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1 Bristol Heart Institute, We have used mitochondrial entrapment of
2-deoxy-D-[3H]glucose (2-DG) to
demonstrate that recovery of Langendorff-perfused rat hearts from
ischemia is accompanied by reversal of the mitochondrial permeability transition (MPT). In hearts loaded with 2-DG before 40 min
of ischemia and 25 min of reperfusion, 2-DG entrapment [expressed as 105 × (mitochondrial
2-[3H]DG dpm per unit
citrate synthase)/(total heart
2-[3H]DG dpm/g wet
wt)] increased from 11.1 ± 1.3 (no ischemia,
n = 4) to 32.5 ± 1.9 (n = 6;
P < 0.001). In other experiments,
2-DG was loaded after 25 min of reperfusion to determine whether some mitochondria that had undergone the MPT during the initial phase of
reperfusion subsequently "resealed" and thus no longer took up
2-DG. The reduction of 2-DG entrapment to 20.6 ± 2.4 units (n = 5) confirmed that this was the
case. Pyruvate (10 mM) in the perfusion medium increased recovery of
left ventricular developed pressure from 57.2 ± 10.3 to 98.9 ± 10.8% (n = 6;
P < 0.05) and reduced entrapment of
2-DG loaded preischemically and postischemically to 23.5 ± 1.5 (n = 4;
P < 0.001) and 10.5 ± 0.5 (n = 4;
P < 0.01) units, respectively. The
presence of pyruvate increased tissue lactate content at the end of
ischemia and decreased the effluent pH during the initial phase
of reperfusion concomitant with an increase in lactate output. We
suggest that pyruvate may inhibit the MPT by decreasing
pHi and scavenging free radicals,
thus protecting hearts from reperfusion injury.
rat; reperfusion injury; apoptosis; deoxyglucose; pHi
THE IRREVERSIBLE INJURY sustained by tissues that have
undergone ischemia and reperfusion has been attributed to a
number of factors, including disturbances of ion homeostasis and free radical production (35). In isolated rat hearts such injury has also
been linked with the mitochondrial permeability transition (MPT), a
process that involves the opening of nonselective pores in the inner
mitochondrial membrane with consequent uncoupling and equilibration of
cytosolic and mitochondrial metabolites (9, 19, 20). Recently, strong
evidence has implicated the MPT as the "central executioner" of
many cells, determining whether they live or die and whether the cell
death pathway is apoptotic or necrotic (26, 35). If MPT pore opening is
extensive and prolonged, mitochondria remain uncoupled and are unable
to generate the ATP required for maintaining ionic homeostasis and
repairing tissue damage. In addition, these uncoupled mitochondria may
actively hydrolyze ATP derived from both glycolysis and residual
oxidative phosphorylation. In the absence of an adequate ATP supply,
tissue damage is exacerbated and leads to cell death by necrosis (9, 19, 20, 35).
The MPT is triggered by high concentrations of
Ca2+ in the mitochondrial matrix,
but the sensitivity to
[Ca2+] is greatly
increased by oxidative stress, adenine nucleotide depletion, and
decreased membrane potential (23, 41). These are all conditions
associated with reperfusion of the heart after ischemia,
suggesting that the MPT might occur under such conditions (9, 19, 20).
The greatly depressed ratio of ATP to ADP (ATP/ADP) and elevated AMP
concentration found in hearts that do not recover during reperfusion
(15, 20) supports this conclusion. A critical role for the pore in
ischemia/reperfusion injury is also implied by the observation
that the immunosuppressant cyclosporin A (CsA), which inhibits the MPT
in isolated mitochondria (10, 21), protects isolated rat hearts and
cardiac myocytes against ischemia/reperfusion damage (15, 16,
31). Direct demonstration that the MPT occurs during reperfusion was
provided by measurements of mitochondrial entrapment of
D-[3H]-2-deoxyglucose
(2-DG; Ref. 16). 2-DG enters cells on the glucose transporter, is
metabolized to 2-deoxyglucose-6-phosphate, and then remains entrapped
in the cytosol. It does not enter the mitochondria unless the MPT pore
opens, whereupon it equilibrates rapidly between the cytosol and
mitochondrial matrix compartments. Thus the extent to which
2-[3H]DG is entrapped
within mitochondria can be used as an indicator of the number of
mitochondria that have undergone the MPT (16). With the use of this
technique it was shown that the MPT does not occur during
ischemia but does do so during reperfusion, reaching a maximal
value after ~5 min of reperfusion (20). However, the technique does
not allow subsequent reversal of the MPT to be determined, because once
inside the mitochondria, 2-DG will remain there when the MPT pores
close. In this paper we use entrapment of 2-DG loaded into the heart
after a period of ischemia and subsequent reperfusion to
determine the extent of pore closure. We use this approach to study the
mechanism by which pyruvate improves the recovery of hearts subjected
to ischemia and reperfusion (6, 7, 12).
