The low oxygen-carrying capacity of Krebs buffer causes a doubling in ventricular wall thickness in the isolated heart.

The buffer-perfused Langendorff heart is significantly vasodilated compared with the in vivo heart. In this study, we employed ultrasound to determine if this vasodilation translated into changes in left ventricular wall thickness (LVWT), and if this effect persisted when these hearts were switched to the "working" mode. To investigate the effects of perfusion pressure, vascular tone, and oxygen availability on cardiac dimensions, we perfused hearts (from male Wistar rats) in the Langendorff mode at 80, 60, and 40 cm H2O pressure, and infused further groups of hearts with either the vasoconstrictor endothelin-1 (ET-1) or the blood substitute FC-43. Buffer perfusion induced a doubling in diastolic LVWT compared with the same hearts in vivo (5.4 +/- 0.2 mm vs. 2.6 +/- 0.2 mm, p < 0.05) that was not reversed by switching hearts to "working" mode. Perfusion pressures of 60 and 40 cm H2O resulted in an increase in diastolic LVWT. ET-1 infusion caused a dose-dependent decrease in diastolic LVWT (6.6 +/- 0.4 to 4.8 +/- 0.4 mm at a concentration of 10(-9) mol/L, p < 0.05), with a concurrent decrease in coronary flow. FC-43 decreased diastolic LVWT from 6.7 +/- 0.5 to 3.8 +/- 0.7 mm (p < 0.05), with coronary flow falling from 16.1 +/- 0.4 to 8.1 +/- 0.4 mL/min (p < 0.05). We conclude that the increased diastolic LVWT observed in buffer-perfused hearts is due to vasodilation induced by the low oxygen-carrying capacity of buffer compared with blood in vivo, and that the inotropic effect of ET-1 in the Langendorff heart may be the result of a reversal of this wall thickening. The implications of these findings are discussed.

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.

Differential uptake of FDG and DG during post-ischaemic reperfusion in the isolated, perfused rat heart.

Fluorine-18 2-fluoro-2-deoxyglucose (FDG) and 2-deoxyglucose (DG) are widely used as tracers of glucose uptake in the myocardium. Although there is agreement that the two analogues behave similarly to glucose under control conditions, there is growing evidence that some interventions (e.g. insulin stimulation or ischaemia/reperfusion) cause differential changes in their behaviour. The addition of a two-surface coil nuclear magnetic resonance (NMR) probe and a dual-perfusion cannula to our recently developed PET and NMR dual-acquisition (PANDA) system allows us to collect PET (FDG) images and phosphorus-31 NMR (2-deoxyglucose-6-phosphate) spectra simultaneously from each independently perfused coronary bed of the heart. We have used this technique to study the effect of regional ischaemia/reperfusion on FDG and DG uptake in the isolated, perfused rat heart. During control perfusion, FDG uptake was almost identical in both coronary beds. When one coronary bed was made ischaemic, FDG uptake ceased on that side but continued on the control side. Reperfusion failed to restore FDG uptake. In contrast, NMR spectra showed that, during reperfusion, the uptake and phosphorylation of DG did not differ between the two coronary beds. The results thus demonstrate that regional myocardial ischaemia/reperfusion has different effects on the uptake of FDG and DG in the isolated, perfused rat heart.

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.

Depressed adenosine and total purine catabolite production in the postischemic rat heart.

The influence of global ischemia on myocardial nucleotide catabolite production and contraction was studied in Langendorff-perfused rat hearts. Hearts in the "ischemic" group were subjected to a 25 minute period of global ischemia at 30 minutes from the start of perfusion, and to two 30 s periods of global ischemia at 20 and 80 minutes of perfusion. Control hearts were subjected to three 30 s periods of ischemia at 20, 45 and 80 minutes of perfusion. Left ventricular developed pressure and concentrations of total nucleotide catabolites and adenosine in the coronary effluent were measured throughout. The concentration of nucleotide catabolites increased transiently by 2.1 +/- 0.5 microM in the control group and 2.2 +/- 0.8 microM in the "ischemic" group, immediately after 30 s ischemia at 20 minutes; while the concentration of adenosine increased transiently by 0.17 +/- 0.08 microM in the control group and 0.13 +/- 0.09 microM in the "ischemic" group. The next 30 s ischemic period in control hearts caused nucleotide catabolites to increase by 1.7 +/- 0.5 microM and adenosine by 0.12 +/- 0.06 microM. In the "ischemic" group, massive purine release was observed after 25 minutes of ischemia, the release decreasing to below pre-ischemic levels after 10 minutes of reperfusion. The increases in effluent nucleotide catabolites and adenosine in response to 30 s ischemia at 80 minutes were 1.4 +/- 0.4 microM and 0.13 +/- 0.1 microM, respectively, in the control group. In contrast, in the "ischemic" group, nucleotide catabolites increased by only 0.3 +/- 0.2 microM and adenosine by 0.011 +/- 0.008 microM after 30 s ischemia at the same time.(ABSTRACT TRUNCATED AT 250 WORDS)

