1458
Active Downregulation of Myocardial Energy
Requirements During Prolonged Moderate
Ischemia in Swine
Andrew E. Arai, George A. Pantely, Cheryl G. Anselone,
James Bristow, and J. David Bristow
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We studied the effects of rapid atrial pacing during the final 10 minutes of a 70-minute, 31%
reduction in coronary blood flow in anesthetized swine to understand the significance of
apparent metabolic improvements during the initial 60 minutes of segmental ischemia. Within
5-10 minutes of ischemia, subendocardial phosphocreatine (PCr) and ATP were depleted to
47% and 63% of control, respectively; lactate accumulated within the subendocardium to 300%
of control; and net arteriovenous lactate production occurred. Despite continued ischemia and
no significant changes in the external determinants of myocardial oxygen consumption, by 60
minutes subendocardial PCr and lactate contents returned to near control levels and there was
net arteriovenous lactate consumption. Ischemic left ventricular wall thickening and ATP levels
remained depressed throughout the experiment. Atrial pacing during the final 10 minutes of
ischemia again resulted in depletion of PCr and lactate production. Since the myocardium was
capable of hydrolyzing PCr in response to atrial pacing at 60 minutes of ischemia, we conclude
it was capable of hydrolyzing PCr during the period of constant ischemia when instead it was
accumulating PCr. We propose the ischemic myocardium downregulates regional energy
requirements below blood flow-limited rates of energy production during ischemia. This
appears to be an active adaptation to ischemia and not a result of passive damage or cellular
injury. (Circulation Research 1991;69:1458-1469)
C onsiderable clinical evidence suggests that
myocardial hypoperfusion produces a
chronic but reversible decrease in myocardial function.12 The cellular mechanisms regulating
myocardial function under these conditions are not
understood. Although the metabolic consequences of
complete coronary occlusions and reperfusion are
well documented, experimental conditions of total
ischemia may exceed the limits to which the myocardium can adapt to reductions in coronary blood flow.
A better understanding of the metabolic and functional response to prolonged incomplete coronary
occlusion may help reveal regulatory processes overwhelmed by more severe ischemia.
From the Heart Research Laboratory, Oregon Health Sciences
University, Portland, Ore.
Supported by grants from the American Heart Association
(86-1278) and its Oregon Affiliate, and a National Institutes of
Health/National Heart, Lung, and Blood Institute National Research Service Award Institutional Training Grant (2 T32 HL07596).
Address for correspondence: J. David Bristow, MD, Division of
Cardiology, L462, Oregon Health Sciences University, 3181 S.W.
Sam Jackson Park Road, Portland, OR 97201-3098.
Received March 20, 1991; accepted June 21, 1991.
Two sets of experiments suggested to us that a
prolonged moderate reduction in coronary blood
flow triggers myocardial adaptations, resulting in a
restoration of energy supply/demand balance despite
ongoing ischemia. In contrast to the depletion of high
energy phosphate (HEP) compounds and eventual
onset of irreversible myocardial damage during sustained total coronary occlusion, we previously demonstrated that, after initial depletion, the myocardium regenerates phosphocreatine (PCr) during 1
hour of a constant partial reduction in coronary
blood flow.3 Despite restoration of PCr content,
regional myocardial function and ATP content remained depressed throughout the ischemic episode.
These experiments suggested that, during constant
subtotal ischemia, either impaired utilization of PCr
occurs or myocardial energy supply/demand balance
improves with time.
Also consistent with an adaptive process, myocardial lactate production does not remain constant
during steady mild to moderate myocardial ischemia.4,5 It is not clear whether this reflects an improvement in myocardial energy balance or a pathological process such as glycogen depletion, gradually
deteriorating myocardium, or other mechanism.4,6
Arai et al Myocardial Energy Requirements During Ischemia
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The current experiments were designed to test the
hypothesis that myocardial energy balance improves
with time, allowing regeneration of PCr during prolonged moderate ischemia. If this is the case, then
additional hemodynamic load at the end of a period
of stable ischemia could upset the new balance
between energy production and demand, once again
causing PCr depletion or lactate production. If PCr
regeneration and decreased lactate production resulted from pathological inability to use PCr and
inability to produce lactate, further metabolic demand should have no further effect on these parameters. To differentiate between these possibilities, we
studied the effects of increasing myocardial energy
needs with rapid atrial pacing during the last 10
minutes of a 70-minute period of moderate ischemia
in open-chest anesthetized pigs. In brief, our results
support a pattern of improving energy balance during
a period of constant moderate ischemia. These findings are not fully explainable by passive damage to
the myocardium and thus seem to represent an active
adaptation to ischemia.
Materials and Methods
Domestic swine of either sex that weighed between
42 and 51 kg were fasted overnight. After premedication with xylazine (2 mg/kg i.m.) and ketamine (10
mg/kg i.m.), anesthesia was induced with a-chloralose (100 mg/kg i.v.) and maintained with 50 mg/kg
i.v. every 2 hours. All animals in protocol 1 also
received a single dose of morphine suifate (0.5-1.5
mg/kg i.v.) after completion of the surgical preparation. Acid-base status was maintained within normal
limits (pH 7.35-7.45, Paco2 35-45 mm Hg, and Pao2
100-150 mm Hg) by titration of mechanical ventilation and administration of intravenous bicarbonate.
The arterial blood gas was always stable for at least
30 minutes before the experiment. Temperature was
maintained with a heating pad as needed.
Catheters were placed in the aorta and inferior
vena cava through femoral access. After median
sternotomy, the heart was suspended in a pericardial
cradle and the proximal left anterior descending
coronary artery (LAD) was dissected. A 2.5-mm
electromagnetic flow probe (Statham) and a polyvinyl hydraulic occluder were placed around the proximal LAD. Catheters (0.6 mm o.d., 0.3 mm i.d.) were
placed below the occluder in the LAD and the
anterior interventricular vein by the Herd-Barger
technique.7 We previously documented the frequency response of the LAD pressure waveform
measured with this system.8 Ultrasonic crystals (5
mHz) were sutured on the epicardial surface and
inserted into the subendocardium to monitor the
ischemic zone wall thickness. Systolic wall thickening
was calculated as the end-systolic thickness minus
end-diastolic thickness difference divided by enddiastolic thickness.9 Catheters were introduced into
the left atrial appendage for radioactive microsphere
injection (11.4±0.1 g.m) and the left ventricular (LV)
apex for LV pressures (catheter tip manometer).
