Postnatal growth restriction alters myocardial mitochondrial energetics in mice.

Postnatal growth restriction (PGR) can increase the risk of cardiovascular disease (CVD) potentially due to impairments in oxidative phosphorylation (OxPhos) within cardiomyocyte mitochondria. The purpose of this investigation was to determine if PGR impairs cardiac metabolism, specifically OxPhos. FVB (Friend Virus B-type) mice were fed a normal-protein (NP: 20% protein), or low-protein (LP: 8% protein) isocaloric diet 2 weeks before mating. LP dams produce ∼20% less milk, and pups nursed by LP dams experience reduced growth into adulthood as compared to pups nursed by NP dams. At birth (PN1), pups born to dams fed the NP diet were transferred to LP dams (PGR group) or a different NP dam (control group: CON). At weaning (PN21), all mice were fed the NP diet. At PN22 and PN80, mitochondria were isolated for respirometry (oxygen consumption rate, J O 2 ${J_{{{\mathrm{O}}_{\mathrm{2}}}}}$ ) and fluorimetry (reactive oxygen species emission, J H 2 O 2 ${J_{{{\mathrm{H}}_{\mathrm{2}}}{{\mathrm{O}}_{\mathrm{2}}}}}$ ) analysis measured as baseline respiration (LEAK) and with saturating ADP (OxPhos). Western blotting at PN22 and PN80 determined protein abundance of uncoupling protein 3, peroxiredoxin-6, voltage-dependent anion channel and adenine nucleotide translocator 1 to provide further insight into mitochondrial function. ANOVAs with the main effects of diet, sex and age with α-level of 0.05 was set a priori. Overall, PGR (7.8 ± 1.1) had significant (P = 0.01) reductions in respiratory control in complex I when compared to CON (8.9 ± 1.0). In general, our results show that PGR led to higher electron leakage in the form of free radical production and reactive oxygen species emission. No significant diet effects were found in protein abundance. The observed reduced respiratory control and increased ROS emission in PGR mice may increase risk for CVD in mice.

The Role of Swelling in the Regulation of OPA1-Mediated Mitochondrial Function in the Heart In Vitro.

Optic atrophy-1 (OPA1) plays a crucial role in the regulation of mitochondria fusion and participates in maintaining the structural integrity of mitochondrial cristae. Here we elucidate the role of OPA1 cleavage induced by calcium swelling in the presence of Myls22 (an OPA1 GTPase activity inhibitor) and TPEN (an OMA1 inhibitor). The rate of ADP-stimulated respiration was found diminished by both inhibitors, and they did not prevent Ca2+-induced mitochondrial respiratory dysfunction, membrane depolarization, or swelling. L-OPA1 cleavage was stimulated at state 3 respiration; therefore, our data suggest that L-OPA1 cleavage produces S-OPA1 to maintain mitochondrial bioenergetics in response to stress.

Crosstalk between adenine nucleotide transporter and mitochondrial swelling: experimental and computational approaches.

Mitochondrial metabolism and function are modulated by changes in matrix Ca2+. Small increases in the matrix Ca2+ stimulate mitochondrial bioenergetics, whereas excessive Ca2+ leads to cell death by causing massive matrix swelling and impairing the structural and functional integrity of mitochondria. Sustained opening of the non-selective mitochondrial permeability transition pores (PTP) is the main mechanism responsible for mitochondrial Ca2+ overload that leads to mitochondrial dysfunction and cell death. Recent studies suggest the existence of two or more types of PTP, and adenine nucleotide translocator (ANT) and FOF1-ATP synthase were proposed to form the PTP independent of each other. Here, we elucidated the role of ANT in PTP opening by applying both experimental and computational approaches. We first developed and corroborated a detailed model of the ANT transport mechanism including the matrix (ANTM), cytosolic (ANTC), and pore (ANTP) states of the transporter. Then, the ANT model was incorporated into a simple, yet effective, empirical model of mitochondrial bioenergetics to ascertain the point when Ca2+ overload initiates PTP opening via an ANT switch-like mechanism activated by matrix Ca2+ and is inhibited by extra-mitochondrial ADP. We found that encoding a heterogeneous Ca2+ response of at least three types of PTPs, weakly, moderately, and strongly sensitive to Ca2+, enabled the model to simulate Ca2+ release dynamics observed after large boluses were administered to a population of energized cardiac mitochondria. Thus, this study demonstrates the potential role of ANT in PTP gating and proposes a novel mechanism governing the cryptic nature of the PTP phenomenon.

