(Circulation. 1995;91:2810-2818.)
© 1995 American Heart Association, Inc.
Articles |
Mechanisms of Ischemic Preconditioning in Rat Myocardium
Roles of Adenosine, Cellular Energy State, and Mitochondrial F1Fo-ATPase
From the Departments of Medical Biochemistry (K.V., P.R., I.E.H., A.A.-R.) and Internal Medicine (K.Y., K.P.), University of Oulu, Oulu, Finland.
Correspondence to Dr Ilmo Hassinen, Department of Medical Biochemistry, University of Oulu, Kajaanintie 52 A, FIN-90220 Oulu 22, Finland.
 |
Abstract |
|---|
 Top Abstract IntroductionMethods Results Discussion References |
|---|
Background Adenosine has been proposed as one mediator for the preconditioning effect in the myocardium of some animals, but recent investigations have shown that this may not be the mechanism in the rat heart, although the effect itself is clearly demonstrable. The cellular energy state has been shown to be better in preconditioned hearts, and the role of ATP consumption has been discussed. The role of inhibition of mitochondrial F1Fo-ATPase as a mechanism for the preservation of ATP in preconditioned hearts remains controversial.
Methods and Results Three-minute global ischemia followed by 9 minutes of reperfusion was used to precondition Langendorff-perfused rat hearts, and control hearts were perfused under normoxic conditions for the same time. The duration of sustained ischemia in both groups of hearts was 21 minutes, after which the hearts were reperfused for 15 minutes to evaluate their mechanical and metabolic recovery. Separate experiments were performed for tissue metabolite determinations, mitochondrial ATPase activity measurements, and 31P nuclear magnetic resonance studies. The recovery of the rate-pressure product was better in the preconditioned group. Three-minute preconditioning ischemia caused inhibition of the mitochondrial ATPase that persisted throughout the 9-minute intervening reperfusion so that at the early stages of sustained ischemia the enzyme activity was still more inhibited in preconditioned hearts. ATP was better preserved in preconditioned hearts than in control hearts during sustained ischemia. The accumulation of adenosine and its degradation products during sustained ischemia was greater in the control group. More lactate and H+ ions accumulated in this group, indicating higher anaerobic glycolysis. Also, inhibition of mitochondrial ATPase by oligomycin slowed ATP depletion during ischemia.
Conclusions The results indicate that preconditioning causes inhibition of rat heart mitochondrial ATPase that persists during reperfusion so that the enzyme is inhibited from the very beginning of the sustained ischemia. This inhibition leads to sparing of high-energy phosphates and improves the time-averaged energy state during ischemia. Although a causal relationship is difficult to prove, this reversible inhibition may contribute to postischemic recovery of the heart.
Key Words: adenosine • myocardium • ischemia
|
Introduction |
|---|
|
TopAbstractIntroductionMethods Results Discussion References |
|---|
The phenomenon of preconditioning was first described in 1986 by Murry et al,1 who discovered the protective effect of a brief ischemic period with regard to the detrimental consequences of subsequent prolonged ischemia. Since then, preconditioning has been shown to limit infarct size2 and to reduce ventricular arrhythmias during sustained ischemia and reperfusion.3 4 5 Brief repetitive periods of ischemia have been shown to retard cardiac energy metabolism during sustained ischemia in dogs,6 and 31P nuclear magnetic resonance (NMR) has been used as a noninvasive method to demonstrate that the decreases in high-energy phosphate concentrations and intracellular pH during prolonged ischemia are attenuated in the preconditioned pig myocardium.7
Adenosine A1 receptor activation has been proposed as a mechanism for preconditioning in some animal species,8 9 but it has recently been reported that postischemic dysfunction in the rat heart is not attenuated by adenosine agonists or exogenous adenosine.10 11 Experiments with isolated rat cardiomyocytes have also failed to demonstrate a beneficial effect of adenosine and A1-selective agonists on cell survival after ischemia.12 Whatever its mechanism, the clinical significance of preconditioning has been emphasized, and some evidence has been found for it in the human myocardium.13 14
The work of Rouslin15 suggests that mitochondrial ATP synthase, a reversible H+-ATPase (F1Fo-ATPase), may be a major ATP consumer in ischemia. ATP synthesis is driven by the proton gradient and membrane potential of the mitochondrial inner membrane (for a review of the structure and regulation of F1Fo-ATPase, see Reference 1616 ). When this electrochemical gradient collapses during ischemia, the reaction catalyzed by the ATP synthetase begins to run in reverse, resulting in ATP hydrolysis. This imposes a need for a physiological inhibitor of F1Fo-ATPase. Indeed, modulation of F1Fo-ATPase by an inhibitory protein (IF1) has been demonstrated in isolated mitochondria and cultured rat cardiomyocytes subjected to a variation in mechanical work load,17 and it has been proposed that the metabolic role of IF1 is to prevent ATP wastage by the backward reaction of ATP synthase in deenergized mitochondria.17 Inhibition of F1Fo-ATPase during ischemia has also been proposed as one mechanism for ATP preservation due to preconditioning,18 although this has never been studied properly, probably because of methodological difficulties.
Despite the tremendous effort put into experimentation, the mechanism of preconditioning remains controversial. We therefore set out to study further the regulation of the mitochondrial F1Fo-ATPase in preconditioned hearts. Langendorff-perfused isolated rat hearts were used as an experimental model; we measured the activity of mitochondrial ATPase during and after preconditioning and during sustained ischemia. Both noninvasive NMR methodology and a conventional biochemical approach were used to evaluate whether metabolic differences exist between preconditioned and control myocardium during sustained global no-flow ischemia and subsequent reperfusion. The rapidity and reversibility of the regulation of F1Fo-ATPase necessitated a new method for rapid tissue fractionation and activity measurement. This allowed us to demonstrate that reversible regulation of the mitochondrial ATP synthase occurs in the rodent heart and that this may be involved in myocardial preconditioning.
|
Methods |
|---|
|
TopAbstractIntroductionMethods Results Discussion References |
|---|
Animals and Perfusion Methods
The work was approved by the local ethical committee in accordance with recognized standards for the care and use of laboratory animals.
Three-month-old male Sprague-Dawley rats from the Department of Physiology, University of Oulu, were used in all of the experiments. They were anesthetized with intraperitoneal sodium pentobarbital (60 mg/kg body wt); 500 IU heparin was injected into the inferior vena cava; and 1 minute later, the hearts were excised and rinsed in phosphate-free ice-cold Krebs-Henseleit perfusion medium containing 120 mmol/L NaCl, 4.7 mmol/L KCl, 2.5 mmol/L CaCl2, 0.25 mmol/L Ca-EDTA, 1.2 mmol/L MgSO4, 25 mmol/L NaHCO3, 10 mmol/L glucose, and 12 IU/L insulin. The hearts were perfused in a modified Langendorff apparatus at a pressure of 90 cm H2O (8.83 kPa) with the medium maintained by thermostat at 37°C and gassed with O2-CO2 (19:1). In the NMR experiments, a 4-m thermostated perfusion line was used to keep the perfusion equipment away from the superconducting magnet of the NMR spectrometer. Total, global ischemia was induced by discontinuation of the perfusate flow.
