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(Circulation. 1996;94:2587-2596.)
© 1996 American Heart Association, Inc.

Articles

Metabolic Adaptation During a Sequence of No-Flow and Low-Flow Ischemia

A Possible Trigger for Hibernation

R. Ferrari, MD, PhD; A. Cargnoni, PhD; P. Bernocchi, BSc; E. Pasini, MD; S. Curello, MD; C. Ceconi, MD; T.J.C. Ruigrok, PhD

the Chair of Cardiology (R.F., S.C., C.C.), University of Brescia, Brescia, Italy; Center of Cardiovascular Pathophysiology (A.C., P.B., E.P.), "S. Maugeri," Foundation, IRCCS, Rehabilitation Institute of Gussago, Brescia, Italy; and Heart Lung Institute (T.J.C.R.), University Hospital, Utrecht, and the InterUniversity Cardiology Institute, the Netherlands.

Correspondence to Prof Roberto Ferrari, Cattedra di Cardiologia, Spedali Civili, P.le Spedali Civili, 1, 25123 Brescia, Italy. E-mail ferrari@master.cci.unibs.it.

	Abstract

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Background Myocardial hibernation is an adaptive phenomenon occurring in patients with a history of acute ischemia followed by prolonged hypoperfusion.

Methods and Results We investigated, in isolated rabbit heart, whether a brief episode of global ischemia followed by hypoperfusion maintains viability. Four groups were studied: group 1, 300 minutes of aerobia; group 2, 240 minutes of total ischemia and 60 minutes of reperfusion; group 3, 10 minutes of total ischemia, 230 minutes of hypoperfusion (90% coronary flow reduction), and 60 minutes of reperfusion; and group 4, 240 minutes of hypoperfusion followed by reperfusion. In group 3, viability was maintained. Ten minutes of ischemia caused quiescence, a fall in interstitial pH (from 7.2±0.01 to 6.1±0.8), creatine phosphate (CP), and ATP (from 54.5±5.0 and 25.0±1.9 to 5.0±1.1 and 15.3±2.5 µmol/g dry wt, P<.01). Subsequent hypoperfusion failed to restore contraction and pH but improved CP (from 5.0±1.1 to 20.1±3.4, P<.01). Reperfusion restored pH, developed pressure (to 92.3%), and NAD/NADH and caused a washout of lactate and creatine phosphokinase with no alterations of mitochondrial function or oxidative stress. In group 4, hypoperfusion resulted in progressive damage. pH fell to 6.2±0.7, diastolic pressure increased to 34±5.6 mm Hg, CP and ATP became depressed, and oxidative stress occurred. Reperfusion partially restored cardiac metabolism and function (47%).

Conclusions A brief episode of total ischemia without intermittent reperfusion maintains viability despite prolonged hypoperfusion. This could be mediated by metabolic adaptation, preconditioning, or both.

Key Words: myocardial hibernation • ischemia • metabolism • preconditioning

	Introduction

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Myocardial ischemia, even if persisting for a prolonged period of time, does not inevitably induce irreversible damage. In some patients, subsequent to successful revascularization, an appreciable metabolic and functional recovery of the previously akinetic areas is possible. This entity has been referred to as "hibernating" myocardium.^{1 2} The condition occurs in the setting of chronic flow limitation and is associated with reduced or abolished function in the hypoperfused region. Thus, the myocardium must be able to adapt to low-flow states and to reset its energy balance for maintaining cell viability. How this is accomplished has not yet been established, nor are the effects of prolonged flow reduction on regulation of cardiac metabolism fully understood.

Hibernation may not be simply the result of adaptation to hypoperfusion but also the consequence of intermittent sequences of no perfusion, hypoperfusion, and normal perfusion. The whole issue is further complicated by the existence of the so-called phenomenon of "ischemic preconditioning." This term describes the capability of a brief episode of ischemia followed by full reperfusion to increase myocardial tolerance to prolonged ischemia.³ Interestingly, the majority of patients with hibernating myocardium have a history of acute ischemic insult followed by hypoperfusion, suggesting a possible mechanistic link between ischemic preconditioning and myocardial hibernation.^{4 5 6}

In this study we tested the hypothesis that the sequence of coronary flow restriction is important for the development of hibernation and that severe and prolonged myocardial hypoperfusion is better tolerated when preceded by a brief episode of no-flow ischemia without intermittent reperfusion. To this end, isolated and perfused hearts were subjected to prolonged (4 hours) and severe (90% coronary flow reduction) hypoperfusion either in the presence or absence of a preceding short period (10 minutes) of total and global ischemia.

