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OBJECTIVES. An acute coronary occlusion and its possible subsequent complications is one of the most common causes of death. One such complication is ventricular fibrillation (VF) due to myocardial ischemia. The severity of ischemia is related to the amount of coronary arterial collateral flow. In dog studies collateral flow has also been shown to be associated with QRS prolongation. The aim of this study was to investigate whether ischemic QRS prolongation (IQP) is associated with impending VF in an experimental acute ischemia dog model. METHODS. Degree of IQP and occurrence of VF were measured in dogs (n=21) during coronary occlusion for 15 minutes and also during subsequent reperfusion (experiments conducted in 1984). RESULTS. There was a significant difference in absolute IQP between dogs which developed VF during reperfusion (47 ±29 ms, mean ±SD) and those which did not (12 ±10 ms; p=0.001). CONCLUSIONS. IQP during acute coronary occlusion is associated with reperfusion VF in an experimental dog model and might therefore be a potential predictor of malignant arrhythmias in patients with acute coronary syndrome.

Supplemental Digital Content is available in the text., Rationale: After acute myocardial infarction (MI), delineating the area-at-risk (AAR) is crucial for measuring how much, if any, ischemic myocardium has been salvaged. T2-weighted MRI is promoted as an excellent method to delineate the AAR. However, the evidence supporting the validity of this method to measure the AAR is indirect, and it has never been validated with direct anatomic measurements. Objective: To determine whether T2-weighted MRI delineates the AAR. Methods and Results: Twenty-one canines and 24 patients with acute MI were studied. We compared bright-blood and black-blood T2-weighted MRI with images of the AAR and MI by histopathology in canines and with MI by in vivo delayed-enhancement MRI in canines and patients. Abnormal regions on MRI and pathology were compared by (a) quantitative measurement of the transmural-extent of the abnormality and (b) picture matching of contours. We found no relationship between the transmural-extent of T2-hyperintense regions and that of the AAR (bright-blood-T2: r=0.06, P=0.69; black-blood-T2: r=0.01, P=0.97). Instead, there was a strong correlation with that of infarction (bright-blood-T2: r=0.94, P

International audience; Ischaemic heart disease and the heart failure that often results, remain the leading causes of death and disability in Europe and worldwide. As such, in order to prevent heart failure and improve clinical outcomes in patients presenting with an acute ST-segment elevation myocardial infarction and patients undergoing coronary artery bypass graft surgery, novel therapies are required to protect the heart against the detrimental effects of acute ischaemia/reperfusion injury (IRI). During the last three decades, a wide variety of ischaemic conditioning strategies and pharmacological treatments have been tested in the clinic-however, their translation from experimental to clinical studies for improving patient outcomes has been both challenging and disappointing. Therefore, in this Position Paper of the European Society of Cardiology Working Group on Cellular Biology of the Heart, we critically analyse the current state of ischaemic conditioning in both the experimental and clinical settings, provide recommendations for improving its translation into the clinical setting, and highlight novel therapeutic targets and new treatment strategies for reducing acute myocardial IRI.

The extent and rate at which necrosis develops in experimental acute myocardial infarction in the dog heart is presented together with an analysis of the role played by protective mechanisms in myocyte death. Preconditioning with ischemia delays but does not prevent myocyte death. Arterial collateral flows exceeding 30% of control flow essentially prevent myocyte death, while lesser amounts of collateral flow delay myocyte death to a variable extent. Flows of

Ischemic preconditioning is a form of intrinsic cardioprotection where an episode of sublethal ischemia protects against subsequent episodes of ischemia. Identifying a clinical biomarker of preconditioning could have important clinical implications, and prior work has focused on the electrocardiographic ST segment. However, the electrophysiology biomarker of preconditioning is increased action potential duration (APD) shortening with subsequent ischemic episodes, and APD shortening should primarily alter the T wave, not the ST segment. We translated findings from simulations to canine to patient models of preconditioning to test the hypothesis that the combination of increased [delta (Δ)] T wave amplitude with decreased ST segment elevation characterizes preconditioning. In simulations, decreased APD caused increased T wave amplitude with minimal ST segment elevation. In contrast, decreased action potential amplitude increased ST segment elevation significantly. In a canine model of preconditioning (9 mongrel dogs undergoing 4 ischemia-reperfusion episodes), ST segment amplitude increased more than T wave amplitude during the first ischemic episode [ΔT/ΔST slope = 0.81, 95% confidence interval (CI) 0.46–1.15]; however, during subsequent ischemic episodes the T wave increased significantly more than the ST segment (ΔT/ΔST slope = 2.43, CI 2.07–2.80) ( P < 0.001 for interaction of occlusions 2 vs. 1). A similar result was observed in patients (9 patients undergoing 2 consecutive prolonged occlusions during elective percutaneous coronary intervention), with an increase in slope of ΔT/ΔST of 0.13 (CI −0.15 to 0.42) in the first occlusion to 1.02 (CI 0.31–1.73) in the second occlusion ( P = 0.02). This integrated analysis of the T wave and ST segment goes beyond the standard approach to only analyze ST elevation, and detects cellular electrophysiology changes of preconditioning.

A selective history of the pathophysiological, structural, and metabolic changes found during an episode of severe myocardial ischemia in the canine heart is presented. The changes that cause ischemic injury to become irreversible are discussed in detail because these changes are the target of any successful therapy designed to prevent ischemic cell death. Of these, the disruption of the sarcolemma, an injury the development of which is accelerated in vivo by the contraction of viable tissue elsewhere in the heart traumatizing the ischemic area, plus the changes in high-energy phosphate and the total adenine nucleotide pool are considered to be the critical events leading to the development of irreversibility. The discovery of preconditioning with ischemia is discussed, together with a brief description of postconditioning. Finally, reperfusion injury is discussed in a summary fashion. The evidence for the fact that myocytes are salvaged by reperfusion is presented, as is the evidence that myocytes become unsalvageable by reperfusion as the duration of ischemia increases. The concept that some of the myocytes that die after successful reperfusion with arterial blood actually are killed by changes initiated by reperfusion, so-called lethal reperfusion injury, is attractive in that prevention of this change would lead to greater salvage; however, the prevalence of this phenomenon in clinical practice remains to be determined.

To commemorate the auspicious occasion of the 30th anniversary of IPC, leading pioneers in the field of cardioprotection gathered in Barcelona in May 2016 to review and discuss the history of IPC, its evolution to IPost and RIC, myocardial reperfusion injury as a therapeutic target, and future targets and strategies for cardioprotection. This article provides an overview of the major topics discussed at this special meeting and underscores the huge importance and impact, the discovery of IPC has made in the field of cardiovascular research. DJH was funded by the British Heart Foundation (Grant Number FS/10/039/28270), the Rosetrees Trust, and is supported by the National Institute for Health Research University College London Hospitals Biomedical Research Centre. HEB was funded by the Danish Council for Independent Research (11-108354), the Danish Council for Strategic Research (11-115818), the Novo-Nordisk Foundation and Trygfonden. PF was funded by the Hungarian Scientific Research Fund (OTKA K 109737 and ANN 107803). HACF is funded by a Startup Grant of the ``Excellence Cluster Cardio-Pulmonary System'' (ECCPS) from the German Research Foundation (DFG, Bonn, Germany) and the ``Peter und Traudl Engelhorn-Stiftung'' (Weilheim, Germany) and in part by the Russian Government Program for competitive growth of Kazan Federal University, Kazan (Russian Federation). GH was supported by the German Research Foundation (He 1320/18-3 and SFB 1116/B8). TM was funded by the Japan Society for the Promotion of Science (Grant-in-Aid for Scientific Research \#23591086, \#2646113). MRS was funded by Novo-Nordisk Foundation. DG-D is supported by the Spanish Institute of Health, Instituto de Salud Carlos III (Grants PIE 13/00027, RETICS-RIC, RD12/0042/0021, and PI14/01431). RS and PF were funded by the European Foundation for the Study of Diabetes (EFSD) New Horizons Collaborative Research Initiative from the European Association for the Study of Diabetes (EASD) and by the European Cooperation in Science and Technology (COST EU-ROS). PF was funded by the Hungarian Scientific Research Fund (OTKA K 109737 and ANN 107803). Sí