Experimental Protocol
The hearts were divided into a
preconditioning group and a
control group for measurement of heart rate, left ventricular systolic
pressure, coronary flow, and oxygen consumption and for collection of
the effluent perfusate samples. All the experiments were preceded by a
15-minute normoxic stabilization period. After a 9-minute normoxic
period, the preconditioning group was subjected to 3 minutes of
preconditioning ischemia, a 9-minute intervening reperfusion period,
and 21 minutes of ischemia followed by a 15-minute reperfusion. The
control group underwent a 21-minute normoxic period and 21 minutes of
ischemia followed by a 15-minute reperfusion. The same protocol was
used in the NMR experiments except the final reperfusion time was 18
minutes.
Two separate sets of experiments with the above perfusion
protocol were
performed to obtain tissue samples for both biochemical determination
of metabolites (Table 1
) and measurement of the activity
of F1Fo-ATPase at various time points (as shown
in Fig 4). When we tested the effects of oligomycin on the
cellular
energetics of perfused or ischemic hearts, the antibiotic was dissolved
in dimethyl sulfoxide (DMSO) and added to the perfusate to give a final
concentration of 3 µg/mL (and 0.3% DMSO). Control perfusions were
performed with the same concentration of DMSO alone.
|
|
An additional
experiment was performed for estimation of the time
course of inhibition of mitochondrial ATPase during sustained ischemia.
Rat hearts were perfused for 36 minutes, and the apex of each was then
cut into three slices. One slice was immediately assayed for ATPase
activity, and the other two were placed into plastic self-sealing bags
that were incubated in a water bath for either 12 or 21 minutes, after
which the ATPase assay was performed. In this way, it was possible to
estimate the time course of inhibition using the heart as its own
control (see Fig 3).
|
Left Ventricular Pressure, Heart Rate, and Oxygen
Consumption
Left ventricular pressure was monitored by inserting a
water-filled latex balloon into the left ventricle and connecting it to
a Statham P231D pressure transducer and Statham SP1400 pressure
monitor. End-diastolic pressure was adjusted to 15 to 20
mm Hg. Heart rate was measured from the pressure curve with a digital
frequency counter. Coronary flow was monitored throughout the
experiments using a drop counter with an analog frequency output.
Oxygen consumption was calculated as the arteriovenous concentration
difference multiplied by the coronary flow.
31P NMR Spectroscopy
A Bruker AM-200 Fourier
transform NMR spectrometer was used to
record the 31P NMR spectra. The field strength of the
superconducting magnet was 4.7 T; phosphorus has a resonance frequency
of 81.02 MHz. The heart and a glass capillary containing
methylenediphosphonic acid (DMPA) to serve as an internal standard were
inserted into a 16-mm tube, which was put into a laboratory-built
probe. Before the experiment, the magnet was shimmed to a line width of
0.15 ppm using an H3PO4 solution. Because the
insertion of the heart with the auxiliary perfusion equipment caused a
minor change in the magnetic field, the homogeneity of the latter was
readjusted to the same line width by reference to the water proton
resonance readily detectable in the phosphorus-tuned probe.
Blocks of 64 free induction decays were accumulated over consecutive 3-minute periods using a pulse width of 20 µs resulting in a 55-degree tilting angle. A pulse interval of 2.7 seconds and an acquisition time of 0.85 second were used. Multiplication by an exponential function resulted in a line broadening of 10 Hz. Partial saturation of inorganic phosphate (Pi) was observed at this pulse-repetition rate. In calculation of concentrations of the phosphorus-containing metabolites, correction for saturation was applied on the basis of a calibration curve obtained with varying pulsing rate. Because all measurements were made in reference to the DMPA internal standard, only saturation factors relative to DMPA saturation were needed; these were 0.781 for Pi, 1.01 for creatine phosphate (CrP), and 1.202 for ATP-ß for the 2.712-second pulse-repetition rate used.
The spectra were manipulated
using Bruker WIN-NMR software
(Bruker Spectrospin AG) on a personal computer, with the areas and
positions of the resonance peaks calculated after phase correction,
baseline correction, and deconvolution. Typical NMR spectra are
presented in Fig 6.
|
The H+ ion concentration in the cytosol was calculated from the chemical shift of Pi by the following equation19 :
 |
where
Pi denotes the chemical shift of
Pi in ppm by reference to CrP. During sustained ischemia,
when the peak area of CrP reached very low values, the DMPA peak (20.9
ppm by reference to CrP) served as a standard for calibration of the
ppm scale.
The relative cytosolic concentrations of ATP, CrP, and Pi were calculated from the area under the ATP-ß-phosphorus, CrP, and Pi peaks, respectively.
Metabolite Measurements
Effluent perfusate samples were
collected over consecutive
1-minute periods for the measurement of adenosine and its degradation
products (inosine, hypoxanthine, and xanthine). The samples were
quenched with perchloric acid (final concentration, 1%) and kept at
-20°C until analyzed. Nucleosides and free purines were measured by
high-pressure liquid chromatography (HPLC), essentially as described by
Raatikainen et al.20
The frozen myocardial tissue samples were pulverized and extracted with 8% (wt/vol) HClO4 in 40% (vol/vol) ethanol precooled to -20°C, and the extraction was repeated with 6% (wt/vol) HClO4. The filtrates were neutralized to pH 6 with 3.75 mol/L K2CO3 containing 0.5 mol/L triethanolamine hydrochloride.21
Lactate and pyruvate were
determined by enzymatic methods, measuring
the appearance or disappearance of NADH in an Aminco DW-2
dual-wavelength spectrophotometer using an 
340 minus
385 value of 5.33 mmol/L per centimeter. Tissue
adenosine and its degradation products were measured as described by
Raatikainen et al.20
Tissue Fractionation and Measurement of
F1Fo-ATPase Activity
After perfusion, the hearts
were immersed in ice-cold buffer
containing 20 mmol/L HEPES, 1 mmol/L MgCl2, and 2
mmol/L EGTA, pH 7.0. The heart was immediately homogenized in an
Ultra-Turrax homogenizer (Ika Werk) for 30 seconds. The homogenate (1.5
mL) was sonicated for a total of 25 seconds in five burst time periods
separated by 7 seconds of cooling in an ice-water bath. The sonication
power and time were optimized to obtain maximal ATPase activity and
retention of the in situ regulatory state of the enzyme. The homogenate
was then centrifuged at 5300 rpm in a Hereus Biofuge A centrifuge for
30 seconds. The supernatant was diluted in the above-described
homogenization-sonication buffer to a protein concentration of
approximately 0.5 mg/mL.
