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Am J Physiol Heart Circ Physiol 292: H1986-H1994, 2007. First published December 15, 2006; doi:10.1152/ajpheart.01214.2006
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Role of oxidative stress in alterations of mitochondrial function in ischemic-reperfused hearts

Zhanna Makazan, Harjot K. Saini, and Naranjan S. Dhalla

Institute of Cardiovascular Sciences, St. Boniface General Hospital Research Centre, and Department of Physiology, Faculty of Medicine, University of Manitoba, Winnipeg, Canada

Submitted 3 November 2006 ; accepted in final form 11 December 2006


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
To study the mechanisms of mitochondrial dysfunction due to ischemia-reperfusion (I/R) injury, rat hearts were subjected to 20 or 30 min of global ischemia followed by 30 min of reperfusion. After recording both left ventricular developed pressure (LVDP) and end-diastolic pressure (LVEDP) to monitor the status of cardiac performance, mitochondria from these hearts were isolated to determine respiratory and oxidative phosphorylation activities. Although hearts subjected to 20 min of ischemia failed to generate LVDP and showed a marked increase in LVEDP, no changes in mitochondrial respiration and phosphorylation were observed. Reperfusion of 20-min ischemic hearts depressed mitochondrial function significantly but recovered LVDP completely and lowered the elevated LVEDP. On the other hand, depressed LVDP and elevated LVEDP in 30-min ischemic hearts were associated with depressions in both mitochondrial respiration and oxidative phosphorylation. Reperfusion of 30-min ischemic hearts elevated LVEDP, attenuated LVDP, and decreased mitochondrial state 3 and uncoupled respiration, respiratory control index, ADP-to-O ratio, as well as oxidative phosphorylation rate. Alterations of cardiac performance and mitochondrial function in I/R hearts were attenuated or prevented by pretreatment with oxyradical scavenging mixture (superoxide dismutase and catalase) or antioxidants [N-acetyl-L-cysteine or N-(2-mercaptopropionyl)-glycine]. Furthermore, alterations in cardiac performance and mitochondrial function due to I/R were simulated by an oxyradical-generating system (xanthine plus xanthine oxidase) and an oxidant (H2O2) either upon perfusing the heart or upon incubation with mitochondria. These results support the view that oxidative stress plays an important role in inducing changes in cardiac performance and mitochondrial function due to I/R.

cardiac performance; oxidative phosphorylation; mitochondrial respiration; oxyradicals; antioxidants

BY VIRTUE OF THEIR ABILITY to carry on the processes of oxidative phosphorylation and electron transport, mitochondria are the major source of energy production in the form of ATP, which is required for cardiac contraction and relaxation (19, 26, 46). Mitochondria are also known to accumulate a substantial amount of Ca2+ and are considered to serve as a sink to maintain the intracellular concentration of free Ca2+ within certain limits (18, 19, 26). However, an excessive amount of intracellular Ca2+ results in the overloading of mitochondria with Ca2+, which impairs mitochondrial energy production under different pathophysiological conditions (18, 19, 28). It has also been demonstrated that mitochondria are capable of producing reactive oxygen species (ROS), including superoxide and hydroxyl radicals as well as H2O2, a well-known oxidant; these ROS promote lipid peroxidation and thus result in disturbance of cation homeostasis and cellular damage (3, 40). Whereas some studies have revealed the participation of mitochondria in cellular apoptosis via the release of cytochrome c (12), others have implicated them in cardioprotection due to the presence of mitochondrial K+ ATP channels (33). Thus mitochondria are multifunctional organelles, which are believed to play diverse roles for the maintenance of cardiac cell activity and structure.

It has now become clear that ischemia-reperfusion (I/R) injury associated with cardiac dysfunction and cellular damage is a consequence of intracellular Ca2+ overload and oxidative stress (6, 7, 14, 21, 34). Recent studies have indicated that changes in sarcolemmal and sarcoplasmic reticular membranes lead to the development of Ca2+-handling abnormalities and intracellular Ca2+ overload due to the occurrence of oxidative stress in the I/R hearts (15, 25, 29, 36, 37, 42, 48). On the other hand, only scattered and conflicting information regarding the status of mitochondrial function with respect to their respiratory and oxidative phosphorylation activities in the I/R hearts is available in the literature (3, 19). It is pointed out that, whereas prolonged ischemia has been reported to depress mitochondrial state 3 respiration and slightly reduce their ability to generate ATP (8, 23), reperfusion of the ischemic heart has been shown to produce no change (1, 45), further depression (17, 22), or increase in the oxidative phopshorylation rate (OPR) (24, 35). In addition, some investigators (13, 16) have shown depressions in both mitochondrial state 3 and state 4 respiration without any change in respiratory control index (RCI), whereas others (9) have reported no change in state 3 respiration but an increase in state 4 respiration in I/R hearts. Depressions in mitochondrial state 3 respiration and RCI due to I/R were also found to be associated with no change in state 4 respiration (43, 44). In addition, an oxyradical-scavenging mixture containing superoxide dismutase (SOD) plus catalase (CAT) was found to prevent the I/R-induced changes in state 3, RCI, and OPR when used in combination with cardioplegic solution but was ineffective when used in the physiological saline solution (39).