1459
Finally, epicardial pacemaker leads were attached to
the appendage of the right atrium.
The following parameters were measured continuously and recorded on an eight-channel Mark 200
Brush recorder: mean LAD flow, phasic LAD flow,
ischemic zone LV wall thickness, electrocardiogram,
mean LAD pressure, phasic LAD pressure, LV pressure, and LV dP/dt. This allowed an observer to
modify pressure in the hydraulic occluder and accurately maintain LAD blood flow or LAD mean
pressure at selected levels. Any animals that developed hemodynamic instability during the experiment
or erratic coronary flow signals were excluded from
analysis.
Four sets of intermittent measurements were obtained from each animal. Obtained over 5 minutes,
each set consisted of blood samples from the aorta
and the anterior interventricular vein, microsphere
blood flow determination, and a transmural LV drill
biopsy (3 mm diameter). The biopsy was always the
last sample in a set to avoid the brief (30-60 seconds)
hemodynamic instability caused by this procedure.
To minimize damage to the ischemic portion of the
ventricle, control biopsies were usually obtained in
the circumflex perfusion zone. Biopsies from the
ischemic zone were obtained in a progression from
distal to more proximal portions of the LV to avoid
biopsy of an area with damaged vascular supply.
Overall metabolic and hemodynamic stability of this
preparation with serial biopsies has been reported,
including comparison of circumflex and LAD zone
biopsies.3
The heart was removed after completion of the
protocol. The right and circumflex coronary arteries
were catheterized close to their origins, and the LAD
was catheterized at the level of the occluder. Colored
dyes were simultaneously injected at constant rate
into each coronary artery to distinguish the ischemic
myocardium from the nonischemic zone. This procedure verified that the ultrasonic crystals and experimental biopsies were all obtained from the ischemic
zone.
The heart was fixed with formalin in preparation for
microsphere blood flow determinations. The six zones
analyzed included the subendocardial, midmyocardial,
and subepicardial portions of the ischemic and nonischemic zones. Border zones between ischemic and
nonischemic ventricle were excluded. The heart was
weighed before and after dye injection and again
after formalin fixation to compensate for a small
(about 10%) but consistent weight change associated
with dye injection and fixation.
Protocol 1
This group constitutes the primary focus of this
paper, evaluating the functional availability of PCr
that reaccumulates during ischemia. After control
measurements were obtained (including an LV transmyocardial drill biopsy, microsphere blood flow, and
blood samples), transmural LAD blood flow was reduced 30% and held constant for 60 minutes. Inter-
1460
Circulation Research Vol 69, No 6 December 1991
mittent measurements were obtained at 5-10 minutes
and 55-60 minutes of ischemia. After the 60-minute
sample, atrial pacing was started at about 50 beats/min
faster than the intrinsic heart rate and distal LAD
pressure was held constant at the level maintained
during ischemia by modifying pressure in the hydraulic
occluder. This allowed LAD flow to stabilize at a new
level. The increment in atrial pacing was reduced if
aortic systolic pressure fell more than 20 mm Hg.
Between about 65 and 70 minutes, the final intermittent measurements were obtained.
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Protocol 2
This group was included to delineate the time
course of lactate production between the 10- and
60-minute biopsies. Hemodynamic data and metabolic data other than lactate contents were previously
reported.3 A complete set of extracts from myocardial biopsies was stored at -70°C for approximately 1
year (n=6). In these animals, transmural microsphere blood flow had been reduced 21% and maintained at that level throughout 1 hour of monitoring.
Intermittent measurements were obtained at control,
5-10 minutes, 25-30 minutes, and 55-60 minutes.
None of these animals received atrial pacing. The
extracts were assayed for lactate content. Arterial
and venous samples were not available.
Protocol 3
This protocol was designed to determine the percentage of collateral flow (non-LAD) contributing to
blood collected from the anterior interventricular
vein. This validation study is important in interpreting the arteriovenous differences of metabolites and
calculating rates of ATP production. We previously
documented the almost complete lack of collateral
coronary arteries in normal swine10"11 but had not
verified that this applied to the venous drainage,
especially during ischemia.
Three animals were anesthetized and surgically
prepared as previously outlined with the following
modifications. A small Silastic catheter was introduced retrograde into the circumflex artery or the
ramus intermedius artery within 5 mm of the left
main coronary artery. This allowed infusion of fluorescein against the direction of blood flow to enhance
mixing. Small Silastic catheters were placed in the
anterior interventricular vein and a vein parallel to
an obtuse marginal branch of the circumflex artery.
The location of the anterior interventricular vein
catheter and technique of insertion were identical to
those used in protocols 1 and 2.
Simultaneous blood samples were obtained from
the aorta, the anterior interventricular vein, and the
vein in the circumflex distribution. Venous samples
were obtained at a rate of 1 ml/min. After control
samples were obtained, fluorescein (1 mg/ml at 1
ml/min) was infused continuously into the circumflex
artery. Simultaneous blood samples were obtained
after 1 minute of infusion and every 3 minutes
thereafter for 13 minutes. The fluorescein infusion
was then stopped. About 30 minutes later, the sampling procedure was repeated except that LAD flow
was reduced by 40% compared with control levels as
determined by the electromagnetic flow probe. After
a stable functional state was reached (less than 2
minutes), the fluorescein infusion was restarted and
blood samples were obtained for 13 minutes in the
same sequence as previously indicated.
If non-LAD blood was supplied to ischemic myocardium or to the anterior interventricular vein at
the level of our venous catheter, then fluorescence
of plasma from this zone would exceed levels that
gradually accumulate in the systemic circulation.
Intensity of fluorescence (exciting 487 nm, emission
515 nm, bandwidth 10 nm) from plasma samples
was measured on a Hitachi spectrofluorometer
(model F-2000). Standard curves in water and
plasma demonstrated linearity over the range of
concentrations collected (intensity versus concentration: r=0.997, n=7). Units reported reflect intensity detected by the photomultiplier.
