[What prospects for the sleep apnea syndrome and connected health?] / Quelles perspectives pour le syndrome d'apnées du sommeil et la santé connectée ?

Connected health is a growing field and can be viewed from different perspectives, particularly in sleep apnea syndrome. The purpose of this review is to show how all these aspects of connected health are already used in the management of sleep apnea syndrome (SAS) and its comorbidities. First, it can give patients a better understanding and a better assessment of their health. It also facilitates their healthcare by allowing them a greater role in their care pathway. For healthcare providers, connected health tools make it possible to set up new procedures for diagnosing and monitoring ambulatory patients, and for the making of joint decisions by health professionals and patients. Finally, for researchers, e-health generates massive amounts of data, thus facilitating the acquisition of knowledge in real life situations and the development of new methodologies for clinical studies that are faster, less expensive and just as reliable. All these considerations are already applicable in the field of sleep apnea, both for proposed treatments and for comorbidities management and for the patient's involvement in his/her care pathway.

Mitochondrial Respiration in Human Colorectal and Breast Cancer Clinical Material Is Regulated Differently.

We conducted quantitative cellular respiration analysis on samples taken from human breast cancer (HBC) and human colorectal cancer (HCC) patients. Respiratory capacity is not lost as a result of tumor formation and even though, functionally, complex I in HCC was found to be suppressed, it was not evident on the protein level. Additionally, metabolic control analysis was used to quantify the role of components of mitochondrial interactosome. The main rate-controlling steps in HBC are complex IV and adenine nucleotide transporter, but in HCC, complexes I and III. Our kinetic measurements confirmed previous studies that respiratory chain complexes I and III in HBC and HCC can be assembled into supercomplexes with a possible partial addition from the complex IV pool. Therefore, the kinetic method can be a useful addition in studying supercomplexes in cell lines or human samples. In addition, when results from culture cells were compared to those from clinical samples, clear differences were present, but we also detected two different types of mitochondria within clinical HBC samples, possibly linked to two-compartment metabolism. Taken together, our data show that mitochondrial respiration and regulation of mitochondrial membrane permeability have substantial differences between these two cancer types when compared to each other to their adjacent healthy tissue or to respective cell cultures.

Simple oxygraphic analysis for the presence of adenylate kinase 1 and 2 in normal and tumor cells.

The adenylate kinase (AK) isoforms network plays an important role in the intracellular energy transfer processes, the maintenance of energy homeostasis, and it is a major player in AMP metabolic signaling circuits in some highly-differentiated cells. For this purpose, a rapid and sensitive method was developed that enables to estimate directly and semi-quantitatively the distribution between cytosolic AK1 and mitochondrial AK2 localized in the intermembrane space, both in isolated cells and tissue samples (biopsy material). Experiments were performed on isolated rat mitochondria or permeabilized material, including undifferentiated and differentiated neuroblastoma Neuro-2a cells, HL-1 cells, isolated rat heart cardiomyocytes as well as on human breast cancer postoperative samples. In these samples, the presence of AK1 and AK2 could be detected by high-resolution respirometry due to the functional coupling of these enzymes with ATP synthesis. By eliminating extra-mitochondrial ADP with an excess of pyruvate kinase and its substrate phosphoenolpyruvate, the coupling of the AK reaction with mitochondrial ATP synthesis could be quantified for total AK and mitochondrial AK2 as a specific AK index. In contrast to the creatine kinase pathway, the AK phosphotransfer pathway is up-regulated in murine neuroblastoma and HL-1 sarcoma cells and in these malignant cells expression of AK2 is higher than AK1. Differentiated Neuro-2a neuroblastoma cells exhibited considerably higher OXPHOS capacity than undifferentiated cells, and this was associated with a remarkable decrease in their AK activity. The respirometric method also revealed a considerable difference in mitochondrial affinity for AMP between non-transformed cells and tumor cells.

Cellular compartmentation of energy metabolism: creatine kinase microcompartments and recruitment of B-type creatine kinase to specific subcellular sites.

