Kinetics of creatine kinase in an experimental model of low
phosphocreatine and ATP in the normoxic heart
V.
Stepanov1,
P.
Mateo1,
B.
Gillet2,
J. C.
Beloeil2,
P.
Lechene1, and
J. A.
Hoerter1
1 U-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 RMN Biologique, Institut de Chimie des Substances
Naturelles, Centre National de la Recherche Scientifique, 91198 Gif sur
Yvette, France
 |
ABSTRACT |
To study the dependence of the forward flux of creatine kinase
(CK) on its substrates and products we designed an acute normoxic model
of steady-state depletion of phosphocreatine (PCr) and adenylate in the
isovolumic acetate-perfused rat heart. Various concentrations of PCr
and ATP were induced by prior perfusion with 2 deoxy-D-glucose in the presence
of insulin. The apparent rate constant
(kf) and the
forward CK flux were measured under metabolic and contractile steady
state by progressive saturation-transfer
31P nuclear magnetic resonance
(NMR). At high adenylate content CK flux was constant for a twofold
reduction in PCr concentration ([PCr]); CK flux was 6.3 ± 0.6 mM/s (vs. 6.5 ± 0.2 mM/s in control) because of a
doubling of kf.
Although, at the lowest ATP concentration and [PCr], CK
flux was reduced by 50%, it nevertheless always remained higher than
ATP synthesis estimated by parallel oxygen consumption measurement.
NMR-measured flux was compared with the flux computed under the
hypothesis of CK equilibrium. CK flux could not be fully predicted by
the concentrations of CK metabolites. This is discussed in terms of
metabolite and CK isozyme compartmentation.
phosphorus-31 nuclear magnetic resonance
magnetization transfer; myocardial energetics; oxygen consumption; free
adenosine 5'-diphosphate; creatine kinase isozymes and kinetics
 |
INTRODUCTION |
CREATINE KINASE (CK) catalyzes the
reversible exchange of high-energy phosphate
(where
Kf and Kr are the forward
and reverse rate constants, respectively, PCr is phosphocreatine, and
Cr is creatine). CK is found as four isozymes, MM-, MB-, BB- and
mitochondrial (mito)-CK in muscle cells. Its function in
muscular energetics has been debated for a long time. Meyer et al. (15)
proposed a quantitative analysis of CK function based on CK as close to
equilibrium at all points of the cell. This implies that the
concentrations of substrates and products of the reaction are identical
at all intracellular localization because of facilitated diffusion. A
corollary of this approach is that, in vivo, CK flux must be governed
by the concentrations of ATP, PCr, Cr, ADP, and
H+. Another approach to CK
function is based on the existence of CK compartmentation by the
localization of CK isozymes close to sites of energy production and
utilization in the cell. MM-CK isozyme bound in the vicinity of
adenosinetriphosphatases (ATPases) affects their apparent kinetics (for
a recent review, see Ref. 26). Such functional coupling is also
evidenced between mito-CK and translocase at the site of mitochondrial
energy production (for a recent review, see Ref. 19), which has led to
the concept of energy channeling by a PCr-Cr-CK system in the
myocardium (for a recent review, see Ref. 27). This approach is in the
form of a general concept in which coupled enzymes can alter the
velocity of a reaction and create in the vicinity of the enzymes a
microcompartment in which the concentration of metabolites may differ
from bulk concentrations, despite the lack of physical barriers (2). A
corollary of this approach is that the function of CK bound to
myofibrils, sarcoplasmic reticulum, or mitochondria should not be
governed by the bulk concentrations of CK metabolites. Therefore, in an
intact muscle, a key question is, does CK (including all isoforms)
function at equilibrium? Alternatively, does the functional coupling of
mito-CK and MM-CK result in a nonequilibrium behavior that can be
observed by nuclear magnetic resonance (NMR)?
In vitro, the known kinetics of CK in dilute solution (11) are
adequately described by NMR magnetization-transfer methods (8). In vivo
CK kinetics have also been extensively studied by NMR. Three main
factors have been proposed to govern the CK flux in vivo: total CK
activity, CK isozymic composition, and concentration of products and
substrates. The importance of isozymic composition was first pointed
out during neonatal development; at constant total CK activity, CK flux
increased together with mito-CK content (18). The dependence of CK
velocity on substrate concentration has been analyzed by comparing
various species during neonatal development (13, 18) or by changing the
size of the guanidine pool by long-term feeding with Cr analogs (22).
However, in all these cases several parameters are affected besides the metabolite concentrations. For example, during perinatal development, in addition to an increase in guanidine pool, a complete reorganization of the cell occurs, as evidenced by the expression of the mito-CK isozyme, the presence of functional coupling of both MM-CK to ATPase
and mito-CK to translocase, and the increase in energetic requirements
(6). Similarly, feeding Cr analogs induces ventricular hypertrophy, a
shift of isomyosin pattern towards slow-type, perturbing the energetic
balance, and mitochondrial adaptation (for review, see Ref. 29). Thus,
in none of these models, because of the surprising plasticity of
the cardiac muscle, could the specific contribution of change in
substrate and product concentrations to CK velocity easily be separated
from cellular remodeling.
Our aim, therefore, was to design an acute experimental model to study
the effects of large changes of CK metabolite concentrations per se on
CK velocity. We took advantage of the model of adenylate depletion
(based on intracellular trapping of phosphorus that we previously
developed; Ref. 7), to design a new experimental model of steady-state
heart contractility and metabolite concentrations with large reductions
in both PCr and adenylate concentrations. We measured the apparent rate
constant (kf) and
the flux of CK in the forward direction (PCr
ATP) by NMR
progressive saturation transfer for a threefold change in PCr content
and a sevenfold decrease in ATP. Up to now there has been to our
knowledge no analysis of myocardial CK velocity under such a wide
change of both guanidylate and adenylate concentrations. In this
normoxic model, contractility is sustained as previously described.
Here we show experimental evidence of sustained cytosolic transfer of
energy at high ATP content. The CK forward flux was unchanged despite a
threefold decrease in PCr content due to an increase in
kf. The
independence of CK flux with regard to PCr concentration is indeed
observed in dilute solution in vitro because of the equilibrium
behavior of CK. However, at variance with the concept of equilibrium,
CK flux decreased at low ATP concentration. The reasons for such
discrepancy are tentatively discussed in terms of the relative
contribution of the CK isozymes to the NMR-measured total CK flux.
 |
MATERIALS AND METHODS |
Physiology
Animal experimentation was performed in accordance with the American
Heart Association's position statement on research animal usage.
Wistar male rats (350-450 g) were anesthetized with ethyl carbamate (2g/kg), and hearts were perfused by the Langendorff technique at a constant flow of 13.5 ml/min. The left ventricle (LV)
was pierced to avoid fluid accumulation. A latex balloon was inserted
in the LV and inflated with 2H2O to
isovolumic conditions of work. LV pressure 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 sodium
acetate, and 20 N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid and was oxygenated with 100%
O2. External pH
(pHo) was adjusted with NaOH to
7.35 at 36.5°C. Contractile parameters were analyzed on-line with home-made software: mean coronary pressure, LV systolic pressure (LVP), end-diastolic pressure (EDP), and spontaneous heart
frequency were measured, allowing the calculation of the rate-pressure
product (RPP; 104
mmHg · beat · min
1).
