AJP - Heart Email Content Delivery
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

Am J Physiol Heart Circ Physiol 276: H134-H140, 1999;
0363-6135/99 $5.00
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Harrison, G. J.
Right arrow Articles by van Beek, J. H. G. M.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Harrison, G. J.
Right arrow Articles by van Beek, J. H. G. M.
Vol. 276, Issue 1, H134-H140, January 1999

CK inhibition accelerates transcytosolic energy signaling during rapid workload steps in isolated rabbit hearts

Glenn J. Harrison, Michiel H. van Wijhe, Bas de Groot, Francina J. Dijk, and Johannes H. G. M. van Beek

Laboratory for Physiology, Institute for Cardiovascular Research, Free University, 1081BT Amsterdam, The Netherlands

    ABSTRACT
Top
Abstract
Introduction
Methods
Results
Discussion
References

The effect of graded creatine kinase (CK) inhibition on the response time of mitochondrial O2 consumption to dynamic workload jumps (tmito) was studied in isolated rabbit hearts. Tyrode-perfused hearts (n = 7/group) were exposed to 15 min of 0, 0.1, 0.2, or 0.4 mM iodoacetamide (IA) (CK activity = 100, 14, 6, and 3%, respectively). Pretreatment tmito was similar across groups at 6.5 ± 0.5 s (mean ± SE). The increase observed over time in control hearts (33 ± 8%) was progressively reversed to 16 ± 6, -20 ± 6 (P < 0.01 vs. control), and -46 ± 6 (P < 0.01 vs. control) % in the 0.1, 0.2 and 0.4 mM IA groups, respectively. The faster response times occurred without reductions in mitochondrial oxidative capacity (assessed in vitro) or myocardial O2 consumption of the whole heart during workload steps. Isovolumic contractile function assessed as rate-pressure product (RPP) and contractile reserve (increase in RPP during heart rate steps) were significantly reduced by IA. We conclude that CK in the myofibrils and/or cytosol does not speed up transfer of the energy-related signal to the mitochondria but rather acts as an energetic buffer, effectively slowing the stimulus between myofibrils/ion pumps and oxidative phosphorylation. This argues against the existence of an obligatory creatine phosphate energy shuttle, because CK is effectively bypassed.

energy transduction; adenosine 5'-diphosphate diffusion; oxygen consumption; contractile reserve

    INTRODUCTION
Top
Abstract
Introduction
Methods
Results
Discussion
References

THE CREATINE KINASE (CK)-catalyzed transfer of high-energy phosphate between ATP and phosphocreatine (PCr) is of central importance in muscle bioenergetics. The role played by CK in the regulation of myocyte energy transfer and signaling remains controversial (see for review Refs. 20, 33, 34). From the accepted temporal ATP/ADP buffering function of the CK/PCr system a progressively more complex model of energy transfer has evolved. The hypothesis of the "PCr shuttle" (3, 11, 33) providing energy transport or "spatial buffering" via PCr/creatine (Cr) and maintenance of optimal ATP-to-ADP ratios also incorporates compartmentation of CK isozymes in mitochondria (8, 18) and myofibrils (13, 32). Moreover, CK has been hypothesized to act as a metabolic control system in the regulation of cellular respiration (20). In opposition to this energy transfer theory remains the concept that the CK reaction operates in equilibrium as a "metabolic capacitor" between mitochondria and myofibrils allowing free diffusion of ATP and ADP, facilitated by PCr and Cr, to act as the energy transfer mechanism (15, 22, 23).

Recent studies in CK-blocked rat hearts (9, 10, 25) and CK knockout mice (29-31) show that normal workloads and high-energy phosphate levels can be maintained without active CK. However, increased performance in both isolated hearts and skeletal muscle is restricted. The loss of contractile reserve is accompanied by decreased free energy of ATP hydrolysis (Delta GATP), which is critical for maintaining the Ca2+-handling capacity of the sarcoplasmic reticulum (25). Two recent isolated heart studies suggest that the predominant effect of CK inactivity is increased ADP, leading to diastolic dysfunction and loss of myofibrillar compliance (12, 24). In human cardiac failure loss of CK activity, changes in CK isozyme distribution, and altered high-energy phosphate metabolite levels coexist (16, 19).

We previously developed a method to assess the delay time (tmito) in mitochondrial ATP synthesis after rapid increases in ATP hydrolysis induced by heart rate steps (28). 31P NMR experiments showed that PCr and Pi change markedly faster than oxidative phosphorylation (7), and we suggested that changes in phosphate metabolite concentrations take place in or near myofibrils/ion pumps before they reach the mitochondria with some delay (27). Thus tmito reflects the transcytosolic signaling speeds between myofibrils/ion pumps and mitochondria at submaximal levels of O2 consumption (7, 27, 28).

The aim of this study was to examine the effect of graded, irreversible CK inhibition [using iodoacetamide (IA) infusion] on the energy transduction speeds and contractile function of isolated rabbit hearts during paced workload steps. On the basis of the PCr shuttle operating as the optimal energy transfer system, it is predicted that blocking CK leads to longer response times (5, 18, 20). Indeed, prolonged response times found in the stunned rabbit heart were hypothesized to be caused by inhibition of CK (36). Our present results during graded inhibition of CK instead show an apparent quickening of ATP hydrolysis to synthesis signal coupling, suggesting that the cytosolic energy transfer function of CK is nonessential and can be bypassed by metabolite diffusion.