	Methods

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Animals
Adult male New Zealand White rabbits (n=138; weight, 2.5 to 3 kg) fed a standard diet were used. Their treatment conformed to the guiding principles of the American Physiological Society and the University of Brescia.

Perfusion of Hearts
The rabbits were stunned by a blow on the neck. The hearts were removed and immersed in an ice-cold modified Krebs-Henseleit solution. The aorta was exposed and suspended on a metal cannula. The hearts were paced and perfused by the nonrecirculating Langendorff technique, with a modified Krebs-Henseleit buffer solution containing 1.5 mmol/L calcium, as previously described.⁷

Left Ventricular Pressure
To obtain an isovolumetrically beating preparation, a latex balloon filled with saline, connected by a catheter to a Statham transducer (P 2306), was inserted into the left ventricle through an atriotomy and secured by a suture around the atrioventricular groove. The balloon was inflated to provide an end-diastolic pressure <1.0 mm Hg.⁷

Interstitial pH
Interstitial pH was monitored by a needle-type pH electrode inserted into the left ventricular wall.⁸ The electrode tip diameter was 0.5 mm. The electrode has a linear output of 57 to 59 mV/pH at 37°C; the mV/pH slope is identical in vivo and in vitro. It is unresponsive to changes in (mmol/L) sodium 100 to 200; potassium 0 to 100; calcium 0 to 10; and magnesium 0 to 10. It is unaffected by osmolarity (300 to 500 mOsm/kg), physical pressure (0 to 300 mm Hg) or PO₂ (6 to 710 mm Hg).⁸ The resistance is 10^-8 . The 95% response time is <300 ms. A calomel reference electrode (Radiometer, K4112) was connected to the tissue via a saline bridge and a fine glass pipette. The signal was amplified and displayed on a pen recorder.

Analysis of the Coronary Effluent and Assay of CPK, Lactate, GSH, and GSSG
During each perfusion, the coronary effluent was collected in chilled glass vials and assayed the same day for CPK, lactate, GSH, and GSSG. CPK activity and lactate concentration were measured by the spectrophotometric methods described by Oliver⁹ and by Hohorst et al,¹⁰ respectively. Methods for glutathione measurement are described later in this section.

Isolation of Mitochondria and Determination of Their Function
Mitochondria were isolated at the end of each perfusion by differential centrifugation, as previously described,¹¹ using the medium described by Sordhal and coworkers,¹² containing 180 mmol/L KCl, 10 mmol/L EDTA, and 0.5% BSA. Rates of oxygen consumption were monitored polarographically at 25°C with a Clark-type electrode, using glutamate (3 mmol/L) as substrate.

Mitochondrial function was assessed in terms of the respiratory control index (RCI), QO₂, and ADP/O. Mitochondria used for the measurements of endogenous calcium and production of ATP were extracted in a medium containing 250 mmol/L sucrose and 5 µmol/L ruthenium red. Mitochondrial ATP production and calcium content were measured as previously described.¹¹

Assay of Proteins
Concentration of proteins in the mitochondria was measured by the method of Bradford,¹³ using BSA as a standard.

Assay of Total Calcium
At the end of each perfusion, duplicate samples of left ventricular muscle were taken and digested in 1 mL HNO₃·g^-1 tissue. The extract was diluted in 25 mL of deionized water; calcium content was assayed by atomic absorption spectrometry.¹¹ Total tissue water was obtained by drying samples to constant weight at 95°C.