ATPase was measured on a Shimadzu-3000 dual-wavelength spectrophotometer at 28°C. The sample (10 µL) was added to a cuvette containing 33 mmol/L Tris acetate, 83 mmol/L sucrose, 10 mmol/L MgCl2, 1 mmol/L KCN, 1 mmol/L EDTA, 2 mmol/L ATP, 1.5 mmol/L phosphoenolpyruvate, 0.17 mmol/L NADH, 6 U pyruvate kinase, and 12 U lactate dehydrogenase at pH 7.2; the decline in the NADH absorbance difference at 340 and 385 nm was observed for 5 minutes, after which oligomycin was added to the reaction cuvette to inhibit F1Fo-ATPase to estimate the contribution of the other ATPase activities.22 Eighty-five percent of the total activity was found to be oligomycin sensitive. The time interval between the end of the perfusion and the beginning of measurement of the enzyme activity varied from 3.5 to 4 minutes. Protein was measured using the Bio-Rad assay.23
Cytochrome c oxidase was assayed essentially according to Cooperstein and Lazarow,24 measuring the oxidation of reduced cytochrome c by the change in the absorbance difference between 550 and 540 nm.
Statistical Analysis
The mechanical function of the hearts
during the final
reperfusion period was evaluated using summary measures,25
ie, the area under curve was integrated in each experiment for the time
period of interest, the average values of the groups were calculated,
and Student's t test for independent mean values was
applied. The same test was also used to evaluate the final mechanical
recovery of the hearts (last data point) and differences in tissue
metabolites and ATPase activities between the preconditioned and
control groups at various time points. In statistical evaluation of the
recovery of mechanical function, fibrillating hearts were included by
giving them a pulse rate value of zero. ANOVA for repeated measurements
followed by paired t test applying the Bonferroni method was
used to evaluate the time course of mitochondrial ATPase inhibition. A
one-way ANOVA followed by the least significant difference test was
used for differences in the ratio of ATPase to cytochrome c
oxidase and in cytochrome c oxidase activity.
An ANOVA for
repeated measurements was used for the data obtained by
NMR spectroscopy using the SPSS program package (SPSS,
Inc). Student's t test served as a post hoc test. Data are
expressed as mean±SEM. A value of P
.05 was considered
statistically significant.
|
Results |
|---|
|
TopAbstractIntroductionMethods Results Discussion References |
|---|
Mechanical Performance, Coronary Flow, and Oxygen Consumption
A rapid reduction in left ventricular systolic pressure occurred during the 3-minute preconditioning ischemia, and the pressure remained depressed for the subsequent 9-minute reperfusion compared with the preischemic values. The hemodynamic parameters during sustained ischemia were similar in both groups, but the systolic pressure was higher in the preconditioned hearts (P=.034 by the summary method) during the entire final perfusion; the heart rate also was higher in these hearts during the same period (P=.019 by the summary method). The data on the mechanical performance of the hearts are summarized in Fig 1
, in which values are
presented in fractional units with reference to the
preischemic basal values. The absolute preischemic heart rate,
systolic pressure, and rate-pressure product values, respectively, were
3.6±0.2 Hz, 118±10 mm Hg, and 424±35 mm Hg · Hz in
the
preconditioned group and 3.5±0.2 Hz, 122±12 mm Hg, and
432±47
mm Hg · Hz in the control group; there were no statistically
significant differences.
|
All of the preconditioned hearts recovered from the sustained ischemia during the final reperfusion, but the response of the control hearts to reperfusion was unpredictable. One heart showed an overshoot in mechanical performance; three recovered in the same way as the preconditioned hearts; five showed only partial recovery; and two started a slow, irregular beat and went into ventricular fibrillation. Two hearts remained in asystole.
The preconditioning ischemia caused a
marked reactive increase in
coronary flow and oxygen consumption during the 9-minute reperfusion,
but the temporal patterns of coronary flow and oxygen consumption
during the final reperfusion period were similar in the two groups (Fig
2).
|
Activity of F1Fo-ATPase
The activity of
the mitochondrial oligomycin-sensitive ATPase
became inhibited in a time-dependent manner during continuous ischemia
(P<.05), as depicted in Fig 3
. ATPase
activities during the ischemia and reperfusion protocol are depicted in
Fig 4. The 3-minute preconditioning ischemia caused an
inhibition of the enzyme activity that persisted throughout the
9-minute reperfusion period so that the activity of the enzyme was
lower in the preconditioned group than in the control group in the
early phase of sustained ischemia. There were no differences in ATPase
activity from 12 minutes of ischemia on, however. During the final
15-minute reperfusion, enzyme activity returned toward preischemic
levels in both groups.
Magnetic Resonance Spectroscopy: High-Energy Phosphates and
Cytosolic pH
The CrP-to-Pi ratio, which qualitatively
reflects the
cellular energy state (cytosolic
[ATP]/[ADP] · [Pi]), decreased
sharply during the
preconditioning ischemia, and an overshoot phenomenon could be seen
during the following reperfusion period (Fig 5). In the
first 3-minute block of sustained ischemia, the CrP-to-Pi
ratio in the preconditioned group was higher than in the control group
(P<.05). The overshoot in the CrP-to-Pi ratio
could also be seen in the preconditioned group during the final
reperfusion period. As shown in Fig 6, this overshoot
was mainly caused by a decrease in the Pi concentration.
Because the changes in high-energy phosphates and Pi are
stoichiometric and the Pi concentration is small compared
with that of CrP or ATP, changes in the cellular energy state mainly
reflect those of Pi, whether expressed as the
CrP-to-Pi or the ATP-to-ADP · Pi
ratio.
|
Despite the decrease in the energy state, the ATP level remained
almost
unchanged during the 3-minute preconditioning ischemia period due to
buffering by the creatine kinase reaction (Fig 5). The ATP
level
appeared to decrease more slowly in the preconditioned group at the
beginning of the sustained ischemia (P.05 in the third and
fourth 3-minute periods). The difference vanished toward the end of the
ischemia period, but the ATP concentration reached higher levels in the
preconditioned group than in the control group during the final
reperfusion.
pH declined to 6.74±0.04 during the preconditioning ischemia. The rate of decrease in cytosolic pH was then similar in both groups for the first 12 minutes of sustained ischemia, but afterward it settled to a steady level in the preconditioned hearts, whereas in the control hearts it declined further, reaching 5.43±0.14 during the final 3 minutes compared with 5.95±0.09 in the preconditioned hearts (mean±SEM, P=.014 for the difference). When perfusion was commenced, the cytosolic pH rapidly returned to the basal level in both groups.
