Mitochondrial nitroalkene formation and mild uncoupling in ischaemic preconditioning: implications for cardioprotection.
ABSTRACT Both mitochondria and nitric oxide (NO*) contribute to cardioprotection by ischaemic preconditioning (IPC). IPC causes mild uncoupling of mitochondria via uncoupling proteins (UCPs) and the adenine nucleotide translocase (ANT), and mild uncoupling per se is cardioprotective. Although electrophilic lipids are known to activate mitochondrial uncoupling, the role of such species in IPC-induced uncoupling and cardioprotection is unclear. We hypothesized that endogenous formation of NO*-derived electrophilic lipids (nitroalkenes such as nitro-linoleate, LNO2) during IPC may stimulate mitochondrial uncoupling via post-translational modification of UCPs and ANT, thus affording cardioprotection.
Hearts from male Sprague-Dawley rats were Langendorff-perfused and subjected to IPC. Nitroalkene formation was measured by HPLC-ESI-MS/MS. The effects of exogenous LNO2 and biotin-tagged LNO2 on isolated heart mitochondria and cardiomyocytes were also investigated.
Nitroalkenes including LNO2 were endogenously generated in mitochondria of IPC hearts. Synthetic LNO2 (<1 microM) activated mild uncoupling, an effect blocked by UCP and ANT inhibitors. LNO2 (<1 microM) also protected cardiomyocytes against simulated ischaemia-reperfusion injury. Biotinylated LNO2 covalently modified ANT thiols and possibly UCP-2. No effects of LNO2 were attributable to NO* release, cGMP signalling, mitochondrial KATP channels, or protective kinase signalling.
Components of a novel signalling pathway are inferred, wherein nitroalkenes formed by IPC-stimulated nitration reactions may induce mild mitochondrial uncoupling via post-translational modification of ANT and UCP-2, subsequently conferring resistance to ischaemia-reperfusion injury.
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ABSTRACT: Inorganic nitrite, a metabolite of endogenously-produced nitric oxide (NO) from NO synthases (NOS), provides the largest endocrine source of directly bioavailable NO. The conversion of nitrite to NO occurs mainly through enzymatic reduction, mediated by a range of proteins, including heme-globins, molybdo-flavoproteins, mitochondrial proteins, cytochrome P450 enzymes, and NOS. Such nitrite reduction is particularly favoured under hypoxia, when endogenous formation of NO from NOS is impaired. Under normoxic conditions, the majority of these nitrite reductases also scavenge NO, or diminish its bioavailability via reactive oxygen species (ROS) production, suggesting an intricate balance. Moreover, nitrite, whether produced endogenously, or derived from exogenous nitrite or nitrate administration (including dietary sources via the Nitrate-Nitrite-NO pathway) beneficially modulates many key cardiovascular pathological processes. In this review, we highlight the landmark studies which revealed nitrite's function in biological systems, and inspect its evolving role in cardiovascular protection. While these effects have mainly been ascribed to the activity of one or more nitrite reductases, we also discuss newly-identified mechanisms, including nitrite anhydration, the involvement of s-nitrosothiols, nitro-fatty acids, and direct nitrite normoxic signalling, involving modification of mitochondrial structure and function, and ROS production. This article is part of a Special Issue entitled 'Redox Signalling in Heart'.Journal of Molecular and Cellular Cardiology 01/2014; · 5.15 Impact Factor
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ABSTRACT: Background and PurposeCardiac ischaemia–reperfusion (IR) injury remains a significant clinical problem with limited treatment options available. We previously showed that cardioprotection against IR injury by nitro-fatty acids, such as nitro-linoleate (LNO2), involves covalent modification of mitochondrial adenine nucleotide translocase 1 (ANT1). Thus, it was hypothesized that conjugation of LNO2 to the mitochondriotropic triphenylphosphonium (TPP+) moiety would enhance its protective properties.Experimental ApproachTPP+-LNO2 was synthesized from aminopropyl-TPP+ and LNO2, and characterized by direct infusion MS/MS. Its effects were assayed in primary cultures of cardiomyocytes from adult C57BL/6 mice and in mitochondria from these cells, exposed to simulated IR (SIR) conditions (oxygen and metabolite deprivation for 1h followed by normal conditions for 1h) by measuring viability by LDH release and exclusion of Trypan blue. Nitro-alkylated mitochondrial proteins were also measured by Western blots, using antibodies to TPP+.Key ResultsTPP+-LNO2 protected cardiomyocytes from SIR injury more potently than the parent compound LNO2. In addition, TPP+-LNO2 modified mitochondrial proteins, including ANT1, in a manner sensitive