Calcium Overload and Mitochondrial Metabolism.

Mitochondria calcium is a double-edged sword. While low levels of calcium are essential to maintain optimal rates of ATP production, extreme levels of calcium overcoming the mitochondrial calcium retention capacity leads to loss of mitochondrial function. In moderate amounts, however, ATP synthesis rates are inhibited in a calcium-titratable manner. While the consequences of extreme calcium overload are well-known, the effects on mitochondrial function in the moderately loaded range remain enigmatic. These observations are associated with changes in the mitochondria ultrastructure and cristae network. The present mini review/perspective follows up on previous studies using well-established cryo-electron microscopy and poses an explanation for the observable depressed ATP synthesis rates in mitochondria during calcium-overloaded states. The results presented herein suggest that the inhibition of oxidative phosphorylation is not caused by a direct decoupling of energy metabolism via the opening of a calcium-sensitive, proteinaceous pore but rather a separate but related calcium-dependent phenomenon. Such inhibition during calcium-overloaded states points towards mitochondrial ultrastructural modifications, enzyme activity changes, or an interplay between both events.

Measuring Mitochondrial Function: From Organelle to Organism.

Mitochondrial energy production is crucial for normal daily activities and maintenance of life. Herein, the logic and execution of two main classes of measurements are outlined to delineate mitochondrial function: ATP production and oxygen consumption. Aerobic ATP production is quantified by phosphorus magnetic resonance spectroscopy (31PMRS) in vivo in both human subjects and animal models using the same protocols and maintaining the same primary assumptions. Mitochondrial oxygen consumption is quantified by oxygen polarography and applied in isolated mitochondria, cultured cells, and permeabilized fibers derived from human or animal tissue biopsies. Traditionally, mitochondrial functional measures focus on maximal oxidative capacity-a flux rate that is rarely, if ever, observed outside of experimental conditions. Perhaps more physiologically relevant, both measurement classes herein focus on one principal design paradigm; submaximal mitochondrial fluxes generated by graded levels of ADP to map the function for ADP sensitivity. We propose this function defines the bioenergetic role that mitochondria fill within the myoplasm to sense and match ATP demands. Any deficit in this vital role for ATP homeostasis leads to symptoms often seen in cardiovascular and cardiopulmonary diseases, diabetes, and metabolic syndrome.

Depletion of mitochondrial inorganic polyphosphate (polyP) in mammalian cells causes metabolic shift from oxidative phosphorylation to glycolysis.

Inorganic polyphosphate (polyP) is a linear polymer composed of up to a few hundred orthophosphates linked together by high-energy phosphoanhydride bonds, identical with those found in ATP. In mammalian mitochondria, polyP has been implicated in multiple processes, including energy metabolism, ion channels function, and the regulation of calcium signaling. However, the specific mechanisms of all these effects of polyP within the organelle remain poorly understood. The central goal of this study was to investigate how mitochondrial polyP participates in the regulation of the mammalian cellular energy metabolism. To accomplish this, we created HEK293 cells depleted of mitochondrial polyP, through the stable expression of the polyP hydrolyzing enzyme (scPPX). We found that these cells have significantly reduced rates of oxidative phosphorylation (OXPHOS), while their rates of glycolysis were elevated. Consistent with this, metabolomics assays confirmed increased levels of metabolites involved in glycolysis in these cells, compared with the wild-type samples. At the same time, key respiratory parameters of the isolated mitochondria were unchanged, suggesting that respiratory chain activity is not affected by the lack of mitochondrial polyP. However, we detected that mitochondria from cells that lack mitochondrial polyP are more fragmented when compared with those from wild-type cells. Based on these results, we propose that mitochondrial polyP plays an important role as a regulator of the metabolic switch between OXPHOS and glycolysis.

Modeling the Effects of Calcium Overload on Mitochondrial Ultrastructural Remodeling.

Mitochondrial cristae are dynamic invaginations of the inner membrane and play a key role in its metabolic capacity to produce ATP. Structural alterations caused by either genetic abnormalities or detrimental environmental factors impede mitochondrial metabolic fluxes and lead to a decrease in their ability to meet metabolic energy requirements. While some of the key proteins associated with mitochondrial cristae are known, very little is known about how the inner membrane dynamics are involved in energy metabolism. In this study, we present a computational strategy to understand how cristae are formed using a phase-based separation approach of both the inner membrane space and matrix space, which are explicitly modeled using the Cahn-Hilliard equation. We show that cristae are formed as a consequence of minimizing an energy function associated with phase interactions which are subject to geometric boundary constraints. We then extended the model to explore how the presence of calcium phosphate granules, entities that form in calcium overload conditions, exert a devastating inner membrane remodeling response that reduces the capacity for mitochondria to produce ATP. This modeling approach can be extended to include arbitrary geometrical constraints, the spatial heterogeneity of enzymes, and electrostatic effects to mechanize the impact of ultrastructural changes on energy metabolism.

