Received: 6 January 2020 | Revised: 13 February 2020 | Accepted: 8 March 2020 DOI: 10.1111/jcmm.15180 REVIEW Cardiac metabolism as a driver and therapeutic target of myocardial infarction Coert J. Zuurbier1 | Luc Bertrand2 | Christoph R. Beauloye2,3 | Ioanna Andreadou4 | Marisol Ruiz-Meana5 | Nichlas R. Jespersen6 | Duvaraka Kula-Alwar7 | Hiran A. Prag7 | Hans Eric Botker6 | Maija Dambrova8 | Christophe Montessuit9 | Tuuli Kaambre10 | Edgars Liepinsh8 | Paul S. Brookes11 | Thomas Krieg7 1 Department of Anesthesiology, Laboratory of Experimental Intensive Care and Anesthesiology, Amsterdam Infection & Immunity, Amsterdam Cardiovascular Sciences, Amsterdam UMC, University of Amsterdam, Amsterdam, The Netherlands 2 Institut de Recherche Expérimentale et Clinique, Pole of Cardiovascular Research, Université catholique de Louvain, Brussels, Belgium 3 Cliniques Universitaires Saint-Luc, Brussels, Belgium 4 Laboratory of Pharmacology, Faculty of Pharmacy, National and Kapodistrian University of Athens, Athens, Greece 5 Department of Cardiology, Hospital Universitari Vall d’Hebron, Vall d’Hebron Institut de Recerca (VHIR), CIBER-CV, Universitat Autonoma de Barcelona and Centro de Investigación Biomédica en Red-CV, Madrid, Spain 6 Department of Cardiology, Aarhus University Hospital, Aarhus N, Denmark 7 Department of Medicine, University of Cambridge, Cambridge, UK 8 Pharmaceutical Pharmacology, Latvian Institute of Organic Synthesis, Riga, Latvia 9 Department of Pathology and Immunology, University of Geneva School of Medicine, Geneva, Switzerland 10 Laboratory of Chemical Biology, National Institute of Chemical Physics and Biophysics, Tallinn, Estonia 11 Department of Anesthesiology, University of Rochester Medical Center, Rochester, NY, USA Correspondence Coert J. Zuurbier, Department of Abstract Anesthesiology, Laboratory of Experimental Reducing infarct size during a cardiac ischaemic-reperfusion episode is still of para- Intensive Care and Anesthesiology, Amsterdam Infection & Immunity, mount importance, because the extension of myocardial necrosis is an important risk Amsterdam Cardiovascular Sciences, factor for developing heart failure. Cardiac ischaemia-reperfusion injury (IRI) is in Amsterdam UMC, University of Amsterdam, Amsterdam, The Netherlands. principle a metabolic pathology as it is caused by abruptly halted metabolism during Email: c.j.zuurbier@amc.uva.nl the ischaemic episode and exacerbated by sudden restart of specific metabolic path- Thomas Krieg, Department of Medicine, ways at reperfusion. It should therefore not come as a surprise that therapy directed University of Cambridge, Cambridge, UK. at metabolic pathways can modulate IRI. Here, we summarize the current knowl- Email: tk382@medschl.cam.ac.uk edge of important metabolic pathways as therapeutic targets to combat cardiac Funding information IRI. Activating metabolic pathways such as glycolysis (eg AMPK activators), glucose European Foundation for the Study of Diabetes (EFSD); ISCIII, Grant/Award oxidation (activating pyruvate dehydrogenase complex), ketone oxidation (increas- Number: PI19-01196; Latvian Council of ing ketone plasma levels), hexosamine biosynthesis pathway (O-GlcNAcylation; ad- Science, Grant/Award Number: LZP-2018/1- 0082; European Cooperation in Science ministration of glucosamine/glutamine) and deacetylation (activating sirtuins 1 or 3; and Technology, Grant/Award Number: administration of NAD+-boosting compounds) all seem to hold promise to reduce CA16225; National Institutes of Health, Grant/Award Number: R01-HL-071158 acute IRI. In contrast, some metabolic pathways may offer protection through di- minished activity. These pathways comprise the malate-aspartate shuttle (in need This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited. © 2020 The Authors. Journal of Cellular and Molecular Medicine published by Foundation for Cellular and Molecular Medicine and John Wiley & Sons Ltd J Cell Mol Med. 2020;24:5937–5954.  wileyonlinelibrary.com/journal/jcmm | 5937 5938 | ZUURBIER et al. 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 ki- nase with mitochondria, or inhibiting destabilization of FOF1-ATPase dimers, prevents mitochondrial damage and thereby reduces cardiac IRI. Currently, the most promis- ing 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. KEYWORDS ischemia, metabolism, mitochondria the heart is to a large extent determined by the amount of substrate 1 | I NTRO D U C TI O N delivered to the heart (ie plasma concentration), the number of spe- cific substrate transporters present in the cell membrane (CD36/Fat Cardiac metabolism changes rapidly during a sudden ischaemic ep- for fatty acids, GLUT1/4 for glucose and MCT1/2 for lactate and isode of the heart, with the oxygen shortage repressing oxidative ketone bodies) and the activities of the metabolic enzymes and sub- metabolism of fatty acids (FA), carbohydrates, ketones and amino strate/products/cofactors present in the enzymatic pathways. 2-4 It acids, and activating anaerobic glycolysis to spare the use of limited should thereby be realized that a high plasma concentration of one oxygen. During reperfusion with the wash-in of oxygen and the substrate usually competes and inhibits the use of other substrates. wash-out of ischaemic metabolites, there is an abrupt normalization For example, high plasma fatty acid levels will impair glycolysis and of intracellular pH and a specific start-up of oxidative metabolism glucose oxidation,4 or increasing plasma lactate levels will impair for the various substrates, with ongoing changes in what is now fatty acid oxidation and glycolysis.5 Although in general fatty acids aerobic glycolysis. These metabolic changes during ischaemia and contribute more than carbohydrates to ATP generation in the heart, early reperfusion are not merely passive bystanders, but determine this depends critically on (patho) physiological, nutritional and hor- to a large extent the actual injury developing in the heart follow- monal state. For example, when insulin, lactate and fatty acids are ing an ischaemic episode. Modulation of these metabolic changes, present at normal physiological concentrations in the ex vivo-per- therefore, offers the opportunity for developing therapy against fused rodent heart, the contribution of carbohydrates can be higher cardiac IRI, as was already shown by Sodi-Pallares in 1962 using than that of fatty acids.6-8 Ketone bodies, when provided at physi- potassium-insulin-glucose administration during myocardial infarc- ological plasma concentrations (<0.3 mmol/L), contribute less than 1 tion. Since these earlier studies, it has become clear that metabolic 5% to cardiac ATP generation.9 Each substrate is finally broken down therapy for IRI has travelled a rather bumpy road, without the de- to acetyl coenzyme A (acetyl-CoA) that feeds the tricarboxylic acid velopment of a proven effective clinical metabolic therapy as of yet. (TCA) cycle to produce reducing equivalents (NADH and FADH2) Here, we review the current literature concerning this topic, going that then feed the electron transport chain to build a proton gradient from the well-known metabolic pathways to novel metabolic targets to drive the FOF1-ATP/synthase to make ATP. During normoxia, ap- that go beyond the general metabolism of glucose and FA, and focus proximately 90% of the ATP produced is derived from mitochondrial on important processes such as acidosis, ketone oxidation, succinate oxidative breakdown of substrates, with cytosolic glycolysis only accumulation, mitochondrial FOF1-ATPase, energy transfer path- contributing ~5%-10% of total ATP production. Only during total ways, protein O-GlcNAcylation and acetylation as novel metabolic ischaemia does glycolysis become the major supplier of ATP, when targets for treating IRI. glycogen stored in the heart starts feeding glycolysis. The malate/ aspartate shuttle (MAS; Figure 1) is critical for maintaining glycolytic rate because it is the main pathway to recycle glycolysis-produced 2 | G E N E R A L A S PEC T S O F C A R D I AC NADH through the mitochondria into cytosolic NAD+ to maintain M E TA B O LI S M I N H E A LTH Y H E A RT glycolytic activity. It should be noted that for cardiac metabolism, it is not only the The healthy heart is a true omnivore in that