Vol. 277, Issue 1, H308-H317, July 1999
Cardiac performance and creatine kinase flux during inhibition
of ATP synthesis in the perfused rat heart
P.
Mateo1,
V.
Stepanov1,
B.
Gillet2,
J.-C.
Beloeil2, and
J. A.
Hoerter1
1 Unité 446, Institut
National de la Santé et de la Recherche Médicale,
Cardiologie Cellulaire et Moléculaire, Université
Paris-Sud, 92296 Chatenay Malabry; and
2 Résonance Magnétique
Nucléaire Biologique, Institut de Chimie des Substances
Naturelles, Centre National de la Recherche Scientifique, 91198 Gif
sur Yvette, France
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ABSTRACT |
To study the relation among mitochondrial energy
supply, cardiac performance, and energy transfer through creatine
kinase (CK), two acute models of inhibition of ATP synthesis were
compared in the isovolumic acetate-perfused rat heart. Similar
impairments of mechanical performance (rate-pressure product, RPP) were
achieved by various stepwise decreases in
O2 supply
(PO2 down to 20% of control) or by
infusing CN (0.15-0.25 mM). The forward CK flux measured by
saturation-transfer 31P NMR
spectroscopy was 6.1 ± 0.4 mM/s in control hearts. Only after
severe hypoxia (PO2 < 40% of
control) did CK flux drop (to 1.9 ± 0.2 mM/s at
PO2 = 25% of control) together with
impaired systolic activity and a rise in end-diastolic pressure. In
contrast, in mild hypoxia CK flux remained constant and similar to
control (5.3 ± 0.5 mM/s, not significant) despite a twofold reduction in systolic activity. Similarly in all CN groups, constant CK
flux was maintained for a threefold reduction in RPP, showing the
absence of a relation between cardiac performance and global NMR-measured CK flux during mild ATP synthesis inhibition.
creatine phosphate shuttle; oxidative phosphorylation inhibition
and energetics; oxygen consumption; creatine kinase kinetics; rigor; magnetization transfer; work
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INTRODUCTION |
THE COMPARTMENTATION of creatine kinase (CK) isozymes
is suggested to play a crucial role in the transfer of energy in the myocardium. Indeed, in the myocardium, 50% of total CK activity is
specifically localized in the vicinity of myofibrillar and sarcoplasmic
reticular ATPases (MM-CK) as well as the adenine translocator (mito-CK)
(for review, see Refs. 20, 29, 31).
In the hypothesis of the creatine (Cr)-phosphocreatine (PCr) shuttle,
the inhibition of ATP synthesis and subsequent contractile failure is
assumed to be associated with an impaired CK flux (provided that all CK
flux is NMR visible). Indeed, such a correlation of CK flux and heart
function is observed in models of reperfusion after ischemia
(17), in hypoxia in the open-chest rat model in vivo (5), and in
certain pathological situations (16). On the other hand, in a normoxic
perfused, beating heart, imposing a threefold increase in ATP synthesis
and workload results only in modest change in CK flux (14). Moreover,
in vivo, pacing or catecholamine stress does not alter CK flux (11, 13,
19). The absence of such a relation between mechanical activity and CK
flux is also observed in skeletal muscle. The latter has been interpreted as evidence of CK being at equilibrium, whereas the relation of CK flux with contractility has been analyzed in the context
of bound CK in support of the CK shuttle (1, 6). As was stressed
recently (30), information resulting from the use of NMR must be
considered in perspective with the abundant literature describing the
high organization and compartmentation of the myocardial cell and the
specific behavior of the subcellular compartments.
Indeed, both in vitro and in subcellular preparations of mitochondria
or myofibrils, the apparent kinetics of an enzyme are known to be
altered by the proximity of another enzyme. For example, in vitro, the
activity of CK in the vicinity of myosin ATPase changes the apparent
kinetics of the myosin ATPase (2) and facilitates cardiac actomyosin
sliding (22). Similarly, in Triton-skinned cardiac fibers, the activity
of MM-CK bound to myofibrils changes the mechanical properties of both
the calcium-activated and the rigor force (28). Considering mito-CK,
located in close vicinity to the adenine nucleotide translocase in the
intermembrane mitochondrial space, the activation of oxidative
phosphorylation has been shown to drive mito-CK out of equilibrium
(21). Thus, from these in vitro and subcellular observations, an
inhibition of ATP synthesis and utilization should result in a
modification of bound CK fluxes. If cytosolic CK is at equilibrium, the
total CK flux is expected to decrease, if NMR detects the activities of
both the cytosolic and the bound CK isoforms.
Three main factors have been proposed to govern the CK flux in vivo:
total CK activity, CK isozymic composition, and the concentration of
products and substrates. In a normoxic model of adenylate depletion [by 2-deoxyglucose (2-DG)], we previously demonstrated (23) that CK flux in the myocardium is largely independent of the
concentration of its substrates and products. In this 2-DG model, the
flux of ATP synthesis and ATP utilization were quasinormal and CK flux was constant for a twofold ATP and PCr decrease. Here we reconsider the
effects of inhibition of ATP synthesis in relation to myocardial performance and CK flux in conditions in which ATP and PCr content, similar to the 2-DG model, should hardly affect the kinetics of the
enzyme. Apparently contradictory results were observed in hearts
submitted to severe hypoxia and in hearts experiencing a mild reduction
of O2 supply or chemical
inhibition of respiratory chain by cyanide. First, no change in CK flux
occurred in the latter models despite marked impairment of systolic
activity. On the other hand, an increase in the severity of hypoxia
resulted in a coordinated decrease in CK flux and systolic activity,
pointing to the absence of a universal relation between NMR-measured CK flux (energy transfer) and contractility (energy demand). Second, in
severe hypoxia, impaired CK flux was associated with a marked rise in
end-diastolic pressure (EDP) for a moderate increase in free ADP,
suggesting a major role for the MM-bound CK in the preservation of
diastolic function.
