Cardiovascular Research 39 (1998) 318–326
Inorganic phosphate content and free energy change of ATP hydrolysis in
regional short-term hibernating myocardium
Claus Martin, Rainer Schulz, Jochen Rose, Gerd Heusch*
¨ Pathophysiologie, Zentrum f ur
¨ Innere Medizin des Universitatsklinikums
¨
, Essen, 45122 Essen, Federal Republic of Germany
Abteilung f ur
Received 14 November 1997; accepted 25 February 1998
Abstract
Objective: Short-term myocardial hibernation is characterized by an adaptation of contractile function to the reduced blood flow, the
recovery of creatine phosphate content and lactate balance back towards normal, whereas ATP content remains reduced at a constant
level. We examined the hypothesis that, despite the absence of ATP recovery, the short-term hibernating myocardium regains an energetic
balance. Methods: An enzymatic method was modified for the measurement of inorganic phosphate (Pi ) in transmural myocardial drill
biopsies (about 5 mg). In 12 anaesthetized swine, moderate ischemia was induced by reduction of coronary inflow into the cannulated left
anterior descending coronary artery to decrease regional myocardial function (sonomicrometry) by 50%. Results: The development of
short-term hibernation was verified by the recovery of creatine phosphate content, the persistence of inotropic reserve in response to
dobutamine and the absence of necrosis (triphenyl tetrazolium chloride). At 5-min ischemia, Pi was increased from 3.660.3 (SD) to
8.161.1 mmol / g wet wt ( p,0.05). The free energy of ATP hydrolysis (DGATP ) was decreased from 257.860.8 to 252.261.4 kJ / mol
( p,0.05). The relationships between function and Pi (r520.81) and DGATP (r520.83), respectively, during control and at 5-min
ischemia became invalid at 90-min ischemia, as myocardial blood flow and function remained reduced at a constant level, but Pi
decreased back to 4.960.9 mmol / g ( p,0.05 vs. control and 5-min ischemia), and DGATP fully recovered back to 257.261.3 kJ / mol
( p,0.05 vs. 5-min ischemia). Conclusions: In short-term hibernating myocardium, myocardial inorganic phosphate content recovers
partially and the free energy change of ATP hydrolysis returns to control values. Contractile function remains reduced by mechanisms
other than an energetic deficit.  1998 Elsevier Science B.V. All rights reserved.
Keywords: Short-term hibernation; Swine; Inorganic phosphate; Free energy of ATP hydrolysis
1. Introduction
Short-term myocardial hibernation is characterized by
perfusion–contraction matching, lack of necrosis, and the
recovery of some metabolic signs of ischemia despite
ongoing hypoperfusion and persistent reduction in contractile function. Following an initial and transient decrease,
the myocardial creatine phosphate content (CP) recovers
back to preischemic values within 60- to 90-min ischemia,
and lactate production is attenuated [1–5]. The content of
ATP tends to decrease in transmural [2,4] and is decreased
more markedly in subendocardial biopsies [1,6]; ATP
content remains reduced at a constant level throughout
ischemia. In contrast to the recovery of lactate balance and
*Corresponding author. Tel.: 149 (201) 723 4480; Fax: 149 (201)
723 4481; E-mail: [email protected]
0008-6363 / 98 / $19.00  1998 Elsevier Science B.V. All rights reserved.
PII: S0008-6363( 98 )00086-8
CP content, the persistent reduction in myocardial ATP
appears inconsistent with the idea that a regulatory reduction in contractile function makes the short-term hibernating myocardium regain its energetic balance. However, it
is not the ATP content but the free energy change of ATP
hydrolysis that determines the energetic state of the
myocardium. Recovery of the myocardial ATP-phosphorylation potential has been previously observed in a
hibernation model subjecting the isolated working, salineperfused guinea pig heart to global low-flow ischemia. In
this model, the entire heart was freeze-clamped for the
measurement of high-energy phosphates and calculation of
the phosphorylation potential [7]. We have now established
a method for biopsy-based sequential determinations of
DGATP from regionally ischemic myocardium in situ and
Time for primary review 25 days.
C. Martin et al. / Cardiovascular Research 39 (1998) 318 – 326
applied this technique to an established pig model of
short-term hibernation [2,8,9]. Since Pi [10–12] and DGATP
[13–15] are considered as potentially causal factors in the
reduction of myocardial contractile work, we tested
whether or not this also holds true in short-term hibernating myocardium in situ. Our results confirm the complete
recovery of DGATP at the end of a 90-min short-term
hibernation protocol and support the idea of a restoration
of the energetic balance in short-term myocardial hibernation [16,17].