Since some of the conflicting results may be due to the duration of ischemia as well as the degree of reperfusion injury, this study was undertaken to investigate alterations in mitochondrial respiration and oxidative phosphorylation by employing I/R hearts in which cardiac performance was assessed to determine the extent of I/R injury. To establish the irreversible state of I/R injury, changes in cardiac performance and mitochondrial function were measured with or without reperfusion of the hearts made ischemic for different times. Alterations in mitochondrial function due to I/R were measured in the presence of low Ca2+ or some Ca2+ antagonists and inhibitors of Na+/Ca2+ exchange to evaluate the role of intracellular Ca2+ overload in I/R hearts. Experiments were also carried out by pretreatment of I/R hearts with SOD plus CAT mixture or some antioxidants to determine the role of oxidative stress in I/R injury. To test whether the effects of I/R on mitochondrial function are simulated by oxidative stress, hearts were perfused or mitochondria were incubated with an oxyradical-generating system as well as H2O2.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
All protocols were approved by the University of Manitoba Animal Care Committee in accordance with the standards of the Canadian Council for Animal Care.

Isolated rat heart preparation. Male Sprague-Dawley rats (250–300 g) were anaesthetized with a mixture of ketamine (90 mg/kg) and xylazine (9 mg/kg). The hearts were quickly excised, mounted on a Langendorff apparatus, and perfused with Krebs-Henseleit buffer gassed with 95% O2-5% CO2 at a constant flow of 10 ml/min (37°C, pH 7.4). The composition of the Krebs-Henseleit solution was (in mM) 120 NaCl, 4.7 KCl, 1.2 KH2PO4, 1.2 MgSO4, 25 NaHCO3, 11 glucose, and 1.25 CaCl2, unless otherwise indicated in the text. The hearts were electrically stimulated at 300 beats/min via a square-wave current of 1.5-ms duration throughout the experiment by using the Phipps and Bird stimulator (Richmond, VA). The left ventricular systolic pressure (LVSP) and the left ventricular end-diastolic pressure (LVEDP) were measured via a transducer (model 1050, BP-Biopac Systems, Goleta, CA), which was connected with a water-filled latex balloon inserted into the left ventricle. At the beginning of the experiment, the LVEDP was adjusted to ~10 mmHg by inflating the balloon; LVDP was taken as the difference between the LVSP and LVEDP. All data were recorded online through an analog-to-digital interface (MP-100, Biopac Systems) and stored in a computer program (Acqknowledge 3.5.3) by using a Biopac Data Acquisition System (Biopac Systems). After stabilization for a period of 20 min, hearts were made globally ischemic for 30 min by stopping the coronary flow, and for reperfusion the flow was restored for 30 min. In some experiments, global ischemia was induced for 20 min followed by 30 min of reperfusion. Antioxidant treatment of I/R hearts was carried out by infusion of a mixture of SOD (5 x 104 U/l; Sigma-Aldrich, Oakville, ON, Canada) plus CAT (7.5 x 104 U/l; Fisher Scientific, Nepean, ON, Canada), N-acetyl-L-cysteine (NAC; 100 µM; Sigma-Aldrich) or N-2-mercaptopropionylglycine (MPG, 300 µM; Sigma-Aldrich) for 10 min before ischemia and during the reperfusion period as described previously (2, 14, 36, 37, 41). The effect of antioxidant treatment with SOD plus CAT, NAC, or MPG on mechanical function and mitochondrial activity of the nonischemic hearts was examined according to the same protocol, except that no global ischemia was induced. It should be mentioned that experiments in our laboratory have revealed that neither SOD nor CAT, when used alone, produces appreciable protective effects on I/R-induced changes in cardiac function in isolated perfused hearts. Furthermore, to determine whether the I/R-induced changes in cardiac performance and mitochondrial function are due to oxidative stress, hearts were perfused with H2O2 (100 µM) for 30 min or xanthine (X; 2 mM; Sigma-Aldrich) plus xanthine oxidase (XO; 60 mU/ml; Boeringer-Mannheim Canada, Laval, Quebec) mixture for 20 min as described previously (42). Control hearts were perfused with Krebs-Henseleit buffer for corresponding periods. Since no significant differences were observed in the cardiac performance and mitochondrial function, the control values in some experiments were grouped together.

