Physiol Rev 94: 909 –950, 2014
doi:10.1152/physrev.00026.2013
MITOCHONDRIAL REACTIVE OXYGEN SPECIES
(ROS) AND ROS-INDUCED ROS RELEASE
Dmitry B. Zorov, Magdalena Juhaszova, and Steven J. Sollott
A. N. Belozersky Institute of Physico-Chemical Biology, Lomonosov Moscow State University, Moscow, Russia;
and Laboratory of Cardiovascular Science, National Institute on Aging, National Institutes of Health, Baltimore,
Maryland
Zorov DB, Juhaszova M, Sollott SJ. Mitochondrial Reactive Oxygen Species
(ROS) and ROS-Induced ROS Release. Physiol Rev 94: 909 –950, 2014;
doi:10.1152/physrev.00026.2013.—Byproducts of normal mitochondrial metabolism and homeostasis include the buildup of potentially damaging levels of reactive
oxygen species (ROS), Ca2⫹, etc., which must be normalized. Evidence suggests that
brief mitochondrial permeability transition pore (mPTP) openings play an important physiological
role maintaining healthy mitochondria homeostasis. Adaptive and maladaptive responses to redox
stress may involve mitochondrial channels such as mPTP and inner membrane anion channel
(IMAC). Their activation causes intra- and intermitochondrial redox-environment changes leading to
ROS release. This regenerative cycle of mitochondrial ROS formation and release was named
ROS-induced ROS release (RIRR). Brief, reversible mPTP opening-associated ROS release apparently constitutes an adaptive housekeeping function by the timely release from mitochondria of
accumulated potentially toxic levels of ROS (and Ca2⫹). At higher ROS levels, longer mPTP openings
may release a ROS burst leading to destruction of mitochondria, and if propagated from mitochondrion to mitochondrion, of the cell itself. The destructive function of RIRR may serve a physiological
role by removal of unwanted cells or damaged mitochondria, or cause the pathological elimination
of vital and essential mitochondria and cells. The adaptive release of sufficient ROS into the vicinity
of mitochondria may also activate local pools of redox-sensitive enzymes involved in protective signaling
pathways that limit ischemic damage to mitochondria and cells in that area. Maladaptive mPTP- or
IMAC-related RIRR may also be playing a role in aging. Because the mechanism of mitochondrial RIRR
highlights the central role of mitochondria-formed ROS, we discuss all of the known ROS-producing sites
(shown in vitro) and their relevance to the mitochondrial ROS production in vivo.
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INTRODUCTION
ROS: GENERAL DEFINITIONS
ROS: FROM SIGNALING TO...
ROS: REDOX STRESS
ROS GENERATION IN MITOCHONDRIA
ROS-INDUCED ROS RELEASE...
RIRR AND Ca2ⴙ
MITOCHONDRIAL COMPARTMENTATION
MITOCHONDRIAL RIRR IN...
IMAC-ASSOCIATED RIRR
RIRR-RELATED PATHOLOGIES: THE...
PRACTICAL ASPECTS OF USING...
CONCLUDING COMMENTS
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I. INTRODUCTION
Photo-activated reactive oxygen species (ROS) may trigger
mitochondrial permeability transition pore (mPTP) induction within individual mitochondria in intact cell systems.
The phenomenon of ROS-triggering of the mPTP associated with further stimulation of ROS formation has been
termed “ROS-induced ROS release” (RIRR) (491). mPTP
opening is a mitochondrial response to an oxidative challenge resulting in an amplified ROS signal, which depending
on ROS levels may result in different outcomes. In addition
to ROS effects in those mitochondria (where the RIRR originated), ROS released into cytosol could trigger a complex
cellular signaling response and/or RIRR in the neighboring
mitochondria. In the latter case, ROS trafficking between
mitochondria could constitute a positive-feedback mechanism resulting in an elevated production of ROS that could
be propagated throughout the cell and may cause perceptible mitochondrial and cellular injury. Although photo-induced formation of ROS could be initially used in the experimental setting as a trigger for more massive, avalanchelike ROS release, this phenomenon is representing a more
fundamental mechanism, e.g., light-independent spontaneous redox transitions associated with the induction of
mPTP or other mitochondrial channel(s) that may occur
under different physiological or pathological conditions
with corresponding impacts on mitochondrial and cellular
physiology. This review will cover the spectrum of RIRRrelated phenomena, both physiological and pathological including the processes of mitochondrial ROS production
0031-9333/14 Copyright © 2014 the American Physiological Society
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ZOROV ET AL.
and scavenging. Ultimately, the imbalance between the inflow, neutralization, and outflow of ROS with corresponding triggers in specific cell signaling pathways may result in
extreme situations such as oxidative and reductive stresses
with the consequent onset of numerous pathologies or even
the cell and organismal death.
and metal-oxygen complexes. These radicals combined
with the previously mentioned ROS form a large and important group of active redox agents playing critical role in
a number of intra- and extracellular processes.
III. ROS: FROM SIGNALING TO
PATHOLOGICAL
II. ROS: GENERAL DEFINITIONS
Eleven years ago this journal published an excellent and
comprehensive review by Droge (117) on free radicals and
their beneficial and detrimental roles in cell physiology and
pathology. Since then, the general interest surrounding the
roles of these species has constantly increased, shifting the
main focus to highly potent oxidants containing oxygen,
called ROS. The term ROS encompasses oxygen free radicals, such as superoxide anion radical (O2·⫺) and hydroxyl
radical (·OH), and nonradical oxidants, such as hydrogen
peroxide (H2O2) and singlet oxygen (1O2).
ROS can be interconverted from one to another (depending
on ⌬G of relevant processes) by enzymatic and nonenzymatic mechanisms. The primary and most abundant ROS
is the superoxide anion radical that has a comparatively
high oxidative capacity [standard redox potential of the
oxygen/superoxide couple ⫽ ⫺0.137 V (337) allowing
single-electron reduction of molecular oxygen by certain
mitochondrial oxidoreductases]. H2O2 is generated
through spontaneous or superoxide dismutase (SOD)catalyzed dismutation of O2·⫺ (143). In mammals, three
SOD isoforms were found in the living cell with precise
compartmentalization: the Cu,Zn-dependent isoform
(Cu,Zn SOD, SOD1) (142) is located in the mitochondrial
intermembrane space and cytosol; the Mn-dependent isoform (Mn SOD, SOD2) (358, 468) is located in the mitochondrial matrix; and Cu,Zn SOD is located in the extracellular space (ecSOD, SOD3) (285).
The most potent and aggressive oxidant primarily responsible for oxidative damage of DNA bases is the hydroxyl
radical, which has a relatively short half-life. ·OH can be
generated through a variety of mechanisms. It is well known
that ·OH is generated from H2O2 and O2·⫺ which is catalyzed by iron ions through the Haber-Weiss reaction (169)
with a specific case of Fe2⫹-mediated decomposition of
H2O2 [the Fenton reaction (130), reviewed in Ref. 237].
Ionizing radiation causes decomposition of H2O, which
also results in forming ·OH and hydrogen atoms. ·OH could
be also generated by photolytic decomposition of alkylhydroperoxides (447).
In addition, a number of other oxygen-containing free radicals are capable of causing oxidation of essential cell components: nitric oxide (NO), peroxynitrite, lipid hydroperoxides (LOOH), alkoxyl radical (RO·), peroxyl radical
(·OOH), nitrogen-centered radical, sulfate radical (SO4·⫺)
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One of our specific aims is to give an overview of a spectrum
of phenomena associated with different ROS serving a “signaling” role (discussed below) which is essential for a large
number of biochemical reactions. In the following sections
we address the significance of reductive and oxidative
stress. Under physiological conditions, the balance between
ROS generation and ROS scavenging is highly controlled.
Depending on circumstances, regulated oxidative stress
could initiate diverse cellular responses ranging from triggering signaling pathways involved in cell protection, initiating coordinated activation of mitochondrial fission and
autophagy to optimize clearance of abnormal mitochondria
and cells to protect spreading the damage to the neighboring mitochondria and cells (100, 117, 490). On the other
hand, unregulated oxidative and reductive stresses could
result in severe cellular damage, unwanted cell death, and
consequently whole organ and organism failure (102, 242,
498). Therefore, adaptive physiological redox stresses, such
as those occurring under the process of a programmed removal of damaged biological systems including mitochondria and other cellular components (“physiological”), must
be differentiated from maladaptive unwanted (“pathological”) oxidative damage.
Under normal physiological conditions, ROS emission (essentially, production minus scavenging) was considered to
account for ⬃2% of the total oxygen consumed by mitochondria (81). [A recent measurement of ROS production
in mitochondria with disabled antioxidant systems revealed
values fluctuating from 0.25 to 11% depending on the animal species and respiration rates (21).] A lower, as well as
a higher, percentage may have deleterious consequences
since ROS, when low, are unable to provide proper cellular
functioning through regulation of a great number of biochemical reactions. When high, they are unable to provide a
controlled regulation. It will require conditions when the
flux of metabolic regulators of ROS level is finely tuned to
respond to the cell demands with mitochondria playing a
critical role. As for many cellular signaling elements, the
principle “multet nocem” (excess is harmful) may become a
key element in a switch operating between “signaling”
(meaning as survival-promoting which is physiologically
required for renovating a biological component) and pathological (meaning undesired death-promoting) modes (216,
218, 219, 232, 491, 493, 497). For example, the ROSmediated ignition of a death of cells designed for long-term
use (postmitotic cells such as cardiac myocytes or neurons),
i.e., occurring under severe ischemic conditions causing pa-
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MITOCHONDRIAL ROS AND ROS-INDUCED ROS RELEASE
thologies such as myocardial infarct or stroke, under any
conditions may be considered a pathological event. In other
cases, apart from their pro-survival signaling role, ROS are
apparently involved in a designated physiological function,
eliminating unwanted mitotic cells or mitochondria and a
significant rise of local ROS level within might be an efficient means to fulfill such functions. Futhermore, ROS have
been shown to play a central role in regulation of the cell
cycle progression (453). Whether the ROS burst can serve a
signaling (survival) function will be discussed in a section
describing the oscillatory behavior of mitochondria. For
clarity, we will stay within definitions of signaling ROS as
of those serving essential pro-survival functions and antisurvival functions including required elimination of unwanted cells (quality control, maintenance of function),
while pathological ROS are considered as those causing
oxidant-induced unwanted changes including unwanted
cell death (loss of function).
IV. ROS: REDOX STRESS
There is an apparent heterogeneity in ROS levels and types
when comparing different cells and organs (54, 173, 271,
342, 373, 401, 477). This is largely due to a heterogeneous
distribution of activities of ROS producing and utilizing
machineries (11, 78, 103, 290, 346). The general consensus
is that overwhelming ROS production when not compensated for with their scavenging by endogenous antioxidants
will lead to the rise of ROS beyond the “normal” or “physiological” threshold level. This results in a process conventionally called “oxidative stress.” Apparently, the definition
of this widely used term (up to the year 2012 this term yields
over 100,000 citations in PubMed) is quite broad and has
“soft borders” considering the scenarios presented above.
This is due to the fact that physiological levels and types of
ROS in different tissues and in different parts of the same
tissue under different physiological conditions are heterogeneous and highly dependent on the energy load that is met
by the cell response. Even within the confines of a single cell,
there are at least eight distinct organellar compartments
(mitochondrial matrix, lysosomes, smooth ER/SR, rough
ER, the Golgi, peroxisomes, the nucleus, the cytosol), each
with its own redox poise (315). Accordingly, the term oxidative stress is often used in the literature in a very general
term to define a state when the levels and types of oxidants
in the cell or the organelle on average significantly exceed
the ground/resting/steady-state level associated with normal homeostatic function. At the opposite end of the redox
spectrum, when the reduced glutathione levels are too high,
“reductive stress” occurs and demonstrates potentially detrimental consequences for the cell (154, 349). Within normal fluctuation of energy load, the productions of ROS and
the ROS levels in mitochondria, cells, and the tissue are safe
to perform normal activity (maintenance of function) of the
particular biological system. ROS signaling and the role of
ROS in vital cellular functions associated with cell prolifer-
ation, differentiation, migration, immune response, cell senescence and death, and number of inherited or acquired
pathologies such as ischemia-related disease, atherosclerosis, neurodegenerative disease, malignant transformation,
diabetes mellitus, rheumatoid arthritis, aging, etc., is described elsewhere (25, 117, 185, 203, 263, 348, 352, 497).
However, under stress, when the ROS levels remain outside
the normal range (either under conditions of enhanced antioxidative pathways associated with reductive stress or of
those characterized by the rise of uncompensated ROS associated with oxidative stress), resultant instability of the
redox environment may develop that could be harmful (unwanted loss of function) if it is not compensated by the
feedback control mechanism.
In summary, considering these scenarios, both high levels of
ROS (oxidative stress) and excessively low levels of ROS
(reductive stress) are deleterious and apparently play a causative role in the pathologies caused by malfunctioning processes related to the dramatic change of redox environment.
Underlying all these arguments, redox homeostasis seems to
be a critical factor for normal functioning of the mitochondrion, cells, and organisms (174). Previously, it has been
argued that the cell normally maintains cytosolic thiols in a
highly reduced redox state, thus not supporting the existence of reductive stress (154). More recently, however, a
profound increase of reduced glutathione concentration
and the ratio of GSH/GSSG in cardiomyopathic animals
carrying the R120G mutation in the ␣B-crystallin molecule
was detected (349). The elevated level of reduced equivalents in these animals was accompanied by the augmented
expression together with increased antioxidative enzymatic
activity of glutathione peroxidase, glutathione reductase,
and catalase, which supports the implication of deleterious
reductive stress (349). The presence of reductive stress in
yeast was also confirmed (440). A more precise term, redox
stress, might be introduced reflecting both the incidence of
oxidative and reductive stresses; however, this review is
focused primarily on the circumstances related to oxidative
stress.
V. ROS GENERATION IN MITOCHONDRIA
In 1961, Jensen was among the first investigators to demonstrate that mitochondria produce ROS (209). He observed that a small portion of the oxygen consumed by
submitochondrial particles oxidizing NADH or succinate
was converted to H2O2 since this consumption was catalase
sensitive. Later, in 1972–1973, a classic, more general
study, was done at the Johnson Research Foundation in
Philadelphia by Britton Chance and co-workers (57, 58)
who initiated the modern era of the mitochondrial ROS
research. Since that time, scientists debated the physiological relevance of data obtained using “artificial” systems
such as isolated mitochondria, inside-out submitochondrial
particles, reconstituted respiratory complexes, and pure en-
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zymes. According to the critics, these are not adequate systems to extrapolate data to the cell, organ, and organism
levels (reviewed in Refs. 340, 480, 492). However, some
counter-arguments support the relevance of these model
systems. We are still lacking a detailed mechanistic knowledge of the architecture of mitochondrial ROS-producing
systems such as of complex I or complex III and detailed
insights on the mechanisms controlling their activities. We
will make an attempt to partially address and clarify this
scientific debate and to present the arguments in support of,
and against, the importance and physiological relevance of
those specific proposed mitochondrial ROS-producing
components. The primary goal of this review is not a comprehensive coverage of this specific issue, but the background that needs to be addressed here to provide a basis
for understanding those cases when an “innocent” molecular site (i.e., normally associated with moderate and physiological ROS production) becomes a “killer,” producing
ROS levels leading to the destruction of the biological system (perhaps through some poorly understood amplification mechanism). Good reviews on the current mechanisms
of ROS production in mitochondria are available elsewhere
for the reader interested in a general background and for
those interested in substantially detailed mechanistic depth
(3, 13, 201, 202, 308, 397, 416).
A. Complex II
1. Under normal conditions
We begin this review of mitochondrial ROS-producing sites
from complex II since succinate is a more frequently used
oxidative substrate to explore the functioning of isolated
mitochondria which became a classical object to study ROS
production.
Complex II, succinate-ubiquinone oxidoreductase (EC
1.3.5.1), commonly known as succinate dehydrogenase
(SDH), is a tetrameric iron-sulfur flavoprotein of the inner
mitochondrial membrane and acts as part of the Krebs cycle
and respiratory chain. SDH catalyzes the conversion of succinate into fumarate, yielding reduced equivalents in the
form of reduced flavin adenine nucleotide (FADH2). This is
followed by a reduction of ubiquinone to ubiquinol. Mammalian Complex II, as well as that from yeast, harbors a
covalently bound FAD, three iron-sulfur clusters, a b-type
heme, and two quinone-binding sites termed Qp and Qd,
standing for proximal or distal sites correspondingly.
