Am J Physiol Cell Physiol Watch the video to learn how APS reaches out to developing nations.
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

Am J Physiol Cell Physiol 294: C460-C466, 2008. First published December 12, 2007; doi:10.1152/ajpcell.00211.2007
0363-6143/08 $8.00
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
294/2/C460    most recent
00211.2007v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Web of Science (9)
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Chen, Q.
Right arrow Articles by Lesnefsky, E. J.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Chen, Q.
Right arrow Articles by Lesnefsky, E. J.

CELLULAR AND MITOCHONDRIAL METABOLISM

Ischemic defects in the electron transport chain increase the production of reactive oxygen species from isolated rat heart mitochondria

Qun Chen,1 Shadi Moghaddas,1 Charles L. Hoppel,2,3 and Edward J. Lesnefsky1,4

1Divisions of Cardiology and 2Clinical Pharmacology, Department of Medicine, 3Department of Pharmacology, School of Medicine, Case Western Reserve University, and 4Medical Service, Louis Stokes Department of Veterans Affairs Medical Center, Cleveland, Ohio

Submitted 22 May 2007 ; accepted in final form 10 December 2007


   ABSTRACT
TOP
 ABSTRACT
METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Cardiac ischemia decreases complex III activity, cytochrome c content, and respiration through cytochrome oxidase in subsarcolemmal mitochondria (SSM) and interfibrillar mitochondria (IFM). The reversible blockade of electron transport with amobarbital during ischemia protects mitochondrial respiration and decreases myocardial injury during reperfusion. These findings support that mitochondrial damage occurs during ischemia and contributes to myocardial injury during reperfusion. The current study addressed whether ischemic damage to the electron transport chain (ETC) increased the net production of reactive oxygen species (ROS) from mitochondria. SSM and IFM were isolated from 6-mo-old Fisher 344 rat hearts following 25 min global ischemia or following 40 min of perfusion alone as controls. H2O2 release from SSM and IFM was measured using the amplex red assay. With glutamate as a complex I substrate, the net production of H2O2 was increased by 178 ± 14% and 179 ± 17% in SSM and IFM (n = 9), respectively, following ischemia compared with controls (n = 8). With succinate as substrate in the presence of rotenone, H2O2 increased by 272 ± 22% and 171 ± 21% in SSM and IFM, respectively, after ischemia. Inhibitors of electron transport were used to assess maximal ROS production. Inhibition of complex I with rotenone increased H2O2 production by 179 ± 24% and 155 ± 14% in SSM and IFM, respectively, following ischemia. Ischemia also increased the antimycin A-stimulated production of H2O2 from complex III. Thus ischemic damage to the ETC increased both the capacity and the net production of H2O2 from complex I and complex III and sets the stage for an increase in ROS production during reperfusion as a mechanism of cardiac injury.

nicotinamide adenine dinucleotide:ubiquinone oxidoreductase (complex I), ubiquinol:cytochrome c oxidoreductase (complex III), cytochrome c; ischemia

MITOCHONDRIAL DYSFUNCTION contributes to myocardial injury during ischemia and reperfusion (11, 35). Cardiac ischemia results in damage to the mitochondrial electron transport chain (ETC) (33, 36, 49). In the isolated, buffer-perfused rat heart, 25 min of global ischemia damages the distal ETC, with decreases in complex III activity, cytochrome c content, and respiration through cytochrome oxidase in both subsarcolemmal (SSM) and interfibrillar mitochondria (IFM) (12, 33). The blockade of mitochondrial respiration at complex I immediately before ischemia with the irreversible inhibitor rotenone (31) or the reversible inhibitor amobarbital (12) protects the distal ETC against ischemic damage. Protection of the ETC during ischemia by the reversible blockade of electron transport with amobarbital markedly decreases myocardial injury measured following reperfusion, supporting the premise that ischemic damage to mitochondria is a key factor in myocardial injury (1, 14). The current study addressed the question if ischemic damage to the ETC was sufficient to increase mitochondrial production and release of reactive oxygen species (ROS). If so, then ischemic damage to the ETC is likely to be a significant source of the ROS produced during reperfusion that can lead to opening of the mitochondrial permeability transition pore (58) and myocyte cell death (10).

The ETC is a major source of ROS, and complex III is the dominant site for production, in the baseline state (15, 22). Inhibition of complex I with rotenone or amobarbital decreases the ROS generation during ischemia in cultured myocytes (7) and in the intact isolated heart (1), suggesting that complex III remains the major site for ROS generation during ischemia. In contrast, inhibition of electron transport at cytochrome oxidase will lead to the accumulation of reducing equivalents at upstream complexes and enhance ROS generation from complex I (13). During ischemia, the internal myocyte milieu becomes altered with hypoxia (7), increased acidification (3), and calcium loading (27), all of which may increase ROS generation in situ in the absence of actual damage to the ETC. Thus, in the present study, we used mitochondria isolated from control and ischemic hearts assessed in the presence of oxygen, physiological pH, and low calcium concentration to test: 1) whether ischemic damage of the ETC alone is sufficient to increase ROS generation and 2) whether the sites of ROS generation in the ETC following ischemia correspond to the sites of ischemic damage.


   METHODS
TOP
 ABSTRACT
 METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Preparation of rat hearts for perfusion. The Animal Care and Use Committees of the Louis Stokes Cleveland Department of Veterans Affairs Medical Center and Case Western Reserve University approved the protocol. Male Fisher rats (6–8 mo of age) were anesthetized with pentobarbital sodium (100 mg/kg ip) and anticoagulated with heparin (1,000 IU/kg ip). Hearts were excised and perfused retrograde via the aorta with modified Krebs-Henseleit (K-H) buffer oxygenated with 95% O2-5% CO2 as previously described (12). Left ventricular developed pressure (LVDP) was measured with a balloon inserted into the left ventricle. Isolated rat hearts initially were perfused for 15 min followed by 25 min global ischemia at 37°C. Hearts in the time control group were perfused for 40 min. The LVDP at 40 min perfusion was maintained at 101 ± 8% of the LVDP at 15 min perfusion (n = 8). Ischemia resulted in cessation of contraction within 5 min and increased diastolic pressure [9 ± 1 mmHg at 15 min equilibration vs. 56 ± 4 mmHg at 25 min ischemia (n = 9) P < 0.01].

