9th Hatter Biannual Meeting: position document on ischaemia/reperfusion injury, conditioning and the ten commandments of cardioprotection.

In the 30 years since the original description of ischaemic preconditioning, understanding of the pathophysiology of ischaemia/reperfusion injury and concepts of cardioprotection have been revolutionised. In the same period of time, management of patients with coronary artery disease has also been transformed: coronary artery and valve surgery are now deemed routine with generally excellent outcomes, and the management of acute coronary syndromes has seen decade on decade reductions in cardiovascular mortality. Nonetheless, despite these improvements, cardiovascular disease and ischaemic heart disease in particular, remain the leading cause of death and a significant cause of long-term morbidity (with a concomitant increase in the incidence of heart failure) worldwide. The need for effective cardioprotective strategies has never been so pressing. However, despite unequivocal evidence of the existence of ischaemia/reperfusion in animal models providing a robust rationale for study in man, recent phase 3 clinical trials studying a variety of cardioprotective strategies in cardiac surgery and acute ST-elevation myocardial infarction have provided mixed results. The investigators meeting at the Hatter Cardiovascular Institute workshop describe the challenge of translating strong pre-clinical data into effective clinical intervention strategies in patients in whom effective medical therapy is already altering the pathophysiology of ischaemia/reperfusion injury-and lay out a clearly defined framework for future basic and clinical research to improve the chances of successful translation of strong pre-clinical interventions in man.

Attenuation of oxidant stress during reoxygenation by AMP 579 in cardiomyocytes.

AMP 579, an adenosine A(1)/A(2) receptor agonist, has a strong anti-infarct effect when administered just before reperfusion. Because oxidative stress has been proposed to contribute to myocardial reperfusion injury, we tested whether AMP 579 can reduce the production of reactive oxidant species (ROS) during reoxygenation in cultured chick embryonic cardiomyocytes. The intracellular fluorescent probe 2',7'-dichlorofluorescin diacetate (DCFH) was used to detect ROS. The cells were subjected to 60 min of simulated ischemia, followed by either 15 min or 3 h of reoxygenation. AMP 579 (0.5 and 1 microM), when started 10 min before reoxygenation, significantly reduced ROS generation from 4.86 +/- 0.30 (arbitrary units) in untreated cells to 2.72 +/- 0.31 and 1.85 +/- 0.14, respectively (P < 0.05). Cell death that was assessed by propidium iodide uptake was markedly reduced by AMP 579 (49.6 +/- 4.7% of control cells vs. 25.4 +/- 2.4%, P < 0.05). In contrast, adenosine did not alter ROS generation or cell death. Attenuation of ROS production by AMP 579 was completely prevented by simultaneous exposure of cells to the selective adenosine A(2) antagonist 8-(13-chlorostyryl) caffeine. These results indicate that AMP 579 directly protects cardiomyocytes from reperfusion injury by a mechanism that attenuates intracellular oxidant stress. Furthermore, adenosine could not duplicate these effects.

Acute alcohol-induced protection against infarction in rabbit hearts: differences from and similarities to ischemic preconditioning.

Recent studies reveal that brief ethanol exposure induces cardioprotection against simulated ischemia in cardiomyocytes by the activation of protein kinase C- epsilon. The present study tests the ability of ethanol to induce protection in rabbit hearts in which infarct size was the end-point and explores the signal transduction pathways involved. In isolated rabbit hearts, 50 m m ethanol infused for 5 min with 10 min of washout prior to 30 min of regional ischemia reduced infarct size (triphenyltetrazolium chloride staining) by 49%. Neither adenosine receptor blockade with 8-(p -sulfophenyl) theophylline nor the free radical scavenger N-2-mercaptopropionyl glycine inhibited the protection triggered by ethanol. In contrast, protein kinase C inhibition with chelerythrine, protein tyrosine kinase inhibition with genistein, and blockade of ATP-sensitive potassium channels (K(ATP)) with either 5-hydroxydecanoate or glibenclamide did abolish protection. Thus, transient ethanol exposure followed by washout prior to ischemia elicits a preconditioning-like effect involving protein kinase C, at least one protein tyrosine kinase, and K(ATP)channels, but neither adenosine nor free radicals.

Amp 579 reduces contracture and limits infarction in rabbit heart by activating adenosine A2 receptors.

