University of Groningen

Selecting heart failure patients for metabolic interventionsBooij, Harmen G.; Koning, Anne M.; van Goor, Harry; de Boer, Rudolf A.; Westenbrink, B.DaanPublished in:Expert review of molecular diagnostics

DOI:10.1080/14737159.2017.1266939

IMPORTANT NOTE: You are advised to consult the publisher's version (publisher's PDF) if you wish to cite fromit. Please check the document version below.

Document VersionPublisher's PDF, also known as Version of record

Publication date:2017

Link to publication in University of Groningen/UMCG research database

Citation for published version (APA):Booij, H. G., Koning, A. M., van Goor, H., de Boer, R. A., & Westenbrink, B. D. (2017). Selecting heartfailure patients for metabolic interventions. Expert review of molecular diagnostics, 17(2), 141-152.https://doi.org/10.1080/14737159.2017.1266939

CopyrightOther than for strictly personal use, it is not permitted to download or to forward/distribute the text or part of it without the consent of theauthor(s) and/or copyright holder(s), unless the work is under an open content license (like Creative Commons).

Take-down policyIf you believe that this document breaches copyright please contact us providing details, and we will remove access to the work immediatelyand investigate your claim.

Downloaded from the University of Groningen/UMCG research database (Pure): http://www.rug.nl/research/portal. For technical reasons thenumber of authors shown on this cover page is limited to 10 maximum.

Download date: 27-08-2020

Full Terms & Conditions of access and use can be found athttp://www.tandfonline.com/action/journalInformation?journalCode=iero20

Download by: [University of Groningen] Date: 30 June 2017, At: 03:45

Expert Review of Molecular Diagnostics

ISSN: 1473-7159 (Print) 1744-8352 (Online) Journal homepage: http://www.tandfonline.com/loi/iero20

Selecting heart failure patients for metabolicinterventions

Harmen G. Booij, Anne M. Koning, Harry van Goor, Rudolf A. de Boer & B.Daan Westenbrink

To cite this article: Harmen G. Booij, Anne M. Koning, Harry van Goor, Rudolf A. de Boer & B.Daan Westenbrink (2017) Selecting heart failure patients for metabolic interventions, Expert Reviewof Molecular Diagnostics, 17:2, 141-152, DOI: 10.1080/14737159.2017.1266939

To link to this article: http://dx.doi.org/10.1080/14737159.2017.1266939

© 2016 The Author(s). Published by InformaUK Limited, trading as Taylor & FrancisGroup

Accepted author version posted online: 30Nov 2016.Published online: 28 Dec 2016.

Submit your article to this journal

Article views: 110

View related articles

View Crossmark data

REVIEW

Selecting heart failure patients for metabolic interventionsHarmen G. Booij*, Anne M. Koning*, Harry van Goor, Rudolf A. de Boer and B. Daan Westenbrink

University Medical Center Groningen, University of Groningen, Groningen, Netherlands

ABSTRACTIntroduction: Heart failure (HF) has become the cardiovascular epidemic of the century and nowimposes an immense burden on health care systems. While our understanding of the pathophysiologyof HF has increased dramatically, the translation of knowledge into clinical practice has been disap-pointing. Metabolic dysfunction in HF has been studied for eight decades, but these efforts have notresulted in effective therapies. This paucity in clinical translation probably results from the variablecontribution of metabolic dysfunction to the underlying heart disease. A major unmet need in cardiacdrug development is therefore the ability to identify a homogeneous subset of patients in whom HF isdriven by a specific mechanism that can be targeted.Areas covered: The available literature was evaluated to describe maladaptive metabolic perturbationsthat occur in failing hearts and may cause metabolic inflexibility, oxidative stress and cardiac energydepletion. Furthermore, the potential utility of various biomarkers and molecular imaging techniques todetect and quantify specific metabolic dysfunctions in HF were compared. Finally, the authors proposeways to utilize these techniques to select patients for specific metabolic interventions.Expert commentary: Metabolic dysfunction is among the most promising therapeutic targets in HF.Meticulous patient-selection with molecular imaging techniques and specific biomarkers appearsindispensable for the effective translation of decades of scientific knowledge into clinical therapeutics.

ARTICLE HISTORYReceived 23 August 2016Accepted 28 November 2016

KEYWORDSBiomarkers; heart failure;magnetic resonancespectroscopy; metabolicdysfunction; oxidative stress;positron emissiontomography

1. Heart failure: the scope of the problem

Over the past four decades, heart failure (HF) patients havederived substantial benefits from the advances in our under-standing of the pathophysiology of this devastating disease.Multiple fundamental discoveries in cardiovascular biologyhave fueled novel treatment paradigms that have successfullybeen translated into clinical applications. As a result, HF hastransformed from a simple descriptive entity treated with pallia-tive measures into a well-defined syndrome that can be treatedwith a vast array of life saving drugs, interventions, and devices[1]. Paradoxically, these advances have done little to reduce thepopulation disease burden, including its economic impact. Forinstance, the rate of hospital admissions for acute HF in the USAhas remained around 1 million per year since the beginning ofthe century. While the prognosis of HF has improved, the 5-yearmortality rate is still worse than for most types of cancer [2]. Infact, HF is currently responsible for one in nine deaths in the USAand Europe [3]. In 2012, HF affected 2% of the US adult popula-tion and 9% of those over 60 years of age, and the associatedhealthcare costs were estimated at 30.7 billion per year [3]. Whilehealthcare systems are barely coping with the immense health-care burden of HF as it is, the prevalence of HF and the associatedcosts are projected to increase by 50% within the next 15 years[3]. HFmay thus be regarded as themost pressing cardiovascularepidemic of this century. New strategies to treat or prevent HFare therefore urgently needed.

2. Heart failure therapy: is it time to transcendneurohormonal blockade?

The evolution of HF treatment is regarded as the epitome ofevidence-based medicine because current pharmacologicalregimens have all survived the scrutiny of randomized con-trolled trials in relatively unselected patient populations. Withsome exceptions, all HF patients are treated with the samedrugs and devices irrespective of the underlying diseasemechanism. The ability to treat such a heterogeneous disorderin such a homogeneous fashion may be explained by the factthat these drugs essentially target the systemic response tocardiac failure. Indeed, while HF drugs target very specificcomponents of the sympathetic nervous system, the renin-angiotensin system or natriuretic peptide biology, they canall be regarded as interventions intended to restore the sys-temic neurohormonal balance. After 15 years without anyprogress in HF therapy, the PARADIGM-HF trial recentlydemonstrated that the combination of a neutral endopepti-dase inhibitor with an angiotensin-receptor blocker was super-ior to the well-established angiotensin converting enzyme-inhibitor enalapril [4]. As both of these drugs target the neu-rohumoral system, the PARADIGM-HF trial clearly indicatesthat there are still viable treatment targets left within theneurohumoral system. However, the increasing time-windowbetween subsequent pharmacological innovations does sug-gest that we are approaching the asymptote for

CONTACT B. Daan Westenbrink b.d.westenbrink@umcg.nl Department of Cardiology, University Medical Center Groningen, Hanzeplein 1, P.O.Box 30001,Groningen, 9700 RB The Netherlands

*These authors contributed equally to this work

EXPERT REVIEW OF MOLECULAR DIAGNOSTICS, 2017VOL. 17, NO. 2, 141–152http://dx.doi.org/10.1080/14737159.2017.1266939

© 2016 The Author(s). Published by Informa UK Limited, trading as Taylor & Francis GroupThis is an Open Access article distributed under the terms of the Creative Commons Attribution-NonCommercial-NoDerivatives License (http://creativecommons.org/licenses/by-nc-nd/4.0/),which permits non-commercial re-use, distribution, and reproduction in any medium, provided the original work is properly cited, and is not altered, transformed, or built upon in any way.

neurohormonal blockade [2]. Accordingly, there is a broadprecedent to refocus drug development efforts from nonse-lective blockade of the systemic consequences of HF, towardinterventions that target the specific underlying diseasemechanisms within the heart [5].

