AJP - Heart Fuel your research with LabChart
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

Am J Physiol Heart Circ Physiol 280: H2770-H2778, 2001;
0363-6135/01 $5.00
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
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 ISI 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 ISI Web of Science (46)
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Lesnefsky, E. J.
Right arrow Articles by Hoppel, C. L.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Lesnefsky, E. J.
Right arrow Articles by Hoppel, C. L.
Vol. 280, Issue 6, H2770-H2778, June 2001

Myocardial ischemia selectively depletes cardiolipin in rabbit heart subsarcolemmal mitochondria

Edward J. Lesnefsky1,4, Thomas J. Slabe4, Maria S. K. Stoll4, Paul E. Minkler4, and Charles L. Hoppel2,3,4

1 Division of Cardiology and 2 Division of Clinical Pharmacology, Department of Medicine, and 3 Department of Pharmacology, Case Western Reserve University, and 4 Geriatric Research, Education, and Clinical Center and Medical Service, Louis Stokes VA Medical Center, Cleveland, Ohio 44106


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Mitochondria contribute to myocyte injury during ischemia. After 30 and 45 min of ischemia in the isolated perfused rabbit heart, subsarcolemmal mitochondria (SSM), located beneath the plasma membrane, sustain a decrease in oxidative phosphorylation through cytochrome oxidase. In contrast, oxidation through cytochrome oxidase in interfibrillar mitochondria (IFM), located between the myofibrils, remains unaffected. Cytochrome oxidase activity in the intact membrane requires an inner mitochondrial membrane lipid environment enriched in cardiolipin. During ischemia, the content of cardiolipin decreased only in SSM, whereas the content of other phospholipids was preserved. Ischemia did not alter the composition of the cardiolipin that remained in SSM. Cardiolipin content was preserved in IFM during ischemia. Thus cardiolipin is a relatively early target of ischemic mitochondrial damage, leading to loss of oxidative phosphorylation through cytochrome oxidase in SSM.

cytochrome oxidase; phospholipids; electron transport chain


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

MITOCHONDRIA SUSTAIN DAMAGE and contribute to myocardial injury during ischemia (2, 7, 9, 44). Ischemia results in progressive damage to the electron transport chain (4, 12, 16, 36, 40, 41, 50). Initially, brief periods of ischemia lead to defects in electron transport complex I (16, 40) and complex V (41) and the adenine nucleotide transporter (4, 12). Mitochondrial oxidative physiology and cardiac contractile function recover after these short durations of ischemia (16). However, as ischemic periods lengthen to 30 min, a time leading to myocardial injury during reperfusion (2, 7, 9, 44), a second defect occurs in the electron transport chain distal to complex I (27, 36, 37, 48). The second defect is due to a decrease in oxidative phosphorylation through cytochrome oxidase (27).

Cardiac mitochondria exist in two functionally distinct populations. Subsarcolemmal mitochondria (SSM) are located beneath the plasma membrane, whereas interfibrillar mitochondria (IFM) are present between the myofibrils (31). These two populations are affected differently in cardiomyopathy (22), aging (15), and ischemia (12, 25, 27, 48). The progression of ischemic damage is more rapid in SSM (12, 27, 48). Forty-five minutes of ischemia decrease oxidative phosphorylation through cytochrome oxidase in SSM (27).

Cytochrome oxidase is a membrane-associated complex of 13 subunits (8) that requires the integrity of catalytic subunits (5), regulatory and structural subunits (3), and an intact inner mitochondrial membrane environment (39, 51) for activity. The decrease in oxidative phosphorylation through cytochrome oxidase in SSM did not occur secondary to damage of a peptide subunit (27). The absence of peptide damage focused attention on damage to mitochondrial membrane phospholipids as a potential mechanism of decreased cytochrome oxidase activity during ischemia. Cytochrome oxidase has a regional membrane microenvironment enriched in cardiolipin, a diphosphatidylglycerol enriched in oxidatively sensitive acyl groups (18, 21, 39, 51). Cytochrome oxidase contains 2 or 3 tightly bound cardiolipin residues (18) and a second less tightly associated group of an additional 20-50 cardiolipin residues, which are required for optimal activity (37, 39, 51).

In the current study, ischemia decreased the content of cardiolipin with a selectivity corresponding to the decrease in oxidative phosphorylation through cytochrome oxidase. The content of cardiolipin was decreased by ischemia only in SSM, whereas the content in IFM remained unchanged even at the prolonged period of 45 min of ischemia. The time course of cardiolipin loss in SSM paralleled the decrease in oxidative phosphorylation through cytochrome oxidase. The composition of the remaining cardiolipin, measured by acyl group composition and individual molecular species of cardiolipin, was similar to the composition of cardiolipin before ischemia. In contrast to cardiolipin, the content of other phospholipids in SSM was preserved during ischemia. Myocardial ischemia selectively depleted cardiolipin in SSM, corresponding to the onset of the decrease in oxidative phosphorylation through cytochrome oxidase, providing a mechanism for the defect in the distal electron transport chain that occurs at 30 min of ischemia.


    MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Materials. All chemicals were of reagent grade and obtained from Sigma (St. Louis, MO). HPLC grade solvents were obtained from Fischer Scientific (Pittsburgh, PA). Phospholipid standards were obtained from Avanti Polar Lipids (Alabaster, AL).

