ANTIOXIDANTS & REDOX SIGNALING
Volume 18, Number 16, 2013
ª Mary Ann Liebert, Inc.
DOI: 10.1089/ars.2012.4729
COMPREHENSIVE INVITED REVIEW
Mitochondria as a Source of Reactive Oxygen and Nitrogen
Species: From Molecular Mechanisms to Human Health
Tiago R. Figueira,1 Mario H. Barros,2,* Anamaria A. Camargo,3,4,* Roger F. Castilho,1,* Julio C.B. Ferreira,5,*
Alicia J. Kowaltowski,6,* Francis E. Sluse,7,* Nadja C. Souza-Pinto,6,* and Anibal E. Vercesi1
Abstract
Mitochondrially generated reactive oxygen species are involved in a myriad of signaling and damaging pathways in different tissues. In addition, mitochondria are an important target of reactive oxygen and nitrogen
species. Here, we discuss basic mechanisms of mitochondrial oxidant generation and removal and the main
factors affecting mitochondrial redox balance. We also discuss the interaction between mitochondrial reactive
oxygen and nitrogen species, and the involvement of these oxidants in mitochondrial diseases, cancer, neurological, and cardiovascular disorders. Antioxid. Redox Signal. 18, 2029–2074.
I. Introduction
A. The respiratory chain: energy and free radicals
B. Superoxide production by the respiratory chain
C. Fate of mitochondrial O2c II. UCP Activity and Regulation
A. Redox regulation of UCPs
B. UCPs: purine nucleotide inhibition and UQ redox state
C. Connecting UQ redox state, O2c - production, UCP activity, and energy conservation
III. Ca2 + Signaling, Mitochondrial ROS Generation, and the Permeability Transition
IV. Mitochondria and Nitric Oxide
A. Mitochondrial generation of NOc
B. NOc effects on mitochondrial electron transport and energy transfer
C. MPT and NOc-mediated cytoprotection
D. NOc-mediated mitochondrial biogenesis
V. Mitochondrial Disorders
A. Nuclear genes in mitochondrial disorders
B. mtDNA alterations in mitochondrial disorders
VI. Redox Imbalance and Cancer
VII. Neuronal Damage and Disorders Associated with Mitochondrially-Generated ROS
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Reviewing Editors: Fernando Antunes, Esther Barreiro, Juan Bolaños, David Booth, Enrique Cadenas, Amadou Camara, Boris Chernyak,
Maria Ciriolo, Melinda Coughlan, Sergey Dikalov, Anne Hamacher-Brady, and Ulrich Hammerling
1
Department of Clinical Pathology, Faculty of Medical Sciences, State University of Campinas, Campinas, Brazil.
Department of Microbiology, Institute of Biomedical Sciences, University of São Paulo, São Paulo, Brazil.
3
Ludwig Institute for Cancer Research, São Paulo, Brazil.
4
Molecular Oncology Center, Sirio-Libanese Hospital, São Paulo, Brazil.
5
Department of Anatomy, Institute of Biomedical Sciences, University of São Paulo, São Paulo, Brazil.
6
Departamento de Bioquı́mica, Instituto de Quı́mica, Universidade de São Paulo, São Paulo, Brazil.
7
Laboratory of Bioenergetics, Department of Life Sciences, Faculty of Sciences, University of Liege, Liege, Belgium.
*These authors contributed equally.
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FIGUEIRA ET AL.
VIII. Redox Imbalance in Cardiovascular Diseases
A. Redox imbalance during ischemia-reperfusion
B. Redox imbalance in hypertension
C. Redox imbalance in heart failure
IX. Final Remarks
I. Introduction
A. The respiratory chain: energy and free radicals
W
hen the Earth’s atmosphere became pro-oxidant,
aerobic organisms evolved and developed enhanced
complexity. Respiration is the main source of energy for
metabolic and housekeeping purposes in these organisms. In
eukaryotic cells, the respiratory chain is located in mitochondria, intracellular organelles that originate from the endosymbiosis of respiring bacteria with proto-eukaryotes. The
respiratory chain constitutes electron carriers that transport
electrons from reduced cofactors, which are reduced during
the catabolism of energy nutrients, to molecular oxygen. At
the expense of oxygen as a final electron acceptor, the respiratory chain produces water and a proton electro-chemical
gradient across the mitochondrial inner membrane. This
comprises the primary energy transformation step. The dual
gradient across the inner mitochondrial membrane, composed of a pH and electrical potential, provides the energy
required (protonmotive force) for many purposes, including
ATP synthesis, metabolite transport, and ion homeostasis.
Mitochondrial energy metabolism is also recognized as the
main source of cellular reactive oxygen species (ROS) in most
eukaryotic cells (50, 51, 515). However, mitochondria also
have the highest antioxidant capacity, making them a player
not only as a superoxide anion (O2c - ) source but also as a
cellular redox sink (138, 281, 413). The initial concept that mitochondrial ROS were essentially an undesirable metabolic
byproduct generated by the mitochondrial respiratory chain
has changed. Based on a large body of experimental evidence, it
is now recognized that, under physiological conditions, mitochondrial ROS generation is a continuous and tightly adjusted
process required for the regulation of many cellular processes
(138, 222, 506). As an example, H2O2, which is a reasonably
stable and diffusible molecule (78), is a cellular signal that
regulates multiple vital processes within and outside mitochondria such as cell cycle, stress response, energy metabolism,
redox balance, and oncogenic transformation (222). Under
normal conditions, cells maintain their redox balance through
the generation and elimination of ROS using different antioxidant systems, as will be discussed next (222, 341, 381).
Our current view about cellular ROS signaling has been
greatly expanded in the last decade. Hamannaka and Chandel
(222) described how multiple inputs such as hypoxia, PI3K,
TNFa, and oncogenes regulate the generation of mitochondrial
ROS and how these ROS activate multiple outputs, including phosphatases and redox-regulated transcription factors,
such as NF-jB, that, in turn, regulate the expression of proinflammatory genes and kinases. In hypoxia, for example, the
stimulation of mitochondrial H2O2 generation can promote
cellular protection via adaptive transcriptional programs regulated by hypoxia inducible transcription factors (HIFs); this
adaptive program consists of a changed expression of genes
regulating erythropoiesis, glycolysis, angiogenesis, cell cycle,
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and survival, as well as lowered energy turnover (93, 94, 122,
158, 547, 548). A physiological example of redox-related processes is that increased mitochondrial ROS in response to
hypoxia are a signal for pulmonary vasoconstriction, which, in
turn, improves ventilation-perfusion matching (548).
It has also been proposed (222) that different classes of
signaling actions may be regulated by different levels of ROS:
(i) low rates of mitochondrial O2c - generation are required for
cellular processes such as proliferation and differentiation; (ii)
moderate cellular stress induces O2c - generation at levels that
activate adaptive programs, including the transcriptional upregulation of antioxidant genes; (iii) at higher levels, ROS
signal for the initiation of senescence and cell death.
The process of O2c - generation by the respiratory chain
seems to be highly regulated, and ROS can function both
adversely and beneficially. However, there is only little evidence regarding differential signaling events behind the
generation of either beneficial or detrimental levels of mitochondrial ROS. In addition to the physiological processes
controlled by mitochondrial ROS, a large body of evidence
indicates that mitochondrial oxidative imbalance is responsible for the development and progression of a series of abnormalities such as cancer, diabetes, inflammatory diseases,
hypertension, neurodegenerative and ischemia-related diseases,
as well as aging (138, 222, 341, 381). In this regard, recent evidence indicates the existence of a cross-talk between mitochondria and NADPH oxidases in which mitochondrial ROS activate
O2c - and H2O2 production by NADPH oxidases that, in turn,
stimulate mitochondrial ROS formation, or alter NAPDH oxidase responses to angiotensin (555). Under some circunstances,
this may generate a feed-forward vicious cycle of ROS generation, which may contribute to the development of cardiovascular
diseases (138, 555). Indeed, scavenging mitochondrial O2c - with
mitochondria-targeted antioxidants interrupts this vicious cycle
and down-regulates NADPH oxidase activity (140). An example
of these interactions is the activation of the PKC-dependent
phagocytic NADPH oxidase by increased mitochondrial O2c levels mediated by matrix Ca2 + (139).
In this comprehensive review, we will approach mechanisms of mitochondrial free radicals generation and discuss
their fate. The role of mitochondrial Ca2 + influx and of reactive oxygen and nitrogen species as signaling agents regulating cellular processess in health and disease will also be
covered. Then, we will focus on selected degenerative diseases in which mitochondrial redox homeostasis is compromised and/or involved in their pathogenesis.
B. Superoxide production by the respiratory chain
Superoxide can be generated in at least five sites within the
respiratory chain (52): the ubiquinone (UQ)-binding sites in
complex I and III, the flavin prosthetic group in complex I, the
electron transferring flavoprotein (ETF), UQ oxidoreductase,
and glycerol 3-phosphate dehydrogenase. Three of these sites
MITOCHONDRIA AND REACTIVE NITROGEN AND OXYGEN SPECIES
are relatively well characterized with regard to the mechanism of O2c - generation (UQo in Complex III and UQ and
flavin in Complex I). For the others, controversies in the literature remain. The topology and relative capacity to produce
O2c - of each site is important, and determines whether the
radical is released toward the matrix side or in the intermembrane space; as well as their contribution toward total
mitochondrial O2c - production (78).
Complex I is believed to produce O2c - at two sites: the
flavin mononucleotide (FMN) binding site (323) and the
UQ-binding site (516). When respiring on NADH-linked
substrates feeding complex I (forward electron transport),
mitochondria produce O2c - at a relatively low rate; rotenone,
a UQ-binding site inhibitor, known to block the electron
flow at complex I, fully reduce the upstream redox centers,
and increase O2c - production (230). This suggests that complex I produces O2c - at a site that is proximal to rotenone
block during forward electron flow from NADH. It has been
proposed that fully reduced flavin in the nucleotide-free
binding site of complex I could react with O2 to produce O2c - .
In addition, reduced FMN is considered an important electron
donor to O2 to produce O2c - at complex I (187). These complex I sites of O2c - generation during forward electron
transfer may comprise a mechanism explaining the link between the mitochondrial NAD pool redox state and O2c production. With regard to reverse electron transfer back to
NADH, we emphasize that succinate, glycerol 3-phosphate,
and acyl-CoAs are physiological substrates which can reduce
the UQ pool and generate protonmotive force through complexes III and IV. In isolated mitochondria under such conditions, electrons can be driven uphill from reduced UQ to
complex I, a process known as reverse electron transfer. If
rotenone is added to mitochondria undergoing reverse electron transfer, a lower production of O2c - is observed at respiratory chain steps that are proximal to rotenone block (the
QH2-binding site) (295). Despite the biochemical characterization of these two sites of O2c - production within complex I
(52), there are controversies about the relative contribution of
these two sites to overall O2c - production and even about the
existence of two separate sites. A recent study shows that
O2c - is produced in complex I only by fully reduced flavin
during both forward and reverse electron transfer in submitochondrial particles (i.e., inside-out preparation of mitochondria) (423). Moreover, the last N2 FeS cluster of complex
I, a structure working distal to FMN during forward electron
transfer, has also been proposed as an electron donor to O2
either directly or indirectly via semiquinone (311).
The production of O2c - at the level of complex III is related to its peculiar mechanism of electron transfer: the Qcycle mechanism (119). Electrons delivered to the respiratory
chain entry sites flow along the lipid-soluble UQ. Complex III
catalyzes the transfer of these electrons from reduced ubiquinone (UQH2) to water-soluble cytochrome c on the outer
surface of the inner membrane, as complex III pumps H + out
of the inner mitochondrial membrane. In the ‘‘classical’’ Q
cycle, UQH2 delivers sequentially the first electron at center o
(outer positive side) to the Rieske iron-sulphur protein (then
to cytochrome c1 and c), along with the release of 2 H + outside
the inner membrane and the formation of an unstable semiubiquinone anion (UQc - ). This radical is quickly oxidized to
UQ by cytochrome bL (cytosolic side of the membrane). The
second electron is then delivered to cytochrome bH (matrix
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side); cytochrome bH is then reoxidized by UQ at center i
(inner negative side), forming another UQc - . The cycle is
completed by the oxidation of a second UQH2 providing one
electron to cytochrome c and one electron to UQc - at center i.
Since the electron transfer from cytochrome bL to cytochrome
bH is slowed down by the electrical gradient acrros inner
mitochondrial membrane, the lifetime of UQc - is prolonged
at site o, allowing the reduction of O2 by UQc - to form O2c (256). Thus, according to this classical Q-cycle model, the
main one-electron donor to O2 is located in center o (external
side of the membrane) and could be the UQc - (353). Indeed,
complex III produces O2c - at a rate that depends on the halflife of UQc - . Inhibitors that increase the half-life of UQc - at
center o (e.g., antimycin) or high protonmotive force across inner
mitochondrial membrane result in higher O2c - production
rates. Indeed, inhibitors that block UQH2 (e.g., myxothiazol)
access to center o, thus decreasing electron delivery to complex
III, lower the rate of O2c - production. Opposingly, it has been
shown that UQc - is formed very transiently at center o and
never accumulates to a significant level in the functionnal
complex III (76), as the bifurcated UQH2 oxidation at center o
occurs in a quasiconcerted reaction (119). There are also other
conflicting data regarding the mechanisms of O2c - production
by complex III: (i) It was recently shown that O2c - production at
center o of the membrane-bound or purified complex III was
stimulated by the presence of oxidized UQ, indicating that one
electron is transferred to O2 in a reverse reaction from reduced
cyt bL via UQ acting as redox mediator (148); (ii) a heme b knockout mutant complex III shows little electron transfer activity but
produces O2c - at a higher rate, indicating that O2c - can also be
formed by a route other than the reaction involving the heme bL
(560). Thus, the precise mechanism of O2c - generation by
complex III still remains elusive. Despite complex III seeming to
release O2c - in an equal amount on both sides of the membrane
(363), due to the strong antioxidant capacity of mitochondrial
matrix, the net amount of ROS could be greater in the intermembrane space side than in the matrix side.
Superoxide generation by glycerol 3-phosphate dehydrogenase, an entry site for respiratory chain electrons, occurs
toward both sides of the membrane (147, 355). The flavin site
appears to be in the intermembrane space, while the UQbinding site is in the membrane and is proposed to be the
main site of O2c - production. Its capacity seems to be lower
than the capacities of UQ sites at complexes I and III. The
ETF:UQ oxido-reductase could produce O2c - into the matrix,
but its mechanism is still poorly characterized (486).
In addition to the respiratory chain, three other mitochondrial O2c - production sites have also been uncovered: the
enzymes 2-oxoglutarate (a-ketoglutarate) and pyruvate dehydrogenases inside the matrix and monoaminoxidase in the
outer mitochondrial membrane; this later source will be discussed in the subsection on redox imbalance in heart failure
(section VIII-C). Although the mechanisms are not fully understood, O2c - production by 2-oxoglutarate and pyruvate
dehydrogenases is enhanced when the physiological electron
acceptor NAD + is unavailable (11, 489, 500, 509). For example, lower NAD + availability (i.e., high NADH:NAD + ratio)
is observed in the liver after alcohol consumption due to the
metabolism of alcohol and acetaldehyde that reduces NAD +
(562). On the other hand, caloric restriction promotes increased NAD + availability and lowers the generation of H2O2
through 2-oxoglutarate dehydrogenase in yeast (500).
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Although it is currently known that mitochondria can
produce O2c - at several sites, we do not precisely know at
which relative rates it occurs under the supply of various
substrates and different energy demands, even in the simplest
system that is, isolated mitochondria (501). It is a methodological challenge to measure mitochondrial O2c - in different
types of mitochondria, and even more so in vivo. Methods
available include the determination of specific hydroethidine
derivatives by HPLC (140, 568). However, it should be noted
that the accumulation of hydroethidine and hydroethidine
conjugated to a triphenylphosphonium (Mito-HE or MitoSOX) in mitochondria is affected by mitochondrial membrane
potentials (67, 78), which are in themselves strong regulators
of O2c - production. A circularly permuted yellow fluorescent
protein used as a mitochondrial O2c - biosensor (545) has been
recently criticized as being sensitive to pH (464). As a result,
many studies that aim at determining rates of mitochondrial
O2c - production do so by indirect measurements such as the
release of O2c - derived H2O2 from isolated mitochondria (78).
Overall, it seems that a large amount of mitochondrial O2c - is
produced when NADH/NAD + is high (e.g., low ATP demand or lack of O2) and/or when protonmotive force is high
(low ATP demand) (365).
A puzzling observation is the increase of mitochondrial ROS
production during hypoxia in cultured cells (213) and in Langendorff perfused hearts during global ischemia, especially in
its late phase, where O2 tension is expected to be the lowest
(264, 434). Conversely, isolated mitochondrial ROS production
is quite independent of O2 concentrations varying between 5
and 250 lM, bearing in mind that O2 concentrations at 5 lM
already limit mitochondrial respiration by *40% under the
tested conditions (233). This suggests an indirect effect of
hypoxia on ROS production, requiring additional factors in
hypoxic cells or tissues. Indeed, such hypoxic bursts of ROS
production seem to be involved in hypoxic adaptive signaling
mediated by the transcription factor HIF1A (233, 421, 544).
An often-repeated quote is that O2c - formation accounts
for 1%–2% of mitochondrial oxygen consumption (11). This
information derives from an extrapolation of early measurements performed in isolated mitochondria under nonphysiological conditions (as clearly stated by the authors):
saturating substrate and O2 concentrations in the presence of
antimycin [a complex III inhibitor that greatly stimulates
mitochondrial O2c - formation (92)] and is, therefore, not
physiologically correct. Knowing how much O2c - is produced by mitochondria in vivo is necessary in order to evaluate its significance in oxidative damage and redox signaling.
However, extrapolations of ROS production by isolated mitochondria to in vivo conditions are questionable because of
several factors, including substrate nature and concentration,
local O2 concentrations, and respiratory states, which may
affect mitochondrial O2c - formation and fate. Thus, even
more realistic extrapolations (0.15% of the oxygen consumption) are not reliable (365). Furthermore, intracellular measurements of mitochondrial ROS production are prone to
artifacts and not quantitative (78).
