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The aim of this study was to measure energy fluxes from mitochondria in isolated permeabilized cardiomyocytes. Respiration of permeabilized cardiomyocytes and mitochondrial membrane potential were measured in presence of MgATP, pyruvate kinase - phosphoenolpyruvate and creatine. ATP and phosphocreatine concentrations in medium surrounding cardiomyocytes were determined. While ATP concentration did not change in time, mitochondria effectively produced phosphocreatine (PCr) with PCr/O(2) ratio equal to 5.68 +/- 0.14. Addition of heterodimeric tubulin to isolated mitochondria was found to increase apparent Km for exogenous ADP from 11 +/- 2 microM to 330 +/- 47 microM, but creatine again decreased it to 23 +/- 6 microM. These results show directly that under physiological conditions the major energy carrier from mitochondria into cytoplasm is PCr, produced by mitochondrial creatine kinase (MtCK), which functional coupling to adenine nucleotide translocase is enhanced by selective limitation of permeability of mitochondrial outer membrane within supercomplex ATP Synthasome-MtCK-VDAC-tubulin, Mitochondrial Interactosome.

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The main focus of this investigation is steady state kinetics of regulation of mitochondrial respiration in permeabilized cardiomyocytes in situ. Complete kinetic analysis of the regulation of respiration by mitochondrial creatine kinase was performed in the presence of pyruvate kinase and phosphoenolpyruvate to simulate interaction of mitochondria with glycolytic enzymes. Such a system analysis revealed striking differences in kinetic behaviour of the MtCK-activated mitochondrial respiration in situ and in vitro. Apparent dissociation constants of MgATP from its binary and ternary complexes with MtCK, Kia and Ka (1.94+/-0.86 mM and 2.04+/-0.14 mM, correspondingly) were increased by several orders of magnitude in situ in comparison with same constants in vitro (0.44+/-0.08 mM and 0.016+/-0.01 mM, respectively). Apparent dissociation constants of creatine, Kib and Kb (2.12+/-0.21 mM 2.17+/-0.40 Mm, correspondingly) were significantly decreased in situ in comparison with in vitro mitochondria (28+/-7 mM and 5+/-1.2 mM, respectively). Dissociation constant for phosphocreatine was not changed. These data may indicate selective restriction of metabolites' diffusion at the level of mitochondrial outer membrane. It is concluded that mechanisms of the regulation of respiration and energy fluxes in vivo are system level properties which depend on intracellular interactions of mitochondria with cytoskeleton, intracellular MgATPases and cytoplasmic glycolytic system.

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In this review, we summarize the main structural and functional data on the role of the phosphocreatine (PCr)--creatine kinase (CK) pathway for compartmentalized energy transfer in cardiac cells. Mitochondrial creatine kinase, MtCK, fixed by cardiolipin molecules in the vicinity of the adenine nucleotide translocator, is a key enzyme in this pathway. Direct transfer of ATP and ADP between these proteins has been revealed both in experimental studies on the kinetics of the regulation of mitochondrial respiration and by mathematical modelling as a main mechanism of functional coupling of PCr production to oxidative phosphorylation. In cells in vivo or in permeabilized cells in situ, this coupling is reinforced by limited permeability of the outer membrane of the mitochondria for adenine nucleotides due to the contacts with cytoskeletal proteins. Due to these mechanisms, at least 80% of total energy is exported from mitochondria by PCr molecules. Mathematical modelling of intracellular diffusion and energy transfer shows that the main function of the PCr-CK pathway is to connect different pools (compartments) of ATP and, by this way, to overcome the local restrictions and diffusion limitation of adenine nucleotides due to the high degree of structural organization of cardiac cells.

