RESUMO
Mitochondria are major producers of reactive oxygen species (ROS) in many cells including cancer cells. However, complex interrelationships between mitochondrial ROS (mitoROS), mitochondrial membrane potential (ΔΨm) and Ca2+ are not completely understood. Using human carcinoma cells, we further highlight biphasic ROS dynamics: - gradual mitoROS increase followed by mitoROS flash. Also, we demonstrate heterogeneity in rates of mitoROS generation and flash initiation time. Comparing mitochondrial and near-extra-mitochondrial signals, we show that mechanisms of mitoROS flashes in single mitochondria, linked to mitochondrial permeability transition pore opening (ΔΨm collapse) and calcium sparks, may involve flash triggering by certain levels of external ROS released from the same mitochondria. In addition, mitochondria-mitochondria interactions can produce wave propagations of mitoROS flashes and ΔΨm collapses in cancer cells similar to phenomena of ROS-induced ROS release (RIRR). Our data suggest that in cancer cells RIRR, activation of mitoROS flashes and mitochondrial depolarization may involve participation of extramitochondrial-ROS produced either by individual mitochondria and/or by neighboring mitochondria. This could represent general mechanisms in ROS-ROS signaling with suggested role in both mitochondrial and cellular physiology and signaling.
Assuntos
Adenocarcinoma/patologia , Sinalização do Cálcio , Potencial da Membrana Mitocondrial , Mitocôndrias/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Antioxidantes/farmacologia , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Neoplasias do Colo/patologia , Ciclosporina/farmacologia , Fluoresceínas/química , Humanos , Microscopia Confocal , Microscopia de Fluorescência , Mitocôndrias/efeitos da radiação , Proteínas de Transporte da Membrana Mitocondrial/fisiologia , Poro de Transição de Permeabilidade Mitocondrial , Estresse Oxidativo , Fotoquímica , Proteínas Recombinantes/metabolismo , Rodaminas/química , Análise de Célula Única , Superóxido Dismutase/metabolismoRESUMO
Adult cardiomyocytes have highly organized intracellular structure and energy metabolism whose formation during postnatal development is still largely unclear. Our previous results together with the data from the literature suggest that cytoskeletal proteins, particularly ßII-tubulin, are involved in the formation of complexes between mitochondria and energy consumption sites. The aim of this study was to examine the arrangement of intracellular architecture parallel to the alterations in regulation of mitochondrial respiration in rat cardiomyocytes during postnatal development, from 1 day to 6 months. Respirometric measurements were performed to study the developmental alterations of mitochondrial function. Changes in the mitochondrial arrangement and cytoarchitecture of ßII- and αIV-tubulin were examined by confocal microscopy. Our results show that functional maturation of oxidative phosphorylation in mitochondria is completed much earlier than efficient feedback regulation is established between mitochondria and ATPases via creatine kinase system. These changes are accompanied by significant remodeling of regular intermyofibrillar mitochondrial arrays aligned along the bundles of ßII-tubulin. Additionally, we demonstrate that formation of regular arrangement of mitochondria is not sufficient per se to provide adult-like efficiency in metabolic feed-back regulation, but organized tubulin networks and reduction in mitochondrial outer membrane permeability for ADP are necessary as well. In conclusion, cardiomyocytes in rat heart become mature on the level of intracellular architecture and energy metabolism at the age of 3 months.
Assuntos
Desenvolvimento Embrionário/genética , Metabolismo Energético , Miócitos Cardíacos/ultraestrutura , Tubulina (Proteína)/metabolismo , Difosfato de Adenosina/metabolismo , Animais , Citoesqueleto , Embrião de Mamíferos/ultraestrutura , Humanos , Mitocôndrias Cardíacas/metabolismo , Mitocôndrias Cardíacas/ultraestrutura , Membranas Mitocondriais/metabolismo , Membranas Mitocondriais/ultraestrutura , Miocárdio/metabolismo , Miocárdio/ultraestrutura , Miócitos Cardíacos/metabolismo , Ratos , Tubulina (Proteína)/químicaRESUMO
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 ßII 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, ßII tubulin. We found that in oxidative soleus skeletal muscle the high apparent Km for ADP is associated with low MOM permeability and high expression of non-polymerized ßII tubulin. Very low expression of non-polymerized form of ßII tubulin in glycolytic muscles is associated with high MOM permeability for adenine nucleotides (low apparent Km for ADP).
