Systems bioenergetics of creatine kinase networks: physiological roles of creatine and phosphocreatine in regulation of cardiac cell function.

Physiological role of creatine (Cr) became first evident in the experiments of Belitzer and Tsybakova in 1939, who showed that oxygen consumption in a well-washed skeletal muscle homogenate increases strongly in the presence of creatine and with this results in phosphocreatine (PCr) production with PCr/O(2) ratio of about 5-6. This was the beginning of quantitative analysis in bioenergetics. It was also observed in many physiological experiments that the contractile force changes in parallel with the alteration in the PCr content. On the other hand, it was shown that when heart function is governed by Frank-Starling law, work performance and oxygen consumption rate increase in parallel without any changes in PCr and ATP tissue contents (metabolic homeostasis). Studies of cellular mechanisms of all these important phenomena helped in shaping new approach to bioenergetics, Molecular System Bioenergetics, a part of Systems Biology. This approach takes into consideration intracellular interactions that lead to novel mechanisms of regulation of energy fluxes. In particular, interactions between mitochondria and cytoskeleton resulting in selective restriction of permeability of outer mitochondrial membrane anion channel (VDAC) for adenine nucleotides and thus their recycling in mitochondria coupled to effective synthesis of PCr by mitochondrial creatine kinase, MtCK. Therefore, Cr concentration and the PCr/Cr ratio became important kinetic parameters in the regulation of respiration and energy fluxes in muscle cells. Decrease in the intracellular contents of Cr and PCr results in a hypodynamic state of muscle and muscle pathology. Many experimental studies have revealed that PCr may play two important roles in the regulation of muscle energetics: first by maintaining local ATP pools via compartmentalized creatine kinase reactions, and secondly by stabilizing cellular membranes due to electrostatic interactions with phospholipids. The second mechanism decreases the production of lysophosphoglycerides in hypoxic heart, protects the cardiac cells sarcolemma against ischemic damage, decreases the frequency of arrhythmias and increases the post-ischemic recovery of contractile function. PCr is used as a pharmacological product Neoton in cardiac surgery as one of the components of cardioplegic solutions for protection of the heart against intraoperational injury and injected intravenously in acute myocardial ischemic conditions for improving the hemodynamic response and clinical conditions of patients with heart failure.

Functional coupling as a basic mechanism of feedback regulation of cardiac energy metabolism.

In this review we analyze the concepts and the experimental data on the mechanisms of the regulation of energy metabolism in muscle cells. Muscular energetics is based on the force-length relationship, which in the whole heart is expressed as a Frank-Starling law, by which the alterations of left ventricle diastolic volume change linearly both the cardiac work and oxygen consumption. The second basic characteristics of the heart is the metabolic stability--almost constant levels of high energy phosphates, ATP and phosphocreatine, which are practically independent of the workload and the rate of oxygen consumption, in contrast to the fast-twitch skeletal muscle with no metabolic stability and rapid fatigue. Analysis of the literature shows that an increase in the rate of oxygen consumption by order of magnitude, due to Frank-Starling law, is observed without any significant changes in the intracellular calcium transients. Therefore, parallel activation of contraction and mitochondrial respiration by calcium ions may play only a minor role in regulation of respiration in the cells. The effective regulation of the respiration under the effect of Frank-Starling law and metabolic stability of the heart are explained by the mechanisms of functional coupling within supramolecular complexes in mitochondria, and at the subcellular level within the intracellular energetic units. Such a complex structural and functional organisation of heart energy metabolism can be described quantitatively by mathematical models.

Metabolic consequences of functional complexes of mitochondria, myofibrils and sarcoplasmic reticulum in muscle cells.