Rabbit heart model of mitochondrial oxidative physiology during ischemia and reperfusion. The isolated buffer-perfused rabbit heart model was used as previously described (27, 28). Two populations of cardiac mitochondria were isolated using the procedure of Palmer et al. (31) except that a modified Chappell-Perry buffer [buffer A; containing (in mM) 100 KCl, 50 MOPS, 1 EGTA, 5 MgSO4 · 7 H2O, and 1 ATP; pH 7.4] was used for mitochondrial isolation (27). Oxygen consumption in intact mitochondria was measured using a Clark-type oxygen electrode at 30°C (27). The rate of oxidative phosphorylation and uncoupled respiration were measured using 1 mM N,N,N',N'-tetramethyl-p-phenylenediamine (TMPD) and 10 mM ascorbate as the substrate (27). Cytochrome c content was measured as the difference between ferricyanide-oxidized and dithionite-reduced spectra as previously described (27). Protein concentrations were measured using the biuret method with BSA as the standard (27).

Separation, quantitation, and characterization of mitochondrial phospholipids. Mitochondrial phospholipids were separated, quantitated, and characterized according to an experimental approach described previously (26). Lipids were extracted from mitochondria using the method of Folch (17) with 50 µM butylated hydroxytoluene (BHT) added as an antioxidant. Organic extracts were stored under N2 at -20°C until subsequent analysis. Lipid classes were serially eluted using solvents of increasing polarity from silica gel columns as previously described with minor modifications (23). Phospholipids and lysophospholipids were eluted together with 8 ml methanol.

The phospholipid fraction was evaporated under N2, dissolved in hexane:2-propanol:water [40:54:6 (vol:vol:vol)], and centrifuged (700 g at 4°C) for 5 min to sediment particulate matter. Normal-phase HPLC separation of phospholipids and lysophospholipids was performed using a HP1100 (quaternary pump, online degasser, autosampler, column heater with switching valve, and variable wavelength detector, Agilent Technologies; Wilmington, DE) with a Hypersil silica 5-µm 250 × 4.6-mm column (Alltech Associates; Deerfield, IL) using a mobile phase of hexane:2-propanol:25 mM potassium acetate (pH 7.0):ethanol:glacial acetic acid [367:490:62:100:0.6 (vol:vol:vol:vol:vol)] with a gradient of hexane:2-propanol:25 mM potassium acetate (pH 7.0):acetonitrile:glacial acetic acid [442:490:62:25:0.6 (vol:vol:vol:vol:vol)] at 30°C over 100 min as previously described (26).

Individual phospholipid and lysophospholipid peaks were identified by comparison of retention time to those of standards. Organic phosphate was measured by the method of Bartlett (6) adapted for use in a multiwell platereader using absorbance at 815 nm (26). The assay was linear from 2.5-50 nmol organic phosphate with a limit of detection of 2.5 nmol. Organic phosphate was measured in 2-ml fractions from normal-phase HPLC. In parallel samples, the cardiolipin peak was collected, and acyl group composition and content were measured. Cardiolipin samples were evaporated under N2, subjected to alkaline hydrolysis, and derivitized to fatty acid methyl esters using 2,2-dimethoxypropane, with gas chromatography and detection using mass spectrometry (MS) total ion current (24).

Individual molecular species of cardiolipin were separated using reverse-phase HPLC and characterized by electrospray ionization/MS (26). The reverse-phase HPLC method of Teng and Smith (46) was adapted for liquid chromotography/MS by substituting ammonium acetate (15 mM; pH 7.4) for 10 mM potassium acetate (pH 7.4). The cardiolipin-containing fraction was collected from normal-phase HPLC, evaporated under N2, and taken up in 110 µl of reverse-phase buffer 1 [acetonitrile:methanol:15 mM ammonium acetate (pH 7.4) 60:30:10 (vol:vol:vol)] for injection. Reverse-phase HPLC-electrospray ionization MS performed with a Hypersil MOS 3-µm column using a gradient of 100% buffer 1 to 100% buffer 2 [acetonitrile:methanol:15 mM ammonium acetate (pH 7.4) 60:38:2 (vol:vol:vol)] over 30 min at 35°C and a flow rate of 0.4 ml/min.

Electrospray ionization MS was performed using Finnegan liquid chromotography MS (San Jose, CA) in the negative ion mode using N2 as the sheath and auxiliary gas. The heated capillary temperature was 250°C, the electrospray voltage was 5.2 kV, and the capillary voltage was set to -4 V. Three scan events were used: 1) 1,000-2,000 mass-to-charge ratio (m/z) full-scan MS, 2) data-dependant full-scan MS2 (MS/MS) on the most intense ion from the MS full spectrum, and 3) data-dependent full-scan MS3 (MS/MS/MS) on the most intense ion from the full scan MS2 spectrum. The MS2 and MS3 relative collision energy was set to 48%. This data-dependent multistage MS fragmentation was used to characterize the individual molecular species of cardiolipin as they eluted from the reverse-phase HPLC system. The first 5 min of effluent flow was diverted to waste.

Statistical analysis. Differences during the time course of ischemia were compared by one-way analysis of variance with post hoc comparisons performed using the Student-Newman-Keuls test of multiple comparisons (46). A difference of P < 0.05 was considered significant.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Rabbit heart model of ischemia. The isolated buffer-perfused rabbit heart was studied at 0 (n = 5), 15 (n = 6), 30 (n = 5), and 45 min (n = 8) of ischemia and at 45 min of nonischemic buffer perfusion (n = 4) as a time control. Heart weights were similar in all groups (data not shown). At the end of the 15-min equilibration period, all groups had similar developed pressure, positive and negative change in pressure over time, and coronary flow (data not shown), similar to previous results in this model (27). Time control hearts maintained 100% of end-equilibration period developed pressure during the additional 45 min of buffer perfusion (data not shown).