it can degrade various flux through the metabolic pathway that regulates cardiac function energy-containing substrates. The major cardiac fuels for respiration and physiology, but also the level of its intermediary metabolites. are fats (triglyceride and long-chain fatty acids), carbohydrates (glu- Typical examples of regulating metabolic intermediates are as fol- cose, lactate and cardiac glycogen) and ketone bodies (acetoacetate lows: a) acetyl-CoA, partly regulating the acetylation status and and β-hydroxybutyrate) (Figure 1). Regulation of substrate use by thereby function of proteins, b) succinate, build-up during ischaemia, ZUURBIER et al. | 5939 Glucose Lactate GLUT1/4 MCT1/2 HEXOSAMINE BIOSYNTHESIS UDP-GlcNAc Pr O-GlcNAc OGT Glycogen Glucose GLYCOLYSIS Pyruvate Lactate NADH NAD+ GLUCOSE OXIDATION Malate HKII MALATE/ ASPARTATE CD36/FAT SHUTTLE MPT Fatty acid Fatty acid HKII CRISTAE Aspartate Pyruvate STRUCTURE CK Acetyl-CoA NAD+ CPT acylcarnitine precursors FATTY ACID METABOLISM TCA Ac Pr PROTEIN Cycle DE/ACETYLATION ROS ATP e- succinate FoF1-ATP SUCCINATE MCT1/2 Acetoacetate β-HB SYNTHASE METABOLISM KETONE Ketones mPTP METABOLISM O2 H2O MITOCHONDRIAL Mitochondrion Cytosol OXYGEN CONSUMPTION F I G U R E 1 Summary of the proposed pathways of cardiac metabolism covered in this review. Many of the discussed pathways show protection against IRI (green arrows) or are protective if blocked (red arrows). CK, creatine kinase; CPT, carnitine palmitoyltransferase; HKII, hexokinase II; MCT, monocarboxylate transporter; MPT, mitochondrial pyruvate transporter; mPTP, mitochondrial permeability transition pore; OGT, O-GlcNAc transferase partly determining reactive oxygen production upon reperfusion, pumps), preventing measurable decreases in ATP during changes in and c) UDP-GlcNAc, as end product of the hexosamine biosynthetic cardiac work.13,14 pathway. Although only a small part of glucose (<0.01% of glycolytic rate)10 is shuttled into this pathway, the generated O-linked attach- ment of N-acetylglucosamine moiety onto proteins affects protein 3 | TA RG E TI N G M E TA B O LI C PATH WAYS function significantly. TO CO M BAT C A R D I AC I R I Finally, spatial and temporal distribution of metabolic enzymes can also be important effectors of cardiac metabolism. One classical 3.1 | Glycolysis example relates to hexokinase II (HKII) that shuttles between mito- chondria and cytosol, depending on nutritional and pathophysiolog- 3.1.1 | Glycolysis during ischaemia ical status. When bound to mitochondria (mtHKII), HKII increases glycolysis, impairs in vivo cardiac oxygen consumption, impairs fatty During ischaemia, glycolysis is increased due to (i) increased glucose acid oxidation, facilitates growth processes and protects mitochon- extraction from the blood (going from 1% during normoxia to 30% dria against injury.7,11,12 Another example is creatine kinase (CK), the extraction during severe low flow), (ii) increased translocation of enzyme making ATP from phosphocreatine (PCr) and thereby func- GLUT transporters to the sarcolemma, (iii) increased glycogenoly- tioning as an important local buffer for ATP breakdown. Cytosolic sis, due to stimulation of glycogen phosphorylase A by diminishing and mitochondrial isoforms of CK form a metabolic signalling net- glucose, ATP, glucose 6-phosphate and increases in AMP and Ca2+, work that is needed for rapid energy (ATP) transfer between ATP (iv) removing citrate-mediated inhibition of the glycolysis-controlling producers (mitochondria) and users (muscle contraction and ion enzyme 6-phosphofructokinase-1 (PFK-1) and (v) AMP-activated 5940 | ZUURBIER et al. protein kinase (AMPK) activation, resulting in both increasing GLUT the increased IRI in these experimental conditions. Mechanisms ex- transport capacity at the membrane and activation of 6-phosphof- plaining increased infarct size with increased accumulation of gly- ructokinase-2 (PFK2), resulting in fructose 2,6-bisphosphate produc- colytic breakdown products during ischaemia are a) detachment of tion, the most potent allosteric stimulator of PFK1.15-18 Activation of hexokinase II from mitochondria (mtHKII) due to high levels of G6P glycolysis during low-flow conditions makes use of all these mecha- and low pH11,33,36 and/or b) increased ischaemic Na+ loading due to nisms, whereas, during no-flow conditions, only mechanisms (iii), (iv) increased proton production by excessive glycogen breakdown, re- and (v) are active. Of note, although most pre-clinical studies have sulting in increased Ca2+ overload upon reperfusion. 23 employed the no-flow ischaemia model, low-flow ischaemia may better reflect the clinical condition of myocardial infarction, where residual flow is often present.19 Following long-lasting glycolytic ac- 3.1.2 | Glycolysis during reperfusion tivity, glycolysis can subsequently be inhibited by accumulation of its products: lactate, protons and NADH.15 As long as glycolysis can pro- The strongest cardioprotective intervention for protecting against ceed and generate ATP during the entire oxygen-limited condition, cardiac IRI, observed across all species, concerns ischaemic pre- it offers protection against IRI. Because during low-flow conditions conditioning (IPC). IPC relates to the application of short, non-lethal lactate and protons can still partly be removed and glucose uptake periods of ischaemia before the long, lethal period of ischaemia, re- proceeds, inhibition of glycolysis is much delayed as compared to sulting in a 75% reduction in infarct size.37 IPC activates glycolysis no-flow conditions. This explains why during even extreme low-flow during normoxia and reperfusion, through activation of AMPK and ischaemia (5% of normal flow), an extended 40-min period of low- Akt, translocation of GLUT4 transporters to the cell membrane and flow induces hardly any IRI (<3% cell death), 20 whereas 40-minute translocation of HKII from cytosol to mitochondria (Figure 1).7,38-43 21 no-flow induces 20x more cell death. Low-flow ischaemia does Interestingly, no protection by IPC is observed in the absence of glu- induce damage when glucose is replaced by another substrate or cose.39 Taken together, the data strongly suggest that IPC protective taken out of the perfusate, supporting the concept that it is the glyc- mechanism is mediated through activated aerobic glycolysis during olytically derived ATP during ischaemia that protects against IRI. 22,23 early reperfusion. An increased glycolysis may mediate protection Increasing glycolysis during low-flow ischaemia through, for exam- through increased activity of mtHKII, knowing that glucose phos- ple, increasing levels of glucose and insulin in the perfusate or boost- phorylation by mtHKII is needed for protection against mitochon- ing endogenous glycogen is associated with delayed contracture drial damage and cell death.44,45 Additionally, older literature has 24 development and protection against IRI. The start of contracture suggested that protection by glycolysis is due to the preferential use development during ischaemia is a read-out of the time when glyco- of glycolytically produced ATP by ion pumps and restoration of ionic lysis stops22 and the free energy of ATP hydrolysis, the ∆GATP, falls homeostasis during reperfusion.46 Finally, increasing glycolysis dur- below a critical level to support the ATPase activity of ion pumps and ing early reperfusion contributes to maintain a low pH, which in turn cross-bridge cycling. 26 Also during no-flow ischaemia activating or exerts multiple protective effects, including the prevention of mito- prolonging glycolysis is most frequently associated with protection chondrial permeability transition pore (mPTP) opening,47 one of the against IRI. In line, AMPK-deficient mouse models are characterized mechanisms proposed for the cardioprotective effect of post-condi- by decreased glucose uptake and glycolysis, increased ATP deple- tioning.48 It is especially an increased ratio of glycolysis over glucose tion during ischaemia, more rapid and severe ischaemic contracture, oxidation that induces this net proton production,49 and may, for increased cell death during reperfusion and poorer post-ischaemic example, explain the decreased infarct size following IR in high-fat contractile recovery. 