Typically, complex II is excluded from the list of potential
candidates for important physiological contributors of
ROS (347, 348, 358). It is partially due to fact that the
succinate level in the tissue is low (in a range of hundreds of
micromoles), while in in vitro experiments with isolated
mitochondria millimolar concentrations are used. Hansford et al. (175) found that while H2O2 production can be
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detected in mitochondria oxidizing succinate in vitro at experimental ambient (5–10 mM) concentrations, they do not
produce significant amounts of peroxide at low, more physiologically relevant succinate concentrations (175).
Some studies point to flavins (flavin adenine nucleotides,
FAD) of SDH rather than other electron-carrying components (such as iron-sulfur clusters or quinones) as the site of
autoxidation responsible for generating ROS (200, 293,
294). Others implicate ubisemiquinone and iron sulfur centers as these sites, although under normal and steady-state
conditions these components are only partially reduced and
short-lived (167, 192, 267), thus giving a low probability to
transfer single electrons directly to oxygen.
2. Redox regulation of ROS production and redox
buffering
During oxidation of succinate in isolated respiring mitochondria, electron flow can bifurcate forming direct (towards cytochrome oxidase) and reverse (toward NAD; rotenone-blocked) transport with the latter requiring energy
input (79, 80, 187). The succinate-driven ROS generation
during reverse electron transport from succinate to NAD
resulting in the formation of NADH is higher when compared with that forming under direct oxidation of NADdependent substrates (456). The observed relationship between ROS formation and the redox state of the couple
NADH/NAD resulted in the proposition that the ROS formation is directly proportional to the level of reduction of
NAD. Possibly, a more generalized rule might be formulated that the more reduced the mitochondrial interior is,
the more probable there will be primary ROS formation.
The redox state of the cellular milieu is mainly determined
by the ratios of reduced/oxidized cofactors and proteins
which carry the bulk of redox-sensitive amino acid residues
and functional groups, NAD(P)H/NAD(P)⫹ and GSH/
GSSG, which all together form a compartmentalized redox
buffer where all components are in a redox equilibrium
under cellular steady-state conditions (reviewed in Ref.
309). This buffer may be an important factor in determining
ROS levels in the compartments such as the mitochondrial
matrix or cytosol (174). High intramitochondrial redox
buffering capacity, only partially represented by 3–5 mM
NAD(P)H and 2–14 mM GSH (416), would resist the shortterm exposure to ROS, while profound sustained ROS exposure would eventually exhaust this buffer, resulting in the
elevation of intramitochondrial ROS levels (353, 416, 460).
Later, we discuss in greater detail the redox dependence of
ROS formation and the role of reducing equivalents and
mitochondrial membrane potential on the net ROS production (see sect. VB5).
The role of complex II in maintaining and modulating the
mitochondrial/cellular redox environment remains undetermined. It is unknown whether in in vivo mitochondria
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MITOCHONDRIAL ROS AND ROS-INDUCED ROS RELEASE
reverse electron transfer from complex II to complex I occurs, and whether under physiological conditions the reverse electron transport could result in substantial ROS
production considering that physiological concentrations
of NADH would significantly attenuate O2·⫺ production
under conditions where reverse electron transport could be
observed in in vitro model systems (165). Thus it remains
questionable under normal conditions if there is a significant contribution of ROS generated in complex II to the net
ROS production.
3. Under pathological conditions
As we discussed previously, the question about complex II
contributing to the net ROS production remains controversial. Although the tissue level of succinate is as low as 200 –
500 ␮M, under oxygen deficiency (hypoxia/ischemia) it
may rise 5- to 10-fold (44, 371, 471). Recently, significantly
increased levels of succinate (to a few millimolar range)
were detected in macrophages exposed to lipopolysaccharide, ultimately identifying succinate as a metabolite in innate immune signaling, which stabilizes HIF-1␣ and enhances interleukin-1␤ production during inflammation
(433). Activation of macrophages is known to be associated
with elevation of their ROS production, but whether succinate triggers this production remains unexplored.
Under some circumstances of drug-induced apoptosis when
intracellular pH becomes significantly acidified, impairment of complex II could correlate with ROS generation
without changes in the SDH enzymatic activity which is a
part of complex II activity (260). This process has been
accompanied by dissociation of the SDHA (flavoprotein
subunit)/SDHB (iron-sulfur protein-containing part) subunits, which encompass the SDH activity, from the membrane-bound components of complex II that are required
for the SQR activity (for details, see FIGURE 1). Such disso-
ciation (see FIGURE 1) might result in a direct single-electron
reduction of oxygen by a reduced iron-sulfur cluster of complex II (113). Consequently, it has been proposed that complex II may function as a general sensor for apoptosis (162,
260). This is an example of a pathological, conformationinduced ROS production which does not happen in intact
complex II.
In a catalytic mechanism, ubiquinone receives electrons
from the [3Fe-4S] center being bound at the Qp site. Since
the ubiquinone is a two-electron acceptor receiving these
electrons in separate steps, the intermediate state of SDH
exists where after receiving a single electron the ubisemiquinone radical must be stabilized to prevent the escape of the
electron to an inappropriate acceptor such as molecular
oxygen. Mutations in the vicinity of the Qp site were shown
(167) to compromise the ability to stabilize ubisemiquinone, and thus its unpaired electron may become more
readily available to react with ambient oxygen producing its
derivative, superoxide anion radical followed by dismutation to form hydrogen peroxide.
There is multiple evidence that impaired electron transport
in SDH, as well as its effect on the levels of NAD(P)H
through the impairment of the Krebs cycle, are the source
and the cause of a substantial amount of ROS determining
the onset of numerous pathologies (e.g., Refs. 32, 204, 481,
482). Malfunctioning of respiratory complexes, including
complex II in brain mitochondria, is a hallmark of Huntington disease (HD), a neurodegenerative genetic disorder that
elicits progressive motor, cognitive, and emotional deficits.
3-Nitropropionic acid, an irreversible inhibitor of SDH,
mimics HD-like pathology and symptoms (68) and evokes
an ROS increase in neurons (270). Leigh syndrome, an infantile-onset progressive neurodegenerative human disease
is suggested to be caused by mutations in the SDHA gene. It
appears that mutations in the SDHB, SDHC, or SDHD
FIGURE 1. Model for the role of specific complex II inhibition for apoptosis induction by various proapoptotic compounds. Mitochondrial matrix, inner membrane (IM), intermembrane space, outer membrane (OM), and cytosolic
space are indicated. A: in healthy cells, complex II serves to
funnel electrons derived from the Krebs cycle to the respiratory chain. SDHA-mediated oxidation of succinate to fumarate by the succinate dehydrogenase activity (SDH), as
part of the Krebs cycle, provides electrons to complex II.
They are transferred to the iron-sulfur centers of the
SDHB subunit and finally to the CoQ reduction site the
succinate CoQ oxidoreductase (SQR). B: proapoptotic
compounds, such as various anticancer drugs, FasL, or
tumor necrosis factor (TNF)-␣, induce cytosolic (pHc) and
mitochondrial (pHM) acidification. These pH changes lead
to the dissociation of the SDHA/B subunits from complex
II and finally to the partial inhibition of the SQR activity
without any impairment of the SDH reaction. This specific
inhibition leads to complex II uncoupling, superoxide production, and apoptosis. [From Lemarie et al. (260). Reprinted by permission from Macmillan Publishers, Ltd.]
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genes can cause paraganglioma (a neuroendocrine, highly
vascularized neoplasm developing tumors in the head, neck,
thorax, or abdomen) (37) or pheochromocytoma (a catecholamine-secreting neuroendocrine tumor occurring in the
medulla of the adrenal glands) (37, 155; reviewed in Ref.
305). Unfortunately, it is impractical so far to estimate the
contribution of the impaired Krebs cycle and reverse electron transport occurring under pathological SDH impairment to the modulating ROS production.
In parasitic worms residing in an anaerobic environment in
a host intestine, the energy partially is obtained from socalled fumarate respiration reflecting a reverse activity of
succinate-ubiquinone reductase of complex II. Apparently,
in their fumarate reductase reaction, ROS are produced in a
FAD site and quinone-binding site as well. Since in the adult
stage, these worms do not have either complex III or IV,
their respiratory chain could serve as a good model to study
the production of ROS in complex II in mitochondria (364,
436). It is noteworthy that this model with a missing cytochrome c oxidase may somehow simulate either hypoxic
conditions or those induced by defective electron transfer
downstream of Complex II.
There is debate on the critical role of the components of
complex I involved in superoxide production. Some consider FMN (247, 256, 287, 345, 455), while others claim
that iron-sulfur clusters N1a and N2 (142, 152, 186, 254),
NAD radical (245), or ubisemiquinone (241) are responsible for O2·⫺ generation in complex I. The last point was
actively challenged by Lenaz (264) who considered only
hydrophilic quinones to be prooxidants, while physiological hydrophobic ubiquinones (such as CoQ10) behave more
as antioxidants rather than prooxidants (264). Therefore,
the question regarding the major source of superoxide in
complex I under physiological conditions remains unresolved.
Recently, the physiological relevance and thus the importance of the production of ROS by complex I was questioned on the basis that NADH-supported complex I-catalyzed superoxide production by submitochondrial particles
shows maximal activity at low NADH concentrations (⬃50
␮M) while at physiological concentrations of NADH (in the
millimolar range) this reaction is severely inhibited (164,
165).
2. Under pathological conditions
B. Complex I
1. Under normal conditions
The association of complex I deficiency with a wide spectrum of pathologies such as cardiomyopathies, cataracts,
Leigh disease, exercise intolerance, mitochondrial encephalomyopathy, lactic acidosis, strokelike episodes (MELAS),
hepatopathy, and tubulopathy has been suggested. Prevailing dogma holds that complex I (NADH-ubiquinone oxidoreductase) is the main source of ROS in mitochondria.
However, the ROS production at complex I depends on
circumstances; consequently, complex I becomes a major
ROS source under pathological conditions rather than being a dominant source under resting and healthy conditions.
When submitochondrial particles or isolated mitochondria
oxidize NAD(P)H or glutamate plus malate, correspondingly, complex I production of superoxide is negligible.
However, supplementation with the inhibitor of complex I,
rotenone, results in robust production of O2·⫺ (350, 456).
This implies that the major site of ROS production in complex I is either upstream of a rotenone-binding site or it is
tightly coupled to the increased level of NAD(P)H after
rotenone supplementation during oxidation of NAD-dependent substrates (175, 444). According to the first alternative, rotenone would induce progressive reduction of the
upstream redox groups (432) including Fe-S clusters, flavin
mononucleotide (FMN), and the tightly bound pool of
ubiquinone (62, 325), which can supply the oxygen molecule with a single electron yielding superoxide anion radical.
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It has been noticed that at least 40% of all mitochondrial
disorders are associated with mutations in subunits of complex I (402). Defects in complex I are associated with a wide
diversity of neurodegenerative pathologies, including Parkinson’s disease (PD) which is characterized by a substantial loss of the dopaminergic neurons and cell bodies of
which are in the substantia nigra pars compacta and nerve
terminals in the striatum. ROS are thought to be highly
involved in PD pathogenesis, triggering the loss of redox
buffers (GSH and proteinaceous thiols) (336) at least partly
caused by dopamine oxidation-related metabolic pathways.
Dopamine in the central nervous system, apart from being a
neuronal neurotransmitter, serves as a precursor of norepinephrine and epinephrine, and is a regulator of movement
(nigrostriatal pathway), and a behavior motivator (mesolimbic pathway) (425). While under normal conditions
oxidative deamination of dopamine by monoamine oxidase
produces hydrogen peroxide (282), it could generate toxic
oxidants through alternative ways of oxidation wherein
mitochondria play a role. In this pathway, dopamine is
oxidized nonenzymatically by superoxide forming dopamine quinone which can be reduced by mitochondrial complex I generating semiquinone followed by a transfer of its
electron to molecular oxygen to form superoxide (488),
completing a vicious oxidative cycle. In addition, PD is
hallmarked by elevated iron levels that may catalyze production of deadly oxidants, possibly in a self-amplifying
mode (411).
PD could be mimicked by the action of complex I inhibitors
such as rotenone, paraquat, and 1-methyl-4-phenyl-1,2,
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MITOCHONDRIAL ROS AND ROS-INDUCED ROS RELEASE
Generation of ROS associated with hypoxia/reoxygenation
is known as one of the most deleterious causes of oxidative
damage. Three potential sources of ROS have been proposed to be responsible for this release: mitochondrial complex I, xanthine oxidase, and NADPH oxidase (2, 471,
491). However, the latter two are probably not involved
since inhibition of these complexes in vivo did not afford
cell protection (84, 129).
3. ROS and hypoxia
The reaction of formation of a primary ROS (superoxide
only) generated in the respiratory chain from molecular
oxygen is of a first order with respect to oxygen concentration. However, paradoxically, generating ROS in mitochondria in the cell remains constant or even increases when
PO2 drops dramatically (i.e., under moderate hypoxic conditions). Robust ROS production under 1.5% of O2 has
been recorded also (314, 374, 462).
Interestingly, in the cell the affinity of molecular oxygen to
ROS-generating modules is higher than to cytochrome oxidase. This obviously takes place under conditions of partial
reduction of cytochrome oxidase, i.e., when the availability
In highly metabolizing tissues, the areas surrounding mitochondria in the cell may have higher ROS levels than remote
areas. Without considerable mitochondrial ROS-quenching
activities, intramitochondrial levels of ROS may potentially
reach very high levels. That may happen in case of an imbalance between the oxygen supply and demand, for example, under conditions of high metabolic needs.
The nature of the tissue oxygen gradient between the source
of oxygen (blood capillary) and the site of its utilization (the
mitochondrion) can be explained by the Krogh cylinder
model which generally serves to analyze capillary tissue
exchange kinetics (246, 266) (FIGURE 3). The effective radius of this cylinder beyond which the tissue hypothetically
is experiencing hypoxia depends in part on O2 consumption. In a tissue with high metabolic rate (such as heart
muscle), capillary density during maximum or moderate
exercise would not be sufficient to supply tissue with oxygen and might potentially produce more ROS in the vicinity
80
120
100
60
80
40
60
40
20
20
H2O2 emission, pmol/min/mg
One of the very specific features of the mammalian NADHubiquinone oxidoreductase is the slow active/deactive state
transition, suggesting gross conformational rearrangements
of complex I, at least in that part which is involved in
rotenone-sensitive ubiquinone reduction [which may be involved in the superoxide production (150, 454)]. It was
found that complex I isolated from the heart which was
exposed to a normoxic perfusion is in a fully active state,
while 30-min anoxic perfusion results in a significant transformation of the enzyme into a deactive state which returns
back to normal after reoxygenation (283). It has been proposed that these conformational transitions can be relevant
to producing ROS by complex I after cardiac tissue is reoxygenated following a coronary occlusion (283). Using
EPR spectroscopy, DeJong et al. (104) showed that NADHcoenzyme Q oxidoreductase undergoes energy-dependent
structural changes in parts determining ubisemiquinone
production (iron-sulfur cluster 2) (104). Thus, under pathological conditions, conformational rearrangements may
be involved in the changes of the efficiency of ROS-producing machinery in complex I.
of oxygen is limiting its utilization. Note that this paradox
is absent in the isolated mitochondrial system (FIGURE 2),
free from extramitochondrial signaling pathways which
confirms that elevated mitochondrial ROS generation in the
cell in response to hypoxia is not intrinsic to the mitochondrial respiratory chain alone but can be attributed to some
involvement of extramitochondrial factors (189). Marshal
et al. (286) indicated that hypoxia-induced superoxide production occurs through activation of NADPH oxidase located in the cell membrane. In addition, under moderate
hypoxia, NO synthesis in mitochondria continues although
being only 5–10% of the normal steady-state level (Km for
oxygen of the mitochondrial NO synthase is 30 – 40 ␮M;
Ref. 8). In turn, NO can partially block cytochrome oxidase
(69, 70, 378), thus reducing mitochondrial electron carriers, increasing its Km for oxygen (91) and favoring generation of superoxide at hypoxic conditions (444).
Respiration rate, nmol o2/min/mg
3,6-tetrahydropyridine (49, 94). Exposure to the latter drug
has shown to produce permanent parkinsonism in humans,
non-human primates, and rodents, by exerting an effect
primarily on the function of mitochondrial complex I. In
patients with Friedreich ataxia, a deficient activity of the
Fe-S cluster-containing subunits of mitochondrial respiratory complexes I, II, III, and aconitase was found (361).