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MATERIALS AND METHODS |
Physiology.
Animal experimentation was performed in accordance with the Helsinki
accords for humane treatment of animals during experimentation. Wistar
male rats (350-450 g) were anesthetized with ethyl carbamate (2 g/kg), and hearts were perfused by the Langendorff technique at a
constant flow of 13.5 ml/min as previously described (23). Briefly, a
latex balloon was inserted into the left ventricle (LV) and inflated
with
2H2O
to isovolumic conditions of work. LV systolic pressure
(LVP) and coronary pressure were recorded with Statham gauges and
continuously monitored on a paper recorder (Brush) and on a computer
(Compaq). The perfusion solution contained (in mM) 124 NaCl, 6 KCl, 1.8 CaCl2, 1 MgSO4, 1.1 mannitol, 10 Na-acetate, and 20 HEPES and was oxygenated with 100%
O2. Extracellular pH
(pHo) was adjusted with NaOH to
7.35 at 36.5°C. Mean coronary pressure, LVP, EDP, and heart
spontaneous frequency were analyzed on-line. The rate-pressure product (RPP, in 104
mmHg · beat · min
1)
was used as an index of contractility reflecting the energetic demand.
Control hearts (n = 7) were perfused
with 100% O2; various levels of
hypoxia were applied by mixing O2
and N2 with flowmeters (Aalborg).
O2 was reduced to 45 (group
H45,
n = 4), 35 (group H35,
n = 3), and 25 (group
H25,
n = 8) % of control. Four additional hearts were perfused with PO2 ranging
from 70 to 20% of control. Partial chemical inhibition of ATP
synthesis was achieved by addition of NaCN
(n = 16) at low concentrations that
induce a decrease in systolic activity similar to that in the hypoxic groups. From pilot experiments three concentrations of CN were selected, 0.15 (group
CNI,
n = 4); 0.20 (group
CNII,
n = 3), and 0.25 (group
CNIII,
n= 6) mM. Because of a marked decrease
in heart rate, three additional hearts perfused at a CN concentration
of 0.25 mM were paced at 170 beats/min (group
CNIV). NaCN
was prepared just before the experiment, and it did not alter
pHo.
NMR.
31P NMR spectra were acquired at
161.93 MHz on a Bruker AM400 wide-bore magnet in 20-mm-diameter tubes.
Magnetic field homogeneity was optimized using the water signal of the
heart and the frequency locked on
2H2O
contained in the LV balloon. We used a pulse angle of 90° measured
on the 
-ATP signal, 4K data point acquisition, and a spectral width
of 10,000 Hz. Spectra were zero-filled to 8K, and line broadening was
20 Hz. Figure
1B shows a
time line of the experimental protocol and acquisition of spectra. The
baseline period acquired after 10 min of equilibration in isovolumic
conditions included four spectra of 64 scans with an interpulse delay
of 2 s and one spectrum of 32 scans with 10-s interpulse delay for equilibrium values. After 10 min of hypoxia or cyanide, four spectra were acquired (interpulse delay 2 s) to quantify the metabolites before
flux determination. Four spectra taken after saturation transfer were
used to check the steady state.
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Fig. 1.
Evolution of NMR spectra. A: typical
spectra obtained during baseline period and just before and just after
saturation-transfer experiment in 2 individual hearts representative of
severe hypoxia [25% of control
O2
(H25)] and 0.25 mM cyanide
(CNIV) groups (no. of scans = 128, pulse interval = 2 s). SP, sugar phosphates; PCr, phosphocreatine;
ppm, parts per million. B: time course
of experimental protocol. After 15 min of equilibration, isovolumic
conditions of work were imposed (arrow, w); 4 baseline spectra (b) were
acquired 5 min later followed by a spectrum acquired at equilibrium
(pulse interval = 10 s). Inhibition of respiration by hypoxia or
cyanide was induced (arrow, i); after 10 min, 4 spectra were acquired
just before saturation period (b.s.). Time-dependent saturation
transfer (TDST) period was followed by 4 spectra (after saturation,
a.s.), and heart was freeze clamped (arrow, f).
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The forward CK rate constant was measured by time-dependent saturation
transfer (TDST) (6) using the delays alternating with nutations for
tailored excitation (DANTE) method. The pulse angle was
1.6-1.8 µs, the delay between pulses was 5.5 ms, and the number
of pulses ranged from 545 to 16,360. The various durations of
saturation were calculated to be equally distributed on the fitting
curve. Each determination of CK rate constant included nine spectra:
one nonsaturated spectrum with a rate of recurrence of 10 s, allowing
an absolute quantification of PCr and ATP content averaged during the
whole saturation procedure; one spectrum saturated for 9 s at the
mirror frequency downfield of PCr to check for eventual spillover of
the irradiation; and seven spectra saturated at the frequency of
-ATP for a duration ranging from 0.3 to 9 s for control conditions.