Over the years, multiple perturbations in the cardiac musclehave been identified that can cause HF or contribute to dis-ease progression, including but not limited to altered cardiacexcitation-contraction (EC) coupling, aberrant activation ofsignal transduction pathways, and dysfunctional myocardialmetabolism [6–11]. While the role of these mechanisms inHF development has been firmly established, their contribu-tion to the underlying disease may vary among HF patients, aswell as during different stages of disease progression withinan individual patient. For instance, the expression and activityof the key calcium cycling molecule sarcoplasmic reticulumcalcium ATPase (SERCA2a) is not uniformly reduced in myo-cardial tissue samples from HF patients and the degree ofmetabolic dysfunction (as specified below) also varies [11,12].An intervention that targets a specific myocardial defect willprobably not be effective or may even be harmful in patientsin whom that particular mechanism is not causal. This may beone of the explanations for the lack-off effect of SERCA2a genetransfer in the CUPID-II trial, as a reduction in myocardialSERCA2a expression was not an entry criterion for participa-tion in this study [13]. Whereas routine interrogation of myo-cardial SERCA2a expression levels may cross ethical andpractical boundaries, there are ample opportunities to evalu-ate metabolic dysfunction in HF that could routinely be incor-porated in trial design for metabolic therapies. In the followingsections, we will therefore discuss major metabolic perturba-tions in HF and the potential utility of biomarkers and mole-cular imaging techniques to pinpoint underlying disease

mechanisms and thereby select patients for specific metabolicinterventions.

3. Metabolic dysfunction in Heart failure

The heart pumps 10 tons of blood around the body each dayfor which it requires up to 30 times its own weight in adeno-sine triphosphate (ATP). The cardiac ATP stores are, however,only sufficient to sustain three heart beats. Cardiac functionand myocardial ATP production are therefore heavily inter-twined and small perturbations in the provision of carbon-based fuels and the efficiency of mitochondrial respiration canhave major consequences for myocardial performance. Thefirst indication that energy reserves are reduced in HF datesback more than eight decades, when Herrmann and Decherddiscovered that creatine levels in failing hearts are reduced[14]. Based on these observations, it was hypothesized thatenergy deprivation leads to cardiac dysfunction, equivalent toan engine running out of fuel [11]. With few exceptions, themajority of the evidence gathered since then has supportedthis hypothesis. In the following paragraphs, we will providean overview of the major metabolic changes that occur in thefailing heart and briefly mention metabolic therapies that arein various stages of clinical development. Current therapeuticconcepts to restore cardiac metabolism are summarized inFigure 1.

3.1. Cardiac substrate preference

The heart has considerable metabolic flexibility as it can trans-form chemical energy stored in various carbon-based substrates,including fatty acids, glucose, lactate, ketones, and amino acids,into ATP through oxidative phosphorylation [15]. In healthy

Figure 1. Therapeutic concepts to restore metabolic dysfunction in heart failure.Simplified schematic depicting cellular energy metabolism from substrate utilization to oxidative phosphorylation and energy transfer to the sites of energyconsumption. Current therapeutic concepts to improve energetic efficiency in heart failure either target proportional substrate use by decreasing fatty acidmetabolism (left panel), increasing glucose oxidation (right upper panel), or augmenting energy transfer (right lower panel). ADP: adenosine diphosphate;ATP: adenosine triphosphate; CPT: carnitine palmitoyltransferase; Cr: free creatine; FFA: free fatty acids; GLUT4: glucose transporter 4; PCr: phosphocreatine;PDH: pyruvate dehydrogenase; MCD: malonyl-CoA decarboxylase; NAD+, NADH: oxidized and reduced forms of nicotinamide adenine dinucleotide; TG: triglycerides;β-ox: beta oxidation.

142 H. G. BOOIJ ET AL.

resting myocardium, 60–90% of the total ATP production isderived from β-oxidation of fatty acids. The second major fuelis pyruvate, which can be generated by glycolysis or lactateoxidation. During exercise as well as the initial stages of mostcardiac pathologies, the heart shifts from fatty acids to glucose asthe primary source of energy. This shift in substrate preference isgenerally considered to be protective as it improves the stoichio-metric ratio of oxygen consumption to ATP production [16]. β-oxidation of fatty acids also promotes uncoupling of the protonmotive force generated by the mitochondrial respiratory chainand ATP synthesis, thereby reducing oxygen utilization efficiencyeven further [10,17]. While the amount of energy generated pergram of substrate is higher for fatty acids compared to carbohy-drates, the oxygen utilization efficiency is much more favorablefor pyruvate- than for β-oxidation [17].

An additional aspect favoring carbohydrate metabolism infailing hearts is that two ATP molecules are generated anae-robically within the cytosol during glycolysis, i.e. the transfor-mation of glucose into pyruvate. Glycolysis is the only sourceof ATP production under hypoxic conditions, but it is veryinefficient as the net ATP yield is about 8% of that obtainedfrom oxidative phosphorylation of pyruvate. In advanced HF,glycolytic enzymes and glycolytic activity are markedlyinduced while the mitochondrial capacity to oxidize pyruvateand other major fuels becomes impaired. This phenomenonresults in uncoupling of glycolysis and glucose oxidation andpromotes anaerobic glycolysis as the primary cardiac route ofATP production [18]. Because anaerobic glycolysis is an ineffi-cient pathway for energy production, augmentation of glyco-lysis further aggravates the myocardial energy deficit.Accordingly, cardiac ATP depletion typically develops whenglucose oxidation diminishes, and increased levels of circulat-ing pyruvate and lactate can be detected in plasma samples ofHF patients at this point [19–21].

The reductions in glucose oxidation in advanced HF are atleast partially caused by mitochondrial damage or dysfunction(discussed below). In addition, a functional block at the levelof the pyruvate dehydrogenase (PDH) complex has alsoemerged as a central driver of this process [17]. PDH is con-sidered the rate-limiting step in glucose oxidation as it trans-forms pyruvate into acetyl-CoA, which subsequently entersthe Krebs cycle. The negative regulator of PDH, PDH kinase,is strongly activated in failing hearts essentially blocking thecardiac capacity to oxidize carbohydrates [22]. It must bestressed, however, that these metabolic adaptations are notexclusive to cardiomyocytes or HF models as they occur invirtually all cell types subjected to hypoxic or metabolic stress[23]. Furthermore, while the data from animal experiments aremore or less consistent, there is a large variation in substrateutilization in patients with cardiac hypertrophy or HF [19,20].Finally, comprehensive metabolomics profiling recently indi-cated that the failing heart relies more on ketone bodies thanon pyruvate, suggesting that advances in biochemical techni-ques may further refine contemporary paradigms [24]. In sum-mary, the initial stages of pathological cardiac stress areaccompanied by a marked augmentation of glycolysis andglucose oxidation, while advanced HF is associated with areduced capacity to metabolize all major fuels and increaseddependence on anaerobic glycolysis.

3.2. Oxidative stress in Heart failure

Oxidative stress is another key element of the pathophysiol-ogy of HF. Since the metabolic changes in HF are inextric-ably linked to the production of reactive oxygen species(ROS) by mitochondria, oxidative stress can be a cause aswell as a consequence of HF. Even under physiologicalconditions, mitochondria produce superoxide (O2

−) as aby-product of respiration [25]. Preferably, this free radicalis broken down to water by manganese-dependent super-oxide dismutase (MnSOD) and nicotinamide adenine dinu-cleotide phosphate (NADPH)-dependent enzymaticreduction [25]. In HF, defective EC coupling and mitochon-drial ion homeostasis impede activation of the Krebs cycle[26]. This not only disturbs regeneration of NADH to supplythe electron transport chain (ETC) with electrons, but alsothat of NADPH required for adequate antioxidant capacity[26]. The consequent oxidative stress leads to a viciouscircle in which oxidative damage to mitochondrial DNAcauses increased electron leakage from the ETC and oxida-tion of proteins contributes to further derangement of ECcoupling [26,27]. Adaptive changes associated with HF,including activation of the sympathetic nervous systemand the renin-angiotensin-aldosterone system, aggravatethis process by increasing cardiac energy supply-and-demand mismatch as well as direct stimulation of ROSproduction [28,29]. Mitochondrial ROS (mtROS) triggersincreased mitochondrial permeability by activation of themitochondrial permeability transition pore, among otherpores and channels, allowing ROS to escape to the cytosol[30]. On emission, mtROS not only induce oxidative damageto non-mitochondrial structures, but also stimulate ROSproduction by other sources, including NADPH oxidase(Nox), uncoupled NO synthase (NOS), and xanthine oxidase(XO) [30]. Reciprocally, ROS produced by these sources pro-mote the production of mtROS [30]. ROS production in HF issummarized in Figure 2.

The relative contribution of different cell types remainsunclear and may well vary based on underlying diseasemechanisms. In fact, comorbidity-related proinflammatorysignaling and consequent coronary endothelial inflamma-tion and ROS production have been implicated in thepathophysiology of HF with a preserved ejection fraction(HF-pEF), whereas in HF with a reduced ejection fraction(HF-rEF) ROS are assumed to primarily originate from cardi-omyocytes [31]. Reflecting the heterogeneous nature of HFpathology, these variations in ROS production emphasizethe need for a personalized approach to improve the treat-ment of patients suffering from this syndrome.

