Effect of Coronary Blood Flow on Glycolytic Flux and
Intracellular pH in Isolated Rat Hearts
By James R. Neely, Jeffrey T. Whitmer, and Michael J. Rovetto
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ABSTRACT
The rate of coronary blood flow was varied in isolated working rat heart preparations to
determine its influence on the rate of glucose utilization, tissue high-energy phosphates,
and intracellular pH. A 60% reduction in coronary blood flow resulted in a 30% reduction
in oxygen consumption, an accelerated rate of glucose utilization, lower
tissue levels of high-energy phosphates, and higher tissue levels of lactate and H + .
Ventricular performance deteriorated as reflected by a decrease in heart rate and peak
systolic pressure. Further reductions in coronary blood flow resulted in inhibition of
glycolysis, a greater decrease in tissue levels of high-energy phosphates, and higher tissue
levels of both lactate and H + . These changes in glycolytic flux, tissue metabolites, and
ventricular performance were proportional to the degree of restriction in coronary blood
flow. The importance of coronary blood flow and washout of the interstitial space in the
maintenance of accelerated glycolytic flux in oxygen-deficient hearts is emphasized. It is
concluded that acceleration of ATP production from glycolysis can occur only in the
marginally ischemic tissue in the peripheral area of tissue supplied by an occluded artery.
The central area of tissue which receives a low rate of coronary blood flow will have a
reduced rate of ATP production due to both a lack of oxygen and an inhibition of
glycolysis.
• The harmful effects of a restriction of coronary
blood flow that leads to a reduction in mechanical function, cell death, and myocardial infarction
may result as much from lack of washout of the
interstitial space as from lower rates of delivery of
oxygen and other substrates to the tissue (1-3).
Alterations in mechanical and metabolic functions
of myocardial cells are generally reversible up to 20
minutes following restriction of coronary blood
flow, but irreversible damage and cell death occur
between 20 and 60 minutes (2-7). The time of onset
of cell death may be directly proportional to the
restriction in coronary blood flow. Several studies
(7-14) have emphasized the heterogeneity of blood
flow in and around an infarcting region. Flow
ranges from near zero in the center of the infarcting
zone to normal or above normal in the surrounding,
tissue. Irreversible cell damage would, therefore, be
expected to occur more rapidly in the region of
tissue receiving the least amount of coronary blood
flow.
From the Department of Physiology, The Milton S. Hershey
Medical Center, The Pennsylvania State University, Hershey,
Pennsylvania 17033.
This work was supported by U. S. Public Health Service
Contract 71-2499 from the National Heart and Lung Institute.
Portions of these data were presented in preliminary form at
the International Study Group for Cardiac Metabolism Meeting
in Quebec, 1974.
Dr. Rovetto's present address is Department of Physiology,
Jefferson Medical College, Philadelphia, Pennsylvania 19107.
Received May 13, 1975. Accepted for publication September
15, 1975.
Circulation Research, Vol. 37, December 1975
The role of coronary blood flow in maintaining a
suitable intracellular environment in addition to
providing the oxygen supply has been emphasized
recently in studies comparing the effects of anoxia
with those of ischemia (15-17). Ischemia has been
shown to be more detrimental to cellular function
and metabolism than is anoxia in which coronary
blood flow is maintained at normal rates. Anoxia
accelerates glycolysis in cardiac muscle (18-19),
and the rate of glucose utilization is increased by a
regional reduction in coronary blood flow (20, 21).
In contrast, glycolysis is reduced in the working rat
heart when coronary blood flow is severely restricted to the whole heart (17). Also, a 60%
reduction in blood flow to the whole heart in a
working pig heart preparation results in reduced
rates of glucose consumption (22). Part of this
discrepancy in results may be due to an incomplete
description of the effects of coronary blood flow on
the metabolic and functional activity of the heart.
