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Effects of fatty acids in isolated mitochondria: implications for ischemic injury and cardioprotection

Cardiovascular Research Laboratory, Departments of Medicine (Cardiology) and Physiology, David Geffen School of Medicine, University of California at Los Angeles, California 90095-1760

Submitted 3 December 2002 ; accepted in final form 27 February 2003

	ABSTRACT

TOP ABSTRACT EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Fatty acids accumulate during myocardial ischemia and are implicated in ischemia-reperfusion injury and mitochondrial dysfunction. Because functional recovery after ischemia-reperfusion ultimately depends on the ability of the mitochondria to recover membrane potential (_m), we studied the effects of fatty acids on _m regulation, cytochrome c release, and Ca²⁺ handling in isolated mitochondria under conditions that mimicked aspects of ischemia-reperfusion. Long-chain but not short-chain free fatty acids caused a progressive and reversible (with BSA) increase in inner membrane leakiness (proton leak), which limited mitochondrial ability to support _m. In comparison, long-chain activated fatty acids promoted 1) a slower depolarization that was not reversible with BSA, 2) cytochrome c loss that was unrelated to permeability transition pore opening, and 3) inhibition of the adenine nucleotide translocator. Together, these results impaired both mitochondrial ATP production and Ca²⁺ handling. Diazoxide, a selective opener of mitochondrial ATP-dependent potassium (K_ATP) channels, partially protected against these effects. These findings indicate that long-chain fatty acid accumulation during ischemia-reperfusion may predispose mitochondria to cytochrome c loss and irreversible injury and identify a novel cardioprotective action of diazoxide.

calcium; adenine nucleotide translocator; membrane potential; cytochrome c; ATP-dependent channel; palmitic acid; palmitoyl-coenzyme A

We focused on how fatty acids affect the ability of mitochondria to regulate membrane potential (_m). As the dominant component of the proton electrochemical gradient, _m represents the interconvertible energy source that is absolutely required for aerobic ATP production in cardiac myocytes. Ischemia-reperfusion causes changes in mitochondrial structure and function that can either be reversible or lead to cell death by necrosis or apoptosis (for reviews, see Refs. 7, 15, 28). The functional significance of these changes is revealed after reoxygenation, at which point mitochondrial viability is determined by the ability to recover _m. During ischemia, the ability of mitochondria to support _m gradually decreases. As this occurs, mitochondria place the highest priority on the maintenance of _m even at the expense of consuming rather than producing ATP. In the progression of ischemic mitochondrial injury, the intermembrane space (35) and complex I (at 15 min) seem to be the most sensitive sites, which are followed by complexes III and IV and decreases in cardiolipin and protein sulfhydryl content (at 30–40 min; Ref. 28). By still-unknown mechanisms, these changes lead to increased inner membrane leakiness, cytochrome c release, and electron transport inhibition, which promote the collapse of _m. Eventually, mitochondria pass a point at which low _m and elevated Ca²⁺ and P_i concentrations ([Ca²⁺] and [P_i], respectively) strongly favor PTP opening, especially during reperfusion (8, 13, 18) when the rapid reversal of the matrix pH to >7.0 and the burst of reactive oxygen species (ROS) dramatically increase PTP open probability (14, 15).

In this study, we show that fatty acids are strong candidates for inducing these changes in membrane leakiness, cytochrome c loss, and electron transport inhibition that precede and make mitochondria increasingly vulnerable to PTP opening. Long-chain but not short-chain fatty acids caused increased membrane leakiness. Long-chain activated fatty acids but not long-chain free fatty acids promoted cytochrome c loss, inhibition of ATP synthesis, and impaired Ca²⁺ uptake in a manner that could not be reversed by chelating fatty acids with BSA. A key finding is that at the lower range of concentrations, these effects occurred independently of cyclosporin-sensitive PTP opening. In addition, the effects of long-chain activated fatty acids on cytochrome c loss were prevented by diazoxide, thereby identifying a novel mechanism by which this drug may exert cardioprotective effects.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Isolation of Mitochondria

Mitochondria were isolated from adult rabbit hearts by enzymatic digestion, homogenization, and differential centrifugation as described previously (22). Isolated mitochondria were resuspended in EGTA-free homogenization buffer (250 mM sucrose, 10 mM HEPES, pH 7.4 with Tris) to yield 30–50 mg/ml of mitochondrial protein. The protein was kept on ice and was normally used within 5 h (in some experiments, within 8 h) after isolation. Freshly isolated mitochondria had coupling ratios ≥8 (in KCl buffer with site I substrates and 0.4 mM ADP). The mitochondria released 0.39 ± 0.04 nmol cytochrome c/mg of protein (n = 4) after hypotonic treatment; this was followed by KCl addition to 200 mM before centrifugation. This indicated that the majority had intact outer membranes or that cytochrome c remained bound to inner membranes.

Experimental Conditions

All measurements except cytochrome c determination were carried out using a fiber-optic spectrofluorometer (Ocean Optics) in a closed, continuously stirred cuvette at room temperature (22–24°C). Mitochondria (0.2–0.6 mg/ml) were added in the cuvette to standard buffer that consisted of 135 mM KCl, 10 mM HEPES, pH 7.4, with KOH. In some experiments, buffer contained 250 mM sucrose in place of KCl, 10 mM HEPES, and pH 7.4 with Tris. Substrates, Ca²⁺, P_i, EGTA, various drugs, and fluorescent indicators were added in the concentrations indicated. For the anoxia-reoxygenation experiments, energized or nonenergized mitochondria were made anoxic by directing a stream of nitrogen through the hole in the cuvette cover and aiming at the buffer (2 ml) so that the stirred buffer had no contact with the air (22). Reoxygenation was accomplished by substituting nitrogen with oxygen (95% O₂-5% CO₂). The PO₂ in the buffer was continuously recorded via a fiber-optic oxygen sensor that was inserted through the same hole.

