Effect of bivalirudin on coagulation in neonatal (cord) and adult human blood in vitro.

INTRODUCTION: Bivalirudin is recommended as an alternative to heparin in cardiac surgery with cardiopulmonary bypass. Although it has been used in infants and children for this indication, there is a paucity of data on the pharmacologic effects of bivalirudin in neonates. Given the immaturity of the hemostatic system in neonates, we hypothesized that coagulation responses to bivalirudin in this population would be different than in adults. METHODS: Blood samples were drawn from placenta-cord units and from healthy adult donors. The study was carried out in two steps. First, bivalirudin was added to cord and adult blood samples at concentrations of 0, 5, 10, 15, and 20 µg/mL. Activated clotting time and thromboelastographic variables were recorded. Next, we used a Chandler loop system to assess the efficacy of bivalirudin in a simple model of cardiopulmonary bypass. The loops were primed with cord or adult blood and were run until thrombus was detected. Plasma bivalirudin concentrations were measured at 1, 15, 30, 45, 60, and 75 min after initiating rotation of the loops using liquid chromatography/mass spectrometry. RESULTS: Bivalirudin elicited a dose-dependent prolongation inhibition of coagulation in both cord and adult blood samples with greater potency in cord blood in comparison to adult blood (activated clotting time: 627 ± 50 vs. 452 ± 22 s at 15 µg/mL bivalirudin, p < .0001). This relative potency was also demonstrated in the Chandler loop system, but interestingly, cord blood appeared to inactivate bivalirudin more rapidly than adult blood with earlier clotting in loops containing cord blood. CONCLUSIONS: This study demonstrates that bivalirudin has greater potency in cord blood in vitro than in adult blood. Plasma degradation appears to proceed more rapidly in cord blood than in adults. Both of these findings should be considered when planning dosing regimens in neonatal patients.

Dabigatran pharmacokinetic-pharmacodynamic in sheep: Informing dose for anticoagulation during cardiopulmonary bypass.

BACKGROUND: The effect of the anticoagulant, dabigatran, and its antagonist, idarucizumab, on coagulation remains poorly quantified. There are few pharmacokinetic-pharmacodynamic data available to determine dabigatran dose in humans or animals undergoing cardiopulmonary bypass. METHODS: Five sheep were given intravenous dabigatran 4 mg/kg. Blood samples were collected for thromboelastometric reaction time (R-time) and drug assay at 5, 15, 30, 60, 120, 240, 480 min, and 24 h. Plasma dabigatran concentrations and R-times were analyzed using an integrated pharmacokinetic-pharmacodynamic model using non-linear mixed effects. The impact of idarucizumab 15 mg/kg administered 120 min after dabigatran 4 mg/kg and its effect on R-time was observed. RESULTS: A 2-compartment model described dabigatran pharmacokinetics with a clearance (CL 0.0453 L/min/70 kg), intercompartment clearance (Q 0.268 L/min/70 kg), central volume of distribution (V1 2.94 L/70 kg), peripheral volume of distribution (V2 9.51 L/70 kg). The effect compartment model estimates for a sigmoid EMAX model using Reaction time had an effect site concentration (Ce50 64.2 mg/L) eliciting half of the maximal effect (EMAX 180 min). The plasma-effect compartment equilibration half time (T1/2keo) was 1.04 min. Idarucizumab 15 mg/kg reduced R-time by approximately 5 min. CONCLUSIONS: Dabigatran reversibly binds to the active site on the thrombin molecule, preventing activation of coagulation factors. The pharmacologic target concentration strategy uses pharmacokinetic-pharmacodynamic information to inform dose. A loading dose of dabigatran 0.25 mg/kg followed by a maintenance infusion of dabigatran 0.0175 mg/kg/min for 30 min and a subsequent infusion dabigatran 0.0075 mg/kg/min achieves a steady state target concentration of 5 mg/L in a sheep model.

Distinct effects of intracellular vs. extracellular acidic pH on the cardiac metabolome during ischemia and reperfusion.

