Regulation of sarcolemmal Na(+)/H(+) exchanger activity by angiotensin II in adult rat ventricular myocytes: opposing actions via AT(1) versus AT(2) receptors.

Increased sarcolemmal Na(+)/H(+) exchanger activity has been implicated as a mediator of the cardiac actions of angiotensin II. We studied the receptor subtypes and signaling pathways involved in the regulation of sarcolemmal Na(+)/H(+) exchanger activity by angiotensin II in adult rat ventricular myocytes. Cells were loaded with the pH-sensitive fluoroprobe carboxy-seminaphthorhodafluor-1, and acid efflux rates estimated during recovery from intracellular acidosis were used to quantify exchanger activity. Sarcolemmal Na(+)/H(+) exchanger activity was not affected by angiotensin II alone but was increased by angiotensin II plus PD123319 (AT(2) antagonist). In contrast, angiotensin II plus losartan (AT(1) antagonist) or CGP42112A (AT(2) agonist) did not affect exchanger activity. The increase in Na(+)/H(+) exchanger activity induced by angiotensin II plus PD123319 was blocked by losartan, PD98059 (extracellular signal-regulated kinase inhibitor), GF109203X (protein kinase C inhibitor), and tyrphostin AG1478 (epidermal growth factor receptor kinase inhibitor). Extracellular signal-regulated kinase phosphorylation and activity, measured by immunoblot analysis and an immune-complex kinase assay, respectively, were increased significantly by angiotensin II plus PD123319; these increases were blocked by losartan and PD98059. The increase in extracellular signal-regulated kinase phosphorylation induced by angiotensin II plus PD123319 was blocked also by GF109203X and tyrphostin AG1478. These data show that AT(1) stimulation increases sarcolemmal Na(+)/H(+) exchanger activity in adult rat ventricular myocytes and that this response requires extracellular signal-regulated kinase activation through a protein kinase C- and epidermal growth factor receptor-mediated mechanism. The positive effect of AT(1) stimulation on Na(+)/H(+) exchanger activity is counteracted by simultaneous AT(2) stimulation through a mechanism that does not involve direct inhibition of the exchanger or attenuation of extracellular signal-regulated kinase activation.

Determinants of infarct size during permanent occlusion of a coronary artery in the closed chest dog.

One hundred nine dogs subjected to experienced experimental coronary occlusion were retrospectively examined in an attempt to identify factors influencing infarct size. A coronary artery was occluded by an embolus. The field of the occluded artery (zone at risk) was determined by subsequent autoradiography of 141-cerium-labeled microspheres that were injected into the left ventricle 2 minutes after embolization. Fifty-eight dogs were analyzed after 24 hours of embolization and 51 after 48 hours. Infarct size (assessed by tetrazolium staining) was directly proportional to the size of the zone at risk in both groups. The percent of the risk zone that developed infarction was independent of risk zone size in both the 24 and 48 hour groups. No differences were seen between male and female animals or between the dogs with left circumflex or left anterior descending artery occlusion. Infarct size was also independent of the heart rate-systolic pressure product at the time of coronary occlusion, and of the time of year. Eighty percent of the variability in the portion of the risk zone that infarcted in this population could be explained by the level of collateral flow in the risk zone 2 minutes after embolization. The linear regression between the percent of the risk zone that developed infarction and collateral flow was the same in slope and intercept between the 24 and 48 hour groups. The correlation between infarct size and collateral flow was slightly better when collateral flow was expressed as a percent of flow in the nonischemic zone than when it was expressed in absolute terms.(ABSTRACT TRUNCATED AT 250 WORDS)

Temporal and spatial characteristics of evolving cell injury during regional myocardial ischemia in the dog: the "border zone" controversy.

An open chest dog heart with multiple coronary ligations was used to define the temporal and spatial characteristics of injury evolving during regional ischemia. With the use of a multiple (40 sample) biopsy device, adjacent transmural biopsy specimens were obtained from the transition zone between normal and ischemic tissue after 5, 30, 45, 60 and 120 minutes of ischemia. The first 1.8 mm of epicardial tissue was taken for the analysis of flow and metabolites. The results confirmed the existence of a sharp interface of flow and metabolism in the epicardial lateral plane at the boundary of the ischemic zone. There was no significant zone of intermediate injury (flow and metabolism being depressed uniformly throughout the ischemic area). Comparison of the distribution of flow determined by radiolabeled gadolinium-153 at onset of ischemia with that indicated by radiolabeled tin-113 microspheres given at the end of various periods of ischemia revealed no change in the position or steepness of the flow interface at any time during the first 2 hours of ischemia. This observation, together with the absence of any major redistribution or enhancement of residual flow to the ischemic zone, indicated that there was little or no significant collateralization between 5 and 120 minutes. Analysis of the adenosine triphosphate (ATP) content revealed a rapid depletion during the first 5 minutes of ischemia; the content then remained essentially unchanged until 30 minutes, after which time a second phase of accelerated ATP depletion was observed until 45 minutes. ATP content then remained relatively constant up to 2 hours.