Regulation of mitochondrial respiration both by endogenous and exogenous ADP in the cells in situ was studied in isolated and permeabilized cardiomyocytes, permeabilized cardiac fibers and 'ghost' fibers (all with a diameter of 10-20 micro m) at different (0-3 micro moll(-1)) free Ca(2+) concentrations in the medium. In all these preparations, the apparent K(m) of mitochondrial respiration for exogenous ADP at free Ca(2+) concentrations of 0-0.1 micro moll(-1) was very high, in the range of 250-350 micro moll(-1), in contrast to isolated mitochondria in vitro (apparent K(m) for ADP is approximately 20 micro moll(-1)). An increase in the free Ca(2+) concentration (up to 3 micro moll(-1), which is within physiological range), resulted in a very significant decrease of the apparent K(m) value to 20-30 micro moll(-1), a decrease of V(max) of respiration in permeabilized intact fibers and a strong contraction of sarcomeres. In ghost cardiac fibers, from which myosin was extracted but mitochondria were intact, neither the high apparent K(m) for ADP (300-350 micro moll(-1)) nor V(max) of respiration changed in the range of free Ca(2+) concentration studied, and no sarcomere contraction was observed. The exogenous-ADP-trapping system (pyruvate kinase + phosphoenolpyruvate) inhibited endogenous-ADP-supported respiration in permeabilized cells by no more than 40%, and this inhibition was reversed by creatine due to activation of mitochondrial creatine kinase. These results are taken to show strong structural associations (functional complexes) among mitochondria, sarcomeres and sarcoplasmic reticulum. Inside these complexes, mitochondrial functional state is controlled by channeling of ADP, mostly via energy- and phosphoryl-transfer networks, and apparently depends on the state of sarcomere structures.

[Analysis of mechanism of work of mitochondrial adenine nucleotide translocase using mathematical models]. / Analiz mekhanizma raboty mitokhondrial'noi adeninnukleotid-translokazy s pomoshch'iu matematicheskikh modelei.

The kinetics of exchange of adenine nucleotides in a system with reconstituted mitochondrial adenine nucleotide translocase (ANT) was simulated mathematically to analyze the basic mechanisms of ANT functioning. Two known alternative kinetic schemes were analyzed, the ping-pong type scheme with single-center substrate binding and the scheme of sequential two-center substrate binding at opposite sides of ANT. According to our modeling, both schemes can explain the experimental data on the adenine nucleotide exchange in the reconstituted ANT system. However, the characteristic kinetic pattern of ADP exchanges in the mono exchange mode was reproduced only by the sequential binding scheme. This scheme is consistent with the data on the tetrameric structure of ANT. On the other hand, only the single-center binding scheme was compatible with recent data on possible translocation of ATP and ADP by the carrier that has no bound adenine nucleotide on its opposite side. Based on the analysis of the literature data on ANT properties, a compromise scheme of ANT operation was proposed. In the framework of this scheme, the ANT dimers function by the single-center binding mechanism: however, in tetramers they are integrated into a substructure with two oppositely oriented binding centers working by the mechanism of sequential substrate binding. Labile bonds between the ANT-forming dimers could allow conformational rearrangements of ANT induced by various influences on mitochondrial membrane structure, including those leading to the induction of permeability transition pores in apoptosis.

Macrocompartmentation of total creatine in cardiomyocytes revisited.

Distribution of total creatine (free creatine + phosphocreatine) between two subcellular macrocompartments--mitochondrial matrix space and cytoplasm--in heart and skeletal muscle cells was reinvestigated by using a permeabilized cell technique. Isolated cardiomyocytes were treated with saponin (50 microg/ml for 30 min or 600 microg/ml for 1 min) to open the outer cellular membrane and release the metabolites from cytoplasm (cytoplasmic fraction, CF). All mitochondrial population in permeabilized cells remained intact: the outer membrane was impermeable for exogenous cytochrome c, the acceptor control index of respiration exceeded 10, the mitochondrial creatine kinase reaction was fully coupled to the adenine nucleotide translocator. Metabolites were released from mitochondrial fraction (MF) by 2-5% Triton X100. Total cellular pool of free creatine + phosphocreatine (69.6 +/- 2.1 nmoles per mg of protein) was found exclusively in CF and was practically absent in MF. When fibers were prepared from perfused rat hearts, cellular distribution of creatine was not dependent on functional state of the heart and only slightly modified by ischemia. It is concluded that there is no stable pool of creatine or phosphocreatine in the mitochondrial matrix in the intact muscle cells, and the total creatine pool is localized in only one macrocompartment--cytoplasm.

Intracellular energetic units in red muscle cells.