Assay of High-Energy Phosphates, Purine, and Pyridine Nucleotides
After each perfusion, the hearts were freeze-clamped with aluminium tongues precooled in liquid nitrogen. Preweighed portions of frozen tissue were ground in a mortar cooled in liquid nitrogen and mixed with either (1) 0.4N HClO₄ (3 mL/500 mg) for extraction of ATP, CP, purine, and oxidized pyridine coenzymes or (2) 0.64 mol/L of phenol buffer solution (3 mL/500 mg) for reduced pyridine nucleotide extraction. The extracts were used for chromatography. The separation and quantification of the metabolites were performed with the use of a reversed-phase, 3-µm C18 column. The mobile phase consisted of a gradient of acetonitril (2.5% to 25% vol/vol) in phosphate buffer solution with the addition of tetrabutylammonium hydrogen sulfate as an ion-pair agent. Detection was performed at 205 nm for creatine phosphate and at 260 nm for nucleotides as previously described.¹⁴

Assay of GSH, GSSG, and SH Groups
Tissue (100 mg) was deproteinized with 3 mol/L HClO₄. The supernatant obtained after centrifugation at 6000g for 15 minutes was neutralized with 2 mol/L K₂CO₃. A sample of the neutralized extract or a proper aliquot of coronary effluent was analyzed for GSH and GSSG as previously described.^{15 16} Protein-SH groups were assayed in the homogenate as described by Sedlack and Lindsay.¹⁷

Protocol
Four groups of hearts were identified: In group 1 (n=27), after 50 minutes of equilibration the hearts were perfused under aerobic condition (coronary flow, 22 mL/min) for 300 minutes. This group constitutes the aerobic control. In group 2 (n=34), after equilibration the hearts were subjected to 240 minutes of total ischemia followed by 60 minutes of aerobic reperfusion at a coronary flow rate of 22 mL/min. This group constitutes the total ischemic reference. In group 3 (n=41), after equilibration the hearts were subjected to 10 minutes of total ischemia followed by 230 minutes of low-flow ischemia (coronary flow, 2.2 mL/min; 10% of the aerobic flow). Thereafter, they were reperfused at full coronary flow (22 mL/min for 60 minutes). This represents the test group. Group 4 hearts (n=39) were subjected to 240 minutes of low-flow ischemia (coronary flow, 2.2 mL/min) followed by 60 minutes of reperfusion at full coronary flow (22 mL/min). This constitutes the low-flow ischemic control.

Tissue temperature was monitored and maintained at 37°C irrespective of coronary flow, as previously described.⁷

Reagents and Statistical Evaluation
All reagents were of grade quality. All enzymes used for the biochemical assays were obtained from Sigma Chemical Co.

Data are mean±SE of n experiments, in which each experiment is an individual perfusion. For statistical evaluation of the results, a multiple-group comparison was performed by ANOVA followed by Student's t test for paired or unpaired data with Bonferroni's corrections. A value of P<.05 was taken as the limit of significance.

	Results

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Mechanical Performance
Fig 1 shows a typical tracing for each group of experiments. The mean results are shown in Fig 2. In group 1, after 300 minutes of aerobic perfusion, developed pressure was 85±0.9% of the initial value. Diastolic pressure did not change. Interstitial pH was 7.2±0.02 (n=13) and was stable to within ±0.009 unit throughout the 300 minutes of aerobic perfusion. Perfusate pH was 7.37 (data not shown).

View larger version (39K): [in this window] [in a new window]   Figure 1. Typical mechanical tracing for each group of experiments. Group 1, 300 minutes of aerobic perfusion at coronary flow of 22 mL/min; group 2, 240 minutes of total ischemia followed by 60 minutes of reperfusion at coronary flow of 22 mL/min; group 3, 10 minutes of total ischemia followed by 230 minutes of low-flow ischemia (coronary flow, 2.2 mL/min) followed by 60 minutes of reperfusion; and group 4, 240 minutes of low-flow ischemia (coronary flow, 2.2 mL/min) followed by 60 minutes of reperfusion (coronary flow, 22 mL/min). LV indicates left ventricular.

View larger version (85K): [in this window] [in a new window]   Figure 2. Developed pressure, interstitial pH, and diastolic pressure over the time course of the experiments in the three groups. Data are expressed as mean±SE of 10 experiments for group 2, 14 for group 3, and 13 for group 4.