Depletion of high-energy phosphate was achieved by addition of
2-deoxy-D-glucose (2DG; Sigma)
in the presence of insulin (4 IU/l; Actrapid Nova). 2DG concentrations
ranged from 1 to 5 mM for times varying from 5 to 20 min depending on
the conditions required (see EXPERIMENTAL
PHYSIOLOGICAL GROUPS). To maintain steady-state metabolite contents, oxygenated perfusate containing 5 mM
2DG without insulin was infused in the main flow perfusion line (final
concentration of infusion ranged from 0.06 to 0.08 mM). The additional
flow, at most 0.2 ml/min, did not change the contractile performances.
Physiological protocols. RATIONALE. To achieve
steady-state situations with various decreased contents in PCr and ATP
we used depletion of high-energy phosphate induced in normoxia by the glucose analog 2DG in the presence of the nonglycolytic substrate acetate (7). Briefly, 2DG is imported in the cell by the glucose transporter. Its phosphorylation by hexokinase consumes ATP and produces
2-deoxy-D-glucose-6-phosphate
(2DG6P) and ADP. ADP is first rephosphorylated to ATP by CK at the
expense of PCr. Later on, the accumulation of ADP activates myokinase,
leading to purine efflux and reducing adenylate content. In the
presence of a nonglycolytic substrate, mitochondrial ATP synthesis is
maintained as well as systolic function. On removal of 2DG,
dephosphorylation of 2DG6P occurs and the phosphorus moiety is
recovered mole-to-mole as PCr. Dephosphorylation of 2DG6P is a
first-order process accelerated by insulin (5). With the 2DG6P
concentration and the time constant of 2DG6P dephosphorylation, the
dephosphorylation rate can be computed. From the known kinetics of
2DG6P phosphorylation in the absence of insulin [maximal CK
activity (Vmax) = 1.8 nmol · min
1 · mg
protein
1, 2DG Michaelis
constant (Km) = 7.6 mM; Ref. 5], we calculated the 2DG concentration needed to
counterbalance the rate of spontaneous 2DG6P dephosphorylation and thus
prevent PCr recovery. This was used in all 2DG protocols to maintain a
steady-state PCr content. When a low ATP content was required
(protocols 2DG-2 and
2DG-2a, see below), massive depletion
of high-energy phosphate compounds was induced by higher concentrations
of 2DG perfusion. On removal of 2DG, PCr was allowed to recover to the
desired content by addition of insulin, which accelerates 2DG6P
dephosphorylation. After leakage of adenine moieties, ATP remains low
because of an extremely slow de novo synthesis. Again, PCr contents
were stabilized by infusion of a minimal concentration of 2DG without
insulin.
EXPERIMENTAL PHYSIOLOGICAL GROUPS.
The experimental design included four protocols: a control group
(n = 6) and three groups with various
PCr concentrations ([PCr]) achieved by 2DG perfusion:
protocol 2DG-1, with intermediate ATP
concentration ([ATP]; i.e., >50%) and
pHo 7.35 (n = 18), and protocols 2DG-2 and
2DG-2a, with low [ATP]
(i.e., <50%) and pHo 7.35 (n = 18) and 7.65 (n = 5), respectively.
In protocol 2DG-1 (variable
[PCr] at medium [ATP]), eight hearts were
perfused with 1 mM 2DG in the presence of 4 IU/L insulin until PCr was
decreased to 50% as checked on the spectrum; the duration of 1 mM 2DG
perfusion was 10.6 ± 1.1 min. On removal of the 2DG + insulin solution, 0.075 mM 2DG without insulin was continuously infused
to prevent PCr recovery. Saturation transfer was performed when this
steady state was achieved. The 2DG-1 group also included 10 additional
hearts in which PCr content was clamped to various levels ranging from
90 to 30% of control PCr value by changing the duration (4-12
min) and the concentration (1-2 mM) of the initial 2DG perfusion.
To maintain a steady state, the concentration of 2DG infusion was
adjusted (0.06-0.08 mM) as a function of 2DG6P content.
Protocol 2DG-2 (variable
[PCr] at low [ATP]) achieved the same range of
PCr contents as in 2DG-1 but at low ATP content. Massive decrease in
high-energy phosphates was induced in the presence of insulin by
perfusion either for 19.4 ± 0.6 min in the presence of 2 mM 2DG (n = 12) or for 15.0 ± 0.5 min in the presence of 5 mM 2DG (n = 6). The two protocols were equivalent, and data were pooled. At this
stage, the PCr peak had disappeared from the NMR spectra. In nine
hearts, PCr was allowed to recover to 50% of its control value by
removal of 2DG in the presence of insulin. As in the previous protocol,
steady-state PCr content was maintained by infusion of 0.075 mM 2DG. In
nine additional hearts, a variation in the duration of 2DG-free insulin
perfusion allowed different levels of PCr recovery, from 30 to 75% of
control. As in protocol 2DG-1, the
concentration of 2DG in the infusion was adjusted for steady state.
Protocols 2DG-1 and
2DG-2 induced intracellular
acidification. To check its influence, we designed a 2DG-2 protocol,
protocol 2DG-2a (same PCr and ATP as
2DG-2), in which intracellular pH
(pHi) was restored to control by
external alkalinization. After a low ATP and a PCr content of 50%
similar to 2DG-2 were reached, pHo
was increased from 7.35 to 7.65; as a consequence,
pHi recovered to its control
value. In these hearts (n = 5),
saturation transfer was performed at both
pHi with 24 scans in each
spectrum.
NMR
31P NMR spectra were acquired at
161.93 MHz on a Brucker AM400 wide-bore magnet in 20-mm-diameter tubes.
Homogeneity was made on the heart water, and the frequency was locked
on 2H2O contained in the LV balloon. We
used a pulse angle of 90° measured on the
-ATP signal,
acquisition of 4,000 data points, a spectral width of 10,000 Hz, and a
line broadening of 20 Hz. The control 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. The signals of 2DG6P,
PCr, and
-ATP were reduced by saturation to 78, 66, and 96%,
respectively, of their equilibrium value. The same NMR parameters
(interpulse delay 2 s) were used to follow PCr depletion by 2DG and to
check the steady state after depletion by comparing four spectra taken
just before and just after saturation transfer was performed. Hearts with >10% change in PCr or ATP content before and after saturation were not included in the study.
The forward CK rate constant was measured by time-dependent saturation
transfer (4) using the Dante method (16). 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 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 2DG and 0.5 to 9 s
for control conditions. The delays between pulses were adjusted to
achieve in each spectrum 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 number of scans for each spectrum = 40-48). This procedure of signal averaging
minimized the possible influence of any change in contractility or
energetics that might occur during the saturation-transfer experiment.
In six 2DG-1 hearts, the flux was measured in the same heart during
control and after PCr depletion. In this case, 24 scans for each
spectrum were used for determination of the control flux and the values
were similar to the control group; all data were pooled.
Metabolite concentrations and NMR-measured forward flux of CK.
Some hearts were freeze clamped at the end of the experiment, and
biochemical analysis of PCA extracts was performed to measure ATP, PCr,
and Cr content as previously described (7). Values were expressed in
nanomoles per milligram of protein. Biochemical determination confirmed
that none of the experimental conditions induced Cr leakage. Cr was
thus calculated from the difference between total Cr plus PCr measured
biochemically at the end of the experiment and PCr measured on each
spectrum.
NMR quantification was performed with a home-made program on the area
of each peak corrected for saturation. Biochemically determined PCr
content of 43 nmol/mg protein for control hearts was used as an
internal standard, and cytosolic volume was taken as 2.72 µl/mg
protein. pHi was determined from
the shift of Pi or 2DG6P peak with
respect to PCr. Free ADP was calculated from the equilibrium of the CK
reaction with the apparent equilibrium constant = 166 × 10
0.87(pH
7) (11).