Because the ATPase measurements suggested that inhibition of the mitochondrial F1Fo-ATPase might be involved in the observed improvement in postischemic survival, the paradigm was tested by infusing oligomycin, an inhibitor of F1Fo-ATPase, into one group of hearts before global ischemia was applied. The 5-minute preischemic infusion of oligomycin did not affect the heart rate or systolic pressure, and the ischemic arrest occurred with a similar time course in oligomycin-treated and control hearts.
NMR measurements showed that
although the cellular energy state began a
decline on oligomycin infusion due to inhibition of ATP synthesis,
further decrease in the ATP concentration during the subsequent
ischemia was slowed (P<.05) by oligomycin (Fig 5).
However,
because the oligomycin inhibition is irreversible, it was not possible
to test the effect of oligomycin on functional and metabolic recovery
during reperfusion. In hearts infused with plain DMSO at the same 0.3%
final concentration, ATP depletion occurred with the same kinetics as
in noninfused hearts, so the observed oligomycin effect was not due to
the vehicle DMSO. An inefficiency of low concentrations of DMSO in
affecting performance or adenine nucleotide content in isolated,
perfused rat hearts has also been reported.26
Tissue Metabolite Content
The lactate-to-pyruvate (L-P)
ratio, which reflects the redox
state of the cytosolic NADH/NAD pool through mediation of the lactate
dehydrogenase reaction, responded to the preconditioning ischemia as
expected, but the values at the end of the intervening reperfusion were
similar in both groups of hearts (Table 1). It is significant
that the
lactate concentration (P=.079) and the L-P ratio
(P<.05) were higher in the nonpreconditioned group at the
end of the sustained ischemia.
Tissue concentrations of adenosine and
adenosine compounds (adenosine
plus inosine plus hypoxanthine plus xanthine) at various time points
are presented in Table 1. Although the expected formation of
adenosine compounds was observed during the 3-minute preconditioning
ischemia, the levels at the end of the 9-minute reperfusion tended to
be lower in the preconditioned hearts. The sustained 21-minute
ischemia caused a marked rise in adenosine and adenosine compounds, but
the increase was smaller in the preconditioned group. There were no
differences in tissue adenosine compounds between the groups at the end
of the final reperfusion.
An enhanced output of adenosine could be observed in effluent perfusate after 3 minutes of preconditioning ischemia (12.2±3.2 versus 1.79±0.5 nmol · min-1 · g dry wt-1), but the adenosine levels in the efflux had returned to preischemic levels (2.1±0.4 nmol · min-1 · g dry wt-1) by the end of the 9-minute reperfusion period. It is surprising that although the tissue levels of adenosine compounds differed significantly after a long period of ischemia, no difference could be seen in the total efflux levels of adenosine compounds (preconditioned, 4.1±0.5 µmol/g dry wt; control, 4.3±0.4 µmol/g dry wt). The slopes of the adenosine output curves were similar during the final reperfusion period (data not shown).
Yield of Mitochondria
As the ATPase activity was measured in
a rather crude tissue
fraction, it was important to determine that the observed activity
changes were not due to variations in the yield of mitochondria. For
this purpose, we measured the activity of cytochrome c
oxidase, a mitochondrial inner membrane enzyme, in the tissue fraction.
Das and Harris27 used NADH oxidase as a mitochondrial
marker enzyme, but since it has been shown that NADH oxidase activity
decreases markedly during the first hour of ischemia,28 we
used cytochrome c oxidase, an intrinsic membrane enzyme that
is fairly stable during ischemia.
Table 2 shows that the
observed changes (on a protein
basis) persist when expressed relative to the inner membrane marker and
that the yield of mitochondria remains fairly constant throughout the
experimental protocol.
|
|
Discussion |
|---|
|
TopAbstractIntroductionMethods Results Discussion References |
|---|
Results from the present study suggest that preconditioning of the myocardium in the rat heart may be mediated by inhibition of F1Fo-ATPase, since inhibition of the mitochondrial ATPase activity resulted in better preservation of the tissue energy state and pH and in better final recovery of the preconditioned hearts. These results are also in accordance with those of Asimakis et al,10 Cave et al,11 and Ganote et al,12 showing that adenosine probably is not the mediator of the preconditioning effect in the rat heart.
Mitochondrial ATP synthase (F1Fo-ATPase) catalyzes a reversible reaction of ATP hydrolysis that is coupled to proton pumping across the mitochondrial inner membrane. Under normoxic conditions, the mitochondrial respiratory chain maintains an electrochemical H+ gradient across the mitochondrial inner membrane sufficient for driving the reaction toward ATP synthesis, but this gradient drops in ischemia and hypoxia, so that the F1Fo-ATPase reaction runs toward ATP hydrolysis.
F1Fo-ATPase is estimated to be a major ATP consumer during ischemia in a number of animal species.29 Mitochondrial ATPase can be actively modulated in submitochondrial particles and intact mitochondria on manipulation of the activity of the mitochondrial respiratory chain.30 31 Rouslin15 was the first to demonstrate an inhibition of mitochondrial ATPase in the ischemic heart. Ischemia-induced ATPase inhibition has been regarded as a property of the cardiac myocytes of large mammals,32 but Das and Harris17 demonstrated that work transitions in cultured rat cardiomyocytes also lead to changes in mitochondrial ATPase when measured after rapid cell disruption.
Although the role of the inhibition of mitochondrial ATPase in preconditioning effect has been discussed by several authors,6 18 33 34 no direct ATPase activity data have been published. In our rat heart model, we found that 3 minutes of ischemia were capable of causing enzyme inhibition that persisted throughout the intervening reperfusion period, so that the enzyme was still inhibited when the sustained ischemia began. The ATPase activity in the preconditioned hearts had decreased to the same level as in the nonpreconditioned hearts after 10 minutes of sustained ischemia.
The time course of ATPase inhibition must be interpreted in the context
of the metabolic energetics and mechanical variables observed. As can
be seen in Fig 5, there is a significant overshoot in the
charging of
the cellular energy reserves after the preconditioning ischemia despite
the inhibition of mitochondrial ATPase. It is most probable that this
exaggerated energization is due to subdued work output (stunning) of
the myocardium (Fig 1), resulting in an increase in the ATP
supply-to-consumption ratio. This overshoot on reperfusion in rat heart
has been observed previously, and its magnitude decreases when the
length of ischemia increases.35 The short, 3-minute
ischemia in the present case produces an appreciable overshoot,
which more than doubles the CrP-to-Pi ratio. Despite the
observed partial inhibition, the capacity of mitochondrial ATP synthase
must still be sufficient to meet metabolic needs in normoxia. This is
in accord with the current views on cardiac energetics. A large body of
evidence indicates that under physiological conditions, the oxidative
phosphorylation in myocardium works in near-equilibrium.36
This is an advantage during normoxia because it contributes to the high
efficiency of oxidative phosphorylation in heart muscle but also means
that the net flux of ATP synthase is far from maximal. During ischemia,
this turns to a disadvantage, because during reversal of the reaction
the extra capacity of the enzyme results in a high rate of ATP wastage.