Spectrofluoremetric Techniques and Other Assays

Mitochondrial _m. Tetramethylrhodamine methyl ester (TMRM, 400 nM) was included in the cuvette solution, and _m was estimated from TMRM fluorescence at 580 nm as described previously (22). The _m was expressed as a percentage of the TMRM fluorescence in the presence of coupled mitochondria and substrates (100%) relative to that after addition of 0.5 µM FCCP to fully depolarize the mitochondria (0%). TMRM fluorescence emission was recorded simultaneously with PO₂.

Mitochondrial Ca²⁺ uptake and efflux. Changes in extramitochondrial [Ca²⁺] were identified by measuring Calcium Green-5N (1 µM, salt form) fluorescence at excitation and emission wavelengths of 475 and 515 nm, respectively. The [Ca²⁺] was calibrated by adding known amounts of Ca²⁺ to the buffer in the presence of mitochondria and FCCP to block Ca²⁺ uptake.

Mitochondrial ATP synthesis. Mitochondrial ATP synthesis was determined as described previously (27) with slight modifications. We used a coupled enzyme assay with continuous monitoring of the reduction of NADP. In this assay, the increase in fluorescence is proportional to the increase in ATP concentration ([ATP]) generated in mitochondria and used in hexokinase/glucose-6-phospate dehydrogenase (G-6-PDH) reactions to generate fluorescent NADPH. Mitochondria were added to KCl buffer that contained 1 mM NADP, 10 mM glucose, 10 U/ml hexokinase, and 5 U/ml G-6-PDH. ATP synthesis was initiated by addition of 100 µM ADP. The _m/ATP synthase-dependent ATP production, relative to that by myokinase, was defined by the extent of inhibition of ATP production with FCCP (0.5 µM) or oligomyocin (10 µM).

Cytochrome c release. Changes in extramitochondrial cytochrome c were recorded as described previously (1) by monitoring the Soret () peak at 414 nm in the spectrum of cytochrome c. Mitochondria (1 mg/ml) were incubated in KCl or sucrose buffer under various experimental conditions. After incubation, mitochondria were centrifuged at 13,000 g for 10 min, and supernatants were filtered through a 0.2-µm Millipore membrane. The optical density of clear supernatants was scanned from 300 to 700 nm using a Shimadzu UV 2101PC spectrophotometer. Release of cytochrome c into the medium was quantified by measurement of the increase in the 414-nm peak over the baseline after addition of a known amount of cytochrome c. In our experiments, cytochrome c concentrations ranged from 0.2 to 1.0 µM, but a linear relationship between optical density and cytochrome c concentration was observed at least up to 10 µM.

Mitochondrial protein. Protein content was determined using the Lowry method.

Chemicals and Data Analysis

Cyclosporin A (CsA) was a generous gift of Ciba-Geigy. Fluorescent dyes were purchased from Molecular Probes, and all other chemicals were from Sigma. Mitochondrial substrates were added as free acids using Tris · HCl to buffer the pH.

Results are presented as original tracings with summary data as means ± SD. Student's t-test was used to assess statistical significance with the Bonferoni correction for more than two groups.

	RESULTS

TOP ABSTRACT EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Long-Chain Free Fatty Acids Cause _m Dissipation That Is Reversible with BSA

Fatty acids are known to accumulate during ischemia (2) and have been implicated in ischemia-reperfusion injury (28). We therefore examined the effects of short-, medium-, and long-chain fatty acids in isolated energized mitochondria. Short- and medium-chain free fatty acids (C ≤ 10), which do not require the carnitine transport system to enter the matrix, had no significant effect on _m even when added in high concentrations to energized mitochondria in KCl buffer (Fig. 1A). As chain length increased, however, free fatty acids started to dissipate _m. Capric acid (C10, 50 µM) induced a small _m dissipation, but lauric acid (C12, 50 µM) caused major _m dissipation (Fig. 1A). The saturated free fatty acid palmitic acid (C16) as well as the polyunsaturated fatty acids linoleic (C18), oleic (C18), arachidonic (C20), and docosahexaenoic (C22) acids all promoted rapid _m dissipation when added to energized mitochondria in low micromolar concentrations (as shown for linoleic acid in Fig. 1B). This response occurred in either KCl or sucrose buffer (Fig. 1C), and _m dissipation was rapidly and fully reversible with BSA (e.g., Fig. 1, A and B).

View larger version (44K): [in this window] [in a new window]   Fig. 1. Medium- and long-chain (but not short chain) fatty acids dissipate membrane potential (m) in a manner readily reversible with BSA. Fresh mitochondria (0.3 mg/ml) were added to KCl (A, B) or sucrose (C) buffer, followed by 5 mM Pi and site I substrates (Mal, Pyr, Glu). Arrows indicate when 100 µM caproic (C-6), 100 µM caprilic (C-8), and 50 µM capric (C-10) acid were added; this was followed by 100 µM caproic acid and had minimal effect on m (A). Addition of 50 µM lauric acid (C-12) promoted rapid and significant m dissipation and further acceleration of O2 consumption (O2) that was reversible with 7 µM BSA pulses. Mitochondria were suspended in either KCl (B) or sucrose (C) buffer and challenged with linoleic acid pulses (1 µM). Pulses resulted in m dissipation and increased O2, which was rapidly reversible by addition of 7 µM BSA. Dissipation of m with linoleoyl-CoA (Lin-CoA) and its reversibility depend on buffer. Fresh mitochondria (0.3 mg/ml) were suspended in KCl (D) or sucrose (E) buffer; these were followed by addition of 5 mM Pi, site I substrates, and Lin-CoA pulses of either 2 (D) or 10 µM (E). With 7 µM BSA in sucrose buffer, m recovered fully; but in KCl buffer, cytochrome c (Cyt c) was also required. Note also the significant increase in O2 after cytochrome c addition. Average changes ± SD for 7 or 8 determinations in 2 different preparations are shown (F).