In group 2, total ischemia caused a rapid fall in interstitial pH and in developed pressure. Interstitial pH fell by 0.23±0.003 unit (n=10) after 60 seconds and was 6.3±0.9 after 10 minutes of ischemia. Developed pressure did not fall below control until after 25 seconds. It fell to 20±2.5% of preischemic values at 1 minute and to 6.4±1.4% at 5 minutes. Ten minutes after the onset of ischemia, all hearts were quiescent and remained so throughout the experiment. Interstitial pH fell continuously during ischemia, to 5.4±1.7. Diastolic pressure began to rise 10 minutes after ischemia to 54±2.2 mm Hg. Reperfusion resulted in a further increase in diastolic pressure to 119±7.9 mm Hg and no recovery of developed pressure or interstitial pH.

In group 3, 10 minutes of total ischemia caused changes in developed pressure and interstitial pH similar to those observed in group 2 after the same period of ischemia. Restoration of low coronary flow for 230 minutes produced the following results: (1) Developed pressure remained significantly depressed (9 hearts were completely quiescent, while the remaining 5 recovered 6±0.7% of the aerobic value). (2) Interstitial pH increased from 6.2±0.8 to 6.8±0.6 (n=14) during the first 50 minutes of low-flow ischemia. Thereafter, it remained constant throughout. (3) The onset and extent of diastolic contracture was delayed and reduced with respect to that of group 2. (4) On reperfusion, there was a prompt recovery of interstitial pH. The most pronounced change in interstitial pH occurred during the first 10 minutes of reperfusion, when developed pressure was rapidly recovering. (5) Recovery of developed pressure at the end of reperfusion was 92±8.5%. (6) The degree and velocity of recovery were independent of development of quiescence. (7) During the first 10 minutes of reperfusion, there was a slight overshoot in diastolic pressure followed by a gradual decline toward the aerobic value.

Hearts (n=13) of group 4 were perfused at low flow (2.2 mL/min) throughout the ischemic period. Interstitial pH and developed pressure fell more slowly and to a lesser extent than in groups 2 and 3. After 10 minutes of ischemia, interstitial pH was 6.8±0.6 (compared with 6.3±0.9 and 6.1±0.7 of groups 2 and 3, P<.05) and continued to gradually fall to reach the value of 6.2±0.7 after 240 minutes of ischemia. After 30 minutes of ischemia, developed pressure was reduced to 7.3±1.1% and remained stable throughout. Four hearts of this group reached quiescence, but only after 210 minutes of hypoperfusion. Diastolic pressure started to increase after 30 minutes of ischemia and reached a peak of 37±5.6 mm Hg at 180 minutes. Reperfusion of these hearts resulted in (1) a further increase in diastolic pressure with a peak 5 minutes after readmission of coronary flow; (2) a slow and incomplete recovery of interstitial pH; and (3) partial recovery of developed pressure (47% of the aerobic value).

Release of Lactate and CPK
Data are shown in Fig 3. In group 1, only small amounts of lactate and CPK were present in the coronary effluent (0.31±0.33 µmol/min per gram of wet wt and 0.133±0.024 U/min per gram of wet wt, respectively). In group 2, lactate and CPK release during ischemia could not be determined. Reperfusion resulted in a marked and sustained release of both lactate and CPK. In group 3, low-flow ischemia caused a minor increase in the rate of lactate release, and reperfusion resulted in a slight washout. In group 4, there was an increase in lactate release throughout the ischemic period, which was exacerbated by reperfusion. Restoration of full flow also resulted in an intensification of CPK release, which was more evident in group 4 than in 3.

View larger version (77K): [in this window] [in a new window]   Figure 3. Lactate and CPK release over the time course of the experiments in the three groups. During total ischemia, lactate and CPK release could not be determined because flow was abolished. This applies to group 2 for the entire ischemic period and to group 3 for the first 10 minutes of ischemia. Data are expressed as mean±SE of 10 experiments for group 2, 14 for group 3, and 13 for group 4.

Mitochondrial Function and Calcium Homeostasis
Aerobic perfusion of group 1 hearts failed to alter mitochondrial function (Table 1). In groups 2, 3, and 4, 10 minutes of either total or low-flow ischemia did not affect mitochondrial function. In group 2, prolongation of total ischemia caused an increase in both tissue and mitochondrial calcium and a progressive deterioration of all indexes of respiratory activity. As expected, reperfusion caused mitochondrial calcium overload and deterioration of their function.