Tissue ischemia results in intracellular pH (pHIN) acidification, and while metabolism is a known driver of acidic pHIN, less is known about how acidic pHIN regulates metabolism. Furthermore, acidic extracellular (pHEX) during early reperfusion confers cardioprotection, but how this impacts metabolism is unclear. Herein we employed LCMS based targeted metabolomics to analyze perfused mouse hearts exposed to: (i) control perfusion, (ii) hypoxia, (iii) ischemia, (iv) enforced acidic pHIN, (v) control reperfusion, and (vi) acidic pHEX (6.8) reperfusion. Surprisingly little overlap was seen between metabolic changes induced by hypoxia, ischemia, and acidic pHIN. Acidic pHIN elevated metabolites in the top half of glycolysis, and enhanced glutathione redox state. Meanwhile, acidic pHEX reperfusion induced substantial metabolic changes in addition to those seen in control reperfusion. This included elevated metabolites in the top half of glycolysis, prevention of purine nucleotide loss, and an enhancement in glutathione redox state. These data led to hypotheses regarding potential roles for methylglyoxal inhibiting the mitochondrial permeability transition pore, and for acidic inhibition of ecto-5'-nucleotidase, as potential mediators of cardioprotection by acidic pHEX reperfusion. However, neither hypothesis was supported by subsequent experiments. In contrast, analysis of cardiac effluents revealed complex effects of pHEX on metabolite transport, suggesting that mildly acidic pHEX may enhance succinate release during reperfusion. Overall, each intervention had distinct and overlapping metabolic effects, suggesting acidic pH is an independent metabolic regulator regardless which side of the cell membrane it is imposed.

Intravenous Dabigatran Provides Adequate Anticoagulation for Cardiopulmonary Bypass Using a Rabbit Model.

BACKGROUND: Heparin anticoagulation has been used successfully for cardiopulmonary bypass (CPB). However, an alternative anticoagulant approach is desirable due to the cases of heparin-induced thrombocytopenia. Dabigatran provides anticoagulation for an in vitro model of simulated CPB. The current analysis tests the hypothesis that dabigatran provides sufficient anticoagulation for CPB in intact rabbits. METHODS: Nonlinear mixed effects models were used to estimate dabigatran parameters for a two-compartment pharmacokinetic model in 10 New Zealand White rabbits. A dabigatran infusion designed to maintain a plasma concentration of 90 µg/ml was run throughout CPB based on the pharmacokinetics. Animals were subjected to sternotomy and anticoagulated with IV dabigatran (six animals) or heparin (four animals). Rabbits were cannulated centrally using the right atrium and ascending aorta and CPB was maintained for 120 min. Measurement of activated clotting time, thromboelastometric reaction time, and blood gases were performed during CPB. Then, the animals were euthanized, and the brain and one kidney were removed for histology. Sections of the arterial filters were inspected using electron microscopy. RESULTS: The observed dabigatran concentrations during CPB were greater than the target concentration, ranging from 137 ± 40 µg/ml at 5 min of CPB to 428 ± 150 µg/ml at 60 min, and 295 ± 35 µg/ml at 120 min. All rabbits completed 2 h of CPB without visible thrombosis. In the two groups, reaction time values were elevated, reaching 10,262 ± 4,198 s (dabigatran group) and 354 ± 141 s (heparin group) at 120 min of CPB. Brains and kidneys showed no evidence of thrombosis or ultrastructural damage. Sections of the arterial line filter showed minimal or no fibrin. There was no significant difference in outcomes between dabigatran- and heparin-treated animals. CONCLUSIONS: In this first-use, proof-of-concept study, the authors have shown that dabigatran provides acceptable anticoagulation similar to heparin to prevent thrombosis using a rabbit CPB model.

Rivaroxaban Reduces the Dabigatran Dose Required for Anticoagulation During Simulated Cardiopulmonary Bypass.