319
ed n 11
WI n 5
O (LVP
n,m
2 LVPn,min ) ? (WTh n,m 2 WTh n,m21 )
ed n
[ed5end-diastole, n5actual cardiac cycle, m5sampling
point within cardiac cycle n at a sampling interval of 5 ms,
LVPn , m 5instantaneous left-ventricular pressure within cardiac cycle n and at sampling point m, LVPmin 5minimum
left-ventricular pressure, WTh5wall thickness]. The maximal work index value during systole is reported as WI [9].
2.3. Regional myocardial blood flow and metabolism
2. Methods
The experimental protocols used in this study were
¨
approved by the local authorities of the district of Dusseldorf, and they conform with the Guide for the Care and
Use of Laboratory Animals published by the US National
Research Council (National Academy Press, Washington,
DC, 1996).
2.1. Experimental model
The experimental model has been described in detail
previously [2,8,9]. In brief, in 12 enflurane / nitrous oxide¨
anaesthetized Gottinger
miniswine, a left-lateral
thoracotomy was performed and a micromanometer (P7,
Konigsberg Instr, Pasadena, Calif) was placed in the left
ventricle through the apex. Ultrasonic dimension gauges
were implanted in the left-ventricular myocardium to
measure the thickness of the anterior and the posterior
(control) walls (System 6, Triton Technologies, San Diego,
Calif). The proximal left anterior descending coronary
artery (LAD) was cannulated and perfused from an
extracorporeal circuit at constant flow. LAD perfusion
pressure was measured from the sidearm of the extracorporeal circuit. The large epicardial vein parallel to the
LAD was dissected and cannulated to sample coronaryvenous blood. Heart rate was controlled throughout the
study by left-atrial pacing (type 215 / T, Hugo Sachs
Elektronik, Hugstetten, FRG).
2.2. Regional myocardial function
End-diastole was defined as the point when left-ventricular dP/ dt started its rapid upstroke after crossing the
zero-line. Regional end-systole was defined as the point of
maximal wall thickness within 20 ms before peak negative
left-ventricular dP/ dt [18]. Systolic wall thickening was
calculated as end-systolic wall thickness minus end-diastolic wall thickness divided by the end-diastolic wall
thickness. In addition, a regional myocardial work index
was determined as the sum of the instantaneous leftventricular pressure–wall thickness product over the time
of the cardiac cycle, using the equation:
Radiolabelled microspheres (NEN, Du Pont, Boston,
Mass) were injected into the coronary perfusion circuit to
determine the regional myocardial blood flow and its
distribution throughout the LAD perfusion bed [19].
Oxygen content was measured using anaerobically sampled blood drawn simultaneously from the cannulated
coronary vein and an artery, and oxygen consumption of
~ 2 ) was calculated by
the anterior myocardial wall (MVO
multiplying the arterial–coronary venous oxygen difference by the transmural blood flow at the crystal site.
Lactate was measured in simultaneously drawn coronaryvenous and arterial blood samples [20], and lactate consumption was calculated by multiplying the arterial–coronary venous difference by transmural blood flow at the
crystal site.
2.4. Inorganic phosphate, high energy phosphates and
DGATP
Transmural drill biopsies (about 5 mg weight) were
taken from the LAD perfusion bed. A 1.5-mm-diameter bit
was used in conjunction with a modified dental drill.
Negative pressure within the drill bit facilitated the taking
of the sample, whereas reversal to positive pressure was
used to rapidly expel the sample into a stainless-steel
mortar cooled with liquid nitrogen. The sample was then
immediately ‘freeze clamped’ using a steel pestle also
cooled with liquid nitrogen. The time from tissue sampling
to clamping was 1–2 s; samples requiring longer time for
acquisition were not used for this analysis. Holes in the
myocardium resulting from the biopsies were closed using
a shallow purse-string suture [2]. The tissue samples were
stored in liquid nitrogen until the subsequent analysis.
2.4.1. Chemicals
Inosine, phosphate-free nucleoside phosphorylase (NP),
phosphate-free xanthine oxidase (XO), creatine kinase,
pyruvate kinase, lactate dehydrogenase and glutamate
pyruvate transaminase were obtained from Boehringer
Mannheim (Mannheim, FRG). 2,6-Dichloroindophenol
(DCIP) was from Sigma Chemie (Deisenhofen, FRG). All
other chemicals were of analytical grade.