3.3. Mitochondrial dysfunction and myocardial energydepletion

Mitochondria from failing hearts exhibit major structural andfunctional defects that diminish their capacity to generate ATPand increase superoxide release from the respiratory chain.The activity of respiratory chain complexes and ATP synthaseis significantly decreased in HF and the sensitivity to endo-genous regulators of oxidative phosphorylation becomes

EXPERT REVIEW OF MOLECULAR DIAGNOSTICS 143

diminished [32]. The contribution of mitochondria to othercellular processes such as cardiac calcium handling, cellularsignaling, and the regulation of cell death also becomesseverely perturbed [33]. Mitochondrial defects are generallyconsidered to result from ROS-mediated damage to mitochon-drial DNA and proteins. However, uncontrolled activation ofmitochondrial signaling pathways, transcriptional mitochon-drial reprogramming, and reduced elimination of damagedmitochondria by defective mitophagy are now increasinglyrecognized as additional causal factors [10,34,35].Nevertheless, current pharmacological strategies to alleviatemitochondrial dysfunction are primarily designed to attenuatemitochondrial oxidative stress with mitochondria-targetedantioxidants, such as Szeto-Schiller peptide (SS-31) andMitoQ [36,37]. Further advances in our understanding of mito-chondrial regulatory mechanisms in HF may provide noveltreatment paradigms that will allow us to specifically targetthe underlying disease mechanisms rather than its conse-quences. For instance, the endogenous regulation of ATPsynthase is still poorly understood and several key regulatorymechanisms have only recently been discovered [38–40].

The transport of ATP from mitochondria to the sites of ATP-consumption within the cell is facilitated through the creatinekinase (CK) shuttle. To allow effective diffusion of energythroughout the cell, one phosphate group is transferred

from ATP to creatine by mitochondrial CK to generate phos-phocreatine. Phosphocreatine rapidly diffuses throughout thecell and ATP is subsequently regenerated from phosphocrea-tine by CK at the sites of ATP consumption. In addition to thecatalysis of ATP transfer, the CK shuttle also acts as a cellularenergy buffer. Whereas cardiac ATP levels only drop in themore advanced stages of HF, an energy deficit expressed asthe phosphocreatine/creatine ratio, already becomes apparentbefore overt cardiac dysfunction develops [41]. Accordingly,the energy status of the heart can be expressed as the phos-phocreatine/creatine ratio. As described above, the energydepletion hypothesis in HF was based on creatine depletionin failing hearts, perhaps making this the best-studied meta-bolic defect in HF. Despite eight decades of research andseveral clinical trials, there are no therapies that can specifi-cally restore cardiac energy levels or ATP transfer [42]. Forinstance, creatine supplementation failed to alleviate HFdevelopment in multiple clinical scenario’s [43,44]. The impor-tance of the cardiac CK shuttle was recently scrutinized in amouse model of systemic creatine knockdown. While thehearts of these mice were fully depleted of creatine, cardiacfunction and exercise performance were not altered [45].Despite the lack of successful interventions in ATP transfermechanisms, and the debate surrounding its physiologicalrelevance, the phosphocreatine/creatine ratio may still prove

Figure 2. Oxidative stress in heart failure: mechanisms, markers and therapeutic concepts.Simplified representation of the mechanisms causing oxidative stress in HF. MtROS production is linked to metabolic dysfunction through defective mitochondrialCa2+ homeostasis. This impedes activation of the Krebs cycle, which in turn disturbs regeneration of NADH to supply the ETC with electrons and NADPH required foradequate antioxidant capacity. The consequent oxidative stress leads to a vicious circle of increased O2

− leakage from the ETC and aggravated metabolicdysfunction. MtROS stimulates ROS production by cytosolic sources (cROS) and vice versa. The contribution of specific cell types, such as endothelial cells (producingeROS) may vary based on underlying disease mechanisms. Plasma and urine markers of oxidative stress are listed in the left panels. Therapeutic targets include O2

−

leakage (lower right panel), XO (middle right panel) and thiol capacity (upper right panel). ADP: adenosine diphosphate; ATP: adenosine triphosphate; Ca2+: calciumions; cROS: cytosolic ROS; eROS: endothelial ROS; ETC: electron transport chain; GSH, GSSG: reduced and oxidized forms of glutathione; H2O: water; H2O2: hydrogenperoxide; H2S: hydrogen sulfide; MDA: malondialdehyde; MnSOD: manganese-dependent superoxide dismutase; MPO: myeloperoxidase; mtROS: mitochondrial ROS;NAC: N-acetylcysteine; NAD+, NADH: oxidized and reduced forms of nicotinamide adenine dinucleotide; NADP+, NADPH: oxidized and reduced forms of nicotinamideadenine dinucleotide phosphate; NO: nitric oxide; NOS: NO synthase; Nox: NADPH oxidase; oxLDL: oxidized low-density lipoprotein; ROS: reactive oxygen species;TMAO: trimethylamine-N-oxide; TRXr, TRXo: reduced and oxidized forms of thioredoxin; O2

−: superoxide; XO: xanthine oxidase; -SH: thiol.

144 H. G. BOOIJ ET AL.

valuable as a biomarker to select patients for metabolicinterventions.

4. Biomarkers for metabolic dysfunction in Heartfailure

4.1. Biomarkers for cardiac substrate preference andenergy depletion

As described above, severe perturbations in cardiac glucoseoxidation in advanced HF are associated with increased circu-lating lactate and pyruvate levels and these biomarkers havebeen used to monitor metabolic interventions in the past [46].Nevertheless, such biomarkers to monitor myocardial sub-strate preference have limited sensitivity and specificity asthey reflect a combination of nutritional intake, gastrointest-inal uptake, and systemic substrate utilization in all organs.Likewise, it will be very hard to untangle cardiac and extra-cardiac contributions to circulating levels of markers for ATPdepletion, such as circulating ADP or AMP levels. Advances inmetabolomics profiling have more recently been adopted todiscover biomarkers with higher specificity for cardiac energyhomeostasis. For instance, long-chain acylcarnitines haveemerged as very promising biomarkers for disrupted β-oxida-tion, but require further validation [47,48]. A more directapproach to evaluate cardiac substrate utilization is the com-parison of metabolite levels in aortic and coronary sinus bloodsamples, which allows more detailed analysis of cardiac sub-strate use, including metabolic substrate flux and ATP produc-tion efficiency [49]. The obvious drawback of this technique isthe invasive nature and this type of analysis is typicallyreserved to monitor the effect of invasive procedures such ascardiac resynchronization therapy.

4.2. Biomarkers for oxidative stress

As extensively described elsewhere, several biomarkers arecommonly used to assess oxidative stress in HF. These includeoxidized low-density lipoprotein, malondialdehyde, and mye-loperoxidase in plasma, and isoprostane in plasma and urine[50]. The gut microbe-generated metabolite trimethylamine-N-oxide (TMAO), which is adversely associated with HF prog-nosis, has also been proposed to be related to oxidative stress[51]. Some of these traditional biomarkers, particularly alde-hydes, have been suggested to further increase ROS forma-tion, although studies on their role in pathophysiologiology ofcardiovascular diseases (CVDs) are conflicting [50]. Aldehydesare formed during many processes including lipid peroxida-tion [52]. During HF, increased ROS production decreases theactivity of aldehyde dehydrogenase 2 [53]. This causes anaccumulation of aldehydes, including malondialdehyde and4-hydroxy-2-nonenal (HNE), which localize to mitochondriaand further increase mitochondrial ROS formation throughreactions with nucleophilic protein residues [52,54,55].Aldehyde levels can be detected in the plasma and severalstudies have indicated that plasma aldehyde levels can serveas biomarkers for oxidative stress. For instance, lipid peroxida-tion has been investigated as a biomarker for artherogenesis[56]. Nevertheless, it should be noted that, regardless of their

pathological significance, aldehydes are products of the reac-tion of ROS with lipids, and thus arise secondarily to theprocess that initiated oxidative stress. Thus, while aldehydesmay serve as biomarkers, it is unlikely that they will serve as anodal point for metabolic interventions [50]. Consequently,these biomarkers can be used as a marker for oxidative stress,but may not for the selection of patients for specific metabolicinterventions.

