The creatine kinase reaction: a simple reaction with functional complexity

The classical role of PCr is seen as a reservoir of high-energy phosphates defending cellular ATP levels under anaerobic conditions, high rates of energy transfer or rapid fluctuations in energy requirement. Although the high concentration of PCr in glycolytic fast-twitch fibers supports the role of PCr as a buffer of ATP, the primary importance of the creatine kinase (CK) reaction may in fact be to counteract large increases in ADP, which could otherwise inhibit cellular ATPase-mediated systems. A primary role for CK in the maintenance of ADP homeostasis may explain why, in many conditions, there is an inverse relationship between PCr and muscle contractility but not between ATP and muscle contractility. The high rate of ATP hydrolysis during muscle contraction combined with restricted diffusion of ADP suggests that ADP concentration increases transiently during the contraction phase (ADP spikes) and that these are synchronized with the contraction. The presence of CK, structurally bound in close vicinity to the sites of ATP utilization, will reduce the amplitude and duration of the ADP spikes through PCr-mediated phosphotransfer. When PCr is reduced, the efficiency of CK as an ATP buffer will be reduced and the changes in ADP will become more prominent. The presence of ADP spikes is supported by the finding that other processes known to be activated by ADP (i.e. AMP deamination and glycolysis) are stimulated during exercise but not during anoxia, despite the same low global energy state. Breakdown of PCr is driven by increases in ADP above that depicted by the CK equilibrium and the current method to calculate ADPfree from the CK reaction in a contracting muscle is therefore questionable.     

Role of phosphocreatine in energy transport in skeletal muscle of bullfrog studied by 31P-NMR

To evaluate the energy-shuttle hypothesis of the phosphocreatine/creatine kinase system, diffusion rates for ATP, phosphocreatine and flux through the creatine kinase reaction were determined by 31P-NMR in resting bullfrog biceps muscle. The diffusion coefficient of phosphocreatine measured by 31P-pulsed gradient NMR was 1.4-times larger than ATP in the muscle, indicating the advantage of phosphocreatine molecules for the intracellular energy transport. The flux of the creatine kinase reaction measured by 31P-saturation transfer NMR was 3.6 mmol/kg wet wt. per s in the resting muscle. The flux is equal to the turnover rate of ATP, ADP, phosphocreatine and creatine molecules, therefore, the life-times of these substrates and the average distance traversed after the life-times by the diffusing molecules were calculated using the diffusion coefficients obtained by 31P-NMR. The mean square length of one-dimensional diffusion was 22 microns in ATP molecules and the minimum diffusion length was 1.8 microns in ADP molecules. The latter was calculated using free ADP concentration, 30 mumol/kg wet wt., obtained from the equilibrium constant of the creatine kinase reaction and the diffusion coefficient assumed to be the same of ATP in muscle. Similar diffusion lengths of ADP were calculated using the reported values for the flux of the creatine kinase reaction in heart and smooth-muscle. The diffusion lengths of all substrates involved in the creatine kinase reaction were larger than the radii of myofibrils. Therefore, in the muscles with an alternating arrangement of mitochondria and myofibrils, such as heart and certain skeletal muscles, ATP and ADP molecules can move freely between myofibrils and mitochondria without the aid of the creatine kinase reaction; thus, we conclude that the energy-shuttle hypothesis is not obligatory for energy transport between the mitochondria and the myofibrils.     

Adenylate kinase: Kinetic behavior in intact cells indicates it is integral to multiple cellular processes

