Download Metabolomics - Circulation: Cardiovascular Genetics

Metabolomics
Citric Acid Cycle Intermediates in Cardioprotection
Gabor Czibik, MD, PhD;* Violetta Steeples, BSc;* Arash Yavari, BSc, MRCP, DPhil;*
Houman Ashrafian, MA, MRCP, DPhil*
Abstract—Over the last decade, there has been a concerted clinical effort to deliver on the laboratory promise that a variety
of maneuvers can profoundly increase cardiac tolerance to ischemia and/or reduce additional damage consequent upon
reperfusion. Here we will review the proximity of the metabolic approach to clinical practice. Specifically, we will focus on
how the citric acid cycle is involved in cardioprotection. Inspired by cross-fertilization between fundamental cancer biology and
cardiovascular medicine, a set of metabolic observations have identified novel metabolic pathways, easily manipulable in man,
which can harness metabolism to robustly combat ischemia-reperfusion injury. (Circ Cardiovasc Genet. 2014;7:711-719.)
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D
of principle, significant reductions in final infarct size (of
≤50%) could be achieved in experimental models.2 Decades
of research have successfully dissected out the molecular
components of IPC, including the time course of protection, triggers evoking the protective signaling and prosurvival kinase cascades (described in recent reviews9,11,12). In
contrast, translation to man has generally been disappointing with no IPC strategy routinely used in clinical practice.
Reasons for this failure have recently been reviewed elsewhere.13–15 Because patients with CAD are often elderly with
numerous other comorbidities and confounding medical
therapies,14 the protective potential of IPC may be compromised, motivating the exploration of new strategies to reduce
ischemic cardiac injury and the associated healthcare and
societal costs.
One such approach has been the application of metabolic
strategies to modify the time course of myocardial injury.
In the 1960s, preceding the pharmacological or mechanical
reperfusion era, Sodi-Pallares proposed using a solution containing glucose, insulin, and potassium to prevent reperfusion
arrhythmias.16 The rationale was to support myocardial energy
generation through augmentation of ischemic glycolytic activity using pharmacological doses of glucose and insulin.17 This
combination reduces circulating free fatty acids that potentially
inhibit glycolysis18 (mitigating the Randle cycle via insulin’s
inhibitory effect on lipolysis) and has been proposed to prevent
arrhythmias through the restoration of intracellular potassium
and stabilisation of membrane potential.16 The majority of
experimental studies applying glucose, insulin, and potassium
treatment have reported reduced infarct size19 with improved
postischemic functional20 and energetic recovery,21 with subsequent successful translation to humans.22,23 Encouraged by
his success and that of the first Diabetes Mellitus InsulinGlucose Infusion in Acute Myocardial Infarction (DIGAMI)
trial,24 the method gained popularity to reduce IR-injury after
espite a dramatic drop in the incidence of acute myocardial infarction over recent decades,1 coronary artery
disease (CAD) is still the foremost cause of death globally,
with >7 million people dying annually.2 In developed countries, reductions in exposure to major risk factors and the
application of evidence-based interventions to patients with
CAD have shifted the epidemiology of CAD toward older
age.3,4 There are concerns that this positive trend may be obviated by the pandemic of obesity and type 2 diabetes mellitus.
Advances in cardiac surgery have led to its application to an
increasingly older subset of patients, with a correspondingly
greater risk of myocardial injury. Sensitive means of detecting myocardial injury have broadened the at-risk population
to include those undergoing major noncardiac surgical procedures (eg, vascular or major abdominal surgery), particularly
in the elderly.5 Much of the effort to reduce CAD-related myocardial injury has focussed on reducing the burden of coronary
atheroma and plaque stability. The delineation of robustly
protective alleles reducing the burden of atherosclerosis (eg,
PCSK96 and APOC37,8) has signposted potential drug targets
to modify the course of CAD. An alternative strategy would
be to focus on identifying approaches to protect the myocardium from disease or iatrogenic injury.
Ischemic myocardium ultimately necroses unless reperfused.9 Paradoxically, reperfusion itself comes at a cost, a phenomenon termed ischemia-reperfusion (IR) injury.2 IR-injury
may manifest in the form of contractile (stunning), vascular
(no-reflow phenomenon), electric (reperfusion arrhythmias)
impairment, or lethal reperfusion injury.2
To mitigate these complications, enthusiasm for a myocardial protection strategy was inspired in 1986 by the description of ischemic preconditioning (IPC) in which brief,
nonlethal episodes of ischemia alternating with episodes
of reperfusion powerfully protected the myocardium from
subsequent otherwise lethal ischemic challenge.10 As proof
From the Division of Cardiovascular Medicine, Radcliffe Department of Medicine, University of Oxford, Oxford, United Kingdom.