The mitochondrial permeability transition phenomenon elucidated by cryo-EM reveals the genuine impact of calcium overload on mitochondrial structure and function.

Mitochondria have a remarkable ability to uptake and store massive amounts of calcium. However, the consequences of massive calcium accumulation remain enigmatic. In the present study, we analyzed a series of time-course experiments to identify the sequence of events that occur in a population of guinea pig cardiac mitochondria exposed to excessive calcium overload that cause mitochondrial permeability transition (MPT). By analyzing coincident structural and functional data, we determined that excessive calcium overload is associated with large calcium phosphate granules and inner membrane fragmentation, which explains the extent of mitochondrial dysfunction. This data also reveals a novel mechanism for cyclosporin A, an inhibitor of MPT, in which it preserves cristae despite the presence of massive calcium phosphate granules in the matrix. Overall, these findings establish a mechanism of calcium-induced mitochondrial dysfunction and the impact of calcium regulation on mitochondrial structure and function.

Identifying Site-Specific Superoxide and Hydrogen Peroxide Production Rates From the Mitochondrial Electron Transport System Using a Computational Strategy.

Mitochondrial reactive oxygen species (ROS) play important roles in cellular signaling; however, certain pathological conditions such as ischemia/reperfusion (I/R) injury disrupt ROS homeostasis and contribute to cell death. A major impediment to developing therapeutic measures against oxidative stress-induced cellular damage is the lack of a quantitative framework to identify the specific sources and regulatory mechanisms of mitochondrial ROS production. We developed a thermodynamically consistent, mass-and-charge balanced, kinetic model of mitochondrial ROS homeostasis focused on redox sites of electron transport chain complexes I, II, and III. The model was calibrated and corroborated using comprehensive data sets relevant to ROS homeostasis. The model predicts that complex I ROS production dominates other sources under conditions favoring a high membrane potential with elevated nicotinamide adenine dinucleotide (NADH) and ubiquinol (QH2) levels. In general, complex I contributes to significant levels of ROS production under pathological conditions, while complexes II and III are responsible for basal levels of ROS production, especially when QH2 levels are elevated. The model also reveals that hydrogen peroxide production by complex I underlies the non-linear relationship between ROS emission and O2 at low O2 concentrations. Lastly, the model highlights the need to quantify scavenging system activity under different conditions to establish a complete picture of mitochondrial ROS homeostasis. In summary, we describe the individual contributions of the electron transport system complex redox sites to total ROS emission in mitochondria respiring under various combinations of NADH- and Q-linked respiratory fuels under varying workloads.

Computationally modeling mammalian succinate dehydrogenase kinetics identifies the origins and primary determinants of ROS production.

Succinate dehydrogenase (SDH) is an inner mitochondrial membrane protein complex that links the Krebs cycle to the electron transport system. It can produce significant amounts of superoxide ([Formula: see text]) and hydrogen peroxide (H2O2); however, the precise mechanisms are unknown. This fact hinders the development of next-generation antioxidant therapies targeting mitochondria. To help address this problem, we developed a computational model to analyze and identify the kinetic mechanism of [Formula: see text] and H2O2 production by SDH. Our model includes the major redox centers in the complex, namely FAD, three iron-sulfur clusters, and a transiently bound semiquinone. Oxidation state transitions involve a one- or two-electron redox reaction, each being thermodynamically constrained. Model parameters were simultaneously fit to many data sets using a variety of succinate oxidation and free radical production data. In the absence of respiratory chain inhibitors, model analysis revealed the 3Fe-4S iron-sulfur cluster as the primary [Formula: see text] source. However, when the quinone reductase site is inhibited or the quinone pool is highly reduced, [Formula: see text] is generated primarily by the FAD. In addition, H2O2 production is only significant when the enzyme is fully reduced, and fumarate is absent. Our simulations also reveal that the redox state of the quinone pool is the primary determinant of free radical production by SDH. In this study, we showed the importance of analyzing enzyme kinetics and associated side reactions in a consistent, quantitative, and biophysically detailed manner using a diverse set of experimental data to interpret and explain experimental observations from a unified perspective.