Monitoring the kinetic behavior of adenylate kinase (AK) and creatine kinase (CK) in intact cells by ¹⁸O-phosphoryl oxygen exchange analysis has provided new perspectives from which to more fully define the involvement of these phosphotransferases in cellular bioenergetics. A primary function attributable to both AK and CK is their apparent capability to couple ATP utilization with its generation by glycolytic and/or oxidative processes depending on cell metabolic status. This is evidenced by the observation that the sum of the net AK- plus CK-catalyzed phosphoryl transfer is equivalent to about 95% of the total ATP metabolic flux in non-contracting rat diaphragm; under basal conditions almost every newly generated ATP molecule appears to be processed by one or the other of these phosphotransferases prior to its utilization. Although CK accounts for the transfer of a majority of the ATP molecules generated/consumed in the basal state there is a progressive, apparently compensatory, shift in phosphotransfer catalysis from the CK to the AK system with increasing muscle contraction or graded chemical inhibition of CK activity. AK and CK appear therefore to provide similar and interrelated functions. Evidence that high energy phosphoryl transfer in some cell types or metabolic states can also be provided by specific nucleoside mono- and diphosphate kinases and by the phosphotransfer capability inherent to the glycolytic system has been obtained. Measurements by ¹⁸O-exchange analyses of net AK- and CK-catalyzed phosphoryl transfer in conjunction with 31P NMR analyses of total unidirectional phosphoryl flux show that each new energy-bearing molecule CK or AK generates subsequently undergoes about 50 or more unidirectional CK-or AK-catalyzed phosphotransfers en route to an ATP consumption site in intact muscle. This evidence of multiple enzyme catalyzed exchanges coincides with the mechanism of vectorial ligand conduction suggested for accomplishing intracellular high energy phosphoryl transfer by the AK and CK systems. AK-catalyzed phosphotransfer also appears to be integral to the transduction of metabolic signals influencing the operation of ion channels regulated by adenine nucleotides such as ATP-inhibitable K⁺ channels in insulin secreting cells; transition from the ATP to ADP liganded states closely coincides with the rate AK-catalyzes phosphotransfer transforming ATP (+AMP) to (2) ADP.     

Altered mitochondrial apparent affinity for ADP and impaired function of mitochondrial creatine kinase in gluteus medius of patients with hip osteoarthritis

The cellular energy metabolism in human musculus gluteus medius (MGM) under normal conditions and hip osteoarthritis (OA) was explored. The functions of oxidative phosphorylation and energy transport systems were analyzed in permeabilized (skinned) muscle fibers by oxygraphy, in relation to myosin heavy chain (MHC) isoform distribution profile analyzed by SDS-PAGE, and to creatine kinase (CK) and adenylate kinase (AK) activities measured spectrophotometrically in the intact muscle. The results revealed high apparent Km for ADP in regulation of respiration that decreased after addition of creatine in MGM of traumatic patients (controls). OA was associated with increased sensitivity of mitochondrial respiration to ADP, decreased total activities of AK and CK with major reduction in mi-CK fraction, and attenuated effect of creatine on apparent Km for ADP compared with control group. It also included a complete loss of type II fibers in a subgroup of patients with the severest disease grade. It is concluded that energy metabolism in MGM cells is organized into functional complexes of mitochondria and ATPases. It is suggested that because of degenerative remodeling occurring during development of OA, these complexes become structurally and functionally impaired, which results in increased access of exogenous ADP to mitochondria and dysfunction of CK-phosphotransfer system.     

Analyzing the functional properties of the creatine kinase system with multiscale 'sloppy' modeling

In this study the function of the two isoforms of creatine kinase (CK; EC 2.7.3.2) in myocardium is investigated. The 'phosphocreatine shuttle' hypothesis states that mitochondrial and cytosolic CK plays a pivotal role in the transport of high-energy phosphate (HEP) groups from mitochondria to myofibrils in contracting muscle. Temporal buffering of changes in ATP and ADP is another potential role of CK. With a mathematical model, we analyzed energy transport and damping of high peaks of ATP hydrolysis during the cardiac cycle. The analysis was based on multiscale data measured at the level of isolated enzymes, isolated mitochondria and on dynamic response times of oxidative phosphorylation measured at the whole heart level. Using 'sloppy modeling' ensemble simulations, we derived confidence intervals for predictions of the contributions by phosphocreatine (PCr) and ATP to the transfer of HEP from mitochondria to sites of ATP hydrolysis. Our calculations indicate that only 15±8% (mean±SD) of transcytosolic energy transport is carried by PCr, contradicting the PCr shuttle hypothesis. We also predicted temporal buffering capabilities of the CK isoforms protecting against high peaks of ATP hydrolysis (3750 μM*s(-1)) in myofibrils. CK inhibition by 98% in silico leads to an increase in amplitude of mitochondrial ATP synthesis pulsation from 215±23 to 566±31 μM*s(-1), while amplitudes of oscillations in cytosolic ADP concentration double from 77±11 to 146±1 μM. Our findings indicate that CK acts as a large bandwidth high-capacity temporal energy buffer maintaining cellular ATP homeostasis and reducing oscillations in mitochondrial metabolism. However, the contribution of CK to the transport of high-energy phosphate groups appears limited. Mitochondrial CK activity lowers cytosolic inorganic phosphate levels while cytosolic CK has the opposite effect.     

