Kalyan C. Vinnakota

Computer modeling of mitochondrial tricarboxylic acid cycle, oxidative phosphorylation, metabolite transport, and electrophysiology

A computational model of mitochondrial metabolism and electrophysiology is introduced and applied to analysis of data from isolated cardiac mitochondria and data on phosphate metabolites in striated muscle in vivo. This model is constructed based on detailed kinetics and thermodynamically balanced reaction mechanisms and a strict accounting of rapidly equilibrating biochemical species. Since building such a model requires introducing a large number of adjustable kinetic parameters, a correspondingly large amount of independent data from isolated mitochondria respiring on different substrates and subject to a variety of protocols is used to parameterize the model and ensure that it is challenged by a wide range of data corresponding to diverse conditions. The developed model is further validated by both in vitro data on isolated cardiac mitochondria and in vivo experimental measurements on human skeletal muscle. The validated model is used to predict the roles of NAD and ADP in regulating the tricarboxylic acid cycle dehydrogenase fluxes, demonstrating that NAD is the more important regulator. Further model predictions reveal that a decrease of cytosolic pH value results in decreases in mitochondrial membrane potential and a corresponding drop in the ability of the mitochondria to synthesize ATP at the hydrolysis potential required for cellular function.

Regulation of ENaC expression at the cell surface by Rab11.

The epithelial Na(+) channel (ENaC) is an essential channel responsible for Na(+) reabsorption. Coexpression of Rab11a and Rab3a small G proteins with ENaC results in a significant increase in channel activity. In contrast, coexpression of Rab5, Rab27a, and Arf-1 had no effect or slightly decreased ENaC activity. Inhibition of MEK with PD98059, Rho-kinase with Y27632 or PI3-kinase with LY294002 had no effect on ENaC activity in Rab11a-transfected CHO cells. Fluorescence imaging methods demonstrate that Rab11a colocalized with ENaC. Rab11a increases ENaC activity in an additive manner with dominant-negative dynamin, which is a GTPase responsible for endocytosis. Brefeldin A, an inhibitor of intracellular protein translocation, blocked the stimulatory action of Rab11a on ENaC activity. We conclude that ENaC channels, present on the apical plasma membrane, are being exchanged with channels from the intracellular pool in a Rab11-dependent manner.

Detailed Enzyme Kinetics in Terms of Biochemical Species: Study of Citrate Synthase

The compulsory-ordered ternary catalytic mechanism for two-substrate two-product enzymes is analyzed to account for binding of inhibitors to each of the four enzyme states and to maintain the relationship between the kinetic constants and the reaction equilibrium constant. The developed quasi-steady flux expression is applied to the analysis of data from citrate synthase to determine and parameterize a kinetic scheme in terms of biochemical species, in which the effects of pH, ionic strength, and cation binding to biochemical species are explicitly accounted for in the analysis of the data. This analysis provides a mechanistic model that is consistent with the data that have been used support competing hypotheses regarding the catalytic mechanism of this enzyme.

Stimulatory effects of calcium on respiration and NAD(P)H synthesis in intact rat heart mitochondria utilizing physiological substrates cannot explain respiratory control in vivo

Mitochondrial TCA cycle dehydrogenase enzymes have been shown to be stimulated by Ca2+ under various substrate and ADP incubation conditions in an attempt to determine and understand the role of Ca2+ in maintaining energy homeostasis in working hearts. In this study, we tested the hypothesis that, at physiological temperature and 1 mm extramitochondrial free magnesium, Ca2+ can stimulate the overall mitochondrial NAD(P)H generation flux in rat heart mitochondria utilizing pyruvate and malate as substrates at both subsaturating and saturating concentrations. In both cases, we found that, in the physiological regime of mitochondrial oxygen consumption observed in the intact animal and in the physiological range of cytosolic Ca2+ concentration averaged per beat, Ca2+ had no observable stimulatory effect. A modest apparent stimulatory effect (22–27%) was observable at supraphysiological maximal ADP-stimulated respiration at 2.5 mm initial phosphate. The stimulatory effects observed over the physiological Ca2+ range are not sufficient to make a significant contribution to the control of oxidative phosphorylation in the heart in vivo.

A database of thermodynamic quantities for the reactions of glycolysis and the tricarboxylic acid cycle.

Analysis of biochemical systems requires reliable and self-consistent databases of thermodynamic properties for biochemical reactions. Here a database of thermodynamic properties for the reactions of glycolysis and the tricarboxylic acid cycle is developed from measured equilibrium data. Species-level free energies of formation are estimated on the basis of comparing thermodynamic model predictions for reaction-level equilibrium constants to previously reported data obtained under different experimental conditions. Matching model predictions to the data involves applying state corrections for ionic strength, pH, and metal ion binding for each input experimental biochemical measurement. By archiving all of the raw data, documenting all model assumptions and calculations, and making the computer package and data available, this work provides a framework for extension and refinement by adding to the underlying raw experimental data in the database and/or refining the underlying model assumptions. Thus the resulting database is a refinement of preexisting databases of thermodynamics in terms of reliability, self-consistency, transparency, and extensibility.