Isolation of mitochondria. Mitochondria were isolated by differential centrifugation in ice-cold isolation medium A containing (in mM) 225 mannitol, 75 sucrose, 10 MOPS, 10 Tris·HCl, and 1 EGTA (pH 7.2) and 1% fatty acids-free bovine serum albumin, as described elsewhere (30) with slight modifications. Briefly, ventricular tissue was homogenized by using a Polytron tissue homogenizer (PT 3000, Brinkmann Instruments, Mississauga, ON, Canada), and the homogenate was centrifuged at 550 g for 5 min. The supernatant was filtered through cheesecloth and centrifuged at 8,800 g for 10 min. The crude mitochondrial pellets were purified by centrifugation through a Percoll discontinuous step gradient of 15%, 26%, and 40% at 31,000 g for 15 min. The purified mitochondria were washed twice and resuspended into a buffer containing 250 mM sucrose and 3 mM Tris·HCl (pH 7.2). The mitochondrial protein content was determined by using a Pierce Coomassie Kit (BioLynx, Brockville, ON, Canada). All buffers were prepared by using chemicals with molecular biological quality in glass double-distilled water.

Mitochondrial oxygen consumption. Oxygen consumption was measured polarographically by using a Clark-type electrode in a 1-ml sealed chamber (Quibit Systems, ON, Canada); the reaction medium was mixed with a magnetic stirring bar at 30°C. The incubation medium B used for measuring mitochondrial respiration contained (in mM) 120 KCl, 10 NaCl, 2 MgCl2, 2 KH2PO4, 20 MOPS, 0.7 CaCl2, and 1 EGTA (pH 7.2). Concentration of Ca2+ in the medium B was calculated to yield Ca2+ free concentration of <1 µM using WEBMAXC STANDARD software (Patton CW, Stanford University, http://www.stanford.edu/~cpatton/webmaxcS.htm, 2004). Respiratory substrate mixture consisted of 5 mM sodium pyruvate and 2 mM sodium malate. Each experiment was initiated by adding 0.5 mg mitochondrial protein. For activation of state 3 respiration, 150 nmol of ADP were used. RCI was calculated as the ratio of state 3 to state 4 respiration. Uncoupled respiration was measured by adding 0.2 mM carbonyl cyanide m-chlorophenylhydrazone.

In vitro effects of ROS on isolated mitochondria. Isolated mitochondria from unperfused hearts were incubated in a reaction medium B containing 0.3 mM X and 11 mU XO for 1, 2, or 3 min at 37°C. For antioxidant treatment, mitochondria were exposed for 2 min in the presence of 50 U/ml SOD and 50 U/ml CAT before exposing to X plus XO for 2 min. To study the effects of H2O2, mitochondria were incubated in medium B with 10, 20, or 30 µM concentration of H2O2 for 3 min. The effect of CAT (8 mU/ml) or mannitol (20 mM) was examined by pretreatment of mitochondria for 2 min before exposing to 20 µM H2O2 for 3 min. All these preparations were washed twice and resuspended in a buffer to measure respiratory activities.

Measurement of GSH and GSSG. Reduced glutathione (GSH) and oxidized glutathione (GSSG) concentrations were determined by a glutathione assay kit (Cayman Chemical, Ann Arbor, MI). Isolated mitochondria were incubated in the presence of Triton X-100 (0.1%) on ice for 10 min, and the samples were deproteinated by addition of an equal volume of freshly made metaphosphoric acid (10% wt/vol). The mixture was incubated at room temperature for 5 min and centrifuged at 5,000 g for 10 min to remove the protein precipitate. The supernatant was collected, and 50 µl of 4 M triethanolamine were added for each milliliter of supernatant to adjust the pH of the sample. For total GSH assay, 50 µl of sample were added to 150 µl of the reaction mixture containing 0.4 M 2-(N-morfolino)ethanesulfonic acid, 0.1 M phosphate (pH 6.0), 2 mM EDTA, 0.24 mM NADPH, 0.1 mM 5,5'-dithiobis(2-nitobenzoic acid), and 0.1 unit glutathione reductase. The reaction was carried out at 37°C for 25 min, and total glutathione was then determined by absorbance at 412 nm using GSSG standards. For the measurement of GSSG, 10 µl of 1 M 2-vinylpyridine were used for 1 ml of the sample to remove GSH, and the remaining GSSG in the sample was then quantified by the total GSH assay. The amount of GSH was obtained by subtracting GSSG from total glutathione; the levels of GSH and GSSG were expressed as mitochondrial protein (in nmol/mg). The ratio of GSH to GSSG was used to indicate the redox status of mitochondria, which represents their antioxidant capacity.

