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Skeletal myoblasts are an attractive cell type for transplantation because they are autologous and resistant to ischemia. However, clinical trials of myoblast transplantation in heart failure have been plagued by ventricular tachyarrhythmias and sudden cardiac death. The pathogenesis of these arrhythmias is poorly understood, but may be related to the fact that skeletal muscle cells, unlike heart cells, are electrically isolated by the absence of gap junctions. Using a novel in vitro model of myoblast transplantation in cardiomyocyte monolayers, we investigated the mechanisms of transplant-associated arrhythmias. Cocultures of human skeletal myoblasts and rat cardiomyocytes resulted in reentrant arrhythmias (spiral waves) that reproduce the features of ventricular tachycardia seen in patients receiving myoblast transplants. These arrhythmias could be terminated by nitrendipine, an l -type calcium channel blocker, but not by the Na channel blocker lidocaine. Genetic modification of myoblasts to express the gap junction protein connexin43 decreased arrhythmogenicity in cocultures, suggesting a specific means for increasing the safety (and perhaps the efficacy) of myoblast transplantation in patients.

In order to investigate the influence of cytoskeletal organization and dynamics on cellular biochemistry, a mathematical model was formulated based on our own experimental evidence. The model couples microtubular protein (MTP) dynamics to the glycolytic pathway and its branches: the Krebs cycle, ethanolic fermentation, and the pentose phosphate (PP) pathway. Results show that the flux through glycolysis coherently and coordinately increases or decreases with increased or decreased levels of polymerized MTP, respectively. The rates of individual enzymatic steps and metabolite concentrations change with the polymeric status of MTP throughout the metabolic network. Negative control is exerted by the PP pathway on the glycolytic flux, and the extent of inhibition depends inversely on the polymerization state of MTP, i.e. a high degree of polymerization relieves the negative control. The stability of the model's steady state dynamics for a wide range of variation of metabolic parameters increased with the degree of polymerized MTP. The findings indicate that the organization of the cytoskeleton bestows coherence and robustness to the coordination of cellular metabolism.

The ultrasensitive response of biological systems is a more sensitive one than that expected from the classical hyperbola of Michaelis-Menten kinetics, and whose physiological relevance depends upon the range of variation of substrate or effector for which ultrasensitivity is observed. Triggering and modulation of the ultrasensitive response in enzymatic and cellular systems are reviewed. Several demonstrations of ultrasensitive behavior in cellular systems and its impact on the amplification properties in signalling cascades and metabolic pathways are also highlighted. It is shown that ubiquitous cytoskeletal proteins may up- or downmodulate ultrasensitivity under physico-chemical conditions resembling those predominant in cells. Fil: Aon, Miguel A.. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Biotecnológicas. Universidad Nacional de San Martín. Instituto de Investigaciones Biotecnológicas; Argentina. University Johns Hopkins; Estados Unidos Fil: Gomez Casati, Diego Fabian. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Biotecnológicas. Universidad Nacional de San Martín. Instituto de Investigaciones Biotecnológicas; Argentina Fil: Iglesias, Alberto Alvaro. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe. Instituto de Agrobiotecnología del Litoral. Universidad Nacional del Litoral. Instituto de Agrobiotecnología del Litoral; Argentina Fil: Cortassa, Sonia del Carmen. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Biotecnológicas. Universidad Nacional de San Martín. Instituto de Investigaciones Biotecnológicas; Argentina

A simple, rapid and reliable procedure for permeabilizing cyanobacterial cells and measuring the glycogen synthetic pathway in situ, is presented. Cells from Anabaena sp. strain PCC 7120 were permeabilized with a mixture of toluene:ethanol (1:4 v/v). Fluorescence microscopy of cells incubated with fluorescein diacetate showed Anabaena non-permeabilized cells as green fluorescents, whereas permeabilized (viable) cells exhibited the intrinsic red fluorescence. Labelled K-1,4-glucan was recovered when permeabilized cells were incubated with the substrates of ADP-glucose pyrophosphorylase or glycogen synthase. The kinetic and regulatory properties of both enzymes could be reproduced in situ. The simplicity of the procedure and the ability to measure in situ glucan fluxes show the methodology as useful for studying the intracellular regulation of storage polysaccharides in a photosynthetic prokaryote. ß 2001 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved.Keywords: Glycogen; Starch synthesis; Cyanobacteria; Anabaena PCC 7120; Glycogen synthase; Permeabilized cell ; Metabolic £ux measurement;ADP-glucose pyrophosphorylase Fil: Gomez Casati, Diego Fabian. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Biotecnológicas. Instituto de Investigaciones Biotecnológicas "Dr. Raúl Alfonsín" (sede Chascomús). Universidad Nacional de San Martín. Instituto de Investigaciones Biotecnológicas. Instituto de Investigaciones Biotecnológicas "Dr. Raúl Alfonsín" (sede Chascomús); Argentina Fil: Aon, Miguel A.. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Biotecnológicas. Instituto de Investigaciones Biotecnológicas "Dr. Raúl Alfonsín" (sede Chascomús). Universidad Nacional de San Martín. Instituto de Investigaciones Biotecnológicas. Instituto de Investigaciones Biotecnológicas "Dr. Raúl Alfonsín" (sede Chascomús); Argentina Fil: Cortassa, Sonia del Carmen. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Biotecnológicas. Instituto de Investigaciones Biotecnológicas "Dr. Raúl Alfonsín" (sede Chascomús). Universidad Nacional de San Martín. Instituto de Investigaciones Biotecnológicas. Instituto de Investigaciones Biotecnológicas "Dr. Raúl Alfonsín" (sede Chascomús); Argentina Fil: Iglesias, Alberto Alvaro. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe. Instituto de Agrobiotecnología del Litoral. Universidad Nacional del Litoral. Instituto de Agrobiotecnología del Litoral; Argentina