C. Fate of mitochondrial O2c Irrespective of the rate of O2c - production in vivo, its formation implies the existence of metabolizing pathways. Superoxide, the primary ROS produced by mitochondria, gives
FIGUEIRA ET AL.
rise to many ROS and reactive nitrogen species (RNS) through
many distinct reactions (256, 281). Superoxide is converted to
H2O2 by the metal-dependant enzyme superoxide dismutase
(Mn-SOD in the matrix and Cu/Zn-SOD in the intermembrane space and in the cytosol, rescepctivelly). However, a
part of O2c - is under the form of its conjugated acid HO2c, a
very reactive species, and some O2c - can react with nitric
oxide (NOc) to form peroxynitrite (ONOO - ). H2O2 is poorly
reactive, can permeate membranes, and is removed by catalase in the cytosol and by glutathione peroxidase (GPx) and
peroxiredoxins (Prx), at the expense of glutathione (GSH) and
thioredoxin-2 (Trx) in the mitochondrial matrix. The oxidized
form of glutathione and Trx are then reduced back by glutathione reductase and thioredoxin reductase (TrxR), respectively, at the expense of NADPH as reducing power. NADPH
is, therefore, of central importance in the removal of mitochondrial H2O2. NADPH is, in turn, regenerated by the protonmotive force-dependent NAD(P)H transhydrogenase
(NNT) and by isocitrate dehydrogenase (IDH2). Given these
properties of mitochondrial ROS metabolism, conditions that
affect protonmotive force or reducing power supply can alter
both O2c - production and H2O2 elimination.
The most reactive oxygen or nitrogen species can damage
proteins (71), DNA (396), and lipids (486). Lipid peroxidation
itself is a source of new radicals, as it forms carbon-centered
radicals in the unsaturated fatty acid chains of phospholipids.
Reactions of carbon-centered radicals with O2 generate peroxyl radicals (ROOc), which react with the side chain of
polyunsaturated fatty acids, yielding phospholipid hydroperoxides (PL-ROOH) and new carbon-centered radicals. PLROOH are cleaved by phospholipase A2 to free fatty acid
hydroperoxides (FAOOH). In the presence of Fe2 + , alkoxyradicals (ROc) are formed and react with polyunsaturated fatty
acids in their vicinity; if not, they generate unsaturated aldehydes. Interestingly, 4-hydroxy-2-nonenal (4-HNE) is formed
by spontaneous cleavage of PL-ROOH (164).
To date, there is little evidence for free-radical chain reactions occurring under physiological conditions, especially in
mitochondria, as an abundance of scavengers abrogates
propagation reactions. However, under pathological conditions, the disruption of intracellular redox signaling facilitates the auto-oxidative deterioration of polyunsaturated
fatty acids, allowing the formation of secondary end products
of lipid peroxidation (440). With the advent of mass spectometry-based lipidomics, new perspectives in the understanding of lipid peroxidation processes in pathophysiology
has emerged.
A characterization of the lipid composition of rat liver
subcellular membranes found that the inner mitochondrial
membrane has a high content of unsaturated fatty acid acyl
chains (440). This unsaturated feature of the mitochondrial
inner membrane renders it susceptible to attacks by free
radicals under pathological conditions, resulting in 4-HNE
generation. 4-HNE is a highly reactive product of peroxidation of arachadonic, linoleic, and linolenic acid acyl chains
(164). Considered a strong electrophile and the most cytotoxic
aldehyde produced by lipid peroxidation, 4-HNE has the
ability to irreversibly modify cellular targets such as proteins,
DNA, and phospholipids (440).
Another process that might contribute to mitochondrial 4HNE generation is the oxidation of mitochondrial cardiolipin.
Mitochondria are rich in cardiolipin, a phospholid located in
MITOCHONDRIA AND REACTIVE NITROGEN AND OXYGEN SPECIES
the inner mitochondrial membrane that contains three glycerol backbones and four acyl chains. Due to its interaction
with electron transport chain complexes and high polyunsaturated fatty acid content, cardiolipin is considered a likely
target for oxidants generated in the mitochondrion. In fact,
stimulating lipid peroxidation in the rat heart and brain reduces both cardiolipin content and cytochrome c oxidase activity (406, 469). In addition, cardiolipin has been shown to be
oxidized by cytochrome c in the presence of H2O2, resulting in
the release of cytochrome c and the initiation of mitochondriamediated apoptosis (260).
RNS can also contribute to oxidative imbalance and/or
damage proteins. NOc can reversibly inhibit cytochrome
c oxidase, as discussed later in section IV-B of this review. This
inhibition may increase the reduced state of electron carriers
in the respiratory chain and, consequently, O2c - production.
Nitrogen dioxide radical (NO2c), a product of NOcoxidation,
can oxidize or nitrate a wide range of biomolecules. Peroxynitrite can oxidize thiol groups, DNA bases, and tyrosine
residues. In mitochondria, excessive ONOO - levels can impair oxidative phosphorylation by inhibiting Complex I,
Complex IV and ATP synthase, and MnSOD activity and
calcium homeostasis (60).
To deal with this cascade of potentially damaging ROS and
RNS, cells employ two strategies: pathways able to metabolize or scavenge these harmful species, and/or systems that
regulate the generation of their common source, O2c - .
Clearly, a tight control of O2c - generation is of paramount
importance in order to maintain the ROS production within a
range that is compatible with redox signaling and homeostasis. High protomotive force is mechanistically linked to a
high level of O2c - production; thus, every process that decreases protonmotive force through its dissipation is likely to
reduce O2c - generation. For instance, less ROS are produced
during phosphorylating respiration (state 3) compared with
resting respiration (state 4), paralleling the changes in protonmotive force. Since a nonlinear relationship (Fig. 1) links ROS
production and the protonmotive force (a small increase or
decrease of protonmotive force induces a large increase or decrease in ROS) (276, 488), a large prevention in ROS production
is achievable without a dramatic decrease in the efficiency of
oxidative phosphorylation (i.e., oxidation-phosphorylation
coupling). This ‘‘mild uncoupling’’ (uncoupling is a process
that decreases the efficiency of ADP phosphorylation through
partial dissipation of protonmotive force) can be achieved by
several processes. A ‘‘futile’’ H + cycle across inner mitochondrial membrane associated with cation homeostasis [e.g., K +
channel plus K + /H + exchanger (165)] and the H + re-entry
mediated by specialized uncoupling proteins (UCPs) can, theoretically, promote this mild uncoupling.
II. UCP Activity and Regulation
UCPs are members of the Mitochondrial Anion Carrier
Family, which transports a large variety of anion metabolites
across the inner mitochondrial membrane (154). There are
around 40 mitochondrial anion carriers, including UCPs, that
are widespread among eukaryotes (479). Despite incompletely understood biochemical mechasnims or relevance for
UCP1 analogues, the general activation of UCPs results in the
re-entry of H + from the intermembrane space back to the
mitochondrial matrix. In doing so, these proteins uncouple
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FIG. 1. Exponential relationship between mitochondrial
membrane potential and H2O2 release rate. Mild uncoupling or ADP-stimulated respiration lowers membrane
potential and diminishes mitochondrial ROS production.
Dashed vertical lines denote mitochondrial membrane potential under phosphorylating (state 3) and resting conditions (state 4); the dotted vertical line represents the decrease
in membrane potential as induced by mild uncoupling via
UCPs or as a result of futile cycle of ions across the inner
mitochondrial membrane (165, 334). This illustrative curve is
based on data from isolated brain mitochondria respiring on
NAD-linked substrates (488). This nonlinear relationship was
first described by Korshunov et al. (276). ROS, reactive oxygen species; UCPs, uncoupling proteins.
mitochondrial oxidative metabolism from ADP phosphorylation by mitochondrial ATP synthase, thus lowering the
ADP:O ratio (i.e., phosphorylation efficiency) (191). Although
free fatty acid anions (FFAs) are considered UCP activators
of/ and purine nucleotides are recognized as inhibitors of
UCPs (480), the exact mechanisms by which these proteins
mediate the control of the H + leak across the inner mitochondrial membrane are still controversial (480). Since the
inhibition by purine nucleotide is considered diagnostic of
UCP activity, UCP1 analogues described after the discovery
of the plant UCPs in 1995 (530) were initially considered a
distinct class of mitochondrial carriers due to the fact that they
lacked purine nucleotides sensitivity (529). However, studies
measuring the ADP:O ratio in phosphorylating mitochondria
(254) revealed FFA-induced uncoupling that was sensitive to
purine nucleotides (i.e., a putative UCP activity) in mitochondria expressing UCP1 analogues (252–254, 373, 479). Interestingly, all UCPs in mitochondria from protists, fungi,
plants, and mammals show this susceptibility to nucleotide
inhibition of FFA-induced uncoupling during phosphorylating respiration (252–254, 373, 479).
A. Redox regulation of UCPs
ROS can either activate or signal for increases in UCP
expression. The direct activation of UCPs by ROS or lipid peroxidation products such as 4-HNE and free polyunsaturated
FAOOH may comprise an immediate response to oxidative
imbalance (151, 152, 248, 432). This ‘‘short-term or fast response’’
downregulates O2c - production without significantly decreasing oxidative phosphorylation efficiency (Figs. 1 and 2). The
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FIGUEIRA ET AL.
FIG. 2. Integrating model connecting
O2c2 production to UCP activity.
Numbers along the arrows indicate four
successive lines of defense that are interrelated: Two lines deal with fast
regulations against acute ROS production, and two lines concern long-term
regulation against chronic ROS production. The first line of defense is the
release of purine nucleotide inhibition
of UCP activity when UQ reduction
levels are high (i.e., high protonmotive
force, low ATP demand, and high reducing power), and the second line of
defense is the ROS-induced deglutathionylation that promotes UCP
activation. The third line is the ROSinduced up-regulation of the expression
of UCP. The fourth line is the upregulation of the expression of enzymes
implicated in ROS elimination. The rationale for this integrative model was
developed throughout section II of this
review. FFA, free fatty acid anion;
4-HNE, 4-hydroxy-2-nonenal; O2c - ,
superoxide anion; UQ, ubiquinone.
second type of activation occurs through UCP protein expression through ROS signaling (14, 375). This adaptation increases
the uncoupling capacity of UCPs and represents a long-term
response (Fig. 2) aimed at preventing increased O2c - production in chronic pathological conditions. The concept that UCPs
are activated by ROS is in line with the role of UCPs in the
regulation of O2c - production, but the mechanisms of UCP
activation by oxidants remain under debate.
GSH is an important redox sensor (377) that may regulate
the activity of UCP2 and UCP3 through reversible Sglutathionylation of thiol residues of these UCPs (334, 335).
Glutathionylation, a process that is favored by high matrix
GSH concentrations and basic pH (242), decreases UCP2/3mediated H + leak, thus increasing protonmotive force and
O2c - formation (334). Oxidative imbalance decreases GSH
availability and promotes deglutathionylation, which activates
UCP2/3-mediated H + leak (334, 335). Of note, UCP1 seems not
to be succeptible to this type of regulation (335). These findings
suggest that oxidative imbalance, H2O2 detoxifying systems,
and UCP activation are mechanistically connected.
Evidence has also been provided that UCP1 analogues
operate as anion carriers promoting the electrophoretic extrusion of fatty-acid hydroperoxide anions from the matrix,
thus protecting the organelle from these harmiful molecules
(205). This concept has been strongly supported by experiments with isolated skeletal muscle mitochondria from UCP3null mice and their wild-type littermates (325) and suggests
that UCP3 is not only involved in mitochondrial uncoupling
but also involved in the protection of mitochondria against
lipid hydroperoxides.
B. UCPs: purine nucleotide inhibition
and UQ redox state
The affinity of UCPs reconstituted into proteoliposomes to
purine nucleotides is in the micromolar range (48, 152, 249,
255, 270, 561), suggesting that UCPs are either fully inhibited
in vivo (milimolar range of purine nucleotides) or that this
inhibition is subjected to some kind of modulation. In this
regard, it has been demonstrated in phosphorylating mitochondria that the inhibition of UCP3 by GTP decreases progressively as the reduced state UQ within respiratory chain
increases (254). This was achieved by modulating the UQ
redox state with malonate during phosphorylating respiration (state 3), while keeping the protonmotive force unchanged (254). According to these data, UCP3 would be
maximally inhibited by GTP during high metabolic demand
(i.e., state 3 respiration), when UQ is in a more oxidized state.
On the other hand, resting-state mitochondria (i.e., highly
reduced UQ) do not display GTP-sensitive FFA-induced uncoupling. Based on these results, muscle UCP3 inhibition by
GTP was proposed to be dependent on UQ redox state, a
metabolic sensor that modulates the inhibition by puridine
nucleotides of FFA-induced UCP activity (480). This hypothesis has been supported by independent studies with many
different UCPs (145, 478, 496). In vivo GTP concentrations
would, therefore, not allow UCPs to operate when energy
demand is high.
Overall, purine nucleotides, UQ redox state, and oxidative
imbalance are modulators of UCPs activity (150), but many
questions remain open with regard to their molecular mechanisms (334).
C. Connecting UQ redox state, O2c - production,
UCP activity, and energy conservation
The data described earlier provides evidence that the main
physiological roles of UCP1 analogues would be the regulation of protonmotive force in order to maintain an optimal
compromise between oxidative phosphorylation efficiency
and O2c - production (480). Indeed, simple in vitro experiments have clearly demonstrated that activation of UCP3 by
FFA is protective against O2c - overproduction during anoxia/
reoxygenation cycles (374). Anoxia leads to a high state of
MITOCHONDRIA AND REACTIVE NITROGEN AND OXYGEN SPECIES
FIG. 3. Prevention of ROS production and damage to
oxidative phosphorylation by UCP3 activation during anoxia/reoxygenation (Anox/Reox) in vitro. (A) In the absence
of FFA during Anox/Reox, superoxide is produced in high
amounts, and, after reoxygenation, a decrease in the yield of
oxidative phosphorylation is observed. Such an alteration is
sensitive to MPT inhibitor cyclosporin A. (B) The presence of
FFA during the event of Anox/Reox atenuates superoxide
production and protects oxidative phosphorylation yield after
reoxygenation. MPT, mitochondrial permeability transition.
reduction of UQ, and reoxygenation leads to its fast oxidation
accompanied by a burst in O2c - production. UCP3 activation
by FFA atenuated O2c - overproduction in mitochondrial
anoxia/reoxygenation, which was accompanied by a lower
degree of mitochondrial permeability transition (MPT) pore
opening and preservation of phosphorylation efficiency. MPT
is a redox-sensitive process that can trigger cell death (many
aspects of MPT will be approached in the section below). Of
note, adenine nucleotide translocator and ATP-sensitive K +
channel also show similar protective effects against anoxia/
reoxygenation (80, 165). Figure 3 sumarizes these findings
linking UCP activity, O2c - generation, and cytoprotection
during the process of anoxia/reoxygenation. The role of UCPs
in cell protection is a concept that has progressively gained
acceptance (55).
With regard to different mechanisms by which UCPs may
be involved in ROS homeostasis and cytoprotection, Figure 2
shows a model integrating the roles of ROS and UCP activity
as a defense mechanism. Four successive lines of defense are
described and connected: Two lines deal with fast regulations
against acute ROS production, and two lines are concerned
with long-term regulation against chronic ROS production.
The first line of defense is the release of purine nucleotide
inhibition of UCP activity when UQ reduction levels are high
(i.e., high protonmotive force, low ATP demand, and high
reducing power), and the second line of defense is the ROSinduced deglutathionylation that promotes UCP activation.
The third line is ROS-induced up-regulation of the expression
of UCP proteins, and the fourth line is the up-regulation of the
expression of enzymes implicated in ROS elimination.
III. Ca21 Signaling, Mitochondrial ROS Generation,
and the Permeability Transition
There is compelling evidence that mitochondrial Ca2 + influx signals for the control of both oxidative phosphorylation
(200) and ROS generation (281). In addition, dysregulation in
cell Ca2 + homeostasis leading to sustained Ca2 + elevation in
the mitochondrial microenvironment is followed by excessive
mitochondrial Ca2 + accumulation that may lead to cell death
via dysregulation in ATP and redox homeostasis (202, 231,
523, 559).
2035
It is well established that Ca2 + is a signaling agent in biological systems due to (i) its binding properties to complex
molecules, (ii) differences in its free concentrations in the
extra-cellular environment and cytosol as well as between the
cytosol and intracellular organelles, and (iii) the existence of a
complex membrane Ca2 + transport system that orchestrates
Ca2 + flux across plasma and intracellular membranes in response to cellular and extra- or sub-cellular signals (41). The
development by Pozzan and Rizzutto (439) of highly sensitive, genetically encoded, Ca2 + probes specifically targeted to
different cellular domains allowed for the demonstration that
transient increases in intracellular free Ca2 + concentrations
promote a variety of specific responses at different sites. These
Ca2 + movements are driven directly or indirectly by ATP
hydrolysis, rendering its signaling functions highly dependent on the energy state of the cell (200). Therefore, deficiencies in mechanisms responsible for cellular ATP supply
lead to deregulation in Ca2 + signaling that may compromise
cell function and survival (231). Experimentally, it is difficult
to differentiate between acute ATP depletion and increased
ROS generation as the primary cause of cell death, because
reduced ATP and increased ROS levels occur concomitantly
with increased cytosolic Ca2 + concentrations and amplify
each other (58, 202). In this regard, distinguishing between the
relative contributions of these events in diseases such as
stroke or cardiac ischemia, for example, will allow for improved strategies for their prevention and/or treatment.
Here, we focus on how cellular, and in particular mitochondrial, Ca2 + homeostasis is related to the integrity of this
organelle. Mitochondrial Ca2 + overload is known to affect
mitochondrial redox state and promote membrane protein
thiol cross-linking, causing mitochondrial permeability transition (MPT) (83, 208, 279, 333). MPT is a condition which is
characterized by the opening of a high conductance, nonspecific proteinaceous pore (241) that leads to mitochondrial
dysfunction (280, 282) and cell death by either apoptosis or
necrosis (39, 268, 309, 341). Activation of MPT, a process first
described by Hunter et al. (241), is considered a major cause of
cell death under a variety of pathophysiological conditions,
including ischemia/reperfusion, neurodegenerative disease,
traumatic brain injury, muscular dystrophy, and drug toxicity
(39, 219, 286, 322, 349, 381, 445, 487, 523). The importance of
the MPT process in mammalian physiology is beginning to be
properly uncovered (157). Inhibition of MPT pore opening
due to cyclophilin D (CypD) ablation impairs mice heart mitochondrial Ca2 + exchange and results in abnormal heart
adaptation to overload stress (157).
Ca2 + signaling for mitochondrial ROS generation occurs
inside the organelle; so the understanding of the mechanisms
of mitochondrial Ca2 + transport is of central importance.
Three different mechanisms have been described for the influx
of Ca2 + into the matrix and two for the efflux of this cation
from mitochondria (210). A uniporter located in the inner
membrane mediates an electrophoretic transport of Ca2 +
down the electrochemical gradient across inner mitochondrial
membrane without coupling Ca2 + transport to the transport
of another ion (Fig. 4). Although this mechanism was discovered in the 1960s (128, 524), the molecular nature of the
channel was only recently identified (34, 125), as a result of the
progress in genome sequencing and knowledge of uniporter
distribution in different eukaryotes, that is occurrence in
vertebrates (210) and kinetoplastids (144) but absence in the
2036
FIGUEIRA ET AL.
