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.

Cardiac metabolism as a driver and therapeutic target of myocardial infarction.

Reducing infarct size during a cardiac ischaemic-reperfusion episode is still of paramount importance, because the extension of myocardial necrosis is an important risk factor for developing heart failure. Cardiac ischaemia-reperfusion injury (IRI) is in principle a metabolic pathology as it is caused by abruptly halted metabolism during the ischaemic episode and exacerbated by sudden restart of specific metabolic pathways at reperfusion. It should therefore not come as a surprise that therapy directed at metabolic pathways can modulate IRI. Here, we summarize the current knowledge of important metabolic pathways as therapeutic targets to combat cardiac IRI. Activating metabolic pathways such as glycolysis (eg AMPK activators), glucose oxidation (activating pyruvate dehydrogenase complex), ketone oxidation (increasing ketone plasma levels), hexosamine biosynthesis pathway (O-GlcNAcylation; administration of glucosamine/glutamine) and deacetylation (activating sirtuins 1 or 3; administration of NAD+ -boosting compounds) all seem to hold promise to reduce acute IRI. In contrast, some metabolic pathways may offer protection through diminished activity. These pathways comprise the malate-aspartate shuttle (in need of novel specific reversible inhibitors), mitochondrial oxygen consumption, fatty acid oxidation (CD36 inhibitors, malonyl-CoA decarboxylase inhibitors) and mitochondrial succinate metabolism (malonate). Additionally, protecting the cristae structure of the mitochondria during IR, by maintaining the association of hexokinase II or creatine kinase with mitochondria, or inhibiting destabilization of FO F1 -ATPase dimers, prevents mitochondrial damage and thereby reduces cardiac IRI. Currently, the most promising and druggable metabolic therapy against cardiac IRI seems to be the singular or combined targeting of glycolysis, O-GlcNAcylation and metabolism of ketones, fatty acids and succinate.

Fndc-1 contributes to paternal mitochondria elimination in C. elegans.

Paternal mitochondria are eliminated following fertilization by selective autophagy, but the mechanisms that restrict this process to sperm-derived organelles are not well understood. FUNDC1 (FUN14 domain containing 1) is a mammalian mitophagy receptor expressed on the mitochondrial outer membrane that contributes to mitochondrial quality control following hypoxic stress. Like FUNDC1, the C. elegans ortholog FNDC-1 is widely expressed in somatic tissues and mediates hypoxic mitophagy. Here, we report that FNDC-1 is strongly expressed in sperm but not oocytes and contributes to paternal mitochondria elimination. Paternal mitochondrial DNA is normally undetectable in wildtype larva, but can be detected in the cross-progeny of fndc-1 mutant males. Moreover, loss of fndc-1 retards the rate of paternal mitochondria degradation, but not that of membranous organelles, a nematode specific membrane compartment whose fusion is required for sperm motility. This is the first example of a ubiquitin-independent mitophagy receptor playing a role in the selective degradation of sperm mitochondria.

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.

Cardiac Function is not Susceptible to Moderate Disassembly of Mitochondrial Respiratory Supercomplexes.

: Mitochondrial respiratory chain supercomplexes (RCS), particularly, the respirasome, which contains complexes I, III, and IV, have been suggested to participate in facilitating electron transport, reducing the production of reactive oxygen species (ROS), and maintaining the structural integrity of individual electron transport chain (ETC) complexes. Disassembly of the RCS has been observed in Barth syndrome, neurodegenerative and cardiovascular diseases, diabetes mellitus, and aging. However, the physiological role of RCS in high energy-demanding tissues such as the heart remains unknown. This study elucidates the relationship between RCS assembly and cardiac function. Adult male Sprague Dawley rats underwent Langendorff retrograde perfusion in the presence and absence of ethanol, isopropanol, or rotenone (an ETC complex I inhibitor). We found that ethanol had no effects on cardiac function, whereas rotenone reduced heart contractility, which was not recovered when rotenone was excluded from the perfusion medium. Blue native polyacrylamide gel electrophoresis revealed significant reductions of respirasome levels in ethanol- or rotenone-treated groups compared to the control group. In addition, rotenone significantly increased while ethanol had no effect on mitochondrial ROS production. In isolated intact mitochondria in vitro, ethanol did not affect respirasome assembly; however, acetaldehyde, a byproduct of ethanol metabolism, induced dissociation of respirasome. Isopropanol, a secondary alcohol which was used as an alternative compound, had effects similar to ethanol on heart function, respirasome levels, and ROS production. In conclusion, ethanol and isopropanol reduced respirasome levels without any noticeable effect on cardiac parameters, and cardiac function is not susceptible to moderate reductions of RCS.