The kinetics of regulation of mitochondrial respiration by endogenous and exogenous ADP in muscle cells in situ was studied in skinned cardiac and skeletal muscle fibres. Endogenous ADP production was initiated by addition of MgATP; under these conditions the respiration rate and ADP concentration in the medium were dependent on the calcium concentration, and 70-80% of maximal rate of respiration was achieved at ADP concentration below 20 microM in the medium. In contrast, when exogenous ADP was added, maximal respiration rate was observed only at millimolar concentrations. An exogenous ADP-consuming system consisting of pyruvate kinase (PK; 20-40 units/ml) and phosphoenolpyruvate (PEP; 5 mM), totally suppressed respiration activated by exogenous ADP, but the respiration maintained by endogenous ADP was not suppressed by more than 20-40%. Creatine (20 mM) further activated respiration in the presence of ATP and PK+PEP. Short treatment with trypsin (50-500 nM for 5 min) decreased the apparent K(m) for exogenous ADP from 300-350 microM to 50-60 microM, increased inhibition of respiration by PK+PEP system up to 70-80%, with no changes in MgATPase activity and maximal respiration rates. Electron-microscopic observations showed detachment of mitochondria and disordering of the regular structure of the sarcomere after trypsin treatment. Two-dimensional electrophoresis revealed a group of at least seven low-molecular-mass proteins in cardiac skinned fibres which were very sensitive to trypsin and not present in glycolytic fibres, which have low apparent K(m) for exogenous ADP. It is concluded that, in oxidative muscle cells, mitochondria are incorporated into functional complexes ('intracellular energetic units') with adjacent ADP-producing systems in myofibrils and in sarcoplasmic reticulum, probably due to specific interaction with cytoskeletal elements responsible for mitochondrial distribution in the cell. It is suggested that these complexes represent the basic pattern of organization of muscle-cell energy metabolism.

Cardioprotection by ischemic preconditioning preserves mitochondrial function and functional coupling between adenine nucleotide translocase and creatine kinase.

M. N. Laclau, S. Boudina, J. B. Thambo, L. Tariosse, G. Gouverneur, S. Bonoron-Adèle, V. A. Saks, K. D. Garlid and P. Dos Santos. Cardioprotection by Ischemic Preconditioning Preserves Mitochondrial Function and Functional Coupling Between Adenine Nucleotide Translocase and Creatine Kinase. Journal of Molecular and Cellular Cardiology (2001) 33, 947-956. This study investigates the effect of ischemic preconditioning on mitochondrial function, including functional coupling between the adenine nucleotide translocase and mitochondrial creatine kinase, which is among the first reactions to be altered in ischemia. Three groups of Langendorff-perfused rat hearts were studied: a control group, a group subjected to 30 min ischemia followed by 15 min reperfusion, and a group subjected to ischemic preconditioning prior to 30 min ischemia and 15 min reperfusion. Ischemic preconditioning significantly delayed the onset and amplitude of contracture during ischemia, decreased enzymatic release, and improved the recovery of heart contractile function after reperfusion. Mitochondrial function was assessed in permeabilized skinned fibers. The protective effect of preconditioning was associated with preservation of mitochondrial function, as evidenced by maintenance of the high K(1/2)for ADP in regulation of mitochondrial respiration and V(max)of respiration, the near absence of respiratory stimulation by exogenous cytochrome c, and preservation of functional coupling between mitochondrial creatine kinase and adenine nucleotide translocase. These data suggest that ischemic preconditioning preserves the structure-function of the intermembrane space, perhaps by opening the mitochondrial ATP-sensitive K(+)channel. The consequence is preservation of energy transfer processes from mitochondria to ATP-utilizing sites in the cytosol. Both of these factors may contribute to cardioprotection and better functional recovery of preconditioned hearts.

Functional complexes of mitochondria with Ca,MgATPases of myofibrils and sarcoplasmic reticulum in muscle cells.