Determination of mitochondrial Mg2+-ATPase activity. Mg2+-ATPase activity was measured by sonicating mitochondria on ice for 30 s by 5-s bursts separated by 30-s cooling periods. Lysed mitochondria (50 µg) were incubated in 0.5 ml of buffer containing (in mM) 100 KCl, 20 Tris·HCl, and 10 MgCl2 (pH 6.8) for 5 min at 37°C. Reaction was initiated by the addition of 4 mM ATP. After 10 min of incubation at 37°C, the reaction was stopped by an addition of an equal volume of 12% (wt/vol) of ice-cold trichloroacetic acid; protein precipitate was removed by centrifugating at 3,000 g for 10 min. The concentration of inorganic phosphate (Pi) was determined in the protein-free supernatant, and the activity of mitochondrial Mg2+-ATPase was expressed as Pi (in µmol) in 10 min/mg of mitochondrial protein.

Measurement of mitochondrial Ca2+-uptake activity. Mitochondrial preparation was loaded with fura-2 AM in a manner similar to that described elsewhere (36). Ca2+ uptake was measured in fura-2 AM-loaded mitochondria in the presence of different concentrations of extracellular Ca2+. The fluorescence intensity was monitored by using a SLM DMX-1100 dual-wavelength spectrofluorometer (SLM Instruments, Urbana, IL). In the SOD plus CAT treatment group, isolated mitochondria were treated with SOD plus CAT mixture for 10 min at room temperature before any measurements were taken. The basal concentration of free Ca2+ in mitochondria ([Ca2+]m) was measured before the addition of extracellular Ca2+, whereas Ca2+-stimulated uptake was determined as an increase in [Ca2+] above the basal value at different times after the addition of various concentrations of Ca2+ in each experiment.

Statistical analysis. Results are expressed as means ± SE. Data were analyzed by a one-way ANOVA followed by a mean comparison using a post hoc Turkey test. Intragroup differences between untreated hearts and those perfused in the presence of various chemical agents were determined by unpaired Student's t-test. All statistical analyses were performed by using Origin 7.5 software (OriginLab). Values of P < 0.05 were considered statistically significant.


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Duration of ischemia and mitochondrial function. To determine whether changes in mitochondrial function due to reperfusion of the ischemic heart are associated with attenuated recovery of cardiac performance, isolated rat hearts were subjected to global ischemia for 20 or 30 min and then reperfused with the normal oxygenated medium for 30 min. The data in Table 1 show that the hearts made ischemic for 20 and 30 min failed to generate any significant LVDP, whereas the LVEDP was markedly increased. Reperfusion of the 20-min ischemic hearts resulted in full recovery of LVDP, but the increased LVEDP was lowered from about 62 to 13.9 mmHg. On the other hand, reperfusion of the 30-min ischemic heart showed about 20% recovery in LVDP, but the LVEDP was further increased from about 44 to 74 mmHg. In contrast to hearts subjected to 20 min of ischemia and 30 min of reperfusion, the mitochondrial protein yield was significantly (P < 0.05) decreased in both 30-min ischemic and 30-min I/R hearts. No changes in mitochondrial state 3 respiration, RCI, ADP-to-O ratio, OPR, and uncoupled respiration were observed in 20-min ischemic hearts, whereas significant (P < 0.05) depressions in state 3 and uncoupled respiration as well as OPR were seen upon reperfusing these hearts (Table 1). Global ischemia for 30 min significantly (P < 0.05) depressed state 3 respiration, RCI, ADP-to-O ratio, and OPR without affecting the uncoupled respiration. The results in Table 1 also show that mitochondrial function, when evaluated with respect to state 3 and uncoupled respiration as well as OPR, was further depressed in 30-min I/R hearts. It may be noted from Table 1 that mitochondrial state 4 respiration was not altered in any of the experimental preparations compared with the control values. In view of the observed alterations in different parameters of mitochondrial function in 30-min I/R hearts, all other experiments in this study were carried out upon reperfusing the 30-min ischemic hearts for a period of 30 min.