The delays between pulses were adjusted to achieve in each spectra a
constant rate of recurrence of 10 s. Free induction decays were
acquired by trains of eight scans cycling five to six times through the
protocol (total no. of scans for each spectra = 40-48; mean
duration of saturation-transfer experiment = 75 ± 2 min). This procedure of signal averaging minimized the
influence of any change in cardiac performance or energetics that might
occur during the saturation transfer experiment. In some hearts (8 in H
groups and 5 in CN groups), the flux was measured in the same heart
during control and after inhibition of ATP synthesis. In this case,
16-24 scans for each spectrum were used for determination of the
control flux; the values were similar to the control group.
Metabolite concentrations and NMR-measured forward flux of CK.
Some control (n = 5), cyanide-perfused
(n = 3), and hypoxic
(n = 7) hearts were freeze clamped at
the end of the experiment. Biochemical analysis of perchloric acid
extracts was performed to measure ATP, PCr, and Cr content as
previously described (9). Values are expressed in
nanomoles per milligram of protein. Biochemical determination confirmed
that none of the experimental conditions induced Cr leakage. Cr and
free ADP were thus calculated from the difference between total Cr + PCr measured biochemically at the end of the experiment and PCr
measured on each spectrum.
NMR quantification was performed with a homemade program on the area of
each peak corrected for saturation. The sum of phosphorylated compounds
(sum P) corresponds to all NMR-visible phosphorus signals. A
biochemically determined PCr content of 43 nmol/mg protein for control
hearts was used as internal standard, and cytosolic volume was taken as
2.72 µl/mg protein. Intracellular pH
(pHi) was determined from the
shift of Pi with respect to PCr.
Free ADP was calculated from the equilibrium of the CK reaction with
the apparent equilibrium constant
(Keq) = 166 × 10 0.87(pH 7).
Quantification of metabolites during flux measurements was made by
averaging four spectra taken just before and just after saturation (corrected to their equilibrium value for each species) and the nonsaturated spectra averaging the whole saturation period.
The forward CK reaction (PCr 
ATP) was analyzed as a
pseudo-first-order rate reaction. The dependence of PCr magnetization (MPCr) as a function of the time
of saturation is described by
where
MoPCr is the intensity of PCr
magnetization in the absence of saturation and
MzPCr is the intensity of PCr
magnetization when -ATP is saturated during
time
t. The fit of the relative PCr
magnetization
(Mz/Mo)
as a function of time of saturation t
allows the determination of
1/
PCr, which is
where
T1PCr is intrinsic relaxation of
PCr and kf is
apparent CK forward rate constant, as described in Ref. 6. The forward CK flux is
expressed
in millimolar per second. T1PCr
values were 3.2 ± 0.3 s (n = 7) in
control; no significant difference was observed among the hypoxic
groups (mean value: 3.4 ± 0.3 s, n = 15) or among the CN groups (mean value: 2.8 ± 0.3 s,
n = 16).
Predicted velocity of CK in myocardium.
We compared for each heart the NMR-measured velocity with the velocity
expected from the well-known equilibrium behavior of MM-CK in dilute
solution in vitro. The steady-state velocity of the reaction
(v) relative to maximal CK activity
(Vmax) is
described by
where
D is a function of the association
(K), dissociation (Ki), and inhibitory
constants (KI) of each metabolite for the various
enzyme complexes
Thus, for each heart, the measured ATP, PCr, and Cr
concentrations were used to predict the reaction velocity expected if myocardial CK isoforms function at equilibrium as CK in dilute solution
in vitro. MgATP was assumed to be equivalent to total ATP because of
the high affinity constant of Mg for ATP. MgADP was calculated from CK
equilibrium. Vmax
was 94.5 mM/s, as estimated from the total CK activity (1,150 IU/g wet
wt measured at 30°C) assuming a
Q10 of 2.4 and a cytosolic volume
of 0.435 ml H2O/g wet weight. The
constants used for prediction, taken from Ref. 1, were (in mM)
association: KADP = 0.167, KPCr = 1.67, KCr = 15.5, KATP=0.4;
dissociation:
Ki,ADP= 0.222, Ki,PCr= 4.73, Ki,Cr= 34.9, Ki,ATP= 0.9; and
inhibitory: KI,Cr = 34.9, KI,PCr = 24. The amount of free
enzyme (i.e., nonsaturated by its substrates) can be computed as
1/D and is expressed as percentage of
total enzyme.
O2 consumption.
Parallel experiments were performed outside the magnet to estimate the
relation between O2 consumption
(
O2)
and mechanical performance in hypoxia
(n = 5) or during cyanide perfusion
(n = 4). Stepwise decrease in
PO2 from 100 to 80, 70, 60, 50, 35 and 25% of control was induced in five hearts and stepwise change in
cyanide concentrations (ranging from 0.1 to 0.25 mM) in four hearts.
Steady-state parameters were measured after 10 min of stabilization in
each condition. "Arterial" PO2 (PaO2) just above the aorta and
"venous" PO2
(PvO2) in the pulmonary artery were
measured in line through two flow cells, Clark electrodes, and
oximeters (Stratkhelvin Inst., Glasgow, UK).
O2 [(PaO2 
PvO2,
in µM
O2 · min1 · g
wet wt1) × O2],
was linearly correlated to RPP. It was described in hypoxia by
O2 = 2.38 × RPP + 0.62 (r 2 = 0.920)
and in cyanide by
O2 = 1.46 × RPP + 2.21 (r2 = 0.899),
with
O2
expressed in micromoles of O2 per
gram of wet weight per minute and RPP expressed in
104 millimeters of mercury times
beats per minute. Some hearts freeze clamped and analyzed for PCr and
ATP contents confirmed the equivalence of the
O2
and the NMR series. Thus the relation between
O2 and RPP was used to estimate
O2
in the NMR-perfused hearts and maximal ATP synthesis assuming a P/O of
3 and a protein content of 160 mg protein/g wet weight.