to the mitochondrial uncoupler carbonylcyanide-p-trifluoromethoxyphenylhydrazone (FCCP) and the ANT1 inhibitor carboxyatractyloside. Similar protein nitro-alkylation was obtained in cells and in isolated mitochondria, indicating the cell membrane was not a significant barrier to TPP+-LNO2.Conclusions and ImplicationsTogether, these results emphasize the importance of ANT1 as a target for the protective effects of LNO2, and suggest that TPP+-conjugated electrophilic lipid compounds may yield novel tools for the investigation of cardioprotection.Linked ArticlesThis article is part of a themed issue on Mitochondrial Pharmacology: Energy, Injury & Beyond. To view the other articles in this issue visit http://dx.doi.org/10.1111/bph.2014.171.issue-8British Journal of Pharmacology 04/2014; 171(8). · 5.07 Impact Factor
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ABSTRACT: Reductions in the blood supply produce considerable injury if the duration of ischemia is prolonged. Paradoxically, restoration of perfusion to ischemic organs can exacerbate tissue damage and extend the size of an evolving infarct. Being highly metabolic organs, the heart and brain are particularly vulnerable to the deleterious effects of ischemia/reperfusion (I/R). While the pathogenetic mechanisms contributing to I/R-induced tissue injury and infarction are multifactorial, the relative importance of each contributing factor remains unclear. However, an emerging body of evidence indicates that the generation of reactive oxygen species (ROS) by mitochondria plays a critical role in damaging cellular components and initiating cell death. In this review, we summarize our current understanding of the mechanisms whereby mitochondrial ROS generation occurs in I/R and contributes to myocardial infarction and stroke. In addition, mitochondrial ROS have been shown to participate in preconditioning by several pharmacologic agents that target potassium channels (e.g., ATP-sensitive potassium (mKATP) channels or large conductance, calcium-activated potassium (mBKCa) channels) to activate cell survival programs that render tissues and organs more resistant to the deleterious effects of I/R. Finally, we review novel therapeutic approaches that selectively target mROS production to reduce postischemic tissue injury, which may prove efficacious in limiting myocardial dysfunction and infarction and abrogating neurocognitive deficits and neuronal cell death in stroke.Redox biology. 01/2014; 2:702-14.
Mitochondrial nitroalkene formation and mild uncoupling
in ischaemic preconditioning: implications for
Sergiy M. Nadtochiy1†, Paul R.S. Baker2†, Bruce A. Freeman2, and Paul S. Brookes1*
1Department of Anesthesiology, Box 604, University of Rochester Medical Center, 601 Elmwood Avenue, Rochester, NY 14642,
USA; and2Department of Pharmacology and Chemical Biology, University of Pittsburgh School of Medicine,
Pittsburgh, PA, USA
Received 21 July 2008; revised 27 October 2008; accepted 20 November 2008; online publish-ahead-of-print 2 December 2008
Time for primary review: 10 days
Aims Both mitochondria and nitric oxide(NO†) contributeto cardioprotectionby ischaemic precondition-
ing (IPC). IPC causes mild uncoupling of mitochondria via uncoupling proteins (UCPs) and the adenine
nucleotide translocase (ANT), and mild uncoupling per se is cardioprotective. Although electrophilic
lipids are known to activate mitochondrial uncoupling, the role of such species in IPC-induced uncoupling
and cardioprotection is unclear. We hypothesized that endogenous formation of NO†-derived electrophi-
lic lipids (nitroalkenes such as nitro-linoleate, LNO2) during IPC may stimulate mitochondrial uncoupling
via post-translational modification of UCPs and ANT, thus affording cardioprotection.
Methods Hearts from male Sprague-Dawley rats were Langendorff-perfused and subjected to IPC.
Nitroalkene formation was measured by HPLC-ESI-MS/MS. The effects of exogenous LNO2and biotin-
tagged LNO2on isolated heart mitochondria and cardiomyocytes were also investigated.
Results Nitroalkenes including LNO2were endogenously generated in mitochondria of IPC hearts. Syn-
thetic LNO2(,1 mM) activated mild uncoupling, an effect blocked by UCP and ANT inhibitors. LNO2
(,1 mM) also protected cardiomyocytes against simulated ischaemia–reperfusion injury. Biotinylated
LNO2covalently modified ANT thiols and possibly UCP-2. No effects of LNO2were attributable to NO†
release, cGMP signalling, mitochondrial KATPchannels, or protective kinase signalling.
Conclusion Components of a novel signalling pathway are inferred, wherein nitroalkenes formed by IPC-
stimulated nitration reactions may induce mild mitochondrial uncoupling via post-translational modifi-
cation of ANTand UCP-2, subsequently conferring resistance to ischaemia–reperfusion injury.