    METHODS
Top
Abstract
Introduction
Methods
Results
Discussion
References

Isolated heart preparation. All experiments were approved by the local animal ethics and experimentation authority (DEC-Free University). Male New Zealand White rabbits (n = 28, 2.86 ± 0.08 kg) were premedicated by an intramuscular injection of 1.5 mg/kg midazolam (Dormicum, Roche The Netherlands) before induction of anesthesia with 0.1 mg/kg fentanyl citrate and 3 mg/kg fluanisone (Hypnorm, Janssen Pharmaceutica), also given intramuscularly. Anesthesia was supplemented if necessary with intramuscular fentanyl (0.03 mg/kg). Animals were artificially ventilated before a median sternotomy, and hearts were cannulated in situ after intravenous infusion of 2,500 IU of heparin. Hearts were perfused in a constant-flow, nonrecirculating Langendorff mode with Tyrode buffer containing (in mM) 128.3 NaCl, 4.7 KCl, 1.36 CaCl2, 1.05 MgCl2, 20.2 NaHCO3, 0.42 NaH2PO4, and 11.0 glucose. Adenosine (10-5 mM) was incorporated in the Tyrode solution to obtain maximal vasodilation in our preparation. The buffer was maintained at 37°C and continuously gassed with 95% O2-5% CO2, yielding pH 7.4 and PO2 ~ 640 mmHg. Spontaneous heart rate was lowered from ~230 to <120 beats/min by crushing the atrioventricular node; hearts were subsequently paced slightly above this intrinsic rate (135 ± 3 beats/min) via bipolar electrodes placed on the right ventricular outflow tract. The right atrium was closed by ligating the caval veins, and venous effluent left the heart by the cannulated pulmonary artery. The left ventricle was drained of thebesian flow with a 22-gauge polyethylene catheter pierced through the apex. Isovolumic contractile function was assessed using a latex balloon secured into the left ventricle with a purse-string suture in the atrial appendage. The balloon was connected to a Gould P23 ID pressure transducer (Gould, Oxnard, CA), and the end-diastolic pressure was adjusted to 5 mmHg by increasing balloon volume. Perfusion flow rate was subsequently adjusted to generate an initial coronary perfusion pressure (CPP) of ~80 mmHg, measured immediately above the heart with a second pressure transducer, and this flow was not altered during experiments. Arterial and venous perfusate samples were continuously drawn through two cuvettes containing custom-made Clark-type O2 electrodes (time constant ~ 1.5 s), calibrated before and after the experiment. All pressure and PO2 data were simultaneously displayed on a chart recorder and digitally stored on a PC (Olivetti Pro). Myocardial O2 consumption (MVO2, in µmol · min-1 · g dry wt-1) was calculated as the product of coronary flow and the arterial-venous O2 concentration difference (O2 solubility = 1.34 µmol O2 · l Tyrode solution-1 · mmHg-1).

Calculation of tmito. Detailed description of the techniques including the O2 transport model and the equations, assumptions, and correction values is found elsewhere (27, 28). Briefly, the venous mean response time (tv) is integrated from the time course of the venous O2 tension (vPO2) during a step to and from a higher heart rate. Previous studies showed that there is an initial overshoot (tRPP) in rate-pressure product (RPP) before steady state is reached and also a small initial increase in vPO2 (tinit) because of transient increases in venous outflow, and hence tv is corrected for these parameters. To correct tv for delays in diffusion and transport of O2 between the mitochondria and the O2 electrode we subtract a mean transport time (ttransport), obtaining the true response time of O2 consumption at the level of the mitochondria during a dynamic step in workload: tmito = tv - ttransport. We therefore use tmito as an index of transcytosolic energy transfer and/or signaling speed during rapid increases in metabolic demand (measured as beat-to-beat RPP) in our isovolumic preparation.

The ttransport is calculated from the response times to a combination of two experimental interventions conducted in series after the heart rate steps (28). First, a small step in arterial concentration (ACS) is made by instantaneous exchange immediately above the heart of 10% of normal, oxygenated Tyrode with identical Tyrode gassed with 95%N2-5%CO2 (PO2 ~ 30 mmHg). Second, intravascular transit time is calculated using an indicator-dilution step with Evans blue bound to albumin (EBS), infused immediately above the heart and detected in a densitometer next to the venous O2 electrode. The ACS and EBS steps are conducted at both intrinsic pacing rate (135 beats/min) and the highest test heart rate (220 beats/min) to assess the effect of heart rate on O2 transport and to check the sensitivity of the preparation to small reductions in O2 supply. The complete calculation of tmito, therefore, involves several tests performed before and after any experimental intervention.

Experimental protocol. The outline of each experiment is schematically represented in Fig. 1. All hearts were equilibrated for 30 min after instrumentation followed by assessment of baseline hemodynamic function (time point 1). During the next 30 min (period 2) the first series of steps to calculate tmito was performed (as outlined in Calculation of tmito), incorporating randomized heart rate steps from basal heart rate (135 beats/min) to 160, 190, and 220 beats/min and back, plus the ACS and EBS steps at 135 and 220 beats/min. Hearts were then randomly assigned to one of four treatment groups (n = 7/group) to receive 0 (control), 0.1, 0.2, or 0.4 mM IA over the next 15 min. Concentrations given are final millimolar values in the perfusing solution after infusion of stock solutions (IA dissolved in H2O) into a side arm of the aortic cannula at ~1.2 ml/min with an infusion pump (Vickers Medical). Control hearts received only vehicle infusion. Time points 3-5 represent hemodynamic measurements made before and after IA infusion. Hearts were allowed 15 min for IA washout and reequilibration before the second series of tmito steps (period 6). At the end of experiments, a piece (~1 g) of left ventricle was rapidly excised into isopentane (precooled in liquid N2) and immediately freeze-dried at -70°C overnight before storage at -80°C for biochemical analysis (time point 7). The remaining heart was trimmed of extraneous tissue and used for blotted wet and dry (48 h at 80°C) weight measurements.