Statistical analysis.
All results are expressed as means ± SE. Differences between groups
were analyzed by variance analysis and Student-Newman-Keuls test.
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RESULTS |
Cardiac performances.
The initial parameters of contractility were similar in all series. The
pooled values were LVP = 143 ± 3 mmHg, frequency = 226 ± 5 beats/min, RPP = 3.2 ± 0.1 × 104
mmHg · beat · min1,
coronary pressure = 70 ± 2 mmHg, and EDP = 6 ± 1 mmHg
(n = 38). Table
1 shows the averaged mechanical performance
developed during the saturation-transfer period in controls, three
groups of hearts submitted to steady-state decrease in
O2 saturation of the perfusate to
45 (H45), 35 (H35), and 25 (H25)% of control, and the four cyanide groups. Hypoxia resulted in the well-known progressive decrease
in systolic pressure and rise in EDP. The same decrease in systolic
activity as in H25 was induced by
partial chemical ATP synthesis inhibition by cyanide in
groups
CNII and
CNIII. Because
cyanide induced a marked decrease in heart rate, an additional series
of experiments were carried out in which hearts were paced in 0.25 mM
CN (CNIV) to develop the same
performance as the H35 group. In
all cyanide groups the rise in EDP was moderate compared with hypoxia
(at most 21 ± 3 mmHg in CNIII
as opposed to 58 ± 8 mmHg in
H25).
Metabolic characteristics.
In hypoxia, the metabolite contents showed the classic decrease in PCr
and ATP together with a rise in Pi
(Table 2). In the absence of external
Pi, total NMR-visible phosphorus
(sum P) significantly decreased in all hypoxic groups compared with
control hearts. pHi was unchanged
by hypoxia. Because of the absence of glycolytic substrate, lactate
production only originates from glycogenolysis and is extruded from the
cell in this constant-flow model. With cyanide, the metabolite contents
show globally the same trends as in hypoxia, a decrease in PCr and ATP
contents and an accumulation of ADP and
Pi. There was, however, no leakage
of phosphorus out of the cell (Table 2). Comparison of hearts
developing similar performance in
groups
CNIV and
H35 shows
similar pHi and ATP contents in
both groups. However, in CNIV, PCr
was lower and Pi and ADP higher
than in H35
(P < 0.05). Similar trends were
observed when H25 and
CNII and
CNIII groups were compared,
suggesting stronger activation of the adenylate kinase and AMP
degradation pathways in hypoxia than in cyanide perfusion.
Biochemical determination of PCr, ATP, and Cr contents performed at the
end of the experiment in some hearts of control, hypoxic, and CN groups
(Table 3) confirmed the absence of creatine
leakage and the full NMR visibility of ATP and PCr in all groups.
CK kinetics.
Spectra obtained during baseline and before and after the period of
saturation are shown for typical hypoxic and cyanide-perfused hearts in
Fig. 1. Comparison of the metabolite levels of spectra taken before and
after the saturation period allowed quantification of the evolution of
PCr and ATP. With cyanide, no significant decrease in PCr content
occurred. However, in hypoxia both PCr and ATP significantly decreased
during the time needed to perform TDST. The rates of PCr decrease were
1.1 ± 0.1 × 103
mM/s in H25 and
H35 and 0.54 ± 0.06 × 103 mM/s in
H45. Because for all groups the
rate of degradation of PCr (and of ATP) is at least three orders of
magnitude slower than the CK flux, all hearts were considered to be in
quasi-steady state for the analysis of CK flux. A typical TDST protocol
is shown in an individual heart in Fig. 2.
Longer saturation of ATP resulted in a progressive decrease in the
observed magnetization of PCr as a result of both CK activity and PCr
relaxation processes (Fig. 2A). The
exponential decrease in the relative PCr magnetization (Mz/Mo)
is plotted as a function of the time of saturation of -ATP in
representative hearts of the control,
H25, and
CNIV groups (Fig.
2B). This decrease was markedly
slowed down by hypoxia and accelerated by cyanide.
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Fig. 2.
Evolution of magnetization of PCr on saturation of -ATP.
A: series of spectra acquired in a
cyanide group heart (CNIV) for a
typical TDST experiment (nos. on right
refer to duration of -ATP saturation in seconds; no. of scans = 40, pulse interval = 10 s). *, Position of saturating pulse.
B: decrease in relative magnetization
of PCr
(MzPCr/MoPCr)
is shown as function of time of saturation of -ATP
(t) in 3 hearts representative of
control, H25, and
CNIV groups. Fit of
Mz/Mo
as a function of t allows
determination of 1/PCr, which
is 1/T1PCr + kf
[T1PCr = intrinsic
relaxation of PCr and
kf = apparent
creatine kinase (CK) forward rate constant].