Free Mg2+ concentration was calculated from the chemical
shifts of
- and
-ATP peaks using the MAGPAC program (28).
Quantification of metabolites during flux measurements was made by
averaging four spectra taken just before and just after saturation
(corrected to 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 1/T1PCr + kf
(T1PCr = intrinsic relaxation of
PCr and
PCr = relaxation time constant) as described
previously (4). The forward CK flux = kf · [PCr] is expressed in millimoles per second. The
T1PCr value was similar in all
groups: control, 3.1 ± 0.2 (n = 12); 2DG-1, 2.9 ± 0.2 (n = 18); 2DG-2, 2.8 ± 0.2 (n = 18); and 2DG-2a, 3.1 ± 0.4 s
(n = 5).
Predicted velocity of CK in myocardium.
To check whether the dependence of CK flux on PCr concentration could
be understood by the hypothesis of all CK isoforms functioning at
equilibrium, we compared for each heart the NMR-measured velocity with
the velocity expected from the well-known equilibrium behavior of MM-CK
in vitro. The forward CK reaction is a rapid equilibrium random
mechanism at neutral pH. The steady-state velocity of the reaction,
v, relative to
Vmax is described
by
where
D is a function of the association
(Km), dissociation (Ki),
and inhibitory (KI) constants of each metabolite
for the various enzyme complexes
Thus,
for each heart, the metabolite concentrations measured by NMR were used
to predict the reaction velocity expected if myocardial CK isoforms
function at equilibrium as CK in dilute solution in vitro. The maximal
CK activity,
Vmax, was
constant in this acute model.
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 H20/g wet weight. The
constants used for prediction were (in mM)
Km: PCr = 1.11, Cr = 3.8; Ki: ADP = 0.135, PCr = 3.9, Cr = 554, ATP = 3.5; and KI: Cr = 58, PCr = 3.9 according to McFarland et al. (14). No correction was applied for
the influence of H+ or Mg2+ (see
RESULTS). The amount of free enzyme,
i.e., nonsaturated by its substrates, can be computed as
1/D and was expressed as percentage of
total enzyme.
Oxygen Consumption
Parallel experiments were performed outside of the magnet to estimate
oxygen consumption
(
O2)
in relation to contractility in the same conditions of perfusion.
"Arterial" PO2 just above the
aorta and "venous" PO2 in the
pulmonary artery were measured in-line through two flow cells, Clark
electrodes, and oxymeters (Stratkhelvin Inst., Glasgow, UK).
O2
("arterial" PO2
"venous" PO2) was expressed in
micromoles of O2 per minute per
gram wet weight. After equilibration, hearts were first submitted to
stepwise increase in balloon volume until isovolumic conditions were
reached to define the relationship between work and
O2
in our control conditions. Next, hearts were submitted to
protocol 2DG-1 or
2DG-2. For protocol
2DG-1, hearts were perfused with 2 mM 2DG in the
presence of insulin for 6, 8, or 10 min
(n = 4, 4, and 5, respectively). For
protocol 2DG-2, five hearts were
perfused for 15 min in 5 mM 2DG in the presence of insulin, followed by
15 min of 2DG-free perfusate containing insulin. Steady state was
obtained for both protocols 10 min after removal of insulin in the
presence of 0.075 mM 2DG. Hearts were freeze clamped for ATP, PCr, and
Cr biochemical determination at the end of the experiment to ensure
that PCr and ATP content were similar to the NMR series. ATP synthesis
of the NMR-perfused hearts was estimated from the relationship between
RPP and oxygen consumption and a phosphate-to-oxygen ratio of
3.
Statistical Analysis
All results are expressed as means ± SE. Differences between groups
were analyzed by analysis of variance and Student-Newman-Keuls test,
except for the effect of acidosis, which was analyzed by paired
t-test.
 |
RESULTS |
Metabolic and Contractile Characteristics of Hearts
The initial contractile performance for each group and the steady-state
contractility during the performance of saturation transfer are
summarized in Table 1 for control hearts
(n = 12) and for 2DG protocols at 50%
decrease in PCr content, including the high-ATP protocol (2DG-1,
n = 8), the low-ATP protocol
(2DG-2, n = 9), and the low-ATP
protocol at constant pHi (2DG-2a,
n = 5). The various 2DG protocols
affected neither EDP nor the coronary pressure (not shown). As
previously described, a massive decrease in ATP and PCr content
resulted in moderate (although significant) change in contractility in
this normoxic model. When PCr was 50% of its control content, RPP was
similar in protocols 2DG-1 and 2DG-2 (80 ± 3% and 77 ± 4 % of initial control value, respectively; Table 1). When PCr varied from
90 to 30%, RPP ranged from 93 to 70% of initial control in
protocol 2DG-1 and from 100 to 60% in
protocol 2DG-2. Recovery
of pHi induced by external
alkalinization after 2DG perfusion restored contractility to its
initial control value (i.e., RPP increased by 37% in 2DG-2a). A
similar increase in contractility was observed on external
alkalinization in two control hearts (+30%, not shown). In all
protocols Pi content remained low,
as previously described (7).
View this table:
[in this window]
[in a new window]
|
Table 1.
Contractile parameters of control and 2DG groups during initial
perfusion and during saturation transfer at 50 and 35% of control
phosphocreatine content
|
|
In some hearts of the 2DG groups with 50% PCr, metabolite contents
were checked biochemically at the end of the experiment (Table
2). Cr leakage occurred in none of the
protocols; the pooled value for PCr + Cr (65 nmol/mg protein) was used
for each NMR heart to calculate Cr and free ADP, assuming CK
equilibrium.
View this table:
[in this window]
[in a new window]
|
Table 2.
Biochemical determination of metabolite contents in selected hearts
from control and 2DG groups at 50% PCr content presented in Table 1
|
|
Validation of Experimental Model: Steady-State Normoxic 50%
Decrease in PCr Induced by 2DG Perfusion (Protocol 2DG-1)
Figure 1 shows a typical 2DG-1 protocol
leading to a steady-state decrease in PCr to 50% of its initial
content and the time course of changes in contractile activity and
phosphorus contents. At the onset of 1 mM 2DG perfusion in the presence
of insulin, a transient biphasic change in contractile activity was
observed. Infusion of 0.075 mM 2DG in the control perfusate medium
stabilized contractility and metabolite contents. The rate of 2DG
phosphorylation used to counterbalance 2DG dephosphorylation (~ 0.016 nmol · mg protein
1 · min
1)
is four orders of magnitude smaller than CK flux and is thus negligible
in terms of ATP consumption. The new steady state in metabolites
allowing steady-state CK flux determination lasted at least 80 min.

View larger version (27K):
[in this window]
[in a new window]
|
Fig. 1.
Protocol 2DG-1 in a representative
heart at 50% phosphocreatine (PCr) content.
A: experimental protocol.
B: contractility index [rate-pressure product (RPP) in
104
mmHg · beat · min 1].
