Mitochondrial dysfunction in cardiac ischemia–reperfusion injury: ROS from complex I, without inhibition

Department of Anesthesiology, University of Rochester, Rochester, New York, United States
Biochimica et Biophysica Acta (Impact Factor: 4.66). 03/2006; 1762(2):223-31. DOI: 10.1016/j.bbadis.2005.10.001
Source: PubMed
A key pathologic event in cardiac ischemia reperfusion (I-R) injury is mitochondrial energetic dysfunction, and several studies have attributed this to complex I (CxI) inhibition. In isolated perfused rat hearts, following I-R, we found that CxI-linked respiration was inhibited, but isolated CxI enzymatic activity was not. Using the mitochondrial thiol probe iodobutyl-triphenylphosphonium in conjunction with proteomic tools, thiol modifications were identified in several subunits of the matrix-facing 1alpha sub-complex of CxI. These thiol modifications were accompanied by enhanced ROS generation from CxI, but not complex III. Implications for the pathology of cardiac I-R injury are discussed.


Available from: Lindsay Burwell, Mar 03, 2015
Mitochondrial dysfunction in cardiac ischemiareperfusion injury:
ROS from complex I, without inhibition
Andrew J. Tompkins
, Lindsay S. Burwell
, Stanley B. Digerness
, Corinne Zaragoza
William L. Holman
, Paul S. Brookes
Department of Anesthesiology, Box 604, University of Rochester Medical Center, 601 Elmwood Avenue, Rochester, NY 14642, USA
Department of Biochemistry and Molecular Biology, University of Rochester Medical Center, Rochester, NY 14642, USA
Department of Surgery, University of Alabama at Birmingham, Birmingham, AL 35294, USA
Received 28 March 2005; received in revised form 27 September 2005; accepted 4 October 2005
Available online 21 October 2005
A key pathologic event in cardiac ischemia reperfusion (I R) injury is mitochondrial energetic dysfunction, and several studies have attributed
this to complex I (CxI) inhibition. In isolated perfused rat hearts, following I R, we found that CxI-linked respiration was inhibited, but isolated
CxI enzymatic activity was not. Using the mitochondrial thiol probe iodobutyl-triphenylphosphonium in conjunction with proteomic tools, thiol
modifications were identified in several subunits of the matrix-facing 1a sub-complex of CxI. These thiol modifications were accompanied by
enhanced ROS generation from CxI, but not complex III. Implications for the pathology of cardiac I R injury are discussed.
D 2005 Elsevier B.V. All rights reserved.
Keywords: ROS; Ischemia; Mitochondria; Complex I; Nitric oxide
1. Introduction
Cardiovascular disease (CVD) is the number 1 cause of
death in the United States, with a large proportion of these
deaths due to myocardial infarction (MI). Despite recent
progress in understanding the origins of CVD, there is still a
lack of understanding on the mechanisms of cell injury in MI.
The amount of injury depends on the length of the ischemic
period and the reperfusion conditions, with short periods of
ischemia being relatively harmless and even eliciting cardio-
protection (vis-a`-vis ischemic preconditionin g). Longer ische-
mic periods result in irreversible injury, often due to hyper-
contracture. The reperfusion phase is also harmful, whereupon
injury is thought to be mediated in part by both reactive oxygen
species (ROS) and reactive nitrogen species (RNS), although
the sources and targets of ROS and RNS are poorly understood
It has been known for some time that mitochon drial
perturbations contribute to cardiac dysfunction in I R injury
(reviewed in [6]). Most of the energetic (ATP) requirements of
the cardiomyocyte are met by mitochondria, and in addition the
organelle plays important roles in Ca
homeostasis [7] and
apoptosis [8]. The precise molecular changes that occur within
mitochondria during I R injury remain unclear, and have been
the focus of much research. It has been reported that respiratory
complexes I, III, IV and V, and many Krebs cycle enzymes are
all affected by I R injury [6,918]. In particular, complex I
(CxI) has been identified as a target for oxidative damage in I R
injury and related pathologies such as autolysis [10 14,18].
In contrast, it was recently shown that in isolated cardiac
mitochondria the activities of CxI, III, and IV were not
inhibited following exposure to 50 AMH
, but the Krebs
cycle enzymes aconitase, a-KGDH, and succinate dehydro-
genase were inhibited [19]. Thus, the precise type and context
of the oxidative insult appears to play an important role in the
pattern of mitochondrial injury.
Complex I is a 900 kDa, >46 subunit enzyme that oxidizes
NADH, passing electrons to ubiquinone, and pumpi ng protons
across the inner membrane. The resulting proton gradient is
then utilized for the production of ATP, and since the heart
relies almost entirely on the CxI-linked oxidation of fatty acids
and glucose [20,21], perturbations to CxI can have a large
impact on cardiac energetics. Early studies in canine myocar-
0925-4439/$ - see front matter D 2005 Elsevier B.V. All rights reserved.
* Corresponding author. Tel.: +1 585 273 1626; fax: +1 585 273 2652.
E-mail address: paul
_ (P.S. Brookes).
Biochimica et Biophysica Acta 1762 (2006) 223 231
Page 1
dium suggested that CxI activity was decreased following I R
injury through the loss of a noncovalently bound FMN [18].
Notably, the FMN site of CxI has been proposed as a site of
ROS generat ion [22,23], although this remains controversial
[24]. In contrast, others have attributed the decrease in CxI
activity foll owing IR to ROS-mediated oxidat ion of cardi-
olipin, the latter being critical for CxI function [10].
Consistently, several laboratories report that ROS or RNS
can inhibit CxI [10,19,2531]. In addition, it has been shown
that CxI inhibition following I R depends on the entry of
into the mitochondrial matrix [12]. Unifying both the
and ROS/RNS data, it has recently been shown that a
combination of Ca
and NO
is required to inhibit CxI in
vitro [25]. Such Ca
dependent CxI dysfunction in I R may
proceed via the known ability of Ca
to enhance mitochon-
drial ROS generation [32,33], leading to secondary generation
from NO
and O
. Alternatively, several studies
have sugges ted tha t CxI modification may occur via s-
nitrosation [25,29,31], although direct molecular evidence for
this is lacking.
Despite these studies, following I R injury both the
molecular defect in CxI and the source of ROS are not well
understood. In order to probe the role of CxI in IR injury both
as a source and target of ROS, we investigated the molecular
nature of the CxI lesion. Surprising ly, we found that 25 min
ischemia plus 30 min reperfusion did not inhi bit CxI activity in
rat hearts, but did lead to thiol modifications within the
complex, and an increase in ROS generation from CxI but not
Cx III. Overall, these data suggest that in IR injury, CxI thiols
may be altered sufficiently to increase ROS production while
not affecting overall enzymatic activity. The implications of
these findings for mitochondrial and cardiac function in IR
injury are discussed.
2. Materials and methods
Male SpragueDawley rats (200 250 g) were obtained from Harlan
and handled in accordance with the procedures of the University Committee
on Animal Research (UCAR). All chemicals were from Sigma (St. Louis
MO) unless otherwise stated. Statistical differences between the control and
I R groups were determined by Student’s t test, with significance set at
P < 0.05.