Guest Editor for this series was Manuel Mayr, MD, PhD.
*Dr Czibik, V. Steeples, A. Yavari, and H. Ashrafian contributed equally to this work.
Correspondence to Houman Ashrafian, MA, MRCP, DPhil, Division of Cardiovascular Medicine, Radcliffe Department of Medicine, West Wing, John
Radcliffe Hospital, Oxford OX3 9DU, United Kingdom. E-mail [email protected]
© 2014 American Heart Association, Inc.
Circ Cardiovasc Genet is available at http://circgenetics.ahajournals.org
711
DOI: 10.1161/CIRCGENETICS.114.000220
712 Circ Cardiovasc Genet October 2014
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acute myocardial infarction.25 The present status of glucose,
insulin, and potassium for cardioprotection is controversial
because large-scale randomized double-blind studies reported
mixed effects.26 Glucose, insulin, and potassium is thus not
routinely used but has motivated the search for more clinically
applicable metabolic approaches to cardioprotection.
For many decades, even before description of the complete
citric acid cycle (CAC), ischemia and hypoxia were recognized to stimulate the degradation of myocardial aspartate and
glutamate resulting in the production of succinate in isolated
hearts.27 It has been proposed that this channelling serves to
generate ATP in the absence of oxygen. These observations
led to the hope that manipulation of the CAC could promote
cardioprotection. As a corollary, in the mid-2000s, germline
heterozygous loss-of-function mutations in the CAC enzyme
fumarate hydratase (FH, catalyzing the hydration of fumarate
to malate)28,29 were found to cause hereditary leiomyomatosis
and renal cell cancer (HLRCC), an autosomal dominant disorder characterized by smooth-muscle tumors of the skin and
uterus, and renal cancer. The resulting accumulation of the
CAC intermediate fumarate has several potentially beneficial
sequlae, including the stabilization of hypoxia-inducible factor 1α (HIF-1α; a master transcription factor coordinating the
response to hypoxia),30 with potentially cytoprotective implications for both cancer biology31 and myocardial protection.32
In this review, we shall start by describing the canonical functions of the CAC with a focus on the myocardium. We will
follow by outlining the CAC's recently explored noncanonical
metabolic (eg, signaling) roles culminating in our experience
with CAC intermediates and their manipulation as promising
cardioprotective therapies.
A
glucose
Traditional Functions of the CAC
The CAC,33 either in its complete or in its modified forms, is a
metabolic hub in all aerobic and some anoxygenic organisms
(eg, green sulfur bacteria). The CAC has 2 principal purposes:
(1) to harness intermediary metabolism to generate ATP (through
oxidative phosphorylation and substrate-level phosphorylation
not requiring a terminal electron acceptor, such as oxygen, eg,
in hypoxia) and (2) to provide precursors for biosynthetic pathways. As traditionally envisaged (eg, in most biochemistry textbooks), the CAC begins with the condensation of oxaloacetate
with acetyl-CoA, consisting of 8 sequential linear irreversible
(eg, citrate synthase and the 2-oxoglutarate dehydrogenase complex) and reversible steps, ending with the regeneration of oxaloacetate (Figure 1A). These CAC steps are generally considered
to take place predominantly in the mitochondrial matrix.
Acetyl-CoA derived from glycolysis (via pyruvate dehydrogenase), from β-oxidation of fatty acids, and from the breakdown of
ketone bodies and amino acids, generate the reducing equivalents
NADH and FADH2 (reduced nicotinamide adenine dinucleotide
and reduced flavin adenine dinucleotide, respectively), which,
via the electron transport chain, synthesise ATP by oxidative
phosphorylation. In addition there is a comparatively small but
significant matrix substrate-level energy generation (guanosine
triphosphate, GTP, convertible to ATP) not requiring oxygen.
However, the CAC is more than a highly efficient sequence
of spatially optimized reactions to degrade acetyl-CoA. Short
sections of the CAC are close to thermodynamic equilibrium
and are, therefore, bidirectional under different conditions.