View larger version (8K):
[in this window]
[in a new window]
  		Fig. 1.   Schematic representing experimental protocol used in present study. Functional measurements [coronary perfusion pressure (CPP), systolic/diastolic ventricular pressure, and O2 consumption] were made at time points 1, 3, 4, 5, and 7. Calculations of response time of mitochondrial O2 consumption (tmito) were made during periods 2 and 6. Biochemical analysis of heart tissue followed experimental completion at time point 7. Iodoacetamide (IA) was infused between time points 3 and 4. HR, heart rate steps; ACS, arterial concentration step; EBS, intravascular indicator (Evans blue) step. For further description see METHODS.

Biochemical assays. Tissue samples (5-10 mg) were cut from the freeze-dried heart sections and homogenized at 4°C for 20 s in 0.1 M potassium phosphate buffer containing 1 mM EDTA and 1 mM beta -mercaptoethanol, pH = 7.4. Aliquots were removed for protein measurement by a modified Lowry method as described by Peterson (17) and is reported as milligrams of protein per milligram of dry heart tissue weight with bovine serum albumin as standard. Triton X-100 was added to the homogenate at a final concentration of 0.1% before measurement of total CK activity coupled to NADH production at 25°C and an absorbance wavelength of 340 nm (1). Adenylate kinase (AK) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) activities were also assayed at 25°C according to methods described by Bergmeyer (1). All enzyme activities are reported as international units (1 IU = µmol/min) per milligram of dry heart tissue weight.

Mitochondrial function. Mitochondria were isolated at the completion of experiments in a subset of control (n = 3) and 0.4 mM IA-treated (n = 3) hearts as described by Mela and Seitz (14). Briefly, 3-5 g of apical left ventricular tissue were excised into 4°C isolation solution containing (in mM) 225 mannitol, 75 sucrose, 1 EDTA, and 10 MOPS with 5 g/l albumin (pH = 7.25) and manually cut into small pieces. After the isolation solution was refreshed, 0.4 g/l protease was added and the tissue was homogenized and centrifuged at 850 g for 5 min. The supernatant was further centrifuged at 6,950 g for 10 min, and the remaining pellet was washed and spun twice more. The final pellet was resuspended without albumin, and the uncontaminated mitochondrial protein content was determined by the Peterson (17) method. Mitochondria (0.5 g/l mitochondrial protein) were added to a 2.0-ml O2 chamber containing (in mM) 225 mannitol, 70 sucrose, 3 KH2PO4, 7 K2HPO4, 1 EDTA, and 5 MOPS with 5 g/l albumin at pH = 7.1 and maintained at 28°C. Glutamate (5 mM) and malate (5 mM) were used as mitochondrial substrates, and rates of O2 consumption were measured using a Clark-type O2 electrode after respiration was stimulated with either 1 mM ADP or a combination of 20 mM Cr and ATP that was varied from 0.5-1.5 mM. CK activity in the mitochondrial fraction was assayed using the same method as for total tissue homogenate CK (1) and is expressed as international units per milligram of mitochondrial protein.

All chemical reagents used were of analytical grade and were obtained from Sigma Chemical (St. Louis, MO) or Boehringer Mannheim (Mannheim, Germany).

Statistical analysis. All data are presented as means ± SE. Comparisons among treatment groups were made using ANOVA with the dose of IA used as a grouping factor. The Newman-Keuls post hoc test was used to examine specific differences between group means. Two-way ANOVA (IA dose and heart rate) was used to analyze changes in tmito before or after treatment. Measurements made before and after treatment within groups were compared using the Student's paired t-test or ANOVA for repeated measures as necessary. A value of P < 0.05 was considered statistically significant for all comparisons.

    RESULTS
Top
Abstract
Introduction
Methods
Results
Discussion
References

IA and hemodynamic function. Baseline contractile function assessed after equilibration (time point 1, Fig. 1) generated values of 93 ± 7 mmHg for systolic left ventricular pressure (SLVP), 4 ± 1 mmHg for end-diastolic pressure (EDP), 85 ± 2 mmHg for CPP, 14 ± 1 ml · min-1 · g wet wt-1 for coronary flow, and 22 ± 1 µmol · min-1 · g dry wt-1 for MVO2. No statistically significant difference was observed between treatment groups in any of these parameters or in the wet (7.46 ± 0.39 g) or dry (1.25 ± 0.06 g) weight of hearts. The effect of IA infusion on SLVP, EDP, CPP, and MVO2 is shown in Fig. 2 as percent changes between time points 3 and 4. There was a small decrease in SLVP in all groups (significant in 0.2 and 0.4 mM IA) and an increase in CPP to a similar degree. The significant rise in EDP that occurred in IA-treated hearts, an increase of 53 ± 9%, was high in relative terms because of the low baseline value of EDP but modest in absolute terms at 2.9 ± 0.4 mmHg in the 0.4 mM IA group. This effect of CK inhibition on EDP is consistent with previous rat heart studies using IA infusion and has been ascribed to increased ADP levels (24, 25). MVO2, like SLVP, decreased slightly in all groups after treatment, with the decrease in the 0.4 mM IA-treated hearts (9.8 ± 1.4%) reaching significance versus controls (4.6 ± 1.1%, P < 0.05). During the 15-min wash-out period contractile parameters did not change further (assessed at time point 5); rather, values were stable at their new levels before the second round of tmito calculations (period 6).


View larger version (23K):
[in this window]
[in a new window]
  		Fig. 2.   Changes in contractile function from baseline in isolated rabbit hearts at 135 beats/min after 15-min infusion with 0 (vehicle infusion, control), 0.1 (IA 0.1), 0.2 (IA 0.2), and 0.4 (IA 0.4) mM IA. Data shown are means ± SE (n = 7/group) and represent percent changes after infusion (time point 4) compared with preinfusion values (time point 3). SLVP, systolic left ventricular pressure; EDP, end-diastolic pressure; MVO2, myocardial O2 consumption. * P < 0.05 vs. control hearts; ** P < 0.01 vs. control hearts.