The dynamics of metabolism has been shown to be involved in the triggering of events that are concurrent with sporulation of the budding yeast Saccharomyces cerevisiae . Indeed, quantitative correlations have been demonstrated between sporulation and the rate of carbon substrate or oxygen consumption, and the fluxes through gluconeogenic and glyoxylate cycle pathways. The results suggest that an imbalance between catabolic and anabolic fluxes influences the occurrence of the differentiation process. The hypothesis that the initiation of sporulation is triggered by the accumulation of an intracellular metabolite is confronted with the notion that intermediary metabolism and the expression of genes involved in sporulation interact to trigger the differentiation process. Several pieces of evidence indicate that derepression of the gluconeogenic pathway is crucial for the initiation of sporulation. One of the possible pathways through which glucose repression hampers sporulation might be the repression of gluconeogenesis as well as that of respiratory activity, in turn modulating the expression of IMEI . The stages defined in the dynamics of sporulating cultures, namely readiness and commitment , are related to metabolic events associated with sporulation. An interpretation in terms of metabolic flux dynamics is given to the reversal of commitment occurring when the normal progression to sporulation is somehow blocked. The quantitative data are here integrated in a model attempting to simulate the dynamics of

Ample evidence shows the role of cytoskeleton mainly in cell division, cell form, and general orientation by the perception of physical forces such as gravity and mechanical ones in plant cells. However, the problem of how cytoskeleton organization and its dynamics at the cellular level in turn affects main metabolic pathways of gene expression and cellular energetics is yet unsolved. The response given by cells to environmental challenges such as stress responses is crucially dependent on the organization of their architecture. Drought, high salinity, and low temperature are sensed by plants as a water stress condition. The latter is known to entrain a series of physiological and metabolic changes at the cellular level. This review hypothesizes that the cytoskeletal network of plant cells and tissues may transduce environmental stress into changes in the organization and dynamics of metabolism and gene expression. Accordingly, experimental evidence concerning the current models of cytoplasmic architecture that have emerged in recent years and the effects of stress on the cytostructure are analyzed.

We have studied the effect of disrupting catabolite (de)repression genes SNF1, SNF4, and MIG1 on the cell cycle behavior of the CEN. PK122 wild type (WT) strain of Saccharomyces cerevisiae by flow cytometry in glucose-limited chemostat cultures or batch growth in the presence of different carbon sources. Through a combination of flow cytometry of propidium iodide-stained cells and mathematical modeling we showed that the deletion of the SNF4 gene provoked a decrease in the length of G1 with respect to the WT strain along with a smaller difference in the cell cycle length of parent and daughter cells. snf1 and mig1 mutants exhibited slightly shorter G1 respect to the WT. Additionally, in the mig1 mutant the cell cycle length of parent and daughter cells was slightly altered. The results obtained are in agreement with the view that the SNF4 gene is involved in the regulation of cell cycle in yeast.