320
C. Martin et al. / Cardiovascular Research 39 (1998) 318 – 326
2.4.2. Analyses
The powdered tissue samples (Mikro Dismembrator, B.
Braun Melsungen, Melsungen, FRG) were extracted with
0.3 mol / l perchloric acid and neutralized with 0.9 mol / l
KOH, 170 mmol / l K 2 SO 4 , 60 mmol / l Tris. Aliquots were
used for the measurements of Pi , high-energy phosphates
and creatine (Cr).
For the measurement of Pi , almost all conventional
colorimetric and enzymatic methods have inherent disadvantages, such as low sensitivity and interference from
other compounds, decomposition of labile organic phosphates or contamination of chemicals and enzyme preparations with Pi [21–25]. We modified an enzymatic assay
based on nucleoside phosphorylase–xanthine oxidase-coupled reactions [26,27]. Pi is transferred to inosine with
phosphorolytic cleavage. The formed hypoxanthine is
oxidized by XO, transferring hydrogen to DCIP which is
reduced to its colourless dihydroderivative. Each molecule
of Pi is equivalent to two molecules of reduced dye, thus
extending the lower detection limit of the assay. DCIP
(0.16 mmol / l) and inosine (5 mmol / l) were dissolved in
0.2 mol / l Tris / HCl buffer (pH 7.2). A volume of 820 ml
of this solution was placed in a cuvette with a 10-mm
optical pathlength tempered to 378C. A volume of 10 ml
each of NP and XO, respectively, dissolved in the above
Tris / HCl buffer were added to a final concentration of 0.2
units / ml. Then, 60 ml of the sample were added and the
absorbance at 606 nm was recorded, until the reaction
endpoint was reached (8452 A Diode Array Spectrophotometer, Hewlett Packard GmbH, Waldbronn, FRG).
External, identically treated Pi -standards were used for
calibration. Pi samples ranging from 0.1 to 12 nmol had a
linear response in the decrease of UV absorbance. The
lower detection limit of about 10 mmol / l sample concentration is equivalent to a tissue content of about 0.6
mmol / g wet wt Pi in a 5-mg tissue sample.
High-energy phosphates were measured using HPLC on
an anion-exchanger column (Protein Pak DEAE 5 PW,
Millipore-Waters GmbH, Eschborn, FRG). The elution
buffer consisted of 3 mmol / l Tris / H 2 SO 4 (pH 9.0) with a
linear gradient from 6 to 290 mmol / l K 2 SO 4 within the
first 7 min of the run; the flow was 1.0 ml / min. UV
absorption was monitored at 214 nm. The retention times
were 8, 10, 14, 16 min for CP, AMP, ADP, ATP,
respectively.
Creatine was determined by the enzymatic method of
Bernt et al. [28].
DGATP was calculated with the use of the mass equilibrium constant of the creatine kinase reaction using the
equation:
[Cr][Pi ]
]]]]
DGATP 5 DG o9
;
ATP 1 RT ln
[CP][H 1 ]K CK
[Cr], [Pi ], [CP] refer to the cytosolic free concentrations,
o’
DG ATP
is the standard free energy change of ATP hy-
drolysis (230.5 kJ / mol) [29], R the universal gas constant,
T the absolute temperature and K CK the equilibrium
constant of the creatine kinase reaction (1.66?10 9 ) [30].
The cytosolic free concentrations were calculated from the
tissue contents according to Giesen and Kammermeier
[31]. The cytosolic pH of 7.2 during normoperfusion, of
6.9 at 5 min and of 7.0 at 90 min of hypoperfusion was
derived from a NMR study in open-chest pigs with a
comparable reduction in regional subendocardial blood
flow [32] (from 0.9660.16 (SEM) to 0.3060.06 in that
study vs. from 0.7960.26 (SD) to 0.2960.12 ml / min / g in
the present study).
2.5. Morphology
At the end of each study, following 120-min reperfusion, the absence of infarcted tissue was verified by the
triphenyl tetrazolium chloride technique as previously
described in our model [9].