FIG. 4. Schematic representation of the mitochondrial-endoplasmatic/sarcoplasmatic reticulum (ER/SR) Ca21 transporting systems and matrix Ca21 signaling actions: On agonist signals, the ER/SR generates sufficient [Ca2 + ] ( > 1 lM) in the
mitochondrial microdomains to permit fast Ca2 + accumulation by mitochondria. At low concentrations, the cation stimulates
ATP production through the activation of matrix dehydrogenases, while mitochondrial Ca2 + overload promotes MPT
through both stimulation of ROS generation and binding to pore membrane sites. Under mitochondrial oxidative imbalance
conditions, p53 accumulates in the mitochondrial matrix and triggers MPT by physical interaction with the pore regulator
cyclophilin D (CypD). SERCA, sarcoplasmic ER Ca2 + -ATPase; VDAC, voltage-dependent anion channel; IP3R, inositol 1,4,5trisphosphate receptor; RyR, ryanodine receptor; MCU, mitochondrial Ca2 + uniporter; MICU1, mitochondrial Ca2 + uniporter regulator; NCLX, Ca2 + /Na + antiporter; LETM1, Ca2 + /H + antiporter; OGDH, oxoglutarate dehydrogenase; IDH,
isocitrate dehydrogenase; PDH, pyruvate dehydrogenase. This figure was partially adapted from Hajnoczky and Csordas
(216) and based on concepts presented elsewhere (121, 208, 216, 280). (To see this illustration in color, the reader is referred to
the web version of this article at www.liebertpub.com/ars.)
yeast S. cerevisiae (210). Using RNAi techniques, the authors
linked the proteins MCU (mitochondrial calcium uniporter)
and MICU1 (mitochondrial Ca2 + uptake 1) to Ca2 + transport.
MCU is a pore-forming subunit containing two transmembrane
helices that are separated by a highly conserved linker facing
the intermembrane space. MICU1, an EF-hand-containing
protein, constitutes a peripheral regulatory partner of MCU. In
addition, recent data reveal a previously unknown role of
MICU1 as a gatekeeper to limit MCU-mediated Ca2 + uptake,
thus preventing mitochondrial Ca2 + overload and associated
stimulation of O2c - generation under resting conditions (339).
Taken together, MCU and MICU1 explain all biochemical and
physiological properties of the putative Ca2 + uniporter (34).
In addition to the uniporter, mitochondria possess two
other systems that mediate Ca2 + influx: a mode of uptake
called the rapid mode or RaM (484) and a Ca2 + uptake
mechanism mediated by a ryanodine receptor, identified in
excitable cells (43).
The ability of isolated mitochondria to take up Ca2 + via the
uniporter and to release it through the recently identified
Ca2 + /Na + exchanger (403) and the putative Ca2 + /H + antiporter was functionally characterized decades earlier (77).
Nevertheless, the low affinity of the Ca2 + uniporter (an apparent Km of 20–30 lM) led to the general idea that mitochondria would not participate in cell Ca2 + homeostasis
under physiological conditions (77). However, the use of intracellular Ca2 + probes (435) demonstrated that endoplasmic
reticulum (ER) calcium release generates sufficient levels of
Ca2 + concentrations in the mitochondrial microdomains to
permit Ca2 + accumulation by mitochondria under in situ
conditions (174, 189, 435–438, 497). Such microdomais were
directly demonstrated in selected regions of contact between
the ER and mitochondria (121, 196).
It is now evident that under physiological conditions, the
uptake of Ca2 + by mitochondria transfers the signal brought
by cytosolic Ca2 + transients to the matrix (200). For example,
tricarboxilic acid cycle activation by Ca2 + increases the rate of
oxidative phosphorylation by using a transient increase in
intracellular and intramitochondrial Ca2 + concentrations
(200). This provides reducing equivalents to the respiratory
chain under increased demand of ATP production. On the
other hand, the close physical contact between mitochondria
and plasma membrane Ca2 + channels (407, 532) and the ER
(217, 218, 438) allows rapid import of large amounts of Ca2 +
MITOCHONDRIA AND REACTIVE NITROGEN AND OXYGEN SPECIES
from these microdomains that may promote MPT (see Fig. 4)
and cell death (286). Therefore, intramitochondrial Ca2 + can
regulate both the rate of ATP generation, required for cell
function and survival, and MPT, which can lead to cell death.
The MPT pore opening is triggered by a synergic combination of high levels of Ca2 + in the mitochondrial matrix and
oxidative imbalance (280) in isolated mitochondria, intact
cells, or in vivo (523). Cyclosporin A potently prevents MPT
pore opening by binding to CypD and displacing it from the
putative complex of proteins assembling the MPT pore [see
Kroemer et al. (286)].
The relevance of MPT was initially questioned due to the
nonphysiological experimental conditions required to trigger
the phenomenon in isolated mitochondria (39, 531). These
included mainly high Ca2 + loading in the matrix (241, 306),
the irreversibility of the mitochondrial alterations associated
with large amplitude swelling (527), and loss of matrix components, including low-molecular-weight proteins (120).
However, the better understanding of the factors controlling
the opening and closing of the MPT pore and numerous observations that MPT blockers, such as cyclosporin A, prevent
cell death under many pathological conditions (133, 225, 322,
517) confirmed the participation of MPT in the pathogenesis
of many diseases.
Despite extensive research since MPT was first described
(241), the structure of the putative pore remains unresolved
and controversial. Literature data suggest that the pore is
composed of an assembly of matrix, inner and outer membrane proteins such as the adenine nucleotide transporter
(ANT), the voltage-dependent anion channel (VDAC), CypD,
aspartate-glutamate and phosphate carriers, hexokinase, and
possibly other proteins (309, 341). However, other studies
demonstrate that some of these proteins are not essential
components of the pore, as they can also occur in inverted
submitochondrial particles, devoid of matrix and outer
membrane (166); in mitoplasts, devoid of outer membrane
(441); and in mitochondria genetically deficient in ANT,
VDAC, or CypD (24, 273, 284). In potato tuber mitochondria,
MPT was showed to be insensitive to cyclosporin A, despite
the fact that cyclosporin A inhibited the isomerase activity of
CypD (181). The results show that although there is a pore
opening under these conditions, some of its properties are
altered. For example, in ANT-deficient mitochondria, atractyloside, a classical ANT ligand, does not promote pore
opening; whereas in CypD-deficient mitochondria, MPT requires larger Ca2 + loads and is not blocked by cyclosporin A
(24). These results suggest that if the pore is indeed composed
of various proteins, it is conceivable that it may still be formed,
with slightly different assembly properties, in the absence of
one or more components.
An aspect that deserves consideration is the understanding
of how Ca2 + and ROS act synergistically in the process of pore
assembly (280). There is a general idea that Ca2 + is essential
and has multiple roles in the process of pore formation (39),
while ROS and other ‘‘pore-inducing agents’’ such as inorganic phosphate (Pi) and thiol oxidants have a facilitating role
and lead to MPT irreversibility (84, 225). Indeed, it is very well
known that Ca2 + alone can induce MPT, but it is also true that
Ca2 + itself stimulates ROS generation by mitochondria (84,
208, 278, 279, 333). In addition, mitochondria are more susceptible to Ca2 + when their antioxidant systems, represented
mainly by NADPH and GSH, are exhausted [reviewed in
2037
(280)]. The first indication for the redox nature of the MPT
arose from experiments showing that isolated liver and heart
mitochondria could not retain accumulated Ca2 + when the
endogenous pool of pyridine nucleotides was shifted to the
oxidized state (306). Further investigations on this mechanism
demonstrated that oxidation of mitochondrial NADPH decreased the mitochondrial antioxidant capacity, leading to
oxidative imbalance associated with a progressive polymerization of inner mitochondrial membrane proteins via thiol
crosslinking (166). Later, a paper by Bernardi’s group (117)
proposed a modulation of the MPT by the redox state of
pyridine nucleotides and thiols at two separate mitochondrial
sites (117). In addition, many prooxidants act as MPT inducers, while many antioxidants prevent or even reverse MPT
(280).
A recent study (523) identified the mitochondrial p53CypD complex as an important contributor to oxidative
imbalance-induced necrosis and implicated the participation
of this protein complex in brain ischemia/reperfusion injury.
The authors provided evidence that p53 accumulates in the
mitochondrial matrix in response to oxidative imbalance
and triggers MPT pore opening and necrosis by interacting
with the MPT regulator CypD. In contrast, reduction of p53
levels or cyclosporin A pretreatment of mice prevents formation of this complex and effectively protects against
stroke.
Literature data provide evidence that Ca2 + stimulates ROS
generation by various mechanisms, including (i) stimulation
of the tricarboxilic acid cycle (58); (ii) activation of ROSgenerating enzymes such as glycerol phosphate and
a-ketoglutarate dehydrogenase (508, 510); (iii) inhibition of
respiration by Ca2 + -induced NOc generation (195); (iv) opening of the MPT pore (224, 333); and (v) alterations in lipid
organization of the inner mitochondrial membrane caused by
Ca2 + binding to cardiolipin, leading to lateral phase separation (208). In addition, (vi) Pi, one of the earliest MPT inducers
known (210), and Ca2 + cooperate in promoting oxidative
imbalance, MPT, and membrane lipid peroxidation (278). This
occurs via generation of triplet state intermediates from lipid
peroxidation, a process probably catalyzed by cytochrome c
(83). Moreover, in in vitro model systems consisting of phosphatidylcholine/diethyl phosphate liposomes, phosphate
and Ca2 + cooperate to promote the propagation of radical
reactions initiated by triplet acetone-generating systems (278).
Finally, (vii) experiments performed with isolated mitochondria demonstrate that high Ca2 + loads promote mobilization
of intramitochondrial Fe2 + followed by oxidative imbalance
and MPT sensitive to the Fe2 + chelator o-phenantroline,
dithiothreitol or exogenous catalase (83). Despite the slow
kinetics of the Fenton reaction (221) and the lack of hydroxyl
radical specificity, these experimental data suggest that under
these conditions, a form of irreversible MPT would be formed
via the attack of protein thiols by the hydroxyl radical. Since
mitochondria are sites of iron uptake and storage (369), as
well as incorporation into heme and electron transfer proteinsulfur iron clusters, this metal is a strong candidate for MPT
stimulation under pathological conditions.
Overall, the above data regarding the redox nature of MPT
provide the basis for a MPT model in which membrane proteins aggregate to assemble a pore with a hydrophilic core that
confers the high conductance of the MPT (166). Although this
form of MPT may occur under certain in vitro conditions, it
2038
does not contemplate other in situ regulators recently discovered such as the p53-CypD complex (523).
IV. Mitochondria and Nitric Oxide
Recent in vivo data indicate that NOc plays a role in the
regulation of cellular metabolic phenotypes (319, 385) and
mitochondrial energy transduction (56, 112, 303, 385, 386,
400), generating a strong interest in understanding the relationship between mitochondria and NOc. On the other hand,
whether mitochondria possess a nitric oxide synthase isoform, the mtNOS, is a disputed point (54, 525, 526). Many
aspects related to the identification, cellular sublocalization,
regulation, and pathophysiology of this putative mtNOS
have been approached in reviews that we indicate the readers
to consult (54, 193, 227, 533). Irrespective of mtNOS existence, there is strong experimental evidence indicating that
NOc and its derivatives affect mitochondrial function (4, 57,
59, 60, 110, 112, 420, 422, 470, 472) and biogenesis (111, 319,
384, 386). Recent studies (22, 23, 299, 300, 302, 303) have
shown that the supplementation of humans with dietary
amounts of inorganic nitrate (which can be converted into
NOc in the body) significantly improves energy metabolism
during exercise. A mitochondrial mechanism has been suggested for this effect (301).
A. Mitochondrial generation of NOc
Nitric oxide synthases catalyze the conversion of L-arginine
into citrulline and NOc:
FIGUEIRA ET AL.
by Giulivi’s group (155, 502). These studies show that mtNOS
is a neuronal NOS (nNOSa) isoform with post-translational
modifications, and that its purified activity required the following substrates and cofactors: L-arginine, NADPH, Ca2 + ,
calmodulin, BH4, and FAD. The high Ca2 + and BH4 requirement for mtNOS activity raised some skepticism in the literature (54, 292, 525, 526). On the other hand, Mann’s group
employed a new approach to evaluate the mitochondrial
proteome (180) that purportedly did not rely on the purification of these organelles. In this proteomic study of white
and brown adipose tissue mitochondria, the authors identified the product of the NOS3 gene (i.e., eNOS) as a true mitochondrial protein (180). It is worth noting that this group
did not find evidence of mtNOS in proteomic studies of mitochondria from other tissues such as heart, liver, and skeletal
muscle, in which they employed a more standard approach
relying on isolated and purified mitochondrial samples (179,
359). Overall, comprehensive studies employing robust techniques to approach mtNOS occurrence, properties, and its
tissue distribution are still necessary.
In addition to being generated by NOS isoforms, NOc and
other bioactive nitrogen molecules have been shown to be
metabolites of dietary inorganic nitrite (NO2 - ) and nitrate
(NO3 - ). Thus, in mammals, both NO2 - and NO3 - are
products of NOc degradation and substrates for NOc generation (Fig. 5). NOc enters the cycle when generated by NOS,
while NO2 - and NO3 - are acquired through the diet (vegetables such as spinach and beetroot are rich in NO3 - ). The
amount of dietary NO3 - has been estimated to exceed the
amount of endogenously produced NOc through NOS
L-arginine + NADPH + O2 + H + /citrulline + NADP + + NOc
This reaction is Ca2 + dependent (except for iNOS) and requires FAD, FMN, BH4, heme, and calmodulin.
Several studies (155, 194, 199, 263, 291, 372, 502, 520) show
that mitochondrial fractions obtained by standard differential centrifugation techniques possess nitric oxide synthase
(NOS) activity, sensitive to specific inhibitors, as identified by
the following assays: (i) radio-labeled L-arginine-citrulline
conversion; (ii) spectrophotometric changes of oxymyoglobin;
and (iii) inhibition of mitochondrial respiration. In addition to
these functional approaches, there are many reports showing
immunoreactivity of mitochondrial fractions with antibodies
against all three known NOS isoforms [nNOS, eNOS, and
iNOS, (291, 292)].
A key point to understand the debate and controversies on
the existence of mtNOS is that mitochondrial isolation techniques generally yield a fraction also containing other cellular
constituents, which may remain as contaminants even when
additional purification steps are conducted (431). Therefore,
classical biochemical assays or proteomic approaches for the
identification of a putative mitochondrial protein are limited
by sample preparation, and the NOS activity identified may
not be mitochondrial. Some studies attempted to purify mitochondria free of other cellular constituents and still recovered
NOS activity in mitochondrial suspensions (199, 291), although
some degree of nonmitochondrial contamination remained
(199). Nonetheless, it may be possible that an NOS is present in
common preparations of isolated mitochondria, not necessarily
comprising an evidence of the putative mtNOS.
An isoform of NOS was purified from isolated rat liver
mitochondria, and its biochemical properties were described
FIG. 5. Mammalian nitrogen oxide cycle. This cycle has
three intermediates: NOc, NO2 - , and NO3 - . NOc and NO3 enter the cycle through NOS isoforms and dietary intake.
The reduction of NO3 - into NO2 - involves enterosalivary
circulation and is mediated by bacteria in the oral cavity.
NO2 - can be reduced to bioactive NOc through a variety of
enzymatic and nonenzymatic reactions, including those
mediated by the respiratory chain. The bioactivity of newly
synthesized NOc, through either NOS or NOS-independent
reactions, is rapidly terminated by its oxidation into NO2 and NO3 - . NO2 - , nitrite; NO3 - , nitrate; NOS, nitric oxide
synthases. (To see this illustration in color, the reader is referred to the web version of this article at www.liebertpub
.com/ars.)
MITOCHONDRIA AND REACTIVE NITROGEN AND OXYGEN SPECIES
isoforms (331, 332). The reduction of NO3 - into NO2 - involves enterosalivary circulation and is mediated by commensal bacteria in the oral cavity. In turn, NO2 - can be
reduced to bioactive NOc through a variety of enzymatic
and nonenzymatic reactions. Notably, deoxyhemoglobin,
deoxymyoglobin, and the mitochondrial respiratory chain
possess nitrite reductase activities (32, 116, 229, 251, 331, 332,
471). The bioactivity of newly synthesized NOc is terminated
by its oxidation into NO2 - and NO3 - . An important feature
of this nitrogen oxide cycle is that hypoxia and/or acidosis
greatly enhance NOc formation from NO2 - , comprising a
possible mechanism for hypoxia-induced vasodilation (116,
123, 229, 301).
In vitro studies have demonstrated that mitochondria are
able to metabolize NO2 - into NOc (32, 283). Specifically, cytochrome c and respiratory chain Complexes III and IV possess nitrite reductase activities that can be stimulated under
hypoxic or acidic conditions (32, 283, 390) (Fig. 6). At these
three sites, electrons are transferred from the electron transport chain to NO2 - , promoting reduction into NOc. A recent
human study indicates that exogenous NO2 - is inert to mitochondria (isolated from human skeletal muscle and incubated in vitro) at a resting physiological pH of 7.2. However, at
a pH of 6.7 (which is promoted intracellularly under conditions such as physical exertion), exogenous NO2 - elicits biochemical effects attributable to NOc (301).
Oxygen desaturated heme proteins are well-characterized
nitrite reductases in mammals (116, 127, 229, 251, 471).
Myoglobin can catalyze NOc formation from NO2 - in the
close vicinity to mitochondria within the skeletal muscle.
Thus, NO3 - and NO2 - seem to be a major sources of NOc.
Indeed, there is evidence that increased NO2 - bioavailability
exerts beneficial effects in vivo (22, 23, 79, 116, 127, 149, 229,
301, 303, 415, 472, 522). NO2 - reduction may even effectively
occur in the absence of acidosis, as acute intravenous administration of NO2 - to healthy humans under resting conditions elicits vasodilation (127).
B. NOc effects on mitochondrial electron transport
and energy transfer
Inhibition of mitochondrial respiration is the best-known
interaction between NOc and mitochondria. Data from a variety of experimental models, ranging from isolated mitochondrial preparations and intact cells to exercising dogs and
FIG. 6. NOc actions modulating
mitochondrial respiration and integrity. Inhibition of mitochondrial respiration by NOc at the level of complex
IV is reversible and competitive with
oxygen. Through S-nitrosylation of
complex I of ETC, NOc partially inhibits its activity and slows down mitochondrial respiration; such inhibition
may have positive effects on mitochondrial redox homeostasis and has
been associated with improved outcomes from ischemia reperfusion. (To
see this illustration in color, the reader
is referred to the web version of this
article at www.liebertpub.com/ars.)