Assuntos
Citoesqueleto/metabolismo , Mitocôndrias/metabolismo , Músculo Estriado/metabolismo , Difosfato de Adenosina/metabolismo , Animais , Western Blotting , Respiração Celular , Proteínas do Citoesqueleto/metabolismo , Metabolismo Energético , Masculino , Análise do Fluxo Metabólico , Microscopia Confocal , Membranas Mitocondriais/metabolismo , Miocárdio/metabolismo , Permeabilidade , Ratos , Ratos Wistar , Tubulina (Proteína)/metabolismoRESUMO
The aim of the present study is to clarify some aspects of the mechanisms of regulation of mitochondrial metabolism in neuroblastoma (NB) cells. Experiments were performed on murine Neuro-2a (N2a) cell line, and the same cells differentiated by all-trans-retinoic acid (dN2a) served as in vitro model of normal neurons. Oxygraphy and Metabolic Control Analysis (MCA) were applied to characterize the function of mitochondrial oxidative phosphorylation (OXPHOS) in NB cells. Flux control coefficients (FCCs) for components of the OXPHOS system were determined using titration studies with specific non-competitive inhibitors in the presence of exogenously added ADP. Respiration rates of undifferentiated Neuro-2a cells (uN2a) and the FCC of Complex-II in these cells were found to be considerably lower than those in dN2a cells. Our results show that NB is not an exclusively glycolytic tumor and could produce a considerable part of ATP via OXPHOS. Two important enzymes - hexokinase-2 and adenylate kinase-2 can play a role in the generation of ATP in NB cells. MCA has shown that in uN2a cells the key sites in the regulation of OXPHOS are complexes I, II and IV, whereas in dN2a cells complexes II and IV. Results obtained for the phosphate and adenine nucleotide carriers showed that in dN2a cells these carriers exerted lower control over the OXPHOS than in undifferentiated cells. The sum of FCCs for both types of NB cells was found to exceed significantly that for normal cells suggesting that in these cells the respiratory chain was somehow reorganized or assembled into large supercomplexes.
Assuntos
Mitocôndrias/metabolismo , Neuroblastoma/metabolismo , Neuroblastoma/patologia , Adenilato Quinase/metabolismo , Animais , Diferenciação Celular/fisiologia , Processos de Crescimento Celular/fisiologia , Creatina Quinase/metabolismo , Metabolismo Energético , Hexoquinase/metabolismo , Microscopia Confocal , Mitocôndrias/enzimologia , Neuroblastoma/enzimologia , RatosRESUMO
This review describes the recent experimental data on the importance of the VDAC-cytoskeleton interactions in determining the mechanisms of energy and metabolite transfer between mitochondria and cytoplasm in cardiac cells. In the intermembrane space mitochondrial creatine kinase connects VDAC with adenine nucleotide translocase and ATP synthase complex, on the cytoplasmic side VDAC is linked to cytoskeletal proteins. Applying immunofluorescent imaging and Western blot analysis we have shown that ß2-tubulin coexpressed with mitochondria is highly important for cardiac muscle cells mitochondrial metabolism. Since it has been shown by Rostovtseva et al. that αß-heterodimer of tubulin binds to VDAC and decreases its permeability, we suppose that the ß-tubulin subunit is bound on the cytoplasmic side and α-tubulin C-terminal tail is inserted into VDAC. Other cytoskeletal proteins, such as plectin and desmin may be involved in this process. The result of VDAC-cytoskeletal interactions is selective restriction of the channel permeability for adenine nucleotides but not for creatine or phosphocreatine that favors energy transfer via the phosphocreatine pathway. In some types of cancer cells these interactions are altered favoring the hexokinase binding and thus explaining the Warburg effect of increased glycolytic lactate production in these cells. This article is part of a Special Issue entitled: VDAC structure, function, and regulation of mitochondrial metabolism.
Assuntos
Creatina Quinase Mitocondrial/metabolismo , Citoesqueleto/metabolismo , Mitocôndrias/metabolismo , Células Musculares/citologia , Células Musculares/metabolismo , Canais de Ânion Dependentes de Voltagem/metabolismo , Animais , Respiração Celular , Humanos , Mitocôndrias/enzimologia , Ligação ProteicaRESUMO
Mitochondria are dynamic structures for which fusion and fission are well characterized for rapidly dividing cells in culture. Based on these data, it has recently been proposed that high respiratory activity is the result of fusion and formation of mitochondrial reticulum, while fission results in fragmented mitochondria with low respiratory activity. In this work we test the validity of this new hypothesis by analyzing our own experimental data obtained in studies of isolated heart mitochondria, permeabilized cells of cardiac phenotype with different mitochondrial arrangement and dynamics. Additionally, we reviewed published data including electron tomographic investigation of mitochondrial membrane-associated structures in heart cells. Oxygraphic studies show that maximal ADP-dependent respiration rates are equally high both in isolated heart mitochondria and in permeabilized cardiomyocytes. On the contrary, these rates are three times lower in NB HL-1 cells with fused mitochondrial reticulum. Confocal and electron tomographic studies show that there is no mitochondrial reticulum in cardiac cells, known to contain 5,000-10,000 individual, single mitochondria, which are regularly arranged at the level of sarcomeres and are at Z-lines separated from each other by membrane structures, including the T-tubular system in close connection to the sarcoplasmic reticulum. The new structural data in the literature show a principal role for the elaborated T-tubular system in organization of cell metabolism by supplying calcium, oxygen and substrates from the extracellular medium into local domains of the cardiac cells for calcium cycling within Calcium Release Units, associated with respiration and its regulation in Intracellular Energetic Units.