Background Previous studies have shown that QRS prolongation is a sign of depressed collateral flow and increased rate of myocardial cell death during coronary occlusion. The aims of this study were to evaluate ischemic QRS prolongation as a biomarker of severe ischemia by establishing the relationship between prolongation and collateral flow experimentally in a dog model, and test if the same pattern of ischemic QRS prolongation occurs in man. Methods Degree of ischemic QRS prolongation was measured using a novel method in dogs (n = 23) and patients (n = 52) during coronary occlusion for 5 min. Collateral arterial flow was assessed in the dogs. Results There was a significant correlation between QRS prolongation and collateral flow in dogs (r = 0.61, p = 0.008). Magnitude and temporal evolution of prolongation during ischemia were similar for dogs and humans (p = 0.202 and p = 0.911). Conclusion Quantification of ischemic QRS prolongation could potentially be used as a biomarker for severe myocardial ischemia.

Coronary heart disease is the largest major killer of American men and women and accounted for 1 of every 6 deaths in the United States in 2007.1 The annual incidence of myocardial infarction in the United States is estimated to be 935 000, with 610 000 new cases and 325 000 recurrent attacks. Survivors have a much higher chance of suffering from congestive heart failure, arrhythmias, and sudden cardiac death. Prognosis after an acute myocardial ischemic injury is primarily dependent on the amount of myocardium that undergoes irreversible injury.2–4 Large transmural infarcts yield a higher probability of cardiogenic shock, arrhythmias, adverse remodeling, and development of late chronic heart failure. Although it has been known since the early 1970s that the size of a myocardial infarction can be modified by various therapeutic interventions,5 early coronary artery reperfusion by fibrinolysis or percutaneous coronary intervention, including balloon angioplasty with or without stenting, remains the only established intervention capable of consistently reducing infarct size in humans. Although reperfusion has led to significant advances in patient care and reduction in hospital mortality, delays in seeking medical attention and inherent limitations in initiating fibrinolysis or percutaneous coronary intervention dictate that additional substantive improvements in morbidity and mortality can be achieved only with the development of new adjunctive therapies coupled with reperfusion. In addition, reperfusion therapy itself may induce reperfusion injury, a phenomenon that may encompass stunned myocardium, no-reflow phenomenon, and lethal myocardial cell death. If this injury could be prevented or minimized by administration of adjunctive therapy, then the net benefit of reperfusion could be enhanced. The problem of acute ischemic injury and myocardial infarction is not limited to patients with acute coronary artery syndrome. It remains a major problem in cardiac surgery as well. It is well documented that the incidence of myocardial necrosis after surgery, as determined by creatine kinase MB enzyme release and troponin levels, ranges somewhere between 40% and 60%, and, depending on its clinical definition, the incidence of myocardial infarction after coronary artery bypass graft surgery may be as high as 19%. The intermediate and long-term implications are considerable. In a recent retrospective analysis of 18 908 patients who underwent coronary artery bypass graft surgery and in whom long-term follow-up was available, it was shown that myocardial enzyme elevation within the first 24 hours of surgery was associated with increasing mortality over the course of months to years. This study confirms earlier reports that even small enzyme elevations after surgery are associated with worse long-term outcomes.4

Three aspects of cardioprotection are discussed in this article. The first is myocyte death as a function of the duration and severity of ischemia in experimental acute myocardial infarction in the dog heart. The short period of time during which reperfusion with arterial blood will salvage myocytes is demonstrated along with data showing that this period diminishes significantly if collateral flow is very low or absent. The second topic is a discussion of potential mechanisms underlying postconditioning. It begins with a review of the changes that lead to irreversible injury during acute ischemia in the dog heart along with a discussion of the genesis of contraction band necrosis and no reflow when myocardium is salvaged by unrestricted reperfusion with arterial blood in order to provide a basis to discuss the potential mechanisms underlying postconditioning, a situation in which reflow is intermittent and restricted. Postconditioning is reported to achieve greater myocyte salvage than unrestricted reflow. Potential explanations for this beneficial effect include: first, sufficient sarcolemmal repair occurring during the intermittent reflow (reoxygenation) to prevent cell death by explosive cell swelling, and second, prevention of the opening of the mitochondrial permeability transition pore, thereby preventing mitochondrial failure and cell death in the reperfused tissue. Since there is no way available to identify and specifically study the myocytes that would have died if not protected by postconditioning, direct demonstration of mechanisms is difficult or impossible. Finally, the third topic in this commentary is an analysis of the obstacles faced by investigators using small rodent hearts to establish cardioprotective mechanisms. Such studies provide valid data but the relationship of the changes and the proposed mechanisms underlying these changes are not necessarily directly transferable to ischemic large animal hearts including the heart of man.

High energy phosphate (HEP) utilization and depletion as well as the production and distribution of catabolic products of adenine nucleotides were studied in ischemic dog hearts in order to characterize the transition from reversible to irreversible cell injury. Severe ischemia, induced by coronary occlusion in vivo was compared to total ischemia, in vitro. Much of the creatine phosphate (CP) was lost within the first one to three minutes. During severe ischemia in vivo, adenosine triphosphate (ATP) was depleted to only 35% of control by 15 minutes (when myocytes are still reversibly injured) and to less than 10% by 40 minutes (at which time irreversible injury has occurred). HEP production from anaerobic glycolysis was estimated from the rate of accumulation of myocardial lactate. HEP production ceased when the ATP was less than 0.4 μmol/g wet weight (control = 5–6). Prior to this time, 80% of the HEP which had been utilized in ischemia had been derived from anaerobic glycolysis whereas 20% came from pre-existing stores of CP and ATP. ATP depletion was paralleled by dephosphorylation of adenine nucleotides. The lost nucleotides were recovered stoichiometrically as adenosine, inosine, hypoxanthine, and xanthine in both models of ischemia. When myocardium was reperfused after 15 minutes of ischemia, these nucleosides and bases were lost to the systemic circulation, and repletion of adenine nucleotides was incomplete for as long as four days, apparently because both de novo and salvage pathways of adenine nucleotide synthesis are slow in myocardium. Further studies were done to assess the nature of the associations between the decreasing ATP of the ischemic tissue and the onset of defects in high energy phosphate regeneration, cell volume and ion regulation, and/or membrane permeability. Slices of control tissue from tissue injured by various periods of total ischemia were incubated in oxygenated Krebs Ringer's phosphate medium containing 14 C-inulin. As long as the ATP of the tissue was not depleted below 5 μmoles/g dry weight prior to incubation, no cellular abnormalities were detected. However, lower ATP levels were associated with depressed high energy phosphate resynthesis and failure of cell volume regulation. Overt membrane damage, as measured by an increased inulin diffusible space, was detected after the tissue ATP content decreased to less than 2.0 μmoles/g dry weight. Thus, cellular viability (reversible injury) was retained despite a modest and persistent ATP depletion, but there was a close assocation between marked ATP depletion and the failure of the damaged tissue to regenerate high energy phosphates and to preserve cell volume and ionic regulation (irreversible injury).