Regulation of mitochondrial respiration in situ in the muscle cells was studied by using fully permeabilized muscle fibers and cardiomyocytes. The results show that the kinetics of regulation of mitochondrial respiration in situ by exogenous ADP are very different from the kinetics of its regulation by endogenous ADP. In cardiac and m. soleus fibers apparent K(m) for exogenous ADP in regulation of respiration was equal to 300-400 microM. However, when ADP production was initiated by intracellular ATPase reactions, the ADP concentration in the medium leveled off at about 40 microM when about 70% of maximal rate of respiration was achieved. Respiration rate maintained by intracellular ATPases was suppressed about 20-30% during exogenous trapping of ADP with excess pyruvate kinase (PK, 20 IU/ml) and phosphoenolpyruvate (PEP, 5 mM). ADP flux via the external PK+PEP system was decreased by half by activation of mitochondrial oxidative phosphorylation. Creatine (20 mM) further activated the respiration in the presence of PK+PEP. It is concluded that in oxidative muscle cells mitochondria behave as if they were incorporated into functional complexes with adjacent ADP producing systems - with the MgATPases in myofibrils and Ca,MgATPases of sarcoplasmic reticulum.

Metabolic control of contractile performance in isolated perfused rat heart. Analysis of experimental data by reaction:diffusion mathematical model.

The intracellular mechanisms of regulation of energy fluxes and respiration in contracting heart cells were studied. For this, we investigated the workload dependencies of the rate of oxygen consumption and metabolic parameters in Langendorff-perfused isolated rat hearts.(31)P NMR spectroscopy was used to study the metabolic changes during transition from perfusion with glucose to that with pyruvate with and without active creatine kinase system. The experimental results showed that transition from perfusion with glucose to that with pyruvate increased the phosphocreatine content and stability of its level at increased workloads. Inhibition of creatine kinase reaction by 15-min infusion of iodoacetamide decreased the maximal developed tension and respiration rates by a factor of two.(31)P NMR data were analyzed by a mathematical model of compartmentalized energy transfer, which is independent from the restrictions of the classical concept of creatine kinase equilibrium. The analysis of experimental data by this model shows that metabolic stability-constant levels of phosphocreatine, ATP and inorganic phosphate-at increased energy fluxes is an inherent property of the compartmentalized system. This explains the observed substrate specificity by changes in mitochondrial membrane potential. The decreased maximal respiration rate and maximal work output of the heart with inhibited creatine kinase is well explained by the rise in myoplasmic ADP concentration. This activates the adenylate kinase reaction in the myofibrillar space and in the mitochondria to fulfil the energy transfer and signal transmission functions, usually performed by creatine kinase. The activity of this system, however, is not sufficient to maintain high enough energy fluxes. Therefore, there is a kinetic explanation for the decreased maximal respiration rate of the heart with inhibited creatine kinase: i.e. a kinetically induced switch from an efficient energy transfer pathway (PCr-CK system) to a non-efficient one (myokinase pathway) within the energy transfer network of the cell under conditions of low apparent affinity of mitochondria to ADP in vivo. This may result in a significant decrease in the thermodynamic affinity of compartmentalized ATPase systems and finally in heart failure.

Rapid spectrophotometric method for quantitation of cytochrome c release from isolated mitochondria or permeabilized cells revisited.

This paper recalls the earlier work by Keilin, Margoliash and others at the beginning of the 20th century and shows how their results can be used for the rapid solution of new problems of modern science. It describes a rapid and simple spectrophotometric method for quantitative determination of cytochrome c release from isolated mitochondria or permeabilized cells induced by proapoptotic proteins. For this, the Soret (gamma) peak at 414 nm in the spectrum of cytochrome c is used. The results of spectrophotometric assay of cytochrome c release are in accord with those of oxygraphic determination of cytochrome c-dependent respiration of isolated mitochondria and permeabilized cardiomyocytes.

Role of the creatine/phosphocreatine system in the regulation of mitochondrial respiration.