Oxidative phosphorylation in SSM and IFM from control and ischemic hearts. The protein yield of SSM and IFM was similar in all groups (Table 1). Oxidative phosphorylation with glutamate as a substrate was similar in both the 15-min equilibration (before ischemia) and 45-min perfusion (time control) nonischemic groups for both SSM and IFM (data not shown). The oxidation of TMPD-ascorbate, an electron donor to cytochrome oxidase via cytochrome c, was also similar in both control groups in SSM and IFM (Table 1).

                              
View this table:
[in this window]
[in a new window]
  		Table 1.   Mitochondrial yield and respiration before and during ischemia

After 45 min of ischemia, the rate of oxidative phosphorylation with glutamate as the substrate was decreased compared with nonischemic controls in SSM, whereas the rate remained unaltered in IFM (data not shown), similar to previously reported results (27). State 4 respiration and the ADP-to-O ratio remained unchanged in both populations compared with nonischemic controls (data not shown). These results support a preserved functional integrity of the inner mitochondrial membrane and phosphorylation apparatus in SSM during the initial 45 min of ischemia. We then studied the time course of the decrease in oxidative phosphorylation through cytochrome oxidase (Table 1). Both ADP-stimulated and dinitrophenol-uncoupled respiration with TMPD-ascorbate as the substrate was decreased at 30 and 45 min of ischemia in SSM (Table 1). The decrease in uncoupled respiration localizes ischemic damage to the electron transport chain. In contrast to the observations in SSM, in IFM the oxidation of TMPD-ascorbate was preserved throughout ischemia (Table 1). Thus, at durations of ischemia of at least 45 min, damage to cytochrome oxidase was selective to SSM.

Quantitation of phospholipids in SSM and IFM during ischemia. The content of phospholipids was measured in SSM and IFM during the time course of ischemia. Lipids were extracted from mitochondria, phospholipids and lysophospholipids were separated from other lipid classes using a silica gel column, and individual phospholipids and lysophospholipids were separated by normal-phase HPLC. The recovery of cardiolipin through the entire procedure totaled 70-80%. Individual phospholipids and lysophospholipids were quantitated by an organic phosphate assay using 2-ml fractions from normal-phase HPLC (Table 2). Total lipid phosphate was measured in the combined phospholipid-lysophospholipid fraction obtained after separation using a silica gel column.

                              
View this table:
[in this window]
[in a new window]
  		Table 2.   Quantitation of mitochondrial phospholipids before and during ischemia

Mitochondria contain phosphatidylethanolamine (PE), phosphatidylinositol (PI), cardiolipin, phosphatidylcholine (PC), and other phospholipids. Time control perfusion did not alter phospholipid content. The recovery of total lipid phosphate as the four individual phospholipids was excellent in both SSM and IFM (Table 2). Total lipid phosphate was accounted for by the four measured phospholipids.

The content of cardiolipin was decreased in SSM at 30 and 45 min of ischemia (Table 2). The content of cardiolipin was preserved after 15 min of ischemia. Ischemia did not decrease the content of PE, PI, or PC in SSM (Table 2). Ischemia did not significantly decrease the content of total lipid phosphate in SSM, further supporting the observed selectivity of cardiolipin loss. The cardiolipin content did not decrease in IFM during ischemia. The recovery of total lipid phosphate as individual phospholipids remained excellent throughout ischemia (Table 2). Thus ischemia led to a selective decrease in cardiolipin content in SSM (Table 2), which occurred concomitant with the decrease in oxidative phosphorylation through cytochrome oxidase (Table 1) during the time course of ischemia.

Hydrolysis by phospholipase A2 removes the acyl group from the sn-2 position, generating lysophospholipids (43). The retention time of dilysocardiolipin was identified in the normal-phase HPLC chromatogram using synthetic dilysocardiolipin (Avanti Polar Lipids). An increase in dilysocardiolipin was not detected at 45 min of ischemia in SSM. In control and 45-min ischemia hearts, the content of dilysocardiolipin remained at the lower limit of detection. The lack of an increase in dilysocardiolipin content after ischemia makes phospholipase A2 hydrolysis unlikely as the sole mechanism of cardiolipin loss.

Characterization of cardiolipin composition in SSM during ischemia. We then asked whether ischemia altered the composition of the remaining cardiolipin in SSM. The acyl group content of cardiolipin was measured after alkaline hydrolysis, derivitization to form fatty acid methyl esters, and separation and quantitation by gas chromatography (24). Cardiolipin from nonischemic rabbit SSM and IFM is composed predominantly of linoleic acid (C18:2) (Table 3). Ischemia did not alter the ratio of C18:2 acyl groups per lipid phosphate in cardiolipin.

                              
View this table:
[in this window]
[in a new window]
  		Table 3.   Composition of cardiolipin before and during ischemia

The composition of cardiolipin in SSM from nonischemic control and ischemic hearts was characterized using an additional approach. Individual molecular species of cardiolipin isolated from normal-phase HPLC were separated by reverse-phase HPLC. Molecular species were characterized by electrospray ionization MS. Two molecular species of cardiolipin were identified in SSM from nonischemic hearts. The m/z of the molecular ion of the major species was 1,448-1,449 and that of the minor species was 1,446 (Fig. 1). The acyl group composition of each molecular species was identified by data-dependent MS3 of the most abundant ion resulting from each collision. The fragmentation products of the major molecular species (m/z 1,448) are shown in Fig. 2. The fragmentation products confirmed the identity of the compound as cardiolipin, with a composition of four C18:2 acyl groups. The MS2 product with a 695 m/z is phosphatidic acid, which contains two C18:2 acyl groups. No other phosphatidic acid residues were identified. Composition was further confirmed by the MS3 fragmentation, generating ions of 415 m/z (lysophosphatidic acid, which contains one C18:2 acyl group) and 279 m/z (C18:2 acyl group). No other acyl groups were identified. In a similar fashion, the minor molecular species (m/z 1,446) was identified as a cardiolipin containing one C18:3 group and three C18:2 acyl groups (Fig. 3). MS2 generated a product with a 693 m/z (phosphatidic acid, which contains one C18:2 group and one C18:3 acyl group). MS3 fragmentation generated a product with 415 m/z (lysophosphatidic acid, which contains one C18:2 acyl group) and 277 m/z (C18:3 acyl group) (Fig. 4).