27,28 Previous studies also clearly showed that diet-induced obesity.50 It is thereby noteworthy that this reduction 29 either increasing pre-ischaemic glycogen or activating glycolysis in infarct size with increased glycolysis uncoupling from glucose through redox control by niacin-induced lowering of NADH/NAD30 oxidation is opposite to the negative effect this uncoupling exerts resulted in delayed ischaemic contracture and reduced IRI. Similarly, on cardiac mechanical function, resulting in a decreased cardiac the pharmacologic overactivation of AMPK reduces cardiomyocyte performance and mechanical efficiency.49 However, the decreased 31,32 death and ameliorates post-ischaemic function recovery. mechanical function upon reperfusion may actually contribute to However, there are experimental conditions where increasing the reduction in infarction, knowing that a slow recovery of energy pre-ischaemic glycogen and/or a delayed contracture development requirement in early reperfusion is cardioprotective51 and that there were actually associated with increased IRI. 23,33,34 Although it is not is often a dichotomy between recovery of mechanical function and completely clear what sets these conditions apart, it could be related cell death following IR.52,53 to excessive glycogen loading (and thus excessive accumulation of Besides ‘conditioning interventions’ to activate aerobic glycoly- glycogen breakdown products and consequently low pH during isch- sis, several pharmacologic cardioprotective agents are also known aemia)23 due to elevated insulin before ischaemia (thereby inhibit- to increase glucose uptake/glycolysis (metformin, insulin, volatile 16 ing non-ischaemic glycogen breakdown) as compared to no insulin anaesthetics, adenosine, NO donors, fructose 1,6-diphosphate, before ischaemia.33 The presence of more insulin before ischaemia nicotinamide mononucleotide (NMN), HIF1α stabilizers and AMPK can also result in impaired activation of the reperfusion injury sal- activators). Some of these agents should ideally be applied at the vage kinase (RISK) pathway during reperfusion,35 possibly explaining end of the ischaemic period or at the onset of reperfusion, in order ZUURBIER et al. | 5941 to prevent excessive glycogen accumulation, resulting in excessive The molecular mechanisms involved in the cardioprotective accumulation of glycogen breakdown products during the ischaemic action of O-GlcNAcylation are not fully understood. It has several episode, thereby offsetting the beneficial effects of an activated gly- effects, including (i) the modification of Ca2+ handling,64 (ii) the in- colysis during early reperfusion. crease in the mitochondrial translocation of the anti-apoptotic Bcl-2 In summary, most studies report that increasing glycolysis during protein,69 (iii) the alteration of P38 MAPK signalling63 and (iv) the at- ischaemia and early reperfusion confers protection against cardiac IRI, tenuation of mitochondrial depolarization and mPTP opening, among making glycolysis a potential important metabolic goal for IRI therapy. others. Importantly, the cardioprotective action of O-GlcNAcylation is lost under diabetic conditions, showing that the action of this post-translational modification could differentially affect cardiac 3.2 | Hexosamine biosynthesis pathway (HBP) function when acutely or chronically induced.54 In summary, most studies report that the acute increase in Beyond glycolysis, glucose can be used by accessory metabolic path- O-GlcNAcylation elicits protection against cardiac IRI. The cardio- ways such as the hexosamine biosynthesis pathway (HBP) (Figure 1). protective action of O-GlcNAcylation could be promoted by acting HBP concludes with O-linked attachment of N-acetylglucosamine pharmacologically on O-GlcNAc enzymes such as OGA or OGT or moiety (O-GlcNAc) onto serine and threonine residues of proteins. by metabolically fuelling HBP via glucosamine and/or glutamine. Similar to other post-translational modifications, O-GlcNAcylation It would be worthwhile to investigate these novel strategies in is a dynamic and reversible molecular process involved in the regu- humans. lation of metabolism, gene expression and protein-protein interac- tion.54,55 The rate of O-GlcNAcylation is governed by (extra)cellular stresses or stimuli but also by nutrient type and availability. Next to 3.3 | The malate-aspartate shuttle glucose, glutamine, but also many other molecules, such as acetyl- CoA and exogenous glucosamine, can enter into HBP to promote In the healthy adult myocardium, the malate-aspartate shuttle (MAS) O-GlcNAcylation.56,57 constitutes the main pathway for transportation of redox products Increases in O-GlcNAcylation level occur and appear del- from glycolysis in the cytosol into the mitochondrial matrix over the eterious under chronic situations such as diabetes and cardiac impermeable inner mitochondrial membrane (Figure 1).70 Under nor- 54,55 hypertrophy. On the other hand, it has been shown that mal conditions, the shuttle capacity is high. The shuttle also meets O-GlcNAcylation is central for the maintenance of cardiovascular elevated glycolytic demand in a variety of physiological and patho- functions. Cardiac-specific deletion of the gene encoding for the physiological processes including cardiac hypertrophy.71 Hence, O-GlcNAc transferase (OGT), the enzyme responsible for the addi- shuttle activity is modifiable. Because of its central role as a regula- tion of O-GlcNAc moiety on proteins, leads to progressive cardiomy- tory mechanism in the energy metabolism of the cardiomyocytes, it opathy.58 More importantly, increases in protein O-GlcNAcylation constitutes a potential target for induction of cardioprotection. are overall protective under acute settings. Using various in vitro, The MAS has gained conceptual interest as a means to modify ex vivo and in vivo animal models, numerous studies demonstrate mitochondrial function, because transient shut down of metabolism that O-GlcNAcylation confers cardioprotection following acute IRI by blocking the cytosolic-mitochondrial crosstalk via the MAS may and other types of cardiac injuries.54,59 Among the in vitro stud- induce cardioprotection (Figure 1). As a proof of concept, transient ies, protocols that mimic IR in isolated cardiomyocytes have been aminooxyacetate (AOA) administration can induce reversible MAS shown to promote protein O-GlcNAcylation, and further increase inhibition. AOA is a non-specific competitive inhibitor of various in O-GlcNAcylation using glucosamine treatment or OGT overex- amino acid transaminases72 but in in situ heart models, AOA primar- 60,61 pression decreases cell death. In line, IR performed in isolated ily leads to reversible inhibition of the MAS.73,74 However, recent perfused hearts increases O-GlcNAc levels and, once again, treat- data also show that AOA directly reacts with alpha-keto acids (py- ment promoting O-GlcNAcylation (glucosamine but also inhibitors ruvate, alpha-ketoglutarate, oxaloacetate, etc) to form stable oximes of the β-N-acetylglucosaminidase OGA, the enzyme responsible for with unknown functional consequences as of yet.75 removing O-GlcNAc moiety) reduces ischaemic contracture and im- Cardioprotection by MAS inhibition using AOA can be induced proves post-ischaemic contractile function recovery.62-65 Notably, both prior to and during an ischaemic insult.76,77 In the normal heart, the treatment was also cardioprotective when applied at the onset MAS inhibition reduces mitochondrial complex I-linked respiration 66 of reperfusion, which increases its clinical relevance. Finally, IPC and glycolysis.76 Even though the effect of MAS inhibition predom- performed in the ex vivo-perfused heart or in vivo by left anterior inantly involves the glycolytic pathway, MAS inhibition also yields descending artery ligation stimulates protein O-GlcNAcylation and cardioprotection in a setting with free FA as additional substrate administration of an OGA inhibitor prior to surgery reduces infarct next to glucose.78 MAS inhibition reduced succinate-induced ROS 66,67 size. This was further validated in humans submitted to remote production (see 3.5.5. for mechanism) by limiting transport of sub- IPC.68 These data suggest that the cardioprotective effect of IPC strate to the succinate build-up.79 The