To determine the mechanism by which AMP 579, an adenosine A1/A2 agonist, administered at reperfusion protects ischemic myocardium, buffer-perfused rabbit hearts were subjected to 30 min of global ischemia and 2 h of reperfusion. AMP 579 (500 nM) was included in the reperfusate for the first 70 min. Average left ventricular diastolic pressure during reperfusion in hearts receiving AMP 579 was lower than that in control hearts (17.9 +/- 2.4 vs. 39.0 +/- 6.5 mm Hg, p < 0.05), indicating attenuation of contracture. Left ventricular developed pressure and coronary flow during reperfusion were also significantly improved with AMP 579 treatment. AMP 579's anti-contracture effect was blocked by the adenosine A2-receptor antagonist 8-(3-chlorostyryl)caffeine (CSC), but not by the A1 antagonist 8-cyclopentyl-1,3-dipropylxanthine (DPCPX). CSC, but not DPCPX, also blocked AMP 579's ability to preserve developed pressure and coronary flow in these hearts. AMP 579 significantly reduced infarction in isolated hearts subjected to regional ischemia. The anti-infarct effect again was abolished by CSC but not by DPCPX. Finally, we tested whether 5'-(N-ethylcarboxamido)adenosine (NECA), another A1/A2 agonist, also administered for the initial 70 min of reperfusion, could duplicate the anti-infarct effect of AMP 579. One-hundred-nanomolar NECA duplicated the protection, but neither 50 nM CGS21680, a selective A2 agonist, nor 100 microM adenosine was protective. Therefore, AMP 579 given at reperfusion reduces contracture and infarction. Anti-contracture and anti-infarct effects require the adenosine A2, but not the A1, receptor suggesting that prevention of contracture and tissue salvage are mechanistically related. Not all A2 agonists were able to duplicate the anti-infarct effect, suggesting something unique about AMP579.

Acetylcholine, bradykinin, opioids, and phenylephrine, but not adenosine, trigger preconditioning by generating free radicals and opening mitochondrial K(ATP) channels.

It has been assumed that all G(i)-coupled receptors trigger the protective action of preconditioning by means of an identical intracellular signaling pathway. To test this assumption, rabbit hearts were isolated and perfused with Krebs buffer. All hearts were subjected to a 30-minute coronary artery occlusion followed by 120 minutes of reperfusion. Risk area was measured with fluorescent particles and infarct size with triphenyltetrazolium chloride staining. Control hearts showed 29.1+/-2.8% infarction of the risk zone. A 5-minute infusion of acetylcholine (0.55 mmol/L) beginning 15 minutes before the 30-minute occlusion resulted in significant protection (9.2+/-2.7% infarction). This protection could be blocked by administration of 300 micromol/L N-2-mercaptopropionyl glycine (MPG), a free radical scavenger, or by 200 micromol/L 5-hydroxydecanoate (5-HD), a mitochondrial K(ATP) antagonist, for 15 minutes beginning 5 minutes before the acetylcholine infusion (35.2+/-3.9% and 27.8+/-2.4% infarction, respectively). Similar protection was observed with other known triggers, ie, bradykinin (0.4 micromol/L), morphine (0.3 micromol/L), and phenylephrine (0.1 micromol/L), and in each case protection was completely abrogated by either MPG or 5-HD. In contrast, protection by adenosine or its analog N(6)-(2-phenylisopropyl) adenosine could not be blocked by either MPG or 5-HD. Therefore, whereas most of the tested agonists trigger protection by a pathway that requires opening of mitochondrial K(ATP) channels and production of free radicals, the protective action of adenosine is not dependent on either of these steps. Hence, it cannot be assumed that all G(i)-coupled receptors use the same signal transduction pathways to trigger preconditioning.

Limitation of infarct size in rabbit hearts by the novel adenosine receptor agonist AMP 579 administered at reperfusion.

The novel A(1)/A(2)adenosine receptor agonist AMP 579 has been reported to reduce myocardial infarct size in pig and dog. The present study tested the effect of AMP 579 in two rabbit models. In open-chest rabbits undergoing 30 min of regional ischemia and 3 h of reperfusion AMP 579 (3 microg/min/kg) reduced infarct size when treatment was started either 10 min before ischemia or 10 min prior to reperfusion from 36.4+/-3.1% of the risk zone in untreated hearts to 11.8+/-4.4 and 12.3+/-1.0%, respectively. To determine whether protection observed when the drug was administered shortly before reperfusion represented a long-lasting effect rather than merely a transient delay of necrosis, the chest wound was closed in layers and the rabbits permitted to recover. After 3 days the hearts were removed to evaluate infarct size. Continued limitation of infarct size after 3 days of reperfusion (8.2+/-2.8% of the risk zone) confirmed that sustained tissue salvage had been conferred by the drug. In isolated, buffer-perfused rabbit hearts undergoing 30 min of regional ischemia and 2 h of reperfusion, AMP 579 again limited infarct size (8.6+/-2.9% of the risk zone) when treatment started 10 min prior to reperfusion, arguing against an anti-leukocyte mechanism of protection. AMP 579's protective effect in this in vitro model was abrogated by 8-(p-sulfophenyl)theophylline, indicating that it was mediated through adenosine receptors. We conclude that AMP 579 given just prior to reperfusion may be an effective anti-infarct intervention.