Starch is the major product of photosynthesis in plants (1). Starch synthesis occurs in chloroplasts (in green tissues) or amyloplasts (reserve tissues) through a pathway which uses ADPG1c as the glucosyl donor, in a similar form as that taking place during glycogen synthesis by bacteria (for reviews see 1-4). The key regulatory step for this biosynthetic route is the production of the sugar-nucleotide, a reaction catalyzed by ADPG1c pyrophosphorylase (AGP; EC 2.7.7.27)

In type 2 diabetes (T2DM), hyperglycemia (HG) and increased sympathetic drive may alter mitochondria energetic/redox properties, decreasing the organelle's functionality. These perturbations sustain basal low-cardiac performance and limited exercise capacity. We have recently reported that improving the redox/energetic balance of T2DM (db/db) murine cardiomyocytes/hearts with GSH or palmitate (Palm) preserves contractile performance under high-energy demand imposed by combined HG and β-adrenergic stimulation. To further understanding the metabolic basis of Palm salutary action, we first applied metabolomics to Langendorff-perfused T2DM murine hearts subjected to energy/redox stress conditions. In the absence of Palm, HG activates polyol pathways (sorbitol, glycerol, xylitol) triggering flux limitations in glycolytic and pentose phosphate (PP) pathways. The metabolite profile under Palm reveals that this fatty acid (FA) reverses the detrimental action of HG by decreasing polyol accumulation and increasing flux through PP, glycolytic, and beta-oxidation pathways, with a concomitant improvement in cardiomyocyte contraction. To confirm this observation, next we inhibited the polyol or activated the PP pathways. Preincubating HG+ISO-treated myocytes with the aldose reductase inhibitor Zopolrestat (1μM) improved the adrenergic inotropic response, in parallel with the re-activation of glycolysis and oxidative phosphorylation. These same effects were observed pre-incubating HG+ISO challenged myocytes with the activator of transketolase benfotiamine (50μM). Present data indicate that in T2DM hearts HG/ISO regimen unveils a status of mitochondrial dysfunction that favors adverse intracellular redox/energetic conditions and a metabolic remodeling that implies a glucose shunt to polyol pathways and an apparent blockade of glycolysis. These changes appear to be relieved by the exogenous administration of the FA Palm.

The temperature-sensitive cell division cycle (cdc) G1 mutants cdc28 and cdc35 show decreased mitochondrial volumes with respect to the wild type strain A364A (WT) at the restrictive temperature. Of the three criteria of mitochondrial biogenesis studied, that is, number of mitochondria per cell, relative area of the cell occupied by mitochondria, or relative area of mitochondria occupied by inner membranes, only the second indicator was significantly lower in cdc mutants than in the WT. The mitochondrial inner membranes development did not compensate for the decrease in the organelles volume. Apparently, the reduced mitochondrial biogenesis was not due to the temperature shift because the relative area of the cell occupied by mitochondria was already significantly lower at 25 degrees C in cdc mutants. The specific fluxes of oxygen consumption confirmed that the respiratory capacity of cdc mutants is largely impaired in respect to the WT. Cdc28 and cdc35 mutants of Saccharomyces cerevisiae had been previously shown to exhibit high respiratory quotients (from 3 to 7) in respect to the WT (RQ approximately 1.0), which correlated with carbon and energy uncoupling probably the result of glucose-induced catabolite repression [Aon MA, Mónaco ME, Cortassa S (1995) Exp Cell Res 217, 42-51; Mónaco ME, Valdecantos PA, Aon MA (1995) Exp Cell Res 217, 52-56].

Several cell division cycle (cdc) mutants of Saccharomyces cerevisiae (cdc28, cdc35, cdc19, cdc21, and cdc17) at the restrictive temperature (37 degrees C) in the presence of 1% glucose and defined medium divert most of the carbon (approximately 50%) to ethanol production with low biomass growth yields (Yglc) that correlate with carbon and energy uncoupling and arrest of cell proliferation. The cdc mutants studied are shown to be glucose-repressed, while this was not the case for the wild-type A364A (WT). At 37 degrees C, in the presence of 1% glycerol, derepressed cdc28 mutant cells did not show arrest of cell division and carbon and energy uncoupling since the Yglc levels measured were similar to those of the WT strain. These results suggest that the increased fermentative ability and carbon and energy uncoupling exhibited in the presence of glucose by cdc mutants with respect to those exhibited by the WT may be due to catabolite repression.

Recent advances in the in vivo control and regulation of glycolysis and oxidative phosphorylation in yeast and tumor cells is revised. New insights are presented from old and new experimental data interpreted in the light of powerful new technologies (e.g. NMR, confocal microscopy) and quantitative techniques combined with mathematical modeling. Those new aspects are mainly concerned with the dynamical organization of glycolysis and oxidative phosphorylation which emerges from the multiple interactions between compartments and processes inside the cells. Those compartments may be of structural origin, e.g. plasma membrane defining the cell boundary, mitochondrial-cytoplasmic, or functional ones such as the alternative association-dissociation of enzymes to subcellular structures (e.g. mitochondria, cytoskeleton) with different kinetic properties in each state. A novel regulatory mechanism concerning polymerization-depolymerization of microtubular protein may add a new dimension to the in vivo physiological properties of cells. One main suggestion coming from the modulatory power of the polymeric status and concentration of cytoskeleton components is that it could function as an intracellular mechanism of synchronization between microscopic (local) to macroscopic (global) processes. How the cell "mixes" or switches on or off those regulatory steps or effectors under different physiological and environmental conditions and for different genetic backgrounds, is a main avenue of systematic research for the future.