Reperfusion-induced injury manifestations, mechanisms, and clinical relevance.

Although reperfusion is an absolute prerequisite for the survival of ischemic tissue, it is not necessarily without hazard. Many (but not all) cardiologists are of the opinion that some component of reperfusion may be detrimental and able to inflict injury over and above that attributable to the ischemia. In this article, we define four sequelae of reperfusion that might be designated as "reperfusion injury," we identify possible underlying mechanisms, and we consider whether any of these forms of reperfusion injury are of clinical relevance.

Microbiopsy metabolite and paired flow analysis: a new rapid procedure for homogenisation, extraction and analysis of high energy phosphates and other intermediates without any errors from tissue loss.

A new procedure is described which allows for the rapid homogenisation, extraction and analysis of the metabolite content of microbiopsy samples (milligram quantities) while completely overcoming the major errors arising as the consequence of the substantial and variable tissue loss associated with conventional procedures. In addition to allowing more accurate and faster analysis of much smaller quantities of tissue the procedure also allows for the coincident paired measurement of flow (radioactive microspheres) in each biopsy. An example of the application of the method to the measurement of flow and high energy phosphate content in multiple microbiopsy samples from normal and ischaemic canine myocardium is provided.

Reperfusion induced injury: manifestations, mechanisms, and clinical relevance.

Although reperfusion is an absolute prerequisite for the survival of ischaemic tissue, it is not necessarily without hazard. Many (but not all) cardiologists are of the opinion that some component of reperfusion may be detrimental and able to inflict injury over and above that attributable to the ischaemia. In this article we define four sequelae of reperfusion which might be designated as "reperfusion injury", we identify possible underlying mechanisms, and consider whether any of these forms of reperfusion injury are of clinical relevance.

Anti-arrhythmic effects of amiodarone and desethylamiodarone on malignant ventricular arrhythmias arising as a consequence of ischaemia and reperfusion in the anaesthetised rat.

The effects of amiodarone and its metabolite desethylamiodarone on arrhythmias induced by ischaemia and reperfusion were studied in vivo in the anaesthetised rat with transient regional ischaemia (7 min of left coronary artery occlusion) and reperfusion (10 min). Amiodarone or desethylamiodarone were administered intravenously either 10 min prior to ischaemia or 2 min prior to reperfusion. Control rats received an equivalent volume of vehicle. Administration of 5.0 mg.kg-1 amiodarone or desethylamiodarone prior to ischaemia reduced the incidence of ventricular tachycardia during the ischaemic period from 67% to 20% (p less than 0.02) and 47% respectively. During reperfusion, mortality was reduced from 53% to 7% and 7% (p less than 0.02) respectively, and reperfusion induced ventricular fibrillation from 73% to 20% (p less than 0.01) and 47% respectively. When the drugs were given prior to ischaemia, the plasma concentrations of amiodarone and desethylamiodarone were 1.03 (SEM 0.18) and 0.22(0.02) micrograms.ml-1 and the myocardial concentrations were 23.43(2.78) and 30.41(1.87) micrograms.g-1 respectively. Similar concentrations were found in plasma and myocardium with drugs given prior to reperfusion. No significant differences in plasma or myocardial concentrations of amiodarone or desethylamiodarone were observed between animals which developed ventricular fibrillation and those which did not. In conclusion, this study demonstrates that desethylamiodarone can, like its parent compound, protect the heart against malignant ventricular arrhythmias arising as a consequence of regional myocardial ischaemia and reperfusion.

Anti-arrhythmic protection by ischaemic preconditioning in isolated rat hearts is not due to depletion of endogenous catecholamines.