Isolation of SSM and IFM mitochondria. At the end of the experiment, hearts were removed from the cannula and placed into buffer A (in mM: 100 KCl, 50 MOPS, 1 EGTA, 5 MgSO4·7H2O, and 1 ATP; pH 7.4) at 4°C. Cardiac mitochondria were isolated using the procedure of Palmer et al. (43) except that trypsin was used as the protease (12, 38). Cardiac tissue was finely minced and placed in buffer A containing 0.2% bovine serum album and homogenized with a polytron tissue processor (Brinkman Instruments, Westbury, NY) for 2.5 s at a rheostat setting of 6.0. The polytron homogenate was centrifuged at 500 g, the supernatant was saved for isolation of SSM, and the pellet was washed. The combined supernatants were centrifuged at 3,000 g to sediment SSM. IFM were isolated by incubation of skinned myofibers, obtained following polytron treatment, with 5 mg/g (wet wt) trypsin for 10 min at 4°C. SSM and IFM were washed twice and then suspended in 100 mM KCl, 50 mM MOPS, and 0.5 mM EGTA. Mitochondrial protein concentration was measured by the Lowry method by using bovine serum album as a standard.

Mitochondrial oxidative phosphorylation. Oxygen consumption by mitochondria was measured using a Clark-type oxygen electrode at 30°C (28, 36). Mitochondria were incubated in 80 mM KCl, 50 mM MOPS, 1 mM EGTA, 5 mM KH2PO4, and 1 mg defatted, dialyzed BSA/ml at pH 7.4. Glutamate (complex I substrate, 20 mM) and succinate (20 mM) plus rotenone (5 µM) (complex II substrate) were used and state 3 (0.2 mM ADP-stimulated), state 4 (ADP-limited) respiration, respiratory control ratio, rate of uncoupled respiration (0.2 mM dinitrophenol), maximal rate of state 3 respiration (2 mM ADP), and the ADP/O ratio were determined. Mitochondria were used within 4 h after isolation from tissue. Endogenous substrates were depleted by addition of 0.1 mM ADP when glutamate was the substrate.

ETC and citrate synthase enzyme activities. The following enzyme activities were measured in detergent-solubilized SSM and IFM using previously described methods (24, 28): NADH-cytochrome c reductase, rotenone sensitive; NADH ferricyanide oxidoreductase (NFR), NADH-decylubiquinone reductase, rotenone sensitive (complex I) and citrate synthase (CS). Enzyme activities were not measured in one heart in the ischemia group due to consumption of the sample by the measurement of other end points.

Measurement of aconitase activity. The coupled aconitase-isocitrate dehydrogenase assay was used (24, 48). The reaction components were 50 mM Tris·HCl (pH 8.1), 1 mM sodium citrate, 1 mM MnCl2, 1 mM NADP+, isocitrate dehydrogenase (1.34 U/ml), and SSM-IFM (50 µg/ml). The production of NADPH was detected by the change in absorbance at 340 nm for 10 min at 37°C.

Detection of H2O2 production. H2O2 production from intact mitochondria was measured using the oxidation of the fluorogenic indicator amplex red in the presence of horseradish peroxidase (15). Glutamate and succinate were used as complex I and complex II substrates, and the concentration of substrates is the same as that used to measure oxidative phosphorylation. Decylubiquinone could not be used as a substrate for complex III due to unacceptable background fluorescence. Cyclosporine A (1 µM) was added in select assays (45).

Measurement of cytochrome content. Cytochrome contents were determined in mitochondria solubilized in 2% deoxycholate in 10 mM sodium phosphate buffer using the difference of sodium dithionite reduced and air-oxidized spectra (34, 59). Cytochrome content was not determined in one heart in ischemia group due to use of the entire sample for other assays.

Statistical analysis. Data are expressed as means ± SE. Differences among groups were compared by two-tailed Student's t-test (Sigmastat 3.5). A difference of P < 0.05 was considered significant.


   RESULTS
TOP
 ABSTRACT
 METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Mitochondrial oxidative phosphorylation. Ischemia markedly decreased the rate of state 3 and increased the rate of state 4 respiration with glutamate substrate compared with mitochondria isolated from time controls (Table 1). Ischemia also decreased succinate-stimulated respiration in both SSM and IFM (Table 1). The ADP/O ratio was not changed by ischemia. Ischemia caused cytochrome c loss from both SSM and IFM. The contents of cytochrome c (in nmol/mg protein) in SSM and IFM after ischemia were 0.14 ± 0.01 (n = 8) and 0.19 ± 0.02 (n = 8), respectively, and were markedly decreased compared with time control SSM (0.27 ± 0.03, n = 8, P < 0.01) and IFM (0.36 ± 0.06, n = 8, P < 0.01). Ischemia did not change the contents of cytochrome c1, b, and aa3 (data not shown). These findings are consistent with the results of previous studies using this model (12, 34).