Long-Chain Activated Fatty Acids Cause Cytochrome c Loss and _m Dissipation That Is Not Reversible with BSA Alone

In contrast with linoleic, arachidonic, and palmitic acids, the activated forms of these free fatty acids, linoleoyl-CoA (Lin-CoA), arachidonoyl-CoA (Ara-CoA), and palmitoyl-CoA (Pal-CoA), also caused _m dissipation; however, it was not fully reversible by BSA either alone or in combination with other additions including succinate (Fig. 1D), EGTA, and CsA. Note also that at low Lin-CoA concentrations ([Lin-CoA]), _m was relatively stable (Fig. 1D) compared to addition of its free fatty acid counterpart linoleic acid (Fig. 1B). After addition of BSA and cytochrome c (5 µM), _m recovered fully in parallel with the increase in O₂ consumption rate (O₂; Fig. 1D). In control experiments, addition of exogenous cytochrome c had no significant effect on O₂ as is generally found to be the case when the outer membrane is intact (26). These results suggest that in KCl medium, activated fatty acids promote changes that lead to cytochrome c loss in addition to _m dissipation. Furthermore, this mode of cytochrome c loss occurred in the absence of PTP opening, because CsA was not required to regenerate _m. In addition, when the above experiment was repeated in sucrose buffer (250 mM sucrose, 10 mM HEPES, and pH 7.4 with Tris · HCl) or in KCl buffer supplemented with polyethylene glycol (mol wt 1,450) to stabilize inner and outer membrane interaction at contact sites (10), BSA alone reversed _m dissipation (Fig. 1E). Also, in the sucrose buffer a much higher [Lin-CoA] was required to dissipate _m (Fig. 1E). Average values of _m recovery after addition of BSA and cytochrome c in KCl buffer and BSA alone in sucrose buffer are presented in Fig. 1F.

Similar effects of long-chain activated fatty acids were observed in mitochondria that respired on endogenous substrates alone. Initial _m dissipation was again more rapid with palmitic acid (Fig. 2A) than the equivalent amount of Pal-CoA (Fig. 2B). However, after _m dissipation by palmitic acid (two 2.5-µM pulses), _m rapidly and fully recovered on the addition of BSA and substrates (Fig. 2A), whereas cytochrome c was required to fully restore the _m dissipated by Pal-CoA (Fig. 2B).

View larger version (29K): [in this window] [in a new window]   Fig. 2. In mitochondria (Mit) respiring on endogenous substrates, 2.5 µM palmitic acid (Palm) pulses were added after 5 mM Pi dissipated m (A). After fatty acids were bound with BSA (7 µM) and site I substrates (I) were added (1.5 mM each of pyruvate, malate, and glutamate), m fully recovered. When m dissipated with palmitoyl-CoA (Pal-CoA pulses, 2.5 µM each), however, it did not fully recover unless cytochrome c (5 µM) was added (B). Other additions that failed to promote m recovery before cytochrome c addition include site I substrates succinate (II, 5 mM) and cyclosporin A (CSA, 1.5 µM). FCCP (0.5 µM) was added at the end for calibration purposes. Ca2+ uptake by mitochondria that respired on endogenous substrates is shown. Mitochondria (0.7 mg/ml) were incubated in KCl buffer with 5 mM Pi after which 5 µM Ca2+ was added. In the absence of Pal-CoA (C), mitochondria took up the added Ca2+. In the presence of Pal-CoA (D; 2.5 µM), mitochondria released Ca2+ after a transient uptake, but addition of BSA (7 µM) and site I substrates restored uptake capacity. In the presence of Pal-CoA (6 µM) sufficient to dissipate m (E), however, Ca2+ uptake was not restored by adding BSA, site I and site IV (IV) substrates, or CsA (1.5 µM) unless cytochrome c (5 µM) was also added. These results were confirmed in three different preparations. [Ca2+]o, extramitochondrial Ca2+ concentration.

Figure 3 documents directly that Pal-CoA induced cytochrome c loss; the distinct peak at 414 nm in the spectrum of cytochrome c was used to monitor its concentration in the buffer (Fig. 3A). Figure 3B shows that although incubation of mitochondria in KCl buffer with 15 µM palmitic acid and P_i (as in Fig. 2A) for 9–10 min caused only a minimal increase in cytochrome c in the buffer, incubation with 15 µM Pal-CoA (as in Fig. 2B) caused a significant increase. However, in sucrose buffer, Pal-CoA promoted only a small release of cytochrome c.

