* Your assessment is very important for improving the workof artificial intelligence, which forms the content of this project
Download Long-chain fatty acids increase basal metabolism
Survey
Document related concepts
Proteolysis wikipedia , lookup
Nucleic acid analogue wikipedia , lookup
Amino acid synthesis wikipedia , lookup
Citric acid cycle wikipedia , lookup
Fluorescence wikipedia , lookup
Specialized pro-resolving mediators wikipedia , lookup
Mitochondrial replacement therapy wikipedia , lookup
Mitochondrion wikipedia , lookup
Biosynthesis wikipedia , lookup
Butyric acid wikipedia , lookup
Biochemistry wikipedia , lookup
Glyceroneogenesis wikipedia , lookup
Basal metabolic rate wikipedia , lookup
Transcript
Am J Physiol Heart Circ Physiol 282: H1495–H1501, 2002. First published January 3, 2002; 10.1152/ajpheart.00696.2001. Long-chain fatty acids increase basal metabolism and depolarize mitochondria in cardiac muscle cells JOHN RAY,1 FRANK NOLL,2 JÜRGEN DAUT,1 AND PETER J. HANLEY1 1 Institut für Normale und Pathologische Physiologie, Universität Marburg, 35037 Marburg; and 2Fachbereich Chemie, Universität Marburg, 35032 Marburg, Germany Received 3 August 2001; accepted in final form 10 December 2001 Ray, John, Frank Noll, Jürgen Daut and Peter J. Hanley. Long-chain fatty acids increase basal metabolism and depolarize mitochondria in cardiac muscle cells. Am J Physiol Heart Circ Physiol 282: H1495–H1501, 2002. First published January 3, 2002; 10.1152/ajpheart.00696.2001.— The effects of long-chain (LC) fatty acids on rate of heat production (heat rate) and mitochondrial membrane potential (⌬⌿) of intact guinea pig cardiac muscle were investigated at 37°C. Heat rate of ventricular trabeculae was measured with microcalorimetry, and ⌬⌿ was monitored in isolated ventricular myocytes with either JC-1 or tetramethylrhodamine ethyl ester (TMRE). Methyl--cyclodextrin was used as fatty acid carrier. Application of 400 M oleate or linoleate increased resting heat rate by ⬃30% and ⬃25%, respectively. When LC fatty acid was supplied as sole metabolic substrate, resting heat rate was decreased by 3-mercaptopropionic acid. In TMRE-loaded myocytes, neither 40–80 M oleate nor 40 M linoleate affected ⌬⌿. At a higher concentration (400 M) both oleate and linoleate increased TMRE fluorescence by ⬃20% of maximum, obtained using 2,4-dinitrophenol (100 M), indicating a depolarization of the inner mitochondrial membrane. We conclude that LC fatty acids, at sufficiently high concentration, increase heat rate and decrease ⌬⌿ in intact cardiac muscle, consistent with a protonophoric uncoupling action. These effects may contribute to the high metabolic rate after reperfusion of postischemic myocardium. important because LC fatty acids are the major metabolic substrates of the heart and, furthermore, they are known to accumulate in high concentrations during ischemia (6, 33). The aims of the present study were to determine the effects of LC fatty acids on both resting heat rate and mitochondrial membrane potential (⌬⌿) of intact cardiac muscle. METHODS IN CARDIAC MUSCLE, the resting (basal) level of metabolism is extraordinarily high, accounting for 25–30% of the metabolic rate of mechanically active muscle (3, 4, 9, 12, 22, 29). Although the origin of this high resting metabolic rate is unknown, it has been shown to be sensitive to metabolic substrate (4, 9). This is well illustrated with pyruvate, which has been shown to increase resting rate of heat production (4) and oxygen consumption (9) of isolated cardiac muscle preparations. In isolated mitochondrial preparations, long-chain (LC) fatty acids have been shown convincingly to uncouple oxidative phosphorylation (27, 31). Whether this uncoupling action manifests in intact cardiac muscle is not known (14, 35). This unresolved issue is Isolation of trabeculae and microcalorimetry. Guinea pigs (200–300 g) were anesthetized with 3–4% isoflurane in oxygen and then decapitated with a guillotine. The heart was rapidly excised and perfused with dissecting solution containing (mM) 105 NaCl, 15 KCl, 2 CaCl2, 1 MgCl2, 1 NaH2PO4, 24 NaHCO3, 20 2,3-butanedione monoxime, and 10 glucose. The solution was equilibrated with 95% O2-5% CO2, and the pH was 7.4. The right ventricle was opened, and free running trabeculae (diameter ⬍400 m) were excised and subsequently transferred to