View this table:
[in this window]
[in a new window]

  		Table 1. Rate of oxidative phosphorylation in subsarcolemmal mitochondria and interfibrillar mitochondria in the presence and absence of ischemia using glutamate (complex I) and succinate (complex II) as substrates

 
ETC enzyme activities. Ischemia decreased complex I activity (NADH:duroquinone reductase) in both SSM and IFM (Table 2). The decreased activity of NADH:cytochrome c reductase (NCR, rotenone sensitive) (Table 2) also largely reflects decreased complex I activity since complex I is the rate-limiting step in this assay (23, 24, 28). NFR reflects the activity of NADH dehydrogenase, the first component of complex I (41, 49). Ischemia did not decrease NFR activity in either SSM or IFM, indicating that the site of ischemic damage to complex I is located distal to the NADH dehydrogenase (Table 2). Decreased complex I activity has been observed in SSM and IFM (14) and in mixed population of mitochondria following ischemia and reperfusion (44, 49, 57). The activity of citrate synthase, a mitochondrial matrix marker enzyme that is unaltered by ischemia (33, 36), was similar in controls and following ischemia in both SSM and IFM (Table 2).


View this table:
[in this window]
[in a new window]

  		Table 2. Complex I-related enzyme activities in subsarcolemmal mitochondria and interfibrillar mitochondria isolated from hearts in the presence or absence of ischemia

 
Production of H2O2 in SSM and IFM isolated at end of ischemia. Since horseradish peroxidase and amplex red do not enter intact mitochondria, only H2O2 that is released from mitochondria (net release of H2O2, pmol·mg–1·30 min–1) is detected by this assay (15). Ischemia increased the net release of H2O2 from both SSM and IFM with glutamate as a substrate in the absence of respiratory inhibitors (Fig. 1). Cyclosporin A, an inhibitor of the opening of the mitochondrial permeability transition pore, did not alter H2O2 generation in SSM and IFM either with or without ischemia (data not shown). When succinate, an electron donor to complex II, was used as substrate, electrons can go either from complex II to complex I (reverse flow) (37) or from complex II to complex III (forward flow). In the presence of rotenone blockade of complex I, reverse flow cannot occur. In mammalian heart mitochondria, complex II appears not to be a site of ROS generation (5). During succinate oxidation in the presence of rotenone, H2O2 is generated from complex III (15, 21, 22). Oxidizing succinate in the presence of rotenone, the amount of H2O2 generated by SSM and IFM following ischemia was much higher compared with control SSM and IFM, indicating that ischemia increases H2O2 generation from complex III (Fig. 1). CSA did not affect the rate of H2O2 generation in SSM and IFM with or without ischemia (data not shown).


Figure 1
View larger version (13K):
[in this window]
[in a new window]

  		Fig. 1. H2O2 generation in subsacrcolemmal mitochondria (SSM) and interfibrillar mitochonrdia (IFM) following ischemia. Ischemia markedly increases the net production of H2O2 from both SSM and IFM with glutamate as a complex I substrate (A) and with succinate plus rotenone as a complex II substrate (B). Data are means ± SE. *P < 0.05 vs. nonischemic time control (n = 8); 25 min ischemia (n = 9).

 
Ischemia also increased net H2O2 generation from SSM and IFM in the presence of rotenone (complex I inhibitor) when compared with that of time controls (Fig. 2), indicating that ischemic damage increases the capacity for the maximal net release of H2O2 from complex I. There was no difference in antimycin A-enhanced H2O2 generation between SSM and IFM from time control (3,025 ± 142 SSM, 3,277 ± 243 IFM, n = 8) and ischemic hearts (2,539 ± 221 SSM, 3,447 ± 235 IFM, n = 9) with glutamate as the substrate. Ischemia increased the antimycin A-stimulated H2O2 generation from SSM with succinate plus rotenone (Fig. 2), and a similar trend to increased antimycin A stimulated H2O2 production was evident in IFM (Fig. 2). Thus ischemic damage also increases capability of complex III to generate ROS.


Figure 2
View larger version (13K):
[in this window]
[in a new window]

  		Fig. 2. Chemical inhibition of complex I and complex III increases H2O2 generation from SSM and IFM following ischemia. Rotenone inhibition increases net H2O2 generation from both SSM and IFM after ischemia with glutamate as substrate, indicating that ischemia increases the capacity for H2O2 generation from complex I (A). In the presence of succinate plus rotenone, antimycin A increases the production of H2O2 in SSM, indicating that ischemia increases the capacity for H2O2 generation from complex III (B). Data are means ± SE. *P < 0.05 ischemia (n = 9) vs. time control (n = 8). IFM in B also exhibits a trend for increased production, but it did not reach the statistical significance.

 
The oxidation of succinate in the absence of rotenone can generate H2O2 from either complex I or complex III. With succinate as the substrate in the absence of rotenone, ischemia did not increase H2O2 generation [SSM: time control 1,023 ± 98 pmol·30 min–1·mg protein–1 (n = 8) vs. ischemia 900 ± 72 (n = 9), P = not significant (NS); IFM: time control 1,610 ± 184 (n = 8) vs. ischemia 1,111 ± 83 (n = 9), P < 0.05 for a decrease]. The rate of H2O2 generated at complex I by reverse flow can be estimated as the difference between production with succinate alone minus the rate of succinate with rotenone (37). Thus ischemia decreased the rate of H2O2 generation due to reverse flow into complex I (Fig. 3) while increasing the rate of production of H2O2 from complex III (Figs. 1 and 2).


Figure 3
View larger version (9K):
[in this window]
[in a new window]

  		Fig. 3. Ischemia decreases the production of reactive oxygen species due to reverse electron flow into complex I from complex II. The difference of net H2O2 production with succinate as the substrate in the presence of rotenone inhibition of complex I (Fig. 1) minus the rate of net H2O2 production with succinate in the absence of rotenone (text) is shown. Ischemia decreased the component of H2O2 production attributed to reverse electron flow from complex II into complex I. Data are means ± SE. *P < 0.05 ischemia (n = 9) vs. time control (n = 8).