Kushnareva et al. (254) claimed that the ratio of NAD(P)H/
NAD(P)⫹, rather than the level of NADH, determines reduction of ROS-producing sites in complex I.
State 3
0
0
0
1
3
5
7
9
11
[O2], µM
FIGURE 2. Effect of the oxygen tension on the rates of phosphorylating respiration and H2O2 emission in rat liver mitochondria. (Note
that there is no ROS production increase under low PO2.) [From
Starkov (416), with permission from John Wiley & Sons.]
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ZOROV ET AL.
Rt
Rc
Z
Flow Direction
FIGURE 3. Geometry of the Krogh cylinder-type model. Inner cylinder represents the capillary. Outer cylinder corresponds to tissue
cylinder. Shaded area: example of hypoxic region under conditions of
high demand. Rt, tissue cylinder radius; Rc, capillary radius; z, distance along the capillary. [From McGuire and Secomb (289).]
and inside of mitochondria/mitochondrial clusters (214).
However, basic theoretical assumptions of the Krogh cylinder model do not consider that the diffusion coefficient for
O2 in muscle tissue may be higher due to the possibility of
facilitated O2 transport by the mitochondrial network (10,
396) and/or by myoglobin molecules (475). In non-muscle
tissues lacking myoglobin, the cytoglobins and neuroglobins may potentially serve as facilitators of oxygen transport (220, 327, 354, 428). Both the extended mitochondrial
network and these various heme-containing proteins may
be responsible for a less steep O2 gradient in the normal
active cell, thus also likely flattening the ROS gradient as
well. Cytoglobin and neuroglobin have been reported to not
only exert an oxygen carrier function, but possibly to be
oxygen sensors and ROS scavengers (220).
Under experimental conditions acutely restricting the oxygen supply to an organ, such as the heart, very steep regional redox transitions have been observed across the borderline of the ischemic area (33), apparently reflecting a
similarly steep oxygen gradient and thus probably the ROS
gradient as well (FIGURE 4). On the basis of the data that
ROS production is directly linked to reduced equivalents
such as NADH (discussed above), hypoxic regions manifesting the highest level of NAD reduction would likely
achieve much higher ROS levels than normoxic regions.
The brain is the organ most vulnerable to the lack of oxygen, immediately responding by the reduction of NAD (78),
thus potentially eliciting ROS within local areas adjacent to
ischemic zones.
tion by the mitochondrial respiratory chain. On the other
hand, the same reduced redox conditions provide more
buffering capacity to quench ROS activity, so it is unclear
under which conditions (more reduced or more oxidized)
the net level of ROS will be higher. It appears that the
steady-state level of ROS in the compartment rather than
the ROS-producing activity determines the level of oxidantinduced biological modifications, many of which are important because of their biological effects, both physiological
and pathological.
Redox steady states of respiratory components responsible
for ROS production should be in redox equilibrium with
adjacent mobile and immobile redox carriers such as
NAD(P)H, GSH, and the thiol groups of the proteins occupying the same compartment.
In 1996 Liu and Huang (275) and in 1997 Korshunov et al.
(240) (FIGURE 5A) found in isolated mitochondria a strong
dependence of mitochondrial ROS production on the level
of the transmembrane potential (⌬␺), supported by succinate oxidation (reviewed in Refs. 272–274). [It should be
emphasized that mitochondrial matrix alkalinization could
be the cause of increased generation of ROS due to the fact
that ⌬pH is an integral part of the ⌬p (381).] While H2O2
production was low at ⌬␺ values at and below the phosphorylating membrane potential [the membrane potential
reached under the state 3 respiration according to B.
Chance’s terminology when mitochondria are supplemented with an excess of ADP (82), i.e., that thermodynamically required to generate ATP from ADP and Pi], it
4. Is the ROS production proton motive force
sensitive?
Proton motive force (pmf, ⌬p) across the inner mitochondrial membrane, with ⌬␺ as a main component, is driving
ATP production (268, 298, 299, 424). Whether ROS production is dependent on this proton motive force/transmembrane potential in mitochondria has become a crucial
question.
As discussed above, a reduced redox intramitochondrial
environment is a prerequisite for high primary ROS forma-
916
FIGURE 4. NADH fluorescence emission from perfused rat heart
with a local ischemic area near the apex (seen as white areas),
caused by ligation of a coronary artery. [From Korshunov et al. (33).
Reprinted with permission from AAAS.]
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MITOCHONDRIAL ROS AND ROS-INDUCED ROS RELEASE
H2O2 production, %
90
80
70
60
50
40
30
20
10
0
50
60
70
80
90
100
ΔΨ, %
110
glutamate + malate
α-ketoglutarate
+ ADP
100
90
80
State 3
B
100
H2O2 production, pmol x min-1 x mg-1
A
70
60
50
40
State 3
30
20
100
120
140
160
180
ΔΨ, %
FIGURE 5. Relationship between mitochondrial ⌬⌿ and H2O2 production supported by succinate or NADHlinked respiratory substrates. A: rat heart mitochondria oxidizing succinate. The ⌬␺ level was varied by adding
different concentrations of uncoupler SF6847 (black squares and solid line), malonate (white squares), or 100
␮M ADP and 5 mM P (triangle). Dashed line, the state 3 ⌬␺ level. [From Korshunov et al. (240), with
permission from Elsevier.] B: rat brain mitochondria oxidizing glutamate ⫹ malate or ␣-ketoglutarate. Differences in ⌬⌿ were generated by adding various concentrations of uncoupler FCCP ranging from 0 to 80 nM.
Alternatively, a decrease in ⌬␺ was induced by adding 0.8 mM ADP to mitochondria. [From Starkov and Fiskum
(419), with permission from John Wiley & Sons.]
rises dramatically above these values proportionally to the
⌬␺ elevation (FIGURE 5). Accordingly, an 18% decrease in
the value of the transmembrane potential inhibits 90% of
mitochondrial ROS production (240) above the phosphorylating membrane potential.
ological state. As discussed previously, regulated moderate
(“mild”) uncoupling of mitochondrial oxidative phosphorylation has been suggested as a feasible therapeutic strategy
(397, 398, 415) for regulation of the intracellular and intramitochondrial ROS level (99).
Subsequently, very steep dependence of H2O2 production
on the values of ⌬␺ exceeding the phosphorylation potential was confirmed in isolated mitochondria oxidizing
NADH-dependent substrates (419) (FIGURE 5B). However,
in another study using NADH-dependent substrates, mitochondrial respiration produced ROS in a membrane potential-independent mode (456).
Mitochondrial uncoupling proteins (UCPs) have been considered as potential mild uncouplers. The relationship between ROS production and UCPs activity was revealed in
1997 in experiments where GDP, an inhibitor of UCP1,
caused an increase of ⌬␺ and ROS production (316). Later,
it was demonstrated that superoxide directly activates
UCPs resulting in a negative feedback controlling both ROS
production and their levels (120).
5. Can ROS production be decreased in mitochondria
without jeopardizing ATP production? Mild uncoupling
as a possible downregulator of ROS production
As was shown in the preceding section (FIGURE 5), moderate lowering of ⌬␺ could result in a lower ROS production
in mitochondria without a significant effect on ATP production. This could be achieved by inducing a small proton leak
through the inner mitochondrial membrane which would
both stimulate oxygen consumption and, in parallel, shift
the level of reduction of mitochondrial ROS-producing sites
to a more oxidized state lowering the probability of ROS
production in the mitochondria. While higher potential
could drive increased ATP production, the higher ⌬␺ could
also result in the production of increased ROS levels and
potentially unwanted oxidative consequences. Therefore,
achieving a reasonable balance between ROS and ATP production by mitochondria is crucial since this reflects the
current energy needs of the cell under the particular physi-
Mild uncoupling may be protective against excitotoxic injury (469) and against injury of dopaminergic neurons in
substantia nigra from mitochondrial poisons such as rotenone (478). Decreasing ROS generation by uncoupling mitochondria increases longevity in healthy animals (74).
Of all the possible mild uncouplers, fatty acids are probably
the most natural ones (240, 398, 476). In their protonated
form they can cross the mitochondrial inner membrane followed by deprotonation in the matrix side, and then the
anionic form of the fatty acid completes the cycle by returning back to the cytosolic side. The rate-limiting step of this
cycle is the transport of anionic form. Different proteins
such as the adenine nucleotide transporter (ANT) and glutamate/aspartate transporter are involved in fatty acid-mediated uncoupling through facilitation of the transport of
the anionic form (12, 368). Thyroid hormones may also be
considered as natural mild uncouplers (177, 397).
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ZOROV ET AL.
Among artificial uncouplers, 2,4-dinitrophenol (DNP) has
been tested as an anti-obesity drug, but it was found to be
too toxic for practical use (90, 99). Additionally, DNP was
found to limit the experimental infarct size in the heart and
brain, and this was interpreted to occur through diminishing the ROS level (238, 359). Recently, mild uncoupling
activity was ascribed to a series of derivatives of cationic
rhodamine (15).
C. Complex III
Complex III (ubiquinol-cytochrome c oxidoreductase) accepts reducing equivalents formed in complexes I and II and
processes them by the Q-cycle operating mechanism. Operation of this cycle is initialized by ubiquinol, which releases
its proton to the intermembrane space and donates one
electron to the Riske iron-sulfur protein (which can bind to,
and be inhibited by, myxothiazol) producing unstable
semiquinone on the outer side of the inner mitochondrial
membrane. The semiquinone serves as an electron donor
for hemes of cytochrome bL, and then of cytochrome bH
which is located close to the inner side of the membrane.
Cytochrome bH reduces ubiquinone in an antimycin A-sensitive way producing ubisemiquinone followed by its further reduction with a second electron and protonation
(442) (FIGURE 6).
Under normal conditions, the probability of existence of
unstable semiquinone (Q_.) is low due to its fast oxidation;
therefore, the probability of donation of one electron to
molecular oxygen in this system is relatively low. Only the
block of the electron flow by antimycin A results in a high
superoxide release apparently due to the reduction of both
hemes of the cytochrome c in parallel with the elevation of
the steady-state level of semiquinone, thus giving a higher
chance of one-electron reduction of oxygen (FIGURE 6).
Among all of the mechanisms presented in the scheme, inhibitors of bc1 complex (antimycin A, myxothiazol, and
stigmatellin), antimycin is the only effective ROS inducer,
although some low level of superoxide production could be
detected in the presence of other inhibitors (418) in the site
of bc1 complex different from that induced by antimycin A.
Therefore, the potential role of complex III as a cause of
gross mitochondrial ROS production under the physiological steady-state mode of operation remains uncertain considering that substantial mitochondrial ROS release occurs
only after application of a drug having no natural analogs in
animal physiology. Noteworthy, as in the case of ROS production in complex I, conformational changes detected in
bcl1 complex after antimycin A binding (45, 95, 193, 355)
may be a prerequisite for dramatic molecular rearrangements in the complex resulting in a marked amount of ROS
production.
Overproduction of ROS by complex III may result from
acquired and genetic defects in the mitochondrial respi-
918
FIGURE 6. Q-cycle model. The mechanism of superoxide formation in complex III (bcl1 complex). The reaction starts from the
oxidation of the CoQ quinol (QH2) in a bifurcated electron transfer
reaction at the Qo site of the complex III. The first electron is transferred to a high reduction potential chain consisting of the iron-sulfur
protein (ISP, aka Rieske protein), cytochrome c1 (cyt c1), cytochrome c (cyt c), and further to cytochrome c oxidase (not shown).
The remaining semiquinone (Qo⫺·) is unstable. It donates the second electron to the low reduction potential chain consisting of two
cytochromes b, cyt bL and cyt bH, which serve as a pathway conducting electrons to the Qi site. There these electrons reduce another CoQ molecule. To provide two electrons required for the complete reduction of CoQ quinone at the Qi-site, the Qo-site oxidizes two
QH2 molecules in two successive steps. The first electron at the
Qi-site generates a stable semiquinone (Qi⫺) that is reduced to a
quinol (QH2) by the second electron. Most frequently used inhibitors
of complex III, stigmatellin and myxothiazol, prevent the transfer of
the first electron to ISP and the binding of the quinol at the Qo site
correspondingly. [Modified from Starkov and Fiskum (418), with
permission from Elsevier.]
ratory chain in close proximity to an antimycin A-binding site. B. Chance’s lab performed a study on a patient
who was diagnosed with a deficiency of cytochrome b in
complex III, resulting in muscle weakness associated with
a ragged-red fiber myopathy and lactic acidosis (122).
The total succinate-cytochrome c reductase activity in
skeletal muscle of this patient was only ⬃5% of normal.
It was already known that menadione can shuttle at least
a portion of electrons over an antimycin-sensitive site
(319). The treatment of this patient with menadione
bridging electrons from coenzyme Q directly to cytochrome c, thus bypassing the defective cytochrome b,
resulted in a significant therapeutic effect with partially
normalized muscle exercise tests. This approach, named
“redox therapy,” demonstrates the importance of de-
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MITOCHONDRIAL ROS AND ROS-INDUCED ROS RELEASE
tailed understanding of mitochondrial redox-related
pathogenesis.
D. Importance of Redox State of NAD(P)H/
NAD(P)ⴙ in Mitochondrial ROS
Production
A comprehensive analysis of the relevance of NADH in
managing mitochondrial production of ROS has been performed by Vinogradov’s group (163, 165). With the use of
submitochondrial particles oxidizing NADH, it has been
found that a substantial amount of superoxide production
took place only when 50 ␮M NADH was used, while in the
presence of 1 mM NADH, the production was remarkably
suppressed. NAD⫹ revealed the same superoxide suppressive ability (165). Considering that physiological concentration of the couple NAD⫹ ⫹ NADH in the mitochondrial
matrix is in range of a few millimolar (474), with a significant fraction of it existing in a free form, the gross generation of ROS mediated by complex I may be almost negligible. Similar experiments with isolated permeabilized mitochondria and the soluble protein fraction of the
mitochondrial matrix showed the same result. The authors
concluded that complex I was not a primary source of ROS
in mitochondria under physiological conditions. Instead,
they hypothesized that some oxidoreductases poised in
equilibrium with NAD(P)/NAD(P)H may be that primary
mitochondrial source of ROS (165).
In isolated permeabilized mitochondria, the same authors
detected quite high NADH-dependent H2O2 production
when they supplemented the system with ammonium salts
(163). The mitochondrial H2O2 release was insensitive to
dicumarol (inhibitor of NADH-quinone oxidoreductase,
D,T diaphorase) and NAD-OH (inhibitor of complex I),
suggesting the matrix localization of H2O2-producing activity. This ROS-generating activity depended on the ratio
of NAD(P)⫹/NAD(P)H. It was concluded that a specific
ammonium-sensitive NADH oxidase activity in the mitochondrial matrix is responsible for this H2O2 production,
but the in vivo relevance of this process is still unknown.
[An alternative explanation for an ammonium effect could
be the hypothesis that mitochondrial matrix alkalinization
(caused by ammonium entry) increases superoxide production by stabilizing semiquinone radical (381).]
Further analysis of the nature of this ammonium-stimulated
enzyme producing primary mitochondrial ROS revealed
that the questioned enzyme possessed NADH:lipoamide
oxidoreductase activity and later was identified as dihydrolipoyl dehydrogenase (224). Dihydrolipoyl dehydrogenase
is an essential component (called E3 component) of two
mitochondrial redox complexes: ␣-ketoglutarate dehydrogenase complex (KGDHC) and pyruvate dehydrogenase
complex (PDHC). This mitochondrial enzyme contains
FAD [which contributes mostly to the overall mitochon-
drial autofluorescence signal originating from cellular flavins (178, 250)] with the redox state in equilibrium with the
environmental NAD(P)H/NAD(P)⫹. It has been found that
E3 is responsible for superoxide and hydrogen peroxide
generation in purified KGDHC and PDHC as well as in
KGDHC operating in mitochondria in vitro (71, 151, 417,
420, 438).
It is not surprising that ammonium has an effect on the
component of ␣-KGDHC since ␣-ketoglutarate, instead of
converting into succinyl CoA in the citric acid cycle (the
Krebs cycle), can be transformed into glutamate by glutamate dehydrogenase. Typically, this reaction does not take
place in mammals, since the equilibrium of the reaction is
shifted toward the reverse direction, but it may occur in
toxic levels of ammonia. Ammonia metabolism is important in all tissues. However, in the brain for which a high
level of ammonium is extremely toxic (59), it becomes a
critical element involved not only in the detoxification process (by astrocytic glutamine synthase and the all-mitochondria-located urea cycle) but also in a number of essential biochemical reactions in the cell as part of the brain
signaling modules (i.e., glutaminase reaction to maintain
optimal cycling of glutamine/glutamate; Refs. 421, 422,
448). With the consideration of results mentioned above,
the ammonium toxicity might be at least partially mediated
by the mitochondria-formed ROS.
