Bistability of Mitochondrial Respiration Underlies Paradoxical Reactive Oxygen Species Generation Induced by Anoxia

Increased production of reactive oxygen species (ROS) in mitochondria underlies major systemic diseases, and this clinical problem stimulates a great scientific interest in the mechanism of ROS generation. However, the mechanism of hypoxia-induced change in ROS production is not fully understood. To mathematically analyze this mechanism in details, taking into consideration all the possible redox states formed in the process of electron transport, even for respiratory complex III, a system of hundreds of differential equations must be constructed. Aimed to facilitate such tasks, we developed a new methodology of modeling, which resides in the automated construction of large sets of differential equations. The detailed modeling of electron transport in mitochondria allowed for the identification of two steady state modes of operation (bistability) of respiratory complex III at the same microenvironmental conditions. Various perturbations could induce the transition of respiratory chain from one steady state to another. While normally complex III is in a low ROS producing mode, temporal anoxia could switch it to a high ROS producing state, which persists after the return to normal oxygen supply. This prediction, which we qualitatively validated experimentally, explains the mechanism of anoxia-induced cell damage. Recognition of bistability of complex III operation may enable novel therapeutic strategies for oxidative stress and our method of modeling could be widely used in systems biology studies

Reactive Oxygen Species Production by Forward and Reverse Electron Fluxes in the Mitochondrial Respiratory Chain

Reactive oxygen species (ROS) produced in the mitochondrial respiratory chain (RC) are primary signals that modulate cellular adaptation to environment, and are also destructive factors that damage cells under the conditions of hypoxia/reoxygenation relevant for various systemic diseases or transplantation. The important role of ROS in cell survival requires detailed investigation of mechanism and determinants of ROS production. To perform such an investigation we extended our rule-based model of complex III in order to account for electron transport in the whole RC coupled to proton translocation, transmembrane electrochemical potential generation, TCA cycle reactions, and substrate transport to mitochondria. It fits respiratory electron fluxes measured in rat brain mitochondria fueled by succinate or pyruvate and malate, and the dynamics of NAD+ reduction by reverse electron transport from succinate through complex I. The fitting of measured characteristics gave an insight into the mechanism of underlying processes governing the formation of free radicals that can transfer an unpaired electron to oxygen-producing superoxide and thus can initiate the generation of ROS. Our analysis revealed an association of ROS production with levels of specific radicals of individual electron transporters and their combinations in species of complexes I and III. It was found that the phenomenon of bistability, revealed previously as a property of complex III, remains valid for the whole RC. The conditions for switching to a state with a high content of free radicals in complex III were predicted based on theoretical analysis and were confirmed experimentally. These findings provide a new insight into the mechanisms of ROS production in RC

Multistationary and Oscillatory Modes of Free Radicals Generation by the Mitochondrial Respiratory Chain Revealed by a Bifurcation Analysis

The mitochondrial electron transport chain transforms energy satisfying cellular demand and generates reactive oxygen species (ROS) that act as metabolic signals or destructive factors. Therefore, knowledge of the possible modes and bifurcations of electron transport that affect ROS signaling provides insight into the interrelationship of mitochondrial respiration with cellular metabolism. Here, a bifurcation analysis of a sequence of the electron transport chain models of increasing complexity was used to analyze the contribution of individual components to the modes of respiratory chain behavior. Our algorithm constructed models as large systems of ordinary differential equations describing the time evolution of the distribution of redox states of the respiratory complexes. The most complete model of the respiratory chain and linked metabolic reactions predicted that condensed mitochondria produce more ROS at low succinate concentration and less ROS at high succinate levels than swelled mitochondria. This prediction was validated by measuring ROS production under various swelling conditions. A numerical bifurcation analysis revealed qualitatively different types of multistationary behavior and sustained oscillations in the parameter space near a region that was previously found to describe the behavior of isolated mitochondria. The oscillations in transmembrane potential and ROS generation, observed in living cells were reproduced in the model that includes interaction of respiratory complexes with the reactions of TCA cycle. Whereas multistationarity is an internal characteristic of the respiratory chain, the functional link of respiration with central metabolism creates oscillations, which can be understood as a means of auto-regulation of cell metabolism. © 2012 Selivanov et al

Oxygen pathway modeling estimates high Reactive oxygen species production above the highest permanent human habitation.