Chapter 2 Multiple Ion Binding Equilibria, Reaction Kinetics, and Thermodynamics in Dynamic Models of Biochemical Pathways

The operation of biochemical systems in vivo and in vitro is strongly influenced by complex interactions between biochemical reactants and ions such as H(+), Mg(2+), K(+), and Ca(2+). These are important second messengers in metabolic and signaling pathways that directly influence the kinetics and thermodynamics of biochemical systems. Herein we describe the biophysical theory and computational methods to account for multiple ion binding to biochemical reactants and demonstrate the crucial effects of ion binding on biochemical reaction kinetics and thermodynamics. In simulations of realistic systems, the concentrations of these ions change with time due to dynamic buffering and competitive binding. In turn, the effective thermodynamic properties vary as functions of cation concentrations and important environmental variables such as temperature and overall ionic strength. Physically realistic simulations of biochemical systems require incorporating all of these phenomena into a coherent mathematical description. Several applications to physiological systems are demonstrated based on this coherent simulation framework.

The computational integrated myocyte: A view into the virtual heart

Abstract: The presentation outlines an integrative approach for developing a computational model of cardiomyocytes. A modular approach is proposed, and strategies of linking the modules (intermediary metabolism, electrophysiology, and mechanics) of the model are presented. A strong recommendation is given toward an integrated system approach backed by experimental validation.

Estrogen maintains mitochondrial content and function in the right ventricle of rats with pulmonary hypertension

The typical cause of death in pulmonary hypertension (PH) is right ventricular (RV) failure, with females showing better survival rates than males. Recently, metabolic shift and mitochondrial dysfunction have been demonstrated in RV failure secondary to PH. In light of evidence showing that estrogen protects mitochondrial function and biogenesis in noncardiovascular systems, we hypothesized that the mechanism by which estrogen preserves RV function is via protection of mitochondrial content and oxidative capacity in PH. We used a well‐established model of PH (Sugen+Hypoxia) in ovariectomized female rats with/without estrogen treatment. RV functional measures were derived from pressure–volume relationships measured via RV catheterization in live rats. Citrate synthase activity, a marker of mitochondrial density, was measured in both RV and LV tissues. Respiratory capacity of mitochondria isolated from RV was measured using oxygraphy. We found that RV ventricular‐vascular coupling efficiency decreased in the placebo‐treated SuHx rats (0.78 ± 0.10 vs. 1.50 ± 0.13 in control, P < 0.05), whereas estrogen restored it. Mitochondrial density decreased in placebo‐treated SuHx rats (0.12 ± 0.01 vs. 0.15 ± 0.01 U citrate synthase/mg in control, P < 0.05), and estrogen attenuated the decrease. Mitochondrial quality and oxidative capacity tended to be lower in placebo‐treated SuHx rats only. The changes in mitochondrial biogenesis and function paralleled the expression levels of PGC‐1α in RV. Our results suggest that estrogen protects RV function by preserving mitochondrial content and oxidative capacity. This provides a mechanism by which estrogen provides protection in female PH patients and paves the way to develop estrogen and its targets as a novel RV‐specific therapy for PH.

Catalytic Coupling of Oxidative Phosphorylation, ATP Demand, and Reactive Oxygen Species Generation

Competing models of mitochondrial energy metabolism in the heart are highly disputed. In addition, the mechanisms of reactive oxygen species (ROS) production and scavenging are not well understood. To deepen our understanding of these processes, a computer model was developed to integrate the biophysical processes of oxidative phosphorylation and ROS generation. The model was calibrated with experimental data obtained from isolated rat heart mitochondria subjected to physiological conditions and workloads. Model simulations show that changes in the quinone pool redox state are responsible for the apparent inorganic phosphate activation of complex III. Model simulations predict that complex III is responsible for more ROS production during physiological working conditions relative to complex I. However, this relationship is reversed under pathological conditions. Finally, model analysis reveals how a highly reduced quinone pool caused by elevated levels of succinate is likely responsible for the burst of ROS seen during reperfusion after ischemia.

Common phenotype of resting mouse extensor digitorum longus and soleus muscles: equal ATPase and glycolytic flux during transient anoxia

Rates of ATPase and glycolysis are several times faster in actively contracting mouse extensor digitorum longus muscle (EDL) than soleus (SOL), but we find these rates are not distinguishable at rest. We used a transient anoxic perturbation of steady state energy balance to decrease phosphocreatine (PCr) reversibly and to measure the rates of ATPase and of lactate production without muscle activation or contraction. The rate of glycolytic ATP synthesis is less than the ATPase rate, accounting for the continual PCr decrease during anoxia in both muscles. We fitted a mathematical model validated with properties of enzymes and solutes measured in vitro and appropriate for the transient perturbation of these muscles to experimental data to test whether the model accounts for the results. Simulations showed equal rates of ATPase and lactate production in both muscles. ATPase controls glycolytic flux by feedback from its products. Adenylate kinase function is critical because a rise in [AMP] is necessary to activate glycogen phosphorylase. ATPase is the primary source of H+ production. The sum of contributions of the 13 reactions of the glycogenolytic and glycolytic network to total proton load is negligible. The stoichiometry of lactate and H+ production is near unity. These results identify a default state of energy metabolism for resting muscle in which there is no difference in the metabolic phenotype of EDL and SOL. Therefore, additional control mechanisms, involving higher ATPase flux and [Ca2+], must exist to explain the well‐known difference in glycolytic rates in fast‐twitch and slow‐twitch muscles in actively contracting muscle.