2.6. Experimental protocol
Coronary inflow was decreased for 90 min to reduce the
regional myocardial work index by approximately 50%;
this degree of ischemia has previously been shown to be
compatible with the development of short-term myocardial
hibernation [2,8,33]. Sets of measurements were performed
under control conditions, at 5- and 90-min ischemia, and in
4 animals additionally after 5 min of dobutamine infusion
(2.5 mg / min) into the LAD perfusion circuit under control
conditions and at 90-min ischemia. These measurements
included the simultaneous withdrawal of pairs of arterial
and coronary-venous blood samples. During the blood
sampling, microspheres were injected into the LAD perfusion system for the measurement of regional myocardial
blood flow, and systemic hemodynamic and regional
dimension data were recorded. Immediately after the
microspheres injection, myocardial biopsies were taken.
2.7. Data analysis and statistics
Hemodynamic parameters reported are left-ventricular
end-diastolic and peak pressures, the maximum and minimum of the first derivative of the LV pressure (LV dP/
dt max , LV dP/ dt min ), and mean coronary-arterial pressure.
Regional wall function is reported as systolic wall thickening and the regional work index of the anterior wall,
and—as a control reference—systolic wall thickening of
the posterior wall. Metabolic parameters include calculated
DGATP , the myocardial contents of ATP, CP and Pi , and the
consumptions of oxygen and lactate (positive values
indicate myocardial uptake).
Changes in systemic hemodynamics, regional myocardial function, blood flow and metabolism over the time
course of the experiment were evaluated using an analysis
of variance for repeated measures, followed by Tukey’s
C. Martin et al. / Cardiovascular Research 39 (1998) 318 – 326
t-test for comparison of single mean values. Linear regression analyses between Pi , DGATP and AWI were performed.
All data are reported as mean values6standard deviation,
and a p-value less than 0.05 was accepted as indicating a
significant difference.
3. Results
In all animals, myocardial necrosis after 90-min ischemia and 2-h reperfusion was absent. Data of systemic
hemodynamics and regional myocardial function are sum-
321
marized in Table 1. Data of blood flow and metabolic
parameters are summarized in Table 2.
3.1. Systemic hemodynamics
Heart rate was kept constant by left-atrial pacing
throughout the protocol. Infusion of dobutamine increased
LV dP/ dt max ( p,0.05), while LV dP/ dt min tended to be
decreased. With the reduction in coronary inflow, leftventricular peak pressure and left-ventricular end-diastolic
pressure were unchanged. LV dP/ dt max was decreased
( p,0.05). There were no further changes in systemic
hemodynamics when ischemia was prolonged to 90 min.
Table 1
Systemic hemodynamics and regional myocardial function
Control (n512)
Control1dobutamine (n54)
5-min
ischemia (n512)
90-min
ischemia (n512)
Ischemia1
dobutamine (n54)
Systemic hemodynamics
HR
10469
LVEDP
563
LVPP
9769
LV dP/ dt max
14186178
LV dP/ dt min
16846391
CAP
121612
101614
762
10068
17996173 a
14066286
108640
10469
863
9369
12136154 a
14756396
4464 a
10469
864
9469
11926198 a
15336328
459 a
104611
965
9867
15516352 b
10726224 a,b
427 a
Regional myocardial function
AWT
3668
AWI
314663
PWT
2569
52615 a
381697 a
2569
2268 a
164656 a
25610
22612 a
170678 a
2167
23616 a
183697 a
2066
HR, heart rate (min 21 ); LVEDP, left-ventricular end-diastolic pressure (mm Hg); LVPP, left-ventricular peak pressure (mm Hg); LV dP/ dt max (LV
dP/ dt min ), maximum (minimum) of first derivative of left-ventricular pressure (mm Hg / s); CAP, mean coronary arterial pressure (mm Hg); AWT, anterior
systolic wall thickening in percent of the end-diastolic wall thickness (%); AWI, anterior myocardial work index (mm Hg?mm); PWT, posterior systolic
wall thickening in percent of the end-diastolic wall thickness (%).
a
p,0.05 vs. control.
b
p,0.05 vs. preceding value.