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Figure 3 summarizes the change in the
dynamic parameters
kf and CK flux
for the various groups. The apparent rate constant of CK forward flux,
kf, was
0.43 ± 0.02 s1
(n = 7) in control. It was similar in
H45 and
H35 [0.48 ± 0.07 (n = 4) and 0.34 ± 0.07 s1
(n = 3), respectively] and
decreased significantly in severe hypoxia [0.28 ± 0.02 in
H25
(n = 8);
P < 0.05]. As a result of changes in both PCr and
kf, CK flux
remained similar to control in moderate hypoxia (5.5 ± 0.4 mM/s in
H45 vs. 6.1 ± 0.4 mM/s in
control). For more severe hypoxic conditions, CK flux progressively decreased. CK flux reached one-half of its control value in
H35 (3.1 ± 0.5 mM/s,
P < 0.01) and one-third in
H25 (1.9 ± 0.2 mM/s, P < 0.001). Paired
analysis of CK flux performed in the same heart in normoxia and hypoxia
(ranging from 20 to 70% of control
O2; n = 8) also illustrates the constancy
of CK flux for a moderate restriction in
O2 supply and its progressive
inhibition in severe hypoxia (Fig. 4). Even
in severe hypoxia, there was no significant magnetization transfer from
- to 
-ATP, a sign of activation of the adenylate kinase exchange.
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Fig. 3.
CK flux during inhibition of ATP synthesis.
A:
kf in control,
hypoxia [45 (H45), 35 (H35), 25 (H25) % of control
O2 in perfusate], and
cyanide perfusion [for various cyanide concentrations
(CNI = 0.15, CNII = 0.20, CNIII = 0.25, CNIV = 0.25 mM) with pacing]
groups. B: CK forward flux
(kf · [PCr],
where [PCr] is PCr concentration) in various groups.
Significantly different from control:
* P < 0.05;
** P < 0.01;
*** P < 0.001.
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Fig. 4.
CK flux measured in individual hearts. A: paired analysis of CK flux at
various level of hypoxia confirmed constancy of CK flux for mild
hypoxia and its progressive decrease in severe
O2 restriction. Each symbol
corresponds to an individual heart (n = 8). PaO2, "arterial"
PO2 measured just above aorta.
B: CK flux was never affected by
cyanide (n = 5 from
groups CNI and
CNIII).
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In all cyanide experiments, the rate constant
kf was
significantly increased to a maximal value of 1.1 ± 0.1 s1 (Fig. 3). As a result of
the opposite change in
kf and PCr
content, CK flux remained constant (6.0 ± 0.4, 6.4 ± 1.1, 5.7 ± 0.3, and 5.8 ± 0.5 mM/s in
CNI-CNIV,
respectively) and similar to control. This constancy was also observed
with paired analysis of CK flux in the same heart in the presence or
absence of cyanide (Fig. 4, n = 5).
Because we focused on the relation between energetic demand (mechanical
performance) and CK flux, we only used low cyanide concentrations. As
expected, higher concentrations (1-2 mM), provoking full
inhibition of oxidative metabolism and cardiac arrest, decreased CK
flux (not shown).
CK flux and metabolite contents.
The dependence of the forward CK flux on the PCr content was analyzed
in individual hearts. In hypoxia, both the apparent rate constant and
CK flux (Fig. 5) progressively decreased
with PCr. With cyanide, however, for the same range of PCr content (from 10.5 to 5.5 mM), CK flux was independent of PCr. The velocity of
CK obviously depends on the concentrations of all CK metabolites. In
the hypothesis of CK equilibrium the theoretical velocity can be
computed for each heart (see MATERIALS AND
METHODS). Table 4 shows
the comparison of the predicted and the NMR-measured velocity in the
various protocols. For control hearts, the predicted flux was in good
agreement with the NMR-measured flux (5.9 ± 0.2 and 6.5 ± 0.2 mM/s, respectively; n = 7 each). In
all treated groups except H45,
predicted flux was significantly higher than measured flux. Neither the
decreased CK flux in severe hypoxia nor the constant flux with cyanide
perfusion can be predicted from the theoretical velocity calculated
from the hypothesis of CK equilibrium. Several hypotheses
could explain this discrepancy: CK activity is modulated by factors
other than the concentration of its metabolites; the concentrations of
these metabolites are not the actual concentrations effective at the
site(s) of CK; or the complexity of compartments in the cell cannot be
accounted for in a simplified equilibrium hypothesis of all CK
isoforms.
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Fig. 5.
Relation between CK flux and PCr content in control, hypoxia, and
cyanide-perfused hearts. CK flux appears linearly related to PCr in
hypoxia (y = 0.59x 2.2, r2 = 0.82, with
y = CK flux in mM/s and
x= PCr in mM) but independent of PCr
with cyanide perfusion
(r2 = 0.02).
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Relation between cardiac performance and CK and ATP synthase fluxes.
Figure 6A
shows the relation between CK flux and RPP for hypoxic and
cyanide-treated hearts. In cyanide a threefold decrease in systolic
function occurred without any change in CK flux. Similarly, in mild
hypoxia, CK flux remained constant for a twofold reduction in RPP. Only
for severe hypoxia could the progressive decrease in cardiac
performance (expressed as RPP) be related to impaired CK flux
[for hypoxia (PO2 
45%), a
linear relation could be observed with a regression coefficient
(r2) = 0.63 calculated from individual hearts]. In this latter case, CK flux
decrease was also associated with a progressive rise in EDP as hypoxia
became more severe (Fig. 6B).
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Fig. 6.
Relation between CK flux and cardiac performance.
A: CK flux as function of
rate-pressure product. CK flux was constant in cyanide groups despite
threefold decrease in systolic activity. Similar contractile
dysfunction induced by severe hypoxia (<40% of control
O2) results in CK flux
impairment. Moderate hypoxia
(H45) does not affect CK flux.