OBJECTIVES: The present study addresses whether the mechanism of anti-arrhythmic protection by ischaemic preconditioning involves depletion of myocardial catecholamines. METHODS: In a randomised series of studies isolated rat hearts, perfused with whole blood, underwent episodes of regional ischaemia and reperfusion induced with a snare around the left coronary artery. Control hearts (Group 1, n = 12) were subjected to 40 min aerobic perfusion, 30 min ischaemia and 10 min reperfusion. Preconditioned hearts (Group 2, n = 12) were subjected to 10 min aerobic perfusion, three cycles of ischaemia and reperfusion (5 min each), 30 min ischaemia and 10 min reperfusion. Cardiac rhythm was recorded continuously and arrhythmias quantified during the final periods of ischaemia and reperfusion. At the end of the experiment samples of right ventricular (RV; non-ischaemic) and left ventricular (LV; ischaemic territory) tissue were separated and frozen. In 5 additional groups (n = 6/group) tissue samples were taken after 10 min aerobic perfusion, after 10 min aerobic perfusion followed by 1, 2 or 3 preconditioning cycles and after 40 min of aerobic perfusion. All tissue samples were analysed for catecholamine content. RESULTS: Preconditioning resulted in reductions in the incidence of ischaemia-induced VF from 67% in Group 1 to 8% in Group 2, the incidence of ischaemia-induced VT from 100% to 17% and the number of ischaemia-induced VPBs from 246 +/- 25 to 59 +/- 19 (each P < 0.05). The mean content of noradrenaline and adrenaline was consistently higher in RV than LV tissue. Within the LV, however, neither preconditioning nor prolonged ischaemia had any significant effect upon tissue catecholamine content at any time in the experimental protocol. CONCLUSIONS: Depletion of myocardial catecholamines is not involved in the mechanism of anti-arrhythmic protection by ischaemic preconditioning in isolated rat hearts.

Species differences in vulnerability to injury by oxidant stress: a possible link with calcium handling?

STUDY OBJECTIVE: The aim was to exploit species- and age dependent differences in calcium handling to assess the role of calcium as a mediator of oxidant stress induced injury in the mammalian heart. DESIGN: Hearts from immature and mature rats and rabbits were perfused aerobically with buffer containing rose bengal which was photoactivated by illumination--a process that generates singlet oxygen and superoxide. This oxidant stress rapidly leads to electrocardiographic abnormalities, ultrastructural injury, and a redistribution of calcium. EXPERIMENTAL MATERIAL: Isolated perfused hearts from mature (60-90 d old) and immature (4-6 d old) rats, and mature (60-90 d old) and immature (7-10 d old) rabbits. MEASUREMENTS AND MAIN RESULTS: In hearts (n = 6 per group) from mature rats, aerobic perfusion and photoactivation for 5 min with buffer containing rose bengal (250 and 1000 nmol.litre-1) had no significant effect upon heart rate (until the onset of arrhythmias) but did result in a dose dependent transient increase in coronary flow. ECG abnormalities appeared within less than 30 s and these deteriorated to ventricular premature beats (VPB) and ventricular tachycardia (VT) in most hearts. By contrast, in identical studies with comparable sized hearts from young rabbits, ECG changes and arrhythmias occurred later, and the incidence of VT and the mean number of VPB were lower. Although arrhythmias were less severe in rabbit hearts, photoactivation of rose bengal caused both coronary flow and heart rate to fall in a dose dependent manner. Increasing the concentration of rose bengal to a value (2500 nmol.litre-1), which would be highly toxic in the rat heart, increased the severity of injury in the rabbit heart, the nature of which differed from that in the rat heart. To assess whether developmental state, as opposed to species, was a factor determining the differences in vulnerability to injury, hearts from immature rats and adult rabbits were perfused with various concentrations of rose bengal (250-2500 nmol.litre-1) with the intensity of illumination (1400-6600 Lux) adjusted to account for the size of the heart. Under these conditions rabbit hearts were found to be less vulnerable to injury and arrhythmias than rat hearts, and hearts from immature rats were found to be less vulnerable than those from adult rats. CONCLUSIONS: The results of this and other studies suggest that species and developmental differences in calcium handling (sarcolemmal v sarcoplasmic reticulum control) might explain the differences in vulnerability to arrhythmias.

Temperature-dependency of nifedipine as a protective agent during cardioplegia in the rat.