BACKGROUND: Heparin is the standard anticoagulant for cardiopulmonary bypass (CPB); however, there are problems with its use that make the development of suitable alternatives desirable. Currently, no ideal alternative exists. We have previously reported that the direct thrombin inhibitor dabigatran can prevent coagulation in simulated CPB at high concentrations. These high concentrations may cause difficulties in achieving the reversal of dabigatran with idarucizumab, given the markedly different pharmacokinetics of the 2 drugs. Herein, we test the hypothesis that the addition of the anti-Xa drug rivaroxaban would provide suitable anticoagulation at a lower concentration of dabigatran given likely synergy between the 2 classes of drugs. The primary goal of the study was to investigate whether the addition of rivaroxaban reduces the concentration of dabigatran necessary to allow 2 hours of simulated CPB. METHODS: The study was performed in sequential steps. Blood collected from consenting healthy donors was used throughout. First, we added graded concentrations of dabigatran and rivaroxaban alone and in combination and assessed inhibition of anticoagulation using thromboelastometry. Using results from this step, combinations of dabigatran and rivaroxaban were tested in both Chandler loop and simulated CPB circuits. Dabigatran and rivaroxaban were added before recalcification, and the circuits were run for 120 minutes. In both models of CPB, 120 minutes of circulation without visible thrombus was considered successful. In the Chandler loop system, idarucizumab was added to reverse anticoagulant effects. In the CPB circuits, the arterial line filters were examined using scanning electron microscope (SEM) to qualitatively assess for fibrin deposition. RESULTS: In vitro analysis of blood samples treated with dabigatran and rivaroxaban showed that dabigatran and rivaroxaban individually prolonged clotting time (CT) in a dose-dependent manner. However, when combined, the drugs behaved synergistically. In the Chandler loop system, dabigatran 2400 and 4800 ng/mL plus rivaroxaban (150 ng/mL) effectively prevented clot formation and reduced the dynamics of clot propagation for 120 minutes. Idarucizumab (250-1000 µg/mL) effectively reversed anticoagulation. In the CPB circuits, dabigatran (2500 ng/mL) and rivaroxaban (200 ng/mL) were successful in allowing 120 minutes of simulated CPB and prevented fibrin deposition. Biomarkers of coagulation activation did not increase during simulated CPB. Heparin controls performed similarly to dabigatran and rivaroxaban. CONCLUSIONS: The dual administration of oral anticoagulant drugs (dabigatran and Rivaroxaban) with different pharmacologic mechanisms of action produced synergistic inhibition of coagulation in vitro and successfully prevented clotting during simulated CPB.

Delayed concentration effect models for dabigatran anticoagulation.

INTRODUCTION: Dabigatran is an anticoagulant with potential use during cardiopulmonary bypass in children and adults. The pharmacokinetic-pharmacodynamic relationship for dabigatran anticoagulation effect was investigated in an intact animal model using rabbits. METHODS: Ten male New Zealand white rabbits were given a novel preparation of intravenous dabigatran 15 mg.kg-1 . Blood samples were collected for activated clotting time, thromboelastometric reaction time, and drug assay at 5, 15, 30, 60, 120, 180, 300, and 420 min. Plasma dabigatran concentrations and coagulation measures were analyzed using an integrated pharmacokinetic-pharmacodynamic model using nonlinear mixed effects. Effects (activated clotting and thromboelastometric reaction times) were described using a sigmoidal EMAX model. Pharmacokinetic parameters were scaled using allometry and standardized to a 70 kg size standard. Pharmacodynamics were investigated using both an effect compartment model and an indirect response (turnover) model. RESULTS: A two-compartment model described dabigatran pharmacokinetics with a clearance (CL 0.135 L.min-1 .70 kg-1 ), intercompartment clearance (Q 0.33 L.min-1 .70 kg-1 ), central volume of distribution (V1 12.3 L.70 kg-1 ), and peripheral volume of distribution (V2 30.1 L.70 kg-1 ). The effect compartment model estimates for a sigmoid EMAX model with activated clotting time had an effect site concentration (Ce50 20.1 mg.L-1 ) eliciting half of the maximal effect (EMAX 899 s) and a Hill coefficient (N 0.66). The equilibration half time (T1/2 keo) was 1.4 min. Results for the reaction time were plasma concentration (Cp50 65.3 mg.L-1 ), EMAX 34 min, N 0.80 with a baseline thromboelastometric reaction time of 0.4 min. The equilibration half time (T1/2 keo) was 2.04 min. CONCLUSIONS: Dabigatran reversibly binds to the active site on the thrombin molecule, preventing thrombin-mediated activation of coagulation factors. The effect compartment model performed slightly better than the turnover model and was able to adequately capture pharmacodynamics for both activated clotting and thromboelastometric reaction times. The equilibration half time was short (<2 min). These data can be used to inform future animal preclinical studies for those undergoing cardiopulmonary bypass. These preclinical data also demonstrate the magnitude of parameter values for a delayed effect compartment model that are applicable to humans.