View larger version (27K): [in this window] [in a new window]   Fig. 3. Effects of fatty acids on cytochrome c loss by isolated mitochondria. Calibration of cytochrome c assay (A). Absorbance spectra (Abs) were recorded after addition of cytochrome c (0.2–1.0 µM) to KCl (135 mM) buffer that contained 5 mM Pi. Plot of optical density (O.D.) at 414 nm against cytochrome c concentration is shown (inset). Similar results were obtained in sucrose buffer. Mitochondria (1 mg/ml) were incubated (B) in KCl buffer with 5 mM Pi and either 15 µM palmitic acid (tracings 1) or 15 µM Pal-CoA (tracings 2) or 15 µM Pal-CoA in sucrose buffer with 5 mM Pi (trace 3). After a 9-min incubation at room temperature, suspensions were centrifuged, filtrated, kept on ice, and scanned. Summary data (means ± SD for the number indicated) for cytochrome c release are shown (inset).

Pal-CoA also affected mitochondrial Ca²⁺ uptake presumably via its effects on _m (Fig. 2, C-E). Figure 2C shows that after incubation with 5 mM P_i, the added 5 µM Ca²⁺ was nearly completely accumulated by mitochondria. When 2.5 µM Pal-CoA was given before the Ca²⁺ pulse, contaminant Ca²⁺ in the buffer decreased consistent with an increase in _m (Fig. 2D). Subsequent addition of Ca²⁺ promoted Ca²⁺ uptake followed by Ca²⁺ release, which recovered when site I substrates were added. When Ca²⁺ was added after 6 µM Pal-CoA (Fig. 2E), however, substrates alone were ineffective, and Ca²⁺ reuptake required both CsA and cytochrome c, which indicates that PTP opening had occurred.

Effect of Fatty Acids on Mitochondrial ATP Production

Figure 4 shows the effects of fatty acids on the rate of ATP synthesis, measured by the rate of NADPH production in a coupled hexokinase/G-6-PDH assay. Under control conditions (Fig. 4A), ATP production by mitochondria suspended in KCl buffer and energized with site I substrates increased rapidly on the addition of ADP and was abolished by FCCP (Fig. 4B); this indicates that _m-dependent ATP production from electron transport rather than adenylate kinase activity was responsible. Caproic acid, a short-chain free fatty acid, had no effect on mitochondrial ATP production even when added in concentrations of 300 µM (Fig. 4C). In contrast, at 10 µM, the long-chain free fatty acid linoleic acid significantly depressed ATP production, which was rapidly reversible with BSA (Fig. 4D). The same concentration of Lin-CoA caused less depression of the ATP synthesis rate, which is consistent with its less-depolarizing effect on _m, but its effects were not reversed by BSA. Further increasing [Lin-CoA] to 15 µM resulted in a marked depression of ATP production that could not be reversed by BSA (Fig. 4F).

View larger version (34K): [in this window] [in a new window]   Fig. 4. Long-chain fatty acids inhibit mitochondrial ATP generation. Mitochondria (0.5 mg/ml) were suspended in KCl buffer and subsequently 5 mM Pi, site I substrates (Pyr, Mal, Glu), and the reaction mixture (R) for enzymatic determination of ATP production, and ADP (100 µM) was added as indicated. Control (A). Addition of FCCP (1 µM) before ADP (B) dissipated m. A small amount of ATP was added to check the reaction mixture response. Short-chain free fatty acid caproic acid (C-6, 300 µM) had no effect on mitochondrial ATP production (C). Long-chain free fatty acid linoleic acid (10 µM) inhibited ATP production, which was subsequently disinhibited by addition of BSA (7 µM; D). Long-chain activated fatty acid Lin-CoA (10 µM) significantly inhibited ATP production, which was not relieved by addition of BSA (7 µM; E). Lin-CoA (15 µM) almost completely inhibited ATP production, which was not reversed by addition of BSA (F). All results in A-F were confirmed in two or three different mitochondrial preparations. Pretreatment with diazoxide (Diaz) partially protected energized mitochondria from inhibition of ATP production by Lin-CoA effect and thereby allowed some recovery after BSA addition (G). Mitochondria (0.4 mg/ml) were added to KCl buffer that contained diazoxide (50 µM). Including 5-hydroxydecanoic acid (5-HD, 200 µM) prevented diazoxide from protecting ATP production (H). Average values of ATP synthesis rate in the presence of diazoxide or diazoxide with 5-HD relative to the rate under control conditions (100%) are as in A for 6 or 7 experiments with two different preparations (I).

Diazoxide Protects against Cytochrome c Loss and Inhibition of ATP Synthesis by Long-Chain Activated Fatty Acids

Diazoxide has been shown to preserve ATP production in mitochondria isolated after anoxia-reoxygenation (32) or ischemia-reperfusion (11). We therefore examined whether diazoxide protected isolated mitochondria against depression of mitochondrial ATP production and cytochrome c loss by long-chain activated fatty acids. Figure 4G shows that in the presence of 50 µM diazoxide, 15 µM Lin-CoA caused comparable suppression of ATP production (compare to Fig. 4F), but BSA was then able to partially reverse the effects of Lin-CoA, which implies that diazoxide attenuated cytochrome c loss or improved ADP/ATP exchange. The protective effect of diazoxide was blocked with 200 µM 5-hydroxydecanoic acid (5-HD; Fig. 4H). The findings are summarized in Fig. 4I (using the average ATP production rate under control conditions as 100%). Thus diazoxide partially protected isolated mitochondria that were exposed to Lin-CoA from reduced ATP production.