Heterogeneous cellular expression of creatine kinase isoenzyme during normal rat heart development

The degree to which developmentally related alterations in cardiac creatine kinase (CK) activity reflect modification of CK isoenzyme gene expression remains uncertain. The present studies addressed this question by assessing multiple aspects of CK in rat heart during the perinatal to adult transition. In addition to whole tissue, isolated and purified muscle and nonmuscle cells were studied, as well as myofibrillar, mitochondrial, and cytosolic subcellular fractions. Whole homogenate CK enzyme specific activity nearly doubled during the weanling to adult developmental period. Muscle cell CK activity increased by a similar magnitude. Nonmuscle cell activity decreased. In the adult heart, both myofibrillar and mitochondrial CK activities were augmented versus the weanling heart. The cytoplasmic fraction activity held constant during development. Electrophoretic isoenzyme analyses of both weanling and adult cardiac muscle cells indicated the presence of mitochondrial CK and MM-CK isoforms. Weanling heart nonmuscle cells contained mitochondrial, MM, MB, and BB isoforms; however, BB isoform was not detected in the adult heart nonmuscle cells. Arrhenius plots provided information regarding heart muscle and nonmuscle cell alterations during development. CK activation energies were also determined for whole tissue, muscle/nonmuscle cells, myofibrils, mitochondria, and cytosol. Results demonstrate that heterogeneous muscle/nonmuscle cellular composition and differential myofibrillar/mitochondrial subcellular composition account for normal, developmentally related changes in heart CK enzyme activity. CK isoenzyme gene expression changes were not detected in cardiac muscle cells, and transition of CK-B to CK-M gene expression is limited to nonmuscle cells during normal, weanling to adult development in the rat heart.     

Phosphotransfer dynamics in skeletal muscle from creatine kinase gene-deleted mice

To assess the significance of energy supply routes in cellular energetic homeostasis, net phosphoryl fluxes catalyzed by creatine kinase (CK), adenylate kinase (AK) and glycolytic enzymes were quantified using 18O-phosphoryl labeling. Diaphragm muscle from double M-CK/ScCKmit knockout mice exhibited virtually no CK-catalyzed phosphotransfer. Deletion of the cytosolic M-CK reduced CK-catalyzed phosphotransfer by 20%, while the absence of the mitochondrial ScCKmit isoform did not affect creatine phosphate metabolic flux. Contribution of the AK-catalyzed phosphotransfer to total cellular ATP turnover was 15.0, 17.2, 20.2 and 28.0% in wild type, ScCKmit, M-CK and M-CK/ScCKmit deficient muscles, respectively. Glycolytic phosphotransfer, assessed by G-6-P 18O-phosphoryl labeling, was elevated by 32 and 65% in M-CK and M-CK/ScCKmit deficient muscles, respectively. Inhibition of glyceraldehyde 3-phosphate dehydrogenase (GAPDH)/phosphoglycerate kinase (PGK) in CK deficient muscles abolished inorganic phosphate compartmentation and redirected high-energy phosphoryl flux through the AK network. Under such conditions, AK phosphotransfer rate was equal to 86% of the total cellular ATP turnover concomitant with almost normal muscle performance. This indicates that near-equilibrium glycolytic phosphotransfer reactions catalyzed by the GAPDH/PGK support a significant portion of the high-energy phosphoryl transfer in CK deficient muscles. However, CK deficient muscles displayed aberrant ATPase-ATPsynthase communication along with lower energetic efficiency (P/O ratio), and were more sensitive to metabolic stress induced by chemical hypoxia. Thus, redistribution of phosphotransfer through glycolytic and AK networks contributes to energetic homeostasis in muscles under genetic and metabolic stress complementing loss of CK function.     