Chemical Analysis
Transmyocardial drill biopsies (3 mm diameter)
were frozen in liquid nitrogen within 1-2 seconds and
stored at -70°C until extracted. The approximately
3 x3 x 12-mm biopsy samples were divided into
subendocardial, midmyocardial, and subepicardial
segments on a stage submerged in liquid nitrogen.
The subepicardium was distinguished from the
subendocardium by a blue dye that had been painted
on the epicardial surface of the heart before the
biopsy was obtained. The frozen samples were extracted and neutralized as previously reported.3
Arterial and venous blood samples were obtained
anaerobically in cold syringes containing heparin
fluoride to inhibit glycolysis. Samples were divided
for oxygen and lactate content and stored on ice until
processed immediately after the experiment. Oxygen
content was measured in duplicate with an IL 382
hemoximeter (Instrumentation Laboratory, Lexington, Mass.). Plasma for lactate content was deproteinated with perchloric acid and neutralized with
potassium hydroxide and imidazole buffer. Plasma
samples were then frozen until enzymatic analysis.
Myocardial metabolite contents (ATP, PCr, and
lactate) and plasma lactate concentrations were determined by the spectrofluorometric NAD/NADH
enzyme-linked methods of Lowry and Passonneau.'2
All samples were run in duplicate. The intra-assay
coefficient of variation for ATP was ±+2.4%, for PCr
+3.4%, and for lactate ±2.8%.
Statistical Analysis
Results are reported as mean ±SD in parentheses
or as error bars. One-way analysis of variance
(ANOVA) with repeated measures followed by
range testing (Tukey's test) was performed for comparisons between groups.13 The tissue lactate content
in the subendocardial and middle layers was analyzed
by the Friedman test (nonparametric equivalent to
Arai et al Myocardial Energy Requirements During Ischemia
1461
TABLE 1. Effect of Partial Ischemia on Microsphere Blood Flow Before and During Atrial Pacing
Ischemia
LAD blood flow
5-10 Minutes
65-70 Minutes (paced)
55-60 Minutes
Control
0.92 (0.28)*
0.73 (0.30)*
1.32 (0.34)
0.92 (0.25)*
Transmural
0.60 (0.15)*
0.33 (0.20)*
1.33 (0.39)
0.63 (0.22)*
Subendocardial
0.68 (0.37)*
0.91 (0.29)*
0.93 (0.26)*
1.39 (0.34)
Midmyocardial
1.23 (0.51)t
1.15 (0.42)t
1.25 (0.33)
1.18 (0.42)t
Subepicardial
0.28 (0.12)*
0.54 (0.24)*
0.58 (0.24)*
1.07 (0.15)
I/O ratio
Results are listed as mean. Numbers in parentheses indicate standard deviation. I/O ratio, subendocardial to
subepicardial blood flow ratio.
*p<<0.001, tNS, vs. control.
(ml/g/min)
the one-way ANOVA with repeated measures) because the data exhibited severe inequality of variance
between groups (p<0.0005, F test). Analysis of covariance was used to compare x-y relations.
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Results
Final results are reported for 18 of the 25 animals
studied (protocol 1, n=9; protocol 2, n=6; and
protocol 3, n =3) Since the purpose of protocol 1 was
to evaluate the physiological significance of PCr
regeneration, we excluded two animals that did not
regenerate PCr to at least 67% of control by the
60-minute biopsy. Adding these two animals to the
nine reported would alter average metabolite contents by less than 10% throughout the experiment. It
is important, however, to exclude them because they
do not meet the experimental criteria of interest.
This also avoids a potential bias toward demonstrating lower average PCr content after pacing. Other
animals were excluded for the following reasons:
congenital ventricular septal defect discovered postmortem (n = 1), abnormally low control ATP and PCr
(n = 1), hemodynamic instability (n = 1), ventricular
fibrillation after biopsy (n = 1), and myocardial infarction of the nonischemic zone likely related to the
circumflex zone biopsy (n = 1).
Protocol 1: Metabolic Effects of Partial Ischemia
Before and During Atrial Pacing
Radioactive microspheres validated the electromagnetic flow probe measurements at four time
periods. After the control measurements, the blood
flow through the LAD was decreased 30% by continuously monitoring the coronary blood flow and manually adjusting pressure in the hydraulic occluder.
Ischemia resulted in a 31% reduction in transmural
microsphere blood flow (p<0.001) as listed in Table
1. LAD blood flow was maintained at this level for 60
minutes, and transmural microsphere blood flow was
essentially the same at 5 and 60 minutes. The subendocardium was more severely affected than the other
layers, resulting in a 55% reduction in microsphere
blood flow to this region (p<0.001) and significant
reductions in the inner/outer blood flow ratio
(p<O0.001). Transmural distribution of blood flow did
not change during the period of constant ischemia
between 5 and 60 minutes (Table 1, Figure 1). There
was a tendency for heart rate to increase and LV
systolic blood pressure to fall during the period of
constant ischemia (not significant versus control)
(Table 2).
Atrial pacing during the final 10 minutes of ischemia increased heart rate by 42% and rate/pressure
product by 35% compared with 60 minutes. Because
mean LAD pressure was held constant during this
time period, transmural myocardial microsphere flow
was reduced 20% from the ischemic level (not significant versus 60 minutes). This resulted in statistically
significant further reductions in LAD inner flow
(p<0.025) and inner/outer flow ratio (p<0.005)
compared with 60 minutes. Thus, after an hour of
constant ischemia, atrial pacing imposed increased
energy requirements in the face of a slight further
decrease in oxygen supply (Tables 1 and 2).
Increases in myocardial oxygen extraction were
unable to maintain oxygen consumption at control
levels with this degree of ischemia (Table 3). Regional systolic LV wall thickening decreased to a new
plateau during ischemia, usually within 30 seconds
1 Rn.
Lov
****p<0.001 vs control
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V.V
Cotrl
CoAtrol
5-dmm
5-10 min
5-6 m
55-60 min
Paced 570m
65-70 min
-
Duration of Ischemia
FIGURE 1. Distribution of myocardial blood flow. Left
anterior descending coronary artery blood flow was manually
reduced 30% for 60 minutes. The degree of ischemia and
distribution ofmicrosphere blood flow remained constant until
rapid atrial pacing imposed added demand and further reduced subendocardial blood flow.
