Oxidative stress and heart failure.

Despite advances in treatment, chronic congestive heart failure carries a poor prognosis and remains a leading cause of cardiovascular death. Accumulating evidence suggests that reactive oxygen species (ROS) play an important role in the development and progression of heart failure, regardless of the etiology. Under pathophysiological conditions, ROS have the potential to cause cellular damage and dysfunction. Whether the effects are beneficial or harmful will depend upon site, source and amount of ROS produced, and the overall redox status of the cell. All cardiovascular cell types are capable of producing ROS, and the major enzymatic sources in heart failure are mitochondria, xanthine oxidases and the nonphagocytic NADPH oxidases (Noxs). As well as direct effects on cellular enzymatic and protein function, ROS have been implicated in the development of agonist-induced cardiac hypertrophy, cardiomyocyte apoptosis and remodelling of the failing myocardium. These alterations in phenotype are driven by redox-sensitive gene expression, and in this way ROS may act a potent intracellular second messengers. Recent experimental studies have suggested a possible causal role for increased ROS in the development of contractile dysfunction following myocardial infarction and pressure overload, however the precise contribution of different cellular and enzymatic sources involved remain under investigation.

Divergent biological actions of coronary endothelial nitric oxide during progression of cardiac hypertrophy.

Coronary endothelial NO synthase expression and NO bioactivity were investigated at sequential stages during the progression of left ventricular hypertrophy. Male guinea pigs underwent abdominal aortic banding or sham operation. Left ventricular contractile function was quantified in isolated ejecting hearts. Coronary endothelial and vasodilator function were assessed in isolated isovolumic hearts in response to boluses of bradykinin (0.001 to 10 micromol/L), substance P (0.01 to 100 micromol/L), diethylamine NONOate (DEA-NO) (0.1 to 1000 micromol/L), N(G)-monomethyl-L-arginine monoacetate (L-NMMA) (10 mmol/L), and adenosine (10 mmol/L). At a stage of compensated left ventricular hypertrophy (3 weeks), left ventricular endothelial NO synthase protein expression was unaltered (Western blot and immunocytochemistry). Vasoconstriction in response to L-NMMA was increased in banded animals compared with sham-operated animals (13.8+/-2.1% versus 6.2+/-1.3%, n=10; P<0.05), but agonist- and DEA-NO-induced vasodilation was similar in the 2 groups. At a stage of decompensated left ventricular hypertrophy (8 to 10 weeks), left ventricular endothelial NO synthase protein expression was significantly lower in banded animals (on Western analysis: banded animals, 7.8+/-0.4 densitometric units; sham-operated animals, 12.2+/-1.7 densitometric units; n=5; P<0.05). At this time point, vasoconstriction in response to L-NMMA was similar in the 2 groups, but vasodilatation in response to bradykinin (30.9+/-2.4% versus 39.7+/-2.2%, n=10; P<0.05), DEA-NO (26.2+/-1.8% versus 34.6+/-1.8%, n=10; P<0.05), and adenosine (24.3+/-2.0% versus 35.7+/-2.0%, n=10; P<0.01) was attenuated in banded animals. These findings indicate that there is an increase in the basal activity of NO (without a significant change in endothelial NO synthase expression) in early compensated left ventricular hypertrophy, followed by a decrease in both endothelial NO synthase expression and NO bioactivity during the transition to myocardial failure.

ATP synthesis during low-flow ischemia: influence of increased glycolytic substrate.

