Cardiovascular Research 72 (2006) 210 – 219
www.elsevier.com/locate/cardiores
Review
Mitochondrial depolarization and the role of uncoupling
proteins in ischemia tolerance
Michael N. Sack ⁎
Cardiology Branch, NHLBI, National Institutes of Health, Building 10-CRC, Room 5-3150, 10 Center Drive, Bethesda, MD 20892-1454, USA
Received 21 March 2006; received in revised form 4 July 2006; accepted 14 July 2006
Available online 21 July 2006
Time for primary review 26 days
Abstract
Modest depolarization of the mitochondrial inner membrane potential is known to attenuate mitochondrial reactive oxygen species
generation. Transient pharmacologic uncoupling of mitochondrial oxidative phosphorylation results in modest depolarization of the
mitochondrial membrane potential and confers protection against subsequent cardiac ischemia–reperfusion injury. Whether cardiac
mitochondria have an innate capacity to temporally self-modulate their membrane potential as a possible adaptive mechanism in the context
of cardiac ischemia and early reperfusion is supported by emerging data and is an intriguing concept that warrants further investigation. The
objective of this review is to explore the various mechanisms whereby mitochondrial depolarization can be evoked in the context of both
cardiac ischemia and reperfusion and in response to the cardioprotective program of ischemic preconditioning. The potential regulatory
pathways orchestrating this biological perturbation of mitochondrial function are explored from the level of signal transduction to potential
transcription-mediated modulations of nuclear-encoded mitochondrial inner membrane proteins, emphasizing the potential function of the
mitochondrial uncoupling proteins.
© 2006 European Society of Cardiology. Published by Elsevier B.V. All rights reserved.
Keywords: Cardiac ischemia; Mitochondrial respiration; Uncoupling proteins; Reactive oxygen species; Mitochondrial depolarization
1. Introduction
“It is a good thing for the entire enterprise that mitochondria and chloroplasts have remained small, conservative,
and stable, since these two organelles are, in a fundamental
sense, the most important living things on earth. Between
them they produce oxygen and arrange for its use. In effect,
they run the place.” This description by the physician scientist
and commentator — Lewis Thomas [1] succinctly illustrates
the centrality of mitochondrial function to sustaining life. The
importance of this organelle is exemplified in the human heart
where ATP in excess of ten times the cardiac mass, is produced and consumed daily [2], in large part produced by
mitochondrial oxidative phosphorylation.
⁎ Tel.: +1 301 402 9259; fax: +1 301 402 0888.
E-mail address: [email protected].
In the last few decades, characterization of the role of
mitochondria in cellular biology has extended above and
beyond oxygen utilization and ATP production. Mitochondria are now recognized as finely tuned rheostats that orchestrate and exquisitely modulate: energy production; reactive
oxygen and nitrogen species homeostasis; calcium regulation; heme metabolism; programmed cell death (apoptosis)
and most recently retrograde signaling to modulate signal
transduction pathways [3] and nuclear regulation [4]. Historically, mitochondria were considered passive ‘victims’ of
cardiac ischemia that contributed to subsequent cell death via
ATP depletion and reactive oxygen species production. In
light of the expanding role of mitochondria, a question posited is whether mitochondria actively modify the biological
response to cardiac ischemia and reperfusion? The hypothesis
explored in this review contends that cardiac ischemia mediated bioenergetic perturbations of mitochondrial oxidative
0008-6363/$ - see front matter © 2006 European Society of Cardiology. Published by Elsevier B.V. All rights reserved.
doi:10.1016/j.cardiores.2006.07.010
M.N. Sack / Cardiovascular Research 72 (2006) 210–219
phosphorylation directly modulate the biological responses
to ischemia and reperfusion. The objective of this manuscript
is to review the current knowledge pertaining to the dynamic
biochemical, cellular and molecular responses of mitochondria
to ischemia and reperfusion injury with respect to ischemia–
reperfusion mediated changes in: oxidative phosphorylation;
the energetic capacity of the mitochondria; the degree of
mitochondrial coupled respiration and the regulation of reactive oxygen species. How these changes modify the final
biological response to ischemia will be discussed. The mitochondrial role in mediating ischemia–reperfusion cell death
via activation of the permeability transition pore, via apoptosis and necrosis have been recently reviewed [5,6] and are not
discussed.