Contractile reserve, defined as the increase in RPP during a step in heart rate from 135 to 220 beats/min, measured before and after treatment was reduced by IA. This increase in RPP, 5,749 ± 524 mmHg/min (or 48 ± 5%), was not different among groups before treatment. It did decline over time in control hearts to 90 ± 2.8% of pretreatment values. This decreased to 88 ± 2.5, 79 ± 2.6 (P < 0.05 vs. control), and 80 ± 2.5 (P < 0.05 vs. control) % in the 0.1 mM, 0.2 mM, and 0.4 mM IA-treated hearts, respectively. The results show that the ability of CK-blocked hearts to tolerate rapid workload increases is compromised.

Biochemical analysis of IA-treated hearts. The activity of CK, AK, and GAPDH enzymes and the protein content in tissue homogenates from the four heart groups appear in Table 1. Total CK activity in control hearts was reduced in a dose-dependent manner by IA to 14, 6, and 3% in 0.1, 0.2, and 0.4 mM IA-treated hearts, respectively. GAPDH, a sulfhydryl group containing glycolytic enzyme, was previously shown to be markedly inhibited by IA (9, 24). Its activity was indeed decreased by 23, 42, and 68%, respectively, in the three treated groups. The AK system may also transfer high-energy phosphoryls in the myocyte (5, 6), and its activity in CK-blocked hearts was therefore examined. AK activity was 55-fold lower than CK activity in control hearts, and this was unchanged by IA treatment at any dose.

                              
View this table:
[in this window]
[in a new window]
  		Table 1.   Biochemical analysis of control and iodoacetamide-treated hearts

To confirm that oxidative phosphorylation was not inhibited in IA-treated hearts, we isolated mitochondria from three control and three 0.4 mM IA-treated hearts. Mitochondrial CK inhibition was verified by direct assay of mitochondrial CK and the lack of Cr/ATP-stimulated respiration. The results of the mitochondrial experiments are shown in Table 1. ADP-stimulated respiration was unaffected by IA treatment with no differences in state 3 or state 4 respiration rate, ADP-to-O ratio (ADP:O), or respiratory control ratio between control and IA hearts. Respiratory stimulation by progressive concentrations of ATP in the presence of 20 mM Cr was, however, significantly lower in the IA-treated hearts (P < 0.05). CK activity in mitochondria isolated from 0.4 mM IA-treated hearts was 2.3% of that in controls, reflecting a similar level of inhibition to mitochondrial CK as to that observed in the total heart homogenate CK (3.2%, see Table 1).

Effect of CK inhibition on energy signaling speeds. As described in Calculation of tmito, our index of the response time of ATP synthesis to a rapid increase in ATP hydrolysis, tmito, is calculated from the time course of the PO2 curve (tv, ~12-14 s) during a heart rate step corrected for tRPP (~1 s), tinit (~0.2 s), and ttransport (~5-6 s). The tmito value for both the upward and the downward step in heart rate was not different in any group, before or after treatment, and therefore the average of both is given. Before IA treatment tmito ranged from 5.4 to 6.9 s, and there were no significant differences between the treatment groups or the three heart rates tested (P > 0.05, 2-way ANOVA). The posttreatment changes in tmito for the four heart groups are shown in Fig. 3. Paired comparisons within groups showed a significant increase over time of tmito in control hearts of 1.5-2 s (33 ± 8%, P < 0.05). This change was progressively reversed by increasing IA concentrations, with tmito increasing by 16 ± 6% in 0.1 mM IA-treated hearts (P < 0.05) but decreasing significantly by 20 ± 6 (P < 0.05) and 46 ± 6 (P < 0.01) % after treatment with 0.2 and 0.4 mM IA, respectively. The changes in these latter two groups were significant compared with changes in the control hearts (P < 0.05) and were independent of the heart rate step used (2-way ANOVA, P > 0.05). These results suggest that CK blockade with IA causes an apparent quickening in the metabolic coupling between oxidative phosphorylation and energy utilization.


View larger version (25K):
[in this window]
[in a new window]
  		Fig. 3.   Posttreatment changes in tmito as a function of 3 test heart rates after infusion of 0 (control), 0.1 (IA 0.1), 0.2 (IA 0.2), and 0.4 (IA 0.4) mM IA, corresponding to 100, 14, 6, and 3% creatine kinase activity, respectively. tmito is calculated from delay in venous PO2 during rapid steps in heart rate from 135 beats/min to those stated and is corrected for lag in O2 diffusion and vascular transport (see METHODS). Data are means ± SE (n = 7/group). ** P < 0.01 vs. control hearts.

Because transport and diffusion delay is subtracted during the tmito calculations, ttransport must be carefully assessed. The ttransport at 4.6 ± 0.4 s was not different between groups before treatment. It increased by 18 ± 5, 11 ± 5, 20 ± 5, and 34 ± 6% in control, 0.1 mM IA, 0.2 mM IA, and 0.4 mM IA groups, respectively (P < 0.05 vs. pretreatment). The increase in 0.4 mM IA-treated hearts was not significantly higher than in controls, and even the extra rise in ttransport (representing ~0.7 s) cannot explain the 3-s difference in tmito between these groups.

The pretreatment correction times for tRPP and tinit during heart rate steps between 135 and 220 beats/min were similar in all groups and were 0.6-1.2 s and 0.1-0.3 s, respectively. These values were not significantly changed over time in the control hearts or affected by IA treatment at any dosage.

