In vivo regulation of mitochondrial respiration in cardiomyocytes: specific restrictions for intracellular diffusion of ADP

Relative diffusivities of ADP and creatine in cardiomyocytes were studied. The isolated rat cardiomyocytes were lysed with saponin (40 micrograms/ml) to perforate or completely disrupt sarcolemma that was evidenced by leakage of 80-100% lactate dehydrogenase. In these cardiomyocytes mitochondria were used as 'enzymatic probes' to determine the average local concentration of substrates exerting acceptor control of respiration--ADP or creatine (the latter activates respiration via mitochondrial creatine kinase reaction)--when their concentrations in the surrounding medium were changed. The kinetic parameters for ADP and creatine in control of respiration of saponin-treated cardiomyocytes were compared with those determined in isolated mitochondria and skinned cardiac fibers. The apparent Km for creatine (at 0.2 mM ATP) was very close and in a range of 6.0-6.9 mM in all systems studied, showing the absence of diffusion difficulties for this substrate. On the contrary, the apparent Km for ADP increased from 18 +/- 1 microM for isolated mitochondria to 250 +/- 59 microM for cardiomyocytes with the lysed sarcolemma and to 264 +/- 57 microM for skinned fibers. This elevation of Km was not eliminated by inhibition of myokinase with diadenosine pentaphosphate. When 25 mM creatine was present, the apparent Km for ADP decreased to 36 +/- 6 microM. These data are taken to indicate specific restrictions of diffusion of ADP most probably due to its interaction with intermediate binding sites in cardiomyocytes. The important role of phosphocreatine-creatine kinase system of energy transport is to overcome the restrictions in regulation of energy fluxes due to decreased diffusivity of ADP.     

Altered mitochondrial sensitivity for ADP and maintenance of creatine-stimulated respiration in oxidative striated muscles from VDAC1-deficient mice

Voltage-dependent anion channels (VDACs) form the main pathway for metabolites across the mitochondrial outer membrane. The mouse vdac1 gene has been disrupted by gene targeting, and the resulting mutant mice have been examined for defects in muscle physiology. To test the hypothesis that VDAC1 constitutes a pathway for ADP translocation into mitochondria, the apparent mitochondrial sensitivity for ADP (Km(ADP)) and the calculated rate of respiration in the presence of the maximal ADP concentration (Vmax) have been assessed using skinned fibers prepared from two oxidative muscles (ventricle and soleus) and a glycolytic muscle (gastrocnemius) in control and vdac1(-/-) mice. We observed a significant increase in the apparent Km((ADP)) in heart and gastrocnemius, whereas the V(max) remained unchanged in both muscles. In contrast, a significant decrease in both the apparent Km((ADP)) and V(max) was observed in soleus. To test whether VDAC1 is required for creatine stimulation of mitochondrial respiration in oxidative muscles, the apparent Km((ADP)) and Vmax were determined in the presence of 25 mm creatine. The creatine effect on mitochondrial respiration was unchanged in both heart and soleus. These data, together with the significant increase in citrate synthase activity in heart, but not in soleus and gastrocnemius, suggest that distinct metabolic responses to altered mitochondrial outer membrane permeability occur in these different striated muscle types.     

Regulation of respiration in brain mitochondria and synaptosomes: restrictions of ADP diffusion in situ, roles of tubulin, and mitochondrial creatine kinase

The role of ubiquitous mitochondrial creatine kinase (uMtCK) reaction in regulation of mitochondrial respiration was studied in purified preparations of rat brain synaptosomes and mitochondria. In permeabilized synaptosomes, apparent Km for exogenous ADP, Km (ADP), in regulation of respiration in situ was rather high (110 +/- 11 microM) in comparison with isolated brain mitochondria (9 +/- 1 microM). This apparent Km for ADP observed in isolated mitochondria in vitro dramatically increased to 169 +/- 52 microM after their incubation with 1 muM of dimeric tubulin showing that in rat brain, particularly in synaptosomes, mitochondrial outer membrane permeability for ADP, and ATP may be restricted by tubulin binding to voltage dependent anion channel (VDAC). On the other hand, in synaptosomes apparent Km (ADP) decreased to 25 +/- 1 microM in the presence of 20 mM creatine. To fully understand this effect of creatine on kinetics of respiration regulation, complete kinetic analysis of uMtCK reaction in isolated brain mitochondria was carried out. This showed that oxidative phosphorylation specifically altered only the dissociation constants for MgATP, by decreasing that from ternary complex MtCK.Cr.MgATP (K (a)) from 0.13 +/- 0.02 to 0.018 +/- 0.007 mM and that from binary complex MtCK.MgATP (K (ia)) from 1.1 +/- 0.29 mM to 0.17 +/- 0.07 mM. Apparent decrease of dissociation constants for MgATP reflects effective cycling of ATP and ADP between uMtCK and adenine nucleotide translocase (ANT). These results emphasize important role and various pathophysiological implications of the phosphocreatine-creatine kinase system in energy transfer in brain cells, including synaptosomes.     

