=5). 13C-Nuclear magnetic resonance spectra were acquired every 1 minute. Kinetic analysis of 13C incorporation into GLU provided both tricarboxylic acid (TCA) cycle flux and the interconversion rate (F1) between the TCA cycle intermediate,
-ketoglutarate (-KG), and the largely cytosolic
GLU. The rate-pressure product in postischemic hearts
was 46% of normal (P<.05). No difference in substrate
utilization occurred between groups, with acetate accounting for 92%
of the carbon units entering the TCA cycle at the citrate synthase
step. TCA cycle flux in postischemic hearts was normal
(normal hearts, 10.7
µmol·min-1·g-1;
postischemic hearts, 9.4
µmol·min-1·g-1),
whereas F1 was 72% lower at 2.9±0.4 versus 10.2±2.5
µmol·min-1·g-1
(mean±SE) in normal hearts (P<.05). From additional hearts
perfused with 2.5 mmol/L [2-13C]acetate plus
supplemental 5 mmol/L glucose, any potential differences in
endogenous carbohydrate availability were proved not to
account for the reduced rate -KG and GLU exchange, which remained
depressed in postischemic hearts. However, specific
activities of the transaminase enzyme, catalyzing chemical exchange of
-KG and GLU, were the same, and transaminase flux was 100
µmol·min-1·g-1
in postischemic hearts versus 68
µmol·min-1·g-1
in normal hearts. Normal transaminase activity and the increased flux
in postischemic hearts are contrary to the reduced
F1. The findings indicate reduced metabolite transport
rates across the mitochondrial membranes of stunned
myocardium, particularly through the reversible
-KG–malate carrier.
Key Words: stunned myocardium • tricarboxylic acid cycle • mitochondria • reperfusion • nuclear magnetic resonance spectroscopy
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A growing body of literature supports the notion that the intermediary metabolism contributes to the level of functional recovery in the postischemic myocardium.1 2 3 4 5 6 7 8 9 Additionally, although the rates of substrate oxidation in postischemic hearts have been examined previously,10 11 12 13 much less is known regarding the regulation of intermediary metabolism and the coordination of flux rates through different pathways of oxidative metabolism. Of course, postischemic contractile dysfunction in the heart, or myocardial stunning, is known to be a complex process, but the regulatory links between oxidative intermediary metabolism in the mitochondria and the physiochemical state of the cytosol are likely to play an important role in managing the metabolic demands for function in postischemic myocardium. For this reason, we have conducted a study using on-line 13C-NMR spectroscopy of the postischemic myocardium in a dynamic fashion to assess the potential for altered subcellular interactions between the mitochondrial and cytosolic compartments of the intact stunned myocardium. The general implication of the present study is that the pathophysiological state of an organ or intact tissue is translated between subcellular compartments via the exchange of intermediary metabolites. Such exchange, as observed here in the intact heart, may provide a level of metabolic regulation not yet extensively studied.
Previous work from our laboratory on isolated rabbit hearts oxidizing 13C-enriched substrates indicated that the rates of carbon turnover within the glutamate pool were slower in postischemic hearts than in normal hearts.1 14 Because the rate of 13C turnover within glutamate is known to be closely related to the rate of labeled substrate entry and recycling within the oxidative TCA cycle,15 16 17 18 one interpretation of this finding was that the depressed function of the stunned myocardium required less energy production from the oxidation of carbon-based fuels. This notion was consistent with the previous observation of reduced flux across the creatine kinase reaction in the postischemic heart.19 However, in the previous 13C-NMR study, the reduced isotope turnover within the glutamate pool was inconsistent with the relatively normal rates of O2 use by the same postischemic hearts. Subsequently, a study by Weiss et al20 confirmed this reduced rate of 13C turnover within the glutamate pool of postischemic hearts. These investigators used an empirical metabolic model to demonstrate the potential for such reduced 13C recycling to occur if the isotope transfer between the observed glutamate pool and the TCA cycle intermediates was delayed.
Despite the consideration that TCA cycle flux may be normal in
postischemic hearts, the reduced interconversion rates
between glutamate and the TCA cycle intermediate, -ketoglutarate,
have yet to be directly related to the activity of the GOT enzyme,
which catalyzes this reaction. The enzyme is not allosterically
regulated and thus does not exist in an active or inactive form. What
also remains to be accounted for in this reduced transfer of label
between the TCA cycle and glutamate is consideration of the exchange of
metabolic intermediates between subcellular
compartments.
The TCA cycle enzymes are located in the mitochondrial matrix, whereas
>90% of the myocardial glutamate pool, which becomes labeled with
13C, is located in the cytosolic space.17 21
We have already shown that flux through GOT in the normal heart is much
too fast, at 20-fold, to account for the observed rates of isotope
turnover in glutamate at normal TCA cycle flux rates.17 A
more recent study from our laboratory then demonstrated that the rate
of 13C incorporation into the glutamate pool of the heart
is influenced by the rate of -ketoglutarate transport from the TCA
cycle across the mitochondrial membrane via the malate-aspartate
shuttle.22 23 Therefore, the possibility exists for the
delayed isotope turnover rates in postischemic hearts to
result from altered metabolite transport and not from changes in TCA
cycle flux or transaminase activity.