the calorimeter. These preparations were shown recently to be composed chiefly of myocytes accompanied by parallel perimysial collagen fibers (13). After a trabecula was mounted in the calorimeter, the solution was then changed to a standard physiological salt solution containing (mM) 121 NaCl, 5 KCl, 2 CaCl2, 0.8 MgCl2, 1 NaH2PO4, 24 NaHCO3, and 10 glucose (pH 7.4). The temperature of the calorimeter was maintained at 37°C. The rate of heat production (heat rate) of trabeculae was measured with the system schematically illustrated in Fig. 1 and described in detail by Daut and Elzinga (3, 4). Drift in the baseline (⬍1 W/h) was checked by repeatedly transferring the trabecula out of the recording chamber and was duly accounted for. In more recent experiments, the mounting procedure was slightly modified. Trabeculae were tied in situ using nylon monofilaments that had a terminal preformed loop of 200-m diameter. The loops were placed over hooks (Fig. 1), which were connected to micromanipulators, and the preparation was positioned in the center of the recording chamber. Isolation of ventricular myocytes. A cannula was attached to the aorta of the isolated heart, and the coronary arteries were perfused with physiological salt solution containing (mM) 115 NaCl, 5.4 KCl, 1.5 MgCl2, 0.5 NaH2PO4, 5 HEPES, 16 taurine, 5 sodium pyruvate, 15 NaHCO3, 1 CaCl2, and 5 glucose (pH 7.4). After 5 min, the heart was perfused for 5 min with nominally Ca2⫹-free solution, followed by solution containing collagenase type I (Sigma), 0.1% bovine serum Address for reprint requests and other correspondence: J. Daut, Institut für Normale und Pathologische Physiologie, Universität Marburg, Deutschhausstrasse 2, 35037 Marburg, Germany (E-mail: [email protected]). 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. mitochondrial membrane potential; microcalorimetry http://www.ajpheart.org 0363-6135/02 $5.00 Copyright © 2002 the American Physiological Society H1495 H1496 LONG-CHAIN FATTY ACIDS INDUCE MITOCHONDRIAL UNCOUPLING Fig. 1. Recording system of the calorimeter. Schematic diagram of a trabecula mounted in the recording chamber of the calorimeter. The preparation was attached to 2 platinum stimulating electrodes by nylon monofilaments and positioned between 6 sets of chromelconstantan thermocouples connected in series (for clarity, only 1 set is shown). The chamber is perfused at a constant rate of 1 l/s, and the voltage difference between positions T1 and T2 (T2 ⫺ T1) is measured. The measured voltage difference is proportional to the rate of heat production by the preparation (70 nV/W for chromelconstantan couples). albumin, and 40–60 M Ca2⫹. After enzymatic digestion (5–7 min), ventricular myocytes were separated by gentle trituration via a wide-bore pipette in solution containing (mM) 45 KCl, 70 K glutamate, 3 MgSO4, 15 KH2PO4, 16 taurine, 10 HEPES, 0.5 EGTA, and 10 glucose (pH, 7.4). After 60 min, myocytes were resuspended in Dulbecco’s modified Eagle’s medium (GIBCO-BRL). Myocytes were placed in a Perspex bath (volume 0.25 ml) located on the stage of an inverted microscope (Diaphot 300; Nikon) and superfused at a rate of 1 ml/min via gravity flow. Solutions were stored in inverted 50-ml plastic syringes, the ends of which marginally protruded through the floor of a custom-built Perspex water jacket heated to 37.5–38°C. Switching of solutions was executed by means of five miniature three-way solenoid valves (LFAA series; Lee). The Perspex bath was placed on an electrically heated aluminum plate that, in turn, was attached to a Perspex microscope stage insert. The temperature of the metal plate was monitored with an embedded thermistor and was maintained at 37°C with a feedback circuit (TC-324A heater controller; Warner Instrument, Hamden, CT). Bath temperature was continuously monitored via a second thermistor. Immediately before entering the bath, solutions passed through an in-line heat exchanger. To minimize heat loss via the oilimmersion objective lens, a major thermal sink, a custommade and tightly fitting brass water jacket was placed over the objective. The in-line heat exchanger, brass water jacket, and Perspex water jacket were perfused with warmed water (37.5–38°C) supplied by a temperature-controlled water bath (B. Braun Melsungen, Melsungen, Germany). Measurement of ⌬⌿ with fluorescent indicators