Diabetes is a syndrome in which mitochondrial alterations may participate in the dysfunction, and ultimately failure of several organs. The diabetic heart in particular can be subjected to both glycemic level oscillations and increased workload (via β-stimulation), events that may further deteriorate its mechanical properties. Here we show that when diabetic heart (db/db) cells are challenged with high levels of extracellular glucose (30 mM) and concomitant β-adrenergic stimulation via isoproterenol (ISO), intracellular GSH is greatly diminished while superoxide and H2O2 generation substantially increased. The typical enhancement in sarcomere shortening and whole Ca2+ transient driven by β-adrenergic stimulation is blunted in db/db cells, along with hampered relaxation and Ca2 reuptake. Mitochondria isolated from db/db hearts subjected to high-glucose/ISO regimen exhibit markedly decreased coupling ratios from respiratory complexes I, II, and IV while ROS emission is greatly increased, under both forward and reverse electron transport. These changes reflect a profound alteration in the crucial (energy supplying) state 4→3 transition that ultimately results in lowered ATP synthesis and in a “non-stop” reactive oxygen species (ROS) emission in the presence of ADP. Incubating high-glucose/ISO treated db/db myocytes with cell-permeable GSH restores intracellular GSH, blunts ROS production and fully rescues contractility/relaxation in these myocytes. These results show the direct role played by an oxidized redox environment in triggering the negative synergy among mitochondrial ROS and energetics leading to the E-C coupling impairment. Our study maps mitochondrial sites, i.e. state 4→3 transition, that in diabetic heart cells account, at least in part, for excess ROS emission and loss in energy supply when metabolically and energetically challenged.

This study uncovers a new mechanism of regulation of DNA supercoiling operative in vivo upon an aerobic-anaerobic transition in Escherichia coli. Exponentially growing aerobic batch cultures were subjected to a shift to anaerobic conditions. The ratio [ATP]/[ADP] remained essentially constant at 8.5 in the aerobic culture and after a transition to anaerobiosis while DNA supercoiling increased noticeably upon anaerobiosis. This result indicated that the mechanism of regulation of DNA supercoiling by the [ATP]/[ADP] ratio was not operative. The increase in DNA supercoiling was followed by a large decrease in the DNA-relaxing activity of topoisomerase I while gyrase activity remained relatively constant. This decrease in the activity of topoisomerase I is likely to be responsible for the increase in DNA supercoiling.

Different levels of organization distinguished by characteristics spatial dimensions, Ec, and relaxation times, Tr, of biological processes ranging from electron transport in energy transduction to growth of microbial and plant cells, are shown to be related through a relation that may be interpreted as allometric and characterized by two different slopes. Processes, at levels of organization occurring in spatial dimensions of micrometers and relaxing in the order of minutes, delimit a 'transition point' between the two curves, that we interpret as a limit for the emergence of macroscopic coherence. The characteristic spatial dimension, Ec, and the relaxation time, Tr, contain dynamical information about the processes occurring at a given level of organization. When a steady state of a biological process at a certain level of organization becomes unstable, the system undergoes a transition to another level of organization. To exemplify the appearance of macroscopic order at levels of organization further from the 'transition point' we present in this report various experimental systems involving many levels of organization allometrically related that exhibit different kinds of self-organized behavior, i.e. bi-stability, oscillations, changes in (a)symmetry.

Progression is the development from bad to worse that is often observed in tumors. It can follow different courses, not only in different organs, but also in the same organ: sometimes there is a smooth, continuous progression from normal to malignant cells, sometimes suddenly a highly malignant tumor arises.

A model simulating oscillations in glycolysis was formulated in terms of nonequilibrium thermodynamics. In the kinetic rate equations every metabolite concentration was replaced with an exponential function of its chemical potential. This led to nonlinear relations between rates and chemical potentials. Each chemical potential was then expanded around its steady-state value as a Taylor series. The linear (first order) term of the Taylor series sufficed to simulate the dynamic behavior of the system, including the damped and even sustained oscillations at low substrate input or high free-energy load. The glycolytic system is autocatalytic in the first half. Because oscillations were obtained only in the presence of that autocatalytic feed-back loop we conclude that this type of kinetic nonlinearity was sufficient to account for the oscillatory behavior. The matrix of phenomenological coefficients of the system is nonsymmetric. Our results indicate that this is the symmetry property and not the linearity of the flow-force relations in the near equilibrium domain that precludes oscillations. Given autocatalytic properties, a system exhibiting liner flow-force relations and being outside the near equilibrium domain may show bifurcations, leading to self-organized behavior.