Background Analysis of ST-segment elevation for assessment of patients with suspected acute coronary occlusion is in widespread use for diagnostic and prognostic purposes. In this study, changes in the QRS complex also were analyzed to determine if these changes that are seldom used clinically can provide additional prognostic information. An acute coronary occlusion canine model, in which direct measurements of myocardial salvage were made, was used to assess whether ST-segment and QRS complex changes during coronary occlusion yielded independent estimates of the amount of salvage provided by reperfusion with arterial blood. Methods and Results Continuous electrocardiographic recordings were obtained from 14 study dogs undergoing a 90-minute period of coronary artery occlusion in which the severity of the ischemia during the occlusion was estimated at 10 and 45 minutes by microsphere injections. After 3 hours of reperfusion, the myocardium at risk and postmortem infarct size was measured. Myocardial salvage correlated inversely with both ST-segment elevation ( r = −0.85; P r = −0.72; P = .003). When dogs were paired so that they had equal amounts of ST elevation but differed with respect to the presence of QRS prolongation, less myocardial salvage was found in those with QRS prolongation. The independent value of QRS prolongation was supported further by the observation that presence of QRS prolongation resulted in a loss of the highly significant correlation between ST elevation and salvage ( r = −0.60; P = .2). Conclusions High magnitudes of ST elevation are correlated significantly with less myocardial salvage. Moreover, for a given magnitude of ST elevation, the presence of concurrent QRS prolongation is associated with even less myocardial salvage.

Acute complete occlusion of a major epicardial coronary artery typically causes sufficient ischemia in the dependent myocardial cells to produce the electrocardiogram change called “epicardial injury”. There is sufficient shift of the ST baseline toward, and of the TP/PR baseline away from the involved region to provide the clinical diagnosis. Relationships between the quantities of this baseline shift and etiologic ischemia have previously been shown to provide clinical value. However, it has now become apparent that potentially reversible ischemia can be so severe, in the absence of protection from collaterals and/or ischemia preconditioning, that it alters myocardial conduction and the resultant QRS complexes. This report presents highlights from two studies in progress to determine the relative abilities of ST segment and QRS changes during coronary occlusion to predict salvage following reperfusion; and the relative effects of collaterals and ischemia preconditioning to produce each of these electrocardiogram changes.

Elucidation of the subcellular mechanism of myocardial ischemic preconditioning should be facilitated by precise knowledge of the biology of the cardioprotective response. Any proposed molecular mechanism for preconditioning must be initiated during the required ischemic stress period. The studies reported in this paper were under-taken to determine whether the infarct-limiting effect of four 5-min episodes of ischemia interspersed by reperfusion can be achieved by a single 5-min episode. Adult openchest mongrel dogs, premedicated with the analgesic butorphanol, and anesthetized with sodium pentobarbital, underwent occlusion of the circumflex coronary artery for 60 min, followed by reperfusion for 3 h. Treated dogs were preconditioned with one, two or four cycles of 5-min occlusion followed by reperfusion. Additional dogs, not premedicated with butorphanol, were either untreated (not preconditioned) or preconditioned with one cycle of ischemia. Infarcts were identified using triphenyl-tetrazolium chloride (TTC) macrochemistry and infarct size (as % of area-at-risk, AAR) was measured and analyzed (using analysis of covariance [ANCOVA]) with respect to coronary collateral blood flow (measured using radio active microspheres). Four 5-min cycles of preconditioning ischemia markedly limited infarct size. Two cycles were as effective as four. In contrast, infarct size was not different from control infarct size after a single episode of preconditioning ischemia. However, when pentobarbital anesthesia was used without premedication with butorphanol, a single 5-min ischemic stress did induce cardioprotection. Thus, the ischemic stress required for myocardial preconditioning in dogs is dependent on the anesthetic and premedication protocol employed. A single 5-min stimulus is effective in dogs anesthetized with pentobarbital. Premedication with the opioid analgesic, butorphanol, increases the threshold for induction of cardioprotection.

Background: The cardioprotective effect of ischemic preconditioning has been hypothesized to occur through one or more signalling mechanisms which activate protein kinase C. Stimulation of α 1-adrenergic receptors by catecholamines released during the preconditioning episodes of ischemia is one of these putative signalling mechanisms. Methods: To determine whether stimulation of α 1-adrenergic receptors before an ischemic challenge can mimic preconditioning, anesthetized dogs were treated with 4 intracoronary infusions of methoxamine HC1 (10 μg/kg/min; n = 8), each 5 min in duration and followed by 5 min of washout. Control dogs ( n = 10) were given similar infusions of 0.9% NaCl. A third group of dogs was preconditioned with 4 cycles of 5 min ischemia, each followed by 5 min of reperfusion ( n = 8). All dogs then underwent 60 min of ischemia (circumflex coronary occlusion) followed by 3 h of reperfusion. Infarct size (expressed as % of area-at-risk) was measured with TTC macrochemistery and analyzed (using analysis of covariance [ANCOVA]) with respect to coronary collateral blood flow (measured using radioactive microspheres). Results: Methoxamine markedly increased systemic arterial and left atrial pressures prior to but not during the ischemic challenge. Baseline predictors of infarct size were not different among the groups. Mean infarct size (adjusted from ANCOVA) did not differ between control and methoxamine-treated groups, 28.3 ± 2.8% vs. 29.7 ± 3.2%, respectively ( P = NS), but was only 12.7 ± 3.2% in the preconditioned group ( P < 0.01 vs. control and methoxamine). Conclusions: A series of methoxamine infusions before an ischemic challenge did not affect infarct size. Thus, stimulation of α 1-adrenergic receptors alone is insufficient to mimic the cardioprotective effect of ischemic preconditioning in this canine model.

The mitochondrial ATPase enzyme accounts for roughly 35 –50 % of the overall energy demand that leads to ATP depletion under conditions of severe myocardial ischemia. In larger mammalian hearts, this energy squandering action of the ATPase is modulated by an endogenous inhibitor protein. The present studies were undertaken to characterize the time course of inhibition of the mitochondrial ATPase in canine myocardium under conditions of severe regional ischemia in vivo . In addition, we determined if the energy sparing effects of ischemic preconditioning (PC) can be explained by persistent inhibition of the mitochondrial ATPase enzyme. The circumflex coronary artery was ligated for 1.5 min ( n =4), 5 min ( n =6), or 15 min ( n =5). In a separate group ( n =7), hearts were preconditioned by four 5-min periods of ischemia each followed by 5 min of reperfusion. Sub-mitochondrial particles were prepared from the sub-endocardial zone of the ischemic and non-ischemic regions and were assayed for oligomycin-sensitive ATPase activity. ATPase activity was reduced to about 79 % at 1.5 min and to approximately 55 % at 5 and 15 min of ischemia, relative to non-ischemic tissue from the same heart. The rate of HEP utilization slowed concurrently with the development of ATPase inhibition. In preconditioned myocardium, ATPase activity was not significantly different from control myocardium from the same heart. We conclude that the early inhibition of the mitochondrial ATPase activity slows the utilization of high energy phosphate and thereby serves as an important endogenous cardioprotective mechanism. Nevertheless, altered activity of the ATPase is not the explanation of the energy sparing effect of ischemic preconditioning.