High-Dose Dabigatran Is an Effective Anticoagulant for Simulated Cardiopulmonary Bypass Using Human Blood.

BACKGROUND: Currently no ideal alternative exists for heparin for cardiopulmonary bypass (CPB). Dabigatran is a direct thrombin inhibitor for which a reversal agent exists. The primary end point of the study was to explore whether Dabigatran was an effective anticoagulant for 120 minutes of simulated CPB. METHODS: The study was designed in 2 sequential steps. Throughout, human blood from healthy donors was used for each experimental step. Initially, increasing concentrations of Dabigatran were added to aliquots of fresh whole blood, and the anticoagulant effect measured using kaolin/tissue factor-activated thromboelastography (rapidTEG). The dynamics of all thromboelastography (TEG) measurements were studied with repeated measures analysis of variance (ANOVA). Based on these data, aliquots of blood were treated with high-concentration Dabigatran and placed in a Chandler loop as a simple ex vivo bypass model to assess whether Dabigatran had sufficient anticoagulant effects to maintain blood fluidity for 2 hours of continuous contact with the artificial surface of the PVC tubing. Idarucizumab, humanized monoclonal antibody fragment, was used to verify the reversibility of Dabigatran effects. Finally, 3 doses of Dabigatran were tested in a simulated CPB setup using a heart-lung machine and a commercially available bypass circuit with an arteriovenous (A-V) loop. The primary outcome was the successful completion of 120 minutes of simulated CPB with dabigatran anticoagulation, defined as lack of visible thrombus. Thromboelastographic reaction (R) time was measured repeatedly in each bypass simulation, and the circuits were continuously observed for clot. Scanning Electron Microscopy (SEM) was used to visualize fibrin formation in the filters meshes during CPB. RESULTS: In in vitro blood samples, Dabigatran prolonged R time and reduced the dynamics of clot propagation (as measured by speed of clot formation [Angle], maximum rate of thrombus generation [MRTG], and time to maximum rate of thrombus generation [TMRTG]) in a dose-dependent manner. In the Chandler Loop, high doses of Dabigatran prevented clot formation for 120 minutes, but only at doses higher than expected. Idarucizumab decreased R time and reversed anticoagulation in both in vitro and Chandler Loops settings. In the A-V loop bypass simulation, Dabigatran prevented gross thrombus generation for 120 minutes of simulated CPB. CONCLUSIONS: Using sequential experimental approaches, we showed that direct thrombin inhibitor Dabigatran in high doses maintained anticoagulation of blood for simulated CPB. Idarucizumab reduced time for clot formation reversing the anticoagulation action of Dabigatran.

Ischaemic accumulation of succinate controls reperfusion injury through mitochondrial ROS.

Ischaemia-reperfusion injury occurs when the blood supply to an organ is disrupted and then restored, and underlies many disorders, notably heart attack and stroke. While reperfusion of ischaemic tissue is essential for survival, it also initiates oxidative damage, cell death and aberrant immune responses through the generation of mitochondrial reactive oxygen species (ROS). Although mitochondrial ROS production in ischaemia reperfusion is established, it has generally been considered a nonspecific response to reperfusion. Here we develop a comparative in vivo metabolomic analysis, and unexpectedly identify widely conserved metabolic pathways responsible for mitochondrial ROS production during ischaemia reperfusion. We show that selective accumulation of the citric acid cycle intermediate succinate is a universal metabolic signature of ischaemia in a range of tissues and is responsible for mitochondrial ROS production during reperfusion. Ischaemic succinate accumulation arises from reversal of succinate dehydrogenase, which in turn is driven by fumarate overflow from purine nucleotide breakdown and partial reversal of the malate/aspartate shuttle. After reperfusion, the accumulated succinate is rapidly re-oxidized by succinate dehydrogenase, driving extensive ROS generation by reverse electron transport at mitochondrial complex I. Decreasing ischaemic succinate accumulation by pharmacological inhibition is sufficient to ameliorate in vivo ischaemia-reperfusion injury in murine models of heart attack and stroke. Thus, we have identified a conserved metabolic response of tissues to ischaemia and reperfusion that unifies many hitherto unconnected aspects of ischaemia-reperfusion injury. Furthermore, these findings reveal a new pathway for metabolic control of ROS production in vivo, while demonstrating that inhibition of ischaemic succinate accumulation and its oxidation after subsequent reperfusion is a potential therapeutic target to decrease ischaemia-reperfusion injury in a range of pathologies.