1462
Circulation Research Vol 69, No 6 December 1991
TABLE 2. Hemodynamic Effects of Partial Ischemia Before and During Atrial Pacing
Ischemia
5-10 Minutes
55-60 Minutes
65-70 Minutes (paced)
Control
103 (16)*
106 (19)*
150 (14)t
102 (13)
HR (beats/min)
118 (9)
111 (12)*
107 (17)*
103 (16)t
LVSP (mm Hg)
11.4 (2.6)*
15.5 (3.0)t
12.1 (1.4)
11.5 (1.9)*
HRxLVSP (x103)
9 (1)
12(4)*
16(3)§
12(3)*
LVEDP(mmHg)
48 (4)t
48 (3)t
95 (10)
46 (4)t
LAD mean pressure (mm Hg)
7% (7)t
15% (9)t
15% (7)t
28% (5)
LV wall thickening
Results are listed as mean. Numbers in parentheses indicate standard deviation. HR, heart rate; LVSP, left
ventricular (LV) systolic pressure; LVEDP, LV end-diastolic pressure; LAD, left anterior descending coronary artery.
*NS, tp<0.001, *p<0.01, §p<0.005, vs. control.
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(data between 0 and 30 seconds not shown). Percent
wall thickening remained stable throughout the period of constant ischemia and decreased further with
the demands of rapid atrial pacing (Table 2).
Ischemia resulted in a graded metabolic response
across the ventricular wall with the most prominent
effects in the subendocardium (Table 3). Subendocardial ATP content fell to 63% of control during
the first 10 minutes of ischemia and to 49% of control
by 60 minutes (Figure 2A, both p<0.001 versus
control). Atrial pacing did not further change ATP
levels.
Subendocardial PCr was transiently depleted to
47% of control during the first 10 minutes of ischemia
(p<0.001) but, during the last 50 minutes of constant
ischemia, was regenerated to 96% of preischemic
levels (Figure 2B, p<0.005 versus 10 minutes). That
the ischemic myocardium can use this regenerated
store of PCr was demonstrated by the subsequent
depletion in response to atrial pacing. The fall in PCr
content was of similar magnitude to the initial fall
after onset of ischemia.
The subendocardial lactate content increased to
approximately 300% of control levels by 5-10 minutes
after the onset of ischemia (Figure 3A). This was a
transient phenomenon, and myocardial lactate levels
were not significantly different from control at 55-60
minutes. Similarly, plasma samples across the ischemic LAD vascular bed demonstrated the transition
from a control lactate-consuming state to a lactateproducing state at 5-10 minutes (Figure 3B). By 60
minutes the ischemic myocardium returned to a net
lactate-consuming state that was not statistically different from control. Atrial pacing caused subendocardial lactate accumulation (p<0.001 versus control)
and net arteriovenous lactate production (p<0.01
versus control). This indicates the myocardium was
capable of producing substantial amounts of lactate at
the end of an hour of ischemia at a time when there
was virtually no net flux through anaerobic glycolysis.
TABLE 3. Metabolic Effects of Partial Ischemia Before and During Atrial Pacing
ATP (,umol/g wet wt)
Subendocardium
Midmyocardium
Subepicardium
Phosphocreatine (gmol/g wet wt)
Subendocardium
Midmyocardium
Subepicardium
Lactate (,umol/g wet wt)
Subendocardium
Midmyocardium
Subepicardium
Lactate consumption (,umol/100 g/min)
Oxygen consumption (ml/100 g/min)
ATP production (,umol/100 g/min)
Control
5-10 Minutes
Ischemia
55-60 Minutes
65-70 Minutes (paced)
3.91 (0.31)
4.43 (0.42)
3.75 (0.58)
2.48 (1.10)*
3.09 (1.19)t
3.73 (0.51)§
1.91 (0.90)*
2.70 (0.88)*
3.73 (0.54)§
1.74 (0.85)*
2.92 (1.01)t
3.61 (0.83)§
6.78 (0.68)
7.90 (0.78)
7.09 (1.25)
3.20 (1.76)*
4.75 (2.37)t
6.79 (1.38)§
6.54 (2.01)§
7.45 (1.60)§
7.92 (1.12)§
3.63 (2.11)t
5.65 (1.72)§
6.48 (1.75)§
2.97 (1.12)
2.83 (1.11)
3.40 (1.19)
115 (86)
11.67 (3.69)
9.60 (5.04)*
7.08 (5.81)t
3.61 (1.10)§
-136 (81)*
8.97 (1.89)*
4.88
3.90
3.97
35
(1.73)§
9.00 (1.78)11
8.48 (5.10)*
5.82 (3.29)§
3.46 (1.15)§
- 15 (41)t
7.09 (2.50)
1,796 (333)11
2 (5)
1,798 (332)11
1,416 (466)*
46 (47)
1,462 (440)*
Aerobic
1,791 (354)11
2,330 (690)
Anaerobic
155 (93)
Negligible
Total
1,945 (406)§
2,330 (690)
Results are listed as mean. Numbers in parentheses indicate standard deviation.
*p<0.001, tp<0.01, tp<O.OOS, §NS, Ilp<O.OS, vs. control.
(2.21)§
(3.24)§
(42)§
Arai et al Myocardial Energy Requirements During Ischemia
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**p<0.01 vs control
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****p<0.001 vs control
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55-60 min
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Duration of Ischemia
5-10 min
55-60 min
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Duration of Ischemia
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Control
5-10 min
Paced
55-60 min
65-70 min
Duration of Ischemia
FIGURE 2. Myocardial high energy phosphate contents.