2039
humans, support the idea that respiratory rates are controlled
by NOc (23, 56, 203, 258, 301–303, 400, 470). Nanomolar levels
of NOc inhibit cytochrome c oxidase (mitochondrial complex
IV) activity reversibly and competitively with O2 (56, 110,
420, 465) (Fig. 6). Indeed, studies employing isolated mitochondria and intact cells clearly show that NOc inhibition of
mitochondrial respiration increases nonlinearly as oxygen
availability decreases (4, 56, 203). Although cytochrome c
oxidase has an apparent excess capacity with regard to
electron flow through the respiratory chain, this enzyme
may be operating much closer to its turnover limit if vicinal
nM levels of NOc are taken into account (12), thus offering an
explanation as to why changes in NOc concentration modulate the rate of mitochondrial oxygen consumption at
physiologically relevant intracellular PO2 [*3–35 mmHg,
depending on energy turnover rates and ambient atmospheric pressures; (4, 433)]. In general, NOc inhibition increases at high mitochondrial respiratory rates and/or low
PO2 (56, 203). With regard to NOc interaction with cytochrome c oxidase, the oxidized form of this enzyme may play
a role in mitochondrial NOc actions and fate, as oxidized
cytochrome c oxidase promotes NOc oxidation into NO2 - at
considerably fast rates (13, 113).
In vitro studies also indicate that other electron transport
chain sites are sensitive to NOc or its derivatives (59). Irreversible inhibition of the activities of mitochondrial respiratory chain complexes has been observed on exposure of
submitochondrial particles and mitochondria to millimolar
levels of NOc or ONOO - (47, 81, 427). Inhibitory mechanisms
include degradation of protein structures, S-nitrosylation, and
oxidation (47, 59, 60, 81, 427). However, some of these findings still require demonstrations of biological relevance or
in vivo confirmations. Mitochondrial complex I protein thiol
modification by NOc (i.e., S-nitrosylation) has been reported
to occur in beating perfused hearts under biologically relevant
conditions (492). By using exogenous S-nitrosothiol targeted
to mitochondria, Prime et al. (422) demonstrated that Snitrosylation of complex I is associated with slower mitochondrial respiration. These authors also found that this
NOc effect only occurs when mitochondria are energized with
complex I-linked substrates, thus indicating a specific inhibition of respiration at the level of complex I (422). Importantly,
S-nitrosylation of complex I has been associated with improved outcomes from heart ischemia reperfusion in animal
models (229, 314, 422, 472).
2040
Boveris’ group (420) perfused ex-vivo beating hearts with
solutions containing NOc donors and found that myocardial
oxygen uptake was decreased up to 50% on increased NOc
availability. Developed left ventricular pressure, an index of
heart mechanical work, remained unchanged (420). These
results are one of the first evidences that the oxygen cost of
mechanical work can be fine-tuned by NOc availability. If on
one hand increased NOc promotes higher mechanical efficiency (i.e., higher mechanical work to oxygen uptake ratio)
(420), decreased NOc availability, by means of pharmacological NOS inhibition, augmented muscle oxygen uptake in
exercising dogs (470). Further studies reinforced this concept
by showing that pharmacological NOS inhibition accelerated
the increase in human skeletal muscle oxidative metabolism
during resting-exercise transitions (258).
More recently, a series of work by two independent research groups uncovered interesting metabolic effects of dietary NO3 - during exercise (22, 258, 300–303, 522). The oxygen
cost of exercise was found to be decreased by acute inorganic
nitrate intake. Since mechanical efficiency is a determinant of
exercise performance, nitrate intake improved performance
after nitrate supplementation (22, 299). The acute intake of a
dietary amount of NO3 - corresponding to *0.5 L of beetroot
juice was enough to increase plasma NO3 - and NO2 - , to
decrease arterial blood pressure, and to elicit the exercise responses described (299–301, 303, 522). While NO3 - intake
lowers ATP turnover during muscle contraction (22), Larsen
et al. (301) found that mitochondrial ADP/O ratios in the
skeletal muscles of healthy humans are improved by NO3 supplementation. Mitochondrial ANT expression was also
decreased, and oxygen dependence of mitochondrial respiration was increased. These two findings may provide
mechanistic support for higher ADP/O ratios: First, ANT can
mediate uncoupling of oxidative phosphorylation; second, a
slight inhibition of mitochondrial respiration at the level of
cytochrome c oxidase may promote positive effects on energy
conservation (112, 204). Cytochrome c oxidase activity can be
limited by very low oxygen tensions or by decreased oxygen
affinity due to the action of NOc (112). In fact, in vitro analyses
of isolated mitochondria show that partial inhibition of mitochondrial respiration by either NOc or cyanide leads to
higher oxidative phosphorylation efficiency (112). Based on
previous studies modulating NOc bioavailability (209, 388)
and on the evidence that NO2 - and NO3 - are sources of
bioactive NOc in living mammals (331, 332), effects of NO3 supplementation on exercise metabolism and mitochondrial
FIGUEIRA ET AL.
energy transfer seem to be mediated by enhanced NOc bioavailability (331, 332).
C. MPT and NOc-mediated cytoprotection
Cell damage or death following MPT opening participates
in the pathophysiology of ischemic diseases, as discussed
earlier. There is also evidence that pharmacological MPT inhibition is of therapeutic value in brain trauma and muscle
dystrophy (198). Adding to the previously demonstrated redox sensitivity of MPT discussed earlier (280, 306, 528), NOc
has been shown to modulate mitochondrial suscebility to
calcium-induced MPT (57, 307, 380). Initial data showed that
incubation of isolated mitochondria with exogenous NOc
donors elicited partial inhibition of calcium-induced MPT
(57). However, parallel measurements of mitochondrial calcium uptake and membrane potential suggested that NOc
inhibition of MPT was secondary to membrane depolarization and calcium uptake failure (57), simply the result of NOc
inhibition of mitochondrial respiration. Since the oxidation of
membrane protein thiols mediates MPT opening (166), Snitrosylation of thiols groups by NOc is presumably a mechanism of protection against thiol oxidation, MPT opening, and
cell damage (Fig. 7). In fact, cytoprotective effects of ischemic
preconditioning are associated with increased S-nitrosylation
of mitochondrial proteins along with lower oxidation of thiol
groups (272).
Our group has recently demonstrated that co-incubation of
isolated mitochondria with NOS inhibitors rendered mitochondria more prone to Ca2 + -induced MPT (307). MPT
stimulation was associated with a decrease in mitochondrial
S-nitrosothiol content (307), building on the concept that reversible modification of thiol groups by NOc plays an important role in the redox regulation of MPT. A very recent study
has demonstrated that cysteine 203 of CypD is a target of Snitrosylation and is of central importance for MPT regulation
(380) (Fig. 7). This work showed that mutation of cysteine 203
of CypD to a serine residue increased mitochondrial Ca2 +
retention capacity to the level presented by CypD null or
cyclosporin A-treated mitochondria. Moreover, exogenous Snitrosoglutathione presumably promotes S-nitrosylation of
CypD and attenuates H2O2-induced MPT opening and cell
death (380). These is strong data revealing the role of protein
thiol S-nitrosylation as a mechanism for protection against
MPT. The peptidyl-prolyl cis-trans isomerase activity of
CypD is not significantly altered by the mutation mentioned
FIG. 7. NOc protection against MPT.
S-nitrosylation of mitochondrial proteins seems to play protective roles.
CypD is the best known protein regulating MPT opening, and its nitrosylation on cysteine residue 203 inhibits
Ca2 + - or Ca2 + plus H2O2-induced
MPT opening. However, excess of NOc
seems to facilitate ONOO - formation,
which is very reactive, and leads to nitrosative stress and MPT opening. (To
see this illustration in color, the reader is
referred to the web version of this article at www.liebertpub.com/ars.)
MITOCHONDRIA AND REACTIVE NITROGEN AND OXYGEN SPECIES
earlier (317). CypD isomerase activity is inhibited by cyclosporin A and also seems to be modulated by acetylation/
deacetylation through mitochondrial Sirt3 activity (474).
Sirt3 promotes deacetylation of CypD and atenuates Ca2 + induced MPT (215). Deacetylated CypD was shown to possess lower isomerase activity (474). Adding to the insights
provided from the mutated CypD studies (317, 380), a previous study by our group showed that cyclosporin A inhibits
the isomerase activity of cyclophilin in isolated mitochondria
from potato tubers, while Ca2 + -induced MPT in this plant
was insensitive to cyclosporin A (181). Therefore, the isomerase activity of CypD is not always associated with MPT
regulation.
In vivo animal studies demonstrate that S-NO or NO2 promotes protection in ischemia reperfusion (131, 149, 259,
422). Improved neurological and cardiac function was observed when mice undergoing cardiac arrest were treated
with NO2 - (131). Interestingly, lower mitochondrial oxidative imbalance and a partial inhibition of complex I were also
documented in these animals (131). Partial complex I inhibition is likely due to S-nitrosylation, as indicated by a study
that pharmacologically targeted S-NO to mitochondria (422).
Mitochondria-targeted S-NO is accumulated within mitochondria, promoting S-nitrosylation and inhibition of complex I (422). Similar to NO2 - and other S-NO donors,
mitochondrially targeted S-NO reduced the extent of tissue
damage in animal models of ischemia reperfusion (422). It is
worth noting that these protective effects are probably not
only confined to NOc interaction with specific mitochondrial
proteins (e.g., CypD or complex I), as S-nitrosylation of other
proteins, such as the transcription factor hypoxia inducible
factor-1a (HIF-1a), seems to be of central importance for tissue
response to hypoxia (314).
S-nitrosylation of complex I and other mitochondrial proteins has been observed after ischemic preconditioning (259,
492). Although the exact mechanisms by which S-nitrosated
proteins protect against ischemia reperfusion are still unclear,
the advantage of inhibited mitochondrial complex I due to Snitrosylation may be a decrease in the likelihood of mitochondrial ROS formation, as discussed earlier. In addition,
lower NADH oxidation by mitochondrial complex I may help
maintain a higher NADH/NAD + ratio, favoring NADP +
reduction at the expense of NADH (306, 528).
Converse to the cytoprotective roles of NOc discussed
earlier, high levels of this oxidant may promote nitrosative
stress and cellular injury (47). Indeed, exposure of mitochondria to high levels of NOc or ONOO - potentiates Ca2 + induced MPT (57, 186, 452).
D. NOc-mediated mitochondrial biogenesis
Peroxisome proliferator-activated receptor coactivator 1 a
(PGC-1a) is a regulator of mitochondrial biogenesis in metabolically active tissues such as heart, skeletal muscle, and
brown adipose tissue, controlling the expression of transcription factors that regulate the expression of a variety of
nuclear and mitochondrial genes encoding mitochondrial
proteins. Genetically modified mice overexpressing skeletal
muscle PGC-1a display a marked increase in mitochondrial
density and exercise capacity (315). NOc is an upstream activator of PGC-1a (385), promoting enhanced mitochondrial
biogenesis and increases in the abundance of mitochondrial
2041
proteins (319, 385, 386) in a manner that is dependent on
cGMP (385). Recently, Lira et al. (319) demonstrated that NOc
interacts with AMPK both in myotubes and in contracting
skeletal muscle, activating NOS and regulating PGC-1a expression.
Mice lacking eNOS have provided important evidence of
NOc-mediated mitochondrial biogenesis and metabolic
health (79, 385, 386). These mice present lower liver and
skeletal muscle mitochondrial density and features of metabolic alterations, despite the expression of other NOS isoforms. Interestingly, dietary supplementation with NO3 - ,
which can be metabolized into bioactive NOc (331), can reverse metabolic abnormalities in eNOS-deficient mice (79).
Mice deficient in eNOS also present a decrease in mitochondrial biogenesis promoted by the limitation of calorie
ingestion (539). Indeed, we have demonstrated that caloric
restriction increases eNOS activity and mitochondrial biogenesis in a mechanism involving enhanced insulin receptor
activation mediated by adiponectin (86, 87, 89). Furthermore,
enhanced NOc and mitochondrial biogenesis may be sufficient to promote some beneficial effects of calorie restriction
such as increased neuronal survival in vitro (87). Interestingly,
calorie restriction increases mitochondrial content (88, 109,
326, 387), but decreases ROS release, possibly due to mitochondrial uncoupling (88, 277, 296).
V. Mitochondrial Disorders
Mitochondrial dysfunction can arise from more than 1200
different gene mutations, toxic agents, or spontaneously
during aging (456, 539). Impairments in mitochondrial oxidative phosphorylation have recently been recognized as the
most common cause of inborn errors of metabolism (118).
That is somewhat expected, considering that oxidative phosphorylation involves at least 90 proteins and more than 300
auxiliary proteins that help them assemble into mature complexes. On the other hand, in addition to ATP synthesis, cellular metabolism depends on mitochondria and the proper
exchange of metabolites across organelle membranes for the
biosynthesis of amino acids, vitamins, lipids, prosthetic
groups, and many other intermediates, which are also required for oxidative phosphorylation and cell viability. Deficiencies in any part of this intricate metabolism may cause
disease. Uncovering of the specific gene mutation involved in
mitochondrial disorders has shed light on the biochemical
function of many human open reading frames and received a
great deal of attention in the last years (37, 136, 141, 288, 402,
511, 513). In the next years, sequencing of a large number of
patients will hopefully overcome most diagnostic limitations,
and the specific gene mutation in a given patient will be more
promptly identified. Since the oxidative phosphorylation
apparatus involves proteins encoded in the nucleus, in addition to 13 subunits encoded in mitochondrial DNA (mtDNA)
and translated in mitochondria (Fig. 8), this section summarizes our current knowledge of the features of mitochondrial
dysfunction and redox imbalance caused by mutations in
nuclear genes that affect oxidative phosphorylation (Tables 1
and 2) and by mutations in the mitochondrial genome.
A. Nuclear genes in mitochondrial disorders
The severity and clinical outcome of a particular loss
of biochemical function may be different in individuals
2042
FIGUEIRA ET AL.
FIG. 8. Biogenesis of oxidative phosphorylation complexes. Biogenesis of respiratory electron transport (complexes I–IV)
chain and ATP synthase (complex V) involves the assembly of subunits encoded by nuclear (green) and mitochondrial
(purple) genomes. The five complexes have altogether 89 subunits. Thirteen are translated inside the organelle, and usually
represent the catalytic core of the respective complex. The number of point mutations, small and large deletions identified in
the mtDNA-encoded genes, is listed in MITOMAP—a Human Mitochondrial Genome Database. (www.mitomap.org, 2011)
(To see this illustration in color, the reader is referred to the web version of this article at www.liebertpub.com/ars.)
expressing the same mutant gene, a typical consequence of
gene interactions in different genetic backgrounds. In addition, mutations in different genes may elicit the same clinical
presentation and, finally, a particular deficiency can have
different prognosis depending on the tissue and organ it is
being expressed in. It is, thus, not unexpectedly remarkable
that tissues with a higher energetic demand are more
promptly affected by lower ATP synthesis due to deficient
mitochondrial metabolism (521). Altogether, there are 502
clinical categories of mitochondrial disorders already described
from 174 gene mutations (456), and neuromuscular-associated
pathologies are the most frequent clinical presentation (456).
Differently from all other cell enzymatic complexes, biogenesis of mitochondrial respiratory complexes depends on two
Table 1. Nuclear Gene Mutations Directly Affecting Mitochondrial Respiratory Chain Components
Biochemical
deficiency
Genes involved
Clinical categories
Complex II structure
NDUFS1,NDUFS2, NDUFS3, NDUFS7,
NDUFS8, NDUFV1, NDUFV2
NDUFAB1,NDUFA1,NDUFA10,
NDUFA11, NDUFS6, NDUFS4
NDUFAF1,NDUFAF2,NDUFAF3,
NDUFAF4, C6orf66, C8orf38, C20orf7,
NUBPC, ACAD9, FOXRED1
SDHA, SDHB, SDHC, SDHD
Complex II assembly
Complex III structure
SDHAF2
UQCRQ, UQBC
Complex III assembly
BCS1L, TTC19
Complex IV structure
Complex IV assembly
COX6B
SURF1, SCO1, SCO2, COX10,
COX15, C2orf64.
Complex V structure
Complex V assembly
CoQ biosynthesis
(primary)
ATP5E
ATP12, TMEM70
COQ2, PDSS1, PDSS2, COQ6,
COQ4, ADCK3, COQ9
Childhood encephalopathy,
cardiomiopathy, Leigh syndrome.
Encephalopathy, cardiomiopathy,
Leigh syndrome
Encephalopathy, cardiomiopathy,
Leigh syndrome, Exercise
intolerance
Leigh syndrome, paranglioma,
pheochromocytoma
Paranglioma.
Dementia, gastroenteritis, Leber
syndrome, exercise intolerance
Tubulopathy, encephalopathy,
liver failure, GRACILE
syndrome.
Leukodystrophy
Leigh syndrome, cardiomyopathy,
encephalopathy, neonatal
hepatic failure,
Encephalopathy
Encephalopathy, cardiomyopathy
Encephalopathy, cerebelar ataxia,
lactic acidosis, renal tubololpathy
Complex I structure
Complex I super
numerary
Complex I assembly
CoQ, Coenzyme Q.
References
(513)
(513)
(513)
(27, 68)
(27)
(37)
(37, 534)
(136)
(28, 136)
(288)
(288, 485)
(159, 294, 511)
MITOCHONDRIA AND REACTIVE NITROGEN AND OXYGEN SPECIES
2043
Table 2. Nuclear Gene Mutations Eliciting Pleiotropic Impairment
of Mitochondrial Oxidative Phosphorylation
Biochemical deficiency
Genes involved
Clinical categories
TCA cycle
DLD, DLAT PDP1, PDHA1,
PDHB, PDHX, FH1
CoQ deficit (secondary)
ETFDH, BRAF, APTX
Cardiolipin biosynthesis
TAZ1
Organelle fission and fusion
OPA1, Mfn2
Deoxinucleotide supply
ANT1, TYMP, DGUOK,
PUS1, MTTK,
SLC25A19, RRM2b
POLG1, POLG2, Twinkle
Vomiting, stroke-like episodes,
Paranglioma, seizures, Leigh
syndrome, Encephalopathy
Ataxia and oculomotor apraxia,
acidosis, Cardiofaciocutaneous
syndrome
Cardiomyopathy, neutropenia,
Barth syndrome
Dominant progressive external
ophtalmoplegia, motor and
sensor neuropathy (CharcotMarie-Tooth disease)
Progressive external ophtalmoplegia,
MNGIE, hepatopathy, MLASA,
microcephaly.
Dominant progressive external
ophtalmoplegia
Friedreich ataxia
Encephalopathy, cardiomypathy,
ataxia.
