importance of the early alterations of energy metabolism in the induction and the disappearance of...

J Mol Cell Cardiol 28, 1671–1682 (1996)

Importance of the Early Alterations ofEnergy Metabolism in the Induction andthe Disappearance of IschemicPreconditioning in the Isolated Rat HeartAnne Garnier, Andre Rossi and Nicole LavanchyLaboratoire de Bioenergetique Fondamentale et Appliquee, Universite Joseph Fourier, BP 53X, 38041Grenoble Cedex, France

(Received 29 January 1996, accepted in revised form 25 March 1996)

A. G, A. R N. L. Importance of the Early Alterations of Energy Metabolism in the Inductionand the Disappearance of Ischemic Preconditioning in the Isolated Rat Heart. Journal of Molecular and CellularCardiology (1996) 28: 1671–1682. The kinetics of alterations in high energy phosphates were studied in isolatedrat hearts during single and multiple ischemic preconditioning (IPC) using [31P]-nuclear magnetic resonance(NMR) spectroscopy. Aortically perfused hearts were subjected to a 25 min sustained ischemia and a 30 minreperfusion. The IPC protocols used a basic pattern of 3 min ischemia plus 6 min reflow, increasing the reflowperiod from 6 to 12 min. Efficient IPC was associated during ischemia with a reduction in ATP degradation, inintracellular acidosis and a maintenance of a residual pool of PCr. Analysis of the IPC phase showed that eachshort ischemia was followed by a vasodilation (40–50%), accompanied by a clear PCr overshoot (115–125%)and a cytosolic Pi undershoot. Thus, the energy producing reactions were swung out of their initial equilibrium.The PCr overshoot remained up to the onset of the sustained ischemia in the efficient protocols, whereas it haspractically vanished in the unefficient ones. In addition, the duration of such a positive imbalance appearedreinforced and prolonged by multiple IPC. It is suggested that an IPC cycle induced a time-dependent positiveimbalance in the mitochondrial oxphosphorylative reactions. The benefit for the heart developed only when theprolonged ischemia was imposed under such conditions, modifying thereby the early dynamics of the energymetabolism processes during the initial phase of the sustained ischemia.

1996 Academic Press Limited

K W: Ischemic preconditioning; Myocardial ischemia; Phosphorylated compounds; Mitochondrialfunction.

several species: dog (Li et al., 1990), pig (Schott etIntroductional., 1990), rabbit (Liu et al., 1991), and rat hearts(Liu and Downey, 1992; Yellon et al., 1992). TheThe expression “ischemic preconditioning” (IPC)

was proposed in 1986 by Murry et al. to describe nature of the protection has also been extended toan attenuation of reperfusion arrythmias (Shikithe seemingly paradoxical finding that multiple

brief episodes of regional myocardial ischemia and and Hearse, 1987) and an improved recovery ofcontractile function (Asimakis et al., 1992) both inreperfusion prior to a sustained ischemic period

reduced infarct size in the intact canine heart. vivo and in isolated rat heart preparations.Attention has been focused on the length of theSince 1986, this phenomenon, based on direct

measurement of infarct size, has been described in transient ischemia, the number of brief ischemia–

Please address all correspondence to: Anne Garnier–Nicole Lavanchy, Laboratoire de Bioenergetique Fondamentale et Appliquee,Universite Joseph Fourier, BP 53X, 38041 Grenoble Cedex, France.

0022–2828/96/081671+12 $18.00/0 1996 Academic Press Limited1671

A. Garnier et al.1672

reperfusion cycles required to elicit an optimal pre- The hearts were subjected to either one or twoischemia–reperfusion cycle(s) 3 and 6 min, re-conditioning and on the duration of the delay

between the transient and sustained ischemia. It spectively, increasing the last reflow period beforesustained ischemia from 6 to 12 min, which per-seems that, in the isolated rat heart model, pre-

conditioning can be produced with ischemic steps mitted in the single step of IPC to observe thevanishement of the preconditioning positive effect.of less than 5 min (Volovsek et al., 1992; Lawson