An isolated working rat heart preparation was used to characterise the temperature-dependency of the anti-ischaemic properties of nifedipine. In this study hearts were subjected to pre-ischaemic infusion with the St Thomas' cardioplegic solution with or without added nifedipine (0.075 mumol X litre-1). Hearts were then rendered globally ischaemic for various periods, (35, 42, 48, 56, 55, 65, 80, 105 or 130 min) at various temperatures (37.0, 35.5, 34.0, 32.5, 31.0, 29.0, 27.0, 24.0 or 20.0 degrees C, respectively). The duration of ischaemia at each temperature was selected to produce a post-ischaemic (37 degrees C) recovery of aortic flow that was approximately 50% of its pre-ischaemic (37 degrees C) control. In addition to functional indices (aortic flow, cardiac output, coronary flow, aortic pressure and heart rate) creatine kinase leakage during reperfusion was measured. At all temperatures at or above 31 degrees C the addition of nifedipine enhanced significantly (maximal value = 43%) the post-ischaemic recovery of aortic flow and other indices of pump function, while at the same time reducing significantly (by up to 56%) enzyme leakage. At ischaemic temperatures below 31 degrees C nifedipine failed to afford any significant additional protection when assessed functionally or enzymatically. It would therefore appear that hypothermia either blocks the action of nifedipine or, by acting on some common mechanism, renders the actions of the drug redundant.

Functional and electrophysiological effects of oxidant stress on isolated ventricular muscle: a role for oscillatory calcium release from sarcoplasmic reticulum in arrhythmogenesis?

STUDY OBJECTIVE: The aim was to investigate the cellular basis of oxidant stress induced arrhythmias by studying the influence of oxidant stress on the contractile and electrophysiological function of isolated cardiac muscle. DESIGN: Oxidant stress was induced by the photoactivation of rose bengal added to the solution superfusing isolated ventricular muscles from a number of species. Measurements of contractile and electrophysiological function were made under control conditions, during exposure to oxidant stress, and under a number of experimental conditions. EXPERIMENTAL MATERIAL: Isolated superfused papillary muscles or trabeculae from rat, rabbit, or frog hearts were used in all studies. MEASUREMENTS AND MAIN RESULTS: The contractile response to oxidant stress was assessed by measuring isometric developed tension and resting tension throughout the experiment, and the electrophysiological response was assessed by recording action potentials using conventional 3 M KCl filled intracellular electrodes. Oxidant stress induced a transient positive inotropy, after-contractions, and eventually contracture. Associated with these contractile changes were prolongation of the action potential, early afterdepolarisations, oscillations in resting membrane potential, and automaticity. These effects were concentration and species dependent and the oscillations in both tension and membrane potential were abolished by inhibition of calcium release from the sarcoplasmic reticulum with caffeine. CONCLUSIONS: The contractile and electrophysiological effects of rose bengal induced oxidant stress are consistent with a cellular calcium overload. The observation that the oscillations in tension and membrane potential were abolished by caffeine and that these effects were species dependent (rat greater than rabbit greater than frog) suggests a role for oscillatory sarcoplasmic reticulum calcium release in these effects. The oscillations in membrane potential and the automaticity induced by rose bengal are likely to underlie the arrhythmias observed in isolated hearts exposed to oxidant stress that have previously been described.

Effects of diltiazem as an additive to St Thomas's Hospital cardioplegic solution in isolated neonatal and adult rabbit hearts.

STUDY OBJECTIVE: The aim was to compare the protective efficacy of diltiazem as an additive to St Thomas's Hospital cardioplegic solution in isolated hearts from neonatal and adult rabbits. DESIGN: The relative responsiveness of the two age groups to diltiazem was first determined in Langendorff perfused hearts (n = 6 per group) by constructing concentration-response curves for the drug's negative inotropic effect. The IC20 and IC50 of diltiazem (the concentrations of diltiazem resulting in 20% or 50% reduction in developed pressure, respectively) were the doses subsequently included in the cardioplegic solution. Isolated working hearts (n = 8 per group) were perfused aerobically (37 degrees C) for 20 min followed by a 2 min infusion of St Thomas's Hospital cardioplegic solution with or without added diltiazem. All hearts were then subjected to global ischaemia (37 degrees C). The durations of ischaemia were 60 min in the neonatal and 45 min in the adult heart. Hearts were then reperfused (15 min Langendorff, 20 min working) before reassessment of function. EXPERIMENTAL ANIMALS: 30 neonatal (7-10 d) and 30 adult (2-3 months) New Zealand white rabbits were used. MEASUREMENTS AND MAIN RESULTS: The IC20 and IC50 of diltiazem were found to be 0.1 and 0.5 mumol.litre-1, respectively, in the neonatal heart, and 0.5 and 2.5 mumol.litre-1, respectively, in the adult heart. Postischaemic recovery of cardiac output was 57.9(SEM 6.7)% in the control group and 64.1(5.0)% (NS) and 47.7(3.8)% (NS) in the 0.5 mumol.litre-1 and 2.5 mumol.litre-1 diltiazem groups, respectively, in the adult hearts. In the neonatal hearts, cardiac output recovered to 55.6(4.8)% in the control group and 59.9(4.2)% (NS) and 62.0(5.6)% (NS) in the 0.1 mumol.litre-1 and 0.5 mumol.litre-1 diltiazem groups, respectively. CONCLUSIONS: The addition of diltiazem, at IC20 or IC50 negative inotropic concentrations, to St Thomas's Hospital cardioplegic solution does not improve postischaemic recovery in either neonatal or adult rabbit hearts. This suggests that the slow calcium channel may not be an important mediator of ischaemia and reperfusion induced injury in the cardioplegically arrested rabbit heart.

