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Am J Physiol Heart Circ Physiol 285: H999–H1006, 2003;
10.1152/ajpheart.00035.2003.
Cariporide preserves mitochondrial proton gradient and delays
ATP depletion in cardiomyocytes during ischemic conditions
Marisol Ruiz-Meana, David Garcia-Dorado, Pilar Pina,
Javier Inserte, Luis Agulló, and Jordi Soler-Soler
Cardiology Department, Hospital Vall d’Hebron, 08035 Barcelona, Spain
Submitted 13 January 2003; accepted in final form 16 April 2003
ischemia; ions; infarction
the Na⫹/H⫹ exchanger (NHE) has a
strong protective effect against cardiomyocyte death
secondary to ischemia-reperfusion (1, 10, 15, 28), but
the exact mechanism of this protection is, however, far
from being fully elucidated. The initial hypothesis that
NHE inhibition prevented cell death by attenuating
INHIBITION OF
Address for reprint requests and other correspondence: D. Garcia-Dorado, Cardiologı́a Experimental, Hospital Vall d’Hebron, Pg. Vall d’Hebron
119-129, 08035 Barcelona, Spain (E-mail: [email protected]).
http://www.ajpheart.org
cytosolic Ca2⫹ overload during reperfusion, associated
to correction of intracellular acidosis, has not been
confirmed by experimental results (26, 27). It has been
suggested that the protective effect of NHE inhibition
is mediated by a slower normalization of intracellular
pH during reperfusion (30). However, the observed
delay in pH recovery is very small, and other studies
(27) have shown that the NaHCO3 cotransporter may
compensate for NHE inhibition, allowing a rapid normalization of intracellular pH during reperfusion. Although with some discrepancies (10), studies in different models have shown that to be effective, NHE inhibition must act during coronary occlusion (6, 16) or
cardioplegia (28), whereas its application at the time of
reperfusion affords little, if any, protection against cell
death. Recent reports (6, 18, 25) indicate that NHE
inhibition delays the progression of ischemic injury as
manifested by ATP depletion or development of rigor
contracture. It has been suggested that NHE1 is markedly inhibited during severe ischemia (23).
More recently, it was proposed that NHE inhibitors
could exert their anti-ischemic effect at the mitochondrial level by favoring the opening of mitochondrial
ATP-sensitive K⫹ channels (20). However, the mechanism by which cariporide could open these channels,
and the link between the ATP-sensitive K⫹ channel
opening and reduced cell death remain elusive. NHE is
not exclusive of plasmatic cell membranes, but it also
exists in the inner mitochondrial membrane (22). H⫹
extrusion from the mitochondrial matrix is coupled to
electron transport during cell respiration and results
in a large H⫹ gradient between both sides of the inner
mitochondrial membrane. This chemical gradient, together with the electrical charge difference across the
inner mitochondrial membrane (⌬⌿m) generates the
protonmotive force that moves H⫹ into the mitochondrial matrix through the H⫹ channel of the F0F1 complex (ATP synthase). H⫹ influx into the mitochondrial
matrix through the mitochondrial NHE (MNHE) constitutes a form of H⫹ leakage not coupled to ATP
synthesis that tends to dissipate the energy stored as
transmitochondrial membrane H⫹ gradient. During
anoxia or ischemia, the normal mechanism of H⫹ extrusion associated to electron transport stops, mitoThe 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.
0363-6135/03 $5.00 Copyright © 2003 the American Physiological Society
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Ruiz-Meana, Marisol, David Garcia-Dorado, Pilar
Pina, Javier Inserte, Luis Agulló, and Jordi Soler-Soler.
Cariporide preserves mitochondrial proton gradient and delays
ATP depletion in cardiomyocytes during ischemic conditions.