et al., 1993; Schjott et al., 1994) and that a singleshort cycle of ischemia–reperfusion is sufficient toinduce protection (Asimakis et al., 1992). The fact Materials and Methodsthat protection is proportional to the number ofpreconditioning cycles remains controversial de- Animalspending on the animal model studied and theend-point used to define efficient ischemic pre- Female Wistar rats, weighing 230–260g and re-conditioning (Li et al., 1990; Van Winkle et al., ceiving a standard commercial diet (Extralabo,1991; Asimakis et al., 1992; Lawson et al., 1993). France), were used for all studies.Murry et al. (1989) first showed the transient nature The investigation conforms with the Guide for theof the cardioprotection afforded by IPC. In fact, they Care and Use of Laboratory Animals published by theobserved, in dogs, an attenuation of the protective US National Institutes of Health (NIH publicationeffects when the reperfusion between the transient No: 85–23, revised 1985).and sustained ischemia was delayed for 2 h. Thecrucial importance of this time period in elicitingefficient preconditioning has thereafter been dem- Perfusiononstrated in several in situ studies and this durationseems to vary from 1 min to hours, depending on The animals were anesthetized with sodium pen-the animal species used (Van Winkle et al., 1991; tobarbital (50 mg/kg, i.p.). After administration ofLi et al., 1992; Alkhulaifi et al., 1993). To the heparin (1500 U/kg, i.v.), the hearts were excisedauthors knowledge, results concerning isolated rat and aortically perfused using a non-recirculatinghearts are not presently available. system. The temperature was fixed at 37°C and the

Although the protective effects of IPC and their perfusion pressure was maintained at 9.81 kPa.transient nature have been well described, the re- The hearts were allowed to beat spontaneously.sponsible mechanism are not completely un- The perfusion medium contained (m): NaClderstood. At the level of energy metabolism, during 120, KCl 5.6, CaCl2 2.4, MgCl2 1.2 and glucose 11.sustained ischemia, proposed mechanisms include: The content in NaHCO3 (20–23 m) was adjusted(1) preservation of myocardial high-energy stores in order to obtain a pH of 7.4 at the level of thewhich might be due to: reduced ATP utilization aorta after bubbling the medium with carbogen(Murry et al., 1986; 1990), overshoot PCr phe- (95% O2/5% CO2).nomenon (Kida et al., 1991; Volovsek et al., 1992; For nuclear magnetic resonance (NMR) meas-Schjott et al., 1994), inhibition of mitochondrial urements, the hearts were placed in a 15-mmATPase (Murry et al., 1990; Vuorinen et al., 1995) diameter Teflon-stoppered tube and immersed in aand/or to decreased myocardial oxygen demand small volume of perfusate, the excess being evac-(Murry et al., 1990; 1991); (2) reduction in cellular uated via a peristaltic pump. The NMR tube wasaccumulation of catabolites (Murry et al., 1986, then placed in the bore of the superconducting1990; Van Wylen 1994); (3) lessened ischemia- magnet. Both temperature and gas content of theinduced intracellular acidosis (Kida et al., 1991; perfusion fluid were maintained constant by usingAsimakis et al., 1992; De Albuquerque et al., 1994); a thermostatically controlled glass column. During(4) attenuation of the rise in cellular calcium ion ischemia, the temperature of the heart was main-influx by a reduced stimulation of Na+/H+ and tained at 37°C by means of a thermostaticallyNa+/Ca2+ exchange (Steenbergen et al., 1993); (5) controlled flux of air introduced into the magnetreduction in the rate of anaerobic glycolysis by a via the base of the NMR probe.depletion of glycogen stores and substrate avail-ability (Murry et al., 1990; Wolfe et al., 1993).

In order to correlate the kinetics of energy meta- Experimental protocolsbolism alterations to the short-lived adaptative re-sponse of the myocardium to IPC, we used [31P]- After a 35, 41, or 47-min perfusion (Controls CI,

CII, CIII), each heart was subjected, via a peristalticNMR spectroscopy in an isolated rat heart model.

Energy Metabolism Alterations in Ischemic Preconditioning 1673

pump, to a 25-min global low-flow ischemia (1.3% phocreatine concentration (PCr), were measuredusing a colorimetric method (Eggelton et al., 1943).of the spontaneous coronary flow measured by

min 20–23 of normoxic perfusion), referred to as Total AMP, ADP, ATP were determined by high-performance liquid chromatography (HPLC), as de-sustained ischemia, and was then reperfused for

30 min. scribed previously by Lavanchy et al. (1984).Four protocols of IPC were carried out. P1s

(short): the hearts were subjected to 26-min nor-moxic perfusion, 3 min global no-flow ischemia