Oxygen supply and oxygen demand in the isolated working rabbit heart perfused with asanguineous crystalloid solution.

STUDY OBJECTIVE: It has been suggested that oxygenation of the isolated, crystalloid perfused, Langendorff rabbit heart is inadequate and that consequent hypoxia limits mechanical performance. The isolated working rabbit heart, which has a higher oxygen requirement than the Langendorff preparation, was used to investigate the relationships between the ability of the heart to perform external work, myocardial oxygen consumption, and tissue high energy phosphate content. DESIGN: Hearts from adult rabbits (1.5-2.2 kg), paced at 200 beats.min-1, were perfused in the working mode with bicarbonate buffer (1.8 mmol.litre-1 Ca2+; PO2 greater than 85 kPa, 630 mm Hg). MEASUREMENTS AND MAIN RESULTS: The effects of (a) increasing preload (10, 14, 18, 22, 26 cm H2O) at fixed afterload (80 cm H2O) and (b) increasing afterload (60, 80, 100, 120 cm H2O) at fixed preload (18 cm H2O) on left ventricular power and myocardial oxygen consumption were determined. Increasing preload (from 10 to 26 cm H2O) resulted in linear increases in left ventricular power [from 1.36(SEM 0.06) to 2.81(0.19) J.min-1] and myocardial oxygen consumption [from 554(37) to 801(59) microliters.g dry wt-1.min-1]; this was made possible by increased oxygen supply secondary to increased coronary flow. Increasing afterload (from 60 to 120 cm H2O) also increased left ventricular power [from 1.76(0.05) to 2.16(0.09) J.min-1] and myocardial oxygen consumption [from 617(48) to 903(30) microliters.g dry wt-1.min-1]. For a given increase in left ventricular power, the increase in myocardial oxygen consumption was greater if the increased workload was due to an increased afterload than if it was due to an increased preload, suggesting that increasing afterload resulted in a reduced external mechanical efficiency. Prolonged perfusion under conditions of low and high workload (80 and 120 cm H2O afterload for 60 min) was associated with comparable myocardial contents of adenosine triphosphate [22.5(1.4) and 21.7(0.4) mumols.g dry wt-1] and creatine phosphate [21.8(4.5) and 23.7(1.9) mumols.g dry wt-1] and comparable rates of lactate efflux [34.7(13.0) and 36.7(12.5) mumols.g dry wt-1.60 min-1]. CONCLUSION: Our observation of increased myocardial oxygen consumption and comparable contents of high energy phosphates with increased workload would suggest that the crystalloid perfused working rabbit heart is adequately oxygenated.

Effects of potassium channel modulation during global ischaemia in isolated rat heart with and without cardioplegia.