C: metabolite contents (in nmol/mg protein). A pulse of 1 mM 2-deoxy-D-glucose (2DG) in
presence of insulin (4 IU/l) induced 2-deoxy-D-glucose-6-phosphate
(2DG6P) accumulation at expense of PCr. On removal of 2DG, infusion of
a small 2DG concentration (0.075 mM) counterbalances spontaneous
dephosphorylation of 2DG6P and prevents recovery of PCr. Original
traces of RPP acquired on-line show initial biphasic change due to
insulin followed by steady-state contractility. Saturation transfer is
started after checking for steady-state metabolites content on 4 spectra. Points in middle of saturation
transfer correspond to metabolite contents measured in nonsaturated
spectra averaging whole saturation-transfer period. , ATP; ,
Pi; , PCr; , 2DG6P.
|
|
Influence of 50% Decrease in PCr Content on Forward Kinetics of CK
Figure 2A
shows spectra representative of the metabolite contents of the initial
control and of the new steady state in three individual hearts of the
control, 2DG-1, and 2DG-2 groups. A saturation-transfer experiment is
shown for a typical 2DG-1 heart at 50% PCr (Fig. 2B). Longer saturation of ATP
resulted in a progressive decrease in the observed magnetization of PCr
as a result of both CK activity and the PCr relaxation process. The
behavior of the relative PCr magnetization
(Mz/Mo)
is plotted as a function of the time of saturation in control and 2DG-1
(Fig. 2C). The decrease in PCr magnetization as a function of time of saturation is markedly accelerated at low PCr content:
kf increased from
0.49 in control to 0.85 s
1
in 2DG-1. Figure 3 summarizes the PCr, ATP,
and free ADP concentrations and
pHi for control hearts and when
PCr was reduced by 50%. In 2DG-1, ATP decreased to 60% of its control
(P < 0.01) and Cr rose from 9.7 ± 0.3 to 16.3 ± 0.4 mM (not shown;
P < 0.001). Consequently, the
calculated free ADP was two times higher in 2DG-1 (72 ± 5 µM)
than in control (38 ± 3 µM, P < 0.001). The kinetics of CK are shown for the same hearts in Fig.
4. The pseudo-first-order apparent rate
constant kf was
two times higher in 2DG-1 than in control (0.82 ± 0.07 vs. 0.46 ± 0.02 s
1;
P < 0.001). As a result, CK flux
remained similar in 2DG-1 and in control (6.3 ± 0.6 and 6.5 ± 0.2 mM/s, respectively). Although, as already described, contractile
activity was significantly reduced compared with each heart initial
value (Table 1), the difference between the absolute contractile value
of the control and that of the 2DG-1 group did not reach significance.

View larger version (23K):
[in this window]
[in a new window]
|
Fig. 2.
Representative spectra and analysis of saturation-transfer experiment.
A: representative spectra of control,
2DG-1, and 2DG-2 protocols corresponding to initial steady state and to
period of saturation transfer. Each trace is sum of 2 spectra (for
saturation, trace is sum of 1 spectrum before and 1 spectrum after
saturation-transfer experiment). B:
progressive saturation-transfer experiments in a representative heart
of 2DG-1 group: -ATP is saturated for various times shown. For each
spectrum, delay was adjusted so that interpulse duration was 10 s (no.
of scans = 40). Intensity of PCr decreases with time of saturation of
-ATP. Saturation at mirror frequency did not affect PCr (not shown).
C: analysis of rate constant
kf. ; 2DG-1;
, control. Magnetization of PCr (Mz) is referred to
magnetization of nonsaturated spectra
(Mo). Each curve is fitted by
1/ = 1/T1 + k, where is
relaxation time constant and T1 is intrinsic relaxation of PCr. For a
representative control, saturation of -ATP was performed for 0, 0.5, 0.8, 1.2, 2.0, 4, and 9 s; T1 = 3.34 s,
k = 0.48 s 1. For 2DG-1 (heart shown
in B), T1 = 2.84 s,
k = 0.85 s 1.
|
|

View larger version (26K):
[in this window]
[in a new window]
|
Fig. 3.
Nuclear magnetic resonance (NMR)-measured metabolite contents in
control (open bars; n = 12) and in 2DG-1 (solid bars;
n = 8), 2DG-2 (striped bars; n = 9), and 2DG-2a
(crosshatched bars; n = 5) protocols at 50% PCr content.
ADPf, free ADP calculated under
hypothesis of CK equilibrium. Difference from control:
** P < 0.01;
*** P < 0.001. Effect of
adenylate depletion, difference from 2DG-1:
# P < 0.05;
### P < 0.001.
|
|

View larger version (28K):
[in this window]
[in a new window]
|
Fig. 4.
Kinetics of forward CK reaction and contractility in control (open
bars; n = 12) and 2DG-1 (solid bars; n = 8),
2DG-2 [striped bars; n = 9; external pH
(pHo) = 7.35], and 2DG-2a (crosshatched bars;
n = 5; pHo = 7.65) protocols. Same hearts as
shown in Fig. 3. Difference from control:
* P < 0.05;
** P < 0.001.
|
|
Influence of Variation in PCr Content on Kinetics of CK Forward Flux
in 2DG
A large range of steady-state levels of PCr (from 95 to 30% of
control) was generated to check the dependence of CK flux on [PCr]. Concomitantly, ATP concentration decreased from 5 to
3.3 mM and free ADP rose from 25 to 116 µM. As [PCr]
decreased, kf increased from 0.38 to 1.29 s
1 (Fig.
5A,a).
The forward CK flux was again independent of [PCr] (Fig.
5A,b);
its mean value, 5.6 ± 0.3 mM/s (n = 18), was similar to control. Thus, because of an increase in
kf, CK flux was
sustained for a threefold change in PCr (and Cr) content.

View larger version (21K):
[in this window]
[in a new window]
|
Fig. 5.
Apparent first-order rate constant
kf and CK forward
flux as a function of PCr concentration.
A: in protocol
2DG-1 at medium ATP content:
a,
kf increased as
PCr was reduced; b, as a result CK
flux was independent of PCr concentration. , Control
(n = 12; ATP = 7.2 ± 0.2 mM, free
ADP = 38 ± 3 µM); , 2DG-1 (n=
18; ATP range: 6.3-3.2 mM, ADP range: 117-25 µM).
B: in protocol
2DG-2 at low ATP content:
a, rise in
kf is moderate
compared with 2DG-1; b, at low
adenylate content CK flux becomes sensitive to decrease in PCr (ATP
range: 3.0-0.8 mM; ADP range: 61-15 µM). , Mean of control hearts shown in A; , 2DG-2
[external pH (pHo) = 7.35, n = 18]; , 2DG-2a
(pHo = 7.65, n = 5).
|
|
Sensitivity of CK Kinetics to Change in Adenylate Content
Because of the CK equilibrium, a modification of [PCr] is
always associated with variations in [ADP] and
[ATP]. To identify the influence of adenylates on CK flux,
we generated an experimental situation (protocol
2DG-2, see EXPERIMENTAL PHYSIOLOGICAL
GROUPS) in which the PCr content was 50% as in 2DG-1
but the ATP was decreased to 27 ± 3% of control, leading to an
[ADP] similar to that found in the control group (Fig. 3).
Figure 4 shows that
kf was still higher than in control
(kf = 0.72 ± 0.08 s
1,
P < 0.05). Although CK flux tended
to decrease (5.3 ± 0.7 mM/s), the difference in flux measured in
control and 2DG protocols did not reach significance. The dependence of
kf and CK flux on
[PCr] at low adenylate content in 10 other hearts is shown
in Fig. 5B. PCr content was varied
from 11 to 4.6 mM and ATP ranged from 3.1 to 0.9 mM and ADP from 61 to
17 µM. As PCr content decreased, the increase in
kf, shown in Fig.
5B,a,
was in most hearts (7 of 10) moderate compared with
protocol 2DG-1. Thus, in this group characterized by low ATP and normal ADP contents, CK flux has a
tendency to decrease together with PCr content (Fig.