Thermodynamic and kinetic calculations were performed in a model of mixed substrate metabolism. The model simulates the catabolic breakdown of a first substrate, glucose (S(1)), in the presence of a second substrate, formate (S(2)), which acts as an additional source of free energy. The principal results obtained with different relative rates of uptake of S(2) allow to predict and interpret the following experimental observations: (1) the existence of increased ATP yields by mixed substrate utilization with a maximum ATP yield and optimum input (or molar) ratio for both substrates; (2) a greater assimilation of S(1) which may be interpreted as a decreasing fraction of energy required for assimilation; (3) a decrease in ATP yields due to increasing energy demand for transport; (4) an increased assimilation of the carbon source (S(1)) as a function of increasing inputs of the additional energy source; (5) thermodynamic efficiency (eta) defined as the ratio between the output power of ATP synthesis and the input catabolic power, increases for S(2)/S(1) ratios ranging between 0.08 and 2 while for ratios higher than two a slight decrease of eta was noticed; and (6) the observed maximum in ATP yield for optimum molar ratio of the two substrates corresponds to high eta predicting that higher biomass yields may be obtained through a variable, high, eta by chanelling fluxes through catabolic pathways with different ATP yields. During oscillatory behavior, maxima in fluxes were almost coincident with maxima in forces. Thus, the pattern of dissipation was not so advantageous as in the single substrate model under starvation conditions.

A dynamic model was developed describing the bio-electromechanochemical changes (Bemchem model) of a plant cell which is concomitantly elongating and changing its surface by cell wall synthesis. The model takes into account the interdependent relationships between mechanical, electrical and biochemical events at the supramolecular-cellular level of organization (cytoplasm-plasmalemma and cell wall). The model simulates, qualitatively and quantitatively, the auxin-induced membrane hyperpolarization. The transcellular MP is coupled to energetic metabolism through its active, i.e. electrogenic (H + pump), component. Model results predict that the value and time-dependent behavior of the MP influences cellulose synthesis both qualitatively and quantitatively. The model also predicts that cell wall synthesis could be elicited prior to an exponential elongation of the cell. The electromechanical changes sensed by the plasma membrane appear to be earlier processes, conditioning future biochemical events, such as cellulose synthesis, associated to plant cell differentiation (secondary cell wall formation).

On the basis of a quantification of the fractal dimension, D, in micrographs of cytoskeleton components or microtrabecular lattice, we propose that the cellular cytoplasm can be described as a percolation cluster, a sort of ‘random fractal’. Our hypothesis deals with: (i) the existence of the percolation threshold — a remarkable property of percolation processes; and (ii) the reactivity increase — when enzymes, or targets, and substrates, or effectors, coexist in the same topological dimension.

Mitochondria can behave as individual oscillators whose dynamics may obey collective, network properties. We have shown that cardiomyocytes exhibit high-amplitude, self-sustained, and synchronous oscillations of bioenergetic parameters when the mitochondrial network is stressed to a critical state. Computational studies suggested that additional low-amplitude, high-frequency oscillations were also possible. Herein, employing power spectral analysis, we show that the temporal behavior of mitochondrial membrane potential (DeltaPsi(m)) in cardiomyocytes under physiological conditions is oscillatory and characterized by a broad frequency distribution that obeys a homogeneous power law (1/f(beta)) with a spectral exponent, beta = 1.74. Additionally, relative dispersional analysis shows that mitochondrial oscillatory dynamics exhibits long-term memory, characterized by an inverse power law that scales with a fractal dimension (D(f)) of 1.008, distinct from random behavior (D(f) = 1.5), over at least three orders of magnitude. Analysis of a computational model of the mitochondrial oscillator suggests that the mechanistic origin of the power law behavior is based on the inverse dependence of amplitude versus frequency of oscillation related to the balance between reactive oxygen species production and scavenging. The results demonstrate that cardiac mitochondria behave as a network of coupled oscillators under both physiological and pathophysiological conditions.