By combining a comprehensive kinetic analysis of
dynamic-mode 13C-NMR spectra of intact functioning hearts
with characterization of the GOT isozyme activities from the
mitochondrial and cytosolic compartments, we have previously
demonstrated the influence of metabolite transport across the
mitochondrial membrane on the rate of isotope turnover within the
NMR-observed glutamate pool of the intact heart.17 22 The
aim of the present study was to extend such analysis to the
stunned myocardium, including evaluation of the GOT
isozymes, to determine the source of reduced carbon turnover, which
appears to be characteristic of oxidative metabolism in
postischemic hearts.1 14 20 The present
study examined both flux through the TCA cycle and the rate of label
exchange between -ketoglutarate and glutamate compared with the
measured activity of GOT in the postischemic heart. The
findings indicate that the exchange of metabolic
intermediates between the mitochondria and cytosol is altered in the
postischemic stunned heart. Evidence is provided for a link
between the pathophysiological state of the
myocardium and the metabolic link between the
cytosol and mitochondria via exchange of metabolites.
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Isolated Heart Preparation
Hearts were excised from Dutch belted rabbits (550 to 700 g) that were heparinized (1000 U) and anesthetized with sodium pentobarbital (100 mg/kg IP injection). Immediately upon excision, the heart was immersed in a solution containing 20 mmol/L KCl and 120 mmol/L NaCl for cardioplegia at 0°C. The aorta was cannulated for retrograde perfusion at 100-cm hydrostatic pressure with a modified Krebs-Henseleit buffer equilibrated with 95% O2/5% CO2 at 37°C. The buffer contained (mmol/L) NaCl 116, KCl 4, CaCl2 1.5, MgSO4 1.2, NaH2PO4 1.2, and NaHCO3 25. During preparation, hearts were perfused with buffer containing 5 mmol/L glucose. This perfusate supply was later changed at the start of each 13C-enrichment protocol to a 450-mL reservoir of Krebs-Henseleit buffer containing 2.5 mmol/L [2-13C]acetate (Isotec Inc) with no glucose. A latex balloon was placed in the left ventricle and connected to a pressure transducer line and physiograph (Gould, Inc). The balloon was inflated with water to create a diastolic pressure of 5 to 10 mm Hg. Left ventricular developed pressure (LVDP) and heart rate (HR) were continually measured and recorded from the intraventricular balloon. RPP (HRxLVDP) was used as an index of mechanical work. O2 content of the perfusion medium and coronary effluent was determined with a blood gas analyzer. During either normal perfusion or reperfusion, M
O2 was determined for each heart from
the difference between the O2 contents of the
perfusate at the aortic cannula and the coronary
effluent, as described elsewhere.16 24 The temperature of
the hearts in the magnet was continuously maintained at 37°C by warm
air flow controlled at the NMR system console, and hearts perfused on
the bench were maintained at 37°C by a water jacket.
Experimental Protocols
NMR experiments were performed on both normal and
postischemic rabbit hearts. Postischemic hearts
were subjected to 10 minutes of zero-flow ischemia at 37°C
before reperfusion. At the start of each protocol, hearts were perfused
(n=6) or reperfused (n=5) with 2.5 mmol/L unlabeled acetate and no
glucose for 10 minutes to ensure metabolic
equilibrium.25 At this time, a "background" spectrum
of naturally abundant (1.1%) 13C was acquired. The
substrate supply was then switched from unlabeled acetate to 2.5
mmol/L [2-13C]acetate, and sequential 13C
spectra were then acquired every 1.25 minutes. Normal hearts and
postischemic hearts were then perfused with
13C-enriched media for an additional 30 minutes. At the end
of each experiment, hearts were rapidly frozen for in vitro NMR
analysis and tissue chemistry. Acetate was chosen as a
substrate in order to compare the results of these experiments with
previously published data on TCA cycle flux measurements with
13C-NMR in stunned hearts. By using similar substrate
delivery protocols, the hypothesis could then be tested as to whether
changes in the transaminase enzyme, which interconverts
-ketoglutarate and glutamate, were the reason that 13C
enrichment is delayed in the postischemic heart, that is
oxidizing acetate.
In order to control for the potential for differences in endogenous carbohydrate availability, which was due to lower glycogen content in the postischemic group, additional hearts were perfused with 2.5 mmol/L [2-13C]acetate plus supplemental 5 mmol/L unlabeled glucose (normal hearts, n=4; postischemic hearts, n=7). In these groups, hearts received 2.5 mmol/L unlabeled acetate and 5 mmol/L glucose for 10 minutes to ensure metabolic equilibrium before delivering [2-13C]acetate and unlabeled glucose. Normal and postischemic hearts receiving supplemental glucose were subjected to the identical protocol in the NMR magnet and then freeze-clamped, as described above. To further examine whether the supplemental glucose contributed to the energy metabolism of the hearts perfused or reperfused with 2.5 mmol/L acetate, additional hearts were perfused with the reverse labeling scheme. Hearts were either perfused (n=3) or reperfused (n=4) with unlabeled 2.5 mmol/L acetate plus 5 mmol/L [1-13C]glucose and then freeze-clamped after 40 minutes for in vitro NMR analysis and tissue chemistry.
At the end point of each NMR experiment, all hearts were rapidly frozen within a liquid nitrogen–cooled clamp. Frozen ventricular tissue was used to determine the tissue concentrations of key metabolites for biochemical assays and in vitro NMR spectroscopy, as described below.
Bench-top experiments were also performed on isolated rabbit hearts to determine the activity of the key enzyme, GOT, in normal (n=5) and postischemic (n=3) myocardium. For characterization of GOT kinetics at the appropriate time point corresponding to the introduction of the carbon isotope, hearts were homogenized fresh after either 10 minutes of normal perfusion or 10 minutes of reperfusion.