Metabolomics reveals critical adrenergic regulatory checkpoints in glycolysis and pentose-phosphate pathways in embryonic heart.

Cardiac energy demands during early embryonic periods are sufficiently met through glycolysis, but as development proceeds, the oxidative phosphorylation in mitochondria becomes increasingly vital. Adrenergic hormones are known to stimulate metabolism in adult mammals and are essential for embryonic development, but relatively little is known about their effects on metabolism in the embryonic heart. Here, we show that embryos lacking adrenergic stimulation have ∼10-fold less cardiac ATP compared with littermate controls. Despite this deficit in steady-state ATP, neither the rates of ATP formation nor degradation was affected in adrenergic hormone-deficient hearts, suggesting that ATP synthesis and hydrolysis mechanisms were fully operational. We thus hypothesized that adrenergic hormones stimulate metabolism of glucose to provide chemical substrates for oxidation in mitochondria. To test this hypothesis, we employed a metabolomics-based approach using LC/MS. Our results showed glucose 1-phosphate and glucose 6-phosphate concentrations were not significantly altered, but several downstream metabolites in both glycolytic and pentose-phosphate pathways were significantly lower compared with controls. Furthermore, we identified glyceraldehyde-3-phosphate dehydrogenase and glucose-6-phosphate dehydrogenase as key enzymes in those respective metabolic pathways whose activity was significantly (p < 0.05) and substantially (80 and 40%, respectively) lower in adrenergic hormone-deficient hearts. Addition of pyruvate and to a lesser extent ribose led to significant recovery of steady-state ATP concentrations. These results demonstrate that without adrenergic stimulation, glucose metabolism in the embryonic heart is severely impaired in multiple pathways, ultimately leading to insufficient metabolic substrate availability for successful transition to aerobic respiration needed for survival.

Cardiac metabolic effects of KNa1.2 channel deletion and evidence for its mitochondrial localization.

Controversy surrounds the molecular identity of mitochondrial K+ channels that are important for protection against cardiac ischemia-reperfusion injury. Although KNa1.2 (sodium-activated potassium channel encoded by Kcn2) is necessary for cardioprotection by volatile anesthetics, electrophysiological evidence for a channel of this type in mitochondria is lacking. The endogenous physiological role of a potential mito-KNa1.2 channel is also unclear. In this study, single channel patch-clamp of 27 independent cardiac mitochondrial inner membrane (mitoplast) preparations from wild-type (WT) mice yielded 6 channels matching the known ion sensitivity, ion selectivity, pharmacology, and conductance properties of KNa1.2 (slope conductance, 138 ± 1 pS). However, similar experiments on 40 preparations from Kcnt2-/- mice yielded no such channels. The KNa opener bithionol uncoupled respiration in WT but not Kcnt2-/- cardiomyocytes. Furthermore, when oxidizing only fat as substrate, Kcnt2-/- cardiomyocytes and hearts were less responsive to increases in energetic demand. Kcnt2-/- mice also had elevated body fat, but no baseline differences in the cardiac metabolome. These data support the existence of a cardiac mitochondrial KNa1.2 channel, and a role for cardiac KNa1.2 in regulating metabolism under conditions of high energetic demand.-Smith, C. O., Wang, Y. T., Nadtochiy, S. M., Miller, J. H., Jonas, E. A., Dirksen, R. T., Nehrke, K., Brookes, P. S. Cardiac metabolic effects of KNa1.2 channel deletion and evidence for its mitochondrial localization.

Cardioprotection by nicotinamide mononucleotide (NMN): Involvement of glycolysis and acidic pH.

