Preconditioning Reduces Infarct Size Research Articles

Myocardial triggers involved in activation of remote ischaemic preconditioning.

What is the central question of this study? Ischaemia-reperfusion of peripheral tissues protects the heart from subsequent myocardial ischaemia-reperfusion injury, a phenomenon referred to as remote ischaemic preconditioning (rIPC). This study evaluated the possible myocardial triggers of rIPC. What is the main finding and its importance? Remote ischaemic preconditioning reduces infarct size through a vagal pathway and a mechanism involving phosphorylation of Akt and endothelial nitric oxide synthase, opening of mitochondrial ATP-dependent K(+) channels and an increase in mitochondrial H2 O2 production. All these phenomena occur before the myocardial ischaemia; hence, they could act as 'triggers' of rIPC. It has been proposed that remote ischaemic preconditioning (rIPC) activates a parasympathetic neural pathway. However, the myocardial intracellular mechanism of rIPC remains unclear. Here, we characterized some of the intracellular signals participating as rIPC triggers. Isolated rat hearts were subjected to 30min of global ischaemia and 120min of reperfusion (Non-rIPC group). In a second group, before the isolation of the heart, an rIPC protocol (three cycles of hindlimb ischaemia-reperfusion) was performed. The infarct size was measured with tetrazolium staining. Expression/phosphorylation of Akt and endothelial nitric oxide synthase (eNOS) and mitochondrial H2 O2 production were evaluated at the end of the rIPC protocol, before myocardial ischaemia-reperfusion. The rIPC significantly decreased the infarct size and induced Akt and eNOS phosphorylation. The protective effect on infarct size was abolished by cervical vagal section, l-NAME (an NO synthesis inhibitor) and 5-hydroxydecanoate (a mitochondrial ATP-dependent K(+) channel blocker). Mitochondrial production of H2 O2 was increased by rIPC, whereas it was abolished by cervical vagal section, l-NAME and 5-hydroxydecanoate. We conclude that rIPC activates a parasympathetic vagal pathway and a mechanism involving the phosphorylation of Akt and eNOS, the opening of mitochondrial ATP-dependent K(+) channels and the release of H2 O2 by the mitochondria. All these phenomena occur before myocardial ischaemia and could act as triggers of rIPC.

Human adaptation to ischemia by preconditioning or unstable angina: involvement of nuclear factor kappa B, but not hypoxia-inducible factor 1 alpha in the heart

Ischemic preconditioning reduces infarct size and improves hemodynamic function. Unstable angina may be a clinical analogue to ischemic preconditioning, and involve activation of gene programs. We hypothesized that preceding unstable angina and/or ischemic preconditioning activated genes regulated by nuclear factor kappa B (NFkappaB) or hypoxia-inducible factor 1 alpha in parallel to improved cardiac function. Patients undergoing coronary artery bypass grafting with stable or unstable angina were subjected to ischemic preconditioning or sham treatment (n=10-11 in each group). Central hemodynamics were monitored. Left ventricular and atrial biopsies were harvested before cardioplegic arrest and after 25 min of reperfusion. Expression of heat shock protein 72 was evaluated by immunoblot, and activation of NFkappaB was detected by electrophoretic mobility shift assay. Real-time PCR was used to quantify expression of genes regulated by NFkappaB (inducible nitric oxide synthase, tumor necrosis factor-alpha, intercellular adhesion molecule 1) or by hypoxia-inducible factor 1 alpha (heme oxygenase-1, glucose transporter-1 and vascular endothelial growth factor-A). Ischemic preconditioning improved postoperative cardiac index and left ventricular stroke work index in both stable and unstable groups on the first postoperative day. Expression of hypoxia-inducible factor 1 alpha regulated genes was not influenced by cardioplegia and reperfusion, ischemic preconditioning or unstable angina. Expression of the NFkappaB-regulated genes increased after cardioplegia and reperfusion, but this was not influenced by ischemic preconditioning in stable patients. Inducible nitric oxide synthase and tumor necrosis factor expression were reduced after ischemic preconditioning in patients with unstable angina. There were no significant differences in gene expression between stable and unstable patients before cardioplegia and ischemic preconditioning. NFkappaB translocation at reperfusion was reduced in stable preconditioned and unstable control patients compared to stable controls. Heat shock protein 72 expression increased after preconditioning of unstable patients. Cardiac function was improved by ischemic preconditioning in both stable and unstable patients. Unstable angina per se had no effect. NFkappaB-regulated genes were influenced by ischemic preconditioning, but hypoxia-inducible genes were not.