Am J Physiol Heart Circ Physiol 285: H999–H1006, 2003;
10.1152/ajpheart.00035.2003.—The mechanism by which inhibition of Na⫹/H⫹ exchanger (NHE) reduces cell death in ischemic-reperfused myocardium remains controversial. This study
investigated whether cariporide could inhibit mitochondrial
NHE during ischemia, delaying H⫹ gradient dissipation and
ATP exhaustion. Mouse cardiac myocytes (HL-1) were submitted to 1 h of simulated ischemia (SI) with NaCN/deoxyglucose
(pH 6.4), with or without 7 ␮M cariporide, and mitochondrial
concentration of Ca2⫹ (Rhod-2), 2⬘,7⬘-bis(2-carboxyethyl)-5(6)carboxyfluorescein (BCECF) and the charge difference across
the mitochondrial membrane potential (⌬⌿m, JC-1) were assessed. ATP content was measured by bioluminescence and
mitochondrial swelling by spectrophotometry in isolated mitochondria. Cariporide significantly attenuated the acidification
of the mitochondrial matrix induced by SI without modifying
⌬⌿m decay, and this effect was associated to a delayed ATP
exhaustion and increased mitochondrial Ca2⫹ load. These effects were reproduced in sarcolemma-permeabilized cells exposed to SI. In these cells, cariporide markedly attenuated the
fall in mitochondrial pH induced by removal of Na⫹ from the
medium. In isolated mitochondria, cariporide significantly reduced the rate and magnitude of passive matrix swelling induced by Na⫹ acetate. In isolated rat hearts submitted to
40-min ischemia at different temperatures (35.5°, 37°, or
38.5°C) pretreatment with cariporide limited ATP depletion
during the first 10 min of ischemia and cell death (lactate
dehydrogenase release) during reperfusion. These effects were
mimicked when a similar ATP preservation was achieved by
hypothermia and were abolished when the sparing effect of
cariporide on ATP was suppressed by hyperthermia. We conclude that cariporide acts at the mitochondrial level, delaying
mitochondrial matrix acidification and delaying ATP exhaustion during ischemia. These effects can contribute to reduce cell
death secondary to ischemia-reperfusion.
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MITOCHONDRIAL EFFECTS OF CARIPORIDE
chondrial matrix is flooded with H⫹, H⫹ gradient collapses, and ATP synthesis ceases (5) and may be eventually replaced by ATP hydrolysis coupled to H⫹
extrusion (4, 5). In the present study, we investigated
the hypothesis that inhibition of MNHE during ischemia slows both acidification of the mitochondrial matrix and ATP hydrolysis and that the resulting delay in
energy depletion significantly contributes to the slowed
progression of ischemic injury and reduced cell death.
METHODS
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This study was performed in HL-1 cells, a culture of
atrial-derived mouse cardiac myocytes (3) plated at 20,000
cells/cm2 density in glass-bottom culture dishes until a 70–
80% confluence was achieved. Additional studies were performed in isolated, perfused rat hearts from male SpragueDawley rats (300–350 g body wt) and in isolated rat heart
mitochondria. The experimental procedures conformed to the
National Institute of Health Guide for the Care and Use of
Laboratory Animals (NIH Publication No. 85-23, Revised
1996) and were approved by the Research Commission on
Ethics of the Hospital Vall d’Hebron.
Simulated ischemia in intact and permeabilized HL-1
cells. Experiments were performed on the stage of an inverted microscope (model IX70, Olympus) at ⫻20 magnification fluorite objective (UplaFL, Olympus). For JC-1 fluorescence measurement a real-time laser confocal microscope
was used (Nipkow system, QLC100, Visitech). Cells were
submitted to 1 h of simulated ischemia at 37°C by incubation
with a glucose-free buffer in the presence or in the absence of
7 ␮M cariporide. The buffer was composed of the following (in
mM): 2 NaCN, 20 2-deoxyglucose (DOG), 140 NaCl, 3.6 KCl,
1.2 MgSO4, 2 CaCl2, 20 HEPES, or, when so stated, 24
NaHCO3, at pH 6.4. Experiments were performed in HEPES
buffer for comparison with studies in membrane-permeabilized cells and replicated in bicarbonate-containing buffer to
rule out the possible influence of the effects of cariporide on
cytosolic pH in the absence of bicarbonate. These protocols
were well tolerated, and, after reexposure to control containing glucose at pH 7.4 for 30 min (simulated reperfusion), the
rate of cell death, assessed by propidium iodide, was ⬍10% in
all groups.
A subset of experiments was performed in myocytes permeabilized by 10-min incubation in an “intracellular-like”
medium containing the following (in mM): 135 KCl, 10 NaCl,
0.5 KH2PO4, 0.5 MgCl2, and 20 HEPES (pH 7.2) as well as 10
␮M digitonin. In these cells, ischemia was simulated by
addition to the “intracellular-like” medium of NaCN (2 mM)
and CaCl2 (10 ␮M) at pH 6.4.