B: CK flux as function of
end-diastolic pressure (EDP). Rise in EDP occurring in cyanide groups
is modest compared with huge contracture developing together with
impaired CK flux in severe hypoxia.
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The O2 and the ATP synthesis
rate can be calculated for each NMR-perfused heart from its RPP
(see MATERIALS AND METHODS) (Table
5). In all cases, CK flux
remained at least threefold higher than ATP synthesis, suggesting that
energy transfer by CK was never limiting.
O2 supply to the hypoxic heart.
To understand the striking differences in the pathway of CK energy
transfer of hearts submitted to mild inhibition of ATP synthesis (CN
and H45) or severe hypoxia
(H35 and
H25), we tested the hypothesis
that severe hypoxia induced a drastic shortage in
O2 supply.
O2
and cardiac performance were analyzed in relation to
PaO2 (inflow) and
PvO2 (effluent) at various levels of
hypoxia. The relation between PvO2 and
PaO2 is shown in Fig.
7A:
PvO2 decreased linearly with aortic
PO2 down to ~40% of the control PaO2 (206 mmHg), and
PvO2 was <20 mmHg for more severe
hypoxic perfusion. Below this threshold, myocardial
O2
(and RPP, not shown) dropped drastically (Fig.
7B) and a marked rise in EDP occurred (Fig. 7C), suggesting a
strong limitation in myocardial O2
supply.
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Fig. 7.
Limitation of heart function and
O2 supply in hypoxia.
A: "venous"
PO2
(PvO2) as function of progressive
decrease in PaO2 from 100 to 20% of
O2 content in perfusate.
B:
O2 consumption
(O2)
as a function of PvO2.
C: rise in EDP as a function of
PvO2. Below a threshold (arrows)
corresponding to reduction of O2
to 40% of control (PaO2 = 206 mmHg),
strong O2 limitation occurs
(PvO2 < 20 mmHg) leading to marked
decrease in
O2
and systolic activity together with a sudden rise in EDP. Parallel
experiments were performed out of magnet
(n = 5).
|
|
| |
DISCUSSION |
Our aim was to reevaluate the relation between energy transfer by CK
and energy supply and demand in the isolated, perfused heart during
impairment of ATP synthesis. Graded inhibition, achieved by decreasing
the O2 content of the perfusate or
by low concentrations of cyanide, was designed to induce similar
impairments in energetic demand. Our main observation was the absence
of a relation between heart function and CK forward flux measured by
saturation-transfer NMR during moderate inhibition of ATP synthesis: CK
flux remained constant despite a two- to threefold decrease in
mechanical performance and
O2.
On the other hand, severe hypoxia induced a parallel decrease in both
performance and CK flux; we discuss here the origin of this impaired CK flux.
Constant CK flux measured by saturation transfer during moderate
inhibition of ATP synthesis.
CK kinetics obviously depend on the concentration of both substrates
and products. In vitro, large changes in concentration are required to
induce changes in the flux of the enzyme; for example the
Km of MM-CK for
PCr is ~2 mM (12). Similarly in the perfused heart, using our
normoxic model of adenylate depletion by 2-DG (9), we demonstrated that
CK flux was in a large range independent of ATP and PCr concentration
(23). Indeed, ATP and PCr must reach concentrations of ~4 mM to start
to impair CK flux in this normoxic model. Here, partial inhibition of
ATP synthesis resulted in minimal concentration of 5.3 and 4.5 mM for
PCr and ATP, respectively (Table 2). Thus, in all our inhibitory
conditions, including severe hypoxia, the mean concentrations of PCr
and ATP did not reach the range necessary to markedly affect the
kinetics of cytosolic CK flux. In the hypothesis of CK equilibrium, and without taking into account CK compartmentation, a constant CK flux is
thus expected from a kinetic point of view.
Independence of CK flux and systolic performance during moderate
inhibition of ATP synthesis.
In a normoxic myocardium, an increase in workload associated with rapid
changes in the rates of ATP synthesis occurs without major changes in
intracellular levels of ATP and PCr (for review, see Ref. 3);
conversely, systolic activity is largely independent of the cytosolic
concentrations of CK metabolites (9). To understand the continuous
balance between ATP synthesis and utilization, two types of mechanisms
have been suggested. The first mechanism is a parallel activation of
ATPases and ATP synthesis pathways by calcium, and the other mechanism
involves signaling between both sites by metabolites of CK. In this
latter hypothesis, one expects a correlation between the rates of ATP
synthesis by oxidative phosphorylation, the rate of energy transfer by
CK, and the rates of ATP utilization by ATPases. CK flux is thus
assumed to be related to ATP synthesis and utilization (6). This is
presumed to be a specificity of the myocardium caused by the high
proportion of bound CK isozymes (~50% of total CK). Indeed, in
skeletal muscle, the 10-fold increase in ATPase rate observed between
rest and maximal activity occurs without any modification of CK flux.
However, this is also observed in the myocardium. In vivo, CK flux
remains constant during a threefold increase in work induced by pacing or catecholamine stress (11, 13, 19). Conversely, in the perfused
heart, a direct 99% inhibition of CK activity by iodoacetamide (IAAM)
does not alter the baseline systolic parameters, although this nearly
abolishes the contractile reserve (26). Moreover, a transition from low
to high work (RPP from 1.5 to 4.5 × 104
mmHg · beat · min1)
doubles ATP synthesis but only moderately affects CK flux (6). Only
during the transition from arrest by KCl to a low systolic activity was
a parallel threefold increase in ATP synthesis and CK flux observed
(6), although such a huge change is not consistently detected (12, 14).