Mohr-Tranebjaerg—deafness
dystonia syndrome
mtDNA metabolism
Iron-sulfur synthesis
Mitochondria translation
Protein import
Frataxin, ABC7
TACO1, LRPPRC,
EFG1, MRPS16
TIMM8A
References
(392, 457)
(159, 511)
(460)
(102, 240)
(232, 403)
(183, 232)
(447)
(188, 447, 552)
(5)
MNGIE, mitochondrial neuro gastrointestinal encephalomyopathy; MLASA, myopathy lactic acidosis sideroblastica anaemia; mtDNA,
mitochondrial DNA.
genomes that create an enormous regulatory complexity required to bring together polypeptides coming from inside and
outside the organelle.
Mitochondrial dysfunction, in turn, leads to different biochemical effects that modulate nuclear gene expression, such
as the over-expression of ROS scavenging enzymes in fibroblasts of patients with progressive external ophtalmoplegia
(329). The amount of ROS produced and the differential
ability of tissues to handle ROS excess are certainly components that exacerbate the clinical heterogeneity of mitochondrial diseases (46). ROS and RNS are components of the crosstalk between mitochondria and nucleus. Elevated levels of
ROS/RNS were shown to increase the mtDNA copy number
and mitochondria abundance, a feedback response to compensate impaired respiration, an adpatation that also occurs
in aged tissues (305). Nevertheless, it is a tautological harmful
cycle, as mitochondria are both generators and targets of ROS.
Elevated ROS can directly damage and mutate mtDNA, a
process 10–20 times more likely than in nuclear DNA (61, 540,
542), as mtDNA is located close to the electron transport
chain, a major source of intracellular ROS, as described earlier.
For instance, high ROS production in mitochondrial diseases
leads to augmented 8-hydroxydeoxy guanosine (8-OHdG)
and lipid peroxidation levels (418), which are, respectively,
related to the process of mtDNA mutagenesis and apoptosis.
Given the important role of ROS in apoptosis, the excess of
ROS generated in mitochondrial diseases can trigger apoptosis through MPT, or by means of cardiolipin oxidation
(163, 192). Apoptosis participates in the pathogenic process
of mitochondrial myopathies and encephalomyopathies
(16, 352), as well as in the degenerative process in hereditary
optic neuropathies (310). As cells are progressively lost
through apoptosis, tissue function declines, leading to the
recrudescence of clinical symptoms. Overall, oxidative imbalance resulting from mitochondrial dysfunction plays an
important role in the pathogenesis and progression of mitochondrial diseases.
mtDNA abnormalities also stem from direct deficiencies in
replication and repair, which depend on nuclear-encoded
proteins. Due to this dependence, mtDNA inheritance and
maintenance are also transmitted as mendelian traits.
Pathogenic mutations in DNA polymerase c (POLG1 and
POLG2) and twinkle DNA-helicase are the major cause for the
multitude of mtDNA deletions in many cases of progressive
external ophtalmoplegia (183). POLG1 codes for the catalytic
core of the mtDNA pol c, and POLG2 codes for the accessory
b-subunit. mtDNA stability is also considerably affected by
deoxynucleotide supply in the organelle. Patients harboring
mutations in genes involved in this process usually present
depletion of mtDNA (232).
The clinical categories of patients with mtDNA deletions
and depletion are very heterogeneous. Their symptoms range
from severe encephalopathy and cardiomyopathy in childhood to late-onset progressive external ophtalmoplegia. There
is also evidence of mtDNA depletion associated with sporadic
Parkinson’s disease, Alzheimer’s, and other neurodegenerative diseases (540).
mtDNA instability can also originate in mitochodria with
defective fusion and fission dynamics (38), which has also
been associated with cell death in neurodegenerative diseases
(102) as well in the pathophysiology of obesity and type-2
diabetes (570). Organelle dynamics is an essential process, influencing mitochondria morphology, biogenesis,
distribution, and metabolism. Morphology modulation of
2044
mitochondrial filaments, for instance, allows mitochondria to
transmit mitochondrial membrane potentials to regions
with low oxygen tension in muscle cells (476). Mitochondrial
fission and fusion depends on guanosine triphosphate
GTPase dynamins, such as dynamin-related protein1 (Drp1),
mitofusins (Mfn1 and Mfn2), presents in the mitochondrial
outter membrane and optic arthropy protein 1 (OPA1),
present in the inner membrane (553). OPA1 dysfuction impairs mitochondrial fusion and is the cause of most cases of
dominant progressive external ophtalmoplegia. (240). On the
other hand, Mfn2 mutations have been reported in patients
with Charcot-Marie-Tooth neuropathy type 2A, a disorder
that is characterized by the gradual degeneration of peripheral neurons (102). OPA1 isoforms are generated in mammalian cells by alternative-splicing and proteolytic processing
by AAA-proteases, such as AFG3L2 and SPG7, also known as
the paraplegin group. Nevertheless, all OPA1 isoforms are
able to interact with Mfn1 and Mfn2 (209). Pathogenic mutations in paraplegin genes were identified in patients with
progressive weakness and spasticity of the lower limbs due to
the degeneration of corticospinal axons. More recently,
pathogenic mutations in the metaloprotease AFG3L2 were
described in patients with dominant cerebellar ataxia, a remarkable feature considering that the autosomal dominant
pattern of inheritance is more frequently associated with
progressive external ophtalmoplegia (132).
In yeast, mtDNA also becomes unstable when the synthesis
of cardiolipin is altered (97). Cardiolipin is usually associated
with the respiratory complexes and other mitochondrial
proteins involved in mitochondrial biogenesis and apoptosis,
being particularly susceptible to ROS-induced lipid peroxidation, a typical feature of neurodegenerative diseases. Cardiolipin biosynthesis occurs inside mitochondria, and depends on
acyl groups remodeled by tafazzin (TAZ1). Impairment in this
step generates aberrant species of cardiolipin, and patients are
commonly diagnosed with Barth syndrome (459).
Deficiency in the synthesis of enzymatic oxidative phosphorylation prosthetic groups associated with metals can also
affect mtDNA stability through increments in ROS release
(182). For instance, in Friedreich ataxia, patients have a specific deficiency in frataxin, a mitochondrial matrix enzyme
that is an iron chaperone functioning in the biosynthesis of
iron-sulfur clusters (429). Patient cells depleted of frataxin
function show multiple Fe-S-dependent respiratory chain
deficiencies, with complexes I, II, and III compromised. This
disease may manifest in adolescence with progressive cerebellar, limb, and gait ataxia, and cardiac hypertrophy. Antioxidant treatment of patients with idebenone has been used in
many trials, and positive effects were observed in the cardiopathic component of the disease (446).
Pathogenic mutations in complex I nuclearly encoded
subunits and assembly factors are the most frequent group of
mitochondrial diseases, responding for 1 out of 3 of all known
oxidative phosphorylation deficiencies (513). Mutations in
genes required for the biogenesis and assembly of complexes
III, IV and V, as well tricarboxilic acid cycle enzymes, are less
frequent.
Complex I is the largest respiratory complex with 45
structural subunits. Approximately 150 patients were already
shown to have pathogenic mutations in 22 genes involved in
complex I structure and assembly (513). Patients with mutations in one of these subunits regularly manifest symptoms of
FIGUEIRA ET AL.
neuromuscular deficiencies. Encephalomyopathy, cardiomiopathy, and Leigh syndrome are more frequently identified during childhood. Interestingly, some complex I
deficiencies and associated pathologies, such as tumorigenesis, can be partially overcome through the heterologous expression of yeast NADH dehydrogenase (NDI1) (21). The
benefits achieved with yeast NDI1 launched it as a putative
gene therapy protocol (558). In fact, Ndi1p delivered by protein transduction results in cardioprotection in models of ischemia/reperfusion (414).
Complex I is an important ROS production site (as discussed
earlier), and studies using cell lines from patients containing
complex I deficiency revealed a correlation between ROS production and mitochondrial morphology (275, 554). Severe
complex I impairment results in higher ROS production and
mitochondrial fragmentation, while mild impairment leads to
discrete ROS elevation and normal elongated mitochondrial
morphology (143). One attractive explanation for this would be
that mitochondrial fragmentation restricts the local effects of
ROS, limiting damage extension (143). The correlation between
oxidative imbalance and mitochondrial dynamics has been
corroborated recently by studies on Drp1 (553). The GTPase
activity of Drp1 is sensitive to NOc (543), while the Snitrosylation of Drp1 (SNO-Drp1) promotes mitochondrial
fragmentation (367). The presence of SNO-Drp1 is increased in
brains of human Alzheimer’s disease patients, and, consequently, may be an important player of the pathologycal development of neurodegeneration (101, 367).
Pathogenic mutations in complex II subunits and fumarate
hydrase have been described as causes of rare late-onset Leigh
syndrome and of tumor formation (68). The mechanisms by
which depressed complex II activity favors tumor formation
are still poorly understood, but one likely hypothesis is that
succinate accumulation induces the hypoxia-response pathway, which activates the transcription of genes related to tumorigenesis and angiogenesis (466).
Nuclearly encoded mutations for complexes III, IV, and V
are rare—only four gene mutations for structural proteins of
these three complexes have been described—but mutations in
another handful of auxiliary proteins involved in the assembly of these complexes have been identified. For instance,
BCS1L plays a role in the synthesis and insertion of the activesite iron-sulfur cluster of the Rieske center in complex III (388).
Patients harboring mutations in BCS1L have been identified
with variable phenotype conditions and with different levels
of neurological degeneration, including GRACILE and
Björnstad syndromes (37).
Together with NADH dehydrogenases in complex I, the
protonmotive Q-cycle in complex III is the major site for
electron leakage and consequent formation of ROS (281).
Therefore, it is somewhat expected that complex-III-deficient
patients have an elevated level of ROS (37). In the case of
BCS1L mutated patients, oxidative imbalance is worse due to
iron overload (534). Nevertheless, in cases of complex III and
IV dysfunction, assembly of complex I is diminished, indicating a possible regulatory attempt to diminish electron
leakage and consequent ROS generation (1, 137).
Primary complex IV deficiency is more commonly identified in patients with mutations in assembly factors of the
holoenzyme (28). Mutations in COX10, COX15, SCO1, SCO2,
and SURF1 promote cardioencephalopathy, hypotonia, lactic
acidosis, hepatic failure, and Leigh syndrome (136). COX10
MITOCHONDRIA AND REACTIVE NITROGEN AND OXYGEN SPECIES
and COX15 mutants are defective in the synthesis of heme a
(28). SCO1 and SCO2 function in the copper delivery route
toward the CuA site of COX2 subunit and in the regulation of
copper efflux under excess conditions (304). The SURF1 gene
product is associated with an early step of cytochrome c oxidase assembly (177). In model organisms, the disruption of
complex IV assembly factors already associated with human
diseases such as COX10, COX15, and SCO1 results in an increment in ROS production (30, 265). Conversely, to our
knowledge, there is no specific indication of ROS increments
in complex IV patient cell lines, perhaps because of compensatory mechanisms such as complex I depletion (137).
Curiously, cytochrome c oxidase is secondarily affected in
patients containing mutations in ETHE1, a sulfur dioxygenase
localized in the matrix and involved in catabolism of sulfide
(134). Deficiency in this enzyme elicits ethylmalonic encephalopathy, an early-onset encephalopathy, normally fatal,
characterized by sulfite accumulation, which is a potent inhibitor of cytochrome c oxidase (134).
Disorders in F1-FO ATP synthase assembly due to nuclear
mutations are very rare (288). Recently, a number of cases of
primary ATP synthase deficiency were elucidated with the
description of mutations in TMEM70, an auxiliary factor of
ATP synthase assembly (485).
Combined deficiencies in the oxidative phosphorylation
system can occur due to defects in iron metabolism, lipid organelle protein import, or mitochondrial translation. Mitochondrial translation defects arise from mtDNA mutations
in tRNA and rRNA genes or nuclear gene mutations. Patients
with mendelian traits that are defective in this process have
been recently identified (173, 552). Considering that yeast has
a considerable number of translational activators for mitochondrially encoded polypeptides and only TACO1, a
COX1 activator, has been identified in humans (511, 552), it is
probable that many cases still remain to be identified.
Finally, the respiratory chain also depends on electron
carriers such as coenzyme Q and cytochrome c. Coenzyme Q
(CoQ, Fig. 9) is responsible for transferring electrons from
NADH and FADH2 dehydrogenases to complex III. Although
lower CoQ levels are identified in many mitochondrial disorders, just recently, primary deficient cases of CoQ have been
described (511). ROS overproduction was detected in cell lines
of patients with primary CoQ deficiencies, and the level of
ROS production correlated with the severity of the CoQ defect, a result also observed in yeast mutants with CoQ dysfunction (69, 294, 425).
The heterogeneity of mitochondrial dysfunction generates
an enormous difficulty to establish therapeutic approaches.
Nevertheless, one therapy regularly applied is the administration of CoQ and its synthetic analogue idebenone. As discussed earlier, idebenone treatment as an antioxidant has also
been effectively employed in Friedreich ataxia (446). CoQ
administration is considered harmless even in treatments
using 2000 mg daily (141), and has improved the clinical
symptoms of many patients, probably by lowering the levels
of ROS being produced in dysfunctional mitochondria as well
shuttling electrons directly from cytossol to complex III, elevating the synthesis of ATP in patients with defective complex I
(161). On the other hand, in patients with primary CoQ deficiency, it is expected that the rapid identification and early
treatment can prevent the irreversible damage to the nervous
system (511).
2045
FIG. 9. Biosynthesis of coenzyme Q in eukaryotic cells
[adapted from Ozeir et al. (397)]. Coq2 prenylates 4hydroxybenzoic acid (4-HB) or para-aminobenzoic acid
(pABA). R stands for the polyprenil tail, and X designates
NH2 or OH depending on the precursor. Patients harboring
mutations in PDSS1, PDSS2, COQ2, COQ6, ADCK3, COQ9,
and COQ4 (unknown function) have already been identified.
It is expected that early detection of primary deficiencies of
coenzyme Q biosynthesis can be treated with supplementary
addition of coenzyme Q analogues. Coq8 (ADCK3) and
Coq10 do not have a direct function in the biosynthesis, but
they are required for Coenzyme Q respiratory activity, Coq8
modulates the stability of Coq3 (468), and Coq10 transports
the mature Coenzyme Q to the respective sites in the respiratory chain (69).
SOD2 allelic variations, which are obviously directly involved with the cellular antioxidant system, were recently
investigated as a susceptibility risk factor in diseases where
oxidative imbalance is considered an important factor (6).
Impairments in MnSOD were detected in C47T polymorphism, the resultant amino acid change (Ala16Val) affects
MnSOD transport into the organelle (495). C47T polymorphism has also been associated with overall coronary artery
disease risk (504), and nonalcoholic fatty liver fibrosis (6).
However, because of the heterogeneity of population studies,
more studies are necessary in order to understand the susceptibility and risk factors in SOD2 polymorphisms in the
cellular antioxidant machinery.
B. mtDNA alterations in mitochondrial disorders
The human mitochondrial genome is extremely streamlined, encoding for only 13 polypeptides, 22 tRNAs, and 2
rRNAs. While the vast majority of genes from the original
bacterial genome were transferred to the nucleus throughout
evolution, these 13 polypeptides were retained in the mtDNA,
likely due to their high hydrophobicity and central location in
2046
the assembled respiratory complexes. The 13 mitochondrially
encoded proteins are essential for proper function and assembly of oxidative phosphorylation complexes I, III, and IV
and of the ATPsynthase (Fig. 8). For example, in the assembly
of Complex I (seven mtDNA-encoded), mutations in three
mtDNA-encoded subunits ND1, ND4, and ND6 result in a
marked decrease in Complex I activity and protein levels (106,
234, 337), while mutations in ND2 cause impaired assembly
and lead to accumulation of assembly intermediates (170).
The hypothesis that alterations in mtDNA could associate
with human diseases was initially put forth in the late 1970s,
from matrilineal inheritance patterns in some forms of ataxia,
such as Frederich’s ataxia and blood diseases (175). However,
it was only after the sequencing of human mtDNA, in 1981 (8),
that a comprehensive search for mtDNA mutations and deletions in human syndromes with clinical features of mitochondrial dysfunction started. In 1988, Holt et al. (235) found
two distinct deletions (4.9 and 5.9 kb) in muscle cells from two
patients with mitochondrial myopathies. However, seven
other patients investigated in this study showed similar, but
not identical, deletions. Other deletions of varying sizes were
identified soon after in patients with different mitochondrial
diseases, from a 0.4 kb deletion in a patient with encephalomyopathy (315) to deletions as large as 7.4 kb, in a
patient with a form of progressive external ophthalmoplegia
(499). It is not unusual for mtDNA molecules with different
deletions to coexist in the same patient, for reasons which will
be discussed later. Moreover, deletions are also detected in
some tissues of ‘‘healthy’’ subjects, and, thus, the diagnostic
value of one particular deletion, taken out of the context of
other markers of mitochondrial dysfunction, is somewhat
limited (350, 503).
A unique aspect of mitochondrial genetics is the fact that, since
each mitochondrion can contain more than one copy of the mitochondrial genome (451), and each cell can contain from a few
dozens to several hundreds of mitochondria, the number of
copies of mtDNA in one single cell can be significant variable.
However, more importantly, these copies can vary slightly from
one to another, both in size (as a consequence of insertions/
deletions) as well as in sequence (as a result of base substitutions). Both processes likely result from mtDNA damage accumulation and incomplete processing of these lesions by DNA
repair pathways (124, 285). The degree of sequence variation
among mtDNA molecules within a single cell is termed heteroplasmy (49, 481). If these different copies have different mutations, and individually would result in impaired gene
expression, a corollary of this feature is that different copies of the
mtDNA can complement each other within an individual organelle (197), as well as within the cell, especially in light of recent
results suggesting a role for mitochondrial fusion and fission in
mtDNA organization (for a review of this topic, see (461)).
In most cases of patients showing mtDNA deletions, the
deleted molecules are highly heteroplasmic, suggesting
de novo events, and enriched in the affected tissue, but not in
unaffected cell types (538). They often do not follow a matrilineal inheritance patterns as well (473). It later became clear
that while mitochondrial dysfunction, and therefore the clinical outcome of the disease, is directly related to the relative
amount of deleted mtDNA molecules (243), these events are
secondary to mutations in nuclear genes whose protein
products are involved in mtDNA maintenance, such as DNA
pol c and the twinkle helicase, as discussed earlier.
FIGUEIRA ET AL.