Anion manipulation, a novel antiarrhythmic approach: mechanism of action.

We recently reported that modulation of anion homeostasis by substitution of extracellular chloride by nitrate prevents ischaemia- and reperfusion-induced ventricular fibrillation (VF) in rat and rabbit in vitro by an unknown mechanism independent of haemodynamic changes but related to widening of QT interval (Ridley and Curtis 1991). In the present study we have examined three possible explanations for the mechanism: modification of membrane anion permeability, alteration of cyclic nucleotide homeostasis and alteration of intracellular pH. In isolated Langerdorff-perfused rat heart (n = 12/group), substitution of chloride in modified Krebs perfusion solution by anion surrogates (methylsulphate, bromide, nitrate or iodide) inhibited left regional ischaemia- and reperfusion-induced arrhythmias only when the membrane permeability of the surrogate was greater than that of chloride (e.g., nitrate, bromide, iodide); the least permeant anion, methylsulphate, was proarrhythmic during ischaemia. Rank order of arrhythmia susceptibility correlated with the relative permeability of each anion, with near abolition of both ischaemia- and reperfusion-induced VF (P < 0.05) by the most permeant anions (iodide and nitrate). Arrhythmia suppression occurring in the iodide and nitrate groups was accompanied by significant widening of QT interval at 90% repolarization, with effects substantially more marked during ischaemia than before ischaemia. In separate studies using the same model we determined cardiac cyclic (c) AMP and cGMP content and their molar ratios by radioimmunoassay of biopsies before, during and after ischaemia. There was no meaningful relation between cyclic nucleotide content and rank order of arrhythmia susceptibility ruling out changes in the former as a contributory mechanism to the latter. In further studies we measured intracellular pH in the isolated perfused rat heart by phosphorus NMR spectroscopy. Nitrate caused a slight intracellular acidosis which was exacerbated when hearts were made globally ischaemic, indicating that its antiarrhythmic activity was not a consequence of alkalinisation (e.g., via inhibition of chloride-bicarbonate exchange). To test for inherent adverse effects on cardiac contractile function we analysed Starling curves in isolated rat hearts perfused under conditions equivalent to those used for arrhythmia studies. There was no relationship between perfusion anion composition and systolic (developed pressure at constant intraventricular volume, and pressure-volume slope) or diastolic function (end-diastolic pressure at constant intraventricular volume). In conclusion, alteration of membrane permeability is a mechanism which may be sufficient to explain modulation of arrhythmias by manipulation of extracellular anion content, appears to be devoid of deleterious effects on contractile function, and may represent a focus for future antiarrhythmic drug development.

Absolute quantification and NMR visibility of glycogen in the isolated, perfused rat heart using 13C NMR spectroscopy.

NMR spectroscopy, possibly, does not detect 100% of large molecules such as glycogen (mol.wt = 10(7)-10(9)). Using both NMR and chemical quantification methods, we have, therefore, determined the NMR visibility of cardiac glycogen (defined as the ratio of the NMR value to the chemical value, expressed as a percentage) in the isolated, perfused heart. Rats (n = 7) were pretreated for 60 min with 0.2 mg/kg isoproterenol (s.c.) to deplete their endogenous myocardial glycogen stores (mainly 12C). The hearts were then aerobically perfused (65 cm H2O, at 37 degrees C) in a double-walled chamber (the annulus contained a standard), for 70 min with Krebs buffer plus 3.5 mM [13C]1-glucose and 5 mM sodium acetate (natural abundance). From 70 to 175 min the sole substrate was natural abundance acetate (5 mM). 13C NMR spectra for glycogen quantification were acquired in two different ways; by applying 896, 90 degree pulses at 0.33 s intervals with 1H decoupling ('fast', practical spectra) and by applying 896, 90 degree pulses at 5 s intervals ('slow', impractical spectra). Hearts were then removed from the magnet, freeze-clamped (-196 degrees C) and analysed chemically. Cardiac glycogen, quantified from the 'fast' spectra (using conversion factors) and the 'slow' spectra was 16.8 +/- 1.1 and 16.1 +/- 1.8 (mean +/- SEM) mumol glucosyl units/heart, respectively. After correction of the chemical value for the residual [12C]glycogen (determined from 1H NMR spectra of the extracted glycogen after hydrolysis), the NMR-visibilities were calculated to be 101 +/- 6 and 109 +/- 7%, for the 'fast' and 'slow' spectra, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)

