[Show abstract][Hide abstract] ABSTRACT: In this review we summarize the main structural and functional data on the role of thephosphocreatine (PCr) – creatine kinase (CK) pathway in the compartmentalized energy-transfer in cardiac cells. Mitochondrial creatine kinase, MtCK, fixed by cardiolipin molecules
in the vicinity of the adenine nucleotide translocator is a key enzyme in this pathway. Direct
transfer of ATP and ADP between these proteins has been revealed both in experimental
studies on the kinetics of the regulation of mitochondrial respiration and by mathematical
modelling as a main mechanism of functional coupling of PCr production to oxidative
phosphorylation. In the cells in vivo or in the permeabilized cells in situ, this coupling is
reinforced by limited permeability of the outer membrane of the mitochondria for adenine
nucleotides due to the contacts with cytoskeletal proteins. Due to these mechanisms, at least
80 % of total energy is exported from mitochondria by PCr molecules. Mathematical
modelling of the intracellular diffusion and energy transfer shows that the main function of
the PCr – CK pathway is to connect different pools (compartments) of ATP and in this way to
overcome the local restrictions of the diffusion of adenine nucleotides due to high degree of
structural organization of cardiac cells.
Systems Biology of Metabolic and Signaling Networks: Energy, Mass and Information Transfer, Edited by Miguel A. Aon, Valdur Saks, Uwe Schlattner, 01/2014: chapter 11) System Level Dynamics in Cardiac Energy Fluxes Via Metabolic Cycles: Role of Creatine, Phosphotransfer Pathways, and AMPK Signaling.: pages 261-323; Springer Heidelberg, New York...., ISBN: 978-3-642-38505-6 (eBook)
[Show abstract][Hide abstract] ABSTRACT: The control of mitochondrial function is a cardinal issue in the field of cardiac bioenergetics, and the analysis of mitochondrial regulations is central to basic research and in the diagnosis of many diseases. Interaction between cytoskeletal proteins and mitochondria can actively participate in mitochondrial regulation. Potential candidates for the key roles in this regulation are the cytoskeletal proteins plectin and tubulin. Analysis of cardiac cells has revealed regular arrangement of β-tubulin II, fully co-localized with mitochondria. β-Tubulin IV demonstrated a characteristic staining of branched network, β-tubulin III was matched with Z-lines, and β-tubulin I was diffusely spotted and fragmentary polymerized. In contrast, HL-1 cells were characterized by the complete absence of β-tubulin II. Comparative analysis of cardiomyocytes and HL-1 cells revealed a dramatic difference in the mechanisms of mitochondrial regulation. In the heart, colocalization of β-tubulin isotype II with mitochondria suggests that it can participate in the coupling of ATP-ADP translocase (ANT), mitochondrial creatine kinase (MtCK), and VDAC (ANT-MtCK-VDAC). This mitochondrial supercomplex is responsible for the efficient intracellular energy transfer via the phosphocreatine pathway. Existing data suggest that cytoskeletal proteins may control the VDAC, contributing to maintenance of mitochondrial and cellular physiology.
[Show abstract][Hide abstract] ABSTRACT: 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.
Journal of Bioenergetics 12/2012; · 1.60 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: 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.
[Show abstract][Hide abstract] ABSTRACT: 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 β2-tubulin coexpressed with mitochondria is highly important for cardiac muscle cells mitochondrial metabolism. Since it has been shown by Rostovtseva et al. that αβ-heterodimer of tubulin binds to VDAC and decreases its permeability, we suppose that the β-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.
Biochimica et Biophysica Acta 01/2012; 1818(6):1545-54. · 4.66 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: A broad spectrum of beneficial effects has been ascribed to creatine (Cr), phosphocreatine (PCr) and their cyclic analogues cyclo-(cCr) and phospho-cyclocreatine (PcCr). Cr is widely used as nutritional supplement in sports and increasingly also as adjuvant treatment for pathologies such as myopathies and a plethora of neurodegenerative diseases. Additionally, Cr and its cyclic analogues have been proposed for anti-cancer treatment. The mechanisms involved in these pleiotropic effects are still controversial and far from being understood. The reversible conversion of Cr and ATP into PCr and ADP by creatine kinase, generating highly diffusible PCr energy reserves, is certainly an important element. However, some protective effects of Cr and analogues cannot be satisfactorily explained solely by effects on the cellular energy state. Here we used mainly liposome model systems to provide evidence for interaction of PCr and PcCr with different zwitterionic phospholipids by applying four independent, complementary biochemical and biophysical assays: (i) chemical binding assay, (ii) surface plasmon resonance spectroscopy (SPR), (iii) solid-state (31)P-NMR, and (iv) differential scanning calorimetry (DSC). SPR revealed low affinity PCr/phospholipid interaction that additionally induced changes in liposome shape as indicated by NMR and SPR. Additionally, DSC revealed evidence for membrane packing effects by PCr, as seen by altered lipid phase transition. Finally, PCr efficiently protected against membrane permeabilization in two different model systems: liposome-permeabilization by the membrane-active peptide melittin, and erythrocyte hemolysis by the oxidative drug doxorubicin, hypoosmotic stress or the mild detergent saponin. These findings suggest a new molecular basis for non-energy related functions of PCr and its cyclic analogue. PCr/phospholipid interaction and alteration of membrane structure may not only protect cellular membranes against various insults, but could have more general implications for many physiological membrane-related functions that are relevant for health and disease.
PLoS ONE 01/2012; 7(8):e43178. · 3.53 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: To study the effects of physical training at mild intensities on skeletal muscle energy metabolism in eight patients with chronic obstructive pulmonary disease (COPD) and eight paired healthy sedentary subjects.
Energy metabolism of patients and controls vastus lateralis muscle was studied before and after 3 months of cycling training at mild exercises intensities.
The total amount of work accomplished was about 4059 ± 336 kJ in patients with COPD and 7531 ± 1693 kJ in control subjects. This work corresponds to a mechanical power set at 65.2 ± 7.5% of the maximum power for patients with COPD and 52 ± 3.3% of the maximum power in control group. Despite this low level of exercise intensities, we observed an improvement in mitochondrial oxidative phosphorylation through the creatine kinase system revealed by the increased apparent K(m) for ADP (from 105.5 ± 16.1 to 176.9 ± 26.5 μm, P < 0.05 in the COPD group and from 126.9 ± 16.8 to 177.7 ± 17.0, P > 0.05 in the control group). Meanwhile, maximal mechanical and metabolic power increased significantly from 83.1 ± 7.1 to 91.3 ± 7.4 Watts (P < 0.05) and from 16 ± 0.8 to 18.7 ± 0.98 mL O(2) kg(-1) min(-1) (P < 0.05) only in the COPD group.
This study shows that physical training at mild intensity is able to induce comparable changes in skeletal muscles oxidative energy metabolism in patients with COPD and sedentary healthy subjects, but different changes of maximal mechanical and metabolic power.
[Show abstract][Hide abstract] ABSTRACT: 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."
Journal of Molecular and Cellular Cardiology 07/2011; 52(2):419-36. · 5.15 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: 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.
Biochimica et Biophysica Acta 03/2011; 1813(6):1144-52. · 4.66 Impact Factor