2. Coupling of electron transfer and ATP generation
The final common pathway for oxidative metabolism,
which generates the bulk of cardiac ATP, is the sequential
passage of electrons from high (NADH or FADH2) to low
(molecular oxygen) redox potentials down the electron
transfer chain (ETC — complexes I through IV). This stepwise electron transfer results in the active pumping of hydrogen ions out of the mitochondrial matrix into the inter-membranous space. The ensuing electrochemical gradient
generated across in the inner mitochondrial membrane facilitates the translocation of protons from the inter-membranous
space through the Fo/F1 ATPase (ATP-synthase) back into the
mitochondrial matrix. This proton translocation is coupled to
the phosphorylation of ADP to generate ATP. Collectively
these reactions constitute oxidative phosphorylation and the
direct synthesis of ATP from electron transfer encapsulates
coupled respiration [7].
Uncoupled oxidative phosphorylation results from electron transfer and proton influx into the mitochondrial matrix
independent of the phosphorylation of ADP and results from
either damage to ATP-synthase or via active or passive increased permeability of the inner-mitochondrial membrane
[8]. Inner mitochondrial membrane proteins such as the uncoupling proteins (UCPs) [9], the adenine nucleotide translocases (ANTs) [10,11] and cytochrome oxidase itself [12]
can facilitate proton leak/slip into the mitochondrial matrix to
promote partial uncoupled respiration. Additionally, ionic
cycling and the phospholipid fatty acids composition of the
inner mitochondrial membrane contribute to a modest component of uncoupled respiration and proton conductance
respectively [13,14]. Together these various mechanisms result in various degrees of depolarization of the inner mitochondrial membrane and possible concurrent de-energization
which can represent regulated physiologic respiratory control
[15] or, if in excess, facilitate mitochondrial and cellular
damage [16].
The physiologic role of uncoupled respiration is best
exemplified by the adaptive non-shivering thermogenesis
induced by uncoupling protein 1 (UCP1) to generate heat in
brown adipocytes [14]. Partial mitochondrial uncoupling has
211
also been implicated as a potential mechanism promoting
enhanced longevity in mice [11] and functions to diminish
reactive oxygen species (ROS) production [7]. Furthermore,
it is suggested that proton leak accounts for up to 20% of
mitochondrial oxygen consumption as an integral component
of the standard metabolic rate [15,17]. Of note, physiologic
uncoupling and modest mitochondrial depolarization are
proposed to concurrently maintain the energetic capacity of
mitochondria and modulate mitochondrial inner membrane
dependent ROS production (reviewed [18]).
3. Effects of ischemia reperfusion on mitochondrial
oxidative phosphorylation
Mitochondrial ultrastructural and functional injury during
cardiac ischemia are both time-dependent and progressive
[8,19]. Fig. 1 schematizes the perturbations of inner mitochondrial membrane protein function in a temporal response
to ischemia. The earliest perturbations in ETC complex
function are evident within 10 min of ischemia where ATPsynthase [20] and ANT activity [21] are diminished. This is
followed by the reduction of activity in complex I [8,20]
within 20 to 30 min of ischemia. In contracting cardiomyocytes the electrochemical gradient is shown to be modestly
diminished in the first 30 min of simulated ischemia [16].
These perturbations are reversible if reperfusion occurs within this timeframe [22]. The time-span of reversibility of
mitochondrial injury is variable and dependent on the heart
rate and on the species being studied [23]. More prolonged
ischemia begins to result in irreversible myocyte damage and
is associated with defects in complexes III and IV [8,24].
Cardiomyocytes exposed to prolonged ischemia demonstrate
a robust reduction in inner mitochondrial membrane potential
[16]. During ischemia, ATP-synthase reverses direction to
facilitate the maintenance of the proton-motive force by ATP
hydrolysis. A higher rate of ATP hydrolysis reduces the time
to irreversible ischemic injury and mitochondrial dysfunction. Interestingly, in slow heart rate animals, an endogenous
antagonist of ATP-synthase activity, ATPase inhibitor factor
[25] is shown to protect mitochondria by delaying ischemic
ATP hydrolysis [26]. Prolonged ischemia and depletion of
ATP function in concert to disrupt mitochondrial oxidative
phosphorylation.