In the regional ischemic models, glucose utilization
by a heterogeneous population of tissue receiving a
variety of coronary blood flows and performing
different levels of mechanical work has been measured (20, 21). Therefore, glycolysis may have been
accelerated in the normal or mildly ischemic tissue
and inhibited in the more severely ischemic cells.
In the global ischemic models, only the control and
one condition of ischemia have been studied (17).
Therefore, the purpose of the present work was to
study the relationship between coronary blood flow
733
734
NEELY, WHITMER. ROVETTO
and glycolysis over a wide range of coronary flow
rates.
Methods
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Hearts were removed from 250-350-g male
Sprague-Dawley rats and perfused in a working heart
apparatus as described previously (23). Ischemia was
induced by placing a one-way valve in the aortic outflow
tract which prevented retrograde perfusion of the coronary arteries during diastole and reduced coronary blood
flow by about 60% (5). Systolic pressure development,
heart rate, and coronary blood flow for hearts perfused by
this method are shown in Figure 1. In hearts which were
electrically paced at 230 beats/min following a 60%
reduction in coronary blood flow, ventricular failure
resulted within 10 minutes; this phenomenon in turn
caused a further reduction in coronary blood flow to 10%
of control rates after 30 minutes. If the hearts were not
paced, ventricular failure was manifest as a smaller
reduction in peak systolic pressure development and a
greater decrease in heart rate. Without pacing, peak
systolic pressure development declined by about 20%
and heart rate decreased from about 250 to 180 beats/
min during 30 minutes of reduced coronary bloodflow.In
these hearts, the rate of coronary blood flow remained at
about 40% of the control rate for up to 1 hour of
perfusion. About 10% of the hearts that were not paced
developed pressure failure similar to that in the paced
hearts, and flow deteriorated. These hearts were not
included in the study. Intermediate flow rates between
10 and 40% of control could be maintained in electrically
paced hearts by providing different levels of aortic
perfusion pressure after ventricular failure had occurred.
Ar.oxia was induced by perfusing the hearts with buffer
equilibidted with a 95% N2-5% CO2 gas mixture,
pH 7.35. Following failure of the ventricle, coronary
blood flow was maintained at control rates by providing a
hydrostatic perfusion pressure of 60 mm Hg. After 10
minutes of anoxic perfusion, coronary blood flow was
reduced to either 2 or 5 ml/min by use of a mechanical
pump connected to the aortic outflow tract, and perfusion at these lower rates was continued for 20 minutes.
The basic perfusate was Krebs-Henseleit-bicarbonate
buffer containing 11 mM glucose and, where indicated,
1.2 mM palmitate bound to 3% albumin. When fatty
acids were used in the perfusate, the fatty acid-albumin
complex was prepared as described previously (24).
The rate of glucose utilization was determined by
measuring the production of 3H2O from 5-3H-n-glucose
as described earlier (25). Oxygen consumption was
calculated from the arterial-venous difference in oxygen
tension (Po2) and the rate of coronary blood flow. The
rate of glucose oxidation was determined by measuring
14
CO2 production from uniformly labeled 14C-glucose.
Intracellular pH was calculated from the distribution
of 14C-5-5-dimethyl-2,4-oxazolidinedione (DMO) (26); 50
mg/100 ml of nonlabeled DMO was included in the
buffer as a carrier. Also, 3H-n-sorbitol and 50 mg/100 ml
of nonlabeled sorbital were included in the buffer for
measurement of the extracellular space. Preliminary
experiments were conducted to determine the time
necessary for final equilibration of the DMO. These
studies indicated that the distribution of DMO within
the tissue was complete within 3 minutes. The intracel-
lular pH calculated by this procedure was 6.70 ± 0.13,
6.91 ± 0.08, 6.99 ± 0.07, 7.04 ± 0.02, 7.02 ± 0.03, and
7.02 ± 0.01 after 1.0, 1.5, 3, 10, 18, and 30 minutes of
perfusion, respectively. In subsequent experiments, intracellular pH was measured after at least 10 minutes of
exposure to DMO.