View this table: [in this window] [in a new window]   Table 1. Mitochondrial Function, ATP Production, and Homeostasis

In group 3, restoration of low coronary flow for 230 minutes maintained mitochondrial function within normal limits, with reperfusion having no further effect.

In group 4, persistent low-flow ischemia failed to preserve oxidative phosphorylation. There was a slow but progressive increase in tissue and mitochondrial calcium concomitant with a deterioration of mitochondrial function less severe than in group 2, reaching a statistical significance only after 240 minutes of ischemia. Reperfusion partially restored mitochondrial function but failed to reduce calcium overload.

High-Energy Phosphates, Purines, and NAD/NADH Ratio
These data are shown in Table 2. Measurements were made after 10, 120, 240, and 300 minutes of perfusion. In group 1, there was a small, nonsignificant decline in CP and ATP and a small decrease in the NAD/NADH ratio (from 9.5 to 8.0). The overall energy charge of the myocardium remained unchanged.

View this table: [in this window] [in a new window]   Table 2. High-Energy Phosphates, Purines, and NAD/NADH Ratio

In groups 2 and 3, 10 minutes of total ischemia caused a severe depletion of CP and a less severe reduction in ATP. ADP and AMP increased proportionally. Energy charge decreased (from 0.9±0.09 to 0.8±0.02). The NAD/NADH ratio dropped from 9.5 to 0.8 (P<.01). Persistence of total ischemia in group 2 resulted in a complete depletion of all high-energy phosphates and in a marked depletion of NAD(H) pool. Reperfusion failed to restore energy metabolism.

In group 3, low coronary flow after the short period of total ischemia resulted in the recovery (although incomplete) of CP (from 5.0±1.1 to 20.1±3.4 µmol/g dry wt; P<.01) and in no significant further depletion of ATP. The energy charge remained unchanged, and the NAD/NADH ratio increased from 0.8 to 2.5. Reperfusion caused no changes in CP and ATP content despite the recovery of developed pressure. There was, however, a further amelioration of the NAD/NADH ratio (from 2.5 to 5.1).

In group 4, persistent low-flow ischemia resulted in a progressive, slow but significant decline in tissue content of ATP and CP. The NAD/NADH ratio declined from 9.5 to 1.6 after 10 minutes of low-flow ischemia. At the end of 230 minutes of hypotension, it further decreased, from 1.6 to 0.4. Reperfusion did not restore ATP content but increased CP levels and the NAD/NADH ratio (from 0.4 to 1.2).

Oxidative Stress
Data are reported in Fig 4 and Table 3. In group 1, there was a small release of GSH and a reduction in the GSH/GSSG ratio, from 113 to 89.

View larger version (76K): [in this window] [in a new window]   Figure 4. GSH and GSSG release over the time course of the experiments in the three groups. During total ischemia, GSH and GSSG release could not be determined because flow was abolished. This applies to group 2 for the entire ischemic period and to group 3 for the first 10 minutes of ischemia. Data are expressed as mean±SE of 10 experiments for group 2, 14 for group 3, and 13 for group 4.

View this table: [in this window] [in a new window]   Table 3. Intracellular Thiol Pool

In groups 2, 3, and 4, 10 minutes of either total or low-flow ischemia caused a small, nonsignificant reduction in GSH/GSSG ratio with almost no changes in protein- and nonprotein-SH groups. In group 2, prolongation of total ischemia produced a marked decline of tissue content of GSH, protein- and nonprotein-SH groups, while GSSG increased from 0.10±0.01 to 0.16±0.02 nmol/mg protein. Reperfusion resulted in an additional higher degree of oxidative stress. There was a significant and persistent release of both GSH and GSSG, an increase in myocardial GSSG, and a further decrease in GSH, yielding a GSH/GSSG ratio as low as 5.6.

In group 3, low coronary flow after 10 minutes of total ischemia completely prevented the occurrence of oxidative stress during both ischemia and reperfusion.

In group 4, persistent low-flow ischemia for 240 minutes failed to protect the hearts against oxidative damage. During ischemia there was a gradual reduction in GSH tissue content with no changes in myocardial GSSG. On reperfusion, there was a release of both GSH and GSSG, myocardial GSSG increased, GSH/GSSG declined from 62 to 29, and protein- and nonprotein-SH groups did not increase.