Panel A: Four transmyocardial drill biopsies were obtained
from each animal. Ischemia produced a gradient of ATP
depletion across the myocardium. Subendocardial ATP contents fell to 63% of control by 10 minutes and only slightly
further between 10 and 60 minutes. Panel B: In contrast to the
persistent derangement observed for A TP contents, phosphocreatine (PCr) was regenerated to near control levels by 60
minutes. The myocardium was capable of using PCr stores
during late ischemia (when PCr was instead accumulating) as
evidenced by the decreased levels after atrial pacing (65-70
minutes).
Protocol 2: Time Course of Metabolic Changes
During Partial Ischemia
This group of pigs was studied during 1 hour of a
steady 21% reduction in transmural LAD blood flow.
The functional and nonlactate metabolic data for
these animals were reported previously.3 These animals demonstrated similar hemodynamics and myocardial blood flow distributions to the animals presented in protocol 1. Ischemic zone systolic wall
thickening was reduced 50% during ischemia and
remained stable. Although absolute lactate content
should be interpreted with some caution considering
these extracts had been frozen for one year, this
should not preclude a qualitative assessment of the
Control
5-10 min 55-60 min 65-70 min
Duration of lschemia
FIGURE 3. Myocardial lactate metabolism. The myocardium was consuming lactate at control. During early ischemia,
the myocardium produces lactate resulting in both intramyocardial accumulation of lactate (panel A) and net arteriovenous production (panel B). These changes are transient
despite continuous ischemia. The myocardium was capable of
producing lactate at 60 minutes as evidenced by the response
to atrial pacing.
time course of lactate accumulation and normalization. Lactate accumulated in the subendocardium
during early ischemia to levels comparable to those
observed in protocol 1 (from control, 2.23+0.97, to
8.95 +4.31 gmol/g wet wt, p<0.005). PCr and lactate
followed reciprocal courses. Lactate levels appear to
plateau between 30 and 60 minutes (4.30±1.74 and
4.20±1.59 ,umol/g wet wt, respectively; neither was
significantly different from control). Thus, anaerobic
glycolysis provides minimal energy production in late
ischemia under these conditions.
Combined Results From Protocols 1 and 2
The relation between subendocardial PCr content
and regional LV wall thickening may provide clues
concerning potential mechanisms regulating myocardial function during ischemia (Figure 4). There is a
linear relation between subendocardial PCr content
Circulation Research Vol 69, No 6 December 1991
1464
A.
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a Early ischemia E
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Subendocardial PCr Content
FIGURE 4. Regional function versus subendocardial phosphocreatine (PCr) content during early and late ischemia.
Data were combined for all animals studied under protocols 1
and 2, including animals that did not fully regenerate PCr. By
comparing early ischemia (control and 5-10-minute data)
with late ischemia (55-60-minute data only), there is a
time-dependent shift in the relation between subendocardial
PCr content and regional function (p<0.0001 by analysis of
covariance). Animals from protocol 1 that do not fully
regenerate PCr (* next to data point) appear to fall along the
same relation as the other animals during late ischemia. This
suggests PCr regeneration during ischemia is a continuous
function, not an "all or none" phenomenon.
Duration of Infusion (minutes)
B.
120,
c
*i
100
0~~~
5
800~~~~~
0
am
CDo
a,
0
.2
LL
40
S~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~
20
a
Protocol 3: Specificity of Anterior Interventricular
Blood Samples in the Swine
Before infusion of fluorescein, no fluorescent emission was detected in the aorta, anterior interventricular
vein, or circumflex vein. Infusion of fluorescein into the
circumflex artery resulted in strong fluorescent emission in the circumflex vein (1,583 ±372 units with basal
LAD blood flow and 1,313+251 units during a 40%
reduction in LAD blood flow). Fluorescein also stained
a sharply demarcated zone on the surface of the heart
that demonstrated the area perfused by the circumflex
artery. Fluorescent activity in the anterior interventricular vein slowly increased over time but on average was
less than 10% of intensities detected in the circumflex
vein (Figure SA). Aortic fluorescence resulting from
recirculation of dye accounted for 93.4% and 95.4% of
the fluorescence detected in the anterior interventricular vein during basal LAD blood flow (r=0.966) and
40% reduced LAD blood flow (r=0.977), respectively
(Figure 5B). Collateral blood flow from the circumflex
artery contributed less than 2% of anterior interventricular vein blood flow (95% confidence limits, 1.8% to
U_
40
80
60
100
120
Fluorescence from Aorta
FIGURE 5. Specificity of blood from the anterior interventricular vein. Panel A: Fluorescein infused into the proximal
circumflex artery results in high fluorescent intensity in plasma
drawn from a vein in the circumflex perfusion zone (Circumflex Vein). Fluorescence from plasma in the anterior interventricular vein (LAD Vein) is no higher than systemic accumulation resultingfiom recirculation (Aorta). Standard deviation
error bars for aorta and LAD vein fluorescence are smaller
than the markers. Panel B: Plasma fluorescence in the LAD
vein is almost entirely due to recirculation and systemic
accumulation, indicating venous samples from this catheter
have high specificity for the ischemic zone. Simultaneous
samples from the aorta and the LAD vein are plotted against
the unity line. This was performed during basal LAD blood
flow (boxes) and a 40% reduction in LAD blood flow
(circles).
0
and LV wall thickening as has been reported by
Schaefer et al.'4 Since PCr regenerates during ischemia
without a significant improvement in regional function,
there is a significant shift in the relation between these
variables when comparing early ischemia (control and
5-10-minute data, y=3.39x+4.11, r=0.72) with late
ischemia (55-60-minute data, y=3.07x-5.70, r=0.84)
by analysis of covariance (p<0.0001).
a
E
20
-1.9% during basal flow and 1.4% to -1.9% during
reduced LAD flow). Thus, blood samples obtained
from the anterior interventricular vein represent drainage from the ischemic myocardium with virtually no
contamination from nonischemic zones.
Calculations
Net rate of ATP production
(gLmol/100 g/min) at a
given time is the sum of the rates of aerobic ATP
production and anaerobic ATP production. Aerobic
ATP production (,umol/100 g/min) is derived from
the oxygen consumption data and assumes a phos-
Arai et al Myocardial Energy Requirements During Ischemia
phorus/oxygen (P:0) ratio of 2.4.15 Although radioisotopic tracer studies have demonstrated that a
small amount of lactate production can occur at a
time of net lactate consumption,16,17 we assume for
these calculations that this contributes a negligible
amount of ATP during the control state.