Intramitochondrial [H⫹], [Ca2⫹], and ⌬⌿m. Changes in
mitochondrial [H⫹] ([H⫹]m), mitochondrial [Ca2⫹] ([Ca2⫹]m)
and ⌬⌿m were monitored by a fluorescence-imaging system
in cells subjected to simulated ischemia either in the presence or in the absence of cariporide. [H⫹]m was measured in
cells loaded with 3 ␮M 2⬘,7⬘-bis(2-carboxyethyl)-5(6)carboxyfluorescein (BCECF; Molecular Probes) (450/490 nm) for 1 h
in medium 199, and the cells were then washed and postincubated. To eliminate the cytosolic component in the overall
signal, the sarcolemma was permeabilized after the loading
procedure. Permeabilized cells were then submitted to 1 h of
simulated ischemia, and alternating excitation of the BCECF
dye at wavelengths of 450 and 505 nm was performed with
the use of a fast-speed monochromator (Visitech). Emitted
light was collected with an air-cooled intensified digital camera, and 450-to-505-nm ratios were calculated for each pixel
at 10-s intervals from background-substracted signal intensities in pairs of images consecutively obtained at the two
wavelengths. Color-coded 450-to-505-nm ratio images were
automatically generated and the average ratio was calculated for regions of interest defined within the cells. Changes
in these average ratio values throughout time were analyzed.
To measure [Ca2⫹]m and ⌬⌿m cells underwent a loading
protocol with 4 ␮M Rhod-2 and 10 ␮M JC-1 for 25 and 10
min, respectively, and were subjected to the same experimental protocol described above except that excitation was performed at 552 nm for Rhod-2 probe and at 488 nm for JC-1
probe (emission fluorescence was recorded at 530 nm and at
590 nm). Results in fluorescence intensity were expressed as
the percentage of change respect to the initial value and for
JC-1 probe as a percentage of change respect to initial 590to-530-nm emission ratio.
Na⫹-dependent changes in [H⫹]m. To confirm that 7 ␮M
cariporide may inhibit NHE at the mitochondrial level,
changes in [H⫹]m were monitored in permeabilized cells
exposed to an abrupt removal of Na⫹ from the intracellularlike buffer in the presence and in the absence of the drug.
Clonazepam (50 ␮M) was added throughout the Na⫹ removal
protocol to block mitochondrial Na⫹/Ca2⫹, thus preventing
Na⫹-dependent changes in [Ca2⫹]m.
Matrix swelling in isolated mitochondria. In a separate
series of experiments, the potential effect of different concentrations of cariporide on mitochondrial matrix swelling was
assessed spectrophotometrically in a suspension of isolated
mitochondria. Rat heart mitochondria were isolated by differential centrifugation, according to the method described
by Holmuhamedov et al. (12). Hearts were rapidly excised
from pentobarbital-anesthetized rats (100 mg/kg) and placed
in an ice-cold isolation buffer containing the following (in
mM): 50 sucrose, 200 mannitol, 5 KH2PO4, 1 EGTA and 5
MOPS and 0.1% BSA (at pH 7.15) adjusted with KOH. After
both atria were removed, ventricular myocardium was homogenized in three 20-s cycles using a Polytron (PT2100).
Homogenized tissue was centrifuged 10 min at 750 g. The
resulting supernatant containing the mitochondrial fraction
was further centrifuged 20 min at 7,000 g, and the pellet was
resuspended in the isolation buffer with no EGTA. Protein
concentration of the mitochondrial suspension was determined by Bradford assay before the experiments.
Increases in mitochondrial matrix volume were determined as changes in the light scattering parameter of mitochondrial suspensions (8) with the use of a spectrophotometer (Lambda20, Perkin-Elmer). Light absorbance of the mitochondrial suspension (at 0.5 mg/ml protein) was measured
at 546 nm. Passive swelling was induced with Na⫹ acetate
solution at pH 7.2 (2). K⫹-acetate was added to mitochondria
from a control group in which matrix swelling was absent.
The effect of cariporide on MNHE at 2, 7, or 20 ␮M and the
effect of 10-min preincubation with cariporide (7 ␮M) were
measured as a reduction of mitochondrial swelling induced
by Na⫹ acetate.