Table 2
Regional myocardial blood flow and metabolism
TMF
ENDO
~ 2
MVO
~ lact
MV
Cr
CP
ATP
ADP
AMP
Pi
pH cv
DGATP
Control (n512)
Control1
dobutamine (n54)
5-min
ischemia (n512)
90-min
ischemia (n512)
Ischemia1
dobutamine (n54)
0.7760.13
0.7960.26
83.8614.6
2.461.5
9.362.3
9.160.8
3.760.5
0.5560.25
0.05560.026
3.660.3
7.3460.05
257.860.8
0.9960.16 a
1.1660.14 a
109.8643.2
2.360.8
10.762.2
8.561.8
3.160.5
0.4960.04
0.05560.037
4.360.8
7.3760.03
256.460.6
0.4360.09 a
0.2960.12 a
a
57.7612.0
21.861.5 a
16.663.6 a
3.761.4 a
3.260.6
0.6260.17
0.07460.026
8.161.1 a
7.2860.06 a
252.261.4 a
0.4460.08 a
0.3460.13 a
a
58.3610.1
0.060.9 a,b
10.963.3 b
8.962.2
3.260.8
0.5760.16
0.05060.010
4.960.9 a,b
7.3260.06
257.261.3 b
0.4260.08 a
0.2360.10 a
a
59.066.0
22.062.2 a,b
16.161.1 a,b
4.360.4 a,b
3.160.4
0.6660.18
0.06760.016
8.060.2 a,b
7.2760.08 a,b
252.960.4 a,b
~ 2 , myocardial oxygen consumption
TMF, transmural myocardial blood flow (ml / min / g); ENDO, subendocardial blood flow (ml / min / g); MVO
~ lact , myocardial lactate consumption (mmol / min / g); Cr, myocardial creatine content (mmol / g wet wt ); CP, myocardial creatine phosphate
(ml / min / g); MV
content (mmol / g wet wt ); ATP, myocardial adenosine triphosphate content (mmol / g wet wt ); ADP, myocardial adenosine diphosphate content (mmol / g wet wt );
AMP, myocardial adenosine monophosphate content (mmol / g wet wt ); Pi , myocardial inorganic phosphate content (mmol / g wet wt ); pH cv , coronary venous pH
(pH units); DGATP , myocardial free energy of ATP hydrolysis (kJ / mol).
a
p,0.05 vs. control.
b
p,0.05 vs. preceding value.
322
C. Martin et al. / Cardiovascular Research 39 (1998) 318 – 326
Infusion of dobutamine at 90-min ischemia increased LV
dP/ dt max and decreased LV dP/ dt min (both p,0.05 vs.
90-min ischemia).
3.2. Coronary-arterial pressure, regional myocardial
blood flow, and regional myocardial function
During normoperfusion, intracoronary dobutamine increased regional blood flow, systolic wall thickening and
the regional work index of the anterior wall (all p,0.05).
With the reduction in coronary inflow, coronary-arterial
pressure and regional myocardial blood flow were reduced
(both p,0.05). At 5-min ischemia, systolic wall thickening and the regional work index of the anterior wall were
significantly decreased to 2268% and 164656 mmHg?
mm, respectively, (both p,0.05), whereas systolic wall
thickening of the posterior wall remained unchanged. With
prolongation of ischemia to 90 min, regional myocardial
blood flow, systolic wall thickening and the work index
did not change further. A close correlation existed between
the regional work index of the anterior wall and transmural
myocardial blood flow during control conditions and
throughout ischemia (Fig. 1).
3.3. Metabolism
With intracoronary dobutamine infusion, myocardial
oxygen consumption tended to be increased, Pi content
tended to be slightly increased and DGATP tended to be
slightly decreased (all nonsignificant vs. the respective
control values). At 5-min ischemia, the myocardial CP
content ( p,0.05) as well as the myocardial oxygen
consumption ( p,0.05) were decreased, the myocardial
ATP content tended to be decreased ( p50.08), and
myocardial lactate consumption was reversed to net lactate
production. Myocardial Pi content was increased from
3.660.3 to 8.161.1 mmol / g wet wt ( p,0.05). The Pi
Fig. 1. Relationship between transmural myocardial blood flow and
anterior work index. The symbols represent data from 12 animals each
under control conditions and after 5- and 90-minutes moderate ischemia.
The regression line ( y5373.6x116.3; n524; r50.80) was obtained
from data under control conditions and at 5-min ischemia.
Fig. 2. Relationship between myocardial inorganic phosphate content and
anterior work index. The symbols represent data from 12 animals each
under control conditions and after 5- and 90-min moderate ischemia. The
regression line ( y5 232.2x1427.1; n524; r5 20.81) was obtained
from data under control conditions and at 5-min ischemia.
content correlated inversely to the regional work index
during control conditions and at 5-min ischemia (Fig. 2).
The free energy change of ATP hydrolysis (DGATP ),
derived from these data, was decreased from 257.860.8
to 252.261.4 kJ / mol ( p,0.05), and DGATP also correlated to regional myocardial function (Fig. 3).