2.1. Heart perfusions and mitochondrial isolation
Isolated rat hearts were retrograde (Langendorff) perfused essentially as
described [15] in constant flow mode (12 ml/min/gram tissue) with oxygenated
(95% O
) Krebs Henseleit (KH) buffer. Hearts were immersed in KH
in a chamber maintained at 37 -C, and cardiac functionality was monitored by a
water-filled left-ventricular latex balloon linked to a pressure transducer with
digital recording (Dataq, Akron OH). In addition, diastolic stiffness was
quantified as the slope of the relationship between diastolic pressure and balloon
volume, by inflating the balloon in 2 Al increments. After 20 min of
equilibration, ischemia was induced for a period of 25 min by stopping flow
and immersing the heart in de-oxygenated KH buffer (bubbled with 95% N
). Reperfusion was then performed for 30 min. For controls, normoxic
perfusion was performed for a matching time period. In a sub-set of experiments,
as part of a separate study [34,35], combination therapy of diazoxide (100 AM)
plus cariporide (10 AM) was infused via a syringe pump just above the perfusion
cannula for a period of 10 min prior to the onset of ischemia. In this case,
ischemia was imposed for 40 min and reperfusion for 30 min.
At the end of all perfusion protocols, hearts were removed onto ice-cold
buffer and mitochondria prepared as described [15]. Protein concentrations
were determined using the Folin-phenol reagent against a standard curve of
bovine serum albumin [36]. The mitochondrial marker enzyme citrate synthase
was measured (see below) to determine mitochondrial yield and purity.
2.2. Mitochondrial respiration and complex assays
Mitochondrial O
consumption was measured at 37 -C using a Clark-type
electrode in a magnetically stirred 0.25 ml chamber. Mitochondria were
incubated at 0.5 mg protein/ml in respiration buffer (RB) comprising sucrose
(300 mM), KCl (50 mM), KH
(5 mM), MgCl
(1 mM), EGTA (5 mM),
and Tris HCl (20 mM), pH 7.35. Complex I linked state 4 respiration was
initiated by addition of glutamate (10 mM) plus malate (2.5 mM), followed
shortly thereafter by addition of ADP (100 AM) to initiate state 3 respiration.
The enzymatic activities of CxI and citrate synthase were measured
spectrophotometrically as previously described [37].
2.3. 4-iodobutyl-triphenylphosphonium (IBTP) labeling
One mg of mitochondrial protein was incubated in 1 ml of RB with the
respiratory substrates glutamate (10 mM), malate (2.5 mM), and succinate (10
mM). After a 1-min equilibration, IBTP (25 AM) was added and mitochondria
incubated for an additional 5 min with mixing to ensure adequate oxygenation.
Mitochondria were then pelleted by centrifugation (5 min at 10,000
g), snap
frozen in liquid N
, and stored at 80 -C until proteomic analysis [38,39].
Samples (1 mg mitochondrial protein) were also prepared without IBTP, for
biotin-maleimide labeling (see below).
2.4. Blue-native electrophoresis, and biotin-maleimide labeling
Blue-native (BN) gels were run essentially as described [39]. One mg
mitochondrial pellets from IBTP labeling (above) were resuspended in 225 Alof
BN-extraction buffer. After 30 min extraction on ice, samples were centrifuged
at 14,000g for 5 min, and 200 Al of supernatant mixed with 12.5 Alof5%
Coomassie blue G in aminocaproate (0.5 M). Forty Al of sample (200 Ag
protein) was loaded into each lane, and gels were run as described [39].
Biotin-maleimide labeling was performed on non-IBTP labeled samples
during the protein extraction step for BN gels. After 10 min on ice, 220 AM
maleimide-PEO-biotin (Pierce, Rockford IL) was added, and samples incubated
for an additional 20 min in the dark at 25 -C. Remaining sample extraction and
BN gel procedures were as described above.
2.5. 2D BN gels and Western blotting
Complex I bands cut from the BN gel were placed between the plates of a
15% SDS-PAGE gel with 5% stacker. A solution of melted agarose (1%), SDS
(1%), and h-ME (0.01%) was poured into the remaining headspace of the plates.
After the agarose had set, a solution of SDS (1%) and h-ME (0.01%) was added
drop-wise to the top edge of the gel and left for 10 min Gels were run at standard
voltages, then either fixed and silver-stained (SilverQuest, Invitrogen, Carlsbad
CA), or transferred to nitrocellulose using semi-dry apparatus. Transfer of
protein was examined by Ponceau S staining, and membranes were then blocked
for 2 h at 25 -C in TBS (Tris buffered saline containing 0.05% Tween-20), plus
5% nonfat dry milk. Primary antibody incubations were for 2 h at 25 -C (anti-
IBTP polyclonal [30] at 1:2000, anti-nitrotyrosine polyclonal (Cayman, Ann
Arbor MI) at 1:2000). The secondary antibody was HRP-linked goat-anti-rabbit
(GE Biosciences, Piscataway, NJ) for 2 h at 25 -C, followed by ECL detection.
Biotin-maleimide blots were probed with streptavidin-HRP (GE Biosciences) at
1:1000 dilution, followed by ECL detection.
2.6. 3D gels (blue-native, IEF, SDS-PAGE)
Complex I bands were cut from the BN gel, homogenized, and serially
extracted in buffer containing urea (7 M), thiourea (2 M), CHAPS (2%), lauryl-
maltoside (0.5%), dithiothreitol (30 mM), tributylphosphine (10 AM), and
Biolytei ampholytes, at 25 -C. Polyacrylamide was removed by centrifuga-
A.J. Tompkins et al. / Biochimica et Biophysica Acta 1762 (2006) 223 231224
Page 2
tion, and supernatants were loaded into ‘IPG-zoom’ cassettes (Invitrogen)
containing IPG strips (linear pH 3 10, BioRad, Hercules CA), sealed and
incubated overnight at 25 -C. Strips were focused at 200 V for 20 min, 450 V
for 20 min, 750 V for 20 min, 2000 V for 1 h. After IEF, strips were
equilibrated in buffer containing urea (6 M), SDS (2%), Tris (0.375 M),
glycerol (20%) and DTT (0.2 mg) for 15 min, then rinsed in SDS-PAGE
running buffer and placed between the plates of a 15% SDS-PAGE gel with 5%
stacker. A solution of melted agarose (1%) was poured into the remaining
headspace of the plates, and gels and Western blots run as described above.
2.7. Peptide mass fingerprinting
Gel spots were cut and de-stained (using reagents in the Invitrogen
SilverQuest kit), then equilibrated in NH
(100 mM), reduced with DTT,
alkylated with IAA, and washed with NH
(100 mM) in acetonitrile
(50%). Trypsin digestion, extraction, and MALDI-TOF peptide mass finger-
printing were performed as described [39,40].
2.8. Measurement of ROS
Mitochondrial ROS production was measured spectrofluorimetrically at 37
-C using the Amplex Red reagent (Molecular Probes, Eugene OR). Cuvets
contained R B (see above), Amplex Red (10 AM), type II horseradish
peroxidase (1 U/ml), Cu/Zn superoxide dismutase (80 U/ml), and mitochondria
(0.5 mg/ml). Glutamate (2.5 mM) and malate (1.25 mM) were added obtain a
basal rate of CxI ROS production, followed by rotenone (2.5 AM) to maximize
ROS from CxI. In separate incubations, succinate (2.5 mM) and rotenone (2.5
AM) were added to obtain a basal rate of ROS production from complex II to III
(rotenone being present to prevent electron back-flow through CxI), followed
by antimycin A (0.5 AM) to maximize generation at complex III. Rates of ROS
generation were calibrated by adding known amounts of H
at the end of
each run.