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  		Table 1. Mitochondrial respiration and oxidative phosphorylation activities, and cardiac performance of rat hearts subjected to 20 or 30 min global ischemia with or without reperfusion for 30 min

 
Calcium, cardiac performance, and mitochondrial function. Since Ca2+ is known to play a critical role in I/R-induced injury to myocardium (18), one set of experiments was carried out by reperfusing the 30-min ischemic hearts with a medium containing different concentrations of Ca2+. The results for changes in cardiac performance and mitochondrial function are given in Table 2. Although the recovery of LVDP was markedly attenuated and the LVEDP was elevated when the ischemic hearts were reperfused with medium containing 0.1, 0.5, 1.25, and 2.5 mM concentrations of Ca2+, the optimal recovery in LVDP and minimal increase in LVEDP were seen in hearts reperfused with 0.5 mM Ca2+. Likewise, varying degrees of changes in different parameters of mitochondrial function were seen upon increasing or decreasing the concentration of Ca2+ in the reperfusion medium, the normal concentration of Ca2+ being 1.25 mM. Maximal depressions in mitochondrial state 3 and uncoupled respiration as well as RCI and OPR were observed when the ischemic hearts were reperfused in the presence of 0.1 mM Ca2+. Increasing the concentration of Ca2+ from 1.25 mM (normal) to 2.5 mM attenuated the depressions in state 3 and uncoupled respiration but produced greater depression in RCI (as a consequence of increased state 4 respiration) without any changes in the ADP-to-O ratio or OPR. Overall, the results given in Table 2 indicate that decreasing the concentration of Ca2+ in the reperfusion medium from the control (1.25 mM Ca2+) did not exert any protective effect on changes in mitochondrial function in I/R hearts.


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  		Table 2. Mitochondrial respiration and oxidative phosphorylation activities, and cardiac performance of rat hearts subjected to 30 min global ischemia followed by 30 min reperfusion in the presence of different concentrations of Ca2+

 
The other set of experiments was carried out by reperfusing the ischemic hearts with some Ca2+ antagonists and blockers of Na+/Ca2+ exchanger and then reperfusing these hearts with medium containing these agents. The results regarding the effects of Ca2+ antagonists verapamil and diltiazem (38), as well as Na+/Ca2+ exchange inhibitors amiloride and KB-R7943 (38), on recovery of cardiac performance and mitochondrial function of the 30-min I/R hearts are shown in Table 3. Although the increased LVEDP in I/R hearts was significantly reduced by all these agents, both verapamil and amiloride further attenuated the recovery of depressed LVDP, whereas diltiazem and KB-R7943 had no effect on the recovery of depressed LVDP due to I/R.


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  		Table 3. Mitochondrial respiration and oxidative phosphorylation activities, and cardiac performance of I/R hearts in the absence and presence of some Ca2+ antagonists or Na+-Ca2+ exchange inhibitors

 
Effect of oxyradical scavenging mixture on I/R hearts. To investigate that oxidative stress in I/R hearts is elicited by the generation of oxyradicals, the ischemic hearts were pre- and postperfused with an oxyradical-scavenging mixture containing SOD plus CAT. The results in Table 4 show that the depression in LVDP and elevation in LVEDP in I/R hearts were markedly attenuated by SOD plus CAT mixture. Furthermore, depressions in mitochondrial state 3 and uncoupled respiration, RCI, ADP-to-O ratio, and OPR in I/R hearts were attenuated or prevented by SOD plus CAT mixture (Table 4). Whereas state 3 respiration was not altered in any experimental group, SOD plus CAT mixture was observed to increase the mitochondrial state 3 and uncoupled respiration as well as RCI and OPR in control hearts. Similar effects of SOD plus CAT mixture in control preparations were also observed with respect to KCl- and ATP-induced increase in intracellular [Ca2+] in cardiomyocytes (36, 37); however, no attempt was made to explain the effects of SOD plus CAT.


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  		Table 4. Effect of oxyradical scavenging mixture containing 5 x 104 U/l SOD and 7.5 x 104 U/l CAT on mitochondrial respiration and oxidative phosphorylation activities, and cardiac performance of rat hearts subjected to 30 min global ischemia followed by 30 min reperfusion

 
The data shown in Table 5 reveal that Ca2+ uptake in mitochondria obtained from ischemic or I/R hearts was depressed compared with control values, when determined at different concentrations of Ca2+ as well as at different times of incubation. Furthermore, these alterations in Ca2+-uptake activities in I/R hearts were not affected by treatment with SOD plus CAT mixture. It can also be seen from Table 5 that the basal [Ca2+]m was not altered in any of the experimental conditions. The characteristics of mitochondrial preparations employed in this study were further examined by measuring the Mg2+-ATPase activity, glutathione contents, and protein composition. The results in Table 6 indicate no change in mitochondrial Mg2+-ATPase activity in ischemic or I/R hearts with or without SOD plus CAT treatment. On the other hand, GSH content and GSH-to-GSSG ratio were depressed in mitochondria from ischemic or I/R hearts; these changes in I/R mitochondria were attenuated by treatment with SOD plus CAT. Although GSSG content was increased in the mitochondria from I/R hearts, this change was not attenuated significantly by treatment with SOD plus CAT. However, the depression in mitochondrial protein in the I/R hearts was attenuated by SOD plus CAT. SDS electrophoretic separation of mitochondrial proteins revealed the appearance of no new protein band or the disappearance of any existing protein band due to ischemia or reperfusion of the ischemic heart in the absence and presence of SOD plus CAT (Fig. 1).