Cardiac ischaemic preconditioning (IPC) is an endogenous
protective mechanism, in which short cycles of non-lethal
ischaemia–reperfusion (IR) elicit protection from sub-
sequent prolonged IR injury.1The mechanisms underlying
IPC-mediated cardioprotection are debated, but a consensus
has emerged that nitric oxide (NO†) and mitochondria play
essential roles.2–6Despite this consensus, links between
NO†signalling and effector mechanisms at the mitochon-
drial level remain elusive. For example, while NO†signalling
via cGMP-dependent protein kinase (PKG) can phosphorylate
several mitochondrial targets of relevance to IPC,7–9the
importance of PKG-independent effects of NO†on mitochon-
dria is less clear.4Similarly, mild uncoupling of mitochondria
is an important IPC-induced protective event,10,11but its
potential upstream regulation by NO†is unclear. The aim
of this study was to elucidate novel mechanisms linking
NO†and mitochondria in IPC.
One unexplored aspect of NO†signalling in IPC is the
nitration of unsaturated fatty acids to yield electrophilic
nitroalkene derivatives (e.g. nitro-linoleate and nitro-oleate,
LNO2and OA-NO2, respectively).12Although the biochemical
mechanisms of lipid nitration are not fully elucidated,13
nitroalkenes are found endogenously in humans14and can
mediate pluripotent cell signalling effects.15These effects
may be mediated by electrophilic reaction of nitroalkenes
with protein thiols, to form covalent ‘nitroalkylation’
ene generation from the abundance of polyunsaturated fatty
acids in mitochondrial membranes.17
include elevated NO†,5,6transient reactive oxygen species
(ROS) generation,18,19acidic pH,13and the activation of both
lipoxygenases and mitochondrial phospholipase A2.20,21In
addition, both peroxynitrite and electrophilic lipids can acti-
vate mitochondrial uncoupling,22,23and mild uncoupling
†Authors contributed equally
*Corresponding author. Tel: þ1 585 273 1626; fax: þ1 585 273 2652.
E-mail address: firstname.lastname@example.org
Published on behalf of the European Society of Cardiology. All rights reserved. & The Author 2008.
For permissions please email: email@example.com.
Cardiovascular Research (2009) 82, 333–340
itself is cardioprotective.11,24Combining these observations
dria during IPC, and may nitroalkylate mitochondrial proteins
thereby activating mild uncoupling, leading to cardioprotec-
tion against IR injury.
Detailed experimental procedures are given in the Supplementary
material online. All chemicals were of the highest grade available
from Sigma (St Louis, MO, USA) unless otherwise stated. LNO2and
biotinylated LNO2(Bt-LNO2) were synthesized, purified, quantified,
and stored as previously,14,25,26with all procedures performed under
subdued light. Non-nitrated linoleic acid (LA) served as a control
Male Sprague-Dawley rats (Harlan, Indianapolis, IN, USA), 200–
250 g body mass, were housed in accordance with the NIH Guide
for the Care and Use of Laboratory Animals (US National Institutes
of Health Publication No. 85–23, revised 1996). All procedures
were also approved by the University of Rochester Committee on
Animal Resources (UCAR, protocols 2003-111 and 2007-087).
Hearts were perfused as previously,10and subjected to either (i)
normoxic perfusion, (ii) IPC, (iii) IPC plus the NO†synthase inhibitor
L-nitro-arginine methyl ester (L-NAME, 100 mM), (iv) ischaemia, or
(v) IPC plus ischaemia.