NMR measurements

and 6 min reperfusion; P1 (long): identical to P1s,[31P]-NMR experiments were performed at

except for the fact that the period of reperfusion101.3 MHz using a Bruker WM 250 spectrometer

was lengthened to 12 min; P2s: a 23-min normoxic(5.9 T field strength). The diameter of the NMR

perfusion was followed by two 3-min global no-probe was 15 mm. The 1.5- and 3-min spectra

flow ischemia interspaced by 6 min reperfusion;were recorded at 5000 Hz spectral width, using a

P2: identical to P2s, except for the fact that the4 K data table, without proton decoupling. A field

last period of reflow was lengthened to 12 min.frequency lock was utilized (D2O in a capillarytube). Each 3-min spectrum of 130 transients wereobtained by applying a 45° flip angle. It includeda downfield reference peak from methylene-Cardiac Function

diphosphonic acid (MDPA in D2O) placed in a ca-The left ventricular developed pressure (LVDP: end- pillary tube. Intracellular pH (pHi) was estimatedsystolic minus end-diastolic pressure) and the heart from the chemical shift of the pH-dependent peakrate (HR) of unpaced hearts were continuously of inorganic phosphate (Pi) relative to the pH-monitored by means of a catheter (PE 160) inserted independent PCr peak. We make use of a personalinto the left ventricle via the atria connected to pH reference curve established from myocardiala Statham pressure transducer and a Gould extracts. ATP, PCr and Pi were calculated from therecorder. The LVDP measured by the 20–23rd min areas under the respective peaks. Integration wasof the stabilization period was 110±2 mm Hg performed using a manual planimeter (Numonics-(mean±..., n=103) and each individual value Phymetron) and an appropriate coefficient was ap-measured at this point served as 100% to normalize plied to the PCr values to correct for the partialthe results from each heart. saturation of the PCr resonance (Lavanchy et al.,

The coronary flow rate (CF) was measured by 1984).timed collection of the returning fluid (ml/min/heart). Heart weights were in the range of530–580 mg. Expression of results and statistical analysis

The amounts of phosphate compounds determinedbiochemically were expressed in lmol/gww. WetBiochemical measurementsweight of cardiac ventricles were corrected for theoedema that developed during perfusion with theThese were performed on freeze-clamped ventriclessaline solution oedema was measured by de-from perfused hearts either after a 20–23-min nor-termining both the tissue protein content of in situmoxic perfusion (reference values), or after a 35-,and perfused hearts (Lowry et al., 1951) and the dry41- or 47-min perfusion with or without IPC (givingto wet weight ratio. The NMR data for phosphatethe values before the onset of prolonged ischemia),compounds (ATP, PCr, free Pi) were normalized byor at the end of the 30-min reperfusion.expressing the results as a percentage of the ATPcontent at min 20–23rd of normoxic perfusion takenas a reference. ATP content, measured on separated

Phosphorylated compounds

The frozen sample of ventricle was crushed to hearts perfused for 20–23 min, was 4.6±0.1 lmol/gww (mean±..., n=20), this value allowedpowder under liquid N2 and homogenized with ice-

cold 0.6 perchloric acid (7 ml/g frozen tissue). conversion of the percentages into lmol/gww.All values are expressed as means ±... Stat-After a 10-min centrifugation (2500 rpm, 0°C), the

supernatant was neutralized with K2CO3 (5 ). istical comparison among basal, preconditioned andrecovery values were made using an analysis ofThe neutralized extract was freed of potassium

percholorate by centrifugation. Free and total cre- variance (ANOVA). Each parameter was evaluatedby one-way or repeated-measures ANOVA for dif-atine (Cr) concentrations, giving thereby the phos-


Table 1 Changes in heart rate (beats/min) in the isolated rat hearts

Group Baseline Before sustained Reperfusion(22th min) ischemia

15th min 25th min

Cl (9) 286±9 285±6 145±44 157±47P1s (9) 229±9 290±5 204±36 202±36Cll (8) 302±12 291±12 146±41 124±44p1 (8) 286±8 266±7 147±41 172±36P2s (8) 274±8 271±8 236±9 247±10Clll (8) 304±6 289±5 299±31 241±32P2 (7) 308±6 296±6 266±5 277±6

Before sustained ischemia, P1s hearts were subjected to a single episode of 3-min global no-flow ischemia followed by 6 min ofreperfusion, P1 hearts to the same protocol but with a longer reperfusion (12 min), P2s hearts to two episodes of global total ischemiainterspaced by 6-min reperfusion and P2 hearts to the same protocol but with the last reperfusion prolonged from 6 to 12 min. Cl,CII and CIII control hearts were submitted to a 35, 41 and 47 min normoxic perfusion respectively. Means ± ... (n).

ferences among the groups. P=0.05 was taken as Then, during the following 3 min, it returned toits baseline value in the four protocols of pre-the limit of significance.conditioning. During the whole reperfusion, the CFof the P2s and hearts were higher than in controls.