3. Results
Cardiac functional damage caused by I R injury is shown
in Fig. 1. The sample trace of left-ventricular pressure in Fig.
1A shows the development of hy per-contracture during
ischemia (arrow). Following reperfusion, diastolic pressure
was increased, systolic pressure decreased, and thus developed
pressure (systolic minus diastolic) was depressed (Fig. 1B).
Diastolic stiffness was also elevated following I R (Fig. 1C).
Previously, elevation in this parameter has been correlated to
mitochondrial dysfunction [15].
3.1. Mitochondrial dysfunction following I R
Inhibition of complex I (NADH)-li nked state 3 respiration
has previously been reported in mitochondria isolated from
hearts subject to IR [18], and this finding is confirmed in
Fig. 2A. This inhibition has previously been attributed to
direct inhibition of complex I itself [9 13]. However, when
CxI activity (normalized to protein) was measured in this
study, no difference was found between control and I R
samples (Fig. 2B).
Since the heart undergoes edema during I R injury, I R
tissue is less dense than control tissue, and thus may yield
different amounts of mitochondria. However, mitochondrial
yields were not different between control and IR samples
(16.8 T 1.3 vs. I R 16.4 T 1.7 mg protein per heart respectively).
Furthermore, the purity/enrichment of the mitochondrial
fraction was not different, since the activity of the mitochon-
drial matrix marker enzyme citrate synthase (CS) was the same
in control and IR groups (Fig. 2C). Thus, even normalizing
CxI activity to CS activity does not reveal an enzymatic defect
at the CxI level (Fig. 2D).
These results are contrary to studies that have demonstrated
CxI inhibition following I R injury and associated pathologies
[913]. Such a variance may originate from differences in the
models of IR injury used (e.g. animal species and age, I R
protocol), as evidenced by a greater than 10-fold variation in the
reported baseline activities of CxI. Notably, it was reported
cardiac IR injury led to a signi ficant decrease CxI activity in
Fig. 1. Langendorff functional data. (A) Typical left ventricular pressure trace (mm Hg) of I R heart, with x axis compressed. (a) and (b) denote the onset of
ischemia and reperfusion respectively, and the arrow indicates ischemic hypercontracture. (B) Left ventricular developed pressure (LVDP) in control perfusion (white
bars) and I R (black bars), both pre- and post- the perfusion protocol. (C) Diastolic stiffness constant in control perfusion (white bars) and I R (black bars). All data
are meansT S.E.M. from 7 independent experiments. *P < 0.01 between control perfusion and I R groups.
A.J. Tompkins et al. / Biochimica et Biophysica Acta 1762 (2006) 223 231 225
Page 3
old rats (28 months), but was without effect in young rats (8
months) [13]. The rats used in this study were relatively young
(2 months). Regarding the I R protocol, we found that
extending the duration of ischemia to 40 min (vs. the 25 min
shown in Fig. 2) elicited no further effect on CxI activity
(control 298 T 27 vs. IR 363 T 16 nmol NADH/min/unit CS).
Notably, none of the previous studies of CxI in IR
normalized CxI acti vity to CS, although it is unli kely that this
accounts for the different findings because we did not find
differences in the mitochondrial yields or specific activities of
CS (see above). It is also possible that variances in CxI assay
method may account for these contrary results. The current
assay uses Co Q
as electron acceptor (vs. ferricyanide in
some other studies [41]) and would not be expected to detect
perturbations in the pathway of electron flux through the
enzyme, or defects in the coupling of H
pumping to e
A lack of knowledge regarding the precise electron flux
pathways within mammalian CxI currently precludes a full
Since i t was foun d that Cx I-linked respiration was
depressed following IR (Fig. 2A), we hypothesized that the
enzymatic defect may lie upstream of CxI in the TCA cycle. It
has previously been reported that several TCA cycle enzymes
are sensitive to oxidative stress [13,19,42,43] and as shown in
Fig. 2E, a-KGDH is indeed inhibited following I R injury.
While the decrease in a-KGDH activity (¨22%) is alone not
sufficient to account for the loss of respiratory activity,
inhibition of other TCA cycle enzymes such as aconitase
and pyruvate dehydrogenase [13,19,42,43] would be sufficient
to elicit such respiratory inhibition. However, the focus of this
investigation was CxI, and owing to the limited amounts of
mitochondria available, we did not pursue this possibility
further by assaying other TCA cycle enzymes.
3.2. Proteomic studies of CxI modification
While it may appear unusual to examine CxI modifications
in a situati on where no enzymatic inhi bition was found, the
Fig. 2. Complex I linked respiration, and complex I activity assays. (A) State 4 and state 3 respiration rates, with complex I linked substrates (glutamate plus malate).
(B) Citrate synthase activity. (C) Complex I activity per mg protein. (D) Complex I activity normalized per unit citrate synthase. (E) a-KGDH activity. Data are
shown for mitochondria isolated from control hearts (white bars), and those subjected to I R injury (black bars). All data are expressed as means T S.E.M. from 7
independent experiments. *P < 0.01 between control perfusion and I R groups.
A.J. Tompkins et al. / Biochimica et Biophysica Acta 1762 (2006) 223 231226
Page 4
proteomic examination of CxI was performed concurrently
with the enzymatic studies. Thus, the prote omic studies had
yielded interesting results well before the conclusion regarding
no CxI inhibition was reached.
3.3. Nitrotyrosine modification of CxI
Complex I has previously been identified as a target for
tyrosine nitration [44], and therefore mitochondrial samples
were resolved on BN gels, the CxI band subjected to SDS-
PAGE to resolve the subunits, and Western blotting for
nitrotyrosine performed. Fig. 3A shows significant nitration
of various subunits in CxI following treatment of isolated
mitochondria with ONOO
. Interestingly however, tyrosine
nitration was not detected in mitochondria isolated from IR
hearts (Fig. 3B), despite over-exposure of the blot membrane
(>24 h ECL development, vs. 20 min for the ONOO
mitochondrial blot). Furthermore, no modification of CxI by
the lipid oxidation product 4-HNE was detected (Western blot,
Calbiochem monoclonal anti-4-HNE Ab, data not shown). In
addition, as shown in Fig. 3C and D, the loss of CxI activity
did not correlate with the degree of nitration, the latter
occurring only at higher levels of ONOO
, beyond those at
which the complex was already inhibited. Owing to limitations
in the antibody detect ion of nitrotyrosine (e.g., epi tope
specificity), quantitative interpretation of such data may be
restricted. Nevertheless, these results suggest that nitration of
CxI can occur with ONOO
treatment, but is probably an
epiphenomenon and not the p rimary mechanism of enzymatic
inhibition. The other reactivities of ONOO
(including but not
limited to oxidation of thiols, tryptophan nitration, or protein
oxidation via secondary OH
formation, [45]) probably account
for the loss of CxI activity in this case.
3.4. Thiol modifications in CxI
Since it was suspected that cysteine thiols in CxI may be
modified in I R injury, mitochondria were labeled with the
mitochondria-specific thiol probe IBTP, and separated on BN
gels. Fig. 4A shows a typical BN gel, indicating that the
physical amount of CxI is not different between control and
IR samp les, and this is confirmed by a Western blot for the
CxI 39 kDa subunit (Fig. 4B).