JC-1 and tetramethylrhodamine ethyl ester. Ventricular myocytes were loaded with JC-1 by incubation at 37°C with solution containing 10 g/ml indicator for 10 min (32). Fluorescence experiments were subsequently performed with a Deltascan 4000 fluorescence system (Photon Technology International; Photomed, Seefeld, Germany) that was coupled to the microscope and software controlled. JC-1 is a positively charged carbocyanine derivative that is driven into mitochondria by ⌬⌿, and when it reaches a critical concentration, J aggregates are formed (24). When excited at 490 nm, the fluorescence emission of JC-1 can be split and simultaneously measured at wavelengths corresponding to its monomer (530 ⫾ 15 nm) and J aggregate (⬎ 590 nm) forms (Fig. 2). In preliminary experiments, we noted that there was a delay between the fall in J aggregate fluorescence and the onset of the AJP-Heart Circ Physiol • VOL rise in the monomer signal when the ⌬⌿ of a JC-1-loaded myocyte was dissipated (Fig. 2). This observation is consistent with the previous report that the monomer form of JC-1 is sensitive to a lower range of ⌬⌿ than the J aggregate form (7). Hence, because JC-1 does not behave as a typical ratioable dye, we used J aggregate fluorescence to monitor ⌬⌿. Myocytes were loaded with tetramethylrhodamine ethyl ester (TMRE) by superfusion with a solution containing 1.2 M TMRE for 10–15 min. TMRE was excited at 555 nm, and fluorescence was detected at ⬎590 nm. Because fluorescent indicators of ⌬⌿ cannot be readily calibrated in intact cells (7, 8), we used relative fluorescence changes to monitor ⌬⌿. The uncoupler 2,4-dinitrophenol (DNP) was used to collapse ⌬⌿ and scale JC-1 and TMRE signals (7, 23). Digitonin-permeabilized myocytes. Myocytes were superfused with Ca2⫹-free solution including 1 mM EGTA for 5 min before being permeabilized by 1- to 2-min exposure to digitonin (15–20 M) (18). Digitonin was directly added to an “intracellular” solution composed of (mM) 10 NaCl, 105 KCl, 5 HEPES, 2 K2ATP, 6 EGTA, 4 Ca-EGTA, 12.7 MgCl2, 5 pyruvate, and 2 malate (pH adjusted to 7.2 with KOH). The calculated free Mg2⫹ concentration ([Mg2⫹]) was 1 mM, and the free [Ca2⫹] was 100 nM. The solution also contained 300 nM JC-1. Mitochondria were rendered orange-red fluorescent by the presence of JC-1, indicating that the mitochondria were well polarized. Chemicals. LC fatty acids were solubilized with methyl-cyclodextrin at a ⬃1:6 molar ratio (Sigma). A major advantage of using methyl--cyclodextrin was that LC fatty acidcontaining solutions did not foam when bubbled with gas. However, at high concentrations (⬎10 mM), cyclodextrins are capable of removing cholesterol and other lipid components from membranes (16). This property, however, is diminished when the cavities of cyclodextrin molecules are occupied (16). In some cases, indicated accordingly, the Na salt form of fatty acids was used. The Na salt form was directly dissolved in solutions and sonicated for 10 min. Sodium hexanoate was added directly. Spectroscopy. Absorption and emission spectra were obtained with aqueous solutions of JC-1 (4 M; 0.3% DMSO) and TMRE (1 M; 0.1% DMSO). The buffer solution contained 150 mM KCl and 10 mM HEPES (pH 8.2 with KOH). To minimize bleaching, a moderately fast scan time was used. Fig. 2. Effect of membrane depolarization on JC-1 fluorescence in an isolated myocyte. The uncoupler 2,4-dinitrophenol (DNP) was used to dissipate the mitochondrial membrane potential while monomer (530 nm) and J aggregate (590 nm) fluorescence was simultaneously measured. 282 • APRIL 2002 • www.ajpheart.org LONG-CHAIN FATTY ACIDS INDUCE MITOCHONDRIAL UNCOUPLING H1497 Fig. 3. Stimulatory effect of oleate on the rate of heat production of a trabecula. Resting heat rate (Hr) was determined by moving the trabecula out of the recording chamber (as shown at the beginning of the recording). Contraction-related heat production was elicited by stimulating the trabecula electrically at 2 Hz (as indicated). When oleate (400 M) was introduced to the preparation, an increase in resting rate of heat production ensued. Statistics. All results are expressed as means ⫾ SE. Statistical significance was determined with ANOVA. A P ⬍ 0.05 was considered significant. The number of preparations (n) from which the data are obtained is indicated in parentheses. RESULTS LC fatty