On the regulation of cellular energetics in health and disease

Very recent experimental data, obtained by using the permeabilized cell technique or tissue homogenates for investigation of the mechanisms of regulation of respiration in the cells in vivo, are shortly summarized. In these studies, surprisingly high values of apparent Km for ADP, exceeding that for isolated mitochondria in vitro by more than order of magnitude, were recorded for heart, slow twitch skeletal muscle, hepatocytes, brain tissue homogenates but not for fast twitch skeletal muscle. Mitochondrial swelling in the hypo-osmotic medium resulted in the sharp decrease of the value of Km for ADP in correlation with the degree of rupture of mitochondrial outer membrane, as determined by the cytochrome c test. Very similar effect was observed when trypsin was used for treatment of skinned fibers, permeabilized cells or homogenates. It is concluded that, in many but not all types of cells, the permeability of the mitochondrial outer membrane for ADP is controlled by some cytoplasmic protein factor(s). Since colchicine and taxol were not found to change high values of the apparent Km for ADP, the participation of microtubular system seems to be excluded in this kind of control of respiration but studies of the roles of other cytoskeletal structures seem to be of high interest.In acute ischemia we observed rapid increase of the permeability of the mitochondrial outer membrane for ADP due to mitochondrial swelling and concomitant loss of creatine control of respiration as a result of dissociation of creatine kinase from the inner mitochondrial membrane. The extent of these damages was decreased by use of proper procedures of myocardial protection showing that outer mitochondrial membrane permeability and creatine control of respiration are valuable indices of myocardial preservation. In contrast to acute ischemia, chronic hypoxia seems to improve the cardiac cell energetics as seen from better postischemic recovery of phosphocreatine, and phosphocreatine overshoot after inotropic stimulation.In general, adaptational possibilities and pathophysiological changes in the mitochondrial outer membrane system point to the central role such a system may play in regulation of cellular energetics in vivo.     

Relevance of fatty acid oxidation in regulation of the outer mitochondrial membrane permeability for ADP.

The present study on saponin-treated rat heart muscle fibers has revealed a new function of the fatty acid oxidation system in the regulation of the outer mitochondrial membrane (OMM) permeability for ADP. It is found that oxidation of palmitoyl-CoA+carnitine, palmitoyl-L-carnitine and octanoyl-L-carnitine (alone or in combination with pyruvate+malate) dramatically decreased a very high value of apparent K(m) of oxidative phosphorylation for ADP. Octanoyl-D-carnitine, as well as palmitate, palmitoyl-CoA, and palmitoyl-L-carnitine were not effective in this respect, when their oxidation was prevented by the absence of necessary cofactors or blocked with rotenone. Our data suggest that oxidation, but not transport of fatty acids into mitochondria, induces an increase in the OMM permeability for ADP.     

Detection of early ischemic damage by analysis of mitochondrial function in skinned fibers

The skinned fibers technique was applied for studies of the effects of global acute ischemia (1 h at 37°C) and long time (15 h) hypothermic (4°C) preservation of isolated rat hearts under different conditions (immersion or low-flow perfusion) on mitochondrial function in the cells in vivo. Skinned fibers were obtained by using saponin for permeabilization of the sarcolemma in separated fiber bundles cut from left ventricle. The experimental protocol of the respiration rate determination included a cytochrome c test to check the intactness of the outer mitochondrial membrane. The apparent K_m for ADP and the effect of creatine on the mitochondrial activity were also evaluated in these permeabilized fibers, taken from different groups of hearts. The preservation of low-flow perfused hearts resulted only in a slight decrease of creatine (20 mM) stimulated respiration at 0.1 mM ADP. The fibers from ischemic hearts or from hearts preserved by immersion showed a decrease of the apparent K_m for ADP, and a complete loss of the stimulatory effect of creatine. In these fibers, we could observe that the outer mitochondrial membrane was damaged. In conclusion, the results of this study show that assessment of mitochondrial parameters sensitive to organelles swelling – intactness of outer membrane and functionally coupled creatine kinase reaction – are the most sensitive indicators of early hypoxic or ischemic damage to mitochondria. Their determination in biopsy samples could be used for evaluation of the efficiency of the cardiac protection in heart surgery. (Mol Cell Biochem 174: 79–85, 1997)     