The results of an IBTP Western blot on CxI subunits are
shown in Fig. 4C (i.e. CxI cut from the BN gel and resolved
into subunits by SDS-PAGE), with a corresponding Coomassie
stained gel in Fig. 4D. The presence of a band on the blot
indicates that a protein at that molecular weight contains a free
thiol capable of reacting with IBTP. Thus, a dimini shed signal
in the I R samples indicates that thiols are no longer able to
react with the IBTP probe either due to oxidation, disulfide
formation, s-nitrosation, or other such modi fications.
Since the resolution of 1D SDS-PAGE gels is somewhat
limited, a 3D gel method was also developed in which CxI
bands cut from BN gels were subjected to IEF and then SDS-
PAGE, thereby constituting 3 dimensions of separation. The
3D gels were Western blotted for IBTP, with the enhanced
resolution of the 3D system affording less overlap between
bands/spots, and thus greater confidence in mass spectrometric
identifications (Fig. 4E and F).
The 3D separation resulted in fewer CxI subunits being
visible on the gel, probably owing to their hydrophobic/basi c
nature and thus incompatibility with IEF. Furthermore, the
separation revealed that fewer CxI subunits were present in the
IR samples, and numerous subunits exhibited small changes
in isoelectric point following I R. The corresponding 3D IBTP
blots also showed significantly fewer spots than the 1D IBTP
blots shown in panel C, but nevertheless, excision of proteins
from either the 2D or 3D gels permitted identification of
several proteins with diminished IBTP labeling in the I R
samples, and these data are shown in Table 1. Notably, all of
the modified proteins were located in the matrix-facing 1a sub-
complex of CxI [46].
3.5. Mitochondrial ROS generation in IR
Complex I has previously been reported as a site of
mitochondrial ROS generation [2224,33], and since the
modified subunits in the 1a sub-complex contain several
electron-transporting moieties, including Fe S centers and the
FMN binding site [46] (Fig. 5A), it was hypothesized that thiol
modifications in this sub-complex might enhance ROS
Fig. 3. Complex I modification by nitrotyrosine. (A) Nitrotyrosine Western blot
of rat heart mitochondria treated with a single bolus addition of 250 AM
. Complex I was isolated by blue-native gel electrophoresis then
separated into its component subunits by SDS-PAGE (see Materials and
methods). Left panel—Coomassie stained gel showing subunits, right panel—
anti-N-Tyr blot. Numbered subunits were excised and identified by MALDI-
TOF as follows: (i) 75 kDa Fe S subunit, (ii) 39 kDa subunit, (iii) ND6
subunit. (B) Nitrotyrosine Western blots of CxI from control and I R hearts.
CxI was separated by 2D blue-native gels as in panel A. Two separate blots are
shown, and ECL exposure was for 24 h in each case. (C) Dose response of rat
heart mitochondria to serial ONOO
additions. Samples were prepared and
analyzed as in panel A. (D) Relationship between nitration and CxI activity.
CxI activity was measured as for Fig. 2, and degree of nitration was determined
by densitometry analysis of the 39 kDa subunit band in panel C (Scion Image
software, Scioncorp, Frederick MD). All data are representative of at least 3
separate experiments.
A.J. Tompkins et al. / Biochimica et Biophysica Acta 1762 (2006) 223 231 227
Page 5
generation. This appears to be the case, since the data in Fig.
5B show that mitochondria from I R heart s exhibited elevated
ROS generation when respiring on CxI-linked substrate, but a
decreased generation of ROS when respiring on CxII-linked
substrates. Notably, no change was seen in the maximally
stimulated rate of ROS generation from CxI (Fig. 5C) in the
presence of rotenone, again suggesting no change in the overall
enzymatic activity of the complex. A faster rate of ROS
generation from Cx III in I R mitochondria vs. controls was
revealed upon addition of antimycin A, which maximizes ROS
from this complex. However, such a supra-physiological ROS
generation condition may not be extrapolated to the in situ
3.6. Pharmacologic cardioprotection and CxI thiols
As part of a series of ongoing studies on cardioprotection by
diazoxide (Dz) and cariporide (Cp), mitochondria were
harvested from hearts subject to I R with or without Dz/Cp
treatment [34,35]. These compounds are proposed to elicit
cardioprotection by convergent mechanisms, where by Dz
opens mitochondrial K
channels, possibly preventing
mitochondrial Ca
overload, and Cp inhibits NHE-1, prevent-
ing cytosolic Na
overload which may subsequently prevent
overload [34,35,47]. As shown in Fig. 6A, administration
of Dz/Cp to hearts elicited a substantial improvement in post-
IR functional recovery. In addition, this recovery was
associated with prevention of CxI thiol modifications (Fig. 6B).
4. Discussion
The m ain findings of this study are as follows: (i)
Mitochondrial CxI activity is not inhibited in I R injury,
despite a substantial decrease in CxI-linked respi ration. (ii)
Tyrosine nitration does not occur at CxI in IR. (iii)
Modification of thiols in the 1a sub-complex elicits an
elevation in CxI-linked ROS generation.
Previously, multiple studies have reported damage to the
mitochondrial oxidative phosphorylation machinery in IR
injury and associated pathologies [6,918], with most of these
studies c oncentrating on respiratory complexes IV. In
addition, a large body of literature exists on the susceptibility
of mitochondria to inhibition by ROS and RNS in a variety of
model systems [19,2532,41 44]. Studies have also shown
that free radical scavengers, especially those targeted to
mitochondria, can ameliorate I R injury [5,48,49].Free
radicals have also been shown to cause contractile dysfunction
and arrhythmias in perfused heart models [50]. Despite these
findings, the molecular events underlying oxidative inhibition
of the respiratory chain and ROS generation in IR are poorly
understood. This is especially true for CxI, since it contains at
least 46 different subunits, and its enzymatic mechanism is still
Fig. 4. Thiol modifications of complex I. (A) First dimension blue-native gel of
mitochondria isolated from control perfusion and I R hearts. (B) Western blot
of the 39-kDa subunit of complex I, performed on a 1D blue-native gel,
showing equal quantities of complex I were present in mitochondria isolated
from control perfused and I R hearts. (C) IBTP Western blot of CxI subunits.
CxI was cut from a gel as in panel A, separated by SDS-PAGE, and Western
blotted for IBTP, as described in the methods. Presence of a band indicates a
protein at that molecular weight with a free thiol available to react with IBTP.
Loss of a band represents modification of a protein thiol at that molecular
weight. The numbers on the Western blot correspond to the bands cut for
identification by MALDI-TOF mass spectrometry (see Table 1). (D) Silver
stained gel of CxI subunits corresponding to the IBTP blot shown in panel C.
(E) Representative silver-stained 3D gels (upper: control perfusion, lower: I R)
in which CxI cut from a blue-native gel was separated by IEF then SDS-PAGE.
(F) Representative IBTP Western blots of 3D gels. In panels A D, each lane
represents an independent perfusion and mitochondrial preparation, and in
addition all gels and blots shown are representative of at least 3 independent
Table 1
MALDI-TOF identification of thiol-modified proteins in CxI
Sample # Protein name Mol.
Wt (kDa)
Accession # MOWSE
1 NADH Dehydrogenase,
Fe S protein 1
79.3 21704020 215
2 NADH Dehydrogenase,
Fe S protein 2
52.6 23346461 158
3 NADH Dehydrogenase,
flavoprotein 1
50.7 34861382 176
4 NADH Dehydrogenase,
24 kDa subunit
26.5 128867 147
5 NADH Dehydrogenase,
1a sub-complex 6 (B14)
15.2 27663138 150
Samples shown in Fig. 4 (sample #) were excised and subjected to MALDI-
TOF peptide mass fingerprinting as described in the methods. Fragment
patterns were matched using Mascot (, and in all
cases shown here significance was obtained with MOWSE scores of >67.