However, a specific mtDNA deletion syndrome has been
identified in which family history indicated the presence of
germ-line alterations. Kearns–Sayre syndrome is a distinctive
type of progressive external ophthalmoplegia that is characterized by pigmentary degeneration of the retina, heart block,
elevated concentration of cerebrospinal fluid protein, and abnormal muscle mitochondria. While several sporadic cases are
attributed to defective mtDNA metabolism, as in patients with
defects in ribonucleotide reductase (419), the identification of
familial cases (459) led to the identification of a large 4.9 kb
germline mtDNA deletion, later known as the common deletion
(360, 563). This deletion has been shown to accumulate with age
as well (115), although to which extent this accumulation is
functionally relevant still remains unclear (114, 237, 411).
Human mtDNA displays an extremely high gene density
(www.mitomap.org) (8, 9). From the 16,568 base pairs, only
around 600 are noncoding, although these constitute the
displacement loop, a control region where the origin of replication of the heavy-strand and transcription promoters for
both the light and heavy strands are located, and therefore are
essential for mtDNA replication (168). Thus, any base substitutions in mtDNA are very likely to reach a coding sequence and have functional consequences. In fact, several of
the known mtDNA polymorphisms in humans, defining the
different mtDNA haplotypes (450), cause slight functional
differences and modify susceptibility to important diseases
such as diabetes, obesity, and neurodegeneration [for review,
see (540)]. In addition, heteroplasmic somatic mutations in the
mtDNA control region have been found in healthy subjects,
and shown to differentially impact aging (443, 564).
From an evolutionary point of view, mutations can be
deleterious, neutral, or advantageous. Since mtDNA encodes
13 essential polypeptides, it is likely that mutations in its
coding sequence will have deleterious consequences. Moreover, in animals, mtDNA mutates at a faster rate than does
nuclear DNA (61, 542). This elevated mutation rate was initially attributed to inefficient DNA repair in mitochondria,
although it is now clear that mitochondria from both lower
and higher eukaryotes are proficient in repairing several types
of lesions, including oxidative DNA modifications, which are
thought to be the most prevalent lesions in mtDNA (347).
Interestingly, a work by Kienhofer et al. (266) has demostrated
that mtDNA colocalizes with antioxidant enzymes, thus indicating the occurrence of a close and integrated mechanism
of protection against mtDNA oxidatiion. Due to the impact of
heteroplasmy, pathological mutations in mtDNA only cause
disease if they accumulate above a tissue-specific threshold.
Thus, the epidemiology of diseases caused by mutations in
mtDNA is quite distinct from that of mitochondrial diseases
caused by mutations in nuclear DNA. In a study with neonatal cord blood samples, looking for de novo mtDNA mutations, Elliott et al. found that at least 1 in 200 individuals
harbor a pathogenic mtDNA mutation (156). Clinically, mitochondrial diseases present in 1 in 10,000 individuals, making them a major group of inherited human diseases (100),
although it should be pointed out that the clinical classifications do not distinguish diseases caused by mutations in
mtDNA from those caused by mutations in nuclear genes.
Nevertheless, the extremely high frequency of mtDNA mutations in the general population also emphasizes the importance of the nuclear background in determining the clinical
outcome of carrying a mtDNA mutation.
MITOCHONDRIA AND REACTIVE NITROGEN AND OXYGEN SPECIES
Mutations in mtDNA can occur at all regions, but diseasecausing mutations are most often found in tRNA genes and in
the protein coding genes. The first mutation in mtDNA
causing a human syndrome was identified in 1988, in a patient
suffering from Leber’s hereditary optic neuropathy (541). This
mutation was a base substitution in a highly conserved arginine in the ND4 subunit of Complex I. Since then, more than
300 pathogenic mutations have been identified in human
mtDNA (207). However, it is noteworthy that most mtDNA
mutations cause variant forms of a few complex syndromes,
with overlapping phenotypes. For an updated and comprehensive review of clinical symptoms of mitochondrial diseases, please refer Greaves et al. (207).
As is the case for mitochondrial diseases caused by mutations in nuclear DNA, therapeutic approaches to diseases of
mtDNA are scarce and often unsuccessful (447, 460, 461).
Pharmacological or nutritional interventions similar to those
discussed earlier have been tried. However, some effort has
also been put into gene replacement therapy. The allotropic
expression of a mammalianized copies (since mitochondria
use a different genetic code from the nucleus) of Leber’s hereditary optic neuropathy-mutated ND4 gene has been successful in restoring respiration in a cellular model of the
disease (212). An efficient allotropic expression of the protein
was also observed in the eyes of mice infected with viral
constructs (211). Other approaches being tested include the
manipulation of heteroplasmic levels of mutated mtDNA by
expressing mitochondrially targeted endonucleases which
recognize only the mutated molecules (18), and manipulation
of signaling pathways that control mitochondrial function,
such as the AMPK/PGC1-a axis (535). Another recent approach was the long-term expression of parkin, an E3ubiquitin ligase that targets mitochondria to degradation (15)
in cells with heteroplasmic COXI mutations (490). The authors
observed a shift in mtDNA heteroplasmy toward the wild-type
genomes, suggesting that the molecular mechanisms which
maintain mitochondrial quality control may also be therapeutic
targets in diseases with high levels of mtDNA mutations in
heteroplasmy. These results are somewhat encouraging, but
the feasibility of such approaches as effective human therapies
remains to be determined. Nonetheless, given the high prevalence of mitochondrial diseases, and implication of mitochondrial dysfunction in more common pathologies, efforts to
devise effective therapies should be a priority.
VI. Redox Imbalance and Cancer
Progressive changes in ROS and RNS production are observed during malignant transformation (498, 507), and experimental, clinical, and epidemiological evidence indicates
that these reactive species are causally involved in malignant
transformation and progression. Indeed, large association
studies report a strong linkage between polymorphisms in
antioxidant enzymes (e.g., GSTs and SOD2) and genetic predisposition to cancer (19, 153). Moreover, increased H2O2 and
O2c - levels have been associated with tumor aggressiveness
and poor disease outcome (289, 409).
The precise pathways leading to oxidative and nitrosative
imbalance in tumor cells remain to be uncovered. Mitochondrial dysfunction and oncogenic stimulation by key oncogenes and tumor suppressor genes are intrinsic factors known
to cause redox imbalance in tumor cells. In addition, extrinsic
2047
factors such as inflammatory cytokines, nutrient deprivation, and the hypoxic tumor environment have also recently
been associated with oxidative imbalance in tumor cells (17,
169, 421).
The intrinsic mechanisms causing increased redox imbalance in tumor cells have been more comprehensively studied
when compared with extrinsic mechanisms. In particular, the
association between oncogenic stimulation and increased
mitochondrially derived ROS has been well investigated.
Activation of key oncogenes and inactivation of tumor suppressor genes (e.g., K-Ras, C-Myc, p53) are known to raise
H2O2 and O2c - levels in tumor cells (245, 448, 518). In a recent
study, Hu et al. (238) showed that K-Ras activation led to
suppressed respiratory chain complex I activity and a decrease in the mitochondrial transmembrane potential, by affecting the cyclosporin A-sensitive MPT, leading to increased
ROS production, decreased respiration, and elevated glycolysis (238). Moreover, gene expression studies on RAStransformed fibroblasts revealed significant changes in the
expression of nuclearly encoded genes involved in mitochondrial biogenesis and function (99). Interestingly, as we
will see next, oncogenic activation also induces a feedback
antioxidant response that enables tumor cells to escape severe
oxidative damage (129).
Mitochondrially generated ROS and RNS have been historically associated with nuclear DNA damage and genome
instability during malignant transformation (90). DNA mutation is a critical step in malignant transformation, and endogenous ROS and RNS account for a significant fraction of
nuclear DNA damage in tumor cells. Elevated levels of oxidative DNA lesions have been noted in various tumors, and
more than 100 oxidative DNA adducts have been identified to
date (40, 340, 342). The most extensively studied oxidative
DNA lesion is the formation of 8-OHdG (430), which has been
considered a potential biomarker of carcinogenesis (519).
Although nuclear DNA is susceptible to ROS-mediated
damage, mtDNA presents a more vulnerable target due to the
lack of protective histones and its close proximity to the
electron transport chain. As mentioned earlier, mitochondria
from both lower and higher eukaryotes are proficient in repairing different oxidative DNA lesions and, recently, Kienhofer et al. (266) demonstrated the co-localization of mtDNA
with both mitochondrial glutathione peroxidase (GPx1) and
manganese superoxide dismutase (SOD2). Based on these
observations, they proposed the existence of an integrated
antioxidant system in the nucleoid structure of the mitochondria to further protect mtDNA from superoxide-induced
oxidative damage (266).
Interestingly, the frequency of random mtDNA mutations
seems to be *70% lower in tumors when compared with
patient-matched normal tissues (162). By examining the
spectrum of random mtDNA mutations in carcinomas, adenomas, and normal colonic samples, Ericson et al. (162) observed that oxidatively mediated mutations, such as C:G to
T:A transitions, occured less frequently in the tumors samples
and proposed that the lower frequency of random mtDNA
mutations was associated with a decrease in ROS production
in the mitochondrial matrix as a result of the metabolic shift
from oxidative phosphorylation to glycolysis—a metabolic
feature of tumors known as the ‘‘Warburg effect.’’ Alternatively, but not mutually exclusively, tumors might also
have a more efficient nucleoid antioxidant system to protect
2048
mtDNA from superoxide-induced oxidative damage, although this remains to be addressed.
In contrast to random mutations, clonally expanded
mtDNA mutations resulting in frameshifts or nonsynonymous substitions have been observed in most cancers. Of
particular interest is the presence of mutations potentially
affecting mitochondrial respiratory chain function in cancer
patients, which can lead to leakage of electrons and the generation of O2c - . Inherited mutations affecting ND5, ND2, and
ND1, CYTb and COX1 genes have been reported in esophageal squamous cell carcinomas and in breast cancer patients
(330). Recently, Ishikawa et al. demonstrated that mitochondrial mutations compromising respiratory function favor
cancer metastasis through the generation of mitochondrial
ROS (246). Mutations affecting mitochondrial respiratory
chain function, whether in the nuclear DNA or in the mtDNA,
may result in increased ROS production, leading to a vicious
cycle of increasing DNA damage and mitochondrial dysfunction (Fig. 10). However, the functional relevance of the
majority of these alterations to tumorigenesis remains to be
determined, as clonally expanded mtDNA mutations have
also been frequently detected in adenomas, suggesting that
expansion can occur before malignancy (162).
FIGUEIRA ET AL.
In addition to DNA damage and genomic instability, in the
last decade, ROS have been shown to act as second-messenger
activating signaling pathways and transcription factors directly involved in tumorigenesis (318, 550). During malignant
transformation, tumor cells escape from senescence and apoptosis, survive as immortalized cells, and undergo uncontrolled proliferation to form primary tumors. At a latter step in
malignant progression, tumor cells induce the formation of
new blood vessels to support tumor growth, invade adjacent
tissues, reach the circulation, and proliferate in distant organs,
forming metastases. The multistep development of human
tumors, thus, requires the acquisition by the tumor cells of
biological capabilities known as the ‘‘hallmarks of cancer’’
(223). ‘‘Hallmarks of cancer’’ include replicative immortality,
resistance to cell death, sustained proliferation, evasion to
growth suppressors, induction of angiogenesis, activation of
invasion and metastasis, deregulation of cellular metabolism,
and evasion of the immune system (223). There has been an
increased focus on the role of redox signaling in malignant
transformation and progression, and evidence exists in the
literature supporting the participation of redox signaling
mediated by H2O2 in the acquisition of most of the hallmarks
of cancer [(404, 549), Fig. 10].
FIG. 10. Redox imbalance and cancer. Intrinsic and extrinsic factors contribute toward the appearance and maintenance of
increased oxidative stress in tumor cells. Elevated ROS and RNS levels contribute to malignant transformation and progression by driving DNA damage and genomic instability and by activating signaling networks (RTKs, PTKs) and redoxsensitive transcription factors (HIF-1, NRF2) that promote tumor cell proliferation, survival, angiogenesis, altered metabolism, and invasiveness. DNA damage and altered metabolism, in turn, promote mitochondrial dysfunction, creating a vicious
cycle of ROS/RNS generation. Tumor cells also develop redox adaptation mechanisms to protect themselves from intrinsic
oxidative stress. These adaptation mechanisms are associated with treatment failure and are being explored to overcome
drug resistance. Compounds that either enhance ROS generation (electron transport chain modulators, redox cyclers) or
abrogate key antioxidant mechanisms (GSH depleting agents, SOD, Trx, and catalase inhibitors) in tumor cells are being used
to selectively kill cancer cells and improve cancer therapy. GSH, reduced glutathione; NRF2, nuclear factor-erythroid 2related factor 2; RNS, reactive nitrogen species; SOD, superoxide dismutase; Trx, thioredoxin-2; RTK, receptor tyrosine
kinases; PTK, protein tyrosine kinases.
MITOCHONDRIA AND REACTIVE NITROGEN AND OXYGEN SPECIES
Increased levels of ROS have been shown to activate different pathways important for tumor cell proliferation and
survival. H2O2 and O2c - accumulate in the cell following the
activation of tyrosine kinase growth factor receptors (EGF,
PDGF, and VEGF) by cognate ligands. Growth-factorstimulated ROS generation can mediate intracellular signaling
pathways by inhibiting protein tyrosine phosphatases (e.g.,
PTEN), activating several nonreceptor protein tyrosine kinases
involved in malignant transformation and progression (e.g., the
Src family, Janus kinase, Mitogen-Activated Protein Kinase
[MAPK], Extracellular Signal-Regulated Kinase and Protein
Kinase B), and regulating redox-sensitive transcription factors
(395, 521, 550). However, in many of the reported studies,
growth-factor-induced ROS generation is considered to be
mainly via NAD(P)H oxidase and a specific role for mitochodrially generated ROS remains to be demonstrated.
Increased levels of ROS have also been shown to activate
different pathways, leading to angiogenesis, deregulation of
cellular energetics, and metastasis. Central to these pathways
are the HIF transcription factors, consisting of HIF1A and
HIF1B subunits (544). The latter is stable under normal oxygen levels, while HIF1A is hydroxylated by prolyl hydroxylases under the same conditions, and subsequently
ubiquitinated by the HIF-specific Von Hippel-Lindau (VHL)
ubiquitin ligase, targeting HIF1A for proteasome-mediated
degradation (247). Mitochondrially derived H2O2 is involved
in the stabilization of HIF1A and activation of HIF transcriptional activity under low oxygen conditions, through the
inactivation of the prolyl hydroxylases, which target HIF1A
for proteasome-mediated degradation (269).
Once stabilized, HIF1A dimerizes with HIF1B and regulates the expression of genes mediating angiogenesis, metastasis, and cellular metabolism (467). HIF1A stabilization is a
major stimulus for increased vascular endothelial growth
factor (VEGF) production and the coordinated expression of
other angiogenic factors by tumor cells. Changes in cell polarity, adhesive properties, and motility have also been associated with HIF transcriptional activity via the up-regulation
of c-Met/HGF-R and the matrix-modifying enzyme lysyl
oxidase. Finally, HIF transcriptional activity is also responsible for increased expression of glucose transporters, glycolytic
enzymes (e.g., hexokinase and lactate dehydrogenase), and
pyruvate dehydrogenase kinase, promoting a global shift in
cellular metabolism from oxidative phosphorylation to glycolysis and providing tumor cells with important metabolic
intermediates (nucleic acids, proteins, and lipids) that are
necessary for cell proliferation and tumor growth (468).
Since tumor cells actively produce high levels of ROS and
are continuously exposed to endogenous oxidants, it is not
surprising that they also develop mechanisms to protect
themselves from intrinsic oxidative stress. Higher levels of
ROS-scavenging enzymes (e.g., SOD, GPx, and Prx) and antioxidant molecules have been observed in tumors compared
with normal tissues (239, 250). Moreover, treatment of normal
epithelial cells with low levels of exogenous oxidants has been
shown to confer cellular resistance to subsequent oxidative
challenges (104). These observations support the idea that
persistent ROS stress may induce an antioxidant response,
enabling cancer cells to escape severe oxidative damage. Recent evidence also suggests that such adaptation contributes
to malignant progression and resistance to anticancer therapy
(297, 412, 463, 556).
2049
ROS levels are tightly controled by an inducible antioxidant
program regulated by the nuclear factor-erythroid 2-related
factor 2 (NRF2) and its cytosolic repressor protein Kelch ECH
associating protein 1 (KEAP1). NRF2 is a transcription factor
that binds to antioxidant response elements and regulates the
expression of antioxidant enzymes and detoxifying enzymes
such as Prx, thioredoxin, catalase, Cu/Zn SOD, glutathione Stransferase A2, and NADPH quinone oxidoreductase 1 (378).
Under normal conditions, NRF2 transcriptional activity is
associated with and suppressed by KEAP1, which binds to
NRF2 and promotes its proteasome-mediated degradation,
keeping antioxidant gene expression tightly regulated.
However, on ROS exposure, KEAP1 undergoes a conformational alteration, freeing NRF2 to translocate to the nucleus
and driveing the expression of antioxidant and detoxifying
enzymes (324). Increased NRF2 activity in tumors can be
promoted by somatic mutations that disrupt NRF2-KEAP1
interaction or by oncogenic activation as previously mentioned. In a recent study, DeNicola et al. (129) observed that
endogenous expression of the KRAS and BRAF oncogenic
alelles increased NRF2 transcription, activating an antioxidant program and lowering intracellular ROS levels.
Since tumor cells have higher levels of ROS when compared with normal cells, they should be, at first sight, more
vulnerable to death by ROS-promoting agents. Therefore,
increasing ROS levels could represent an effective way to selectively kill cancer cells without causing significant toxicity to
normal cells and undesirable side effects. Indeed, many
commonly used anti-cancer drugs such as doxorubicin, paclitaxel, and platinum-based drugs promote ROS-mediated
cell killing. However, redox adaptation, through the activation of anti-oxidants mechanisms, confers tolerance to exogenous stress and contributes to the drug-resistant phenotype
of tumor cells [(297, 412, 463), Fig. 10]. The thioredoxin system, for example, is up-regulated in tumor cells, and elevated
levels of this thiol-based antioxidant system correlate with
poor prognosis and drug resistance (85). Thus, the use of
compounds that abrogate the antioxidant response in tumor
cells represents an alternative strategy to selectively kill cancer cells. This concept has recently been proved by Raj et al.
(428) in a search for small molecules that selectively kill cancer
cells. Using small-molecule screening and quantitative proteomics, they found that Piperlongumine, a natural product
isolated from the plant species Piper longum L., increased ROS
levels and apoptotic cell death in cancer cells. Piperlongumine
also exhibited in vivo antitumor and antimetastatic effects in a
mouse model (428).