Heart mitochondria were isolated and protein determined as pre-
viously.10Mitochondrial respiration and uncoupling were measured
as previously.10Optional additions to incubations were: 1–5 mM
LNO2, 1 mM guanosine diphosphate (GDP), or 50 mM Genipin
(Wako, Richmond, VA, USA)27to inhibit uncoupling proteins (UCPs)
or UCP-2, respectively; 5 mM carboxyatractyloside (CATr, Calbio-
chem, San Diego, CA, USA) to inhibit adenine nucleotide translocase
(ANT); 20 mM ethanethiol (E-SH) to reverse thiol modifications;
30 mM 2-(4-carboxyphenyl)-4,4,5,5-tetramethyl-imidazoline-1-oxyl-
3-oxide (c-PTIO, Axxora, San Diego, CA, USA) to scavenge NO†; or
1 mM 2,6-di-tert-butyl-4-methyl-phenol (BHT), a lipid-soluble anti-
oxidant. Mitochondrial permeability transition (PT) pore opening
was measured as previously.28
Adult rat ventricular cardiomyocytes were isolated and state 4
respiration plus mitochondrial membrane potential (Dcm) were
measured as previously.29Cells were subjected to simulated ischae-
mia–reperfusion (SIR) injury as previously,29with cell viability
measured by Trypan blue exclusion. This model eliminates the
potentially confounding vascular effects of LNO2.15Briefly, SIR com-
prised 1 h hypoxia in glucose-free buffer at pH 6.5, then 30 min
reoxygenation in glucose-replete buffer at pH 7.4. The following
were optionally present: LNO2 (0.25–1 mM), LA (0.5–1 mM), the
soluble guanylate cyclase (sGC) inhibitor 1H-[1,2,4]oxadiazole-
[4,3-a]quinoxalin-1-one (ODQ, 10 mM), the mitochondrial KATP
channel antagonists 5-hydroxydecanoate (5-HD, 300 mM) or glyben-
clamide (2 mM), the extracellular signal regulated kinase (ERK)
inhibitor UO126 (10 mM), or the phosphoinositide 3 kinase (PI3K)
inhibitor wortmannin (100 nM).
Biotinylated proteins were immunoprecipitated from Bt-LNO2-
treated mitochondria or cardiomyocytes using neutravidin-agarose
as previously.10Immunoprecipitated samples or whole extracts
were western blotted as previously,10using antibodies against
biotin, ANT, or UCP-2.
Lipids were extracted from 5 mg mitochondrial protein as pre-
viously.30A synthetic [13C18]-LNO2internal standard was added to
correct for extraction losses. Nitroalkenes were detected by HPLC
electrospray ionization tandem mass spectrometry (HPLC-ESI-MS/
MS) as previously.14Biologically derived nitroalkenes were identified
by retention time, precursor ion mass, MS/MS fragmentation
pattern, and thiol reactivity,14and were quantified relative to the
All experiments were performed three to eight times, each
n representing an independent mitochondrial, cell, or heart
preparation (separate animal). Significance between groups was
established by ANOVA.
In the current study, we investigated whether fatty acid
nitration could occur in cardiac mitochondria during IPC.
Mitochondrial lipid extracts from perfused hearts were ana-
lysed by HPLC-ESI-MS/MS using a multiple reaction monitor-
ing (MRM) transition of 324/46 in negative ion mode. The
chromatograms (Figure 1A) revealed that mitochondria
from IPC hearts contained elevated levels of an LNO2
nitroalkene derivative, with positional isomers eluting at
identical times to a synthetic LNO2nitroalkene standard.
Concomitant product ion analysis (Figure 1B) revealed a
fragmentation pattern for the IPC-derived nitroalkene con-
sistent with the previously reported LNO2structure.14Quan-
concentration in IPC mitochondria to be 619+137 fmol/
mg mitochondrial protein (Figure 1C). The NOS inhibitor
L-NAME attenuated the IPC-induced increase in mitochon-
drial LNO2by ?60% (P ¼ 0.06 vs. IPC), although it is not
known if the L-NAME insensitive fraction of LNO2is due to
incomplete NOS inhibition or represents LNO2 generation
from other reactive nitrogen species (RNSs) such as NO2
Ischaemia alone generated a miniscule amount of LNO2,
but notably in mitochondria from hearts subjected to IPC
plus ischaemia, LNO2 levels dropped to ?16% of those
seen in IPC alone, suggesting rapid LNO2degradation. Mito-
chondria also contained OA-NO2, but its levels did not
change in IPC (215+74 vs. 245+41 fmol/mg protein in
control vs. IPC, respectively). In addition nitroalkenes
were detected in other subcellular compartments (data
not shown). Due to space restrictions the current study
focuses on mitochondrial IPC samples, and a more complete
characterization of cardiac nitroalkenes during IPC and IR,
including their metabolism by mitochondrial b-oxidation, is
anticipated to be the subject of a subsequent manuscript.
Next, the potential for exogenous LNO2to protect against
SIR injury was tested in isolated cardiomyocytes. Figure 2A
shows that LNO2significantly improved post-SIR cardiomyo-
cyte viability, with maximal protection at 0.5 mM LNO2. Non-
nitrated LA was without effect, and the mito-KATPchannel
antagonists 5-HD or glybenclamide31did not reverse the
effect of LNO2, suggesting no role for this channel in LNO2-
mediated protection. Notably in this system, 5-HD did
block protection by the mito-KATPchannel agonist diazoxide,
indicating appropriate 5-HD efficacy (not shown). In
addition, the sGC inhibitor ODQ,4–7the ERK inhibitor
UO-126,32and the PI3K inhibitor wortmannin32did not
affect LNO2-mediated protection, indicating no role for clas-
sical NO†/cGMP/PKG signalling, or ERK/PI3K signalling. Fur-
thermore, post-SIR mitochondrial function (intracellular
Dcm) correlated well with cell viability and benefited from
LNO2treatment (Figure 2B).