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.

Cardioprotection by the mitochondrial unfolded protein response requires ATF5.

The mitochondrial unfolded protein response (UPRmt) is a cytoprotective signaling pathway triggered by mitochondrial dysfunction. UPRmt activation upregulates chaperones, proteases, antioxidants, and glycolysis at the gene level to restore proteostasis and cell energetics. Activating transcription factor 5 (ATF5) is a proposed mediator of the mammalian UPRmt. Herein, we hypothesized pharmacological UPRmt activation may protect against cardiac ischemia-reperfusion (I/R) injury in an ATF5-dependent manner. Accordingly, in vivo administration of the UPRmt inducers oligomycin or doxycycline 6 h before ex vivo I/R injury (perfused heart) was cardioprotective in wild-type but not global Atf5-/- mice. Acute ex vivo UPRmt activation was not cardioprotective, and loss of ATF5 did not impact baseline I/R injury without UPRmt induction. In vivo UPRmt induction significantly upregulated many known UPRmt-linked genes (cardiac quantitative PCR and Western blot analysis), and RNA-Seq revealed an UPRmt-induced ATF5-dependent gene set, which may contribute to cardioprotection. This is the first in vivo proof of a role for ATF5 in the mammalian UPRmt and the first demonstration that UPRmt is a cardioprotective drug target.NEW & NOTEWORTHY Cardioprotection can be induced by drugs that activate the mitochondrial unfolded protein response (UPRmt). UPRmt protection is dependent on activating transcription factor 5 (ATF5). This is the first in vivo evidence for a role of ATF5 in the mammalian UPRmt.

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.

Krebs cycle metabolites and preferential succinate oxidation following neonatal hypoxic-ischemic brain injury in mice.

BackgroundReverse electron transport (RET) driven by the oxidation of succinate has been proposed as the mechanism of accelerated production of reactive oxygen species (ROS) in post-ischemic mitochondria. However, it remains unclear whether upon reperfusion, mitochondria preferentially oxidase succinate.MethodsNeonatal mice were subjected to Rice-Vannucci model of hypoxic-ischemic brain injury (HI) followed by assessment of Krebs cycle metabolites, mitochondrial substrate preference, and H2O2 generation rate in the ischemic brain.ResultsWhile brain mitochondria from control mice exhibited a rotenone-sensitive complex-I-dependent respiration, HI-brain mitochondria, at the initiation of reperfusion, demonstrated complex-II-dependent respiration, as rotenone minimally affected, but inhibition of complex-II ceased respiration. This was associated with a 30-fold increase of cerebral succinate concentration and significantly elevated H2O2 emission rate in HI-mice compared to controls. At 60 min of reperfusion, cerebral succinate content and the mitochondrial response to rotenone did not differ from that in controls.ConclusionThese data are the first ex vivo evidence, that at the initiation of reperfusion, brain mitochondria transiently shift their metabolism from complex-I-dependent oxidation of NADH toward complex II-linked oxidation of succinate. Our study provides a critical piece of support for existence of the RET-dependent mechanism of elevated ROS production in reperfusion.

Moving Forwards by Blocking Back-Flow: The Yin and Yang of MI Therapy.