	Discussion

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These data highlight the importance of residual coronary flow to maintain viability during prolonged ischemia in the isolated rabbit heart. In group 2, total ischemia for 4 hours caused irreversible damage. In groups 3 and 4, low-flow ischemia maintained a certain degree of viability. The sequence of coronary flow restriction plays an important role in the final outcome on reperfusion. In group 3, a brief episode of no-flow ischemia increased the tolerance to the subsequent, prolonged period of low-flow ischemia.

We designed these experiments on the basis of the clinical experience of coronary artery disease patients with hibernating myocardium. In the history of these patients there is often evidence of an acute ischemic insult either in the form of a transmural myocardial infarction or prolonged ischemic pain. Thus, to mimic the acute ischemic insult, we submitted the isolated heart to a period of 10 minutes of no-flow ischemia. In addition, patients with hibernating myocardium show signs of chronic hypoperfusion, as demonstrated by positive late rest redistribution with thallium.^{5 6} For this reason, we underperfused the isolated hearts for 230 minutes immediately after the 10 minutes of total ischemia.

The setting up of low coronary flow after 10 minutes of total ischemia failed to restore mechanical activity but prevented the rise in diastolic pressure, thus suggesting that calcium homeostasis was maintained. This was confirmed by the absence of tissue and mitochondrial calcium overload. During low-flow ischemia there was no further degradation of ATP and a gradual (although incomplete) recovery of CP and NAD/NADH ratio. This suggests that the reduced amount of oxygen provided by the residual coronary flow was sufficient to support a degree of oxidative metabolism which, most likely, accounted for the partial recovery of CP. Occurrence of oxidative metabolism during low-flow ischemia is confirmed by the limited release of lactate and by the preservation of mitochondrial function. The NAD(H) pool was preserved, suggesting that viability was maintained. Furthermore, the quiescent state of the myocardium substantially reduced ATP utilization and contributed to the maintenance of myocardial energy charge. Therefore, a resetting of cardiac metabolism, with a substantial downregulation of both energy need and production, did occur in these hearts. The prompt recovery of function on reperfusion in the absence of CPK release, oxidative stress, and calcium overload supports the concept that myocytes of these hearts remained viable.

To answer the question of whether the brief period of ischemia did play a role in these results, we repeated the experiment, submitting the hearts to 4 hours of low-flow ischemia in the absence of 10 minutes of total ischemia (group 4). In this group, although the onset and the degree of myocardial damage were considerably delayed and reduced with respect to group 2, damage was not prevented as in group 3. We must conclude, therefore, that 10 minutes of total ischemia preceding low-flow ischemia results in significant cardioprotection and maintenance of viability. There are several explanations for this finding.

A Brief Period of No-Flow Ischemia Causes Metabolic Adaptation
There is evidence that ischemia initiates mechanisms (which are not yet fully understood) resulting in a loss of regional contractility in relation to the loss of blood flow.^{18 19 20} The results of group 4 indicate that coronary flow reduction was matched by a proportional energy demand reduction, which nonetheless was inadequate to maintain viability in the long term, since there was a depletion in high-energy phosphates with prolongation of ischemia. In this group, developed pressure was not completely abolished, at least initially, but was severely reduced.

A metabolic adaptation to a severe reduction in flow, however, occurred in the group 3 hearts, and it is likely that the preceding 10-minute no-flow ischemia is at least in part responsible for such adaptation. In this group, the early decline in contractile function was more pronounced and significantly faster than in group 4. All hearts in group 3 stopped beating (quiescence) within 5 minutes after the onset of ischemia, while the majority (9 out of 14) remained quiescent throughout the experiment. It is possible that this faster decrease in contractile function and, consequently, in metabolic need accounts for the better preservation of myocardial energy stores, allowing a new and more favorable balance between energy supply and energy demand. The hearts that became quiescent showed a tendency toward a better recovery of function with respect to the 5 hearts that developed some degree of contraction. Because of the high variability and the low number of experiments, the difference did not reach statistical significance. The decline in contractile function during no-flow ischemia was accompanied by a greater decrease in interstitial pH, and quiescence probably was due to a faster development of myocardial acidosis. Interestingly, interstitial pH during subsequent low-flow ischemia was not restored to the preischemic value of 7.2 but remained constantly reduced at 6.8. This might have contributed to downregulation of contraction during low-flow ischemia.