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The aim of this study was to investigate the mechanism of cellular regulation of mitochondrial respiration in permeabilized cardiac cells with clearly different structural organization: (i) in isolated rat cardiomyocytes with very regular mitochondrial arrangement, (ii) in HL-1 cells from mouse heart, and (iii) in non-beating (NB HL-1 cells) without sarcomeres with irregular and dynamic filamentous mitochondrial network. We found striking differences in the kinetics of respiration regulation by exogenous ADP between these cells: the apparent Km for exogenous ADP was by more than order of magnitude (14 times) lower in the permeabilized non-beating NB HL-1 cells without sarcomeres (25+/-4 microM) and seven times lower in normally cultured HL-1 cells (47+/-15 microM) than in permeabilized primary cardiomyocytes (360+/-51 microM). In the latter cells, treatment with trypsin resulted in dramatic changes in intracellular structure that were associated with 3-fold decrease in apparent Km for ADP in regulation of respiration. In contrast to permeabilized cardiomyocytes, in NB HL-1 cells creatine kinase activity was low and the endogenous ADP fluxes from MgATPases recorded spectrophotometrically by the coupled enzyme assay were not reduced after activation of mitochondrial oxidative phosphorylation by the addition of mitochondrial substrates, showing the absence of ADP channelling in the NB HL-1 cells. While in the permeabilized cardiomyocytes creatine strongly activated mitochondrial respiration even in the presence of powerful competing pyruvate kinase-phosphoenolpyruvate system, in the NB HL-1 cells the stimulatory effect of creatine was not significant. The results of this study show that in normal adult cardiomyocytes and HL-1 cells intracellular local restrictions of diffusion of adenine nucleotides and metabolic feedback regulation of respiration via phosphotransfer networks are different, most probably related to differences in structural organization of these cells.

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The present study discusses the role of structural organization of cardiac cells in determining the mechanisms of regulation of oxidative phosphorylation and interaction between mitochondria and ATPases. In permeabilized adult cardiomyocytes, the apparent K(m) (Michaelis-Menten constant) for ADP in the regulation of respiration is far higher than in mitochondria isolated from the myocardium. Respiration of mitochondria in permeabilized cardiomyocytes is effectively activated by endogenous ADP produced by ATPases from exogenous ATP, and the activation of respiration is associated with a decrease in the apparent K(m) for ATP in the regulation of ATPase activity compared with this parameter in the absence of oxidative phosphorylation. It has also been shown that a large fraction of the endogenous ADP stimulating respiration remains inaccessible for the exogenous ADP trapping system, consisting of pyruvate kinase and phosphoenolpyruvate, unless the mitochondrial structures are modified by controlled proteolysis. These data point to the endogenous cycling of adenine nucleotides between mitochondria and ATPases. Accordingly, the current hypothesis is that in cardiac cells, mitochondria and ATPases are compartmentalized into functional complexes (ie, intracellular energetic units [ICEUs]), which appear to represent a basic pattern of organization of energy metabolism in these cells. Within the ICEUs, the mitochondria and ATPases interact via different routes: creatine kinase-mediated phosphoryltransfer; adenylate kinase-mediated phosphoryltransfer; and direct ATP and ADP channelling. The function of ICEUs changes not only after selective proteolysis, but also during contraction of cardiomyocytes caused by an increase in cytosolic Ca(2+) concentration up to micromolar levels. In these conditions, the apparent K(m) for exogenous ADP and ATP in the regulation of respiration markedly decreases, and more ADP becomes available for the exogenous pyruvate kinase-phosphoenolpyruvate system, which indicates altered barrier functions of the ICEUs. Thus, structural changes transmitted from the contractile apparatus to mitochondria clearly participate in the regulation of mitochondrial function due to alterations in localized restriction of the diffusion of adenine nucleotides. The importance of strict structural organization in cardiac cells emerged drastically from experiments in which the regulation of mitochondrial respiration was assessed in a novel cardiac cell line, that is, beating and nonbeating HL-1 cells. In these cells, the mitochondrial arrangement is irregular and dynamic, whereas the sarcomeric structures are either absent (in nonbeating HL-1 cells) or only rarely present (in beating HL-1 cells). In parallel, the apparent K(m) for exogenous ADP in the regulation of respiration was much lower than that in permeabilized primary cardiomyocytes, and trypsin treatment exerted no impact on the low K(m) value for ADP, in contrast to adult cardiomyocytes where it caused a marked decrease in this parameter. The HL-1 cells were also characterized by the absence of direct exchange of adenine nucleotides. The results further support the concept that the ICEUs in adult cardiomyocytes are products of complex structural organization developed to create the most optimal conditions for effective energy transfer and feedback between mitochondria and ATPases.