Krebs emphasized this reversibility by observing (albeit with
relatively nonspecific inhibitors, such as malonic acid) that
the enzymes interconverting succinate↔fumarate↔malate↔
B
Pyruvate
NAD+ + CoA-SH
Pyruvate dehydrogenase
NA DH + H+ + CO2
fatty acids
Pyruvate
CoA-SH
Acetyl-CoA
Citrate synthase
Oxaloacetate
H20
Acetyl-CoA
cis-Aconitate
Citrate
H20
Aconitase
Malate dehydrogenase
Isocitrate
Isocitrate dehydrogenase
Malate
fatty acids
acetyl-CoA
Aconitase
NA DH + H+
NA D+
malonyl-CoA
H20
Citrate
CO2
proline
arginine
histidine
Isocitrate
aspartate
NA DH + H+
Fumarate hydratase
H20
cis-Aconitate
Oxaloacetate
NA D +
Oxaloacetate
Malate
asparagine
2-oxoglutarate
glutamate
2-oxoglutarate
2-oxoglutarate dehydrogenase
Fumarate
Succinate dehydrogenase
QH2
Succinyl-CoA
Succinyl-CoA synthetase
Q
Succinate
GDP + Pi
Fumarate
NAD+ + CoA-SH
NA DH + H+ + CO2
phenylalanine
tyrosine
Succinyl-CoA
Succinate
glutamine
heme
odd-chain fatty acids
methionine
valine
isoleucine
CoA-SH + GTP
Figure 1. The citric acid cycle (CAC). A, An overview of CAC reactions. Observe that apart from 3 irreversible steps (citrate synthase,
isocitrate dehydrogenase, and 2-oxoglutarate dehydrogenase), the rest of the CAC reactions are reversible, allowing reverse flux under
certain conditions (such as in anoxia). B, Anaplerosis and cataplerosis of the CAC. Anaplerosis replenishes CAC intermediates, whereas
cataplerosis allows production of various biosynthetic precursors making the CAC as a central metabolic hub, connecting diverse metabolic pathways. CoA-SH indicates reduced coenzyme A; FADH2, reduced flavin adenine dinucleotide; GTP, guanosine triphosphate;
NAD+, oxidized nicotinamide adenine dinucleotide; NADH, reduced nicotinamide adenine dinucleotide; and QH2, reduced coenzyme Q.
Czibik et al CAC in Cardioprotection 713
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oxaloacetate are reversible. This reversibility facilitates the
generation of precursors for glucose synthesis (from oxaloacetate via the pyruvate recycling pathway), fatty acid and
cholesterol synthesis (from citrate), amino acid anabolism,
nucleotides (via 2-oxoglutarate [2-OG]), and heme biosynthesis (via succinyl-CoA). In contrast to its perceived role as
a perpetual rotor degrading acetyl-CoA, the CAC represents
a highly versatile collection of reactions that can be concatenated in its entirety or in part to generate energy (oxidatively
or without oxygen) and as a biosynthetic toolbox.
Exemplifying the versatility of the CAC as a whole and individual reaction steps per se, dominant mutations in 2 of the 3
isocitrate dehydrogenases (IDH1 and IDH2) have been found
to promote glioma, acute myeloid leukemia, chondrosarcoma,
and other tumours. In the traditional conception, acetyl-CoA is
thought to progress unidirectionally through the CAC. Instead,
IDH1 (primarily cytoplasmic and NADP+ dependant) and
IDH2 (primarily mitochondrial and NADP+ dependant) along
with IDH3 promote the conventional oxidative decarboxylation of isocitrate to 2-OG but may also promote the reverse
(reductive carboxylation). Reductively metabolized glutamine
has been proposed to be a cellular carbon source for fatty acid
synthesis during hypoxia or impaired mitochondrial respiration
(eg, FH deficiency).34 Although these observations, adduced
from ex vivo cancer cell preparations, remain controversial
and potentially confounded by technical aspects of metabolic
tracer studies,35 there is little doubt that mutated IDH isoforms
also catalyze a neomorphic reaction converting 2-OG to the
oncometabolite 2-hydroxyglutarate. This example confirms the
complexity of a simple single CAC step and the consequences
of its modification. The same flexibility provides potential therapeutic opportunities to mitigate hypoxia/ischemia.36
To maintain its activity and to compensate for metabolite
leakage from the mitochondria,37 CAC flux can only be maintained if this cataplerotic activity constituting the removal
of intermediates from the CAC is balanced by anaplerotic
reactions replenishing CAC intermediates (Figure 1B).37,38
Breakdown of amino acids replenish 2-OG, succinyl-CoA,
and oxaloacetate; odd-chain fatty acid oxidation generates
succinyl-CoA; carboxylation of pyruvate by pyruvate carboxylase and malic enzyme produces oxaloacetate and malate,
respectively. (Figure 1B).37
Adequate anaplerosis is especially important because
the steady state concentrations of CAC intermediates are
typically low in relation to the fluxes through the CAC.37–39
These uneven CAC intermediate pools are explained, in
part, by the differential turnover rate of individual metabolic
reactions, the differing thermodynamic/kinetic properties of
each CAC step and differential mitochondrial inner membrane permeability. The measurement of both concentration and flux is technically challenging, even with dynamic
tracer studies (for flux).38 For example, citrate, with the largest pool size (≈200 nmol/g), has a turnover time of minutes, whereas oxaloacetate with the smallest pool size (5–10
nmol/g) has a turnover of the order of 100× per minute.38
Consonant with metabolic control analysis, in the CAC, flux
considerations dominate over steady-state thermodynamic
principles per se.38
mitochondrial matrix
2-oxoglutarate
NAD+
malate
NADH
Mdh2
oxaloacetate
Got2
intermembrane space/cytosol
2-oxoglutarate
Slc25a11
NAD+
malate
NADH
Mdh1
oxaloacetate
glycolysis
aspartate
glutamate
Slc1a3
aspartate
Got1
glutamate
Figure 2. The malate–aspartate shuttle. NADH (reduced nicotinamide adenine dinucleotide) formed during glycolysis needs
reoxidizing in the cytosol, and the malate–aspartate shuttle acts
as the major cardiac mechanism to transport the protons and
electrons to the mitochondrial matrix where they, via the citric
acid cycle and electron transport chain, are oxidized to water.