Additional sets of hearts were frozen at additional time periods for comparison of glutamate levels in postischemic hearts with levels in heart tissue frozen at the end of the protocol performed in the NMR magnet. Four additional groups of hearts were freeze-clamped (1) after perfusion with 5 mmol/L glucose (n=3), (2) immediately after 10 minutes of ischemia with no reperfusion (n=3), (3) at 10 minutes of reperfusion with 2.5 mmol/L acetate (n=3) to correspond to the introduction of 13C-enriched acetate during the NMR experiments, and (4) at 30 minutes of reperfusion with acetate (n=3). These hearts allowed the metabolite content to be measured after the introduction of acetate immediately after ischemia, at the time point corresponding to the introduction of carbon isotope during the NMR protocol, and at a 30-minute time point corresponding to steady state isotope enrichment. Biochemical assays of tissue metabolites were performed on acid extracts of frozen ventricle as described below. These measurements allowed comparison of tissue metabolite levels between the early and late phases of reperfusion to ensure that the key tissue metabolite compartments for the kinetic analysis were not changing over the course of the reperfusion period.
Isotope-Labeling Scheme
Over the course of perfusion with 13C-enriched
substrate, incorporation of label into the glutamate pool was detected
by 13C-NMR. The appearance of resonance peaks that
correspond to 13C enrichment at specific carbon positions
within the glutamate pool have been described in detail by
others.14 15 17 Detection of the 13C-NMR
signal from glutamate is based on the relatively high concentration of
intracellular glutamate that is in constant exchange with the TCA cycle
via -ketoglutarate. The pattern of pre–steady state isotope
enrichment of the carbon positions of the TCA cycle intermediates and
glutamate is shown in Fig 1
. Initial incorporation of
label from the oxidation of [2-13C]acetate into the
glutamate pool occurs at the 4-carbon position. The recycling of the
label places 13C at either the 2- or 3-carbon of glutamate.
The labeling at the 2- and 3-carbon of glutamate is of equal
probability because of the symmetry of succinate molecule. Since
glutamate is largely located in the cytosol, whereas the TCA cycle
occurs in the mitochondria, 13C-enriched
-keto-glutarate is transported out of the mitochondria before
chemical exchange occurs between -ketoglutarate and
glutamate.21
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Secondary
enrichment sites at the 2- and 3-carbon positions, as label from
4-carbon position is recycled through the TCA cycle.
Dynamic 13C-NMR Spectroscopy
Perfused hearts were placed within a sample tube in a 20-mm
broad-band NMR probe (Bruker Instruments), which was situated in a
vertical-bore superconducting NMR magnet operating at a field strength
of 9.4 T (Bruker Instruments). NMR data were collected using a Bruker
400-MSL NMR spectrometer. Before each experiment, the magnetic field
homogeneity was optimized by shimming on the proton signal of water in
the sample to a line width of 15 to 30 Hz.
13C-NMR spectra from intact hearts were acquired at 101 MHz with a 45° pulse angle and 2-second recycle time over 30 scans (1.25 minutes). Bilevel broad-band decoupling at 0.5 W (1.8 seconds) and 7.0 W (17 microseconds) was applied to eliminate carbon-proton coupling and induce nuclear Overhauser enhancement without sample heating. The free induction decay was acquired with an 8 kiloword data set. Changes in relative signal intensities due to nuclear Overhauser enhancement or relaxation effect were negligible under these pulsing conditions.26 27 Natural-abundance 13C signal was digitally subtracted, and the raw signal was processed by exponential filtering with a line broadening of 20 Hz to enhance the signal-to-noise ratio before being converted into frequency domain by Fourier transformation. Peak assignments were referenced to the known resonance of the exogenous 13C-enriched substrate (2-carbon of acetate at 24.1 ppm) and the well-documented glutamate signals.15 28 NMR signal intensities were determined for all spectra by curve-fitting each resonance peak with a lorentzian curve and integrating the area under the fitted curve with NMR-dedicated software (NMR1, Tripos Associates, Inc).
In Vitro 13C-NMR Spectroscopy
High-resolution 13C-NMR spectra of tissue
extracts reconstituted in 0.5 mL D2O were obtained with a
5-mm 13C probe (Bruker Instruments). In vitro
13C spectra were collected over 3K data points (45°
pulse, 1.8-second recycle time) with broad-band proton decoupling. The
free induction decay was acquired over 32K data points and zero-filled
to 64K to improve spectral resolution. The signal was processed by 2-Hz
exponential filtering, followed by Fourier transformation. The
multiplet structure of the glutamate carbon signal allowed
Fc and the ratio of anaplerotic flux to citrate synthase
(y) to be calculated.29 The amount of glutamate labeled at
the 4-carbon position was determined by comparing the signal intensity
from glutamate 4-carbon resonance peak within each 13C
spectrum to a standard 100 mmol/L (1.1 mmol/L 13C
natural abundance) solution of glutamate. Total 13C-labeled
glutamate at the 4-carbon position was then divided by total tissue
concentration of glutamate from enzymatic assay to obtain the
fractional enrichment at the 4-carbon position of
glutamate.16 17 27
Tissue Biochemistry
Perchloric acid extracts were obtained from
ventricular muscle of hearts as previously
described.16 Glutamate, -ketoglutarate, aspartate, and
citrate contents in myocardium were determined by
spectrophotometric or fluorometric techniques.30 31
Extracts were then lyophilized and reconstituted in 0.5 mL
D2O. High-resolution 13C spectra of
reconstituted extract material were obtained.