Indeed, compounds that either enhance ROS generation or
abrogate key antioxidant mechanisms in tumor cells have
been used in combination with radio- and chemotherapy to
improve therapeutic response and to circumvent chemotherapy resistance, with a significant clinical impact [(357, 463),
Fig. 10]. Several compounds that promote ROS generation
such as mitochondrial electron transport chain modulators
(e.g., arsenic trioxide) and redox-cycling compounds (e.g.,
motexafin gadolinium), or which abrogate antioxidant
mechanisms such as GSH-depleting agents (e.g., buthionine
sulphoximine) and SOD inhibitors (e.g., 2-methoxyestradiol),
are currently being used in clinical trials for different types of
cancer (357). Motexafin gadolinium is being used in phase II
clinical trials for treating refractory chronic lymphocytic leukemia and brain metastases (316). The combination of arsenic
2050
trioxide and ascorbic acid-mediated GSH depletion was also
shown to improve treatment efficacy in refractory multiple
myeloma (20), and phase I trials combining the use of Imexon—a GSH-depleting compound—and docetaxel are ongoing for lung, breast, and prostate cancer (362). Finally,
brusatol, a compound found in plant extracts, increases ubiquitination of NRF2, inhibiting its activity and rendering xenografts more sensitive to the chemotherapeutic agent
cisplatin (432).
Improving therapeutic selectivity is a major goal in the
development of novel anti-cancer drugs, and exploration of
the biochemical differences between normal and tumor cells
leads to the emergence of several molecular-targeted drugs
(e.g., Herceptin, Avastin, Gleevec), with enhanced therapeutic
activity and fewer side effects (214). As we saw in this section,
increased levels of ROS, coupled to redox adaptation to protect tumor cells from intrinsic oxidative imbalance, are responsible for the appearance and maintenance of the cancer
phenotype. Targeting these biochemical properties of tumor
cells with redox-modulating compounds will likely prove in
the near future to be an effective way to selectively kill tumor
cells and to circumvent drug resistance.
VII. Neuronal Damage and Disorders Associated
with Mitochondrially-Generated ROS
Since neurons are longlived and highly dependent on oxidative metabolism, they are uniquely predisposed to accumulate injury by endogenously produced ROS. Even under
nonpathological conditions, human brain tissue presents cumulative effects of ROS, described early on in anatomopathological evaluations of elderly brains as accumulation of
granules of lipofuscin, a yellowish-brown pigment composed
of protein and lipid oxidation products and localized within
the lysosomes of cells [for review, see (62)]. An important role
for mitochondrially generated ROS under nonpathological
conditions in the accumulation of brain oxidative products
was recently demonstrated in mice chronically treated with
the mitochondrial uncoupler dinitrophenol (72). A decreased
accumulation of protein carbonyls and 8-OHdG in brain tissue was observed after 1 or 5 months of continuous treatment
of mice with dinitrophenol in the drinking water. Although an
increased survival was observed in dinitrophenol-treated
mice (72), future studies seek to evaluate whether these mice
will perform better in behavioral tests.
An indication for mitochondrial uncoupling as an important regulatory mechanism in the central nervous system was
the characterization of three mitochondrial uncoupling proteins (UCP2, UCP4, and UCP5) in brain tissue. Furthermore,
rats submitted to a 3 min period of sublethal ischemia, which
leads to ischemic preconditioning, showed an increased expression of UCP2 in hippocampal CA1 field (346), indicating a
role of UCP2 in neuroprotection. In fact, several studies
demonstrated that UCP2 overexpression is associated with
neuroprotection in experimental models of ischemia (35, 126,
346), seizures (135, 491), and trauma (346). In addition, mice
overexpressing UCP2 present decreased 1-methyl-4-phenyl1,2,5,6 tetrahydropyridine (MPTP)-induced nigral dopaminergic cell loss (10). UCP4 (320, 549) and UCP5 (267, 290) have
also been associated with neuroprotection. In this framework,
mitochondrial redox imbalance has been implicated in neural
cell death associated with various disorders. Next, we will
FIGUEIRA ET AL.
present evidence for the involvement of mitochondrial redox
imbalance in stroke and Parkinson’s disease.
Brain lesion after stroke is not an immediate process.
Within the penumbra region, cell death becomes prominent
between 24 and 72 h after reperfusion after a 30 min period of
middle cerebral artery occlusion in mice (142, 160). Redox
imbalance may be one of the events that determine neuronal
cell death in the penumbra region. In fact, early experimental
evidence indicates that some antioxidants, such as lazaroids
and N-tert-butyl-alpha-phenylnitrone (220, 416), are potent
protective agents in animal models of brain ischemia.
In addition to different systems for oxidant generation,
such as NOS, NADPH oxidase, and the cyclooxygenase and
lipoxygenase pathways, mitochondria are also a source of
ROS in stroke. In fact, hippocampal mitochondria isolated
after transient global ischemia show an increased production
of ROS at time points that precede neuronal death (185). In
addition, some reports have provided in vivo evidence of increased mitochondrial ROS generation in experimental
models of brain ischemia using microdialysis and the salicylate trap (417) or hydroethidine oxidation (268) techniques.
Mitochondrial aconitase, a citric acid cycle enzyme inactivated by O2c - (190), has also been found to be inhibited in
cortical neuron cultures subject to oxygen-glucose deprivation (312).
An impairment of the respiratory complexes may be the
underlying cause of higher mitochondrial production of ROS
in stroke [for a review, see (361); Fig. 11]. There are several
studies indicating dysfunction of mitochondrial respiratory
chain complexes I-IV and ATP synthase in experimental
models of brain ischemia/reperfusion (7, 376, 426, 475). Respiratory chain complexes may be the target of RNS or ROS
(356, 361). Activation of nonmitochondrial ROS generation
during brain ischemia or reperfusion may lead to the formation of these reactive species.
One of the early triggers of cellular and mitochondrial
dysfunction during stroke is excitotoxicity (444) (Fig. 11), a
central nervous system process in which an increased glutamate release due to energy deprivation results in neuronal
necrosis or apoptosis (176). This glutamate-mediated neuronal cell death is promoted mainly by activation of N-methylD-aspartate receptors, resulting in Ca2 + and Na + influx (103,
444). The involvement of redox imbalance in excitotoxicity
was demonstrated by several studies showing the protective
effect of antioxidants in in vitro (293, 410) or in vivo experimental models (368, 462). Under excitotoxic conditions, mitochondria are the main organelles responsible for Ca2 +
sequestration (as discussed earlier), an event associated with
neuronal cell death as long as inner membrane potentials are
maintained by respiration or ATP hydrolysis (82, 382).
As previously discussed in this review, increased Ca2 +
concentrations in the mitochondrial matrix may induce mitochondrial redox imbalance and MPT (Fig. 4). Schild and
Reiser (458) reported that brain mitochondria subjected to
hypoxia/reoxygenation in the presence of low micromolar
Ca2 + concentrations generated higher levels of H2O2 during
reoxygenation. Mitochondrial Ca2 + accumulation may also
result in a reversible inhibition of H2O2 elimination (509). In
addition, Vaseva et al. (523) have shown that p53 accumulates
in mitochondria and trigggers MPT in response to redox imbalance during brain ischemia. In fact, neuronal death after
hypoglycemia and brain ischemia is prevented by MPT
MITOCHONDRIA AND REACTIVE NITROGEN AND OXYGEN SPECIES
2051
FIG. 11. Scheme summarizing events associated with
brain mitochondrial redox
imbalance that may contribute to neuronal damage after
brain ischemia and reperfusion.
inhibitors (184, 344). However, the participation of MPT in
excitotoxicity is controversial (63, 82, 313, 333). A possible
explanation for the lack of or limited participation of MPT in
several experimental models of excitotoxicity is the presence
of high levels of endogenous inhibitors of this phenomenon,
such as ADP and Mg2 + (449).
Finally, mitochondrial ROS production can also comprise a
protective signal for brain organelles in stroke. We showed
that ROS produced at the respiratory chain activate brain
mitochondrial ATP-sensitive K + channels, a process associated with protection against excitotoxicity (178).
Parkinson’s disease is characterized by rigidity, resting
tremor, bradykinesia, and postural instability, accompanied
by predominant degeneration of dopaminergic neurons in the
substantia nigra. Most cases of Parkinson’s disease are considered idiopathic and sporadic. In addition to altered protein
handling and inflammation, mitochondrial dysfunction
and redox imbalance have been indicated as important factors
in the pathogenesis of Parkinson’s disease [for review, see
(74, 453)].
Postmortem studies have provided evidence of a partial
inhibition (20%–40%) of mitochondrial respiratory chain
complex I (454, 455) and oxidative damage (130, 167) in brains
from idiopathic Parkinson’s disease patients. Respiratory
complex I inhibition was also observed in peripheral tissues
(45, 408). Although the origin of respiratory chain complex I
deficiency is unknown, it may be related to cumulative
mtDNA deletions (36). The importance of complex I inhibition
in Parkinson’s disease pathology is also supported by the fact
that humans exposed to MPTP, whose metabolite 1-methyl-4phenylpyridine (MPP + ) inhibits complex I (383), develop
parkinsonism (298). In addition, Greenamyre’s group also
showed that systemic treatment of rodents with the classical
complex I inhibitor rotenone leads to anatomical, neurochemical, behavioral, and neuropathological features of Parkinson’s disease (42, 75).
The mechanism involved in complex I inhibition-mediated
dopaminergic neurodegeneration probably involves increased mitochondrial ROS generation [(29, 453, 488); Fig. 12].
Complex I inhibition in isolated brain mitochondria results in
a marked stimulation of mitochondrial H2O2 production
through a mechanism linked to an increased NAD(P)H/
NAD(P) + ratio (488). In addition, a partial inhibition of
complex I activity can contribute toward excitotoxicity in a
mechanism that involves impaired mitochondrial oxidative
phosphorylation (557).
Why dopaminergic neurons are more sensitive to complex I
inhibition is still unknown; of note, dopaminergic and nondopaminergic presynaptic synaptosomes present similar
bioenergetic capacities (105). One possibility would be that an
increased mitochondrial ROS production due to complex I
inhibition may lead to dopamine redistribution from vesicles
to the cytosol (546). Dopamine is readily oxidized in the cytosol into H2O2, O2c - and various dopamine metabolites
(327). The higher susceptibility of substantia nigra pars compacta dopaminergic neurons to degeneration may also be related to increased cytosolic Ca2 + concentrations due to Ca2 +
influx via L-type calcium channels [(91, 494); Fig. 12]. In this
regard, Ca2 + and complex I inhibition synergistically increase
brain mitochondrial ROS release and neuronal death (482,
483), in a mechanism associated with MPT induction (537).
Parkinsonism can also be associated with disturbance in
mitochondrial quality control (371). Mitochondrial membrane
depolarization leads to an increased expression level of
PINK1 on the outer mitochondrial membrane (343, 371, 536)
and accumulation of PINK1 signals toward the recruitment of
parkin from the cytosol to the impaired (depolarized) mitochondria (370). Parkin, an E2 ubiquitin ligase, promotes the
ubiquitinization of outer membrane proteins, ultimately
leading to degradation of the organelle by autophagy (371). A
failure in this process may explain neurodegeneration observed in the familiar form of Parkinson’s disease due to lossof-function mutations within the PARK2 locus, which encodes
parkin. In addition, Johnson et al. (257) have shown that
parkin has an antiapoptotic role by ubiquitinating and, consequently, inactivating the proapoptotic protein BAX in the
cytosol.
VIII. Redox Imbalance in Cardiovascular Diseases
Cardiovascular complications are a major public health
problem; acute myocardial infarction and heart failure are
2052
FIGUEIRA ET AL.
FIG. 12. Putative mechanisms involving redox imbalance and degeneration of substantia nigra pars
compacta (SNpc) dopaminergic neurons in the sporadic form of Parkinson’s disease. Inflammation and
altered intracellular protein handing
are additional mechanisms proposed
to take part in the degeneration of
SNpc dopaminergic neurons.
leading causes of morbidity and death worldwide. According
to the World Health Organization, more than 7 million people
die of ischemic heart disease every year. Consequently, fundamental mechanisms responsible for the establishment and
progression of cardiovascular diseases, as well as the generation of novel pharmacological and nonpharmacological interventions, should be extensively studied. In this section, we
describe the critical role of mitochondrial metabolism and
redox signaling in cardiac pathophysiology.
Mitochondria have emerged over the last decades as the
critical organelle in maintaining redox balance and in regulating survival and death pathways in cardiomyocytes (236).
Considering their high demand for ATP synthesis and elevated oxygen uptake rate, cardiac cells have the highest volume density of mitochondria in the entire body (512)—
mitochondria constitute 30% of myocardial mass (98). In
cardiovascular diseases, mitochondria in the heart (i.e., cardiomyocytes and endothelial cells) are thought to be the major
source of ROS and RNS (351, 391, 399). Specific forms of ROS
and RNS generated during acute (ischemia-reperfusion injury) or chronic (hypertension and heart failure) cardiovascular
diseases by dysfunctional mitochondria include H2O2, O2c - ,
NOc, and ONOO - . These species can react with membrane
phospholipids, DNA, and proteins, which, in turn, exacerbate
mitochondrial dysfunction, oxidative stress, and accumulation of lipid reactive species (i.e., 4-HNE) in a feed-forward
loop. This cyclic process results in protein aggregation, cellular collapse, and decreased cardiac viability (107, 401).
A. Redox imbalance during ischemia-reperfusion
Acute myocardial infarction is characterized by changes in
biochemical properties during ischemia and reperfusion. The
heart can survive a short period of ischemia by reducing
myocardial contractility, increasing glucose uptake, and
switching metabolism to glycolysis. However, considering that
the heart is one of the most energy-demanding tissues in the
body, sustained oxygen and nutrient deprivation results in irreversible damage. Thus, reperfusion of the ischemic heart is a
prerequisite for survival. Paradoxically, reperfusion can further
increase the myocardial damage that occurs during ischemia.
The severity of reperfusion injury depends on the duration of
the preceding ischemia and the effectiveness of blood flow
during reperfusion. Several lines of evidence demonstrate that
reperfusion injury is directly associated with cardiac mito-
chondrial dysfunction and increased ROS and RNS generation
(95, 96). Hearse et al. (228) noted that reperfusion of isolated
hearts after ischemia resulted in abrupt cardiomyocyte death.
Following this paper, several studies showed that ischemia
reperfusion is associated with a burst of H2O2, O2c - , NOc, and
ONOO - , but the exact mechanism of their generation is debated. Although some ROS may be generated by NADPH
oxidase and xanthine oxidase, it is likely that complexes I and
III of the mitochondrial respiratory chain are the main sources
of ROS during myocardial ischemia reperfusion (95, 405). In
fact, studies using mitochondrial respiratory inhibitors show
that the electron leak along the oxidative phosphorylation most
likely occurs at the Fe-S centers of complex I and at some
components of complex III (70, 323, 389). During the early
stages of reperfusion, ROS generation levels increase drastically
(477). Interestingly, low amounts of ROS generated by mitochondria during brief and intermittent episodes of ischemia,
termed ischemic preconditioning, significantly protect the
heart against prolonged ischemia (364).
Ischemic preconditioning-mediated cardioprotection is
driven by activation of cytosolic, nuclear, and mitochondrial
signaling pathways. Data indicate that many of these intracellular signals converge to the mitochondria, resulting in
lower mitochondrial oxidative damage during prolonged ischemia and reperfusion events (364). Interestingly, ischemic
preconditioning benefits are lost by a co-intervention with
antioxidants, an evidence of its redox signaling nature. Several studies have demonstrated that avoiding exacerbated
ROS and RNS generation by preconditioning is a crucial step
in protecting mitochondrial key proteins against oxidation
during ischemia and reperfusion (73, 364). Although the
functional benefits of ischemic preconditioning are well described, the underlying molecular mechanisms are still uncertain. Of interest, activation and further mitochondrial
translocation of specific cytosolic kinases such as PKCe, ERK,
and AKT seems to play a key role in priming organs against
sustained ischemic events (66, 354).
Over the last decade, oxidative protein post-translational
modifications such as oxidation and nitration have been associated with cardiac ischemia-reperfusion-related damage
(514, 565). Conversely, NOc-mediated protein S-nitrosylation
plays a pivotal role in preconditioning-mediated cardioprotection (399, 505). During a prolonged ischemia-reperfusion
episode, exacerbated ROS generation oxidizes key proteins
involved in cellular metabolism, contractility, and protein
MITOCHONDRIA AND REACTIVE NITROGEN AND OXYGEN SPECIES
quality control in the heart, favoring cell dysfunction and
death (399). Moreover, O2c - reacts with NOc, forming
ONOO - , a key effector of cardiomyocyte cell death (328).
Peroxynitrite affects cardiomyocyte viability through different mechanisms, including mitochondrial dysfunction, DNA
damage, activation of poly(ADP-ribose) polymerase, and
protein inactivation (reacting with tyrosine and thiol groups)
(328, 398). However, at low concentrations, even ONOO - has
been reported to be cardioprotective through the reaction of
its subproducts NO2 - and NO3 - with glutathione, forming Snitrosoglutathione (73).
Excessive ROS and RNS production can induce MPT and
cell death (551). ROS-induced MPT collapses the mitochondrial membrane potential, increases ROS and RNS release,
triggers mitophagy, and results in cardiomyocyte death,
characterizing a positive feedback loop of ROS-induced ROS
release [(287); Fig. 13]. In this case, mitophagy functions as an
early cardioprotective response, removing damaged mitochondria. Zorov et al. (569) have shown that intracellular
photoactivation of tetramethylrhodamine compounds triggers exacerbated ROS release in isolated cardiomyocytes,
which results in mitochondrial depolarization, along with
prolonged MPT opening and a consequent burst of ROS and
RNS from mitochondria.
2053
Despite the increased knowledge regarding the molecular
basis of mitochondrial ROS and RNS generation and redox
balance in cardiovascular diseases, there is still uncertainty in
the literature regarding the cellular node/transducer molecules responsible for communicating mitochondrial and cytosolic signals during pathological conditions such as
ischemia-reperfusion injury. The serine threonine protein kinase C family (PKC) is a good candidate for mediating the
cross-talk between mitochondria and the cytosol signaling
under cardiac redox imbalance conditions, as PKC contains
domains that are susceptible to oxidative modification, including zinc-binding and cysteine-rich motifs (206). Among
different PKC isozymes expressed in the heart, dPKC seems to
play a key role in ischemia-reperfusion injury (Fig. 13).