The mechanism of metabolic regulation of mitochondrial respiration in cardiac muscle cells was studied experimentally in the permeabilized heart fibres of mice and by computer modelling in silico. The experiments showed that the rate of mitochondrial respiration could be controlled by local production of ADP by mitochondrial creatine kinase in the intermembrane space of mitochondria. The spatially inhomogenous reaction-diffusion model of compartmentalized energy transfer was used to analyse which metabolite level in cytoplasm may be important for regulation of respiration. At low and moderate workloads, up to VO2 equal to 70 micromol min-1 g-1 dry weight, the only factor to which respiration responded was inorganic phosphate. At the values of VO2 higher than 70 micromol min-1 g-1 dry weight, the respiration rate responded mostly to changes in creatine, phosphocreatine and then time-averaged (over the contractile cycle) ADP concentrations in the cytoplasm. These results are taken to show that under conditions of moderate workloads, creatine kinase activity at given physiological creatine and phosphocreatine concentrations (apparent maximal activity achievable under these conditions) is in excess to oxidative phosphorylation rate, which is controlled by Pi concentration changes starting from very low values of the latter. At higher workloads mi-CK should be upregulated by increasing creatine and decreasing phosphocreatine concentrations, and only at very high workloads the ADP diffusion flux should be increased to upregulate oxidative phosphorylation. Thus, on the basis of the study in silico of compartmentalized energy transfer by phophocreatine/creatine system, the authors conclude that there exist multiple parallel regulatory factors controlling the rate of oxygen consumption in dependence of the workload. If creatine kinase is inhibited (and there is no myokinase activity), respiration requires high diffusive flux of ADP back into mitochondria, which is the sole regulator of respiration. This needs, however, increased ADP concentrations in the cytoplasm, which in turn result in inhibition of contraction.

Permeabilized cell and skinned fiber techniques in studies of mitochondrial function in vivo.

In this chapter we describe in details the permeabilized cell and skinned fiber techniques and their applications for studies of mitochondrial function in vivo. The experience of more than 10 years of research in four countries is summarized. The use of saponin in very low concentration (50-100 microg/ml) for permeabilisation of the sarcolemma leaves all intracellular structures, including mitochondria, completely intact. The intactness of mitochondrial function in these skinned muscle fibers is demonstrated in this work by multiple methods, such as NADH and flavoprotein fluorescence studies, fluorescence imaging, confocal immunofluorescence microscopy and respiratory analysis. Permeabilized cell and skinned fiber techniques have several very significant advantages for studies of mitochondrial function, in comparison with the traditional methods of use of isolated mitochondria: (1) very small tissue samples are required; (2) all cellular population of mitochondria can be investigated; (3) most important, however, is that mitochondria are studied in their natural surrounding. The results of research by using this method show the existence of several new phenomenon--tissue dependence of the mechanism of regulation of mitochondrial respiration, and activation of respiration by selective proteolysis. These phenomena are explained by interaction of mitochondria with other cellular structures in vivo. The details of experimental studies with use of these techniques and problems of kinetic analysis of the results are discussed. Examples of large-scale clinical application of these methods are given.

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

Octamer formation and coupling of cardiac sarcomeric mitochondrial creatine kinase are mediated by charged N-terminal residues.

Mitochondrial creatine kinases form octameric structures composed of four active and stable dimers. Octamer formation has been postulated to occur via interaction of the charged amino acids in the N-terminal peptide of the mature enzyme. We altered codons for charged amino acids in the N-terminal region of mature sarcomeric mitochondrial creatine kinase (sMtCK) to those encoding neutral amino acids. Transfection of normal sMtCK cDNA or those with the mutations R42G, E43G/H45G, and K46G into rat neonatal cardiomyocytes resulted in enzymatically active sMtCK expression in all. After hypoosmotic treatment of isolated mitochondria, mitochondrial inner membrane-associated and soluble sMtCK from the intermembranous space were measured. The R42G and E43G/H45G double mutation caused destabilization of the octameric structure of sMtCK and a profound reduction in binding of sMtCK to the inner mitochondrial membrane. The other mutant sMtCK proteins had modest reductions in binding. Creatine-stimulated respiration was markedly reduced in mitochondria isolated from cells transfected with the R42G mutant cDNA as compared with those transfected with normal sMtCK cDNA. We conclude that neutralization of charges in N-terminal peptide resulted in destabilization of octamer structure of sMtCK. Thus, charged amino acids at the N-terminal moiety of mature sMtCK are essential for octamer formation, binding of sMtCK with inner mitochondrial membrane, and coupling of sMtCK to oxidative phosphorylation.

Chronic exposure of rats to hypoxic environment alters the mechanism of energy transfer in myocardium.