Stimulation of the cytosolic NAD+ dependent deacetylase SIRT1 is cardioprotective against ischemia-reperfusion (IR) injury. NAD+ precursors including nicotinamide mononucleotide (NMN) are thought to induce cardioprotection via SIRT1. Herein, while NMN protected perfused hearts against IR (functional recovery: NMN 42 ±â€¯7% vs. vehicle 11 ±â€¯3%), this protection was insensitive to the SIRT1 inhibitor splitomicin (recovery 47 ±â€¯8%). Although NMN-induced cardioprotection was absent in Sirt3-/- hearts (recovery 9 ±â€¯5%), this was likely due to enhanced baseline injury in Sirt3-/- (recovery 6 ±â€¯2%), since similar injury levels in WT hearts also blunted the protective efficacy of NMN. Considering alternative cardiac effects of NMN, and the requirement of glycolysis for NAD+, we hypothesized NMN may confer protection in part via direct stimulation of cardiac glycolysis. In primary cardiomyocytes, NMN induced cytosolic and extracellular acidification and elevated lactate. In addition, [U-13C]glucose tracing in intact hearts revealed that NMN stimulated glycolytic flux. Consistent with a role for glycolysis in NMN-induced protection, hearts perfused without glucose (palmitate as fuel source), or hearts perfused with galactose (no ATP from glycolysis) exhibited no benefit from NMN (recovery 11 ±â€¯4% and 15 ±â€¯2% respectively). Acidosis during early reperfusion is known to be cardioprotective (i.e., acid post-conditioning), and we also found that NMN was cardioprotective when delivered acutely at reperfusion (recovery 39 ±â€¯8%). This effect of NMN was not additive with acidosis, suggesting overlapping mechanisms. We conclude that the acute cardioprotective benefits of NMN are mediated in part via glycolytic stimulation, with the downstream protective mechanism involving enhanced ATP synthesis during ischemia and/or enhanced acidosis during reperfusion.

Potential mechanisms linking SIRT activity and hypoxic 2-hydroxyglutarate generation: no role for direct enzyme (de)acetylation.

2-Hydroxyglutarate (2-HG) is a hypoxic metabolite with potentially important epigenetic signaling roles. The mechanisms underlying 2-HG generation are poorly understood, but evidence suggests a potential regulatory role for the sirtuin family of lysine deacetylases. Thus, we hypothesized that the acetylation status of the major 2-HG-generating enzymes [lactate dehydrogenase (LDH), isocitrate dehydrogenase (IDH) and malate dehydrogenase (MDH)] may govern their 2-HG-generating activity. In vitro acetylation of these enzymes, with confirmation by western blotting, mass spectrometry, reversibility by recombinant sirtuins and an assay for global lysine occupancy, yielded no effect on 2-HG-generating activity. In addition, while elevated 2-HG in hypoxia is associated with the activation of lysine deacetylases, we found that mice lacking mitochondrial SIRT3 exhibited hyperacetylation and elevated 2-HG. These data suggest that there is no direct link between enzyme acetylation and 2-HG production. Furthermore, our observed effects of in vitro acetylation on the canonical activities of IDH, MDH and LDH appeared to contrast with previous findings wherein acetyl-mimetic lysine mutations resulted in the inhibition of these enzymes. Overall, these data suggest that a causal relationship should not be assumed between acetylation of metabolic enzymes and their activities, canonical or otherwise.

Acidic pH Is a Metabolic Switch for 2-Hydroxyglutarate Generation and Signaling.

2-Hydroxyglutarate (2-HG) is an important epigenetic regulator, with potential roles in cancer and stem cell biology. The d-(R)-enantiomer (d-2-HG) is an oncometabolite generated from α-ketoglutarate (α-KG) by mutant isocitrate dehydrogenase, whereas l-(S)-2-HG is generated by lactate dehydrogenase and malate dehydrogenase in response to hypoxia. Because acidic pH is a common feature of hypoxia, as well as tumor and stem cell microenvironments, we hypothesized that pH may regulate cellular 2-HG levels. Herein we report that cytosolic acidification under normoxia moderately elevated 2-HG in cells, and boosting endogenous substrate α-KG levels further stimulated this elevation. Studies with isolated lactate dehydrogenase-1 and malate dehydrogenase-2 revealed that generation of 2-HG by both enzymes was stimulated severalfold at acidic pH, relative to normal physiologic pH. In addition, acidic pH was found to inhibit the activity of the mitochondrial l-2-HG removal enzyme l-2-HG dehydrogenase and to stimulate the reverse reaction of isocitrate dehydrogenase (carboxylation of α-KG to isocitrate). Furthermore, because acidic pH is known to stabilize hypoxia-inducible factor (HIF) and 2-HG is a known inhibitor of HIF prolyl hydroxylases, we hypothesized that 2-HG may be required for acid-induced HIF stabilization. Accordingly, cells stably overexpressing l-2-HG dehydrogenase exhibited a blunted HIF response to acid. Together, these results suggest that acidosis is an important and previously overlooked regulator of 2-HG accumulation and other oncometabolic events, with implications for HIF signaling.