NMR visibility of Pi in perfused rat hearts is affected by changes in substrate and contractility.

Nuclear magnetic resonance (NMR) spectroscopy does not always detect the total metabolite content in isolated perfused rat hearts. Alterations in NMR peak areas therefore could be caused by a change either in the metabolite content per se or in its NMR visibility. We have therefore compared the ATP, phosphocreatine (PCr), and Pi content of hearts, as determined by 31P-NMR spectroscopy, with the total, chemically determined ATP, PCr, and Pi contents of the same hearts to determine the fractions, if any, that are NMR invisible under different perfusion conditions. The three perfusion buffers used contained 1) glucose, 2) glucose plus high K+, and 3) pyruvate. Fully relaxed 31P-NMR spectra were collected during the final 10 min of perfusion in each group, and the ATP, PCr, and Pi contents were quantified using methylene diphosphonate as an external standard. The hearts were then freeze-clamped and chemically assayed for ATP, PCr, and Pi. Under all three conditions, the NMR-determined ATP and PCr contents were almost identical to the chemically determined values. However, only a portion of the chemically determined Pi was NMR visible. During perfusion with glucose-containing buffer, 39 +/- 8% of the total Pi was NMR visible, and this decreased to 12 +/- 2% (P less than 0.01) during K+ arrest and to 9 +/- 5% (P less than 0.01) during perfusion with pyruvate-containing buffer. In conclusion, whereas the total cellular content of ATP and PCr is always NMR visible during normoxic perfusion, alterations in substrate and contractile status can change the fraction of NMR-visible Pi.

Magnetic resonance spectroscopy.

The aims of this article are to introduce the reader to the basic principles of nuclear magnetic resonance spectroscopy and to review the biochemical and clinical applications of the technique. The theoretical section, dealing with the basic principles, can be avoided if necessary without jeopardizing the understanding of the remainder!

Evidence that mitochondrial phosphate is visible in 31P NMR spectra of isolated, perfused rat hearts.

We have previously found (P. B. Garlick, T. R. Brown, R. H. Sullivan and K. Ugurbil, J. Mol. Cell Cardiol. 15, 855-858 (1983)) that two peaks could be observed in the phosphate region of NMR spectra of isolated, perfused rat hearts. Upon valinomycin treatment, an increase in the peak at 2.8 ppm in the phosphate region (phosphocreatine set at -2.52 ppm) had been observed. We have now confirmed our hypothesis that this peak originates from the mitochondrial phosphate by: (i) determination of myocardial mitochondrial phosphate contents using density gradient centrifugation in non-aqueous solvents; and (ii) quantitative electron microscopy of the heart tissue. Thus, we conclude that mitochondrial and cytosolic phosphate can be distinguished from each other in 31P NMR spectra of isolated, perfused rat hearts.

NMR-visible ATP and Pi in normoxic and reperfused rat hearts: a quantitative study.

Nuclear magnetic resonance (NMR) spectroscopy detects only free, unbound metabolites. We have therefore compared the free high-energy phosphate content of isolated perfused rat hearts (determined by 31P-NMR) with the total high-energy phosphates of the same hearts (determined by chemical analysis) to determine the fractions, if any, that are NMR invisible. Aerobic perfusion (40 min at 37 degrees C, Pi-free Krebs buffer) was followed by 10, 14, or 18 min total global ischemia and 30 min reperfusion (n = 6 in each group). Fully relaxed 31P-NMR spectra (40 scans using 90 degrees pulses at 15-s intervals) were collected at various times throughout the protocol, and the signal intensities of the beta-phosphate of ATP, phosphocreatine (PCr), and Pi were quantified using methylenediphosphonate as an external standard. Hearts were freeze clamped either before ischemia or at the end of reperfusion and were chemically assayed for ATP, PCr, and Pi. After 40 min of normoxia, the ATP and PCr contents determined by NMR were almost identical to the values determined by chemical analysis. However, only 39 +/- 8% of the total Pi was NMR visible. After reperfusion, after 14 or 18 min of ischemia, the proportion of NMR-visible ATP had decreased to 64 +/- 9% (P less than 0.005). After reperfusion after 18 min ischemia, the proportion of NMR-visible Pi had increased to 76 +/- 10% (P less than 0.05). In conclusion, whereas the total cellular content of PCr is always NMR visible, ischemia-reperfusion can alter the fraction of NMR-visible ATP and Pi.