E. Other Mitochondrial ROS-Producing Sites
1. NADPH-oxidase
The prototypic NADPH-oxidase (Nox) has been found in
the plasma membrane of phagocytes and B lymphocytes,
and it is involved in the phagocytic activity by ejecting superoxide radical which is a primary element igniting antibacterial defense. Its membrane domain is represented by a
protein gp91PHOX (PHOX for phagocyte oxidase) which
can be organized as a heterodimer in combination with
other cytosolic proteins from the PHOS family which all
together form flavocytochrome b558 complex (6, 24).
So far, seven isoforms of Nox (Nox1–5, Duox1–2) have
been identified, with all having distinct catalytic domains.
The above-mentioned form is called Nox2. It has been
found not only in the cell membrane, but also in the cell
interior. Other isoforms reside in different specialized tissues and different intracellular loci. Nox4 is the only member of the family found to have a mitochondrial localization
[in cultured mesangial cells (55), rat kidney cortex (55) and
cardiac myocytes (251)]. Nox4 differs from the other members of the Nox family in that it preferentially produces
H2O2 rather than O2·⫺ (356). The discovery of its mitochondrial localization conflicts with data that shows that
the natural cytosolic partner of Nox4 complex,
p22PHOX, whose presence affords NADPH oxidation
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ZOROV ET AL.
was not found in mitochondria, and furthermore, that
the specific activity of Nox in mitochondria is not measurable (111). Considering the high importance of NADPH-oxidase, specifically Nox4, in angiogenesis (483) and
pathogenesis of atherosclerosis, diabetic injury (279) and
other pathologies (280) including aging (4), further experimental research is needed to resolve this apparent conflict
and gain a better understanding of the relevant aspects of
Nox-mediated redox signaling.
An interesting mode of interaction between Nox and mitochondria was shown recently in cancer cells where glucose
deprivation provoked a signaling-based positive-feedback
loop that amplifies ROS levels above a toxicity threshold
resulting in cell death (159). This positive-feedback loop
involved the complex integration of homeostatic control
mechanisms for metabolism (particularly, redox balance
established by Nox and mitochondria) and tyrosine kinase
signaling through regulation of protein tyrosine phosphatases. According to the authors, glucose withdrawal activates supraphysiological phosphotyrosine signaling and
ROS-mediated cell death. In cancer cells that are highly
dependent on glucose for survival, glucose and pyruvate
deprivation induces oxidative stress driven by Nox and mitochondria. This oxidative stress provokes a positive-feedback loop in which Nox and mitochondria generate ROS
and inhibit tyrosine phosphatases by oxidation. With the
negative regulators turned off, tyrosine kinase activates
Nox, further amplifying ROS generation and provoking
cell death.
2. Monoaminoxidase
Monoaminoxidase (MAO) resides in the outer mitochondrial membrane and serves as a marker there. This flavoenzyme has two isoforms A and B with different substrate
specificity and sensitivity to inhibitors (121). Their substrates are biogenic amines whose oxidation yields in the
generation of corresponding aldehydes, H2O2, and ammonium base. MAO activity has special importance in the
brain where peroxidase and catalase activities are low to
fully decompose H2O2 formed during oxidative deamination of neurotransmitters (e.g., dopamine, serotonin), thus
significantly depleting the endogenous pool of reduced glutathione (370).
The H2O2-generating activity of MAO might be the highest
among all mitochondrial ROS generators. Isolated rat brain
mitochondria produce H2O2 during oxidation of the exogenous amine, tyramine (at supraphysiological 2 mM concentration) at a rate 45.2 ␮M/s (179), while H2O2 production during succinate oxidation in the presence of antimycin
(considered to be the “gold standard” method for mitochondrial ROS production) is 0.95 ␮M/s (179, 334), i.e.,
MAO activity is 48 times more H2O2-generating than complex III. Oxidation of tyramine by brain mitochondria results in oxidative damage of mitochondrial DNA which is
920
abolished by MAO inhibitors (179). Compared with normal conditions, MAO produces much more H2O2 during
ischemia/reperfusion of the brain (389), the kidney (249),
and the heart (50, 221). MAO activity in cardiac mitochondria of 24-mo-old rats was about eight times higher than
that in 1-mo-old rats, demonstrating that MAO may be an
important source of ROS in the aging heart (109, 288).
However, the basic MAO activity in normal tissue is quite
low due to the limitation of the availability of endogenous
substrates of oxidation (such as serotonin, epinephrine,
norepinephrine, dopamine, and others present in the brain
in only nanomolar concentrations). On the other hand,
chemical inhibitors of MAO elevate ROS production in
cells (50 –52). Paradoxically, the ablation of MAO causes a
very slight rise of its endogenous substrates in the brain
tissue (386). Because the levels remain in the nanomolar
range, it is doubtful that these substrates could contribute
significantly to overall ROS production in tissue. However,
close proximity to the sites where biologically active amines
are formed and released (such as synapses and extraneuronal compartments such as astrocytes and glial cells) and the
high mitochondrial density in and around these areas, may
render mitochondrial MAO an important component in
both inactivation of amines and the local rather than overall
ROS production in such areas. It has been speculated that
under normal physiological activity, ROS produced by
MAO in these areas performs metabolic and signaling functions in the brain (31). In addition to ROS, the by-products
of biologically active amine conversion by MAO may play a
direct role in degenerative processes, e.g., the dopamine
molecule after entering MAO reaction produces a reactive
quinone that could modify and damage cellular components (425).
3. p66shc
On the basis of the observation that p66shc-deficient mice
display extended life span, remarkably reduced levels of
ROS, and increased tolerance to oxidative stress, it has been
suggested that p66shc could have an important role in ROS
production and aging (295, 328, 439). Normally p66shc
resides in the cytosol, while under oxidative stress (e.g.,
under ischemia/reperfusion insult), it could be translocated
in the mitochondria in a PKC␤-dependent way (341) where
it serves as an important source of ROS (109). In mitochondria, this adapter molecule has been suggested to function
as a redox enzyme possibly oxidizing cytochrome c and
generating H2O2 in the amino-terminal portion of p66shc
containing sequence similar to that of certain redox enzymes (156).
It was hypothesized that p66shc in mitochondria exists
within a high-molecular-weight complex which includes
mtHSP70 and TIM-TOM complex. Inside of such a complex, p66shc is inactive. After propagation of apoptotic signal, the complex is dissociated resulting in the release of free
p66shc, which becomes activated and capable of participat-
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MITOCHONDRIAL ROS AND ROS-INDUCED ROS RELEASE
ing in the electron transport which generates H2O2. Mutations in the redox active sequence of p66shc abolish its
pro-apoptotic activity apparently through the inability to
interact with cytochrome c (156). Recent data show that
p66shc-generated ROS regulate insulin signaling (48), T-cell
and B-cell signaling pathways (133), and expression and
activity of the ROS-generating enzyme NADPH oxidase
(NOX4) as well as activate NF-␬B (226, 291), thereby amplifying oxidative stress and inflammation.
comes from ETF which accepts electrons from different
dehydrogenases including those involved in ␤-oxidation
(41) and transfers them to the ubiquinone pool in the inner
mitochondrial membrane by a reaction catalyzed by ETFubiquinone oxidoreductase (ETF-QO) residing in the matrix side of the inner mitochondrial membrane (153). In
␤-oxidation, the sequence of reactions is as follows: acyl
CoA dehydrogenase ¡ ETF ¡ ETF-QO ¡ Ubiquinone ¡
complex III.
4. ␣-Glycerophosphate dehydrogenase
ETF-QO contains flavin (FAD) and a [4Fe-4S] cluster which
makes it vulnerable to ROS (363), and it serves as a convergence point for electrons flowing from nine flavoprotein
acyl-CoA dehydrogenases and two N-methyl dehydrogenases (153, 227). Recently, it was found that oxidation in
muscle mitochondria of long-chain fatty acids in physiological (low) concentration is associated with higher rates of
ROS formation than oxidation of NADH-linked substrates
while exhibiting relatively low dependence on the mitochondrial membrane potential (380). The authors suggest
that this enzymatic activity may be responsible for ⌬␺-independent ROS production.
Another potential source of ROS in mitochondria could be
␣-glycerophosphate dehydrogenase that occupies the outer
surface of the inner mitochondrial membrane, but its activity is relatively low in the liver, heart, and brain but high in
brown adipose tissue (304). However, isolated mitochondria supplemented with ␣-glycerophosphate in the presence
of antimycin A produce hydrogen peroxide which, when
normalized to an enzymatic activity, exceeds that originating from complexes I or II (116). This source could represent one of the most efficient ROS generators in mitochondria. It is almost insensitive to the presence of an uncoupler
or rotenone, implying that ROS generation by ␣-glycerophosphate oxidation is not dependent on mitochondrial ⌬␺
contrary to, e.g., succinate oxidation (304). The detailed
mechanism of ROS production by this enzyme is not defined yet.
In addition to previously mentioned sources of mitochondrial ROS production, it was shown that cytochrome b5
reductase (470) and dihydroorotate dehydrogenase (112,
140, 276) produce ROS on the outer surface of the inner
membrane. However, the significance of these enzymes in
the total ROS production remains questionable.
5. Electron transfer flavoprotein (ETF) and ETF
quinone oxidoreductase (ETF dehydrogenase)
In 1972 Boveris et al. (58) found that rat liver mitochondria
can produce a substantial amount of hydrogen peroxide
when oxidizing palmitoyl carnitine or octanoate. This production was ceased in the presence of an uncoupler. The
fatty acid-induced ROS generation was comparable to that
in the presence of glutamate plus malate and was slightly
lower than with succinate (58). Thirty years later, similar
results were obtained with skeletal muscle and heart mitochondria (414). Increased lipid metabolism was correlated
with upregulated UCPs expression/activities (73, 369), possibly to salvage the system from excessive ROS production
(414). Since ROS generation was almost insensitive to external SOD, it has been proposed that the fatty acid ␤-oxidation may result in ROS generation at a distinct mitochondrial matrix site which is different from o-center of complex
III. In addition, it has been proposed that a significant contribution to ROS production during fatty acid oxidation
Deficiency of ETF-QO in most cases is caused by single
point mutations around the FAD-ubiquinone interface
(326) and results in a human genetic disorder known as
multiple acyl-CoA dehydrogenase deficiency (MADD) or
glutaric acidemia type II (141). This is characterized by
impaired fat and protein metabolism. It may be associated
with acidosis or hypoglycemia and accompanied by other
symptoms such as general weakness, liver enlargement, increased risk of heart failure, and carnitine deficiency, and
could result in a fatal metabolic crisis (144, 390, 443).
6. Aconitase
Aconitase catalyzes transformation of citrate to isocitrate in
the Krebs cycle. It contains a cubane-type [4Fe-4S] center
with three iron atoms interacting with cysteine residues and
inorganic sulfur atoms, while the fourth iron, Fe-␣, is exposed to the solvent that allows the catalytic dehydration of
citrate to form the intermediate cis-aconitate, as well as the
subsequent hydration of cis-aconitate to form isocitrate
(42, 138). The prosthetic group of aconitase is highly susceptible to inactivation by superoxide anion radical yielding
an inactive [3Fe-4S] form, Fe2⫹-␣ and H2O2 (138, 267,
452). Interaction of the latter two ignites Fenton’s reaction
resulting in the release of ·OH radical (452). It has been
proposed that aconitase would be an ideal sensor for ROS
in cells (147, 148). Subsequently, superoxide toxicity in
mitochondria could be explained by enhanced aconitase
inactivation and related processes. For example, doxorubicin cardiotoxicity was explained mainly by an aconitase
inactivation accompanied by hydroxyl radical release
(296). Aconitase inactivation is an example of a “ROS
cross-talk” where one type of ROS (superoxide) is inducing
the release of another, potentially more damaging, ROS
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921
ZOROV ET AL.
(the hydroxyl radical). An alternative point of view is that
aconitase inactivation may serve a protective role by diminishing electron flow through the ROS-generating respiratory chain. In addition, the accumulation of citrate as a
result of a decreased Krebs cycle flux promotes chelation of
Fe2⫹ which is then irreversibly oxidized to a more stable
complex citrate-Fe3⫹, thus preventing catalysis of Fenton’s
reaction by free Fe2⫹ (400).
VI. ROS-INDUCED ROS RELEASE
ASSOCIATED WITH THE mPTP
A. Fundamentals of the Discovery
The importance of cellular redox homeostasis in progression of inherited and acquired pathologies, including those
associated with an aggressive oxidative environment, was
postulated elsewhere (reviewed in Refs. 72, 174). As we
discussed earlier, the redox homeostasis is determined by
the balance between ROS generation matching metabolic
needs and ROS quenching capacity. Undoubtedly, tipping
the balance in favor of increased ROS production within the
cellular microenvironment can severely alter the cellular
redox equilibrium, potentially resulting in oxidative stress
which when mild can cause oxidation of essential mitochondrial components. In extreme cases it can irreversibly
damage these components resulting in a cell death. Within
the great diversity of types of cell death, which to date
comprise as much as 13 types (145), at least some of them
could be associated with induction of the mPTP.
mPTP opening is a phenomenon known in the field of mitochondrial research for many decades, but for the first time
described in details in a set of three consecutive papers by
Haworth and Hunter in 1979 (182, 195, 196). This phenomenon [also recognized as an opening of a megachannel
(228, 229, 430)], originally studied in isolated mitochondria, represents a sudden change in the permeability of the
inner mitochondrial membrane allowing not only protons
but also other ions and solutes of a size up to ⬃1.5 kDa to
go through this membrane. There are many reviews on the
tentative nature and identity of the mPTP (64, 489, 494)
with details which are far beyond the scope of the present
review. Previously, many candidates were considered to
serve as the core of the pore [i.e., mitochondrial VDAC,
cyclophilin D (cyPD), ANT (108, 170, 217, 351, 457, 489,
494)], but largely they have been all dismissed because of
various reasons, but still leaving for them important poremodulating functions. Recent evidence suggests that a
dimer of mitochondrial ATP-synthase is essential to form a
core of the mitochondrial pore (157) and c subunit of the
mitochondrial ATP synthase complex may be required for
mPTP-dependent mitochondrial fragmentation and cell
death (56).
922
Although mPTP induction is typically referred to as a pathological event very often resulting in the degradation of
mitochondria or the cell (which will be discussed later),
there is multiple evidence and assumptions that in fact it can
also serve physiological functions. It has been postulated
that mPTP could serve as a release valve for quick release of
cations constantly leaking into the mitochondrial matrix
due to the mitochondrial membrane potential. A flickering
mode of mPTP may serve this purpose with the mPTP opening for a time not sufficient for the onset of complete mitochondrial depolarization (239). Another support of a physiological role of the mPTP was obtained in CyPD knockout
mice which demonstrated an obvious maladaptive phenotype in their hearts (124). While the role of CyPD as a core
of the mPTP has been dismissed, there is a general consensus that CyPD can serve a modulatory role in the
process of the mPTP induction. It was shown that CyPD
knockout mice exhibit substantially greater cardiac hypertrophy, fibrosis, and reduction in myocardial function
in response to pressure overload stimulation than control
mice while cardiomyocyte-specific transgene expression
of CyPD in these mice helped to rescue from the named
pathologies. Also, in mice lacking CyPD ischemic preconditioning was augmented (239) while mPTP openings
in wild-type mitochondria were much more frequent
than in mitochondria of knockout mice. This supports
the notion that the mPTP might have an important physiological role, possibly through regulation of intramitochondrial Ca2⫹.
The role of Ca2⫹ in the induction of the mPTP has been
already mentioned above; similarly, the role of oxidants in
generation of the mPTP pore is essential too, although both
resulting in the same phenomenological outcome (255,
277). It has been recognized that the mPTP induction represents a highly complex phenomenon (322, 409). Particularly, isolated mitochondria exposed to Ca2⫹ plus Pi
demonstrate the collapse of ⌬␺ preceding mitochondrial
swelling, suggesting that the generation of a smaller, lowconductance pore occurs with permeability for ions but not
for solutes prior to induction of the mPTP (full size) pore (5,
67, 244, 339). Oxidants such as hydrogen peroxides, organic peroxides, and some other inducers generate both
pores, but the insensitivity of induction of low-conductance
pore to Ca2⫹ and insensitivity of fully induced mPTP to
conventional inhibitor, cyclosporine A and sometimes to
EGTA (65, 243, 262, 284), makes them different from classical pore inducers (255; reviewed in Ref. 489). Therefore,
the mitochondrial pore phenomenon appears to be multifaceted.