4.3. Serum free thiols

Recently, the serum free thiol level regained interest as anindicator of oxidative stress in the context of chronic HF.Reactive species can oxidize their targets by reacting withthiols, i.e. functional groups composed of a sulfur and a hydro-gen atom. Because free (or reduced) thiols are readily oxidizedby reactive species, their level may be seen as a direct reflec-tion of the balance between oxidants and antioxidant capa-city, and thus the overall level of oxidative stress [57,58]. Whatmakes free thiols different from other biomarkers is that apartfrom reflecting the overall level of oxidative stress, thiols arecritical active components of the antioxidant defense, whichmay be receptive to therapeutic modulation. So far, moststudies looking into antioxidant machinery have focused onlow-molecular-weight thiols, in particular glutathione andcysteine. For example, a recent study has shown an associa-tion between higher cystine (i.e. oxidized cysteine) and lowerglutathione levels and an increased mortality risk of coronaryartery disease patients [59]. However, whereas these low-molecular-weight thiols are key actors in intracellular antiox-idant defense, in the extracellular compartment they arestrongly outnumbered by protein thiols [60]. In fact, the singlesulfhydryl group of albumin, the most abundant serum pro-tein, accounts for the majority of thiols in serum [60]. Hence,compared to the concentration of low-molecular-weightthiols, the total free thiol level may be a more relevant circu-latory biomarker of oxidative stress.

Depletion of circulatory free thiols has been shown inpatients with CVD, including acute myocardial infarction[57,61,62]. Also, thiol oxidation has been linked to establishedCVD risk factors, such as aging, smoking, and obesity [63].Moreover, our group has recently reported a positive associa-tion between protein-adjusted serum free thiols and a favor-able disease outcome in a small cohort of 101 stable chronicHF patients [64]. This finding suggests that serum free thiolsprovide a robust reflection of redox status and warrants con-firmation in a larger cohort.

4.4. Gaseous signaling molecules NO and H2S

Reversible oxidative modifications of protein thiols by severalsmall molecules may protect proteins from irreversible oxida-tive damage and, in some cases, alter protein function [58].Among these small molecules are gasotransmitters nitric oxide(NO) and hydrogen sulfide (H2S).

NO, also known as endothelial-derived relaxing factor, haslong been acknowledged for its versatile role in cardiac phy-siology [65]. As the alias suggests, it is an important regulatorof vascular tone, among numerous other bodily processes

EXPERT REVIEW OF MOLECULAR DIAGNOSTICS 145

[65]. In HF, NO bioavailability is assumed to be reduced[66,67]. At the same time, increased levels of NO metabolites,nitrite and nitrate (NOx) have been described in HF, possiblyreflecting impaired renal excretion [68,69]. More recently, H2Shas been recognized as a cardiovascular signaling molecule,similar to NO. Like NO, H2S is involved in the regulation ofvarious (patho)physiological processes and features vasodila-tory, antioxidant, and anti-inflammatory properties [70].

Unfortunately, reliable methods for direct detection of NOand H2S in biological samples are lacking. Alternatively, quan-tification of their metabolites – i.e. NOx for NO and thiosulfateand sulfate for H2S – may be used to evaluate changes in themetabolism of these gaseous signaling molecules. Naturally,other factors that may be of influence, such as intake andrenal function, should be considered in this process. To date,very few studies have looked into concentrations of NO andH2S metabolites in relation to outcome of CVD. One paperdescribes a higher plasma concentration of NOx 24 hours afteronset of ST-segment elevation myocardial infarction to beassociated with an increased risk of one-year all-cause mortal-ity or rehospitalization [71]. Another study has found bothurinary thiosulfate and sulfate concentrations of renal trans-plant recipients to be positively associated with a favorablecardiovascular risk profile and patient survival [72].

5. Molecular imaging of cardiac metabolism

5.1. Positron emission tomography

Positron emission tomography (PET) is a three-dimensionalimaging technique that can monitor biological processes invivo through the use of molecules that have been labeled withpositron emitting radionucleotides. PET already has a centralrole in clinical cardiac diagnostics, for the evaluation of myo-cardial perfusion and viability in ischemic heart disease, thedifferential diagnostics of cardiomyopathies, and the diagnosisand risk stratification of endocarditis. PET also holds greatpromise for cardiac drug development as virtually every mole-cule can be modified to be detectable by PET [73]. The mostabundantly used clinical PET tracer, 18F-FDG, was specificallydesigned to monitor glycolytic activity and tracers for β-oxida-tion have also been developed for clinical application. Thesetracers accurately monitor changes in glycolysis and β-oxida-tion in experimental animals, as well as in patients with HF[74–77]. In particular, 18F-FDG PET is a sensitive marker forcardiac glucose uptake, and appears to have reasonable accu-racy for the prediction of a treatment response to variousinterventions [78–80]. More studies are, however, required tofurther define the utility of PET as a biomarker for cardiacsubstrate utilization in HF.

Monitoring and quantification of focal oxidative stress intissues has several potential clinical applications and severalPET probes have been developed for the detection of hypoxiaor ROS production, mostly for oncological or for cardiovascularapplications [81–83]. Unfortunately, the cardiac uptake ofthese tracers has been disappointing as they provide insuffi-cient signal intensity to detect the mild hypoxia or ROS pro-duction in HF. There are, however, several promising noveltracers in development and these may prove more reliable for

application in HF [84,85]. Of particular interest for ROS mon-itoring in HF is radiolabeled vitamin C ([11C]Ascorbic acid),which exhibits ROS-dependent cellular accumulation thatcould result in significant augmentation of signal intensity.Accordingly, PET holds great promise for the selection andmonitoring of patients for metabolic interventions.

5.2. Magnetic resonance spectroscopy

Cardiovascular magnetic resonance imaging (MRI) has emergedas the primary method for myocardial tissue characterization inclinical cardiology, but few people realize that MRI is in fact amolecular imaging technique. Traditional MRI employs the com-bination of a high magnetic field to align hydrogen nuclei, themost abundant MR active nuclei in the body, and radio wavesthat causes these nuclei to resonate. When hydrogen nuclei relaxfrom their resonance, they emit radio waves that can bedetected. Differences in relaxation time in various tissues resultin differences in resonance intensity that can be transformed tocreate images. These images can be used to evaluate myocardialtissue characteristics, such as the presence of fibrosis or edema,and also allows for detailed quantification of cardiac volumesand function in HF. Nuclear magnetic resonance spectroscopy(MRS) utilizes the same hardware and radio waves, but ratherthan creating an image from the signal that it receives, it detectsdistortions in magnetic resonance frequency within the tissue.This so-called chemical shift results from the interactions amongneighboring nuclei and electrons in the tissue. As specific mole-cules generate a specific shift in resonance frequency, detailedanalysis of the frequencies can be used to quantify the concen-trations of various abundant metabolites in tissues.(Figure 3)MRS can be used to measure the concentrations of moleculesrich in protons (1H) but also less abundant nuclei such as phos-phorus (31P), carbon (13C), or sodium (23Na). While several meta-bolites can be detected with MRS, for human applications, 1HMRS has primarily been used to detect cardiac creatine andtriglyceride content, 31P MRS to detect cardiac ATP, phospho-creatine, and phosphocreatine/creatine ratios and 23Na to moni-tor cardiac sodium content (Figure 3) [86]. Despite its vastpotential, cardiac MRS is a challenging technique as measure-ments need to be adjusted for cardiac and respiratory motions.The time required tomeasureMRS spectra varies between 30 sec-onds and 20 minutes for 1H MRS and 20 and 30 minutes for 31PMRS, depending on the gating technique and the pulsesequences employed. Even though it can be performed onmost commercially available scanners, currently, MRS is primarilyused as a research tool in specialized centers. The most com-monly studied MRS-derived parameters are myocardial triglycer-ide content and phosphocreatine/creatine ratios [86]. The latterappears to have remarkable specificity for changes in cardiacenergy stores. For instance, a study in type 2 diabetic patientswas capable of detecting a transient, 12% reduction in cardiacphosphocreatine/creatine ratio during exercise [87]. MRS hasbeen used to monitor cardiac energy levels for more than25 years and can detect changes in high-energy phosphatelevels during treatment [88]. As such, it could be regarded asthe gold standard for the evaluation of energy stores.

The application of MRS as a tool to study cardiac ROS islimited, although myocardial oxygen levels can be measured

146 H. G. BOOIJ ET AL.

with MRI using blood oxygen level-dependent sequences [89]. Intheory, 13C MRS holds a vast potential for metabolic imagingbecause cellular energy is derived from carbon-based fuels.Unfortunately, the clinical application of native 13CMRS is limitedby the low abundance and low sensitivity of 13C nuclei to MRpulses. To circumvent this limitation, a hyperpolarization techni-que has been developed that can increase the signal of specific13C-containing molecules by more than 10,000-fold, using acombination of free radicals and intense freezing under highmagnetic field strengths. These hyperpolarized metabolites aresubsequently injected into the bloodstream and their cardiacuptake and transformation into downstream metabolites canbe monitored. Hyperpolarization has a short half-life and cannotbe used to enhance native molecules within the heart. It can beused to monitor metabolic processes in vivo, including the PDHflux, β-oxidation, and the Krebs cycle [90]. In experimental ani-mals, hyperpolarized 13C MRS can effectively monitor changes insubstrate utilization during the progression of HF and the effectsof interventions in cardiac substrate utilization [20,91,92].Regulatory approval of hyperpolarized 13C MRS for applicationin humans is pending and, considering its potential, eagerlyanticipated.