Cellular ATP content. ATP content was measured in cells
frozen in liquid N2 by means of the Bioluminescent Somatic
Cell Assay (Sigma Aldrich) after different periods of simulated ischemia.
Studies in isolated perfused rat heart. The relationship
between the effect of cariporide on ATP depletion during
ischemia and its protective effect against cell death induced
by ischemia-reperfusion was studied in isolated, perfused
hearts (n ⫽ 56). After intraperitoneal injection of pentobarbital sodium (100 mg/kg), the hearts were excised and perfused with a modified Krebs-Henseleit bicarbonate buffer
composed of (in mM) 140 NaCl, 24 NaHCO3, 2.7 KCl, 0.4
MITOCHONDRIAL EFFECTS OF CARIPORIDE
KH2PO4, 1 MgSO4, 1.8 CaCl2, and 11 glucose and equilibrated with 95% O2-5% CO2 (pH 7.4) at 10 ml/min. The
temperatures of the buffer and the heart chamber were
digitally controlled. Left ventricular pressure was monitored
with a water-filled latex balloon as previously described (7).
After 30 min of normoxic perfusion, the hearts were subjected to global nonflow ischemia at either 35.5°, 37°, or
38.5°C. Within each temperature group, hearts were allocated to receive 7 ␮M cariporide or vehicle during the last 10
min before ischemia. Four hearts from each of the resulting 6
groups were frozen in liquid N2 after 10 min of ischemia for
determination of myocardial ATP content. The remaining
hearts from each group (n ⫽ 4–5) were reperfused for 30 min
after 40 min of ischemia. Lactate dehydrogenase (LDH) activity in the coronary effluent was spectrophotometrically
measured as described (7). Myocardial ATP content was
determined as described for cell cultures.
Statistical analysis. The homogeneity between groups was
tested by ANOVA test; the effect of treatments on changes
along time were studied by means of the MANOVA test; and
the relationship between ATP content, time of onset of rigor
contracture and LDH release was assessed by regression
analysis (SPSS for Windows 8.0). To compare curves with
complex and highly variable forms, as those for [Ca2⫹]m, a
functional ANOVA test for functional data was used in which
curve functions rather than isolated numerical observations
are compared. A critical P value of 0.05 was used for all tests.
All values are expressed as means ⫾ SE.
membrane-permeabilized cells to media with different
and known pHs in the presence of 30 ␮M of the mitochondrial membrane protonophore dinitrophenol
(DNP) under identical acquisition conditions. The average BCEF ratio observed after 10 min of simulated
ischemia corresponded to a mitochondrial pH of 6.7 in
control cells and of 7.0 in the cariporide group.
Contribution of NHE to [Ca2⫹]m overload during
simulated ischemia. [Ca2⫹]m started to rise very early
after the initiation of simulated ischemia, a kinetics
that clearly differs from that obtained in cytosolic compartment, in which Ca2⫹ starts to rise after a more
prolonged time gap. Blockade of NHE during simulated ischemia with cariporide was associated to a
more sustained [Ca2⫹]m overload. At the end of simulated ischemia, Rhod-2 fluorescence was 199 ⫾ 29% of
the preischemic value in control cells and 238 ⫾ 30% in
cariporide-treated cells (functional ANOVA test, P ⫽
0.004; Fig. 2). In permeabilized cells, the initial rise in
[Ca2⫹]m was followed by a plateau and a subsequent
decay, probably reflecting Rhod-2 leakage in deenergized mitochondria and/or a net Ca2⫹ extrusion in the
absence of further influx. In these cells, the relative
increase in Rhod-2 fluorescence was attenuated respect
to what was obtained in intact cells, probably because
part of the fluorescent signal in intact cells comes from
cytosolic compartment. As in intact cells, cariporide
increased the overall amount of [Ca2⫹]m during simulated ischemia in permeabilized cells, and Rhod-2 fluorescence at the end of ischemia was 86 ⫾ 10% of
preischemic value in control cells and 127 ⫾ 17% in
cariporide-treated cells, P ⫽ 0.004 (Fig. 2). To assess
whether the increase in [Ca2⫹]m was mediated by reverse operation of the mitochondrial Na⫹/Ca2⫹ exchanger, additional experiments were performed, in
which this exchanger was inhibited by addition of
clonazepam (50 ␮M) during simulated ischemia. This
intervention did not result in a prevention of mitochondrial Ca2⫹ influx, but rather in an increase of the total
mitochondrial Ca2⫹ load.