attenuated succinate oxida- may be mediated at least in part by an increase in OGT expression tion during reperfusion reduces reverse electron flow at complex and activity.67 I and hence reduces oxidative stress and cellular damage. This key 5942 | ZUURBIER et al. mechanism is further supported by the ability of MAS inhibition to cardiomyocyte SIRT1 resides mostly in the cytosol91 and is thus posi- 77 preserve post-ischaemic complex I respiration. tioned to interact with the cardioprotective signalling machinery. However, the mechanism does not explain that MAS inhibition Inhibition of SIRT1 either pharmacologically91 or genetically39,90 also reduces infarct size when administrated in the late phase of an prevents cardioprotection via ischaemic pre-conditioning (IPC). ischaemic event, when it is unlikely to reduce succinate substantially.77 Conversely, activation of SIRT1 either pharmacologically95-97 or ge- MAS inhibition may activate other mechanisms, depending on the tim- netically39 is sufficient to confer cardioprotection against acute IRI. ing of administration. The main effect of AOA is inhibition of aspartate Furthermore, a decline in SIRT1 activity is proposed to play a role in aminotransferase, which is the pivotal enzyme in the MAS. The aspar- the loss of IPC protection with ageing.98 In the context of metabolism, tate aminotransferase uses oxaloacetate as substrate, and inhibition it was shown that the metabolic remodelling that occurs in acute IPC is by AOA may increase the intramitochondrial concentration of this tri- critically dependent on SIRT1.99 SIRT1 is also proposed to be an inter- carboxylic acid cycle intermediate. Because oxaloacetate serves as a mediate signal in cardioprotection via other stimuli such as phosphodi- strong inhibitor of succinate dehydrogenase thereby limiting post-isch- esterase (PDE) inhibitors.10 Given the original discovery of the sirtuins aemic oxidation of succinate, it is of interest to study whether the en- as putative mediators of the longevity effects of caloric restriction dogenously formed oxaloacetate conveys protection through reduced (CR)11 it has also been posited that the cardioprotective benefits of CR succinate dehydrogenase activity during reperfusion. may be mediated via SIRT1.12 Similar cardioprotective benefits have The MAS constitutes an important regulatory mechanism in the also been described for mitochondrial SIRT3,92,93 with its pharmaco- cardiomyocyte. The MAS may serve as a target for cardioprotection. logic activation also shown to confer acute cardioprotection.13 Among However, drug toxicity remains a challenge and development of new the more prominent SIRT3 deacetylation targets is the mPTP regulator reversible MAS inhibitors is necessary. cyclophilin D (CypD), with deacetylation at lysine 166 (mouse) required for the cardioprotective effects of SIRT3.14 Although activation of SIRT1 or SIRT3 may appear attractive as 3.4 | Lysine acetylation of cardiac proteins a pharmacologic protective strategy, it should be cautioned that the specificity of many SIRT-activating drugs is uncertain, with stilbenes The adduction of acyl groups to protein lysine residues (acylation) such as resveratrol plus several commercial SIRT1-activating drugs is a biologically significant post-translational modification. Although called into question.15 In addition, inactivation of the NAD+-recycling many such acylations exist (eg succinylation, glutarylation and mal- enzyme NAMPT phenocopies SIRT1 inhibition in preventing car- onylation), here we focus on the most widely studied, acetylation. dioprotection,93,106 suggesting that NAD+ availability is a critical Early studies on the cardiac acetyl-proteome revealed a preponder- determinant for the cardioprotective efficacy of SIRT1. However, ance of metabolic and mitochondrial targets,80,81 leading to specu- while the potential of boosting NAD+ bioavailability via delivery of lation that lysine acetylation is an important metabolic regulator. precursors such as nicotinamide mononucleotide (NMN) or nicotin- However, recent studies in hyperacetylation models have revealed amide riboside (NR) has shown considerable promise in pre-clinical a minimal impact on bioenergetics,82 consistent with studies at the studies,95,107 the wide variety of non-SIRT metabolic pathways that molecular level.83 An important consideration in this regard is acety- rely on the NAD+/NADH redox couple18 suggests that side effects lation stoichiometry, with little understood about the precise rela- of such compounds may outweigh their cardioprotective benefits.19 tionship between site occupancy and enzyme activity. Interestingly, it may well be that part of the cardioprotective effects While there are thousands of kinases and phosphatases in a typ- of NAD+-boosting strategies mainly involves redox-controlled ac- ical cell, enzymes that regulate acetylation number far fewer. Most tivation of glycolysis.19 In line, we recently showed that increased work on acetyltransferases has focused on those in the nucleus that protein acetylation by metabolic over-fuelling dramatically reduced 84 regulate histones (see Ref. for recent review). However, more both insulin- and AMPK-mediated glucose uptake in cardiomyo- recently other acetylation pathways have been uncovered. In par- cytes, presuming the importance of reducing protein acetylation ticular, GCN5L1 was identified as a mitochondrial lysine acetyltrans- for promoting glucose metabolism during IRI.110,111 Overall, while 85 ferase expressed in the heart, and its deletion confers a number promoting deacetylation, for example by targeting SIRT1 activation, of cardiac pathologic phenotypes.86 Furthermore, direct non-enzy- is an attractive avenue for cardioprotection, considerable effort is matic lysine acetylation from acetyl-CoA (and corresponding acyla- required for the development of specific drugs to achieve this end. tion from other acyl-CoAs) has been found to occur in the cell,87 such that the concept of ‘acyl-carbon stress’ is now well established to be partly mediated by excessive lysine acetylation.88,89 3.5 | Mitochondrial metabolism On the deacetylation side, in addition to the histone deacetylases (HDACs), the sirtuin (SIRT) class of NAD+-dependent lysine deacylases 3.5.1 | Oxygen consumption has emerged as key mediators of cardioprotection (Figure 1). Of the 7 mammalian SIRTs, SIRT1 and SIRT3 are robustly expressed in the heart A general characteristic of irreversible ischaemia, that is long and have been shown to play a direct role in several cardioprotective (≥25 minutes) periods of ischaemia with cell death occurring, is the paradigms.39,90-94 Unlike other tissues where SIRT1 is mostly nuclear, fast and complete recovery of oxygen consumption and therefore ZUURBIER et al. | 5943 metabolic recovery during early reperfusion. At the same time, the or through overexpression of the GLUT1 transporter.132 Indirectly, recovery of mechanical function is lagging behind and still severely inhibition of FA oxidation133-135 or uptake136,137 may prevent inhi- depressed. Thus, there is metabolic recovery that is uncoupled from bition of glucose oxidation by reversing the Randle mechanism. mechanical recovery. In contrast, with reversible ischaemia, thus Importantly, IPC, the most robust cardioprotective intervention, was short (≤20 minutes) periods of ischaemia without cell death, recovery also shown to increase glucose oxidation during reperfusion,138,139 of oxygen consumption is much slower and still coupled to mechani- although other studies found no changes.140 112-114 cal function following reversible ischaemia. This fast recovery A major limitation of ex vivo experiments is that they do not allow of post-ischaemic metabolism was associated with increased mito- assessment of long-term post-ischaemic myocardial salvage. Indeed, chondrial Ca2+ in early reperfusion with irreversible ischaemia.112-115 the improved recovery of function observed during the short reper- The fast recovery of oxygen consumption can possibly be explained fusion period (usually one hour) might reflect only speeding up of the by Ca2+ activation of pyruvate dehydrogenase, resulting in increased mechanical recovery, associated with the improved re-energization carbohydrate (glucose, lactate, pyruvate) oxidation