Figure 5 shows that the preservation of ATP production by diazoxide could be attributed at least in part to prevention of cytochrome c loss. In Fig. 5A, mitochondria energized with site IV substrates exposed to Ara-CoA pulses developed progressive _m dissipation that could not be reversed with BSA alone and required 5 µM cytochrome c for full recovery. When the same experiment was repeated in the presence of diazoxide (Fig. 5B), _m dissipation was attenuated and it then recovered completely with BSA alone, which suggests that cytochrome c loss had been averted. In contrast, 5-HD had no significant effect on _m in energized mitochondria, but accelerated the Ara-CoA-induced _m dissipation (Fig. 5C). Diazoxide was similarly protective against _m dissipation and cytochrome c loss induced by Lin-CoA in site I energized mitochondria. In the presence of 5-HD, the time to reach half-maximal _m dissipation in response to 10 µM Lin-CoA was 47 ± 19% (n = 6) of that in the presence of diazoxide.

View larger version (32K): [in this window] [in a new window]   Fig. 5. Dissipation of m and loss of cytochrome c by long-chain activated fatty acids are significantly inhibited by diazoxide or SOD with catalase. Mitochondria (0.3 mg/ml) were suspended in KCl buffer and 5 mM Pi and site IV substrates that consisted of ascorbic acid (AA, 2 mM) and N,N,N',N'-tetramethyl-p-phenylenediamine (TMPD, 0.2 mM) were added, which was followed by addition of arachidonoyl-CoA (Ara-CoA, in 0.5-µM pulses), BSA (1.5 µM/1 µM Ara-CoA), and cytochrome c (5 µM) where indicated. At the end, FCCP was added for calibration. Dissipation of m occurred after six Ara-CoA pulses, and full recovery required both BSA and cytochrome c (A). Diazoxide (100 µM) was added as indicated, and mitochondria were challenged with six Ara-CoA pulses (B). Again, almost full recovery was achieved with BSA alone. Addition of 5-HD (200 µM) as indicated was followed by three Ara-CoA pulses; 5 µM cytochrome c as well as BSA were required for m recovery (C). Buffer contained SOD (150 U/ml) and catalase (200 U/ml); mitochondria challenged with eight Ara-CoA pulses showed minimal m dissipation and recovered fully with BSA alone (D). Buffer contained SOD and catalase (Cat), and 5-HD (200 µM) was added as indicated followed by eight Ara-CoA pulses (E). Full recovery was then achieved with BSA alone. Bar graph summarizes m recovery (F) after BSA in the presence of 5-HD (C), diazoxide (B), and 5-HD with SOD and catalase (E) in two different preparations.

Role of ROS in Cytochrome c Loss Induced by Long-Chain Activated Fatty Acids

Nonesterified long-chain polyunsaturated fatty acids, particularly arachidonic acid, are known to promote ROS generation (5) and have been previously shown to induce cytochrome c release in isolated mitochondria in association with (but not in the absence of) PTP opening that was only partially sensitive to CsA (9). In addition, ROS have been shown to be required for release of the cytochrome c pool that is bound to cardiolipin in the inner membrane (34). We therefore investigated the role of ROS in _m dissipation and PTP-independent cytochrome c loss induced by long-chain activated fatty acids. In Fig. 5D, the protocol in Fig. 5A was repeated in the presence of SOD and catalase. Exposure to pulses of Ara-CoA resulted in much less _m dissipation, and _m recovered with BSA, which indicates that cytochrome c loss was averted. The protective effects of SOD with catalase were not prevented by 5-HD, which indicates that the ROS acted downstream to mitochondrial K_ATP (mitoK_ATP) channels in the prevention of cytochrome c loss (Fig. 5E). The average recovery amounts of _m after Ara-CoA pulses in the presence of 5-HD, diazoxide, and ROS scavengers are summarized in Fig. 5F.

These findings implicate enhanced ROS production in the mechanism of cytochrome c loss and _m dissipation by long-chain activated fatty acids. In addition, they may explain the protective effect of diazoxide, because diazoxide has been reported to decrease mitochondrial ROS production during reoxygenation of isolated mitochondria (32). Diazoxide also caused a modest depolarizing effect on _m, which is known to inhibit ROS production (23, 24).

Cytochrome c Loss Induced by Long-Chain Activated Fatty Acids Involves Adenine Nucleotide Translocator Inhibition

In Figs. 1 and 4, _m dissipated less rapidly during exposure to long-chain activated fatty acids than to the free fatty acid counterparts. To examine the underlying mechanism as well as its relationship to cytochrome c loss, Fig. 6 shows _m recorded in freshly isolated mitochondria that were respiring on endogenous substrates alone. After P_i addition, O₂ was removed until _m dissipated, which was followed by reoxygenation and addition of BSA (7 µM). Subsequent addition of two pulses of Pal-CoA (1.5 µM) led to almost full _m recovery with only a slight additional increase after site I substrates were added (1.5 mM each). There was no increase in O₂ after the Pal-CoA pulses (Fig. 6A, bottom trace), although changes in O₂ below the O₂ electrode sensitivity cannot be excluded due to the high affinity of cytochrome oxidase for O₂ (22). Indeed, a small acceleration of electron transport was sufficient to recover dissipated _m under these conditions, because a small pulse of pyruvate (1.5 µM) also led to _m recovery without a detectable increase in O₂ (Fig. 6B). In contrast, palmitic acid induced practically no recovery of _m under the same conditions, although full recovery was achieved with site I substrates (Fig. 6C). Similar responses were also observed with Lin-CoA and linoleic acid.

View larger version (19K): [in this window] [in a new window]   Fig. 6. Long-chain activated fatty acids increase m after it has been dissipated by anoxia. Mitochondria (0.25 mg/ml) were suspended in KCl buffer, followed by the addition of 5 mM Pi. Electron transport, maintained by oxidation of endogenous substrates, was inhibited with N2, which resulted in m dissipation. Mitochondria were then reoxygenated, and BSA (7 µM) was added. Addition of Pal-CoA pulses (A; 1.5 µM) or pyruvate (B; 1.5 µM) led to m recovery without detectable increases in O2 consumption (PO2; bottom trace). In contrast, palmitic acid pulses (C; 1.5 µM) failed to increase m, which required addition of site I substrates for recovery.