5B,b). This was confirmed by comparing two groups of hearts at 35% PCr content in protocols 2DG-1 and
2DG-2. In 2DG-1 (PCr = 5.3 ± 0.3 mM, ATP = 4.3 ± 0.47 mM, and ADP = 95 ± 7 µM), CK flux was similar to
control (5.1 ± 0.5 mM/s, n = 4).
However, in 2DG-2 (identical PCr concentration = 5.2 ± 0.2 mM
but ATP = 1.6 ± 0.2 mM and ADP = 34 ± 16 µM), CK flux (3.1 ± 0.2 mM/s, n= 6) appeared
significantly decreased in comparison to both control and 2DG-1
(P < 0.05). Thus at a low adenylate
content a decrease in PCr impaired CK flux.
Influence of pH on CK Kinetics at Low ATP
2DG induced intracellular acidosis in protocols
2DG-1 and 2DG-2 (Fig.
3). We checked the eventual influence of this acidosis on CK kinetics
in protocol 2DG-2 by changing the
pHi. At steady-state metabolite contents (50% PCr at low
ATP) pHi was 6.98 ± 0.01 (n = 5). By increasing
pHo to 7.65, we restored
pHi to the control value, 7.14 ± 0.01. Despite the increased contractility (Table 1),
neither PCr and ATP contents nor
kf changed
significantly. CK flux was thus similar at
pHo 7.35 and 7.65 (4.9 ± 0.7 and 4.8 ± 0.5 mM/s, respectively;
n = 5). Similar results were observed in two control hearts perfused at
pHo 7.65 (not shown). Thus this degree of acidosis does not account for the decrease in CK flux observed at low adenylate content.
Relationship Between ATP Synthesis and CK Flux
To estimate ATP synthesis flux, we determined the relation between
O2
and contractility in parallel experiments. All metabolite contents were
similar to those of the NMR series (shown in Fig. 3). For example, in
protocol 2DG-2, PCr, ATP, and Cr
contents measured at the end of the
O2
experiment were 22.6 ± 1.3, 7.1 ± 0.4, and 42.8 ± 2.3 nmol/mg protein, respectively (n = 5).
O2 and contractility are given in Table 3 for
each series for control perfusion and at the new steady state. None of
the 2DG perfusion protocols affected the relationship between
O2
and RPP, which was described by y=
2.377x + 0.625, r2 = 0.920 (where
y =
O2
in µmol
O2 · min-1 · g
wet wt-1 and
x = RPP in
104 mmHg · beat · min
1).
From this relation we calculated
O2
and ATP synthesis for each NMR-perfused heart. In the control group the
calculated ATP synthesis was 5.6 ± 0.4 nmol · mg
protein
1 · s
1.
CK flux measured in the same hearts (17.1 ± 0.5 nmol · mg
protein
1 · s
1)
was 3.3 ± 0.2 times higher than the ATP synthesis flux. The ratio
of CK flux to ATP synthesis was similar to control in 2DG-1 and 2DG-2
groups (Table 4). Although
this ratio tended to decrease in the low-ATP groups, the difference did
not reach significance. Thus, in all protocols, CK fluxes
exceeded ATP synthesis rate, showing that CK flux was never limiting
for ATPase activities.
View this table:
[in this window]
[in a new window]
|
Table 3.
Oxygen consumption and contractility during control and steady-state
metabolite depletion in protocols 2DG-1 and 2DG-2
|
|
Comparison of NMR-Measured Flux and Flux Predicted in Hypothesis of
All CK Isoforms at Equilibrium
The velocity v of CK was predicted for
each heart from the in vitro kinetic constants of the enzyme, the
NMR-measured concentrations of PCr, ATP, and
H+, and the free ADP calculated by
the CK-equilibrium hypothesis (see Metabolite
concentrations and NMR-measured forward flux of CK). Table
5 shows the comparison of the predicted and
the NMR-measured velocity in the various protocols. For control hearts,
the predicted v was 14.5 ± 0.6 (n= 12) and the NMR-measured
value was 6.5 ± 0.2 mM/s. This difference, although highly
significant, should be interpreted with caution because it relies on
several assumptions (see Dependence of CK Flux on
Concentrations of CK Substrates and Products at Low Adenylate
Content). The deviation from prediction markedly
increased in all 2DG groups (Table 5). The predicted flux
was threefold higher than actually measured by NMR in
the 2DG-1 group and even fivefold higher at low PCr and
adenylate content (2DG-2 35% PCr group).
 |
DISCUSSION |
Using an acute normoxic model of adenylate and PCr depletion, with
long-term steady state of contractile activity and metabolite contents,
we have measured in the isovolumic perfused heart the kinetics of the
CK reaction under a wide range of concentrations of its substrates and
products. Furthermore, the experiment was designed to separate the
respective influence of the adenylate and guanidylate pools on CK
velocity. The main importance of this study was to provide an acute
model in which the dependence of CK kinetics on PCr and ATP
concentrations can be analyzed, without change in the other factors
that are known to influence CK activity in the pathophysiological
cardiac muscle. These include total CK activity, isozymic CK
distribution, and remodeling of the myocardial cell.
Characteristics of Model
The experimental evidence of a sustained CK flux despite wide
variations of its substrates confirms the hypothesis we put forward to
explain sustained contractility in our normoxic model of adenylate
depletion (7). As previously discussed, sustained ATP synthesis is
expected from the presence of abundant oxygen and nonglycolytic
substrate. The decrease in ATP-to-ADP ratio and/or the rise in
ADP would promote rather than inhibit respiration of isolated
mitochondria. ATP does not limit ATPase activity, because even at its
lower cytosolic concentration it is still two orders of magnitude
higher than the reported
Km of the
ATPases. Thus sustained myofibrillar and mitochondrial functions were
understood, and we hypothesized that transcytosolic transport of
substrate should also be normal. Here we demonstrate experimentally the validity of this hypothesis. Wide variations in CK substrate
concentrations induced minimal changes in CK flux, which in all cases
remained much higher than the ATP synthesis rate and should not limit
energy transfer in the cell.
We can apply these findings to the understanding of other physiological
perturbations. For example, in the isolated perfused heart, severe
hypoxia induces the same variation in metabolite contents as in
protocol 2DG-1, namely a moderate
change in ATP, a marked decrease in PCr, and a rise in Cr and ADP
contents. However, it results in a strong impairment of CK flux (24).
Because CK flux in 2DG-1 is normal, the hypoxic low CK flux cannot be
explained by kinetic factors resulting from changes in CK metabolite
contents. Moreover, in vivo, when the animal breathes hypoxic mixtures, a similar reduction in CK flux occurs even in the absence of
significant alterations in ATP and PCr contents (3). In the absence of kinetic factors, the origin of hypoxic impairment of CK flux is still
not fully understood. Further work is needed to study the respective
roles of an inhibition of mitochondrial CK flux, of a defect in
oxygenation leading to cellular heterogeneity in the myocardium, or of
the accumulation of some unknown cytosolic factor inhibiting CK.
Insensitivity of CK Flux to Variation in Concentration of CK
Substrates at High Adenylate Content
The insensitivity of myocardial forward CK flux to a wide variation in
PCr, Cr and ADP at high adenylate content (Fig. 4) is in agreement with
the known behavior of MM-CK in dilute solution in vitro (9). In
skeletal muscle a threefold change in PCr content at constant ATP can
be induced by a transition from rest to high stimulation rates. CK flux
remained constant despite a 10-fold increase in ATP synthesis rate.