Incubation of a bovine retina membrane preparation with micromolar amounts of UDP-[14C]glucose resulted in the incorporation of [14C]glucose into endogenous (1----4)-alpha-glucan, insoluble in trichloroacetic acid, and acid-soluble ethanol-insoluble glycogen. The trichloroacetic-acid-insoluble glucan fraction of retina migrated in 2.6-3% acrylamide gels when subjected to sodium dodecyl sulfate/polyacrylamide gel electrophoresis (SDS-PAGE) and was rendered acid-soluble by digestion with pronase. The solubility of the acid-insoluble glucan in acidified organic solvent was different from that of amylose or glycogen and similar to membrane proteins and glycoproteins. The glycogen fraction of retina contained 1.5-2.0 micrograms protein/100 micrograms glucose. When this fraction was analyzed by SDS-PAGE only one band, which moved near the top of 3% acrylamide gels, was stained with periodic acid Schiff reagent and Coomassie blue. The protein nature of the Coomassie-blue-stainable material was demonstrated by iodination of the glycogen fraction with [131I]iodide and identification of labeled monoiodotyrosine and diiodotyrosine. The bulk of the label comigrated with carbohydrate near the top of gels in SDS-PAGE and treatment with alpha- amylse decreased the molecular size of both labeled and stainable material. Physical dissociative conditions (7.5 M urea/0.83% SDS/0.83% mercaptoethanol) and the following chemical treatments failed to dissociate the iodinated protein from glycogen: (a) 0.1 M NaOH/0.1 M NaBH4 at room temperature for 24 h; (b) 1 M HCl in methanol at 50 degrees C for 10 min; (c) trifluoroacetic acid at 50 degrees C for 6 min. 131I-labeled glycogenpeptide was isolated after 131I-labeled protein-bound glycogen had been subjected to digestion with papain/pronase and passed through a Sepharose column. The results suggest that at least part of glycogen in bovine retina is firmly combined to protein as a single proteoglycogen molecule. Furthermore some of the proteoglycogen might be present as a trichloroacetic-acid-precipitable proteoglucan owing to its lower glucose content.

Metabolic or oxidative stress can trigger the abrupt collapse or oscillation of mitochondrial membrane potential, which activates KATP current and alters the cardiac action potential. This mechanism can introduce both temporal and spatial dispersion of electrical excitability in the form of a “metabolic sink”, leading to heterogeneous conduction in the tissue, reentrant tachyarrhythmias, or fibrillation. To quantitatively study this mechanism, a 2D monodomain model of the myocardium (5×5cm2; 200 micron resolution) was developed, comprised of 63,000 nodes, each representing an integrated cellular model of cardiac excitation-contraction coupling, mitochondrial energetics, and ROS-induced ROS release (ECME-RIRR). Oxidative stress was initiated in a central circular zone of the tissue by increasing the fractional mitochondrial ROS production (shunt) during oxidative phosphorylation from 2% to 10%. Model simulations show that mitochondrial dynamics bifurcate during stimulation at 1 Hz and complete depolarization of αΨm ensues in the sink zone. Within the metabolic sink, sarcolemmal KATP currents increase, action potentials dramatically shorten, and the refractory period is abbreviated. These effects are enhanced by increasing the KATP density. In addition, fast and irregular electric activity (ventricular fibrillation) in the electrically paced tissue is observed when an S2 stimulus is introduced within or near the border of the metabolic sink, but only when the sink exceeds a critical size (r>0.4cm). Phase singularity analysis indicates that the fibrillatory activity is initiated at sites close the border zone. The results emphasize the power of integrating cellular electrophysiological, Ca2+ handling, and metabolic subsystems into a multiscale model to simulate emergent macroscopic phenomena in the heart. Moreover, the results provide a proof-of-concept of the metabolic sink hypothesis and a new tool to study its role in arrhythmogenesis and sudden cardiac death.

Ionic equilibria are known to be dramatically altered in failing hearts, as well as during and after ischemic injury. Ion transport across the mitochondrial inner membrane has been shown to modulate the energetic performance of mitochondria. Consequently, it is critical to thoroughly understand the interrelationship between ion fluxes and energetics. With this aim in mind, here we continue to develop our computational model of mitochondrial energetics to account for pH regulation, Na+/H+ cotransport, and the Pi carrier, and study their effects on mitochondrial energy production and Ca2+ handling mediated by the Ca2+ uniporter and Na+/Ca+ exchanger. Since time-dependent behavior represents the most stringent criterion of model validation, we perform direct comparisons of simulation results with experimental data obtained after challenging isolated mitochondria from guinea pig hearts with substrate and Ca2+ additions in the presence of different Na+ concentrations. Experimentally, we measured NADH and mitochondrial membrane potential (ratiometrically determined with TMRM) to monitor mitochondrial energetics. The model is able to reproduce the time course of NADH and membrane potential upon addition of 5mM glutamate plus malate followed by 2mM Pi and 1mM ADP. Moreover, the model recapitulates the NADH recovery profile after Ca2+ addition (0.1 to 0.5uM) during state 3 or state 4 respiration in the presence of either 5 or 15mM Na+. The results indicate that the computational model employed is able to account for the response of mitochondrial energetics to all experimental conditions tested. This work is supported by NIH grant R33HL87345.