RESULTS
Effect of cariporide on [H⫹]m during simulated ischemia. Simulated ischemia at pH 6.4 induced a progressive increase in [H⫹]m (reflecting a pH decline) that
reached its maximum after 10 min, and 7 ␮M cariporide markedly attenuated this increase (Fig. 1). This
effect reflected a blockade of NHE at the mitochondrial
membrane because results were obtained in permeabilized cells in which cytosolic composition was kept
constant. To provide an idea of the magnitude of the
effect of cariporide on mitochondrial pH, the BCECF
ratio was calibrated by the exposure of additional
AJP-Heart Circ Physiol • VOL
Fig. 2. Changes in mitochondrial [Ca2⫹] as assessed by fluorescence
imaging induced by SI in membrane-permeabilized and intact cardiomyocytes loaded with Rhod-2 (n ⫽ no. of cells) are shown.
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Fig. 1. Effect of 7 ␮M cariporide on mitochondrial matrix acidification as assessed by ratiofluorescence induced by simulated ischemia
(SI) in membrane-permeabilized cardiomyocytes loaded with 2⬘,7⬘bis(2-carboxyethyl)-5(6)-carboxyfluorescein (BCECF). An increase in
the 450-to-490-nm ratio indicates an increase in [H⫹].
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MITOCHONDRIAL EFFECTS OF CARIPORIDE
Mitochondrial depolarization during energy deprivation. The cells exposed to simulated ischemia experienced a rapid fall in the ⌬⌿m, as measured by the
decline in the 590/530 JC-1 emitted fluorescence. Mitochondrial depolarization reached its minimum level
at 10–15 min, a time coincident with ATP exhaustion
in this model. The addition of cariporide during simulated ischemia did not preserve ⌬⌿m or significantly
delayed its decline (Fig. 3).
Effect of cariporide on intracellular ATP depletion
during simulated ischemia. Intracellular ATP content
started to decline immediately after the onset of sim-
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Fig. 3. A: sequence of cardiomyocytes exposed to simulated ischemia in the absence (a) and in the presence of
cariporide (b). Images were obtained by a real-time laser confocal microscopy (⫻40) and represent the decline in
mitochondrial membrane potential (⌬⌿m) as assessed by a progressive decrease in JC-1 red fluorescence and an
increase in JC-1 green fluorescence during the initial minutes of simulated ischemia (0, 4, and 10 min), when the
maximal depolarization takes place, and after addition of 100 ␮M dinitrophenol (DNP) to promote complete
mitochondrial depolarization. B: average curve of mitochondrial depolarization during 60 min of simulated
ischemia assessed as the 590-to-530-nm ratio of JC-1 emitted fluorescence and after addition of 100 ␮M DNP at the
end of the experiment. C: ATP content in cells exposed to 5, 10, or 15 min of simulated ischemia.
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MITOCHONDRIAL EFFECTS OF CARIPORIDE
H1003
creased the degree of mitochondrial matrix swelling
induced by Na⫹-acetate and the rate at which it occurred, measured as the slope of the curve, whereas at
2 ␮M did not have any effect. Preincubation with 7 ␮M
cariporide 10 min before the addition of Na⫹-acetate
did not prevent matrix swelling development (Fig. 5).
Contribution of ATP preservation to protective effect
of NHE inhibition against ischemic-reperfusion cell
death. In isolated rat hearts exposed to nonflow ischemia at 37°C, pretreatment with cariporide significantly
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Fig. 4. Effect of cariporide on mitochondrial matrix acidification
induced by acute withdrawal of Na⫹ from intracellular-like medium
in membrane-permeabilized cardiomyocytes loaded with BCECF.
[Na⫹]o, extracellular Na⫹ concentration. A: time course of changes in
450-to-490-nm ratio. An increase in ratio indicates an increase in
[H⫹]. B: representative ratiofluorescence images obtained before (A1
and B1) and after acute removal of Na⫹ in the absence (A2) and in
the presence (B2) of 7 ␮M cariporide.
ulated ischemia. In NaHCO3 buffer, the ATP fall
reached 0.6 ⫾ 0.1% of its initial level after 15 min,
whereas in HEPES buffer, the ATP fall was significantly slower (data not shown). The presence of cariporide significantly delayed the rate of ATP exhaustion
(Fig. 3).