Hearts used for lactate, adenosine triphosphate
(ATP), and creatine phosphate analysis were rapidly
frozen by clamping them with aluminum tongs cooled in
liquid nitrogen. The frozen tissue was powdered and
extracted in 6% perchloric acid, and the neutralized
1
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FIGURE 1
Effects of ischemia on ventricular function. Peak systolic
pressure, heart rate, and coronary flow for control hearts are
shown by the open circles. These hearts had a left atrial filling
pressure of 10 cm H2O and an afterload of 60 mm Hg of
hydrostatic pressure. At zero time in the figure, coronary blood
flow was reduced by about 60% by use of a one-way value in the
aortic outflow tract. The effect of reducing coronary blood flow
in nonpaced hearts is shown by the solid triangles. In a second
group of ischemic hearts, heart rate was maintained at about
230 beats/min by electrical pacing as shown by the open
triangles. The data points represent means ± SE for 8-12 hearts
in this and all subsequent figures.
Circulation Research, Vol. 37, December (975
CORONARY BLOOD FLOW. GLYCOLYSIS, AND pH
735
extract was used to determine tissue levels of metabolites. Lactate was assayed by the lactate dehydrogenase
procedure, and ATP and creatine phosphate were assayed by the hexokinase, glucose-6-P-dehydrogenase
procedure as described in Bergmeyer (27).
Results
EFFECTS OF CORONARY BLOOD FLOW ON GLYCOLYTIC FLUX AND
GLUCOSE OXIDATION
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Figure 2 shows the rate of glucose utilization as a
function of perfusion time at three different rates of
coronary blood flow. In the control hearts, coronary
blood flow averaged about 15 ml/min and was
maintained throughout the perfusion period. In
these hearts, the rate of exogenous glucose utilization was about 4.5 Mmoles/g dry tissue min" \ When
the rate of coronary blood flow was reduced and
allowed to decline as ventricular failure occurred,
glycolysis was inhibited. The rate of utilization was
not limited by glucose availability (17) but represented inhibition within the glycolytic pathway.
However, if coronary blood flow was maintained at
about 2 ml/min, utilization of exogenous glucose
was accelerated to above the control rate after a lag
period of about 10 minutes. When the rate of
coronary blood flow was maintained at about 5
ml/min, glycolysis was increased even further. At
these flows, the glycolytic rate was the same
whether the hearts were electrically paced or not.
The apparent lag in the increased rate of exogenous
glucose utilization probably reflects a rapid breakdown of glycogen and dilution of the specific
activity of the 3H-glucose-6-P pool. The tissue
content of glycogen was essentially depleted during
the first 16 minutes of perfusion under ischemic
conditions (17). Glycogen levels decreased rapidly
in the first 4-8 minutes of ischemic perfusion and
then at a slower rate. These data indicated that
glucose from glycogen was preferentially utilized as
a substrate for glycolysis and that acceleration of
exogenous glucose utilization occurred only after
the tissue stores of glycogen were essentially depleted.
The effect of ischemia on glucose utilization in
hearts perfused with a combination of glucose and
fatty acid is illustrated in Figure 3. The control rate
of utilization in these hearts was somewhat lower
than that in hearts perfused with glucose as the
only exogenous substrate (compare Figs. 2 and 3),
illustrating the well-known inhibitory effect of
fatty acid oxidation on glycolysis in aerobic hearts.
When coronary blood flow was reduced, the steadystate rate of glucose utilization was essentially the
same as that found in ischemic hearts perfused
with glucose as the only substrate. In addition, the
Circulation Research, Vol. 37, December 1975
0
5
10
15
20
25
PERFUSION TIME (min)
FIGURE 2
Effect of coronary blood flow and perfusion time on the rate of
glucose utilization. The perfusate contained 11 mM 5-3H-glucose
and was bubbled with a 95% O2-5% CO2 gas mixture. The rate
of glucose utilization in control hearts is shown by the solid
squares. Ischemia was induced at zero time by decreasing the
rate of coronary blood flow from the control level of 14.8 to about
6 ml/min in each case. Continued perfusions of ischemic hearts
at three different rates of coronary blood flow are shown. In one
group, the rate of coronary blood flow was allowed to decline to
0.6 ml/min as ventricular failure occurred (open triangles). In
the second group, the rate of coronary flow was maintained at 2
ml/min (open circles). In the third group, the rate of flow was
maintained at between 5 and 6 ml/min (solid triangles).
rate of utilization was roughly proportional to
coronary blood flow in the ischemic hearts.