Enzymology
The exchange of carbon isotope from the TCA cycle to glutamate
occurs through a transamination reaction catalyzed by the GOT enzyme.
Two isoforms of GOT exist, mitochondrial and cytosolic.32
The Km values for both -ketoglutarate and
aspartate of both GOT isozymes have been determined in a previous study
from our laboratory.17 In the present study, the
specific activities of the GOT in normal versus
postischemic hearts were compared. From the measured tissue
concentrations of the substrates for this enzyme, -ketoglutarate and
aspartate, flux through GOT could then be determined for both normal
and postischemic hearts. Tissue was harvested at the
10-minute mark of normal perfusion or reperfusion, with the time point
corresponding to the introduction of label in the
13C-enrichment protocol.
Isolated rabbit hearts were perfused or reperfused, as described in the protocols above, with MSEE buffer (mmol/L: mannitol 225, sucrose 75, MOPS 5, EDTA 0.1, and EGTA 0.2 at 37°C and pH 7.0). From homogenized tissue, the cytosolic and mitochondrial fractions were separated by differential centrifugation.33 Measurement of protein content per milliliter in the total homogenate and in the supernatant after high-speed centrifugation (14 000g) yielded the percent of supernatant (cytosolic) present in the total rabbit heart homogenate. Measurements of citrate synthase activity34 in the total homogenate and the isolated mitochondrial fraction allowed determination of the percent mitochondrial protein in the total homogenate. The fraction of mitochondrial protein was determined to be 31%. The contamination of the supernatant fraction with mitochondrial matrix enzymes was determined by measuring citrate synthase in the supernatant. This value was used to correct the GOT activity in the supernatant for any contaminating mitochondrial GOT isozyme.
The specific activity of GOT for the synthesis of glutamate and
oxaloacetate was measured at 37°C in (mmol/L) KCl 150, MOPS 25, Tris
10, NaCl 5, and NADH 0.15, along with 2 U/mL malate dehydrogenase at pH
7.2. The reaction was executed in the presence of 10 mmol/L
aspartate and 10 mmol/L -ketoglutarate. Enzymatic activity
in the forward and reverse directions was determined by the change in
absorbance at 340 or 280 nm, respectively. Mitochondrial GOT activity
was determined at 37°C and at pH 7.7 to mimic the mitochondrial
matrix.35 Aliquots of mitochondria were preincubated with
0.05% Triton X-100 to permeabilize the mitochondria.
The activity of citrate synthase was measured at 412 nm in 50
mmol/L Tris-HCl, 0.1 mmol/L 5,5'-dithiobis-(2-nitrobenzoic acid),
0.5 mmol/L acetyl-CoA, and 0.05% Triton X-100, pH 8.0 at 37°C.
The reaction was started by the addition of 1 mmol/L oxaloacetate.
Kinetic Model and Data Analysis
Analysis of metabolic activity was based on
observations of pre–steady state 13C-NMR spectra of
glutamate, reflecting the dynamic labeling pattern described above. A
kinetic model was used to investigate 13C labeling within
key metabolic compartments, as described elsewhere in
greater detail.17 The original model is derived from the
simplified metabolic compartment model, which includes key
rate-limiting tricarboxylate pools as well as the major contributing
amino acid pools, glutamate and aspartate. Analysis was
performed at steady state flux through the TCA cycle and with constant
metabolite pool sizes. Effects of both the exogenous 13C
and endogenous substrate entry into the TCA cycle at the
citrate synthase reaction along with unlabeled anaplerosis are
accounted for by incorporating data measured from high-resolution
13C-NMR as parameters into the model. This
model can be used with or without
MO2 as a input parameter
to improve the accuracy of the output variables. Previous
analysis has demonstrated that only a 10% difference was
generated in the VTCA determined from this model when
MO2 was not used.
As previously described, the kinetic model is composed of nine
differential equations and was derived by the principle of mass
conservation among the metabolic compartments or pools to
characterize both VTCA and the interconversion rates
between metabolite pools.17 18 22 The entire compartment
model could be simplified by focusing on the 13C enrichment
at the 4-carbon and 2-carbon positions of glutamate and eliminating
equations that describe 13C enrichment at the 3-carbon site
due to the symmetry of 2- and 3-carbon labeling. The resulting
equations that were applied for this analysis are as
follows:
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KG, GLU, MAL, OAA, and ASP denote the metabolites
citrate, -ketoglutarate, glutamate, malate, oxaloacetate, and
aspartate, respectively. F1 and F2, the fluxes
of interconversion between -ketoglutarate and glutamate and between
aspartate and oxaloacetate, respectively, include both transamination
and membrane transport. Pool sizes are expressed as µmol/g dry
wt; flux values, as
µmol·min-1·g dry
wt-1.
The detected signals in this model are the 2- and 4-carbon–enrichment
sites of glutamate. Acetyl-CoA enrichment (Fc) and the
ratio of anaplerosis to citrate synthase (y) were determined from the
high-resolution NMR spectrum of tissue extracts. Glutamate, aspartate,
-ketoglutarate, and citrate concentrations were measured by
enzymatic assays. Other metabolite concentrations whose values are
relatively stable to the changes in VTCA were taken from
literature on hearts perfused under similar substrate conditions:
malate, 0.60 µmol·g dry wt-1;
oxaloacetate, 0.04 µmol·g dry
wt-1.36 37 As shown by extensive
sensitivity analysis, the model is relatively insensitive to
changes in these metabolite pools values taken from the
literature.17 18
Since alanine and pyruvate concentrations in all four experimental
groups were low, alanine aminotransferase activity was minimal.