This study conforms with the Guide for the Care and Use of
Laboratory Animals [DHEW Publication No. (NIH) 85-23,
Revised 1985, Office of Science and Health Reports, DRR/NIH, Bethesda,
MD 20205].
Heart perfusion. The procedures were
essentially the same as described previously (15, 16). Hearts (~0.7
g) were removed from male Wistar rats (235-275 g) and immediately
arrested in ice-cold Krebs-Henseleit buffer. The aorta was rapidly
cannulated and perfused in the Langendorff mode using Krebs-Henseleit
buffer containing (in mM) 118 NaCl, 25 NaHCO3, 4.8 KCl, 1.2 KH2PO4,
1.2 MgSO4, 11 glucose, and 1.2 CaCl2. The buffer was bubbled with 95% O2-5%
CO2 at 37°C (pH 7.4), and
perfusion was performed at a constant flow rate of 10 ml/min. It was
confirmed that this flow rate was in excess of that required to
maintain maximal rates of oxygen uptake. A water-filled balloon was
inserted into the left ventricle for continuous monitoring of developed
pressure. Balloon volume was set to give an initial end-diastolic
pressure (EDP) of 2.5-5 mmHg. Global normothermic ischemia
was induced by switching off the pump and immersing the heart in buffer
maintained at 37°C. In all experiments, the ischemic period was 40 min. When present, pyruvate was added 10 min before the onset of
ischemia and throughout the reperfusion phase. Coronary flow
was maintained at 10 ml/min during reperfusion without a significant
change in aortic pressure, implying that perfusion remained homogeneous.
2-[3H]DG
loading before ischemia. This method and the
calculation of the units of 2-DG uptake have previously been described (16). In outline, after a 20-min flow-through period for stabilization, hearts were perfused in recirculating mode with 40 ml of Krebs buffer
containing 0.5 mM
2-[3H]DG (0.1 µCi/ml) for 30 min. Perfusion was then returned to flow-through (nonrecirculating) mode with normal buffer for 10 min in the absence or
presence of 10 mM pyruvate before induction of ischemia as described above. This washes out extracellular
2-[3H]DG from the
heart while both cytosolic and mitochondrial
2-[3H]deoxyglucose-6-phosphate
remain entrapped.
2-[3H]DG
loading after ischemia. Hearts were perfused
for 60 min in flow-through mode; where required 10 mM pyruvate was
present for the final 10 min. Total ischemia was then induced
for 40 min before reperfusion of hearts. This was continued until full
functional recovery was achieved after ~25 min, at which time hearts
were loaded with
2-[3H]DG for 30 min as
described above. Excess label was washed out by 5 min of perfusion with
normal buffer, and then mitochondria were prepared as above.
Isolation of mitochondria and measurement of
2-[3H]DG
entrapment. After the perfusion protocol, the
ventricles were rapidly cut away, weighed, and homogenized with a
Polytron homogenizer in 5 ml of ice-cold sucrose buffer (in mM: 300 sucrose, 10 Tris · HCl, 2 EGTA; pH 7.4). The volume
was made up to 40 ml with buffer containing 5 mg/ml BSA, and a sample
was retained for measurement of total
3H after protein precipitation by
addition of 5% (wt/vol) perchloric acid (PCA). Heart mitochondria were
prepared from the remainder of the homogenate by centrifugation for 2 min at 2,000 g in a benchtop
centrifuge to remove cell debris, followed by centrifugation of the
supernatant at 10,000 g for 5 min to
sediment the mitochondria. The pellet was then washed two
times at 10,000 g in 40 ml of BSA-free sucrose buffer and finally resuspended in 0.5 ml of
sucrose buffer. A 100-µl sample of the suspension was retained for
assay of citrate synthase to correct for mitochondrial recovery, while
an equal volume of 5% PCA was added to the remainder to release the
entrapped 3H. Protein was
precipitated by centrifugation at 10,000 g for 2 min, and the resultant
supernatant was assayed for radioactivity in 10 ml scintillant (Packard
Emulsifier-Safe). Mitochondrial entrapment of 2-DG was calculated as
previously described (16) and is expressed as
105 times mitochondrial
2-[3H]DG dpm per unit
citrate synthase divided by total heart
2-[3H]DG dpm per gram
wet weight. This method of calculation corrects for variation in
loading of the hearts with 2-DG and recovery of mitochondria (16). If
all mitochondria undergo the MPT, the predicted value for the
mitochondrial 2-DG entrapment is 110 units.
Tissue metabolite measurements.