Got1 and 2 indicates glutamic-oxaloacetatic transaminase 1 and
2; Mdh1 and 2, malate dehydrogenase 1 and 2; Slc1a3, glutamate/aspartate transporter; and Slc25a11, mitochondrial
2-oxoglutarate/malate carrier protein.
To maintain locally high metabolite concentrations, CAC
reactions are compartmentalized within the mitochondria.
There may be further spatial subcompartmentalization in
the vicinity of individual CAC enzymes to maximize local
effective substrate concentrations and reaction efficiency.
Specialized carrier proteins catalyze the transport of nucleotides, amino acids, inorganic ions, fatty acids, keto acids,
and cofactors across the impermeable mitochondrial inner
membrane. These transport steps are important to maintain a
separate inner mitochondrial pool and micromilieu for ATP
production, for amino acid breakdown, for macromolecular
and heme biosynthesis, and for heat generation. An example
of such a mitochondrial carrier mechanism is the malateaspartate shuttle (MAS). MAS is a key metabolic shuttle,
active in the heart, which transfers electrons and protons from
glycolysis-derived cytosolic NADH (to which the inner mitochondrial membrane is impervious) into the matrix for use by
the electron transport chain using malate as an electron carrier. This involves a series of reactions involving the cytosolic
reduction of oxaloacetate to malate, transport of malate into
the mitochondrial matrix (in exchange for 2-OG), oxidation of
malate to regenerate oxaloacetate with reduction of mitochondrial NAD+ to NADH. Oxaloacetate then undergoes transamination using glutamate as nitrogen donor to generate aspartate
(and 2-OG), which exits the matrix via a glutamate–aspartate
transporter40 (Figure 2). Although the influence of MAS in the
heart in ischemia is complex and requires further clarification,
its manipulation is likely to be important in ischemia-reperfusion injury.41
The MAS is the most important although not the only means
to transport net protons and electrons from the cytoplasm to
the mitochondrial matrix for oxidation. Like the MAS, the
glycerol-3-phosphate shuttle helps reoxidise cytosolic NADH
to NAD+, making it available for glycolysis. Should this turnover fail, accumulating cytosolic NADH would act as a brake
to glycolysis in the face of a diminishing reducible NAD+ pool.
Reducing equivalents generated during glycolysis and more
substantially by the CAC are converted to ATP through the
transfer of electrons to molecular O2 by the electron transport
chain. Any lack of oxygen increases the NADH/NAD+ ratio,
substrate inhibits glycolysis/CAC, and substantially disrupts
714 Circ Cardiovasc Genet October 2014
energy production. Accordingly, during hypoxia, anaerobic
regeneration of NAD+ continues to occur, in part, through
reoxidation of NADH by lactate dehydrogenase, which catalyzes electron transfer to pyruvate, rather than O2, to yield
lactate. As oxidative phosphorylation and the closely coupled
CAC flux are arrested, NADH accumulates. In the absence of
molecular oxygen, even glycolytic acceleration may be inhibited by incomplete myocardial NADH reoxidation limiting
the regeneration of NAD+ essential for continued glycolysis.
As opposed to skeletal muscle, lactate dehydrogenase in the
heart is predominantly the H-type, with increased susceptibility to form the abortive ternary complex lactate dehydrogenase-NAD-enol that competes with NADH.42 Thus, without
alternative means to deplete NADH, the hypoxic metabolism
would slow because of redox imbalance (increased NADH).
As a corollary, modalities promoting NAD+ regeneration
would potentially improve energetics.43,44
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Noncanonical Functions of the CAC:
Succinate Production in Hypoxia
Ischemic cell death likely results from a rapid decline in
intracellular ATP concentration, [ATP]IC, that is irreconcilable with viability.45 Maintenance of adequate cellular energy
production is thus a fundamental and continual requirement
for all tissues, with its disruption leading to significant clinical consequences. Metabolic studies in isolated hearts and
muscle preparations and in natural states of extreme physiology (eg, diving mammals) have yielded important insights
into cellular mechanisms, specifically emphasizing the CAC
components, which maintain [ATP]IC and have broadened our
understanding of the CAC.