In summary, PET is a well-established technique that couldbe regarded as the gold standard to monitor cardiac substrateutilization and is a promising technique for myocardial ROSdetection. Systemic redox status can be measured with circu-lating and urinary biomarkers, although cardiac specificityremains limited. MRS is the gold standard for the detection

of cardiac energy levels and holds great promise for thedetection of cardiac substrate utilization through hyperpolar-ization techniques, but clinical validation is still pending.Accordingly, we propose that PET should be employed forpatient selection and monitoring when interventions in car-diac substrate utilization are considered, whereas biomarkers,such as total serum free thiols, should be used to selectpatients for antioxidant treatments. Finally, MRS could beused to monitor interventions in cardiac ATP production, butmay also serve to monitor the efficacy of other metabolicinterventions. A scheme depicting the potential value of bio-markers, PET, and MRS to select HF patients for metabolicinterventions during various stages of disease progression issummarized in Figure 4.

6. Therapeutic interventions in cardiac metabolism

6.1. Therapeutic interventions in cardiac substratepreference

As the shift from fatty acid to glucose oxidation is generallyconsidered a physiological adaptation to stress, and severaltherapeutic strategies have been developed that aim to pro-mote glucose oxidation in failing hearts [16]. These drugseither promote cellular glucose uptake, augment glucose oxi-dation by restoring PDH complex functionality or inhibit fattyacid β-oxidation (as summarized in Figure 1 and extensivelyreviewed in ref. 17) [17]. While there have been some encoura-ging improvements in symptoms and exercise performance

Figure 3. Cardiac magnetic resonance spectroscopy.A, Four chamber and short axis magnetic resonance image of a human heart. The white square depicts the spectroscopic volume that is typically placed in theintraventricular septum to reduce motion artifacts. B, Typical 1H spectrum of the intraventricular septum. Because water is the most abundant substance, the imagesare acquired after a water suppression pulse. C, Typical 31P magnetic resonance spectrum of the left ventricle. D, Typical 13C spectrum of the pig heart obtained afterinfusion of a hyperpolarized 1-13C pyruvate tracer. ATP: adenosine triphosphate; 2,3 DPG: 2,3-diphosphoglycerate; PDE: phosphodiesters; PCr: phosphocreatine.Adapted by permission from BMJ Publishing Group Limited. [Metabolic imaging of the human heart: clinical application of magnetic resonance spectroscopy, HJHeart Br. Card. Soc., Bizino MB, Hammer S, Lamb, 100:881–890, 2014.] [86].

EXPERT REVIEW OF MOLECULAR DIAGNOSTICS 147

with these drugs such as dicholoracetate and etomoxir, phaseIII clinical trials have been neutral or were stopped early dueto drug toxicity or increased HF events [17,93–96]. While thismight be regarded as proof that reductions in glucose oxida-tion reflect an adaptive mechanism or that the therapeuticinterventions were poorly designed, it should be borne inmind that defective cardiac substrate utilization was not aninclusion criterion for these studies. In selected patients, theoutcomes of these studies may have been positive.

6.2. Therapeutic interventions to alleviate myocardialoxidative stress

As outlined above, excess ROS production is a key factor in thepathophysiology of HF. At the same time, redox signaling is anintegral part of physiology and lower levels of ROS areinvolved in all kinds of beneficial cellular processes, includingimmunity, cell growth, and apoptosis [97]. Besides, recurrentepisodes of oxidative stress have been found to upregulateendogenous antioxidant mechanisms, which in turn may pro-mote health and longevity [97]. Thus, disturbance of the

physiological functions of ROS is likely to have contributedto the contradicting findings of studies evaluating uncon-trolled and untargeted antioxidant therapies, includingN-acetylcysteine (NAC) and vitamins, in cardiac disease[98,99]. Also, when a specific ROS producing component isdisarmed, this may adversely affect other parts of the redoxnetwork. For example, this may in part explain the disappoint-ing results of treatment with an XO inhibitor in HF, as thissubstance prevents XO from producing O2-, but also decreasesNO production [100]. Perhaps, combination therapy of an XOinhibitor and an NO donor can resolve this problem. Moreover,physiological regulatory processes may prevent administeredantioxidants from reaching adequate bioavailability, unlessthere is a deficiency to overcome [97]. Finally, oxidation ofan antioxidant substance before or during ingestion may notonly abate its efficacy, but in fact induce oxidative stress [97].

Despite these challenges, in patients with aberrant endo-genous antioxidant capacity, antioxidant therapies may well bebeneficial. For example, restoring redox status by therapeuticmodulation of free thiols may hold promise to improve diseaseoutcome in HF. Indeed, in multiple clinical trials, the cysteinederivative NAC has been shown to both directly reduce dis-ulfide bonds and act as a glutathione precursor [101]. Otherthiol compounds, with similar modes of action, have also beendescribed [102]. Although therapeutic results have been incon-sistent, these compounds may prove to be effective in selectedpatients with low free thiol concentrations. Alternatively, phar-macological stimulation of molecules that induce reversibleprotein modifications, including NO and H2S, may offer oppor-tunities to influence the amount of free thiols, as well asfavorably steer protein function [58,65,70]. One H2S releasingcompound, AP39, may be of particular interest as it is specifi-cally targeted to mitochondria, one of the most importantsources of ROS in HF. In experimental models, AP39 has beenshown to attenuate H2O2-induced cytotoxicity and to posi-tively affect vascular tone and intracellular calcium homeosta-sis [103]. However, further research is needed to prove itsclinical applicability. Other mitochondria-targeted antioxi-dants, including MitoQ and SS-31, have also shown promisein experimental HF. Moreover, SS-31 is currently investigated ina clinical trial focused on myocardial reperfusion injury and atrial studying this compound it the context of HF will soonbegin [36,104].

7. Conclusions

Metabolic dysfunction is among the most promising therapeu-tic targets in HF. Meticulous patient selection with molecularimaging techniques and specific biomarkers appears indispen-sable for the effective translation of decades of scientificknowledge into clinical therapeutics.

8. Expert commentary

Although HF patients are treated with up to 5 differentclasses of drugs, their mode of action is roughly similar asthey can all be regarded as interventions that restore thesystemic neurohormonal balance. Despite continuousrefinements in this strategy, multiple lines of evidence

Figure 4. Pathological changes and applicability of markers during heart failuredevelopment.Scheme illustrating the pathological changes and applicability of PET, MRS andbiomarkers to select patients for metabolic interventions during various stagesof HF development. ATP: adenosine triphosphate; HF: heart failure;MRS: magnetic resonance spectroscopy; PET: Positron Emission Tomography;ROS: reactive oxygen species.

148 H. G. BOOIJ ET AL.

indicate that we are approaching the asymptote for addedbenefit of neurohormonal interventions. Rather than focus-ing on the secondary effects of HF, we believe that weshould refocus drug development efforts toward interven-tions that target specific pathologic changes within theheart. Despite tremendous advances in our understandingof the molecular perturbations that occur within the failingheart, the translation of this knowledge into clinical prac-tice has been disappointing. The most likely explanation isthat the contribution of a given defect to the underlyingheart disease is highly variable, highlighting the need toselect a homogeneous subset of patients in whom HF isdriven by a specific mechanism that can be targeted. Thisis particularly true for myocardial metabolism, which hasbeen studied for the better part of a century, but has notproven to be amendable by specific therapeutic interven-tions. Perhaps the most daunting challenge for metabolicdrug development is the paucity of specific tools to studycardiac metabolism in an individual patient. Metabolomicanalysis of plasma lacks cardiac specificity and isolation ofcardiac muscle may identify specific metabolic defects, butcannot be applied to intervention trials, as it requiresventricular biopsies. Cardiac PET is a highly versatile tech-nique that allows virtually every molecule to be modifiedfor detection by PET. Nevertheless, the technique is limitedby variations in signal to noise, the inability to distinguishtracers from metabolites, and by the fact that it exposessubjects to ionizing radiation. Another key limitation inmetabolic research in HF is the fact that there is no evi-dence to support that the level of energy depletion iscausing cardiac dysfunction. It is possible that the meta-bolic changes in the myocardium reflect beneficial cardiacadaptation to stress or even an epiphenomenon.