Structural and functional adaptations of striated muscles to CK deficiency

In adult mammalian muscle cells, energy consuming processes are mainly localized to the sarcolemma, sarcoplasmic reticulum (SR) and myofibrillar compartments, while energy production occurs within mitochondria or glycolytic complexes. Due to the restricted diffusion of adenine nucleotides near the active sites of ATPases involved in contractile activity and calcium homeostasis, there are multiple local systems that can locally rephosphorylate ADP and provide ATP. The creatine kinase (CK) system, with specific isoenzymes localized within each compartment, efficiently controls local adenylate pools and links energy production and utilization. However, mice lacking one or both of the MM-CK and mi-CK isoforms (CK-/-) are viable and develop almost normal cardiac and skeletal muscle function under the conditions of moderate workload, suggesting adaptations or other mechanisms that may ensure efficient energy transfer. While fixed CK is essentially important, other systems could also be involved as well, such as bound glycolytic enzymes or adenylate kinase. We have shown that, additionally, a direct functional interplay exists between mitochondria and sarcoplasmic reticulum, or between mitochondria and myofilaments in muscle cells, that catalyzes direct energy and signal transfer between organelles. In cardiac cells of CK-/- mice, marked cytoarchitectural modifications were observed, and direct adenine nucleotide channeling between mitochondria and organelles was very effective to rescue SR and myofilament functions. In fast skeletal muscles, increased oxidative capacity also indicates compensatory mechanisms. In mutant mice, mitochondrial capacity increases and a direct energy channeling occurs between mitochondria on one hand and ATP consuming sites on the other. However, these systems appear to be insufficient to fully compensate for the lack of CK at high workload. It can be concluded that local rephosphorylation of ADP is a crucial regulatory point in highly differentiated and organized muscle cells to ensure contractile diversity and efficiency and that the CK system is important to control energy fluxes and energy homeostasis.     

Creatine kinase and mitochondrial respiration in hearts of trout,cod and freshwater turtle

The importance of the creatine kinase system in the cardiac muscle of ectothermic vertebrates is unclear. Mammalian cardiac muscle seems to be structurally organized in a manner that compartmentalizes the intracellular environment as evidenced by the substantially higher mitochondrial apparent K_m for ADP in skinned fibres compared to isolated mitochondria. A mitochondrial fraction of creatine kinase is functionally coupled to the mitochondrial respiration, and the transport of phosphocreatine and creatine as energy equivalents of ATP and ADP, respectively, increases the mitochondrial apparent ADP affinity, i.e. lowers the K_m. This function of creatine kinase seems to be absent in hearts of frog species. To find out whether this applies to hearts of ectothermic vertebrate species in general, we investigated the effect of creatine on the mitochondrial respiration of saponin-skinned fibres from the ventricle of rainbow trout, Atlantic cod and freshwater turtle. For all three species, the apparent K_m for ADP appeared to be substantially higher than for isolated mitochondria. Creatine lowered this K_m in trout and turtle, thus indicating a functional coupling between mitochondrial creatine kinase and respiration. However, creatine had no effect on K_m in cod ventricle. In conclusion, the creatine kinase-system in trout and turtle hearts seems to fulfil the same functions as in the mammalian heart, i.e. facilitating energy transport and communication between cellular compartments. In cod heart, however, this does not seem to be the case.Abbreviations ACR acceptor control ratio - CK creatine kinase - PCr creatine phosphate - V_ADP ADP-stimulated respiration rate - V_max maximal respiration rate - V₀ respiration rate in the absence of ADPCommunicated by: G. Heidmaier     

The creatine kinase system in smooth muscle

Despite the energetic flux being much lower in smooth muscle compared to striated muscles (such as the heart and skeletal muscle) creatine kinase (CK) has been found present and active in all smooth muscles studied to date. A complete CK circuit has been identified, with CK found in the mitochondria, contractile elements, membrane pumps and the cytoplasm. CK isoenzymes are coupled to many cellular energetic processes and appears to be involved in energy production and consumption by acting as an energy transducer. The CK system responds to pathological insults and development (e.g. hypertrophy and gestation respectively) by changes in sub-cellular distribution localization, isoenzymes, and specific activity. The conclusion from these observations is that creatine kinase is intimately involved in the energetic system of smooth muscle.Abbreviations CK creatine kinase - Mi-CK mitochondrial creatine kinase - Cr creatine - PCr phosphocreatiner - NMR nuclear magnetic resonance - SHR spontaneously hypertensive rat - -GPA -guanidinopropionic acid     