OBJECTIVE: The opening of potassium (K+) channels during regional ischaemia may, by inducing rapid contractile arrest, be an intrinsic energy sparing mechanism. Thus K+ channel openers (for example, lemakalim) exert significant anti-ischaemic effects, whereas glibenclamide exacerbates ischaemic contracture and limits postischaemic functional recovery. The aim of the study was to investigate the ability of these compounds to influence ischaemic injury when used either alone or in combination with rapid arrest induced by a high K+ cardioplegic solution. METHODS: Contractile function of isolated Langendorff perfused rat hearts was assessed using an intraventricular balloon. Hearts were subjected to normothermic global ischaemia (20 min) or cardioplegic arrest (35 min) with and without glibenclamide or lemakalim. Lemakalim (10 mumol.litre-1) or glibenclamide (10 mumol.litre-1) was given, in the presence or absence of cardioplegia, for 2 min immediately prior to the onset of ischaemia. The rate of ischaemia induced contractile failure, the severity of ischaemic contracture, and the degree of postischaemic functional recovery were all measured. RESULTS: In the absence of cardioplegia, the time to contractile arrest in control hearts was 133 (SEM 4) s. This was increased by glibenclamide, to 145(6) s, and decreased by lemakalim, to 112(6) s. The time to onset of ischaemic contracture [8(1) min] was accelerated by glibenclamide [4(1) min] and delayed by lemakalim [14(1) min]. Lemakalim significantly improved the recovery of left ventricular developed pressure from 49(7)% in control hearts to 65(3)%, and left ventricular end diastolic pressure from 41(3) to 21(4) mm Hg. Hearts pretreated with glibenclamide showed similar recoveries to control hearts. During reperfusion, lemakalim exerted a transient vasodilator effect whereas glibenclamide caused a transient vasoconstriction. When either glibenclamide or lemakalim was added to a high K+ cardioplegic solution and hearts rendered ischaemic for 35 min, the ability of both compounds to influence ischaemic contracture and postischaemic functional recovery was lost. In additional studies the effect of lemakalim on the relative times to ischaemia induced mechanical failure and electrical arrest was assessed. In control hearts the time to contractile failure was 128(5) s and the time to electrical arrest was 241(30) s, while in the lemakalim treated hearts the values were 103(2) s and 509(161) s, respectively. In the lemakalim group all the hearts showed sustained ventricular arrhythmias; in the control group there were no arrhythmias. CONCLUSIONS: Lemakalim can exert a significant anti-ischaemic effect when given alone. This effect is lost when it is used in combination with high K+ cardioplegia. The anti-ischaemic properties of lemakalim may thus be limited to its ability to accelerate contractile arrest.

Rate of reflow and reperfusion induced arrhythmias: studies with dual coronary perfusion.

OBJECTIVE: The aim was to determine the relationship between the rate of reflow and vulnerability to reperfusion induced arrhythmias. METHODS: Isolated rat hearts, in which left and right coronary arteries were perfused independently, were subjected to transient (10 min) cessation of flow to the left coronary bed. During the subsequent 10 min of reperfusion, flow in the left coronary bed was regulated to create five different reflow profiles (n = 12 per group): (1) immediate restoration of 100% flow which was maintained throughout reperfusion (control); (2) stepwise restoration of flow with 10% flow from 0-1 min, 20% flow from 1-2 min, 40% flow from 2-3 min, 60% flow from 3-4 min, 80% flow from 4-5 min, and 100% flow from 5-10 min; (3) 10% flow from 0-5 min and 100% flow from 5-10 min; (4) 20% flow from 0-5 min and 100% flow from 5-10 min; and (5) 40% flow from 0-5 min and 100% flow from 5-10 min. RESULTS: There were no significant differences between the groups in the incidences of reperfusion induced ventricular tachycardia or fibrillation, which were 100% and 58-92%, respectively. However, the time to onset of reperfusion induced ventricular fibrillation was delayed (p less than 0.05) from 15 (SEM 1) s in group (1) to 191(39), 210(38), and 77(29) s in groups 2, 3, and 4, respectively. No significant delay was observed in group 5. There were no significant differences between the groups in size of ischaemic zone or heart rate. CONCLUSIONS: In crystalloid perfused hearts, restricted reflow delays the time to onset of reperfusion induced ventricular fibrillation but does not reduce the incidence of reperfusion induced ventricular tachycardia or fibrillation.

Photoactivation of porphyrins: studies of reactive oxygen intermediates and arrhythmogenesis in the aerobic rat heart.

STUDY OBJECTIVE: In situ production of reactive oxygen intermediates (singlet oxygen and superoxide) during the photoactivation of rose bengal can induce arrhythmias in the aerobically perfused rat heart. The present study was undertaken (1) to assess whether these effects occur with other photosensitizers; (2) to identify the injurious intermediates; (3) to probe the site of action of these phenomena. DESIGN - The study involved the use of meso-tetra-(4-sulphonatophenyl)-porphine (TPPS), a porphyrin which, in contrast to rose bengal, promotes the production of singlet oxygen alone when illuminated. After 10 min of TPPS free perfusion, rat hearts (n = 6 per group) were perfused aerobically with TPPS (1, 5, 10 or 50 mumol.litre-1) for 25 min; during the last 20 min, the hearts were illuminated (3600 lux). In additional studies, TPPS (50 mumol.litre-1) was washed out before illumination. EXPERIMENTAL MATERIAL: Hearts from 30 male Wistar rats, weighing 220-280 g, were excised and perfused retrogradely. MEASUREMENTS AND MAIN RESULTS: Cardiac function was unaffected with TPPS alone. Upon illumination, electrocardiographic changes (increase in QT interval and/or T wave changes) and arrhythmias developed in a dose dependent manner. At the highest dose, electrocardiographic changes occurred within 7.0(SEM 0.4) s; all hearts exhibited ventricular premature beats and complete atrioventricular block; 67% developed ventricular tachycardia and 17% ventricular fibrillation. During illumination, hearts also exhibited a dose and time dependent decrease in coronary flow. In additional studies, despite the absence of TPPS in the perfusate, all hearts exhibited complete atrioventricular block, 67% developed ventricular premature beats and 33% ventricular tachycardia; none exhibited ventricular fibrillation. CONCLUSIONS: The results suggest that singlet oxygen, as opposed to superoxide, is responsible for the injury which occurs at tissue surfaces to which photosensitizer is bound.