BACKGROUND: Our goals were to (1) simulate the degree of low-flow ischemia and mixed anaerobic and aerobic metabolism of an acutely infarcting region; (2) define changes in anaerobic glycolysis, oxidative phosphorylation, and the creatine kinase (CK) reaction velocity; and (3) determine whether and how increased glycolytic substrate alters the energetic profile, function, and recovery of the ischemic myocardium in the isolated blood-perfused rat heart. METHODS AND RESULTS: Hearts had 60 minutes of low-flow ischemia (10% of baseline coronary flow) and 30 minutes of reperfusion with either control or high glucose and insulin (G+I) as substrate. In controls, during ischemia, rate-pressure product and oxygen consumption decreased by 84%. CK velocity decreased by 64%; ATP and phosphocreatine (PCr) concentrations decreased by 51% and 63%, respectively; inorganic phosphate (P(i)) concentration increased by 300%; and free [ADP] did not increase. During ischemia, relative to controls, the G+I group had similar CK velocity, oxygen consumption, and tissue acidosis but increased glycolysis, higher [ATP] and [PCr], and lower [P(i)] and therefore had a greater free energy yield from ATP hydrolysis. Ischemic systolic and diastolic function and postischemic recovery were better. CONCLUSIONS: During low-flow ischemia simulating an acute myocardial infarction region, oxidative phosphorylation accounted for 90% of ATP synthesis. The CK velocity fell by 66%, and CK did not completely use available PCr to slow ATP depletion. G+I, by increasing glycolysis, slowed ATP depletion, maintained lower [P(i)], and maintained a higher free energy from ATP hydrolysis. This improved energetic profile resulted in better systolic and diastolic function during ischemia and reperfusion. These results support the clinical use of G+I in acute MI.

A new system for the metabolic investigation of the isolated, perfused rat heart.

NMR spectroscopy is an invaluable technique in metabolic investigations of isolated, perfused hearts. Most studies employ global perfusion methods together with an NMR coil that surrounds the heart and thus detects signals from its entirety. The present report describes the construction and testing of a novel, two surface-coil probe, in combination with a dual-perfused heart preparation, that enables spectra to be collected independently from the two coronary beds of the rat heart.(31)P NMR spectra of perfused rat hearts in which the septum and right ventricle have been made ischaemic, while the free left ventricular wall is fully perfused, demonstrate the powerful potential of this new system.

Is a functional sarcoplasmic reticulum necessary for preconditioning?

Several studies have shown that the protective effect of ischemic preconditioning (PC) is associated with decreased calcium release from the sarcoplasmic reticulum (SR). However, no study has yet demonstrated whether these changes are essential in the mechanism of PC. In order to investigate whether a functional SR was necessary for PC, we manipulated SR calcium handling using (i) 0.1microM ryanodine (RY), a concentration known to lock the SR calcium release channel in the open state and (ii) 50microM cyclopiazonic acid (CPA), a specific inhibitor of the SR calcium ATPase. Initial experiments confirmed that both RY and CPA eliminated the ability of the SR to accumulate calcium. Isolated rat hearts (n=6-7/group) were perfused normoxically for 30 min prior to either a further 40 min of perfusion [control (C)] or 4x[5 min ischemia (I) + 5 min reperfusion (R)] (PC). All hearts were then subjected to a further 40 min I + 40 min R. The C and PC protocols were then repeated in the presence of RY or CPA, introduced after 10 min of perfusion.(31)P-NMR was used to measure ATP, PCr, P(i)and intracellular pH. RY and CPA decreased developed pressure (DP) by 75% and 59%, respectively. Percentage recovery of LVDP was significantly higher in PC (72+/-8%), PC+RY (72+/-7%) and PC+CPA (49+/-7%) groups compared with their respective controls (43+/-7%, 47+/-7% and 10+/-4%) (P<0.05). Thus, PC remains protective in the presence of a SR unable to accumulate calcium, suggesting that the changes in SR calcium release are not essential in the mechanism of preconditioning.

Ischemic preconditioning does not protect against contractile dysfunction in the presence of residual flow: studies in the isolated, blood-perfused rat heart.