9. Five-year view

Eight decades of metabolic research in HF have not providedus with reliable metabolic interventions for this devastatingdisease. Over the next 5 years, we anticipate that severalmetabolic interventions will be tested in clinical trials. Oneof the most promising and timely interventions is the mito-chondria-targeted antioxidant SS-31 (also known asBendavia), which has been established as safe and well tol-erated in patients after an acute myocardial infarction [104].While a conference abstract indicated that Bendavia is effec-tive in HF patients as well, appropriately sized trials have notstarted yet. We expect that the success of these interventionsdepend on the individual degree of mitochondrial dysfunc-tion and oxidative stress. Accordingly, we believe that thebiomarkers described above should be employed for patientselection and monitoring. Furthermore, we expect thathyperpolarized MRS will replace PET as the gold standardfor the assessment of cardiac substrate utilization, as it canlink metabolism to function, differentiate between tracersand metabolites, and avoids ionizing radiation. Whether bio-marker-based patient selection will illuminate pathways for-ward for metabolic interventions in HF remains to beestablished.

Key issues

● A major unmet need in drug development for heart failureis the ability to identify a homogeneous subset of patientsin whom cardiac dysfunction is driven by a specificmechanism.

● The contribution of metabolic dysfunction and aberrantproduction of reactive oxygen species (ROS) tot the under-lying disease mechanism is highly variable, underscoringthe need for meticulous patient selection.

● Molecular imaging techniques such as magnetic resonancespectroscopy and positron emission tomography are reli-able tools to detect a variety of metabolic perturbations inHF and monitor the effect of interventions.

● Mitochondrial ROS production and the resulting oxidativestress are both a cause and a consequence of heart failurethat are potentially amendable by therapeuticinterventions.

● Biomarkers for oxidative stress, such as plasma free thiols,may serve to select patients for antioxidant therapies.

Acknowledgments

The authors would like to thank Else Koning for the artwork she hascreated for this article.

Funding

This paper was not funded.

Declaration of interest

The authors have no relevant affiliations or financial involvement with anyorganization or entity with a financial interest in or financial conflict withthe subject matter or materials discussed in the manuscript. This includesemployment, consultancies, honoraria, stock ownership or options, experttestimony, grants or patents received or pending, or royalties.

ORCID

Rudolf A. de Boer http://orcid.org/0000-0002-4775-9140

References

Papers of special note have been highlighted as either of interest (•) or ofconsiderable interest (••) to readers.

1. Ponikowski P, Voors AA, Anker SD, et al. 2016 ESC guidelines forthe diagnosis and treatment of acute and chronic heart failure: thetask force for the diagnosis and treatment of acute and chronicheart failure of the European Society of Cardiology (ESC).Developed with the special contribution of the heart failure asso-ciation (HFA) of the ESC. Eur J Heart Fail. 2016;18:891–975.

• Most recent, most comprehensive, and most innovative HFguidelines to date.

2. Udelson JE, Stevenson LW. The future of heart failure diagnosis,therapy, and management. Circulation. 2016;133:2671–2686.

•• Inspiring viewpoint editorial that highlights the great achieve-ments in HF care and also provides guidance for the dauntingchallenge to cope with the impeding HF epidemic.

3. Mozaffarian D, Benjamin EJ, Go AS, et al. Executive summary: heartdisease and stroke statistics–2016 update: a report from theAmerican heart association. Circulation. 2016;133:447–454.

EXPERT REVIEW OF MOLECULAR DIAGNOSTICS 149

4. McMurray JJV, Packer M, Desai AS, et al. Angiotensin-neprilysininhibition versus enalapril in heart failure. N Engl J Med.2014;371:993–1004.

5. Gheorghiade M, Larson CJ, Shah SJ, et al. Developing new treat-ments for heart failure: focus on the heart. Circ Heart Fail. 2016;9:e002727.

6. Bers DM. Cardiac excitation-contraction coupling. Nature.2002;415:198–205.

7. Westenbrink BD, Edwards AG, McCulloch AD, et al. The promise ofCaMKII inhibition for heart disease: preventing heart failure andarrhythmias. Expert Opin Ther Targets. 2013;17:889–903.

8. Mudd JO, Kass DA. Tackling heart failure in the twenty-first century.Nature. 2008;451:919–928.

9. Shah AM, Mann DL. In search of new therapeutic targets andstrategies for heart failure: recent advances in basic science.Lancet Lond Engl. 2011;378:704–712.

10. Westenbrink BD, Ling H, Divakaruni AS, et al. Mitochondrial repro-gramming induced by CaMKIIδ mediates hypertrophy decompen-sation. Circ Res. 2015;116:e28–e39.

11. Neubauer S. The failing heart–an engine out of fuel. N Engl J Med.2007;356:1140–1151.

12. Hasenfuss G. Alterations of calcium-regulatory proteins in heartfailure. Cardiovasc Res. 1998;37:279–289.

13. Greenberg B, Butler J, Felker GM, et al. Calcium upregulation bypercutaneous administration of gene therapy in patients with car-diac disease (CUPID 2): a randomised, multinational, double-blind,placebo-controlled, phase 2b trial. Lancet Lond Engl.2016;387:1178–1186.

14. Herrmann G, Decherd G. The chemical nature of heart failure. AnnIntern Med. 1939;12:1233–1244.

• The paper describes the discovery of metabolic dysfunction inHF that inspired eight decades of research.

15. Stanley WC, Recchia FA, Lopaschuk GD. Myocardial substrate meta-bolism in the normal and failing heart. Physiol Rev. 2005;85:1093–1129.

16. Ardehali H, Sabbah HN, Burke MA, et al. Targeting myocardialsubstrate metabolism in heart failure: potential for new therapies.Eur J Heart Fail. 2012;14:120–129.

17. Doehner W, Frenneaux M, Anker SD. Metabolic impairment in heartfailure: the myocardial and systemic perspective. J Am Coll Cardiol.2014;64:1388–1400.

•• Exceptional review of the systemic metabolic impairment inHF, with an online supplement that provides a detailed yetpalatable summary of cardiac metabolism.

18. Akhmedov AT, Rybin V, Marín-García J. Mitochondrial oxidativemetabolism and uncoupling proteins in the failing heart. HeartFail Rev. 2015;20:227–249.

19. Sorokina N, O’Donnell JM, McKinney RD, et al. Recruitment ofcompensatory pathways to sustain oxidative flux with reducedcarnitine palmitoyltransferase I activity characterizes inefficiencyin energy metabolism in hypertrophied hearts. Circulation.2007;115:2033–2041.

20. Schroeder MA, Lau AZ, Chen AP, et al. Hyperpolarized (13)C mag-netic resonance reveals early- and late-onset changes to in vivopyruvate metabolism in the failing heart. Eur J Heart Fail.2013;15:130–140.

•• An elaborate study on the prospective changes in cardiacmetabolism during HF progression using state-of-the-arthyperpolarized magnetic resonance spectroscopy.

21. Alexander D, Lombardi R, Rodriguez G, et al. Metabolomic distinc-tion and insights into the pathogenesis of human primary dilatedcardiomyopathy. Eur J Clin Invest. 2011;41:527–538.

22. Fillmore N, Lopaschuk GD. Targeting mitochondrial oxidative meta-bolism as an approach to treat heart failure. Biochim Biophys Acta.2013;1833:857–865.

23. Kim J, Tchernyshyov I, Semenza GL, et al. HIF-1-mediated expres-sion of pyruvate dehydrogenase kinase: a metabolic switchrequired for cellular adaptation to hypoxia. Cell Metab.2006;3:177–185.

24. Aubert G, Martin OJ, Horton JL, et al. The failing heart relies onketone bodies as a fuel. Circulation. 2016;133:698–705.

25. Balaban RS, Nemoto S, Finkel T. Mitochondria, oxidants, and aging.Cell. 2005;120:483–495.

26. Kohlhaas M, Maack C. Interplay of defective excitation-contractioncoupling, energy starvation, and oxidative stress in heart failure.Trends Cardiovasc Med. 2011;21:69–73.

• Comprehensively describes the link between metabolic dys-function and (mitochondrial) oxidative stress in heart failure.

27. Ide T, Tsutsui H, Hayashidani S, et al. Mitochondrial DNAdamage and dysfunction associated with oxidative stress infailing hearts after myocardial infarction. Circ Res. 2001;88:529–535.

28. Galougahi KK, Liu -C-C, Gentile C, et al. Glutathionylation mediatesangiotensin II-induced eNOS uncoupling, amplifying NADPH oxi-dase-dependent endothelial dysfunction. J Am Heart Assoc. 2014;3:e000731.