Na⫹-dependent changes in [H⫹]m. Removal of Na⫹
from the intracellular-like buffer induced an abrupt
increase in [H⫹]m in permeabilized cells. This increase
was markedly attenuated by 7 ␮M cariporide (Fig. 4).
Effect of cariporide on mitochondrial swelling. The
addition of Na⫹-acetate solution to a suspension of
isolated rat heart mitochondria was immediately followed by passive matrix swelling, as determined spectrophotometrically by a decrease in the 546-nm absorbance. Cariporide (7 and 20 ␮M) significantly deAJP-Heart Circ Physiol • VOL
Fig. 5. Effect of cariporide on the prevention of mitochondrial matrix
swelling in isolated rat heart mitochondria. A: changes in light
absorbance (arbitrary units) in isolated rat heart mitochondria submitted to passive matrix swelling in Na⫹ acetate-based medium. A
decrease in light absorbance reflects mitochondrial matrix swelling.
Each curve represents the average of five experiments performed in
the absence (control) or in the presence of 7 ␮M cariporide in the
Na⫹-acetate medium. B: changes in the slope of the absorbance
curves in isolated rat heart mitochondria during the initial 5 s of the
addition of different treatments as a parameter of the rate of mitochondrial swelling development. Prevention of mitochondrial swelling in a Na⫹-acetate medium was observed with cariporide at 7 and
20 ␮M, but not at 2 ␮M. Preincubation with cariporide at 7 ␮M
during 10 min before the addition of Na⫹-acetate did not attenuate
mitochondrial swelling. K⫹ acetate was used as a control group, in
which mitochondrial swelling was absent. a.u., Arbitrary units of
light absorbance.
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MITOCHONDRIAL EFFECTS OF CARIPORIDE
DISCUSSION
This study demonstrates that NHE inhibition with
cariporide exerts a protective effect at the mitochondrial level, delaying acidification of the mitochondrial
matrix in cultured cardiomyocytes during simulated
ischemia without modifying ⌬⌿m. This effect is associ-
Fig. 6. Correlation between lactate dehydrogenase (LDH) release
during reperfusion and time of onset of ischemic rigor contracture (A)
or ATP content after 10 min of ischemia (B) in isolated rat hearts
submitted to 40 min of nonflow ischemia at different temperatures.
Both correlations are very high and not influenced by treatment with
cariporide.
AJP-Heart Circ Physiol • VOL
ated to slowed ATP depletion and is not a mere reflection of changes in cytosolic cation concentration or pH,
because it is observable in permeabilized cells. The
protective effect of cariporide against ATP depletion is
also observable in isolated rat hearts submitted to
nonflow ischemia, and regression analysis indicates
that it fully accounts for the reduction in cardiomyocyte death during subsequent reperfusion. These results are consistent with the hypothesis that NHE
inhibitors may protect myocardium against cell death
secondary to ischemia-reperfusion by acting on mitochondrial membrane, an effect that results in less H⫹
entry into the matrix, and preservation of transmembrane H⫹ gradient and ATP concentration.
Effect of cariporide on mitochondrial matrix acidification. Cariporide is an amiloride-related compound
with a potent and highly selective inhibitory effect on
NHE, in particular on the NHE1 isoform. The hypothesis that cariporide inhibits MNHE implies that it can
cross cell membranes and reach mitochondria and that
the mitochondrial exchanger can be inhibited by the
drug. To our knowledge, no published study has measured the permeability of cell membranes to cariporide
or other NHE inhibitors. However, calculation of the
logarithmic number of partition coefficient for cariporide yields a value of 0.1 at a pH higher than its pKa
(pH ⬎ 4.42). This predicts that cariporide can penetrate cell membranes and reach MNHE. The observation that cariporide markedly attenuates both the decrease in mitochondrial pH induced by removal of Na⫹
in permeabilized cells and passive matrix swelling induced by Na⫹ acetate in isolated mitochondria clearly
indicates that the drug can inhibit MNHE in cardiomyocytes.