The production of reactive oxygen species (ROS) from the inner mitochondrial membrane is one of many fundamental processes governing the balance between health and disease. It is well known that ROS are necessary signaling molecules in gene expression, yet when expressed at high levels, ROS may cause oxidative stress and cell damage. Both hypoxia and hyperoxia may alter ROS production by changing mitochondrial Po2 (). Because depends on the balance between O2 transport and utilization, we formulated an integrative mathematical model of O2 transport and utilization in skeletal muscle to predict conditions to cause abnormally high ROS generation. Simulations using data from healthy subjects during maximal exercise at sea level reveal little mitochondrial ROS production. However, altitude triggers high mitochondrial ROS production in muscle regions with high metabolic capacity but limited O2 delivery. This altitude roughly coincides with the highest location of permanent human habitation. Above 25,000 ft., more than 90% of exercising muscle is predicted to produce abnormally high levels of ROS, corresponding to the "death zone" in mountaineering

Unveiling a key role of oxaloacetate-glutamate interaction in regulation of respiration and ROS generation in nonsynaptic brain mitochondria using a kinetic model.

Glutamate plays diverse roles in neuronal cells, affecting cell energetics and reactive oxygen species (ROS) generation. These roles are especially vital for neuronal cells, which deal with high amounts of glutamate as a neurotransmitter. Our analysis explored neuronal glutamate implication in cellular energy metabolism and ROS generation, using a kinetic model that simulates electron transport details in respiratory complexes, linked ROS generation and metabolic reactions. The analysis focused on the fact that glutamate attenuates complex II inhibition by oxaloacetate, stimulating the latter's transformation into aspartate. Such a mechanism of complex II activation by glutamate could cause almost complete reduction of ubiquinone and deficiency of oxidized form (Q), which closes the main stream of electron transport and opens a way to massive ROS generating transfer in complex III from semiquinone radicals to molecular oxygen. In this way, under low workload, glutamate triggers the respiratory chain (RC) into a different steady state characterized by high ROS generation rate. The observed stepwise dependence of ROS generation on glutamate concentration experimentally validated this prediction. However, glutamate's attenuation of oxaloacetate's inhibition accelerates electron transport under high workload. Glutamate-oxaloacetate interaction in complex II regulation underlies the observed effects of uncouplers and inhibitors and acceleration of Ca2+ uptake. Thus, this theoretical analysis uncovered the previously unknown roles of oxaloacetate as a regulator of ROS generation and glutamate as a modifier of this regulation. The model predicted that this mechanism of complex II activation by glutamate might be operative in situ and responsible for excitotoxicity. Spatial-time gradients of synthesized hydrogen peroxide concentration, calculated in the reaction-diffusion model with convection under a non-uniform local approximation of nervous tissue, have shown that overproduction of H2O2 in a cell causes excess of its level in neighbor cells

A Computational Model of Reactive Oxygen Species and Redox Balance in Cardiac Mitochondria

AbstractElevated levels of reactive oxygen species (ROS) play a critical role in cardiac myocyte signaling in both healthy and diseased cells. Mitochondria represent the predominant cellular source of ROS, specifically the activity of complexes I and III. The model presented here explores the modulation of electron transport chain ROS production for state 3 and state 4 respiration and the role of substrates and respiratory inhibitors. Model simulations show that ROS production from complex III increases exponentially with membrane potential (ΔΨm) when in state 4. Complex I ROS release in the model can occur in the presence of NADH and succinate (reverse electron flow), leading to a highly reduced ubiquinone pool, displaying the highest ROS production flux in state 4. In the presence of ample ROS scavenging, total ROS production is moderate in state 3 and increases substantially under state 4 conditions. The ROS production model was extended by combining it with a minimal model of ROS scavenging. When the mitochondrial redox status was oxidized by increasing the proton permeability of the inner mitochondrial membrane, simulations with the combined model show that ROS levels initially decline as production drops off with decreasing ΔΨm and then increase as scavenging capacity is exhausted. Hence, this mechanistic model of ROS production demonstrates how ROS levels are controlled by mitochondrial redox balance

Computational modeling analysis of mitochondrial superoxide production under varying substrate conditions and upon inhibition of different segments of the electron transport chain.