Accession numbers, protein names, and molecular weights are from the Mascot
A.J. Tompkins et al. / Biochimica et Biophysica Acta 1762 (2006) 223 231228
Page 6
under debate [46]. Thus, we sought to apply proteomic and
other tools to investigate CxI in I R injury.
Surprisingly, despite observing a decrease in CxI-linked
respiration following I R, no decrease was seen in the
enzymatic activity of CxI itself. One reason for this lack of
effect may be that only ‘healthy’ mitochondria were isolated,
thereby removing any differences present in the intact tissue.
However, two pieces of evidence suggest this was not the case:
firstly, respiration was inhibited, suggesting mitoch ondria
isolated from I R heart s were damaged (Fig. 2A). Secondly,
the IBTP labeling data (Fig. 4C) clearly show modification of
mitochondrial proteins was occurring. Thus, it was unlikely
that we missed the inhibition of CxI due to poor experimental
design or assay methods.
If CxI activity was not inhibited in I R injury, why was
CxI-linked respiration inhibited? The data in Fig. 2E showing
inhibition of the Krebs cycle enzyme a-KGDH, coupled with
several reports that Krebs cycle enzymes are sensitive to
oxidative stress [13,19,42,43], suggests that the Krebs cycle
and not the respiratory chain is a major site for oxidative
damage during IR injury. While the degree of inhibition of a-
KGDH is not sufficient to account for all of the inhibition of
CxI-linked respiration, it is expected that inhibition of the other
Krebs cycle enzymes is also occurring, leading to a cumulative
effect on respiration.
Regarding the disparity of our data with the many previous
reports of CxI inhibition in I R and associated pathologies
[6,9 18], notable differences between previ ous and present
investigations could include: (i) the experimental model
(perfused hearts vs. cardiomyocytes), (ii) the animal species
and the age of animals used, (iii) normalization of data to
mitochondrial marker enzymes such as CS, (iv) the duration of
ischemia and reperfusion, (v) the use of volatile anesthetics that
are known to precondition the heart [9], (vi) gender differences
in the response to I R injury, or (vii) dietary factors. While it is
beyond the scope of this discussion to elucidate each of these
differences, it can be summarized that further investigation is
required, especially in humans, to determine whether enzy-
matic inhibition of CxI is an important factor in cardiac IR
injury. Most notably, age appears to be a critical facto r since it
has previously been reported that following I R, CxI
inhibition occurs only in old rats, not in young rats like those
used in the current study [13].
Temporally, despite the lack of inhibition of CxI in the
current IR model, investigations into the post-translational
modification of CxI were already underway, and yielded some
interesting results. While ONOO
-treated mitochondria exhib-
ited nitration of CxI subunits (Fig. 3A and C), no such
modification was seen in IR mitochondria (Fig. 3B),
suggesting that CxI nitration is n ot significant in IR .
Furthermore, the mismatch between CxI nitration and loss of
activity in ONOO
treatment (Fig. 3D) suggests that ONOO
may inhibit CxI by mechanisms other than nitration.
One possible mechanism of CxI inhibition that can be
mediated by ONOO
and other ROS/RNS is the modification
of cysteine thiols, including but not limited to oxidation,
nitrosation, nitration, glutathionylation, or disulfide formation
[45]. To investigate thiol modifications, we used the mito-
chondrial thiol probe IBTP, and demonstrated that thiol groups
in the 1a sub-complex are modified following IR injury. The
Fig. 6. Cardioprotection by diazoxide and cariporide. Combination therapy of
diazoxide (Dz) plus cariporide (Cp) was administered as described in the
methods, and has previously been shown to be cardioprotective in I R injury
[34,35]. (A) Left ventricular developed pressure, pre- and post-I R, in normal
and drug-treated gr oups. Data are meansT S.E.M. from 3 indepe ndent
experiments. (B) Representative anti-IBTP Western blot of CxI subunits, in
mitochondria isolated from control hearts, those subjected to I R alone, or I R
in the presence of the combination Dz/Cp therapy. Data are representative of at
least 3 independent experiments.
Fig. 5. Complex I ROS generation. (A) Schematic diagram of complex I, showing arrangement of subcomplexes, and highlighting the subunits in the 1a sub-
complex. (B/C) Rates of ROS production by mitochondria isolated from control perfusion (white bars) and I R hearts (black bars), measured spectrofluorimetrically
at 37 -C using the Amplex Red reagent (see methods). Panel B shows data obtained in the presence of complex I-linked substrates (glutamate plus malate), or
complex II-linked substrates (succinate plus rotenone). Panel C shows data in which ROS generation from either complex I or complex III was maximized by further
addition of rotenone or antimycin A respectively. All data are means T S.E.M. from 6 independent experiments, normalized to CS activity. *P < 0.01 between control
perfusion and I R groups.
A.J. Tompkins et al. / Biochimica et Biophysica Acta 1762 (2006) 223 231 229
Page 7
precise molecular nature of the thiol modification leading to
loss of IBTP labeling is not yet known.
Since the accumulation of IBTP into mitochondria depends
on Dw, and it is known that Dw is lower in mitochondria from
IR hearts [51,52], it could be argued that the loss of IBTP
labeling in certain CxI subunits in I R mitochondria may
simply be due to less uptake of IBTP in these mitochondria.
However, several factors suggest this is not the case: Firstly,
IBTP labeling is only lost in certain subunits of CxI, not all
subunits as would occur if IBTP uptake was diminished.
Seco ndly, even if Dw was diminished significantly, the
Nernstian accum ulation of IBTP would not drastically affect
its intra-mitochondrial level. For examp le a drop of Dw from
160 mV down to 130 mV would cause the intra-mitochon-
drial concentration of IBTP (at an external concentration of
25 AM) to fall from 21 mM to 7 mM, which is still above the
threshold needed for adequate labeling over a time course of
minutes [38]. Thirdly, experiments performed with biotin-
maleimide labeling, which is not sensitive to Dw, gave similar
results (not shown). Furthermore it should be noted that
several CxI sub-units that are not in the 1a sub-complex were
labeled by IBTP (Fig. 4), and that such labeling was not lost
following I R, thereby suggesting specific modifications to
the 1a sub-complex.
The 1a sub-co mplex of CxI consists of the NADH-binding
51 kDa subunit, the FMN-binding site, the 24 and 10 kDa
subunits, the tightly bound 75 kDa subunit, and numerous Fe
S centers [46]. The presence of so many electron carrying
moieties, and the documented generation of ROS by CxI [22
24], led us to hypothesize that thiol modifications within the 1a
sub-complex may elevate ROS generation by the complex.
Indeed, as Fig. 5B shows, CxI-linked but not CxII-III-linked
ROS generation was significantly elevated in I R mitochon-
dria. Notably, no difference between control and I R
mitochondria was seen when CxI was maximally stimulated
to generate ROS by adding rotenone (Fig. 5C). This suggests
that the generation of ROS by CxI is not due to different
amounts of CxI, which is in agreement with the data in Figs.
