Südamelihase rakkude struktuuri olulisus rakuhingamise regulatsioonis

Viimastel aastatel on järjest selgemaks saanud seos raku energeetilise ainevahetuse ja südamehaiguste vahel, mistõttu on oluline uurida seda mõjutavaid tegureid. Töös uuriti südamelihase rakkude mitokondriaalse hingamise regulatsiooni väga erineva rakustruktuuriga preparaatides: 1) permeabiliseeritud kardiomüotsüütides, kus mitokondrid on regulaarselt organiseeritud; 2) südamelihase fenotüübiga sarnastes kontraheeruvates HL-1 (B HL-1) rakkudes ja 3) HL-1 mittekontraheeruvates (NB HL-1) rakkudes. Nende preparaatide vahel esines suur erinevus mitokondriaalse hingamise regulatsioonis. Selline tulemus näitab raku struktuuri ja funktsiooni vaheliste seoste tähtsust südamelihase rakkudes ning võimaldab paremini mõista protsesse nii terves kui ka patoloogilises südamelihases. Eesti Arst 2008; 87(1):19−2

Etude du contrôle de la respiration mitochondriale des cellules musculaires (rôle de la membrane externe)

GRENOBLE1-BU Sciences (384212103) / SudocSudocFranceF

Control of cellular respiration in vivo by mitochondrial outer membrane and by creatine kinase. A new speculative hypothesis: possible involvement of mitochondrial-cytoskeleton interactions.

International audienceThe current problems of regulation of myocardial energy metabolism and oxidative phosphorylation in vivo are considered. With this purpose, retarded diffusion of ADP in cardiomyocytes was studied by analysis of elevated apparent Km for this substrate in regulation of respiration of saponin-skinned cardiac fibers, as compared to isolated mitochondria. Recently published data showing the importance of the outer mitochondrial membrane were compared with new experimental results on the proteolysis of skinned fibers and tissue homogenates. In both cases 10 min incubation and 0.125 mg/ml of trypsin resulted in a decrease of apparent Km for ADP from 297 +/- 35 and 228 +/- 16 to 109 +/- 2 and 36 +/- 16, respectively. Thus, the permeability of the outer mitochondrial membrane for ADP may be controlled by some unknown cytoplasmic protein(s), probably related to the cytoskeleton, which are separated from mitochondria during their isolation. The extent of expression of this protein(s) depends on the energy state and type of muscle. Activation of mitochondrial creatine kinase reaction coupled to oxidative phosphorylation overcomes the diffusion difficulties of ADP by amplifying the stimulatory effect of ADP on respiration. It is concluded that both cytoplasmic and mitochondrial creatine kinases, adenylate kinase and cytoplasmic factor controlling outer membrane permeability may participate in metabolic feedback regulation of respiration in muscle cells

Permeabilized cell and skinned fiber techniques in studies of mitochondrial function in vitro

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

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

International audienceIn saponin-skinned muscle fibers from adult rat heart and m. soleus the apparent affinity of the mitochondrial oxidative phosphorylation system for ADP (Km = 200-400 microM) is much lower than in isolated mitochondria (Km = 10-20 microM). 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

Striking differences between the kinetics of regulation of respiration by ADP in slow-twitch and fast-twitch muscles in vivo.

International audienceThe kinetics of in vivo regulation of mitochondrial respiration by ADP was studied in rat heart, slow-twitch skeletal muscle (soleus) and fast-twitch skeletal muscle (gastrocnemius, plantaris, quadriceps and tibialis anterior) by means of saponin-skinned fibres. Mitochondrial respiratory parameters were determined in the absence and presence of creatine (20 mM), and the effect of proteolytic enzymes (trypsin, chymotrypsin or elastase) on these parameters was investigated in detail. The results of these experiments confirm the observation of Veksler et al. [Veksler, V.I., Kuznetsov, A. V., Anflous, K., Mateo, P., van Deursen, J., Wieringa, B. & Ventura-Clapier, R. (1995) J. Biol. Chem. 270, 19921-19929], who studied muscle fibres from normal and transgenic mice, that the kinetics of respiration regulation in muscle cells is tissue specific. We found that in rat cardiac and soleus muscle fibres the apparent K(m) for respiration regulation was 300-400 microM and decreased to 50-80 microM in the presence of creatine. In contrast, in skinned fibres from gastrocnemius, plantaris, tibialis anterior and quadriceps muscles, this value was initially very low, 10-20 microM, i.e. the same as that is in isolated muscle mitochondria, and the effect of creatine was not observable under these experimental conditions. Treatment of the fibres with trypsin, chymotrypsin or elastase (0.125 micrograms/ml) for 15 min decreased the apparent K(m) for ADP in cardiac and soleus muscle fibres to 40-98 microM without significant alteration of Vmax or the intactness of outer mitochondrial membrane, as assessed by the cytochrome c test. In fibres from gastrocnemius, trypsin increased the apparent K(m) for ADP transiently. The effects of trypsin and chymotrypsin were studied in detail and found to be concentration dependent and time dependent. The effects were characterised by saturation phenomenon with respect to the proteolytic enzyme concentration, saturation being observed above 1 microM enzyme. These results are taken to show that in cardiac and slow-twitch skeletal muscle, the permeability of the outer mitochondrial membrane to adenine nucleotides is low and controlled by a cytoplasmic protein that is sensitive to trypsin and chymotrypsin. This protein may participate in feedback signal transduction by a mechanism of vectorial-ligand conduction. This protein factor is not expressed in fast-twitch skeletal muscle, in which cellular mechanism of regulation of respiration is probably very different from that of slow-twitch muscles

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

International audienceThe 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

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

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

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

International audienceRegulation 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

On the regulation of cellular energetics in health and disease.

International audienceVery 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 mitochondria 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 or 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