 
Aconitase is an enzyme located in the mitochondrial matrix and is sensitive to inactivation by superoxide (25). Aconitase is used as an index of increased net superoxide generation directed into the mitochondrial matrix. Ischemia did not decrease the activity of aconitase in SSM and IFM following 25 min ischemia compared with nonischemic time controls [SSM: time control 101 ± 13 mU/mg protein (n = 3) vs. ischemia 93 ± 14 (n = 3), P = NS; IFM: time control 120 ± 12 (n = 3) vs. ischemia 142 ± 10 (n = 3), P = NS]. To confirm that the measurement of aconitase activity was a sensitive indicator of superoxide generation directed into the mitochondrial matrix, SSM from nonischemic time control hearts were incubated in the presence or absence of rotenone with glutamate as the substrate for 20 min at 37°C. In SSM without ischemic damage, rotenone inhibition of complex I increases superoxide generation directed toward the matrix (15, 22). Incubation in the presence of rotenone decreased aconitase activity by 61 ± 6% (n = 3, P < 0.05), confirming the sensitivity of the aconitase assay for matrix superoxide exposure. These results support that the increased H2O2 production from complex I following the onset of ischemic damage is directed toward the intermembrane space, rather than into the mitochondrial matrix, consistent with the results observed using H2O2 production.


   DISCUSSION
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
In the present study, we found that ischemic damage to the ETC increased the net production of H2O2 from complex I and complex III. Ischemia decreased complex I activity in both SSM and IFM, supporting that damage occurs to both the proximal ETC (current study) as well as the distal ETC (14, 33). Ischemia also increased the capacity for ROS production from both complex I and complex III. Since mitochondrial damage mainly occurs during ischemia (14, 32), the increased production and release of ROS following ischemic damage to the ETC observed in the current study sets the stage for the increased ROS generation during the reoxygenation of early reperfusion (6, 11, 40).

Ischemia decreased complex I activity without alternation of NFR. Ferricyanide accepts electrons from the flavin mononucleotide (FMN) cofactor of complex I (Fig. 4) (41). Preserved NADH dehydrogenase activity during ischemia localizes the site of damage distal to FMN (Fig. 4). However, in blocks of canine myocardium incubated in plastic bags at 37°C, both complex I activity and NFR decreased (49). The inconsistency between our study and this previous study may be due to the different model of ischemia used in that study, as well as the different species used.


Figure 4
View larger version (40K):
[in this window]
[in a new window]

  		Fig. 4. Depiction of the complexes of the electron transport chain and the potential sites for the production of reactive oxygen species. Complex I is NADH:ubiquinone oxidoreductase, and NADH dehydrogenase is the first component. When succinate was used as substrate, electrons can flow from complex II to complex I (reverse flow, solid dashed arrow) as well as forward from complex II to Q and to complex III (open arrow). Rotenone inhibits at quinone acceptor site of complex I. Antimycin A inhibits complex III at cytochrome b of the Qi site, the quinol binding site located on the matrix ("inside") aspect of the inner mitochondrial membrane. Complex I (FMN site, Fe-S N2 center) and the Qi site site of complex III release reactive oxygen species directed toward mitochondrial matrix. In contrast, the Qo site of complex III, the quinol binding site located on the outer aspect (cytosol side) of the inner mitochondrial membrane, releases reactive oxygen species toward the intermembrane space.

 
Complex I is one of the sites for ROS generation from the ETC, but the exact site for ROS generation within complex I remains uncertain (5, 13, 15, 20, 41). FMN in NADH dehydrogenase (29, 30), iron sulfur cluster N2, and the two tightly bound ubiquinone located distal in the path of electron flow through complex I (20, 41) are all potential sites for ROS generation. In the baseline state, the inhibition of NADH dehydrogenase decreases ROS generation from complex I, indicating that the site for ROS generation in complex I is distal to FMN center (41, 50), though other investigators previously assigned the production of ROS to NADH dehydrogenase (42, 56). In the present study, ischemia increased ROS generation from complex I with unaltered NADH dehydrogenase activity, localizing the site for ROS generation distal to FMN (20, 41, 50). Rotenone blocks electron transport at the ubiquinone acceptor site (9). Rotenone inhibition increased ROS generation from complex I in the mitochondria from ischemic hearts, bracketing the site for ROS generation proximal to the ubiquinone binding site (41) (Figs. 2 and 4). These observations suggest that the N2 iron-sulfur site or the tightly bound quinones within complex I are the loci for production of ROS following ischemia. In contrast, when succinate was the substrate, ischemia decreased ROS generated by reverse electron flow (Figs. 3 and 4). This finding suggests that the site of ischemic damage is close to the distal portion of complex I because it likely limits reverse electron flow into complex I. Taken together, ischemia appears to have decreased complex I activity most likely by altering the portion of the complex that contains the two tightly bound quinones (Fig. 4). There are two tightly bound quinones in complex I:, Qnf (fast relaxing ubisemiquinone) and Qns (slow relaxing ubisemiquinone)(41). Based on the use of forward and reverse electron flow to localize the defect within complex I (41), the site of net H2O2 production from complex I following ischemia appears likely to lie within the iron-sulfur clusters of the complex, immediately proximal to the likely site of ischemic damage at the tightly bound quinones. Further experimental studies are needed to challenge this proposal.