Assuntos
Respiração Celular/fisiologia , Mitocôndrias Cardíacas/metabolismo , Miócitos Cardíacos/metabolismo , Animais , Cálcio/metabolismo , Metabolismo Energético , Microscopia Confocal , Dinâmica Mitocondrial , Miócitos Cardíacos/citologiaRESUMO
The permeabilized cells and muscle fibres technique allows one to study the functional properties of mitochondria without their isolation, thus preserving all of the contacts with cellular structures, mostly the cytoskeleton, to study the whole mitochondrial population in the cell in their natural surroundings and it is increasingly being used in both experimental and clinical studies. The functional parameters (affinity for ADP in regulation of respiration) of mitochondria in permeabilized myocytes or myocardial fibres are very different from those in isolated mitochondria in vitro. In the present study, we have analysed the data showing the dependence of this parameter upon the muscle contractile state. Most remarkable is the effect of recently described Ca(2+)-independent contraction of permeabilized muscle fibres induced by elevated temperatures (30-37°C). We show that very similar strong spontaneous Ca(2+)-independent contraction can be produced by proteolytic treatment of permeabilized muscle fibres that result in a disorganization of mitochondrial arrangement, leading to a significant increase in affinity for ADP. These data show that Ca(2+)-insensitive contraction may be related to the destruction of cytoskeleton structures by intracellular proteases. Therefore the use of their inhibitors is strongly advised at the permeabilization step with careful washing of fibres or cells afterwards. A possible physiologically relevant relationship between Ca(2+)-regulated ATP-dependent contraction and mitochondrial functional parameters is also discussed.
Assuntos
Contração Muscular/fisiologia , Trifosfato de Adenosina/metabolismo , Animais , Cálcio/metabolismo , Técnicas In Vitro , Mitocôndrias Cardíacas/metabolismo , Mitocôndrias Musculares/metabolismo , Fibras Musculares Esqueléticas/fisiologia , Músculo Esquelético/fisiologia , Contração Miocárdica/fisiologia , Miócitos Cardíacos/fisiologia , RatosRESUMO
The aim of this study was to investigate the possible role of tubulin ßII, a cytoskeletal protein, in regulation of mitochondrial oxidative phosphorylation and energy fluxes in heart cells. This isotype of tubulin is closely associated with mitochondria and co-expressed with mitochondrial creatine kinase (MtCK). It can be rapidly removed by mild proteolytic treatment of permeabilized cardiomyocytes in the absence of stimulatory effect of cytochrome c, that demonstrating the intactness of the outer mitochondrial membrane. Contrary to isolated mitochondria, in permeabilized cardiomyocytes (in situ mitochondria) the addition of pyruvate kinase (PK) and phosphoenolpyruvate (PEP) in the presence of creatine had no effect on the rate of respiration controlled by activated MtCK, showing limited permeability of voltage-dependent anion channel (VDAC) in mitochondrial outer membrane (MOM) for ADP regenerated by MtCK. Under normal conditions, this effect can be considered as one of the most sensitive tests of the intactness of cardiomyocytes and controlled permeability of MOM for adenine nucleotides. However, proteolytic treatment of permeabilized cardiomyocytes with trypsin, by removing mitochondrial ßII tubulin, induces high sensitivity of MtCK-regulated respiration to PK-PEP, significantly changes its kinetics and the affinity to exogenous ADP. MtCK coupled to ATP synthasome and to VDAC controlled by tubulin ßII provides functional compartmentation of ATP in mitochondria and energy channeling into cytoplasm via phosphotransfer network. Therefore, direct transfer of mitochondrially produced ATP to sites of its utilization is largely avoided under physiological conditions, but may occur in pathology when mitochondria are damaged. This article is part of a Special Issue entitled ''Local Signaling in Myocytes''.