It has been shown that metabolic acidosis, with a reduction in perfusate pH from 7.4 to 6.8 or 6.9, reduces hypoxic damage in the isolated heart by a mechanism related to a reduction in energy expenditure, while a further reduction in perfusate pH results in further damage.²¹

Recently, Ito²² showed that downregulation of myocardial metabolism with gradually decreased flow to severe levels results in reduced myocardial injury for a given period of low-flow. Interestingly, this was associated with less decline in pH. Thus, discrete alteration of myocardial pH might play a role in the adaptation of the isolated heart to ischemia by initiating and maintaining a reduction in contractile function, which in turn diminishes energy demand. Recently, Schulz et al²³ carried out experiments in anesthetized pigs, following a protocol similar to ours. They also found that a brief episode of no-flow ischemia without intermittent reperfusion increases the tolerance to sustained low-flow ischemia but concluded that changes in pH are not responsible for the changes in contractile function. They measured coronary venous pH which, because of the high buffering capacity of blood, is only a rather crude measure of myocardial pH. In a study on isolated rat hearts from us, the protocol for groups 3 (with 5 minutes of no-flow ischemia) and 4 was followed with the use of ³¹P NMR spectroscopy, which provided information on intracellular pH.²⁴ During control perfusion, pH amounted to 7.05. In the experiments performed following the protocol for group 3, mean intracellular pH decreased to 6.55 during no-flow ischemia and increased to 6.80 during the subsequent first 5-minute period of low-flow ischemia. Because intracellular pH values were average values of 5-minute periods, the final pH after 5 minutes of no-flow ischemia must have been well below 6.55. In the experiments according to group 4, intracellular pH decreased from 7.05 to 6.86 and 6.81 during the first two 5-minute periods of low-flow ischemia.

A Brief Period of No-Flow Ischemia Preconditions the Heart
Ischemic preconditioning refers to the reduction in infarct size resulting from prolonged and severe myocardial ischemia by one or more preceding short episodes of ischemia and reperfusion.³ This phenomenon has been confirmed in a number of animal species, including rabbit^{25 26 27} and, more recently, in humans.²⁸ The sequence of events of group 3, however, cannot be considered a classic ischemic preconditioning protocol because the brief period of total ischemia was not followed by full reperfusion but by a low-flow perfusion. Ischemic preconditioning without intermittent reperfusion is a controversial issue. In anesthetized pigs, infarct size after a 60-minute coronary artery occlusion was reduced by a preceding 30-minute period of partial coronary stenosis, enough to decrease coronary blood flow by 70%, when compared with pigs undergoing only 60 minutes of coronary artery occlusion.²⁹ In contrast, in anesthetized dogs in the absence of intermittent reperfusion, 15 minutes of partial coronary stenosis did not reduce infarct size after subsequent coronary artery occlusion for 1 hour.³⁰ In isolated buffer-perfused rabbit hearts, 10 minutes of global hypoxia decreased infarct size after a subsequent 30-minute period of regional no-flow ischemia, indicating that intermittent reoxygenation is not required for ischemic preconditioning.³¹ In our study, 10 minutes of no-flow ischemia resulted in a long-lasting (at least 4 hours) protection. The mechanism(s) of preconditioning-mediated protection are not yet fully understood. It has been suggested that activation of protein kinase C mediated by either adenosine release³² or activation of ATP-dependent potassium channels³³ might be involved. Alternatively, preconditioning might increase the resistance of the myocardium to oxygen free radical–mediated damage³⁴ or reduce the rate of ATP consumption.³⁵ Usually, ischemic preconditioning is a short-lived phenomenon, with protective benefits waning within 2 hours. Recently, however, a delayed (24 hours) and perhaps longer-lasting second phase of protection has been described.³⁶ The mechanism(s) of this "second window" of preconditioning remain speculative.

We have not investigated all these mechanisms, as this was not the aim of our work. However, it is interesting to recall that in the hearts of group 3, oxidative stress did not occur and high-energy phosphates were maintained.