Studies on the effects of antioxidants and inhibitors of radical generation on free radical production in the reperfused rat heart using electron spin resonance spectroscopy.

Reperfusion of the heart after a period of ischaemia can precipitate ventricular arrhythmias and lead to an exacerbation of tissue injury. Direct evidence to suggest the involvement of free radicals has been obtained using electron spin resonance (esr) spectroscopy and the spin trap N-tert. butyl-alpha-phenyl nitrone (PBN). In the present study, we have used esr spectroscopy and PBN to examine the individual effects of superoxide dismutase (SOD), catalase, allopurinol or desferal on radical production in the isolated, reperfused rat heart. A burst of radical production was observed in the control group during the first 5 minutes of reperfusion; the peak occurred during the first minute, when signal intensity had increased by almost 300%, but returned to the baseline by 15 minutes of reperfusion. The esr signals were consistent with the trapping of either alkoxyl or carbon-centered radicals (aN = 13.6 and aH = 1.56 G). In the desferal-treated group, a burst of radical production was observed during the first five minutes of reperfusion; this was maximal during the second minute, when signal intensity had increased by almost 200%, but had returned to the baseline value by 30 minutes of reperfusion. In the SOD-treated group, a burst of radical production was observed during the first 10 minutes of reperfusion; signal intensity was maximal during the tenth minute of reperfusion, when signal intensity had increased by almost 200%, but had returned to the baseline value by 30 minutes of reperfusion. In the allopurinol- and catalase-treated groups, no significant burst of radical production could be detected. These data further support the concept that cytotoxic, oxygen-derived species are formed upon reperfusion and that hydrogen peroxide and/or hydroxyl radicals, are likely to be involved.

Myocardial tissue preparation for ESR spectroscopy: some methods may cause artifactual generation of signals.

It has been suggested that some techniques of tissue preparation for esr spectroscopy may artifactually generate radicals. We have investigated this, together with the possibility that the susceptibility of the tissue to preparation artifacts may be altered by ischaemia and reperfusion. Three different methods of tissue processing have been assessed: (i) freeze-clamping (-196 degrees C), using grooved, aluminium tongs which produce frozen cylinders of tissue (3 mm diameter) which fit directly into esr tubes; (ii) grinding of freeze-clamped tissue with a porcelain pestle and mortar; (iii) lyophilization of ground, freeze-clamped, tissue. Isolated rat hearts (n = 7 or n = 5/group) were subjected to aerobic perfusion (10 min, 37 degrees C), total, global ischaemia (15 min) and reperfusion (30 sec). Hearts were freeze-clamped at the end of each period. Tissue was prepared by each of the three methods and esr spectra recorded at -100 degrees C. In spectra from tissue which had been freeze-clamped only, broad high- and low-spin iron III signals (g = 1.9, g = 2.2-2.9 and g = 4.6) were seen together with a narrow, well-defined signal (g = 2.005), possibly from a semiquinone radical.(ABSTRACT TRUNCATED AT 250 WORDS)

The synthesis, release and action of leukotrienes in the isolated, unstimulated, buffer-perfused rat heart.

