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.

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.

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.

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.

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, overall mitochondrial ROS generation was only 56% of that seen with succinate alone, and only 52% of this ROS was assignable to Cx-I RET. The residual non-RET ROS could be partially assigned to complex III (Cx-III) with the remainder 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.

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.

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.

Inhibiting Succinate Release Worsens Cardiac Reperfusion Injury by Enhancing Mitochondrial Reactive Oxygen Species Generation.

Background The metabolite succinate accumulates during cardiac ischemia. Within 5 minutes of reperfusion, succinate returns to baseline levels via both its release from cells and oxidation by mitochondrial complex II. The latter drives reactive oxygen species (ROS) generation and subsequent opening of the mitochondrial permeability transition (PT) pore, leading to cell death. Targeting succinate dynamics (accumulation/oxidation/release) may be therapeutically beneficial in cardiac ischemia-reperfusion (IR) injury. It has been proposed that blocking MCT1 (monocarboxylate transporter 1) may be beneficial in IR injury, by preventing succinate release and subsequent engagement of downstream inflammatory signaling pathways. In contrast, herein we hypothesized that blocking MCT1 would retain succinate in cells, exacerbating ROS generation and IR injury. Methods and Results Using the mitochondrial ROS probe mitoSOX and a custom-built murine heart perfusion rig built into a spectrofluorometer, we measured ROS generation in situ during the first moments of reperfusion. We found that acute MCT1 inhibition enhanced mitochondrial ROS generation at reperfusion and worsened IR injury (recovery of function and infarct size). Both of these effects were abrogated by tandem inhibition of mitochondrial complex II, suggesting that succinate retention worsens IR because it drives more mitochondrial ROS generation. Furthermore, using the PT pore inhibitor cyclosporin A, along with monitoring of PT pore opening via the mitochondrial membrane potential indicator tetramethylrhodamine ethyl ester, we herein provide evidence that ROS generation during early reperfusion is upstream of the PT pore, not downstream as proposed by others. In addition, pore opening was exacerbated by MCT1 inhibition. Conclusions Together, these findings highlight the importance of succinate dynamics and mitochondrial ROS generation as key determinants of PT pore opening and IR injury outcomes.

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.

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.

Modified Blue Native Gel Approach for Analysis of Respiratory Supercomplexes.

In mitochondrial oxidative phosphorylation (Ox-Phos), individual electron transport chain complexes are thought to assemble into supramolecular entities termed supercomplexes (SCs). The technique of blue native (BN) gel electrophoresis has emerged as the method of choice for analyzing SCs. However, the process of sample extraction for BN gel analysis is somewhat tedious and introduces the possibility for experimental artifacts. Here we outline a streamlined method that eliminates a centrifugation step and provides a more representative sampling of a population of mitochondria on the final gel. Using this method, we show that SC composition does not appear to change dynamically with altered mitochondrial function.

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.

ALKBH7 mediates necrosis via rewiring of glyoxal metabolism.

Alkb homolog 7 (ALKBH7) is a mitochondrial α-ketoglutarate dioxygenase required for DNA alkylation-induced necrosis, but its function and substrates remain unclear. Herein, we show ALKBH7 regulates dialdehyde metabolism, which impacts the cardiac response to ischemia-reperfusion (IR) injury. Using a multi-omics approach, we find no evidence ALKBH7 functions as a prolyl-hydroxylase, but we do find Alkbh7-/- mice have elevated glyoxalase I (GLO-1), a dialdehyde detoxifying enzyme. Metabolic pathways related to the glycolytic by-product methylglyoxal (MGO) are rewired in Alkbh7-/- mice, along with elevated levels of MGO protein adducts. Despite greater glycative stress, hearts from Alkbh7-/- mice are protected against IR injury, in a manner blocked by GLO-1 inhibition. Integrating these observations, we propose ALKBH7 regulates glyoxal metabolism, and that protection against necrosis and cardiac IR injury bought on by ALKBH7 deficiency originates from the signaling response to elevated MGO stress.

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.

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.

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.

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.

Cyclophilin D Knock-Out Mice Show Enhanced Resistance to Osteoporosis and to Metabolic Changes Observed in Aging Bone.

Pathogenic factors associated with aging, such as oxidative stress and hormone depletion converge on mitochondria and impair their function via opening of the mitochondrial permeability transition pore (MPTP). The MPTP is a large non-selective pore regulated by cyclophilin D (CypD) that disrupts mitochondrial membrane integrity. MPTP involvement has been firmly established in degenerative processes in heart, brain, and muscle. Bone has high energy demands and is therefore expected to be highly sensitive to mitochondrial dysfunction. Despite this, the role of mitochondria and the MPTP in bone maintenance and bone pathology has not been elucidated. Our goal was to determine whether mitochondria are impaired in aging bone and to see if protecting mitochondria from MPTP opening via CypD deletion protects against bone loss. We found that bone mass, strength, and formation progressively decline over the course of 18 months in C57BL/6J mice. Using metabolomics and electron microscopy, we determined that oxidative metabolism is impaired in aging bone leading to a glycolytic shift, imbalance in nucleotides, and decreased NAD+/NADH ratio. Mitochondria in osteocytes appear swollen which is a major marker of MPTP opening. CypD deletion by CypD knockout mouse model (CypD KO) protects against bone loss in 13- and 18-month-old mice and prevents decline in bone formation and mitochondrial changes observed in wild type C57BL/6J mice. Together, these data demonstrate that mitochondria are impaired in aging bone and that CypD deletion protects against this impairment to prevent bone loss. This implicates CypD-regulated MPTP and mitochondrial dysfunction in the impairment of bone cells and in aging-related bone loss. Our findings suggest mitochondrial metabolism as a new target for bone therapeutics and inhibition of CypD as a novel strategy against bone loss.