The restoration of blood flow restores oxygen and substrate supply and serves to remove residual metabolites that
are detrimental to mitochondrial and myocytes recovery
[27]. Consistent with the temporal recovery of mitochondrial
function, brief ischemic periods result in the rapid restoration
of oxidative phosphorylation at reperfusion [22]. Conversely
once ischemic damage to complexes III and IV is evident
energetic recovery is limited. Reperfusion, at this point may
exacerbate injury including further disruption of oxidative
phosphorylation [28].
The exact mechanisms underlying ETC complex dysfunction and the degree and rate of recovery during ischemia
and reperfusion are not well established. However, ETC
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M.N. Sack / Cardiovascular Research 72 (2006) 210–219
Fig. 1. Temporal effects of ischemia and reperfusion on mitochondrial ETC function and respiratory coupling.
complex protein modifications via: redox or phosphorylation
signaling; perturbations in electrolyte concentrations; dysregulation of substrate supply and following depletion of
cardiolipin content of the inner mitochondrial membrane all
contribute to ischemia–reperfusion mediated disruption of
the functional integrity of electron transfer [8,29–31]. Interestingly, following an ischemic insult of sufficient duration
to result in persistent modest contractile dysfunction, coupling of mitochondrial oxidative phosphorylation to oxygen
consumption is restored to baseline ratios 20 min into reperfusion, in the isolated perfused heart [32].
Additional dysregulation in energetics is also operational
during cardiac ischemia and reperfusion, including modulations in substrate or metabolic pathway utilization [27] and in
extra-mitochondrial high-energy phosphocreatine transduction [33–35]. Moreover, perturbations in high-energy phosphocreatine transduction and ATP utilization are also integral
to persistent contractile dysfunction following the acute
perturbations associated with early reperfusion [32].
4. Ischemia–reperfusion and the generation of reactive
oxygen species
In the heart, the electron transfer chain is the main source
of reactive oxygen species (ROS) production. It is generally
believed that the superoxide radical anion (O2−) is produced
by the transfer of an unpaired electron from complex I and
from ubisemiquinone of complex III to molecular oxygen
[4,18]. Minimal to no ROS is generated under anoxic conditions [36], but myocardial ischemia is associated with a
progressive reduction in tissue oxygen levels and then a
rapid restoration and/or overshoot of oxygen levels at reperfusion [37]. During ischemia free radicals are generated in
the myocardium [16,38]. At cardiac reperfusion, following a
short ischemic stress, up to a 6-fold induction of ROS generation can occur [38,39]. As prolonged myocardial ischemia irreversibly disrupts electron transfer chain activity
additional extra-mitochondrial sources of ROS probably
become the major source of reperfusion ROS generation.
One such source results from the infiltration of neutrophils
into the infarcting tissue [40]. Additional sources include
oxyradical generation from xanthine oxidase, activation of
the arachidonate cascade, autoxidation of catecholamines,
activation of various NAD(P)H oxidases and from reactive
nitrogen species [41].
Additionally mitochondrial matrix Ca2+ overload is postulated to enhance ETC ROS generation during cardiac ischemia and reperfusion [42]. This hypothesis is less well
established, with some discordant data as discussed below. As
background, the primary effect of mitochondrial Ca2+ is the
stimulation of oxidative-phosphorylation via allosteric activation of tricarboxylic acid cycle and ETC enzymes [43]. Under
normoxic circumstances Ca2+ influx into the mitochondrial matrix is augmented in response to energetic demand
with Ca2+ uptake primarily driven by the electrochemical
gradient established by the mitochondrial membrane potential
and by the relatively low intramitochondrial Ca2+ concentration [44]. In response to cardiac ischemia and reperfusion,
Ca2+ influx into the mitochondrial matrix is facilitated, in part
by ROS mediated calcium release from the sarcoplasmic
M.N. Sack / Cardiovascular Research 72 (2006) 210–219
reticulum [45]. Evidence to support a role for Ca2+ influx in the
generation of additional ROS includes: 1) a direct correlation
between Ca2+ levels and ROS generation when complex I is
pharmacologically inhibited [46,47]; 2) similarly, in isolated
heart mitochondria ROS generation is calcium concentration
dependent following complex III inhibition [48] and 3) mitochondria extracted from hypoxic hearts demonstrate a direct
correlation between ROS generation and the Ca2+ concentration [49]. Contrary data, which fails to demonstrate Ca2+dependent mitochondrial ROS production is evident under
different experimental conditions where complex I was inhibited by rotenone [48,49].