To understand the mechanisms involved in the control and regulation of mitochondrial reactive oxygen species (ROS) levels, a two-compartment computational mitochondrial energetic-redox (ME-R) model accounting for energetic, redox, and ROS metabolisms is presented. The ME-R model incorporates four main redox couples (NADH/NAD+, NADPH/NADP+, GSH/GSSG, Trx(SH)2/TrxSS). Scavenging systems—glutathione, thioredoxin, superoxide dismutase, catalase—are distributed in mitochondrial matrix and extra-matrix compartments, and transport between compartments of ROS species (superoxide: O2⋅−, hydrogen peroxide: H2O2), and GSH is also taken into account. Model simulations are compared with experimental data obtained from isolated heart mitochondria. The ME-R model is able to simulate: i), the shape and order of magnitude of H2O2 emission and dose-response kinetics observed after treatment with inhibitors of the GSH or Trx scavenging systems and ii), steady and transient behavior of ΔΨm and NADH after single or repetitive pulses of substrate- or uncoupler-elicited energetic-redox transitions. The dynamics of the redox environment in both compartments is analyzed with the model following substrate addition. The ME-R model represents a useful computational tool for exploring ROS dynamics, the role of compartmentation in the modulation of the redox environment, and how redox regulation participates in the control of mitochondrial function. Fil: Kembro, Jackelyn Melissa. University Johns Hopkins; Estados Unidos. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina Fil: Aon, Miguel A.. University Johns Hopkins; Estados Unidos Fil: Winslow, Raimond L.. Institute for Computational Medicine; Estados Unidos Fil: O'Rourke, Brian. University Johns Hopkins; Estados Unidos Fil: Cortassa, Sonia del Carmen. University Johns Hopkins; Estados Unidos. Institute for Computational Medicine; Estados Unidos. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina

Respiring mitochondria continually produce hydrogen peroxide (H2O2). When production exceeds scavenging capacity, increased H2O2 emission will occur, thus transforming potential signaling into overt oxidative stress and cellular dysfunction. Glutathione and thioredoxin (Trx) are two major systems providing reducing equivalents, yet their relative contribution in keeping proper mitochondrial redox balance is unclear. Here we hypothesize that under physiological forward electron transport (FET) mode Trx2 is a major ROS buffering system in mitochondria. Thioredoxin reductase 2 (TrxR2) reconverts oxidized Trx2 into its reduced, active form, in mitochondria. Utilizing the highly specific TrxR2 inhibitor Auronofin (AF) we measured H2O2 flux from isolated mitochondria and ventricular myocytes obtained from C57/BL6 mice or guinea pig hearts. In isolated mitochondria, H2O2 emission and the redox status of Trx2 were analyzed in parallel in the absence or presence of AF, under states 4 and 3 of respiration triggered by Glutamate/Malate (G/M, 5mM) and 1mM ADP, respectively. Mitochondria preincubation with AF resulted in 20-fold or 14-fold rise in H2O2 levels with K0.5 of 4.8±0.5 or 5.8±0.6 nM in states 4 or 3 respiration, respectively, In energized and actively phosphorylating mitochondria we measured a 56±8% rise in reduced Trx2 abundance, equivalent to an increase in reductive potential from −330±4 to −348±6 mV, which could be fully prevented by preincubation with 100nM AF. Importantly, AF had no effect on mitochondrial membrane potential, NADH, or GSH levels, reinforcing the high selectivity of AF while demonstrating the major H2O2 scavenging role played by the Trx2 system. In keeping with the results observed in isolated mitochondria, administering AF to cardiomyocytes provoked a rise in total cellular O2.- and H2O2 levels, resulting in reduced cell contractility. Our data indicate that the Trx2 system controls mitochondrial redox balance, H2O2 emission, and ultimately cardiomyocyte function.

Type 2 diabetes mellitus (T2DM) is characterized by obesity, hyperglycemia and insulin resistance; these traits are recapitulated by the Zucker Diabetic Fatty (ZDF) rat model. Although fatty acid oxidation is known to be augmented in the heart, it is unclear how substrate fuel selection and redox behavior affect optimization of cardiac work output in T2DM. We studied the mechanical, and energetics/redox behavior of heart trabeculae from ZDF rats and their lean controls perfused with either normal (euglycemia, EG, 5mM) or high glucose (HG, 30mM), in the absence or presence of Palmitate (Palm). Contractile performance, measured as developed force and area under the muscle tension curve (contractile work), increased with frequency, as did oxygen consumption rate (VO2). The ratio of contractile work over VO2 was defined as an index of the energy yield for contractile work (Ycw). Unlike in lean animals, ZDF heart trabeculae showed a significant increase in Ycw under HG in the presence of Palm. On the contrary, ZDF trabeculae perfused under EG in the presence of Palm exhibited lower Ycw than in lean animals, in agreement with published data. The improved function with Palm under HG was associated with improved redox status in ZDF trabeculae, but not in lean controls. Indeed, MCB fluorescence, an indicator of cellular GSH levels, and the probe of oxidative stress, CM-DCF, revealed more oxidizing redox conditions under HG that could be reverted by the addition of Palm in ZDF trabeculae. In contrast with the behavior under EG, Palm elicited an increased reliance of contractility on glycolysis in conjunction with oxidative phosphorylation under HG in ZDF but not in lean trabeculae. As a result, Palm induces a redox-dependent higher contractile work yield from respiration in muscles from diabetic rat hearts.