Net ATP production=
aerobic ATP production+
anaerobic ATP production
1465
l-
0)
0
0
c0
m
1-1
(1)
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1 mol ATP
Anaerobic production=1 mol lactatex
(arteriovenous lactate production+
lactate accumulation)
(2)
Anaerobic ATP production (,umol/100 g/min) is
calculated from arteriovenous lactate production and
transmural accumulation of lactate. Between 1.0 and
1.5 moles of ATP are produced per minute for each
mole of lactate produced or accumulated per minute,
depending on the relative proportion of glucose and
glycogen serving as substrate. To avoid overestimating anaerobic ATP production and in keeping with
the fact that even the subendocardium maintains
approximately 50% of basal myocardial blood flow in
these experiments, we assume glucose is the primary
substrate for anaerobic glycolysis and 1.0 mole of
ATP is produced per mole of lactate.17
Because of the time delays inherent to each measurement, blood samples for oxygen and lactate were
obtained at 5, 55, and 65 minutes of ischemia, and LV
biopsies were obtained at 10, 60, and 70 minutes. If
one assumes a linear accumulation of lactate between 0 and 10 minutes, the rate of lactate accumulation at 5 minutes can be calculated from the
difference in lactate contents between control and 10
minutes and dividing by 10 minutes. Similar calculations provide ATP production at 55 and 65 minutes
except that lactate accumulation is negligible between 30 and 60 minutes (protocol 2).
Calculated rates of aerobic and anaerobic ATP
production from the eight animals in protocol 1 with
complete oxygen consumption data are shown in
Figure 6 and Table 3. Control rates of ATP production indicate the total HEP pool turns over about
twice per minute at this workload. During early ischemia, anaerobic lactate production contributes 7.8%
of total ATP production. Although total ATP production during early ischemia is not statistically different
from control, this is likely a type II statistical error
since anaerobic ATP production accounts for less
than 30% of the decrement in aerobic ATP production between control and 5 minutes. The increase in
anaerobic ATP production is significantly less than the
decrease in aerobic ATP production between control
and 5 minutes (two-tailed, paired t test, p<0.05). By
55 minutes of ischemia, anaerobic ATP production
accounts for 0.1% of total ATP production. Thus, the
new homeostasis, characterized by persistently re-
0
t
~00
0(
Control
5 min
55 min
65 min
Duration of Ischemia
FIGURE 6. ATP production at four points of the experiment
was calculated from myocardial oxygen consumption (aerobic
production, shaded areas) and myocardial arteriovenous lactate production and transmural accumulation of lactate (anaerobic production, black areas). There is minimal anaerobic
ATPproduction at 60 minutes of ischemia despite reduced total
ATP production compared with control and residual capacity
to upregulate anaerobic glycolysis, as evidenced by pacing.
duced wall thickening and regenerated PCr, occurs at
a time when the myocardium produces 23% less ATP
per minute than at control (p<0.025) and no longer
requires anaerobic glycolysis.
Discussion
We have previously reported that the myocardium
regenerates PCr during 60 minutes of moderate
hypoperfusion.3 These data, along with evidence for
a decrease in arteriovenous lactate production,4,5 are
consistent with an improvement in the myocardial
metabolic state during prolonged partial ischemia.
However, these results could also be compatible with
pathological abnormalities as noted in each of these
articles. Subcellular compartmentation may have allowed accumulation of PCr in a location inaccessible
to use by the myofibrils. Alternatively, PCr regeneration might have been due to selective inhibition of
cytosolic creatine kinase. Either situation would limit
the availability or rate of utilization of PCr and could
lead to paradoxical accumulation of PCr during
ischemia. Similarly, inhibition of anaerobic glycolysis
caused by intracellular acidosis or lack of substrate,
such as glycogen, might explain decreasing arteriovenous lactate production during late ischemia.
The current experiments were designed to differentiate whether these dynamic metabolic changes represent energetic improvements during ischemia or progressive deterioration. Our current results confirm our
earlier findings of PCr regeneration in the majority of
animals undergoing 60 minutes of moderate myocardial
ischemia. We add several important observations suggesting the metabolic changes during 60-minute reductions in coronary blood flow represent overall improve-
1466
Circulation Research Vol 69, No 6 December 1991
ments in myocardial energetics despite ongoing
ischemia. The decrease in arteriovenous lactate production parallels decreased subendocardial lactate content. That the subendocardium is capable of producing
lactate in response to the added stress of atrial pacing
demonstrates that substrate availability does not limit
lactate production during late ischemia. Since the myocardium is capable of using PCr stores in response to
atrial pacing, we conclude the myocardium is capable
of using these stores during late ischemia when it
instead reaccumulates the high energy compound.
Taken one step further, we can now conclude PCr
regeneration represents accumulation of useful high
energy stores during ischemia. Thus, the metabolic
response to atrial pacing is critical to understanding the
importance of PCr regeneration during the period of
stable ischemia. This logic forms the crux of our thesis
that myocardial energy balance is improving during
moderate ischemia.
Downloaded from http://circres.ahajournals.org/ by guest on July 31, 2017
Downregulation of Energy Requirements
During Ischemia
We propose that the myocardium actively downregulates regional energy requirements during ischemia, allowing reaccumulation of PCr during ongoing
ischemia. This hypothesis may be an extension of the
observation that the normally perfused myocardium
must precisely balance energy production to meet
increased energy requirements. It is widely accepted
that ATP production must increase to meet the
demand of higher myocardial workloads under normal conditions. This has been demonstrated in the
isolated perfused rat heart by direct assessment of
the creatine kinase reaction rate.18 Furthermore, the
well-perfused myocardium rapidly increases ATP
production to meet the demands of increased workload without depleting PCr or ATP.19,20
Ischemia clearly interferes with the balance between energy utilization and production and results
in an ATP and PCr depleting phase. This has been
well described under experimental conditions of anoxia, hypoxia, no blood flow ischemia, and partial
reductions in coronary blood flow.21-25 If the absolute
difference between the rates of ATP utilization and
production remained static, the rate of depletion
would remain constant and eventually the total HEP
pool would be depleted. Quantification of the rate at
which the total adenine nucleotide pool is depleted
demonstrates that the rate of depletion slows after
the rapid phase during early total ischemia.26-28
During constant partial reductions in coronary flow,
ATP content eventually stabilizes, suggesting that a
new balance between utilization and production has
been established.29 Studying the relation between
regional function and coronary blood flow per beat,
Gallagher et al30 concluded myocardial energy requirements and function might be gradually decreased during ischemia until a "new equilibrium
between (limited) supply and (reduced) demand"
was established.