5. Strategic control points of mitochondrial respiration
to modulate cardiac ischemia tolerance
In general mitochondrial ROS generation increases with
a more negative inner mitochondrial membrane potential
[18,50]. When the mitochondrial membrane potential is high,
superoxide production increases as a result on non-specific
single-electron reduction of molecular oxygen at complexes I
and III of the ETC [18]. In contrast, modest reduction in
mitochondrial membrane potential (mitochondrial depolarization), more tightly associates electron transfer to the ETC
complexes, thereby limiting random electron disassociation
and superoxide production [9]. A second proposed mechanism
of modest depolarization-mediated reduction in ROS generation is via decreased membrane potential dependent Ca2+
influx into the mitochondria [51]. This reduction in mitochondrial Ca2+ may attenuate ischemia-mediated ROS production.
The concept that the mitochondrial membrane potential is
regulated as a component of the cellular biological program
modulating cellular tolerance to ischemic injury is actively
being explored. Direct evidence supporting post-translational
modulation of ETC complex proteins, with subsequent
changes in their respiratory activity during cardiac ischemia
and reperfusion is not well established. However, accruing
evidence demonstrates that numerous signaling pathways
govern ETC flux and may play a role in modulating mitochondrial energetics and membrane potential. Complex IV
activity appears to be the most susceptible to post-translational
modification of various complex protein moieties. Tyrosine
phosphorylation of the subunit I of cytochrome oxidase results
in inactivation of the enzyme and with a reduction in overall
cellular ATP levels [52]. Protein kinase A also inhibits complex IV via phosphorylation of multiple subunits within the
cytochrome oxidase complex [53]. Conversely, protein kinase
Cε activates cytochrome oxidase via phosphorylation of subunit IV [54]. Whether any of these modifications are operational during ischemia and reperfusion require investigation.
The most extensive characterization of signal transduction mediated modulation in electron transfer chain flux is
underscored by the regulation of cytochrome oxidase activity by nitric oxide (NO) [55]. NO competitively inhibits
cytochrome oxidase potently and reversibly and reduces the
affinity of this enzyme for O2. This inhibitory effect of NO is
213
inversely dependent on O2 levels, with greater inhibition of
cytochrome oxidase activity at lower oxygen concentrations
[56,57]. Furthermore, the temporal inhibitory effect of NO
has diverse phenotypic outcomes. Short-term exposure to
NO in mitochondria results in transient and reversible, mitochondrial depolarization [58]. In contrast, persistent inhibition of respiration [59], or high concentrations of NO [60]
result in the collapse of the mitochondrial membrane potential, ATP depletion, and cell death. The potential biological
relevance of NO mediated control of mitochondrial respiration is evident where NO donors confer protection against
ischemia–reperfusion injury in cardiomyocytes in parallel
with modest mitochondrial depolarization and reduced
mitochondrial Ca2+ uptake [61]. This control of ETC flux
by NO supports the concept of modest mitochondrial depolarization as an adaptive mechanism against ischemia–
reperfusion injury. Interestingly, modest mitochondrial respiratory inhibition using pharmacologic inhibitors similarly
demonstrates enhanced tolerance to cardiac and skeletal
muscle ischemic injury [62–65].
Together, these data identify potential signaling pathways
that may contribute to the modulation of ETC activity during
ischemia and reperfusion. Direct inhibition of ETC flux using
pharmacologic agents that are known to diminish the inner
mitochondrial membrane potential confers tolerance against
ischemic injury [61,66,67]. This concept is further supported
where modest depolarization by the administration of low
concentrations of uncoupling agents such as carbonyl cyanide p-(trifluoromethoxy) phenylhydrazone (FCCP) or 2,4
dinitrophenol evokes protection against ischemic damage in
the intact heart [68], in cardiomyocytes [69] and in the brain
[70] in parallel with a diminution in mitochondrial ROS
production. Interestingly, the potassium channel opening
compounds that promote cardioprotection also partially uncouple mitochondrial respiration [13].