The results demonstrate that, among the fourResults

protocols of IPC, only three of them (P1s, P2s and )Cardiac functionwere efficient in improving residual contractilecapabilities on reperfusion, without a previousmarked reduction in function. Two episodes of3 min ischemia did not appear to further enhance

Heart rate

In our model, HR was subjected to slight variations,in particular during the preconditioning phase, as significantly the recovery of post-ischemic con-seen in Table 1, while LVDP appeared to undergo tractile function obtained with a single episode,larger changes. although the early contraction resumed at a higher

level in P2 than in P1s hearts and although theimprovement in coronary reperfusion rate clearlyLVDPpersisted throughout the reperfusion.

Figure 1 shows the time course of changes in LVDP These results emphasize the key importance offor the preconditioned hearts and their respective the duration of the reperfusion period prior to thecontrols. During the preconditioning phase, LVDP prolonged ischemia, since, in the single ischemicwas decreased in the second minute of reflow, after step protocol doubling the reflow period (from 6 toeach brief ischemic episode of the P1 and P2 groups. 12 min) results in the disappearance of the pre-However, this reduction appeared to be transient, conditioning effect. The same lengthening of thesince at the onset of the sustained ischemia, no last reflow period (preceding sustained ischemia)significant difference between preconditioned and appears of no significance in the multiple ischemiccontrol hearts was observed, except in P2L hearts. steps protocol which remains operative, as if theOn reperfusion, LVDP was significantly reduced to “memory” of the transiently induced processes25, 35 and 48% of baseline values in the three underlying preconditioning was also lengthened.groups of control hearts CI, CII, CIII. LVDP of P1s,P2s and L hearts was significantly higher (two- tothree-fold) than the respective values in controls. Alterations in phosphate compound concentrations and

pHiNo significant difference in LVDP being detectedbetween P1L and CII control hearts, preconditioning

Before sustained ischemia, during the preconditioningwas not elicited using this protocol.phase

Coronary flow (CF)ATP content. A significant decrease in ATP contentwas detected in preconditioned hearts before theDuring the second minute after any short ischemic

episode, the CF markedly increased by 50% when onset of prolonged ischemia: 10% for P1, 20% forP2 hearts (Fig. 3).compared to control and initial values (Fig. 2).


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Figure 2 Changes in coronary flow rate. Control hearts:open symbols, preconditioned hearts: closed symbols. The

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four ischemic preconditioning protocols are defined inFigure 1. Vertical dotted lines indicate the short 3-minFigure 1 Changes in left ventricular developed pressureischemic episode(s). Means± ... (n), in ml/min/heart.(LVDP). Each heart was subjected to a 25-min sustained∗P<0.05 v respective controls. For key see legend toischemia and a 30-min reperfusion. Four different pro-Figure 1.tocols of ischemic preconditioning were developed.

Shaded areas indicate ischemic periods. The LVDP isexpressed as a percentage of the reference value takenat the 23rd min of normoxic perfusion (CI hearts: which were quantitatively correlated in our short112±7 mmHg, P1s: 100±9, CII: 107±8, P1: 116±4, ischemic step model. One very interesting ob-P2s: 104±10, CIII: 110±8 and P2: 104±5). Means servation was that, on the following reflow, PCr±...(n). ∗P<0.05 v respective controls. Key: Top panel

regained higher values than the initial ones andCI (9)(Β), P1s (9) (Χ); second panel CII (8) (Φ), P1exhibited a significant “overshoot” (around 110–(8) (Ε); third panel CII (8) (Α), P2 (8)(Μ); bottom

panel CIII (8) (Η), P2 (7) (Ο). 115% of the baseline value), the maximum oc-curring in the 3–4th min of reflow, in parallel withthe coronary overflow (Fig. 2). Such a significantincrease in PCr was observed during the first reflowPCr and Pi contents (Figs 4, 5, 6). During each short

ischemic step, NMR spectroscopy revealed the well period in the four IPC protocols. However, in the(unoperative) single model P1, PCr had regaineddocumented hydrolysis of PCr and increase in Pi


early in reflow, PCr had not yet regained initialvalues, whilst cytosolic Pi resonance had totallydisappeared (from 1.5 as well as from 3-min spec-tra). The most likely explanation is that Pi hasbecome NMR-invisible due to its rapidly enteringinto the mitochondria.