Opening of mitochondrial K(ATP) channels triggers the preconditioned state by generating free radicals.

The critical time for opening mitochondrial (mito) K(ATP) channels, putative end effectors of ischemic preconditioning (PC), was examined. In isolated rabbit hearts 29+/-3% of risk zone infarcted after 30 minutes of regional ischemia. Ischemic PC or 5-minute exposure to 10 micromol/L diazoxide, a mito K(ATP) channel opener, reduced infarction to 3+/-1% and 8+/-1%, respectively. The mito K(ATP) channel closer 5-hydroxydecanoate (200 micromol/L), bracketing either 5-minute PC ischemia or diazoxide infusion, blocked protection (24+/-3 and 28+/-6% infarction, respectively). However, 5-hydroxydecanoate starting 5 minutes before long ischemia did not affect protection. Glibenclamide (5 micromol/L), another K(ATP) channel closer, blocked the protection by PC only when administered early. These data suggest that K(ATP) channel opening triggers protection but is not the final step. Five minutes of diazoxide followed by a 30-minute washout still reduced infarct size (8+/-3%), implying memory as seen with other PC triggers. The protection by diazoxide was not blocked by 5 micromol/L chelerythrine, a protein kinase C antagonist, given either to bracket diazoxide infusion or just before the index ischemia. Bracketing preischemic exposure to diazoxide with 50 micromol/L genistein, a tyrosine kinase antagonist, did not affect infarction, but genistein blocked the protection by diazoxide when administered shortly before the index ischemia. Thus, although it is not protein kinase C-dependent, the protection by diazoxide involves tyrosine kinase. Bracketing diazoxide perfusion with N:-(2-mercaptopropionyl) glycine (300 micromol/L) or Mn(III)tetrakis(4-benzoic acid) porphyrin chloride (7 micromol/L), each of which is a free radical scavenger, blocked protection, indicating that diazoxide triggers protection through free radicals. Therefore, mito K(ATP) channels are not the end effectors of protection, but rather their opening before ischemia generates free radicals that trigger entrance into a preconditioned state and activation of kinases.

Ischemic preconditioning: from basic mechanisms to clinical applications.

When the heart is subjected to a transient nonlethal period of ischemia, it quickly adapts itself to become resistant to infarction from a subsequent ischemic insult. This adaptation is called preconditioning. This cardioprotection has been shown to be mediated by stimulation of receptors linked to protein kinase C (PKC) (adenosine, bradykinin, opioids, etc.), and these receptors protect by activating PKC. PKC appears to be the first element of a complex kinase cascade that is activated during the prolonged ischemia in the preconditioned heart. Recent studies imply that p38 mitogen-activated protein kinase carries the signal from PKC to the mitochondrial K(ATP) channels, causing them to open and thus protect the heart. The cardioprotection of preconditioning occurs in all species tested to date, and possibly also humans. It is expected that as the mechanism of preconditioning is more thoroughly understood, pharmacological preconditioning will become practical for clinical use.

Ischemic preconditioning: from adenosine receptor to KATP channel.