The paradigm being established is that transient modest
mitochondrial depolarization, whether via modulation in
ETC flux or via pharmacologic uncoupling, confers protection against ischemia–reperfusion injury. In an analogous
fashion to innate regulatory control of ETC flux, the question
arises as to whether endogenous modulation of mitochondrial uncoupling may be a component of the regulatory
machinery governing cellular ischemia tolerance. This
question is explored below where the role of mitochondrial
uncoupling proteins function as a mediator of proton leak in
the heart is discussed.
6. A proposed role for cardiac enriched mitochondrial
uncoupling proteins
Mitochondrial uncoupling proteins are integral membrane
proteins localized to the inner mitochondrial membranes.
Uncoupling protein 1 (UCP1), is the most extensively characterized member of this family and plays a central role in
adaptive thermogenesis in brown adipocytes [71]. UCP1
allows the influx of protons back into the mitochondrial
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M.N. Sack / Cardiovascular Research 72 (2006) 210–219
matrix thereby uncoupling mitochondrial respiration from
ATP-synthesis. This in turn, leads to more rapid respiration
and heat production. This prototype of the uncoupling
proteins is directly activated by fatty acids and intrinsically
inhibited by purine nucleotides (reviewed [9]).
Two UCP1 homologues UCP2 and UCP3, which are
present in the heart [72], share high amino acid sequence
identity (73%) and are similar to UCP1 [9]. As these inner
mitochondrial membrane carrier proteins are most closely
aligned to UCP1, it is suspected that they may have similar
biochemical functions.
The abundance of UCP2 gene expression in the heart
appears to be species specific with relatively higher levels in
mice compared to humans [73]. Interestingly the presence of
UCP2 protein in the murine heart has been questioned [74],
however, it is present in human and rat myocardium [72,75].
UCP3, has a more restricted tissue distribution, and is found
predominantly in skeletal muscle, with lower levels in the
heart [76].
The functional roles of UCP2 and UCP3 are less well
characterized than that of UCP1. In general it is accepted that
these proteins are not constitutive uncouplers of oxidative
phosphorylation and that they are not crucial for non-shivering
thermogenesis, except in response to specific pharmacologic
activation [77]. The functional characterization of the UCP
homologues has been complicated by experimental artifact
which has arisen in overexpression studies. Overexpression of
UCP2 and UCP3 in the inner mitochondrial membrane can
compromise the inner membrane integrity [78] with a resultant
excess uncoupling of oxidative phosphorylation [79–81].
Furthermore, tissue specific functions are operational in that
UCP2 overexpression demonstrates divergent cell-type specific activities [9,82].
In rat neonatal cardiomyocytes, UCP2 overexpression
does not have intrinsic ETC uncoupling activity, but does
confer tolerance to oxidative stress via diminished mitochondrial Ca2+ overload and reduced ROS generation [83].
This lack of constitutive effect on mitochondrial coupling is
consistent with the revelation that UCP2 and UCP3 are
functionally quiescent until activated by either ROS signaling [72,84], by fatty acid peroxidation products [85] or via
reactive aldehydes [86]. Interestingly, ectopic overexpression of UCP1 in cardiac derived H9c2 cells [87] and in the
murine heart [88] mirrors the cytoprotective effect of UCP2
overexpression against ischemia and reperfusion injury.
Genetic depletion of the uncoupling proteins should give
greater insight into their role in protection against ischemic
injury. No studies to date have explored this in the intact
heart. However, RNA interference studies where UCP2 and
or UCP3 was partially depleted in cardiac derived H9c2
cells demonstrate diminished tolerance to anoxic stress in
association with excess ROS production [72]. In this study
the hierarchical effects of these homologues were established where UCP2 plays a functionally greater role than
UCP3 in promoting tolerance to anoxia and reoxygenation
injury [72].