To get additional insight into the mechanism of _m polarization by long-chain activated fatty acids, we reinvestigated this effect in another setting (Fig. 7). When ADP was added to freshly isolated mitochondria that were respiring on endogenous substrates alone in the presence of P_i, _m rapidly dissipated. At low ADP concentration ([ADP], 5–30 µM), _m dissipation was concentration dependent and transient and it recovered without any detectable change in O₂ (i.e., within O₂-electrode sensitivity). However, when ≥50 µM ADP was added (Fig. 7A), _m remained dissipated unless exogenous substrates were also added to enhance electron transport (data not shown). Dissipation of _m was related to ADP/ATP uptake via the adenine nucleotide translocator (ANT), because it was completely prevented by the selective ANT inhibitor atractyloside (Fig. 7B). After ADP-induced _m dissipation, addition of Pal-CoA (2.5 µM) significantly increased _m (Fig. 7A). Conversely, when Pal-CoA was added before ADP, ADP failed to induce _m dissipation (Fig. 7C). The ability of Pal-CoA to increase _m required electron transport albeit with low activity, because the effects of Pal-CoA were reversed by inhibition of electron transport with antimycin A and then recovered by addition of exogenous site IV substrates beyond the site of antimycin A inhibition (Fig. 7A). In contrast, Pal-CoA added after antimycin A had no effect on _m, whereas _m rapidly recovered with site IV substrates (Fig. 7D).

View larger version (30K): [in this window] [in a new window]   Fig. 7. Pal-CoA promotes recovery of m by inhibiting the adenine nucleotide translocator (ANT). In isolated (0.4 mg/ml) mitochondria (A) incubated in KCl buffer and respiring on endogenous substrates only, 5 mM Pi increased m, which was then decreased by addition of ADP (50 µM). PO2 slightly increased after ADP addition (bottom trace). However, a more significant ADP-promoted increase was observed when the amount of mitochondria was greater (data not shown). Subsequent addition of Pal-CoA (2.5 µM) increased m; this was reversed by addition of antimycin A (0.5 µM), which indicates that electron transport at a low level was required. Addition of site IV substrates (2 mM AA and 0.2 mM TMPD) beyond the site of antimycin A inhibition rapidly increased m and O2 consumption. Preincubation with atractyloside (Atr, 10 µM) prevented ADP (10, 20, 40, and 50 µM) from dissipating m (B). Addition of Pal-CoA (2.5 µM) before ADP (50 µM) also prevented m dissipation similar to atracytoside (C). After ADP-induced m dissipation, addition of antimycin A (0.5 µM) prevented Pal-CoA from regenerating m (D). Similar to Pal-CoA, 10 µM atractyloside also increased m after its dissipation by ADP in a manner dependent on oxidation of endogenous substrates (E, F). After ADP-induced m dissipation, higher doses of Pal-CoA (two 2.5-µM pulses) led to transient m recovery followed by dissipation (G). Addition of BSA (7 µM) and site IV substrates had led to only partial m recovery, which required cytochrome c (5 µM) for full recovery. Summary data for 3–5 preparations of isolated mitochondria (H). When added before ADP, Pal-CoA (2.5 µM) or atractyloside (10 µM) both prevented ADP-induced m dissipation (with dissipation induced by ADP taken as 100%; left). When added after ADP, Pal-CoA or atractyloside almost completely reversed ADP-induced dissipation except when antimycin A was present (right). Numbers of determinations (n) are indicated.

These results indicate that the Pal-CoA-induced _m increase requires a low level of electron transport to be maintained using either endogenous substrates or Pal-CoA. In the absence of exogenous L-carnitine, however, Pal-CoA is not expected to reach the matrix and so should not be able to increase _m by directly accelerating electron transport. Therefore, an effect of Pal-CoA on membrane permeability must also be considered, because at low levels of electron transport, _m is highly sensitive to inner membrane permeability. Because ANT is known to be inhibited by long-chain activated fatty acids (36), we examined the effects of atractyloside under the same conditions. Figure 7, E and F, show that atractyloside mimicked the effects of Pal-CoA on _m, and that the _m increase after atractyloside addition depended similarly on residual electron transport. In addition, the effects of atractyloside were not artifacts of TMRM quenching, because atractyloside had no effect when it was added after FCCP (Fig. 7E). Similar effects were also obtained with the ANT inhibitor bongkrekic acid (4 µM, data not shown). Summary data for the modulation of ADP-induced changes in _m by Pal-CoA and atractyloside are presented in Fig. 7F.

These results indicate that stimulation of ANT by exogenous ADP was sufficient to dissipate _m, which did not recover for [ADP] ≥ 50 µM if electron transport capability was limited by substrate availability. Our findings suggest that Pal-CoA prevents _m dissipation under these conditions by inhibiting ANT activity (ADP/ATP exchange). This hypothesis is in line with previous observations in which cytochrome c loss was induced by ANT inhibitors with (37) or without (30) PTP opening. The effect of Pal-CoA when _m was low (due to ADP-promoted dissipation) depended both on its concentration and the availability of endogenous substrates to support electron transport. As more Pal-CoA was added, _m polarization was converted to _m dissipation, and cytochrome c was required for full _m recovery (Fig. 7G).