Volatile Anesthetic Preconditioning Attenuates Myocardial Apoptosis in Rabbits after Regional Ischemia and Reperfusion via Akt Signaling and Modulation of Bcl-2 Family Proteins

We tested whether isoflurane preconditioning inhibits cardiomyocyte apoptosis and evaluated the role of the phosphatidylinositol-3-kinase (PI3K)/Akt pathway in anesthetic preconditioning and determined whether PI3K/Akt signaling modulates the expression of pro- and antiapoptotic proteins in anesthetic preconditioning. Six-month-old New Zealand rabbits subjected to 40 min of myocardial ischemia followed by 180 min of reperfusion were assigned to the following groups: ischemia-reperfusion (I/R), isoflurane preconditioning and isoflurane plus PI3K inhibitors, wortmannin and 2-(4-morpholinyl)-8-phenyl-4H-l-benzopyran-4-one (LY294002) (0.6 and 0.3 mg/kg i.v., respectively). Sham-operated, wortmannin+I/R, wortmannin+sham, LY294002+I/R, and LY294002+sham groups were also included. Infarct size was assessed by triphenyltetrazolium chloride staining. Apoptosis was evaluated by terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling and activated caspase-3 assays. Akt phosphorylation, Bax, Bcl-2, Bad, and phosphorylated Bad (phospho-Bad) expression was assessed by immunoblotting. Isoflurane preconditioning reduced infarct size compared with the I/R group: 22+/-4 versus 41+/-5% (p<0.05). The percentage of apoptotic cells decreased in the isoflurane group (3.8+/-1.2%) compared with the I/R group (12.4+/-1.6%; p<0.05). These results were also confirmed by the activated caspase-3 assay. Wortmannin and LY294002 inhibited the effects of isoflurane. Myocardial infarction increased to 44+/-3 and 45+/-2% and the percentage of apoptotic cells was 11.9+/-2.1 and 11.7+/-3.3%, respectively. Akt phosphorylation and Bcl-2 and phospho-Bad expression increased after isoflurane preconditioning, whereas Bax expression decreased. These effects were inhibited by wortmannin and LY294002. The data indicate that isoflurane preconditioning reduces infarct size and myocardial apoptosis after I/R. Activation of PI3K and modulation of the expression of pro- and antiapoptotic proteins may play a role in isoflurane-induced myocardial protection.

How to use the paradigm of ischemic preconditioning to protect the heart?

Ischemic preconditioning affords the most powerful protection to a heart submitted to a prolonged ischemia-reperfusion. During the past decade, a huge amount of work allowed to better understand the features of this protective effect as well as the molecular mechanisms. Ischemic preconditioning reduces infarct size and improves functional recovery; its effects on arrhythmias remain debated. Triggering of the protection involves cell surface receptors that activate pro-survival pathways including protein kinase C, PI3-kinase, possibly Akt and ERK1/2, whose downstream targets remain to be determined. Much attention has been recently focused on the role of mitochondrial K(+)ATP channels and the permeability transition pore that seem to play a major role in the progression toward irreversible cellular injury. Based on these experimental studies attempts have been made to transfer preconditioning from bench to bedside. Human experimental models of ischemic preconditioning have been set up, including cardiac surgery, coronary angioplasty or treadmill exercise, to perform pathophysiological studies. Yet, protecting the heart of CAD (coronary artery disease) patients requires a pharmacological approach. The IONA trial has been an example of the clinical utility of preconditioning. It helped to demonstrate that chronic administration of nicorandil, a K(+)ATP opener that mimics ischemic preconditioning in experimental preparations, improves the cardiovascular prognosis in CAD patients. Recent experimental studies appear further encouraging. It appears that "postconditioning" the heart (i.e. performing brief episodes of ischemia-reperfusion at the time of reperfusion) is as protective as preconditioning. In other words, a therapeutic intervention performed as late as at the time of reflow can still significantly limit infarct size. Further work is needed to determine whether this may be transferred to the clinical practice.