Lipid hydroperoxide modification of proteins during myocardial ischaemia.

OBJECTIVE: Lipid hydroperoxides (LOOH) are lipid peroxidation products formed during oxidative stress. A component of their cytotoxicity is mediated by the direct modification of proteins. OBJECTIVES: (i) To assess whether ischaemia and reperfusion in the isolated rat heart generates LOOH-protein (ii) to characterise the extent, time-course and subcellular localization of any protein adducts formed. METHODS: Using a well-characterised antibody which binds to LOOH-modified proteins and densitometry of Western blots, we quantified the amounts of LOOH-protein in control aerobically perfused rat hearts and those subjected to ischaemia with and without reperfusion. RESULTS: Hearts (n=3/4 group), analysed after various periods (0, 5, 10, 20 and 30 min) of zero-flow global ischaemia, exhibited a time-dependent increase in the LOOH-mediated modification of a number of proteins. In hearts subjected to 30 min of ischaemia and then reperfused for various times (0, 5, 10, 20, 30 or 60 min) no changes in LOOH-protein content achieved during the proceeding ischaemic episode were detected. Reperfusion after short periods of ischaemia (5 or 10 min) also did not result in reperfusion-induced LOOH-protein formation. Administration of mercaptopropionylglycine (1 mM) to hearts for 5 min before the onset of 30 min ischaemia efficiently attenuated the formation of LOOH-protein, maintaining the modified proteins at control levels. These Western immunoblot results were confirmed by additional in situ immunofluorescent studies which showed marked LOOH-protein immunostaining in ischaemic tissue around the sarcolemmal membrane. CONCLUSIONS: We conclude that the modification of proteins (particularly those associated with sarcolemmal membranes) by LOOH during ischaemia may contribute to the pathophysiology of ischaemic injury. In addition, these modifications may be initiators of oxidant-induced signal transduction pathways. These findings are consistent with an oxidant stress occurring during ischaemia which is not exacerbated or reduced during the first 60 min of reperfusion.

Metabolic and functional effects of the nucleoside transport inhibitor R75231 in the ischaemic and blood reperfused rabbit heart.

OBJECTIVE: The ability of R75231, a nucleoside transport inhibitor, to influence adenine nucleotide metabolism and enhance postischaemic functional recovery was assessed in the blood perfused rabbit heart. METHODS: Hearts (n = 8 per group) from donor animals were excised and perfused with blood at 37 degrees C from a support rabbit. After 20 min of aerobic perfusion hearts were arrested with St Thomas' Hospital cardioplegic solution (2 min at 37 degrees C) and rendered globally ischaemic for 60 min. This was followed by 60 min of reperfusion. R75231 (0.1 mg.kg-1, intravenously) was given to donor and support rabbits 1 h before the experiment, control rabbits receiving the same volume of vehicle. RESULTS: Treatment with R75231 resulted in a 45% reduction in coronary vascular resistance in aerobically perfused control hearts, an effect that was absent during postischaemic reperfusion. Thus, before ischaemia, coronary flow was greater in R75231 treated hearts [6.6(SEM 0.8) ml.min-1] than in controls [4.3(0.6) ml.min-1; p < 0.05] but during reperfusion no significant difference was observed [4.0(0.6) v 3.6(0.3) ml.min-1]. The mean time to onset and extent of contracture during ischaemia was similar in R75231 treated and control groups, at 42(4) v 41(4) min and 27(3) v 26(6) mm Hg, respectively. Left ventricular developed pressure recovered to approximately 50% of its preischaemic value during the first 40 min of reperfusion in both groups; however, after longer durations of reperfusion, it tended to deteriorate in the R75231 treated group whereas it was maintained at a constant level in the controls [37(10) v 53(6) mm Hg, respectively; NS]. At the end of reperfusion, tissue adenosine content was 13-fold greater in the R75231 treated group, at 0.40(0.09) v 0.03(0.01) mumol.g-1 dry wt in controls; p < 0.05; the nucleotide pool, nicotinamide adenine dinucleotide phosphate content, and the energy charge potential were similar in groups. CONCLUSIONS: R75231 decreased coronary vascular resistance and increased coronary flow during aerobic perfusion in control hearts, an effect that was lost after ischaemia and reperfusion. R75231 also increased greatly the tissue content of adenosine but, despite this, failed to improve either the recovery of cardiac contractile function or the replenishment of the adenine nucleotide pool.