For decades, it has been recognized that hypoxia/ischemia confers substantial changes in CAC metabolites, most
prominently succinate. For example, in 1970, while investigating the ability of CAC metabolites to improve cardiac
performance in anoxia, Penney and Cascarano46 observed
that perfusion of anoxic rat hearts with glucose and a combination of fumarate+malate+glutamate or oxaloacetate+2OG ­(2-oxoglutarate) enhanced glycogen and ATP levels over
glucose perfusion alone, without increased glycolysis. In
both CAC metabolite treatment groups, succinate levels were
found consistently elevated. The authors concluded that mitochondrial energy production took place even during anoxia;
however, the precise nature of this mitochondrial contribution to anoxic energy generation was undefined.46 Similarly,
glutamate and aspartate were recognized to lead to increased
succinate levels in skeletal muscle before the discovery of the
CAC itself.47
The importance of this mechanism was implied by its
evolutionary conservation. Hochachka et al48 observed that
in diving mammals, blood concentrations of aspartate and
2-OG (2-oxoglutarate) levels decreased, whereas those of lactate, alanine, and succinate increased after diving (a hypoxic
manoeuvre). They proposed a model of 2 interlinked, oxygenindependent pathways (Figure 3),48 whereby NADH produced
during glycolysis and through anterograde carbon flow through
2-OG dehydrogenase generating GTP and succinate is coupled to NADH depletion through reducing aspartate-derived
Glucose-6P
Aspartate
Fructose-6P
2-oxoglutarate
Fructose-1,6DP
NAD+
Triose-P
Pi
NADH
NAD+
NADH
NADH
NAD+
Oxaloacetate
Glutamate
Malate
Diphosphoglycerate-1,3P
Phosphoenolpyruvate
NADH
Pyruvate
Glutamate
2-oxoglutarate
NAD+
Lactate
Alanine
Malate
2-oxoglutarate
CO2
Fumarate
NAD+
FPred
NADH
FPox
CoA-SH
Succinyl-CoA
GDP + Pi
cytoplasm
mitochondrion
ADP + Pi
ATP
Succinate
CoA-SH
GTP
Succinate
Figure 3. Modified adaptation of Hochacka’s model of energy
production by the citric acid cycle (CAC) in hypoxia.48 In this
model, anoxic succinate accumulation was suggested through
an oxidative (via pyruvate, 2-oxoglutarate, and succinyl-CoA)
and reductive (via aspartate, oxaloacetate, malate, and fumarate)
pathway, with CAC-dependent energy generation by substratelevel phosphorylation in addition to glycolytic ATP generation.
CoA-SH indicates reduced coenzyme A; FPox, oxidized flavoprotein; FPred, reduced flavoprotein; and GTP, guanosine
triphosphate.
oxaloacetate to malate. The malate is converted to fumarate
and ultimately succinate contrasting with the traditional conception of carbon flow in the CAC (because in the context of
lacking oxygen normal CAC flux ceases).48 Other mechanisms
contributing to NADH depletion may include mitochondrial
diaphorases.49 Increased succinate production has also been
observed in hypoxic cardiac myocytes.27
Although there are controversies about the pertinence of this
model to IR injury,43,50–53 at least under some circumstances,
loading of the CAC using glutamate, fumarate, and aspartate
have generally been successful but may not be clinically tractable. This strategy is also not without jeopardy—the energetic
benefit of channelling amino acids to succinate may be mitigated by the release of ROS at reperfusion resulting from a
burst of FADH2 in the context of too sudden forward flow of
succinate through succinate dehydrogenase (complex II).