Ischemic preconditioning is a phenomenon whereby exposure of the myocardium to a brief episode of ischemia and reperfusion markedly reduces tissue necrosis induced by a subsequent prolonged ischemia. It is hoped that elucidation of the mechanism for preconditioning will yield therapeutic strategies capable of reducing myocardial infarction. In the rabbit, the brief period of preconditioning ischemia and reperfusion releases adenosine, bradykinin, opioids, and oxygen radicals. The combined effect of the release of these substances on G proteins and the cell's phospholipases induces the translocation and activation of the epsilon isozyme of protein kinase C. Protein kinase C appears to be the first element of a complex kinase cascade that is activated during the prolonged ischemia in preconditioned hearts. Current evidence indicates that this cascade contains at least one tyrosine kinase and ultimately leads to the activation of p38 mitogen-activated protein kinase. p38 Mitogen-activated protein kinase phosphorylates mitogen-activated protein kinase-activated protein kinase 2. Mitogen-activated protein kinase-activated protein kinase 2 phosphorylates HSP27, a 27-kDa heat shock protein that controls actin filament polymerization, and, therefore, affects the integrity of the cytoskeleton. Finally, mitochondrial adenosine 5'-triphosphate-sensitive K+ channels open, and the latter may be the final mediator of protection for ischemic preconditioning. The protective pathway has many built-in redundancies, perhaps creating a safety factor. These redundancies may also explain some of the species-related differences seen in ischemic preconditioning in which one redundant pathway may predominate over another.

Ischemic preconditioning activates MAPKAPK2 in the isolated rabbit heart: evidence for involvement of p38 MAPK.

Recent studies suggest that p38 mitogen-activated protein kinase (MAPK) may be involved in ischemic preconditioning (PC). To further test this possibility, the regulation of MAPK-activated protein kinase 2 (MAPKAPK2), a kinase immediately downstream from p38 MAPK, and the activity of c-Jun NH(2)-terminal kinase (JNK), a second MAPK, were examined in preconditioned hearts. Isolated, perfused rabbit hearts were subjected to 20 to 30 minutes of global ischemia. Ventricular biopsies before treatment and after 20 minutes of ischemia were homogenized, and the activities of MAPKAPK2 and JNK were evaluated. For the MAPKAPK2 experiments, 7 groups were studied, as follows: control hearts; preconditioned hearts; hearts treated with 500 nmol/L R(-) N(6)-(2-phenylisopropyl) adenosine (PIA), an A(1)-adenosine receptor agonist; preconditioned hearts pretreated with 100 micromol/L 8-(p-sulfophenyl) theophylline (SPT), an adenosine receptor antagonist; preconditioned hearts also treated with SB 203580, a potent inhibitor of p38 MAPK activation; hearts treated with 50 ng/mL anisomycin (a p38 MAPK/JNK activator); and hearts treated with both anisomycin (50 ng/mL) and the tyrosine kinase inhibitor genistein (50 micromol/L). MAPKAPK2 activity was not altered in control hearts after 20 minutes of global ischemia. By contrast, there was a 3.8-fold increase in activity during ischemia in preconditioned hearts. Activation of MAPKAPK2 in preconditioned hearts was blocked by both SPT and SB 203580. MAPKAPK2 activity during ischemia increased 3.5-fold and 3.3-fold in hearts pretreated with PIA or anisomycin, respectively. MAPKAPK2 activation during ischemia in hearts pretreated with anisomycin was blocked by genistein. In separate hearts, anisomycin mimicked the anti-infarct effect of PC, and that protection was abolished by genistein. JNK activity was measured in control and preconditioned hearts. There was a comparable, modest decline in activity during 30 minutes of global ischemia in both groups. As a positive control, a third group of hearts was treated with anisomycin before global ischemia, and in these, JNK activity increased by 290% above baseline. These results confirm that the p38 MAPK/MAPKAPK2 pathway is activated during ischemia only if the heart is in a preconditioned state. These data further support p38 MAPK as an important signaling component in ischemic PC.

Adenosine A1 agonist at reperfusion trial (AART): results of a three-center, blinded, randomized, controlled experimental infarct study.

Adenosine A1 receptor agonists given prior to myocardial ischemia limit ischemic injury in several species. However, the ability of adenosine receptor agonists to limit infarct size when given at reperfusion has proved controversial. We designed a three-center experimental study using a blinded, randomized treatment protocol to test the hypothesis that adenosine A1 receptor activation during early reperfusion can attenuate lethal reperfusion injury, thereby reducing infarct size. Sixty anesthetized rabbits (20 in each laboratory) underwent 30 minutes coronary artery occlusion followed by 120 minutes reperfusion. The selective adenosine A1 receptor agonist GR79236 (10.5 microg/kg, a dose shown to limit infarction in this model when given before ischemia) or vehicle were administered IV 10 minutes before reperfusion. Infarct size was assessed by tetrazolium staining and, after the randomization code was revealed, data from the three laboratories were pooled for statistical analysis. Infarct size was not modified by administration of GR79236. In the vehicle-treated group, the infarct-to-risk ratio was 28.9 +/- 2.7% (n = 24) compared with 31.9 +/- 2.6% (n = 26) in the GR79236-treated group (not significant). Risk zone volume was similar in the two groups (1.06 +/- 0.05 cm3 vs 1.00 +/- 0.05 cm3, respectively). A modest reduction in rate-pressure product was noted following the administration of GR79236, but this effect was transient. The same dose of GR79236 was found to limit infarct size when given prior to coronary artery occlusion. We conclude that A1 receptor activation does not modify lethal reperfusion injury in myocardium.