The rate of oxygen consumption in control hearts
averaged about 30 jtmoles/g dry weight min"' (Fig.
4). This rate decreased by about 30% when coro-
10
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t
15
20
PERFUSION TIME (mm)
FIGURE 3
Effect of ischemia on glucose utilization in hearts perfused with
fatty acids and glucose as exogenous substrates. These hearts
were perfused as described for Figure 2 except that 1.0 mM
palmitate bound to 3% albumin was included in the perfusate in
addition to II mM 5-3H-glucose. The control rate of glucose
utilization is shown by the solid line. Ischemic hearts in which
the rate of coronary blood flow was allowed to decline to 1
ml/min as ventricular failure progressed are shown by the solid
circles, and ischemic hearts in which the rate of coronary blood
flow was maintained at between 5 and 6 ml/min are shown by
the solid triangles.
736
NEELY, WHITMER, ROVETTO
utilization increased to about 14 ^moles/g dry
tissue min" 1 in anoxic hearts when coronary blood
flow was maintained at control levels (Fig. 6).
However, when coronary blood flow was reduced,
the rate of glycolysis declined to about the same
extent as it did in oxygenated hearts receiving a
comparable coronary blood flow. These data indicate that simple oxygen deficiency with maintenance of coronary blood flow accelerated glycolysis
but that reduced coronary blood flow inhibited this
source of ATP.
EFFECTS OF CORONARY BLOOD FLOW ON TISSUE LEVELS OF HIGHENERGY PHOSPHATES. LACTATE. AND H*
1 2
3
4
5
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CORONARY FLOW ml/min
FIGURE 4
Effect of coronary blood flow on oxygen consumption. Hearts
were perfused with buffer containing glucose as the only
substrate at the coronary blood flows indicated. Oxygen consumption was measured after 10 minutes of perfusion at each
flow rate. These rates were maintained for at least 30 minutes in
each case. Each point represents the mean of five to eight
determinations.
nary blood flow was reduced to 5 ml/min, and at
lower flows oxygen consumption decreased in direct proportion to the reduction in coronary blood
flow and oxygen delivery. When the rate of coronary blood flow was held at any of the intermediate
levels, the rate of oxygen consumption decreased
immediately, but the reduced rate was maintained
for at least 30 minutes of perfusion. Oxidation of
glucose averaged about 2 ^moles/min in the control
hearts (Fig. 5). In ischemic hearts receiving 5 ml/
min of flow, glucose oxidation was reduced by
about 50% initially, but subsequently increased to
above the control rate. This reduction in the first
10 minutes of perfusion correlates with the lag in
acceleration of 3H2O production from exogenous
glucose (Fig. 2) and probably also represents dilution of the specific activity of the glucose-6-P pool
by breakdown of glycogen. Oxidation of glucose
accounted for about 40% of the total oxygen consumed in control hearts, and presumably oxidation
of endogenous lipid accounted for the remainder.
In the ischemic hearts, oxidation of exogenous
glucose accounted for about 90% of the total oxygen consumed after 20 minutes of perfusion.
Although the rate of glycolysis was accelerated
by ischemia when coronary blood flow was maintained at either 2 or 6 ml/min, the maximum rate
observed in ischemic hearts was only about 60% of
that found in anaerobic hearts. The rate of glucose
Tissue levels of high-energy phosphates were low
in both anoxic and ischemic hearts, but the levels
of ATP and creatine phosphate decreased in proportion to the restriction in coronary blood flow and
oxygen delivery in hearts perfused with oxygenated
perfusate (Table 1). The levels of creatine phosphate were somewhat higher than those of ATP in
control hearts, especially when palmitate was present, but they appeared to be more sensitive to
changes in coronary blood flow. In anoxic hearts,
the levels of creatine phosphate were low regardless
of coronary blood flow, but the levels of ATP were
maintained somewhat at the higher flow rates.