Light is known to be a potent oxidant inducer when it
interacts with photosensitizing agents, and such a property
is successfully utilized in photodynamic therapy (114, 317).
It is based on the ability of the excited fluorophore to generate primary ROS which after release inside of the biolog-
Physiol Rev • VOL 94 • JULY 2014 • www.prv.org
MITOCHONDRIAL ROS AND ROS-INDUCED ROS RELEASE
ical sample may become destructive for cellular components. In principle, many biological molecules and cell compartments can be fluorescently labeled and oxidatively
modified after interaction with excitation light. The mitochondrion is an easy target for such modification since it
can accumulate fluorescent cations to a great extent using
its intrinsic proton motive force, namely, its ⌬␺ (211, 298).
Mitochondrial ⌬␺ provides selectivity for photodynamic
action localized exclusively in mitochondria without potential impact on other intracellular compartments (301, 303,
366, 367, 403). After exposure to light, the photosensitizer,
e.g., tetramethyl rhodamine methyl ester (TMRM) which is
a conventional probe for mitochondrial membrane potential widely used for visualization of energized mitochondria
in the cell, generates various ROS in water including very
strong oxidants such as superoxide anion radical and hydrogen anion radical (491).
Because mitochondria are critical intracellular loci of ROS
production, together with the fact that ROS exposure can
lead to the mPTP, it was hypothesized that under certain
circumstances the mPTP could become self-amplifying and
unstable (491). This hypothesis was tested in cardiac myocytes, taking advantage of the unique organization of mitochondria between myofilaments in an ordered three-dimensional latticelike array forming straight lines thus allowing
specific applications of line-scan confocal microscopy to
address this question (491, 493). For this purpose, rat cardiac myocytes are double stained with the probe for mitochondrial membrane potential (TMRM or ethyl derivative,
TMRE having excitation maximum in a green region of the
spectrum) and the ROS probe, 2,7-dichlorodihydorfluorescein diacetate (DCF-H2). DCF-H2 itself is nonfluorescing
and unreactive toward oxidants; however, after base hydrolysis of the ester bonds, the resulting nonfluorescing com-
CONTROL
DIAZOXIDE
pound becomes reactive toward ROS, and after oxidation it
is transformed into fluorescing DCF with maximum excitation in the blue region of the spectrum. The first scan of a
cardiac myocyte previously unexposed to light frequently
reveals the area(s) in which mitochondria are expected to
occupy, but unstained with TMRM (suggesting that mitochondrial ⌬␺ had collapsed in these mitochondria) but also
demonstrating high DCF fluorescence. The size of the area
is dependent on the physiological status of the cell (FIGURE 7),
possibly reflecting the cellular level of tolerance to ROS.
Therefore, isolated adult rat cardiac myocytes are a convenient model to study cellular oxidative stress.
B. mPTP and Ischemia/Reperfusion Injury
For the last 50 years it has been well recognized that coronary reperfusion of infarcted myocardium is associated
with increased necrotic death of irreversibly injured cardiac
myocytes. Jennings et al. (208) were first to report harmful,
both structural and functional, changes associated with reperfusion. Therefore, early reperfusion while crucial for preserving ventricular function, preventing infarct expansion
and potential development of heart failure, may also contribute to the pathogenesis of reperfusion arrhythmias and
myocardial stunning manifested by reversible contractile
dysfunction. The biological basis of reperfusion injury has
been extensively studied since then and consequently the
notion of reperfusion as a double-edged sword was formulated in 1985 by Braunwald and Kloner (63). In 1986
ischemic preconditioning was described as a means to render the heart more resistant to ischemia/reperfusion injury
(311). The importance and the clinical potential of these
discoveries has prompted the research community to focus
on deciphering the molecular mechanisms that underlie rep-
HYPOXIC-PC
20 µm
(no PC)
(no PC)
HYPOXIC-PC + 5HD
FIGURE 7. ⌬␺ loss in a significant fraction
of mitochondria, caused by hypoxia/reoxygenation. Depolarized mitochondria (red-fluorescence ”holes“; bottom panels) are associated with increased ROS (green; bottom
left panel). Hypoxic PC or pharmacological
preconditioning (PC), represented by diazoxide (Dz), prevents mitochondrial depolarization, and 5-hydroxydecanoate (5HD) accentuates the loss. [From Hoffman et al. (189).]
TMRM ΔΨ
DCF (ROS)
Physiol Rev • VOL 94 • JULY 2014 • www.prv.org
923
ZOROV ET AL.
erfusion-induced cellular injury as well as cardioprotection.
Thousands of research papers have already been published,
and a detailed picture has emerged, e.g., recently reviewed
in References 181, 205, 207, 375. The significant role of
so-called oxygen paradox was recognized (183) early on
and was based on the finding that substantial injury occurs
when molecular oxygen is reintroduced into ischemic tissue. A burst of ROS production on reperfusion has been
detected (149, 500). Immediately after exposure of the
ischemic organ to oxygen, superoxide anion radicals dominate among the emerging ROS in the effluent perfusate,
with further formation of hydroxyl radical (499) pointing
to the occurrence of iron-mediated Fenton chemistry. This
can be explained by the likelihood that in oxygen-depleted
medium the iron ions are mostly reduced (redox potential of
the couple Fe2⫹/Fe3⫹ ⫽ ⫹0.77 V), but since hydrogen peroxide is absent, the Fenton reaction does not yet occur.
Reperfusion ignites the formation of superoxide anions
which dismutate resulting in formation of H2O2 and immediately reacting with iron ion [still in a reduced form
(Fe2⫹)], thus generating highly reactive ·OH (400).
mitochondrial Ca2⫹ uptake and consequent induction of
mPTP (96, 97). This results in mitochondrial uncoupling
and activation of ATP hydrolysis by F1Fo-ATP-synthase
(96, 97, 118).
Mitochondria have been implicated as a potential source as
well as a target of the generated ROS resulting in an observed loss in mitochondrial function during ischemia/reperfusion and consequent irreversible cellular injury (9, 101,
329). It has been noted that not only these radicals play a
significant role in the tissue damage observed following
ischemia/reperfusion but that this injury can be mitigated
by oxygen radical scavengers (e.g., Ref. 213). In the early
1990s, Crompton and colleagues demonstrated in isolated
mitochondria that the derangement of mitochondrial bioenergetics that develop on reoxygenation, when resting cytosolic Ca2⫹ is high and ATP low, could lead to excessive
C. RIRR: Experimental Demonstration
TMRM FLUORESCENCE
200
100
ΔΨm
175
CALCEIN
150
50
125
MPT
100
0
0
5
10
15
TIME (sec)
2 µm
FIGURE 8. RIRR and MPT induction. A: confocal microscope fluorescence imaging of cardiac myocytes
loaded with TMRM (red) and DCF-H2 (green). A, top: fluorescence image of TMRM-loaded cardiac myocyte.
B: enlarged portion of the TMRM-loaded cardiac myocyte. Line drawn on image shows position scanned for
experiment in bottom panel. C: row of mitochondria were line-scan imaged at 2 Hz with excitation at both 488
for DCF and 543 nm for TMRM and collecting simultaneous fluorescence emission at 510 –550 nm and ⬎560
nm, respectively. The sudden loss of ⌬␺ and rise of ROS generation in individual mitochondria are indicated by
the loss of TMRM fluorescence and increase in DCF fluorescence. B: cells dual-loaded with TMRM (⌬␺) and
calcein-AM (the latter loaded under conditions that results in a cytosolic distribution initially in excess over that
in mitochondria); line-scan imaging at 2 Hz. For details, see Refs. 491, 493.
924
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CALCEIN FLUORESCENCE
B
50 sec
To further explore this puzzling phenomenon, confocal microscopy was employed in line-scan mode allowing repeated excitation of a row of mitochondria along the selected line in cardiac myocyte (FIGURE 8, A–C). Within the
light-exposed mitochondria, a sudden loss of ⌬␺ with corresponding rise of ROS generation was revealed, suggesting mPTP induction. Although these transitions were
modestly sensitive to cyclosporine A, they were dramatically delayed by another mPTP inhibitor, bongkrekic
acid, and by a ROS scavenger, Trolox. Furthermore, re-
D
A
C
The cardiomyocytes exposed to the hypoxia-reoxygenation
cycle have much larger areas occupied with fully deenergized mitochondria and high levels of ROS, and these levels
were diminished and ⌬␺ was regained in most mitochondria after ischemic or pharmacological preconditioning of
the cell (FIGURE 7) (218). Based on the previously expressed
assumption that the cardiac ischemia-reperfusion injury is
associated with the induction of the mPTP (97, 161), the
speculation was put forth that the found abnormal subcellular loci in cardiac myocytes were indeed occupied by mitochondria which had undergone permeability transition.
The most striking detail was that mitochondrial deenergization, if caused by the mPTP opening, was associated with
higher, rather than lower, ROS production as follows from
FIGURE 5.
MITOCHONDRIAL ROS AND ROS-INDUCED ROS RELEASE
distribution of the inert fluorescent probe calcein (mol wt
620) from cytosol to mitochondria at the moment of
mPTP induction also confirmed opening of a pore permeable to a 620-Da compound (FIGURE 8B). Thus it was
concluded that the collapse of the mitochondrial ⌬␺ occurs due to the mPTP pore opening.
Fine analysis of the ROS burst kinetics associated with the
mPTP induction showed that ROS generation in the affected mitochondrion proceeds in two distinct phases: the
initial, slow rise due to the accumulation of a photochemistry-associated production of ROS (called “trigger ROS”)
and the subsequent ROS burst associated with dissipation
of the mitochondrial membrane potential. This biphasic
process was named “ROS-induced ROS release” (abbreviated RIRR). In addition to the ROS burst and the mPTP
induction, it was accompanied by the burst of nitric oxide
production; thus the term ROS-induced ROS/RNS release
where RNS stands for reactive nitrogen species is also relevant (491).
The identical phenomenon using the same instrumentation
as in original RIRR study (491) was later described in cells
infected by adenovirus carrying mitochondria targeted circularly permuted yellow fluorescent protein (cpYFP) as the
ROS sensor. The term superoxide flashes was used to describe the RIRR in transfected cardiac myocytes (461). Recently, this approach was reexamined and challenged by
observation that the cpYFP is very sensitive to pH changes,
therefore rendering this interpretation inconclusive (376,
377, 466).
The complex I inhibitor rotenone (at low concentrations
not affecting TMRM sequestration) significantly decreased
the ROS burst magnitude, confirming the mitochondrial
nature of the ROS burst during mPTP-associated RIRR
(491). The few second delay of NADH oxidation following
the collapse of the mitochondrial ⌬⌿ suggests that NADH
is the redox-energy store driving the electron donor necessary to support the single-electron reduction of molecular
oxygen that produces superoxide as an initiating radical for
generation of primary ROS followed by the mPTP induction. As we have already mentioned, the ROS burst accompanying ⌬⌿ collapse is against the existing “dogma” that
ROS production is inversely related to the mitochondrial
membrane potential magnitude (FIGURE 5). This dogma
seems to be true when the classical uncoupling is considered, i.e., the drop of ⌬⌿ due to the enhanced proton conductance of the inner mitochondrial membrane. It seems to
be irrelevant to the extreme dysequilibrium situation when
a megachannel is opened in this membrane. The enhanced
ROS production in mitochondria undergoing mPTP opening has been confirmed in vitro when isolated mitochondria
were supplemented with NADH to compensate for its loss
as a result of opening a megachannel (35).
The paradoxical aberration of established relations between ROS production and mitochondrial transmembrane
potential (240, 275) may have the same nature that we
suggested when discussing the ROS production in complexes I and II. We concluded that, at least partially, it is
determined by unusual conformational changes within
these complexes. It is known that at least some components
demonstrate conformational changes within mitochondria
undergoing mPTP (171, 426, 427, 463, 464). Such conformational changes have been described, e.g., for ANT. It was
shown that the c-conformation of ANT (when ADP is
bound to the cytosolic side) is more preferred for the open
state of the mPTP than the m-conformation (when ATP is
bound to the matrix side), and it corresponds to the closed
state of the pore. Accordingly, atractylates stabilize the cconformation of the ANT and promote the mPTP while
bongkrekic acid (184) stabilizes ANT in the m-conformation and inhibits the mPTP (172, 233, 320). However, ANT
was proposed to be a pore modulator (494) rather than a
component of the core of the pore (235).
Mitochondrial ROS rise simultaneously with the mPTPinduced drop of ⌬⌿. However, quite often it has been possible to observe a very brief phase of mitochondrial hyperpolarization coinciding with the onset of the mPTP opening. The analysis proved that the hyperpolarization phase
also coincided with the start of the excessive ROS generation (493) (FIGURE 9). Although a hyperpolarization spike
has not been observed in all recorded mPTP opening events
in the cardiac myocyte, the flicker could be sufficiently brief
and be below the kinetic resolution of the fluorescent signal
from TMRM belonging to the class of “slow-response”
(distributed or accumulated) probes for the mitochondrial
⌬⌿ (458, 459). The flickering mitochondrial hyperpolarization
mode was found to be a frequent phenomenon during photo-excitation of TMRM in loaded cardiac myocytes observed in the confocal microscope during line-scanning.
Not every flicker could ignite the mPTP opening; nevertheless, each flicker initiated a small burst of ROS production that was insensitive to the mPTP inhibitor bongkrekic acid. Also these hyperpolarization flickers were
not associated with the entry of the inert probe calcein
from the cytosol into the mitochondria, suggesting these
flickers do not involve a long opening of the mPTP. So,
apparently there were at least two different modes of
RIRR, one of which was accompanied by the large-conductor mPTP opening, while another was too brief to
establish the conductance. It is noteworthy that the flicker-induced ROS production associated with mitochondrial hyperpolarization theoretically obeyed the conventionally established relations between ROS production
and mitochondrial transmembrane potential (FIGURE 5)
(240, 275), while the mPTP-associated ROS production
did not. Both were caused by the triggering ROS formed
during the photoexcitation process.
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925
ZOROV ET AL.
A
B
C
D
Transient
hyperpolarization
*
TMRM Intensity (arb)
300
250
200
150
100
MPT
50
5 µm
0
0
5
10
15
20
25
Time (sec)
10 sec
ΔΨ
F
ROS
H
2 µm
E
1 sec
1 2 3 4
G
70
190
60
50
30
40
20
TMRM
DCF
10
1
2
Time (sec)
3
1 2 3
140
4
90
56 7
30
20
0
DCF
40
TMRM
70
50
TMRM
I
80
60
0
56 7
240
40
50
100
150
200
250
300
350
Time (sec)
FIGURE 9. Transient ⌬␺ hyperpolarization and depolarization (flickering) preceding MPT induction can be
observed. A: overlay showing the 20-Hz linescan image in a cardiomyocyte loaded with TMRM (red) and DCF
(green). B: the TMRM channel from A; arrows indicate examples of transient hyperpolarization immediately
preceding ⌬␺ loss (MPT induction). C: the directional derivative of the TMRM intensity with respect to time, to
enhance the identification of intensity transients (transient hyperpolarization prior to MPT induction); arrows
indicate examples of transient hyperpolarization (positive slope maxima) immediately preceding MPT. D: intensity
plot of the TMRM responses of the mitochondrial pair indicated by the asterisk in B; inset shows an enlargement
of the area inside the red box and shows transient (relative) hyperpolarization immediately preceding MPT. [From
Zorov et al. (493), with permission from Elsevier.] E–G: ROS production (green) occurs during transient mitochondrial hyperpolarization flickering (red image, arrows in E). H–I: 2-Hz line scan image in a cardiomyocyte loaded with
TMRM. Transient hyperpolarization and depolarization spikes are seen as white and black vertical lines (shown by
the arrows from 1 to 7). TMRM fluorescence intensity (⌬␺) in the region of the cardiomyocyte denoted by the
arrow-bracket in H (⬃6 ␮m in width, consisting of 3 sarcomere-associated pairs of mitochondria) within which
mitochondria display concerted flickerings of the membrane potential. Note that these transient depolarizations,
during the time interval between arrows 1–7, are accompanied by progressive mitochondrial swelling (as seen by
the lateral displacement of adjacent mitochondria) consistent with repeated transient MPT-induction episodes.
