Identifying functional metabolic shifts in heart failure with the integration of omics data and a heart-specific, genome-scale model.

In diseased states, the heart can shift to use different carbon substrates, measured through changes in uptake of metabolites by imaging methods or blood metabolomics. However, it is not known whether these measured changes are a result of transcriptional changes or external factors. Here, we explore transcriptional changes in late-stage heart failure using publicly available data integrated with a model of heart metabolism.

Predicting changes in renal metabolism after compound exposure with a genome-scale metabolic model.

The kidneys are metabolically active organs with importance in several physiological tasks such as the secretion of soluble wastes into the urine and synthesizing glucose and oxidizing fatty acids for energy in fasting (non-fed) conditions. Once damaged, the metabolic capability of the kidneys becomes altered. Here, we define metabolic tasks in a computational modeling framework to capture kidney function in an update to the iRno network reconstruction of rat metabolism using literature-based evidence.

Genome-Scale Model-Based Identification of Metabolite Indicators for Early Detection of Kidney Toxicity.

Identifying early indicators of toxicant-induced organ damage is critical to provide effective treatment. To discover such indicators and the underlying mechanisms of toxicity, we used gentamicin as an exemplar kidney toxicant and performed systematic perturbation studies in Sprague Dawley rats. We obtained high-throughput data 7 and 13 h after administration of a single dose of gentamicin (0.

Genome-Scale Characterization of Toxicity-Induced Metabolic Alterations in Primary Hepatocytes.

Context-specific GEnome-scale metabolic Network REconstructions (GENREs) provide a means to understand cellular metabolism at a deeper level of physiological detail. Here, we use transcriptomics data from chemically-exposed rat hepatocytes to constrain a GENRE of rat hepatocyte metabolism and predict biomarkers of liver toxicity using the Transcriptionally Inferred Metabolic Biomarker Response algorithm. We profiled alterations in cellular hepatocyte metabolism following in vitro exposure to four toxicants (acetaminophen, carbon tetrachloride, 2,3,7,8-tetrachlorodibenzodioxin, and trichloroethylene) for six hour.

Mechanistic identification of biofluid metabolite changes as markers of acetaminophen-induced liver toxicity in rats.

Acetaminophen (APAP) is the most commonly used analgesic and antipyretic drug in the world. Yet, it poses a major risk of liver injury when taken in excess of the therapeutic dose. Current clinical markers do not detect the early onset of liver injury associated with excess APAP-information that is vital to reverse injury progression through available therapeutic interventions.

Network Modeling of Liver Metabolism to Predict Plasma Metabolite Changes During Short-Term Fasting in the Laboratory Rat.

The liver-a central metabolic organ that integrates whole-body metabolism to maintain glucose and fatty-acid regulation, and detoxify ammonia-is susceptible to injuries induced by drugs and toxic substances. Although plasma metabolite profiles are increasingly investigated for their potential to detect liver injury earlier than current clinical markers, their utility may be compromised because such profiles are affected by the nutritional state and the physiological state of the animal, and by contributions from extrahepatic sources. To tease apart the contributions of liver and non-liver sources to alterations in plasma metabolite profiles, here we sought to computationally isolate the plasma metabolite changes originating in the liver during short-term fasting.

A simplified metabolic network reconstruction to promote understanding and development of flux balance analysis tools.

GEnome-scale Network REconstructions (GENREs) mathematically describe metabolic reactions of an organism or a specific cell type. GENREs can be used with a number of constraint-based reconstruction and analysis (COBRA) methods to make computational predictions on how a system changes in different environments. We created a simplified GENRE (referred to as iSIM) that captures central energy metabolism with nine metabolic reactions to illustrate the use of and promote the understanding of GENREs and constraint-based methods.

Metabolic network-based predictions of toxicant-induced metabolite changes in the laboratory rat.