Although the O2 consumption of the whole heart was marginally reduced by IA infusion (see Fig. 2), the increases in MVO2 for steps from 135 to 160, 190, and 220 beats/min (11, 20, and 29%, respectively) were not significantly different between or within groups before and after IA. When arterial O2 concentration was decreased by 8.3 ± 0.4%, MVO2 did not decrease significantly at 135 beats/min (0.7 ± 0.8%) but was 2.1 ± 0.5% lower at 220 beats/min (P < 0.05). These changes were similar in all groups and were not significantly altered after CK inhibition. From these results we chose to use the transport time derived from the arterial concentration step at 135 beats/min, at which MVO2 was stable during supply reduction, to correct the vPO2response time (tv) for transport delay.

    DISCUSSION
Top
Abstract
Introduction
Methods
Results
Discussion
References

The present study was designed to test the acute effects of graded CK inhibition on the response times of myocyte energy signaling during rapid submaximal workload shifts in the intact rabbit heart. The results demonstrate accelerated signal speeds to mitochondria for increased oxidative phosphorylation with progressively higher blockade of CK activity. This finding was obtained without changes in mitochondrial oxidative capacity or substrate limitation. However, the observed reduction in contractile reserve at high workloads in previous (12, 25) and present studies suggests that although energy transfer by CK from oxidative phosphorylation can be effectively bypassed (given the quick mitochondrial response), ADP and ATP buffering near ion pumps and myofibrillar ATPases by CK is essential during enhanced myocardial performance.

The specificity of IA to block CK activity must be critically examined. Although IA was previously used in isolated rat heart studies as a specific CK inhibitor (9, 10, 12, 24), it is known to cause alkylation of other sulfhydryl enzymes, as shown by GAPDH inhibition in the present study and by others (9, 24). However, glycolytic flux (24), AK (Ref. 10 and present study), and myofibrillar ATPase (10) activities remain unaffected by IA. Moreover, Tian and Ingwall (25) observed some heterogeneity in the blockade pattern of myocardial CK isozymes after infusion of IA. In the present study we also observed a small but nonsignificant difference in the inhibition rates of mitochondrial and total tissue CK activities. Therefore, without the current availability of isozyme-specific CK inhibitors and as yet no way to measure our dynamic oxidative response times in the CK "knockout" mice (29-31), we conclude that IA infused slowly at set concentrations is an effective means of studying the acute effects of CK inactivity in isolated hearts. Importantly, acute inhibition of CK with IA has the advantage over knockout of a gene in that no compensatory mechanisms involving altered gene expression play a role (29).

Effects of CK inhibition on contractile function. IA did have significant effects on contractile function during infusion of the higher concentrations, similar to those seen in rat hearts (25). Prominent was the 53% rise in EDP, which presumably reflects elevated free ADP concentration. Indeed, Tian et al. (24) recently used IA to cause a threefold increase in EDP that was matched by ADP levels without changing ATP concentration, Pi concentration, or intracellular pH. The ability of hearts to still maintain low-to-moderate cardiac workloads after severe reductions in CK reaction velocity has now been demonstrated by a number of groups using chemical inhibition of CK (9, 10, 24) and animals fed with Cr pool analogs (22).

Loss of contractile reserve in response to inotropic (10, 25) or pressure-volume (9, 12) work stimulation is also a finding common to hearts treated with IA. Reductions of 35-72% in functional reserve in rat hearts with <10% of CK activity have been observed during high-calcium perfusion (10, 24). The modest (but significant) 10% decreases in contractile reserve we observed in heart groups with 3 and 6% remaining CK activity perhaps reflects the higher energy cost of pressure (as used in Refs. 9, 10, 12, 25) versus rate work as used in the present study (2).

CK activity and response times. ADP-stimulated O2 consumption in isolated mitochondria indexed as ADP:O, respiratory control ratios, and maximal state 3 VO2 were unchanged in IA-treated hearts (Ref. 10 and present study). Moreover, we recently reported that up to 50% reductions in mitochondrial aerobic capacity do not change the response times (tmito) in normoxic rabbit hearts with full CK activity (4, 36). Furthermore, in the present experiments no differences in the response of hearts to small reductions in arterial O2 concentration were observed after inhibition of CK, suggesting unaltered sensitivity to O2 supply.

The progressive shortening of tmito paralleled the level of inhibition of CK activity such that in hearts with only 3% CK, energy signaling in response to a heart rate step was ~3 s faster than in controls. This graded effect on tmito is similar to the effect on contractile reserve seen during progressive CK inhibition in the present study and in rat hearts (25). Contractile reserve and tmito were maintained until CK velocity fell below 15% of control, and then they fell sharply, highlighting the functional reserve of the CK system. During myocardial stunning secondary to ischemia and hypoxia tmito was increased by ~40% (36). The hypothesis that this was caused by inhibition of CK by reactive oxygen species is dispelled by the findings in the present study. With the quicker response times in the current experiments being independent of any changes in mitochondrial oxidative capacity, we suggest that cytosolic ATP synthesis to hydrolysis coupling is accelerated in the absence of CK buffering. This finding contradicts the essential transport role of the CK/PCr shuttle (3, 11, 33) and the concept of PCr/Cr rather than ATP/ADP metabolite signal transduction (18, 35), instead supporting the concept of cytosolic CK being a high-capacity temporal buffer (15, 23) that normally delays the response of mitochondria to rapid energy demand increases. Such a system would allow temporary cytosolic uncoupling of energy production from its utilization, especially during large cardiac workload transitions (20).

The increase in response times over time in control hearts (33 ± 8%) is similar to that observed in the previous study of Zuurbier and van Beek (36) using an analogous heart preparation and time protocol. Although a clear explanation cannot be given, we speculate that such factors as interstitial edema, intracellular swelling, and the potential for oxygen free radical buildup in the high-PO2 crystalloid-perfused hearts may contribute to increased intracellular diffusion distances and inhibition of cytosolic enzymes, respectively. In addition, the exhaustion of endogenous substrates and the consequent transition and reliance on exogenous supplies over time may also play a role, because we observed variations in response times when exogenous substrates were varied among glucose, pyruvate, and lactate (27). When pyruvate (2 mM) was added in addition to glucose (unpublished pilot experiments, n = 6), the increase in tmito over time in control hearts was reduced. Because of this gradual change in response time, the effect of IA infusion was carefully compared with a time-control group.