Complex III is the dominant site for the net production of ROS from intact mitochondria in the baseline state (15, 21, 22, 39, 51). The increased production of H2O2 in mitochondria following ischemia with succinate as the substrate in the presence of rotenone indicates that complex III remains a key site for ROS generation during ischemia. Most of the ROS generated at complex III are from the Qo center (quinol binding and oxidation site located on the cytosolic aspect of the inner mitochondrial membrane) (Fig. 4), whereas the Qi center (quinol binding site located on the matrix aspect of the inner mitochondrial membrane) also can be a site for ROS generation, especially when electron flow into the Qo center of complex III is limited (41, 47). The previously reported ischemia-induced dysfunction of the iron-sulfur center in complex III (33) coupled with the loss of a portion of the cytochrome c (12, 33) could limit electron flow into complex III and potentially decrease ROS release from complex III (13). If this were the case, ischemia should decrease antimycin A-stimulated H2O2 generation (13, 55), which was not observed. Ischemia increased antimycin A-stimulated H2O2 production in SSM in the presence of succinate plus rotenone. Thus ischemic damage to complex III allows adequate residual electron flow into the complex for ROS production.

Complex III exists as a dimer. Electrons communicate between the b hemes located at the Qi centers of each monomer by electron tunneling (16) that favors the production of ROS from these centers (47, 54). The inactivation of a portion of the Rieske iron-sulfur centers of complex III that occurs during ischemia (33) would be expected, on a statistical basis, to inactivate one Qo center in many complex III dimers. The loss of cytochrome c further limits electron flow into the Qo center (30). The combined effects of inactivation of a portion of the Qo site iron-sulfur peptides coupled with the partial loss of cytochrome c is expected to block a reasonable portion of Qo sites, consistent with the decrease in duroquinol-stimulated respiration previously observed following ischemia (33). Inactivation of a portion of the Qo sites may facilitate ROS generation from complex III Qi centers, as shown in studies in yeast (16).

An increase in ROS production from complex I also can occur due to blockade of the distal ETC in the absence of complex I damage (13, 15, 55) due to increased relative reduction and electron leak from complex I (15, 41, 55). The decrease in complex III activity and cytochrome c loss with ischemia will increase ROS production from upstream complex I (13), augmenting the impact of direct ischemic damage to complex I.

In intact mitochondria, complexes I and III produce superoxide and release it to both matrix and intermembrane space aspects of the inner membrane (15, 51, 55). In the baseline state, chemical inhibition of complex I with rotenone did not increase net ROS generation with a complex I substrate, supporting superoxide production directed toward the matrix (15, 47). In the current study, rotenone inhibition increased net ROS release from complex I after ischemia, suggesting a shift in the site for ROS generation to the N2/quinol site that now favors release toward the intermembrane space. The lack of a decrease in aconitase activity during in situ ischemia also favors ROS production oriented away from the mitochondrial matrix.

Superoxide generated in the matrix cannot cross the inner membrane unless an ion channel is opened (22, 41). The mitochondrial permeability transition pore (MPTP) opening is a well-known mechanism to induce leakage across both the inner and outer membranes (17, 18). Ischemia and reperfusion increase the probability of MPTP opening (58). However, in the isolated mitochondria studies, results do not support MPTP as a mechanism for superoxide egress since mitochondria are isolated in the presence of EGTA and ATP, which favor MPTP closure, and cyclosporin A did not block the increased H2O2 production from mitochondria after ischemia. Superoxide may traverse the inner membrane via the inner membrane anion channel (IMAC) (4, 8). Unfortunately 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid disodium salt hydrate (DIDS), which blocks the IMAC channel (4), quenched fluorescence in our system and could not be used to evaluate the contribution of IMAC. Ischemia does not substantially inactivate glutaredoxin or glutathione peroxidase in isolated SSM and IFM (53). The content of manganese superoxide dismutase in the mitochondria is unaltered in mouse heart mitochondria following ischemia and reperfusion (26). Thus,changes in matrix antioxidant capacity during ischemia are an unlikely explanation for the observed increases in the net production of H2O2 after ischemia.

In the isolated heart model, superoxide production occurs during ischemia (1, 27). Perfusion of amobarbital, a complex I inhibitor that blocks electron flux into complex III (12, 14), decreases superoxide generation during ischemia (1), suggesting that ROS were generated by the ETC and that complex III is the dominant site. Blockade of electron transport during ischemia protects mitochondria against damage, supporting that the ETC itself contributes to the genesis of ischemic mitochondrial damage (12, 31). Ischemic damage increases ROS generation from both complex I and complex III as shown in the present study, in turn providing a mechanism for increased ROS production with the reoxygenation of early reperfusion. In support of this mechanism, reperfusion of myocardium when mitochondria are protected from ischemic damage to the ETC (12) markedly decreased H2O2 production and substantially limited infarct size (14). Approaches to modulate mitochondrial metabolism during reperfusion that include postconditioning (19, 52, 60), direct chemical inhibition of respiration (2), and hypoxic reperfusion (46) minimize myocardial injury following ischemia and reperfusion, likely by decreasing ROS production from mitochondria that were damaged by the antecedent ischemia.


   GRANTS
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
This work was supported by the National Institutes of Health (2PO1AG-15885), by the American Heart Association (0425307B), and by the Office of Research and Development, Medical Research Service, Department of Veterans Affairs.


   ACKNOWLEDGMENTS
 
The technical assistance of Edwin Vasquez, Sarah Stewart, and Dr. William Parland is appreciated.


   FOOTNOTES
 

Address for reprint requests and other correspondence: E. J. Lesnefsky, Cardiology Section, Medical Service 111(W), Louis Stokes VA Medical Center, 10701 East Blvd., Cleveland, OH 44106 (e-mail: EXL9{at}cwru.edu)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


   REFERENCES
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
1. Aldakkak M, Stowe DF, Chen Q, Lesnefsky EJ, Camara AK. Inhibited mitochondrial respiration by amobarbital during cardiac ischemia improves redox state and reduces matrix Ca2+ overload and ROS release. Cardiovasc Res, 2007. Aug 23, epub.