Therapies Modifying CAC Intermediates
as Potential Therapies of IR
Recognizing the potential value of modifying, specifically
augmenting, CAC intermediates, several strategies have been
investigated. One readily translatable is the indirect augmentation of CAC intermediates using glutmate/glutamine.54
Czibik et al CAC in Cardioprotection 715
Glutamate/glutamine has exhibited variable promise in animal
and human models of myocardial IR, especially as a constituent of cardioplegia in coronary surgery.55–58 Another approach
is to augment CAC intermediates using anaplerotic precursors directly. Despite successfully increasing myocardial CAC
intermediates, dipropionylcysteine ethyl ester and heptanoate
failed to protect the myocardium,59,60 in part, perhaps because
of the differing protocols of drug infusion and myocardial
ischemia. In contrast, dipyruvyl-acetyl-glycerol decreases
myocardial infarct size in the pig.61 This is consistent with
the observation that fumarate-enriched cardioplegia results in
complete functional recovery of immature myocardium.62
Signaling Roles of the CAC:
Insights From Renal Tumours
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Notwithstanding the controversies surrounding the energetic
merits of succinate generation through mitochondrial substrate-level phosphorylation, increased succinate production
has been consistently identified in different tissues, in different models in hypoxia/IR. In addition to its metabolic role,
elevated succinate seen ubiquitously in ischemia is likely to
activate its cognate cell surface receptor—G-protein–coupled
receptor 91 (Gpr91).63 Succinate acting through this receptor
contributes to retinal neovascularization and acting through
prostaglandin E receptor 2–prostaglandin E receptor 4 modifies post–hypoxia-ischemic injury in the brain64, emphasizing
A Normoxia
Hydroxylation
PHD
HIFα
the pleotropic contributions of CAC metabolites to IR
injury.63,64
The discovery of normoxic HIF-1α stabilization by elevated fumarate levels in hereditary HLRCC and elevated
succinate in hereditary paraganglioma with pheochromocytomas resulting from deficient FH and SDH (succinate
dehydrogenase)28,29,65 respectively, potentially indicates
another signaling role for CAC metabolites in the response
to IR. The mechanism underlying normoxic HIF-1α stabilization in these tumors is thought to be substrate competition of the accumulating CAC metabolites with 2-OG. The
latter is a key cofactor for prolyl hydroxylase domain 1 to
3 proteins66,67 that regulate HIF-1α by hydroxylation under
normoxic conditions, promoting its proteosomal destruction
(Figure 4A).
This mechanism is supported by the finding that addition
of excess 2-OG is able to out-compete fumarate, thereby
reactivating prolyl hydroxylase domains and hydroxylating
HIF-1α.68 The possibility that different metabolic states—
signaled by varying levels of CAC intermediates—can regulate the central transcriptional coordinator of the cellular
response to hypoxia, resonates with the observation that CAC
metabolites are channelled toward succinate during ischemia
and has important consequences beyond cancer, in other
states of hypoxia/ischemia. Analogous HIF-1α augmentation
by succinate may be germane to cardiac ischemia because
mice overexpressing HIF-1α in the heart, when subjected to
Proteasomal
Degradation
VHL
HO-
HIFα
2-oxoglutarate
Pseudohypoxia
Fumarate and Succinate
Hydroxylation
HIFα
PHD
HIFα
HIFα
HIF-target
gene expression
2-oxoglutarate
B At Baseline
Electrophiles
e.g Fumarate
Cytoplasmic
Sequestration
KEAP1
and
Proteasomal
Degradation
NRF2
In the presence of Electrophiles
Electrophiles
e.g Fumarate
KEAP1
MAF
NRF2
NRF2
NRF2-target
gene expression
Figure 4. Signaling functions of citric acid cycle
(CAC) intermediates. A, The hypoxia-inducible factor (HIF)-1α-hypoxia pathway. The key in regulation
of HIF-1α stability is an O2- and 2-oxoglutaratedependent proline hydroxylation. When in excess,
fumarate and succinate can out-compete 2-oxoglutarate and can stabilize HIF-1α protein in normoxia.
B, The Nrf2-antioxidant pathway. The critical event
in Nrf2 activation is when specific cysteine residues on Keap1 react with electrophiles, such as
fumarate, and consequently Keap1 releases Nrf2.
KEAP1 indicates Kelch-like ECH-associated protein
1; NRF2, nuclear factor (erythroid-derived 2)-like
2; PHD, prolyl hydroxylase domain; and VHL, von
Hippel-Lindau tumor suppressor.
716 Circ Cardiovasc Genet October 2014
coronary artery ligation, exhibited a reduction in infarct size
and improved postischemic recovery.69 Succinate-augmented
HIF-1α levels may contribute to IPC because mice with
reduced HIF-1α cannot be preconditioned.70,71
Fumarate Is Cardioprotective via Activation
of the Nrf2-Antioxidant Pathway
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As with succinate, the cardioprotective influence of fumarate
has been recognized in both immature62 and adult hearts;72
however, it is likely that the contribution of fumarate to the
coupled reductive pathway originally proposed by Hochachka
et al48 would not suffice to account for the cardioprotective
effect completely.72 Furthermore, the cardioprotective influence observed with exogenous nonesterified fumarate in
adult rat hearts was relatively modest,72 raising the possibility
of inadequate intracellular penetration of fumarate from the
perfusate.32 Analogous to its role in normoxic HIF-1α augmentation, we wondered whether CAC metabolites, such as
fumarate, may activate other cytoprotective transcriptional
programmes.