The role of lactate accumulation in lethal ischemic myocardial cell injury was assessed by partially depleting hearts of glycogen before ischemia by using glucagon. Isolated adult rat hearts were perfused with glucose-free Krebs-Henseleit buffer containing acetate as substrate. After stabilization, treated hearts were perfused briefly (3 min) with buffer containing 2 micrograms/ml glucagon to reduce tissue glycogen stores, followed by 10 min of perfusion with control buffer, and 60 or 90 min of global ischemia. Before the onset of ischemia, glucagon-treated hearts contained 40% less glycogen than untreated hearts, but myocardial function and tissue levels of high-energy phosphates, lactate, and glucose 6-phosphate were similar. Lactate production during ischemia in the glucagon-treated hearts was 50% less than in untreated hearts. However, there was no decrease in the amount of creatine kinase release during reperfusion after either 60 or 90 min of ischemia. Thus although partial glycogen depletion reduced lactate accumulation during ischemia, this did not decrease the amount of lethal myocardial cell injury.

Objectives: Cardioprotective adaptation to brief periods of ischemia and reperfusion is termed ischemic preconditioning (PC). Limitation of infarct size by preconditioning is associated with marked slowing of ischemic metabolism. The cause of metabolic slowing has not been determined but may involve either pro- or anti-adrenergic mechanisms. Hypothetically, adrenergic stimulation could signal the adaptive response. Alternatively, metabolic slowing during the sustained ischemic challenge could occur through a reduction in β-adrenergic stimulation. This study was designed to test the role of cardiac norepinephrine (NE) in PC. Methods: The effect of PC on myocardial infarct size was studied in control dogs and dogs depleted of catecholamines by pretreatment with reserpine (RES; 0.25 mg/kg i.V.). PC was induced by four cycles of 5 min of ischemia and 5 min of reperfusion. Infarcts were produced by 60 min of ischemia and 3 h of reperfusion. Cardiac NE depletion was verified by radioimmunoassay of tissue samples and by absence of hemodynamic response to a tyramine bolus (1.4 mg/kg) administered at the end of each experiment. Infarct size, expressed as percent of area at risk, was controlled for variation in collateral blood flow using analysis of covariance (ANCOVA). Results: Adjusted mean infarct size was 25.5 ± 3.2% in untreated controls vs. 19.1 ± 3.3% in RES-treated controls ( P = NS). PC limited infarct size in untreated dogs (7.4 ± 1.8 vs. 25.5 ± 3.2%; PC vs. control; P < 0.01) but not in RES-treated dogs (15.7 ± 3.0% vs. 19.1 ± 3.3%; RES + PC vs. RES; P = NS). Infarct size was larger in dogs with RES + PC than with PC alone, even though there was a trend toward a slight beneficial effect with RES alone. Conclusions: The Cardioprotective effect of ischemic preconditioning cannot be explained entirely as an anti-adrenergic effect. On the contrary, adrenergic receptor stimulation may be required for the full expression of ischemic preconditioning in canine myocardium.

Objective: Previous studies have shown that cardiac protection by ischaemic preconditioning wanes before contractile function recovers; thus stunning is insufficient to cause preconditioning. To test whether reduced contractile effort is necessary for preconditioning induced protection, the effect on myocardial infarct size of restoring contractile function with dobutamine was examined in preconditioned and control dogs. Methods: In two experimental groups (groups P and P+D), preconditioning was produced by four 5 min occlusions of the left anterior descending coronary artery, each separated by 5 min of reperfusion. Contractile function was assessed by sonomicrometry 5 min after completion of the preconditioning protocol. In group P+D, dobutamine (average dose = 5 μg·kg−1·min−1) was then infused intravenously to restore systolic shortening to baseline. The artery then was reoccluded for 40 min of sustained ischaemia followed by 4 d of reperfusion. Two additional groups of non-preconditioned control dogs (groups C and C+D) also underwent 40 min of coronary occlusion and 4 d of reperfusion. Group C+D received a dobutamine infusion beginning 15 min before and during the 40 min occlusion to match the dobutamine received in group P+D, whereas group C received normal saline. Results: Preconditioning caused mild postischaemic contractile dysfunction (50% decrease in systolic shortening) which was easily reversed by dobutamine treatment. Dobutamine also increased both the rate-pressure product and the left ventricular dP/dt in both treated groups (C+D and P+D). Histological infarct size was 12.3(SEM 2.0)% of the area at risk in the untreated control group (n=ll), and was reduced to 4.4(1.7)% in the untreated preconditioning group (n=8; p

The effect of inhibition of the mitochondrial ATPase with oligomycin on the rate of ATP depletion and anaerobic glycolysis was studied in the totally ischemic dog heart. An oxygenated, buffered crystalloidal solution containing 10 μ m oligomycin and 12 m m glucose was delivered at 100 mmHg pressure to the circumflex bed of the excised cooled heart. Buffered solution without oligomycin was delivered simultaneously to the anterior descending bed of the same heart. Little metabolic evidence of ischemia developed until the heart was made totally ischemic by incubating it in a sealed plastic bag at 37°C. Successful inhibition of the mitochondrial ATPase was confirmed by the absence of both mitochondrial ATPase activity and the loss of respiratory control in mitochondria isolated from treated tissue. ATP, glycolytic intermediates and catabolites of the adenine nucleotide pool were measured in the control and treated beds at various intervals during 120 min of ischemia. Inhibition of the ATPase resulted in slowing of the rates of ATP depletion and anaerobic glycolysis (estimated by lactate accumulation). Also, degradation of the adenine nucleotide pool occurred more slowly in the inhibited group. These data establish that about 35% of the ATP utilization observed during the first 90 min of total ischemia in the canine heart is due to mitochondrial ATPase activity.

Myocardium which has been preconditioned by one or several brief episodes of ischemia has much slower energy utilization during a subsequent sustained episode of ischemia. Since preconditioned tissue also is 'stunned', the reduced energy utilization of preconditioned tissue may be due to reduced contractile effort. This study was done to assess whether differences in energy utilization persisted or disappeared under conditions of total ischemia, in vitro, when contractile activity was abolished in both control and preconditioned regions by hyperkalemic cardiac arrest. Preconditioned myocardium was produced in open-chest anesthetized dogs by exposing the circumflex bed to four 5-min episodes of ischemia each followed by 5 min of arterial reperfusion. Non-preconditioned anterior descending bed was used as control myocardium. Hearts were arrested with hyperkalemia after the last reperfusion period in order to reduce or eliminate the effects of contractile activity. Metabolite content was measured in sequential biopsies of the tissue. Large differences in the rate of energy metabolism of the two regions were noted during the first 15 minutes of ischemia. During this time, the preconditioned tissue utilized less glycogen, and produced less lactate, glucose-6-phosphate (G6P), glucose-1-phosphate (G1P), and alpha-glycerol phosphate (alpha GP), than did control myocardium. Moreover, there was a much smaller decrease in net tissue ATP in the preconditioned than in the control tissue. Thus, the decrease in the demand of preconditioned tissue for energy, which has been observed in vivo, persisted despite the elimination of differences in contractile effort between control and preconditioned myocardium. Although the cause of this decrease in energy demand in preconditioned myocardium remains unknown, the present results suggest that it is not due to concomitant stunning.