Mitochondrial reactive oxygen species production has emerged as an important pathological mechanism in myocardial ischemia/reperfusion injury. Attempts at targeting reactive oxygen species by scavenging using antioxidants have, however, been clinically disappointing. This review will provide an overview of the current understanding of mitochondrial reactive oxygen species in ischemia/reperfusion injury. We will outline novel therapeutic approaches designed to directly target the mitochondrial respiratory chain and prevent excessive reactive oxygen species production and its associated pathology. This approach could lead to more effective interventions in an area where there is an urgent need for new treatments.

The Slo(w) path to identifying the mitochondrial channels responsible for ischemic protection.

Mitochondria play an important role in tissue ischemia and reperfusion (IR) injury, with energetic failure and the opening of the mitochondrial permeability transition pore being the major causes of IR-induced cell death. Thus, mitochondria are an appropriate focus for strategies to protect against IR injury. Two widely studied paradigms of IR protection, particularly in the field of cardiac IR, are ischemic preconditioning (IPC) and volatile anesthetic preconditioning (APC). While the molecular mechanisms recruited by these protective paradigms are not fully elucidated, a commonality is the involvement of mitochondrial K+ channel opening. In the case of IPC, research has focused on a mitochondrial ATP-sensitive K+ channel (mitoKATP), but, despite recent progress, the molecular identity of this channel remains a subject of contention. In the case of APC, early research suggested the existence of a mitochondrial large-conductance K+ (BK, big conductance of potassium) channel encoded by the Kcnma1 gene, although more recent work has shown that the channel that underlies APC is in fact encoded by Kcnt2 In this review, we discuss both the pharmacologic and genetic evidence for the existence and identity of mitochondrial K+ channels, and the role of these channels both in IR protection and in regulating normal mitochondrial function.

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.

SMG-1 kinase attenuates mitochondrial ROS production but not cell respiration deficits during hyperoxia.

PURPOSE: Supplemental oxygen (hyperoxia) used to treat individuals in respiratory distress causes cell injury by enhancing the production of toxic reactive oxygen species (ROS) and inhibiting mitochondrial respiration. The suppressor of morphogenesis of genitalia (SMG-1) kinase is activated during hyperoxia and promotes cell survival by phosphorylating the tumor suppressor p53 on serine 15. Here, we investigate whether SMG-1 and p53 blunt this vicious cycle of progressive ROS production and decline in mitochondrial respiration seen during hyperoxia. MATERIALS AND METHODS: Human lung adenocarcinoma A549 and H1299 or colon carcinoma HCT116 cells were depleted of SMG-1, UPF-1, or p53 using RNA interference, and then exposed to room air (21% oxygen) or hyperoxia (95% oxygen). Immunoblotting was used to evaluate protein expression; a Seahorse Bioanalyzer was used to assess cellular respiration; and flow cytometry was used to evaluate fluorescence intensity of cells stained with mitochondrial or redox sensitive dyes. RESULTS: Hyperoxia increased mitochondrial and cytoplasmic ROS and suppressed mitochondrial respiration without changing mitochondrial mass or membrane potential. Depletion of SMG-1 or its cofactor, UPF1, significantly enhanced hyperoxia-induced mitochondrial but not cytosolic ROS abundance. They did not affect mitochondrial mass, membrane potential, or hyperoxia-induced deficits in mitochondrial respiration. Genetic depletion of p53 in A549 cells and ablation of the p53 gene in H1299 or HCT116 cells revealed that SMG-1 influences mitochondrial ROS through activation of p53. CONCLUSIONS: Our findings show that hyperoxia does not promote a vicious cycle of progressive mitochondrial ROS and dysfunction because SMG-1-p53 signaling attenuates production of mitochondrial ROS without preserving respiration. This suggests antioxidant therapies that blunt ROS production during hyperoxia may not suffice to restore cellular respiration.

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.

Cardiac Slo2.1 Is Required for Volatile Anesthetic Stimulation of K+ Transport and Anesthetic Preconditioning.