On the other hand, a noticeable percentage of inhibition of
F1Fo-ATPase would be beneficial in ischemia but
still tolerable during the early stages of reperfusion because of the
basal surplus capacity, which decreases the metabolic control
coefficient of the enzyme.37 During sustained ischemia,
the high-energy phosphate concentration (CrP plus ATP) declines at the
same pace in the preconditioned and control hearts. The energy state,
which is determined by the ratio of [ATP]f to
[ADP]f · [Pi]f, is
higher in preconditioned hearts than in control hearts during the first
few minutes of ischemia and throughout the final reperfusion period, as
has also been reported by others.38 Moreover, during the
final reperfusion, the preconditioned hearts showed both an increased
energy state and enhanced mechanical performance compared with the
control hearts. Because the degrees of inhibition of ATPase were
similar in both groups at the beginning and end of the final
reperfusion period, it is obvious that the state of the ATPase in the
early stages of ischemia is most important for recovery.
31P NMR is a rather insensitive method, and some of the
phosphate compounds that are the main determinants of the cellular
energy state are present in very low concentrations, below the
detection limit. Therefore, although not apparent in Fig 5, the
energy
state was probably higher in the preconditioned hearts than in the
control hearts when estimated on the basis of the data presented in
Table 1, which shows a significant diminution in the output of
adenosine and its degradation products. It is known that adenosine
formation in the heart is regulated by the concentration of the
precursor molecule AMP, which is in turn connected with the cellular
energy state through the adenylate kinase reaction.39 If
this holds good in the present case, the time-averaged energy state
during the sustained ischemia must have been higher in the
preconditioned hearts.
The fact that the tissue lactate concentration and L-P ratio were lower
in the preconditioned hearts at the end of the sustained ischemia
indicates retardation of glycolysis. This also fits with the higher
cytosolic pH at this time point (Fig 5). The ATPase reaction is
also an
H+ producer, and the ATPase inhibition may have contributed
to the maintenance of the H+ balance. The reason for the
decrease in the glycolytic rate is not evident, but the preconditioning
ischemia-induced depletion of glycogen may contribute to it along with
the effect of energy sparing by ATPase inhibition.6
Preservation of cytosolic pH by preconditioning has also been shown by
others.7 40
The concept of ATPase inhibition during ischemia in small rodent hearts
has been under debate, because the IF1 content of the heart
of an animal with a fast heart rate has been considered to be
low.32 According to Das and Harris,27 no
reliable estimates of the IF1 content of rat mitochondrial
membranes are available, and the conjectures regarding their low
IF1 content have been based on preparations that have not
shown downregulation of
F1Fo-ATPase.32 The present
data show a clear, reversible downregulation of
F1Fo-ATPase in rat heart during ischemia. Its
mechanism remains unknown, but its reversibility excludes ischemic
autolysis. The F1Fo-ATPase values in Table
2
are expressed in reference to cytochrome oxidase, an intrinsic membrane
enzyme. The catalytic site of F1Fo-ATPase is in
an external arm of the enzyme and can be removed by urea
treatment.41 Therefore, it is possible that the observed
effect is due to differential recovery of the enzyme in the
submitochondrial particles used in the enzyme assay. Also, the
reversibility of the phenomenon makes this less probable. It is
significant that Rouslin and Broge42 have reported
that they have not been able to observe ATPase activity modulation in
Langendorff-perfused rat hearts on pacing, perfusate
Ca2+ elevation, or isoproterenol stimulation in
experiments (unpublished data) where ATPase activity measurements were
begun 5 minutes after the conclusion of perturbation. This lack of
effect is difficult to explain, although it is true that the method
they used for ATPase assay used acid extraction and the determination
of Pi,43 which means that the
oligomycin sensitivity of the reaction must be tested with a separate
sample. The coupled-enzyme assay of Rosing et al22 was
used in the present study, so the oligomycin sensitivity could be
tested in the same cuvette. The time delay between finishing the
biological experiment and beginning the enzyme assay is critical and
was reduced here to only 3 minutes. The present data argue that
mitochondrial ATPase inhibition by ischemia or oligomycin contributes
to ATP sparing in small rodent hearts, although this has been
previously demonstrated only in dog hearts.29
Thus, there is a discrepancy between the data reported by Rouslin32 and those reported by Das and Harris17 from a study in cultured cells and reported in the present study in isolated hearts. Although a difference certainly exists between the fast-beating hearts of small rodents and the slow-beating hearts of larger mammals, it would be strange if this were to represent an all-or-none phenomenon despite the presence of a low but measurable amount of IF1 in the former. If the proton conductance of the mitochondrial inner membrane were low and stable during ischemia, the hydrolysis of ATP by F1Fo-ATPase would be self-limiting even in the absence of inhibition by IF1. The physiological significance of IF1 in humans has been elegantly proven by the recent demonstration of its absence in Luft's disease,44 a condition that results in euthyroid hypermetabolism caused by loosely coupled mitochondria with high ATPase activity.45 The symptoms of Luft's disease can thus be interpreted as meaning that IF1 is needed to maintain the appropriate level of proton conductance in the mitochondrial inner membrane or the stoichiometry of the proton-pumping ATPase. This mechanism may also be important for cell survival in ischemia.
The role of adenosine in the preconditioning effect remains controversial. It has been suggested by several authors that mechanisms of preconditioning may be species specific, and our experiments show that the formation of adenosine and its degradation products was enhanced by 3 minutes of preconditioning ischemia, as estimated from tissue adenosine and effluent perfusate adenosine measurements, but all the adenosine products had been washed away before the sustained ischemia began. In light of the latter observation and some previous studies,10 12 it therefore appears that adenosine does not play a significant role in the phenomenon, as seen in the rat heart.
It is also interesting that brief low-flow ischemia, zero-flow ischemia, and hypoxia improve the recovery of rat heart from subsequent, more prolonged ischemia to the same extent.46 47 All three conditions would result in mitochondrial deenergization, which is known to lead to ATPase inhibition.17 It is therefore possible that the protection is linked to the inhibition of F1Fo-ATPase in these cases, too.