The cardioprotective compound cloxyquin uncouples mitochondria and induces autophagy.

Mitochondrial quality control mechanisms have been implicated in protection against cardiac ischemia-reperfusion (IR) injury. Previously, cloxyquin (5-chloroquinolin-8-ol) was identified via phenotypic screening as a cardioprotective compound. Herein, cloxyquin was identified as a mitochondrial uncoupler in both isolated heart mitochondria and adult cardiomyocytes. Additionally, cardiomyocytes isolated from transgenic mice expressing green fluorescent protein-tagged microtubule-associated protein light chain 3 showed increased autophagosome formation with cloxyquin treatment. The autophagy inhibitor chloroquine abolished cloxyquin-induced cardioprotection in both cellular and perfused heart (Langendorff) models of IR injury. Finally, in an in vivo murine left anterior descending coronary artery occlusion model of IR injury, cloxyquin significantly reduced infarct size from 31.4 ± 3.4% to 16.1 ± 2.2%. In conclusion, the cardioprotective compound cloxyquin simultaneously uncoupled mitochondria and induced autophagy. Importantly, autophagy appears to be required for cloxyquin-induced cardioprotection.

Metabolomic profiling of the heart during acute ischemic preconditioning reveals a role for SIRT1 in rapid cardioprotective metabolic adaptation.

Ischemic preconditioning (IPC) protects tissues such as the heart from prolonged ischemia-reperfusion (IR) injury. We previously showed that the lysine deacetylase SIRT1 is required for acute IPC, and has numerous metabolic targets. While it is known that metabolism is altered during IPC, the underlying metabolic regulatory mechanisms are unknown, including the relative importance of SIRT1. Thus, we sought to test the hypothesis that some of the metabolic adaptations that occur in IPC may require SIRT1 as a regulatory mediator. Using both ex-vivo-perfused and in-vivo mouse hearts, LC-MS/MS based metabolomics and (13)C-labeled substrate tracing, we found that acute IPC altered several metabolic pathways including: (i) stimulation of glycolysis, (ii) increased synthesis of glycogen and several amino acids, (iii) increased reduced glutathione levels, (iv) elevation in the oncometabolite 2-hydroxyglutarate, and (v) inhibition of fatty-acid dependent respiration. The majority (83%) of metabolic alterations induced by IPC were ablated when SIRT1 was acutely inhibited with splitomicin, and a principal component analysis revealed that metabolic changes in response to IPC were fundamentally different in nature when SIRT1 was inhibited. Furthermore, the protective benefit of IPC was abrogated by eliminating glucose from perfusion media while sustaining normal cardiac function by burning fat, thus indicating that glucose dependency is required for acute IPC. Together, these data suggest that SIRT1 signaling is required for rapid cardioprotective metabolic adaptation in acute IPC.

SIRT3 deficiency exacerbates ischemia-reperfusion injury: implication for aged hearts.

Ischemia-reperfusion (IR) injury is significantly worse in aged hearts, but the underlying mechanisms are poorly understood. Age-related damage to mitochondria may be a critical feature, which manifests in an exacerbation of IR injury. Silent information regulator of transcription 3 (SIRT3), the major mitochondrial NAD(+)-dependent lysine deacetylase, regulates a variety of functions, and its inhibition may disrupt mitochondrial function to impact recovery from IR injury. In this study, the role of SIRT3 in mediating the response to cardiac IR injury was examined using an in vitro model of SIRT3 knockdown (SIRT3(kd)) in H9c2 cardiac-derived cells and in Langendorff preparations from adult (7 mo old) wild-type (WT) and SIRT3(+/-) hearts and aged (18 mo old) WT hearts. SIRT3(kd) cells were more vulnerable to simulated IR injury and exhibited a 46% decrease in mitochondrial complex I (Cx I) activity with low O2 consumption rates compared with controls. In the Langendorff model, SIRT3(+/-) adult hearts showed less functional recovery and greater infarct vs. WT, which recapitulates the in vitro results. In WT aged hearts, recovery from IR injury was similar to SIRT3(+/-) adult hearts. Mitochondrial protein acetylation was increased in both SIRT3(+/-) adult and WT aged hearts (relative to WT adult), suggesting similar activities of SIRT3. Also, enzymatic activities of two SIRT3 targets, Cx I and MnSOD, were similarly and significantly inhibited in SIRT3(+/-) adult and WT aged cardiac mitochondria. In conclusion, decreased SIRT3 may increase the susceptibility of cardiac-derived cells and adult hearts to IR injury and may contribute to a greater level of IR injury in the aged heart.