Developmental changes in regulation of mitochondrial respiration by ADP and creatine in rat heart in vivo

In saponin-skinned muscle fibers from adult rat heart and m. soleus the apparent affinity of the mitochondrial oxidative phosphorylation system for ADP (K_m = 200-400 M) is much lower than in isolated mitochondria (K_m = 10-20 M). This suggests a limited permeability of the outer mitochondrial membrane (OMM) to adenine nucleotides in slow-twitch muscle cells. We have studied the postnatal changes in the affinity of mitochondrial respiration for ADP, in relation to morphological alterations and expression of mitochondrial creatine kinase (mi-CK) in rat heart in vivo. Analysis of respiration of skinned fibers revealed a gradual decrease in the apparent affinity of mitochondria to ADP throughout 6 weeks post partum that indicates the development of mechanism which increasingly limits the access of ADP to mitochondria. The expression of mi-CK started between the 1st and 2nd weeks and reached the adult levels after 6 weeks. This process was associated with increases in creatine-activated respiration and affinity of oxidative phosphorylation to ADP thus reflecting the progressive coupling of mi-CK to adenine nucleotide translocase. Laser confocal microscopy revealed significant changes in rearrangement of mitochondria in cardiac cells: while the mitochondria of variable shape and size appeared to be random-clustered in the cardiomyocytes of 1 day old rat, they formed a fine network between the myofibrils by the age of 3 weeks. These results allow to conclude that in early period of development, i.e. within 2-3 weeks, the diffusion of ADP to mitochondria becomes progressively restricted, that appears to be related to significant structural rearrangements such as formation of the mitochondrial network. Later (after 3 weeks) the control shifts to mi-CK, which by coupling to adenine nucleotide translocase, allows to maximally activate the processes of oxidative phosphorylation despite limited access of ADP through the OMM.     

Heterogeneity of ADP diffusion and regulation of respiration in cardiac cells

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.     

Compartmented coupling of chicken heart mitochondrial creatine kinase to the nucleotide translocase requires the outer mitochondrial membrane

The kinetic coupling of mitochondrial creatine kinase (MiMi-CK) to ADP/ATP translocase in chicken heart mitochondrial preparations is demonstrated. Measuring the MiMi-CK apparent Km value for MgATP2- (at saturating creatine) gives a value of 36 microM when MiMi-CK is coupled to oxidative phosphorylation. This Km value is threefold lower than the Km for enzyme bound to mitoplasts or free in solution. The nucleotide translocase Km value for ADP decreases from 20 to 10 microM in the presence of 50 mM creatine only with intact mitochondria. Similar experiments with mitoplasts do not give decreased Km values. The observed Km differences can be used to calculate the concentration of ATP and ADP under steady-state conditions showing that the observed differences in the kinetic constants accurately reflect the enzyme activities of MiMi-CK under the different conditions. The behavior of the Km values provides evidence for what we term compartmented coupling. Therefore, like the rabbit heart system (S. Erickson-Viitanen, P. Viitanen, P. J. Geiger, W. C. T. Yang, and S. P. Bessman (1982) J. Biol. Chem. 257, 14395-14404) compartmented coupling requires an intact outer mitochondrial membrane. The apparent Km values for normal or compartmentally coupled systems can be used to calculate steady-state values of ATP and ADP by coupling enzyme theory. Hence, the overall kinetic parameters accurately reflect the behavior of the enzymes whether free in solution or in the intermembrane space.     

