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Biochimica et Biophysica Acta

Review

Mitochondrial dysfunction in cardiac ischemia–reperfusion injury:

ROS from complex I, without inhibition

Andrew J. Tompkins a, Lindsay S. Burwell b, Stanley B. Digerness c, Corinne Zaragoza c,

William L. Holman c, Paul S. Brookes a,*

a Department of Anesthesiology, Box 604, University of Rochester Medical Center, 601 Elmwood Avenue, Rochester, NY 14642, USAb Department of Biochemistry and Molecular Biology, University of Rochester Medical Center, Rochester, NY 14642, USA

c 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

Abstract

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 preconditioning). 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

[1–5].

It has been known for some time that mitochondrial

perturbations contribute to cardiac dysfunction in I–R injury

(reviewed in [6]). Most of the energetic (ATP) requirements of

0925-4439/$ - see front matter D 2005 Elsevier B.V. All rights reserved.

doi:10.1016/j.bbadis.2005.10.001

* Corresponding author. Tel.: +1 585 273 1626; fax: +1 585 273 2652.

E-mail address: paul_brookes@urmc.rochester.edu (P.S. Brookes).

the cardiomyocyte are met by mitochondria, and in addition the

organelle plays important roles in Ca2+ 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 [6,9–18]. 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 AM H2O2, 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 pumping 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-

1762 (2006) 223 – 231

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A.J. Tompkins et al. / Biochimica et Biophysica Acta 1762 (2006) 223–231224

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 generation [22,23], although this remains controversial

[24]. In contrast, others have attributed the decrease in CxI

activity following I–R to ROS-mediated oxidation of cardi-

olipin, the latter being critical for CxI function [10].

Consistently, several laboratories report that ROS or RNS

can inhibit CxI [10,19,25–31]. In addition, it has been shown

that CxI inhibition following I–R depends on the entry of

Ca2+ into the mitochondrial matrix [12]. Unifying both the

Ca2+ and ROS/RNS data, it has recently been shown that a

combination of Ca2+ and NOSis required to inhibit CxI in

vitro [25]. Such Ca2+ dependent CxI dysfunction in I–R may

proceed via the known ability of Ca2+ to enhance mitochon-

drial ROS generation [32,33], leading to secondary generation

of ONOO� from NOSand O2

S�. Alternatively, several studies

have suggested that 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 I–R injury both

as a source and target of ROS, we investigated the molecular

nature of the CxI lesion. Surprisingly, we found that 25 min

ischemia plus 30 min reperfusion did not inhibit 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 I–R 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 I–R

injury are discussed.

2. Materials and methods

Male Sprague–Dawley 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% O2, 5% CO2) 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% N2/5%

CO2). 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 O2 consumption was measured at 37 -C using a Clark-type

O2 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), KH2PO4 (5 mM), MgCl2 (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 N2, 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 Al ofBN-extraction buffer. After 30 min extraction on ice, samples were centrifuged

at 14,000�g for 5 min, and 200 Al of supernatant mixed with 12.5 Al of 5%Coomassie blue G in aminocaproate (0.5 M). Forty Al of sample (200 Agprotein) 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 AMmaleimide-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) 223–231 225

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 NH4HCO3 (100 mM), reduced with DTT,

alkylated with IAA, and washed with NH4HCO3 (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 RB (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 H2O2 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 hyper-contracture during

ischemia (arrow). Following reperfusion, diastolic pressure

Fig. 1. Langendorff functional data. (A) Typical left ventricular pressure trace (mm

ischemia and reperfusion respectively, and the arrow indicates ischemic hypercontrac

bars) and I–R (black bars), both pre- and post- the perfusion protocol. (C) Diastolic s

are meansTS.E.M. from 7 independent experiments. *P <0.01 between control per

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)-linked state 3 respiration

has previously been reported in mitochondria isolated from

hearts subject to I–R [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 I–R samples

(16.8T1.3 vs. I–R 16.4T1.7 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 I–R 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

[9–13]. Such a variance may originate from differences in the

models of I–R 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 I–R injury led to a significant decrease CxI activity in

Hg) of I–R heart, with x axis compressed. (a) and (b) denote the onset of

ture. (B) Left ventricular developed pressure (LVDP) in control perfusion (white

tiffness constant in control perfusion (white bars) and I–R (black bars). All data

fusion and I–R groups.