Therefore, the interconversion rate between -ketoglutarate and
glutamate and between oxaloacetate and aspartate were set equal, ie,
F1=F2. As performed in an earlier application
of the model, the effect of possible variations or errors in these pool
sizes was evaluated by sensitivity analysis with a 3-fold
change in pool sizes.17 18 The TCA cycle flux, given as
VTCA, and the interconversion rate between TCA cycle
intermediates and glutamate, given as F1, were determined
by nonlinear least-squares fitting of the model to
13C-enrichment data from NMR spectra using the
Levenberg-Marquardt method (MATLAB, The MathWorks Inc) on a computer
workstation (Sun Microsystems).
Statistical Analysis
Comparison of intragroup data sets was performed with Student's
paired two-tailed t test. Testing for differences between
multiple groups or data sets was performed using ANOVA.
Analysis of multiple data sets obtained from the same group
over time was performed using repeated-measures ANOVA. Differences in
mean values were considered statistically significant at
P<.05.
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Contractile Function and M
O2RPP values indicated a level of contractile dysfunction in the postischemic group that was consistent with previous observations of postischemic dysfunction in isolated hearts.38 39 40 Fig 2A
displays the
values of RPP over the course of perfusion and illustrates the reduced
capacity for mechanical function in postischemic hearts
oxidizing acetate compared with control hearts oxidizing acetate.
Before the 10-minute period of ischemia in the
postischemic group, RPP (mean±SD) was 20 389±2490
bpm·mm Hg, a value similar to the RPP value of 19 274±6660
bpm·mm Hg observed before the start of the control perfusion with
acetate (initial perfusion with glucose). Also consistent with
previous observations,14
MO2 was not statistically different
between normal and postischemic hearts (19.4±3.7
µmol·min-1·g dry
wt-1 for normal hearts and 17.4±3.6
µmol·min-1·g dry
wt-1 for postischemic hearts)
despite the large sustained differences in mechanical function between
both groups.
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represents values from normal hearts; 
, values from
postischemic hearts. Postischemic hearts
displayed significantly reduced RPP throughout the reperfusion period
and reduced RPP compared with normal hearts (P<.05). B, RPP
during normal perfusion or reperfusion is shown for oxidation of
acetate in the presence of supplemental glucose (mean±SE).
represents values from normal hearts; 
, values
from postischemic hearts. Postischemic hearts
displayed significantly reduced RPP throughout the reperfusion period
(P<.05). The presence of supplemental glucose had no
significant effect on RPP compared with that from hearts oxidizing
acetate alone in panel A.
Fig 2B
displays the values of RPP over the course of perfusion in
hearts receiving both [2-13C]acetate and supplemental
glucose (5 mmol/L) to control for differences in carbohydrate
availability between normal and postischemic hearts. Again,
mean RPP values were consistently depressed in the
postischemic group (P<.05). RPP values were not
affected by the presence of glucose in this protocol and were similar
to those for hearts oxidizing acetate alone, as shown in Fig 2A.
MO2 was also not significantly affected
by the addition of supplemental glucose (normal hearts, 17.5±0.67
µmol·min-1·g dry
wt-1; postischemic hearts,
17.0±1.3 µmol·min-1·g dry
wt-1) compared with
MO2 in hearts oxidizing acetate
alone.
Metabolite Content, In Vitro NMR, and GOT Kinetics
Steady state metabolite contents for all experimental groups are
tabulated in Table 1. Of note is the lower level of
glutamate in postischemic hearts compared with normal
hearts that were oxidizing acetate (P<.05). Fig 3 displays the results of glutamate assays performed on
myocardium sampled at various times during the
ischemia/reperfusion protocol with acetate alone. As observed
in a previous study, glutamate levels did not drop during the brief
(10-minute) period of ischemia compared with
preischemic perfusion with glucose.7 (All
hearts in this study received glucose as sole substrate before either
normal perfusion or reperfusion with 13C-enriched acetate.)
During reperfusion, the mean glutamate levels (11.1 to 15.2
µmol/g dry wt) were not as elevated as in the normal hearts that were
perfused with acetate, which had a mean glutamate content of 29.1
µmol/g dry wt (Table 1). Instead, the relatively low glutamate level
that was present in the heart during perfusion with glucose
remained unchanged throughout 10 minutes of ischemia and 40
minutes of reperfusion. Therefore, the glutamate pool did not change
over time, enabling kinetic analysis of the 13C
turnover in glutamate. Reduced glutamate content in
postischemic hearts did not result from glutamate loss
during ischemia but resulted from a lack of increased glutamate
in response to reperfusion with acetate. Postischemic
hearts did not respond to acetate by elevating tissue glutamate
content, as did the normal hearts in this study (reported as control in
Fig 3) and those reported elsewhere for earlier periods of
perfusion.17 20 In view of our results, this apparent
discrepancy suggests that altered transporter function within the
malate-aspartate shuttle has induced a difference balance of carbon
mass among the intermediates of oxidative metabolism in
postischemic hearts.