29. Lijnen PJ, van Pelt JF, Fagard RH. Downregulation of manganesesuperoxide dismutase by angiotensin II in cardiac fibroblasts ofrats: association with oxidative stress in myocardium. Am JHypertens. 2010;23:1128–1135.

30. Daiber A. Redox signaling (cross-talk) from and to mitochondriainvolves mitochondrial pores and reactive oxygen species. BiochimBiophys Acta. 2010;1797:897–906.

31. Paulus WJ, Tschöpe C. A novel paradigm for heart failure withpreserved ejection fraction: comorbidities drive myocardial dys-function and remodeling through coronary microvascularendothelial inflammation. J Am Coll Cardiol. 2013;62:263–271.

• Introduces the concept of distinct mechanisms underlyingheart failure with a preserved and heart failure with a reducedejection fraction.

32. Casademont J, Miró O. Electron transport chain defects in heartfailure. Heart Fail Rev. 2002;7:131–139.

33. Aimo A, Borrelli C, Vergaro G, et al. Targeting mitochondrial dys-function in chronic heart failure: current evidence and potentialapproaches. Curr Pharm Des. 2016;22:4807-4822.

34. Joiner M-LA, Koval OM, Li J, et al. CaMKII determines mitochondrialstress responses in heart. Nature. 2012;491:269–273.

35. Oka T, Hikoso S, Yamaguchi O, et al. Mitochondrial DNA thatescapes from autophagy causes inflammation and heart failure.Nature. 2012;485:251–255.

36. Dai D-F, Chen T, Szeto H, et al. Mitochondrial targeted antioxidantpeptide ameliorates hypertensive cardiomyopathy. J Am CollCardiol. 2011;58:73–82.

37. Yancey DM, Guichard JL, Ahmed MI, et al. Cardiomyocyte mito-chondrial oxidative stress and cytoskeletal breakdown in the heartwith a primary volume overload. Am J Physiol Heart Circ Physiol.2015;308:H651–H663.

38. Long Q, Yang K, Yang Q. Regulation of mitochondrial ATPsynthase in cardiac pathophysiology. Am J Cardiovasc Dis.2015;5:19–32.

39. García-Bermúdez J, Sánchez-Aragó M, Soldevilla B, et al. PKA phos-phorylates the ATPase inhibitory factor 1 and inactivates its capa-city to bind and inhibit the mitochondrial H(+)-ATP synthase. CellRep. 2015;12:2143–2155.

40. Wang S-B, Murray CI, Chung HS, et al. Redox regulation of mito-chondrial ATP synthase. Trends Cardiovasc Med. 2013;23:14–18.

41. Beer M, Seyfarth T, Sandstede J, et al. Absolute concentrations ofhigh-energy phosphate metabolites in normal, hypertrophied, andfailing human myocardium measured noninvasively with (31)P-SLOOP magnetic resonance spectroscopy. J Am Coll Cardiol.2002;40:1267–1274.

42. Carley AN, Taegtmeyer H, Lewandowski ED. Matrix revisited:mechanisms linking energy substrate metabolism to the functionof the heart. Circ Res. 2014;114:717–729.

43. Cornelissen VA, Defoor JGM, Stevens A, et al. Effect of creatinesupplementation as a potential adjuvant therapy to exercise train-ing in cardiac patients: a randomized controlled trial. Clin Rehabil.2010;24:988–999.

150 H. G. BOOIJ ET AL.

44. Carvalho APPF, Rassi S, Fontana KE, et al. Influence of creatinesupplementation on the functional capacity of patients with heartfailure. Arq Bras Cardiol. 2012;99:623–629.

45. Lygate CA, Aksentijevic D, Dawson D, et al. Living withoutcreatine: unchanged exercise capacity and response to chronicmyocardial infarction in creatine-deficient mice. Circ Res.2013;112:945–955.

46. Aquilani R, Opasich C, Gualco A, et al. Adequate energy-proteinintake is not enough to improve nutritional and metabolic status inmuscle-depleted patients with chronic heart failure. Eur J HeartFail. 2008;10:1127–1135.

47. Ahmad T, Kelly JP, McGarrah RW, et al. Prognostic implications oflong-chain acylcarnitines in heart failure and reversibility withmechanical circulatory support. J Am Coll Cardiol. 2016;67:291–299.

48. Hunter WG, Kelly JP, McGarrah RW, et al. Metabolomic profilingidentifies novel circulating biomarkers of mitochondrial dysfunc-tion differentially elevated in heart failure with preserved versusreduced ejection fraction: evidence for shared metabolic impair-ments in clinical heart failure. J Am Heart Assoc. 2016;5:e003190.

49. Obrzut S, Tiongson J, Jamshidi N, et al. Assessment of metabolicphenotypes in patients with non-ischemic dilated cardiomyopathyundergoing cardiac resynchronization therapy. J Cardiovasc TranslRes. 2010;3:643–651.

50. Ho E, Karimi Galougahi K, Liu -C-C, et al. Biological markers ofoxidative stress: applications to cardiovascular research and prac-tice. Redox Biol. 2013;1:483–491.

• Evaluates oxidative stress biomarkers commonly used in car-diovascular research and practice.

51. Tang WHW, Wang Z, Fan Y, et al. Prognostic value of elevatedlevels of intestinal microbe-generated metabolite trimethylamine-N-oxide in patients with heart failure: refining the gut hypothesis. JAm Coll Cardiol. 2014;64:1908–1914.

52. Roul D, Recchia FA. Metabolic alterations induce oxidative stress indiabetic and failing hearts: different pathways, same outcome.Antioxid Redox Signal. 2015;22:1502–1514.

53. Sun A, Zou Y, Wang P, et al. Mitochondrial aldehyde dehydrogen-ase 2 plays protective roles in heart failure after myocardial infarc-tion via suppression of the cytosolic JNK/p53 pathway in mice. JAm Heart Assoc. 2014;3:e000779.

54. Landar A, Zmijewski JW, Dickinson DA, et al. Interaction of electro-philic lipid oxidation products with mitochondria in endothelialcells and formation of reactive oxygen species. Am J PhysiolHeart Circ Physiol. 2006;290:H1777–87.

55. Srivastava S, Chandrasekar B, Bhatnagar A, et al. Lipid peroxidation-derived aldehydes and oxidative stress in the failing heart: role ofaldose reductase. Am J Physiol Heart Circ Physiol. 2002;283:H2612–9.

56. Salonen JT, Nyyssönen K, Salonen R, et al. Lipoprotein oxidationand progression of carotid atherosclerosis. Circulation.1997;95:840–845.

57. Banne AF, Amiri A, Pero RW. Reduced level of serum thiols inpatients with a diagnosis of active disease. J Anti-Aging Med.2003;6:327–334.

58. Chung HS, Wang S-B, Venkatraman V, et al. Cysteine oxidativeposttranslational modifications: emerging regulation in the cardio-vascular system. Circ Res. 2013;112:382–392.

59. Patel RS, Ghasemzadeh N, Eapen DJ, et al. Novel biomarker ofoxidative stress is associated with risk of death in patients withcoronary artery disease. Circulation. 2016;133:361–369.

60. Turell L, Radi R, Alvarez B. The thiol pool in human plasma: thecentral contribution of albumin to redox processes. Free Radic BiolMed. 2013;65:244–253.

61. Belch JJ, Bridges AB, Scott N, et al. Oxygen free radicals andcongestive heart failure. Br Heart J. 1991;65:245–248.

62. Kundi H, Ates I, Kiziltunc E, et al. A novel oxidative stress marker inacute myocardial infarction; thiol/disulphide homeostasis. Am JEmerg Med. 2015;33:1567–1571.

63. Go Y-M, Jones DP. Cysteine/cystine redox signaling in cardiovascu-lar disease. Free Radic Biol Med. 2011;50:495–509.

64. Koning AM, Meijers WC, Pasch A, et al. Serum free thiols in chronicheart failure. Pharmacol Res. 2016;111:452–458.

•First report on the association of total serum free thiols anddisease outcome.

65. Massion PB, Feron O, Dessy C, et al. Nitric oxide and cardiacfunction: ten years after, and continuing. Circ Res. 2003;93:388–398.

66. Silberman GA, Fan T-HM, Liu H, et al. Uncoupled cardiac nitricoxide synthase mediates diastolic dysfunction. Circulation.2010;121:519–528.

67. Figueira TR, Barros MH, Camargo AA, et al. Mitochondria as asource of reactive oxygen and nitrogen species: from molecularmechanisms to human health. Antioxid Redox Signal.2013;18:2029–2074.