The metabolic changes associated with the sudden onset of ischemia caused by occlusion of a major coronary artery include (a) cessation of aerobic metabolism, (b) depletion of creatine phosphate (CP), (c) onset of anaerobic glycolysis, and (d) accumulation of glycolytic products, such as lactate and alpha glycerol phosphate (alpha GP), and catabolites of the nucleotide pools in the tissue. These changes are associated with contractile failure and electrocardiographic alterations. Since the demand of the myocardium for high-energy phosphate (approximately P) exceeds the available supply, the net amount of ATP in tissue decreases. Eighty percent of the supply of approximately P utilized by severely ischemic tissue comes from anaerobic glycolysis using glycogen as the principal substrate. Early in ischemia, contractile activity utilizes ATP, but much of the continuing utilization of ATP by the ischemic tissue is energy wasted via the mitochondrial ATPase. A lesser quantity of ATP is used by ion transport ATPases. Metabolic changes slow as the duration of ischemia increases. Irreversibly injured myocytes exhibit (a) very low levels of ATP (less than 10% of control); (b) cessation of anaerobic glycolysis; (c) high levels of H+, AMP, INO, lactate, and alpha GP; (d) a greatly increased osmolar load; (e) mitochondrial swelling and formation of amorphous matrix densities; and (f) disruption of the sarcolemma. The latter event is generally recognized as lethal, but its pathogenesis remains to be established. Most severely ischemic myocytes are dead in regional ischemia in the anesthetized open-chest dog heart after only 60 minutes of ischemia. Less severely ischemic myocytes in the mid- and subepicardial myocardium survive for as long as six hours. Virtually all myocytes destined to die in a zone of ischemia are irreversibly injured after six hours of ischemia have passed. Certain changes exhibited by myocytes injured by severe ischemia and reperfused late in the reversible phase of injury do not return to the control conditions for a period of days, while others rebound in only seconds to minutes. The adenine nucleotide pool still is not fully restored after four days of reperfusion. Stunning disappears after one to two days of reflow. The preconditioning effect is partially lost after two hours of reperfusion. The timing of its disappearance has not been fully established. Aerobic metabolism is restored after only a few minutes of reperfusion. Thus, reperfusion salvages injured myocardium and restores its structure and function to the control state at a variable rate.

Study objective – Although timely reperfusion limits myocardial infarct size, it has been postulated that reperfusion itself may lull some myocytes which were alive at the end of an episode of ischaemia (lethal reperfusion injury). The aim of this study was to test the hypothesis that ischaemic catabolites may “prime” myocardium for such injury and that preliminary hypoxic washout of such catabolites, prior to arterial reperfusion, would limit myocardial infarct size. Design – Dogs underwent a 40 min occlusion of the left circumflex coronary artery, followed by 4 d reperfusion. In a treated group, a 5 min episode of coronary artery perfusion with hypoxic buffer was instituted at the end of this ischaemic episode, before blood reperfusion was restored. Control dogs received a similar volume of hypoxic buffer intravenously. Systemic fluid overload was attenuated by haemofiltration. The effect of this preliminary hypoxic washout on myocardial infarct size was assessed. Experimental material – 18 anaesthetised, open chest dogs were used. After the acute study they recovered from surgery for 4 d and were then killed for further study. Measurements and main results – Infarct size, determined by microscopic evaluation, was not significantly different in the two groups, at (control) 31.3 (SEM 6.2)% v (hypoxic reperfusion) 25.8(3.9)% of the vascular area at risk. In control dogs, infarct size was inversely related to the amount of collateral blood flow (measured using microspheres); hypoxic reperfusion did not shift this relation (analysis of covariance, F = 0.236, NS). Conclusions – The washout of ischaemic catabolites by hypoxic perfusate prior to reoxygenation did not limit infarct size.

We have shown previously that preconditioning myocardium with four 5-minute episodes of ischemia and reperfusion dramatically limited the size of infarcts caused by a subsequent 40-minute episode of sustained ischemia. The current study was undertaken to assess whether the same preconditioning protocol slowed the loss of high energy phosphates, limited catabolite accumulation, and/or delayed ultrastructural damage during a sustained ischemic episode. Myocardial metabolites and ultrastructure in the severely ischemic subendocardial regions were compared between control and preconditioned canine hearts. Hearts (four to 10 per group) were excised after 0, 5, 10, 20, or 40 minutes of sustained ischemia. All groups had comparable collateral blood flow. Preconditioned hearts developed ultrastructural injury more slowly than controls; evidence of irreversible injury was observed after 20 minutes in controls but not until 40 minutes in preconditioned hearts. Furthermore, after 40 minutes of ischemia, irreversible injury was homogeneous in controls but only focal in preconditioned myocardium. Preconditioning reduced starting levels of ATP by 29%. Nevertheless, it also slowed the rate of ATP depletion during the episode of sustained ischemia, so that after 10 minutes of ischemia, preconditioned hearts had more ATP than controls. However, after 40 minutes, ATP contents were not significantly different between groups. Preservation of ATP resulted from reduced ATP utilization and was not due to increased ATP production. Accumulation of purine nucleosides and bases (products of adenine nucleotide degradation) was limited in preconditioned myocardium. Accumulation of glucose-1-phosphate, glucose-6-phosphate, and lactate also was reduced markedly by preconditioning, due to reduced rates of glycogen breakdown and and anaerobic glycolysis. We propose that preconditioning reduces myocardial energy demand during ischemia, which results in a reduced rate of high energy phosphate utilization and a reduced rate of anaerobic glycolysis. Either preservation of ATP or reduction of the cellular load of catabolites may be responsible for delaying ischemic cell death.

Abundant evidence now is available that free radicals are produced in excess when myocardium is reperfused following an episode of ischemia and that free radicals can injure myocytes and endothelial cells. Free radicals may contribute to either reversible or irreversible manifestations of cell injury from ischemia and reperfusion. Several investigators have observed that postischemic contractile dysfunction (myocardial stunning) can be attenuated by a variety of anti-free radical therapies, and there seems to be general agreement that free radical injury contributes to stunning. Whether free radicals are an important cause of lethal myocyte injury (“lethal reperfusion injury”) remains controversial. Using similar interventions and animal models, both positive and negative results have been reported from a growing number of studies done to test the effect of anti-free radical therapies on infarct size. Proposed explanations include differences in: 1) dose of drug and onset or duration of treatment, 2) duration of occlusion or reperfusion, 3) methods of measuring infarct size or area at risk, and 4) failure of some studies to control for baseline variation in the major determinants of infarct size, e.g., collateral blood flow. At present, none of these explanations seems sufficient to resolve the question.

The changes in ion content and H2O detectable in vivo in the intact canine heart in reversible and irreversible ischemic injury are described, with emphasis on the role Ca2+ movements may play in causing ischemic injury. Changes in extracellular ion concentrations and pH revealed by ion-specific electrodes in ischemia are reviewed, as are the contributions of nuclear magnetic resonance measurements of ionized Ca2+ to our understanding of Ca2+ ion homeostasis in ischemia.