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In vitro analysis provided data on the fractional enrichments of key metabolites for the model. No significant differences in the fractional 13C enrichment of metabolites occurred between normal and postischemic hearts. From in vitro 13C-NMR spectroscopy of tissue extracts, the end-point enrichment of glutamate in both groups was not different, ranging between 95% and 100%. Fc and the ratio of anaplerotic flux to citrate synthase activity (y) calculated from high resolution 13C-NMR spectra were determined. For normal hearts, values (mean±SD) were Fc=0.92±0.07 and y=9±4%. Values in the postischemic group were similar at Fc=0.92±0.09 and y=10±6%. Values from hearts oxidizing acetate in the presence of supplemental glucose were not significantly different for normal hearts (Fc=0.86±0.07, y=6±2%) and postischemic hearts (Fc=0.87±0.09, y=10±6%), owing to the expected inhibition of glucose oxidation by acetate or short chain fatty acids.21 However, values of Fc are not of significance and y exerts very little influence in determining the flux rates via kinetic analysis of these 13C-NMR data.17 18
Reversing the labeling of the substrate combination to use unlabeled acetate and 13C-enriched glucose demonstrated no differences in glucose metabolism between normal and postischemic hearts. 13C-NMR spectra of extracts of hearts perfused or reperfused with unlabeled acetate and the 13C-enriched glucose generally showed no NMR signal from the metabolites of glucose. This lack of signals indicates that little, if any, glucose metabolism was occurring in the presence of acetate. Thus, irrespective of substrate availability, no differences in substrate competition or utilization occurred between normal and postischemic hearts, whereas metabolic flux rates were different, as shown below.
Specific activity levels of GOT isozymes from both the cytosol and
mitochondria of normal and postischemic hearts oxidizing
acetate are presented in Table 2. As shown, the
specific activity of either isozyme of GOT, which catalyzes the
interconversion between -ketoglutarate and glutamate, was
essentially the same in normal as in postischemic
myocardium. With the same amount and activity of the GOT
enzyme, which is not allosterically regulated, the actual reaction rate
was then determined using the measured Vmax of the
cytosolic isozyme (Table 2) and previously determined
Km values for the cardiac GOT17 in
the double-displacement reaction equation.17 From these
parameters, the calculated rates for the GOT reaction in
the cytosol, where 90% of the glutamate resides, of normal and
postischemic hearts were found to be 68
µmol·min-1·g dry
wt-1 in normal hearts and 100
µmol·min-1·g dry
wt-1 in postischemic hearts. These
values are also presented in Fig 6 for comparison with the
observed rate of interconversion between -ketoglutarate and
glutamate (F1), which, in contrast to flux through the GOT
enzyme, was much lower in the postischemic
myocardium. Note that because of differences in the
aspartate content (Table 1), the postischemic heart
actually demonstrates a faster reaction rate than that of the normal
heart. Thus, delayed 13C turnover in the glutamate pool of
postischemic hearts is not due to reduced transaminase
activity.
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13C-NMR Spectroscopy, Isotope Kinetics, and
Metabolic Flux
Fig 4 shows representative
sequential 13C-NMR spectra acquired from a normal (Fig 4A)
and a postischemic (Fig 4B) heart oxidizing
[2-13C]acetate. Isotopic enrichment of the glutamate pool
was significantly delayed in reaching steady state in
postischemic hearts compared with normal hearts. This delay
in the 13C enrichment of glutamate in
postischemic hearts is evident in the spectra shown in Fig 4. For illustrative purposes, Fig 5 graphically displays
the time course of 13C enrichment of glutamate (mean±SD)
at 2- and 4-carbons from all acquired spectra of hearts in each group
shown along with the least-squares fitting of the kinetic model. Note
the closeness of fit, which corresponded to a correlation coefficient
of at least .98 between the model and the experimental data from either
group. Values of VTCA and F1 were the same for
each experimental group when least-squares fitting was performed either
to the mean enrichment curves for all hearts, as shown in Fig 5, or on
each of the curves from individual hearts to provide statistical mean
and standard deviations to allow comparison between the two groups.
Output from this model provided measures of TCA cycle flux and the
interconversion rate between -ketoglutarate and glutamate, which are
graphically displayed in Fig 6.
|
|
As shown in Fig 6, mean VTCA was not significantly
different in the postischemic group of hearts compared with
normal hearts. However, VTCA in postischemic
hearts was not proportional to the large reduction in RPP, as is
consistent with previous observations of respiratory rate in
stunned myocardium.1 16 38 39 40 The rate of
interconversion between -ketoglutarate and glutamate
(F1) was 72% lower in postischemic hearts than
in normal hearts, despite the much faster chemical exchange via GOT
flux in postischemic hearts, as described above. Therefore,
in postischemic hearts, the reduced 13C
labeling kinetics of glutamate and the 3-fold-lower rate of
interconversion between -ketoglutarate and glutamate were not due to
reduced flux across the GOT reaction (nor VTCA). Therefore,
the rate of the cytosolic GOT enzyme was not reduced in
postischemic hearts and could not account for the reduced
isotope exchange between -ketoglutarate and glutamate.