BACKGROUND: We have previously demonstrated that ischemic preconditioning (PC) does not protect when oxygen deprivation is accompanied by a high level of perfusion (hypoxia). Since clinical ischemia can vary from mild to severe, we wished to determine whether PC could protect against injury arising from low-flow ischemia. METHODS AND RESULTS: Functional recovery after 30 minutes of reperfusion was assessed in isolated, blood-perfused rat hearts (n=6 per group) subjected to (A) 30 minutes of zero-flow ischemia, (B) 30 minutes of zero-flow ischemia preceded by 3xPC (PC=5 minutes of ischemia+5 minutes of reperfusion), (C) 90 minutes of low-flow ischemia at 10% of baseline coronary flow (0.31+/-0.02 mL/min per gram wet wt), (D) 90 minutes of low-flow ischemia at 10% of baseline coronary flow (0.29+/-0.02 mL/min per gram wet wt) preceded by 3xPC. PC significantly protected against injury resulting from zero-flow ischemia (developed pressure recovered to 67+/-6% versus 31+/-12% in B and A, respectively; P<.05) but not resulting from low-flow ischemia (recovery of developed pressure was 40+/-8% versus 37+/-7% in C and D, respectively). Protein kinase C (PKC) is widely considered to be involved in the mechanism of PC such that prior activation and translocation of PKC by the PC protocol allows phosphorylation of the end-effector protein early during the subsequent ischemic insult, before loss of adenosine triphosphate occurs. However, because adenosine triphosphate content falls slowly during low-flow ischemia, PKC may be activated and translocated early enough to be active during this insult. If so, inhibition of PKC should decrease functional recovery in the control group. However, functional recovery in control groups was not decreased in the presence of the PKC inhibitor polymyxin B (50+/-6%), suggesting that if activation of PKC occurred during low-flow ischemia, it was not protective. CONCLUSIONS: PC does not protect against contractile dysfunction in the rat when a low level (10% of baseline flow) of ischemic perfusion remains during the prolonged insult.

PET and NMR dual acquisition (PANDA): applications to isolated, perfused rat hearts.

Positron emission tomography and nuclear magnetic resonance spectroscopy are non-invasive techniques that allow serial metabolic measurements to be obtained in a single subject. Significant advantages could be obtained if both types of scans could be acquired with a single machine. A small-scale PET scanner, designed to operate in a high magnetic field, was therefore constructed and inserted into the top half of a 7.3 cm bore, 9.4 T NMR magnet and its performance characterized. The magnetic field did not significantly affect either the sensitivity (approximately 3 kcps/MBq) or the spatial resolution (2.0 mm full width at half maximum, measured using a 0.25 mm diameter line source) of the scanner. However, the presence of the PET scanner resulted in a small decrease in field homogeneity. The first, simultaneous 31P NMR spectra (200, 80 degrees pulses collected at 6 s intervals) and PET images (transverse, mid-ventricular slices at the level of the mitral value) from isolated, perfused rat hearts were acquired using a specially designed NMR probe inserted into the bottom half of the magnet. The PET images were of excellent quality, enabling the left ventricular wall and interventricular septum to be clearly seen. In conclusion, we have demonstrated the simultaneous acquisition of PET and NMR data from perfused rat hearts; we believe that the combination of these two powerful techniques has tremendous potential in both the laboratory and the clinic.

Ischemic preconditioning and intracellular pH: a 31P NMR study in the isolated rat heart.

The aim of these studies was to investigate whether manipulation of intracellular pH affects preconditioning in isolated buffer-perfused rat hearts. Control and preconditioned [PC; 3 min of ischemia (I) + 3 min of reperfusion (R) + 5 min of I + 5 min of R or 4 x (5 min of I + 5 min of R)] hearts were subjected to two different protocols expected to alter intracellular pH during the sustained ischemic insult: 1) increased extracellular buffering capacity with the addition of 25 mM N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES) to the buffer to alleviate acidosis and 2) increased preischemic glycogen content to exacerbate acidosis. All hearts were subjected to 40 min of I + 40 min of R. 31P nuclear magnetic resonance was used to measure ATP, phosphocreatine, Pi, and intracellular pH. Despite a significantly better recovery of function in all PC groups, there were no significant differences in intracellular pH (rate-pressure product = 60 +/- 5, 66 +/- 10, 42 +/- 5, and 57 +/- 8% of baseline in PC, 4 x 5 PC, PC + HEPES, and PC fasted hearts, respectively, compared with 36 +/- 9, 17 +/- 7, and 20 +/- 10% of baseline in control, control + HEPES, and control fasted hearts, respectively; pH at end ischemia: control, 6.31 +/- 0.02; PC, 6.35 +/- 0.03; 4 x 5 PC, 6.35 +/- 0.04; control + HEPES, 6.40 +/- 0.10; PC + HEPES, 6.56 +/- 0.07: control fasted, 6.46 +/- 0.03; PC fasted, 6.43 +/- 0.01). No significant differences were observed among groups in ATP, phosphocreatine, or Pi on reperfusion. Thus the mechanism of preconditioning in glucose-perfused hearts does not depend on an alleviation of intracellular acidosis during the sustained ischemic period. Furthermore, under the conditions of this study, intracellular pH during ischemia did not predict functional recovery on reperfusion.