During the sustained ischemia

ATP content. As outlined above, before the sustainedischemia, ATP content was significantly reducedin all hearts subjected to preconditioning. Duringprolonged ischemia, only the efficiently pre-conditioned hearts (P1s, P2s–) exhibited a slowing-down in the degradation of ATP content (Fig. 3).Indeed, the net variation in ATP content was mark-edly reduced in these hearts, whereas it was roughlythe same as in controls in P1 hearts (CI: 3.1±0.3,P1s: 2.1±0.2, CII: 2.8±0.4, P1: 2.5±0.5, P2s:1.6±0.1, CIII: 3.0±0.4, P2: 1.1±0.2 lmol/gww,mean±...). Thus, the intensity and the kineticsof ATP production and/or utilization were clearlydifferent in hearts subjected to preconditioning pro-cesses.

PCr and Pi contents. Although PCr content decreasedsharply during the first minutes of sustained isch-emia, the PCr resonance never completely dis-appeared throughout the whole ischemia in P1sand P2s– hearts and could be easily measured(Fig. 4). Conversely in P1 and in all control hearts,the PCr resonance could not be detected at all in

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NMR spectra recorded after the rapid fall of the firstFigure 3 Changes in myocardial ATP content. Control

few minutes. Furthermore, in hearts that elicitedhearts: open symbols, preconditioned hearts: closed sym-preconditioning, the accumulation of cytosolic Pibols. See Figure 1 for definitions of the four different

protocols of ischemic preconditioning carried out. Means was significantly reduced after its initial increase,± ... (n), in lmol/g. ∗Significantly different from whereas the kinetics of the increase in Pi wererespective controls, P<0.05. For key see legend to Figure identical to controls in P1 hearts (Fig. 5).1.

Intracellular pH. A significant reduction in in-tracellular acidification was observed in heartsits initial values at the onset of ischemia: PCr

overshoot had vanished. When there was a second where preconditioning was operating (P1s, P2s–)when compared to controls (6.4–6.5 v 6.0–6.2).IPC step after 6 min of reflow, PCr exhibited a new

overshoot in the second reflow period which was P1 hearts exhibited the same acidosis as in controls(Fig. 7).higher than the first one and lasted longer, because

it remained significant during 12 min of reperfusion(125–128%, Fig. 4).

During the 30-min reperfusionIn correlation with the PCr overshoot, a Pi un-

dershoot was observed (Fig. 5). Cytosolic Pi res-onance disappeared from NMR spectra (or could ATP, PCr and Pi contents. In the efficiently pre-

conditioned hearts P1s and P2s–, the improvednot be confidently attributed, Fig. 6) and appearedagain clearly with time only in hearts when the rephosphorylation of creatine (Fig. 4) was correlated

with a significant decrease in cytosolic Pi levelspreconditioning effect was disappeared, i.e. in P1

hearts. In addition, it has to be emphasized that, as compared with the respective controls (Fig. 5).


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Figure 4 Time course changes in myocardial phosphocreatine content (PCr). Control hearts: open symbols, pre-conditioned hearts: closed symbols. Four different protocols of ischemic preconditioning were used: a 3-min episode oftotal ischemia followed by 6 (P1s) or 12 min of reperfusion (P1); two 3-min episodes of total ischemia separated bya 6 min reperfusion and followed by a 6 (P2s) or 12 min reperfusion (P2). Means ± ... (n), in lmol/g. ∗P<0.05v respective controls.

Despite an identical loss of total creatine, the PCr in control hearts this dominant value was ac-companied by more acidic side-pHs correspondingto free Cr ratio was significantly enhanced on re-to shoulders on the main Pi peak in NMR spectra,perfusion in hearts eliciting preconditioning whenindicating the presence of more heterogeneouslycompared to controls (75–80% in P1s and P2s–

injured tissue.v 43–47% in control hearts and P1 hearts).Thus, on the reperfusion following the sustainedNMR data revealed no significant difference in

ischemia, the preconditioned hearts present ATPATP content between preconditioned and controlcontents roughly identical to the control ones. Long-hearts, although there was some tendency forlasting (more than a few minutes) higher re-higher ATP in preconditioned hearts (Fig. 3).phosphorylation of creatine on reperfusion clearly