The difference in F1 between normal and reperfused hearts
was not due to differences in carbohydrate availability due to glycogen
depletion during ischemia. Values for VTCA and
F1 were not affected by the presence of glucose to control
for glycogen depletion in postischemic groups. For
comparison with values shown in Fig 6, values from normal hearts
perfused with acetate plus glucose were VTCA=8.9±0.8
µmol·min–1·g–1 and
F1=9.8±3.4
µmol·min–1·g–1, and values from
postischemic hearts reperfused with acetate plus glucose
were VTCA=8.3±0.9
µmol·min–1·g–1 and
F1=2.1± 1.1
µmol·min–1·g–1. Again, in the
second protocol, in which hearts received supplemental glucose to
control for glycogen loss during ischemia, F1 was
significantly depressed in the postischemic group
(P<.05). A significant rate-determining step in carbon
isotope turnover between the citric acid cycle intermediates and
glutamate has been shown to be the transport of metabolic
intermediates between the mitochondria (TCA cycle intermediates) and
the cytosolic space (glutamate) via the malate-aspartate
shuttle.41 In the presence of normal GOT activity, the
lower F1 value reported here for postischemic
hearts must result from reduced rates of metabolite transport via this
shuttle.
|
Discussion |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
In the present study, concurrent measurements of TCA cycle flux and M
O2 were mismatched to the
impaired contractile performance of postischemic
hearts. TCA cycle flux and O2 belied the reduction in the
RPP in postischemic hearts. These metabolic
observations reported in the present study are consistent
with previous findings from both our own laboratory1 14
and those of other investigators.13 20 More important,
these experiments demonstrated that the carbon isotope turnover in the
intramyocardial glutamate pool, as detected with 13C-NMR,
was significantly reduced and that chemical exchange between metabolite
pools alone did not account for this reduction. Indeed, the present
study indicates that the specific activity of the cardiac GOT enzyme,
which catalyzes this interconversion, is normal in the
postischemic heart. Therefore, the reduced turnover of
13C in the glutamate pool must instead be due to other
factors that influence the exchange of isotope between glutamate and
-ketoglutarate. A principal factor, recently shown to directly
influence 13C turnover within the glutamate of the heart,
is the rate of metabolite transport between the TCA cycle in the
mitochondria and the glutamate pool in the cytosol.17 22
In documenting normal transaminase activity despite reduced
interconversion between mitochondrial and cytosolic metabolite pools in
postischemic hearts, the results of the present study
indicate altered exchange of metabolic intermediates across
the mitochondrial membrane of the stunned myocardium.
A series of studies has led to the present understanding that three
principal factors predominate the rate of 13C enrichment of
glutamate in the heart. These factors are as follows: (1) TCA cycle
flux16 17 20 42 43 ; (2) the rate of the equilibrium
reaction between -ketoglutarate and glutamate, which is catalyzed by
the GOT enzyme17 44 ; and (3) physical exchange of
metabolic intermediates between the mitochondria, where the
TCA cycle enzymes are located, and the cytosol, where >90% of the
glutamate pool resides.17 22 42 Each of these factors must
then be considered in examining the reduced 13C kinetics
observed in the postischemic heart. The present set of
experiments was designed to address each factor with the benefit of the
understanding that has been gained from previous 13C-NMR
investigations.
After an initial report of reduced 13C turnover rates
within the glutamate pool of postischemic hearts from our
laboratory,14 a subsequent study by Weiss et
al20 offered the important finding that the observed
changes need not be due to reduced TCA cycle flux alone. However, in
the present study, the analysis of GOT clearly shows normal
levels of the enzyme activity in the postischemic heart.
Thus, the delay in isotope turnover cannot be attributed to impaired
enzyme action in catalyzing the chemical interconversion between the
NMR-detected glutamate pool and the TCA cycle intermediates. In
contrast, a recent study has demonstrated that the
13C-enrichment rates of glutamate are strongly influenced
by the activity of the malate-aspartate shuttle,22 a
mechanism by which the labeled -ketoglutarate from the mitochondria
gains access to the cytosol for exchange with the bulk of the glutamate
pool via the cytosolic GOT.17 21 41 In that study, the
rate of 13C turnover within glutamate was accelerated
simply by stimulating metabolite transport across the mitochondrial
membrane with increases in cytosolic redox state and without changes in
TCA cycle flux. In view of this previous result and the present
data showing normal GOT activity in postischemic hearts,
the pronounced delay in 13C enrichment of glutamate in
postischemic hearts reflects neither TCA cycle flux nor
flux through GOT. Therefore, the delay in isotope kinetics indicates a
reduced rate of metabolite transport across the mitochondrial membrane
of postischemic myocytes.
Net forward flux through the malate-aspartate shuttle involves the
concerted activity of a unidirectional glutamate-aspartate exchanger
and a reversible -ketoglutarate–malate exchanger, both on the
mitochondrial membrane.21 23 However, the exchange of
isotope labeled -ketoglutarate from the mitochondria with the
cytosolic glutamate pool does not necessarily require net forward flux
through the malate-aspartate shuttle. Instead, of the two transporters
that constitute this shuttle, the reversible -ketoglutarate–malate
exchanger need be available only to enable the interconversion of
labeled -ketoglutarate with cytosolic glutamate. Therefore, the
transport of metabolites across the mitochondrial membrane may play a
significant role in coordinating TCA cycle flux with cytosolic
metabolism in the absence of significant activity through
the malate-aspartate shuttle.
Experiments with 13C-enriched acetate and supplemental
glucose controlled for potential differences in the availability of
endogenous carbohydrates due to the depletion of glycogen
during ischemia. These experiments demonstrated that any
differences in carbohydrate availability for cytosolic NADH
production did not account for the differences in transport
rates (F1) between normal and postischemic
hearts, because supplemental glucose had no effect on F1.
Also, negligible glycolytic activity for cytosolic NADH
production was in evidence from experiments with
[1-13C]glucose plus acetate, which suggests that net
forward flux through the entire malate-aspartate shuttle is not likely
to have produced the observed changes in metabolite transport. The data
suggest very little activity through the malate-aspartate shuttle. In
contrast, only the reversible, -ketoglutarate–malate exchanger is
likely to have facilitated the measured exchange of -ketoglutarate
with glutamate. Thus, the efflux of labeled -ketoglutarate from the
mitochondria was delayed in the postischemic hearts, most
likely without involvement of the unidirectional glutamate-aspartate
exchanger and net transport of reducing equivalents into the
mitochondria.