tection and hypothesized that LNO2, like other electrophilic
lipids,23may uncouple mitochondria, which itself is known to
be cardioprotective.10,11,24Consistent with this hypothesis,
Figure 2C shows that LNO2 stimulated cellular state 4
respiration (a surrogate marker for uncoupling), while LA was
without effect. Such respiratory stimulation could be due to
S.M. Nadtochiy et al.
therefore, we next assayed the direct effects of LNO2on
uncoupling in isolated mitochondria. Titration curves of state
4 respiration vs. Dcm(Figure 3) showed that 1 mM LNO2stimu-
lated uncoupling, as indicated by a left shift of the curve (a
chain activity to maintain a given Dcm
effect. Figure 3B and C shows that LNO2-induced uncoupling
was inhibited by the ANT inhibitor CATr and the UCP inhibitor
GDP. Neither inhibitor affected baseline function in the
absence of LNO2(Figure 3D).
Toxicity studies (Figure 3E) revealed that LNO2.10 mM
both inhibited respiration and dropped DCm. This respiratory
inhibition was not due to complex I inhibition (result not
shown), as has been shown for other electrophilic lipids.33
Furthermore, similar to other electrophilic lipids,34LNO2
.10 mM induced large-scale mitochondrial swelling indica-
tive of PT pore opening (Figure 4). This effect was insensi-
tive to the PT pore inhibitor cyclosporin A (CsA), indicating
a possible role for the ‘unregulated’ PT pore resulting
from membrane protein aggregation.35PT pore opening
was not induced by 5 mM LNO2(Figure 4), suggesting this
was not the mechanism of uncoupling induced by 1 mM
Experiments to elucidate the mechanism of LNO2-induced
uncoupling (Figure 5) employed oligomycin-clamped state 4
mitochondrial respiration as a surrogate marker for uncou-
pling.10LNO2-induced uncoupling was not inhibited by BHT
or c-PTIO, respectively, indicating no role for secondary
lipid oxidation or NO†released from LNO236. However, LNO2-
induced uncoupling was sensitive to the UCP-2 inhibitor
10), while LAwas without
hearts, and analysed by HPLC ESI-MS/MS in MRM mode using m/z 324/46 tran-
sition to identify LNO2. Blank solvent extract and synthetic standards were ana-
lysed by the same methods. Insets to chromatograms highlight the co-elution of
LNO2derived from IPC mitochondria with thesynthetic LNO2standard. Data are
representative of n ¼ 8 samples. (B) Product ion analysis of LNO2derived from
IPC-treated heart mitochondria shows the major fragment ions generated
after collision-induced dissociation. Fragments at m/z 324, 306, 293, 288, and
277 are [M–H]2, [M–H2O]2, [M–HNO]2, [M–2H2O]2, and [M–HNO2]2, respect-
ively. The major product ion, m/z 46 is the ionized nitro group (NO2
mentation pattern of IPC mitochondria-derived LNO2 is the same as that
generated from synthetic LNO225. (C) Quantitation of LNO2in mitochondrial
samples, achieved by spiking mitochondria prior to lipid extraction with 500 pg
of [13C18]LNO2as internal standard (m/z 342). The relative peak areas of
amount of mitochondrial protein. Data are means+SEM, n ¼ 4. *P , 0.05 vs.
control.#P , 0.05 vs. IPC alone. Treatment groups are detailed in the methods.
Endogenous LNO2formation in mitochondria during IPC. (A) Lipid
2). The frag-
myocyte respiration. (A) Post-SIR cell viability. Cardiomyocytes were sub-
jected to SIR injury in the presence of indicated concentrations of LNO2or
LA, added 20 min before ischaemia. Where indicated, the mKATPantagonists
5-HD or glybenclamide, the sGC inhibitor ODQ, the ERK inhibitor UO126, or
the PI3K inhibitor wortmannin was present from the beginning of incubations.
(B) Effects of LNO2on post-SIR intracellular mitochondrial membrane poten-
tial, measured using TMRE fluoresence. Treatment groups were as in
Figure 2A. (C) Effects of LNO2or LA on cellular state 4 respiration rate
(clamped with oligomycin). All data are means+SEM, n . 5. *P , 0.05 vs.
SIR alone in (A) and (B), or *P , 0.05 vs. control in (C).