Assuntos
Metabolismo Energético/fisiologia , Mitocôndrias Cardíacas/metabolismo , Miócitos Cardíacos/metabolismo , Tubulina (Proteína)/metabolismo , Difosfato de Adenosina/metabolismo , Animais , Respiração Celular , Creatina Quinase Mitocondrial/metabolismo , Masculino , Microscopia Confocal , Microscopia de Fluorescência , Membranas Mitocondriais/metabolismo , Consumo de Oxigênio , Transporte Proteico , Ratos , Ratos WistarRESUMO
This review describes developments in historical perspective as well as recent results of investigations of cellular mechanisms of regulation of energy fluxes and mitochondrial respiration by cardiac work - the metabolic aspect of the Frank-Starling law of the heart. A Systems Biology solution to this problem needs the integration of physiological and biochemical mechanisms that take into account intracellular interactions of mitochondria with other cellular systems, in particular with cytoskeleton components. Recent data show that different tubulin isotypes are involved in the regular arrangement exhibited by mitochondria and ATP-consuming systems into Intracellular Energetic Units (ICEUs). Beta II tubulin association with the mitochondrial outer membrane, when co-expressed with mitochondrial creatine kinase (MtCK) specifically limits the permeability of voltage-dependent anion channel for adenine nucleotides. In the MtCK reaction this interaction changes the regulatory kinetics of respiration through a decrease in the affinity for adenine nucleotides and an increase in the affinity for creatine. Metabolic Control Analysis of the coupled MtCK-ATP Synthasome in permeabilized cardiomyocytes showed a significant increase in flux control by steps involved in ADP recycling. Mathematical modeling of compartmentalized energy transfer represented by ICEUs shows that cyclic changes in local ADP, Pi, phosphocreatine and creatine concentrations during contraction cycle represent effective metabolic feedback signals when amplified in the coupled non-equilibrium MtCK-ATP Synthasome reactions in mitochondria. This mechanism explains the regulation of respiration on beat to beat basis during workload changes under conditions of metabolic stability. This article is part of a Special Issue entitled "Local Signaling in Myocytes."
Assuntos
Metabolismo Energético/fisiologia , Miócitos Cardíacos/metabolismo , Animais , Permeabilidade da Membrana Celular , Citoesqueleto/metabolismo , Humanos , Espaço Intracelular/metabolismo , Mitocôndrias Cardíacas/metabolismo , Modelos Teóricos , Tubulina (Proteína)/metabolismoRESUMO
Mitochondria play central roles in cell life as a source of energy and in cell death by inducing apoptosis. Many important functions of mitochondria change in cancer, and these organelles can be a target of chemotherapy. The widely used anticancer drug doxorubicin (DOX) causes cell death, inhibition of cell cycle/proliferation and mitochondrial impairment. However, the mechanism of such impairment is not completely understood. In our study we used confocal and two-photon fluorescence imaging together with enzymatic and respirometric analysis to study short- and long-term effects of doxorubicin on mitochondria in various human carcinoma cells. We show that short-term (<30 min) effects include i) rapid changes in mitochondrial redox potentials towards a more oxidized state (flavoproteins and NADH), ii) mitochondrial depolarization, iii) elevated matrix calcium levels, and iv) mitochondrial ROS production, demonstrating a complex pattern of mitochondrial alterations. Significant inhibition of mitochondrial endogenous and uncoupled respiration, ATP depletion and changes in the activities of marker enzymes were observed after 48 h of DOX treatment (long-term effects) associated with cell cycle arrest and death.
Assuntos
Apoptose/efeitos dos fármacos , Cálcio/metabolismo , Doxorrubicina/farmacologia , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Mitocôndrias/efeitos dos fármacos , Trifosfato de Adenosina/metabolismo , Antibióticos Antineoplásicos/farmacologia , Carbonil Cianeto p-Trifluormetoxifenil Hidrazona/farmacologia , Ciclo Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Respiração Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Citrato (si)-Sintase/metabolismo , Relação Dose-Resposta a Droga , Complexo I de Transporte de Elétrons/metabolismo , Células HT29 , Humanos , Microscopia Confocal , Mitocôndrias/metabolismo , Mitocôndrias/fisiologia , Oxirredução/efeitos dos fármacos , Espécies Reativas de Oxigênio/metabolismo , Fatores de Tempo , Desacopladores/farmacologiaRESUMO
The aim of our study was to analyze a distribution of metabolic flux controls of all mitochondrial complexes of ATP-Synthasome and mitochondrial creatine kinase (MtCK) in situ in permeabilized cardiac cells. For this we used their specific inhibitors to measure flux control coefficients (C(vi)(JATP)) in two different systems: A) direct stimulation of respiration by ADP and B) activation of respiration by coupled MtCK reaction in the presence of MgATP and creatine. In isolated mitochondria the C(vi)(JATP) were for system A: Complex I - 0.19, Complex III - 0.06, Complex IV 0.18, adenine nucleotide translocase (ANT) - 0.11, ATP synthase - 0.01, Pi carrier - 0.20, and the sum of C(vi)(JATP) was 0.75. In the presence of 10mM creatine (system B) the C(vi)(JATP) were 0.38 for ANT and 0.80 for MtCK. In the permeabilized cardiomyocytes inhibitors had to be added in much higher final concentration, and the following values of C(vi)(JATP) were determined for condition A and B, respectively: Complex I - 0.20 and 0.64, Complex III - 0.41 and 0.40, Complex IV - 0.40 and 0.49, ANT - 0.20 and 0.92, ATP synthase - 0.065 and 0.38, Pi carrier - 0.06 and 0.06, MtCK 0.95. The sum of C(vi)(JATP) was 1.33 and 3.84, respectively. Thus, C(vi)(JATP) were specifically increased under conditions B only for steps involved in ADP turnover and for Complex I in permeabilized cardiomyocytes within Mitochondrial Interactosome, a supercomplex consisting of MtCK, ATP-Synthasome, voltage dependent anion channel associated with tubulin ßII which restricts permeability of the mitochondrial outer membrane.