The very low number of calcium-induced permeability transition pores in the single mitochondrion.

Mitochondrial permeability transition (PT) is a phenomenon of stress-induced increase in nonspecific permeability of the mitochondrial inner membrane that leads to disruption of oxidative phosphorylation and cell death. Quantitative measurement of the membrane permeability increase during PT is critically important for understanding the PT's impact on mitochondrial function. The elementary unit of PT is a PT pore (PTP), a single channel presumably formed by either ATP synthase or adenine nucleotide translocator (ANT). It is not known how many channels are open in a single mitochondrion during PT, which makes it difficult to quantitatively estimate the overall degree of membrane permeability. Here, we used wide-field microscopy to record mitochondrial swelling and quantitatively measure rates of single-mitochondrion volume increase during PT-induced high-amplitude swelling. PT was quantified by calculating the rates of water flux responsible for measured volume changes. The total water flux through the mitochondrial membrane of a single mitochondrion during PT was in the range of (2.5 ± 0.4) × 10-17 kg/s for swelling in 2 mM Ca2+ and (1.1 ± 0.2) × 10-17 kg/s for swelling in 200 µM Ca2+. Under these experimental conditions, a single PTP channel with ionic conductance of 1.5 nS could allow passage of water at the rate of 0.65 × 10-17 kg/s. Thus, we estimate the integral ionic conductance of the whole mitochondrion during PT to be 5.9 ± 0.9 nS for 2 mM concentration of Ca2+ and 2.6 ± 0.4 nS for 200 µM of Ca2+. The number of PTPs per mitochondrion ranged from one to nine. Due to the uncertainties in PTP structure and model parameters, PTP count results may be slightly underestimated. However, taking into account that each mitochondrion has ∼15,000 copies of ATP synthases and ANTs, our data imply that PTP activation is a rare event that occurs only in a small subpopulation of these proteins.

Regulation of lipid metabolism in pancreatic beta cells by interferon gamma: A link to anti-viral function.

Interferons (IFN) have been shown to alter lipid metabolism in immune and some non-hematopoietic cells and this affects host cell response to pathogens. In type 1 diabetes, IFNγ acts as a proinflammatory cytokine that, along with other cytokines, is released during pancreatic beta cell autoinflammation and contributes to immune response and beta cell dysfunction. The hypothesis tested herein is that IFN modifies beta cell lipid metabolism and this is associated with enhanced anti-viral response and beta cell stress. Treatment of INS-1 cells with IFNγ for 6 to 24 h led to a dynamic change in TAG and lipid droplet (LD) levels, with a decrease at 6 h and an increase at 24 h. The later accumulation of TAG was associated with increased de novo lipogenesis (DNL), and impaired mitochondrial fatty acid oxidation (FAO). Gene expression results suggested that IFNγ regulates lipolytic, lipogenic, LD and FAO genes in a temporal manner. The changes in lipid gene expression are dependent on the classical Janus kinase (JAK) pathway. Pretreatment with IFNγ robustly enhanced anti-viral gene expression induced by the viral mimetic polyinosinic: polycytidylic acid (PIC), and this potentiating effect of IFNγ was markedly attenuated by inhibitors of DNL. The IFNγ-induced accumulation of lipid, however, was insufficient to cause endoplasmic reticulum (ER) stress. These studies demonstrated a non-canonical effect of IFNγ in regulation of pancreatic beta cell lipid metabolism that is intimately linked with host cell defense and might alter cellular function early in the progression to type 1 diabetes.

Calcium overload decreases net free radical emission in cardiac mitochondria.

Elevated calcium and reactive oxygen species (ROS) are responsible for the bulk of cell death occurring in a variety of clinical settings that include acute coronary events, cerebrovascular accidents, and acute kidney injury. It is commonly believed that calcium and ROS participate in a viscous cycle during these events. However, the precise feedback mechanisms are unknown. We quantitatively demonstrate in this study that, on the contrary, calcium does not stimulate free radical production but suppresses it. Isolated mitochondria from guinea pig hearts were energized with a variety of substrates and exposed to calcium concentrations designed to induce moderate calcium overload conditions associated with ischemia/reperfusion injury but do not elicit the well-known mitochondrial permeability transition phenomenon. Metabolic function and free radical emission were simultaneously quantified using high-resolution respirometry and fluorimetry. Membrane potential, high amplitude swelling, and calcium dynamics were also quantified in parallel. Our results reveal that calcium overload does not lead to excessive ROS emission but does decrease ADP stimulated respiration rates for NADH-dependent pathways. Moreover, we developed an empirical model of mitochondrial free radical homeostasis to identify the processes that are different for each substrate and calcium condition. In summary, we show that in healthy guinea pig mitochondria, calcium uptake and free radical generation do not contribute to a viscous cycle and that the relationship between net free radical production and oxygen concentration is hyperbolic. Altogether, these results lay out an important foundation necessary to quantitatively determine the role of calcium in IR injury and ROS production.