Background During acute myocardial infarction, both ST elevation and QRS distortion on the initial electrocardiogram (ECG) have been correlated with poorer prognosis. Studies in dogs and humans suggest that these ECG markers provide information about myocardial protection from both collateral blood flow and ischemic preconditioning. Methods In a protocol designed to precondition the heart with ischemia, we examined both ST-segment elevation and QRS complex prolongation in lead II of the ECG in 23 mongrel dogs during the first and fourth episode of 5 minutes of left circumflex artery occlusion. Myocardial collateral flow was measured during each of these episodes by injection of radioactive microspheres 2.5 minutes into the episode of ischemia. Results During ischemia, the degree of elevation of the ST segments was reduced markedly in hearts preconditioned with ischemia and/or in hearts with the greatest amounts of collateral arterial flow. During the first episode of ischemia, the ST segments increased to a similar extent in severe and moderate ischemia, but less in hearts in which the ischemia was mild. However, marked QRS prolongation was present only in hearts with severe ischemia, and decreased when the hearts were preconditioned. In addition, large ischemic beds exhibited the most marked QRS prolongation, whereas small but even severely ischemic beds showed little or no change in QRS duration. Conclusion Both ST elevation and QRS prolongation are reduced by the presence of collateral flow and ischemic preconditioning. The QRS complex merits further study as an important marker of the degree of myocardial protection during human acute myocardial ischemia/infarction.

Background. – Actomyosin ATPase is one of the major ATP consuming enzymes in the myocardium. We tested whether 2,3-butanedione monoxime (BDM), a reversible inhibitor of actomyosin ATPase, given before coronary occlusion, limits infarct size in anesthetized open-chest dogs. Methods and results. – After circumflex artery catheterization using fluoroscopic guidance, BDM (125 mM) or buffer vehicle was infused (12.0 ml/min) for 20 min (BDM-20, n = 5 and Buffer-20, n = 6) or for 5 min (BDM-5, n = 6 and Buffer-5, n = 6) prior to 60 min of ischemia and 3ho freperfusion. BDM administration increased subendocardial blood flow 271% above baseline flow (radioactive microspheres), and systolic wall thickening was converted to wall bulging (wall thickening by sonomicrometry was ‐27 ± 29% and ‐22 ± 13% of baseline in BDM-20 and BDM-5, respectively). Adjusted mean infarct size (% area-at-risk) was 11.0 ± 2.8% and 11.9 ± 2.6% in BDM-20 and BDM-5 vs. 20.2 ± 2.5% and 20.5 ± 2.5% in Buffer-20 and Buffer-5 (ancova, P < 0.05 for each BDM vs. Buffer group). Measurement of glycolytic metabolites and the adenine nucleotide pool of myocardium paced electronically at 150 beats per minute during total ischemia at 37 °C following BDM showed a metabolic response similar to that seen in ischemic preconditioning. ATP depletion, nucleoside production, and lactate accumulation were slowed in ischemic tissue treated with BDM. Conclusion. – BDM given before the onset of ischemia markedly limited infarct size and reduced energy demand after the onset of ischemia. The explanation for the reduced infarct size induced by BDM treatment is hypothesized to be the persistent reduction in energy demand found in ischemic BDM treated myocardium.

The phosphocreatine (PCr) overshoot is a well-documented phenomenon and is readily observable by 31P MRS. In addition, a second 31P MRS observation during ischemia with reperfusion is a diminution in ATP levels. Combining these two as the 'PCr Overshoot' the PCr/ATP ratio may provide an index of viability. However little information is available regarding the duration of this 'overshoot'. For this approach to be useful clinically, the duration of this phenomenon must be ascertained. An open chest canine model of 12 min of ischemia followed by reperfusion (6h) was used. A 2 cm surface coil was sutured to the myocardium and spectra were acquired at 4.7 T. Gated spectra were acquired in

In experimental studies in the dog, total proximal coronary artery occlusions of up to 15 minutes result in reversible injury, meaning that the myocytes survive this insult. The 15 minutes of ischemia, however, induce numerous changes in the myocardium, including certain monuments to the brief episode of ischemia that may persist for days. One of these monuments is stunned myocardium, which represents “prolonged postischemic contractile dysfunction of myocardium salvaged by reperfusion.” The mechanism of stunning involves generation of oxygen radicals as well as alteration in calcium homeostasis and possibly alteration in contractile protein structure. Stunning has been observed in several clinical scenarios, including after percutaneous transluminal coronary angioplasty, unstable angina, stress-induced ischemia, after thrombolysis, and after cardiopulmonary bypass. Oxygen radical scavengers and calcium channel blockers have been shown to enhance function of stunned myocardium in experimental studies, and in a few clinical studies, calcium channel blockers have been shown to ameliorate stunning. Although brief periods of ischemia can contribute to prolonged left ventricular dysfunction and even heart failure, they paradoxically play a cardioprotective role. Episodes of ischemia as short as 5 minutes, followed by reperfusion, protect the heart from a subsequent longer coronary artery occlusion by markedly reducing the amount of necrosis that results from the test episode of ischemia. This phenomenon, called ischemic preconditioning, has been observed in virtually every species in which it has been studied and is a powerful cardioprotective effect. The mechanism of ischemic preconditioning involves both triggers and mediators and involves complex second messenger pathways that appear to involve such components as adenosine, adenosine receptors, the epsilon isoform of protein kinase C, the ATP-dependent potassium channels, as well as others, including a paradoxical protective role of oxygen radicals. Both an early and a late phase of preconditioning have been described, and the mechanisms underlying their induction are under investigation. That preconditioning may occur in humans is suggested by the observations that repetitive balloon inflations in the coronary artery are associated with progressively less chest pain, ST-segment elevation, lactate production, the protective effects of preinfarction angina, the anginal “warm-up phenomenon,” and studies performed on human cardiac biopsies that show metabolic properties suggesting preconditioning. Development of pharmacological agents that stimulate second messenger pathways thought to be involved in preconditioning, but without causing ischemia, could result in novel approaches to treating ischemia. Hence, on one hand, brief episodes of ischemia can have a negative effect on the heart: stunning; and on the other hand, they have a protective effect: preconditioning. The future challenge is how to minimize the stunning phenomenon and maximize the preconditioning phenomenon in clinical practice.

Ischemic preconditioning is associated with slower destruction of the adenine nucleotide pool and a slower rate of anaerobic glycolysis during subsequent ischemic stress. Whether this association is causal is uncertain. Using metabolite levels found at baseline and after a 15 min test episode of ischemia, this study tested for concordance, or lack thereof, between the presence or absence of metabolic features v the presence or absence of the preconditioned state. Dogs were assigned to one of four groups: non-preconditioned control (C), full preconditioning (PC) caused by 10 min ischemia (I)+10 min reperfusion (R), dissipated PC (DPC) caused by 10 min I and 180 min R, or reinstated PC in which PC was reinstated in DPC hearts by another 10 min I and 10 min R. At baseline, PC and RPC hearts had a 25% or more decrease in the adenine nucleotide pool (summation operatorAd), a substantial creatine phosphate (CP) overshoot, and a 4-6 times elevation in tissue glucose (G). Of these changes, the decreased summation operatorAd and the CP overshoot persisted during DPC, whereas only G returned to control. Thus, increased G was the only baseline feature, which was concordant with the preconditioned state. The response to ischemic stress in PC and RPC tissue included less lactate production and much less degradation of the summation operatorAd pool to nucleosides and bases than in the C or DPC groups. Thus, slower destruction of the summation operatorAd pool and slower lactate production during ischemia also were concordant with the PC state. The results support the hypothesis that a reduction in energy demand is an essential component of the mechanism of cardioprotection in preconditioned myocardium. However, the mechanism through which ischemic preconditioning results in lower energy demand remains to be established.