Further evidence to support tissue specific cytoprotective
effects of UCP2 is evident in the brain where UCP2 is shown
to play a similar role in modulating neuronal tolerance to
ischemic stress (reviewed [89]). The ROS modulating effects
of UCP2 are also evident in UCP2 knockout mice which
exhibit: (1) increased ROS production in response to infectious agents; (2) excess ROS in response to organ injury and
(3) demonstrate increased susceptibility to ROS-mediated
atherosclerosis (reviewed [90]).
An additional proposed role for UCP3 is as a conduit to
export fatty acid anions from the mitochondria to prevent
accumulation of fatty acid metabolic by-products in the
mitochondria. This hypothesis, although attractive, is currently supported predominantly by associational studies
which cannot distinguish between primary fatty acid export
and ROS-mediated effects (see recent reviews [9,90]). Studies
designed to directly test this is are an active area of investigation. Interestingly, the ischemic heart is exposed to elevated
levels of free fatty acids [27] and an innate mechanism to
attenuate lipotoxic effects via cardiac UCP3 activation is an
attractive hypothesis that deserves exploration.
7. Mitochondrial uncoupling, uncoupling proteins and
ischemic preconditioning
It has been shown that the heart can be protected from
ischemic injury through activation of innate pro-survival
programs. Tolerance against otherwise lethal ischemia is
most powerfully induced following repetitive transient nonlethal periods of myocardial ischemia and reperfusion termed
ischemic preconditioning (reviewed [91]). This preconditioning effect is evident in two temporally distinct phases
which correlate with their cognate regulatory control mechanisms. Classic preconditioning is an acute adaptive phase
evident within minutes to 2–4 h following ischemic preconditioning and is regulated at the post-translational level.
Delayed-preconditioning is an ischemia-tolerant phase
evoked 12–72 h following the preconditioning ischemia
and is primarily regulated at the transcriptional level [92]. The
veracity of mitochondrial coupling as an integral component
in modulating cardiac tolerance to ischemia would be reinforced if mitochondria from preconditioning mirror the
mitochondrial cytoprotective phenotype described above.
The initial description of the classical preconditioning
phenomenon conforms with the central paradigm presented
in this review in that immediately prior to an index ischemic
event, preconditioned hearts are modestly depleted in ATP
levels (1/3 reduction) compared to non-preconditioned
control hearts [19]. Further studies in cell culture show that
acute preconditioned mitochondria in situ are uncoupled as
evident by a lower inner membrane potential, reduced ATP
levels and greater oxygen consumption versus non-preconditioned control cells [93]. The association of classic preconditioning with proton leak has been directly confirmed by
measuring concurrent oxygen consumption and mitochondrial membrane potential in mitochondria extracted from
M.N. Sack / Cardiovascular Research 72 (2006) 210–219
preconditioned rat hearts prior to the onset of a prolonged
ischemia–reperfusion insult [94]. The putative modifiers that
abolish this phenotype implicate activation of the uncoupling
proteins and of ANT as this proton leak is blocked by GDP
and carboxyattractyloside respectively [94].
The mitochondrial bioenergetic milieu of hearts following
delayed preconditioning has been less well characterized,
however it is known that the basal metabolic function of
delayed preconditioned mitochondria does not exhibit increased proton leak [92]. However, in parallel with the
acutely preconditioned mitochondria [95], these mitochondria are more tolerant of an anoxia–reperfusion insult compared to non-preconditioned mitochondria [92]. Interestingly,
the transcriptional activator peroxisome proliferator activated
receptor co-activator 1 alpha (PGC1α) is upregulated as a
component of the delayed preconditioning program [92].
Transient activation of PGC1α in skeletal muscle cells upregulates UCP2 and UCP3, in concert with increased mitochondrial proton leak [96]. Although, the direct link between
PGC1α activation and proton leak in delayed preconditioning has not been established, upregulation of UCP2 and
UCP3 is evident in the delayed preconditioned rat heart [72].
This regulation is associated with increased GDP-sensitive
proton leak in the presence of ROS and these mitochondria
generate less ROS at reperfusion following a prolonged
anoxic insult [72]. This prerequisite of ROS intermediates to
induce mitochondrial uncoupling is consistent with the requirement of signaling intermediates to activate UCP2 and
UCP3.