during the first observed.132-133,136 Nevertheless, several ex vivo studies observed hour of reperfusion.116 At high FA levels (>1.0 mmol/L), increased a reduction of myocardial necrosis biomarkers associated with in- 117 glucose oxidation is not observed, likely because high FA impair creased glucose oxidation in response to glucose addition141 or FA pyruvate dehydrogenase (PDH) activation. Thus, fast metabolic re- uptake blockade.136 covery uncoupled from mechanical function is a signature of damag- Fewer in vivo studies have addressed this question; yet, they ing irreversible IR, although it is unknown whether this is causal to, have consistently reported a reduction in infarct size associated with or just an epiphenomenon of, IRI. When causal, it offers the pos- stimulation of glucose oxidation by various mechanisms. Thus, one sibility that imposing slower metabolic recovery in early reperfusion to several days after reperfusion following 30 minutes of coronary will protect from IRI. Indeed, interventions directed at an attenu- occlusion, the infarct size was reduced in response to acute admin- ated recovery of blood flow,118 or impaired mitochondrial activity by istration prior to reperfusion of dichloroacetate,142 phosphonate rotenone or mitochondrial complex I by nitrosating/NO agents are compounds143 or reconstituted high-density lipoproteins (HDL),144 119-121 51 protective, supporting the notion of slow metabolic wake-up which all stimulated glucose oxidation. Although administration of as a primary mechanism to combat IRI. GLP-1 analogue albiglutide145 or rosiglitazone days before the isch- aemic event in diabetic rats146 also increased post-ischaemic myo- cardial glucose oxidation and reduced infarct size, such approach is 3.5.2 | Glucose oxidation less relevant for the emergency room situation where patients can- not be treated beforehand. Glucose oxidation, which actually is oxidation of the end product of Thus, most studies concur that stimulation of glucose oxidation glycolysis, that is pyruvate, occurs in the mitochondria. The flux from during reperfusion improves the recovery of function and reduces glycolysis to glucose oxidation is mostly controlled by the activity of infarct size. Conversely, in situations with reduced glucose oxida- the PDH complex located in the mitochondrial matrix. Glucose oxi- tion, such as in pathologically hypertrophied hearts,147 a lesser re- dation is completely shut down during ischaemia; its degree of reac- covery of function and/or larger infarct size is observed. There are, tivation during reperfusion depends, for example, on the availability however, a few discordant observations, with studies indicating that of FA for oxidation because FA compete with glucose for oxidation. stimulating PDH activity or reversing the Randle cycle may not be During early reperfusion, FA oxidation is increased due to activa- sufficient to limit post-ischaemic injury.131,148 Also, administration tion of AMPK by the ischaemia.122,123 FA oxidation reduces glucose of Ruthenium Red, an inhibitor of mitochondrial Ca2+ uptake, after oxidation because of the Randle cycle, in which acetyl-CoA derived severe IR reduced PDH activation but improved the recovery of from FA oxidation inhibits PDH.124 However, activation of PDH by function and reduced creatine kinase release.125 Possibly, following 2+ the mitochondrial Ca overload occurring at reperfusion may over- severe ischaemia inducing massive post-ischaemic Ca2+, the nefar- come this inhibition and result in higher glucose oxidation than in ious effects of mitochondrial Ca2+ overload overcome the positive 125 normoxic conditions. Thus, glucose oxidation is reduced during effects of stimulating glucose oxidation. early reperfusion following moderate ischaemia, but may increase Mainly two hypotheses are invoked to explain the positive im- following more severe or longer ischaemia.114,126 pact of stimulating glucose oxidation on post-ischaemic recovery of Several ex vivo (ie in isolated perfused hearts) and in vivo stud- function and myocardial salvage: 1. efficiency of oxygen use and 2. ies have shown that stimulation of glucose oxidation during reper- reduction of proton overload. 1. Depending on the activity of the fusion is associated with a better recovery of myocardial function MAS, the complete oxidation of one glucose yields 31-33 ATP, with and a lesser reperfusion injury. In the ex vivo setting, interventions a P/O ratio (ie moles of ATP produced divided by the moles of oxy- at reperfusion that increase glucose oxidation and improve the re- gen atoms used) of 2.6-2.8.149 For complete oxidation of palmitate covery of myocardial function may directly target the PDH flux, by or oleate, the P/O ratio is only 2.45-2.47. This 5% to 15% better activating PDH activity with dichloroacetate,127-130 or by the law of oxygen efficiency of glucose oxidation over FA oxidation may not mass action, provisioning extra pyruvate129 or increasing glucose up- seem impressive, but may become critical when oxygen supply is take by administration of high glucose/high insulin at reperfusion131 limited or when mitochondrial function is deteriorated. 2. At least 5944 | ZUURBIER et al. TA B L E 1 FAO inhibitors for IRI Compound Mechanism/target Activity in MI models Model Ref Sulfo-N- Inhibition of sarcolemmal FAT/CD36 Prevented cardiac Isolated diabetic and control [21] succinimidyl dysfunction after ischaemia male Wistar rat hearts oleate (SSO) CBM-301940 Inhibition of malonyl-CoA decarboxylase Improved cardiac function Isolated rat hearts [22,23] CBM-300864 during and after ischaemia Pigs in vivo Methyl-GBB Decreased accumulation of long-chain Decreased MI size, improved Ligation of LAD, rats [24] acylcarnitines survival Isolated perfused rat hearts Trimetazidine Long-chain 3-ketoacyl-CoA thiolase inhibitor AMPK and ERK signalling pathways Reduced MI size and In vivo regional ischaemia and [25] oxidative stress reperfusion, mice Carvedilol Adrenergic receptor blocker; modulator of MI size reduction, improved Ligation of LAD, mice [26] cardiac AMPK signalling pathway cardiac functions Abbreviations: LAD, left anterior descending coronary artery; MI, myocardial infarction. in isolated perfused hearts, the rate of glycolytic flux exceeds the next step, carnitine palmitoyltransferase 1 (CPT-1) catalyses syn- rate of glucose oxidation, by up to one order of magnitude in the thesis of LC acylcarnitine which is necessary for transportation of presence of FA.128,130 Although complete glucose metabolism, that fatty acid intermediates through the inner mitochondrial membrane is glycolysis + oxidation, is proton neutral, glycolysis not followed (Figure 1).153 LC acylcarnitine synthesis rate is the FA metabolism by pyruvate oxidation generates two protons.150 During reperfusion, rate-limiting step which is regulated by concentrations of malonyl- the glycolysis/oxidation uncoupling worsens, aggravating the proton CoA, an endogenous inhibitor of CPT-1.154 Malonyl-CoA is synthe- + 2+ overload, which drives the Na and Ca overload leading to cardio- sized by acetyl-CoA carboxylase (ACC) from acetyl-CoA using biotin myocyte necrosis and/or opening of the mPTP.151 Stimulation of glu- and ATP as cofactors. ACC activity is regulated by its phosphoryla- cose oxidation without a concomitant stimulation of glycolysis, such tion (inactivation) and dephosphorylation (activation). Accordingly, as achieved with dichloroacetate, for example, reduces uncoupling stimulation of fatty acid metabolism by AMPK is achieved by phos- and the generation of protons, thereby possibly limiting reperfusion phorylation of ACC, yielding inhibition of malonyl-CoA synthesis to injury. This interpretation puts uncoupled glycolysis as a driver of ensure rapid LC acylcarnitine synthesis by CPT-1.155 In contrast, acti- myocardial reperfusion injury, seemingly in contradiction with what vation of insulin signalling prevents ACC phosphorylation, stimulates was said in the previous section. Thus, stimulation of glycolysis could malonyl-CoA synthesis and results in CPT-1 inhibition.156 Another perhaps be a double-edged sword, but the self-harming edge can be enzyme, malonyl-CoA decarboxylase (MCD), catalyses the reverse blunted by concomitant stimulation of glucose oxidation. reaction