Alternative mechanisms of faster energy signaling. An explanation for the decrease of tmito in CK-inhibited hearts is that higher ADP levels alter diffusion. Given that PCr is decreased quickly during upward steps in heart rate in our preparation (7), CK action may delay the local increase in ADP. However, alternative explanations should be considered.

A possible reason for the faster tmito could be that as CK is blocked, energy transfer via the AK shuttle, which is normally low, is upregulated (6, 21). In rat diaphragm in which 1-fluoro-2,4-dinitrobenzene was used to block CK activity by 98%, the contribution of AK flux to the total phosphoryl transfer (~7%) increased reciprocally (as CK flux fell) to levels approaching the preinhibition CK flux (~88%) (5). Our results and those of others (10) have shown no increase in in vitro AK activity in hearts with chemically inhibited CK activity. Given that the total phosphate group transfer was still decreased in these rat diaphragm experiments, indicating incomplete compensation by the AK shuttle, it is unlikely that enhanced AK flux would explain the dramatically quickened energetic signaling speeds observed in our studies in hearts.

Further contributions to the observed faster response times to rapid ATP hydrolysis may come from glycolytic buffering. We recently suggested that glycolysis may also play an important energy transfer function in the myocyte and that glycolytic buffering, especially in and near myofibrils, may also retard the oxidative signal to the mitochondria (26, 27); the work of Dzeja et al. (5) points in a similar direction. We observed a 68% reduction in activity of the key glycolytic enzyme GAPDH at the highest level of CK inhibition (97%), consistent with previous IA infusion experiments in rat hearts (9, 24). Recent work by the group of Ingwall (10, 24) in IA-treated rat hearts showed no 31P NMR-detectable increases in glycolytic intermediates, and measured glycolytic rate was unchanged in hearts with only 20% GAPDH activity remaining, which reflects the fact that GAPDH activity normally exceeds glycolytic rate >50-fold. Furthermore, in a series of pilot experiments (n = 6, results not shown), we added pyruvate (2 mM) to the Tyrode buffer before inhibition of CK with 0.4 mM IA. The tmito measurements again decreased (independent of the test heart rate) by ~44% from pretreatment levels (3.4 ± 0.5 to 1.9 ± 0.4 s, P < 0.05), thereby removing the possibility that restricted glycolysis confounded our original results.

In conclusion, we found that graded inhibition of CK by IA infusion in isolated rabbit hearts caused an increase in the cytosolic signaling speed for ATP synthesis, indicated by a quicker response of mitochondrial O2 consumption during rapid pacing-induced workload jumps. The acceleration in the response time of oxidative phosphorylation was dose dependent and not related to changes in mitochondrial oxidative capacity or changes in O2 diffusion or transport delays to the myocyte. These results go against the theory of cytosolic CK operating as an essential energy transport shuttle. CK may instead act as a high-capacity temporal buffer localized in the myofibrils and the cytosol effectively slowing the signal to the mitochondria. The loss of this CK-mediated buffering of ADP and Pi near the myofibrils and ion pumps is reflected by decreased contractile reserve of hearts during rapid increases in cardiac performance.

    ACKNOWLEDGEMENTS

The authors thank Wim Gerrissen for animal care and preparation and Dr. Coert Zuurbier for stimulating discussion and criticism of the manuscript.

    FOOTNOTES

This study was supported by Netherlands Hearts Foundation Grant 94.099 and Established Investigator Grant D94.016 to J. H. G. M. van Beek.

This work was presented in part at the XXXIII International Congress of Physiological Sciences, St. Petersburg, Russia, June 30-July 5, 1997.

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

Address for reprint requests: J. H. G. M. van Beek, Laboratory for Physiology ICaR-VU, Van der Boechorststraat 7, Free University, 1081 BT Amsterdam, The Netherlands.

Received 18 May 1998; accepted in final form 24 September 1998.

    REFERENCES
Top
Abstract
Introduction
Methods
Results
Discussion
References

1.   Bergmeyer, H. U. (Editor). Methods of Enzymatic Analysis (3rd ed.). Weinheim, Germany: Verlag Chemie, 1983, vol. IIBergmeyer, H. U. (Editor). Methods of Enzymatic Analysis (3rd ed.). Weinheim, Germany: Verlag Chemie, 1983, vol. II.

2.   Berne, R. M., and M. N. Levy (Editors). Cardiovascular Physiology. St. Louis, MO: Mosby Year Book, 1992, p. 228Berne, R. M., and M. N. Levy (Editors). Cardiovascular Physiology. St. Louis, MO: Mosby Year Book, 1992, p. 228.

3.   Bessman, S. P., and P. G. Geiger. Transport of energy in muscle: the phosphorylcreatine shuttle. Science 211: 448-452, 1981[Abstract/Free Full Text].

4.  De Groot, B., F. J. Dijk, and J. H. G. M. van Beek. Response of cardiac respiration to steps in heart rate is not slowed after reduction of mitochondrial capacity in normal and pressure overloaded hearts (Abstract). Proceedings of the XXXIII International Congress of Physiological Sciences, St. Petersburg, Russia, 1997, p. 063.06.

5.   Dzeja, P. P., R. J. Zeleznikar, and N. D. Goldberg. Suppression of creatine kinase-catalyzed phosphotransfer results in increased phosphoryl transfer by adenylate kinase in intact skeletal muscle. J. Biol. Chem. 271: 12847-12851, 1996[Abstract/Free Full Text].