View larger version (22K):
[in this window]
[in a new window]
  		Fig. 1.   Content of cardiolipin was unchanged ater 15 min of ischemia (I) and was decreased after 30 and 45 min of ischemia in subsarcolemmal mitochondria (SSM; A). In contrast, cardiolipin content was preserved in interfibrillar mitochondria (IFM; B) during myocardial ischemia.



View larger version (15K):
[in this window]
[in a new window]
  		Fig. 2.   Characterization of the major cardiolipin molecular species present in SSM using reverse-phase HPLC (top) and data-dependent mass spectroscopy (MS; bottom). Two molecular species of cardiolipin were identified. Species I elutes at 14 min and has a mass-to-charge ratio (m/z) of the molecular ion of 1,448 Da. Species II elutes at 11 min and has a m/z of the molecular ion of 1,446 Da. Further characterization of the cardiolipin molecular species using MS3 is shown in Figs. 3 and 4. MS technique is described in the text. Insets: molecular ion mass spectra of the peaks in the total ion current. UV, ultraviolet; AU, arbitrary units.



View larger version (16K):
[in this window]
[in a new window]
  		Fig. 3.   Left: characterization of cardiolipin species I using data-dependent MS3 on the most intense ion (m/z 1,448) obtained at a retention time of 14 min. Two data-dependent full-scan MS readings (MS/MS) of the most intense ion from the MS spectrum (MS2) fragmentation yielded m/z of 695 and 831, which correspond to the fragments shown in the accompanying structural diagram (right). MS3 yielded additional fragments, which correspond to lysophosphatidic acid [containing one linoleic acid (C18:2) residue (m/z 415)] and linoleic acid (C18:2; m/z 279). Structures corresponding to each fragmentation product are shown (right).



View larger version (17K):
[in this window]
[in a new window]
  		Fig. 4.   Left: characterization of cardiolipin species II using data-dependent MS3 on the most intense ion (m/z 1,446) obtained at a retention time of 11 min. MS2 fragmentation yielded m/z of 693 and 829, which correspond to the fragments shown in the accompanying structural diagram (right). MS3 yielded additional fragments, which correspond to lysophosphatidic acid [containing one C18:2 residue (m/z 415)] and linolenic acid (C18:3; m/z 277). Structures corresponding to each fragmentation product are shown (right).

After both 30 and 45 min of ischemia, the same molecular species were identified in SSM. New molecular species of cardiolipin were not observed after ischemia. Thus identification and characterization of individual molecular species of cardiolipin by MS do not suggest that ischemia alters the composition of cardiolipin.

Cytochrome c content in SSM and IFM during ischemia. Myocardial ischemia decreased the content of cytochrome c in SSM at 30 and 45 min of ischemia, with preserved content of cytochrome c at 15 min of ischemia (Table 4). The content of cytochrome c was preserved in IFM during the time course of ischemia, consistent with previous results (27).

                              
View this table:
[in this window]
[in a new window]
  		Table 4.   Content of cytochrome c before and during ischemia


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

The content of cardiolipin decreased in SSM after 30 and 45 min of myocardial ischemia in the isolated rabbit heart. Cardiolipin was the only phospholipid that decreased during ischemia in SSM. While ischemia decreased the content of cardiolipin in SSM, the composition of the remaining cardiolipin was unaltered. In contrast, the content and composition of cardiolipin was unaffected throughout the course of ischemia in IFM. In concert with the decrease in the content of cardiolipin, the rate of oxidative phosphorylation through cytochrome oxidase decreased in SSM after 30 and 45 min of ischemia. Oxidative phosphorylation through cytochrome oxidase and the content of cardiolipin were maintained in SSM at 15 min of ischemia. The rate of oxidative phosphorylation through cytochrome oxidase remained unaltered in IFM during ischemia.

The decrease in cardiolipin content and oxidation through cytochrome oxidase occurred in the setting of preserved myocyte and mitochondrial morphology previously observed after 45 min of ischemia in this model (27). At 45 min of ischemia, oxidative phosphorylation in SSM remained well coupled without an increase in state 4 respiration, providing functional evidence of the integrity of inner mitochondrial membrane (27). Thus, by both functional and morphological criteria, the loss of cardiolipin occurred in SSM that retained their integrity despite ischemic stress.

Cardiolipin is a phospholipid that is unique to mitochondria and resides predominantly in the inner mitochondrial membrane (21). Cardiolipin is enriched in oxidatively sensitive acyl residues, containing 90-95% linoleic acid (C18:2). Electron transport complexes (14, 19), especially cytochrome oxidase (11, 18, 39, 51), require cardiolipin for optimal activity. In addition to content, the activity of cytochrome oxidase is a function of the acyl group composition of cardiolipin. Cardiolipin that contains unsaturated acyl groups provides optimal cytochrome oxidase activity (1, 11, 51).