acids increase resting heat rate. The rate of heat production of small isolated cardiac muscle preparations, an indicator of basal metabolism, was measured at high resolution with a microcalorimetric technique. Figure 3 shows a representative example of the effect of oleate (C18:1) on resting heat rate of an isolated trabecula. Resting heat rate was determined by transferring the preparation out of the recording chamber (Fig. 1), whereas contraction-related heat production was elicited by stimulating the trabecula at a rate of 2 Hz (Fig. 3). Application of 400 M oleate, solubilized with methyl--cyclodextrin, increased resting heat rate, whereas the same concentration of methyl-cyclodextrin alone had no effect on resting heat rate (n ⫽ 10), as illustrated in Fig. 4. On average, 400 M oleate increased heat rate by 29.3 ⫾ 0.9% (n ⫽ 26). When oleate was supplied as sole metabolic substrate, the introduction of the -oxidation blocker 3-mercaptopropionic acid (2 mM; Ref. 26) reduced resting heat rate by 27 ⫾ 2.6% (n ⫽ 6; not shown). Resting heat rate was unaffected by 3-mercaptopropionic acid when glucose was provided as metabolic substrate. The effect of various concentrations of the LC fatty acids oleate and linoleate (C18:2) on resting heat rate are summarized in Fig. 5. Trabeculae were superfused with fatty acid solutions for at least 10 min. At concentrations of 100 and 200 M, neither oleate nor linoleate increased resting heat rate. At 300 M, oleate and linoleate increased resting heat rate by 5.1 ⫾ 0.8% (n ⫽ 20) and 4.8 ⫾ 0.6% (n ⫽ 10), respectively. At the highest concentration tested (400 M), oleate increased resting heat rate by ⬃30% and linoleate produced a 25.8 ⫾ 0.9% (n ⫽ 20) increase. For comparison with LC fatty acids, we examined the effect of the short-chain fatty acid hexanoate (C6:0) on heat rate. Hexanoate (1 mM) reversibly increased heat rate by 39.9 ⫾ 7.3% (n ⫽ 4; not shown). In light of previous work with isolated mitochondrial preparations, the most likely mechanism for the increase in resting heat rate evoked by LC fatty acids is a protonophoric uncoupling action. We therefore examined whether oleate and linoleate depolarized the mitochondrial membrane of isolated myocytes under similar experimental conditions (37°C). LC fatty acids depolarize inner mitochondrial membrane. ⌬⌿ was initially measured in ventricular myocytes with the fluorescent indicator JC-1. To reduce photobleaching, JC-1 was intermittently excited (as illustrated in Fig. 6). Cyclodextrin had no significant effect on ⌬⌿. However, when the superfusate was switched to a solution containing oleate (solubilized with the same concentration of cyclodextrin), a decrease in ⌬⌿ was observed (Fig. 6A). Similar results were observed in four other myocytes. At the end of experiments, we used DNP to dissipate ⌬⌿ and scale the JC-1 fluorescence signals. On average, oleate decreased JC-1 fluorescence to 45.3 ⫾ 5.6% (n ⫽ 5) of the maximal response produced by DNP. Hexanoate (1 mM; C6:0) had no effect on ⌬ (Fig. 6B). Fig. 4. Effect of cyclodextrin on resting heat rate. The trabecula was superfused with cyclodextrin for 30 min. No effect of 2.4 mM cyclodextrin on resting heat rate was observed. For clarity, only the first 8 min of exposure are shown. After washout of cyclodextrin, the amplitude of contraction-related heat rate was similar to the preexposure value. AJP-Heart Circ Physiol • VOL 282 • APRIL 2002 • www.ajpheart.org H1498 LONG-CHAIN FATTY ACIDS INDUCE MITOCHONDRIAL UNCOUPLING Fig. 7. Effects of oleoyl-CoA on J aggregate fluorescence in a digitonin-permeabilized ventricular myocyte. Application of oleoyl-CoA (10 M) had no effect on J aggregate fluorescence, an index of ⌬⌿. Fig. 5. Summary of dose-dependent effects of oleate (E) and linoleate (‚) on resting heat rate. An increase in resting heat rate was only observed at concentrations of 300 and 400 M. Digitonin-permeabilized myocytes. Although the most likely explanation for the decrease in ⌬⌿ produced by oleate is that it exerts a protonophoric uncoupling action, activation of mitochondrial ATP-sensitive K⫹ (KATP) channels by its CoA derivative (oleoyl-CoA) could also play a role. We recently showed (21) that oleoyl-CoA potently activates the cardiac sarcolemmal Fig. 6. Effects of oleate and hexanoate on J aggregate fluorescence in JC-1-loaded myocytes. A: oleate (400 M), solubilized with methyl-cyclodextrin, produced a decrease in J aggregate fluorescence, whereas cyclodextrin alone had no effect. DNP (100 M) was used to collapse mitochondrial membrane potential (⌬⌿). B: the short-chain fatty acid hexanoate had no effect on J aggregate fluorescence. AJP-Heart Circ Physiol • VOL KATP channel. Because oleoyl-CoA is membrane impermeant, we examined its effects on JC-1 fluorescence in permeabilized myocytes. Figure 7 shows that a high concentration of oleoyl-CoA (10 M) did not depolarize the mitochondria. Comparable results were obtained in four other permeabilized myocytes. To confirm that metabolism of oleoyl-CoA was not masking a decrease in ⌬⌿ (14), we also found that oleoyl-CoA did not decrease ⌬⌿ in the presence of 3-mercaptopropionic acid. Hence, it is unlikely that oleoyl-CoA-mediated activation of mitochondrial KATP channels contributes to the observed membrane depolarization evoked by oleate. In vitro effects of LC fatty acids on indicators JC-1 and TMRE. We tested whether the large decrease in J aggregate fluorescence produced by oleate could reflect, at least in part, a direct interaction of oleate with the indicator. Figure 8A shows an absorption scan of JC-1 (4 M; 0.3% DMSO) in 0.15 M KCl solution (pH 8.2). The absorption spectrum shows two peaks that correspond to the monomer (peak 500 nm) and J aggregate (peak 595 nm) forms of JC-1. Addition of 40 M Na oleate produced a dramatic change in the fluorescence spectrum (excitation wavelength 488 nm). The loss of distinct peaks and resulting broad-based spectrum are probably due to the disruption of J aggregates and the formation of various JC-1 oligomers. The corresponding emission spectra are shown in Fig. 8B. Under control conditions, a peak is seen at 591 nm, which corresponds to J aggregate fluorescence. When 40 M Na oleate was added, J aggregate fluorescence intensity was decreased to 7% of the control value and a peak occurred at 534 nm, probably due to either JC-1 monomers or a predominant oligomer. Similar to oleate, linoleate (40 M) decreased J aggregate fluorescence to 20% of control (not shown). The carboxyl group of LC fatty acids is unlikely to be responsible for this effect because addition of acetic acid (40 M) had no effect on JC-1 fluorescence. When Na oleate was added to JC-1 at a ratio of 1:1, the decrease in fluorescence was ⬍10%. Hence, between a oleate-to-JC-1 ratio of 1:1 and a ratio of 10:1 a large decrease in J aggregate fluorescence occurs. Although we do not know the mitochondrial concentration of JC-1 in myocytes, the critical concentration for J aggregate formation was pre- 282 • APRIL 2002 • www.ajpheart.org LONG-CHAIN FATTY ACIDS INDUCE MITOCHONDRIAL UNCOUPLING H1499 Fig. 8. In vitro effects of oleate on JC-1 (A and B) and tetramethylrhodamine ethyl ester (TMRE; C and D) spectra. The ordinate is expressed in arbitrary units (a.u.). Addition of 40 M oleate (⫹oleate) modified the JC-1 absorption spectrum (A) and decreased J aggregate fluorescence (B). TMRE peak absorption was red-shifted by 8 nm by addition of 40 M oleate (C), and its fluorescence emission intensity was reduced (D) (excitation wavelength 550 nm). Cyclodextrin alone (⫹cyclo) and cyclodextrin-oleate (400 M; ⫹cyclooleate) reduced TMRE fluorescence emission to the same extent (D). viously estimated to be 0.26 M under in vitro conditions (pH 7.2; 37°C) and 0.16 M for suspended cardiac mitochondria (24). Because our in vitro observations complicated the interpretation of in vivo JC-1 experiments, we tested whether oleate affected the spectral properties of the rhodamine-based indicator TMRE. Figure 8C shows the absorption spectrum of 1 M TMRE. Addition of 40 M oleate shifted the absorption peak from 552 to 560 nm. The corresponding emission spectrum (excitation wavelength 550 nm) shows that oleate also reduced peak fluorescence (Fig. 8D). Linoleate (40 M) had effects comparable to those of oleate. We also found that cyclodextrin-solubilized oleate (400 M) decreased TMRE fluorescence; however, this effect could be accounted for by cyclodextrin alone (Fig. 8D). In conclusion, LC fatty acids are capable of decreasing the fluorescence intensity of the ⌬⌿ indicators JC-1 and TMRE. Effects of LC fatty acids on ⌬⌿ in myocytes assessed with TMRE. In myocytes loaded with TMRE, fluorescence has been reported either