926
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MITOCHONDRIAL ROS AND ROS-INDUCED ROS RELEASE
Consequently, “X-induced X release” may be a general
mechanism for any determined (such as RIRR and CICR)
and still undetermined signaling amplification loops. Meanwhile, reciprocal amplification of ROS and Ca2⫹ signals or
any other “X-induced Y” release processes representing solitary events or additional complex processes within a signaling cascade, probably also occur, e.g., in the case when ROS
generated in mitochondria may be a source of Ca2⫹ unitary
releases from ryanodine receptor of SR (Ca2⫹-sparks) (491)
(see FIGURE 10). The process of the spontaneous ROS-induced Ca2⫹ release by the SR ryanodine receptor described
in Reference 491 has been recently analyzed in a model of
sustained mitochondrial ⌬⌿ oscillations driven by the mitochondrially produced ROS. Dynamic changes in mitochondrial energy state resulted in altered frequency and
properties of the Ca2⫹ sparks (485).
50
Ca2+ Sparks
(FLUO-3)
40
8
7
6
5
4
3
2
1
0
30
25
Control
30
MPTProximity
20
20
15
10
ΔΨm
(TMRM)
0
0
0.5
1
1.5
2
2.5
3
FLUO-3 Fluorescence
Although there have been some attempts in the past to
ascribe mitochondrial mPTP to the existence of mitochondrial CICR (194) or even strontium-induced strontium release (190), eventually the essential role of ROS has been
established in this process. In fact, mitochondria have quite
a high Ca2⫹-buffering capacity (maximal calcium uptake),
explaining the fact that a number of consecutive Ca2⫹
pulses could be sequestered and tolerated by isolated mitochondria or permeabilized cells until mPTP opening occurs
resulting in a robust release from mitochondria of both
sequestered and intrinsic amounts of Ca2⫹ (or Sr2⫹) (77,
166, 321, 331). Such mitochondrial ability to sequester
Ca2⫹ could be modified by a number of factors (7, 30, 132,
306, 465).
Interestingly, calcium ions target and activate those mitochondrial enzymes (pyruvate dehydrogenase and ␣-ketoglutarate dehydrogenase) that are considered to be a key
source of ROS in mitochondria (see above). However, while
the crucial role of Ca2⫹ in mitochondrial metabolism is
established (reviewed in Refs. 166, 434) with recent molecular identification of the Ca2⫹-uniporter (36, 105) and
Ca2⫹/2Na⫹ exchanger (332), the role of Ca2⫹ in ROS production in mitochondria remains controversial. In isolated
mitochondria, the mPTP is opened by elevated Ca2⫹. This
led to the conventional assumption that mPTP induction by
mitochondrial Ca2⫹ loading may be the cause of many
EVENT RATE
(100 µm · s)-1
RIRR was named for the analogy with the process of Ca2⫹induced Ca2⫹ release (with acronym CICR) which has been
known since the 1970s (125, 127, 128, 139) and which
plays a critical role in excitation-contraction coupling. In
this process, an action potential depolarizes the cell membrane providing a small influx of Ca2⫹ through the plasma
membrane, which by itself is insufficient to provide contractile activation. Moreover, since the contractile apparatus is
at a distance from the sites of inward Ca2⫹ transport, Ca2⫹induced muscle fibers contraction would be retarded if it
depended only on Ca2⫹ diffusion within the muscle cell.
CICR serves two goals. First, it significantly facilitates the
propagation of the Ca2⫹ signal in the cell, and second, it
amplifies Ca2⫹ concentration to reach Ca2⫹ levels in the
vicinity of contractile apparatus essential to provide a contraction. Then, both RIRR and CICR contain signaling amplification loops to reach the threshold necessary for the
transition. CICR and RIRR are also similar in spatial terms;
CICR includes different intracellular compartments and
covers the space between the plasma membrane Ca2⫹-channels and ER/SR and fibers, and RIRR can span between
mitochondria and thus spread across and between cells.
Isolated mitochondria possess an ability to sequester very
large amounts of Ca2⫹ from the medium (85, 106, 259).
Although proton motive force potentially drives cations to
negatively charged mitochondrial matrix forcing cations to
be accumulated inside of mitochondria, it has been found
that at resting conditions in rat cardiac myocytes the intramitochondrial free Ca2⫹ level is ⬍100 nM. Mitochondrial Ca2⫹ concentration is maintained by inward and outward Ca2⫹-transporting systems in the inner mitochondrial
membrane. One of these, the uniporter (231, 372, 450,
451), has low affinity but high capacity for transporting
Ca2⫹, whereas Ca2⫹/2H⫹ and Ca2⫹/2Na⫹ exchangers (60,
61, 75, 98, 136, 137) have much lower capacity. In stimulated cells at higher pacing rates, the increased cytosolic
Ca2⫹ will steadily raise the [Ca2⫹]m. Over the course of
many contractions, [Ca2⫹]m can rise up to 600 nM (300)
and potentially results in activation of mitochondrial dehydrogenases. Pyruvate and ␣-ketoglutarate dehydrogenases
show the greatest dependence on [Ca2⫹]m (107, 176, 278,
302). Such activation of the key mitochondrial dehydrogenases results in activation of respiration and ATP synthesis
to meet rising energy demands under increased work load.
TMRM Fluorescence
VII. RIRR AND Ca2ⴙ
10
3.5
Time (sec)
FIGURE 10. Induction of Ca2⫹ sparks after the mPTP. Cell is
dual-loaded with TMRM (⌬␺) and fluo-3 (Ca2⫹) and line-scan imaged
at 230 Hz. Representative example showing the dissipation of
TMRM fluorescence from a single mitochondrion and a cluster of
Ca2⫹ sparks in the immediate vicinity, within seconds of MPT induction. Inset: comparison of Ca2⫹ spark rate in proximity of the mPTP
occurrence, i.e., within the sarcomere containing the involved mitochondria and within 3 s after the mPTP occurrence. [From Zorov et
al. (491).]
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927
ZOROV ET AL.
tMPT (normalized)
A
1.4
1.2
1.0
0.8
0.6
0.4
0.2
0.0
1 Ca2+ (rest)
5 Ca2+
(10 HzTet)
100 nM Ca2+
B
500 nM Ca2+
Perm SL
Intact SL
1.2
1.0
tMPT (normalized)
types of cell death, such as those induced by ischemia-reperfusion injury and chemical toxins, in the heart and brain.
In intact cardiomyocytes and neurons, however, normal
excitability achieves Ca2⫹ elevations to levels comparable
to those believed to induce the mPTP based on in vitro data.
This suggests a serious paradox that will have to be reconciled regarding the role of Ca2⫹ in mPTP induction in vitro
with the empirical evidence of health and longevity of these
postmitotic cells in vivo (218, 494). Although Ca2⫹ is a
typical tool for induction of permeability transition in isolated mitochondria (182, 195, 196; reviewed in Ref. 489),
experiments with intact cardiac myocytes and neurons
demonstrated that the mPTP is largely insensitive to increased cytosolic Ca2⫹ (see supplements to Ref. 218). In
these experiments, however, after cell permeabilization, the
Ca2⫹ sensitivity of the mPTP is unmasked and is similar to
that observed in mitochondria isolated from these cells (FIGURE 11, A AND B). Probably, some cytosolic factors that are
lost after permeabilization and mitochondrial isolation are
responsible for Ca2⫹ insensitivity of the mPTP in the intact
excitable cell such as the cardiac myocyte or neuron. These
facts themselves put into question the role of Ca2⫹ in cell
death correlated with mPTP induction after, e.g., hypoxia/
reoxygenation which may be overestimated due to limitation of the biological model (212) (see FIGURE 11C).
0.8
0.6
0.4
0.2
0.0
1 Ca2+ (pre Sr2+)
1 Sr2+ (1 hr)
1 Sr2+ (6 hr)
1 Ca2+ (post Sr2+)
C 100
90
80
% CELLS DEAD
There is also a puzzling discrepancy between a very high
apparent Km (250 –350 ␮M) for endogenous ADP in the
control of mitochondrial respiration in permeabilized muscle cells and that in isolated mitochondria where the apparent Km equals 15–20 ␮M (248). One of the possible explanations for this inconsistency is that isolated mitochondria
are stripped from cytoskeletal proteins that maintain mitochondrial integrity within the intracellular energetic unit,
which might be involved in fine regulation of enzymatic
parameters in the live cell and be responsible for observed
differences (22).
70
60
50
40
30
20
10
0
O2
--
Bapta
O Ca2+
N2
2⫹
VIII. MITOCHONDRIAL
COMPARTMENTATION
The reviewed data raise the question about the adequacy of
isolated mitochondria or permeabilized cells to the actual
processes taking place in an intact live cell, as all three
objects have different levels of structural and organizational
complexity. One of the structural parameters missing in
isolated mitochondria is a close proximity of mitochondria
to ER/SR found in the cell (119, 379, 384), thus making
easier shuttling of small signaling elements (e.g., ROS, H⫹,
or Ca2⫹) and tightening the spatial and functional compartmentation of two organelles (357). The loss of spatial compartmentation due to the isolation procedure may dramatically change kinetics and, thus, enzymatic parameters of
interactive systems. The compartmentalized system might
928
FIGURE 11. Ca
dependence of the mPTP in rat adult cardiac
myocytes. A: mPTP ROS threshold during a “clamp” of intracellular
Ca2⫹ ⬎500nM for ⬎20 min (using 10-Hz electrical tetanization in 5
mM bathing Ca2⫹ in the presence of thapsigargin or cyclopiazonic
acid to disable the sarcoplasmic reticulum) is identical to that in
unstimulated cells (with a cytoplasmic Ca2⫹ of ⬃100 nM). B: complete equimolar replacement of Ca2⫹ for Sr2⫹ (for 6 h) in intact
cardiac myocytes resulted in the same mPTP ROS threshold as seen
in cells with normal Ca2⫹. C: buffering intracellular Ca2⫹ (with
BAPTA) or limiting Ca2⫹ influx (in nominally Ca2⫹-free buffer) does
not limit cardiac myocyte death after hypoxia/reoxygenation. [From
Juhaszova et al. (218). Republished with permission from the American Society for Clinical Investigation; permission conveyed through
Copyright Clearance Center, Inc.]
greatly diminish the diffusion-controlled step(s) when the
product of one enzymatic reaction almost immediately becomes a substrate of the second enzyme without the step of
a product release in the bulk phase followed by extraction
from this phase.
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MITOCHONDRIAL ROS AND ROS-INDUCED ROS RELEASE
It is beyond the scope of this review, but the dramatic difference between two theories of oxidative phosphorylation,
one proposed by Peter Mitchell and another by Robert
Williams, deserves mentioning. Both theories were
named chemiosmotic with one principal difference: while
Mitchell’s theory considered the delocalized proton ejected
from a proton pump into the bulk phase to be a substrate
for ATP synthase complex (298, 299), Williams considered
a localized proton to be consumed by ATP synthase without
an intermediate step of going into the bulk phase (472,
473). Although there are some more or less sketchy observations supporting the second mechanism which apparently requires organization of supramolecular protein complexes (reviewed in Ref. 131), the first mechanism was generally accepted, and its founder was awarded the Nobel
prize in 1978.
The problem of intramitochondrial compartmentation has
many supporters (e.g., Refs. 412, 413; reviewed in Ref.
265). However, recent data has led to an interpretation
highly critical of the organization of mitochondrial respiratory chain into supramolecular complexes (organized protein assemblies) (441), leaving the compartmentation issue
still rather controversial.
Another important issue is the intermitochondrial compartmentation. The possibility that mitochondrial units may
work in concert unifying energy transmission and signaling
has been highly explored. Concerted work of individual
mitochondria in striated muscles interconnected by electrically permeable junctions (28) was proposed (394) and later
confirmed for cardiac myocytes (10). Such electrical unification of mitochondria was postulated to adequately allow
fast transportation of energy along the mitochondrial reticulum to all cellular regions remote from the initial energy
source. Later the mitochondrial matrix lumen continuity
was confirmed by using photoactivated GFP (445), supporting the idea of mitochondrial organization in networks
(115, 199, 395) which play an important role in intracellular signaling (46, 215, 395). Cooperation of mitochondria
in terms of synchronous response to oxidative challenge as
part of the RIRR process seems to open a new door for
exploring alternative roles of mitochondria in the cell apart
from their energetic function (491, 493).
IX. MITOCHONDRIAL RIRR IN
OSCILLATING MODE
It is noteworthy that fluctuations of ROS within the cell in
the vicinity of mitochondria can elicit instability of the intracellular redox state which, as we pointed out earlier, is
greatly maintained by homeostatic mitochondrial functioning.
FIGURE 12 demonstrates ROS-induced fluctuations of the
mitochondrial ⌬␺ (one recorded in adult and another in
neonatal cardiac myocyte) showing that mPTP in these cells
can be reversed and the mitochondrial inner membrane be
FIGURE 12. Oscillations of ⌬␺ in individual mitochondria over time
in a portion of a cardiac myocyte induced by 10 ␮M clotrimazole.
Panels of consecutive fluorescence frames with a different interval
indicated in each plane 10 s acquired from either a 18 ⫻ 15 ␮m
region of an adult cardiac myocyte [A; from Zorov et al. (493), with
permission from Elsevier.] or neonatal cardiac myocytes loaded with
TMRM (B). Arrows indicate individual mitochondria or mitochondrial
clusters displaying periodic changes in the membrane potential.
sealed and ⌬␺ regained with many repetitive cycles of the
mPTP induction and closure. The first demonstration in
intact cells of such an oscillatory mode of the mPTP triggered by photodynamically induced ROS accompanied by
the RIRR was given in 2000 (491) and further explored in
2006 (493).
The regulation of mitochondrial ROS production and their
levels is exerted by a number of factors, such as the redox
state of respiratory components, oxygen tension, ionic environment and the activity of redox buffers, etc. However,
the mitochondrial environment may become unstable,
which may cause the instability of mitochondrial functioning, and potential episodes of fluctuating mitochondrial
ROS production are not an exception. Several researchers
described synchronized mitochondrial oscillations including rhythmic changes of ion fluxes, respiration, and mitochondrial volume in vitro in suspensions of isolated mitochondria (particularly, under conditions of energized ion
transport) (23, 83, 158, 168, 198, 312, 330). Typically,
these oscillations had the form of damped sinusoidal rhythmic changes. Under these conditions, within each cycle, the
mitochondrial shrinkage phase was associated with increased respiration, a more highly oxidized state of pyridine
nucleotides, a stimulation of ATP hydrolysis, an inhibition
of proton release, and stimulation of cation release. The
shrinkage phase is followed by a swelling phase, and when
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929
ZOROV ET AL.
these changes were reversed, it also showed evidence of
damping. It was concluded that the oscillatory states of
electron and energy transfer pathways might be under the
control of mitochondrial swelling and ion transport by a
feedback mechanism.
Later, such oscillatory behavior of different mitochondrial parameters including those induced by ROS has
been detected in intact cells and tissues (18, 92, 126, 281,
297, 360, 491, 493) showing that structural peculiarities
in organization of mitochondria within the cell do not
play the most important role in induction and propagation of oscillations.
X. IMAC-ASSOCIATED RIRR
Another model of RIRR was generated and explored in B.
O’Rourke’s lab at Johns Hopkins University. The study was
preceded by an investigation of oscillations in the sarcolemmal current in cardiac myocytes caused by substrate deprivation resulting in spontaneous fluctuations of ATP-sensitive K⫹ current in parallel with changes of the redox state of
NADH/NAD (324). This implies the critical role of energy
in generating oscillations. Since metabolic oscillations were
in concert with the shortening and suppression of the action
potential, it has been concluded that these metabolic oscillations may be relevant to pathologies such as arrhythmias
as a result of ischemia/reperfusion insult (reviewed in Refs.
20, 323). Later, it was found that a local two-photon excitation of cardiac myocyte covering a few mitochondria
loaded with a mitochondrial membrane potential-sensitive
fluorescent dye triggered oscillation of the membrane potential within these mitochondria. It also triggered the oscillations in intramitochondrial redox potential measured
by a ratio of reduced-to-oxidized flavins. The igniting impulse was triggered by ROS generated by the local excitation resulting in production of primary ROS. The oscillations spread in three dimensions along all interconnected
mitochondria forming a lattice with the primary oscillators
consisting of only a few mitochondria. These could later
spread over the entire mitochondrial network and possibly
extend beyond the boundaries of the single cell.