The antiarrhythmic action of ischaemic preconditioning in rat hearts does not involve functional Gi proteins.

OBJECTIVE: Ischaemic preconditioning has been reported to be mediated by inhibitory G (Gi) proteins in rabbits; however, the mechanism of preconditioning in rats appears to differ from that in other species. The aim of this study was to determine whether functional Gi proteins are required for the antiarrhythmic action of preconditioning in rats. METHODS: Donor rats were randomised to receive pertussis toxin (25 micrograms.kg-1 intravenously) or saline. After 48 h the hearts were isolated and Langendorff perfused with blood from untreated support rats. Cardiac rhythm was recorded continuously. Ischaemia and reperfusion were induced by occluding and releasing a snare around the left coronary artery. Following 10 min of aerobic perfusion hearts were further randomised to: (1) control groups (n = 12 per group) which underwent a further 30 min of aerobic perfusion, or (2) preconditioned groups (n = 12 per group) which were subjected to three cycles of 5 min of ischaemia and 5 min of reperfusion. All hearts subsequently underwent 30 min of regional ischaemia and 10 min of reperfusion during which arrhythmias were quantified. RESULTS: In hearts not pretreated with pertussis toxin, preconditioning limited the severity of ischaemia induced arrhythmias. The incidence of ventricular tachycardia was reduced from 100% to 33% and the mean number of ventricular premature beats from 164(SEM 42) to 23(14) (each p < 0.05). Although pretreatment with pertussis toxin completely abolished the bradycardic response to both acetylcholine and adenosine (indicating functional blockade of Gi proteins), it did not significantly influence the degree of antiarrhythmic protection afforded by preconditioning. In pretreated hearts preconditioning reduced the incidence of ventricular tachycardia from 83% to 33% and the mean number of ventricular premature beats from 267(66) to 62(32) (each p < 0.05). CONCLUSIONS: The antiarrhythmic action of preconditioning in isolated blood perfused rat hearts does not require functional Gi proteins.

Species variation in the coronary collateral circulation during regional myocardial ischaemia: a critical determinant of the rate of evolution and extent of myocardial infarction.

To determine residual flow to ischaemic tissue, which is the primary determinant of the rate of development and ultimate size of the myocardial infarct resulting from coronary artery occlusion, the coronary collateral circulation was quantified during acute myocardial ischaemia in eight species in vivo using the radiolabelled microsphere technique. In each case, a prominent branch of the left coronary artery was ligated, and within 5 min microspheres (141Ce labelled, 15 micron diameter) were injected intra-atrially. Hearts were then excised, frozen, and sliced perpendicular to the septum. Using autoradiograms as a guide, tissue samples were obtained from non-ischaemic and ischaemic tissue and the radioactivity of the ischaemic samples measured and expressed as a percentage of the activity in the non-ischaemic myocardium. In the guinea pig heart, despite ligation of a major artery, no zone of significant underperfusion was detected. In the hearts from other species, coronary collateral flow (as a percentage (mean(SEM)) of non-ischaemic flow) was: dog 15.9(1.8) (n = 6); cat 11.8(1.1) (n = 16); rat 6.1(0.7) (n = 6); ferret 2.4(0.6) (n = 6); baboon 2.1(0.3) (n = 6); rabbit 2.0(0.5) (n = 9); pig 0.6(0.2) (n = 6). The dog and cat hearts both possessed transmural gradients of collateral flow with greatest delivery in the epicardium. The patterns of flow distribution in the guinea pig heart were further examined in a Langendorff perfused preparation. Blue dye was injected into the coronary circulation and its distribution over 5 s recorded on cine film. After ligation of the left anterior descending or circumflex arteries, or both, the perfusion field of these arteries was seen to fill retrogradely within seconds. It is concluded that a wide spectrum of collateral flow exists between various mammalian species, a fact that should be taken into account in the study of the pathophysiology and control of regional ischaemia and myocardial infarction.