Assuntos
Respiração Celular/fisiologia , Metabolismo Energético/fisiologia , Mitocôndrias/metabolismo , Miócitos Cardíacos/metabolismo , Trifosfato de Adenosina/biossíntese , Trifosfato de Adenosina/metabolismo , Animais , Antimicina A/análogos & derivados , Antimicina A/metabolismo , Atractilosídeo/análogos & derivados , Atractilosídeo/metabolismo , Creatina Quinase Mitocondrial/metabolismo , Dinitrofluorbenzeno/metabolismo , Inibidores Enzimáticos/metabolismo , Masculino , Mersalil/metabolismo , Translocases Mitocondriais de ADP e ATP/metabolismo , ATPases Mitocondriais Próton-Translocadoras/metabolismo , Modelos Teóricos , Miócitos Cardíacos/citologia , Consumo de Oxigênio , Ratos , Ratos Wistar , Rotenona/metabolismo , Cianeto de Sódio/metabolismo , Desacopladores/metabolismoRESUMO
Mitochondria-cytoskeleton interactions were analyzed in adult rat cardiomyocytes and in cancerous non-beating HL-1 cells of cardiac phenotype. We show that in adult cardiomyocytes ßII-tubulin is associated with mitochondrial outer membrane (MOM). ßI-tubulin demonstrates diffused intracellular distribution, ßIII-tubulin is colocalized with Z-lines and ßIV-tubulin forms microtubular network. HL-1 cells are characterized by the absence of ßII-tubulin, by the presence of bundles of filamentous ßIV-tubulin and diffusely distributed ßI- and ßIII-tubulins. Mitochondrial isoform of creatine kinase (MtCK), highly expressed in cardiomyocytes, is absent in HL-1 cells. Our results show that high apparent K(m) for exogenous ADP in regulation of respiration and high expression of MtCK both correlate with the expression of ßII-tubulin. The absence of ßII-tubulin isotype in isolated mitochondria and in HL-1 cells results in increased apparent affinity of oxidative phosphorylation for exogenous ADP. This observation is consistent with the assumption that the binding of ßII-tubulin to mitochondria limits ADP/ATP diffusion through voltage-dependent anion channel of MOM and thus shifts energy transfer via the phosphocreatine pathway. On the other hand, absence of both ßII-tubulin and MtCK in HL-1 cells can be associated with their more glycolysis-dependent energy metabolism which is typical for cancer cells (Warburg effect).
Assuntos
Citoesqueleto/metabolismo , Mitocôndrias/metabolismo , Miócitos Cardíacos/metabolismo , Tubulina (Proteína)/metabolismo , Animais , Linhagem Celular Tumoral , CamundongosRESUMO
The aim of this study was to analyze quantitatively cellular respiration in intraoperational tissue samples taken from human breast cancer (BC) patients. We used oxygraphy and the permeabilized cell techniques in combination with Metabolic Control Analysis (MCA) to measure a corresponding flux control coefficient (FCC). The activity of all components of ATP synthasome, and respiratory chain complexes was found to be significantly increased in human BC cells in situ as compared to the adjacent control tissue. FCC(s) were determined upon direct activation of respiration with exogenously-added ADP and by titrating the complexes with their specific inhibitors to stepwise decrease their activity. MCA showed very high sensitivity of all complexes and carriers studied in human BC cells to inhibition as compared to mitochondria in normal oxidative tissues. The sum of FCC(s) for all ATP synthasome and respiratory chain components was found to be around 4, and the value exceeded significantly that for normal tissue (close to 1). In BC cells, the key sites of the regulation of respiration are Complex IV (FCC = 0.74), ATP synthase (FCC = 0.61), and phosphate carrier (FCC = 0.60); these FCC(s) exceed considerably (~10-fold) those for normal oxidative tissues. In human BC cells, the outer mitochondrial membrane is characterized by an increased permeability towards adenine nucleotides, the mean value of the apparent K(m) for ADP being equal to 114.8 ± 13.6 µM. Our data support the two-compartment hypothesis of tumor metabolism, the high sum of FCC(s) showing structural and functional organization of mitochondrial respiratory chain and ATP synthasome as supercomplexes in human BC.