More recently, the KCl-arrested heart has been recognized to be a
puzzling situation in which the set of kinetic constants that predicts
CK flux in a beating heart cannot be used (15). Thus, apart from this
specific case, the evidence of a relation between work and CK flux
appears rather weak in the normoxic myocardium both in vivo and in the
perfused heart. Our results with cyanide and moderate hypoxia agree
with this theory: a threefold change in ATP synthesis and utilization
pathway occurs without any change in the flux of energy transfer by CK as measured by NMR saturation transfer.
CK flux and diastolic properties.
In severe hypoxia, the decrease in CK flux was associated with a marked
rise in EDP (Fig. 6B). We favor the
hypothesis that this is caused by the formation of strongly attached
cross bridges (rigor type contracture): low CK activity impairing ADP
rephosphorylation that would result in an inhibition of myosin ATPase.
In Triton-skinned fibers, such rigor force is evidenced when MgATP
concentration is not sufficient to allow the detachment of myosin from
actin; half-maximum rigor force occurs for an apparent concentration of
MgATP of ~300 µM in the absence of CK and 10 µM when MM-bound CK
is functional and locally rephosphorylates the ADP produced by the
ATPase (for review, see Ref. 29). Even in severe hypoxia, the cytosolic
ATP concentration (~4 mM), which was at least one order of magnitude
higher than the apparent
Km of the ATPase
for MgATP, should not be responsible for a marked rise in EDP. On the
other hand, the cytosolic free ADP concentration (30-120 µM) is
in the range likely to influence the ATPase and to induce rigor development (29). Indeed, in the perfused heart, increase in free ADP
induced by a direct inhibition of CK by IAAM causes a dose-dependent
increase in EDP that has been demonstrated to be caused by rigor (25).
A rise in EDP was observed here in all conditions of inhibition of
respiration. However, at variance with the IAAM model, no general
relation could be observed between the free ADP calculated from the CK
equilibrium and the rise in EDP. With cyanide, the free ADP
concentration was two times higher than in severe hypoxia, but the rise
in EDP was modest, three times lower than in
H25 (Tables 1 and 2). This is
compatible with the fact that in skinned fibers rigor is prevented,
even in the presence of 20 mM ADP in the bulk solution, if MM-bound CK
is functional (28). This suggests that in cyanide the high CK activity
is sufficient to rephosphorylate the ADP in the vicinity of ATPases and
prevent their inhibition despite the high cytosolic free ADP as
calculated using the equilibrium constant. By contrast, the impaired CK
flux in severe hypoxia could not prevent a local rise in ADP and
subsequent myofibrillar ATPase inhibition. This points again to the
importance of bound CK for the myocardium and further suggests that a
rise in calculated cytosolic free ADP does not automatically reflect
change in ADP at the sites of interest in the subcompartments.
Impaired CK flux in severe hypoxia: hypothesis of myocardial
heterogeneity.
The decreased CK flux observed for severe hypoxia is in agreement with
previous reports both in the isolated heart perfused with 20%
O2 (8) and, in vivo, in the
open-chest rat respiring mixtures with
O2 reduced from 21 to 8% (5). As
discussed in Constant CK flux during moderate
inhibition of ATP synthesis, the concentration of CK metabolites
induced by severe hypoxia is not expected to decrease CK flux, as
confirmed by the calculation of the theoretical CK flux (Table
4). Because CK velocity also depends on the concentration
of active enzyme (i.e., saturated with substrate) an increasing
proportion of free enzyme would decrease CK flux. This was not the
case, because the increase in free enzyme did not reach significance.
Alternatively, as suggested in longer-lasting ischemia, a loss
of CK molecules or their inactivation by free radicals could be
responsible for the impaired hypoxic flux. We do not favor these
hypothesis because both the leakage of cytoplasmic enzymes (CK and
lactate dehydrogenase) and the inactivation of CK occur only during the
phase of reoxygenation (4).
In our model, in the absence of glucose, the
O2 content of the cardiac effluent
became negligible and the heart function was severely impaired when the
PO2 in the perfusate was decreased below 40% of control (~200 mmHg, Fig. 7). As a result of myocardial cell
O2,
marked O2 gradients are well known
to occur along the pathway of capillaries. Even at the level of one
cell, increasing O2
results in a radial gradient of O2
whereas cyanide inhibition does not induce such heterogeneity (24). At
the organ level, decreasing O2
supply below a "critical PO2"
results in a patchwork of areas with different metabolic
characteristics (for review, see Ref. 18). Obviously, this critical
PO2 depends on the working conditions
and the type of substrate available. Indeed, in the isolated heart
using glucose as substrate, this critical
PO2 is lower (data not shown). Such
would also be the case in vivo because mixed substrates are available.
Thus we suggest that in mild hypoxia all cells receive adequate
O2 supply and/or are able to
downregulate their ATP utilization and ATP synthesis (7). As hypoxia
becomes more severe, an increasing number of cells do not receive
adequate O2 supply, stop
developing systolic activity, and enter the state of rigor. In these
anoxic cells, one (or several) of the CK metabolites may reach
concentrations inhibiting CK. The linear relation existing between
impaired mechanical performance and CK flux in severe hypoxic hearts
(in the range of 40 to 20% O2)
would thus result from a progressive increase in the proportion of
CK-inhibited cells. Such a correlation is also consistently observed,
in the same range of cardiac performances as found here, in another
model of tissular heterogeneity, reperfusion after long-term
ischemia (17). Interestingly, a decrease in CK flux is also
observed in vivo when rats inspire air with reduced O2 content (5). This suggests that
the impairment of CK flux in severe hypoxia is not the consequence of
the known limitation in oxygenation of the crystalline
solution-perfused heart but reflects a more common feature linked to
the imbalance in O2 supply and demand.