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  		Table 5. Basal [Ca2+]m and mitochondrial Ca2+ uptake at different times in the presence of various concentrations of Ca2+ in hearts subjected to ischemia, I/R, and I/R treated with SOD and CAT

 

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  		Table 6. Mitochondrial protein yield and Mg2+-ATPase activity as well as content of GSH and GSSG in mitochondrial preparations from ischemic, I/R, and I/R hearts treated with antioxidant mixture containing SOD and CAT

 

Figure 1
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  		Fig. 1. Representative gel showing bands by SDS-electrophoretic separation of mitochondrial protein samples from the control (A), ischemic (B), ischemic-reperfused (I/R; C), and I/R hearts treated with antioxidant mixture containing superoxide dismutase and catalase (D). Values show molecular mass (in Da) obtained from gel for standard proteins.

 
Effect of some antioxidants I/R hearts. To further test whether changes in cardiac performance and mitochondrial function due to I/R are due to oxidative stress, the ischemic hearts were pre- and postperfused with some well-known antioxidants such as NAC and MPG (2, 41). The data in Tables 7 and 8 indicate that the depression in LVDP in I/R hearts was attenuated, whereas the elevated LVEDP was markedly lowered by both NAC and MPG. Likewise, depressions in mitochondrial state 3 and uncoupled respiration, as well as RCI, ADP-to-O ratio, and OPR in I/R hearts, were either attenuated or prevented by NAC and MPG (Tables 7 and 8). These agents were found to exert no effect on cardiac performance or mitochondrial function in control hearts. The mitochondrial state 4 respiration was not affected by these agents in both control and experimental preparations.


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  		Table 7. Effect of 100 µM NAC on mitochondrial respiration and oxidative phosphorylation activities, and cardiac performance of rat hearts subjected to 30 min global ischemia followed by 30 min reperfusion

 

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  		Table 8. Effect of 300 µM antioxidant MPG on mitochondrial respiration and oxidative phosphorylation activities, and cardiac performance of rat hearts subjected to 30 min global ischemia followed by 30 min reperfusion

 
Effects of perfusion with oxyradical-generating system or H2O2. To test whether changes in mitochondrial function due to I/R are simulated by oxyradicals and oxidant, the hearts were perfused with X plus XO as well as H2O2. The results regarding cardiac performance and mitochondrial function are given in Table 9. Both X plus XO and H2O2 depressed LVDP, elevated LVEDP, and decreased the yield of mitochondrial proteins. Perfusion of hearts with X plus XO decreased mitochondrial state 3 and uncoupled respiration, RCI, and ADP-to-O ratio without any change in the state 4 respiration. On the other hand, perfusion with H2O2 increased mitochondrial state 4 respiration and decreased RCI, ADP-to-O ratio, as well as mitochondrial state 3 and uncoupled respiration.


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  		Table 9. Mitochondrial respiration and oxidative phosphorylation activities, and cardiac performance of rat hearts perfused with (X + XO) or H2O2

 
In vitro effect of oxyradical-generating system and H2O2. In view of the observed difference in the effect of perfusion with X plus XO and H2O2 on mitochondrial function, the actions of these interventions on mitochondria were examined under in vitro conditions. Incubation of mitochondria in the presence of X plus XO for 1, 2, and 3 min indicated that state 3 and uncoupled respiration, as well RCI and ADP-to-O ratio, were depressed, except that ADP-to-O ratio at 1 min incubation was unaltered (Table 10). Furthermore, incubation of mitochondria with 20 or 30 µM H2O2 for 3 min, unlike 10 µM H2O2, produced depression in mitochondrial state 3 and uncoupled respiration as well as RCI and ADP-to-O ratio (Table 10). No change in state 4 respiration was evident upon incubating mitochondria with either X plus XO or H2O2. The data in Table 11 show that changes in mitochondrial function due to X plus XO were attenuated or prevented by the presence of SOD plus CAT, whereas those by H2O2 were attenuated by the presence of CAT plus mannitol but not by CAT alone.