S-T segment voltage during sequential coronary occlusions is an unreliable marker of preconditioning.

During coronary angioplasty, a stair-step decrease in peak S-T segment elevation from the first to the second coronary occlusion has been assumed to indicate a preconditioning (PC) effect. This association was evaluated with myocardial electrograms in rabbits, which revealed that two sequential 5-min coronary occlusions resulted in a marked decrease in the area under the S-T segment voltage-time curve (P < 0.05) with no change during a third occlusion. Pretreatment with either 5-hydroxydecanoate, a mitochondrial ATP-sensitive potassium (K(ATP)) channel blocker, or anisomycin, an activator of stress-activated protein kinases, had no effect on the stair-step decline in the S-T segment voltage between the first two occlusions. HMR-1883, a potent closer of sarcolemmal K(ATP) channels, abolished changes in S-T segment elevation after brief coronary occlusions but had no effect on the infarct-sparing property of the two preconditioning 5-min occlusions. Interestingly, HMR-1883 blocked myocardial protection from diazoxide, raising doubt that the latter opens only mitochondrial channels. Therefore, myocardial protection and S-T segment changes during ischemia are dissociated. These data suggest that it is the mitochondrial K(ATP) channel that protects the myocardium, and it is the sarcolemmal channel that is responsible for changes in S-T elevation. Therefore, it cannot always be inferred that changes in S-T segment elevation reflect the state of myocardial protection.

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.

Signal transduction in ischemic preconditioning: the role of kinases and mitochondrial K(ATP) channels.

Ischemic preconditioning is a phenomenon whereby exposure of the myocardium to a brief episode of ischemia and reperfusion markedly reduces tissue necrosis induced by a subsequent prolonged ischemia. Therefore, it is hoped that elucidation of the mechanism of preconditioning will yield therapeutic strategies capable of reducing myocardial infarction. In the rabbit, the brief period of preconditioning ischemia and reperfusion releases adenosine, bradykinin, opioids, and oxygen radicals that summate to induce the translocation and activation of protein kinase C (PKC). PKC appears to be the first element of a complex kinase cascade that is activated during the prolonged ischemia in preconditioned hearts. Current evidence indicates that PKC activates a tyrosine kinase that leads to the activation of p38 mitogen-activated protein (MAP) kinase or JNK, or possibly both. The stimulation of these stress-activated protein kinases ultimately induces the opening of mitochondrial K(ATP) channels that may be the final mediator of protection by ischemic preconditioning.

Ischemic preconditioning depends on interaction between mitochondrial KATP channels and actin cytoskeleton.

Both mitochondrial ATP-sensitive K+ (KATP) channels and the actin cytoskeleton have been proposed to be end-effectors in ischemic preconditioning (PC). For evaluation of the participation of these proposed end effectors, rabbits underwent 30 min of regional ischemia and 3 h of reperfusion. PC by 5-min ischemia + 10-min reperfusion reduced infarct size by 60%. Diazoxide, a mitochondrial KATP-channel opener, administered before ischemia was protective. Protection was lost when diazoxide was given after onset of ischemia. Anisomycin, a p38/JNK activator, reduced infarct size, but protection from both diazoxide and anisomycin was abolished by 5-hydroxydecanoate (5-HD), an inhibitor of mitochondrial KATP channels. Isolated adult rabbit cardiomyocytes were subjected to simulated ischemia by centrifuging the cells into an oxygen-free pellet for 3 h. PC was induced by prior pelleting for 10 min followed by resuspension for 15 min. Osmotic fragility was assessed by adding cells to hypotonic (85 mosmol) Trypan blue. PC delayed the progressive increase in fragility seen in non-PC cells. Incubation with diazoxide or pinacidil was as protective as PC. Anisomycin reduced osmotic fragility, and this was reversed by 5-HD. Interestingly, protection by PC, diazoxide, and pinacidil could be abolished by disruption of the cytoskeleton by cytochalasin D. These data support a role for both mitochondrial KATP channels and cytoskeletal actin in protection by PC.