We conclude that preconditioning ischemia induced a slowly reversible inhibition of mitochondrial ATPase in the rat myocardium and that this may play an important role in the preservation of high-energy phosphates during sustained ischemia.
|
Acknowledgments |
|---|
This study was supported by grants from the Medical Research Council of the Academy of Finland; the Sigrid Juselius Foundation, Helsinki, Finland; and the Ida Montin Foundation, Finland. We thank Maija-Leena Lehtonen for her technical assistance.
Received October 31, 1994; accepted December 13, 1994.
|
References |
|---|
|
TopAbstractIntroductionMethods Results Discussion References |
|---|
1. Murry CE, Jennings RB, Reimer KA. Preconditioning with ischemia: a delay of lethal cell injury in ischemic myocardium. Circulation. 1986;74:1124-1136.
2. Li Y, Whittaker P, Kloner RA. The transient nature of the effect of ischemic preconditioning on myocardial infarct size and ventricular arrhythmia. Am Heart J. 1992;123:346-353. [Medline] [Order article via Infotrieve]
3.
Shiki K, Hearse DJ. Preconditioning of ischemic myocardium:
reperfusion-induced arrhythmias. Am J Physiol. 1987;253:H1470-H1476.
4.
Osada M, Sato T, Komori S, Tamura K. Protective effect of
preconditioning on reperfusion induced arrhythmias of isolated rat
hearts. Cardiovasc Res. 1991;25:441-444.
5. Vegh A, Komori S, Szekeres L, Parratt JR. Antiarrhythmic effects of preconditioning in anaesthetised dogs and rats. Cardiovasc Res. 1992;26:487-495. [Medline] [Order article via Infotrieve]
6.
Murry CE, Richard VJ, Reimer KA, Jennings RB. Ischemic
preconditioning slows energy metabolism and delays ultrastructural
damage during a sustained ischemic episode. Circ
Res. 1990;66:913-931.
7.
Kida M, Fujiwara H, Ishida M, Kawai C, Ohura M, Miura I,
Yabuuchi Y. Ischemic preconditioning preserves creatine phosphate and
intracellular pH. Circulation. 1991;84:2495-2503.
8.
Liu GS, Thornton J, Van Winkle DM, Stanley AWH, Olsson RA,
Downey JM. Protection against infarction afforded by preconditioning is
mediated by A1 adenosine receptors in rabbit heart.
Circulation. 1991;84:350-356.
9.
Thornton JD, Liu GS, Olsson RA, Downey JM. Intravenous
pretreatment with A1-selective adenosine analogues protects
the heart against infarction. Circulation. 1992;85:659-665.
10.
Asimakis GK, Inners-McBride K, Conti VR. Attenuation of
postischaemic dysfunction by ischaemic preconditioning is not mediated
by adenosine in the isolated rat heart. Cardiovasc Res. 1993;27:1522-1530.
11.
Cave AC, Collis CS, Downey JM, Hearse DJ. Improved functional
recovery by ischaemic proconditioning is not mediated by adenosine in
the globally ischaemic isolated rat heart. Cardiovasc Res. 1993;27:663-668.
12.
Ganote CE, Armstrong S, Downey JM. Adenosine and
A1 selective agonists offer minimal protection against
ischaemic injury to isolated rat cardiomyocytes. Cardiovasc
Res. 1993;27:1670-1676.
13.
Deutsch E, Berger M, Kussmaul WG, Hirshfeld JW Jr, Herrmann
HC, Laskey WK. Adaptation to ischemia during percutaneous transluminal
coronary angioplasty: clinical, hemodynamic, and metabolic features.
Circulation. 1990;82:2044-2051.
14. Yellon DM, Alkhulaifi AM, Pugsley WB. Preconditioning the human myocardium. Lancet. 1993;342:276-277. [Medline] [Order article via Infotrieve]
15.
Rouslin W. Protonic inhibition of the mitochondrial
oligomycin-sensitive adenosine 5'-triphosphatase in ischemic and
autolyzing cardiac muscle. J Biol Chem. 1983;258:9657-9661.
16.
Senior AE. ATP synthesis by oxidative phosphorylation.
Physiol Rev. 1988;68:177-231.
17. Das AM, Harris DA. Reversible modulation of the mitochondrial ATP synthase with energy demand in cultured rat cardiomyocytes. FEBS Lett. 1989;256:97-100. [Medline] [Order article via Infotrieve]
18. Jennings RB, Reimer KA, Steenbergen S. Effect of inhibition of the mitochondrial ATPase on net myocardial ATP in total ischemia. J Mol Cell Cardiol. 1991;23:1383-1395. [Medline] [Order article via Infotrieve]
19.
Katz LA, Swain JA, Portman MA, Balaban RS. Intracellular pH
and inorganic phosphate content of heart in vivo: a 31P-NMR
study. Am J Physiol. 1988;255:H189-H196.
20. Raatikainen MJP, Peuhkurinen KJ, Hassinen IE. Cellular source and role of adenosine in isoproterenol-induced coronary vasodilation. J Mol Cell Cardiol. 1991;23:1137-1148. [Medline] [Order article via Infotrieve]
21. Williamson JR, Corkey B. Assays of intermediates of citric acid cycle and related compounds by fluorometric enzymate methods. In: Colowick SP, Kaplan NO, eds. Methods in Enzymology, vol 13. New York, NY: Academic Press; 1969:439.
22. Rosing J, Harris DA, Kemp A, Slater EC. Nucleotide-binding properties of native and cold-treated mitochondrial ATPase. Biochim Biophys Acta. 1975;376:13-26. [Medline] [Order article via Infotrieve]
23. Bradford MM. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem. 1976;72:248-254. [Medline] [Order article via Infotrieve]
24. Cooperstein SJ, Lazarow A. A microspectrophotometric method for the determination of cytochrome oxidase. J Biol Chem. 1951; 189:665-670.
25. Matthews JMS, Altman DG, Campbell MJ, Royston P. Analysis of serial measurements in medical research. Br Med J. 1990; 300:230-235.
26. Griffiths EJ, Halestrap AP. Protection by cyclosporin A of ischemia/reperfusion-induced damage in isolated rat hearts. J Mol Cell Cardiol. 1993;25:1461-1469. [Medline] [Order article via Infotrieve]
27. Das AM, Harris DA. Regulation of the mitochondrial ATP synthase in intact rat cardiomyocytes. Biochem J. 1990;266:355-361. [Medline] [Order article via Infotrieve]
28. Hiltunen JK, Saukko P, Hirvonen J. Correlations between enzyme histochemical reactions and respective enzyme activities in global ischaemic rat hearts. Br J Exp Pathol. 1985;66:743-752. [Medline] [Order article via Infotrieve]
29. Rouslin W, Erickson JL, Solaro RJ. Effects of oligomycin and acidosis on rates of ATP depletion in ischemic heart muscle. Am J Physiol. 1986;250:H503-H508.