Tissue levels of lactate were slightly higher in
aerobic hearts perfused with palmitate when coro-
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PERFUSION TIME (min)
FIGURE 5
Effect of ischemia on glucose oxidation. These hearts were
perfused as described for Figure 2 with glucose as the onlyexogenous substrate. The rate of glucose oxidation in control
hearts is shown by the solid line. Glucose oxidation was
estimated by the rate of "CO2 production. Ischemic hearts
(broken lines) were perfused with a coronary blood flow of
between 5 and 6 ml/min throughout the 20 minutes of perfusion.
Circulation Research, Vol. 37. December 1975
737
CORONARY BLOOD FLOW. GLYCOLYSIS. AND pH
in these hearts resulted in accumulation of lactate
to levels similar to those found in oxygenated
hearts.
A decrease in cellular pH is generally thought to
be associated with an accumulation of metabolic
products such as lactate and C0 2 . In the present
study, changes in intracellular pH in relationship
to the restriction in coronary blood flow and oxygen
availability were estimated by the DMO procedure
(Table 2). Calculation of intracellular pH from the
tissue distribution of DMO is based on the theory
that weak acids penetrate the cell membrane in the
protonated form and that the distribution of acid
across the membrane depends on the concentration
gradient of H + (26). Therefore, accumulation of
DMO in the intracellular space will depend on the
pH in the extracellular compartment, and this pH
must be known before intracellular pH can be
calculated. Obviously, the pH in the interstitial
space adjacent to the myocardial cells should be
used in the calculation, but this pH is impossible to
obtain. In the present study, the extracellular pH
used for the calculation was the pH measured in
the coronary venous perfusate. The arterial perfusate pH was maintained at 7.35. The decrease in
pH measured in the coronary, effluent as the
perfusate passed through the vascular bed would
be expected to represent only the direction of
change in interstitial pH and not the absolute
change. Coronary effluent pH may be higher than
the true interstitial pH which would cause an
overestimation of intracellular pH. Therefore, the
changes in both extracellular and intracellular pH,
reported in Table 2, are minimum changes.
CD
"5
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CORONARY FLOW (ml/min)
FIGURE 6
Effects of reducing coronary blood flow in anoxic hearts on the
rate of glucose utilization. The hearts received a 10-minute
preliminary perfusion prior to starting anoxic perfusion with
perfusate containing 11 mu glucose and bubbled with a 95%
N2-5% CO, gas mixture. The rate of flow in some of the anoxic
hearts was maintained at the control rate by providing a 60-mm
Hg hydrostatic perfusion pressure. Flow was reduced to the
lower rates by decreasing the hydrostatic perfusion pressure.
Rates of glucose utilization were determined after 10 minutes of
anoxic perfusion at each coronary blood flow.