2. Ambrosio G, Zweier JL, Duilio C, Kuppusamy P, Santoro G, Elia PP, Tritto I, Cirillo P, Condorelli M, and Chiariello M. Evidence that mitochondrial respiration is a source of potentially toxic oxygen free radicals in intact rabbit hearts subjected to ischemia and reflow. J Biol Chem 268: 18532–18541, 1993.[Abstract/Free Full Text]

3. An J, Varadarajan SG, Camara A, Chen Q, Novalija E, Gross GJ, Stowe DF. Blocking Na+/H+ exchange reduces [Na+]i and [Ca2+]i load after ischemia and improves function in intact hearts. Am J Physiol Heart Circ Physiol 281: H2398–H2409, 2001.[Abstract/Free Full Text]

4. Aon MA, Cortassa S, Marban E, O'Rourke B. Synchronized whole cell oscillations in mitochondrial metabolism triggered by a local release of reactive oxygen species in cardiac myocytes. J Biol Chem 278: 44735–44744, 2003.[Abstract/Free Full Text]

5. Barja G. Mitochondrial free radical production and aging in mammals and birds. Ann NY Acad Sci 854: 224–238, 1998.[CrossRef][Web of Science][Medline]

6. Becker LB. New concepts in reactive oxygen species and cardiovascular reperfusion physiology. Cardiovasc Res 61: 461–470, 2004.[Abstract/Free Full Text]

7. Becker LB, vanden Hoek TL, Shao ZH, Li CQ, Schumacker PT. Generation of superoxide in cardiomyocytes during ischemia before reperfusion. Am J Physiol Heart Circ Physiol 277: H2240–H2246, 1999.[Abstract/Free Full Text]

8. Brady NR, Hamacher-Brady A, Westerhoff HV, Gottlieb RA. A wave of reactive oxygen species (ROS)-induced ROS release in a sea of excitable mitochondria. Antioxid Redox Signal 8: 1651–1665, 2006.[CrossRef][Web of Science][Medline]

9. Chance B, Williams GR, Hollunger G. Inhibition of electron and energy transfer in mitochondria. I. Effects of Amytal, thiopental, rotenone, progesterone, and methylene glycol. J Biol Chem 238: 418–431, 1963.[Free Full Text]

10. Chen J, Henderson GI, Freeman GL. Role of 4-hydroxynonenal in modification of cytochrome c oxidase in ischemia/reperfused rat heart. J Mol Cell Cardiol 33: 1919–1927, 2001.[CrossRef][Web of Science][Medline]

11. Chen Q, Camara AK, Stowe DF, Hoppel CL, Lesnefsky EJ. Modulation of electron transport protects cardiac mitochondria and decreases myocardial injury during ischemia and reperfusion. Am J Physiol Cell Physiol 292: C137–C147, 2007.[Abstract/Free Full Text]

12. Chen Q, Hoppel CL, Lesnefsky EJ. Blockade of electron transport before cardiac ischemia with the reversible inhibitor amobarbital protects rat heart mitochondria. J Pharmacol Exp Ther 316: 200–207, 2006.[Abstract/Free Full Text]

13. Chen Q, Lesnefsky EJ. Depletion of cardiolipin and cytochrome c during ischemia increases hydrogen peroxide production from the electron transport chain. Free Radic Biol Med 40: 976–982, 2006.[CrossRef][Web of Science][Medline]

14. Chen Q, Moghaddas S, Hoppel CL, Lesnefsky EJ. Reversible blockade of electron transport during ischemia protects mitochondria and decreases myocardial injury following reperfusion. J Pharmacol Exp Ther 319: 1405–1412, 2006.[Abstract/Free Full Text]

15. Chen Q, Vazquez EJ, Moghaddas S, Hoppel CL, Lesnefsky EJ. Production of reactive oxygen species by mitochondria: Central role of complex III. J Biol Chem 278: 36027–36031, 2003.[Abstract/Free Full Text]

16. Covian R, Trumpower BL. Regulatory interactions between ubiquinol oxidation and ubiquinone reduction sites in the dimeric cytochrome bc1 complex. J Biol Chem 281: 30925–30932, 2006.[Abstract/Free Full Text]

17. Di Lisa F, Canton M, Menabo R, Dodoni G, Bernardi P. Mitochondria and reperfusion injury. The role of permeability transition. Basic Res Cardiol 98: 235–241, 2003.[Web of Science][Medline]

18. Di Lisa F, Menabo R, Canton M, Barile M, Bernardi P. Opening of the mitochondrial permeability transition pore causes depletion of mitochondrial and cytosolic NAD+ and is a causative event in the death of myocytes in postischemic reperfusion of the heart. J Biol Chem 276: 2571–2575, 2001.[Abstract/Free Full Text]

19. Gateau-Roesch O, Argaud L, Ovize M. Mitochondrial permeability transition pore and postconditioning. Cardiovasc Res 70: 264–273, 2006.[Abstract/Free Full Text]

20. Genova ML, Ventura B, Giuliano G, Bovina C, Formiggini G, Parenti Castelli G, Lenaz G. The site of production of superoxide radical in mitochondrial Complex I is not a bound ubisemiquinone but presumably iron-sulfur cluster N2. FEBS Lett 505: 364–368, 2001.[CrossRef][Web of Science][Medline]

21. Gille L, Nohl H. The ubiquinol/bc1 redox couple regulates mitochondrial oxygen radical formation. Arch Biochem Biophys 388: 34–38, 2001.[CrossRef][Web of Science][Medline]

22. Han D, Antunes F, Canali R, Rettori D, Cadenas E. Voltage-dependent anion channels control the release of the superoxide anion from mitochondria to cytosol. J Biol Chem 278: 5557–5563, 2003.[Abstract/Free Full Text]