Assuntos
Trifosfato de Adenosina/biossíntese , Neoplasias da Mama/enzimologia , Complexo de Proteínas da Cadeia de Transporte de Elétrons/metabolismo , Mitocôndrias/enzimologia , Proteínas de Neoplasias/metabolismo , Consumo de Oxigênio , Idoso , Neoplasias da Mama/patologia , Transporte de Elétrons , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Mitocôndrias/patologiaRESUMO
The aim of this review is to analyze the results of experimental research of mechanisms of regulation of mitochondrial respiration in cardiac and skeletal muscle cells in vivo obtained by using the permeabilized cell technique. Such an analysis in the framework of Molecular Systems Bioenergetics shows that the mechanisms of regulation of energy fluxes depend on the structural organization of the cells and interaction of mitochondria with cytoskeletal elements. Two types of cells of cardiac phenotype with very different structures were analyzed: adult cardiomyocytes and continuously dividing cancerous HL-1 cells. In cardiomyocytes mitochondria are arranged very regularly, and show rapid configuration changes of inner membrane but no fusion or fission, diffusion of ADP and ATP is restricted mostly at the level of mitochondrial outer membrane due to an interaction of heterodimeric tubulin with voltage dependent anion channel, VDAC. VDAC with associated tubulin forms a supercomplex, Mitochondrial Interactosome, with mitochondrial creatine kinase, MtCK, which is structurally and functionally coupled to ATP synthasome. Due to selectively limited permeability of VDAC for adenine nucleotides, mitochondrial respiration rate depends almost linearly upon the changes of cytoplasmic ADP concentration in their physiological range. Functional coupling of MtCK with ATP synthasome amplifies this signal by recycling adenine nucleotides in mitochondria coupled to effective phosphocreatine synthesis. In cancerous HL-1 cells this complex is significantly modified: tubulin is replaced by hexokinase and MtCK is lacking, resulting in direct utilization of mitochondrial ATP for glycolytic lactate production and in this way contributing in the mechanism of the Warburg effect. Systemic analysis of changes in the integrated system of energy metabolism is also helpful for better understanding of pathogenesis of many other diseases.
Assuntos
Mitocôndrias/metabolismo , Nucleotídeos de Adenina/metabolismo , Animais , Respiração Celular , Creatina Quinase Mitocondrial/metabolismo , Citoesqueleto/metabolismo , Metabolismo Energético , Retroalimentação Fisiológica , Humanos , Cinética , Mitocôndrias Cardíacas/metabolismo , Mitocôndrias Musculares/metabolismo , Modelos Biológicos , Fibras Musculares Esqueléticas/metabolismo , Miócitos Cardíacos/metabolismo , Fosfocreatina/metabolismo , Tubulina (Proteína)/metabolismo , Canais de Ânion Dependentes de Voltagem/metabolismoRESUMO
Regulation of mitochondrial outer membrane (MOM) permeability has dual importance: in normal metabolite and energy exchange between mitochondria and cytoplasm and thus in control of respiration, and in apoptosis by release of apoptogenic factors into the cytosol. However, the mechanism of this regulation, dependent on the voltage-dependent anion channel (VDAC), the major channel of MOM, remains controversial. A long-standing puzzle is that in permeabilized cells, adenine nucleotide translocase (ANT) is less accessible to cytosolic ADP than in isolated mitochondria. We solve this puzzle by finding a missing player in the regulation of MOM permeability: the cytoskeletal protein tubulin. We show that nanomolar concentrations of dimeric tubulin induce voltage-sensitive reversible closure of VDAC reconstituted into planar phospholipid membranes. Tubulin strikingly increases VDAC voltage sensitivity and at physiological salt conditions could induce VDAC closure at <10 mV transmembrane potentials. Experiments with isolated mitochondria confirm these findings. Tubulin added to isolated mitochondria decreases ADP availability to ANT, partially restoring the low MOM permeability (high apparent K(m) for ADP) found in permeabilized cells. Our findings suggest a previously unknown mechanism of regulation of mitochondrial energetics, governed by VDAC and tubulin at the mitochondria-cytosol interface. This tubulin-VDAC interaction requires tubulin anionic C-terminal tail (CTT) peptides. The significance of this interaction may be reflected in the evolutionary conservation of length and anionic charge in CTT throughout eukaryotes, despite wide changes in the exact sequence. Additionally, tubulins that have lost significant length or anionic character are only found in cells that do not have mitochondria.