These studies were undertaken to determine the duration of protection against myocardial infarction provided by ischemic preconditioning in the canine heart, and to learn if cardioprotection can be restored by another preconditioning stimulus when the initial effect is lost. Control and four preconditioning groups of anesthetized, open-chest dogs were compared. All underwent a test 60 min episode of ischemia, induced by occlusion of the anterior descending (LAD) artery, followed by 3 h of reperfusion. Preconditioning was induced by one 10 min LAD occlusion, followed by either 10 min, 2, 3, or 5 h of reperfusion. In order to test whether preconditioning could be reinstated, another group of dogs with preconditioning plus 3 h reperfusion underwent a second 10 min preconditioning stimulus with 10 min reperfusion before the 60 min test-occlusion. Infarct size (as percent of area-at-risk) was analyzed (using analysis of covariance) with respect to coronary collateral blood flow measured with radioactive microspheres. Infarct size was limited markedly by preconditioning (23+/-6 v 6+/-2%, P0.05) but the protective effect was dissipated partially after 2 h reperfusion and was dissipated completely after 3 h reperfusion (20+/-4%, non-significant v Control and significant P0.05 v preconditioning). Protection was restored in three of six dogs with preconditioning +5 h reperfusion, suggesting that the second window of protection appears early in some canine hearts. When preconditioning was repeated after 3 h reperfusion, cardioprotection was reinstated fully (7+/-2%, P0.05 v Control and NS v preconditioning). The results show that maximal preconditioning cardioprotection is present in the dog heart after 10 min of reperfusion and is dissipated totally following 3 h of reperfusion. However, a second preconditioning stimulus of 10 min of ischemia followed by 10 min of reperfusion to the dissipated preconditioned heart reinstates full preconditioning. Thus, this model provides a system to test for theoretical causes of the preconditioned state. Final mediators should be present when preconditioning is present and absent when preconditioning is dissipated. It is noteworthy that a second window of protection appeared in 50% of dogs when the period of reperfusion was extended to 5 h.

The PKC hypothesis and its variants are attractive but unproved. The chief difficulty is the lack of an objective means to assess whether or not PKC has been activated in hearts preconditioned in vivo with ischemia or in hearts preconditioned by pharmacologic means. The strongest evidence is supporting the PKC hypothesis in vivo is the prevention of preconditioning with antagonists such as staurosporine. However, these data are weakened by the failure of these agents to eliminate PC in large animal hearts. Since hearts of virtually all mammalian species can be preconditioned, it seems likely that the general mechanism causing it is similar in each and should respond to a strong inhibitor. More work needs to be done.

Background Cardiac failure remains an important problem after heart transplantation and may be associated with events that occur during brain death (BD) before transplantation. In this study, cardiac function is studied after BD, and biochemical evaluation of myocardial high-energy phosphates and the β-adrenergic receptor system is presented. Methods and Results The hearts of 17 mongrel dogs (23 to 31 kg) were instrumented with flow probes, micromanometers, and ultrasonic dimension transducers to measure ventricular pressure and volume relationships. In a validated canine BD model, systolic right and left ventricular (RV/LV) function was analyzed by load-insensitive measurements during caval occlusion (preload-recruitable stroke work, PRSW). The β-adrenergic receptor (BAR) density, adenylate cyclase (AC) activity, and myocardial ATP and creatine phosphate (CP) were measured before and 6 to 7 hours after BD. Results are expressed as mean±SEM (* P t test). Myocardial function deteriorated significantly from baseline PRSW (RV, 22±1 erg×10 3 ; LV, 75±4 erg×10 3 ) by 37±10% for the RV ( P P Conclusions BD causes significant systolic biventricular dysfunction. The loss of ventricular function after BD was more prominent in the right ventricle and may contribute to early postoperative RV failure in the recipient. These injuries occurred despite BAR system upregulation after BD. Global myocardial ischemia is unlikely, since ATP and CP remained normal before and after BD.

In the early 1950s, the leadership of the American Heart Association recognized that much more fundamental knowledge of basic cardiovascular function was required if one was going to improve the treatment and diagnosis of heart disease. They believed that a journal dedicated to basic research on the circulation would foster an increased understanding of the cardiovascular system and founded Circulation Research . In 1953, when volume 1 of Circulation Research was published, the basic sciences relevant to cardiovascular research such as physiology, biochemistry, pharmacology, and pathology all had journals that were devoted to these disciplines in which there were scattered studies on the heart and circulation. However, these studies were not always read by basic scientists in other disciplines, and the field lacked a journal devoted primarily to understanding how the cardiovascular system worked in health and disease. Circulation Research provided a forum where studies on the heart and cardiovascular system could be published and has been eminently successful in fostering the application of the basic medical sciences to understanding the function of the cardiovascular system. At the time the journal was founded, basic research support was beginning to be available from the National Institutes of Health, and basic knowledge of biology was being developed at an enormous rate, but from meager foundations. Mitochondria had moved from objects difficult to discern by histological techniques to organelles of considerable interest to biochemists. Moreover, methods of isolating them from heart and studying their metabolism were under development. Energy metabolism in general, the use of electron microscopy to establish the ultrastructure of cardiac muscle, the isolation of …

Myocardium that is exposed to a mild, sublethal stress is capable of undergoing adaptive changes that enable it to better tolerate a more severe, potentially lethal exposure to the same (or different) form of stress. This response may be rapid (minutes) or delayed (hours). For example, when animals are exposed to heat stress, the myocardium responds by synthesizing a family of heat shock proteins (HSPs) [1,2], which, 24 hours later, are associated with an increased tolerance to various insults, including heat stress and ischemia [3, 4, 5, 6] (see Chapter 6).

In the last 10 years our understanding of the heart’s response to ischemic injury has changed dramatically. Until the mid-1980s, prevailing opinion held that reversibly injured myocardium was more vulnerable to the effects of a subsequent period of ischemia [1,2]. Cardiac biologists considered tissue injured by, for example, a 15-minute period of ischemia, to remain near the brink of cell death for many hours after it was salvaged by reperfusion. This notion turned out to be wrong; in fact, the exact opposite is true. Paradoxically, myocardium that has been reversibly injured by ischemia is more tolerant of a subsequent episode of ischemia. This phenomenon has been termed ischemic preconditioning [3]. In this chapter we shall describe the studies that led to our original report of the preconditioning phenomenon and review the effects of preconditioning on myocardial infarct size. We shall then review the effects of repeated, brief ischemic insults in other organs and compare their responses with that of the heart.