Mitochondrial inhibitors such as rotenone and bongkrekic
acid suppressed mPTP-associated RIRR and ROS to the
level below the threshold implicating the source of ROS as
mitochondrial (491). However, these inhibitors as well as
others including cyclosporine A, cyanide, myxothiazol, nigericin, and oligomycin did not block RIRR associated with
transient mitochondrial hyperpolarizations (mitochondrial
membrane potential flickers described above) (FIGURE 9)
(493). In contrast to mPTP-associated RIRR, in the RIRR
mode associated with hyperpolarization flickers, the brief
changes of the membrane potential were not accompanied
by significant redistribution of an inert fluorescent agent of
620 Da (calcein) between mitochondria and cytosol. There-
930
fore, the mPTP is either too transient for sufficient movement of calcein to be measured, or of low conductance, or
possibly even not involved in this type of oscillatory mode.
In a comprehensive search for ion channels responsible for
the various types of non-mPTP-related mitochondrial oscillations, the focus moved to the so-called IMAC because the
oscillations were blocked by the IMAC inhibitor DIDS (38 –
40). In addition, these oscillations were significantly attenuated by ligands of the mitochondrial (peripheral) benzodiazepine receptor (PBR) such as Ro5– 4864 and PK11195
(18, 323). It has been speculated that PBR (which presumably consists of ANT, VDAC, and 18-kDa protein of the
outer mitochondrial membrane, TSPO) could be responsible for IMAC (20) since some ligands of PBR can block the
mitochondrial inner membrane 107-pS channel (230)
which has moderate anion selectivity (410). However, PBR
ligands (230) as well as the mPTP inhibitor cyclosporine A
(430) both block mitochondrial giant MCC (multiple conductance channel), implying that mPTP might be related to
the functional state of PBR. However, this speculation
needs additional experimental support with elucidation of
the molecular identity of IMAC.
Thus, in addition to mPTP-associated RIRR, another independent mode was proposed for RIRR which involves
IMAC. According to this model, mitochondrial respiratory chain is the main oscillatory source of ROS which
can be released to the cytosol through IMAC due to its
permeability for superoxide (449) produced in complex
III (FIGURE 13).
Recently, the ⌬␺ oscillations and RIRR induced by local
oxidative stress (401) and by perfusion with hydrogen peroxide (53) have been detected in live, intact myocardium.
Perfusion of rat hearts with hydrogen peroxide, depending
on the concentration of the oxidant, elicited two peaks of
superoxide in live myocardium measured by optical mapping of the fluorescent ROS probe, with the second peak
being much more intense than the first one (53). Spatiotemporal ROS mapping during the second ROS peak revealed a
propagation of superoxide signal within the cardiac tissue
with a velocity ⬃20 ␮m/s. The hearts with the second peak
displayed a much greater arrhythmia compared with those
where the second peak was absent. PBR ligand, Ro5– 4864,
and superoxide dismutase mimetic but not cyclosporine A
abolished RIRR and ventricular tachycardia and ventricular fibrillation, suggesting IMAC involvement. These findings further extended the concept of RIRR as a key factor in
the incidence of postischemic arrhythmias.
A computational model with superoxide as a trigger of
mitochondrial ⌬␺ depolarization has been developed (484,
486). The model is based on the percolation theory to explain how neighbor-to-neighbor interaction defines propagation of the signals along the spatially organized excitable
matrix, arising from a spanning cluster of oxidatively
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MITOCHONDRIAL ROS AND ROS-INDUCED ROS RELEASE
stressed mitochondria united in the network (19, 20). In
addition, the dynamic spatiotemporal properties of individual mitochondrial oscillators within stress-induced synchronized oscillatory clusters of cardiac mitochondrial network have been characterized by applying wavelet-based
analysis (252, 253). Individual oscillating mitochondria
within clusters have been identified and their frequency correlated with the size of the cluster. As the cluster grew
larger, they required more time to achieve full synchronization of all mitochondria within the cluster. Additionally, the
percentage of cluster size has been inversely correlated with
the percentage of amplitude. Nonlinear characteristics of
the mitochondrial network, particularly in the heart, make
mitochondria prone to the local disturbances including
those involving oxidative stress. Once these local disturbances are formed, they spread over the network causing
loss of its normal functioning. They cause a mismatch of the
finely tuned balance of available energy production to energy expenditures (93, 365, 435, 479), resulting in an inability of mitochondrial network to completely repolarize
between oscillations. Finally, it reaches a point of no return
that results in pathologies and ultimately cell death.
XI. RIRR-RELATED PATHOLOGIES: THE
IMPORTANCE OF ⌬␺ HOMEOSTASIS
Mitochondria are not only the energy source but also one of
the primary sources of a vast number of deadly pathologies.
As to cardiovascular problems, a striking example of “misbehavior” of a mitochondrial network extending over the
whole heart and affecting the whole organism is ischemia/
reperfusion-induced oxidative injury which in extreme
cases results not only in cell death, but also organ failure,
and eventually organismal death. Experiments with single
cardiac myocytes exposed to transient hypoxia, followed by
a reoxygenation phase, demonstrated substantial mito-
chondrial instability caused by the introduction of oxygen
into the system. In these cells, transient mitochondrial hyperpolarization was followed by a progressive rise of the
ROS level in deteriorating mitochondria to the point of
mPTP introduction and accompanying large ROS burst
(FIGURE 14A), i.e., typical of RIRR. The incidence of cardiac
cell death depended on the proportion of damaged mitochondria having developed mPTP to their total number
in the cell and on the physiological status of the cell (218)
(FIGURE 14B).
To assess the degree of system readiness to resist an oxidative challenge, a simple approach was developed using time
as a factor. Time quantifies the titrated amount of ROS
(delivered incrementally by successive photoactivated exposures during linescan imaging) required to achieve the
threshold for mPTP induction in a particular mitochondrion (218, 491). Apparently, mitochondria with higher
resistance to oxidative stress require a longer time (i.e.,
higher total oxidant exposure level) to open the pore while
those with a low tolerance require shorter time (i.e., lower
total oxidant exposure level) for the mPTP induction. The
timing of mPTP opening (tmPTP) is an integral factor, depending on the rate of oxidant quenching (the level of intraand extramitochondrial antioxidants, e.g., glutathione) and
the physicochemical state of the system responsible for the
generation of the mPTP as an event. While the molecular
identity of the mPTP needs to be validated in detail (157,
217, 494), the presence of bcl-2/bcl-xL or phosphorylated
glycogen synthase kinase 3␤ (P-GSK-3␤) in the vicinity of
the pore or attachment of hexokinase II desensitize the pore
to oxidants (86, 217, 218) and extend the time necessary to
overcome the desensitization and open the pore. The conventional line-scan confocal microscopy has been employed
as a model to determine the ROS threshold for inducing the
mPTP. For this purpose, an ideal experimental object is a
FIGURE 13. Hypothetical scheme of the
IMAC-associated ROS-induced ROS release
mechanism underlying the synchronization
of mitochondrial activity during the oscillations. [Modified from Aon et al. (18), with
permission from The American Society for
Biochemistry and Molecular Biology.]
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ZOROV ET AL.
FIGURE 14. ROS are involved in mitochondria and cell deterioration. A: hypoxia/reoxygenation-induced
mitochondrial hyperpolarization leads to increased ROS. Mitochondria stained with TMRM (⌬␺, red) and DCF
(ROS, green) were laser line-scanned (2 Hz) during hypoxia and the reoxygenation phase. The ROS burst is
delayed after reoxygenation and starts at the maximum ⌬␺. Mitochondrial hyperpolarization lasts for ⬃2 min,
followed by loss of ⌬␺. B: cell survival after constant-energy photoexcitation of a 25 ⫻ 25 ␮m2 region. Right
panels show TMRM-stained cardiomyocyte (red) immediately after, and 1 h after, irradiation. Survival is
inversely related to the fraction of mitochondria (mito) undergoing mPTP induction and is improved by ROS
scavenger (Trolox), NO donor (SNAP), and the KATP channel opener diazoxide (Dz) and is impaired by 5-hydroxydecanoate (5HD), the blocker of KATP channel. [From Juhaszova et al. (218). Republished with permission
from the American Society for Clinical Investigation; permission conveyed through Copyright Clearance Center,
Inc.]
cardiac myocyte with mitochondria arranged in regular latticelike arrays rendering the line-scan instrumentation
straightforward (491, 493, 496) (FIGURE 15A). Furthermore, the same approach has been successfully applied to
neuronal mitochondria in situ and cultured neonatal cardiac myocytes (217) and potentially could be applied to
mitochondria in other cell types with cell motility or intracellular mitochondrial motion during the line-can procedure being carefully considered.
It is not surprising that the mPTP threshold is highly
dependent on ambient molecular oxygen, since ROS production in the system is a reaction of the first order with
respect to molecular oxygen. In cardiac myocytes incu-
bated in media with low O2, tmPTP was much greater than
in mitochondria incubated in normoxic or hyperoxic media (FIGURE 15B). Consequently, adequate oxygenation
of cellular mitochondria is important for unambiguous
interpretation of tmPTP.
The value of tmPTP determined by the above-mentioned
approach, the inherent resistance of mitochondria to
mPTP induction by ROS, and can be significantly prolonged by ischemic or pharmacological preconditioning.
This approach has been applied for the detailed elucidation of the architecture of the signaling pathways engaged by ischemic/pharmacological preconditioning
(218, 385).
FIGURE 15. Linescan technique applied to mitochondria in rat cardiac myocytes. A: definition of the mPTP
ROS threshold as an average time over population of mitochondria (shown by a dashed red line) in myocyte
stained with fluorescent mitochondrial probe for the membrane potential to induce the mPTP (shown by arrow)
(491, 493). B: dependence of the mPTP induction in the cell on ambient molecular oxygen.
932
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MITOCHONDRIAL ROS AND ROS-INDUCED ROS RELEASE
MPT with long
mean open time
A
tMPT
tMPT
MPT with short
mean open time
MPT in oscillation
mode
B
C
FIGURE 16. Basic mitochondrial redox states
(see explanation in the text). A: unstressed mitochondria with high tolerance to ROS (high survival). B: stressed mitochondria with low tolerance
to ROS (low survival). C: stressed mitochondria
with high tolerance to ROS (unknown survival).
DEATH
The term mitochondrial criticality was coined (16, 17) for
the situation when the mitochondrial network in the cardiac myocyte becomes highly sensitive to the changes of
ambient/intramitochondrial ROS, for example, by responding as synchronized oscillatory depolarizations of the mitochondrial ⌬␺ which could be scaled to the whole cell or
cluster of myocytes (401). When the mitochondrial system
reaches the point of criticality, oscillations of ⌬␺ are ignited,
eventually creating spatial and temporal heterogeneity of
excitability in a heart exposed to oxidative challenge, e.g.,
under hypoxia/reoxygenation. This inflicts dramatic pathological changes to the heart involving lethal cardiac arrhythmias possibly followed by cardiac arrest and an individual’s death.
Based on the discussion above, we would tentatively suggest
the existence of specific in vivo mitochondrial states that
may contribute to the fate of the cell (see FIGURE 16). The
relatively stable state (state A) has a comparatively high
tolerance to oxidative stress and is able to sustain the ⌬␺ for
a longer time (with longer tmPTP). This is characteristic of
the healthy cell where the mPTP formation is delayed. States
B and C characterize mitochondria which have been
stressed (prior to experimental procedure of the mPTP-induction protocol has been employed) and thus are unable to
sustain the ROS challenge and to maintain their ⌬␺ for an
extended period. In both cases (B and C), the first mean
tmPTP value is similar, characterized by a shorter tmPTP compared with mitochondria in state A, but there are some
obvious and significant differences between the states B and
C. While in state B mitochondria remain depolarized after
mPTP induction, mitochondria in state C are characterized
by dynamic instability revealed by ⌬␺ oscillations which
progressively deteriorate and ultimately cease. These differences probably reflect not only differences in the state of the
antioxidant defense system in B versus C but also intrinsic
differences in the function/activities of the pore complex
subunits itself triggered in response to posttranslational
modifications, the signaling status of the immediate pore
modulators, and/or changes in the pore complex arrangement at the protein level (FIGURE 16).
A preponderance of data point to the fact that mitochondrial membrane potential is an essential attribute of mitochondria, and its homeostasis is a prerequisite for the health
of mitochondria and the preservation of normal cell and
tissue function. Under hypoxic conditions, when mitochondria are incapable of sustaining ⌬⌿ driven by respiration,
the mitochondrial membrane potential is maintained at the
expense of cytosolic ATP hydrolysis by mitochondrial
ATPase (110). Sufficient buffering of the mitochondrial ⌬␺
by cytosolic ATP may explain why the intracellular concentration of ATP (a few mM) exceeds the Km for intracellular
ATP-utilizing enzymes by orders of magnitude. Thus, under
physiological conditions, these enzymes are fully saturated
and do not depend on ATP concentration for their function.
The homeostasis of mitochondrial ⌬␺ is critical for the mitochondrial function. Only small vital variations in a range
(approximately between 160 and 220 mV) were tolerated in
isolated mitochondria at states 3 and 4 according to the
terminology of B. Chance (82). These small variations pre-
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933
ZOROV ET AL.
FIGURE 17. Tentative model of regulation of the mitochondrial quality control by ⌬␺. Parkin is an E3 ubiquitin
ligase located in the cytosol, while PINK1 is a mitochondrial kinase carrying mitochondrial import sequence to
be accumulated inside of mitochondria followed by degradation by PARL. ⌬␺ drives PINK1 into mitochondria
where it is processed by mitochondrial processing protease (MMP). When mitochondrial membrane potential
is dissipated, PINK1 accumulates on the outer mitochondrial membrane, where it can recruit Parkin to
impaired mitochondria followed by its ubiquitination as a starting point for mitophagy. OM, outer membrane;
IM, inner membrane. TOM and TIM are transport outer and inner membrane proteins correspondingly. (From
Ref. 210: Copyright 2010 Jin et al. Originally published in Journal of Cell Biology. 191: 933–942.
doi:10.1083/jcb.201008084.)
serve the minimal energy that may be sufficient for phosphorylation of ADP by mitochondrial ATP synthase but
may dramatically influence ROS production within mitochondria (see FIGURE 5). In case of a drop of the membrane
potential below the phosphorylating membrane potential,
especially when it is nulled after induction of mPTP, mitochondria could initiate a process of self-destruction involving Ca2⫹-independent degrading hydrolases and phospholipases (66, 261, 495, 496). This process is called mitophagy
(reviewed in Refs. 88, 234, 258) and further substantiates
the vital importance of maintaining ⌬␺ homeostasis to keep
mitochondria structurally and functionally intact. The
questions regarding a mitochondrial ⌬␺ sensor, or which
processes in mitochondria could be considered as vital, the
cessation of which will result in mitochondrial degradation,
934
remain unknown. One possible candidate for such a process
is the energy (⌬␺)-dependent transport of peptides into mitochondria (reviewed in Ref. 338). This machinery is highly
important for the quality control of mitochondria (362)
through the ⌬␺-dependent ubiquitin labeling of the impaired mitochondrion resulting in the initiation of mitophagy (see explanations in the FIGURE 17; Ref. 210).
XII. PRACTICAL ASPECTS OF USING
MITOCHONDRIAL ⌬␺ AS A DRIVER
AND REPORTER
The mitochondrial membrane potential drives natural cationic substances into mitochondria, and this property could
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MITOCHONDRIAL ROS AND ROS-INDUCED ROS RELEASE
be utilized for intramitochondrial transport of man-made
drugs.