A model of anoxic preconditioning in the isolated rat cardiac myocyte. Importance of adenosine and insulin.

OBJECTIVE: Ischemic or hypoxic preconditioning has been shown, in multicellular preparations, to reduce post-ischemic injury. In the present study, we attempted to develop a model of preconditioning in isolated rat myocytes in order to facilitate investigation into the mechanism of preconditioning. METHODS: The protective effect of a short period (10 min) of anoxia and reoxygenation against a subsequent longer period of anoxia was studied in single, electrically stimulated (0.2 Hz, 37 degrees C) adult rat cardiac myocytes. The control group received only the long period of anoxia. Three protocols were tested: Protocol 1 in which octanoate was the only substrate; Protocol 2 in which only glucose was present during all normoxic phases and during the preconditioning anoxia and octanoate alone during the prolonged period of anoxia and; Protocol 3 in which protocol 2 was repeated with the addition of adenosine (100 microM) and insulin (15 microU/ml) during the prolonged anoxic period. The end-point of assessment was loss of cell morphology, i.e., hypercontracture (death) or relengthening (survival) on reoxygenation following the prolonged anoxic period. Membrane integrity was also examined at the end of each protocol by observing if the cells excluded trypan blue. RESULTS: No protective effect of preconditioning on cell survival was observed in protocols 1 or 2. In contrast, in protocol 3, a significant protection was observed in the preconditioned versus control group (58% vs 27% survival respectively; p < 0.001). However, in the absence of preconditioning, adenosine and insulin provided no additional protection in the control group. No significant differences in trypan blue exclusion were observed between the groups in any protocol. CONCLUSIONS: These results suggest that preconditioning cannot protect against a subsequent period of anoxia where the accumulation of metabolic products, e.g., adenosine is prevented. However, that protection can be re-instated by the presence of adenosine and insulin during the period of prolonged anoxia. Furthermore, this study suggests that the preconditioning by anoxia may induce a change in the A1-receptor or its second messenger system such that adenosine is able to provide protection.

Polymyxin B, a protein kinase C inhibitor, abolishes preconditioning-induced protection against contractile dysfunction in the isolated blood perfused rat heart.

The aims of this study were (1) to determine the characteristics of preconditioning against contractile dysfunction in a blood perfused isolated heart model in the presence of a physiologic combination of substrates, and (2) to determine if protein kinase C (PKC) is involved in preconditioning in this model. In order to investigate these aims, isolated isovolumic, blood perfused rat hearts (balloon-in-LV, n = 6/group) were perfused normoxically for 30 min and then divided into three groups and subjected to: (1) a further 30 min of perfusion (control group) (2) a further 20 min of perfusion + 5 min of ischaemia and 5 min of reperfusion (1 x preconditioned group) and (3) 3 x (5 min of ischaemia+5 min of reperfusion) (3 x preconditioned group). All hearts were then subjected to 30 min of ischaemia and 30 min of reperfusion. Contractile function, myocardial oxygen consumption (MVO2), lactate release and creatine kinase release were all assessed. To determine if PKC is involved in the mechanism of preconditioning in this model, the control and 3 x preconditioned group experiments were repeated in the presence of polymyxin B (50 microM), a relatively specific PKC inhibitor. Final recovery of LVDP was 31 +/- 12, 67 +/- 6 and 60 +/- 5% in the control, 1 x and 3 x preconditioned groups, respectively. Protection of contractile function was accompanied by both a preservation of diastolic function and the ratio of MVO2 to contractile function (ratio of metabolic:mechanical efficiency). However, lactate release was decreased only in the 3 x preconditioned group. Polymyxin B abolished preconditioning-induced protection against contractile and diastolic dysfunction and the protection of the ratio of MVO2 to contractile function. Lactate release was still however reduced in the polymyxin B-preconditioned group. Thus, preconditioning-induced protection against contractile dysfunction appears to be accompanied by a preservation of both diastolic function and the metabolic: mechanical efficiency and is effective in the presence of a physiologic combination of substrates. However, limitation of glycolysis during ischaemia, as assessed by lactate release, appears to be an epiphenomenon of the preconditioning protocol and is not consistently related to protection. PKC activation appears to be pivotal to the mechanism of protection against contractile dysfunction, since administration of polymyxin B abolished any protection.