In order to provide timely treatment for organ damage initiated by therapeutic drugs or exposure to environmental toxicants, we first need to identify markers that provide an early diagnosis of potential adverse effects before permanent damage occurs. Specifically, the liver, as a primary organ prone to toxicants-induced injuries, lacks diagnostic markers that are specific and sensitive to the early onset of injury. Here, to identify plasma metabolites as markers of early toxicant-induced injury, we used a constraint-based modeling approach with a genome-scale network reconstruction of rat liver metabolism to incorporate perturbations of gene expression induced by acetaminophen, a known hepatotoxicant.

Systems-level computational modeling demonstrates fuel selection switching in high capacity running and low capacity running rats.

High capacity and low capacity running rats, HCR and LCR respectively, have been bred to represent two extremes of running endurance and have recently demonstrated disparities in fuel usage during transient aerobic exercise. HCR rats can maintain fatty acid (FA) utilization throughout the course of transient aerobic exercise whereas LCR rats rely predominantly on glucose utilization. We hypothesized that the difference between HCR and LCR fuel utilization could be explained by a difference in mitochondrial density.

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.

Influence of metabolic dysfunction on cardiac mechanics in decompensated hypertrophy and heart failure.

Alterations in energetic state of the myocardium are associated with decompensated heart failure in humans and in animal models. However, the functional consequences of the observed changes in energetic state on mechanical function are not known. The primary aim of the study was to quantify mechanical/energetic coupling in the heart and to determine if energetic dysfunction can contribute to mechanical failure.

Mitochondrial structure and function are not different between nonfailing donor and end-stage failing human hearts.

During human heart failure, the balance of cardiac energy use switches from predominantly fatty acids (FAs) to glucose. We hypothesized that this substrate shift was the result of mitochondrial degeneration; therefore, we examined mitochondrial oxidation and ultrastructure in the failing human heart by using respirometry, transmission electron microscopy, and gene expression studies of demographically matched donor and failing human heart left ventricular (LV) tissues. Surprisingly, respiratory capacities for failing LV isolated mitochondria (n = 9) were not significantly diminished compared with donor LV isolated mitochondria (n = 7) for glycolysis (pyruvate + malate)- or FA (palmitoylcarnitine)-derived substrates, and mitochondrial densities, assessed via citrate synthase activity, were consistent between groups.

Improving the physiological realism of experimental models.

The Virtual Physiological Human (VPH) project aims to develop integrative, explanatory and predictive computational models (C-Models) as numerical investigational tools to study disease, identify and design effective therapies and provide an in silico platform for drug screening. Ultimately, these models rely on the analysis and integration of experimental data. As such, the success of VPH depends on the availability of physiologically realistic experimental models (E-Models) of human organ function that can be parametrized to test the numerical models.

Feedback Regulation and Time Hierarchy of Oxidative Phosphorylation in Cardiac Mitochondria.

To determine how oxidative ATP synthesis is regulated in the heart, the responses of cardiac mitochondria oxidizing pyruvate to alterations in [ATP], [ADP], and inorganic phosphate ([Pi]) were characterized over a range of steady-state levels of extramitochondrial [ATP], [ADP], and [Pi]. Evolution of the steady states of the measured variables with the flux of respiration shows that: (1) a higher phosphorylation potential is achieved by mitochondria at higher [Pi] for a given flux of respiration; (2) the time hierarchy of oxidative phosphorylation is given by phosphorylation subsystem, electron transport chain, and substrate dehydrogenation subsystems listed in increasing order of their response times; (3) the matrix ATP hydrolysis mass action ratio [ADP] × [Pi]/[ATP] provides feedback to the substrate dehydrogenation flux over the entire range of respiratory flux examined in this study; and finally, (4) contrary to previous models of regulation of oxidative phosphorylation, [Pi] does not modulate the activity of complex III.

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.

Open-Loop Control of Oxidative Phosphorylation in Skeletal and Cardiac Muscle Mitochondria by Ca(2.).