Separate heart perfusions to those used for the determination of 2-DG
entrapment were performed for these measurements. Hearts were perfused
in flow-through mode for 60 min in the absence or presence of 10 mM
pyruvate for the final 10 min before ischemia. At the required
time in the ischemia/reperfusion cycle, the ventricles were cut
from the heart and immediately freeze-clamped in tongs precooled in
liquid nitrogen and stored at Changes in effluent pH and lactate
content. A single sample of effluent (2 ml over a 12-s
period) was collected 1 min before ischemia, and seven
additional samples (2 ml) were collected between 0 and 12, 24 and 30, 36 and 48, 90 and 102, 150 and 162, and 600 and 612 s of reperfusion.
The pH of each was measured within 10 s of collection, during which
time it remained stable. Samples were retained and stored at
Analysis of results. Results are
expressed as means ± SE. The statistical differences between groups
were calculated by two-tailed Student's
t-test.
Effect of pyruvate on contractile recovery and adenine
nucleotide content. The effect of pyruvate on
contractile function was investigated using hearts perfused for 60 min
before ischemia. This time period was chosen to correspond to
the time of preperfusion required to load hearts with
2-[3H]DG (see below).
Contractile function in control hearts and those treated with pyruvate
(10 mM) before and after ischemia and reperfusion is shown in
Table 1. Values are given for hearts loaded
with 2-DG before ischemia (such preloading involves 30 min in
recirculating mode) and those loaded with 2-DG after
ischemia-reperfusion (postloading involves no recirculation).
We have previously demonstrated that 0.5 mM 2-DG has no effect on heart
function (16), although the present data (Table 1) suggest that hearts
loaded with 2-DG before ischemia have a slightly higher EDP and
depressed left ventricular developed pressure (LVDP) on reperfusion
than postloaded hearts. However, this effect was shown to be due to the
period of recirculation required to load with 2-DG rather than the
presence of 2-DG itself (data not shown). It may reflect accumulation
of metabolites in the effluent during recirculation. During the
preischemic perfusion, pyruvate transiently depressed contractile
function (not shown) but LVDP recovered and did not significantly
differ from control hearts at the end of the preischemic perfusion
(Table 1). The tissue content of adenine nucleotides was also unchanged
by pyruvate during the preischemic period, although there was a
small but significant increase in creatine phosphate (Table
2). During the ischemic phase (after
preperfusion without prior recirculation), pyruvate increased the time
taken to reach the onset of ischemic contracture from 5.2 ± 0.7 to
17.2 ± 1.6 min (P < 0.001;
n = 4) and also appeared to decrease
the rate of glycogen use during ischemia, which presumably
reflects a reduced rate of glycolysis. Thus, before ischemia,
control and pyruvate-treated hearts had similar glycogen contents
[88 ± 8 (n = 7) and
93 ± 5 (n = 5) µmol glycosil
units/g dry wt, respectively; P > 0.05]. At the onset of ischemic contracture, the glycogen content
of control hearts was 22 ± 4 µmol glycosil units/g dry wt
(n = 6), whereas after the same period
of ischemia, pyruvate-treated hearts (which had not yet
initiated contracture) had significantly higher glycogen levels
(57 ± 11 µmol glycosil units/g dry wt;
n = 4;
P < 0.05). At the onset of
contracture the glycogen content of pyruvate-treated hearts (26 ± 3
µmol glycosil units/g dry wt; n = 4)
was similar to that of control hearts. On reperfusion, the recovery of
LVDP was significantly increased from 36-57 to 100%
(P < 0.05) by the presence of
pyruvate (Table 1). However, this increased recovery of heart function
was not accompanied by any significant changes in the tissue content of
adenine nucleotides or creatine phosphate measured either during the
initial reperfusion phase (after 5 min) or once full recovery had been
attained (25 min of reperfusion) as shown in Table 2.
ABSTRACT
Top
Abstract
Introduction
Methods
Results
Discussion
References
INTRODUCTION
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Abstract
Introduction
Methods
Results
Discussion
References
METHODS
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Abstract
Introduction
Methods
Results
Discussion
References
70°C until required. Tissue
metabolites were extracted by grinding of frozen hearts in liquid
N2 and homogenization in 5% PCA,
followed by neutralization with 5 M KOH containing 0.5 3-(N-morpholino)propanesulfonic acid and 20 mM EDTA. Samples were then centrifuged, and
the supernatant was assayed for glycogen, lactate, adenine
nucleotides, and creatine phosphate as described elsewhere (15,
32).
70°C for subsequent assay of lactate.
RESULTS
Top
Abstract
Introduction
Methods
Results
Discussion
References
Table 1.
Effect of pyruvate on functional recovery in perfused rat hearts
Table 2.