(10, 14, 21). Thus in skeletal muscle the stability of CK flux, which
is expected from the kinetics of CK in vitro, has been interpreted as a
proof of the equilibrium of CK at all points of the cell, in agreement with the predominance of the buffering role of CK in this type of
muscle (15).
In myocardium it is not as straightforward to study the dependence of
flux towards CK metabolites because the cardiac specificity, as opposed
to skeletal muscle, is to adjust its contractility with minor changes
in high-energy phosphate concentrations. Two experimental models have
been previously used, transition from the KCl-arrested heart to maximal
work and a chronic model of creatine depletion induced by feeding the
animal analogs of Cr [e.g., guadinopropionic acid (GPA)].
In the former, on transition from arrest to maximal systolic activity,
[PCr] decreased by at most 30%, and the CK forward flux
was enhanced by 30-50% (4, 9, 12). However, as seen from analysis
of Fig. 3 in Bittl and Ingwall (4), the main increase in CK flux occurs
during the transition from arrest to low-work conditions. Since that report it has been recognized that the KCl-arrested heart is a puzzling
case in which the set of kinetic constants that predict CK flux in
hearts performing work cannot be used (13, 16). In the chronic GPA-fed
animal model, the massive decrease of both Cr and PCr occurs without
major change in CK flux (22). Our deoxyglucose model confirms these
results in an acute model in which the influence of Cr and PCr content
on CK velocity can be analyzed in the absence of the numerous
adaptations in glycolytic pathway, mitochondrial function, isomyosin
profile, and thus in the economy of contraction found in chronic
GPA-fed animals.
Dependence of CK Flux on Concentrations of CK Substrates and
Products at Low Adenylate Content
In none of the previous models of cardiac or skeletal muscle could the
influence of adenylate content on CK velocity be studied because ATP
levels remained high. The low adenylate content consistently observed
in models of ischemia and on reperfusion is due to imbalance of ATP
synthesis and utilization and is often associated with a detachment of
mito-CK or a decrease in its activity due to a reduced oxidative
phosphorylation. Our 2DG model offers a unique opportunity to study the
influence of wide change in adenylate content independently of an
alteration of ATP synthesis flux. CK flux remained constant for a
twofold reduction in ATP content (protocol
2DG-1) in agreement with in vitro analysis (9);
however, the impaired CK flux observed at lower adenylate content
(protocol 2DG-2, Fig.
5B) was not previously observed. In
the hypothesis of the CK equilibrium, such sensitivity should result
from a change in the concentrations of PCr, Cr, ATP, and ADP or in
ionic content of H+ or
Mg2+. Let us compare the
metabolite and ionic contents in the 2DG-1 and 2DG-2 groups at 50% PCr
content (Fig. 3). The design of the experiment excludes the role of PCr
and Cr because their contents were chosen to be identical. In vitro CK
flux is well known to be modulated by
H+ and
Mg2+ concentration (9, 11). The CK
flux is identical in 2DG-2 and 2DG-2a, in which all metabolite contents
except H+ are similar. This
insensitivity of CK to small pH changes (0.2 pH unit) agrees with
previous data (11). In ischemia, ATP depletion is associated with an
accumulation of free Mg2+; such is
not the case in our deoxyglucose model, most probably because of the
preservation of mitochondrial function and ionic gradients. Free
Mg2+ was similar in control,
2DG-1, and 2DG-2 groups [0.31 + 0.01 (n = 9), 0.35 + 0.01 (n = 12), and 0.31 + 0.02 mM
(n = 10), respectively]. Marked
alterations of free ADP (from 70 µM in 2DG-1 to 30 µM in 2DG-2) are
in the range expected to influence CK flux (see
Predicted velocity of CK in
myocardium). However, because in
control conditions maximal CK flux was obtained for an ADP content of
38 µM, i.e., similar to 2DG-2, the decreased ADP content should not
account for the impairment of CK flux observed in 2DG-2. This is
confirmed by the comparison of the NMR-measured velocity and the
theoretical velocity predicted from the in vitro kinetics of CK in
dilute solution. In the absence of cellular compartmentation of CK
metabolites and if all CK isoforms function at equilibrium, the
velocity of myocardial CK must be governed by the NMR-measured
concentrations of ATP, PCr, Cr,
H+, and
Mg2+ and by the free ADP
concentration calculated assuming CK equilibrium. In control perfusion,
the NMR-measured flux was 50% lower than predicted from the in vitro
kinetic analysis (Table 5) using the set of constants of McFarland et
al. (14). It should be noted that the different sets of CK kinetic
constants (1, 9, 13, 14) result in a wide range of CK theoretical
velocity (for our control hearts from 6 to 20 mM/s). However, for any
set of constants the discrepancy between measured and theoretical velocity consistently increased at low PCr and adenylate content. Several alternative hypothesis could explain this disparity. First, in
vivo CK cellular activity might be modulated by cytosolic factors other
than CK metabolites; second, the flux of the CK-bound isozymes may not
be governed by the bulk cytosolic metabolite concentrations; and third,
the concentration of active enzyme might vary. On the basis of
modeling, planar anions (mainly chloride and bicarbonate) have been
suggested to decrease in vivo CK by stabilizing the dead-end complexes
of the "enzyme · Cr · MgADP"
(14, 17). This hypothesis, based purely on modeling, definitely
requires experimental validation, because such inhibition would
potentially be a very important regulatory mechanism of cellular CK
flux in view of the relative insensitivity of CK to its substrate and products. However, we do not believe an increase in "planar anion inhibition of CK" to be a major determinant of the impaired CK flux
in our 2DG model, because respiration, contractility, and electrical
activities are close to normal.
Alternatively, the NMR-measured metabolite content might not represent
the concentration at the active sites of the enzyme(s). In other words,
the velocity of CK-bound isozymes could be controlled by metabolite
concentrations different from the bulk cytosol concentrations. Even
without any physical compartmentation the vicinity of two enzymes is
known to affect their kinetics, as illustrated in vitro by the
alterations of the apparent kinetic properties of myosin when
coimmobilized with CK on artificial membranes (2). Similarly, in
skinned fiber preparations, the apparent kinetics of myofibrillar ATPase are changed by the activity of MM-CK (26), and oxidative phosphorylation drives mito-CK out of equilibrium (20). Until now we
considered the CK at equilibrium at any point in the cell and neglected
the implication for cell function of CK bound to intracellular
structures. We showed that the velocity of the enzyme cannot be fully
predicted in this simplified theory, and it is therefore worth
examining the hypothesis of metabolite compartmentation. Can the
analysis of the NMR data give any information for this question? The
design of the NMR determination of CK flux considers CK as a
pseudo-first-order reaction. CK is well known to be a higher-order
reaction. It follows that, first, the NMR-measured apparent rate
constant, kf,
does not directly measure the rate constant of the CK reaction
(Kf). Second,
kf values should
depend on the concentration of ADP, the other substrate of CK forward flux; kf indeed
varied in the various protocols (Table 5, Figs. 4 and 5). Figure
6A shows
the dependence of
kf on the free
ADP concentration calculated in the hypothesis of CK equilibrium. For
the control and 2DG-1 groups,
kf is linearly
related to ADP. For each heart one can compute
kf' = kf · [ADP]
1,
which should be proportional to the true rate constant of the enzyme.
As expected,
kf' is
indeed a constant independent of ADP (Fig.