In cardiac muscle, mitochondrial ATP synthesis is driven by demand for ATP through feedback from the products of ATP hydrolysis. However, in skeletal muscle at higher workloads there is an apparent contribution of open-loop stimulation of ATP synthesis. Open-loop control is defined as modulation of flux through a biochemical pathway by a moiety, which is not a reactant or a product of the biochemical reactions in the pathway.

Characterization of the kinetics of cardiac cytosolic malate dehydrogenase and comparative analysis of cytosolic and mitochondrial isoforms.

Because the mitochondrial inner membrane is impermeable to pyridine nucleotides, transport of reducing equivalents between the mitochondrial matrix and the cytoplasm relies on shuttle mechanisms, including the malate-aspartate shuttle and the glycerol-3-phosphate shuttle. These shuttles are needed for reducing equivalents generated by metabolic reactions in the cytosol to be oxidized via aerobic metabolism. Two isoenzymes of malate dehydrogenase (MDH) operate as components of the malate-aspartate shuttle, in which a reducing equivalent is transported via malate, which when oxidized to oxaloacetate, transfers an electron pair to reduce NAD to NADH.

Determination of the catalytic mechanism for mitochondrial malate dehydrogenase.

The kinetics of malate dehydrogenase (MDH) catalyzed oxidation/reduction of L-malate/oxaloacetate is pH-dependent due to the proton generated/taken up during the reaction. Previous kinetic studies on the mitochondrial MDH did not yield a consensus kinetic model that explains both substrate and pH dependency of the initial velocity. In this study, we propose, to our knowledge, a new kinetic mechanism to explain kinetic data acquired over a range of pH and substrate concentrations.

Analysis of the kinetics and bistability of ubiquinol:cytochrome c oxidoreductase.

Ubiquinol:cytochrome c oxidoreductase, bc1 complex, is the enzyme in the respiratory chain of mitochondria responsible for the transfer reducing potential from ubiquinol to cytochrome c coupled to the movement of charge against the electrostatic potential across the mitochondrial inner membrane. The complex is also implicated in the generation of reactive oxygen species under certain conditions and is thus a contributor to cellular oxidative stress. Here, a biophysically detailed, thermodynamically consistent model of the bc1 complex for mammalian mitochondria is developed.

Modeling to link regional myocardial work, metabolism and blood flows.

Given the mono-functional, highly coordinated processes of cardiac excitation and contraction, the observations that regional myocardial blood flows, rMBF, are broadly heterogeneous has provoked much attention, but a clear explanation has not emerged. In isolated and in vivo heart studies the total coronary flow is found to be proportional to the rate-pressure product (systolic mean blood pressure times heart rate), a measure of external cardiac work. The same relationship might be expected on a local basis: more work requires more flow.

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 Ca(2+) under various substrate and ADP incubation conditions in an attempt to determine and understand the role of Ca(2+) in maintaining energy homeostasis in working hearts. In this study, we tested the hypothesis that, at physiological temperature and 1 mM extramitochondrial free magnesium, Ca(2+) 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 Ca(2+) concentration averaged per beat, Ca(2+) had no observable stimulatory effect.

Identification of the catalytic mechanism and estimation of kinetic parameters for fumarase.

The enzyme fumarase catalyzes the reversible hydration of fumarate to malate. The reaction catalyzed by fumarase is critical for cellular energetics as a part of the tricarboxylic acid cycle, which produces reducing equivalents to drive oxidative ATP synthesis. A catalytic mechanism for the fumarase reaction that can account for the kinetic behavior of the enzyme observed in both isotope exchange studies and initial velocity studies has not yet been identified.

Kinetic analysis and design of experiments to identify the catalytic mechanism of the monocarboxylate transporter isoforms 4 and 1.

Transport of lactate, pyruvate, and other monocarboxylates across the sarcolemma of skeletal and cardiac myocytes occurs via passive diffusion and by monocarboxylate transporter (MCT) mediated transport. The flux of lactate and protons through the MCT plays an important role in muscle energy metabolism during rest and exercise and in pH regulation during exercise. The MCT isoforms 1 and 4 are the major isoforms of this transporter in skeletal and cardiac muscle.