In the current study, ischemia decreased the content of cardiolipin only in SSM. The decrease in cardiolipin was selective to this phospholipid, because the content of the other phospholipids and total lipid phosphate were unaltered. The recovery of total lipid phosphate was accounted for as the sum of the four phospholipids measured, making the presence of additional phosphorous-containing compounds unlikely. The content of lysocardiolipin did not increase during ischemia. Potential mechanisms of cardiolipin loss in SSM during ischemia include phospholipase A2 hydrolysis (43) and oxidative damage (30, 35). The lack of an increase in the content of dilysocardiolipin does not suggest that phospholipid hydrolysis mediated by phospholipase A2 contributes to the observed ischemic damage to cardiolipin in SSM.

Cardiolipin, due to the presence of a high content of oxidatively sensitive C18:2 acyl groups, may sustain oxidative damage during ischemia as a mechanism of the decrease in content. Despite the low oxygen content, mitochondria produce reactive oxygen species during ischemia (7). Cardiolipin is located in proximity to electron transport complexes, including tightly bound association with electron transport complexes I (19), III (14), and IV (18). We examined the composition of cardiolipin after ischemia to answer whether an altered composition of cardiolipin, due to either the loss of oxidatively sensitive C18:2 acyl groups or the formation of peroxidized acyl groups, occurred during ischemia. The composition of cardiolipin that remains during ischemia is similar to that observed in nonischemic SSM based on both the C18:2-to-phosphate ratio and the component molecular species. The ratio of cardiolipin C18:2 groups to phosphate was similar, making the presence of appreciable amounts of undetected acyl groups unlikely. Oxidatively altered acyl groups were also not detected in cardiolipin during ischemia. However, oxidatively generated peroxy groups are unstable and may not be available for detection. Decomposition of lipid peroxides in cardiolipin can lead to the direct destruction of cardiolipin (30). Furthermore, oxidative alteration of phospholipids generates acyl residues that react directly with proteins, leading to covalent phospholipid-protein complexes that render the phospholipid no longer extractable by organic solvents (35). Thus oxidative processes can decrease cardiolipin content without the generation of lysocardiolipin or other detectable intermediates. As ischemia damages cardiolipin, the altered cardiolipin is degraded and lost, and the acyl group composition of the remaining cardiolipin remains unchanged.

Myocardial ischemia altered the phospholipid composition of mitochondrial membranes (13, 25, 29, 34, 45, 49). Mitochondrial cardiolipin content decreased during global ischemia in the isolated rat heart (13, 34) and during in vivo ischemia in the canine heart (49). These studies used a single population of cardiac mitochondria of uncertain regional origin. Cardiolipin content decreased in subsarcolemmal mitochondria but not in interfibrillar mitochondria after 60 min of regional ischemia in the canine heart (25). Mitochondrial cardiolipin content decreased after 60 min of ischemia in the brain (29) and 120 min of ischemia in the kidney (45). Similar to our findings in SSM, cytochrome oxidase activity decreased during cerebral ischemia at the time of the decrease in cardiolipin content (29).

Cytochrome oxidase activity requires the integrity of peptide subunits (8) and a lipid microenvironment in the inner membrane enriched in cardiolipin (11, 18, 27, 39, 51). A decrease in cardiolipin content decreases cytochrome oxidase activity in isolated membrane systems (1, 11, 39, 51). The content of cardiolipin decreased concomitant with the onset of decreased oxidative phosphorylation through cytochrome oxidase during the time course of ischemia in SSM. In IFM, both oxidative phosphorylation through cytochrome oxidase and cardiolipin content remained unaltered. These findings strongly suggest that cardiolipin loss is the mechanism of the defect in intact mitochondria. The outer lipid environment of cytochrome oxidase is exchangeable with detergents that can replete activity (11, 39, 51). The relief of the ischemic defect in cytochrome oxidase in SSM provided by detergent solubilization of SSM in our previous study (27) provides additional functional data supporting cardiolipin as the mechanism of the defect in cytochrome oxidase in SSM. Cytochrome oxidase activity also decreased after ischemia and reperfusion in the isolated rat heart (13). In the rat heart, fusion of mitochondria with cardiolipin-containing liposomes repleted cytochrome oxidase activity (34). Thus multiple lines of evidence support that ischemic damage to cardiolipin is the likely mechanism of the observed decrease in cytochrome oxidase activity in SSM.

The content of cytochrome c decreased in concert with the content of cardiolipin during the time course of ischemia in SSM (Table 4). Cardiolipin interacts with positively charged cytochrome c via the negatively charged head groups of the phospholipid (42). The negatively charged head groups of cardiolipin help to localize cytochrome c at membranes (42). The decrease in cardiolipin content during ischemia may lead to a decreased localization of cytochrome c at the inner mitochondrial membrane, perhaps contributing to the decreased content of cytochrome c observed in SSM during ischemia. Loss of cytochrome c, in turn, is likely to facilitate the execution of apoptotic programs in the myocyte (20).

SSM sustain a more rapid onset of ischemic damage than IFM (12, 25, 27, 48). The increased damage may occur secondary either to their location in the myocyte or due to an inherent susceptibility to damage during ischemia. Isolated SSM have a decreased capacity for calcium accumulation compared with IFM (32). Moreover, SSM release cytochrome c in response to calcium exposure, whereas IFM do not (32). This finding raises the possibility that SSM may be intrinsically more sensitive to ischemic damage than IFM.

Cardiac mitochondria contribute to myocyte injury by producing reactive oxygen species (2, 7, 9, 44). The observed decrease in cardiolipin content is a likely mechanism of the decrease in oxidative phosphorylation through cytochrome oxidase after 30 min of ischemia. A loss of cytochrome oxidase activity predisposes production of reactive oxygen species by the electron transport chain (10, 33, 38). During reperfusion after 30 min of ischemia in the rabbit heart, the electron transport chain generated reactive oxygen species, leading to oxidative tissue damage and contractile dysfunction (2). Cardiolipin loss in SSM represents a potential link between ischemic damage to the electron transport chain and mitochondrial-derived oxidative injury during reperfusion. SSM, via either oxidant generation or cytochrome c release, are likely the population of mitochondria that participate in mitochondrial-driven myocyte injury.