Sea lamprey cardiac mitochondrial bioenergetics after exposure to TFM and its metabolites.

Population control of invasive sea lamprey relies heavily on lampricide treatment of infested streams. The lampricide 3-trifluoromethyl-4-nitrophenol (TFM) is thought to impair mitochondrial ATP production through uncoupling oxidative phosphorylation. However, the effect of TFM on the entire electron transport chain (complexes I to V) in the mitochondria is not clear. In addition, TFM is reduced in phase I metabolism by sea lamprey at higher levels than in other fish species. The effects of these TFM reductive metabolites on mitochondria have not been explored. In this study, we sought to examine the effects of TFM and its reductive metabolite amino-TFM (TFMa) on cardiac mitochondrial oxygen consumption and membrane potential to delineate potential mechanisms for toxicity. To determine if molecules with similar structure also exhibit similar effects on mitochondria, we used 4-nitro-3-methylphenol (NMP) and its reductive metabolites 4-amino-3-methylphenol (NMPa) and 4-nitroso-3-methylphenol (NMPn) for comparisons. We found that mitochondrial bioenergetics was heavily affected with increasing concentrations of TFM, NMP, and NMPa when complexes I and II of the electron transport chain were examined, indicating that the toxic action of these compounds was exerted not only by uncoupling complex V, but also affecting complexes I and II.

Quantification of Mitochondrial Oxidative Phosphorylation in Metabolic Disease: Application to Type 2 Diabetes.

Type 2 diabetes (T2D) is a growing health concern with nearly 400 million affected worldwide as of 2014. T2D presents with hyperglycemia and insulin resistance resulting in increased risk for blindness, renal failure, nerve damage, and premature death. Skeletal muscle is a major site for insulin resistance and is responsible for up to 80% of glucose uptake during euglycemic hyperglycemic clamps. Glucose uptake in skeletal muscle is driven by mitochondrial oxidative phosphorylation and for this reason mitochondrial dysfunction has been implicated in T2D. In this review we integrate mitochondrial function with physiologic function to present a broader understanding of mitochondrial functional status in T2D utilizing studies from both human and rodent models. Quantification of mitochondrial function is explained both in vitro and in vivo highlighting the use of proper controls and the complications imposed by obesity and sedentary lifestyle. This review suggests that skeletal muscle mitochondria are not necessarily dysfunctional but limited oxygen supply to working muscle creates this misperception. Finally, we propose changes in experimental design to address this question unequivocally. If mitochondrial function is not impaired it suggests that therapeutic interventions and drug development must move away from the organelle and toward the cardiovascular system.

Skeletal muscle energetics are compromised only during high-intensity contractions in the Goto-Kakizaki rat model of type 2 diabetes.

Type 2 diabetes (T2D) presents with hyperglycemia and insulin resistance, affecting over 30 million people in the United States alone. Previous work has hypothesized that mitochondria are dysfunctional in T2D and results in both reduced ATP production and glucose disposal. However, a direct link between mitochondrial function and T2D has not been determined. In the current study, the Goto-Kakizaki (GK) rat model of T2D was used to quantify mitochondrial function in vitro and in vivo over a broad range of contraction-induced metabolic workloads. During high-frequency sciatic nerve stimulation, hindlimb muscle contractions at 2- and 4-Hz intensities, the GK rat failed to maintain similar bioenergetic steady states to Wistar control (WC) rats measured by phosphorus magnetic resonance spectroscopy, despite similar force production. Differences were not due to changes in mitochondrial content in red (RG) or white gastrocnemius (WG) muscles (cytochrome c oxidase, RG: 22.2 ± 1.6 vs. 23.3 ± 1.7 U/g wet wt; WG: 10.8 ± 1.1 vs. 12.1 ± 0.9 U/g wet wt; GK vs. WC, respectively). Mitochondria isolated from muscles of GK and WC rats also showed no difference in mitochondrial ATP production capacity in vitro, measured by high-resolution respirometry. At lower intensities (0.25-1 Hz) there were no detectable differences between GK and WC rats in sustained energy balance. There were similar phosphocreatine concentrations during steady-state contraction and postcontractile recovery (τ = 72 ± 6 s GK versus 71 ± 2 s WC). Taken together, these results suggest that deficiencies in skeletal muscle energetics seen at higher intensities are not due to mitochondrial dysfunction in the GK rat.