When the perfusion medium of an isolated, non-recirculating, Langendorff rat heart is changed from Krebs buffer to coronary effluent, a significant vasoconstriction (23%, P less than 0.005) is observed. In this study we have investigated the involvement of leukotrienes in this phenomenon. We have extracted and quantified leukotrienes C4, D4 and E4 in samples of coronary effluent taken at different times during the first 2 h of perfusion; the total amounts released during this time were 9, 5 and 32 pmol of LTC4, LTD4 and LTE4 respectively. We have used two different methods to prevent the action of the effluent leukotrienes on the heart. Firstly, we have blocked the leukotriene receptors in the heart, with FPL 55712 (3.8 microM), during perfusion with effluent and, secondly, we have perfused with coronary effluent which was collected in the presence of a leukotriene synthesis inhibitor, AA861 (1 microM). The addition of FPL 55712 to the effluent decreased the normally observed vasoconstriction such that after 30 min the coronary flow rate (CFR) was 114 +/- 3% (n = 6) compared with 66 +/- 1% (n = 7) with effluent alone (P less than 0.005). Effluent collected in the presence of AA861 also caused a decrease in the normally observed vasoconstriction such that by 30 min the CFR was still 88 +/- 2% (n = 6, P less than 0.005 compared to controls). We have confirmed the proposed involvement of leukotrienes in the effluent-induced vasoconstriction by investigating the effect of a mixture of the synthetic leukotrienes C4, D4 and E4, when each of them was present at the same concentration as measured in the coronary effluent; the vasoconstriction observed was superimposable upon that seen with effluent. This vasoactive effect of the leukotriene mixture was not secondary to a change in contractility, since this only decreased to 97 +/- 5% (n = 9) during the 30 min of the leukotriene infusion. Finally, we have studied the effects of the same two leukotriene blockers in normal, buffer-perfused hearts after an initial perfusion of either 30 or 120 min. Application of either AA861 or FPL 55712 resulted in a dramatic vasodilatation (25 to 45% increase), a larger effect always being observed after the shorter initial period of perfusion. Our conclusions are two-fold. Firstly, isolated, buffer-perfused rat hearts synthesize leukotrienes C4, D4 and E4 in considerable amounts and release them into the coronary effluent and secondly, the coronary flow rates of isolated, buffer-perfused rat hearts are partly controlled by the action of internally produced leukotrienes.

Manipulation of myocardial alpha-tocopherol levels fails to affect reperfusion arrhythmias or functional recovery following ischemic challenge in the rat heart.

Antioxidants that act as free radical scavengers have the potential to inhibit lipid peroxidation. It has previously been proposed that a relationship exists between free radicals, lipid peroxidation and reperfusion-induced arrhythmias. We have therefore examined the ability of the lipid-soluble antioxidant, alpha-tocopherol, to decrease the incidence of reperfusion-induced arrhythmias. We have shown that the myocardial alpha-tocopherol content can be significantly increased from its control value of 65.3 +/- 5.6 nmoles/g wet wt of heart to 115.0 +/- 15.6 nmoles/g wet wt of heart (p less than 0.01) by chronic intraperitoneal pretreatment and that it can be decreased, to 21.1 +/- 3.7 nmoles/g wet wt of heart (p less than 0.01), by chronic dietary manipulation. Rat hearts isolated from either of these groups, or from placebo-treated control animals, were subjected to 5 or 10 min coronary artery occlusion and were subsequently reperfused; there were no significant differences between the incidence and duration of VF and VT or the incidence and number of VPBs in these three groups. The effect of alpha-tocopherol manipulation on metabolic and functional recovery of working hearts subjected to 20 min global ischemia was subsequently examined and no significant changes were observed. Cardiac output recovered to 82 +/- 4, 81 +/- 6 and 76 +/- 5% of its preischemic value in the control, enriched and depleted groups, respectively. In conclusion, myocardial alpha-tocopherol content appears to bear no relation to the severity of reperfusion-induced arrhythmias or to the resistance of the heart to ischemic injury.

Direct detection of free radicals in the reperfused rat heart using electron spin resonance spectroscopy.

The purpose of this study was to use a direct method, that of electron spin resonance (ESR) spectroscopy, to demonstrate that reperfusion after a period of ischemia results in a sudden increase in the production of free radicals in the myocardium. The isolated buffer-perfused rat heart was used with N-tert-butyl-alpha-phenylnitrone (PBN) as a spin-trapping agent. Samples of coronary effluent were taken and extracted into toluene for detection of radical adducts by ESR spectroscopy. After 15 minutes of total, global ischemia, aerobic reperfusion resulted in a sudden burst of radical formation that peaked at 4 minutes. When hearts were reperfused with anoxic buffer, no dramatic increase in radical production was observed. Subsequent reintroduction of oxygen, however, resulted in an immediate burst of radical production of a similar magnitude to that seen in the wholly aerobic reperfusion experiments. The ESR signals obtained (aN = 13.60 G, aH = 1.56 G) are consistent with the spin-trapping by PBN of either a carbon-centered species or an alkoxyl radical, both of which could be formed by secondary reactions of initially-formed oxygen radicals with membrane lipid components.