nary blood flow was maintained at control levels. In
the ischemic hearts, however, lactate accumulated
in proportion to the restriction in flow regardless of
the substrates present. Anoxia elevated the level of
lactate at control flow rates, but restriction of flow
TABLE 1
Effects of Coronary Blood Flow on High-Energy Phosphates and Lactate in Aerobic and Anoxic Hearts
Coronary
blood flow
(ml/min)
ATP
Substrate
15
6
2.6
1
13
5
0.6
Glucose
13
6
2
Glucose
Glucose and palmitate
(moles/g dry wt)
Creatine
phosphate
(moles/g dry wt)
Lactate
(moles/g dry wt)
Aerobic Hearts
22 ± 0.8
16 ± 0.8
15 ± 1.5
10 ± 0.6
20 ± 0.7
20 ± 1.6
11 ± 0.4
Anoxic Hearts
23 ± 2
14 ± 0.5
10 ± 1.3
3 ±0.7
26 ± 1
14 ± 1.4
4 ±0.3
3 ±0.9
18 ±3.0
33 ± 2
59 ± 6
9 ± 1.8
27 ± 2.9
54 ±2.0
12 ± 2
6.3 ± 1.2
5 ± 1.0
2.3 ± 0.7
30 ± 5
41 ± 7
8±1
Hearts were perfused for 20 minutes with coronary blood flow maintained at the levels indicated in the table. The perfusate was
bubbled with a 95% O2-5% CO, gas mixture for aerobic hearts and a 95% N,-5% CO, gas mixture for anoxic hearts; the perfusate contained 11 mM glucose in both experiments. When present, palmitate (1.0 mM) was bound to 3% bovine serum albumin. Each value reppresents the mean ± SE for six to eight hearts.
Circulation Research, Vol. 37, December 1975
738
NEELY, WHITMER, ROVETTO
TABLE 2
Effect of Coronary Blood Flow and Oxygen Supply on Tissue pH
pH
Coronary
blood flow
(ml/min)
15
15
15
4
2
1
0.5
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15
6
2
13
15
15
rcflUbiUH
time
(minutes)
Coronary
effluent
Intracellular
Control Hearts
7.02 ± 0.03
7.25
7.25
7.01 ±0.01
Ischemic Hearts without Maintained Flow
7.04 ± 0.02
0
7.25
6
6.96 ± 0.03
7.05
16
6.87 ± 0.02
6.87
6.83 ± 0.02
26
6.84
36
6.80 ± 0.02
6.79
Ischemic Hearts with Maintained Flow
20
7.27 ± 0.02
20
7.18 ±0.03
20
7.00 ± 0.05
Anoxic Hearts
0
7.01 ± 0.01
7.27 ± 0.02
2
6.97 ± 0.04
7.11 ±0.03
30
7.00 ± 0.02
7.25 ± 0.03
0
35
blood flow whether flow was maintained constant
for 20 minutes or allowed to decline as ventricular
failure occurred. Similarly, intracellular pH declined in proportion to the restriction in flow. The
decline in extracellular pH was larger than that in
intracellular pH at all flow rates studied, indicating that the intracellular space is better buffered
than the extracellular space in hearts perfused with
Krebs-Henseleit-bicarbonate buffer. When the extracellular pH was below 7.0, the pH of both spaces
was equal. This observation held whether the
extracellular pH was decreased by ischemia or
adjusted with various buffers in aerobic control
hearts (data not shown). In contrast to ischemia,
anoxia caused only a small decrease in pH in the
first 2 minutes of perfusion, and the pH had returned to control levels after 30 minutes.
Discussion
Hearts were perfused for the times indicated with perfusate
containing 11 mm glucose and bubbled with a 95% O2-5% CO2
gas mixture for control and ischemic hearts and a 95% N2-5%
CO2 gas mixture for anoxic hearts. In one group of ischemic
hearts, perfusion was continued for 36 minutes, and coronary
blood flow was allowed to decline as ventricular failure occurred
corresponding to the electrically paced hearts in Figure 1
(ischemic without maintained flow). In the ischemic hearts with
maintained flow, ischemia was induced and flow was maintained
at either 6 or 2 ml/min for 20 minutes. The pH of the coronary
effluent was the same throughout the 20 minutes, and only the
last values are shown. Each value represents the mean ± SE for
6-12 hearts.
