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.     

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.     

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.     

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.     

What controls the outer mitochondrial membrane permeability for ADP: facts for and against the role of oncotic pressure

In our study 10% of bovine serum albumin was added to the physiological incubation medium to mimic the oncotic pressure of the cellular cytoplasm and to test for its effect on the respiration of isolated rat heart mitochondria, saponin- or saponin plus crude collagenase (type IV)-treated heart muscle fibers and saponin-treated rat quadriceps muscle fibers. Pyruvate and malate were used as substrates. We found that albumin slightly decreased the maximal ADP-stimulated respiration rate only for saponin-treated heart muscle fibers. The apparent Km ADP of oxidative phosphorylation increased significantly, by 70-100%, for isolated heart mitochondria, saponin plus collagenase-treated heart muscle fibers and for saponin-treated quadriceps muscle fibers but remained unchanged for saponin-treated heart muscle fibers. The saponin-treated heart muscle fibers were characterized by a very high control apparent Km ADP value (234+/-24 microM ADP) compared with other preparations (14-28 microM ADP). The results suggest that in vivo the oncotic pressure is not the relevant factor causing the low outer mitochondrial membrane permeability for ADP in cardiomyocytes, in contrast to quadriceps muscle cells. It is likely that the outer mitochondrial membrane-bound protein(s) which is supposed to remain in saponin-treated heart muscle fibers is responsible for this property of the membrane.     

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.     

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.     

Mitochondrial creatine kinase in contact sites: interaction with porin and adenine nucleotide translocase, role in permeability transition and sensitivity to oxidative damage

The creatine/phosphocreatine circuit provides an efficient energy buffering and transport system in a variety of cells with high and fluctuating energy requirements. It connects sites of energy production (mitochondria, glycolysis) with sites of energy consumption (various cellular ATPases). The cellular creatine/phosphocreatine pool is linked to the ATP/ADP pool by the action of different isoforms of creatine kinase located at distinct subcellular compartments. Octameric mitochondrial creatine kinase (MtCK), together with porin and adenine nucleotide translocase, forms a microcompartment at contact sites between inner and outer mitochondrial membranes and facilitates the production and export into the cytosol of phosphocreatine. MtCK is probably in direct protein-protein contact with outer membrane porin, whereas interaction with inner membrane adenine nucleotide translocase is rather mediated by acidic phopholipids (like cardiolipin) present in significant amounts in the inner membrane. Octamer-dimer transitions of MtCK as well as different creatine kinase substrates have a profound influence on controlling mitochondrial permeability transition (MPT). Inactivation by reactive oxygen species of MtCK and destabilization of its octameric structure are factors that contribute to impairment of energy homeostasis and facilitated opening of the MPT pore, which eventually lead to tissue damage during periods of ischemia/reperfusion.     

Desmin cytoskeleton linked to muscle mitochondrial distribution and respiratory function

Ultrastructural studies have previously suggested potential association of intermediate filaments (IFs) with mitochondria. Thus, we have investigated mitochondrial distribution and function in muscle lacking the IF protein desmin. Immunostaining of skeletal muscle tissue sections, as well as histochemical staining for the mitochondrial marker enzymes cytochrome C oxidase and succinate dehydrogenase, demonstrate abnormal accumulation of subsarcolemmal clumps of mitochondria in predominantly slow twitch skeletal muscle of desmin-null mice. Ultrastructural observation of desmin-null cardiac muscle demonstrates in addition to clumping, extensive mitochondrial proliferation in a significant fraction of the myocytes, particularly after work overload. These alterations are frequently associated with swelling and degeneration of the mitochondrial matrix. Mitochondrial abnormalities can be detected very early, before other structural defects become obvious. To investigate related changes in mitochondrial function, we have analyzed ADP-stimulated respiration of isolated muscle mitochondria, and ADP-stimulated mitochondrial respiration in situ using saponin skinned muscle fibers. The in vitro maximal rates of respiration in isolated cardiac mitochondria from desmin-null and wild-type mice were similar. However, mitochondrial respiration in situ is significantly altered in desmin-null muscle. Both the maximal rate of ADP-stimulated oxygen consumption and the dissociation constant (K(m)) for ADP are significantly reduced in desmin-null cardiac and soleus muscle compared with controls. Respiratory parameters for desmin-null fast twitch gastrocnemius muscle were unaffected. Additionally, respiratory measurements in the presence of creatine indicate that coupling of creatine kinase and the adenine translocator is lost in desmin-null soleus muscle. This coupling is unaffected in cardiac muscle from desmin-null animals. All of these studies indicate that desmin IFs play a significant role in mitochondrial positioning and respiratory function in cardiac and skeletal muscle.     

Interaction of creatine kinase with phosphorylating rabbit heart mitochondria and mitoplasts

This paper demonstrates that the mitochondrial isoenzyme of creatine kinase (CKm) can be solubilized from rabbit heart mitochondria, the outer membrane of which has been removed or at least broken by a digitonin treatment or a short hypotonic exposure, but which has retained an important part of the capacity to phosphorylate ADP. Phosphate, ADP, or ATP, at concentrations which are used to study oxidative phosphorylation and creatine phosphate synthesis, solubilize CKm; the same is true with MgCl2 and KCl. The effect of adenine nucleotides does not seem to be due to their interaction with the adenine nucleotide translocase. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis shows that CKm is the main protein released in the described conditions; however, it does not amount to more than 1% of the total protein content of the mitoplasts. When the apparent Km for ATP of CKm was estimated by measuring creatine phosphate synthesis, the values obtained using water-treated mitochondria (0.21 mM) were slightly higher than those of intact mitochondria (0.12 mM) but the difference was not significant. In the former preparation 77% of CKm was in a soluble state. If we can extrapolate these results to intact mitochondria and suppose that in this case a fraction of CKm is also soluble in the intermembrane space, this does not support the theory of functional association between CKm and the adenine nucleotide translocase.     