In principle, the delivery of drugs to the mitochondrial interior can be executed by using two targeting vehicles: 1)
mitochondrial targeting signal sequences and 2) mitochondriaphillic compounds. While the first approach implies
specific transport of the vehicle through the TIM/TOM mitochondrial protein transport system (318), the second uses
positively charged lipophilic compounds, for example, constructed in a way to delocalize the charge over the set of
coupled double bonds (268). Due to negative charge of the
mitochondrial interior, the latter nonspecifically accumulates these cations to the thermodynamically permissive
level which theoretically exceeds extramitochondrial levels
by about three orders of magnitude. The idea of driving
such charged compounds by virtue of the electrogenic nature of mitochondrial energetics is fully consistent with
Mitchell’s chemiosmotic theory (268, 298, 299). Nowadays, after establishment of this mechanism (1), the approach started to serve two experimental missions: to deliver to the mitochondrial interior a desired compound
which is bound to a mitochondriatropic vehicle (225, 393,
407) and, on the other hand, to discriminate between the
mitochondrial and nonmitochondrial nature of some process under study. In this context, mitochondriatropic antioxidants are not only potentially beneficially targeted drugs
to quench unwanted excessive ROS and other reactive species in mitochondria, but they may also serve as excellent
reporters providing an answer as to whether a given mechanism involves mitochondrial ROS. In the first case, in vitro
data and animal experiments have suggested the possible
beneficial effect of mitochondria-targeted antioxidants
(such as MitoQ, SkQ1, SkQR1, and others) in treatment of
a great number of model systems associated with oxidative
stress. These include stroke, arrhythmias, ischemia/reoxygenation, chemical toxicity, infection, inflammation, and
some inherited and acquired age-related and unrelated diseases (such as Alzheimer’s, diabetes, hepatitis, metabolic
syndrome, hearing loss, etc.) (14, 27, 146, 206, 223, 236,
269, 292, 307, 343, 388, 391, 399, 404 – 406, 408).
Apart from the practical issue of using mitochondria-targeted chimeric compounds as potential therapeutic agents,
their specific effects uncovered the fundamental role of mitochondria and mitochondrial ROS in the onset and propagation of different pathologies. What we have learned is
that ROS originating from mitochondrial rather than from
other intracellular sources, when they exceed the homeostatic level, are among the most pathogenic factors (222,
382, 383, 437, 467). In this context, targeted normalization
of ROS levels (specifically in mitochondria) may be beneficial in preventing a number of ROS-related pathologies including that involving RIRR (310, 392). The requirement of
mitochondria-derived ROS for the propagation of RIRR
together with its concomitant pathological cell death has
received support from experiments wherein 1) the primary
extramitochondrial ROS source induced a secondary intramitochondrial ROS release, and 2) both of these processes were prevented by specific mitochondria-targeted antioxidants (344).
XIII. CONCLUDING COMMENTS
ROS-induced ROS release is a fundamental process originating in mitochondria (18, 491, 493) responding to an
increased oxidative stress by a positive feedback loop resulting in a regenerative, autocatalytic cascade which in a vast
majority of cases is terminated, presumably by the local
redox environment together with a change of functional
properties of the “release valve.” Indeed, for this mechanism to serve a physiological role, its underlying properties
of chain-reaction-like propagation must be efficiently terminated to prevent the unwanted spread and widespread
destruction of mitochondria risking unintended and unwanted loss of the cell. These considerations are entirely
analogous to the control of a nuclear chain reaction in
power plants (e.g., by control rods) or that of CICR in
excitable cells by intrinsic ryanodine receptor- and SR-related mechanisms (423). When RIRR is appropriately not
terminated, it may lead to the wanted removal of a mitochondrion or cell, e.g., damaged beyond repair. When
RIRR is inappropriately not terminated, it may lead to unwanted cell loss such as after stroke or myocardial infarction.
We propose that RIRR may result in a cellular response that
can be considered either adaptive (e.g., promoting signaling
resulting in the elevation of the tolerance to oxidative stress
or the removal of unwanted organelles and cells) or maladaptive (e.g., the pathological destruction and death of
functional mitochondria and vital, essential cells). Adaptive
cell elimination may involve cell death executed by necrotic
or apoptotic mechanisms (for example, elimination of nonfunctional or damaged cells). It remains an open question
whether RIRR mechanisms would be recruited to serve the
adaptive elimination of cells necessary to achieve proper
organ size, shape and functions related to embryogenesis,
self-antigen-recognizing immune cells that would be dangerous, cells no longer needed (e.g., nonlactating breast involution after pregnancy), activated lymphocytes after infection is resolved, or pre-cancer and cancer cells.
The RIRR phenomenon provides an opportunity to see the
mitochondrion as machinery designed not only to trigger
but also to facilitate important signaling mechanisms, further extending the concept of the mitochondrion as an excitable organelle capable of generating and conveying redox, electrical (10), and Ca2⫹ (199) signals. Therefore,
mitochondria are not only efficient cellular energy powerhouses, they are significant signal transmitters and signal
resonators, by which we mean the mitochondrial ability to
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ZOROV ET AL.
respond to and to enhance, internal and external signals, in
a manner analogous to the propagation, and Ca2⫹-activated reaction triggering of CICR (125, 127, 128, 139).
We propose that the mPTP may be involved in at least four
conceptual modes of pathophysiological function. The first
role is to serve as a potential release valve which transiently
opens a gate for controlled release of accumulated ions or
newly formed low-molecular-weight toxic substances including elevated ROS and Ca2⫹. This likely maintains mitochondrial (and cellular) redox homeostasis, which is a
critical point in normal cell functioning. The characteristics
of this mode include brief and reversible episodes of the
mPTP pore opening or activation of some other mitochondrial pores/channels/leaks constituting a low-conductance
state of the mPTP which have been demonstrated in in vitro
systems (5, 29, 244, 255). We conceive that this serves a
housekeeping function, and at low energy demand (rest), it
is likely relatively inactive. At high stress/energy demand
conditions, this function plays a significant role (123). This
is borne out by the study (124) whereby the genetic loss of
CyPD (Ppif⫺/⫺) and acute cyclosporine A treatment (presumably causing mPTP-desensitization to ROS-induction)
result in elevated resting levels of Ca2⫹ in the mitochondrial
matrix of cardiomyocytes and an increase in the time of
decay to baseline following continuous pacing. Also,
Ppif⫺/⫺ mice display increased mortality and heart failure in
pathological and physiological models of hypertrophy
(124) presumably due to an inappropriate high “set-point”
for the physiological activation of transient mPTP-related
RIRR events leading to the inappropriate mitochondrial
accumulation of toxic levels of Ca2⫹ and ROS.
The second mode may also include mPTP openings that
prevent the accumulation of ROS and Ca2⫹ (etc.) inside
mitochondria that would cause damage to that organelle
if not released into the cytosol where a large buffer pool
might better deal with that stress factor. In this scenario,
mPTP openings, although much more frequent, are still
brief enough not to cause irreversible mitochondrial or
cellular changes. These brief episodes of mPTP [or some
other channel(s)] opening may coincide with the flickering mode of the mitochondrial membrane potential
which when integrated may cause a small albeit significant rise of ROS in the immediate vicinity of mitochondria, thus activating local pools of redox-sensitive enzymes, such as protein kinase C (218). In this mode, for
example, the levels of released ROS are enough to precondition the cell to upcoming more drastic oxidative
challenge (e.g., long-lasting ischemic insult; Refs. 180,
218, 335). The potential protective significance of the second
mode of the mPTP is to adaptively desensitize the mPTP pore
complex and to delay the transition to the third and fourth
destructive modes of mPTP associated with much higher levels
of oxidative stress.
936
The third mode constitutes a persistent, generally irreversible mPTP-associated large conductance of the inner mitochondrial membrane sufficient to activate the mechanisms
of destruction of mitochondria destined for elimination (for
example, by mitophagy, because they do not meet the necessary mitochondrial “quality control” standards). Apparently, the existence of impaired mitochondria could be a
threat for the proper functioning of the whole mitochondrial population (because they are connected in a functional
network), and opening of the mPTP may be interpreted as
the switch leading to programmed mitochondrial elimination (mitophagy) (66, 76, 88, 210, 234, 258, 261, 446, 495,
496). The signal initiating the process of mitochondrial execution may arise from the consistent and long-lasting drop
of the mitochondrial membrane potential (210) (FIGURE
17), which may reinforce the transition to irreversible
mPTP opening (i.e., the point of no return) (88, 188). The
timing to reach this point is controlled in part by the depletion of the intramitochondrial redox buffer caused by the
flux of external (or internally produced) oxidants (491).
This point may correspond to the term mitochondrial criticality (17) by reaching a redox crisis in an oxidatively
stressed cell after which the behavior of mitochondrial energetics becomes unstable. By this principle, criticality determines ROS-induced organization of an excitable mitochondrial cluster spreading oscillations of ⌬␺ over the cell.
The fourth mode of the mPTP is the terminal destructive
step resulting in the elimination of the cell (which could be
wanted or unwanted). As for mitochondria, impaired cells
can be programmed for the destruction with the mPTP playing a critical role in that process. In general, this mechanism
of cell elimination would encompass a large fraction of
mitochondria, undergoing the process described above, involving significant releases of ROS from mitochondria undergoing mPTP (218).
Schematically, FIGURE 18 represents a hierarchical model of
the proposed pathophysiological roles and functions of the
mPTP. According to this model, the stable state with the
closed mPTP can undergo a transition to a metastable (transient) state of the mPTP. By analogy to an energy barrier
(threshold) necessary to overcome in order to undergo a
physico-chemical transition, the reaction probability and
rate is determined by a redox threshold (process I) that
normally is lower than the threshold of process II determining the transition to the fully open, stable mPTP. Such a
metastable state of the pore (preceding its potential transition to a long open state) occurs as a result of intra- and
extramitochondrial changes of the redox buffer still compromising the dynamic balance between the influx/production of oxidants and antioxidants, and the stability of the
“gating architecture” of the mPTP complex. As discussed
above, the RIRR-mPTP process may serve to provide an
adaptive release of excess Ca2⫹ and ROS from mitochondria. The latter may be responsible for redox-activated pro-
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MITOCHONDRIAL ROS AND ROS-INDUCED ROS RELEASE
ROS-Threshold
(“Energy Barrier”)
Protection
signaling
I.
Stable
Closed
mPTP
I.
ΔΨm
Physiology
II.
Transientstate
mPTP
Stable
Open
mPTP
(repeated. . .)
ROS
mPTP state
II.
Pathology
ROS
Burst
ΔΨm
ROS
mPTP state
FIGURE 18. Two-ROS thresholds scheme for the induction of the mPTP. Resting state (shown by a pink ball)
is a state with a stable closed mPTP. After depletion of a redox buffer, the pore may undergo a transition to a
metastable flickering state (I) (shown by yellow color). The flickering mode of the pore is accompanied by a small
release of ROS from mitochondria, and when low it serves a physiological housekeeping purposes as a release
valve and when higher it is enough to precondition the system for better output after the following, more
extensive oxidative challenge (180). If the oxidative stress is severe, the phase I is changed by a state II (shown
by red) characterized by a stable open mPTP accompanied by a ROS burst. The pathological ROS burst results
in a programmed elimination of unwanted organelles or cells. The critical height of the ROS threshold is
determined by a balance between ROS production and quenching within mitochondria. It can be raised by
protective signaling [through preconditioning (218), cyclosporin A (180, 218), sanglofehrin (89), acidosis
(47), cyclophilin D acetylation or its knockout (26, 34, 313, 387)] shown by a green dotted line. Low redox
buffering (491), low SIRT3 activity (387), hexokinase II detachment from mitochondria (86), as well as high age
(216, 487) drive the threshold II down (shown by a brown dotted line). Note that one of the most important
elements of the scheme is that the long occupancy in state I hardens the onset of the state II.
tective signaling. The stable-open mPTP state is a result of
the redox transition to the point of no return (long-lasting
mPTP-opening) (188) leading to the destruction of mitochondria (210), and when involving a sufficient fraction of
the entire mitochondrial population, it results in the death
of the cell. The height of both ROS thresholds (FIGURE 18)
is under redox control as well as the sensitivity of the mPTPgating mechanism (which is regulated by certain enzymatic
posttranslational modifications), both of which may be targets for pharmacological intervention. It has been shown
that the height of the second threshold is age dependent,
being higher in mitochondria of young, and lower in mitochondria of old animals (216, 257, 487). Various mPTPrelated cytoprotective signaling cascades (e.g., through preconditioning, activation of PKC, receptor triggers including
adenosine, opioid and ␤-receptors, mitochondrial ATP-de-
pendent potassium channels, inhibition of glycogen synthase kinase-3␤, and other effectors; described in Ref. 218)
as well as more enhanced redox buffering (491), high
deacetylase activity, specifically resulting in deacetylation
of CyPD (387), attachment of hexokinase II to the mPTP
complex (86, 333) restore or even drive this threshold up,
but this protective signaling also fails with aging (reviewed
in Ref. 216). Therefore, it is possible that the beneficial
“anti-aging” effect of increased deacetylation (191) (particularly by mitochondrial SIRT3–5; Refs. 43, 160, 429)
of critical mPTP-regulatory elements such as CyPD (172)
may partially be explained by a relative desensitization of
the mPTP complex to ROS induction which could restore
the age-dependent decline of mPTP ROS threshold (i.e.,
“aged” state) (257) back towards to the basal level
(“young” state).
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ZOROV ET AL.
There is new information concerning the identification of
ATP synthase complex as the core structure necessary to
form the mPTP that is certain to be relevant to the RIRR
mechanism. The important role of mitochondrial ATP synthase complex (complex V) in the mPTP functioning has
been proposed in a number of studies over more than two
decades (56, 87, 197, 322), in part based on the fact that
oligomycin prevents mPTP in isolated mitochondria (321).
However, this fact was explained by a probable indirect
effect of changed levels of adenine nucleotides rather than
by a direct effect on the pore structure itself. Recent evidence indicates that a dimer of complex V is necessary and
sufficient to form the mitochondrial pore with the apparent
modulating role of CyPD (157). It is tempting to speculate
how this could potentially confer the gating mechanism of
the mPTP and explain bimodal behavior of ATP synthase in
terms of induction of the mPTP. Such bimodality (opened/
closed pore) may correlate with a bimodality of ATP synthase (dimer/monomer and higher order oligomers). Quick
and sometimes reversible transitions from closed to open
state of the pore (mPTP oscillations or flickers) may be
speculated to stem from a bimodal output of CyPD enzymatic activity which is a rotamase (peptidyl prolyl cis-trans
isomerase; Refs. 135, 431) determining two modes of conformation of proline residues in the targeted protein(s)
(134) (e.g., such as ATP synthase complex dimer), forming
the mitochondrial pore. Additionally, this may also explain
why CyPD acetylation critically appears to be affecting this
process, for example, during aging (387).
In addition to the mPTP-associated RIRR, the important
contribution of the IMAC-associated mode of RIRR functioning (20, 323), especially in the mitochondrial network
propagation properties, and its possible role in arrhythmogenesis, needs to be established. Resolving the molecular
identity of the IMAC will enable us to better appreciate its
role in physiology and pathology. It seems that IMAC-associated redox instability during pathological stress is quite
different from a safe, physiological flickering mode of the
mPTP, and it is possible that IMAC-related mechanisms
may contribute to a transitional state between mPTP-associated signaling and pathological modes of RIRR.
In conclusion, mPTP and IMAC are the main known players in the mechanism of RIRR which is a multifaceted process important in the physiology and pathology of the cell.
The mitochondrial role in this mechanism is above and
beyond its well-recognized energy-producing activity, but
includes signaling potentially regulating both positive and
negative destructive roles. Mitochondrially produced ROS
are elements that may activate/deactivate a diverse set of
signals igniting as well as terminating signaling cascades.
Apparently, the “fine-tuning” regulation of ROS levels in
mitochondria is an essential function of RIRR, and mitochondria are important players in the propagation of ROS
signals within a cell. Hypoxia/ischemia raises basal produc-
938
tion of ROS which may activate the mPTP and/or IMAC
and, if not appropriately terminated, is involved in the conversion of signaling to pathological ROS. The primary ROS
signal to ignite RIRR may originate in as well as outside of
mitochondria and may in turn be amplified by the RIRR
mechanism. The ROS amplification site may coincide with
one or more already known mitochondrial sites of ROS
production. The full understanding of the RIRR mechanism
will require a more comprehensive and detailed mechanistic
knowledge of the ROS-producing sites operating in situ and
in vivo. It is certain that at least some of the mechanisms of
mitochondrial ROS production presented in this review are
relevant to both physiological and pathological ROS production in the cell.
ACKNOWLEDGMENTS
D. B. Zorov and S. J. Sollott contributed equally to this
paper.
Address for reprint requests and other correspondence:
D. B. Zorov, A. N. Belozersky Institute of Physico-Chemical Biology, Lomonosov Moscow State University, Moscow, Russia 119992 (e-mail: zorov@genebee.msu.su).
GRANTS
This work was supported by the Intramural Research Program of the National Institutes of Health, National Institute
on Aging, Russian Foundation of Basic Research (14-0400542) and Russian Science Foundation (14-15-00147, 1424-00107). The funders had no role in data collection and
analysis, decision to publish, or preparation of the manuscript.
DISCLOSURES
No conflicts of interest, financial or otherwise, are declared
by the authors.
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