In control hearts, perfusate pH decreased from
7.35 to 7.25 on one passage through the heart. In
ischemic hearts, the effluent pH generally decreased in proportion to the restriction in coronary
Since occlusion of a coronary artery results in a
heterogeneous pattern of blood flow in and around
the area of tissue supplied by the occluded artery
(8), it was of interest to study the rates of energy
metabolism that might be expected in the peripheral central areas of the ischemic tissue. One would
expect the rate of oxygen consumption and ATP
production from oxidative metabolism to be proportional to coronary blood flow and to also occur
in a heterogeneous pattern. Anaerobic production
of ATP normally represents less than 5% of the
total, and even under conditions of hypoxia or
anoxia acceleration of glycolysis can produce only
about 20% of the ATP required by aerobic tissue
(28-30). In the present study, aerobic hearts produced about 190 Mmoles ATP/g min" 1 (Table 3). If
an equivalent amount of ATP were to be produced
from glycolysis, the rate would have to be about 90
TABLE 3
Effects of Coronary Blood Flow on ATP Production from Glycolysis and Oxidative Metabolism in
Perfused Rat Hearts
ATP from
Condition
Control
Ischemic
Anoxic
Coronary
blood flow
(ml/g min"')
Tntal ATP
produced
(jimoles/g min"1)
Glycolysis
Oxidative
metabolism
14
4.7
1.7
0.5
13
2.0
189
138
70
31
34
8
9
18
13
6
34
8
180
120
57
25
0
0
The perfusate contained 11 mM glucose. Glucose oxidation was estimated by measuring 14CO2
production from U-"C-glucose. Rates of ATP production were calculated after 20 minutes of perfusion under the conditions indicated. Total ATP production was calculated from glycolytic flux and
oxygen consumption, assuming that two ATP molecules are generated per glucose molecule from
glycolysis and a P-0 ratio of 3.
Circulation Research, Vol. 37, December 1975
CORONARY BLOOD FLOW. GLYCOLYSIS. AND pH
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/imoles glucose/g min" 1 or about 20 times the
control rate. The most rapid rates of glycolysis
occur under anoxic conditions when glycogen is
rapidly broken down (17, 28, 31). However, tissue
glycogen is essentially depleted within 4 minutes
(31), and the steady-state rate of glycolysis from
exogenous glucose represents only about 20% of
that required for normal aerobic production of ATP
(Table 3). A reduction in coronary blood flow of
60% accelerated glycolytic production of ATP
(about 18 /imoles ATP/g min' 1 ), but this rate was
less than 10% of the normal aerobic rate. Oxidative
metabolism using the residual amount of oxygen
consumption (20 jtmoles O2/g min"') could account
for another 120 jtmoles of ATP, leaving the tissue
energy deficient by about 50 /imoles ATP/g min" 1 .
This deficit resulted in a decreased energy expenditure as indicated by a slower heart rate and a
small reduction in peak systolic pressure.
With further reductions in coronary blood flow,
oxygen consumption declined proportionally. The
rate of glycolysis was inhibited, tissue levels of ATP
and creatine phosphate declined in proportion to
the restriction in flow, and ventricular failure was
more severe. At the lowest rates of coronary blood
flow studied, total ATP production from both
oxidative and glycolytic processes averaged about
30 /imoles/g min"1, only about 15% of the normal
aerobic rate. It, therefore, appears that the central
area of tissue supplied by an occluded artery does
not receive enough coronary blood flow to maintain
even the low anaerobic rate of ATP production.
From these studies, it appears that maximum
stimulation of glycolysis is able to supplement
energy production from oxidative sources to near
the normal rate in the peripheral area of infarcting
tissue where coronary blood flow is adequate to
supply about 85% of the normal oxygen consumed.
As flow decreases from this level, glycolysis, even if
it is maximally stimulated, is not capable of
making up the deficit between oxidative ATP
production and the normal ATP requirements. To
complicate this lack of energy production, glycolysis becomes progressively inhibited as coronary
blood flow is decreased.