A Brief Period of Ischemia Permits the Development of Hibernation
In designing the experiment of group 3, we thought to study a sequence of ischemic events that may occur in some patients with reversible left ventricular dysfunction. Although the data obtained with isolated and perfused rabbit hearts cannot be transferred to the clinical condition, it is at least tempting to speculate that in the isolated heart, an initial stimulus of severe ischemia is required to permit the development of a condition similar to that occurring during myocardial hibernation. In experimental hibernation, myocardial CP content, after an initial reduction, recovers toward control values,³⁷ lactate production is reduced over time,³⁸ the ischemia-induced metabolic alterations are attenuated,³⁹ and there is a recovery of function on reperfusion.⁴⁰ The hearts of group 3 fulfill all these criteria. These features, however, are also typical of the ischemic preconditioned heart, raising the possibility of a link between the two phenomena. We believe that these data mimic, although in an extremely simplified form, a sequence of events likely to occur in coronary artery disease patients independent of whether this should be named "hibernation" or "preconditioning." These terms are a useful shorthand but might also cause confusion, especially considering that strict definitions do not apply to the extremely complex situation arising from myocardial ischemia in humans.

Limitations of the Study
Our experiments were performed in isolated and buffer-perfused rabbit hearts. This model allows the study of many different aspects of heart metabolism and to precisely control degree and composition of coronary flow, heart rate, temperature, and myocardial function in the absence of systemic and neuroendocrine changes. The data obtained from this model, however, have several limitations and cannot be extrapolated to the in situ heart. For instance, oxygen content of the buffer perfusate is low, with consequent high coronary flow. Glucose is usually the sole energy source. Therefore, the relative importance of glycolysis may influence the metabolic changes during ischemia. Because free fatty acids are quite harmful by accumulation of toxic derivatives, the absence of free fatty acids might decrease ischemia-reperfusion injury. Furthermore, the absence of cells from crystalloid buffer eliminates the influence of leukocyte- and platelet-mediated injury during ischemia and reperfusion.

We measured interstitial pH by means of a pH-sensitive electrode. Although interstitial pH correlates well with ischemic left ventricular dysfunction,⁴¹ it does not provide information about the changes in intracellular or cytosolic pH. Intracellular pH can be monitored by nuclear magnetic resonance techniques, which also offer the advantage of providing continuous information on high-energy phosphates and inorganic phosphate, which might be involved in the downregulation of contraction. Data on intracellular pH obtained with ³¹P NMR spectroscopy in rats subjected to an identical protocol proved similar to those reported here.²⁴

Coronary flow was reduced and kept constant as long as 4 hours. This is unlikely to occur in the ischemic myocardium in vivo, where coronary flow is expected to fluctuate and myocytes to be exposed to different, repetitive sequences of no perfusion, hypoperfusion, and normal perfusion. Under these circumstances, hibernation may be the result of repetitive stunning.

Despite all limitations, we hope that the results of a study like this will provide clues for other studies in larger animal models that may provide insights into mechanisms potentially common to myocardial preconditioning and hibernation.

Conclusions
In the isolated perfused rabbit heart, the sequence of coronary flow restriction strongly affects recovery on reperfusion. A brief period of no-flow ischemia is important to maintain viability despite severe low-flow ischemia for 4 hours. The cardioprotective effect may be the result of a resetting of cardiac metabolism with downregulation of both energy need and production or of ischemic preconditioning without intermittent reperfusion or both. Similar sequences of coronary flow restriction might occur during development of hibernation.

	Selected Abbreviations and Acronyms

 

CP = creatine phosphate CPK = creatine phosphokinase GSH = reduced glutathione GSSG = oxidized glutathione SH = sulfhydryl

	Acknowledgments

 
This work was supported by the National Research Council (C.N.R.) targeted project "Prevention and Control Disease Factors" No. 93.00656. PF 41/115 and the "New Ischaemic Syndromes" European Commission project No. PL 95/0838. The authors thank Alessandro Colizzi and Roberta Bonetti for editorial and secretarial assistance in preparing the manuscript.

Received December 28, 1995; revision received June 5, 1996; accepted June 10, 1996.

	References

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