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The relationships between cardiac cell structure and the regulation of mitochondrial respiration were studied by applying fluorescent confocal microscopy and analysing the kinetics of mitochondrial ADP-stimulated respiration, during calcium-induced contraction in permeabilized cardiomyocytes and myocardial fibers, and in their 'ghost' preparations (after selective myosin extraction). Up to 3 microm free calcium, in the presence of ATP, induced strong contraction of permeabilized cardiomyocytes with intact sarcomeres, accompanied by alterations in mitochondrial arrangement and a significant decrease in the apparent K(m) for exogenous ADP and ATP in the kinetics of mitochondrial respiration. The V(max) of respiration showed a moderate (50%) increase, with an optimum at 0.4 microm free calcium and a decrease at higher calcium concentrations. At high free-calcium concentrations, the direct flux of ADP from ATPases to mitochondria was diminished compared to that at low calcium levels. All of these effects were unrelated either to mitochondrial calcium overload or to mitochondrial permeability transition and were not observed in 'ghost' preparations after the selective extraction of myosin. Our results suggest that the structural changes transmitted from contractile apparatus to mitochondria modify localized restrictions of the diffusion of adenine nucleotides and thus may actively participate in the regulation of mitochondrial function, in addition to the metabolic signalling via the creatine kinase system.

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A method for the in situ measurement of the kinetics of ATP metabolic transformation using capillary electrophoresis (CE) has been developed. The depletion of ATP and formation of ADP were monitored in situ by using saponin-permeabilized muscle fibers. The method of micellar electrokinetic chromatography, employing reversed electroosmotic flow by cationic surfactant and reversed-polarity mode, provided an efficient and reproducible separation of nucleotides and enabled kinetic analysis of the reaction to be performed in a large range of nucleotide concentrations that approaches physiological concentrations of ATP in the muscle cells, without the need for precipitation of proteins prior to sample application. The analytes were detected at a nM level with a reproducibility of about 7%. This reproducibility enabled the comparison of different competing kinetic models of ATP conversion to ADP and the results show that the MgATPase activity in the fast-twitch gastrocnemius muscle followed biphasic kinetics that corresponds to the allosteric character of regulation of the enzyme(s) activity at physiological ATP concentrations. The results also confirmed that the combination of minimal sample volume requirements, rapid measurement and reproducibility makes the micellar CE a valuable tool for the analysis of biological fluids and understanding the processes of biological interest.

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Applications of permeabilized cell and skinned fiber techniques in combination with methods of mathematical modelling for studies of mitochondrial function in the cell are critically evaluated. Mathematical models may be useful tools for explaining biological phenomena, but only if they are selected by fitting the computing results with real experimental data. Confocal microscopy has been used in experiments with permeabilized cardiomyocytes and myocardial fibers to determine the maximal diffusion distance from medium to the core of cells, which is shown not to exceed 8-10 microm. This is a principal index for correctly explaining high apparent Km for exogenous ADP (200-300 microM) in regulation of mitochondrial respiration in oxidative muscle cells in situ. The best fitting of the results of in silico studies may be achieved by using of the compartmentalized energy transfer model. From these results, it may be concluded that in cardiac muscle cells the mitochondria and ATPases are organized into intracellular energetic units (ICEUs) separated from the bulk phase of cytoplasm by some barriers which limit the diffusion of adenine nucleotides. In contrast, alternative models based on the concept of the cell as homogenous system do not explain the observed experimental phenomena and have led to misleading conclusions. The various sources of experimental and conceptual errors are analyzed.