Mathematical model of compartmentalized energy transfer: Its use for analysis and interpretation of 31P-NMR studies of isolated heart of creatine kinase deficient mice

A mathematical model of the compartmentalized energy transfer in cardiac cells is described and used for interpretation of novel experimental data obtained by using phosphorus NMR for determination of the energy fluxes in the isolated hearts of transgenic mice with knocked out creatine kinase isoenzymes. These experiments were designed to study the meaning and importance of compartmentation of creatine kinase isoenzymes in the cells in vivo. The model was constructed to describe quantitatively the processes of energy production, transfer, utilization, and feedback between these processes. It describes the production of ATP in mitochondrial matrix space by ATP synthase, use of this ATP for phosphocreatine production in the mitochondrial creatine kinase reaction coupled to the adenine nucleotide translocation, diffusional exchange of metabolites in the cytoplasmic space, and use of phosphocreatine for resynthesis of ATP in the myoplasmic creatine kinase reaction. It accounts also for the recently discovered phenomenon of restricted diffusion of adenine nucleotides through mitochondrial outer membrane porin pores (VDAC). Practically all parameters of the model were determined experimentally. The analysis of energy fluxes between different cellular compartments shows that in all cellular compartments of working heart cells the creatine kinase reaction is far from equilibrium in the systolic phase of the contraction cycle and approaches equilibrium only in cytoplasm and only in the end-diastolic phase of the contraction cycle.Experimental determination of the relationship between energy fluxes by a ³¹P-NMR saturation transfer method and workload in isolated and perfused heart of transgenic mice deficient in MM isoenzyme of the creatine kinase, MM -/- showed that in the hearts from wild mice, containing all creatine kinase isoenzymes, the energy fluxes determined increased 3-4 times with elevation of the workload. By contrast, in the hearts in which only the mitochondrial creatine kinase was active, the energy fluxes became practically independent of the workload in spite of the preservation of 26% of normal creatine kinase activity. These results cannot be explained on the basis of the conventional near-equilibrium theory of creatine kinase in the cells, which excludes any difference between creatine kinase isoenzymes. However, these apparently paradoxical experimental results are quantitatively described by a mathematical model of the compartmentalized energy transfer based on the steady state kinetics of coupled creatine kinase reactions, compartmentation of creatine kinase isoenzymes in the cells, and the kinetics of ATP production and utilization reactions. The use of this model shows that: (1) in the wild type heart cells a major part of energy is transported out of mitochondria via phosphocreatine, which is used for complete regeneration of ATP locally in the myofibrils - this is the quantitative estimate for PCr pathway; (2) however, in the absence of MM-creatine kinase in the myofibrils in transgenic mice the contraction results in a very rapid rise of ADP in cytoplasmic space, that reverses the mitochondrial creatine kinase reaction in the direction of ATP production. In this way, because of increasing concentrations of cytoplasmic ADP, mitochondrial creatine kinase is switched off functionally due to the absence of its counterpart in PCr pathway, MM-creatine kinase. This may explain why the creatine kinase flux becomes practically independent from the workload in the hearts of transgenic mouse without MM-CK. Thus, the analysis of the results of studies of hearts of creatine kinase-deficient transgenic mice, based on the use of a mathematical model of compartmentalized energy transfer, show that in the PCr pathway of intracellular energy transport two isoenzymes of creatine kinase always function in a coordinated manner out of equilibrium, in the steady state, and disturbances in functioning of one of them inevitably result in the disturbances of the other component of the PCr pathway. In the latter case, energy is transferred from mitochondria to myofibrils by alternative metabolic pathways, probably involving adenylate kinase or other systems.     