Considerations on NMR-measured CK flux and its physiological
interpretation.
The absence of modification of CK flux observed here for a threefold
change in heart function could be directly discussed, as previously
suggested, as evidence for CK functioning at equilibrium everywhere in
the cell. However, this observation is difficult to reconcile with the
abundant literature describing the function of the CK isozymes in
myofibrillar and mitochondria subcellular preparations (2, 20-22,
28-31).
NMR is generally assumed to reflect all unidirectional CK fluxes
originating from the cytosolic and the bound isoenzymes. Indeed,
mito-CK flux can be detected in isolated mitochondria (10) and in
transgenic mice in which the homodimeric MM-CK gene has been knocked
out (27). The NMR-detected forward CK flux thus represents the
contribution of forward fluxes of cytosol, myofibrils, and
mitochondria. For various working conditions, the total forward CK flux
would be expected to be constant if all cytosolic and bound CKs are at
equilibrium but also if opposite changes occurred in the forward
mitochondrial and myofibrillar flux. Such precise tuning of both
myofibrillar and mitochondrial CK activity is indeed a prerequisite of
the continuous balance of energy production and utilization in the
hypothesis of the PCr-Cr shuttle and of the intracellular signaling
role of CK metabolites. This aspect needs careful reevaluation before
any definitive conclusion can be drawn from the NMR data in terms of
physiological importance of the CK in the whole organ.
In conclusion, during inhibition of ATP synthesis, heart function is
not related to NMR-measured CK flux except during severe hypoxia. Our
results suggest that the progressive impairment of CK flux in severe
hypoxia results from an increasing number of cells with nonfunctional
CK, a situation also likely to occur in pathological cases such as
infarct or ischemic insult.
| |
ACKNOWLEDGEMENTS |
The authors thank P. Lechene for the programs of analysis, R. Ventura-Clapier, R. Fischmeister for continuous scientific support, and
V. Saks for stimulating discussions and acknowledge the valuable help
of E. Boehm for correction of the manuscript.
| |
FOOTNOTES |
V. Stepanov was supported by a 12-month grant from the French Ministry
of Research and Technology (Réseau Formation Recherche) and by an
International Association for the Promotion of Cooperation with
Scientists from the Independent States of the Soviet Union grant (94 4738).
Present address of V. Stepanov: Institute of Cardiology, Moscow, Russia.
The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement"
in accordance with 18 U.S.C. §1734 solely to indicate this fact.
Address for reprint requests and other correspondence: J. A. Hoerter,
U-446 INSERM, Cardiologie Cellulaire et Moléculaire,
Université Paris Sud, 92296 Chatenay Malabry, France (E-mail:
jacqueline.hoerter{at}cep.u-psud.fr).
Received 16 November 1998; accepted in final form 9 March 1999.
| |
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Am J Physiol Heart Circ Physiol 277(1):H308-H317
0002-9513/99 $5.00
Copyright © 1999 the American Physiological Society
TOP
ABSTRACT
INTRODUCTION
![<IT>F</IT> = <IT>k</IT><SUB>f</SUB> ⋅ [PCr]](/content/vol277/issue1/fulltext/H308/img003.gif)
![<IT>v</IT> = <IT>V</IT><SUB>max</SUB> ⋅ [([MgADP] ⋅ [PCr]/<IT>K</IT><SUB>i,ADP</SUB> ⋅ <IT>K</IT><SUB>PCr</SUB>)/<IT>D</IT>]](/content/vol277/issue1/fulltext/H308/img004.gif)
![<IT>D</IT> = 1 + <FR><NU>[MgADP]</NU><DE><IT>K</IT><SUB>i,ADP</SUB></DE></FR> + <FR><NU>[PCr]</NU><DE><IT>K</IT><SUB>i,PCr</SUB></DE></FR> + <FR><NU>[Cr]</NU><DE><IT>K</IT><SUB>i,Cr</SUB></DE></FR> + <FR><NU>[MgATP]</NU><DE><IT>K</IT><SUB>i,ATP</SUB></DE></FR>](/content/vol277/issue1/fulltext/H308/img005.gif)
![+ <FR><NU>[MgADP] ⋅ [PCr]</NU><DE><IT>K</IT><SUB>i,ADP</SUB> ⋅ K<SUB>PCr</SUB></DE></FR> + <FR><NU>[MgATP] ⋅ [Cr]</NU><DE><IT>K</IT><SUB>Cr</SUB> ⋅ K<SUB>i,ATP</SUB></DE></FR>](/content/vol277/issue1/fulltext/H308/img006.gif)
![+ <FR><NU>[MgADP] ⋅ [Cr]</NU><DE><IT>K</IT><SUB>I,Cr</SUB> ⋅ <IT>K</IT><SUB>i,ADP</SUB></DE></FR> + <FR><NU>[MgATP] ⋅ [PCr]</NU><DE><IT>K</IT><SUB>I,PCr</SUB> ⋅ <IT>K</IT><SUB>i,ATP</SUB></DE></FR>](/content/vol277/issue1/fulltext/H308/img007.gif)