With prolongation of ischemia to 90 min, regional
myocardial blood flow remained unchanged. Net lactate
production was no longer present ( p,0.05 vs. 5-min
ischemia). The myocardial ATP content remained unchanged at its slightly reduced level ( p50.06 vs. control),
whereas the myocardial contents of Pi and CP recovered
towards their preischemic values: the Pi content decreased
back to 4.960.9 mmol / g wet wt ( p,0.05 vs. control and vs.
5-min ischemia) and the CP content increased back to
8.962.2 mmol / g wet wt (ns vs. control, p,0.05 vs. 5-min
ischemia). DGATP recovered to 257.261.3 kJ / mol (ns vs.
control, p,0.05 vs. 5-min ischemia). Most single data and
the means of the data for Pi content vs. function and DGATP
vs. function were outside the 95% confidence intervals of
the respective correlations at 90-min ischemia (Figs. 2 and
3).
Fig. 3. Relationship between free energy change of ATP hydrolysis and
anterior work index. The symbols represent data from 12 animals each
under control conditions and after 5- and 90-min moderate ischemia. The
regression line ( y5 228.5x21336.6; n524; r5 20.83) was obtained
from data under control conditions and at 5-min ischemia.
C. Martin et al. / Cardiovascular Research 39 (1998) 318 – 326
At 90-min ischemia, intracoronary dobutamine infusion
once more increased the Pi content and decreased DGATP to
an extent comparable to that during early ischemia (both
nonsignificant vs. 5-min ischemia).
4. Discussion
In short-term hibernating myocardium, myocardial inorganic phosphate is decreased back towards control values
and the free energy change of ATP hydrolysis is restored,
despite persistently reduced myocardial blood flow and
contractile function.
4.1. Critique of methods
Short-term hibernation was verified by recovery of CP
and lactate balance, increased contractile function during
inotropic stimulation with dobutamine at the expense of
metabolic recovery, and the absence of TTC-detectable
necrosis. In TTC positive and thus viable myocardium, no
signs of irreversible tissue damage could be demonstrated
by light microscopy in a previous set of experiments [2].
DGATP was calculated from simultaneous measurements
of CP, creatine and Pi in single transmural biopsies taken
from the LAD bed of anaesthetized swine during normoand hypoperfusion. Thus, the calculated DGATP reflects the
regional transmural value of the free energy change of
ATP hydrolysis. Regional transmural myocardial work was
estimated as a work index by multiplying wall thickening
by the instantaneous left-ventricular chamber pressure; this
product was integrated throughout ventricular systole.
Thus, unidimensional excursion against a load during
systole was used as an estimate of regional myocardial
work. It could obviously not be measured in the same
region in which biopsies were taken. Therefore, the
relation of DGATP to the work index assumes comparable
degrees of ischemia in both regions. Indeed, analysis of
transmural blood flow in adjacent pieces of myocardium
within and along different heart slices of the LAD-perfused
territory revealed a coefficient of variation below 5%. The
mean coefficient of variation in the entire series of
experiments averaged 2.3661.63% (control conditions:
2.9061.46%, 5-min ischemia: 2.3562.41%, 90-min ischemia:
2.1761.02%,
dobutamine
stimulation:
1.8561.17%) in a total of 272 pieces with an average
weight of 1.4160.63 g.
DGATP was calculated from the presumed cytosolic free
concentrations of Pi , CP and Cr. In biopsies such as in the
present study, only the total cardiac content of these
metabolites, but not their myocardial cytosolic free concentrations can be measured. This limitation appears to be
less relevant for Cr and CP, since compartmentation and
binding of these metabolites are negligible [34]. Their
cytosolic concentrations were calculated in proportion to
their cytosolic space distribution [31]. The myocardial
323
tissue content of Pi was transferred into a cytosolic
concentration by correction for bound Pi and compartmentation of Pi in the extracellular and mitochondrial
space, according to Giesen and Kammermeier [31]. These
authors also introduced an approximation of cytosolic Pi
with reference to the pH gradient across the inner mitochondrial membrane, which is related to myocardial oxygen consumption and derived from an empirical equation
[35]. This additional approximation, however, was unnecessary in our calculations for the recovery of DGATP
with prolonged hypoperfusion as myocardial oxygen consumption remained unchanged throughout the ischemic
period. The cytosolic H 1 concentration which also contributes to the calculation of DGATP could not be measured in
our experimental setting. It was therefore derived from a
NMR spectroscopy study in open-chest pigs with a comparable reduction in regional myocardial flow [32]. The
cytosolic free ADP concentration was calculated by means
of the equilibrium constant of the creatine kinase reaction
[30].