Functional changes in the mitochondrial site of adenylate kinase and creatine kinase systems of energy transport induced by myocardial ischemia and adriablastin

Under effects of myocardial ischemia (30 min), the activities of the intermembrane enzymes of rabbit heart mitochondria, i.e., adenylate kinase and creatine kinase, are inhibited by 20% and 23%, respectively. Consequently, the creatine- and AMP-activated respiration of mitochondria diminishes by 52% and 39%, respectively. An inhibitory analysis of ADP-, AMP- and creatine-activated mitochondrial respiration performed in the presence of atractyloside has demonstrated that ischemia (30 min), adriblastin (0.688 mM) and succinate (10 mM) cause alterations in the functional coupling of adenylate kinase and creatine kinase with the adenine nucleotide translocator. These alterations lead to the diminution of the rate and efficiency of energy transfer from mitochondria to hexokinase, as an arbitrary site of energy consumption. An addition of cytochrome c to ischemic heart mitochondria results in an increase in the rate of ATP synthesis; however, the efficiency of this process is lowered. The toxic effect of the anticancer drug--adriblastin on heart mitochondria respiration is enhanced in the presence of creatine in the bathing solution.     

Kinetic characterization of human heart and skeletal muscle CK isoenzymes

The purpose of this study was to investigate the kinetic properties of human creatine kinase (CK) isoenzymes partially purified from heart and skeletal muscle. Utilizing the backward CK-catalyzed reaction of creatine phosphate + ADP in equilibrium creatine + ATP, Km values for heart and skeletal muscle CK MM (3.7 mmol/l) were significantly (p less than 0.05) greater than CK MB (2.1 mmol/l) which were significantly (p less than 0.05) greater than mitochondrial CK (1.8 mmol/l) at variable creatine phosphate and fixed ADP concentrations. However, Km values for similar isoenzymes from the two different tissues, i.e., CK MB from heart vs. skeletal muscle, were not different. These results show that kinetic analysis of CK isoenzymes cannot differentiate the tissue source of elevated blood CK isoenzymes after the acute stress of long distance running or after acute myocardial infarction.     

Lack of dystrophin is associated with altered integration of the mitochondria and ATPases in slow-twitch muscle cells of MDX mice

The potential role of dystrophin-mediated control of systems integrating mitochondria with ATPases was assessed in muscle cells. Mitochondrial distribution and function in skinned cardiac and skeletal muscle fibers from dystrophin-deficient (MDX) and wild-type mice were compared. Laser confocal microscopy revealed disorganized mitochondrial arrays in m. gastrocnemius in MDX mice, whereas the other muscles appeared normal in this group. Irrespective of muscle type, the absence of dystrophin had no effect on the maximal capacity of oxidative phosphorylation, nor on coupling between oxidation and phosphorylation. However, in the myocardium and m. soleus, the coupling of mitochondrial creatine kinase to adenine nucleotide translocase was attenuated as evidenced by the decreased effect of creatine on the Km for ADP in the reactions of oxidative phosphorylation. In m. soleus, a low Km for ADP compared to the wild-type counterpart was found, which implies increased permeability for that nucleotide across the mitochondrial outer membrane. In normal cardiac fibers 35% of the ADP flux generated by ATPases was not accessible to the external pyruvate kinase-phosphoenolpyruvate system, which suggests the compartmentalized (direct) channeling of that fraction of ADP to mitochondria. Compared to control, the direct ADP transfer was increased in MDX ventricles. In conclusion, our data indicate that in slow-twitch muscle cells, the absence of dystrophin is associated with the rearrangement of the intracellular energy and feedback signal transfer systems between mitochondria and ATPases. As the mechanisms mediated by creatine kinases become ineffective, the role of diffusion of adenine nucleotides increases due to the higher permeability of the mitochondrial outer membrane for ADP and enhanced compartmentalization of ADP flux.     