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 meansTS.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

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 298T27 vs. I–R 363T16 nmol NADH/min/unit CS).

Notably, none of the previous studies of CxI in I–R

normalized CxI activity to CS, although it is unlikely 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–Q1 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� flux.

A lack of knowledge regarding the precise electron flux

pathways within mammalian CxI currently precludes a full

explanation.

Since it was found that CxI-linked respiration was

depressed following I–R (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 situation where no enzymatic inhibition was found, the

A.J. Tompkins et al. / Biochimica et Biophysica Acta 1762 (2006) 223–231 227

proteomic examination of CxI was performed concurrently

with the enzymatic studies. Thus, the proteomic 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 I–R

hearts (Fig. 3B), despite over-exposure of the blot membrane

(>24 h ECL development, vs. 20 min for the ONOO�-treated

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 detection of nitrotyrosine (e.g., epitope

Fig. 3. Complex I modification by nitrotyrosine. (A) Nitrotyrosine Western blot

of rat heart mitochondria treated with a single bolus addition of 250 AMONOO�. 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.

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 primary mechanism of enzymatic

inhibition. The other reactivities of ONOO� (including but not

limited to oxidation of thiols, tryptophan nitration, or protein

oxidation via secondary OHI 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

I–R samples, 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 diminished 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 modifications.

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/basic

nature and thus incompatibility with IEF. Furthermore, the

separation revealed that fewer CxI subunits were present in the

I–R 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 I–R

Complex I has previously been reported as a site of

mitochondrial ROS generation [22–24,33], 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. 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

experiments.

Table 1

MALDI-TOF identification of thiol-modified proteins in CxI

Sample # Protein name Mol.

Wt (kDa)

Accession # MOWSE

score

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 (http://www.matrixscience.com), 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

database.

A.J. Tompkins et al. / Biochimica et Biophysica Acta 1762 (2006) 223–231228

generation. This appears to be the case, since the data in Fig.

5B show that mitochondria from I–R hearts 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

condition.

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, whereby Dz

opens mitochondrial K+ATP channels, possibly preventing

mitochondrial Ca2+ overload, and Cp inhibits NHE-1, prevent-

ing cytosolic Na+ overload which may subsequently prevent

Ca2+ overload [34,35,47]. As shown in Fig. 6A, administration

of Dz/Cp to hearts elicited a substantial improvement in post-

I–R functional recovery. In addition, this recovery was

associated with prevention of CxI thiol modifications (Fig. 6B).

4. Discussion

The main 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 respiration. (ii)

Tyrosine nitration does not occur at CxI in I–R. (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 I–R

injury and associated pathologies [6,9–18], with most of these

studies concentrating on respiratory complexes I–V. 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 [19,25–32,41–44]. 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 I–R are poorly

understood. This is especially true for CxI, since it contains at

least 46 different subunits, and its enzymatic mechanism is still

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 meansTS.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

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 mitochondria

isolated from I–R hearts 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

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 groups. Data are meansTS.E.M. from 3 independent

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.

damage during I–R 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 previous 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 I–R

injury. Most notably, age appears to be a critical factor 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 I–R 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 I–R mitochondria (Fig. 3B),

suggesting that CxI nitration is not significant in I–R.

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 I–R injury. The

A.J. Tompkins et al. / Biochimica et Biophysica Acta 1762 (2006) 223–231230

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

I–R 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.

Secondly, even if Dw was diminished significantly, the

Nernstian accumulation of IBTP would not drastically affect

its intra-mitochondrial level. For example 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-complex 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, I–R mito-

chondria generated significantly 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 I–R [6].

Comparison of CxI activity (nmols NADH/min) with rates

of ROS generation (pmols H2O2/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 I–R. 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, would 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

system.

In summary, the current investigation further implicates

mitochondrial ROS generation and ROS-mediated post-trans-

lational modifications in the pathology of I–R, 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.

Acknowledgements

We thank Michael Murphy (Cambridge, UK) for the kind

gift of IBTP and antibodies against it. We also thank Victor

Darley-Usmar (Birmingham AL), Shey-Shing Sheu and Jim

Robotham (Rochester NY) for invaluable discussions on this

topic.

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