There is an increasing body of evidence for local circuits of ATP generation and consumption that are largely independent of global cellular ATP levels. These are mostly based on the formation of multiprotein(-lipid) complexes and diffusion limitations existing in cells at different levels of organization, e.g., due to the viscosity of the cytosolic medium, macromolecular crowding, multiple and bulky intracellular structures, or controlled permeability across membranes. Enzymes generating ATP or GTP are found associated with ATPases and GTPases enabling the direct fueling of these energy-dependent processes, and thereby implying that it is the local and not the global concentration of high-energy metabolites that is functionally relevant. A paradigm for such microcompartmentation is creatine kinase (CK). Cytosolic and mitochondrial isoforms of CK constitute a well established energy buffering and shuttling system whose functions are very much based on local association of CK isoforms with ATP-providing and ATP-consuming processes. Here we review current knowledge on the subcellular localization and direct protein and lipid interactions of CK isoforms, in particular about cytosolic brain-type CK (BCK) much less is known compared to muscle-type CK (MCK). We further present novel data on BCK, based on three different experimental approaches: (1) co-purification experiments, suggesting association of BCK with membrane structures such as synaptic vesicles and mitochondria, involving hydrophobic and electrostatic interactions, respectively; (2) yeast-two-hybrid analysis using cytosolic split-protein assays and the identifying membrane proteins VAMP2, VAMP3 and JWA as putative BCK interaction partners; and (3) phosphorylation experiments, showing that the cellular energy sensor AMP-activated protein kinase (AMPK) is able to phosphorylate BCK at serine 6 to trigger BCK localization at the ER, in close vicinity of the highly energy-demanding Ca(2+) ATPase pump. Thus, membrane localization of BCK seems to be an important and regulated feature for the fueling of membrane-located, ATP-dependent processes, stressing again the importance of local rather than global ATP concentrations.

Restrictions in ATP diffusion within sarcomeres can provoke ATP-depleted zones impairing exercise capacity in chronic obstructive pulmonary disease.

BACKGROUND: Chronic obstructive pulmonary disease (COPD) is characterized by the inability of patients to sustain a high level of ventilation resulting in perceived exertional discomfort and limited exercise capacity of leg muscles at average intracellular ATP levels sufficient to support contractility. METHODS: Myosin ATPase activity in biopsy samples from healthy and COPD individuals was implemented as a local nucleotide sensor to determine ATP diffusion coefficients within myofibrils. Ergometric parameters clinically measured during maximal exercise tests in both groups were used to define the rates of myosin ATPase reaction and aerobic ATP re-synthesis. The obtained parameters in combination with AK- and CK-catalyzed reactions were implemented to compute the kinetic and steady-state spatial ATP distributions within control and COPD sarcomeres. RESULTS: The developed reaction-diffusion model of two-dimensional sarcomeric space identified similar, yet extremely low nucleotide diffusion in normal and COPD myofibrils. The corresponding spatio-temporal ATP distributions, constructed during imposed exercise, predicted in COPD sarcomeres a depletion of ATP in the zones of overlap between actin and myosin filaments along the center axis at average cytosolic ATP levels similar to healthy muscles. CONCLUSIONS: ATP-depleted zones can induce rigor tension foci impairing muscle contraction and increase a risk for sarcomere damages. Thus, intra-sarcomeric diffusion restrictions at limited aerobic ATP re-synthesis can be an additional risk factor contributing to the muscle contractile deficiency experienced by COPD patients. GENERAL SIGNIFICANCE: This study demonstrates how restricted substrate mobility within a cellular organelle can provoke an energy imbalance state paradoxically occurring at abounding average metabolic resources.

The impact of cardiac ischemia/reperfusion on the mitochondria-cytoskeleton interactions.

Cardiac ischemia-reperfusion (IR) injury compromises mitochondrial oxidative phosphorylation (OxPhos) and compartmentalized intracellular energy transfer via the phosphocreatine/creatine kinase (CK) network. The restriction of ATP/ADP diffusion at the level of the mitochondrial outer membrane (MOM) is an essential element of compartmentalized energy transfer. In adult cardiomyocytes, the MOM permeability to ADP is regulated by the interaction of voltage-dependent anion channel with cytoskeletal proteins, particularly with ß tubulin II. The IR-injury alters the expression and the intracellular arrangement of cytoskeletal proteins. The objective of the present study was to investigate the impact of IR on the intracellular arrangement of ß tubulin II and its effect on the regulation of mitochondrial respiration. Perfused rat hearts were subjected to total ischemia (for 20min (I20) and 45min (I45)) or to ischemia followed by 30min of reperfusion (I20R and I45R groups). High resolution respirometry and fluorescent confocal microscopy were used to study respiration, ß tubulin II and mitochondrial arrangements in cardiac fibers. The results of these experiments evidence a heterogeneous response of mitochondria to IR-induced damage. Moreover, the intracellular rearrangement of ß tubulin II, which in the control group colocalized with mitochondria, was associated with increased apparent affinity of OxPhos for ADP, decreased regulation of respiration by creatine without altering mitochondrial CK activity and the ratio between octameric to dimeric isoenzymes. The results of this study allow us to highlight changes of mitochondrial interactions with cytoskeleton as one of the possible mechanisms underlying cardiac IR injury.