Effects of pyruvate on the adenine nucleotide and creatine phosphate
content of hearts before and after ischemia-reperfusion
Mitochondrial pore opening during reperfusion. The optimal conditions for loading hearts with 2-[3H]DG before ischemia have been described previously (16). Mitochondrial 2-DG entrapment can be used to determine pore opening, because 2-deoxyglucose-6-phosphate (formed from 2-DG in the cytosol) can only enter the mitochondria when the pores are open. The kinetics of solute entry through open pores are such that for any mitochondrion, once a pore opens, equilibration of the 2-deoxyglucose-6-phosphate between its matrix and the cytosol occurs almost instantaneously (9, 19). 2-Deoxyglucose-6-phosphate may then be trapped within the mitochondria during their preparation by chelating calcium with EGTA, which rapidly closes the pores. Thus determination of 3H in subsequently isolated mitochondria acts as a measure of the number of mitochondria that have undergone the MPT at any time during the perfusion/reperfusion period. The method of calculation uses recovery of citrate synthase to correct for variation in recovery of mitochondria and total homogenate 2-[3H]DG to correct for differential loading of hearts with 2-DG (16). It was confirmed that the presence of pyruvate had no effect on either parameter (data no shown).
As shown in Fig. 1, in hearts that had been
perfused for 60 min after loading (i.e., no ischemia or
reperfusion), the mitochondrial 2-DG uptake (in the units defined under
METHODS) was 11.1 ± 1.3 (n = 4). This basal level of 2-DG
entrapment is believed to reflect a slow MPT-independent entry of 2-DG
into mitochondria in situ or during mitochondrial isolation (16, 20).
If all mitochondria undergo the MPT, the predicted value for the
mitochondrial 2-DG entrapment is 110 units. The uptake of 2-DG
increased approximately threefold on subsequent reperfusion for a
period of 20 min after 40 min ischemia to 32.5 ± 1.9 units
(n = 6;
P < 0.001). In hearts loaded with
2-DG before ischemia and treated with pyruvate, the improved
recovery of LVDP function was associated with a significant decrease in
mitochondrial 2-DG uptake ratio to 23.5 ± 1.5 (n = 4;
P < 0.01).
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Mitochondrial pores reseal during recovery of the heart from ischemia-reperfusion. Once 2-DG has entered the mitochondria during reperfusion, it will not leave again when the pore closes. Thus, to determine whether the mitochondrial pores remain open during the reperfusion phase after maximum functional recovery, hearts were loaded with 2-[3H]DG after 25 min of reperfusion. Data are shown in Fig. 1. The entrapment of 2-DG in postischemically loaded hearts was 20.6 ± 2.4 (n = 5), which is significantly lower than that in preischemically loaded hearts subjected to the same period of ischemia and reperfusion (P < 0.01). 2-DG uptake was further reduced to 10.5 ± 0.5 (n = 4) in hearts pretreated with pyruvate (P < 0.01). This is almost identical to the value found in hearts not subjected to ischemia-reperfusion (11.1 ± 1.3) and is associated with almost total recovery of LVDP (Table 1). If it is assumed that the values for entrapment of 2-DG loaded preischemically and postischemically can be directly compared, our data imply that some pore closure has occurred during partial recovery of heart function in the absence of pyruvate, whereas in the presence of pyruvate complete closure accompanies total recovery of the heart. Such an assumption appears to be valid. The methodology corrects for any differential loading of the hearts with 2-DG by expressing mitochondrial loading as a percentage of total heart loading. Differences in recovery of mitochondria are accounted for by measurement of citrate synthase; ruptured mitochondria that have lost their citrate synthase will also lose their 2-deoxyglucose-6-phosphate. It could be argued that the time between loading mitochondria and their subsequent isolation is longer for the preloaded hearts than for the postloaded hearts because of the 30-min period of ischemia in the former case. However, we have previously shown that a 30-min period of ischemia does not produce an increase in 2-DG entrapment (16), and we have also confirmed that there is no significant increase in mitochondrial 2-DG entrapment when hearts are perfused for increasing periods of time after 2-DG loading. Thus the mitochondrial 2-DG entrapment of control hearts perfused for 3 and 75 min in flow-through mode after 30 min of 2-DG loading was, respectively, 10.4 ± 1.3 (n = 7) and 8.3 ± 1.3 units (n = 4).
Changes in effluent pH and lactate concentrations in
ischemic/reperfused hearts. The pH of the effluent of
untreated and pyruvate-treated hearts during the preischemic period was
the same (Fig. 2). In untreated
ischemic/reperfused hearts, the effluent pH fell to 7.21 ± 0.04 (n = 6) in the first 12 s of reperfusion and rose steadily over the next 1 min, returning to
control values after 1 min of reperfusion. The effluent pH in the first
sample of buffer after ischemia from pyruvate-treated hearts
was 7.02 ± 0.03 (n = 4;
P < 0.01), significantly lower than
control ischemic/reperfused hearts, and remained lower in all samples
taken (P < 0.05) even after 10 min
of reperfusion. Figure 2 also shows that the lactate output in the
effluent from control and pyruvate-treated hearts was low during the
preischemic perfusion. After reperfusion, lactate output from untreated
hearts rose to 3.60 ± 0.44 µmol
(n = 6) in the first 2-ml sample and
declined with each subsequent sample, reaching control levels after 10 min. For pyruvate-treated hearts the lactate content of the first 2-ml
sample on reperfusion was more than double that of the untreated hearts
(8.23 ± 0.29 µmol; n = 4; P < 0.001) and remained
significantly higher at all time points, except for samples taken after
48-60 and 90-102 s of reperfusion. Hearts treated with
pyruvate also showed a small increase in tissue lactate content at the
end of the ischemic period (108.0 ± 17.8 and 129.5 ± 6.6 µmol/g dry wt in the absence and presence of pyruvate, respectively;
n = 4;
P > 0.05). As expected, these values
were much greater than the preischemic value (5.9 ± 0.7 µmol/g
dry wt; n = 4).