Can ischemic preconditioning protect against hypoxia-induced damage? Studies of contractile function in isolated perfused rat hearts.

Ischemic preconditioning in the rat significantly improves functional recovery following global ischemia by undefined mechanisms. It has been suggested that preconditioning protects by altering the tissue metabolic milieu during ischemia, either by increasing ischemic tissue accumulation of a beneficial substance (e.g. adenosine), or inhibiting tissue accumulation of a malefic component (e.g. protons). If this is the case, we hypothesized that no protection should be afforded by preconditioning against a prolonged period of hypoxia, since the continued coronary flow would prevent the accumulation of any metabolic products in the myocardium. To test this hypothesis, isolated buffer-perfused rat hearts were preconditioned by 5 min of ischemia + 5 min of reperfusion and then subjected to 30 min of ischemia, or 25 min of substrate-free hypoxia, or 60 or 90 min of hypoxia with substrate. Function was re-assessed after reperfusion/reoxygenation for a further 30 min and compared to non-preconditioned controls. Ischemic preconditioning improved functional recovery following 30 min of global ischemia (% recovery of developed pressure (LVDP) in control v preconditioned hearts was 31 +/- 4 v 66 +/- 6%; P < 0.05). Importantly, this protection was achieved almost entirely via a better preservation of diastolic function (end diastolic pressure = 78 +/- 3 mmHg in control and 40 +/- 5 mmHg in preconditioned hearts following 30 min of reperfusion; P < 0.05). However, no preconditioning-induced protection was observed following either substrate-free hypoxia or hypoxia with substrate (% recovery of LVDP in control v preconditioned hearts was 31 +/- 4 v 34 +/- 4% after 25 min of substrate-free hypoxia, 48 +/- 3 v 53 +/- 6% after 60 min of hypoxia + substrate and 25 +/- 5 v 30 +/- 6% after 90 min of hypoxia + substrate respectively). Furthermore, no protection by preconditioning against hypoxia-induced diastolic dysfunction was observed. We conclude that preconditioning protects against ischemic injury, but not hypoxic injury. Although hypoxia-induced injury may differ from that induced by ischemia, the results are consistent with the hypothesis that the continued presence of flow with hypoxia abolishes the protective effect of preconditioning. Furthermore, the results support the concept that preconditioning of the ischemic myocardium requires the accumulation of a factor in the ischemic myocardium, either to exert the preconditioning protective effect, or as a factor of injury against which preconditioning affords protection.

Thromboxane A2 mediates pulmonary hypertension after cardiopulmonary bypass in the rabbit.