Despite these observations, the concept of downregulation of myocardial energy requirements during
ischemia is not well accepted. To further illustrate
that myocardial energy utilization must be downregulated during ischemia, the metabolic consequences
of three different models of myocardial downregulation will be discussed. In each of these examples, the
time course of ATP production is extrapolated from
the four determinations made in protocol 1 and the
fact that subendocardial lactate levels were close to
control levels by 30 minutes in protocol 2. Three
different time courses of ATP utilization are compared with the same interpolated ATP production
curve. The difference between the integral of ATP
production and ATP utilization determines the net
accumulation or depletion of HEP contents.
The rate of ATP utilization for an ischemic myocardium with no capacity for downregulating regional
energy requirements should remain at baseline levels
while ATP production falls during ischemia. Because
ATP utilization exceeds production at all times during the ischemic episode, the myocardium should
continuously deplete HEP stores. With a 25% decrease in myocardial oxygen consumption and an
HEP turnover rate of two to four times per minute,3'
total depletion of ATP and PCr should occur between 1 and 2 minutes. This degree of depletion does
not even occur with complete occlusions of much
longer duration. Thus, a model stating that regional
myocardial energy requirements are determined
largely by external factors and are not locally regulated is incompatible with virtually every study measuring HEP contents during ischemia.
Broadly defined, passive downregulation of local
myocardial energy requirements could encompass
conditions ranging from metabolite depletion and
undetectable structural damage to myocardial infarction. In each case the primary mechanism altering
regional energy requirements involves injury to the
energy-utilizing machinery. Because of this unspecified damage, energy requirements gradually fall until
a new balance is reached between ischemic ATP
production and utilization (Figure 7A). Since energy
production falls more rapidly, there is a transient
depletion of HEP compounds. Later ischemia is
characterized by a new homeostasis, and HEP contents could remain stable indefinitely if the pathological process that initiated these changes was not
progressive. Whether or not the myocardium continues to produce lactate during late ischemia may
depend on the severity of the blood flow reduction.
This model is consistent with the findings of Neill et
a129 during moderate ischemia and Gallagher et al.30
Because the rate of ATP utilization never falls below
the rate of ATP production, this model does not
predict an energy-accumulating state during late
ischemia as demonstrated in our experiments.
If regional myocardial energy requirements could
be actively downregulated during ischemia, then
ATP utilization could fall with time without necessarily resulting in myocardial damage. HEP depletion
would occur only during the initial phase of ischemia
until utilization equals production. The important
Arai et al Myocardial Energy Requirements During Ischemia
A. Passively Downregulated Model
25Mu
L
m
_pv
c
0
0
b.
oL
C-
=e2000
o9
0
o-
1-
t~~~~~~
-
1500
,, ,) 10001
DHpEPtO
Depletion
Stable HEP Derangement
I....
20 ...
50 60,
Duration of lschemia (minutes)
-10' 6i010o
...
......--
B. Actively Downregulated Model
2500
r-i P -
cj:
+ r' 2000-
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0
00)
0 .o 0
X 0 1500-
.N
0.
._
`_i
o
_1 t=
c
--i
CL CL =
[L
1000
_
-10
HEP
PCr Regeneration
DeptetioHEP
0. 10 20 30 40 50
60
Duration of lschemia (minutes)
FIGURE 7. Models of myocardial downregulation of energy
requirements during ischemia. ATP utilization (triangles) for
each model is plotted against the same ATP production curve
(open circles), which is extrapolated from Figure 5. In both
models, high energy phosphate (HEP) depletion occurs during
early ischemia while ATP utilization exceeds ATP production.
Panel A: A model of passive downregulation can explain
stabilization of a derangement in ATP metabolism once
production equals utilization. Panel B: Only an actively
downregulated system lowers utilization below flow-limited
ATP production, explaining why phosphocreatine (PCr) can
accumulate during late ischemia. The difference between ATP
utilization and production is exaggerated in these diagrams for
clarity. See text for further discussion.
distinction between this model and a passively regulated system involves the logical extension to the
reparative phase of ischemia. An actively regulated
system could decrease local energy requirement
somewhat below the flow-limited rate of energy
production (Figure 7B). This model is not incompatible in any aspect with the findings of Neill et a129 but
provides an explanation for our observed regeneration of PCr despite ongoing ischemia. To what extent
this process can continue remains to be defined.
Some of the animals studied exhibited higher than
control levels of PCr by the end of 60 minutes of
ischemia ("PCr overshoot"). Whether or not ATP
content will eventually improve is unclear. The failure to regenerate ATP during the period of energy
surplus can likely be explained by extracellular loss of
adenine nucleotides and their precursors.32 The slow
rate and high energetic cost of de novo adenine
nucleotide synthesis would require days before ATP
content normalized.33
1467
ATP Production During Ischemia
There are several possible mechanisms to explain
why the rate of ATP production appears to decrease
slightly between 10 and 60 minutes of stable ischemia. A gradual reduction in energy requirements
between 5 and 60 minutes, as postulated in our
model of actively downregulated energy requirements during ischemia, could account for decreased
anaerobic ATP production during late ischemia. The
transition back to a lactate-consuming state might
therefore be a marker for the transition to a PCraccumulating state or vice versa. Additional possibilities are considered in the following paragraphs.