30. Van de Stadt RJ, Van Dam K. The equilibrium between the mitochondrial ATPase (F1) and its natural inhibitor in submitochondrial particles. Biochim Biophys Acta. 1974;347:240-252. [Medline] [Order article via Infotrieve]
31. Ferguson SJ, Harris DA, Radda GK. Influence of the inhibitor on adenosine triphosphate-dependent reactions. Biochem J. 1977; 162:351-357.
32.
Rouslin W. The mitochondrial adenosine 5'-triphosphatase in
slow and fast heart rate hearts. Am J Physiol. 1987;252:H622-H627.
33.
Liu Y, Downey JM. Ischemic preconditioning protects against
infarction in rat heart. Am J Physiol. 1992;263:H1107-H1112.
34.
Yellon DM, Alkhulaifi AM, Browne EE, Pugsley WB. Ischaemic
preconditioning limits infarct size in the rat heart. Cardiovasc
Res. 1992;26:983-987.
35. Bailey IA, Seymour AM. The effect of reperfusion on the 31P n.m.r. spectrum of ischemic rat hearts. Biochem Soc Trans. 1981;9:234-235.
36. Hassinen IE. Mitochondrial respiratory control in the myocardium. Biochim Biophys Acta. 1986;853:135-151. [Medline] [Order article via Infotrieve]
37. Brown GC. Control of respiration and ATP synthesis in mammalian mitochondria and cells. Biochem J. 1992;284:1-13.
38.
Miyamae M, Fujiwara H, Kida M, Yokota R, Tanaka M, Katsuragawa
M, Hasegava K, Ohura M, Koga K, Yabuuchi Y, Sesayama S. Preconditioning
improves energy metabolism during reperfusion but does not attenuate
myocardial stunning in porcine hearts.
Circulation. 1993;88:223-234.
39. Kiviluoma KT, Peuhkurinen KJ, Hassinen IE. Role of cellular energy state and adenosine in the regulation of coronary flow during variation in contraction frequency in an isolated perfused heart. J Mol Cell Cardiol. 1986;18:1133-1142. [Medline] [Order article via Infotrieve]
40. Albuquerque CP, Gerstenblith G, Weiss RG. Importance of metabolic inhibition and cellular pH in mediating preconditioning contractile and metabolic effects in rat hearts. Circ Res. 1994; 74:139-150.
41. Kagawa Y, Racker E. Partial resolution of the enzymes catalyzing oxidative phosphorylation, IX: reconstruction of oligomycin-sensitive adenosine triphosphatase. J Biol Chem. 1966; 241:2467-2474.
42. Rouslin W, Broge CW. Factors affecting the species-homologous and species-heterologous binding of mitochondrial ATPase inhibitor, IF1, to the mitochondrial ATPase of slow and fast heart-rate hearts. Arch Biochem Biophys. 1993;303:443-450. [Medline] [Order article via Infotrieve]
43. Tzagoloff A, Byington KH, MacLennan DH. Studies of the mitochondrial adenosine triphosphatase system, II: the isolation and characterization of an oligomycin-sensitive adenosine triphosphatase from bovine heart mitochondria. J Biol Chem. 1968; 243:2405-2412.
44. Yamada EW, Huzel NJ. Distribution of the ATPase inhibitor proteins of mitochondria in mammalian tissues including fibroblasts from patient with Luft's disease. Biochim Biophys Acta. 1992; 1139:143-147.
45. Di Mauro S, Bonille E, Lee CP, Schotland DL, Scarpe A, Conn H, Chance B. Luft's disease: further biochemical and ultrastructural studies of skeletal muscle in the second case. J Neurol Sci. 1976; 27:217-232.
46. Shizukuda Y, Mallet RT, Lee S-C, Downey HF. Hypoxic preconditioning of ischaemic canine myocardium. Cardiovasc Res. 1992; 26:534-542.
47. Zhai X, Lawson CS, Cave AC, Hearse DJ. Preconditioning and post-ischaemic contractile dysfunction: the role of impaired oxygen delivery vs extracellular metabolite accumulation. J Mol Cell Cardiol. 1993;25:847-857.[Medline] [Order article via Infotrieve]
This article has been cited by other articles:
 |
|
 |
|
  T. Kamota, T.-S. Li, N. Morikage, M. Murakami, M. Ohshima, M. Kubo, T. Kobayashi, A. Mikamo, Y. Ikeda, M. Matsuzaki, et al. Ischemic pre-conditioning enhances the mobilization and recruitment of bone marrow stem cells to protect against ischemia/reperfusion injury in the late phase. J. Am. Coll. Cardiol., May 12, 2009; 53(19): 1814 - 1822. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 T. Nguyen, M. Ogbi, and J. A. Johnson Delta Protein Kinase C Interacts with the d Subunit of the F1F0 ATPase in Neonatal Cardiac Myocytes Exposed to Hypoxia or Phorbol Ester: IMPLICATIONS FOR F1F0 ATPase REGULATION J. Biol. Chem., October 31, 2008; 283(44): 29831 - 29840. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 A. B. Gustafsson and R. A. Gottlieb Heart mitochondria: gates of life and death Cardiovasc Res, January 15, 2008; 77(2): 334 - 343. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J. R. Gledhill, M. G. Montgomery, A. G. W. Leslie, and J. E. Walker Mechanism of inhibition of bovine F1-ATPase by resveratrol and related polyphenols PNAS, August 21, 2007; 104(34): 13632 - 13637. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. A. Aon, S. Cortassa, C. Maack, and B. O'Rourke Sequential Opening of Mitochondrial Ion Channels as a Function of Glutathione Redox Thiol Status J. Biol. Chem., July 27, 2007; 282(30): 21889 - 21900. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. A. Moses, P. D. Addison, P. C. Neligan, H. Ashrafpour, N. Huang, S. E. McAllister, J. E. Lipa, C. R. Forrest, and C. Y. Pang Inducing late phase of infarct protection in skeletal muscle by remote preconditioning: efficacy and mechanism Am J Physiol Regulatory Integrative Comp Physiol, December 1, 2005; 289(6): R1609 - R1617. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. A. Moses, P. D. Addison, P. C. Neligan, H. Ashrafpour, N. Huang, M. Zair, A. Rassuli, C. R. Forrest, G. J. Grover, and C. Y. Pang Mitochondrial KATP channels in hindlimb remote ischemic preconditioning of skeletal muscle against infarction Am J Physiol Heart Circ Physiol, February 1, 2005; 288(2): H559 - H567. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
C. Penna, P. Pagliaro, R. Rastaldo, F. Di Pancrazio, G. Lippe, D. Gattullo, D. Mancardi, M. Samaja, G. Losano, and I. Mavelli F0F1 ATP synthase activity is differently modulated by coronary reactive hyperemia before and after ischemic preconditioning in the goat Am J Physiol Heart Circ Physiol, November 1, 2004; 287(5): H2192 - H2200. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