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  		Table 10. In vitro effects of X + XO at different times of incubation as well as various concentrations of H2O2 on mitochondrial respiration and oxidative phosphorylation activities

 

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  		Table 11. Modification of the X + XO or H2O2 effects on mitochondrial respiration and oxidative phosphorylation activities by incubation in the presence of SOD, CAT and mannitol

 

   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
In this study, we have shown that hearts, subjected to 20 or 30 min of global ischemia, failed to develop LVDP and exhibited a marked increase in LVEDP. Furthermore, 30-min ischemic hearts, unlike 20-min ischemic hearts, showed depressions in mitochondrial state 3 respiration, RCI, ADP-to-O ratio, and OPR. Since no changes in state 4 and uncoupled respiration were observed in both 20- and 30-min ischemic hearts, it appears that the functional integrity of inner mitochondrial membrane is preserved (27). Prolonged myocardial ischemia has also been shown to affect the mitochondrial function (3, 16, 19). However, 20-min ischemic hearts showed full recovery of depressed LVDP and a marked reduction in the elevated LVEDP upon perfusion, whereas the 30-min I/R hearts exhibited markedly attenuated recovery of the depressed LVDP while LVEDP was further increased. Although varying degrees of defects in different parameters of mitochondrial respiratory and oxidative phosphorylation activities were observed in 20- and 30-min I/R hearts, the changes in 30-min I/R hearts were greater than those in 20-min I/R hearts. These observations reveal that alterations of cardiac performance and mitochondrial function become irreversible within 20 to 30 min of global ischemia and are in agreement with the findings of others (3, 16, 19). However, it should be pointed out that the observed changes in mitochondrial respiratory and oxidative phosphorylation due to I/R are not due to a generalized defect in mitochondria because Mg2+-ATPase activity in the ischemic or I/R heart was not altered. Furthermore, a 20- to 30-min period of global ischemia has been reported to produce irreversible injury with respect to change in cardiac performance and cardiomyocytes Ca2+-handling abnormalities in I/R hearts.

The results in this study indicate optimal recovery in LVDP and minimal increase in LVEDP in ischemic hearts reperfused with 0.5 mM Ca2+ compared with 0.1, 1.25, and 2.5 mM extracellular Ca2+. Although the possible reasons for this bell-shaped curve for the effect of Ca2+ concentrations in perfusate are not clear, 0.5 mM concentration of Ca2+ may be critical for maintaining cardiac performance in the I/R hearts. Since intracellular Ca2+ overload is considered to produce I/R injury (15, 18), it is possible that the irreversible state of the mitochondrial function in 30-min I/R hearts may be a consequence of the development of intracellular Ca2+ overload in the myocardium. However, this does not seem to be the case, because decreasing the extracellular concentration of Ca2+ from 1.25 to 0.1 mM, which markedly reduced the elevated LVEDP in I/R hearts and is expected to decrease the intracellular Ca2+ overload, did not protect alterations in mitochondrial respiration and oxidative phosphorylation due to I/R. In addition, pretreatment of I/R hearts with Ca2+ antagonists (verapamil and diltiazem) as well as Na+/Ca2+ exchange inhibitors (amiloride and KB-R7943), which lowered the elevated LVEDP and are known to depress the entry of Ca2+ into the myocardium (18), also failed to attenuate changes in mitochondrial respiration and oxidative activities. In fact, verapamil and amiloride further decreased the recovery of depressed LVDP in I/R hearts in contrast to diltiazem and KB-R7943. Such differences in the effects of these two types of drugs may be due to their nonspecific actions on other sites in cardiomyocytes at the concentrations employed in this study. Nonetheless, these results indicate that the increased entry of Ca2+ due to I/R via sarcolemmal L-type Ca2+ channels and Na+/Ca2+ exchanger may not be associated with mitochondrial abnormalities in I/R hearts. However, these experiments do not rule out the participation of intracellular Ca2+ overload in the genesis of mitochondrial dysfunction as a consequence of defects in the sarcoplasmic reticulum due to I/R (42, 48). In fact, overloading of mitochondria with Ca2+ has been reported to depress mitochondrial respiratory and oxidative phosphorylation activities (4, 18), and the occurrence of mitochondrial Ca2+ overload has been shown during ischemia as well as the initial phase of reperfusion (18). Our inability to show an increase in Ca2+-uptake activity in mitochondria from 30-min I/R hearts may be due to a defect in the mitochondrial Ca2+-transport system at this late stage of I/R; this view is supported by an observation that Ca2+ uptake by mitochondria from 30-min I/R hearts was depressed. Furthermore, the basal concentration of free Ca2+ in mitochondria from I/R hearts was not different from control, and this may be due to the leakage of varying amounts of Ca2+ from mitochondrial preparations during the isolation and purification procedures. It is pointed out that the basal concentration of free Ca2+ in mitochondria should not be confused with total Ca2+ content because this parameter was not measured under the experimental conditions employed in this study.