Specific limitations for intracellular diffusion of ADP in cardiomyocytes

Isolated cardiomyocytes and bundles of cardiac fibers were studied after lysis of their sarcolemma by saponin (40-50 micrograms/ml). 60-70% of cardiomyocytes were rod-like and Ca2(+)-tolerant. The kinetics of stimulation of oxidative phosphorylation by ADP and creatine via the mitochondrial creatine kinase reaction: MgATP + creatine----MgADP + phosphocreatine, was investigated after perforation of sarcolemma. The criterion for sarcolemmal perforation was an almost complete (80-100%) leakage of lactate dehydrogenase. It was shown that the Km values for ADP during stimulation of oxidative phosphorylation in cardiomyocytes are 250 +/- 39 microM (264 +/- 57 microM in cardiac bundles) which exceeds by one order of magnitude the Km value for ADP in isolated mitochondria (18 +/- 5 microM). On the contrary, Km for creatine is the same for all preparations studied (6-6.9 mM). The data obtained suggest the absence of diffusion difficulties for creatine inside the cells. In contrast, intracellular diffusion of ADP is restricted, most probably, dye to its binding to intracellular structures. These data emphasize the crucial role of the creatine kinase system in energy transfer processes. In the presence of 25 mM creatine Km for ADP is decreased to 36 +/- 6 mM due to a manyfold use of ADP in the coupled creatine kinase-oxidative phosphorylation reaction occurring in mitochondria.     

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.     

Early ischemia-induced alterations of the outer mitochondrial membrane and the intermembrane space: A potential cause for altered energy transfer in cardiac muscle?

Our aim was to carefully analyse the time-dependent changes that affect the mitochondrial function of myocardial cells during and after an ischemic episode. To this end, variables characterizing mitochondrial function have been evaluated on myocardial samples from isolated rat hearts subjected to different conditions of ischemia. The technique of permeabilized fibers was used in order to evaluate the mitochondrial function whilst retaining intracellular structure.The earliest alteration that could be detected was a decrease in the stimulatory effect of creatine on mitochondrial respiration. This alteration became more pronounced as the severity (or duration) of the ischemia increased. Afterwards, a significant decrease in the apparent K_m of mitochondrial respiration for ADP also appeared, followed by a diminution of the maximal respiration rate which was partly restored by adding cytochrome c. Finally, for the most severe conditions of ischemia, the basal respiratory rate also increased. These observations are indicative of a sequence of alterations affecting first the intermembrane space, then the outer mitochondrial membrane, and finally the inner membrane. The discussion is focused on the very early alterations, that could not be detected using the conventional techniques of isolated mitochondria. We postulate that these alterations to the intermembrane space and outer mitochondrial membrane can induce disturbances both in the channelling of energy from the mitochondria, and on the signalling towards the mitochondria. The potential consequences on the regulation of the production of energy (ATP, PC) by the mitochondria are evoked.     

Different kinetics of the regulation of respiration in permeabilized cardiomyocytes and in HL-1 cardiac cells. Importance of cell structure/organization for respiration regulation

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.     

Diffusion barriers for ADP in the cardiac cell

The regulation of mitochondrial respiration in the intact heart may differ from that of isolated mitochondria if intracellular diffusion is restricted. Here we consider which factors may hinder diffusion in vivo and, based on computational analysis, design a reverse engineering approach to estimate the role of diffusional resistance in mitochondrial regulation from an experiment on the intact heart. Computational analysis of respiration measurements on skinned heart fibers shows that the outer mitochondrial membrane does not hinder diffusion enough to cause ADP gradients of tens of micromolars. A diffusion model further shows that the mesoscale structure of the myofibrillar space also does not hinder diffusion appreciably. However, ADP gradients are suggested by the measured activation time of oxidative phosphorylation and may be caused by diffusion restriction of other intracellular structures or the in vivo microstructure of networks of physically interacting proteins. Based on computational modeling we propose an experiment on the intact heart that allows to estimate the effective diffusion restriction between ATP producing and consuming sites in the cardiac cell.     

Creatine kinase of rat heart mitochondria. The demonstration of functional coupling to oxidative phosphorylation in an inner membrane-matrix preparation