The role of uncoupling proteins in augmenting tolerance
to delayed ischemic preconditioning is also evident in the
brain. Here sub-lethal ischemia upregulates UCP2 expression and confers protection to the brain against a delayed
ischemia injury [97]. Moreover, overexpression of UCP2
confers protection against neuronal ischemia injury and is
proposed to mediate these salutary effects through control of
ROS generation and possibly via the mediation of redox
signaling [97,98].
The direct demonstration that ROS signaling activates
UCPs and results in subsequent attenuation in subsequent
ROS levels has begun to be explored during delayed preconditioning [72]. The role of ROS activation of UCPs during
acute preconditioning has not to my knowledge been
investigated. However, the real time demonstration that superoxide levels are necessary in ischemic preconditioning
signal transduction and that subsequent superoxide levels are
markedly attenuated during ischemia and reperfusion following preconditioning compared to non-preconditioned
ischemia–reperfusion controls [99] is compatible with the
paradigm of ROS-mediated UCP activation resulting in
diminution of subsequent ROS generation. In this study by
Stowe and colleagues [99] the preponderance of the attenuation of ROS generation following preconditioning occurs in
the latter half of the ischemic period and even more robustly
during reperfusion. Whether this aligns with the regulatory
control of UCP activation has not been established.
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8. Mitochondrial uncoupling/depolarization and the
maintenance of oxidative phosphorylation capacity
The premise underlying the cardioprotective role of
transient activation of uncoupling proteins predicates that
mitochondrial depolarization can occur to a sufficient level to
attenuate ETC ROS generation without further disrupting the
capacity to generate cardiac ATP production during ischemia
and reperfusion. The stoichiometric efficiency of oxidative
phosphorylation is defined by the P/O ratio, or the amount of
inorganic phosphate incorporated into ATP per amount of
oxygen consumed. In classical equilibrium thermodynamics
the maximal efficiency of energy conversion is achieved
when the energy transduction systems are fully coupled [18].
In contrast, biological systems exhibit variable levels of
efficiency that display non-ohmic characteristics [100]. This
efficiency variation is orchestrated by numerous extrinsic
and intrinsic factors that modulate coupling between the
proton-motive force and the rate of respiration (reviewed by
Kadenbach [18]). A concept posited by Kadenbach [18,101]
which possibly relates to the mechanism of action of uncoupling proteins is that: i) a relatively minor alterations of the
inner mitochondrial membrane potential at high physiologic
inner mitochondrial membrane potential significantly alter
ETC ROS generation and ii) the maximal rate of ATPsynthase energy generating capacity occurs at low physiologic mitochondrial membrane potential with no augmentation with further increasing membrane potential. Continuous
temporal analysis of these mitochondrial perturbations during
ischemic preconditioning has not been undertaken. However,
acute ischemic preconditioning is associated with both increased proton leak immediately following the preconditioning ischemia and with increased efficiency of cardiac oxygen
consumption to the degree of contractile recovery, as a measure of preserved energetics, compared to non-preconditioned ischemic control hearts [94]. Although UCP2 and
UCP3 have been associated with these mitochondrial alterations as discussed in this review, there is no direct demonstration that UCP2 and or UCP3 can both increase proton leak
and concurrently maintain oxidative phosphorylation. This
potential mechanism of action of the uncoupling proteins
under ischemic stress conditions requires investigation to
definitively prove the hypothesis presented here.
9. Limitations in the data supporting the mitochondrial
uncoupling hypothesis in augmenting resilience to
ischemic stress
Insertion of exogenous proteins into the mitochondrial
inner membrane may give rise to artifactual uncoupling, as
shown following the insertion of multiple UCP homologues
[79–81,102] and by the insertion of other related mitochondrial carrier proteins [103]. This insertion of proteins into the
inner mitochondrial membrane may give rise to improper
insertion or folding or to abnormal interaction with native
proteins, thereby conferring unphysiologic action. This is
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M.N. Sack / Cardiovascular Research 72 (2006) 210–219
supported where overexpression of UCP3 does not exhibit
superoxide mediated proton conductance [78] and where
robust overexpression of UCP2 promotes mitochondrial uncoupling without the need for redox activation [104]. Thus,
although the uncoupling protein overexpression data presented is one component of the data supporting modest uncoupling as a mechanism to diminished ROS-mediated
cellular sensitivity to ischemic injury, we should remain
cognizant of the potential experimental artifact that can contribute to this phenotype.