Several investigators have demonstrated that oxidative stress due to the formation of oxyradicals and accumulation of different oxidants plays a critical role in the development of I/R injury (6, 14, 15, 21, 34). From the experiments described in this study, it is evident that oxidative stress in I/R hearts may be intimately involved in producing alterations in mitochondrial respiration and oxidative phosphorylation. This view is attested by our observations that pretreatment of I/R hearts with an oxyradical-scavenging mixture containing SOD plus CAT improved cardiac performance and attenuated changes in mitochondrial respiratory and oxidative phosphorylation activities. Since SOD plus CAT treatment did not fully reverse I/R-induced changes, it is likely that non-ROS-dependent pathway may also be involved in causing defects in cardiac and mitochondrial functions. However, this effect of SOD plus CAT was specific because depression in Ca2+ uptake by mitochondria obtained from I/R hearts was not affected by this intervention. The depression of oxidative stress in mitochondria from I/R hearts by SOD plus CAT treatment was also evident from our observation that decreases in mitochondrial GSH content as well as the GSH-to-GSSG ratio, which is an excellent index of oxidative stress (10), were attenuated by this intervention. Furthermore, antioxidants such as NAC and MPG were found to attenuate changes in both cardiac performance and mitochondrial respiration and oxidative phosphorylation in I/R hearts. Both NAC and MPG have been reported to attenuate some changes in mitochondrial function due to I/R (10, 41). It should also be pointed out that the effects of I/R on cardiac function as well as mitochondrial respiration and oxidative phosphorylation were simulated by perfusing the hearts with oxyradical-generating system (X plus XO) and an oxidant (H2O2), except that mitochondrial state 4 respiration was increased upon perfusing the hearts with H2O2. The exact reason for this difference between the action of X plus XO mixture and H2O2 is not clear at present because incubation of mitochondria with both X plus XO mixture and H2O2 produced similar effects on mitochondrial respiration and oxidative phosphorylation. Nonetheless, the mechanisms for the actions of X plus XO mixture and H2O2 on mitochondrial function seem to be different from each other, because the effects of X plus XO mixture were attenuated by SOD plus CAT, whereas the effects of H2O2 were prevented by CAT and mannitol, the mixture of which is required for scavenging hydroxyl radicals. Both X plus XO and H2O2 have been reported to impair cardiac performance and to induce Ca2+-handling abnormalities in cardiomyocytes (36, 37). Taken together, this study provides comprehensive evidence that oxyradicals and oxidants generated during I/R may produce abnormalities in mitochondrial respiration and oxidative phosphorylation.

Although I/R, an oxyradical generating system, and H2O2 were observed to produce mitochondrial dysfunction, the exact site affected by these interventions within mitochondria remain to be established. Some investigators have reported a defect at the level of the electron transport chain (20), whereas others have indicated that changes in mitochondrial membrane potentials (5) and mitochondrial permeability (11) may induce the I/R-induced changes in mitochondrial function. Lipid peroxidation of mitochondrial membrane (32) and a decrease in cardiolipin content (27, 31) as a consequence of oxidative stress have also been shown to serve as mechanisms for changes in mitochondrial respiration and oxidative phosphorylation. Exposure of mitochondria to both superoxide and hydroxyl radicals as well as H2O2 was found to inactivate various mitochondrial proteins to varying degrees (47); however, defects in cytochrome-c oxidase (32) and ADP/ATP translocase have been suggested to play a critical role in I/R-induced changes in mitochondrial function. Nonetheless, irrespective of the exact site of defect in mitochondria, the observed decrease in energy production as a consequence of oxidative stress can be seen to contribute toward the depression in cardiac performance of the I/R hearts.


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 ABSTRACT
 MATERIALS AND METHODS
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This work was supported by a grant from the Canadian Institutes of Health Research.


   ACKNOWLEDGMENTS
 
H. K. Saini is a predoctoral fellow of the Heart and Stroke Foundation of Canada.


   FOOTNOTES
 

Address for reprint requests and other correspondence: N. S. Dhalla, Inst. of Cardiovascular Sciences, St. Boniface General Hospital Research Centre, 351 Tache Ave., Winnipeg, Manitoba R2H 2A6, Canada (e-mail: nsdhalla{at}sbrc.ca)

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
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