23. Hatefi Y, Galante YM, Stiggall DL, Ragan CI. Proteins, polypeptides, prosthetic groups, and enzymic properties of complexes I, II, III, IV, and V of the mitochondrial oxidative phosphorylation system. Methods Enzymol 56: 577–602, 1979.[Medline]

24. Hoppel CL, Kerr DS, Dahms B, Roessmann U. Deficiency of the reduced nicotinamide adenine dinucleotide dehydrogenase component of complex I of mitochondrial electron transport. Fatal infantile lactic acidosis and hypermetabolism with skeletal-cardiac myopathy and encephalopathy. J Clin Invest 80: 71–77, 1987.[Web of Science][Medline]

25. Inarrea P, Moini H, Han D, Rettori D, Aguilo I, Alava MA, Iturralde M, Cadenas E. Mitochondrial respiratory chain and thioredoxin reductase regulate intermembrane Cu,Zn-superoxide dismutase activity: implications for mitochondrial energy metabolism and apoptosis. Biochem J 405: 173–179, 2007.[Web of Science][Medline]

26. Jin ZQ, Zhou HZ, Cecchini G, Gray MO, Karliner JS. MnSOD in mouse heart: acute responses to ischemic preconditioning and ischemia-reperfusion injury. Am J Physiol Heart Circ Physiol 288: H2986–H2994, 2005.[Abstract/Free Full Text]

27. Kevin LG, Camara AK, Riess ML, Novalija E, Stowe DF. Ischemic preconditioning alters real-time measure of O2 radicals in intact hearts with ischemia and reperfusion. Am J Physiol Heart Circ Physiol 284: H566–H574, 2003.[Abstract/Free Full Text]

28. Krahenbuhl S, Chang M, Brass EP, Hoppel CL. Decreased activities of ubiquinol:ferricytochrome c oxidoreductase (complex III) and ferrocytochrome c:oxygen oxidoreductase (complex IV) in liver mitochondria from rats with hydroxycobalamin[c-lactam]-induced methylmalonic aciduria. J Biol Chem 266: 20998–21003, 1991.[Abstract/Free Full Text]

29. Kudin AP, Bimpong-Buta NY, Vielhaber S, Elger CE, Kunz WS. Characterization of superoxide-producing sites in isolated brain mitochondria. J Biol Chem 279: 4127–4135, 2004.[Abstract/Free Full Text]

30. Kushnareva Y, Murphy AN, Andreyev A. Complex I-mediated reactive oxygen species generation: modulation by cytochrome c and NAD(P)+ oxidation-reduction state. Biochem J 368: 545–553, 2002.[CrossRef][Web of Science][Medline]

31. Lesnefsky EJ, Chen Q, Moghaddas S, Hassan MO, Tandler B, Hoppel CL. Blockade of electron transport during Ischemia protects cardiac mitochondria. J Biol Chem 279: 47961–47967, 2004.[Abstract/Free Full Text]

32. Lesnefsky EJ, Chen Q, Slabe TJ, Stoll MS, Minkler PE, Hassan MO, Tandler B, Hoppel CL. Ischemia, rather than reperfusion, inhibits respiration through cytochrome oxidase in the isolated, perfused rabbit heart: role of cardiolipin. Am J Physiol Heart Circ Physiol 287: H258–H267, 2004.[Abstract/Free Full Text]

33. Lesnefsky EJ, Gudz TI, Migita CT, Ikeda-Saito M, Hassan MO, Turkaly PJ, Hoppel CL. 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: 117–128, 2001.[CrossRef][Web of Science][Medline]

34. Lesnefsky EJ, Gudz TI, Moghaddas S, Migita CT, Ikeda_Saito M, Turkaly PJ, Hoppel CL. Aging decreases electron transport complex III activity in heart interfibrillar mitochondria by alteration of the cytochrome c binding site. J Mol Cell Cardiol 33: 37–47, 2001.[CrossRef][Web of Science][Medline]

35. Lesnefsky EJ, Moghaddas S, Tandler B, Kerner J, Hoppel CL. Mitochondrial dysfunction in cardiac disease: ischemia-reperfusion, aging, and heart failure. J Mol Cell Cardiol 33: 1065–1089, 2001.[CrossRef][Web of Science][Medline]

36. Lesnefsky EJ, Tandler B, Ye J, Slabe TJ, Turkaly J, Hoppel CL. Myocardial ischemia decreases oxidative phosphorylation through cytochrome oxidase in subsarcolemmal mitochondria. Am J Physiol Heart Circ Physiol 273: H1544–H1554, 1997.[Abstract/Free Full Text]

37. Miwa S, St-Pierre J, Partridge L, Brand MD. Superoxide and hydrogen peroxide production by Drosophila mitochondria. Free Radic Biol Med 35: 938–948, 2003.[CrossRef][Web of Science][Medline]

38. Moghaddas S, Stoll MS, Minkler PE, Salomon RG, Hoppel CL, Lesnefsky EJ. Preservation of cardiolipin content during aging in rat heart interfibrillar mitochondria. J Gerontol A Biol Sci Med Sci 57: B22–B28, 2002.[Abstract/Free Full Text]

39. Nohl H. Generation of superoxide radicals as byproduct of cellular respiration. Ann Biol Clin (Paris) 52: 199–204, 1994.[Medline]

40. O'Rourke B, Cortassa S, Aon MA. Mitochondrial ion channels: gatekeepers of life and death. Physiology Bethesda 20: 303–315, 2005.[CrossRef][Medline]

41. Ohnishi ST, Ohnishi T, Muranaka S, Fujita H, Kimura H, Uemura K, Yoshida K, Utsumi K. A possible site of superoxide generation in the complex I segment of rat heart mitochondria. J Bioenerg Biomembr 37: 1–15, 2005.[CrossRef][Web of Science][Medline]