6.   Dzeja, P. P., R. J. Zeleznikar, and N. D. Goldberg. Adenylate kinase: kinetic behaviour in intact cells indicates it is integral to multiple cellular processes. Mol. Cell. Biochem. 184: 169-182, 1998[Medline].

7.   Eijgelshoven, M. H. J, J. H. G. M. van Beek, I. Mottet, M. G. J. Nederhoff, C. J. A. van Echteld, and N. Westerhof. Cardiac high-energy phosphates adapt faster than oxygen consumption to changes in heart rate. Circ. Res. 75: 751-759, 1994[Abstract/Free Full Text].

8.   Erickson-Viitanen, S., P. Vitanen, P. J. Geiger, W. C. T. Yang, and S. P. Bessman. Compartmentation of mitochondrial creatine phosphokinase. I. Direct demonstration of compartmentation with the use of labelled precursors. J. Biol. Chem. 257: 14395-14404, 1982[Abstract/Free Full Text].

9.   Fossel, E. T., and H. Hoefeler. Complete inhibition of creatine kinase in isolated perfused rat hearts. Am. J. Physiol. 252 (Endocrinol. Metab. 15): E124-E130, 1987[Abstract/Free Full Text].

10.   Hamman, B. L., J. A. Bittl, W. E. Jacobus, P. D. Allen, R. S. Spencer, R. Tian, and J. S. Ingwall. Inhibition of the creatine kinase reaction decreases the contractile reserve of isolated rabbit hearts. Am. J. Physiol. 269 (Heart Circ. Physiol. 38): H1030-H1036, 1995[Abstract/Free Full Text].

11.   Kammermeier, H. Why do cells need phosphocreatine and a phosphocreatine shuttle? J. Mol. Cell. Cardiol. 19: 115-118, 1987[Medline].

12.   Kapelko, V. I., V. L. Lakomkin, O. V. Korchazhkina, and O. I. Pisarenko. Cardiac pump function of the isolated rat heart at two modes of energy deprivation and the effect of adrenergic stimulation. Mol. Cell. Biochem. 163/164: 131-136, 1996.

13.   Krause, S. M., and W. E. Jacobus. Specific enhancement of the cardiac myofibrillar ATPase by bound creatine kinase. J. Biol. Chem. 267: 2480-2486, 1992[Abstract/Free Full Text].

14.   Mela, L., and S. Seitz. Isolation of mitochondria with emphasis on heart mitochondria from small amounts of tissue. Methods Enzymol. 55: 39-46, 1979[Medline].

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/Free Full Text].

16.   Nascimben, L., J. S. Ingwall, P. Pauletto, J. Friedrich, J. K. Gwathmey, V. Saks, A. C. Pessina, and P. D. Allen. Creatine kinase system in failing and nonfailing human myocardium. Circulation 94: 1894-1901, 1996[Abstract/Free Full Text].

17.   Peterson, G. L. Determination of total protein. Methods Enzymol. 91: 95-105, 1983[Medline].

18.   Saks, V. A., Z. A. Khuchua, E. V. Vasilyeva, O. Yu Belikova, and A. V. Kuznetsov. Metabolic compartmentation and substrate channelling in muscle cells. Role of coupled creatine kinases in in vivo regulation of cellular respiration---a synthesis. Mol. Cell. Biochem. 133/134: 155-192, 1994.

19.   Saks, V., T. Tiivel, L. Kay, V. Novel-Chate, Z. Daneshrad, A. Rossi, E. Fontaine, C. Keriel, X. Leverve, R. Ventura-Clapier, K. Anflous, J.-L. Samuel, and L. Rappaport. On the regulation of cellular energetics in health and disease. Mol. Cell. Biochem. 160/161: 195-208, 1996.

20.   Saks, V. A., R. Ventura-Clapier, and M. K. Aliev. Metabolic control and metabolic capacity: two aspects of creatine kinase functioning in the cells. Biochim. Biophys. Acta 1274: 81-88, 1996[Medline].

21.   Savabi, F. Interaction of creatine kinase and adenylate kinase systems in muscle cells. Mol. Cell. Biochem. 133/134: 145-152, 1994.

22.   Shoubridge, E. A., 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 beta -guanidinopropionic acid. Biochim. Biophys. Acta 847: 25-32, 1985[Medline].

23.   Sweeney, H. L. The importance of the creatine kinase reaction: the concept of metabolic capacitance. Med. Sci. Sports Exerc. 26: 30-36, 1994[Medline].

24.   Tian, R., M. E. Christe, M. Spindler, J. C. A. Hopkins, J. M. Halow, S. A. Camacho, and J. S. Ingwall. Role of MgADP in the development of diastolic dysfunction in the intact beating rat heart. J. Clin. Invest. 99: 745-751, 1997[Medline].

25.   Tian, R., and J. S. Ingwall. Energetic basis for reduced contractile reserve in isolated rat hearts. Am. J. Physiol. 270 (Heart Circ. Physiol. 39): H1207-H1216, 1996[Abstract/Free Full Text].

26.   Van Beek, J. H. G. M., M. H. J. Eijgelshoven, X. Tian, and C. J. van Echteld. Effect of glycolytic buffering on myocardial transcytosolic energy signaling (Abstract). Circulation 96: I-573, 1997.

27.   Van Beek, J. H. G. M., X. Tian, C. J. Zuurbier, B. de Groot, C. J. A. van Echteld, M. H. J. Eijgelshoven, and J. B. Hak. The dynamic regulation of myocardial oxidative phosphorylation: analysis of the response time of oxygen consumption. Mol. Cell. Biochem. 184: 321-344, 1998[Medline].

28.   Van Beek, J. H. G. M., and N. Westerhof. Response time of cardiac mitochondrial oxygen consumption to heart rate steps. Am. J. Physiol. 260 (Heart Circ. Physiol. 29): H613-H625, 1991[Abstract/Free Full Text].