    ACKNOWLEDGEMENTS

We appreciate the technical assistance of Edwin Vazquez and Colleen King.


    FOOTNOTES

This material is based on work supported by the Office of Research and Development, Medical Research Service, Department of Veterans Affairs, and by National Institute of Aging Grant 1PO1 AG-15885 and Research Career Development Award 1K04 AG-00676 (to E. J. Lesnefsky).

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

Received 17 August 2000; accepted in final form 31 January 2001.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

1.   Abramovitch, DA, Marsh D, and Powell GL. Activation of beef-heart cytochrome c oxidase by cardiolipin and analogues of cardiolipin. Biochim Biophys Acta 1020: 34-42, 1990[Medline].

2.   Ambrosio, G, Zweier JL, Duilio C, Kuppusamy P, Santoro G, Elia PP, Tritto I, Crillo P, Condorelli M, Chiariello M, and Flaherty JT. 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.   Anthony, G, Reimann A, and Kadenbach B. Tissue-specific regulation of bovine heart cytochrome c oxidase activity by ADP via interaction with subunit VIa. Proc Natl Acad Sci USA 90: 1652-1656, 1993[Abstract/Free Full Text].

4.   Asimakis, GK, and Conti VR. Myocardial ischemia: correlation of mitochondrial adenine nucleotide and respiratory function. J Mol Cell Cardiol 16: 439-448, 1984[ISI][Medline].

5.   Babcock, GT, and Varotsis C. Discrete steps in dioxygen activation-the cytochrome oxidase/O2 reaction. J Bioenerg Biomembr 25: 71-80, 1993[ISI][Medline].

6.   Bartlett, GR. Phosphorous assay in column chromatography. J Biol Chem 234: 466-468, 1959[Free Full Text].

7.   Becker, LB, Vanden Hoek TL, Shao ZH, Li CQ, and 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.   Cooper, CE, Nichols P, and Freedman JA. Cytochrome c oxidase: structure, function, and membrane topology of the polypeptide subunits. Biochem Cell Biol 69: 586-607, 1990.

9.   Das, DK, George A, Liu X, and Rao PS. Detection of hydroxyl radical in the mitochondria of ischemic-reperfused myocardium by trapping with salicylate. Biochem Biophys Res Commun 165: 1004-1009, 1989[ISI][Medline].

10.   Dawson, TL, Gores GL, Nieninen AL, Herman B, and Lemasters JJ. Mitochondria as a a source of reactive oxygen species during reductive stress in rat hepatocytes. Am J Physiol Cell Physiol 264: C961-C966, 1993[Abstract/Free Full Text].

11.   DeCuyper, M, and Joniau M. Behaviour of beef-heart cytochrome c oxidase in reconstituted proteovesicles. Eur J Biochem 104: 397-405, 1980[ISI][Medline].

12.   Duan, J, and Karmazyn M. Relationship between oxidative phosphorylation and adenine nucleotide translocase activity of two populations of cardiac mitochondria and mechanical recovery of ischemic hearts following reperfusion. Can J Physiol Pharmacol 67: 704-709, 1989[ISI][Medline].

13.   Edoute, Y, van der Merwe E, Sanan D, Kotze JCN, Steinmann C, and Lochner A. Normothermic ischemic cardiac arrest of the isolated working rat heart. Circ Res 53: 663-678, 1983[Abstract/Free Full Text].

14.   Gomez, B, and Robinson NC. Phospholipase digestion of bound cardiolipin reversibly inactivitates bovine cytochrome bc1. Biochemistry 38: 9031-9038, 1999[Medline].

15.   Fannin, SW, Lesnefsky EJ, Slabe TJ, Hassan MO, and Hoppel CL. Aging selectively decrease oxidative capacity in rat heart interfibrillar mitochondria. Arch Biochem Biophys 373: 399-407, 1999.

16.   Flameng, W, Andres J, Ferdinande P, Mattheussen M, and Van Belle H. Mitochondrial function in myocardial stunning. J Mol Cell Cardiol 23: 1-11, 1991[ISI][Medline].

17.   Folch, J, Lees M, and Sloane Stanley GH. A simple method for the isolation and purification of total lipids from animal tissues. J Biol Chem 226: 497-509, 1957[Free Full Text].

18.   Fry, M, Blondin GA, and Green DE. The localization of tightly bound cardiolipin in cytochrome oxidase. J Biol Chem 255: 9967-9970, 1980[Abstract/Free Full Text].

19.   Fry, M, and Green DE. Cardiolipin requirement for electron transfer in complex I and III of the mitochondrial respiratory chain. J Biol Chem 256: 1874-1880, 1981[Abstract/Free Full Text].

20.   Green, DR, and Reed JC. Mitochondria and apoptosis. Science 281: 1309-1316, 1998[Abstract/Free Full Text].

21.   Hoch, FL. Cardiolipins and biomembrane function. Biochim Biophys Acta 1113: 71-133, 1992[Medline].

22.   Hoppel, CL, Tandler B, Parland W, Turkaly JS, and Albers LD. Hamster cardiomyopathy: a defect in oxidative phosphorylation in the cardiac interfibrillar mitochondria. J Biol Chem 257: 1540-1548, 1982[Free Full Text].