We have investigated the effect of chronic exposure of rats to an hypoxic environment (10% O2; 3 weeks), on the first step of the intracellular energy transfer process in the myocardium, i.e. the transfer at mitochondrial level of high energy bonds from ATP to creatine. In the left ventricles from rats adapted to normobaric hypoxia, we observed, using the permeabilized fiber technique, that the stimulatory effect of creatine on the mitochondrial respiration in presence of a low ADP concentration (0.1 mM) was attenuated when compared to control. Furthermore, the creatine-induced decrease of the apparent K(m) for ADP of the mitochondrial respiration, which is observed in control, was significantly reduced. Both the basal and maximal respiratory rates of the fibers were unchanged by the hypoxic exposure of the rats. A significant decrease of the total creatine kinase activity from 755 to 630 IU/g wet weight (for control and hypoxic rats, respectively) was detected and was accompanied by a 25% decrease in mitochondrial isoform activity (mitoCK) and in the mitoCK/citrate synthase ratio. In the right ventricles, identical alterations in the effect of creatine on apparent K(m) for ADP were observed while we did not detect any changes in CK activity. The decrease in mitoCK activity and the fall in the reactivity of respiration to creatine could be interpreted as a mechanism for downregulating oxygen demand during chronic hypoxia. The consequences of such alterations on energy metabolism of cardiomyocytes under conditions of reduced oxygen supply are discussed.

Comparative study of respiration kinetics and protein composition of skinned fibers from various types of rat muscle.

The respiration parameters of mitochondria from rat heart muscle and from fast-twitch and slow-twitch skeletal muscle skinned fibers were comparatively analyzed. Electrophoretic patterns of fiber protein composition were also compared. It was found that fibers with low affinity of mitochondria for ADP (i.e., heart and slow-twitch skeletal muscle soleus) contain a 27.5-kD protein that is absent from the fibers that exert high affinity for ADP (i.e., fast-twitch skeletal muscle gastrocnemius). Partial proteolysis, which increases the affinity of mitochondria of the heart and slow-twitch skeletal muscles for ADP, results in the disappearance of this protein. The results suggest that this protein may be an intracellular factor that controls the permeability of the outer mitochondrial membrane for ADP.

Study of regulation of mitochondrial respiration in vivo. An analysis of influence of ADP diffusion and possible role of cytoskeleton.

The purpose of this work was to investigate the mechanism of regulation of mitochondrial respiration in vivo in different muscles of normal rat and mice, and in transgenic mice deficient in desmin. Skinned fiber technique was used to study the mitochondrial respiration in the cells in vivo in the heart, soleus and white gastrocnemius skeletal muscles of these animals. Also, cardiomyocytes were isolated from the normal rat heart, permeabilized by saponin and the "ghost" (phantom) cardiomyocytes were produced by extraction of myosin with 800 mM KCl. Use of confocal immunofluorescent microscopy and anti-desmin antibodies showed good preservation of mitochondria and cytoskeletal system in these phantom cells. Kinetics of respiration regulation by ADP was also studied in these cells in detail before and after binding of anti-desmine antibodies with intermediate filaments. In skinned cardiac or soleus skeletal muscle fibers but not in fibers from fast twitch skeletal muscle the kinetics of mitochondrial respiration regulation by ADP was characterized by very high apparent Km (low affinity) equal to 300-400 microM, exceeding that for isolated mitochondria by factor of 25. In skinned fibers from m. soleus, partial inhibition of respiration by NaN3 did not decrease the apparent Km for ADP significantly, this excluding the possible explanation of low apparent affinity of mitochondria to ADP in these cells by its rapid consumption due to high oxidative activity and by intracellular diffusion problems. However, short treatment of fibers with trypsin decreased this constant value to 40-70 microM, confirming the earlier proposition that mitochondrial sensitivity to ADP in vivo is controlled by some cytoplasmic protein. Phantom cardiomyocytes which contain mostly mitochondria and cytoskeleton and retain the normal shape, showed also high apparent Km values for ADP. Therefore, they are probably the most suitable system for studies of cellular factors which control mitochondrial function in the cells in vivo. In these phantom cells anti-desmin antibodies did not change the kinetics of respiration regulation by ADP. However, in skinned fibers from the heart and m. soleus of transgenic desmin-deficient mice some changes in kinetics of respiration regulation by ADP were observed: in these fibers two populations of mitochondria were observed, one with usually high apparent Km for ADP and the second one with very low apparent Km for ADP. Morphological observations by electron microscopy confirmed the existence of two distinct cellular populations in the muscle cells of desmin-deficient mice. The results conform to the conclusion that the reason for observed high apparent Km for ADP in regulation of oxidative phosphorylation in heart and slow twitch skeletal muscle cells in vivo is low permeability of mitochondrial outer membrane porins but not diffusion problems of ADP into and inside the cells. Most probably, in these cells there is a protein associated with cytoskeleton, which controls the permeability of the outer mitochondrial porin pores (VDAC) for ADP. Desmin itself does not display this type of control of mitochondrial porin pores, but its absence results in appearance of cells with disorganised structure and of altered mitochondrial population probably lacking this unknown VDAC controlling protein. Thus, there may be functional connection between mitochondria, cellular structural organisation and cytoskeleton in the cells in vivo due to the existence of still unidentified protein factor(s).