The importance of NHE at the inner mitochondrial
membrane has been known for almost three decades.
In higher eukaryotes, studies have found that MNHE
is encoded by the NHE6 gene and has a molecular
structure similar but not identical to sarcolemmal
NHE (22), although recent reports question the unequivocal identification of NHE6 to the MNHE (21).
MNHE can be effectively inhibited by sulfonylurea
derivatives, including amiloride and more recently developed inhibitors of sarcolemmal NHE (2, 13, 14).
Under normal conditions, a gradient of H⫹ across the
inner mitochondrial membrane, with the mitochondrial matrix more alkaline than the intermembrane or
cytosolic spaces, is maintained by the active extrusion
of H⫹ coupled with electron transport along the respiratory chain, as was first postulated by Mitchell in his
chemiosmotic theory (19). During ischemia H⫹ extrusion stops, leading to matrix acidification and a progressive dissipation of H⫹ gradient. The results of the
present study support the hypothesis that H⫹ influx
into the mitochondrial matrix via MNHE significantly
contributes to matrix acidification during ischemia.
The attenuated matrix acidification observed in the
presence of cariporide cannot be the indirect consequence of changes in cytosolic pH because it is observed
for a constant pH in permeabilized cells. Moreover,
previous studies have demonstrated a lack of effect of
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delayed the onset of rigor contracture by ⬃3 min,
increased myocardial ATP content observed after 10
min of ischemia by 41% (P ⬍ 0.01), and reduced by
⬎50% (P ⬍ 0.01) the extent of reperfusion-induced
hypercontracture, as assessed by peak left ventricular
diastolic pressure, and cell death, as assessed by LDH
release. Reducing myocardial temperature during ischemia had per se a strong effect on myocardial ATP
content, and did mimic the protection afforded by cariporide. Increasing myocardial temperature accelerated ATP consumption during ischemia and attenuated the effects of the drug. In the whole series of
hearts, a close relationship was observed between the
rate of ATP depletion during the first 10 min of ischemia and the time of onset of ischemic rigor (r ⫽ 0.85).
Both variables were closely correlated with LDH release during reperfusion (r ⫽ 0.99) (Fig. 6). Nevertheless, these relationships were not modified by cariporide. Multiple regression analysis showed that time of
rigor onset accurately predicted the extent of LDH
release during reperfusion, and that inclusion of treatment allocation in the regression model did not significantly improve its predictive value.
MITOCHONDRIAL EFFECTS OF CARIPORIDE
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cardial temperature within the physiological range
(35.5–38.5°C) allowed us to evaluate the contribution
of the effect of cariporide on ATP content to its protective effect against ischemia-reperfusion injury. Regression analysis demonstrated that the extent of cell
death during reperfusion could be predicted by the
degree of ATP depletion after 10 min of ischemia or by
the time of onset of ischemic rigor contracture (an
index of severe ATP depletion). This is consistent with
studies showing that rigor contracture marks the onset
of progressive cytosolic Ca2⫹ rise and the development
of cell fragility (17) and with data showing that the
time elapsed between development of rigor and reoxygenation or reperfusion, rather than total duration of
hypoxia or ischemia, determines the outcome of reenergization (29). Cariporide shifted rigor onset and LDH
release along the regression line without modifying it,
and inclusion of the drug treatment in the model did
not improve its predictive value. The protective effect
of cariporide against cell death secondary to ischemiareperfusion was, therefore, fully explained in this
model by its effect on ATP decay during the ischemic
period. This result is consistent with previous studies
showing that cariporide needs to be present during the
ischemic period to be protective (6, 16).
Study limitations. Because the present study was
focused on the potential role of mitochondria in the
protective effect of cariporide during ischemia, a cell
system most adequate to this purpose was used.
HL1 cardiomyocyte cells accurately reproduce the
changes in cytosolic composition induced by simulated
ischemia in freshly isolated cardiomyocytes, but do not
develop the changes in cell shape associated to ischemic rigor contracture and reenergization-induced hypercontracture. These changes in cell shape create
important artifacts in nonratiometric fluorescent signals, including those from several important mitochondrial probes. On the other hand, these cells present the
disadvantage of being more tolerant than freshly isolated cardiomyocytes to energy deprivation. This precluded any attempt to establish a direct correlation
between the mitochondrial effects of cariporide during
simulated ischemia and its protective effect against
cell death, which occurs during reenergization. However, the direct correlation observed in intact rat
hearts between the magnitude of the effect on ATP and
the reduction of cell death suggests that the mitochondrial effects observed in cell cultures participate in the
protection.