The role of ADAM protease in the tyrosine kinase-mediated trigger mechanism of ischemic preconditioning

The aim of this study was to determine the role of an a disintegrin and metalloprotease (ADAM) in tyrosine kinase-mediated mechanisms of ischemic preconditioning (PC). In isolated rabbit hearts, PC was performed with two cycles of 5 min ischemia/5 min reperfusion and infarction was induced by 30 min global ischemia/2 h reperfusion. Translocation of protein kinase C- (PKC-) and tyrosine phosphorylation in the tissue and TNF-alpha in coronary effluent were determined by immunoblotting. PC reduced infarct size from 55.1+/-6.8% of the left ventricle to 24.4+/-5.2%, and this protection was mimicked by pretreatment with 100 nM angiotensin II. Both the PC effect and angiotensin II-induced protection were abolished by genistein and by 10 microM KB-R7785 (KBR), an inhibitor of ADAM12/17, but not by a lower ADAM12-selective dose (1 microM) of KBR. AG1478, an inhibitor of EGF receptor tyrosine kinase, did not inhibit protection afforded by PC. PC provoked release of TNF-alpha into the coronary effluent, which was abolished by 10 microM KBR but not by 1 microM of KBR or calphostin C, a PKC inhibitor. PKC- translocation by PC was not affected by KBR. PC induced tyrosine phosphorylation of 60 and 90 kDa proteins, and this phosphorylation was abolished by 10 microM KBR but not by calphostin C. Pretreatment with TNF-alpha limited infarct size to 16.7+/-3.7% and induced tyrosine phosphorylation of a 60 kDa protein. The results support the hypothesis that an ADAM contributes to the triggering of a tyrosine kinase-mediated and PKC-independent pathway of PC. The ADAM responsible for this tyrosine kinase-mediated pathway is likely to be ADAM17, which sheds TNF-alpha.

Is microvascular protection by cariporide and ischemic preconditioning causally linked to myocardial salvage?

Two independent cardioprotective interventions, Na(+)/H(+) exchange inhibition and ischemic preconditioning (PC), were investigated with respect to differential effects on microvascular and myocardial salvage in anesthetized rabbits (30 min of ischemia, 180 min of reperfusion). Cariporide (Car, 300 microg/kg) administered before occlusion and PC reduced infarct size (IS) as measured by triphenyltetrazolium staining [control, 46.0 +/- 4.2% of risk area (RA); Car, 17.6 +/- 3.7% (P < 0.01); PC, 27.5 +/- 4.1% (P < 0.01)] and concomitantly decreased the area of anatomic no reflow (ANR) as measured by thioflavin S staining [control, 40.4 +/- 3.7%; Car, 19.0 +/- 2.9% (P < 0.01); PC, 26.9 +/- 3.4% (P < 0.05)]. Regional myocardial blood flow (RMBF, measured by radioactive microspheres) in the RA, which deteriorated between 30 and 180 min of reperfusion (control, from 79 +/- 6 to 26 +/- 2% of nonischemic flow), was shifted to higher values with both treatments [Car, from 110 +/- 12 to 49 +/- 7% (P < 0.05); PC, from 109 +/- 8 to 38 +/- 6% (P < 0.05)]. However, neither intervention uncoupled the close relationship between IS and ANR (r = 0.92-0.95) or RMBF. Car given at reperfusion did not alter IS, ANR, RMBF, or the close interrelationships. Because size and spatial distribution of no reflow and myocardial necrosis remained closely coupled with independent cardioprotective interventions, a potential causal connection between microvascular and myocardial salvage is discussed.

Expression of Active α1B-Adrenergic Receptors in the Heart does not Alleviate Ischemic Reperfusion Injury

X.-M. Gao, B.-H. Wang, E. Woodcock and X.-J. Du. Expression of Activeα1B -Adrenergic Receptors in the Heart does not Alleviate Ischemic Reperfusion Injury. Journal of Molecular and Cellular Cardiology (2000) 32, 1679–1686. Ischemic preconditioning reduces infarct size and improves cardiac function in various species, including mice. The mechanism for ischemic preconditioning protection is not entirely clear and activation of α1B-adrenergic receptors (AR) is believed to be involved. Transgenic mice expressing constitutively active mutant α1B-AR in the heart have enhanced α1B-AR activity and therefore can be used to test the role of α1B-AR in ischemic preconditioning. Wild-type and transgenic mice were subjected to 30- or 40-min periods of left coronary artery occlusion followed by 60-min reperfusion, or ischemic preconditioning prior to sustained ischemia–reperfusion. Risk and infarct zones were determined by staining with Evans blue and triphenyltetrazolium, respectively, and quantitated digitally. Infarct zone and infarct size were not different between wild-type and transgenic mice, nor was the extent of reduction in infarct size by preconditioning ischemia (wild-type mice: 45±3 to 18±3%, transgenic mice: 46±3 to 19±2% of the left ventricle, bothP <0.01). Ventricular function was similar between wild-type and transgenic mice with or without ischemia–reperfusion injury. In conclusion, enhanced α1B-AR activity by cardiac-specific expression of constitutively active mutant α1B-AR in mice does not mimic ischemic preconditioning to protect against ischemia–reperfusion injury.