to increase or to decrease after membrane depolarization, the difference probably reflecting the extent of dye loading (10). We found that depolarization of the mitochondrial membrane by DNP consistently elicited a large and reversible increase in fluorescence after cells had been loaded by 10- to 15-min incubation with 1.2 M TMRE. Thus any changes in membrane potential induced by LC fatty acids may, in principle, be underestimated because of direct interaction of matrix fatty acids with the dye (Fig. 8). However, this confounding effect would be minimal because the matrix free concentration of fatty acid is probably in the low micromolar range. AJP-Heart Circ Physiol • VOL Neither oleate (40–80 M) nor linoleate (40 M), whether supplied as the Na salt (n ⫽ 4) or in the presence of methyl--cyclodextrin (n ⫽ 3), affected ⌬⌿ in myocytes. At higher concentration, however, cyclodextrin-solubilized oleate (400 M) increased TMRE fluorescence by 22.1 ⫾ 2% (n ⫽ 5) of maximum, consistent with a decrease in ⌬⌿. A representative example is shown in Fig. 9A. Cyclodextrin alone had no effect (not shown), whereas it decreased TMRE fluorescence in vitro (Fig. 8). This may be explained by the fact that cyclodextrin is membrane impermeant. Similar to oleate, application of linoleate (400 M; Fig. 9B) increased TMRE fluorescence by 17.8 ⫾ 1.8% of maximum (n ⫽ 5). DISCUSSION We have shown that LC fatty acids increase basal metabolism (resting heat rate) and depolarize the inner mitochondrial membrane of intact cardiac muscle. These stimulatory effects were observed at total fatty acid concentrations of 300–400 M, within the range of 200–1,000 M reported for biological fluids (19, 33). In agreement with our calorimetric results, Challoner and Steinberg (2) previously reported that palmitate (C16:0), solubilized with albumin, enhances the rate of oxygen consumption of isolated rat hearts arrested by an increase in extracellular potassium concentration. When methyl--cyclodextrin is used as carrier, we do not know the rate of cellular uptake of LC fatty acids, which is thought to depend largely on the unbound concentration (1). However, when cyclodextrin-solubilized oleate was the sole source of substrate for ventricular trabeculae, addition of the -oxidation inhibitor 282 • APRIL 2002 • www.ajpheart.org H1500 LONG-CHAIN FATTY ACIDS INDUCE MITOCHONDRIAL UNCOUPLING Fig. 9. Measurement of ⌬⌿ in a TMRE-loaded myocyte. A: depolarization of ⌬⌿ with DNP produced a reversible increase in TMRE fluorescence. Application of oleate (400 M) evoked an increase in TMRE fluorescence, consistent with a decrease in ⌬⌿. B: linoleate (400 M) similarly increased TMRE fluorescence. The membrane could be further depolarized by a second application of DNP. 3-mercaptopropionic acid decreased heat rate, indicating that the fatty acid was indeed taken up. During prolonged ischemia (⬎20 min) and after reperfusion, the intracellular concentration of LC fatty acids is known to increase significantly (6, 33), probably because of lipase-catalyzed release of acyl groups from phospholipids and endogenous triacylglycerols. For example, in the glucose-perfused heart, tissue fatty acid content has been shown to increase from ⬃70 nmol/g dry wt (preischemia) to ⬃290 nmol/g dry wt after 30-min ischemia and to remain elevated during reperfusion (6). When fatty acid content exceeded a threshold value of ⬃400 nmol/g dry wt, reperfused hearts did not recover any contractile function. Our results suggest that the high rate of energy metabolism that follows reperfusion of postischemic myocardium (20) is due, at least in part, to the accompanying increase in intracellular LC fatty acid concentration. The ability of LC fatty acids to stimulate resting heat rate of intact cardiac muscle is probably due to a protonophoric uncoupling mechanism, as suggested by work with isolated mitochondria. Hütter and Soboll (15) have also speculated that the increase in oxygen consumption evoked by LC fatty acids in the intact heart is related to futile cycles such as extramitochondrial -oxidation or uncoupling of oxidative phosphorylation. The latter effect, uncoupling, is thought to involve cyclic mitochondrial influx of fatty acid (R-COOH) and efflux of its anionic (R-COO⫺) form (27, 31). Translocation of the less membrane-permeant fatty acid anion across the AJP-Heart Circ Physiol • VOL inner membrane is rate limiting and has been deduced to be catalyzed by the ADP/ATP carrier and/or other inner mitochondrial membrane proteins such as uncoupling protein (5, 17, 30). The LC fatty acids probably flip-flop between the inner and outer face of the bilayer, delivering protons to the matrix (11), as schematically illustrated in Fig. 10. Whether such an uncoupling mechanism occurs in the intact cell has been a source of controversy (14, 35). However, consistent with uncoupling, we have shown that LC fatty acids decrease ⌬⌿ in intact cardiac muscle when supplied at sufficiently high concentration. The mechanism by which hexanoate increases heat rate without affecting ⌬⌿ is probably via a futile intramitochondrial ATP-consuming cycle of hexanoyl-CoA synthesis and hydrolysis (28). We also revealed that LC fatty acids directly affect the fluorescence properties of the commonly used indicators JC-1 and TMRE. If not recognized, this interaction might lead to misinterpretation of their fluorescence signals. The mechanism by which LC fatty acids disrupt J aggregates in vitro (Fig. 8) is probably comparable to the recently described effects of cationic and anionic surfactants on the carbocyanine derivative C803 (34). It should also be noted that Rottenberg and Hashimoto (25) have speculated that positively charged lipophilic dyes, which include JC-1, may directly complex with fatty acid anions (R-COO⫺) and thereby catalyze their cyclic uncoupling action. This is unlikely to be the case, because it is improbable that the carboxyl group of fatty acids complex with carbocyanine derivatives, and, moreover, we were unable to demonstrate any in vitro effect of acetic acid (CH3-COOH) on JC-1 spectra. In conclusion, our calorimetric and photometric measurements suggest that LC fatty acids, at sufficiently high concentration, depolarize the inner mitochondrial membrane and stimulate basal metabolism of intact cardiac muscle. These observations may partially explain the increased energy expenditure of reperfused postischemic myocardium because LC fatty acid concentration increases markedly during ischemia. Fig. 10. Schematic diagram showing the proposed mechanism by which long-chain fatty acids dissipate the proton gradient of the inner mitochondrial membrane and thereby produce heat. Translocation of the fatty acid anion is thought to be facilitated by uncoupling protein (UCP; as shown) and/or the ADP/ATP carrier (not shown). 282 • APRIL 2002 • www.ajpheart.org LONG-CHAIN FATTY ACIDS INDUCE MITOCHONDRIAL UNCOUPLING We thank R. Graf, L. Krapp, E. Reisinger, G. Schlichthörl, and K. Schneider for technical help. This study was supported by the Deutsche Forschungsgemeinschaft (Grant Da177/7–3) and the P. E. Kempkes Stiftung. 19. REFERENCES 20. 1. Berk PD and Stump DD. Mechanisms of cellular uptake of long chain free fatty acids. Mol Cell Biochem 192: 17–31, 1999. 2. Challoner DR and Steinberg D. Effect of free fatty acid on the oxygen consumption of perfused rat heart. Am J Physiol 210: 280–286, 1966. 3. Daut J and Elzinga G. Heat production of quiescent ventricular trabeculae isolated from guinea-pig heart. J Physiol (Lond) 398: 259–275, 1988. 4. Daut J and Elzinga G. Substrate dependence of energy metabolism in isolated guinea-pig cardiac muscle: a microcalorimetric study. J Physiol (Lond) 413: 379–397, 1989. 5. Dedukhova VI, Mokhova EN, Skulachev VP, Starkov AA, Arrigoni-Martelli E, and Bobyleva VA. Uncoupling effect of fatty acids on heart muscle mitochondria and submitochondrial particles. FEBS Lett 295: 51–54, 1991. 6. De Groot MJM, Coumans WA, Willemsen PHM, and van der Vusse GJ. Substrate-induced changes in the lipid content of ischemic and reperfused myocardium. Its relation to hemodynamic recovery. Circ Res 72: 176–186, 1993. 7. Di Lisa F, Blank PS, Colonna R, Gambassi G, Silverman HS, Stern MD, and Hansford RG. Mitochondrial membrane potential in single living adult rat cardiac myocytes exposed to anoxia or metabolic inhibition. J Physiol (Lond) 486: 1–13, 1995. 8. Farkas DL, Wei M, Febbroriello P, Carson JH, and Loew LM. Simultaneous imaging of cell and mitochondrial membrane potentials. Biophys J 56: 1053–1069, 1989. 9. Gibbs CL. Cardiac energetics. Physiol Rev 58: 174–254, 1978. 10. Griffiths EJ. Mitochondria—potential role in cell life and death. Cardiovasc Res 46: 24–27, 2000. 