	DISCUSSION

TOP ABSTRACT EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
In the present study, we investigated the mechanisms by which fatty acids cause mitochondrial dysfunction in isolated mitochondria in a context generally relevant to cardiac ischemia-reperfusion injury. This dysfunction precedes and leaves mitochondria vulnerable to PTP opening and was prevented by pharmacological preconditioning with the cardioprotective agent diazoxide. We have defined mitochondrial dysfunction in this study as the inability of mitochondria to recover _m, because _m recovery is ultimately required for the functional recovery of a cell. Two major factors that determine the ability of the mitochondria to recover _m under conditions such as ischemia are the state of inner membrane leakiness and the cytochrome c content in the crista and intermembrane space. Our findings suggest that fatty acid accumulation during ischemia compromises both factors. In isolated mitochondria respiring on exogenous or endogenous substrates, long-chain fatty acids (C > 10) but not short- or medium-chain fatty acids promoted _m dissipation by increasing inner membrane leakiness in a manner that was reversible with BSA. Furthermore, long-chain activated fatty acids induced cytochrome c loss from the intermembrane space as indicated by direct measurement of cytochrome c content in the buffer as well as by the requirement for exogenous cytochrome c to reestablish _m after exposure to these compounds. The major novel finding reported here is that cytochrome c loss occurred in the absence of PTP opening in the high conductance mode, because CsA was not required to reestablish _m. However, we cannot exclude the possibility that PTP opened in a low-conductance, proton-permeable mode that was insensitive to CsA, ADP, or BSA (3), although how this would promote cytochrome c loss is unclear. Long-chain activated fatty acids also made mitochondria more susceptible to PTP opening in response to a Ca²⁺ load (see Fig. 2) and completely inhibited ATP synthesis in mitochondria energized with exogenous substrates (see Fig. 4). These findings extend previous observations in isolated liver mitochondria (12), in which long-chain activated fatty acids induced both cytochrome c loss and PTP opening. Our results clearly establish that the cytochrome c loss precedes PTP opening rather than being caused by it as previously postulated (12). Furthermore, we show that the mechanism by which long-chain activated fatty acids induce cytochrome c loss and impair ATP-production capability involves both ROS and ANT inhibition, because ROS scavengers prevented those effects and atractyloside mimicked them. In addition, the cardioprotective agent diazoxide attenuated both loss of cytochrome c and inhibition of ATP production by long-chain activated fatty acids. We present a unifying mechanism to explain these observations (Fig. 8).

View larger version (19K): [in this window] [in a new window]   Fig. 8. Schematic summary of the proposed actions of the long-chain free fatty acid palmitic acid (Pal) and its activated counterpart Pal-CoA. Palmitic acid enters the matrix in protonated form and is then pumped out in deprotonated form via the ANT and other anion transporters. Net entry of protons dissipates m. Pal-CoA enters the intermembrane space (IMS) via porin and inhibits ANT, thereby decreasing IM leak and transiently hyperpolarizing m. With carnitine present, Pal-CoA also enters the matrix via carnitine-acylcarnitine translocase (CACT) and stimulates electron transport, which further hyperpolarizes m and stimulates reactive oxygen species (ROS) production. ROS production induces cytochrome c mobilization from the inter-cristal space (ICS) and its release through the outer membrane (OM) via an as-yet undefined mechanism (dashed line) as shown (bottom, right). Diazoxide may prevent the increased ROS stimulation by modestly dissipating m via mitochondrial ATP-dependent (KATP) channels or nonselective actions.

Differences between Long-Chain Free and Activated Fatty Acids

Long-chain free fatty acids had distinctly different effects than long-chain activated fatty acids. Dissipation of _m by long-chain free fatty acids was rapidly reversible in mitochondria respiring on endogenous or exogenous substrates. Fatty acids are uncouplers; their protonophoric effect in mitochondria is well documented (41). As shown by Skulachev (38), long-chain free fatty acids enter mitochondria predominantly in the nonpolar protonated form (see Fig. 8). Once inside the mitochondrium, the proton dissociates, and the deprotonated fatty acid anion is transported out by anion carriers including the ATP/ADP and aspartate/glutamate antiporters, dicarboxylate carriers, and possibly the uncoupling proteins. This process results in a net proton influx. For long-chain free fatty acids, this cycling seemed to have no harmful effects on isolated mitochondria during short time intervals as was indicated by the full _m recovery with BSA.

In contrast, long-chain activated fatty acids, which cannot cycle via anion carriers, had biphasic effects on _m. The first phase, which was manifested only at low concentrations, increased _m, whereas the second phase dissipated _m. Activated fatty acids can move into the intermembrane space via porin (39) where they can interact with inner membrane components including ANT (Ref. 36; Fig. 8). The increase in _m could be due to the entry of activated fatty acids into the matrix as substrates with acceleration of electron transport. However, without the presence of L-carnitine, the extent to which activated fatty acids are able to enter the matrix is questionable. An alternative mechanism of hyperpolarization may be related to activated fatty acids causing decreased inner membrane permeability and thus allowing the same low level of endogenous substrate oxidation to maintain a higher _m. Consistent with this possibility, long-chain activated fatty acids are known to inhibit ANT (36), and we found that the ANT inhibitors atractyloside and bongkrekic acid promoted similar _m increases under the same conditions. Our finding that Pal-CoA, when added to isolated mitochondria, had an initial transient hyperpolarizing effect followed by _m dissipation was noted before (12), although the mechanism was not explored. In these previous experiments, _m dissipation was accompanied by cytochrome c release that was attributed to PTP opening, and these changes were inhibited by L-carnitine. ANT inhibition was also found to produce cytochrome c loss in association with PTP opening (37) and in addition was shown to induce cytochrome c release from isolated mitochondria without PTP opening via an unknown mechanism (30). ANT has a relatively wide porelike domain (19), and it may be that when ADP/ATP exchange is actively occurring, other ions shunt through the pore and effectively increase the inner membrane permeability. Ca²⁺-induced pore formation has been demonstrated with reconstituted purified ANT by single-channel current measurements (4). The mechanism of the second phase, _m dissipation by long-chain activated fatty acids, is unclear but could be related to the detergent-like effects of long-chain activated fatty acids at higher concentrations.

Role of ROS in Cytochrome c Loss Induced by Long-Chain Activated Fatty Acids

Our experiments with ROS scavengers indicate that ROS production is involved in the mechanism by which long-chain activated fatty acids caused cytochrome c loss, inhibition of ATP synthesis, and impaired Ca²⁺ handling preceding and thereby promoting PTP opening. Long-chain activated fatty acid-induced ANT inhibition appears to be more important than the effects on inner membrane leakiness, because free fatty acids induced similar leakiness without these consequences. Also, under the appropriate conditions (e.g., see Fig. 7 with presence of ADP), long-chain activated fatty acids or atractyloside induced _m hyperpolarization rather than depolarization. In some cases, apoptotic cell death has been preceded by an early _m increase (6) connected with inhibition of ADP/ATP exchange and/or adenine nucleotide import via porin (40). One possible unwanted effect of hyperpolarization is increased ROS generation (24), which is known to be promoted by long-chain fatty acids (5). In isolated heart mitochondria respiring in state 4, H₂O₂ production decreased markedly when _m was dissipated by only 15–20% (24). By inhibiting ANT, long-chain activated fatty acids may preserve _m and enhance ROS generation to a greater extent than long-chain free fatty acids. Our finding that ROS scavengers prevented long-chain activated fatty acids from inducing cytochrome c loss supports this hypothetical scenario (see Fig. 8).

The molecular mechanism by which enhanced ROS production might trigger cytochrome c loss is unknown but may involve release of cytochrome c that is anchored to the inner membrane cardiolipin (31) and/or protection of contact sites between the outer and inner mitochondrial membranes (Ref. 10; see Fig. 8). These contact sites (at which ANT protein is enriched) are important for a variety of functions, and together with porin, these sites facilitate long-chain activated fatty acid transport (17). ANT contains four critical cysteine thiol groups (20) and could be a primary target of ROS. Experiments with isolated mitochondria suggest that protein-protein interactions at contact sites are responsible for generation of a specific release pathway for cytochrome c that is independent of PTP opening. This is supported by studies that show that macromolecules, which decrease the volume of intermembrane space and increase the number of contact sites, protect against cytochrome c release (10). We observed that replacing KCl with sucrose in the buffer also protected mitochondria from cytochrome c loss by long-chain activated fatty acids. This effect is not likely to be due to ionic strength, although cytochrome c binding to the inner membrane is known to decrease at higher ionic strengths. Doran and Halestrap (10) have shown that even at low ionic strength, the osmotic effects of sucrose remained critical for preventing cytochrome c and adenylated kinase loss in isolated rat liver mitochondria.

Mitochondrial Protection by Diazoxide

We previously showed that under conditions relevant to ischemia-reperfusion, diazoxide protected isolated mitochondria respiring on endogenous substrates by dissipating _m in the presence of elevated Ca²⁺ and P_i, thereby reducing Ca²⁺ uptake and inhibiting PTP opening (21). In the present study, we have identified another novel protective effect of diazoxide that is relevant to ischemia-reperfusion; namely, prevention of fatty acid-induced cytochrome c loss before PTP opening. This idea is in line with evidence from Garlid's group (25, 26) that diazoxide protects mitochondria by preserving intermembrane structure and function, and is also congruent with the recent finding (32) that diazoxide markedly reduces mitochondrial ROS production at reoxygenation, and this effect is antagonized by 5-HD. We further speculate that the modest dissipation of _m caused by diazoxide, whether mediated via mitoK_ATP channels or nonspecific actions (16, 29), could be sufficient to reduce ROS production by counteracting the initial hyperpolarizing effects of long-chain activating fatty acids (see Fig. 8). On the other hand, under normal energized aerobic conditions in the absence of elevated long-chain activated fatty acids, diazoxide stimulated ROS production, which was required to trigger cardioprotection (33).

Limitations

In this study, we analyzed the effects of fatty acids on the function of isolated mitochondria to understand how mitochondrial damage develops and to provide insight into how this damage might be reduced during ischemia-reperfusion. A major limitation in experiments with isolated mitochondria is the inability to exactly mimic the complex in vivo environment that mitochondria experience during real ischemia-reperfusion. A major challenge is to evaluate mitochondrial protection by pharmacological or ischemic preconditioning in the cellular environment, an approach that so far has been limited by technical difficulties. Nevertheless, studies in isolated mitochondria may be useful for understanding mitochondrial pathophysiology and guiding therapeutic interventions in more realistic settings.

	ACKNOWLEDGMENTS

 
The authors thank Tan Duong for technical assistance.

This work is supported by National Institutes of Health Specialized Centers of Research in Sudden Cardiac Death (Grant P50 HL-52319), the Laubisch Fund, and Kawata Endowments.

	FOOTNOTES

 

Address for reprint requests and other correspondence: P. Korge, Dept. of Physiology, 3641 MRL Bldg., UCLA School of Medicine, Los Angeles, CA 90095 (E-mail: pkorge{at}mednet.ucla.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.

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