Clinically, it is well established that cardiopulmonary bypass results in pulmonary dysfunction. Using a recently developed preparation for cardiopulmonary bypass in the rabbit, we have been able to mimic a similar, but more severe, condition. We found that, despite normal histologic structure of the myocardium, hearts could not be weaned from bypass because of a serious increase in pulmonary vascular resistance. Histologic studies of the lungs showed severe intravascular neutrophil aggregation and marked vasoconstriction. To identify the nature and origin of the mediator responsible for the changes in the pulmonary vasculature, we subjected groups of rabbits (n = 4 per group) to bypass with cooling to 18 degrees C, circulatory arrest for 1 hour, and rewarming on bypass to 33 degrees C. Pulmonary vascular resistance was measured at the same temperature before and after bypass. Four groups were studied: group I were untreated controls; group II received the cyclooxygenase inhibitor, indomethacin (0.2 mg/kg intravenously), before operation; group III received the thromboxane A2 synthetase inhibitor, Dazmegral (5 mg/kg intravenously), before operation together with the thromboxane A2 receptor blocker GR 32191B (2 mg/kg per 30 minutes intravenously); and group IV were treated with mustine hydrochloride (1.75 mg/kg intravenously) 3 days before the experiment to deplete the neutrophils by 90%. During circulatory arrest, the heart was protected with an initial infusion (10 ml at 4 degrees C over 1 minute) of St. Thomas' Hospital cardioplegic solution. At the end of the experiment, the heart and lungs were histologically examined. In the control group, a significant increase (+395% when compared with the value recorded before bypass) in pulmonary vascular resistance was observed after bypass. However, in none of the treated groups did pulmonary vascular resistance increase significantly (percentage changes in groups II, III, and IV were -24%, 0%, and +33%, respectively). Pulmonary histologic characteristics were normal in all treated groups, and all animals were successfully weaned from bypass. These results indicate that the increase in pulmonary vascular resistance that arises as a consequence of bypass in rabbits is primarily a result of the production of thromboxane A2, a process in which the neutrophil plays a pivotal role.

Preconditioning and post-ischaemic contractile dysfunction: the role of impaired oxygen delivery vs extracellular metabolite accumulation.

The aim of the present study was to identify components of ischaemia involved in the induction of preconditioning. Isolated rat hearts (n = 8 per group) were perfused with bicarbonate buffer. Following 10 min aerobic perfusion they were randomised and subjected to 5 min periods during which the perfusion conditions were: (i) normal aerobic perfusion (controls); (ii) zero flow ischaemia; (iii) low flow ischaemia (10% of control O2 delivery); (iv) hypoxia (10% of control O2 delivery); or (v) acidosis (pH 6.4). After these periods of "preconditioning", all hearts underwent 5 min aerobic perfusion followed by 40 min zero flow global ischaemia and 35 min reperfusion. Contractile function was measured at the beginning and at the end of the experiment. Despite profound differences in coronary flow during preconditioning, substantial and similar protection was observed in all groups preconditioned by transiently limiting oxygen delivery. Recovery of cardiac output was 66.7 +/- 6.3%, 58.7 +/- 5.1% and 62.6% +/- 3.3% in the zero flow, low flow and hypoxic groups, respectively, vs 31.0 +/- 3.0% in controls (all P < 0.05). In hearts subjected to acidosis there was no protection (recovery of cardiac output 38.1 +/- 2.7%). Impairment of oxygen delivery appears to be the principle component of ischaemia responsible for the induction of preconditioning. Metabolite accumulation appears to play no significant role.

Improved functional recovery by ischaemic preconditioning is not mediated by adenosine in the globally ischaemic isolated rat heart.

OBJECTIVE: A brief period of ischaemia (5 min) and reperfusion (5 min), prior to a longer period of ischaemia and reperfusion, has been shown to reduce the extent of injury (necrosis, arrhythmias, or postischaemic contractile malfunction) caused by a subsequent longer period of ischaemia and reperfusion. Adenosine has been identified as a factor in the protection afforded against regional tissue necrosis by such preconditioning. The aim of this study was to assess the role of adenosine in preconditioning induced protection of postischaemic function in the globally ischaemic isolated rat heart. METHODS: The ability of global ischaemia to precondition against postischaemic contractile malfunction was first confirmed in the isolated ejecting rat heart preparation. Hearts (n = 6 per group) were perfused aerobically (37 degrees C, paced at 350 beats.min-1) for 20 min, at the end of which contractile function was measured. This was followed by 10 min of Langendorff perfusion (control group) or 5 min of global ischaemia plus 5 min of Langendorff reperfusion (preconditioned group). The hearts were then subjected to 20 min of global ischaemia (37 degrees C) and 35 min of reperfusion (15 min Langendorff and 20 min ejecting); function was then reassessed. RESULTS: Postischaemic recovery of aortic flow was 26(SEM 8)% in the control group v 57(4)% in the preconditioned group (p < 0.05). To assess whether exogenous adenosine could mimic this protection, the experiments were repeated with the 5 min period of ischaemic preconditioning replaced by 5 min of aerobic Langendorff perfusion with adenosine-containing buffer (100, 50, or 10 mumol.litre-1). No protection of postischaemic function was observed in any of the adenosine treated groups. In further experiments, we assessed whether ischaemic preconditioning persisted in the presence of the A1/A2 adenosine antagonist, 8 (p-sulphophenyl) theophylline (8-SPT). Since pacing was not used in these studies, the ability of ischaemia to precondition the myocardium was again confirmed; the protocol was then repeated with 8-SPT (10 mumol.litre-1) present in the perfusate throughout. Although 8-SPT depressed recovery in both control and preconditioned hearts it failed to abolish the protective effects of ischaemic preconditioning. CONCLUSIONS: There is no evidence from these results to support the involvement of adenosine to any major extent in preconditioning induced protection of postischaemic contractile function in the isolated rat heart.