BACKGROUND: Anesthetic preconditioning (APC) is a clinically important phenomenon in which volatile anesthetics (VAs) protect tissues such as heart against ischemic injury. The mechanism of APC is thought to involve K channels encoded by the Slo gene family, and the authors showed previously that slo-2 is required for APC in Caenorhabditis elegans. Thus, the authors hypothesized that a slo-2 ortholog may mediate APC-induced cardioprotection in mammals. METHODS: A perfused heart model of ischemia-reperfusion injury, a fluorescent assay for K flux, and mice lacking Slo2.1 (Slick), Slo2.2 (Slack), or both (double knockouts, Slo2.x dKO) were used to test whether these channels are required for APC-induced cardioprotection and for cardiomyocyte or mitochondrial K transport. RESULTS: In wild-type (WT) hearts, APC improved post-ischemia-reperfusion functional recovery (APC = 39.5 ± 3.7% of preischemic rate × pressure product vs. 20.3 ± 2.3% in controls, means ± SEM, P = 0.00051, unpaired two-tailed t test, n = 8) and lowered infarct size (APC = 29.0 ± 4.8% of LV area vs. 51.4 ± 4.5% in controls, P = 0.0043, n = 8). Protection by APC was absent in hearts from Slo2.1 mice (% recovery APC = 14.6 ± 2.6% vs. 16.5 ± 2.1% in controls, P = 0.569, n = 8 to 9, infarct APC = 52.2 ± 5.4% vs. 53.5 ± 4.7% in controls, P = 0.865, n = 8 to 9). APC protection was also absent in Slo2.x dKO hearts (% recovery APC = 11.0 ± 1.7% vs. 11.9 ± 2.2% in controls, P = 0.725, n = 8, infarct APC = 51.6 ± 4.4% vs. 50.5 ± 3.9% in controls, P = 0.855, n = 8). Meanwhile, Slo2.2 hearts responded similar to WT (% recovery APC = 41.9 ± 4.0% vs. 18.0 ± 2.5% in controls, P = 0.00016, n = 8, infarct APC = 25.2 ± 1.3% vs. 50.8 ± 3.3% in controls, P < 0.000005, n = 8). Furthermore, VA-stimulated K transport seen in cardiomyocytes or mitochondria from WT or Slo2.2 mice was absent in Slo2.1 or Slo2.x dKO. CONCLUSION: Slick (Slo2.1) is required for both VA-stimulated K flux and for the APC-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.

Meclizine inhibits mitochondrial respiration through direct targeting of cytosolic phosphoethanolamine metabolism.

We recently identified meclizine, an over-the-counter drug, as an inhibitor of mitochondrial respiration. Curiously, meclizine blunted respiration in intact cells but not in isolated mitochondria, suggesting an unorthodox mechanism. Using a metabolic profiling approach, we now show that treatment with meclizine leads to a sharp elevation of cellular phosphoethanolamine, an intermediate in the ethanolamine branch of the Kennedy pathway of phosphatidylethanolamine biosynthesis. Metabolic labeling and in vitro enzyme assays confirmed direct inhibition of the cytosolic enzyme CTP:phosphoethanolamine cytidylyltransferase (PCYT2). Inhibition of PCYT2 by meclizine led to rapid accumulation of its substrate, phosphoethanolamine, which is itself an inhibitor of mitochondrial respiration. Our work identifies the first pharmacologic inhibitor of the Kennedy pathway, demonstrates that its biosynthetic intermediate is an endogenous inhibitor of respiration, and provides key mechanistic insights that may facilitate repurposing meclizine for disorders of energy metabolism.

Physiological consequences of complex II inhibition for aging, disease, and the mKATP channel.

In recent years, it has become apparent that there exist several roles for respiratory complex II beyond metabolism. These include: (i) succinate signaling, (ii) reactive oxygen species (ROS) generation, (iii) ischemic preconditioning, (iv) various disease states and aging, and (v) a role in the function of the mitochondrial ATP-sensitive K(+) (mKATP) channel. This review will address the involvement of complex II in each of these areas, with a focus on how complex II regulates or may be involved in the assembly of the mKATP. This article is part of a Special Issue entitled: Respiratory complex II: Role in cellular physiology and disease.