Intracellular pH. After the first 5 min of reperfusion, indicates higher mitochondrial function. The res-the pHi regained values of around 7.0 in all ex- toration rate of PCr appears to be a more appropriate

index to describe the dynamics of functional im-perimental groups (Fig. 7). It should be added that


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Figure 5 Time course changes in myocardial free inorganic phosphate (Pi). Open symbols are used for control heartsand closed symbols for preconditioned ones. Shaded areas indicate the 25-min sustained ischemia. The four protocolsof ischemic preconditioning (P1s, P1, P2s, P2) are explained in Figure 1. Means± ... (n), in lmol/gww. ∗P<0.05v respective controls.

provements than crude ATP content. It leads to a on reperfusion after the sustained ischemia, anhigher PCr:free Cr ratio, which indicates that the earlier and greater resumption of creatine re-cytosolic ATP:free ADP ratio is also higher and that phosphorylation and a lesser residual accumulationthe mitochondrial respiration operates at a higher of cytosolic free Pi; during sustained ischemia, arate in the efficiently preconditioned hearts. slowing down in the decrease in ATP content and

in the development of intracellular acidosis. Theseresults agree with the most commonly observedmetabolic features of preconditioning (Murry et al.,Discussion1990; Kida et al., 1991; Asimakis et al., 1992;Volovsek et al., 1992). However, the persistenceDespite the publication of an increasing number ofof significant amounts of phosphocreatine is lessreports on the phenomenon of preconditioning, thecommonly observed, except in the study of Schjottrole played by the changes of energy metabolismet al., (1994), probably due to the largely used no-in the determinism of such a composite process isflow conditions of prolonged ischemia.not yet clearly demonstrated. The aim of this study

As for the IPC phase, the present results dem-was therefore to follow carefully the time course ofonstrate that a single 3-min episode of total globalalterations in phosphorylated compound contents ofischemia was sufficient to elicit a protection againstthe isolated rat heart subjected to several protocolsthe mechanical and metabolic alterations inducedeliciting or not preconditioning, in order to detectby a further sustained ischemia. These observationsthe transient energy metabolism-linked mech-are also in accordance with data published beforeanisms associated with efficient preconditioning.(Asimakis et al., 1992; De Albuquerque et al., 1994;When IPC, as evaluated by an improvementSchjott et al., 1994; Vuorinen et al., 1995). Fur-of function, was effective under our experimental

conditions, the associated metabolic events were: thermore, the results highlight the crucial import-


PCr

ATPCII

P2s

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Pi

io

io

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Figure 6 Effects of ischemic preconditioning on perfusedrat hearts as assessed by 31P NMR spectroscopy. Theseries of 3-min spectra show the last 21 min of normoxicperfusion before the sustained ischemia in Cll controlhearts, P2s hearts two steps of 3 min total ischemia andin P1 hearts 3 min ischemia followed by a 12-minreperfusion. Each spectrum is the sum of the individualspectra from four hearts, recorded on a Bruker WM 250operating at 101.3 MHz (see Materials and Methods).

ance of the duration of the reperfusion period beforethe prolonged ischemia, i.e. following the (last)ischemic step. To the authors’ knowledge, theseexperiments are the first to analyse carefully thetime-dependence of energy metabolism alterations

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under both operative and unoperative conditionsFigure 7 Time course changes in intracellular pH. The

of IPC. Indeed, the positive effect of IPC using a pHi is evaluated on each 31-P NMR spectrum from thesingle ischemic step has disappeared when the re- chemical shift of the Pi peak relative to that of the PCr

peak (see methods). Rat hearts were divided into fourflow period was increased from 6 to 12 min. Thegroups according to the ischemic preconditioning pro-same lengthening of this delay did not supress thetocol used (see Fig. 1). Control hearts: open symbols,preconditioning positive effect when IPC consistedpreconditioned hearts: closed symbols. Means ± ...

in two steps of short ischemia. (n). ∗P<0.05 v respective controls. For key see legend toWhen the time course of changes in phos- Figure 1.