and converts malonyl-CoA into acetyl-CoA. MCD activity In conclusion of this section, glucose oxidation appears to be a is inhibited by SIRT4-mediated deacetylation of the enzyme ensur- promising cardioprotection target that seems to have been over- ing high malonyl-CoA concentrations and facilitated LC acylcarnitine looked by clinical studies. A search of the ClinicalTrials.gov database synthesis.157 LC acylcarnitines are further transferred from the in- with the keywords ‘myocardial infarction’ or ‘reperfusion injury’ and termembrane space into mitochondrial matrix and are converted to ‘glucose oxidation’ failed to retrieve a single study. Acute stimulation acetyl-CoA by CPT-2 to enter β-oxidation in mitochondria. of glucose oxidation at reperfusion with well-tolerated agents such The shift towards LC acylcarnitine accumulation in the mito- as dichloroacetate or reconstituted HDL would be worth trying in a chondria is a result of unbalanced AC synthesis and mitochondrial clinical context. oxidation rates. As a consequence, CPT-1 generates LC acylcar- nitines at amounts which mitochondria cannot fully metabolize. During cardiac ischaemia, mitochondrial malfunction combined 3.5.3 | Fatty acid metabolism with energy deficiency-driven activation of CPT-1 results in even higher content of LC acylcarnitines. At high levels, LC acylcarni- The energy metabolism in heart heavily relies on fat oxidation. tines inhibit oxidative phosphorylation (OXPHOS), which in turn Transmembrane proteins such as CD36 and fatty acid transport pro- induces mitochondrial membrane hyperpolarization and stimu- teins (FATPs) are involved in transport of nonesterified (free) long- lates the production of reactive oxygen species (ROS) in cardiac chain FA from circulation to cardiac tissues (Figure 1). Following mitochondria (Figure 1).158,159 Therefore, FA metabolism regula- cellular uptake, FA can either be stored in the form of triglycerides tion approaches aiming at cardioprotection in IR settings should or undergo metabolism in mitochondria. Long-chain (LC) FA metabo- be carefully evaluated for effects on overall energy homeostasis lism proceeds through multiple steps ensuring transfer of LC acyl and possible cardiotoxicity because of harmful fatty acid inter- groups into mitochondria. The first step in this process is synthe- mediate accumulation (ie ‘acyl-carbon stress’). Some knockout sis of acetyl-CoA in the outer mitochondrial membrane.152 In the mouse models altering transporters or enzymes involved in FA ZUURBIER et al. | 5945 metabolism have resulted in disturbances in cardiac function.160 reducing myocardial infarct size, either by increased levels during This highlights the importance of FA as essential substrates for fasting or when they were administered exogenously. Because of the heart energy production to maintain normal cardiac function concentration-dependent dynamics, increases in ketone bodies under chronic conditions. Nevertheless, the use of small molecular during fasting can elevate the rate of BHB utilization.182 It has been inhibitors of enzymes in fatty acid transport and metabolism path- reported that fasting increased the myocardial BHB/AcAc ratio ways has demonstrated that inhibition of FA oxidation in general reflecting altered mitochondrial redox state and fasting of rats for reduces damage induced by IR160-167 (Table 1). It should be noted, only 24 hours improved the post-ischaemic recovery of contractile however, that effects of FA on cardiac IRI are critically dependent function and reduced the lactate dehydrogenase release in isolated on FA concentrations, with detrimental effects commonly only ob- hearts subjected to global IR.183 In another study, short-term fasting 168 served at rather high levels (>0.8 mmol/L) of FA. Although older increased the concentration of BHB and BHB/AcAc ratio compared literature has reported much higher FA plasma levels in cardiac to controls, limited the infarct size and reduced the total number patients, these high levels were likely the result of ongoing lip- of premature ventricular complexes and the duration of ventricular olysis in the test tube due to the use of heparin in these patients; tachycardia occurring at early reperfusion.184 However, low concen- preventing test tube lipolysis shows that these high FA levels are tration of endogenous ketone bodies failed to preserve the myocar- usually not observed during human in vivo IR episodes.169 Finally, dial ATP levels whereas exogenous supplementation (to 40 times FA may also contribute to cardiac IRI due to its dislodging effects the original concentration) prevented the loss of ATP by ischaemic on hexokinase II-mitochondrial binding in the heart.170 injury.185 Administration of exogenous BHB 60 minutes before the start of ischaemia reduced in vivo myocardial infarct size and apoptosis 3.5.4 | Ketones in rats subjected to IR.186 It was recently demonstrated that starting in vivo BHB treatment at reperfusion and continuing administration Beta-hydroxybutyrate (BHB) and acetoacetate (AcAc) represent the for the next 24 hours of reperfusion using minipumps reduced in- two main ketone bodies.171 BHB, which is produced by degradation farct size, attenuated apoptosis in myocardium and preserved cardiac of FA in liver and then transported to extrahepatic tissues (including function of IR in mice. The above-mentioned beneficial effects were the heart), is one of the important metabolic substrates for energy attributed to reduced mitochondrial formation of ROS, enhanced 172 production during fasting. BHB is not only a metabolic intermedi- ATP production, attenuated mitochondrial swelling and partly re- ate, but also possesses a variety of signalling functions.173 It has been stored mitochondrial membrane potential in myocardium.187 shown to inhibit Class I histone deacetylases (HDACs), increasing In summary, in the experimental IRI context, ketone bodies may therefore histone acetylation, and thereby induces the expression of confer cardioprotective effects possibly due to altered mitochon- genes that restrain oxidative stress.174 BHB also suppresses sympa- drial redox state resulting from increased ketogenesis, up-regulation thetic nervous system activity and reduces total energy expenditure of crucial OXPHOS mediators and reduction of oxidative stress. and heart rate by inhibiting short-chain fatty acid signalling through G protein-coupled receptor 41 (GPR41).175 The myocardium is the highest ketone body consumer per unit mass and under physiologi- 3.5.5 | Succinate and ROS cal conditions oxidizes ketone bodies in proportion to their delivery, at the expense of fatty acid and glucose oxidation.176,177 Ketone bod- A new mechanism is emerging whereby the production of mi- ies have an intermediate energetic efficiency, yielding more ATP per tochondrial ROS is considered a highly orchestrated, metabo- molecule of oxygen used (P/O ratio) in comparison with fatty acid lite-driven process early in IRI. The citric acid cycle metabolite, oxidation, but less when compared to glucose.178,179 Additionally, succinate, is extensively accumulated during ischaemia and is the oxidation of ketone bodies also yields potentially higher energy rapidly oxidized upon reperfusion.79,188,189 In vivo, succinate likely than fatty acid oxidation, keeping ubiquinone oxidized, which raises accumulates via the reduction of fumarate by succinate dehydro- redox span in the electron transport chain (ETC) and makes more genase (SDH or complex II) reversal. A lack of the terminal electron energy available to synthesize ATP. A recent ex vivo cardiac study acceptor, oxygen, maintains a reduced CoQ pool, and additionally, employing direct measurements of cardiac efficiency showed no the pH of ischaemic tissue is lowered. 