A cell-based phenotypic assay to identify cardioprotective agents.

RATIONALE: Tissue ischemia/reperfusion (IR) injury underlies several leading causes of death such as heart-attack and stroke. The lack of clinical therapies for IR injury may be partly due to the difficulty of adapting IR injury models to high-throughput screening (HTS). OBJECTIVE: To develop a model of IR injury that is both physiologically relevant and amenable to HTS. METHODS AND RESULTS: A microplate-based respirometry apparatus was used. Controlling gas flow in the plate head space, coupled with the instrument's mechanical systems, yielded a 24-well model of IR injury in which H9c2 cardiomyocytes were transiently trapped in a small volume, rendering them ischemic. After initial validation with known protective molecules, the model was used to screen a 2000-molecule library, with post-IR cell death as an end point. Po2 and pH monitoring in each well also afforded metabolic data. Ten protective, detrimental, and inert molecules from the screen were subsequently tested in a Langendorff-perfused heart model of IR injury, revealing strong correlations between the screening end point and both recovery of cardiac function (negative, r2=0.66) and infarct size (positive, r2=0.62). Relationships between the effects of added molecules on cellular bioenergetics and protection against IR injury were also studied. CONCLUSIONS: This novel cell-based assay can predict either protective or detrimental effects on IR injury in the intact heart. Its application may help identify therapeutic or harmful molecules.

Reactive oxygen species generation by reverse electron transfer at mitochondrial complex I under simulated early reperfusion conditions.

Ischemic tissues accumulate succinate, which is rapidly oxidized upon reperfusion, driving a burst of mitochondrial reactive oxygen species (ROS) generation that triggers cell death. In isolated mitochondria with succinate as the sole metabolic substrate under non-phosphorylating conditions, 90 % of ROS generation is from reverse electron transfer (RET) at the Q site of respiratory complex I (Cx-I). Together, these observations suggest Cx-I RET is the source of pathologic ROS in reperfusion injury. However, numerous factors present in early reperfusion may impact Cx-I RET, including: (i) High [NADH]; (ii) High [lactate]; (iii) Mildly acidic pH; (iv) Defined ATP/ADP ratios; (v) Presence of the nucleosides adenosine and inosine; and (vi) Defined free [Ca2+]. Herein, experiments with mouse cardiac mitochondria revealed that under simulated early reperfusion conditions including these factors, total mitochondrial ROS generation was only 56 ± 17 % of that seen with succinate alone (mean ± 95 % confidence intervals). Of this ROS, only 52 ± 20 % was assignable to Cx-I RET. A further 14 ± 7 % could be assigned to complex III, with the remainder (34 ± 11 %) likely originating from other ROS sources upstream of the Cx-I Q site. Together, these data suggest the relative contribution of Cx-I RET ROS to reperfusion injury may be overestimated, and other ROS sources may contribute a significant fraction of ROS in early reperfusion.

Coordinated metabolic responses to cyclophilin D deletion in the developing heart.

In the embryonic heart, the activation of the mitochondrial electron transport chain (ETC) coincides with the closure of the cyclophilin D (CypD) regulated mitochondrial permeability transition pore (mPTP). However, it remains to be established whether the absence of CypD has a regulatory effect on mitochondria during cardiac development. Using a variety of assays to analyze cardiac tissue from wildtype and CypD knockout mice from embryonic day (E)9.5 to adult, we found that mitochondrial structure, function, and metabolism show distinct transitions. Deletion of CypD altered the timing of these transitions as the mPTP was closed at all ages, leading to coupled ETC activity in the early embryo, decreased citrate synthase activity, and an altered metabolome particularly after birth. Our results suggest that manipulating CypD activity may control myocyte proliferation and differentiation and could be a tool to increase ATP production and cardiac function in immature hearts.