The free energy change for ATP hydrolysis (dG/
dE) represents the maximal amount of chemical work
available from hydrolysis of the ATP pool. As an
exergonic reaction, dG/dE is a negative quantity,
indicating hydrolysis of ATP tends to occur spontaneously and can supply chemical energy to drive
other reactions. The concentrations of ATP, free
ADP, and Pi influence dG/dE as shown in Equation 3,
where AG0 is the standard free energy change of the
reaction, R is the gas constant, and T is the absolute
temperature:
dG/dE=AWG- RT ln
[ADP]LP])
(3)
Although the absolute value of dG/dE likely does not
decrease sufficiently to explain the observed decrease
in regional wall thickening,34 these changes may, however, be large enough to account for why ATP production is higher during early ischemia than late ischemia.
In essence, if the magnitude of dG/dE decreases transiently, less chemical work could be derived from the
ATP pool during early ischemia compared with 60
minutes. Thus, if mechanical function and other determinants of ischemic zone energy requirements remained stable throughout the first hour of ischemia, a
transient decrease in the magnitude of dG/dE would
require a transient increase in ATP turnover, which is
consistent with our observations.
Transient decreases in the efficiency of mitochondrial oxygen utilization could also explain why ATP
production appears higher in early ischemia than at
60 minutes. Partial uncoupling of oxidative phosphorylation would mean our estimate of aerobic ATP
production is falsely high since we assume a constant
P: 0 ratio of 2.4. If partial uncoupling of oxidative
phosphorylation occurred for a limited time, this
could also explain the brief need for lactate production. A decrease in the P: 0 ratio associated with a
change in mitochondrial substrate would have similar
effects.35
Conversely, increases in the efficiency of mitochondrial function during ischemia could mean our estimates of ATP production are falsely low. An increase
in the P: 0 ratio from 2.4 to 3.0 would be sufficient to
balance ATP production to control levels with the
degree of ischemia studied. When analyzed by layers
1468
Circulation Research Vol 69, No 6 December 1991
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across the LV wall, the P: 0 ratio in the subendocardium would need to increase even further to account
for the 55% decrease in subendocardial blood flow. To
date, there is no experimental work to suggest such a
mechanism is possible. We feel that such a mechanism
is less likely than local downregulation of energy
requirements in light of the marked impairment in
regional wall thickening throughout ischemia.
With the exception of an increase in mitochondrial
efficiency during ischemia, none of the mechanisms
described eliminates the conceptual need for active
local downregulation of energy requirements during
ischemia as an explanation for the regeneration of
PCr during ischemia.
mechanisms allowing accumulation of PCr despite
ongoing ischemia. The animals failing to regenerate
PCr by 60 minutes appeared to follow the same PCr
versus function relation as the other animals during
late ischemia (Figure 4). So, although these animals
appear to have downregulated regional wall thickening during late ischemia, this was not a sufficient
adaptation to allow PCr reaccumulation. Factors such
as size of the ischemic zone or baseline oxygen consumption or factors we cannot yet identify may determine the limits to which the myocardium can actively
downregulate regional energy requirements and the
rate of PCr regeneration during ischemia. Resolution
of these issues will require further study.
Limitations
The greatest limitations of these methods are
inherent to tissue extraction and enzymatic analysis.
The inability to measure free [ADP] is not unique to
chemical determinations. In experiments by nuclear
magnetic resonance spectroscopy, free [ADP] is generally calculated from measurements of other reactants in the creatine kinase equilibrium.36 Similarly,
our inability to measure intracellular concentrations
of inorganic phosphorus, free intracellular calcium
(Ca2), and pH requires circumstantial analysis.
The relation between regional function and myocardial PCr content shifts between early ischemia
and late ischemia (Figure 4). These relations may
also shift during early reperfusion but in the opposite
direction.3738 The mechanisms responsible for the
time-dependent modification in the PCr versus function relation cannot be determined by our data.
However, assuming intracellular pH improves on a
similar time course to the myocardial lactate content,
normalization of pH would have been predicted to
improve contractile function for a given energetic
state, opposite the trend in Figure 4. Similarly,
intracellular Pi should have decreased as PCr is
regenerated, which should improve function during
late ischemia. Thus, we hypothesize that Pi and pH
cannot be the sole regulators of myocardial function
during ischemia as is also suggested by From et al.37
Alterations in Ca2' transient size39 or altered myofibril-Ca2' interactions may play an important role
during late ischemia.
The experimental model used to evaluate these
systems may be important. Our in situ model of
regional ischemia functions under relatively low
workloads with enough nonischemic ventricle to support global ventricular function. The myocardium
may be able to adapt to much more severe ischemic
insults than we have studied but at the cost of
markedly depressed function that would not be tolerated in situ and at the cost of continuous myocardial lactate production.40
Although the majority of animals regenerate PCr
during continuous moderate ischemia, two animals
from protocol 1 and three animals from protocol 2 did
not regenerate PCr to at least 67% of control levels by
60 minutes. These animals may provide clues to the
Implications and Conclusions
Despite certain limitations, these studies provide
considerable insight into the dynamic changes that
occur during the course of an hour of myocardial
ischemia. This work indicates the ischemic myocardium actively downregulates myocardial energy utilization beyond that necessary to achieve energy balance. This allows the myocardium to accumulate PCr
and eliminate anaerobic glycolysis despite ongoing
ischemia. Although reduced function and energy requirements during prolonged subtotal ischemia are
important characteristics of the hibernating myocardium, extrapolation from our observations to the
clinically described situation remains indirect. Studies
of anoxia or complete coronary occlusion may exceed
the capability of these regulatory mechanisms. Further
study of the relation between myocardial function and
metabolism during prolonged partial ischemia may
provide important insight into the mechanisms that
regulate myocardial function and into the common
clinical correlates to these problems.
Acknowledgment
The authors wish to acknowledge the advice provided by William J. Thoma, PhD, in the preparation
of this manuscript.
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KEY WoRDSs ATP * phosphocreatine * lactate * metabolism
* hibernation
Active downregulation of myocardial energy requirements during prolonged moderate
ischemia in swine.
A E Arai, G A Pantely, C G Anselone, J Bristow and J D Bristow
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Circ Res. 1991;69:1458-1469
doi: 10.1161/01.RES.69.6.1458
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