A computational mechanistic model of superoxide (O2•-) formation in the mitochondrial electron transport chain (ETC) was developed to facilitate the quantitative analysis of factors controlling mitochondrial O2•- production and assist in the interpretation of experimental studies. The model takes into account all individual electron transfer reactions in Complexes I and III. The model accounts for multiple, often seemingly contradictory observations on the effects of ΔΨ and ΔpH, and for the effects of multiple substrate and inhibitor conditions, including differential effects of Complex III inhibitors antimycin A, myxothiazol and stigmatellin. Simulation results confirm that, in addition to O2•- formation in Complex III and at the flavin site of Complex I, the quinone binding site of Complex I is an additional superoxide generating site that accounts for experimental observations on O2•- production during reverse electron transfer. However, our simulation results predict that, when cytochrome c oxidase is inhibited during oxidation of succinate, ROS production at this site is eliminated and almost all superoxide in Complex I is generated by reduced FMN, even when the redox pressure for reverse electron transfer from succinate is strong. In addition, the model indicates that conflicting literature data on the kinetics of electron transfer in Complex III involving the iron-sulfur protein-cytochrome bL complex can be resolved in favor of a dissociation of the protein only after electron transfer to cytochrome bH. The model predictions can be helpful in understanding factors driving mitochondrial superoxide formation in intact cells and tissues

Concerning the mechanisms of influence of redox-active nanoparticles based on rare-earth elements on the redox-processes in mitochondria

Using the kinetic computation model, which demonstrates the dependence of generation of reactive oxygen species (ROS) by complexes I and III of mitochondrion respiration chain on the values of mitochondrial potential, pH of matrix, presence of different respiratory substrates and inhibitors, and data obtained experimentally, the basic mechanisms of the influence of nanoparticles (NPs) on the basis of rare earth elements on the bioenergetic processes in the mitochondria have been proposed. The mitochondrial potential, the processes of respiration and oxidative phosphorylation, as well as the ability of NPs to have an influence on the generation of ROS in the respiratory chain of mitochondria have been investigated. We propose two main mechanisms of the influence of the redox-active NPs based on the rare earth elements on the redox-processes in the mitochondria: the first mechanism involves the inhibition of electron transport and activation of the formation of superoxide and hydrogen peroxide, the second - integration of NPs into the electron-transport processes in the mitochondria as donors or acceptors of electron

Edelfosine-induced metabolic changes in cancer cells that precede the overproduction of reactive oxygen species and apoptosis

Background: Metabolic flux profiling based on the analysis of distribution of stable isotope tracer in metabolites is an important method widely used in cancer research to understand the regulation of cell metabolism and elaborate new therapeutic strategies. Recently, we developed software Isodyn, which extends the methodology of kinetic modeling to the analysis of isotopic isomer distribution for the evaluation of cellular metabolic flux profile under relevant conditions. This tool can be applied to reveal the metabolic effect of proapoptotic drug edelfosine in leukemia Jurkat cell line, uncovering the mechanisms of induction of apoptosis in cancer cells. Results: The study of 13C distribution of Jukat cells exposed to low edelfosine concentration, which induces apoptosis in ¿5% of cells, revealed metabolic changes previous to the development of apoptotic program. Specifically, it was found that low dose of edelfosine stimulates the TCA cycle. These metabolic perturbations were coupled with an increase of nucleic acid synthesis de novo, which indicates acceleration of biosynthetic and reparative processes. The further increase of the TCA cycle fluxes, when higher doses of drug applied, eventually enhance reactive oxygen species (ROS) production and trigger apoptotic program. Conclusion: The application of Isodyn to the analysis of mechanism of edelfosine-induced apoptosis revealed primary drug-induced metabolic changes, which are important for the subsequent initiation of apoptotic program. Initiation of such metabolic changes could be exploited in anticancer therapy