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Heterogeneity of ADP diffusion and regulation of respiration were studied in permeabilized cardiomyocytes and cardiac fibers in situ and in silico. Regular arrangement of mitochondria in cells was altered by short-time treatment with trypsin and visualized by confocal microscopy. Manipulation of matrix volumes by changing K(+) and sucrose concentrations did not affect the affinity for ADP either in isolated heart mitochondria or in skinned fibers. Pyruvate kinase (PK)-phosphoenolpyruvate (PEP) were used to trap ADP generated in Ca,MgATPase reactions. Inhibition of respiration by PK-PEP increased 2-3 times after disorganization of regular mitochondrial arrangement in cells. ADP produced locally in the mitochondrial creatine kinase reaction was not accessible to PK-PEP in intact permeabilized fibers, but some part of it was released from mitochondria after short proteolysis due to increased permeability of outer mitochondrial membrane. In in silico studies we show by mathematical modeling that these results can be explained by heterogeneity of ADP diffusion due to its restrictions at the outer mitochondrial membrane and in close areas, which is changed after proteolysis. Localized restrictions and heterogeneity of ADP diffusion demonstrate the importance of mitochondrial functional complexes with sarcoplasmic reticulum and myofibrillar structures and creatine kinase in regulation of oxidative phosphorylation.

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The origin of significant differences between the apparent affinities of heart mitochondrial respiration for exogenous ADP in isolated mitochondria in vitro and in permeabilized cardiomyocytes or skinned fibres in situ is critically analysed. All experimental data demonstrate the importance of structural factors of intracellular arrangement of mitochondria into functional complexes with myofibrils and sarcoplasmic reticulum in oxidative muscle cells and the control of outer mitochondrial membrane permeability. It has been shown that the high apparent K(m) for exogenous ADP (250-350 mM) in permeabilized cells and in ghost cells (without myosin) and fibres (diameter 15-20 mm) is independent of intrinsic MgATPase activity. However, the K(m) may be decreased significantly by a selective proteolytic treatment, which also destroys the regular arrangement of mitochondria between sarcomeres and increases the accessibility of endogenous ADP to the exogenous pyruvate kinase-phosphoenolpyruvate system. The confocal microscopy was used to study the changes in intracellular distribution of mitochondria and localization of cytoskeletal proteins, such as desmin, tubulin and plectin in permeabilized cardiac cells during short proteolytic treatment. The results show the rapid collapse of microtubular and plectin networks but not of desmin localization under these conditions. These results point to the participation of cytoskeletal proteins in the intracellular organization and control of mitochondrial function in the cells in vivo, where mitochondria are incorporated into functional complexes with sarcomeres and sarcoplasmic reticulum.

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The effects of Bax (full-length, FL, and C-terminal truncated, DeltaC) on respiration rate, membrane potential, MgATPase activity and kinetics of regulation of respiration were studied in isolated rat heart mitochondria and permeabilized cardiomyocytes. The results showed that while both Bax-FL and Bax-DeltaC permeabilized the outer mitochondrial membrane, released cytochrome c and reduced the respiration rate, the latter could be fully restored by exogenous cytochrome c only in the case of Bax-DeltaC, but not in presence of Bax-FL. In addition, Bax-FL but not Bax-DeltaC increased the MgATPase activity, and their effects on the mitochondrial membrane potential were quantitatively different. None of these effects was sensitive to cyclosporin A (CsA). It is concluded that Bax-FL affects both the outer and the inner mitochondrial membranes by: (1) opening large pores in the outer membrane; (2) inhibiting some segments of the respiratory chain in the inner membrane; and (3) uncoupling the inner mitochondrial membrane by increasing proton leak without opening the permeability transition pore (PTP).

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