2D and 4A. Interestingly, when antimycin A was added to
maximally stimulate CxIII-linked ROS generation, IR mito-
chondria generated signifi cantly more ROS than those from
control hearts. This artificially high ROS rate may be more
representative of a diminished antioxidant status in I R hearts
than due to any enzymatic differences in complex III, although
the latter has been demonstrated in IR [6].
Comparison of CxI activity (nmols NADH/min) with rates
of ROS generation (pmols H
/min) reveals that the
percentage of electron flux through CxI diverted to ROS
generation increases from ¨0.01% in control conditions to
¨0.015% following IR. Thus, while CxI enzymatic activity is
not inhibited by I R injury, modification of thiol groups may
result in diversion of more electrons towards ROS generation.
It remains unknown whether such a small increase in ROS
generation can cause pathologic effects within the mitochon-
drion, but notably a recent hypothesis paper has suggested that
inhibition of TCA cycle enzymes by ROS may be a protective
strategy, since it would diminish NADH generation by the
TCA cycle, thereby slowing electron flow into the respiratory
chain, and limiting downstream ROS production [53]. Thus,
ROS generation at CxI and subsequent inhibition of the Krebs
cycle by ROS, w ould appear to form a feed-back loop
mechanism. The proximity of the 1a sub-complex of CxI to
the matrix TCA cycle enzymes would greatly facilitate such a
In summary, the current investigation further implicates
mitochondrial ROS generation and ROS-mediated post-trans-
lational modifications in the pathology of IR, providing a
framework for understanding the molecular events at CxI. It is
expected that targeted therapeutics which can prevent such
modifications may be beneficial in I R injury.
We thank Michael Murphy (Cambridge, UK) for the kind
gift of IBTP and antibodies agains t it. We also thank Victor
Darley-Usmar (Birmingham AL), Shey-Shing Sheu and Jim
Robotham (Rochester NY) for invaluable discussions on this
[1] G. Ambrosio, J.L. Zweier, J.T. Flaherty, The relationship between oxygen
radical generation and impairment of myocardial energy metabolism
following post-ischemic reperfusion, J. Mol. Cell. Cardiol. 23 (1991)
1359 1374.
[2] J.L. Zweier, J.T. Flaherty, M.L. Weisfeldt, Direct measurement of free
radical generation following reperfusion of ischemic myocardium, Proc.
Natl. Acad. Sci. U. S. A. 84 (1987) 1404 1407.
[3] R. Bolli, E. Marban, Molecular and cellular mechanisms of myocardial
stunning, Physiol. Rev. 79 (1999) 609 634.
[4] M.M. Lalu, W. Wang, R. Schulz, Peroxynitrite in myocardial ischemia
reperfusion injury, Heart Fail. Rev. 7 (2002) 359 369.
[5] S.R. Powell, A.J. Tortolani, Recent advances in the role of reactive oxygen
intermediates in ischemic injury: I. Evidence demonstrating presence of
reactive oxygen intermediates; II. Role of metals in site-specific formation
of radicals, J. Surg. Res. 53 (1992) 417 429.
[6] E.J. Lesnefsky, S. Moghaddas, B. Tandler, J. Kerner, C.L. Hoppel,
Mitochondrial dysfunction in cardiac disease: ischemia reperfusion,
aging, and heart failure, J. Mol. Cell. Cardiol. 33 (2001) 1065 1089.
[7] T. Anmann, M. Eimre, A.V. Kuznetsov, T. Andrienko, T. Kaambre, P. Sikk,
E. Seppet, T. Tiivel, M. Vendelin, E. Seppet, V.A. Saks, Calcium-induced
contraction of sarcomeres changes the regulation of mitochondrial
respiration in permeabilized cardiac cells, FEBS J. 272 (2005) 3145 3161.
[8] M. Crompton, The mitochondrial permeability transition pore and its role
in cell death, Biochem. J. 341 (1999) 233 249.
[9] W. Rouslin, Mitochondrial complexes I, II, III, IV, and V in myocardial
ischemia and autolysis, Am. J. Physiol. 244 (1983) H743 H748.
[10] G. Paradies, G. Petrosillo, M. Pistolese, N. Di Venosa, A. Federici, F.M.
Ruggiero, Decrease in mitochondrial complex I activity in ischemic/re-
perfused rat heart: involvement of reactive oxygen species and cardiolipin,
Circ. Res. 94 (2004) 53 59.
[11] D.L. Hardy, J.B. Clark, V.M. Darley-Usmar, D.R. Smith, Reoxygenation
of the hypoxic myocardium causes a mitochondrial complex I defect,
Biochem. Soc. Trans. 18 (1990) 549.
[12] D.L. Hardy, J.B. Clark, V.M. Darley-Usmar, D.R. Smith, D. Stone,
Reoxygenation-dependent decrease in mitochondrial NADH:CoQ reduc-
tase (Complex I) activity in the hypoxic/reoxygenated rat heart, Biochem.
J. 274 (1991) 133 137.
[13] D.T. Lucas, L.I. Szweda, Declines in mitochondrial respiration during
cardiac reperfusion: age-dependent inactivation of alpha-ketoglutarate
dehydrogenase, Proc. Natl. Acad. Sci. U. S. A. 96 (1999) 6689 6693.
A.J. Tompkins et al. / Biochimica et Biophysica Acta 1762 (2006) 223 231230
Page 8
[14] W. Rouslin, R.W. Millard, Mitochondrial inner membrane enzyme defects
in porcine myocardial ischemia, Am. J. Physiol. 240 (1981) H308 H313.
[15] P.S. Brookes, S.B. Digerness, D.A. Parks, V.M. Darley-Usmar, Mitochon-
drial function in response to cardiac ischemia reperfusion after oral
treatment with quercetin, Free Radical Biol. Med. 32 (2002) 1220 1228.
[16] E.J. Lesnefsky, B. Tandler, J. Ye, T.J. Slabe, J. Turkaly, C.L. Hoppel,
Myocardial ischemia decreases oxidative phosphorylation through cyto-
chrome oxidase in subsarcolemmal mitochondria, Am. J. Physiol. 273
(1997) H1544 H1554.
[17] E.J. Lesnefsky, T.I. Gudz, C.T. Migita, M. Ikeda-Saito, M.O. Hassan, P.J.
Turkaly, C.L. Hoppel, Ischemic injury to mitochondrial electron transport
in the aging heart: damage to the iron sulfur protein subunit of electron
transport complex III, Arch. Biochem. Biophys. 385 (2001) 117 128.
[18] W. Rouslin, R.W. Millard, Canine myocardial ischemia: defect in
mitochondrial electron transfer complex I, J. Mol. Cell. Cardiol. 12
(1980) 639 645.
[19] A.C. Nulton-Persson, L.I. Szweda, Modulation of mitochondrial function
by hydrogen peroxide, J. Biol. Chem. 276 (2001) 23357 23361.
[20] W.C. Stanley, G.D. Lopaschuk, J.L. Hall, J.G. McCormack, Regulation of
myocardial carbohydrate metabolism under normal and ischemic condi-
tions, Cardiovasc. Res. 33 (1997) 243 257.
[21] H. Taegtmeyer, Energy substrate metabolism as target for pharmacother-
apy in ischemic and reperfused heart muscle, Heart Metab. 1 (1998) 5 9.
[22] Y. Liu, G. Fiskum, D. Schubert, Generation of reactive oxygen species
by the mitochondrial electron transport chain, J. Neurochem. 80 (2002)
780 787.