68. Winlaw DS, Smythe GA, Keogh AM, et al. Increased nitric oxideproduction in heart failure. Lancet Lond Engl. 1994;344:373–374.

69. Osorio JC, Xu X, Vogel T, et al. Plasma nitrate accumulation duringthe development of pacing-induced dilated cardiac myopathy inconscious dogs is due to renal impairment. Nitric Oxide Biol ChemOff J Nitric Oxide Soc. 2001;5:7–17.

70. Polhemus DJ, Lefer DJ. Emergence of hydrogen sulfide as anendogenous gaseous signaling molecule in cardiovascular disease.Circ Res. 2014;114:730–737.

71. Parenica J, Kala P, Pavkova MG, et al. Natriuretic peptides, nitrite/nitrate and superoxide dismutase have additional value on topof the GRACE score in prediction of one-year mortality andrehospitalisation for heart failure in STEMI patients - Multiplebiomarkers prospective cohort study. Int J Cardiol.2016;211:96–104.

72. van den Berg E, Pasch A, Westendorp WH, et al. Urinary sulfurmetabolites associate with a favorable cardiovascular risk profileand survival benefit in renal transplant recipients. J Am SocNephrol JASN. 2014;25(6):1303–1312.

73. Papadimitriou L, Smith-Jones PM, Sarwar CMS, et al. Utility ofpositron emission tomography for drug development for heartfailure. Am Heart J. 2016;175:142–152.

74. Nakatani K, Watabe T, Masuda D, et al. Myocardial energy provisionis preserved by increased utilization of glucose and ketone bodiesin CD36 knockout mice. Metabolism. 2015;64:1165–1174.

75. Cannon MV, Silljé HHW, Sijbesma JWA, et al. Cardiac LXRα protectsagainst pathological cardiac hypertrophy and dysfunction byenhancing glucose uptake and utilization. EMBO Mol Med.2015;7:1229–1243.

76. Dávila-Román VG, Vedala G, Herrero P, et al. Altered myocardialfatty acid and glucose metabolism in idiopathic dilated cardiomyo-pathy. J Am Coll Cardiol. 2002;40:271–277.

77. Slart RHJA, Tio RA, van der Vleuten PA, et al. Myocardial perfu-sion reserve and contractile pattern after beta-blocker therapyin patients with idiopathic dilated cardiomyopathy. J NuclCardiol Off Publ Am Soc Nucl Cardiol. 2010;17:479–485.

78. Nielsen R, Jorsal A, Iversen P, et al. Heart failure patients withprediabetes and newly diagnosed diabetes display abnormalitiesin myocardial metabolism. J Nucl Cardiol Off Publ Am Soc NuclCardiol. 2016. DOI: 10.1007/s12350-016-0622-0.

79. van Campen CMC, Visser FC, van der Weerdt AP, et al. FDG PET as apredictor of response to resynchronisation therapy in patients withischaemic cardiomyopathy. Eur J Nucl Med Mol Imaging.2007;34:309–315.

80. Winter JL, Castro PF, Quintana JC, et al. Effects of trimetazidine innonischemic heart failure: a randomized study. J Card Fail.2014;20:149–154.

81. Handley MG, Medina RA, Nagel E, et al. PET imaging of cardiachypoxia: opportunities and challenges. J Mol Cell Cardiol.2011;51:640–650.

82. Scherer DJ, Psaltis PJ. Future imaging of atherosclerosis: molecularimaging of coronary atherosclerosis with (18)F positron emissiontomography. Cardiovasc Diagn Ther. 2016;6:354–367.

83. Medina RA, Mariotti E, Pavlovic D, et al. 64Cu-CTS: a promisingradiopharmaceutical for the identification of low-grade cardiachypoxia by PET. J Nucl Med Off Publ Soc Nucl Med. 2015;56:921–926.

84. Carroll VN, Truillet C, Shen B, et al. (11)C.Ascorbic and (11)C.dehy-droascorbic acid, an endogenous redox pair for sensing reactive

EXPERT REVIEW OF MOLECULAR DIAGNOSTICS 151

oxygen species using positron emission tomography. ChemCommun Camb Engl. 2016;52:4888–4890.

85. Huang X, Gao Y, Qin J, et al. The role of miR-34a in the hepa-toprotective effect of hydrogen sulfide on ischemia/reperfusioninjury in young and old rats. Lesnefsky EJ, editor. Plos ONE.2014;9:e113305.

86. Bizino MB, Hammer S, Lamb HJ. Metabolic imaging of the humanheart: clinical application of magnetic resonance spectroscopy.Heart Br Card Soc. 2014;100:881–890.

87. Levelt E, Rodgers CT, Clarke WT, et al. Cardiac energetics, oxygena-tion, and perfusion during increased workload in patients with type2 diabetes mellitus. Eur Heart J. 2015. DOI: 10.1093/eurheartj/ehv42.

88. Fragasso G, Perseghin G, De Cobelli F, et al. Effects of metabolicmodulation by trimetazidine on left ventricular function and phos-phocreatine/adenosine triphosphate ratio in patients with heartfailure. Eur Heart J. 2006;27:942–948.

89. Dass S, Holloway CJ, Cochlin LE, et al. No evidence of myocardialoxygen deprivation in nonischemic heart failure. Circ Heart Fail.2015;8:1088–1093.

90. Schroeder MA, Clarke K, Neubauer S, et al. Hyperpolarized mag-netic resonance: a novel technique for the in vivo assessment ofcardiovascular disease. Circulation. 2011;124:1580–1594.

91. Westenbrink BD, Dorn GW. Imaging the cardiac diet. Eur J HeartFail. 2013;15:123–124.

92. Josan S, Park JM, Hurd R, et al. In vivo investigation of cardiacmetabolism in the rat using MRS of hyperpolarized 1-13C. and2-13C.pyruvate. NMR Biomed. 2013;26:1680–1687.

93. Bersin RM, Wolfe C, Kwasman M, et al. Improved hemodynamicfunction and mechanical efficiency in congestive heart failure withsodium dichloroacetate. J Am Coll Cardiol. 1994;23:1617–1624.

94. Wilson JR, Mancini DM, Ferraro N, et al. Effect of dichloroacetate onthe exercise performance of patients with heart failure. J Am CollCardiol. 1988;12:1464–1469.

95. Home PD, Pocock SJ, Beck-Nielsen H, et al. Rosiglitazone evaluatedfor cardiovascular outcomes in oral agent combination therapy fortype 2 diabetes (RECORD): a multicentre, randomised, open-labeltrial. Lancet Lond Engl. 2009;373:2125–2135.

96. Holubarsch CJF, Rohrbach M, Karrasch M, et al. A double-blindrandomized multicentre clinical trial to evaluate the efficacy andsafety of two doses of etomoxir in comparison with placebo inpatients with moderate congestive heart failure: the ERGO (eto-moxir for the recovery of glucose oxidation) study. Clin Sci LondEngl. 1979;2007(113):205–212.

97. Goszcz K, Deakin SJ, Duthie GG, et al. Antioxidants in cardiovasculartherapy: panacea or false hope? front. Cardiovasc Med. 2015;2:29.

98. Aitio M-L. N-acetylcysteine – passe-partout or much ado aboutnothing? Br J Clin Pharmacol. 2006;61:5–15.

99. Ye Y, Li J, Yuan Z. Effect of antioxidant vitamin supplementation oncardiovascular outcomes: a meta-analysis of randomized controlledtrials. Plos One. 2013;8:e56803.

100. Tamariz L, Hare JM. Xanthine oxidase inhibitors in heart failure:where do we go from here? Circulation. 2015;131:1741–1744.

101. Atkuri KR, Mantovani JJ, Herzenberg LA, et al. N-Acetylcysteine–asafe antidote for cysteine/glutathione deficiency. Curr OpinPharmacol. 2007;7:355–359.

• Explains the potential benefit of therapeutic modulation of theserum free thiol level in selected patients.

102. Dröge W. Aging-related changes in the thiol/disulfide redox state:implications for the use of thiol antioxidants. Exp Gerontol.2002;37:1333–1345.

103. Chatzianastasiou A, Bibli S-I, Andreadou I, et al. Cardioprotection byH2S donors: nitric oxide-dependent and -independent mechan-isms. J Pharmacol Exp Ther. 2016;358:431–440.

104. Gibson CM, Giugliano RP, Kloner RA, et al. EMBRACE STEMI study: aPhase 2a trial to evaluate the safety, tolerability, and efficacy of intra-venous MTP-131 on reperfusion injury in patients undergoing primarypercutaneous coronary intervention. Eur Heart J. 2016;37:1296–1303.

152 H. G. BOOIJ ET AL.