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DISCUSSION |
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Top
Abstract
Introduction
Methods
Results
Discussion
References
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Protection from reperfusion injury correlates with opening of MPT pores. During reperfusion, Ca2+ that has accumulated in the cytosol during hypoxia is accumulated by mitochondria as they are reenergized and transport Ca2+ in preference to ATP synthesis. In conjunction with the depletion of adenine nucleotides and generation of oxygen-free radicals, the conditions are now optimal for opening of the MPT pore (see introduction). It has been shown previously that pyruvate can protect hearts and hepatocytes against ischemia-reperfusion and anoxia/reoxygenation injury (5-7, 12, 38), and this has been attributed to beneficial metabolic alterations (6, 7, 38) and attributed to protection from free radical production (8, 12). An additional effect of pyruvate might be to reduce mitochondrial pore opening. Data in Fig. 1 confirm that significantly less pore opening does occur on reperfusion in the presence of pyruvate. Furthermore, once the hearts have reestablished maximal LVDP, loading with 2-DG shows that the pores are now closed. Thus those pores that had opened on reperfusion must have resealed. Some resealing was also apparent after reperfusion in the absence of pyruvate, but recovery did not reach preischemic values. These data are the first direct evidence that mitochondrial pore opening can reverse when hearts damaged by reperfusion recover. Although we cannot exclude the possibility that mitochondrial pore opening is secondary to irreversible cell damage, rather than a primary cause of it, we can be confident that pore opening does not follow breakdown of the plasma membrane permeability barrier. If this were to occur, 2-DG would be lost from the cell before it could enter the mitochondria and no increase in mitochondrial 2-DG would be measured.
Possible mechanisms by which pyruvate decreases MPT pore opening. We have shown that pyruvate has no direct effect on the MPT (unpublished data), and thus its effects on the MPT in the heart are likely to be through indirect means. Three potential mechanisms would appear likely. First, pyruvate is known to act as an efficient free radical scavenger (5, 8, 12). This will lead to the accumulation of fewer oxygen free radicals during reperfusion, thus impairing the ability of matrix [Ca2+] to induce the MPT. Second, pyruvate acts as an excellent fuel for mitochondrial respiration and this may help to maintain the mitochondrial membrane potential that inhibits pore opening (2). Third, mitochondrial pore opening is greatly inhibited as the pH drops below 7 (3, 18, 29) and thus pyruvate may act by decreasing pHi. Monocarboxylates such as pyruvate and lactate are transported into the cell with a proton by the monocarboxylate transporter (34), whose activity also serves as a major efflux H+ pathway during reperfusion in conjunction with the Na+/H+ antiporter and chloride/bicarbonate exchange mechanisms (22, 34, 39). Thus pyruvate uptake by the heart would be predicted to increase the acid load that the heart experiences and so decrease pHi. Direct evidence for this has been obtained using nuclear magnetic resonance in a low-flow model of ischemia (11). In the present study this is reflected in the greater drop in effluent pH of pyruvate-treated hearts on reperfusion than that of control hearts (Fig. 2), suggesting that pHi was significantly lower at the end of ischemia and during the reperfusion phase. A lower pHi during ischemia would also inhibit glycolysis and glycogen use, and thus may account for the longer time period taken for glycogen depletion and contracture in pyruvate-treated hearts. The rise in effluent pH over the first 2 min of reperfusion may also be significant, because pore opening is thought to be inhibited during ischemia as a result of the low pHi. However, this inhibition will be released as the pH rises on reperfusion, and we have previously reported that MPT pores open between 2 and 5 min after the onset of reperfusion (20), the same time period over which pHi returns to normal (39). Several reports have confirmed that low pH can protect hearts against reperfusion injury (24, 28) and isolated myocytes against reoxygenation injury (1, 4). Although there may be other mechanisms involved in this effect, inhibition of the MPT is likely to be important.