Compartmentalized energy transfer in cardiomyocytes: use of mathematical modeling for analysis of in vivo regulation of respiration.

The mathematical model of the compartmentalized energy transfer system in cardiac myocytes presented includes mitochondrial synthesis of ATP by ATP synthase, phosphocreatine production in the coupled mitochondrial creatine kinase reaction, the myofibrillar and cytoplasmic creatine kinase reactions, ATP utilization by actomyosin ATPase during the contraction cycle, and diffusional exchange of metabolites between different compartments. The model was used to calculate the changes in metabolite profiles during the cardiac cycle, metabolite and energy fluxes in different cellular compartments at high workload (corresponding to the rate of oxygen consumption of 46 mu atoms of O.(g wet mass)-1.min-1) under varying conditions of restricted ADP diffusion across mitochondrial outer membrane and creatine kinase isoenzyme "switchoff." In the complete system, restricted diffusion of ADP across the outer mitochondrial membrane stabilizes phosphocreatine production in cardiac mitochondria and increases the role of the phosphocreatine shuttle in energy transport and respiration regulation. Selective inhibition of myoplasmic or mitochondrial creatine kinase (modeling the experiments with transgenic animals) results in "takeover" of their function by another, active creatine kinase isoenzyme. This mathematical modeling also shows that assumption of the creatine kinase equilibrium in the cell may only be a very rough approximation to the reality at increased workload. The mathematical model developed can be used as a basis for further quantitative analyses of energy fluxes in the cell and their regulation, particularly by adding modules for adenylate kinase, the glycolytic system, and other reactions of energy metabolism of the cell.

Alteration in the control of mitochondrial respiration by outer mitochondrial membrane and creatine during heart preservation.

OBJECTIVE: This study aimed to evaluate the nature and extent of mitochondrial alterations during heart preservation. METHODS: Rat hearts, isolated after cardioplegia in situ, were preserved for 6 or 15 h at 4 degrees C either by immersion in cardioplegic solution or by low-flow perfusion (0.3 ml/min) with cardioplegic solution. The energy state of hearts at the end of preservation was determined by 31P-NMR spectroscopy and functional recovery was evaluated on reperfusion. Variables of mitochondrial respiration (maximal rate of respiration in the presence of ADP = delta Vmax, apparent Km for ADP, effect of creatine) were evaluated on skinned fibers and compared with those determined in controls and in hearts subjected to 1 hour of ischemia at 37 degrees C. RESULTS: Serious mitochondrial alterations were detected in fibers from 15 h immersed hearts: decrease of delta Vmax and of apparent Km for ADP, loss of the stimulatory effect of creatine, and disruption of the outer mitochondrial membrane. The extent of alternations was more accentuated in fibers from normothermic ischemic hearts, in which some damage of the inner mitochondrial membrane also occurred. In fibers from hearts preserved for 6 h, no significant changes in mitochondrial variables could be detected. When the hearts were preserved under low-flow perfusion for 15 h, only the stimulatory effect of creatine on respiration was significantly decreased. The extent of the loss of the stimulatory effect of creatine paralleled the accumulation of inorganic phosphate (Pi) during preservation and the decrease in left ventricular function on reperfusion. CONCLUSIONS: Alterations related to the outer membrane and the intermembrane space are among the earliest signs of damage to mitochondrial function during heart preservation. These alterations could be attributed to the swelling of mitochondria under the effect of Pi. The determination of mitochondrial parameters in biopsy samples could allow a simple and rapid evaluation of energy-producing and transfer capacities of the myocardium.