29.   Van Deursen, J., A. Heerschap, F. Oerlemans, W. Ruitenbeek, P. Jap, H. ter Laak, and B. Wieringa. Skeletal muscles of mice deficient in muscle creatine kinase lack burst activity. Cell 74: 621-631, 1993[Medline].

30.   Van Dorsten, F. A., T. Reese, J. F. Gellerich, C. J. A. van Echteld, M. G. J. Nederhoff, H. J. Muller, G. van Vliet, and K. Nicolay. Fluxes through cytosolic and mitochondrial creatine kinase, measured by P-31 NMR. Mol. Cell. Biochem. 174: 33-42, 1997[Medline].

31.   Veksler, V. A., A. V. Kuznetsov, K. Anflous, P. Mateo, J. van Deursen, B. Wieringa, and R. Ventura-Clapier. Muscle creatine kinase-deficient mice. II. Cardiac and skeletal muscles exhibit tissue-specific adaptation of the mitochondrial function. J. Biol. Chem. 270: 19921-19929, 1995[Abstract/Free Full Text].

32.   Ventura-Clapier, R., V. Veksler, and J. A. Hoerter. Myofibrillar creatine kinase and cardiac contraction. Mol. Cell. Biochem. 133/134: 125-144, 1994.

33.   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 and fluctuating energy demands: the "phosphocreatine circuit" for cellular energy homeostasis. Biochem. J. 281: 21-40, 1992.

34.   Wyss, M., J. Smeitink, R. A. Wevers, and T. Wallimann. Mitochondrial creatine kinase: a key enzyme of aerobic energy metabolism. Biochim. Biophys. Acta 1102: 119-166, 1992[Medline].

35.   Zeleznikar, R. J., and N. D. Goldberg. Kinetics and compartmentation of energy metabolism in intact skeletal muscle determined from 18O labeling of metabolite phosphoryls. J. Biol. Chem. 266: 15110-15119, 1991[Abstract/Free Full Text].

36.   Zuurbier, C. J., and J. H. G. M. van Beek. Mitochondrial response to heart rate steps in isolated rabbit heart is slowed after myocardial stunning. Circ. Res. 81: 69-75, 1997[Abstract/Free Full Text].

Am J Physiol Heart Circ Physiol 276(1):H134-H140
0002-9513/99 $5.00 Copyright © 1999 the American Physiological Society


This article has been cited by other articles:


Home page
 Am. J. Physiol. Heart Circ. Physiol.Home page
E. Takahashi
Anoxic cell core can promote necrotic cell death in cardiomyocytes at physiological extracellular PO2
Am J Physiol Heart Circ Physiol, June 1, 2008; 294(6): H2507 - H2515.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Physiol. Cell Physiol.Home page
J. H. G. M. van Beek
Adenine nucleotide-creatine-phosphate module in myocardial metabolic system explains fast phase of dynamic regulation of oxidative phosphorylation
Am J Physiol Cell Physiol, September 1, 2007; 293(3): C815 - C829.
[Abstract] [Full Text] [PDF]

Home page
 J. Appl. Physiol.Home page
C. A. Kindig, R. A. Howlett, C. M. Stary, B. Walsh, and M. C. Hogan
Effects of acute creatine kinase inhibition on metabolism and tension development in isolated single myocytes
J Appl Physiol, February 1, 2005; 98(2): 541 - 549.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Physiol. Heart Circ. Physiol.Home page
L. A. Gustafson and J. H. G. M. Van Beek
Activation time of myocardial oxidative phosphorylation in creatine kinase and adenylate kinase knockout mice
Am J Physiol Heart Circ Physiol, June 1, 2002; 282(6): H2259 - H2264.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Physiol. Heart Circ. Physiol.Home page
L. A. Gustafson and J. H. G. M. Van Beek
Measurement of the activation time of oxidative phosphorylation in isolated mouse hearts
Am J Physiol Heart Circ Physiol, December 1, 2000; 279(6): H3118 - H3123.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Physiol. Cell Physiol.Home page
M. Vendelin, O. Kongas, and V. Saks
Regulation of mitochondrial respiration in heart cells analyzed by reaction-diffusion model of energy transfer
Am J Physiol Cell Physiol, April 1, 2000; 278(4): C747 - C764.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Physiol. Heart Circ. Physiol.Home page
J. H. G. M. van Beek, M. H. van Wijhe, M. H. J. Eijgelshoven, and J. B. Hak
Dynamic adaptation of cardiac oxidative phosphorylation is not mediated by simple feedback control
Am J Physiol Heart Circ Physiol, October 1, 1999; 277(4): H1375 - H1384.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Physiol. Heart Circ. Physiol.Home page
B. de Groot, C. J. Zuurbier, and J. H. G. M. van Beek
Dynamics of tissue oxygenation in isolated rabbit heart as measured with near-infrared spectroscopy
Am J Physiol Heart Circ Physiol, May 1, 1999; 276(5): H1616 - H1624.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Physiol. Heart Circ. Physiol.Home page
L. A. Gustafson and J. H. G. M. Van Beek
Activation time of myocardial oxidative phosphorylation in creatine kinase and adenylate kinase knockout mice
Am J Physiol Heart Circ Physiol, June 1, 2002; 282(6): H2259 - H2264.
[Abstract] [Full Text] [PDF]

Home page
 Circ. Res.Home page
A. Kaasik, V. Veksler, E. Boehm, M. Novotova, A. Minajeva, and R. Ventura-Clapier
Energetic Crosstalk Between Organelles: Architectural Integration of Energy Production and Utilization
Circ. Res., July 20, 2001; 89(2): 153 - 159.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Harrison, G. J.
Right arrow Articles by van Beek, J. H. G. M.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Harrison, G. J.
Right arrow Articles by van Beek, J. H. G. M.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Visit Other APS Journals Online