The normal activity of the GOT enzyme that is documented here is not
surprising, given that the duration of global ischemia was
relatively short and that the myocardium retained
viability. Without much cell lysis, significant loss of the
transaminase was unlikely to occur. In fact, this was the case upon
reperfusion, when the myocardium was harvested at a time
that would otherwise have allowed for ample washout of extracellular
enzymes that may have leached out of the myocyte. In addition, the GOT
enzyme is not allosterically regulated and would not be expected to
display altered kinetics in the reperfused myocardium.
Rather, the GOT activity is more dependent on the concentration of
substrate for the reaction, given that the amount of the enzyme
present has not changed, which was the case in the present
study. Therefore, the evidence points to normal chemical
interconversion rates between -ketoglutarate and glutamate in
postischemic hearts with altered metabolite transport to
account for the discrepancy between the observed exchange of label
between the two metabolites and calculated flux through the GOT
enzyme.
Such altered metabolite exchange is reflected by the shift in the
glutamate pool sizes that were observed in postischemic
hearts compared with normal hearts. In normal hearts, oxidation of
acetate induces relatively high concentrations of glutamate in the
myocardium.16 27 The significantly lower
glutamate pool in postischemic hearts did have an impact on
the net flux of the isotope through the rate-determining metabolite
compartments. This finding of lower glutamate content in
postischemic hearts is consistent with normal flux
through the TCA cycle enzyme, -ketoglutarate dehydrogenase, and our
observation of reduced exchange with the cytosolic glutamate pool.
However, if either the substrate affinity (Km)
or level of activity of -ketoglutarate dehydrogenase is increased,
as may occur at altered pH or elevated calcium
concentration,45 the mitochondrial -ketoglutarate pool
becomes less available for efflux to the cytosol.21 The
reduced exchange of metabolites during reperfusion that have been
observed in the present study may then result from either changes
in the exchange proteins or changes that are intrinsic to the TCA cycle
enzymes.
In the present study, the lack of an increase in glutamate content during reperfusion after 10 minutes of ischemia is also consistent with the previous observation of reduced glutamate levels in rat hearts that were reperfused with acetate. This previous study20 in rat hearts was performed with a concentration of acetate that was double the concentration used in the present study. However, in that previous investigation, hearts were also perfused with 5 mmol/L acetate before ischemia, which elevates the level of glutamate well above the level observed in the hearts that were perfused with glucose before ischemia, as performed in this present set of experiments. Additionally, values for glutamate are reported here per gram of dry weight tissue to eliminate the potential confounding effects of increased wet weight that occurs during reperfusion of the isolated heart that is reperfused with crystalloid medium that does not contain colloidal material. Therefore, although glutamate values were observed to be lower in the reperfused hearts that were examined in both the present study and the previous study by Weiss et al,20 the mechanisms for lower glutamate content may also differ because of the distinctions between the two different protocols. Despite the reduced glutamate concentration in postischemic hearts, which would tend to increase the turnover of label, a consistent feature of such studies on postischemic hearts is the significant reduction in 13C kinetics.
In summary, the findings of this investigation again document the
respiratory inefficiency of contractile performance in
postischemic hearts, as shown by the apparent mismatch
between mechanical work and MO2. TCA
cycle flux rates were normal in postischemic hearts,
confirming this inefficiency at the level of intermediary
metabolism. The reduced rates of isotope turnover in the
glutamate pools of postischemic hearts have been shown here
to occur despite normal activity of the associated transaminase enzyme,
GOT, in both mitochondria and cytosol. Reduced efflux of
-ketoglutarate across the mitochondrial membrane is then left to
account for the slower exchange of label between the mitochondrial
metabolites and cytosolic glutamate in postischemic
myocardium. The findings of the present study indicate
that the interconversion between -ketoglutarate and glutamate is
reduced in postischemic myocardium, as a result
of reduced -ketoglutarate efflux rates from the mitochondria. The
results also indicate that the altered exchange of labeled
-ketoglutarate with cytosolic glutamate is most likely to
have been mediated by reduced flux across the reversible
-ketoglutarate–malate exchanger without involvement of the entire
malate-aspartate shuttle system. In either case, the altered metabolite
exchange between subcellular compartments in the stunned
myocardium that is reported in the present study may
prove to be a factor in the metabolic inefficiency of
postischemic myocardium. As a consequence, the
role of metabolite exchange across the mitochondrial membrane in
coordinating the metabolic support of
physiological function warrants further
investigation in normal and diseased tissues.
|
Selected Abbreviations and Acronyms |
|---|
|
|
Acknowledgments |
|---|
This study was supported by National Heart, Lung, and Blood Institute grants RO1 HL-49244 and RO1 HL-56178 (Dr Lewandowski) and by Grant-in-Aid 13-522-912 from the American Heart Association, Massachusetts Affiliate, Inc (Dr Lewandowski) and was performed during the tenure of an Established Investigator Award from the American Heart Association to Dr Lewandowski.
|
Footnotes |
|---|
This manuscript was sent to Ketty Schwartz, Associate Editor, for review by expert referees, editorial decision, and final disposition.
Presented in part in abstract form at the 68th Scientific Sessions of the American Heart Association, Anaheim, Calif, November 13-16, 1995.
Received September 3, 1996; accepted May 6, 1997.
|
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