Relationship between the MPT and short- and long-term recovery of the heart. The data we present in this paper are consistent with the extent of MPT pore opening and subsequent resealing being directly related to the recovery of the heart on reperfusion. However, the lack of any dramatic effect of pyruvate on tissue adenine nucleotide and creatine phosphate concentrations might be taken as an argument against this, implying rather that the relationship is not one of "cause and effect." In this sense the protective effects of pyruvate differ from those of CsA, where protection is associated with increased tissue ATP/ADP and decreased AMP concentrations (15). A likely explanation of the difference between CsA and pyruvate is that the latter probably exerts its effects on the MPT pores indirectly through its ability to lower pHi and decrease in oxidative stress. These factors are also likely to have additional protective effects on the heart, enabling greater contractile activity (reflected in the increased LVDP) and thus increased ATP demand. Parallel protection of the mitochondria from the MPT would enable this increased ATP demand to be met without causing a decrease in ATP/ADP. In hearts perfused in the Langendorff mode, the demand on the mitochondria will not be great in comparison with the working heart, where protection of mitochondria from the MPT might be expected to have greater effects on cellular energetics.
It is now known that in cells subject to insults, such as oxidative stress, growth factor removal, or exposure to cytokines (e.g., tumor necrosis factor), mitochondria release cytochrome c from the intermembrane space. This then activates cytosolic caspases, which in turn stimulate the apoptotic cascade (29, 30, 40). It has been shown that the mitochondrial swelling and outer membrane rupture that follows the MPT may be one mechanism of such cytochrome c release and thus provides the MPT with a critical role in the regulation of apoptosis as well as in necrosis (17, 27). Apoptosis is an ATP-requiring process and thus is inconsistent with MPT pores remaining open during reperfusion. However, a major significant finding of our present data is that pores can open and then close again. This would cause mitochondrial swelling and cytochrome c release to occur initially, while maintaining tissue ATP concentrations sufficient to enable apoptosis during subsequent stages of reperfusion. Indeed, in the failing heart and hearts damaged by reperfusion injury some cells have been shown to undergo apoptotic cell death rather than necrosis. This is particularly pronounced in areas surrounding a myocardial infarct, i.e., areas that experience a less pronounced ischemic insult than that which leads to necrosis (13, 14, 33, 37). It can be envisaged that in these areas the insult to cells is sufficiently modest to allow transient pore opening with subsequent closure, whereas in the necrotic areas the MPT pores might remain open. Thus the ability of pyruvate to decrease the MPT may not only have implications for the short-term recovery of the heart, but also for the fate of those cells that initially recover from a modest reperfusion insult but are subsequently at risk of dying by apoptosis.
In summary, our data demonstrate that opening of MPT pores occurs in the initial phase of reperfusion of the heart after a period of ischemia. However, some subsequent resealing of the pores may take place, the extent to which this occurs correlating with the ability of the heart to recover its functional integrity. The presence of 10 mM pyruvate throughout ischemia and reperfusion greatly improves functional recovery and this is associated with less initial pore opening during reperfusion and almost total resealing of the pore subsequently.
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ACKNOWLEDGEMENTS |
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We thank Dr. Elinor Griffiths for helpful discussions.
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FOOTNOTES |
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This work was supported by the British Heart Foundation.
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. §1734 solely to indicate this fact.
Address for reprint requests: A. P. Halestrap, Dept. of Biochemistry, University of Bristol, University Walk, Bristol BS8 1TD, UK.
Received 18 May 1998; accepted in final form 21 October 1998.
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REFERENCES |
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Abstract
Introduction
Methods
Results
Discussion
References
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1.
Atsma, D. E.,
E. M. L. Bastiaanse,
L. Vander-Valk,
and
A. Vander-Laarse.
Low external pH limits cell death of energy-depleted cardiomyocytes by attenuation of Ca2+ overload.
Am. J. Physiol.
270 (Heart Circ. Physiol. 39):
H2149-H2156,
1996
2.
Bernardi, P.
Modulation of the mitochondrial cyclosporin-A-sensitive permeability transition pore by the proton electrochemical gradient
evidence that the pore can be opened by membrane depolarization.
J. Biol. Chem.
267:
8834-8839,
1992
3.
Bernardi, P.,
S. Vassanelli,
P. Veronese,
R. Colonna,
I. Szabo,
and
M. Zoratti.
Modulation of the mitochondrial permeability transition poreeffect of protons and divalent cations.
J. Biol. Chem.
267:
2934-2939,
1992
4.
Bond, J. M.,
E. Chacon,
B. Herman,
and
J. J. Lemasters.
Intracellular pH and Ca2+ homeostasis in the pH paradox of reperfusion injury to neonatal rat cardiac myocytes.
Am. J. Physiol.
265 (Cell Physiol. 34):
C129-C137,
1993
5.
Borle, A. B.,
and
R. T. Stanko.
Pyruvate reduces anoxic injury and free radical formation in perfused rat hepatocytes.
Am. J. Physiol.
270 (Gastrointest. Liver Physiol. 33):
G535-G540,
1996
6.
Bunger, R.,
R. T. Mallet,
and
D. A. Hartman.
Pyruvate-enhanced phosphorylation potential and inotropism in normoxic and post-ischemic isolated working heart.