23.   Ingalls, ST, Kriaris MS, Xu Y, DeWulf DW, Tserng KY, and Hoppel CL. Method for isolation of non-esterified fatty acids and several other classes of plasma lipids by column chromatography on silica gel. J Chromatogr A 619: 9-19, 1993.

24.   Ingalls, ST, Xu Y, and Hoppel CL. Determination of plasma non-esterified fatty acids and triglyceride fatty acids by gas chromatography of their methyl esters after isolation by column chromatography on silica gel. J Chromatogr B Biomed Appl 666: 1-12, 1995[ISI][Medline].

25.   Kayiyama, K, Pauly DF, Hughes H, Yoon BY, Entman ML, and McMillin-Wood JB. Protection by verapamil of mitochondrial glutathione equilibrium and phospholipid changes during reperfusion of ischemic canine myocardium. Circ Res 61: 301-310, 1987[Abstract/Free Full Text].

26.   Lesnefsky, EJ, Stoll MSK, Minkler PE, and Hoppel CL. Separation and quantitation of phospholipids and lysophospholipids by high performance liquid chromatography. Anal Biochem 285: 256-254, 2000.

27.   Lesnefsky, EJ, Tandler B, Ye J, Slabe TJ, Turkaly J, and 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].

28.   Lesnefsky, EJ, and Ye J. Exogenous intracellular, but not extracellular, iron augments myocardial oxidative injury. Am J Physiol Heart Circ Physiol 266: H384-H392, 1994[Abstract/Free Full Text].

29.   Nakahara, I, Kikuchi H, Take W, Nishi S, Kito M, Yonekawa Y, Goto Y, and Ogata N. Degradation of mitochondrial phospholipids during experimental cerebral ischemia in rats. J Neurochem 57: 839-844, 1991[ISI][Medline].

30.   O'Brien, PJ. Intracellular mechanisms for the decomposition of a lipid peroxide. I. Decomposition of a lipid peroxide by metal ions, heme compounds, and nucleophiles. Can J Biochem 47: 485-499, 1969[ISI][Medline].

31.   Palmer, JW, Tandler B, and 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].

32.   Palmer, JW, Tandler B, and Hoppel CL. Heterogeneous response of subsarcolemmal heart mitochondria to calcium. Am J Physiol Heart Circ Physiol 250: H741-H748, 1986[Abstract/Free Full Text].

33.   Papa, S, Guerrieri F, and Capitanio N. A possible role of slips in cytochrome c oxidase in the antioxygen defense system of the cell. Biosci Rep 17: 23-31, 1997[ISI][Medline].

34.   Paradies, G, Petrosillo G, Pistolese M, DiVenosa N, Serena D, and Ruggiero FM. Lipid peroxidation and alterations to oxidative metabolism in mitochondria isolated from rat heart subjected to ischemia and reperfusion. Free Radic Biol Med 27: 42-50, 1999[ISI][Medline].

35.   Parinandi, NL, Zwizinski CW, and Schmid HHO Free radical-induced alterations of myocardial membrane proteins. Arch Biochem Biophys 289: 118-123, 1991[ISI][Medline].

36.   Piper, HM, Sezer O, Schleyer M, Schwartz P, Hutter JF, and Spieckermann PG. Development of ischemia-induced damage in defined mitochondrial subpopulations. J Mol Cell Cardiol 17: 885-896, 1985[ISI][Medline].

37.   Powell, GL, Knowles PF, and Marsh D. Spin-label studies on the specificity of interaction of cardiolipin with beef heart cytochrome oxidase. Biochemistry 26: 8138-8145, 1987[Medline].

38.   Qian, T, Nieminen AL, Herman B, and Lemasters JJ. Mitochondrial permeability transition in pH-dependent reperfusion injury to rat hepatocytes. Am J Physiol Cell Physiol 273: C1783-C1792, 1997[Abstract/Free Full Text].

39.   Robinson, NC, Strey F, and Talbert L. Investigation of the essential boundary layer phospholipids of cytochrome c oxidase using Triton X-100 delipidation. Biochemistry 19: 3656-3661, 1980[Medline].

40.   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.

41.   Rouslin, W, Broge CW, and Grupp IL. ATP depletion and mitochondrial functional loss during ischemia in slow and fast heart-rate hearts. Am J Physiol Heart Circ Physiol 259: H1759-H1766, 1990[Abstract/Free Full Text].

42.   Rytomaa, M, and Kinnunen PKJ Evidence for two distinct acidic phospholipid-binding sites in cytochrome c. J Biol Chem 269: 1770-1774, 1994[Abstract/Free Full Text].

43.   Sevanian, A, and Kim E. Phospholipase A2 dependent release of fatty acids from peroxidized membranes. Free Radic Biol Med 1: 263-271, 1985.

44.   Shlafer, M, Gallagher KP, and Adkins S. Hydrogen peroxide generation by mitochondria isolated from regionally ischemic and nonischemic dog myocardium. Basic Res Cardiol 85: 318-329, 1990[ISI][Medline].

45.   Smith, MW, Collan Y, Kahng MW, and Trump BF. Changes in mitochondrial lipids of rat kidney during ischemia. Biochim Biophys Acta 618: 192-201, 1980[Medline].

46.   Steele, RGD, and Torrie JH. Principles and Procedures of Statistics. New York: McGraw-Hill, 1960.

47.   Teng, JI, and Smith LL. High-performance liquid chromatography of cardiolipin. J Chromatogr A 339: 35-44, 1985.

48.   Ueta, H, Ogura R, Sugiyama M, Kagiyama A, and Shin G. Spin trapping on cardiac submitochondrial particles isolated from ischemic and non-ischemic myocardium. J Mol Cell Cardiol 22: 893-899, 1990[ISI][Medline].