To augment myocardial fumarate levels reliably, we generated a model of cardiac FH deficiency by crossing mice bearing LoxP sequences flanking exons 2 to 3 of the Fh1 gene
with mice carrying Cre recombinase under the ventricular
cardiomyocyte-specific Mlc2v promoter. By interrupting the
conversion of fumarate to malate, the resulting mice represented a genetic model of augmented cardiac fumarate concentrations. Mice with cardiac Fh1 deficiency were viable and
healthy (despite a disrupted CAC) until ≈3 to 4 months.32 The
phosphocreatine to ATP ratio (a readout of energetics) was
unaltered when compared with control hearts consistent with
their viability. Successful Fh1 knockout in the cardiac myocytes was confirmed at both mRNA and protein level. These
hearts exhibited increased fumarate levels when compared
with control (Fh1 fl/fl, no Cre) hearts. The increases were significant and consistent though modest in magnitude (×1.6 fold
of controls in total heart preparations).
When subject to ex vivo IR (40 minutes of global ischemia
followed by 60 minutes of reperfusion), Fh1 knockout hearts
exhibited a marked reduction in mean infarct size from 37% in
control hearts to 17% in Fh1 knockout hearts. Assessment of
markers of myocardial injury corroborated the robust cardioprotection in Fh1 knockout hearts. A time-course of interstitial metabolite profiling detected reduced ischemic release of
succinate, glutamate, and adenosine in Fh1 knockout hearts,
a pattern that has been associated with attenuated cardiac
injury.73,74
In other models of fumarate augmentation, either using
FH-deficient tumors or increased cellular fumarate through
the application of cell permeable fumarate analogues, activation of the Keap1-Nrf2-antioxidant pathway has been
observed (Figure 4B).31,75 Nrf2 is a transcription factor that
coordinates the defense against oxidative stress by activating
antioxidant response element genes.76 In unstimulated cells,
Nrf2 is sequestered in the cytoplasm by Keap1, promoting
Nrf2’s proteosomal destruction.77 On cellular overload with
ROS or electrophiles, reactive cysteine residues on Keap1 are
modified, resulting in release of Nrf2, its translocation to the
nucleus, and heterodimerization with small Maf proteins to
transactivate antioxidant response element genes.77 By acting
as an electrophile via its unsaturated bond, fumarate (but not
succinate which is saturated) binds to the reactive cysteine
residues of Keap1, forming an irreversible chemical modification of S-(2-succinyl)-cysteine (2SC), termed succination.78
Proteomic assessment of FH-deficient murine embryonic
fibroblasts found a variety of proteins altered by this novel
post-translational modification, many of which had metabolic
functions and were mitochondrial in location.79 Although the
functionality of succination remains generally undefined, the
effect of succinated cysteine residues binding an obligatory
[Fe4S4]2+ cluster in the CAC enzyme aconitase 2 has been
shown to be a dose-dependent inhibition of aconitase activity.79 The iron–sulfur cluster of aconitase 2 is also highly sensitive to oxidation by superoxide, acting as a mitochondrial
sensor of oxidative stress.80
Systematic expression profiling of our FH-deficient hearts,
consistent with the cancer literature,31,75 supported the observation that fumarate augmentation upregulated several canonical Nrf2 target and coregulated genes, including glutathione
S-transferases, methylenetetrahydrofolate dehydrogenase,
NAD(P)H:quinone oxidoreductase 1 and, perhaps importantly, heme oxygenase 1 (Hmox1). Examination of succination in Fh1 knockout hearts with an anti-2SC antibody
revealed substantially increased succination. This supports
the hypothesis that succination of Keap1 may be the likely
mechanism for the observed reductions in Keap1 protein and
the enhanced stabilization/translocation of Nrf2.
Substantial evidence supports the hypothesis that Hmox1
activation is robustly cardioprotective. This inducible cytoprotective enzyme catabolizes the pro-oxidant heme to free
iron, carbon monoxide (CO) and biliverdin, with the latter then converted to bilirubin.81 Bilirubin itself is a potent
antioxidant, whereas carbon monoxide is a vasodilator with
additional anti-inflammatory and antiapoptotic properties.81,82
Both CO and bilirubin protect against cardiac IR injury; the
former via activation of p38 MAPK and Akt kinases,2,83 the
latter likely via an antioxidant mechanism.84 Although mice
heterozygous for Hmox1 were found to be susceptible to IR
injury,85 cardiac-restricted overexpression of Hmox1 has been
shown to attenuate infarct size both ex vivo and in vivo, with
reduced oxidative stress, inflammatory cell infiltration,81 left
ventricular dysfunction, and apoptosis.86
Upregulation of Hmox1, coupled with heme resulting from
the cataplerosis of succinyl-CoA predicted by our own computational modeling32 and that of others,87 raised the possibility of
Hmox1 contributing to the cardioprotection. To address this,
we injected control and Fh1 knockout animals with vehicle or
zinc deuteroporpyrin 2-4-bis ethylenglycol (ZnBG), a specific
heme oxygenase inhibitor88,89 before ex vivo IR. Pretreatment
with ZnBG had no effect on infarct size in control hearts, but
negated the cardioprotection observed in Fh1 knockout hearts.