11. Hamilton JA. Fatty acid transport: difficult or easy? J Lipid Res 39: 467–481, 1998. 12. Hanley PJ, Cooper PJ, and Loiselle DS. Effect of hyperosmotic perfusion on rate of oxygen consumption of isolated guinea pig and rat hearts during cardioplegia. Cardiovasc Res 28: 485– 493, 1994. 13. Hanley PJ, Young AA, LeGrice IJ, Edgar SG, and Loiselle DS. 3-Dimensional configuration of perimysial collagen fibres in rat cardiac muscle at resting and extended sarcomere lengths. J Physiol (Lond) 517: 831–837, 1999. 14. Hermesh O, Kalderon B, and Bar-Tana J. Mitochondria uncoupling by a long chain fatty acyl analogue. J Biol Chem 273: 3937–3942, 1998. 15. Hütter JF and Soboll S. Role of fatty acid metabolites in the development of myocardial ischemic damage. Int J Biochem 24: 399–403, 1992. 16. Irie T and Uekama K. Pharmaceutical applications of cyclodextrins. III. Toxicological issues and safety evaluation. J Pharm Sci 86: 147–162, 1997. 17. Jezek P, Modrianský M, and Garlid KD. A structure-activity study of fatty acid interaction with mitochondrial uncoupling protein. FEBS Lett 408: 166–170, 1997. 18. Köhnke D, Schramm M, and Daut J. Oxidative phosphorylation in myocardial mitochondria “in situ”: a calorimetric study on 21. AJP-Heart Circ Physiol • VOL 22. 23. 24. 25. 26. 27. 28. 29. 30. 31. 32. 33. 34. 35. H1501 permeabilized cardiac muscle preparations. Mol Cell Biochem 174: 101–113, 1997. Kotani A, Fuse T, and Kusu F. Determination of plasma free fatty acids by high-performance liquid chromatography with electrochemical detection. Anal Biochem 284: 65–69, 2000. Lerch R. Oxidative substrate metabolism during postischemic reperfusion. Basic Res Cardiol 88: 525–544, 1993. Liu GX, Hanley PJ, Ray J, and Daut J. Long-chain acylcoenzyme A esters and fatty acids directly link metabolism to KATP channels in the heart. Circ Res 88: 918–924, 2001. Loiselle DS and Gibbs CL. Species differences in cardiac energetics. Am J Physiol Heart Circ Physiol 237: H90–H98, 1979. Minezaki KK, Suleiman MS, and Chapman RA. Changes in mitochondrial function induced in isolated guinea-pig ventricular myocytes by calcium overload. J Physiol (Lond) 476: 459– 471, 1994. Reers M, Smith TW, and Chen LB. J-aggregate formation of a carbocyanine as a quantitative fluorescent indicator of membrane potential. Biochemistry 30: 4480–4486, 1991. Rottenberg H and Hashimoto K. Fatty acid uncoupling of oxidative phosphorylation in rat liver mitochondria. Biochemistry 25: 1747–1755, 1986. Sabbagh E, Cuebas D, and Schulz H. 3-Mercaptopropionic acid, a potent inhibitor of fatty acid oxidation in rat heart mitochondria. J Biol Chem 260: 7337–7342, 1985. Schönfeld P, Wieckowski MR, and Wojtczak L. Long-chain fatty acid-promoted swelling of mitochondria: further evidence for the protonophoric effect of fatty acids in the inner mitochondrial membrane. FEBS Lett 471: 108–112, 2000. Schönfeld P, Wojtczak AB, Geelen MJH, Kunz W, and Wojtczak L. On the mechanism of the so-called uncoupling effect of medium- and short-chain fatty acids. Biochim Biophys Acta 936: 280–288, 1988. Schramm M, Klieber HG, and Daut J. The energy expenditure of actomyosin-ATPase, Ca2⫹-ATPase and Na⫹,K⫹-ATPase in guinea-pig cardiac ventricular muscle. J Physiol (Lond) 481: 647–662, 1994. Simonyan RA and Skulachev VP. Thermoregulatory uncoupling in heart muscle mitochondria: involvement of the ATP/ ADP antiporter and uncoupling protein. FEBS Lett 436: 81–84, 1998. Skulachev VP. Uncoupling: new approaches to an old problem of bioenergetics. Biochim Biophys Acta 1363: 100–124, 1998. Smiley ST, Reers M, Mottola-Hartshorn C, Lin M, Chen A, Smith TW, Steele GD, and Chen LB. Intracellular heterogeneity in mitochondrial membrane potentials revealed by a Jaggregate-forming lipophilic cation JC-1. Proc Natl Acad Sci USA 88: 3671–3675, 1991. Van der Vusse GJ, Glatz JFC, Stam HCG, and Reneman RS. Fatty acid homeostasis in the normoxic and ischemic heart. Physiol Rev 72: 881–940, 1992. Von Berlepsch H, Böttcher C, Ouart A, Regenbrecht M, Akari S, Keiderling U, Schnablegger H, Dähne S, and Kirstein S. Surfactant-induced changes of morphology of Jaggregates: superhelix-to-tubule transformation. Langmuir 16: 5908–5916, 2000. Wojtczak L and Schönfeld P. Effect of fatty acids on energy coupling processes in mitochondria. Biochim Biophys Acta 1183: 41–57, 1993. 282 • APRIL 2002 • www.ajpheart.org