A model of oxidative phosphorylation in mammalian skeletal muscle

A dynamic computer model of oxidative phosphorylation in oxidative mammalian skeletal muscle was developed. The previously published model of oxidative phosphorylation in isolated skeletal muscle mitochondria was extended by incorporation of the creatine kinase system (creatine kinase plus phosphocreatine/creatine pair), cytosolic proton production/consumption system (proton production/consumption by the creatine kinase-catalysed reaction, efflux/influx of protons), physiological size of the adenine nucleotide pool and some additional minor changes. Theoretical studies performed by means of the extended model demonstrated that the CK system, which allows for large changes in P(i) in relation to isolated mitochondria system, has no significant influence on the kinetic properties of oxidative phosphorylation, as inorganic phosphate only slightly modifies the relationship between the respiration rate and [ADP]. Computer simulations also suggested that the second-order dependence of oxidative phosphorylation on [ADP] proposed in the literature refers only to the ATP synthesis flux, but not to the oxygen consumption flux (the difference between these two fluxes being due to the proton leak). Next, time courses of changes in fluxes and metabolite concentrations during transition between different steady-states were simulated. The model suggests, in accordance with previous theoretical predictions, that activation of oxidative phosphorylation by an increase in [ADP] can (roughly) explain the behaviour of the system only at low work intensities, while at higher work intensities parallel activation of different steps of oxidative phosphorylation is involved.     

Compartmentation of energy metabolism in atrial myocardium of patients undergoing cardiac surgery

The parameters of oxidative phosphorylation and its interaction with creatine kinase (CK)- and adenylate kinase (AK)-phosphotransfer networks in situ were studied in skinned atrial fibers from 59 patients undergoing coronary artery bypass surgery, valve replacement/correction and atrial septal defect correction. In atria, the mitochondrial CK and AK are effectively coupled to oxidative phosphorylation, the MM-CK is coupled to ATPases and there exists a direct transfer of adenine nucleotides between mitochondria and ATPases. Elimination of cytoplasmic ADP with exogenous pyruvate kinase was not associated with a blockade of the stimulatory effects of creatine and AMP on respiration, neither could it abolish the coupling of MM-CK to ATPases and direct transfer of adenine nucleotides. Thus, atrial energy metabolism is compartmentalized so that mitochondria form functional complexes with adjacent ATPases. These complexes isolate a part of cellular adenine nucleotides from their cytoplasmic pool for participating in energy transfer via CK- and AK-networks, and/or direct exchange. Compared to atria in sinus rhythm, the fibrillating atria were larger and exhibited increased succinate-dependent respiration relative to glutamate-dependent respiration and augmented proton leak. Thus, alterations in mitochondrial oxidative phosphorylation may contribute to pathogenesis of atrial fibrillation. (Mol Cell Biochem 270: 49–61, 2005)     

Alterations of the bioenergetics systems of the cell in acute and chronic myocardial ischemia

The aim of the works presented here is to analyze the alterations induced by acute ischemia-reperfusion and chronic ischemia on mitochondrial function, in relation to alterations on heart function. Parameters of mitochondrial function were assessed on skinned fibers coming from isolated perfused rat hearts. The effects of chronic ischemia were studied on a rat model of left descending coronary artery stenosis. Two key events observed after acute ischemia-reperfusion and chronic ischemia are the decrease (or the loss) of the stimulatory effect of creatine and the alteration of outer mitochondrial permeability to cytochrome c and ADP. Taken together, these effects indicate the alteration of the intermembrane space architecture leading to the loss of intracellular adenine nucleotides compartmentation and possibly of functional coupling of mitochondrial creatine kinase and adenine nucleotide translocase. These alterations result in the impairment of intracellular energy transfer (channeling) from mitochondria to ATP-utilizing sites located in the cytosol. This may play a significant role in ischemic injury and alterations in heart function. We show that these effects were prevented by effective cardioprotective strategies like ischemic preconditioning or pharmacological preconditioning by perfusion of mitochondrial ATP-sensitive potassium channel openers. We hypothesize that an open mitochondrial ATP-sensitive potassium channel during ischemia maintains the tight structure of the intermembrane space that is required to preserve the normal low outer membrane permeability to ADP and ATP.     