phorylated compounds during this reflow period isconsidered, it appears that the transient “memory”of preconditioning is associated with a PCr over-shoot (and a Pi undershoot). This overshoot is and seems to be one of the notable metabolic

differences between preconditioned and controlshort-lived after a single preconditioning step andit is reinforced and prolonged upon repetition of myocardium. The preconditioning results of De Al-

buquerque et al. (1994) on the isolated rat heartthe preconditioning short ischemia. Such a PCrovershoot phenomenon after the short ischemic are puzzling because short periods of metabolic

inhibition (induced by cyanide exposure) wereperiod(s) has been described in various in situ andin vitro preconditioning experiments in hearts from clearly followed by a PCr overshoot, whereas short

ischemic periods were not. Nevertheless, it has todifferent species (Kida et al., 1991; Asimakis et al.,1992; Volovsek et al., 1992; Steenbergen et al., be noticed that the reflow period between brief and

sustained ischemia was two-fold shorter (5 v 101993; Schjott et al., 1994; Vuorinen et al., 1995)


min) in the ischemia-preconditioned hearts com- mechanism does not seem to be preponderant inmyocardium of rats via an ATPase inhibitory factorpared to cyanide-preconditioned hearts. This could

result in an incomplete development of PCr over- because myocardium of rodents with fast heart ratescontains only 20–30% as much of the endogenousshoot in ischemia-preconditioned hearts. Fur-

thermore, PCr values were given through 5-min inhibitor (IF1) as mammals with slower heart rates(Rouslin et al., 1990). However, in a recent paper,intervals, summing probably phenomena and at-

tenuating thereby the PCr overshoot intensity. Vuorinen et al. (1995) claimed that a 3-min isch-emic step induces an inhibition of rat heart mito-In our experiments, the PCr overshoot developed

in synchrony with the hyperemic response. This chondrial ATPase activity, which remainsthroughout the intervening 9-min reperfusionobservation is of great interest if we consider that

the preconditioning effect seems to be linked to the period, so that the enzyme activity is decreased atthe very beginning of the sustained ischemia.repayment of the flow debt in the perfused rat heart

(Schjott et al., 1994) and seems to develop fully It can be suggested that the events occurringduring the very early phase of ischemia are criticalonly when reflow is complete in the in-situ dog

heart (Ovize et al., 1992). A minimum time of and determinant in the further dynamics of energymetabolism. Indeed, during the sustained ischemia,reflow is required for the protective effects of a

preceding 5 min ischemia to develop in in situ rat the half-life of the metabolic alterations are in therange of 5 min or less for PCr, Pi and pHi, steady-heart (Alkhulaifi et al., 1993). Furthermore, the

comparative studies of Liu et al. (1991, 1992) state levels of PCr and pHi being completelyachieved in less than 12–15 min. The t50 of theconfirmed the necessity of such a repayment of the

flow debt: blocking the adenosine receptors in the ATP content decrease during ischemia is longer(10 min), but the slope of the linearlike evolutionisolated rabbit heart completely abolished the hemo-

dynamic effect and suppressed preconditioning of this variable is determined very early. Theseconsiderations are consistent with those of Murrybeneficial effects, while the same dose of inhibitor

only attenuated the adenosine hemodynamic effect et al. (1990) and Jennings et al. (1991) whichled to the conclusion that the larger metabolicin the rat heart and could not block preconditioning.

It seems reasonable to postulate that the mech- differences between preconditioned hearts and con-trols were observed during the first 10 min of isch-anisms underlying the PCr overshoot participate in

the mechanisms involved in preconditioning. The emia of in situ dog hearts. It can be hypothesizedtherefore that the transient energization state ofovershoot of PCr (and the undershoot of Pi) is

indicative of a transient positive energy imbalance the cell induced by preconditioning is crucial duringthe first minutes of sustained ischemia and, con-and it is tempting to consider that this transient

state puts the myocardial cell in a favourable state sequently, for the alterations occuring thereafter.After the early phase of the prolonged ischemia,during a short period of time at the onset of the

sustained ischemia. it was observed, under the present experimentalconditions, that significant amounts of PCr persistThe reasons for such an energy imbalance can

be diverse. A depression of mechanical activity is at an almost constant concentration. This indicatesobviously the establishment of a new energy bal-always present shortly after the short pre-

conditioning episode of ischemia (Miura et al., 1991; ance. Such a steady-state indicates that, at least fora period of time, the ischemic processes do notMurry et al., 1991), resulting in an increase in the