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

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

Calcium-induced contraction of sarcomeres changes the regulation of mitochondrial respiration in permeabilized cardiac cells

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.     

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.     

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

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

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.     

Role of mitochondria–cytoskeleton interactions in respiration regulation and mitochondrial organization in striated muscles

The aim of this work was to study the regulation of respiration and energy fluxes in permeabilized oxidative and glycolytic skeletal muscle fibers, focusing also on the role of cytoskeletal protein tubulin βII isotype in mitochondrial metabolism and organization. By analyzing accessibility of mitochondrial ADP, using respirometry and pyruvate kinase–phosphoenolpyruvate trapping system for ADP, we show that the apparent affinity of respiration for ADP can be directly linked to the permeability of the mitochondrial outer membrane (MOM). Previous studies have shown that MOM permeability in cardiomyocytes can be regulated by VDAC interaction with cytoskeletal protein, βII tubulin. We found that in oxidative soleus skeletal muscle the high apparent K_m for ADP is associated with low MOM permeability and high expression of non-polymerized βII tubulin. Very low expression of non-polymerized form of βII tubulin in glycolytic muscles is associated with high MOM permeability for adenine nucleotides (low apparent K_m for ADP).     

Inhibition of the mitochondrial permeability transition by creatine kinase substrates. Requirement for microcompartmentation

Mitochondria from transgenic mice, expressing enzymatically active mitochondrial creatine kinase in liver, were analyzed for opening of the permeability transition pore in the absence and presence of creatine kinase substrates but with no external adenine nucleotides added. In mitochondria from these transgenic mice, cyclosporin A-inhibited pore opening was delayed by creatine or cyclocreatine but not by beta-guanidinopropionic acid. This observation correlated with the ability of these substrates to stimulate state 3 respiration in the presence of extramitochondrial ATP. The dependence of transition pore opening on calcium and magnesium concentration was studied in the presence and absence of creatine. If mitochondrial creatine kinase activity decreased (i.e. by omitting magnesium from the medium), protection of permeability transition pore opening by creatine or cyclocreatine was no longer seen. Likewise, when creatine kinase was added externally to liver mitochondria from wild-type mice that do not express mitochondrial creatine kinase in liver, no protective effect on pore opening by creatine and its analog was observed. All these findings indicate that mitochondrial creatine kinase activity located within the intermembrane and intercristae space, in conjunction with its tight functional coupling to oxidative phosphorylation, via the adenine nucleotide translocase, can modulate mitochondrial permeability transition in the presence of creatine. These results are of relevance for the design of creatine analogs for cell protection as potential adjuvant therapeutic tools against neurodegenerative diseases.     

Direct measurement of energy fluxes from mitochondria into cytoplasm in permeabilized cardiac cells <Emphasis Type="Italic">in situ:</Emphasis> some evidence for mitochondrial interactosome

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/O₂ 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 μM to 330 ± 47 μM, but creatine again decreased it to 23 ± 6 μM. 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.     

Metabolic compartmentation and substrate channelling in muscle cells

The published experimental data and existing concepts of cellular regulation of respiration are analyzed. Conventional, simplified considerations of regulatory mechanism by cytoplasmic ADP according to Michaelis-Menten kinetics or by derived parameters such as phosphate potential etc. do not explain relationships between oxygen consumption, workload and metabolic state of the cell. On the other hand, there are abundant data in literature showing microheterogeneity of cytoplasmic space in muscle cells, in particular with respect to ATP (and ADP) due to the structural organization of cell interior, existence of multienzyme complexes and structured water phase. Also very recent experimental data show that the intracellular diffusion of ADP is retarded in cardiomyocytes because of very low permeability of the mitochondrial outer membrane for adenine nucleotidesin vivo. Most probably, permeability of the outer mitochondrial membrane porin channels is controlled in the cellsin vivo by some intracellular factors which may be connected to cytoskeleton and lost during mitochondrial isolation. All these numerous data show convincingly that cellular metabolism cannot be understood if cell interior is considered as homogenous solution, and it is necessary to use the theories of organized metabolic systems and substrate-product channelling in multienzyme systems to understand metabolic regulation of respiration. One of these systems is the creatine kinase system, which channels high energy phosphates from mitochondria to sites of energy utilization. It is proposed that in muscle cells feed-back signal between contraction and mitochondrial respiration may be conducted by metabolic wave (propagation of oscillations of local concentration of ADP and creatine) through cytoplasmic equilibrium creatine and adenylate kinases and is amplified by coupled creatine kinase reaction in mitochondria. Mitochondrial creatine kinase has experimentally been shown to be a powerful amplifier of regulatory action of weak ADP fluxes due to its coupling to adenine nucleotide translocase. This phenomenon is also carefully analyzed.It is easier to explain biochemistry in terms of transport than it is to explain transport in terms of biochemistry. P. Mitchell The Ninth Sir Hans Krebs Lecture, Dresden, July 2, 1978.     

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.