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Am J Physiol Cell Physiol 273: C1397-C1408, 1997;
0363-6143/97 $5.00
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Vol. 273, Issue 4, C1397-C1408, October 1997

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
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

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
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

CREATINE KINASE (CK) catalyzes the reversible exchange of high-energy phosphate
PCr + MgADP + H<SUP>+</SUP> <AR><R><C><IT>K</IT><SUB>f</SUB></C></R><R><C>⇄</C></R><R><C><IT>K</IT><SUB>r</SUB></C></R></AR> Cr + MgATP
(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 right-arrow 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
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

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 gamma -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 beta -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 gamma -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 alpha - and beta -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 right-arrow 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
dM<SUB>PCr</SUB>/d<IT>t</IT> = M<SUB>o<SUB>PCr</SUB></SUB>/T1<SUB>PCr</SUB> − M<SUB>z<SUB>PCr</SUB></SUB>/&tgr;<SUB>PCr</SUB>
where MoPCr is the intensity of PCr magnetization in the absence of saturation and MzPCr is the intensity of PCr magnetization when gamma -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/tau PCr, which is 1/T1PCr + kf (T1PCr = intrinsic relaxation of PCr and tau 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
<IT>v</IT> = <IT>V</IT><SUB>max</SUB> · [([MgADP] · [PCr]/<IT>K</IT><SUB>i<SUB>ADP</SUB></SUB> · <IT>K</IT><SUB>m<SUB>PCr</SUB></SUB>)/<IT>D</IT>]
where D is a function of the association (Km), dissociation (Ki), and inhibitory (KI) constants of each metabolite for the various enzyme complexes
<IT>D</IT> = 1 + <FR><NU>[MgADP]</NU><DE><IT>K</IT><SUB>i<SUB>ADP</SUB></SUB></DE></FR> + <FR><NU>[PCr]</NU><DE><IT>K</IT><SUB>i<SUB>PCr</SUB></SUB></DE></FR> + <FR><NU>[Cr]</NU><DE><IT>K</IT><SUB>i<SUB>Cr</SUB></SUB></DE></FR> + <FR><NU>[MgATP]</NU><DE><IT>K</IT><SUB>i<SUB>ATP</SUB></SUB></DE></FR>
+ <FR><NU>[MgADP] · [PCr]</NU><DE><IT>K</IT><SUB>i<SUB>ADP</SUB></SUB> · <IT>K</IT><SUB>i<SUB>PCr</SUB></SUB></DE></FR> + <FR><NU>[MgATP] · [Cr]</NU><DE><IT>K</IT><SUB>m<SUB>Cr</SUB></SUB> · <IT>K</IT><SUB>i<SUB>ATP</SUB></SUB></DE></FR>
+ <FR><NU>[MgADP] · [Cr]</NU><DE><IT>K</IT><SUB>I<SUB>Cr</SUB></SUB> · <IT>K</IT><SUB>i<SUB>ADP</SUB></SUB></DE></FR> + <FR><NU>[MgATP] · [PCr]</NU><DE><IT>K</IT><SUB>I<SUB>PCr</SUB></SUB> · <IT>K</IT><SUB>i<SUB>ATP</SUB></SUB></DE></FR>
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 (QO2) 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). QO2 ("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 QO2 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
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

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).

                              
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  		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.

                              
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  		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.


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  		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. triangle , ATP; square , Pi; black-square, PCr; open circle , 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.


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  		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: gamma -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 gamma -ATP. Saturation at mirror frequency did not affect PCr (not shown). C: analysis of rate constant kf. black-square; 2DG-1; bullet , control. Magnetization of PCr (Mz) is referred to magnetization of nonsaturated spectra (Mo). Each curve is fitted by 1/tau  = 1/T1 + k, where tau  is relaxation time constant and T1 is intrinsic relaxation of PCr. For a representative control, saturation of gamma -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.


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  		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.


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  		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.


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  		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. open circle , Control (n = 12; ATP = 7.2 ± 0.2 mM, free ADP = 38 ± 3 µM); square , 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). open circle , Mean of control hearts shown in A; triangle , 2DG-2 [external pH (pHo) = 7.35, n = 18]; black-down-triangle , 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 QO2 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 QO2 experiment were 22.6 ± 1.3, 7.1 ± 0.4, and 42.8 ± 2.3 nmol/mg protein, respectively (n = 5). QO2 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 QO2 and RPP, which was described by y= 2.377x + 0.625, r2 = 0.920 (where y = QO2 in µmol O2 · min-1 · g wet wt-1 and x = RPP in 104 mmHg · beat · min-1). From this relation we calculated QO2 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.

                              
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  		Table 3.   Oxygen consumption and contractility during control and steady-state metabolite depletion in protocols 2DG-1 and 2DG-2

                              
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  		Table 4.   Creatine kinase and ATP synthesis fluxes

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).

                              
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  		Table 5.   Comparison of measured and predicted velocity of CK forward reaction

    DISCUSSION
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

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).


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  		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. open circle , Control; square , 2DG-1; triangle , 2DG-2; black-down-triangle , 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 gamma -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 gamma -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
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

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AJP Cell Physiol 273(4):C1397-C1408
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