51,79,190 Upon reperfusion, effects of ketones on cardiac efficiency.9 However, increasing the succinate is rapidly oxidized to fumarate, and together with ETC supply of ketones to the heart did increase total ketone body oxida- activity restarting, the reduced CoQ pool provides electrons for tion without a decrease in any other metabolic pathway.9 Therefore, the ETC complex to proton pump, establishing a large proton mo- ketone bodies are considered an alternative and efficient energy tive force (Δp). The large Δp and highly reduced CoQ pool, to- source in myocardium, especially in failing hearts.180 Increased circu- gether with depleted adenine nucleotides, drive reverse electron lating ketone bodies have been previously reported in patients with transport (RET) through mitochondrial complex I, resulting in the congestive heart failure.181 production of superoxide at the flavin mononucleotide (FMN) In addition to the beneficial effects of ketone bodies in heart site (Figure 1).191,192 The mitochondrial ROS produced, together failure, they have been shown to have beneficial effects in IRI by with impaired calcium handling, activate downstream pathways, 5946 | ZUURBIER et al. resulting in mitochondrial permeability pore formation and, ulti- that governs the life of all organisms. It contains an H+ channel do- mately, cell death. main (Fo) embedded within the inner mitochondrial membrane and Not all of the succinate that has accumulated at ischaemia is oxi- a catalytic domain (F1) protruding towards the mitochondrial matrix, dized by SDH, as it has been suggested that a proportion is released interconnected by central and peripheral stalks.198 High-resolution from the cell.75 Succinate was significantly elevated in the blood of cryoelectron microscopy of native mitochondrial membranes has 193 patients with an acute ST-elevation myocardial infarct suggesting revealed that FoF1-ATP/synthase self-associates into long rows of its release into the bloodstream upon reperfusion. This opens the in- dimers that shape the cristae of mitochondria of all eukaryotic cells triguing possibility that changes in mitochondrial metabolites during into elongated tubular cristae.199 Adequate shaping of mitochondrial IRI could be involved in paracrine signalling, complementary to the cristae determines the respiratory fitness. 20 The FoF1-ATP/synthase 194 signalling role succinate plays in the immune system. is extremely efficient in generating ATP (30 kg/day in a healthy Accumulation of succinate, with its derivative succinyl-CoA, heart), but when cardiomyocytes are challenged by an anoxic epi- also leads to protein succinylation.195 The Sirt5, which has limited sode, the catalytic subunit may paradoxically reverse into an energy- deacylase activity, also catalyses the removal of succinyl groups dissipating machine, favouring H+ extrusion at the expense of ATP from proteins. In line, it has been shown that the increase in IRI in hydrolysis. 21 Sirt5-deficient mouse heart can be reversed by preventing succinate Under physiological conditions, a fraction of FoF1-ATP/synthase accumulation.196 remains blocked by the inhibitory factor 1 (IF1), a 12 kDa protein Blocking succinate metabolism during ischaemia or reperfusion that translocates exclusively to the mitochondria. Remarkably, these with malonate, a competitive SDH inhibitor, has been found to be tissues with high-energy demand, like the heart, have the highest protective in multiple pre-clinical models of cardiac IRI in mouse, rat content of IF1, probably because in these tissues a relevant fraction or pig.51,188 Therefore, targeting succinate metabolism through SDH of the FoF1-ATP/synthase only becomes activated upon demand, inhibition is twofold, either by preventing the succinate rise during acting as a reservoir for ATP synthesis. Expression of IF1 varies be- ischaemia or by reducing the oxidation upon reperfusion, highlight- tween species, and as a general rule, it is higher in animals with a ing a potential therapeutic target.197 high rate of heart contraction, like mice, and lower in species with At present, the metabolic source for succinate in vivo appears to low rate of heart contraction, like humans. 22 Under ischaemic condi- be fumarate. However, a key question remains as to the mechanism tions, reversion of FoF1-ATP/synthase into a hydrolase precipitates of fumarate production during ischaemia. Firstly, adenosine mono- energy exhaustion and rigour contracture in cells already jeopar- phosphate (AMP) build-up during ischaemia can be broken down to dized by the lack of oxygen. 23 Therefore, species-dependent expres- fumarate via the purine nucleotide cycle (PNC). Secondly, the con- sion levels of IF1 can affect the susceptibility of cardiomyocytes to version of aspartate to oxaloacetate and then reduction to malate in ischaemic damage. 24 Moreover, IF1 has been involved in metabolic 51,112 the MAS could provide the fumarate required. Alternatively, a reprogramming: By impeding ATP synthesis, it can inhibit OXPHOS recent mechanism has suggested that canonical TCA cycle activity and drive the cell towards a more glycolytic metabolism. may result in succinate accumulation, by aminotransferase anaple- Cardiomyocytes from aged mice exhibit a partial failure of FoF1- rosis, as opposed to SDH reversal.75 Differences in the models used ATP/synthase to revert its catalytic mode of operation34; this alteration to investigate metabolic changes may be resulting in discrepancy as delays the development of ischaemic-rigour contracture secondary to to which direction of the TCA cycle contributes most to ischaemic ATP exhaustion but accelerates (ΔΨm) decline during ischaemia and succinate accumulation. Further work is required to elucidate accu- impairs (ΔΨm) recovery upon reperfusion in the aged cardiomyocytes, mulation pathways. However, the mechanistic insights produced by a response that is paralleled by more pronounced mPTP opening, hy- further investigating succinate in IRI will provide key targets for the percontracture and cardiomyocyte death.34 Indeed, recent evidence design of cardioprotective drugs, thus providing many lucrative ave- suggests that FoF1-ATP/synthase could be the true molecular entity nues for future therapies in many pathologies. of the mPTP (Figure 1), the opening of which has been consistently Importantly, the role of succinate as a proximal source of elec- associated with the extension of myocardial necrosis during isch- trons for ROS generation during reperfusion is undisputed. As such, aemia-reperfusion injury (IRI) in different experimental models.25 A the acute delivery of complex II inhibitors at the onset of reperfu- structural alteration in the C-ring within FoF1-ATP/synthase26 or in the sion, regardless of succinate accumulation, appears to be a poten- molecule dimerization27 has been proposed to act as ‘death channel’, tially promising clinical approach for treatment of MI in the acute with the latter receiving a greater consensus and more experimental setting (eg during PCI, percutaneous coronary intervention). support.28 An intermediate model includes the dissociation of FoF1- ATP/synthase dimers followed by the rearrangement of the C-ring.29 The concept that FoF1-ATP/synthase is the anatomical support for H+ 3.5.6 | FOF1-ATPase during ischaemia dissipation and cell death integrates other well-accepted mPTP regu- lators,210 including CypD, which has been recently proposed to reduce Mitochondrial ATP synthase or FoF1-ATP/synthase transforms mito-ATP synthase supramolecular assembly, thereby increasing mPTP the electrical power generated during respiration (ΔΨm) into ATP- opening probability.211 Mass spectrometry methods have detected containing chemical energy, following the chemiosmotic principle several types of post-translational modifications in different subunits ZUURBIER et al. | 5947 of FoF1-ATP/synthase,212 some of them, that is, phosphorylation of intracellular compartmentalized energy transfer and the loss of β-subunit, have been detected in response to adenosine-induced probability of interaction between mitochondria and BetaII-tubulin. conditioning strategy.213 Nevertheless, to date, the impact of FoF1- In adult cardiomyocytes, octameric mitochondrial creatine kinase ATP/synthase post-translation modifications on the susceptibility of (MtCK) binds electrostatically to the negatively charged cardiolipins cardiomyocytes to undergo mPTP and death upon IRI has not been of the mitochondrial inner membrane sharing the same cardiolipin established. Because mPTP is increasingly recognized as a prominent patches with ANT. 221 It is possible that the unaltered octameric iso- therapeutic target in the context of myocardial IRI, the elucidation of enzyme of MtCK ensures the stability of its molecular interaction the role of FoF1-ATP/synthase as a modulator of cardiomyocyte death with ANT after IR. 220 Alternatively, the IR-induced alteration of mi- and survival may help to identify new pharmacological strategies for tochondrial increased MOM permeability might influence kinetic cardioprotection. properties of the MtCK. 220 The predominant HK isoform in adult heart, HK2, dynamically shuttles between the mitochondria and cytoplasm, resulting in in- 3.6 | Energy transfer pathways creased glycolysis when bound to mitochondria. 