ATP supply-to-consumption ratio. But it could also evolve rapidly towards irreversible alterations. Theadenine nucleotide pools in several subcellularbe argued that, due to the transient ischemia and

the induced changes in free ADP and free creatine, microcompartments could thus be maintained inan energized state through the various creatinean increase in mitochondrial ATP production oc-

curs, contributing to the charging of the cellular kinases described inside the cell (for a review seeSaks et al., 1994). Such a state could probably limitenergy reserves. Another factor that can be de-

terminant during the same short period is a re- the constitution of rigor-bonds and result in a lessdamaged cellular ionic homeostasis. These ex-duction in ATP wastage due to ATPase activity at

the mitochondrial level. Indeed, the mitochondrial planations could account for the improved re-sumption of mechanical performance early onATPase begins to work in reverse during ischemia

when the electrochemical gradient collapses, re- reperfusion (Fig. 1) associated with reduced post-ischemic diastolic dysfunction (Fig. 8) in pre-sulting in ATP hydrolysis. This process could be a

major ATP consumer in ischemia. A possible effect conditioned hearts. In fact, after 25 min of ischemia,hearts efficiently preconditioned showed a reducedof preconditioning could be an inhibition of the

mitochondrial ATPase (Murry et al., 1990). Such a left ventricular end-diastolic pressure. During re-


In conclusion, an accurate analysis of the timecourse alterations in phosphorylated compoundconcentrations brings to light the fact that theenergy balance of the myocardium could play acrucial role in IPC. The energization state of thecardiomyocyte is enhanced by IPC just prior thesustained ischemia and thereby a new steady-statebalance in energy metabolism is reached in pre-conditioned hearts during sustained ischemia. It ishypothesized that the mitochondrial function playsan important role in the phenomenon of IPC via(1) an inhibition of mitochondrial ATPase inducedby the IPC episode and (2) a protection duringsustained ischemia of the functional coupling of

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mitochondrial creatine kinase to the other en-Figure 8 Time course changes in left ventricular end zymatic systems of the mitochondria.diastolic pressure (LVEDP). Control hearts: open symbols,preconditioned hearts: closed symbols. The ischemic pre-conditioning protocols used one or two cycles of 3 minischemia followed by 6 min reperfusion (P1s, P2s), in- Referencescreasing the last reflow period from 6 to 12 min (P1,P2). All hearts were then submitted to a 25-min sus- A AM, P WB, Y DM, 1993. Thetained global partial ischemia, and finally reperfused influence of the time period between preconditioningduring 30 min. Means ± ... in mmHg. ischemia and prolonged ischemia on myocardial pro-

tection. Cardioscience 4: 163–169.A GK, I-MB K, M G, C VR,

1992. Ischemic preconditioning attenuates acidosisperfusion, control hearts exhibited reperfusion con-and postischemic dysfunction in isolated rat heart. Amtracture, whereas preconditioned hearts showedJ Physiol 263: H887–H894.lower end-diastolic pressures, especially those sub-

D A CP, G G, W RG, 1994.jected to a multiple IPC protocol (Fig. 8). Importance of metabolic inhibition and cellular pH in

On reperfusion following the sustained ischemia, mediating preconditioning contractile and metaboliceffects in rat hearts. Circ res 74: 139–150.it is also important to make a distinction between

E P, E SR, G N, 1943. Estimation ofa very early phase and a later one. The eventscreatine and of diacethyl. Biochem J 37: 526–529.occurring during the first minutes of reperfusion

H N, D L M, 1984. The effect of inorganic phos-also determine the future level of the new energy phate on creatine kinase in respiring rat heart mito-steady-state. Following 25 min of ischemia, the chondria. Arch Biochem Biophys 229: 477–482.

J RB, M CE, R KA, 1991. Energy meta-phosphorylation capabilities of the mitochondriabolism in preconditioned and control myocardium:are probably virtually intact (Saks et al., 1989;Effect of total ischemia. J Mol Cell Cardiol 23: 1449–Piper et al., 1994), but the efficiency of production1458.

of PCr is different in control and preconditioned K M, F H, I M, K C, O M,hearts. Indeed, the rate of phosphorylation of cre- M I, Y Y, 1991. Ischemic preconditioning

preserves creatine phosphate and intracellular pH. Cir-atine is faster in preconditioned than in controlculation 84: 2495–2503.hearts. This means that the functional coupling of

L N, M J, R A, 1984. Graded globalmitochondrial creatine kinase to oxidative phos-ischemia and reperfusion of the isolated perfused rat

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L GC, V JA, G KP, L BR, 1990.a decoupling, it is well known that a high inorganicMyocardial protection with preconditioning. Circulationphosphate concentration plays a major role (Vial et82: 609–619.al., 1979; Hall et al., 1984). Under our experimental

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