The quantization of modest ‘physiologic’ depolarization
versus robust ‘pathologic’ depolarization is not well characterized, nor reviewed in this manuscript. The physiologic
mitochondrial membrane potential is in the order of 200 mV
and is subject to some debate due to experimental limitations
in its direct measurement [105]. Interestingly, the maximal
attenuation of mitochondrial membrane potential with robust
overexpression of UCP2 approaches 15 mV [104]. Thus, the
degree of mitochondrial uncoupling, that is termed modest,
is most likely to be a modulation of less than 10% and
probably much less than this under physiologic conditions.
However, the absolute modulation of the inner mitochondrial
membrane potential has not been determined in the studies
presented in this review.
Additional inner membrane carrier proteins and channels
may play a role in modulating the inner mitochondrial membrane potential in response to ischemia injury. Examples of
this include the ANT [106] and the ‘elusive’ mitochondrial
KATP channel [107]. However, these and other potential
membrane potential modulatory proteins have not been
discussed in this review.
Finally, although numerous groups have demonstrated
that ‘preconditioned’ mitochondria exhibit enhanced recovery from anoxia and re-oxygenation [92,95], extra-mitochondrial energy transduction also contributes to overall
cellular protection [108] and hence modulation of mitochondrial biology itself, is only part of the overall metabolic
program modulating ischemia tolerance.
10. Conclusions
The preponderance of evidence supports modest mitochondrial depolarization as both an early and reversible event
during cardiac ischemia. This probably occurs in part due to
modest inhibition of electron transfer and possibly via the
activation of endogenous inner membrane proteins including
the uncoupling proteins. These modest changes in the rate of
coupled oxidative phosphorylation would reduce electron
transfer to molecular oxygen and thereby diminish ROS
production. An additional function of modest mitochondrial
depolarization includes a reduction in mitochondrial Ca2+
uptake. Interestingly, modulation of mitochondrial coupling is
also evident in ischemic preconditioning, the biological
phenomenon known to maximally augment cell survival
programming in the heart. Hence, short-lived modest mitochondrial depolarization is associated with the delay and the
attenuation of subsequent ischemia–reperfusion mediated cell
death. The postulated mechanisms are schematized in Fig. 2.
Fig. 2. Mediators of modest mitochondrial depolarization and potential adaptive responses to this depolarization.
M.N. Sack / Cardiovascular Research 72 (2006) 210–219
The regulatory mechanisms orchestrating the modulation in
coupled respiration are complex, but appear to span the
regulatory spectrum from transcriptional regulation (in delayed preconditioning), to post-translational signal transduction which directly modulates ETC complex protein activities
and which may activate inner mitochondrial membrane
transport proteins such as the uncoupling proteins. The complexity and role of the mitochondria as both a recipient and a
mediator in signal transduction is further underscored where
retrograde signaling from de-energized mitochondria signals
the nucleus to modulate additional adaptive responses [4,109].
Overall these data demonstrate that the mitochondrion is
truly a finely tuned and highly regulated organelle which
functions as a multi-dimensional rheostat controlling biological responses including the cellular capacity to respond
to pathophysiologic insults such as cardiac ischemia and
reperfusion injury. Our understanding of the complex regulatory mechanisms governing mitochondrial function and their
role in retrograde signaling to the cytosol and nucleus is in its
infancy. The further dissection of regulatory control of mitochondrial function and the role of mitochondria in molecular
regulation should identify novel targets to mediate cardiac
tolerance to oxidative stress.
Acknowledgements
I would like to thank Toren Finkel, M.D., Ph.D. and Paul
Hwang, M.D., Ph.D. for their critical review of this manuscript. Funding for my laboratory is generously provided by
the National Heart Lung and Blood Institute, Division of
Intramural Research of the National Institutes of Health, USA.
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