LNO2 protects cardiomyocytes from SIR injury and stimulates
Nitro-fatty acids, mitochondrial uncoupling and IPC 335
genipin27and was also reversed by E-SH suggesting protein
thiol modification as a possible mechanism.16Neither BHT,
GDP, genipin, nor E-SH affected baseline state 4 respiration.
To define mitochondrial targets of LNO2, biotin-tagged
chondrial uncoupling in the same GDP- and CATr-sensitive
manner as native LNO2(Figure 6A, B). Following Bt-LNO2
addition to mitochondria, biotinylated proteins were immu-
noprecipitated and western blotted with anti-biotin or
anti-ANT antibodies. Figure 6C (upper panel) shows that
26was employed. Importantly, Bt-LNO2induced mito-
Bt-LNO2adducted several mitochondrial proteins, including
a prominent band at ?32 kDa which was identified as ANT
(6C, lower panel). Furthermore, Bt-LNO2labelled several
(Figure 6D), thus indicating that Bt-LNO2, similar to other
electrophilic lipids, can enter cells and target mitochon-
dria.37Full characterization of the nitroalkene-reactive pro-
teome is anticipated to be the subject of subsequent
studies. Nevertheless, as detailed in the Supplementary
material online, several other mitochondrial and non-
mined as previously.10The upper-right point in each curve represents state 4 respiration, with the remaining curve resulting from titration with the complex II
inhibitor malonate. An up/left shift in the curve indicates more uncoupled mitochondria. (A) Effect of 1 mM LA or LNO2. In (B) and (C), respectively, 5 mM CATr
(ANT inhibitor) or 1 mM GDP (UCP inhibitor) was present where indicated, for 1 min before LNO2addition. (D) Lack of effect of GDP or CATr on baseline (non-LNO2
stimulated) function. (E) Dose response to LNO2of mitochondrial respiration and Dcm. The solid point represents state 4, with subsequent LNO2additions (0.5, 1,
3, 5 mM) resulting in an up/left shift indicating uncoupling. Later LNO2additions (10, 20 mM) resulted in a down/left shift indicating respiratory inhibition. All
data are means+SEM, n . 6.
LNO2stimulates mitochondrial uncoupling, in a manner sensitive to inhibitors of ANTand UCPs. Isolated mitochondrial uncoupling (Hþleak) was deter-
S.M. Nadtochiy et al.
mitochondrial proteins were also identified as nitroalkyla-
As shown in Figure 6E, two conformations of the ANT can
be enforced by different inhibitors. Furthermore, in the
CATr-induced c-conformation, a redox-sensitive thiol (C57)
is inaccessible, whereas this thiol is exposed in the bong-
krekic acid (BKA)-induced m-conformation.38Support for
this thiol as a potential target of LNO2 is provided by
observations that CATr and E-SH inhibited Bt-LNO2ANT modi-
fication, whereas BKA did not (Figure 6C). These results are
consistent with the ability of both CATr and E-SH to inhibit
LNO2-induced uncoupling (Figures 3B and 5). c-PTIO was
without effect on ANT modification, indicating no role for
NO†release. To quantify ANT modification, the fraction of
ANT immunoprecipitated (i.e. ANT in pellet vs. supernatant)
was examined. Figure 6F shows a pellet/supernatant pair
from a biotin immunoprecipitation, western blotted for
ANT. Densitometry on several such blots revealed that
upon Bt-LNO2treatment (1 mM) 48.2+4.1% of ANT disap-
peared from the supernatant and appeared in the pellet.
Since LNO2-induced Hþleak was also sensitive to UCP-2
inhibitors (Figures 3C and 5), nitroalkylation of UCP-2 was
studies similar to those performed for ANT revealed no
UCP-2 pull-down (data not shown). Subsequently, mitochon-
dria were treated with native LNO2 in the presence or
absence of the UCP inhibitor GDP, followed by western blot-
ting for UCP-2. Figure 6G shows that UCP-2 progressively dis-
appeared from the blot with increasing LNO2doses, and this
disappearance was attenuated by GDP. We hypothesized this
may be due to nitroalkylation increasing the hydrophobicity
of UCP-2 and preventing its SDS-PAGE migration. Supplemen-
tary material online, Figure S1D provides support for this
hypothesis; blotting the entire gel including stacker plus
comb revealed that high-LNO2treatment resulted in aggre-
gation of UCP-2 immunoreactivity at the stacker/separating
The major findings of this study are: (i) Electrophilic nitroalk-
ene derivatives are formed endogenously in mitochondria
during IPC; (ii) Exogenous LNO2 protects cardiomyocytes
from SIR injury; (iii) LNO2stimulates mitochondrial uncou-
pling, via ANT and UCP-2 dependent mechanisms; (iv) LNO2
nitroalkylates ANT and possibly UCP-2.