Ischaemic preconditioning and contractile function: studies with normothermic and hypothermic global ischaemia.

A significant reduction in the extent of cell necrosis or the incidence of reperfusion-induced arrhythmias can be achieved with ischaemic preconditioning. If preconditioning was also found to be effective in protecting against global ischaemia, then this may have significant implications for the preservation of the heart during cardiac surgery. We therefore investigated this phenomenon in relation to recovery of contractile function after global ischaemia in the isolated rat heart. Isolated working rat hearts (n = 6 per group) were perfused aerobically at 37 degrees C for 20 min and contractile function recorded. This was followed by 10 min of aerobic Langendorff perfusion (control hearts) or 5 min global ischaemia (37 degrees C) + 5 min Langendorff reperfusion (preconditioned hearts). The hearts were then subjected to 10, 15, 20 or 25 min of global ischaemia (37 degrees C) and reperfusion (15 min Langendorff + 20 min working) after which function was again assessed. Preconditioning improved functional recovery after all durations of ischaemia. Thus aortic flow after 10, 15, 20 and 25 min of ischaemia and 35 min of reperfusion recovered to 84, 58, 16 and 5%, respectively, in controls and 88, 74, 55 and 20%, respectively, in the preconditioned groups. To assess whether preconditioning was effective in a surgically relevant model of hypothermic ischaemia, the experiments were repeated with longer periods (45, 70, 90, 115, 135 and 160 min) of ischaemia at 20 degrees C. Under these conditions, normothermic preconditioning increase the post-ischaemic recovery of aortic flow after 115, 135 and 160 min of ischaemic (from 36, 20 and 10%, respectively, in controls to 57, 39 and 26%, respectively, in preconditioned hearts). There was no consistent correlation between tissue high energy phosphate content and enhanced post-ischaemic recovery. Thus, we have demonstrated that ischaemic preconditioning can improve contractile function after global ischaemia in the isolated rat heart, we have defined the duration of ischaemia for which it is operative, and we have shown that this protection is additive to that of hypothermia-induced protection during ischaemia. This may have clinical implications for cardiac surgery.

Mechanical unloading and infarct size: studies with the heterotopically transplanted rabbit heart.

The possibility that unloading the heart during regional ischemia and/or reperfusion may limit infarct size was investigated by inducing regional ischemia in both a heterotopically transplanted heart (unloaded) and the recipient's heart (loaded control). In this way, the extent of myocardial infarction was compared in paired hearts in the same animal under similar experimental conditions. Hearts excised from donor rabbits, were arrested with St. Thomas' Hospital cardioplegic solution and maintained at 15 degrees C for 1 hour during which time they were transplanted into the necks of recipient rabbits. 24 hours later, rabbits were reanesthetized and the left circumflex coronary artery ligated in both the transplanted and the recipient's hearts. After 1 hour of regional ischemia hearts were reperfused for 3 hours. The transplanted heart was paced at 205 beats per minute (bpm) throughout the experiment. Similar values for infarct size were obtained in both the loaded and unloaded hearts (73 +/- 4% vs 75 +/- 6%, respectively). No significant differences were seen in any other parameters. In conclusion, our results suggest that during regional ischemia the amount of work performed by the heart does not appear to be a major factor in determining the ultimate size of an infarct.