The role of tubulin in the mitochondrial metabolism and arrangement in muscle cells.

Tubulin, a well-known component of the microtubule in the cytoskeleton, has an important role in the transport and positioning of mitochondria in a cell type dependent manner. This review describes different functional interactions of tubulin with cellular protein complexes and its functional interaction with the mitochondrial outer membrane. Tubulin is present in oxidative as well as glycolytic type muscle cells, but the kinetics of the in vivo regulation of mitochondrial respiration in these muscle types is drastically different. The interaction between VDAC and tubulin is probably influenced by such factors as isoformic patterns of VDAC and tubulin, post-translational modifications of tubulin and phosphorylation of VDAC. Important factor of the selective permeability of VDAC is the mitochondrial creatine kinase pathway which is present in oxidative cells, but is inactive or missing in glycolytic muscle and cancer cells. As the tubulin-VDAC interaction reduces the permeability of the channel by adenine nucleotides, energy transfer can then take place effectively only through the mitochondrial creatine kinase/phosphocreatine pathway. Therefore, closure of VDAC by tubulin may be one of the reasons of apoptosis in cells without the creatine kinase pathway. An important question in tubulin regulated interactions is whether other proteins are interacting with tubulin. The functional interaction may be direct, through other proteins like plectins, or influenced by simultaneous interaction of other complexes with VDAC.

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 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).

Metabolic control analysis of respiration in human cancer tissue.

Bioenergetic profiling of cancer cells is of great potential because it can bring forward new and effective therapeutic strategies along with early diagnosis. Metabolic Control Analysis (MCA) is a methodology that enables quantification of the flux control exerted by different enzymatic steps in a metabolic network thus assessing their contribution to the system's function. Our main goal is to demonstrate the applicability of MCA for in situ studies of energy metabolism in human breast and colorectal cancer cells as well as in normal tissues. We seek to determine the metabolic conditions leading to energy flux redirection in cancer cells. A main result obtained is that the adenine nucleotide translocator exhibits the highest control of respiration in human breast cancer thus becoming a prospective therapeutic target. Additionally, we present evidence suggesting the existence of mitochondrial respiratory supercomplexes that may represent a way by which cancer cells avoid apoptosis. The data obtained show that MCA applied in situ can be insightful in cancer cell energetic research.

Cytoskeleton and regulation of mitochondrial function: the role of beta-tubulin II.

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 ß-tubulin II, fully co-localized with mitochondria. ß-Tubulin IV demonstrated a characteristic staining of branched network, ß-tubulin III was matched with Z-lines, and ß-tubulin I was diffusely spotted and fragmentary polymerized. In contrast, HL-1 cells were characterized by the complete absence of ß-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 ß-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.

Matters of the heart in bioenergetics: mitochondrial fusion into continuous reticulum is not needed for maximal respiratory activity.

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.

Mysterious Ca(2+)-independent muscular contraction: déjà vu.

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.

Regulation of respiration in muscle cells in vivo by VDAC through interaction with the cytoskeleton and MtCK within Mitochondrial Interactosome.

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.

Studies of the role of tubulin beta II isotype in regulation of mitochondrial respiration in intracellular energetic units in cardiac cells.

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''.

Intracellular Energetic Units regulate metabolism in cardiac cells.

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."

High efficiency of energy flux controls within mitochondrial interactosome in cardiac intracellular energetic units.

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.

Mitochondria-cytoskeleton interaction: distribution of ß-tubulins in cardiomyocytes and HL-1 cells.

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).

Molecular system bioenergics of the heart: experimental studies of metabolic compartmentation and energy fluxes versus computer modeling.

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.

Application of the principles of systems biology and Wiener's cybernetics for analysis of regulation of energy fluxes in muscle cells in vivo.

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.