In conclusion, this study demonstrates that NHE
inhibitors may have effects at the mitochondrial level,
delaying mitochondrial matrix acidification and slowing down ATP depletion during ischemia. These results
provide a new mechanism for the beneficial effect of
NHE inhibitors against cell death secondary to ischemia-reperfusion and stresses the importance of mitochondria as targets for novel therapeutic approaches.
We are indebted to Prof. Antonio Cuevas for performing the
functional ANOVA statistics and to Dr. Alberto Fernández for calculation of the logarithmic number of partition coefficient for caripo-
285 • SEPTEMBER 2003 •
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cariporide on cytosolic pH when other mechanisms of
pH correction, like NaHCO3 cotransporter, are active.
The effect of cariporide should be an enhancement of
cytosolic acidosis that would not explain the reduced
acidification of mitochondrial matrix. Because the H⫹
gradient across the inner mitochondrial membrane is
one of the two components of the protonmotive force
responsible for H⫹ flow through the F0 channel of the
ATP synthase, and its reduction may determine ATP
hydrolysis by the F1 unit of ATP coupled to H⫹ extrusion, delayed matrix acidification during simulated ischemia should be expected to result in attenuated rate
of ATP depletion, as observed.
To our knowledge, no previous study has analyzed
the effect of NHE inhibition on the increase in [Ca2⫹]m
induced by ischemia. In a recent study (31), pretreatment with the selective NHE inhibitor SM-20550 was
associated with attenuated Ca2⫹ overload in mitochondria obtained from rat hearts submitted to 40 min of
ischemia and 20 min of reperfusion, a result in agreement with the protective effect of NHE inhibition
against cell necrosis secondary to ischemia-reperfusion,
but [Ca2⫹]m during ischemia was not measured. Our
observation that cariporide, at concentrations that
have been consistently found cardioprotective in isolated cells (25, 27) and intact myocardium in vivo (6,
16), enhanced mitochondrial Ca2⫹ accumulation during simulated ischemia may appear surprising. Although increased [Ca2⫹]m has been shown to have
detrimental effects (4), there is a lack of information on
the consequences of mitochondrial Ca2⫹ overload during ischemia. It has been previously shown that mitochondria with severe Ca2⫹ overload secondary to hypoxia or simulated ischemia may rapidly recover metabolic competence upon reoxygenation (9). Also, Ca2⫹
sequestration into mitochondria may attenuate cytosolic Ca2⫹ overload during ischemia and reperfusion.
Moreover, extracellular acidification, a condition
known to be strongly protective against hypoxic injury,
is associated to enhanced increase of [Ca2⫹]m induced
by hypoxia (see below).
Several recent reports have described the ability of
mitochondria to accumulate Ca2⫹, acting as a local
spatial buffering system often in close association with
sarcoplasmic reticulum (24). The presence of 50 ␮M
clonazepam during simulated ischemia did not result
in a prevention of mitochondrial Ca2⫹ influx, but
rather in an increase of the total mitochondrial Ca2⫹
load suggesting that reverse-mode operation of the
mitochondrial Na⫹/Ca2⫹ exchanger is not responsible
for mitochondrial Ca2⫹ influx during prolonged ischemia and may in fact represent a route of Ca2⫹ efflux.
Slowed ATP depletion during ischemic conditions.
The present observations in cultured cardiomyocytes
exposed to simulated ischemia and in intact myocardium submitted to zero-flow ischemia are in agreement
with previous studies showing that NHE inhibition
slows-down the rate of ATP depletion during ischemia
and delays the onset of rigor contracture (6, 11, 25).
Moreover, in the present study, modulation of the rate
of ATP depletion during ischemia by modifying myo-
H1005
H1006
MITOCHONDRIAL EFFECTS OF CARIPORIDE
ride, and for collaboration in experiments with isolated mitochondria. Cariporide was a kind gift from Aventis Pharma.
DISCLOSURES
This work was supported in part by Spanish Ministry of Science
and Technology Grant Comisión Interministerial de Ciencia y Tecnologı́a/99-102.
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