Assuntos
Respiração Celular/fisiologia , Mitocôndrias/metabolismo , Tubulina (Proteína)/metabolismo , Canais de Ânion Dependentes de Voltagem/metabolismo , Sequência de Aminoácidos , Animais , Eletrofisiologia , Evolução Molecular , Humanos , Ativação do Canal Iônico , Bicamadas Lipídicas/química , Modelos Moleculares , Dados de Sequência Molecular , Fosforilação Oxidativa , Peptídeos/química , Peptídeos/genética , Peptídeos/metabolismo , Ligação Proteica , Conformação Proteica , Ratos , Alinhamento de Sequência , Tubulina (Proteína)/química , Tubulina (Proteína)/genética , Canais de Ânion Dependentes de Voltagem/genéticaRESUMO
In this review we analyze the recent important and remarkable advancements in studies of compartmentation of adenine nucleotides in muscle cells due to their binding to macromolecular complexes and cellular structures, which results in non-equilibrium steady state of the creatine kinase reaction. We discuss the problems of measuring the energy fluxes between different cellular compartments and their simulation by using different computer models. Energy flux determinations by (18)O transfer method have shown that in heart about 80% of energy is carried out of mitochondrial intermembrane space into cytoplasm by phosphocreatine fluxes generated by mitochondrial creatine kinase from adenosine triphosphate (ATP), produced by ATP Synthasome. We have applied the mathematical model of compartmentalized energy transfer for analysis of experimental data on the dependence of oxygen consumption rate on heart workload in isolated working heart reported by Williamson et al. The analysis of these data show that even at the maximal workloads and respiration rates, equal to 174 µmol O(2) per min per g dry weight, phosphocreatine flux, and not ATP, carries about 80-85% percent of energy needed out of mitochondria into the cytosol. We analyze also the reasons of failures of several computer models published in the literature to correctly describe the experimental data.
Assuntos
Metabolismo Energético , Mitocôndrias Cardíacas/metabolismo , Modelos Cardiovasculares , Miocárdio/metabolismo , Trifosfato de Adenosina/metabolismo , Animais , Creatina Quinase/química , Creatina Quinase/metabolismo , Humanos , Miocárdio/citologiaRESUMO
The main focus of this investigation is steady state kinetics of regulation of mitochondrial respiration in permeabilized cardiomyocytes in situ. Complete kinetic analysis of the regulation of respiration by mitochondrial creatine kinase was performed in the presence of pyruvate kinase and phosphoenolpyruvate to simulate interaction of mitochondria with glycolytic enzymes. Such a system analysis revealed striking differences in kinetic behaviour of the MtCK-activated mitochondrial respiration in situ and in vitro. Apparent dissociation constants of MgATP from its binary and ternary complexes with MtCK, Kia and Ka (1.94+/-0.86 mM and 2.04+/-0.14 mM, correspondingly) were increased by several orders of magnitude in situ in comparison with same constants in vitro (0.44+/-0.08 mM and 0.016+/-0.01 mM, respectively). Apparent dissociation constants of creatine, Kib and Kb (2.12+/-0.21 mM 2.17+/-0.40 Mm, correspondingly) were significantly decreased in situ in comparison with in vitro mitochondria (28+/-7 mM and 5+/-1.2 mM, respectively). Dissociation constant for phosphocreatine was not changed. These data may indicate selective restriction of metabolites' diffusion at the level of mitochondrial outer membrane. It is concluded that mechanisms of the regulation of respiration and energy fluxes in vivo are system level properties which depend on intracellular interactions of mitochondria with cytoskeleton, intracellular MgATPases and cytoplasmic glycolytic system.
Assuntos
Respiração Celular/fisiologia , Creatina Quinase Mitocondrial/metabolismo , Mitocôndrias Cardíacas/metabolismo , Miócitos Cardíacos/metabolismo , Trifosfato de Adenosina/farmacologia , Animais , Cardiotônicos/farmacologia , Cromatografia Líquida de Alta Pressão , Creatina/farmacologia , Ativação Enzimática/efeitos dos fármacos , Cinética , Mitocôndrias Cardíacas/efeitos dos fármacos , Miócitos Cardíacos/efeitos dos fármacos , Consumo de Oxigênio/efeitos dos fármacos , Fosfocreatina/farmacologia , Fosfoenolpiruvato/metabolismo , Piruvato Quinase/metabolismo , Ratos , Ratos WistarRESUMO
Doxorubicin (DXR) belongs to the most efficient anticancer drugs. However, its clinical application is limited by the risk of severe cardiac-specific toxicity, for which an efficient treatment is missing. Underlying molecular mechanisms are not sufficiently understood so far, but nonbiased, systemic approaches can yield new clues to develop targeted therapies. Here, we applied a genome-wide transcriptome analysis to determine the early cardiac response to DXR in a model characterized earlier, that is, rat heart perfusion with 2 muM DXR, leading to only mild cardiac dysfunction. Single-gene and gene set enrichment analysis of DNA microarrays yielded robust data on cardiac transcriptional reprogramming, including novel DXR-responsive pathways. Main characteristics of transcriptional reprogramming were 1) selective upregulation of individual genes or gene sets together with widespread downregulation of gene expression; 2) repression of numerous transcripts involved in cardiac stress response and stress signaling; 3) modulation of genes with cardiac remodeling capacity; 4) upregulation of "energy-related" pathways; and 5) similarities to the transcriptional response of cancer cells. Some early responses like the induction of glycolytic and Krebs cycle genes may have compensatory function. Only minor changes in the cardiac energy status or the respiratory activity of permeabilized cardiac fibers have been observed. Other responses potentially contribute to acute and also chronic toxicity, in particular, those in stress-responsive and cardiac remodeling transcripts. We propose that a blunted response to stress and reduced "danger signaling" is a prime component of toxic DXR action and can drive cardiac cells into pathology.