Mitochondrial affinity for ADP is twofold lower in creatine kinase knock-out muscles. Possible role in rescuing cellular energy homeostasis

Adaptations of the kinetic properties of mitochondria in striated muscle lacking cytosolic (M) and/or mitochondrial (Mi) creatine kinase (CK) isoforms in comparison to wild-type (WT) were investigated in vitro. Intact mitochondria were isolated from heart and gastrocnemius muscle of WT and single- and double CK-knock-out mice strains (cytosolic (M-CK-/-), mitochondrial (Mi-CK-/-) and double knock-out (MiM-CK-/-), respectively). Maximal ADP-stimulated oxygen consumption flux (State3 Vmax; nmol O2 x mg mitochondrial protein(-1) x min(-1)) and ADP affinity (K50ADP; microM) were determined by respirometry. State 3 Vmax and of M-CK-/- and MiM-CK-/- gastrocnemius mitochondria were twofold higher than those of WT, but were unchanged for Mi-CK-/-. For mutant cardiac mitochondria, only the of mitochondria isolated from the MiM-CK-/- phenotype was different (i.e. twofold higher) than that of WT. The implications of these adaptations for striated muscle function were explored by constructing force-flow relations of skeletal muscle respiration. It was found that the identified shift in affinity towards higher ADP concentrations in MiM-CK-/- muscle genotypes may contribute to linear mitochondrial control of the reduced cytosolic ATP free energy potentials in these phenotypes.     

Impaired intracellular energetic communication in muscles from creatine kinase and adenylate kinase (M-CK/AK1) double knock-out mice

Previously we demonstrated that efficient coupling between cellular sites of ATP production and ATP utilization, required for optimal muscle performance, is mainly mediated by the combined activities of creatine kinase (CK)- and adenylate kinase (AK)-catalyzed phosphotransfer reactions. Herein, we show that simultaneous disruption of the genes for the cytosolic M-CK- and AK1 isoenzymes compromises intracellular energetic communication and severely reduces the cellular capability to maintain total ATP turnover under muscle functional load. M-CK/AK1 (MAK=/=) mutant skeletal muscle displayed aberrant ATP/ADP, ADP/AMP and ATP/GTP ratios, reduced intracellular phosphotransfer communication, and increased ATP supply capacity as assessed by 18O labeling of [Pi] and [ATP]. An analysis of actomyosin complexes in vitro demonstrated that one of the consequences of M-CK and AK1 deficiency is hampered phosphoryl delivery to the actomyosin ATPase, resulting in a loss of contractile performance. These results suggest that MAK=/= muscles are energetically less efficient than wild-type muscles, but an apparent compensatory redistribution of high-energy phosphoryl flux through glycolytic and guanylate phosphotransfer pathways limited the overall energetic deficit. Thus, this study suggests a coordinated network of complementary enzymatic pathways that serve in the maintenance of energetic homeostasis and physiological efficiency.     

Quantitative studies of enzyme-substrate compartmentation,functional coupling and metabolic channelling in muscle cells

Some historical aspects of development of the concepts of functional coupling, metabolic channelling, compartmentation and energy transfer networks are reviewed. Different quantitative approaches, including kinetic and mathematical modeling of energy metabolism, intracellular energy transfer and metabolic regulation of energy production and fluxes in the cells in vivo are analyzed. As an example of the system with metabolic channelling, thermodynamic aspects of the functioning the mitochondrial creatine kinase functionally coupled to the oxidative phosphorylation are considered. The internal thermodynamics of the mitochondrial creatine kinase reaction is similar to that for other isoenzymes of creatine kinase, and the oxidative phosphorylation process specifically influences steps of association and dissociation of MgATP with the enzyme due to channelling of ATP from adenine nucleotide translocase. A new paradigm of muscle bioenergetics - the paradigm of energy transfer and feedback signaling networks based on analysis of compartmentation phenomena and structural and functional interactions in the cell is described. Analysis of the results of mathematical modeling of the compartmentalized energy transfer leads to conclusion that both calcium and ADP, which concentration changes synchronously in contraction cycle, may simultaneously activate oxidative phosphorylation in the muscle cells in vivo. The importance of the phosphocreatine circuit among other pathways of intracellular energy transfer network is discussed on the basis of the recent data published in the literature, with some experimental demonstration. The results of studies of perfused rat hearts with completely inhibited creatine kinase show significantly decreased work capacity and respectively, energy fluxes, in these hearts in spite of significant activation of adenylate kinase system (Dzeja et al. this volume). These results, combined with those of mathematical analysis of the energy metabolism of hearts of transgenic mice with switched off creatine kinase isoenzymes confirm the importance of phosphocreatine pathway for energy transfer for cell function and energetics in mature heart and many other types of cells, as one of major parts of intracellular energy transfer network and metabolic regulation.