To assess whether our findings in Fh1 knockout mice were
translatable, we used dimethyl fumarate (DMF), an orally
available and cell-permeable version of fumarate67 already
in clinical use to treat patients with psoriasis and multiple
sclerosis.76 Treatment of atrial cardiomyocyte-derived HL-1
cells with DMF induced nuclear translocation of Nrf2. When
Czibik et al CAC in Cardioprotection 717
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administered orally to wild-type mice, dimethylfumarate
recapitulated much of the Nrf2 target profiling observed in
untreated Fh1 knockout hearts. When subject to IR, hearts
from DMF-pretreated wild-type mice displayed a ≈75%
reduction in infarct size to 9.3% when compared with 36.9%
in vehicle pretreated wild-type hearts, accompanied by an
improvement in postischemic coronary flow. To determine
whether DMF's infarct-sparing effect translated to an in vivo
model, wild-type mice pretreated with oral DMF or vehicle
underwent left anterior descending coronary artery ligation
with subsequent evaluation of infarct size. A significant reduction in infarct size was observed in the DMF-treated group.
This protective effect of DMF was absent in Nrf2 knockout
mice, indicating the critical role played by Nrf2 in mediating
DMF-conferred cardioprotection.32
These findings do not preclude the possibility that the cardioprotective potential of fumarate is also dependent on other
pathways. Motivated by findings in the cancer literature, we
examined whether increased fumarate was sufficient to activate the HIF hypoxia pathway but found no evidence to support
this.32 There are several possibilities for the apparent discrepancy: (1) Fumarate levels of Fh1 knockout murine embryonic fibroblasts are 100-fold higher than in wild-type murine
embryonic fibroblasts,79 suggesting that an estimated 2.2-fold
increase in fumarate observed in Fh1 knockout cardiac myocytes (1.6-fold in whole hearts) might be insufficient to outcompete 2-OG; (2) prolyl hydroxylase domain 2, the major
HIF-1α hydroxylase,90 has a much higher Kd for fumarate than
for 2-OG; accordingly, fumarate levels have to exceed 2-OG
greatly for binding to prolyl hydroxylase domain 266; (3) as an
alternative mechanism to substrate competition, Sullivan et al91
proposed that elevated levels of fumarate resulted in increased
succination and inactivation of the antioxidant glutathione
to form GSF, which—by acting as an inhibitory substrate to
glutathione reductase—enhances mitochondrial ROS levels,
serving to stabilize HIF-1α. In Fh1 knockout cardiomyocytes,
the modestly elevated fumarate is unlikely to have altered
superoxide levels directly. Notwithstanding these considerations, endogenous antioxidant protection via activation of the
Nrf2-dependent arsenal of cytoprotective genes is desirable
in the setting of reperfusion injury, where oxidative stress is
recognized as a major pathogenic contributor. The preclinical
evidence for cardioprotection using the orally available, cellpermeable, and clinically safe DMF provides a tangible agent
for clinical testing in the setting of acute coronary syndromes
or predictable myocardial injury (eg, cardiac surgery).5,32
Conclusions and Future Directions
As outlined above, there is substantial evidence, accumulating over almost a century, that CAC intermediates change dramatically during the course of IR injury. The recognition that
these changes, most prominently succinate production, may
be beneficial has been substantially enhanced by the recognition that succinate and related metabolites have additional
and perhaps more potent effects than ATP generation. These
include cell surface receptor activation (eg, Gpr91), HIF-1α
activation, and Nrf2 liberation from Keap1. However, relatively simple strategies to exploit the metabolic benefits of
CAC intermediate (eg, modification through the provision
of feedstock amino acids) have proved clinically challenging
with variable efficacy. Alternative metabolic strategies using
similar CAC-related products that will augment Hmox1 activity (either through increasing its substrate haem or increasing
enzymatic activity) may be germane and are the subject of
investigation.
Sources of Funding
This work was supported by the British Heart Foundation. A.Yavari
is supported by the UK Department of Health's National Institute for
Health Research.
Disclosures
H. Ashrafian has received an unrestricted research grant from SBIALA, Japan. The other authors report no conflicts.
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Key Words: cardioprotection ◼ citric acid cycle ◼ fumaric acid ◼ oxidationreduction ◼ nuclear factor (erythroid-derived 2)-like 2 ◼ reperfusion injury
Citric Acid Cycle Intermediates in Cardioprotection
Gabor Czibik, Violetta Steeples, Arash Yavari and Houman Ashrafian
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Circ Cardiovasc Genet. 2014;7:711-719
doi: 10.1161/CIRCGENETICS.114.000220
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