The inability to measure cytosolic free ADP concentrations is not unique to chemical determinations; its
approximation via the CK equilibrium is usually performed
in NMR spectroscopy based measurements, too. Both the
chemical analysis as well as the NMR technique use
further approximations, the one in calculating cytosolic
free concentrations from measured tissue contents and
assuming cytosolic H 1 concentrations, the other in transforming peak areas into absolute concentrations of the
respective compounds and estimating creatine concentrations. Thus, both methods are complementary.
The course of the intracellular pH values as derived
from data in the literature [32] shows an early decrease
followed by a slight increase at 90-min ischemia. A
corresponding—though less-pronounced course of the
coronary-venous pH values was measured in the present
study (Table 2). Nevertheless, minor deviations of the
assumed from the actual intracellular pH values are not
critical for the major results of the present study: a
deviation of 0.1 pH units results in a less than 1.5% (mean
for all measurements 1.0660.05%) error in the calculated
DGATP . From the differences in the total amount of
creatine (Cr1CP) and phosphates (CP1ATP1Pi ) (Table
2), potential errors of about 10% can be assumed for the
measurements of the respective tissue contents. The resulting potential error in the calculation of DG is, however,
considerably lower than the potential errors in the measurements of single tissue contents, as the tissue contents
in the calculation appear, after conversion into cytosolic
free concentrations, on a logarithmic scale in the formula
of DG. The potential ‘worst case’-error in the calculated
DGATP (Cr and Pi : 110%; CP: 210%) is below 2% (mean
for all measurements: 1.6360.09%) and thus unlikely to
affect the observed changes in DGATP of about 10%.
The calculation of DGATP by means of the equilibrium
constant of the CK reaction is based on near equilibrium
324
C. Martin et al. / Cardiovascular Research 39 (1998) 318 – 326
conditions for this reaction in the cytosol. In the course of
myocardial hibernation, however, the ratio of CP to ATP
content is altered dramatically. Nevertheless, there is good
evidence that, despite this altered CP/ATP ratio, the CK
reaction still runs at equilibrium: in an isolated rat-heart
model of short-term hibernation, the ratio of the pseudo
first order rate constants of the foreward and reverse CK
reaction remained unchanged throughout control conditions and ischemia, indicating the maintenance of the CK
equilibrium [36]. Furthermore, increased energy utilization
with pacing [37] or inotropic stimulation [2,9,33] after 60
or 90 min of moderate ischemia results in rapid breakdown
of CP, as also seen in the present study. Hence, the CP
accumulated during short-term hibernation is still available
for energy production. Its breakdown during inotropic
stimulation at 90-min ischemia is comparable to that at
5-min ischemia (from 8.962.2 to 4.360.4 vs. from
9.160.8 to 3.761.4 mmol / g wet wt ) indicating an unchanged, high activity of cytosolic CK. Thus, the most
plausible explanation for the reduction of ATP with
normalized CP results from the equilibrium conditions,
where the primary alteration is a loss of total adenine
nucleotides, as reported previously [38–41].
Our biopsy technique sampled transmurally since tissue
samples became highly distorted by the ‘freeze-clamping’
and could therefore not be further divided. Thus, our
technique attempted to freeze tissue as rapidly as possible
but sacrificed the possibility of dividing it transmurally.
Since swine do not have an extensive collateral circulation,
the gradient of subendocardial to subepicardial blood flow
is smaller as compared to species with an extensive
collateral circulation such as the dog. Thus, despite some
transmural perfusion heterogeneity, the metabolic response
to ischemia appears to be transmurally more uniform
[2,42], thereby minimizing the underestimation of the
metabolic consequences of ischemia due to our examining
only transmural samples.
One limitation of all animal experiments that have
studied myocardial hibernation is the limited observation
period [43]. Myocardial hibernation, as introduced by
Rahimtoola [16], is a clinical situation of contractile
dysfunction in patients with coronary-artery disease that
exists for months and longer but is fully reversible on
reperfusion.
4.2. Implications
The Pi content was increased by 125% at 5-min ischemia and was then decreased back towards preischemic
values in regional short-term hibernating myocardium.