To define more clearly the interactions between mitochondrial creatine kinase and the adenine nucleotide translocase, the outer membrane of rat heart mitochondria was removed by digitonin, producing an inner membrane-matrix (mitoplast) preparation. This mitoplast fracton was well-coupled and contained a high specific activity of mitochondrial creatine kinase. Outer membrane permeabilization was documented by the loss of adenylate kinase, a soluble intermembrane enzyme, and by direct antibody inhibition of mitochondrial creatine kinase activity. With this preparation, we documented four important aspects of functional coupling. Kinetic studies showed that oxidative phosphorylation decreased the value of the ternary enzyme-substrate complex dissociation constant for MgATP from 140 to 16 microM. Two approaches were used to document the adenine nucleotide translocase specificity for ADP generated by mitochondrial creatine kinase. Exogenous pyruvate kinase (20 IU/ml) could not readily phosphorylate ADP produced by creatine kinase, since added pyruvate kinase did not markedly inhibit creatine + ATP-stimulated respiration. Additionally, when ADP was produced by mitochondrial creatine kinase, the inhibition of the translocase required 2 nmol of atractyloside/mg of mitoplast protein, while only 1 nmol/mg was necessary when exogenous ADP was added. Finally, the mass action ratio of the mitochondrial creatine kinase reaction exceeded the apparent equilibrium constant when ATP was supplied to the creatine kinase reaction by oxidative phosphorylation. Overall, these results are consistent with much data from intact rat heart mitochondria, and suggest that the outer membrane plays a minor role in the compartmentation of adenine nucleotides. Furthermore, since the removal of the outer membrane does not alter the unique coupling between oxidative phosphorylation and mitochondrial creatine kinase, we suggest that this cooperation is the result of protein-protein proximity at the inner membrane surface.     

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.     

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.     

The quantitation of ADP diffusion gradients across the outer membrane of heart mitochondria in the presence of macromolecules

We have previously provided evidence that diffusion of metabolites across the porin pores of mitochondrial outer membrane is hindered. A functional consequence of this diffusion limitation is the dynamic compartmentation of ADP in the intermembrane space. These earlier studies were done on isolated mitochondria suspended in isotonic media without macromolecules, in which intermembrane space of mitochondria is enlarged. The present study was undertaken to assess the diffusion limitation of outer membrane in the presence of 10% (w/v) dextran M20, in order to mimic the action of cytosolic macromolecules on mitochondria. Under these conditions, mitochondria have a more native, condensed configuration.Flux-dependent concentration gradients of ADP were estimated by measuring the ADP diffusion fluxes across the porin pores of isolated rat heart mitochondria incubated together with pyruvate kinase (PK), both of which compete for ADP regenerated by mitochondrial creatine kinase (mtCK) within the intermembrane space or by yeast hexokinase (HK) extramitochondrially. From diffusion fluxes and bulk phase concentrations of ADP, its concentrations in the intermembrane space were calculated using Fick's law of diffusion. Flux-dependent gradients up to 23 microM ADP (for a diffusion rate of J(Dif)=1.9 micromol ADP/min/mg mitochondrial protein) were observed. These gradients are about twice those estimated in the absence of dextran and in the same order of magnitude as the cytosolic ADP concentration (30 microM), but they are negligibly low for cytosolic ATP (5 mM). Therefore, it is concluded that the dynamic ADP compartmentation is of biological importance for intact heart cells.If mtCK generates ADP within the intermembrane space, the local ADP concentration can be clearly higher than in the cytosol resulting in higher extramitochondrial phosphorylation potentials. In this way, mtCK contributes to ensure optimal kinetic conditions for ATP-splitting reactions in the extramitochondrial compartment.     

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.     

The effects of ischemia and experimental conditions on the respiration rate of cardiac fibers

The mitochondrial respiratory parameters were measured in situ, i.e. in saponin-skinned rabbit cardiac fibers and in fibers treated with saponin + collagenase. It was found that the decrease of maximal ADP-stimulated respiration rate of saponin-skinned fibers with pyruvate + malate under the conditions of total ischemia (0.5–1.5 h) is less pronounced as compared to isolated mitochondria. Maximal succinate oxidation rate (+ADP), however, was not different from control (1 h ischemia) but it exceeded the control level when measured in the medium supplemented with cytochrome c. It was also demonstrated that treatment of fibers with collagenase alone or in combination with saponin significantly (almost 2 fold) enhanced the maximal ADP-stimulated respiration rate if compared with saponin-skinned fibers. The data obtained suggest that mitochondrial respiration in saponin-skinned rabit cardiac fibers is not completely revealed, most probably, due to insufficient permeabilization of sarcolemma by saponin and, thus, inadequate accessibility of mitochondria to exogenous substrates, ADP in particular. These parameters can be improved by pre-treatment of fibers with collagenase. (Mol Cell Biochem 174: 87–90, 1997)