Calcium phosphate precipitation inhibits mitochondrial energy metabolism.

Early studies have shown that moderate levels of calcium overload can cause lower oxidative phosphorylation rates. However, the mechanistic interpretations of these findings were inadequate. And while the effect of excessive calcium overload on mitochondrial function is well appreciated, there has been little to no reports on the consequences of low to moderate calcium overload. To resolve this inadequacy, mitochondrial function from guinea pig hearts was quantified using several well-established methods including high-resolution respirometry and spectrofluorimetry and analyzed using mathematical modeling. We measured key mitochondrial variables such as respiration, mitochondrial membrane potential, buffer calcium, and substrate effects for a range of mitochondrial calcium loads from near zero to levels approaching mitochondrial permeability transition. In addition, we developed a computer model closely mimicking the experimental conditions and used this model to design experiments capable of eliminating many hypotheses generated from the data analysis. We subsequently performed those experiments and determined why mitochondrial ADP-stimulated respiration is significantly lowered during calcium overload. We found that when calcium phosphate levels, not matrix free calcium, reached sufficient levels, complex I activity is inhibited, and the rate of ATP synthesis is reduced. Our findings suggest that calcium phosphate granules form physical barriers that isolate complex I from NADH, disrupt complex I activity, or destabilize cristae and inhibit NADH-dependent respiration.

Micro-respirometry of whole cells and isolated mitochondria.

Oxygen consumption is a key metric of metabolism in aerobic organisms. Current respirometric methods led to seminal discoveries despite limitations such as high sample demand, exchange with atmospheric O2, and cumulative titration protocols leading to limited choice of useable tissue, complex data interpretation, and restricted experimental design. We developed a sensitive and customizable method of measuring O2 consumption rates by a variety of biological samples in microliter volumes without interference from the aerobic environment. We demonstrate that O2 permeability of the photopolymer, VeroClear, is comparable to that of polyetheretherketone (0.125 vs. 0.143 barrer, respectively) providing an efficient barrier to oxygen ingress. Optical transparency of VeroClear, combined with high resolution 3D printing, allows for optode-based oxygen detection in enclosed samples. These properties yield a microrespirometer with over 100× dynamic range for O2 consumption rates. Importantly, the enclosed respirometer configuration and very low oxygen permeability of materials makes it suitable, with resin pre-conditioning, for quantitative assessment of O2 consumption rates at any desired [O2], including hyperbaric, physiological or hypoxic conditions as necessary for each cell type. We characterized two configurations to study soluble enzymes, isolated mitochondria, cells in suspension, and adherent cells cultured on-chip. Improved sensitivity allows for routine quantitative detection of respiration by as few as several hundred cells. Specific activity of cell suspensions in the microrespirometer was in close agreement with that obtained by high-resolution polarographic respirometry. Adherent cell protocols allowed for physiologically relevant assessment of respiration in retinal pigment epithelial cells, ARPE-19, which displayed lower metabolic rates compared with those in suspension. By exchanging medium composition, we demonstrate that cells can be transiently inhibited by cyanide and that 99.6% of basal O2 uptake is recovered upon its removal. This approach is amenable to new experimental designs and precision measurements on limited sample quantities across basic research and applied fields.

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. To test this hypothesis and to investigate mechanisms of fuel selection, we used a constraint-based kinetic analysis of whole-body metabolism to analyze transient exercise data from these rats. Our model analysis used a thermodynamically constrained kinetic framework that accounts for glycolysis, the TCA cycle, and mitochondrial FA transport and oxidation. The model can effectively match the observed relative rates of oxidation of glucose versus FA, as a function of ATP demand. In searching for the minimal differences required to explain metabolic function in HCR versus LCR rats, it was determined that the whole-body metabolic phenotype of LCR, compared to the HCR, could be explained by a ~50% reduction in total mitochondrial activity with an additional 5-fold reduction in mitochondrial FA transport activity. Finally, we postulate that over sustained periods of exercise that LCR can partly overcome the initial deficit in FA catabolic activity by upregulating FA transport and/or oxidation processes.