Central roles in IPC signalling are played by mitochon-
dria,2–4NO†,4–6and ROS,18,19,24. However, the relationships
between these key players are not fully understood. One
unexplored mechanism for the interaction of ROS, NO†,
and mitochondria in IPC may be the generation of electro-
philic lipids such as nitroalkenes. Oxidative lipid derivatives
are known to be cardioprotective39–41and can induce mito-
chondrial uncoupling.23Furthermore, mild mitochondrial
uncoupling itself is cardioprotective.10,11,24Therefore, the
current data together with these previous findings suggest
that electrophilic nitroalkenes may be endogenously gener-
ated in mitochondria during IPC and may induce mitochon-
drial uncoupling, thereby contributing to cardioprotection.
Regarding the contribution of this pathway to the overall
cardioprotective effects of IPC, if a mitochondrial volume
of 0.65 ml/mg protein is assumed,10the amount of LNO2
in IPC mitochondria (Figure 1C) translates to an intra-
mitochondrial concentration of 0.95 mM, which is the same
level of exogenous LNO2(1 mM) that stimulated uncoupling
in isolated mitochondria (Figure 3). Thus, the mitochondrial
concentration of LNO2 generated in IPC is theoretically
capable of inducing uncoupling.
Considering the mechanism of acyl chain nitration in IPC,
the finding that L-NAME only partially inhibited IPC-induced
LNO2 formation (assuming efficient NOS inhibition by
Typical PT pore swelling traces28are shown. LNO2or LA was added at the
arrow. Where indicated, the PT pore inhibitor cyclosporin A (CsA, 2 mM)
was present from the beginning of the experiment. (B) Quantitation of swel-
ling magnitude at 10 min, from traces of the type shown in (A). Dose response
to LNO2is shown, with CsA effect shown for the 20 mM LNO2dose (effect of
CsA was similar at all LNO2 doses). Positive control for CsA-sensitive PT
pore opening (to prove CsA was working appropriately) is shown on the
right (open bars). Ca2þwas added at the arrow as shown in (A), instead of
LNO2. All data are means+SEM, n . 5.
Induction of unregulated PT pore opening at high [LNO2] (A)
Oligomycin-clamped state 4 mitochondrial respiration was used as a surrogate
for uncoupling (see Methods). Indicated concentrations of reagents were
added prior to LNO2, except E-SH, which was added after LNO2to reverse
the latter’s effects. Open bars represent effects of reagents in the absence
or presence of native (non-nitrated) LA. Filled bars are in the presence of
indicated concentrations of LNO2. GDP is a UCP inhibitor, genipin a UCP-2
inhibitor, E-SH a thiol reducing agent, c-PTIO a NO†scavenger, and BHTa lipo-
philic antioxidant. Data are means+SEM, n . 6. *P , 0.05 vs. control.
The effect of various reagents on LNO2-induced uncoupling.
Nitro-fatty acids, mitochondrial uncoupling and IPC 337
L-NAME in this system) suggests that non-NOS sources of RNS
may be involved. In this regard, an observed ?40% increase
in mitochondrial NO2
Brookes, unpublished) suggests NO2
lipid nitration under the acidic conditions of IPC. The
overall role of NOS in IPC is somewhat controversial,42
since in vivo studies suggest NOS is essential for IPC,43
whereas in vitro studies have found that L-NAME does not
block IPC.44,45The mechanism by which nitro fatty acids
are liberated from membranes may involve PLA2, which is
known to liberate linoleate and arachidonate, but not
oleate, during IPC.46This is consistent with our finding
that mitochondrial OA-NO2 levels did not change in IPC,
indicating some specificity in fatty-acid liberation.
2levels during IPC (Nadtochiy and
2may contribute to
Regardless the mechanism of nitroalkene formation or
liberation in IPC, exogenous LNO2 was protective in a
cardiomyocyte model of SIR injury. The signalling pathways
by which nitroalkenes elicit protection could include
PPARg activation47–49and subsequent HO-1 up-regulation.50
However, such gene transcription effects unlikely account
for the immediate short-term (20 min) effects of LNO2
observed herein. Thus, we chose to focus on the short-term
direct effects of LNO2on mitochondrial function as a poten-
tial mechanism of protection and demonstrated that LNO2
induces mild mitochondrial uncoupling.