Smaller infarct after preconditioning does not predict extent of early functional improvement of reperfused heart.

We evaluated the ability of ischemic preconditioning to restore function to salvaged myocardium in rabbits. Although ischemic preconditioning reduces infarct size, few investigators studying recovery of function after coronary occlusions lasting >/=30 min have reported any mechanical benefit in preconditioned hearts. However, because myocardial function was seldom evaluated beyond 5 h after reperfusion stunning may have masked the benefit. Accordingly, rabbits were chronically instrumented with a pneumatic occluder around a branch of the left coronary artery, a pair of 1-mm ultrasonic crystals in the myocardial territory destined to become ischemic, and electrocardiogram (ECG) leads. One week after surgery the ECG and segment length tracing were recorded at rest, during 30-min occlusion and 1 h of reflow, and again at 24, 48, and 72 h. In ischemically preconditioned rabbits, 5-min coronary occlusion and 10-min reperfusion preceded the long occlusion. The beginning and end of systole were determined by recording the first and second heart sounds with a hand-held precordial microphone. Postmortem infarct size was measured with triphenyltetrazolium chloride. During the 30-min coronary occlusion all segments became nearly akinetic or bulged during systole. After 60 min of reflow there was little return of function in either group. Between 24 and 72 h there was minimal recovery in the control group (segment shortening equals 13.3 +/- 4.1% of baseline), whereas function was much better in preconditioned hearts (44.2 +/- 7.4% of baseline, P < 0.02). Infarct size as a percentage of risk zone was much smaller in preconditioned hearts (10.2 +/- 1.4 vs. 29.7 +/- 1.8%, P < 0.001). Thus there is a gradual recovery of systolic function of reperfused myocardium after a coronary occlusion. Although early mechanical recovery is significantly better after preconditioning, it is much less than would be predicted by the reduction of infarct size.

Preconditioning does not attenuate cardiac dysfunction after global ischaemia in the guinea-pig.

Ischaemic preconditioning reduces infarct size, but the effects on cardiac function after global ischaemia are more controversial. Additionally, species differences may exist. The present study investigates the effects of preconditioning on cardiac performance in the globally ischaemic, Langendorff-perfused guinea-pig heart. Hearts were stabilized for 25 min, and divided into the following groups: (1) (n = 8) control perfusion for 16 min before 30-min global ischaemia and 30-min reperfusion, (2) (n = 7) two episodes of 3-min ischaemia and 5-min reperfusion before global ischaemia, (3) (n = 7) 5-min ischaemia and 10-min reperfusion before ischaemia, (4) (n = 8) control perfusion before 40-min ischaemia and 30-min reperfusion, (5) (n = 8) Preconditioning as group 2 before ischaemia as group 4, (6) (n = 9) Control perfusion before 50-min ischaemia and 30-min reperfusion, (7) (n = 10) Preconditioning as group 2 before ischaemia as group 6. A dose-dependent reduction of left ventricular systolic pressure, and increase of end-diastolic pressure was observed during reperfusion after 30-, 40- and 50-min ischaemia. Preconditioning did not influence these changes, nor did it attenuate the incidence of severe reperfusion arrhythmias or reduction of coronary flow. In conclusion, ischaemic preconditioning does not improve cardiac function during reperfusion of the globally ischaemic, isolated guinea-pig heart.

Preconditioning improves cardiac function after global ischemia, but not after cold cardioplegia