The heart from a diabetic animal exhibits dysfunction when exposed to challenging energetic and tissue redox conditions. We showed that when cardiomyocytes from type-2 diabetes (db/db) mice are exposed to high glucose (HG) and β-adrenergic stimulation (via isoproterenol, ISO), these cells show blunted β-contractile reserve and increased oxidative stress. Treating these cells with the fatty acid (FA) palmitate (Palm) offsets those changes, an effect likely due to its ability of generating more reducing equivalents. Yet whether Palm infusion may benefit contractile performance and vascular tone of db/db hearts subjected to metabolic/redox stress is unclear. Using a Langendorff approach, we perfused wild type (WT) and db/db mice with HG (30mM glucose) + ISO (10nM), in absence or presence of Palm. WT hearts were infused with 0.2mM Palm and db/db ones with 0.4mM Palm to mimic higher circulating FA content in diabetic animals. Under HG, coronary perfusion pressure (CPP) was higher in db/db hearts (92±8 vs. 63±7 mmHg, p

K+ movement across mitochondrial membranes is involved in volume regulation and may play a role in cardioprotection. The Ca2+-dependent K+ (KCa) channel has been proposed as a contributor to mitochondrial K+ uniport activity, but its functional role is not well understood. To investigate the impact of KCa channels on mitochondrial energetics, we measured K+ fluxes in parallel with ΔΨm and light scattering in isolated mitochondria from guinea pig hearts. We first analyzed the role of different anions on K+ fluxes. Mitochondria loaded with the K+-sensitive fluorescent probe PBFI were incubated with 5mM glutamate-Na+/malate-Na+ in isotonic sucrose medium and subjected to pulses containing different concentrations of KCl, KAc or KH2PO4. K+ fluxes saturated at ≈10mM regardless of the anion, attaining maximal rates (nmol K+/min/mg) of 172±17 (KCl), 84±2.4 (KAc), and 74±3.8 (KH2PO4), with similar K0.5 in all three cases. We then analyzed the effect of NS11021, a novel activator of KCa channels, on the maximal K+ uptake rate. In the presence of KH2PO4 or KAc, 20-50nM of NS11021 increased mitochondrial volume and K+ flux by ∼2.5-fold whereas KCl increased K+ uptake by 30% with little change in volume. ΔΨm was minimally affected in this concentration range. The NS11021 effect was blocked by 200nM charybdotoxin, a KCa channel blocker. At 50nM NS11021, the respiratory control ratio of the mitochondria increased 2.5-fold in the presence of KH2PO4, but not KCl, indicating that a regulatory volume increase is required to improve oxidative phosphorylation. At higher concentrations of the compound (≥1μM) substantial effects on ΔΨm and state 4 respiration were observed, which were not inhibited by Chtx. The findings indicate that activation of K+ fluxes through KCa channels, coupled with swelling without loss of ΔΨm, improves mitochondrial energetic performance.

International audience; Complete metabolic maps are currently available for a number of important bacteria. Even when these maps are not experimentally confirmed, the topology of the metabolic network can be reconstructed from the genome sequence. Despite this extensive information, we still lack a good understanding of metabolic adaptations, the interactions of metabolism with gene regulation and tools for predicting the metabolic consequences of modifying the metabolic or genetic regulatory network of a bacterium. This chapter will briefly review current methods for analyzing bacterial metabolism from topological models and steady state techniques, such as flux balance analysis, to dynamical models using ordinary differential equations. Even though still incomplete, these models can predict the metabolic behavior of modified organisms. Using these tools, we can create novel metabolic pathways or optimize the yield of a desired metabolite. Focusing on Escherichia coli, we present examples of successful metabolic engineering using such systems-wide, rational approaches, integrating modeling and experiments. The conjunction of systems biology to metabolic engineering yields new insights into the fundamental functioning of the cell and opens the path to the biological production of a large variety of commodity chemicals.