G. J. Grover, K. S. Atwal, P. G. Sleph, F.-L. Wang, H. Monshizadegan, T. Monticello, and D. W. Green Excessive ATP hydrolysis in ischemic myocardium by mitochondrial F1F0-ATPase: effect of selective pharmacological inhibition of mitochondrial ATPase hydrolase activity Am J Physiol Heart Circ Physiol, October 1, 2004; 287(4): H1747 - H1755. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 K. Imahashi, M. D. Schneider, C. Steenbergen, and E. Murphy Transgenic Expression of Bcl-2 Modulates Energy Metabolism, Prevents Cytosolic Acidification During Ischemia, and Reduces Ischemia/Reperfusion Injury Circ. Res., October 1, 2004; 95(7): 734 - 741. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
E. Murphy Primary and Secondary Signaling Pathways in Early Preconditioning That Converge on the Mitochondria to Produce Cardioprotection Circ. Res., January 9, 2004; 94(1): 7 - 16. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
P. D. Addison, P. C. Neligan, H. Ashrafpour, A. Khan, A. Zhong, M. Moses, C. R. Forrest, and C. Y. Pang Noninvasive remote ischemic preconditioning for global protection of skeletal muscle against infarction Am J Physiol Heart Circ Physiol, October 1, 2003; 285(4): H1435 - H1443. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 S. A Javadov, S. Clarke, M. Das, E. J Griffiths, K. H H Lim, and A. P Halestrap Ischaemic preconditioning inhibits opening of mitochondrial permeability transition pores in the reperfused rat heart J. Physiol., June 1, 2003; 549(2): 513 - 524. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. V. Haist, C. N. Hirst, and M. Karmazyn Effective protection by NHE-1 inhibition in ischemic and reperfused heart under preconditioning blockade Am J Physiol Heart Circ Physiol, March 1, 2003; 284(3): H798 - H803. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 A. Babsky, S. Hekmatyar, S. Wehrli, N. Doliba, M. Osbakken, and N. Bansal Influence of Ischemic Preconditioning on Intracellular Sodium, pH, and Cellular Energy Status in Isolated Perfused Heart Experimental Biology and Medicine, July 1, 2002; 227(7): 520 - 528. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
R. Schulz, M. V Cohen, M. Behrends, J. M Downey, and G. Heusch Signal transduction of ischemic preconditioning Cardiovasc Res, November 1, 2001; 52(2): 181 - 198. [Full Text] [PDF]
|
|
|
|
 |
|
 C.-Y. Xiao, A. Hara, K.-i. Yuhki, T. Fujino, H. Ma, Y. Okada, O. Takahata, T. Yamada, T. Murata, S. Narumiya, et al. Roles of Prostaglandin I2 and Thromboxane A2 in Cardiac Ischemia-Reperfusion Injury: A Study Using Mice Lacking Their Respective Receptors Circulation, October 30, 2001; 104(18): 2210 - 2215. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
N. Sasaki, T. Sato, E. Marban, and B. O'Rourke ATP consumption by uncoupled mitochondria activates sarcolemmal KATP channels in cardiac myocytes Am J Physiol Heart Circ Physiol, April 1, 2001; 280(4): H1882 - H1888. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. Tani, Y. Honma, H. Hasegawa, and K. Tamaki Direct activation of mitochondrial KATP channels mimics preconditioning but protein kinase C activation is less effective in middle-aged rat hearts Cardiovasc Res, January 1, 2001; 49(1): 56 - 68. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 W. E. Johnston Preconditioning the Brain and Heart: Implications for Cardiac Surgery Seminars in Cardiothoracic and Vascular Anesthesia, July 1, 2000; 4(2): 70 - 79. [Abstract] [PDF]
|
|
|
|
|
|
E. Kevelaitis, A. Oubenaissa, J. Peynet, C. Mouas, and P. Menasche Preconditioning by Mitochondrial ATP-Sensitive Potassium Channel Openers : An Effective Approach for Improving the Preservation of Heart Transplants Circulation, November 9, 1999; 100 (2009): II-345 - II-350. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. Takeo and Y. Nasa Role of energy metabolism in the preconditioned heart - a possible contribution of mitochondria Cardiovasc Res, July 1, 1999; 43(1): 32 - 43. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
G. J. Harrison, R. J. Willis, and J. P. Headrick Extracellular adenosine levels and cellular energy metabolism in ischemically preconditioned rat heart Cardiovasc Res, October 1, 1998; 40(1): 74 - 87. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
X.-H. Ning, C.-S. Xu, Y. C. Song, Y. Xiao, Y.-J. Hu, F. M. Lupinetti, and M. A. Portman Hypothermia preserves function and signaling for mitochondrial biogenesis during subsequent ischemia Am J Physiol Heart Circ Physiol, March 1, 1998; 274(3): H786 - H793. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
L. A. Gustafson and K. Kroll Downregulation of 5'-nucleotidase in rabbit heart during coronary underperfusion Am J Physiol Heart Circ Physiol, February 1, 1998; 274(2): H529 - H538. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
D. W. Green, H. N. Murray, P. G. Sleph, F.-L. Wang, A. J. Baird, W. L. Rogers, and G. J. Grover Preconditioning in rat hearts is independent of mitochondrial F1F0 ATPase inhibition Am J Physiol Heart Circ Physiol, January 1, 1998; 274(1): H90 - H97. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 L. P. Perrault, P. Menasche, K. V. Ylitalo, and K. J. Peuhkurinen Ischemic Preconditioning Before Normothermic Retrograde Cardioplegia Ann. Thorac. Surg., December 1, 1997; 64(6): 1874 - 1876. [Full Text]
|
|
|
|
|
|
S. Schaefer and R. Ramasamy Glycogen utilization and ischemic injury in the isolated rat heart Cardiovasc Res, July 1, 1997; 35(1): 90 - 98. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
K.-i. Yabe, Y. Nasa, M. Sato, R. Iijima, and S. Takeo Preconditioning preserves mitochondrial function and glycolytic flux during an early period of reperfusion in perfused rat hearts Cardiovasc Res, March 1, 1997; 33(3): 677 - 685. [Abstract] [PDF]
|
|
|
|
|
|
P. Menasche, C. Mouas, and C. Grousset Is Potassium Channel Opening an Effective Form of Preconditioning Before Cardioplegia? Ann. Thorac. Surg., June 1, 1996; 61(6): 1764 - 1768. [Abstract] [Full Text]
|
|
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||