Assuntos
Doxorrubicina/farmacologia , Perfilação da Expressão Gênica , Coração/fisiologia , Transcrição Gênica/efeitos dos fármacos , Trifosfato de Adenosina/metabolismo , Animais , Ciclo do Ácido Cítrico/efeitos dos fármacos , Ciclo do Ácido Cítrico/genética , Regulação para Baixo/efeitos dos fármacos , Glicólise/efeitos dos fármacos , Glicólise/genética , Coração/efeitos dos fármacos , Masculino , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/fisiologia , Quinases de Proteína Quinase Ativadas por Mitógeno/genética , Hibridização de Ácido Nucleico , Análise de Sequência com Séries de Oligonucleotídeos , Consumo de Oxigênio/efeitos dos fármacos , Fosfocreatina/metabolismo , Reação em Cadeia da Polimerase , RNA/genética , RNA/isolamento & purificação , RNA Mensageiro/genética , Ratos , Ratos Wistar , Regulação para Cima/efeitos dos fármacosRESUMO
We studied possible connections of tubulin, microtubular system, and microtubular network stabilizing STOP protein with mitochondria in rat and mouse cardiac and skeletal muscles by confocal microscopy and oxygraphy. Intracellular localization and content of tubulin was found to be muscle type-specific, with high amounts in oxidative muscles, and much lower in glycolytic skeletal muscle. STOP protein localization and content in muscle cells was also muscle type-specific. In isolated heart mitochondria, addition of 1 microM tubulin heterodimer increased apparent K(m) for ADP significantly. Dissociation of microtubular system into free tubulin by colchicine treatment only slightly decreased initially high apparent K(m) for ADP in permeabilized cells, and diffusely distributed free tubulin stayed inside the cells, obviously connected to the intracellular structures. To identify the genes that are specific for oxidative muscle, we developed and applied a method of kindred DNA. The results of sequencing and bioinformatic analysis of isolated cDNA pool common for heart and m. soleus showed that in adult mice the beta-tubulin gene is expressed predominantly in oxidative muscle cells. It is concluded that whereas dimeric tubulin may play a significant role in regulation of mitochondrial outer membrane permeability in the cells in vivo, its organization into microtubular network has a minor significance on that process.
Assuntos
Proteínas Associadas aos Microtúbulos/metabolismo , Microtúbulos/metabolismo , Mitocôndrias Musculares/metabolismo , Fibras Musculares Esqueléticas/metabolismo , Tubulina (Proteína)/metabolismo , Animais , Respiração Celular/genética , Respiração Celular/fisiologia , Biblioteca Gênica , Camundongos , Camundongos Endogâmicos C57BL , Mitocôndrias Cardíacas/metabolismo , Fibras Musculares Esqueléticas/ultraestrutura , Miocárdio/metabolismo , Miocárdio/ultraestrutura , Miócitos Cardíacos/metabolismo , Consumo de Oxigênio/genética , Consumo de Oxigênio/fisiologia , Ligação Proteica , Ratos , Ratos WistarRESUMO
The mechanisms of regulation of respiration and energy fluxes in the cells are analyzed based on the concepts of systems biology, non-equilibrium steady state kinetics and applications of Wiener's cybernetic principles of feedback regulation. Under physiological conditions cardiac function is governed by the Frank-Starling law and the main metabolic characteristic of cardiac muscle cells is metabolic homeostasis, when both workload and respiration rate can be changed manifold at constant intracellular level of phosphocreatine and ATP in the cells. This is not observed in skeletal muscles. Controversies in theoretical explanations of these observations are analyzed. Experimental studies of permeabilized fibers from human skeletal muscle vastus lateralis and adult rat cardiomyocytes showed that the respiration rate is always an apparent hyperbolic but not a sigmoid function of ADP concentration. It is our conclusion that realistic explanations of regulation of energy fluxes in muscle cells require systemic approaches including application of the feedback theory of Wiener's cybernetics in combination with detailed experimental research. Such an analysis reveals the importance of limited permeability of mitochondrial outer membrane for ADP due to interactions of mitochondria with cytoskeleton resulting in quasi-linear dependence of respiration rate on amplitude of cyclic changes in cytoplasmic ADP concentrations. The system of compartmentalized creatine kinase (CK) isoenzymes functionally coupled to ANT and ATPases, and mitochondrial-cytoskeletal interactions separate energy fluxes (mass and energy transfer) from signalling (information transfer) within dissipative metabolic structures - intracellular energetic units (ICEU). Due to the non-equilibrium state of CK reactions, intracellular ATP utilization and mitochondrial ATP regeneration are interconnected by the PCr flux from mitochondria. The feedback regulation of respiration occurring via cyclic fluctuations of cytosolic ADP, Pi and Cr/PCr ensures metabolic stability necessary for normal function of cardiac cells.