[23] A.P. Kudin, N.Y.-B. Bimpong-Buta, S. Vielhaber, C.E. Elger, W.S. Kunz,
Characterization of superoxide-producing sites in isolated brain mito-
chondria, J. Biol. Chem. 279 (2004) 4127 4135.
[24] J. St-Pierre, J.A. Buckingham, S.J. Roebuck, M.D. Brand, Topology of
superoxide production from different sites in the mitochondrial electron
transport chain, J. Biol. Chem. 277 (2002) 44784 44790.
[25] A. Jekabsone, L. Ivanoviene, G.C. Brown, V. Borutaite, Nitric oxide and
calcium together inactivate mitochondrial complex I and induce cyto-
chrome c release, J. Mol. Cell. Cardiol. 35 (2003) 803 809.
[26] A. Orsi, B. Beltran, E. Clementi, K. Hallen, M. Feelisch, S. Moncada,
Continuous exposure to high concentrations of nitric oxide leads to
persistent inhibition of oxygen consumption by J774 cells as well as
extraction of oxygen by the extracellular medium, Biochem. J. 346 (2000)
407 412.
[27] F. Schopfer, N. Riobo, M.C. Carreras, B. Alvarez, R. Radi, A. Boveris, E.
Cadenas, J.J. Poderoso, Oxidation of ubiquinol by peroxynitrite: implica-
tions for protection of mitochondria against nitrosative damage, Biochem.
J. 349 (2000) 35 42.
[28] N.A. Riobo, E. Clementi, M. Melani, A. Boveris, E. Cadenas, S.
Moncada, J.J. Poderoso, Nitric oxide inhibits mitochondrial NADH:ubi-
quinone reductase activity through peroxynitrite formation, Biochem. J.
359 (2001) 139 145.
[29] G.C. Brown, V. Borutaite, Inhibition of mitochondrial respiratory complex
I by nitric oxide, peroxynitrite and S-nitrosothiols, Biochim. Biophys.
Acta 1658 (2004) 44 49.
[30] A. Cassina, R. Radi, Differential inhibitory action of nitric oxide and
peroxynitrite on mitochondrial electron transport, Arch. Biochem.
Biophys. 328 (1996) 309 316.
[31] E. Clementi, G.C. Brown, M. Feelisch, S. Moncada, Persistent inhibition
of cell respiration by nitric oxide: crucial role of S-nitrosylation of
mitochondrial complex I and protective action of glutathione, Proc. Natl.
Acad. Sci. U. S. A. 95 (1998) 7631 7636.
[32] F.R. Gadelha, L. Thomson, M.M. Fagian, A.D. Costa, R. Radi, A.E.
Vercesi, Ca2+-independent permeabilization of the inner mitochondrial
membrane by peroxynitrite is mediated by membrane protein thiol cross-
linking and lipid peroxidation, Arch. Biochem. Biophys. 345 (1997)
243 250.
[33] A.A. Starkov, B.M. Polster, G. Fiskum, Regulation of hydrogen peroxide
production by brain mitochondria by calcium and Bax, J. Neurochem. 83
(2002) 220 228.
[34] S.B. Digerness, P.S. Brookes, S.P. Goldberg, C.R. Katholi, W.L. Holman,
Modulation of mitochondrial adenosine triphosphate-sensitive potassium
channels and sodium-hydrogen exchange provide additive protection from
severe ischemia reperfusion injury, J. Thorac. Cardiovasc. Surg. 125
(2003) 863 871.
[35] J.E. Davies, S.B. Digerness, S.P. Goldberg, C.R. Killingsworth, C.R.
Katholi, P.S. Brookes, W.L. Holman, Intra-myocyte ion homeostasis during
ischemia reperfusion injury: effects of pharmacologic preconditioning
and controlled reperfusion, Ann. Thorac. Surg. 76 (2003) 1252 1258.
[36] O.H. Lowry, N.J. Rosebrough, A.L. Farr, R.J. Randall, Protein measure-
ment with the Folin phenol reagent, J. Biol. Chem. 193 (1951) 265 275.
[37] P.S. Brookes, J.M. Land, J.B. Clark, S.J. Heales, Peroxynitrite and brain
mitochondria: evidence for increased proton leak, J. Neurochem. 70
(1998) 2195 2202.
[38] T.K. Lin, G. Hughes, A. Muratovska, F.H. Blaikie, P.S. Brookes, V.
Darley-Usmar, R.A. Smith, M.P. Murphy, Specific modification of
mitochondrial protein thiols in response to oxidative stress: a proteomics
approach, J. Biol. Chem. 277 (2002) 17048 17056.
[39] P.S. Brookes, A. Pinner, A. Ramachandran, L. Coward, S. Barnes, H.
Kim, V.M. Darley-Usmar, High throughput two-dimensional blue-native
electrophoresis: a tool for functional proteomics of mitochondria and
signaling complexes, Proteomics 2 (2002) 969 977.
[40] B.A. van Montfort, B. Canas, R. Duurkens, J. Godovac-Zimmermann, G.T.
Robillard, Improved in-gel approaches to generate peptide maps of integral
membrane proteins with matrix-assisted laser desorption/ionization time-
of-flight mass spectrometry, J. Mass. Spectrom. 37 (2002) 322 330.
[41] C.S. Powell, R.M. Jackson, Mitochondrial complex I, aconitase, and
succinate dehydrogenase during hypoxia-reoxygenation: modulation of
enzyme activities by MnSOD, Am. J. Physiol. 285 (2003) L189 L198.
[42] U. Andersson, B. Leighton, M.E. Young, E. Blomstrand, E.A. News-
holme, Inactivation of aconitase and oxoglutarate dehydrogenase in
skeletal muscle in-vitro by superoxide anions and/or nitric oxide,
Biochem. Biophys. Res. Commun. 249 (1998) 512 516.
[43] K.M. Humphries, L.I. Szweda, Selective inactivation of alpha-ketogluta-
rate dehydrogenase and pyruvate dehydrogenase: reaction of lipoic acid
with 4-hydroxy-2-nonenal, Biochemistry 37 (1998) 15835 15841.
[44] J. Murray, S.W. Taylor, B. Zhang, S.S. Ghosh, R.A. Capaldi, Oxidative
damage to mitochondrial complex I due to peroxynitrite: identification of
reactive tyrosines by mass spectrometry, J. Biol. Chem. 278 (2003)
37223 37230.
[45] B. Alvarez, R. Radi, Peroxynitrite reactivity with amino acids and
proteins, Amino Acids 25 (2003) 295 311.
[46] L.A. Sazanov, S.Y. Peak-Chew, I.M. Fearnley, J.E. Walker, Resolution of
the membrane domain of bovine complex I into subcomplexes: implica-
tions for the structural organization of the enzyme, Biochemistry 39
(2000) 7229 7235.
[47] P.S. Brookes, Y. Yoon, J.L. Robotham, M.W. Anders, S.S. Sheu, Calcium,
ATP, and ROS: a mitochondrial love hate triangle, Am. J. Physiol. 287
(2004) C817 C833.
[48] V.J. Adlam, J.C. Harrison, C.M. Porteous, A.M. James, R.A. Smith, M.P.
Murphy, I.A. Sammut, Targeting an antioxidant to mitochondria decreases
cardiac ischemia reperfusion injury, FASEB J. 19 (2005) 1088 1095.
[49] K. Zhao, G.M. Zhao, D. Wu, Y. Soong, A.V. Birk, P.W. Schiller, H.H.