Eur. J. Biochem.
180:
221-233,
1989[Medline].
7.
Cavallini, L.,
M. Valente,
and
M. P. Rigobello.
The protective action of pyruvate on recovery of ischemic rat heartcomparison with other oxidizable substrates.
J. Mol. Cell. Cardiol.
22:
143-154,
1990[Medline].
8.
Constantopoulos, G.,
and
J. A. Barranger.
Nonenzymatic decarboxylation of pyruvate.
Anal. Biochem.
139:
353-358,
1984[Medline].
9.
Crompton, M.
The role of Ca2+ in the function and dysfunction of heart mitochondria.
In: Calcium and the Heart, edited by G. A. Langer. New York: Raven, 1990, p. 167-198.
10.
Crompton, M.,
H. Ellinger,
and
A. Costi.
Inhibition by cyclosporin A of a Ca2+-dependent pore in heart mitochondria activated by inorganic phosphate and oxidative stress.
Biochem. J.
255:
357-360,
1988[Medline].
11.
Cross, H. R.,
K. Clarke,
L. H. Opie,
and
G. K. Radda.
Is lactate-induced myocardial ischaemic injury mediated by decreased pH or increased intracellular lactate?
J. Mol. Cell. Cardiol.
27:
1369-1381,
1995[Medline].
12.
Deboer, L. W. V.,
P. A. Bekx,
L. H. Han,
and
L. Steinke.
Pyruvate enhances recovery of rat hearts after ischemia and reperfusion by preventing free radical generation.
Am. J. Physiol.
265 (Heart Circ. Physiol. 34):
H1571-H1576,
1993
13.
Fliss, H.,
and
D. Gattinger.
Apoptosis in ischemic and reperfused rat myocardium.
Circ. Res.
79:
949-956,
1996
14.
Gottlieb, R. A.,
K. O. Burleson,
R. A. Kloner,
B. M. Babior,
and
R. L. Engler.
Reperfusion injury induces apoptosis in rabbit cardiomyocytes.
J. Clin. Invest.
94:
1621-1628,
1994.
15.
Griffiths, E. J.,
and
A. P. Halestrap.
Protection by cyclosporin A of ischemia reperfusion-induced damage in isolated rat hearts.
J. Mol. Cell. Cardiol.
25:
1461-1469,
1993[Medline].
16.
Griffiths, E. J.,
and
A. P. Halestrap.
Mitochondrial non-specific pores remain closed during cardiac ischaemia, but open upon reperfusion.
Biochem. J.
307:
93-98,
1995.
17.
Halestrap, A. P.
Calcium-dependent opening of a non-specific pore in the mitochondrial inner membrane is inhibited at pH values below 7implications for the protective effect of low pH against chemical and hypoxic cell damage.
Biochem. J.
278:
715-719,
1991.
18.
Halestrap, A. P.
The nature of the stimulation of the respiratory chain of rat liver mitochondria by glucagon pretreatment of animals.
Biochem. J.
204:
37-47,
1982[Medline].
19.
Halestrap, A. P.
Interactions between oxidative stress and calcium overload on mitochondrial function.
In: Mitochondria: DNA, Proteins and Disease, edited by V. Darley-Usmar,
and A. H. V. Schapira. London: Portland, 1994, p. 113-142.
20.
Halestrap, A. P.,
C. P. Connern,
E. J. Griffiths,
and
P. M. Kerr.
Cyclosporin A binding to mitochondrial cyclophilin inhibits the permeability transition pore and protects hearts from ischaemia/reperfusion injury.
Mol. Cell. Biochem.
174:
167-172,
1997[Medline].
21.
Halestrap, A. P.,
and
A. M. Davidson.
Inhibition of Ca2+-induced large amplitude swelling of liver and heart mitochondria by cyclosporin A is probably caused by the inhibitor binding to mitochondrial matrix peptidyl-prolyl cis-trans isomerase and preventing it interacting with the adenine nucleotide translocase.
Biochem. J.
268:
153-160,
1990[Medline].
22.
Halestrap, A. P.,
X. Wang,
R. C. Poole,
V. N. Jackson,
and
N. T. Price.
Lactate transport in heart in relation to ischemia.
Am. J. Cardiol.
80:
17A-25A,
1997[Medline].
23.
Halestrap, A. P.,
K. Y. Woodfield,
and
C. P. Connern.
Oxidative stress, thiol reagents, and membrane potential modulate the mitochondrial permeability transition by affecting nucleotide binding to the adenine nucleotide translocase.
J. Biol. Chem.
272:
3346-3354,
1997
24.
Harada, K. A.,
R. G. Franklin,
W. Johnson,
W. Grossman,
and
J. P. Morgan.
Acidemia and hypernatremia enhance postischemic recovery of exitation-contraction coupling.