42. Okun JG, Lummen P, Brandt U. Three classes of inhibitors share a common binding domain in mitochondrial complex I (NADH:ubiquinone oxidoreductase). J Biol Chem 274: 2625–2630, 1999.[Abstract/Free Full Text]

43. Palmer JW, Tandler B, Hoppel CL. Biochemical properties of subsarcolemmal and interfibrillar mitochondria isolated from rat cardiac muscle. J Biol Chem 252: 8731–8739, 1977.[Abstract/Free Full Text]

44. Paradies G, Petrosillo G, Pistolese M, Di Venosa N, Federici A, Ruggiero FM. Decrease in mitochondrial complex I activity in ischemic/reperfused rat heart: involvement of reactive oxygen species and cardiolipin. Circ Res 94: 53–59, 2004.[Abstract/Free Full Text]

45. Petrosillo G, Casanova G, Matera M, Ruggiero FM, Paradies G. Interaction of peroxidized cardiolipin with rat-heart mitochondrial membranes: induction of permeability transition and cytochrome c release. FEBS Lett 580: 6311–6316, 2006.[CrossRef][Web of Science][Medline]

46. Petrosillo G, Di Venosa N, Ruggiero FM, Pistolese M, D'Agostino D, Tiravanti E, Fiore T, Paradies G. Mitochondrial dysfunction associated with cardiac ischemia/reperfusion can be attenuated by oxygen tension control. Role of oxygen-free radicals and cardiolipin. Biochim Biophys Acta 1710: 78–86, 2005.[Medline]

47. Raha S, McEachern GE, Myint AT, Robinson BH. Superoxides from mitochondrial complex III: the role of manganese superoxide dismutase. Free Radic Biol Med 29: 170–180, 2000.[CrossRef][Web of Science][Medline]

48. Rose IA, O'Connell EL. Mechanism of aconitase action. I. The hydrogen transfer reaction. J Biol Chem 242: 1870–1879, 1967.[Abstract/Free Full Text]

49. Rouslin W. Mitochondrial complexes I, II, III, IV, and V in myocardial ischemia and autolysis. Am J Physiol Heart Circ Physiol 244: H743–H748, 1983.[Abstract/Free Full Text]

50. Seo BB, Marella M, Yagi T, Matsuno-Yagi A. The single subunit NADH dehydrogenase reduces generation of reactive oxygen species from complex I. FEBS Lett 580: 6105–6108, 2006.[CrossRef][Web of Science][Medline]

51. St-Pierre J, Buckingham JA, Roebuck SJ, Brand MD. Topology of superoxide production from different sites in the mitochondrial electron transport chain. J Biol Chem 277: 44784–44790, 2002.[Abstract/Free Full Text]

52. Staat P, Rioufol G, Piot C, Cottin Y, Cung TT, L'Huillier I, Aupetit JF, Bonnefoy E, Finet G, Andre-Fouet X, Ovize M. Postconditioning the human heart. Circulation 112: 2143–2148, 2005.[Abstract/Free Full Text]

53. Starke DW, Chen Y, Bapna CP, Lesnefsky EJ, Mieyal JJ. Sensitivity of protein sulfhydryl repair enzymes to oxidative stress. Free Radic Biol Med 23: 373–384, 1997.[CrossRef][Web of Science][Medline]

54. Sun J, Trumpower BL. Superoxide anion generation by the cytochrome bc1 complex. Arch Biochem Biophys 419: 198–206, 2003.[CrossRef][Web of Science][Medline]

55. Turrens JF. Mitochondrial formation of reactive oxygen species. J Physiol 552: 335–344, 2003.[Abstract/Free Full Text]

56. Turrens JF, Boveris A. Generation of superoxide anion by the NADH dehydrogenase of bovine heart mitochondria. Biochem J 191: 421–427, 1980.[Web of Science][Medline]

57. Veitch K, Hombroeckx A, Caucheteux D, Pouleur H, Hue L. Global ischaemia induces a biphasic response of the mitochondrial respiratory chain. Anoxic pre-perfusion protects against ischaemic damage. Biochem J 281: 709–715, 1992.[Web of Science][Medline]

58. Weiss JN, Korge P, Honda HM, Ping P. Role of the mitochondrial permeability transition in myocardial disease. Circ Res 93: 292–301, 2003.[Abstract/Free Full Text]

59. Williams JN Jr. A method for the simultaneous quantitative estimation of cytochromes A, B, C1, and C in mitochondria. Arch Biochem Biophys 107: 537–543, 1964.[CrossRef][Web of Science][Medline]

60. Yellon DM, Hausenloy DJ. Realizing the clinical potential of ischemic preconditioning and postconditioning. Nat Clin Pract Cardiovasc Med 2: 568–575, 2005.[CrossRef][Web of Science][Medline]




This article has been cited by other articles:


Home page
 J. Biol. Chem.Home page
D. L. Hoffman and P. S. Brookes
Oxygen Sensitivity of Mitochondrial Reactive Oxygen Species Generation Depends on Metabolic Conditions
J. Biol. Chem., June 12, 2009; 284(24): 16236 - 16245.
[Abstract] [Full Text] [PDF]

Home page
 Circ. Res.Home page
P. Korge, P. Ping, and J. N. Weiss
Reactive Oxygen Species Production in Energized Cardiac Mitochondria During Hypoxia/Reoxygenation: Modulation by Nitric Oxide
Circ. Res., October 10, 2008; 103(8): 873 - 880.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
294/2/C460    most recent
00211.2007v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Web of Science (9)
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Chen, Q.
Right arrow Articles by Lesnefsky, E. J.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Chen, Q.
Right arrow Articles by Lesnefsky, E. J.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Visit Other APS Journals Online
Copyright © 2008 by the American Physiological Society.