Keith Arnold Reimer, MD, PhD, a well-known experimental cardiologist and cardiac pathologist and a Professor of Pathology at Duke University died on March 15, 2002, at the age of 56. The cause of death was metastatic carcinoma of the kidney. His disease was advanced when it was diagnosed in September of 2001, and it essentially incapacitated him for the remainder of his life. Keith was born in Beatrice, a small town in Nebraska, on April 10, 1945. He graduated from high school in a class of 7 students. Typical of Keith is the fact that by the time he had graduated, he had taken every course offered by the school except Home Economics. After graduating from high school, he attended Bethel College in North Newton, Kansas, from which he graduated in 1967. While at Bethel, he met and married Susan Stuckey. Although this educational history seems an unlikely prelude to a career in science, he became a very successful scientist.⇓ I first met Keith on a spring day 34 years ago, when a young medical student with a red beard turned up in my laboratory at Northwestern University Medical School. At this time, he was finishing his second year in medical school and was searching for a laboratory in which to pursue graduate work. His aim was to enter the MD, PhD program at Northwestern. The goal of this program was to produce physicians trained as scientists. In Keith’s case, the science was experimental pathology. I questioned him carefully about why he wanted to follow a course of study that would lead him to become an expert in the field of cell injury, simply because research in cell injury was unpopular in 1968. Few physicians and even fewer scientists cared why cardiac muscle cells …

Objective: Studies in rabbits suggest that the cardioprotective effects of adenosine against lethal cell injury may be related to production of adenosine and subsequent activation of adenosine A1, receptors. However, it is not known whether intracoronary adenosine therapy can mimic the metabolic sparing effects of preconditioning in rabbits or dogs. The purpose of this study was to determine the effect of intracoronary adenosine on ischaemic metabolism in totally ischaemic canine myocardium. Methods: Dog hearts (n=13) were excised and the coronary arteries were perfused with an oxygenated Krebs' buffer containing glucose. Adenosine was added to the buffer perfusing the circumflex (treated) region. Following perfusion, control and treated beds from each heart were subjected to 90 min total ischaemia at 37°C. Tissue levels of ATP and glycolytic intermediates were determined at several time points during the ischaemic incubation. Results: Adenosine significantly slowed the rate of ATP depletion, glycogen utilisation, and lactate accumulation during the first 20 minutes of total ischaemia. Conclusions: The results suggest that adenosine is capable of slowing ischaemic metabolism and they are consistent with the hypothesis that adenosine may mediate ischaemic preconditioning. Cardiovascular Research 1993; 27 :669-673

Studies to test whether superoxide dismutase (SOD), with or without catalase, limits myocardial infarct size have produced conflicting results. Positive results following short periods of reperfusion vs negative results following longer periods of reperfusion could be explained if either: (1) myocytes, initially salvaged by SOD, are killed by continued production of free radicals after the administered SOD have been excreted, or (2) false positive results occur because SOD transiently preserves the TTC reaction, despite loss of cellular viability. To evaluate these two possibilities, we measured infarct size after 90 min of ischemia and 4 h of reperfusion in SOD+catalase treated and untreated dogs. Treated dogs received a 60 min intra-arterial infusion of SOD (15,000 U/kg) plus catalase (CAT) (55,000 U/kg) beginning 25 min before reperfusion. Infarct size was measured using triphenyl tetrazolium (TTC) macrochemistry and was compared with the extent of necrosis assessed semi-quantitatively by light microscopy. Mean infarct size was similar in the control and treated groups. In addition, there was a positive linear correlation (r = 0.95) between the extent of necrosis estimated by microscopy and that estimated by TTC in both groups, and treatment did not alter the regression line. These current results were compared with results from the control dogs from our previous study (Richard et al., 1988) in which 90 min of ischemia was followed by 4 days of reperfusion. TTC-based infarct size at 4 days of reperfusion was similar to that observed in both groups at 4 h. These data indicate that oxygen free radicals, accessible to intravascular SOD and catalase, are not a cause of myocyte death detectable by measurement of infarct size after 4 h of reperfusion. Moreover, neither an "early protection, delayed death" hypothesis nor a specific preservation of the TTC reaction explain the positive results of other studies. TTC macrochemistry provides reliable estimates of myocardial infarct size, provided that sufficient magnification is used to permit resolution of interdigitating peninsulas of viable and necrotic tissue.

BACKGROUND Polymorphonuclear neutrophils (PMNs) accumulate in postischemic myocardium and may cause injury to myocardium or to vessels by production of oxygen free radicals or by release of proteases and lipases. PMN accumulation is dependent on adherence to endothelium, which is mediated by a family of glycoproteins on the PMN surface, each of which has a common beta-subunit (CD18). The purpose of this study was to determine whether an antibody (IB4) against the CD18 protein could attenuate PMN accumulation and limit myocardial infarct size. METHODS AND RESULTS F(ab')2 fragments of a mouse monoclonal antibody to human adherence-promoting leukocyte glycoprotein (CD18) were used. Infarct size after 90 minutes of ischemia and 3 hours of reperfusion was compared in dogs with (n = 8) and without (n = 8) the anti-CD18 treatment. Myocardial PMN accumulation was assessed with 111In-labeled autologous PMNs. Anti-CD18 treatment significantly reduced the number of PMNs in the ischemic region (19,123 +/- 5,352/mg versus 5,204 +/- 927/mg in the control and treated groups, respectively; p < 0.05). In addition, the ratio of myocardial blood flow (ischemic/nonischemic wall) at 45 minutes into reperfusion was higher in the treated than in the control group (1.18 +/- 0.18 versus 0.69 +/- 0.09; p < 0.05). Nevertheless, infarct size was similar between the control and treated groups (40.5 +/- 7.4% versus 48.5 +/- 4.4% of the area at risk; p = NS). Transmural mean collateral blood flow to the ischemic myocardium was similar between the two groups, and the inverse relation between infarct size and collateral blood flow was not shifted by anti-CD18 therapy. CONCLUSIONS Although PMN accumulation contributed to reduced postischemic microvascular perfusion, it caused insufficient additional myocardial cell death to measurably affect infarct size in this model.

The sudden onset of severe ischaemia in previously healthy myocytes sets into motion a series of events that continue until the myocytes die. These changes begin seconds after the vessel is occluded and occur because the supply of 02 is insufficient to support oxidative phosphorylation. Within 8–10 s, the myocardium converts to anaerobic glycolysis (AG) as its only source of new high-energy phosphate (˜P). Because the demand of the ischaemic tissue for ˜P exceeds the supply provided by the reserve supplies of ˜P, and by anaerobic glycolysis, net myocardial adenosine triphosphate (ATP) decreases rapidly (Figure 2.1). Simultaneously, as a result of the marked reduction in arterial flow, tissue pH decreases as large quantities of lactate and other glycolytic intermediates accumulate in the tissue. By the time 40 min of severe ischaemia has passed, little ATP remains (Figure 2.1), and the tissue contains a large osmotic load of lactate, alpha glycerol phosphate (aGP), creatine, phosphate (Pi), H+, etc. (Figure 2.2).

O ur concepts of myocardial injury have undergone considerable revision in recent years. Traditionally, myocardium reversibly injured by an episode of ischemia was thought to be more vulnerable to the effects of a subsequent episode; an extension of this view was that brief repeated ischemic episodes might cumulatively cause infarction. Multiple studies have now shown that this is not the case. Paradoxically, brief episodes of ischemia increase the heart's tolerance to a subsequent sustained period of ischemia, such that the rate of cell death is slowed substantially. Induction of tolerance in this manner has been termed ischemic preconditioning.1 In the current issue of Circulation, Liu et a12 have provided new evidence for a role for adenosine in mediating this ischemic tolerance. In this editorial comment we shall review what is known about the basic biology of preconditioning and its potential mechanisms, including the adenosine hypothesis, and address what is unknown and in need of future study.