Ischemic preconditioning reduces infarct size and cardiac dysfunction during reperfusion. Preconditioning may offer myocardial protection in open heart operations. The effect of preconditioning before ischemia and cardioplegia was investigated in Langendorff-perfused rat hearts in the following groups. First, group 1 received two episodes of 3-minute ischemia and 5-minute reperfusion before 25 minutes of global (37 degrees C) ischemia and 60 minutes of reperfusion. Group 2 served as ischemic controls to group 1. Groups 3, 5, and 7 were preconditioned as described, before 3.5, 4, or 5 hours of cold (6 degrees to 8 degrees C) St. Thomas' II cardioplegia and 1 hour of reperfusion (37 degrees C). Groups 4, 6, and 8 were cardioplegic controls to groups 3, 5, and 7 (n = 17 in groups 1 and 2, and n = 10 in groups 3 to 8). Preconditioning before warm ischemia attenuated the ischemia-induced increase of left ventricular end-diastolic pressure (3 +/- 1 versus 17 +/- 4 mm Hg; p < 0.01) (mean +/- standard error of the mean), the reduction of coronary flow (14 +/- 1 versus 9 +/- 0.5 mL/min; p < 0.001) and heart rate (252 +/- 19 versus 198 +/- 18 beats/min; p < 0.04), and the incidence of ventricular fibrillation (2 of 17 versus 10 of 17 hearts; p < 0.04) at the start of reperfusion. However, preconditioning did not influence postischemic cardiac function or the release of lactate dehydrogenase in any of the cardioplegia groups. Ischemic preconditioning improved post-ischemic cardiac function after warm global ischemia, but did not protect cold cardioplegic hearts, perhaps because of the time span used.

Reduction in infarct size by ischemic preconditioning persists in a chronic rat model of myocardial ischemia-reperfusion injury.

Ischemic preconditioning (PC) has been consistently observed to reduce infarct size in models of regional myocardial ischemia. However, it is also known to render the heart resistant to injury for only a finite period of time (< 2 h). Myocardial adenosine is widely believed to be one of the mediators of PC and may produce myoprotection in part through an anti-neutrophil effect during the early reperfusion period. When infarct size is assessed following a relatively short period of reperfusion (< 3 h) PC hearts may appear protected although reperfusion injury in the myocardium may be ongoing. Thus, infarct expansion may occur as the effects of PC fade. To substantiate that PC produces a sustained reduction in myocardial necrosis, 27 male Sprague-Dawley rats were anesthetized with pentobarbital and instrumented for regional coronary occlusion (30 min) and reperfusion (7 days). Animals were randomized to a control group (n = 16) or PC (n = 11), which consisted of 2 cycles of 5 min of ischemia and 5 min of reperfusion immediately prior to coronary occlusion. Successful reperfusion was confirmed visually and the occluding suture was left in the chest during recovery. Seven days later, staining for risk area was made by the injection of Evans blue dye while the occluder was in place and necrosis was detected with triphenyltetrazolium chloride staining. Planimetry was performed by a blinded investigator who found the risk area to be 27.2 +/- 1.6 and 33.6 +/- 1.7% of the left ventricle (p = NS) in PC and controls, respectively. All hemodynamic measurements were comparable between groups at all times during ischemia and reperfusion. PC reduced infarct size from 43.3 +/- 2.0% of area at risk to 20.6 +/- -2.1%, a 48% reduction (p < 0.01), and eliminated transmural necrosis which was common in the control group. From these studies we conclude that ischemic PC results in a permanent reduction in infarct size rather than a transient reduction in infarct size in the context of a gradually evolving infarction due to reperfusion injury.

Different effects of an adenosine A 1 analogue and ischemic preconditioning in isolated rabbit hearts

Ischemic preconditioning reduces infarct size, but its effects on postischemic function are variable. Adenosine, which is thought to play a role in ischemic preconditioning, reduces both infarct size and postischemic dysfunction. The purpose of this study was to compare the cardioprotective effects of ischemic preconditioning and an adenosine A1 receptor agonist on recovery of function and infarct size in isolated rabbit hearts. Krebs buffer-perfused hearts (at least 7 per group) were subjected to 60 minutes of global ischemia (37 degrees C) and 60 minutes of reperfusion. Ventricular function was assessed by measuring left ventricular developed pressure, and infarct size (percentage of the left ventricle) was determined by tetrazolium staining. Control hearts exhibited 34% +/- 6% infarct size and 56% +/- 4% recovery of preischemic left ventricular developed pressure. Ischemic preconditioning reduced infarct size to 13% +/- 1% but had no effect on recovery of function (65% +/- 5%). Hearts treated with the adenosine A1 agonist R-phenylisopropyladenosine for 5 minutes immediately before ischemia exhibited both reduced infarct size (10% +/- 2%) and enhanced postischemic recovery of left ventricular developed pressure (86% +/- 3%). Termination of the R-phenylisopropyladenosine treatment before ischemia eliminated its beneficial effects. The adenosine A1 receptor antagonist DPCPX blocked both of the effects of R-phenylisopropyladenosine but did not block ischemic preconditioning. These results demonstrate fundamental differences between the cardioprotective effects of adenosine A1 receptor activation and ischemic preconditioning.