49.   Vasdev, SC, and Biro GP. Membrane changes induced by early myocardial ischemia in the dog. Can J Biochem 58: 1112-1119, 1980[ISI][Medline].

50.   Veitch, KA, Hombroeckx A, Caucheteux D, Pouleur H, and Hue L. Global ischemia induces a biphasic response of the mitochondrial respiratory chain. Biochem J 281: 709-715, 1992.

51.   Vik, SB, and Capaldi RA. Lipid requirements for cytochrome c oxidase activity. Biochemistry 16: 5755-5759, 1977[Medline].

Am J Physiol Heart Circ Physiol 280(6):H2770-H2778


This article has been cited by other articles:


Home page
 J. Lipid Res.Home page
G. C. Sparagna, A. J. Chicco, R. C. Murphy, M. R. Bristow, C. A. Johnson, M. L. Rees, M. L. Maxey, S. A. McCune, and R. L. Moore
Loss of cardiac tetralinoleoyl cardiolipin in human and experimental heart failure
J. Lipid Res., July 1, 2007; 48(7): 1559 - 1570.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Physiol. Heart Circ. Physiol.Home page
P. A. Watson, J. E. B. Reusch, S. A. McCune, L. A. Leinwand, S. W. Luckey, J. P. Konhilas, D. A. Brown, A. J. Chicco, G. C. Sparagna, C. S. Long, et al.
Restoration of CREB function is linked to completion and stabilization of adaptive cardiac hypertrophy in response to exercise
Am J Physiol Heart Circ Physiol, July 1, 2007; 293(1): H246 - H259.
[Abstract] [Full Text] [PDF]

Home page
 J. Biol. Chem.Home page
E. Robin, R. D. Guzy, G. Loor, H. Iwase, G. B. Waypa, J. D. Marks, T. L. V. Hoek, and P. T. Schumacker
Oxidant Stress during Simulated Ischemia Primes Cardiomyocytes for Cell Death during Reperfusion
J. Biol. Chem., June 29, 2007; 282(26): 19133 - 19143.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Physiol. Heart Circ. Physiol.Home page
Z. Makazan, H. K. Saini, and N. S. Dhalla
Role of oxidative stress in alterations of mitochondrial function in ischemic-reperfused hearts
Am J Physiol Heart Circ Physiol, April 1, 2007; 292(4): H1986 - H1994.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Physiol. Cell Physiol.Home page
A. J. Chicco and G. C. Sparagna
Role of cardiolipin alterations in mitochondrial dysfunction and disease
Am J Physiol Cell Physiol, January 1, 2007; 292(1): C33 - C44.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Physiol. Cell Physiol.Home page
Q. Chen, A. K. S. Camara, D. F. Stowe, C. L. Hoppel, and E. J. Lesnefsky
Modulation of electron transport protects cardiac mitochondria and decreases myocardial injury during ischemia and reperfusion
Am J Physiol Cell Physiol, January 1, 2007; 292(1): C137 - C147.
[Abstract] [Full Text] [PDF]

Home page
 J. Lipid Res.Home page
G. C. Sparagna, C. A. Johnson, S. A. McCune, R. L. Moore, and R. C. Murphy
Quantitation of cardiolipin molecular species in spontaneously hypertensive heart failure rats using electrospray ionization mass spectrometry
J. Lipid Res., June 1, 2005; 46(6): 1196 - 1204.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Physiol. Heart Circ. Physiol.Home page
S. Ghosh, D. Qi, D. An, T. Pulinilkunnil, A. Abrahani, K.-H. Kuo, R. B. Wambolt, M. Allard, S. M. Innis, and B. Rodrigues
Brief episode of STZ-induced hyperglycemia produces cardiac abnormalities in rats fed a diet rich in n-6 PUFA
Am J Physiol Heart Circ Physiol, December 1, 2004; 287(6): H2518 - H2527.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Physiol. Heart Circ. Physiol.Home page
E. J. Lesnefsky, Q. Chen, T. J. Slabe, M. S. K. Stoll, P. E. Minkler, M. O. Hassan, B. Tandler, and C. L. Hoppel
Ischemia, rather than reperfusion, inhibits respiration through cytochrome oxidase in the isolated, perfused rabbit heart: role of cardiolipin
Am J Physiol Heart Circ Physiol, July 1, 2004; 287(1): H258 - H267.
[Abstract] [Full Text] [PDF]

Home page
 Circ. Res.Home page
G. Paradies, G. Petrosillo, M. Pistolese, N. Di Venosa, A. Federici, and F. M. Ruggiero
Decrease in Mitochondrial Complex I Activity in Ischemic/Reperfused Rat Heart: Involvement of Reactive Oxygen Species and Cardiolipin
Circ. Res., January 9, 2004; 94(1): 53 - 59.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Physiol. Heart Circ. Physiol.Home page
J. Levraut, H. Iwase, Z.-H. Shao, T. L. Vanden Hoek, and P. T. Schumacker
Cell death during ischemia: relationship to mitochondrial depolarization and ROS generation
Am J Physiol Heart Circ Physiol, February 1, 2003; 284(2): H549 - H558.
[Abstract] [Full Text] [PDF]

Home page
 J. Biol. Chem.Home page
D. B. Ostrander, G. C. Sparagna, A. A. Amoscato, J. B. McMillin, and W. Dowhan
Decreased Cardiolipin Synthesis Corresponds with Cytochrome c Release in Palmitate-induced Cardiomyocyte Apoptosis
J. Biol. Chem., October 5, 2001; 276(41): 38061 - 38067.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
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 ISI 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 ISI Web of Science (46)
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Lesnefsky, E. J.