Szeto, Cell-permeable peptide antioxidants targeted to inner mitochondrial
membrane inhibit mitochondrial swelling, oxidative cell death, and
reperfusion injury, J. Biol. Chem. 279 (2004) 34682 34690.
[50] M.A. Aon, S. Cortassa, E. Marban, B. O’Rourke, Synchronized whole cell
oscillations in mitochondrial metabolism triggered by a local release of
reactive oxygen species in cardiac myocytes, J. Biol. Chem. 278 (2003)
44735 44744.
[51] J. Minners, L. Lacerda, J. McCarthy, J.J. Meiring, D.M. Yellon, M.N.
Sack, Ischemic and pharmacological preconditioning in Girardi cells and
C2C12 myotubes induce mitochondrial uncoupling, Circ. Res. 89 (2001)
787 792.
[52] D.A. Berkich, G. Salama, K.F. LaNoue, Mitochondrial membrane
potentials in ischemic hearts, Arch. Biochem. Biophys. 420 (2003)
279 286.
[53] J.S. Armstrong, M. Whiteman, H. Yang, D.P. Jones, The redox regulation
of intermediary metabolism by a superoxide-aconitase rheostat, BioEssays
26 (2004) 894 900.
A.J. Tompkins et al. / Biochimica et Biophysica Acta 1762 (2006) 223 231 231
Page 9
  • Source
    • "Changes to the Krebs cycle metabolites α-ketoglutarate, fumarate , malate, and oxaloacetate were not observed with BTPP treatment, indicating that the anti-anaplerotic effects are dependent on mitochondrial thiol modification. Previous studies by our lab and others using isolated mitochondria in other cell types have shown that IBTP forms adducts with several proteins involved in the Krebs cycle [24,25,28,29] . In this study, we showed that aconitase enzymatic activity and GAC protein levels were decreased by IBTP (Figs. 8–10). "
    [Show abstract] [Hide abstract] ABSTRACT: Many cancer cells follow an aberrant metabolic program to maintain energy for rapid cell proliferation. Metabolic reprogramming often involves the upregulation of glutaminolysis to generate reducing equivalents for the electron transport chain and amino acids for protein synthesis. Critical enzymes involved in metabolism possess a reactive thiolate group, which can be modified by certain oxidants. In the current study, we show that modification of mitochondrial protein thiols by a model compound, iodobutyl triphenylphosphonium (IBTP), decreased mitochondrial metabolism and ATP in MDA-MB 231 (MB231) breast adenocarcinoma cells up to 6 days after an initial 24h treatment. Mitochondrial thiol modification also depressed oxygen consumption rates (OCR) in a dose-dependent manner to a greater extent than a non-thiol modifying analog, suggesting that thiol reactivity is an important factor in the inhibition of cancer cell metabolism. In non-tumorigenic MCF10A cells, IBTP also decreased OCR; however the extracellular acidification rate was significantly increased at all but the highest concentration (10µM) of IBTP indicating that thiol modification can have significantly different effects on bioenergetics in tumorigenic versus non-tumorigenic cells. ATP and other adenonucleotide levels were also decreased by thiol modification up to 6 days post-treatment, indicating a decreased overall energetic state in MB231 cells. Cellular proliferation of MB231 cells was also inhibited up to 6 days post-treatment with little change to cell viability. Targeted metabolomic analyses revealed that thiol modification caused depletion of both Krebs cycle and glutaminolysis intermediates. Further experiments revealed that the activity of the Krebs cycle enzyme, aconitase, was attenuated in response to thiol modification. Additionally, the inhibition of glutaminolysis corresponded to decreased glutaminase C (GAC) protein levels, although other protein levels were unaffected. This study demonstrates for the first time that mitochondrial thiol modification inhibits metabolism via inhibition of both aconitase and GAC in a breast cancer cell model.
    Full-text · Article · Jan 2016
  • Source
    • "The energetic requirements of cardiac myocytes are mainly met by mitochondria. This organelle also plays important roles in maintaining intracellular Ca 2+ homeostasis and in regulating cell death [14]. With ischemia, the loss of O 2 and consequent decrease in ATP levels disrupts cardiac myocyte ionic homeostasis resulting in depolarization and cytoplasmic Ca 2+ accumulation [15]. "
    [Show abstract] [Hide abstract] ABSTRACT: Background: Targeting the mitochondria during ischemia/reperfusion (IR) can confer cardioprotection leading to improved clinical outcomes. The cardioprotective potential of (-)-epicatechin (EPI) during IR via modulation of mitochondrial function was evaluated. Methods and results: Ischemia was induced in rats via a 45 min occlusion of the left anterior descending coronary artery followed by 1 h, 48 h, or 3 week reperfusion. EPI (10 mg/kg) was administered IV 15 min prior to reperfusion for the single dose group and again 12 h later for the double dose group. Controls received water. Experiments also utilized cultured neonatal rat ventricular myocytes (NRVM) and myoblasts. A single dose of EPI reduced infarct size by 27% at 48 h and 28% at 3 week. Double dose treatment further decreased infarct size by 80% at 48 h, and 52% by 3 weeks. The protective effect of EPI on mitochondrial function was evident after 1h of reperfusion when mitochondria demonstrated less respiratory inhibition, lower mitochondrial Ca2+ load, and a preserved pool of NADH that correlated with higher tissue ATP levels. Mechanistic studies in NRVM revealed that EPI acutely stimulated maximal rates of respiration, an effect that was blocked by inhibitors of the mitochondrial pyruvate carrier, nitric oxide synthase, or soluble guanylyl cyclase. In myoblasts, knockdown of components of the mitochondrial pyruvate carrier blocked EPI-induced respiratory stimulation. Conclusions: IV EPI confers cardioprotection via preservation of mitochondrial function potentially through enhanced substrate provision. These provocative results document a novel mechanism of a natural product with potential clinical utility.
    Full-text · Article · May 2014 · International Journal of Cardiology
  • Source
    • "These data suggest that mitochondrial oxidative stress plays a critical role in AngII-induced gap junction remodeling and arrhythmia. As mitochondrial ROS are increased in cardiac diseases such as cardiac hypertrophy [48], myocardial ischemia [50,210], and heart failure [51,52], conditions that are known to be associated with RAS activation, ventricular Cx43 downregulation, and increased risk of arrhythmias, it would be of great interest to test if treatment with mitochondria-targeted antioxidant can normalize Cx43 expression and prevent life-threatening arrhythmias in these pathological conditions. "
    [Show abstract] [Hide abstract] ABSTRACT: Mitochondria are essential to providing ATP thereby satisfying the energy demand of the incessant electrical activity and contractile action of cardiac muscle. Emerging evidence indicates that mitochondrial dysfunction can adversely impact cardiac electrical functioning by impairing the intracellular ion homeostasis and membrane excitability through reduced ATP production and excessive reactive oxidative species (ROS) generation, resulting in increased propensity to cardiac arrhythmias. In this review, the molecular mechanisms linking mitochondrial dysfunction to cardiac arrhythmias are discussed with an emphasis on the impact of increased mitochondrial ROS on the cardiac ion channels and transporters that are critical to maintaining normal electromechanical functioning of the cardiomyocytes. The potential of using mitochondria-targeted antioxidants as a novel anti-arrhythmia therapy is highlighted.
    Full-text · Article · Apr 2014 · Free Radical Biology and Medicine
Show more