Initially during ischemia, the Pi content correlated inversely to regional function, consistent with the idea that Pi is
important in decreasing myocardial contractile force at the
onset of ischemia [10–12]. An increase in Pi has been
suggested to depress maximal Ca 21 -activated force in
early ischemia [44,45]. In a previous study from our
laboratory [9], short-term hibernating myocardium has
been characterized by a decrease in calcium responsiveness
without significant desensitization to calcium. However,
with the development of short-term hibernating, calcium
responsiveness and contractile function, on the one hand,
and Pi content, on the other hand, obviously diverge: while
calcium responsiveness and contractile function remain
reduced at a constant level, the Pi content recovers towards
preischemic values, yet remains increased by 36% above
its control value ( p,0.05). It remains unclear whether or
not the remaining increase in Pi content is sufficient to
maintain the reduction in contractile function at 90-min
ischemia. If so, the initial early ischemic increase in Pi
would be overshooting for the observed decrease in
contractile function, an idea not consistent with the close
relation between Pi and contractile function during early
ischemia reported by He et al. [12] and observed in the
present study. Alternatively, an initial short bout of
substantially increased Pi content could induce more longlasting effects on the contractile machinery to maintain the
reduction of contractile function. Most probably, however,
increased Pi content per se is not responsible for the
reduced contractile function in hibernating myocardium.
In the concept of myocardial hibernation, the observed
recovery of CP and lactate is assumed to indicate an active
downregulation of energy demand somewhat below energy
supply, thus restoring the energetic balance [1,37,46,47].
However, the recovery of single metabolic parameters can
certainly suggest but not prove the restoration of energetic
balance. In contrast, DGATP integrates all single metabolites relevant for the energy available from ATP hydrolysis
to various cellular processes, including contractile function.
A close relation between global myocardial function and
global DGATP has been reported from studies in isolated
hearts with graded hypoxia or graded hypoperfusion
[14,48,49]. Recovery of the myocardial phosphorylation
potential during ongoing moderate ischemia has been
reported by Gao et al. [7]: isolated working, saline-perfused guinea pig hearts were subjected to global low-flow
ischemia, and the experiments were terminated by freeze
stop either during control or at distinct times of ischemia.
At 60-min ischemia, the myocardial phosphorylation potential had completely recovered to preischemic values. In
the present study, we have confirmed the recovery of
DGATP during ongoing moderate ischemia and extended
the finding of Gao et al. [7] to an in situ model of regional
short-term hibernation in pigs with sequential, biopsybased measurements of high energy phosphates. In this
model, neither the initial decrease in DGATP nor its
recovery with the development of short-term hibernation
were accompanied by equivalent changes in the myocardial ATP content: the ATP content decreased only slightly
and remained at this reduced level during short-term
hibernation, consistent with previous studies from our
laboratory [2,8,9,33]. The initial slight decrease in myocar-
C. Martin et al. / Cardiovascular Research 39 (1998) 318 – 326
dial ATP content points to an initial imbalance in ATP
demand and supply. However, myocardial mitochondria
possess a tremendous reserve capacity for the production
of ATP and the maintenance of various energetic steady
states depending on demand [50,51]. When ATP production is reduced by a slow and progressive reduction of
blood flow, ATP synthesis and consumption still remain
tightly linked: no sustained fall in the CP/ATP ratio and no
or minimal lactate production are observed, suggesting an
actively regulated reduction of ATP consumption in response to the gradual reduction of ATP production [52].
The results of the present study support the idea of an
active downregulation of energy demand since DGATP
recovered while oxidative phosphorylation, as indicated by
~ 2 , remained stable and anaerobic glycolysis, as
MVO
indicated by lactate production, was attenuated during
ongoing ischemia. There is no evidence for an impaired
flux of energy to the contractile machinery through the CK
reaction [53,54] in the present study. On the contrary, the
recovery of CP together with its availability for energy
production almost exclude transport problems or changes
in the overall CK reaction. The recovery of CP demonstrates the availability of ATP for the phosphorylation of
creatine although the myocardial ATP content remains
unchanged at its reduced level. Loss of adenine nucleotides
via the 59 nucleotidase and myokinase reactions is responsible for the persistent reduction of ATP content; moreover, any decrease in cytosolic free ADP content shifts the
equilibrium of the CK reaction towards CP formation and
increases DGATP despite an unchanged, reduced ATP
content [41].
The present study demonstrates complete restoration of
the energetic balance in regionally short-term hibernating
myocardium in situ, supporting the regulatory nature of
decreased contractile function as a hallmark of the concept
of myocardial hibernation.
Acknowledgements
This research was supported by the Hans und Gertie
¨
Fischer-Stiftung. We thank Petra Gres and Ursula Pragler
for their technical support.
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