Since RNSs are known to have a number of other effects on
mitochondria including the modulation of many proteins
implicated in IPC,4control experiments were performed
and CATr. Data are means+SEM, n ¼ 4. (C) Biotin labelled proteins were immunoprecipitated from Bt-LNO2treated mitochondria and then western blotted with
anti-biotin (upper panel) or anti-ANT (lower panel) antibodies. Ctrl. indicates mitochondria without Bt-LNO2. In all other lanes Bt-LNO2(1 mM) was added. Where
indicated 5 mM CATr, 20 mM BKA, 50 mM c-PTIO, or 20 mM E-SH was present. Inhibitors were added before LNO2, except E-SH, which was added after LNO2. Blots
are representative of at least four experiments. (D) Similar experiment to (C), but with intact cardiomyocytes treated with Bt-LNO2, instead of mitochondria. (E)
Schematic indicating the effects of ANT inhibitors CATr and BKA on ANTconformation. c cytosolic facing, m matrix facing. (F) Representative ANTwestern blot on
a supernatant plus pellet pair from biotin immunoprecipitation of the type shown in (C). Upper panel is the IP supernatant (i.e. non-modified proteins which
escaped pull-down) and lower panel is the pellet (i.e. Bt-LNO2modified proteins pulled down by neutravidin). Data representative of four independent experi-
ments. (G) Mitochondria were treated with indicated concentrations of non-biotinylated LNO2in the absence or presence of the UCP inhibitor GDP (1 mM), and
western blotted for UCP-2 (no immunoprecipitation). Blot is shown for an anti-N-terminal UCP antibody (Santa-Cruz), with similar results obtained using an
anti-C-terminal UCP antibody (ADI, not shown). Representative blot from at least four independent experiments. Densitometry values (means+SEM, as % of
control) for the prominent UCP-2 band at 32 kDa are shown below the blot.
LNO2nitroalkylates ANTand UCP-2. (A) Bt-LNO2induced uncoupling similar to LNO2(c.f. Figure 3), and (B) this effect of Bt-LNO2was sensitive to GDP
S.M. Nadtochiy et al.
to exclude the involvement of NO†released from LNO2, sGC,
ERK, PI3K, or mKATPchannels in LNO2-mediated protection.
Furthermore, even though the mKATPchannel has been pro-
posed to uncouple mitochondria,18,31,51the magnitude of
mKATPflux is insufficient to account for IPC-induced uncou-
pling,10and the current lack of a molecular identity for
mKATPprecludes its identification as an LNO2target.
Having established that LNO2 uncoupled mitochondria,
we next investigated the mechanism of uncoupling. One possi-
bility is that cycling of protonated/deprotonated nitro-fatty-
acids across the membrane, as observed for nitro-aromatics
However, such uncoupling should not occur with Bt-LNO2in
which the carboxylic acid group is blocked by biotin. The data
in Figure 6A and B thus precludes this mechanism. Rather,
based on evidence from biotin-tagged LNO2(Figure 6), the
likely mechanism of LNO2-induced uncoupling is the nitro-
alkylation of ANT and UCP-2. Although the exact mechanism
of Hþtransport by these proteins is unknown,52we speculate
that nitroalkylation of cysteine residues may result in struc-
tural/conformation changes that cause uncoupling.
Mild mitochondrial uncoupling is thought to be cardiopro-
tective via inhibition of mitochondrial Ca2þoverload2–4or
ROS generation,53although we consider it unlikely54that a
direct role for UCPs in mitochondrial Ca2þtransport55exists.
Regarding ROS, it has been shown that uncoupling-induced
cardioprotection is blocked by antioxidants, suggesting a
role for ROS downstream of uncoupling.24These apparently
inconsistent findings are reconciled by the paradigm that
low levels of ROS generated during IPC (prior to index ischae-
mia) may activate signalling processes that inhibit large-
scale ROS generation during subsequent IR injury.10,19
In summary, the current work has elucidated components
of a potential signalling pathway, in which nitroalkenes are
generated in mitochondria during IPC and activate mito-
chondrial uncoupling via nitroalkylation of proteins such as
ANT. These studies advance our understanding of the biologi-
cal roles of nitroalkenes and also suggest that nitroalkenes
may be useful cardioprotective pharmacologic agents. It is
also possible that some of the cardioprotective benefits of
or mitochondrially targeted NO†
involve nitroalkene generation, and that some cardioprotec-
tive benefits of the ‘Mediterranean diet’ may be due to
intra-gastric nitroalkene generation.57
Supplementary material is available at Cardiovascular
Conflict of interest: BAF acknowledges financial interest in
This work was supported by grants from the US National Institutes of
Health (RO1 HL071158 to P.S.B. and HL58115 and HL64937 to B.A.F.)
and the American Diabetes Association (ADA 7-06-JF-06 to P.R.S.B).
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