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.

Experimental and in silico studies of regulation of mitochondrial respiration in permeabilized muscle fibers have revealed functional complexes of mitochondria with myofibrils and sarcoplasmic reticulum-intracellular energetic units-as basic pattern of organization of cell energy metabolism in oxidative muscle cells (Saks et al. Biochem. J. 356, 643-657, 2001). In this paper we review new data showing that in the cells in vivo there are at least two major factors of the control of mitochondrial function which are lost in experiments in vitro: mitochondrial position in the cell with respect to other cellular structures, and the tightly controlled mitochondrial outer membrane. Because of this, the local concentration of ADP available in the vicinity of ATP/ADP translocase is strictly controlled by the organized metabolic networks of energy transfer including the creatine kinase and adenylate kinase systems within the intracellular energetic units. Mathematical modelling of energy fluxes within these units satisfactorily explains the linear relationship between heart work and oxygen uptake under conditions of metabolic stability, and allows to quantitatively analyse the mechanism of the regulation of mitochondrial function in the cells in vivo. The knowledge of metabolic changes within the functional complexes of mitochondria with MgATPases in normal and pathological conditions may help to explain the mechanism of acute ischemic cardiac failure.

SUMMARY 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 μm) at different (0–3 μmoll-1) free Ca2+ concentrations in the medium. In all these preparations, the apparent Km of mitochondrial respiration for exogenous ADP at free Ca2+ concentrations of 0–0.1μ moll-1 was very high, in the range of 250–350μ moll-1, in contrast to isolated mitochondria in vitro (apparent Km for ADP is approximately 20μ moll-1). An increase in the free Ca2+ concentration (up to 3 μmoll-1, which is within physiological range), resulted in a very significant decrease of the apparent Km value to 20–30 μmoll-1, a decrease of Vmax 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 Km for ADP (300–350 μmoll-1) nor Vmax of respiration changed in the range of free Ca2+ 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.

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.

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 mum) at different (0-3 mumol l(-1)) free Ca2+ concentrations in the medium. In all these preparations, the apparent K., of mitochondrial respiration for exogenous ADP at free Ca2+ concentrations of 0-0.1 mumol l(-1) was very high, in the range of 250-350 mumol l(-1), in contrast to isolated mitochondria in vitro (apparent K. for ADP is approximately 20 mumol l(-1)). An increase in the free Ca2+ concentration (up to 3 mumol l(-1), which is within physiological range), resulted in a very significant decrease of the apparent K-m value to 20-30 mumol l(-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 mumol l(-1)) nor V-max of respiration changed in the range of free Ca2+ 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.

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).

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 20μM 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; 20–40units/ml) and phosphoenolpyruvate (PEP; 5mM), totally suppressed respiration activated by exogenous ADP, but the respiration maintained by endogenous ADP was not suppressed by more than 20–40%. Creatine (20mM) further activated respiration in the presence of ATP and PK+PEP. Short treatment with trypsin (50–500nM for 5min) decreased the apparent Km for exogenous ADP from 300–350μM to 50–60μM, 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 Km 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.

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 Km for exogenous ADP in regulation of respiration was equal to 300–400 μM. However, when ADP production was initiated by intracellular ATPase reactions, the ADP concentration in the medium leveled off at about 40 μM 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.

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 (γ) 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.