222 Besides that, mitochondrially bound HK2 is an important player in the field of Mitochondria are central players of cellular energy metabolism, es- voltage-dependent anion channel (VDAC) interactions with reg- pecially in striated oxidative muscles and heart. This is much more ulatory proteins and its functional coupling with OXPHOS. It was complicated than only production of ATP via OXPHOS, located on recently shown that IR disrupts interactions between VDAC, ANT mitochondrial inner membrane (MIM). Mitochondria are also source and HK2 through nitration of tyrosine residues in VDAC and ANT, of ROS, proapoptotic factors; they synthesize different metabolites, contributing to mitochondrial and cellular dysfunction following regulate cellular redox potential and play an important role in ion IR. 223 Contact sites between MIM and MOM have a substantial homeostasis regulation and thermogenesis. Severe myocardial in- role in transporting ATP, generated within the mitochondria, to the farction leads to heart failure due to a marked loss of functional ac- cytosol as PCr. Binding between CK and HK2 may stabilize these tivities of cardiomyocytes, where reorganization of energy transport contact sites, and loss of HK2 during ischaemia leads to contact site pathways is an important component. breakage and decreased rates of extramitochondrial PCr synthe- Studies in the last decades have led to an understanding that in sis.36,224,225 The interplay between energy transfer pathways and cardiomyocytes, the cellular energy metabolism is a precisely orga- different binding sites for tubulin and hexokinase to VDAC may be nized system where mitochondria and ATPases are linked to each one of the targets of IPC. other by specialized energy transfer pathways formed by isoen- The connections of mechanisms of energy transfer pathways zymes of creatine kinase (CK) and adenylate kinase (AK) and gly- and cardioprotection are not clear yet; one component participat- colytic enzymes like hexokinase (HK). In addition to the regulation ing in this system is the AK system together with K ATP channel. The of cellular respiration by calcium homeostasis, CK and AK energy AK-catalysed phosphotransfer system would promote K ATP chan- transfer pathways ensure precise feedback signalling between con- nel opening primarily by accelerating conversion of ATP to ADP, traction workload and oxygen consumption in mitochondria. Each whereas CK systems would predominantly facilitate conversion of sarcomere has its own corresponding mitochondrion, which to- ADP to ATP and K ATP channel closure. 226 gether with phosphotransfer system and feedback metabolic signal- The alterations in MOM permeability for adenine nucleotides ling creates the intracellular energy unit (ICEU). 214-216 seem to be an important feature of cardiac ischaemic and IR injuries, An important characteristic of the heart is its metabolic stabil- as it regulates the energy transfer pathways. IPC induced redistri- ity, as reflected by the apparent invariability of intracellular con- bution of high-energy phosphoryl transfer and increased phospho- centration of ATP and phosphocreatine (PCr) in spite of the variable transfer reactions (creatine kinase, glycolysis), leading to improved workloads, corresponding rates of ATP oxidative synthesis and my- intracellular metabolic communication and preservation of cellular ofibrillar hydrolysis. 217 Under conditions of total ischaemia, the PCr ATP synthesis and ATP consumption following IR. 227 These phos- concentration falls rapidly and heart contraction ceases, but ATP phoryl fluxes correlated tightly with post-ischaemic functional re- concentration stays almost stable decreasing only by 10% at the end covery227 and pre-conditioning-induced energetic remodelling, 217 of the first minute of ischaemia. Colocalization of BetaII-tubulin improving contractile performance following IR. The study of intra- and VDAC in heart muscle is functionally related to the ability of cellular phosphotransfer reactions is still not fully unravelled and will creatine to stimulate OXPHOS due to functional coupling between need more sophisticated studies, before it can be used in the devel- mitochondrial creatine kinase (MtCK) and adenine nucleotide trans- opment of an injury-tolerant state in cardiomyocytes. locase (ANT). 218 Key events observed after acute ischaemia-reper- fusion (IR) and chronic ischaemia are the decrease (or loss) in the stimulatory effect of creatine and decrease in diffusion restrictions 4 | CO N C LU S I O N for ATP and ADP at the level of the mitochondrial outer membrane (MOM), which is mediated by BetaII-tubulin219,220 The disruption of We have outlined some of the important metabolic changes oc- mitochondrial interactions with cytoskeleton will result in decreased curring during ischaemia and subsequent reperfusion. While many 5948 | ZUURBIER et al. aspects happen in the entire cell, the mitochondria are a clear perfused working rat hearts. Am J Physiol Heart Circ Physiol. 2004;286:H257-H266. focus within many of these metabolic changes. Such changes in- 7. Nederlof R, Denis S, Lauzier B, et al. Acute detachment of hexoki- clude alterations in fatty acid and succinate metabolism, along with nase II from mitochondria modestly increases oxygen consump- FOF1-ATP/synthase activity, resulting in increased mitochondrial tion of the intact mouse heart. Metabolism. 2017;72:66-74. ROS production and subsequent opening of mPTP. Additionally, 8. Van Weeghel M, Abdurrachim D, Nederlof R, et al. Increased fatty acid oxidation in a mouse model with decreased malonyl-CoA sen- glycolysis, hexosamine biosynthesis, glucose oxidation ketone me- sitivity of CPT1B. Cardiovasc Res. 2018;114:1324-1334. tabolism and the malate/aspartate shuttle are all processes which 9. Ho KL, Zhang L, Wagg C, et al. Increased ketone body oxidation directly affect metabolite levels and mitochondrial pathways. provides additional energy for the failing heart without improving All of which have shown to elicit cardioprotective effects when cardiac efficiency. Cardiovasc Res. 2019;115:1606-1616. altered. 10. Olson AK, Bouchard B, Zhu WZ, et al. First characterization of glu- cose flux through the hexosamine biosynthesis pathway (HBP) in While many of the outlines metabolic processes are ideal drug ex vivo mouse heart. J Biol Chem 2020;295:2018-2033.. https:// targets for ischaemia/reperfusion injury, further studies are nec- doi.org/10.1074/jbc.RA119.010565. essary to fully understand underlying mechanisms and establish 11. Nederlof R, Eerbeek O, Hollmann MW, et al. Targeting hexoki- potential therapies. In this context, it is vital to test possible phar- nase II to mitochondria to modulate energy metabolism and reduce ischemia-reperfusion injury in heart. Brit J Pharmacol. macologic interventions at a clinically relevant time-point at the 2014;171:2067-2079. end of ischaemia as well as in pre-diseased and aged models. 228 12. Nederlof R, Gurel-Gurevin E, Eerbeek O, et al. Reducing mitochon- Some of the outlined mechanisms have already been shown to be drial bound hexokinase II mediates transition from non-injurious relevant in man193 and targeted successfully in many species, in- into injurious ischemia/reperfusion of the intact heart. J Physiol Biochem. 2016;73:323-333. cluding large animals.189,197 Others are still awaiting a relevant drug 13. Saks V, Dzeja P, Schlattner U, et al. Cardiac system bioen- target suitable for translation in patient with an acute MI. Recent ergetics: metabolic basis of the Frank-Starling law. J Physiol. methodological advances in detecting metabolic changes within 2006;571:253-273. the heart will make these efforts easier to achieve. Furthermore, 14. van Beek JH, Tian X, Zuurbier CJ, et al. 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