6B); its value was 12.5 ± 0.9 s
1 · mM
1
in control hearts and was similar for all 2DG-1 hearts (11.8 + 0.9 s
1 · mM
1,
n = 18). As previously observed (16),
the influence of H+ on the rate
constant is minor compared with that of ADP (not shown). However, at
low ATP content (2DG-2), the measured rate constant
kf is higher than
expected from the ADP concentration: most hearts are outside the 95%
confidence interval (Fig. 6A). Estimation of
kf'
in both 2DG-2 and 2DG-2a groups also yields higher values than found in
control conditions [23.7 ± 2.4 (n = 18) and 19.1 ± 2.5 s
1 · mM
1
(n = 5), respectively]. This
deviation is clearly seen in Fig. 6B.
Such a failure to observe a constant
kf'
has already been reported in the failing cardiomyopathic hamster (16).
The discrepancy of both
kf and
kf' in the
ATP-depleted groups suggests that the ADP calculated from the CK
equilibrium might not adequately reflect the ADP in vicinity of the
enzyme(s). Why would this phenomenon influence CK flux only at low
cytosolic adenylate concentration? We hypothesize that at high
cytosolic ATP, the function of bound CKs, whether or not they are at
the same chemical potential, might not visibly influence the global
NMR-measured CK flux, which is dominated by the equilibrium behavior of
cytosolic CK. However, this might not be the case at low cytosolic
adenylate content. Indeed, the distribution of ATP and ADP between
cytosol and mitochondria should markedly differ from control in
protocol 2DG-2. Thus we propose that
at low adenylate content, CK bound to intracellular structures exposed
to metabolite concentrations different from bulk concentrations could
become predominant in the NMR-measured flux. This hypothesis is
currently under investigation by measuring, by cell fractionation (23),
the distribution of metabolites between cytosol and mitochondria and by
computing the flux using a model of mito-CK coupled to translocase (1).

View larger version (14K):
[in this window]
[in a new window]
|
Fig. 6.
Dependence of forward rate constant on ADP concentration.
A: relationship between apparent
pseudo-first-order rate constant kf measured by
NMR and ADPf in control, 2DG-1,
2DG-2, and 2DG-2a (same hearts as shown in Fig. 5).
kf increased with
ADP in control and 2DG-1. This dependency can be described by
y = 0.0044x + 0.16, r2= 0.640. However, at low ATP content, in 2DG-2 and 2DG-2a most hearts are
outside 95% confidence interval (thin lines).
B: relationship of calculated apparent
rate constant
kf' = kf · [ADP] 1
and ADPf. In control and 2DG-1,
slope of this relationship is not different from zero;
kf'
is constant from 22 to 120 µM ADP. Again, data of 2DG-2 and 2DG-2a
groups are not fitted by this relationship; at low ATP content
kf'
is higher than expected. , Control; , 2DG-1; , 2DG-2; ,
2DG-2a.
|
|
Finally, because CK reaction velocity depends on the active enzyme
concentration (i.e., the amount of enzyme saturated with substrates),
an increasing proportion of free enzyme would decrease CK flux. In
heart the total CK concentration is 7.5 IU/mg protein and mito-CK
accounts for 30% of total CK. Thus with a specific activity of ~300
IU/mg CK, the concentration of nonmitochondrial CK is ~150 µM,
i.e., highly concentrated compared with ADP. One cannot currently
exclude the possibility that, as adenylate content decreases, an
increasing proportion of free cytosolic CK might not be fully saturated
by ADP. Furthermore, in this case, CK might not be governed by
classical Michaelis-Menten kinetics. Free enzyme (see
Predicted velocity of CK in
myocardium) was estimated to be 4.3 ± 0.1% of
total enzyme in controls and 5.9 ± 0.2% in DG-1. The
increase in free enzyme in DG-2 (12 ± 1%) was, however, not sufficient to fully account for the impairment of CK flux.
Methodological Considerations
In dilute solution, in vitro, the rate of exchange of phosphoryl group
catalyzed by MM-CK is the rate-determining step in the CK reaction and
thus the exchange rate between PCr and
-ATP adequately represents
the CK flux (8). In vivo, because PCr is the substrate only for CK, the
determination of the forward rate by conventional one-site saturation
(ST) avoids the contamination by concurrent reactions occurring when
the reverse flux is measured. ST, as a steady-state technique, allows
equilibration among the exchanging spins during the saturation pulse
and should detect exchange between all metabolite pools. Such might not
be the case when rapid labeling methods are used, because they have
been suggested to minimize the contribution of small intracellular CK
pools (14). Several questions still arise in relation with the
subcellular compartments of metabolites and CK isozymes. First, the
calculation of CK flux from magnetization-transfer experiments assumes
that all reactants are NMR visible and have spatially invariant T1. Such is the case for PCr; however, two pools of ATP with widely different T1 values have been observed in the isolated perfused heart
(25). Thus the saturation of
-ATP under standard
magnetization-transfer conditions might not be as efficient in all
subcellular compartments. As previously suggested (30), the calculation
of CK flux would be more complex than generally assumed. Finally,
because of the poor sensitivity of NMR, flux originating from small
intracellular compartments might not be detected. It is likely that in
skeletal muscle mito-CK, which represents 2-6% of total CK, would
be beyond the limit of NMR detection. The situation is more favorable
in the myocardium because mito-CK represents ~30% of total CK.
Because our limit of flux detection is ~6% (SE/mean in Table 3) the
function of bound CKs, if fully NMR visible, should be detected in the myocardium.
 |
ACKNOWLEDGEMENTS |
We thank R. Ventura-Clapier, R. Fischmeister, and V. A. Saks for
continuous support, V. Veksler and E. Boehm for helpful criticism of
the manuscript, T. Keller for advice in spectroscopy, and R. Wiseman
and M. Kushmerick for very stimulating discussions.
 |
FOOTNOTES |
V. Stepanov was supported by a 12-month grant from the French Ministry
of Research and Technology (Réseau Formation Recherche) and by
Grant 94-4738 from INTAS (an international association for the
promotion of cooperation with scientists from the independent states of
the Soviet Union).
Present address of V. Stepanov: Institute of Cardiology, Moscow,
Russia.
Address for reprint requests: J. A. Hoerter, U-446 INSERM, Cardiologie
Cellulaire et Moléculaire, Faculté de Pharmacie, 5 rue
J. B. Clément, F-92296 Chatenay Malabry, France.
Received 5 February 1997; accepted in final form 6 June 1997.
 |
REFERENCES |
1.
Aliev, M. K.,
and
V. A. Saks.
Quantitative analysis of the "phosphocreatine shuttle." I. A probability approach to the description of phosphocreatine production in the coupled creatine kinase-ATP/ADP translocase-oxidative phosphorylation in heart mitochondria.
Biochim. Biophys. Acta
1143:
291-300,
1993[Medline].
2.
Arrio-Dupont, M.,
J. J. Bechet,
and
A. d'Albis.
A model system of coupled activity of co-immobilized creatine kinase and myosin.
Eur. J. Biochem.
207:
951-955,
1992[Abstract].
3.
Bittl, J. A.,
J. A. Balschi,
and
J. S. Ingwall.
Contractile failure and high energy phosphate turnover during hypoxia: 31P NMR surface coil studies in living rat.
Circ. Res.
60:
871-878,
1987[Abstract].
4.
Bittl, J. A.,
and
J. S. Ingwall.
Reaction rates of creatine kinase and ATP synthesis in the isolated rat heart. A 31P NMR magnetization transfer study.
J. Biol. Chem.
260:
3512-3517,
1985[Abstract].
5.
Hoerter, J. A.,
D. Dormont,
M. Girault,
M. Guéron,
and
A. Syrota.
Insulin increases the rate of degradation of 2-deoxy-glucose-6-phosphate in the perfused rat heart: a 31P NMR study.