Inhibition of glycolysis in ischemic hearts was
associated with increased tissue levels of lactate
and a lowering of both the extracellular and the
intracellular pH. The importance of coronary blood
flow in maintaining accelerated glycolytic rates is
emphasized by the observations that in anoxic
hearts with normal tissue pH (after a slight transient decrease in the first 2 minutes of perfusion)
tissue lactate did not accumulate to anything like
Circulation Research, Vol. 37, December 1975
739
the same extent as it did in ischemic hearts and
glycolysis proceeded at a rapid rate. Reducing
coronary blood flow in the anoxic hearts inhibited
glycolysis. It can be concluded from these observations that washout of the interstitial space with
removal of lactate and maintenance of cellular pH
are important factors in maintaining accelerated
glycolytic flux and that lack of washout of the
interstitial space complicates energy production in
ischemic tissue. Calculated intracellular pH using
DM0 as a marker may represent minimum values
since the interstitial pH cannot be measured directly, but the procedure appears to be useful for
estimating relative pH changes. The decrease in
coronary effluent and tissue pH measured in ischemic hearts in the present study agrees with the
decrease (about 0.6 units) in interstitial pH measured with microelectrodes following ligation of a
coronary artery (23).
Isolated rat hearts perfused with Krebs-Henseleit-bicarbonate buffer have coronary blood flows
ranging from 10 to 15 ml/g wet tissue. This rate of
flow is much higher than that which has been
observed in hearts from large animals perfused
with blood. Estimates of in vivo coronary flow rates
in the rat are not available, but it can be assumed
that, since the rat heart beats at about three times
the rate of large animal hearts and develops about
the same peak systolic pressure, energy requirements and, therefore, coronary blood flow will be
higher per gram of tissue in the rat heart and may
approach 3.0 ml/g wet weight as compared with 1
ml/g in larger animals. Thus, a 60% reduction in
the flow rate in isolated rat hearts was at least
twice the in vivo flow rate. However, this in vitro
flow rate was insufficient to provide adequate
oxygen and to prevent lactate accumulation, decreased pH, and ventricular failure, indicating that
an ischemic condition was induced. Since coronary
flow rates less than 2 ml/g wet weight min" 1 in the
rat heart resulted in glycolytic inhibition, it is
unlikely that glycolysis can be stimulated in a
blood-perfused heart in which coronary blood flow
is decreased sufficiently to limit oxygen supply. In
this regard, a 50 or 60% reduction in coronary blood
flow in the swine heart (normal coronary flow about
1 ml/g wet weight min"1) results in inhibition of
glycolysis, a large increase in tissue lactate, and
ventricular failure (22). Therefore, maximum stimulation of glycolysis is unlikely in larger blood-perfused hearts even in the peripheral area of infarcting tissue. The increased glucose utilization observed with regional ischemia in larger in situ
hearts (20, 21) is more likely due to increased
740
NEELY, WHITMER, ROVETTO
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metabolism in the nonischemic tissue. Both the
rate of coronary blood flow and the contractile force
have been reported to be increased in tissue immediately adjacent to the area served by an occluded
vessel (9, 33).
Cardiac glycogen appears to be utilized very
rapidly in anoxic (31) and arrested hearts receiving
no coronary blood flow (34-36). In anoxic hearts,
rapid utilization of glycogen results from activation
of phosphorylase b by increased tissue levels of
adenosine monophosphate (AMP) and inorganic
phosphate and decreased levels of ATP and glucose-6-P (31). In addition phosphorylase b conversion to phosphorylase a occurs (31, 34, 35). The rate
of glycogenolysis, although greatly accelerated,
appears to occur at a slower rate in ischemic than
in anoxic hearts (17, 36). In potassium-arrested
hearts, the rate of glycogenolysis appears to decrease as tissue lactate accumulates (35). In the
isolated heart, acceleration of glycogenolysis under
ischemic conditions may result from increased
levels of inorganic phosphate due to breakdown of
creatine phosphate and increased levels of 5'-AMP
(5, 17).
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Circulation Research, Vol. 37, December 1975
Effect of coronary blood flow on glycolytic flux and intracellular pH in isolated rat hearts.
J R Neely, J T Whitmer and M J Rovetto
Downloaded from http://circres.ahajournals.org/ by guest on October 1, 2016
Circ Res. 1975;37:733-741
doi: 10.1161/01.RES.37.6.733
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