Circ. Res.
74:
1197-1209,
1994
25.
Haworth, R. A.,
and
D. S. Hunter.
The Ca2+-induced membrane transition in mitochondria. II. Nature of the Ca2+ trigger site.
Arch. Biochem. Biophys.
195:
460-467,
1979[Medline].
26.
Hirsch, T.,
I. Marzo,
and
G. Kroemer.
Role of the mitochondrial permeability transition pore in apoptosis.
Biosci. Rep.
17:
67-76,
1997[Medline].
27.
Kantrow, S. P.,
and
C. A. Piantadosi.
Release of cytochrome c from liver mitochondria during permeability transition.
Biochem. Biophys. Res. Commun.
232:
669-671,
1997[Medline].
28.
Kitakaze, M.,
S. Takashima,
H. Funaya,
T. Minamino,
K. Node,
Y. Shinozaki,
H. Mori,
and
M. Hori.
Temporary acidosis during reperfusion limits myocardial infarct size in dogs.
Am. J. Physiol.
272 (Heart Circ. Physiol. 41):
H2071-H2078,
1997
29.
Kluck, R. M.,
E. Bossy-Wetzel,
D. R. Green,
and
D. D. Newmeyer.
The release of cytochrome c from mitochondria: a primary site for Bcl-2 regulation of apoptosis.
Science
275:
1132-1136,
1997
30.
Kluck, R. M.,
S. J. Martin,
B. M. Hoffman,
J. S. Zhou,
D. R. Green,
and
D. D. Newmeyer.
Cytochrome c activation of CPP32-like proteolysis plays a critical role in a Xenopus cell-free apoptosis system.
EMBO J.
16:
4639-4649,
1997[Medline].
31.
Nazareth, W.,
N. Yafei,
and
M. Crompton.
Inhibition of anoxia-induced injury in heart myocytes by cyclosporin-A.
J. Mol. Cell. Cardiol.
23:
1351-1354,
1991[Medline].
32.
Neely, J. R.,
R. M. Denton,
P. J. England,
and
P. J. Randle.
The effects of increased heart work on the tricarboxylate cycle and its interaction with glycolysis in the perfused rat heart.
Biochem. J.
128:
147-159,
1972[Medline].
33.
Olivetti, G.,
F. Quaini,
R. Sala,
C. Lagrasta,
D. Corradi,
E. Bonacina,
S. R. Gambert,
E. Cigola,
and
P. Anversa.
Acute myocardial infarction in humans is associated with activation of programmed myocyte cell death in the surviving portion of the heart.
J. Mol. Cell. Cardiol.
28:
2005-2016,
1996[Medline].
34.
Poole, R. C.,
and
A. P. Halestrap.
Transport of lactate and other monocarboxylates across mammalian plasma membranes.
Am. J. Physiol.
264 (Cell Physiol. 33):
C761-C782,
1993
35.
Reimer, K. A.,
and
R. B. Jennings.
Myocardial ischemia, hypoxia and infarction.
In: The Heart and Cardiovascular System (2nd ed.), edited by H. A. Fozzard,
R. B. Jennings,
E. Huber,
A. M. Katz,
and H. E. Morgan. New York: Raven, 1992, p. 1875-1973.
36.
Susin, S. A.,
N. Zamzami,
M. Castedo,
E. Daugas,
H. G. Wang,
S. Geley,
F. Fassy,
J. C. Reed,
and
G. Kroemer.
The central executioner of apoptosis: multiple connections between protease activation and mitochondria in Fas/APO-1/CD95- and ceramide-induced apoptosis.
J. Exp. Med.
186:
25-37,
1997
37.
Umansky, S. R.,
and
L. D. Tomei.
Apoptosis in the heart.
Adv. Pharmacol.
41:
383-407,
1997.
38.
Van Bilsen, M.,
G. J. van der Vusse,
L. H. E. H. Snoeckx,
T. Arts,
W. A. Coumans,
P. H. M. Willemsen,
and
R. S. Reneham.
Effects of pyruvate on post-ischemic myocardial recovery at various workloads.
Pflügers Arch.
413:
167-173,
1988[Medline].
39.
Vandenberg, J. I.,
J. C. Metcalfe,
and
A. A. Grace.
Mechanisms of intracellular pH recovery following global ischaemia in the perfused heart.
Circ. Res.
72:
993-1003,
1993
40.
Yang, J.,
X. S. Liu,
K. Bhalla,
C. N. Kim,
A. M. Ibrado,
J. Y. Cai,
T. I. Peng,
D. P. Jones,
and
X. D. Wang.
Prevention of apoptosis by Bcl-2: release of cytochrome c from mitochondria blocked.
Science
275:
1129-1132,
1997
41.
Zoratti, M.,
and
I. Szabo.
The mitochondrial permeability transition.
Biochim. Biophys. Acta
1241:
139-176,
1995[Medline].
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