J. Mol. Cell. Cardiol.
23:
1101-1115,
1991[Medline].
6.
Hoerter, J. A.,
A. Kuznetsov,
and
R. Ventura-Clapier.
Functional development of the creatine kinase system in perinatal rabbit heart.
Circ. Res.
69:
665-676,
1991[Abstract].
7.
Hoerter, J. A.,
C. Lauer,
G. Vassort,
and
M. Guéron.
Sustained function of normoxic hearts depleted in ATP and phosphocreatine: a 31P-NMR study.
Am. J. Physiol.
255 (Cell Physiol. 24):
C192-C201,
1988[Abstract/Free Full Text].
8.
Kupriyanov, V. V.,
N. V. Lyulina,
A. Y. Steinschneider,
M. Y. Zueva,
and
V. A. Saks.
Creatine kinase catalysed ATP phosphocreatine exchange. Comparison of 31P saturation transfer technique and radioisotope tracer methods.
FEBS Lett.
208:
89-93,
1986[Medline].
9.
Kupriyanov, V.,
A. Y. Steinschneider,
E. K. Ruuge,
V. I. Kapelko,
M. Y. Zueva,
V. L. Lakomkin,
V. N. Smirnov,
and
V. A. Saks.
Regulation of energy flux through the creatine kinase reaction in vitro and in perfused rat heart.
Biochim. Biophys. Acta
805:
319-331,
1984[Medline].
10.
Kushmerick, M. J.,
R. A. Meyer,
and
T. R. Brown.
Regulation of oxygen consumption in fast-twitch and slow-twitch muscle.
Am. J. Physiol.
263 (Cell Physiol. 32):
C598-C606,
1992[Abstract/Free Full Text].
11.
Lawson, J. W.,
and
R. L. Veech.
Effects of pH and free Mg2+ on the Keq of the creatine kinase reaction and other phosphate hydrolyses and phosphate transfer reactions.
J. Biol. Chem.
254:
6528-6537,
1979[Abstract].
12.
Matthews, P. M.,
J. L. Bland,
D. G. Gadian,
and
G. K. Radda.
A 31P NMR saturation transfer study of the regulation of creatine kinase in the rat heart.
Biochim. Biophys. Acta
721:
312-320,
1982[Medline].
13.
McAuliffe, J. J.,
S. B. Perry,
E. E. Brooks,
and
J. S. Ingwall.
Kinetics of the creatine kinase reaction in neonatal rabbit heart: an empirical analysis of the rate equation.
Biochemistry
30:
2585-2593,
1991[Medline].
14.
McFarland, E. W.,
M. J. Kushmerick,
and
T. S. Moerland.
Activity of creatine kinase in a contracting mammalian muscle of uniform fiber type.
Biophys. J.
67:
1912-1924,
1994[Abstract].
15.
Meyer, R. A.,
H. L. Sweeney,
and
M. J. Kushmerick.
A simple analysis of the "phosphocreatine shuttle."
Am. J. Physiol.
246 (Cell Physiol. 15):
C365-C377,
1984[Abstract].
16.
Morris, G. A.,
and
R. Freeman.
Selective excitation in Fourier transform nuclear magnetic resonance.
J. Magn. Reson.
29:
433-462,
1978.
17.
Nascimben, L.,
J. Friedrich,
R. L. Liao,
P. Pauletto,
A. C. Pessina,
and
J. S. Ingwall.
Enalapril treatment increases cardiac performance and energy reserve via the creatine kinase reaction in myocardium of syrian myopathic hamsters with advanced heart failure.
Circulation
91:
1824-1833,
1995[Abstract/Free Full Text].
18.
Perry, S. B.,
J. McAuliffe,
J. A. Balschi,
P. R. Hickey,
and
J. S. Ingwall.
Velocity of the creatine kinase reaction in the neonatal rabbit heart: role of mitochondrial creatine kinase.
Biochem. J.
27:
2165-2172,
1988.
19.
Saks, V. A.,
Z. A. Khuchua,
E. V. Vasilyeva,
O. Y. Belikova,
and
A. V. Kuznetsov.
Metabolic compartmentation and substrate channeling in muscle cells. Role of coupled creatine kinase in vivo regulation of cellular respiration: a review.
Mol. Cell. Biochem.
133/134:
155-192,
1994.
20.
Saks, V. A.,
A. V. Kuznetsov,
V. V. Kupriyanov,
M. V. Miceli,
and
W. E. Jacobus.
Creatine kinase of rat heart mitochondria. The demonstration of functional coupling to oxidative phosphorylation in an inner membrane matrix preparation.
J. Biol. Chem.
260:
7757-7764,
1985[Abstract/Free Full Text].
21.
Shoubridge, E. A. J.,
J. L. Bland,
and
G. K. Radda.
Regulation of creatine kinase during steady state isometric twitch contraction in rat skeletal muscle.
Biochim. Biophys. Acta
805:
72-78,
1984[Medline].
22.
Shoubridge, E. A. J.,
F. M. H. Jeffry,
J. M. Keogh,
G. K. Radda,
and
A.-M. L. Seymour.
Creatine kinase kinetics, ATP turnover, and cardiac performance in hearts depleted of creatine with the substrate analogue
-guanidinopropionic acid.
Biochim. Biophys. Acta
847:
25-32,
1985[Medline].
23.
Soboll, S.,
and
R. Bünger.
Compartmentation of adenine nucleotides in the isolated working guinea pig heart.
Hoppe-Seyler's Z. Physiol. Chem.
362:
125-132,
1981[Medline].
24.
Stepanov, V.,
P. Mateo,
B. Gillet,
J.-C. Beloeil,
and
J. A. Hoerter.
Myocardial flux of creatine kinase in phosphocreatine depleted perfused rat heart.
J. Mol. Cell. Cardiol.
27:
A175,
1995.
25.
Suzuki, E.,
M. Munehiro,
S. Kuki,
M. C. Steward,
H. Takami,
Y. Seo,
M. Murakami,
and
H. Watari.
Adenosine triphosphate compartmentation in the rat heart: a 31P spin lattice relaxation study.
J. Biochem. (Tokyo)
107:
559-562,
1990[Abstract].
26.
Ventura-Clapier, R.,
V. Veksler,
and
J. A. Hoerter.
Myofibrillar creatine kinase and cardiac contraction.
Mol. Cell. Biochem.
133/134:
125-144,
1994.
27.
Wallimann, T.,
M. Wyss,
D. Brdiczka,
K. Nicolay,
and
H. M. Eppenberger.
Intracellular compartmentation, structure and function of creatine kinase isoenzymes in tissues with high fluctuating energy demand: the phosphocreatine circuit for cellular energy homeostasis.
Biochem. J.
281:
21-40,
1992[Medline].
28.
Williams, G. D.,
T. J. Mosher,
and
M. B. Smith.
Simultaneous determination of intracellular magnesium and pH from the three 31P NMR chemical shifts of ATP.
Anal. Biochem.
214:
458-467,
1993[Medline].
29.
Wyss, M.,
and
T. Wallimann.
Creatine metabolism and the consequences of creatine depletion in muscle.
Mol. Cell. Biochem.
133/134:
51-66,
1994.
30.
Zahler, R.,
J. A. Bittl,
and
J. S. Ingwall.
Analysis of compartmentation in skeletal and cardiac muscle using 31P NMR saturation transfer.
Biophys. J.
51:
883-893,
1987[Abstract].
AJP Cell Physiol 273(4):C1397-C1408
0363-6143/97 $5.00
Copyright © 1997 the American Physiological Society