Loss of myocardial protection after preconditioning correlates with the time course of glycogen recovery within the preconditioned segment.

Although previous investigators have demonstrated that myocardial preconditioning reduces infarct size, the mechanisms of cardioprotection associated with preconditioning are not completely understood. To test the hypothesis that preconditioning (four 5-minute episodes of ischemia each followed by 5 minutes of reperfusion) reduces infarct size by depleting cardiac glycogen stores and attenuating the degree of intracellular acidosis during subsequent prolonged left coronary artery occlusion, preconditioned and control rats were subjected to 45 minutes of left coronary artery occlusion and 120 minutes of reflow immediately after preconditioning (groups 1P and 1C, respectively) or after 30 minutes (groups 2P+30m and 2C), 1 hour (groups 3P+60m and 3C), or 6 hours (groups 4P+360m and 4C) of nonischemic recovery after preconditioning but before prolonged ischemia. In each group, cardiectomy was performed in selected rats immediately before prolonged ischemia for cardiac glycogen assay. In selected animals, 31P magnetic resonance spectroscopy was performed to monitor intracellular pH and measure high-energy phosphate levels during ischemia and reperfusion. Group 1P rats demonstrated marked glycogen depletion after preconditioning compared with controls (0.72 +/- 0.39 [n = 9] versus 5.67 +/- 1.73 [n = 12] mg glucose/g wet wt; p < 0.001 versus group 1C) that was associated with attenuation of intracellular acidosis during ischemia, as measured by 31P magnetic resonance spectroscopy (6.8 +/- 0.3 [n = 11] versus 6.2 +/- 0.3 [n = 9] pH units; p < 0.01), and marked infarct size reduction (0.3 +/- 0.6% [n = 7] versus 38.1 +/- 11.3% [n = 7], infarct size divided by risk area; p < 0.0001). During ischemia, there were no differences in myocardial ATP or phosphocreatine levels or in any hemodynamic determinant of myocardial oxygen demand between groups 1P and 1C. In preconditioned rats that were allowed to recover before ischemia (groups 2P+30m, 3P+60m, and 4P+360m), the time course of glycogen repletion paralleled the loss of protection from ischemic injury. Glycogen depletion and the attenuation of intracellular acidosis during ischemia appear to be important factors in delaying irreversible injury and reducing infarct size in this animal model of myocardial preconditioning.

Ischemic preconditioning reduces infarct size in swine myocardium.

We evaluated the hypothesis that stunning swine myocardium with brief ischemia reduces oxygen demand in the stunned region and increases tolerance of myocardium to longer periods of ischemia. Wall function was quantified with ultrasonic crystals aligned to measure wall thickening, and stunning was achieved with two cycles of left anterior descending coronary artery (LAD) occlusion (10 minutes) and reperfusion (30 minutes), after which the LAD was occluded for 60 minutes and reperfused for 90 minutes. Infarct size (as a percent of risk region) was then determined by incubating myocardium with para-nitro blue tetrazolium. Regional oxygen demand was measured as myocardial oxygen consumption before the 60-minute LAD occlusion in the stunned region; tracer microspheres were used to determine blood flow, and blood from the anterior interventricular vein and left atrium was used to calculate oxygen saturations. After the second reperfusion period, wall thickening in the stunned region was reduced to 1.4 +/- 2.4% compared with 36.7 +/- 2.5% (mean +/- SEM) before ischemia (p less than 0.001). Regional myocardial oxygen consumption after stunning (3.1 +/- 0.7 ml O2/min/100 g) was no different from regional myocardial oxygen consumption before stunning (3.7 +/- 0.6 ml O2/min/100 g). In the nine pigs "preconditioned" by stunning, infarct size was 10.4 +/- 6.3% of the risk region compared with 48.0 +/- 12.7% in the six control pigs subjected to 60 minutes of ischemia without prior stunning (p less than 0.005). The risk regions were similar (14.4 +/- 1.5% vs. 14.6 +/- 1.9% of the left ventricle, preconditioned vs. control pigs, respectively). We conclude that stunning swine myocardium with two cycles of a 10-minute LAD occlusion followed by reperfusion increases ischemic tolerance but that changes in regional demand in stunned myocardium do not predict the marked reduction in infarct size that follows a subsequent 60-minute period of ischemia.