Vol. 282, Issue 4, H1495-H1501, April 2002
Long-chain fatty acids increase basal metabolism and
depolarize mitochondria in cardiac muscle cells
John
Ray1,
Frank
Noll2,
Jürgen
Daut1, and
Peter J.
Hanley1
1 Institut für Normale und Pathologische Physiologie,
Universität Marburg, 35037 Marburg; and 2 Fachbereich
Chemie, Universität Marburg, 35032 Marburg, Germany
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ABSTRACT |
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 µM 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
µM oleate nor 40 µM linoleate affected . At a higher
concentration (400 µM) both oleate and linoleate increased TMRE
fluorescence by ~20% of maximum, obtained using 2,4-dinitrophenol
(100 µM), 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.
mitochondrial membrane potential; microcalorimetry
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INTRODUCTION |
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 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.
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METHODS |
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 µm) 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 µW/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-µm 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.
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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 chromel-constantan
thermocouples connected in series (for clarity, only 1 set is shown).
The chamber is perfused at a constant rate of 1 µl/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/µW for chromel-constantan couples).
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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 albumin, and 40-60
µM 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 oil-immersion objective lens, a major
thermal sink, a custom-made 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 µg/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 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 .
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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.
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Myocytes were loaded with tetramethylrhodamine ethyl ester (TMRE) by
superfusion with a solution containing 1.2 µM 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 µM) (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 acid-containing 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 µM; 0.3% DMSO) and TMRE (1 µM; 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.
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.
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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 µM 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 µM 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.
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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 µM) was introduced to the preparation,
an increase in resting rate of heat production ensued.
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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.
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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 µM, neither oleate nor linoleate increased resting heat rate.
At 300 µM, 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 µM), 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).
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Fig. 5.
Summary of dose-dependent effects of oleate
( ) and linoleate ( ) on resting heat
rate. An increase in resting heat rate was only observed at
concentrations of 300 and 400 µM.
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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).
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Fig. 6.
Effects of oleate and hexanoate on J aggregate
fluorescence in JC-1-loaded myocytes. A: oleate (400 µM),
solubilized with methyl--cyclodextrin, produced a decrease in J
aggregate fluorescence, whereas cyclodextrin alone had no effect. DNP
(100 µM) was used to collapse mitochondrial membrane potential
(). B: the short-chain fatty acid hexanoate had no
effect on J aggregate fluorescence.
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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 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 µM) 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.
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Fig. 7.
Effects of oleoyl-CoA on J aggregate fluorescence in a
digitonin-permeabilized ventricular myocyte. Application of oleoyl-CoA
(10 µM) had no effect on J aggregate fluorescence, an index of
.
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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 µM; 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 µM 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 µM 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 µM) 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 µM) 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 previously estimated to be 0.26 µM under in
vitro conditions (pH 7.2; 37°C) and 0.16 µM for suspended cardiac
mitochondria (24).
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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 µM 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 µM
oleate (C), and its fluorescence emission intensity was
reduced (D) (excitation wavelength 550 nm). Cyclodextrin
alone (+cyclo) and cyclodextrin-oleate (400 µM; +cyclo-oleate)
reduced TMRE fluorescence emission to the same extent (D).
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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 µM TMRE. Addition of 40 µM
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 µM) had effects comparable to those of oleate. We also
found that cyclodextrin-solubilized oleate (400 µM) 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 µM
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.
Neither oleate (40-80 µM) nor linoleate (40 µM), 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 µM) 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 µM; Fig. 9B) increased TMRE fluorescence by 17.8 ± 1.8% of maximum (n = 5).
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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 µM) evoked
an increase in TMRE fluorescence, consistent with a decrease in .
B: linoleate (400 µM) similarly increased TMRE
fluorescence. The membrane could be further depolarized by a second
application of DNP.
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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 µM, within the range of
200-1,000 µM 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 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 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).
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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).
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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.
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ACKNOWLEDGEMENTS |
We thank R. Graf, L. Krapp, E. Reisinger, G. Schlichthörl,
and K. Schneider for technical help.
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FOOTNOTES |
This study was supported by the Deutsche Forschungsgemeinschaft (Grant
Da177/7-3) and the P. E. Kempkes Stiftung.
Address for reprint requests and other correspondence: J. Daut,
Institut für Normale und Pathologische Physiologie,
Universität Marburg, Deutschhausstrasse 2, 35037 Marburg, Germany
(E-mail: daut{at}mailer.uni-marburg.de).
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.
First published January 3, 2002;10.1152/ajpheart.00696.2001
Received 3 August 2001; accepted in final form 10 December 2001.
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