The first evidence for hydrogen peroxide-induced dPKC
translocation from the cytoplasm to the mitochondria was
provided by Majumder et al. (336). In fact, mitochondrial
translocation of dPKC was associated with the loss of mitochondrial membrane potential and the release of cytochrome c. The biological relevance of this phenomenon was
supported by the demonstration that H2O2-induced apoptosis
was blocked by the inhibition of dPKC translocation to mitochondria. Since then, several publications have described
the role of dPKC in aspects of mitochondrial biology, such as
FIG. 13. dPKC activation-mediated mitochondrial dysfunction during ischemia-reperfusion injury. Increased ROS
during reperfusion activates dPKC through direct oxidation of redox-sensitive residues within the isozyme. Active dPKC
translocates into mitochondria and regulates mitochondrial function by phosphorylating/inhibiting (P) complexes I, III, and
V. These events result in exacerbated ROS generation. Further increases in ROS will result in positive feedback to amplify
dPKC activation. In addition, oxidation of phospholipids in the inner mitochondrial membrane results in destabilization and
releasing of cytochrome c (cyt c). Under this condition, MPT pore opening is likely to occur. Finally, dPKC-mediated ROS
generation results in mtDNA damage. All these events contribute to cell death. Thus, dPKC could be defined as a redox
node/transducer modulating a tunable system during ischemia-reperfusion injury. (To see this illustration in color, the reader
is referred to the web version of this article at www.liebertpub.com/ars.) PKC, protein kinase C family.
2054
ROS generation, cytochrome c release, and phosphorylation.
Given the effects of dPKC on mitochondrial function, it could
be targeted in therapeutic interventions against ischemiareperfusion injury. Interestingly, selective inhibition of dPKC
using the peptide inhibitor dV1-1 reduces ischemia-reperfusion
injury in cardiac myocytes, ex vivo Langendorff-perfused
hearts, and in animal models of acute myocardial infarction
(244). Moreover, intracoronary delivery of dV1-1 during cardiac reperfusion may promote myocardial protection in patients with acute ST-elevation myocardial infarction (33).
Reperfusion leads to accumulation of dPKC within the
mitochondria (sixfold increase), which results in reduced ATP
levels, increased mitochondrial Bad/Bcl-2 ratios, elevated
proapoptotic cytochrome c release, PARP cleavage, and DNA
fragmentation (107, 366). Inhibition of dPKC translocation
during cardiac reperfusion leads to a faster recovery of ATP
levels in the heart (244). Nguyen et al. (379) demonstrated that
dPKC inhibits F1Fo activity via an interaction with the ‘‘d’’
subunit of F1Fo-ATP synthase in neonatal cardiomyocytes.
Treatment of cardiomyocytes with selective peptides that
block the interaction between dPKC and F1Fo-ATP synthase
abolishes the phorbol ester (a nonselective PKC activator)induced inhibition of F1Fo-ATP synthase activity (379).
dPKC also regulates mitochondrial morphology through
regulation of the Drp1, a large GTPase protein required for
mitochondrial fission in mammalian cells. A number of
stimuli lead to the translocation of Drp1 from the cytosol to
the mitochondria, where it binds to Fis1, a protein located in
the mitochondrial outer membrane. Cell culture studies
demonstrated that excessive mitochondrial fission is associated with mitochondrial dysfunction and apoptosis in neurons (31). Activation of dPKC induces aberrant mitochondrial
fragmentation and mitochondrial dysfunction in neuronal
cells and in a rat model of hypertension-induced encephalopathy (424). During oxidative imbalance, dPKC promotes
mitochondrial fission by interacting with and phosphorylating Drp1 (424), resulting in further increased Drp1 GTPase
activity. Importantly, inhibition of dPKC using either pharmacologic or molecular tools reduced mitochondrial fission
and fragmentation and conferred protection in cultured
neurons and in the brain (424). During cardiac ischemia–
reperfusion, mitochondria also undergo excessive fragmentation, which is associated with decreased mitochondrial
membrane potential, opening of MPT pore, and apoptosis
(394). However, the role of dPKC in this process remains to be
clarified.
Important insights into ROS-mediated dPKC activation
have been derived from findings that ROS induces dPKC
phosphorylation (Tyr-512 and Tyr-523) in a lipid cofactorindependent manner (274). Moreover, S-cysteinylation or
glutathione depletion results in dPKC activation-induced
apoptosis in response to H2O2. Recently, Zhao et al. (566)
demonstrated that both lipid agonist binding and oxidation
release the zinc-finger centers of PKC, which contributes to its
activation (566). This process seems to coordinate mitochondrial dPKC activation through a dPKC/p66Shc/vitamin A/
cytochrome c complex (2). Therefore, ROS-mediated dPKC
activation may be a key factor in reperfusion injury. In addition, selective activation of dPKC stimulates ROS generation.
Inhibition of dPKC translocation to mitochondria completely
abolishes ROS generation in acute renal failure (271). In the
heart, inhibition of dPKC translocation to mitochondria
FIGUEIRA ET AL.
during the first minutes of reperfusion blocks dPKC-mediated
impaired mitochondrial function and increased ROS production (458). In dPKC knockout mice, ischemic preconditioning fails to produce ROS and shows exacerbated
postischemic damage, which is related to a decreased antioxidant capacity in these mice (308, 348). These findings
suggest that the redox-sensitive dPKC works in a feed forward loop to regulate mitochondrial ROS production and
may provide an important switch, allowing the system to
respond to ischemia reperfusion in a graded fashion (Fig. 13).
To date, cardiac mitochondrial ROS-generating targets for
dPKC have not been clearly identified. There are several interesting candidates, including the NADPH oxidase-like activity of complex I (567), pyruvate dehydrogenase kinase 2,
and cytochrome c (321). Overall, translocation of dPKC to
mitochondria is a hallmark of cardiac injury after ischemia
reperfusion. At the early stages of reperfusion, ROS levels
quickly increase within the cardiomyocyte, which can activate
dPKC through oxidative modifications. dPKC translocates to
mitochondria, impairs mitochondrial metabolism, and amplifies ROS generation through the following mechanisms:
activation of pyruvate dehydrogenase kinase 2, which can
block complex I activity (108); activation of the NADPH oxidase-like activity of complex I; and activation of the phospholipid scramblase 3, which increases cardiolipin oxidation
and cytochrome c derangement and release. These changes
would be expected to increase cellular oxidative imbalance,
further activating dPKC and resulting in a positive feedback
loop. Thus, dPKC could be defined as a redox node/transducer, modulating a tunable system in which cell damage
only occurs if the initial stimulus is large enough to evoke a
prolonged response.
Another protein kinase C isozyme (ePKC) has been considered a key regulator of cellular signal transduction
involved in ischemic preconditioning-mediated cardioprotection. Analysis of ePKC subcellular distribution in rodents
overexpressing a constitutively active cardiac ePKC revealed
that ePKC forms signaling complexes with a number of mitochondrial proteins (25). Further, in wild-type mice hearts,
ePKC was colocalized in the mitochondrial vicinity; whereas
in constitutively active ePKC mice or as a result of ischemic
preconditioning, ePKC was present in the mitochondrial
matrix (64). Several mitochondrial targets of ePKC have been
described over the last years, including those regulating ion
transport, MPT, the electron transport chain, and ROS generation (25, 65, 95, 171). Finally, one of the cardioprotective
mechanisms mediated by ePKC is an increased aldehyde dehydrogenase 2 (ALDH2) activity during ischemia, which increases the capacity to metabolize the excess of cytotoxic
reactive aldehydes (i.e., 4-HNE) derived from mitochondrial
dysfunction-mediated ROS generation (95).
B. Redox imbalance in hypertension
Although ROS work as intracelular messengers in the
healthy vasculature by promoting short-term signaling
events, they are also involved in the establishment of diseases
related to vascular dysfunction, such as hypertension and
atherosclerosis (26).
Hypertension is a degenerative disease characterized by
endothelial dysfunction and neurohumoral disruption of
vascular tone. Irrespective of its etiology, hypertension has
MITOCHONDRIA AND REACTIVE NITROGEN AND OXYGEN SPECIES
been associated with increased H2O2, O2,c - and ONOO generation along with lower NOc bioavailability in the vasculature. At the cellular level, this redox status is closely related to activation of protein kinases (dPKC, MAPK, and
tyrosine kinases), transcription factors (AP-1 and HIF1), and
proinflammatory genes (358), which further contribute to elevated peripheral resistance and increased blood pressure.
Since recent studies have implicated a role for H2O2 and
O2c - in the pathogenesis of hypertension (138, 146), an extensive interest in finding the main source of ROS in the cells
of vasculature wall has emerged. Among different nonenzymatic and enzymatic systems that generate free radicals,
hypertension-related ROS production is mainly associated
with NADPH oxidase, xanthine oxidoreductase, mitochondrial enzymes, and uncoupled NOcsynthase (138). Haque and
Majid (226) have recently shown that mice lacking the gene for
gp91PHOX, a subunit of NADPH oxidase, are more resistant to
high salt-diet-induced hypertension. Moreover, Doughan
et al. (146) and Wosniak et al. (555) demonstrated a cross-talk
between NADPH oxidase and mitochondrial O2c - production
in angiotensin-II-exposed endothelial cells. Elucidating data on
the role of mitochondrial oxidative stress in hypertension were
provided by Dikalova et al. (140). These authors showed that
in vivo treatment with mitoTEMPO (a mitochondrially targeted
antioxidant) or overexpression of SOD2 reduced mitochondrial
O2c - generation and NADPH oxidase activity in hypertensive
animals. These changes were followed by increased vascular
NOcavailability, improved endothelial-dependent vasorelaxation, and promoted a 30 mmHg drop in blood pressure (140).
Administration of TEMPOL (a nontargeted antioxidant) did
not promote the same effects.
2055
C. Redox imbalance in heart failure
Heart failure is a clinical syndrome characterized by cardiac dysfunction and neurohumoral activation, which further
leads to low cardiac output, fluid retention, and decreased
survival. A broad variety of defects in respiratory chain machinery and components of oxidative phosphorylation complexes has been related to the progression of heart failure (442).
In fact, reduction of mitochondrial oxidative phosphorylation
is associated with an increase in electron leakage and O2c generation in complexes I and III, as discussed earlier. Recent
clinical and experimental studies have demonstrated that mitochondrial H2O2 and O2c - generation directly contributes to
the pathophysiology of cardiac remodeling and heart failure (3,
53). Sustained increases in H2O2, O2,c - and ONOO - generation in failing cardiomyocytes are catastrophic to the heart, as
they result in mtDNA damage, activation of a broad variety of
kinases, and transcriptional factors related to cardiac hypertrophy and disruption of excitation-contraction coupling (401)
(Fig. 14). Generation of excessive H2O2 stimulates mast cell
degranulation, cardiac inflammatory response, and fibroblast
proliferation, along with activation of matrix metalloproteinases (401). These processes are tightly related to the development and progression of pathological cardiac remodeling
and failure.
In the setting of haemodynamic overload, Oka et al. (393)
recently demonstrated that damaged mitochondria which
escape from autophagy are capable of inducing cardiac inflammation and dilated cardiomyopathy, linking mitochondrial damage to other features of heart faillure. The
mechanism behind this process is very interesting, as it
FIG. 14. A scheme depicting mitochondrial dysfunction-induced lipid peroxidation in cardiovascular diseases. Acute
(i.e., ischemia-reperfusion) and chronic (i.e., heart failure) cardiovascular events result in mitochondrial dysfunction and
increased ROS generation, which triggers lipid peroxidation and the accumulation of reactive aldehydes, such as 4-HNE.
Aldehydes induce inactivation of a number of macromolecules, including cytosolic proteins, components of the electron
transport chain, mtDNA, and inactivation of aldehyde dehydrogenase 2 itself. Excessive mitochondrial lipid peroxidation
may lead to a catastrophic cycle of mitochondrial functional decline, further ROS generation, and cardiomyocyte collapse. (To
see this illustration in color, the reader is referred to the web version of this article at www.liebertpub.com/ars.)
2056
involves mtDNA (leaked from damage mitochondria) activation of Toll-like receptor 9. Worth noting, due to the endosymbiotic origin of mitochondria, mtDNA is similar to
bacterial DNA and can trigger a sterile inflammation. So,
disrupted mitochondria could be involved in the genesis of
chronic inflammation in failing hearts (393).
Overexpression of antioxidant enzymes such as SOD or
GPx confers resistance against myocardial oxidative damage
and heart failure in mice (345). These effects were related to
the attenuation of cardiomyocyte hypertrophy, inflammation,
and interstitial fibrosis, which contributes to improved survival in animals. Similarly, overexpression of either GPx or
SOD reduced the severity of diabetic cardiomyopathy in animals (391, 512). Conversely, cardiac-specific SOD-deficient
mice developed heart failure associated with defects in mitochondrial respiration and excess O2c - formation (391).
Although early efforts focused on the harmful effects of
ROS and RNS in the failing myocardium, recent work revealed that accumulation of cytotoxic lipid peroxides derived
from ROS-mediated stress can impair cardiac function [(97);
Fig. 14]. Excessive peroxidation of phospholipids localized
within the inner mitochondrial membrane (i.e., cardiolipin)
changes the composition of the cardiac mitochondrial membrane and results in a drastic decline in the rate of oxidative
phosphorylation and ATP synthesis in heart failure (442).
Heart failure patients present increased circulating lipid peroxide levels, and the concentration of circulating lipid peroxides level such as 4-HNE and malondialdehyde inversely
correlates with left ventricular function (338).
Beside its effect on mitochondrial membrane composition,
augmented ROS-mediated lipid peroxidation during heart
failure results in extensive generation and accumulation of
toxic aldehydes, which can form adducts with lipids, DNA,
and proteins, and negatively affect the function of these
macromolecules (171). Selective activation of the mitochondrial enzyme responsible for removing these aldehydes,
ALDH2, protects the heart against pathological remodeling
and failure (493).
Recent evidence highlights monoamine oxidases as another important source of mitochondrial H2O2 in cardiovascular diseases, including ischemia-reperfusion injury,
cardiac hypertrophy, and heart failure (73, 261). Monoamine
oxidases are a class of flavoenzymes located in the outer
mitochondrial membrane responsible for oxidative deamination of neurotransmitters such as epineprine, norepinephrine, and dopamine. The products of monoamine
oxidases are aldehydes, ammonia, and H2O2 (261). In experimental animals, the pharmacological inhibition of
monoamine oxidase or its genetic ablation protects the heart
against acute (i.e., ischemia-reperfusion injury) and chronic
(i.e., heart failure) insults (44, 262). The likely benefits of
monamine oxidase inhibiton under such conditions may
also include higher cathecolamine availability in addition to
lower H2O2 formation (261).
Altogether, these findings highlight the central role played
by mitochondria in regulating different forms of cardiovascular diseases. Mitochondria do not only adapt their metabolism to cope with the demand for energy and substrates, but
also influence cellular morphology, function, and survival in
an ATP-independent manner, through the excessive generation of oxidants in the damaged heart. Finally, the abnormal
generation of ROS and RNS, as well as their byproducts,
FIGUEIRA ET AL.
clearly contributes to the establishment and progression of
cardiovascular diseases in both animal models and humans.
IX. Final Remarks
Overall, we discussed here a wealth of mechanisms that
promote and regulate mitochondrial oxidant production, as
well as the consequences of this production on mitochondrial
and cellular signaling events and within diseases. Given the
enormous progress in this filed in the last few years, it will
certainly be exciting to see what future studies will uncover in
this field.
Acknowledgments
Financial support has been rendered by Fundação de
Amparo à Pesquisa do Estado de São Paulo (FAPESP), Conselho Nacional de Desenvolvimento Cientı́fico e Tecnológico
(CNPq), Instituto Nacional de Ciência e Tecnologia de Processos Redox em Biomedicina (INCT Redoxoma), Núcleo de
Apoio à Pesquisa de Processos Redox em Biomedicina (NAP
Redoxoma), and The John Simon Guggenheim Memorial
Foundation. T.R.F. is currently supported by a postdoctoral
FAPESP fellowship.
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Address correspondence to:
Dr. Anibal E. Vercesi
Departamento de Patologia Clı́nica
Faculdade de Ciências Médicas
Universidade Estadual de Campinas
Rua Cinco de Junho, 350
NMCE Campus Unicamp (Lab. de Bioenergética)
Cidade Universitária
13083-877-Campinas
Brazil
E-mail: [email protected]
Date of first submission to ARS Central, June 1, 2012; date of
final revised submission, December 11, 2012; date of acceptance, December 17, 2012.
Abbreviations Used
4-HNE ¼ 4-hydroxy-2-nonenal
8-OHdG ¼ 8-hydroxydeoxy guanosine
ALDH2 ¼ aldehyde dehydrogenase 2
ANT ¼ adenine nucleotide transporter
CoQ ¼ Coenzyme Q
CypD ¼ cyclophilin D
Drp1 ¼ dynamin-related protein1
ER ¼ endoplasmic reticulum
ERK1/2 ¼ extracellular signal-regulated kinase
ETF ¼ electron transferring flavoprotein
FAOOH ¼ fatty acid hydroperoxides
FFA ¼ free fatty acid anion
FMN ¼ flavin mononucleotide
GPx ¼ glutathione peroxidase
GSH ¼ reduced glutathione
HIFs ¼ hypoxia inducible transcription
factors
KEAP1 ¼ Kelch ECH associating protein 1
MAPK ¼ mitogen-activated protein kinase
MPP+ ¼ 1-methyl-4-phenylpyridine
MPT ¼ mitochondrial permeability
transition
MPTP ¼ 1-methyl-4-phenyl-1,2,5,6
tetrahydropyridine
mtDNA ¼ mitochondrial DNA
mtNOS ¼ mitochondrial nitric oxide synthase
isoform
NDI1 ¼ NADH dehydrogenase
NOc ¼ nitric oxide
NO2 - ¼ nitrite
NO2 c ¼ nitrogen dioxide radical
NO3 - ¼ nitrate
NOS ¼ nitric oxide synthases
NRF2 ¼ nuclear factor-erythroid 2-related
factor 2
O2 c- ¼ superoxide anion
OHc ¼ hydroxyl radical
ONOO- ¼ peroxynitrite
OPA1 ¼ optic arthropy protein 1
PGC-1a ¼ proliferator-activated receptor
coactivator 1 a
Pi ¼ inorganic phosphate
PKC ¼ protein kinase C family
PL-ROOH ¼ phospholipid hydroperoxides
POLG1 and POLG2 ¼ DNA polymerase c
Prx ¼ peroxiredoxin
RNS ¼ reactive nitrogen species
ROc ¼ alkoxyradicals
ROOc ¼ peroxyl radicals
ROS ¼ reactive oxygen species
SODs ¼ superoxide dismutases
TAZ1 ¼ tafazzin
Trx ¼ thioredoxin-2
TrxR ¼ thioredoxin reductase
UCPs ¼ uncoupling proteins
UQ ¼ ubiquinone
UQc- ¼ semi-ubiquinone anion
VDAC ¼ voltage dependent anion channel
VEGF ¼ vascular endothelial growth factor
VHL ¼ Von Hippel-Lindau