© 1999 by European Society of Cardiology
Copyright © 1999, European Society of Cardiology
Inhibitors of poly (ADP-ribose) synthetase protect rat cardiomyocytes against oxidant stress
William Harvey Research Institute, St Bartholomew's and the Royal London School of Medicine and Dentistry, Charterhouse Square, London, EC1M 6BQ, UK
* Corresponding author. Tel.: +44-171-982-6119; fax: +44-171-251-1685; e-mail: cthiemermann@mds.qmw.ac.uk
Received 18 March 1998; accepted 27 May 1998
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Abstract |
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Objective: Inhibitors of poly (ADP-ribose) synthetase (PARS) activity reduce the infarct size caused by regional myocardial ischaemia and reperfusion in the rabbit and rat in vivo. The mechanism of action of these inhibitors is unclear. Here we investigate the effects of the PARS inhibitor 3-aminobenzamide (3-AB) on infarct size caused by ischaemia and reperfusion of the isolated, perfused heart of the rat. We also investigate the role of PARS in the hydrogen peroxide-mediated cell injury/necrosis in rat cardiac myoblasts. Methods: Rat isolated hearts perfused at constant pressure (80 mmHg) were subjected to 35 min of regional ischaemia and 2 h of reperfusion. Infarct size was determined at the end of the experiment using nitro-blue tetrazolium. 3-AB (300 µM) or 3-aminobenzoic acid (3-ABA, 300 µM) were infused during the reperfusion period. Rat cardiac myoblasts (H9c2 cells) were preincubated with the PARS inhibitors, 3-AB, nicotinamide (Nic) or 1,5-dihydroxyisoquinoline (ISO) or the inactive analogues 3-ABA or nicotinic acid (NicA) prior to exposure with hydrogen peroxide (1 mM). Cell injury was assessed by measuring mitochondrial respiration and cell necrosis by measuring the release of LDH. PARS activity was determined by measuring the incorporation of NAD into nuclear proteins. Results: Regional ischaemia and reperfusion of the isolated rat heart resulted in an infarct size of 54% which was reduced by 3-AB, but not by 3-ABA. Exposure of rat cardiac myoblasts to hydrogen peroxide caused an increase in PARS activity and cell injury/necrosis which was attenuated by pretreatment with the PARS inhibitors. Conclusion: Inhibition of the activity of PARS attenuates the cell death associated with oxidant stress in rat cardiac myoblasts and heart.
KEYWORDS Rat; Heart; H9c2 cells; Reperfusion injury; Reactive oxygen species
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1 Introduction |
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There is good evidence that reperfusion of the previously ischaemic heart is associated with the generation of reactive oxygen species (ROS) such as superoxide anions (O2–), hydroxyl radicals (OH·), and hydrogen peroxide (H2O2) as well as peroxynitrite (ONOO–) [1–3]. All of these ROS have been directly identified using the techniques of electron paramagnetic resonance spectroscopy [4], chemiluminescence [5] and spin trapping [6]. There is a burst-like release of ROS peaking in the first 2 min of reperfusion followed by continued release for at least 2 h [6]. The generation of these ROS correlates with indices of reperfusion injury such as reperfusion arrhythmias, post-ischaemic contractile dysfunction and lethal cell injury (necrosis) [6, 7]. Strategies aimed at reducing the generation, or the damaging effects, of ROS exert beneficial effects in a variety of models of ischaemia-reperfusion injury of the heart [8–11]. ROS cause cell injury by peroxidation of membrane lipids, denaturation of proteins such as enzymes and ion channels, and DNA injury. For instance, exposure of cultured cells to ROS including H2O2, ONOO– or O2– results in single strand breaks in DNA and subsequent activation of the DNA repair enzyme, poly (ADP-ribose) synthetase (PARS) [12]. Once activated, PARS catalyses the transfer of poly (ADP-ribose) groups from nicotinamide adenine dinucleotide (NAD) to nuclear proteins with concomitant formation of nicotinamide. Under conditions of oxidant stress, DNA injury causes excessive activation of the PARS enzyme resulting in a fall in the intracellular levels of its substrate NAD [13]. As NAD is necessary for mitochondrial respiration, depletion of NAD leads to a fall in the intracellular levels of ATP. In addition, the nicotinamide formed when PARS is activated can be recycled to NAD in a reaction that consumes ATP [14]. A decline in the intracellular levels of ATP results in severe cellular dysfunction and ultimately cell death [15]. This mechanism of cell death is termed the ‘PARS Suicide Hypothesis’ [16]. Inhibition of the activity of PARS e.g. with 3-aminobenzamide (3-AB), prevents the NAD and ATP depletion caused by oxidant stress, and improves cell survival [13].
We have recently discovered that administration of PARS inhibitors just prior to reperfusion, reduces the degree of infarction caused by myocardial ischaemia–reperfusion in the anaesthetised rabbit [17]. We have proposed that the activation of PARS contributes to ischaemia–reperfusion injury of the heart (and other organs) [17]. Our hypothesis has recently been supported by the finding that the PARS inhibitor 3-AB reduces infarct size caused by regional myocardial ischaemia and reperfusion in the anaesthetised rat in vivo [18]. The mechanism of the reduction in reperfusion injury by PARS inhibitors is unclear, but may involve direct protection of cardiomyocytes against the cell injury caused by reactive oxygen radicals [17] as well as inhibition of the infiltration of the previously ischaemic myocardium with polymorphonuclear leukocytes (PMNs) [18].
The overall goal of this study was to elucidate whether inhibition of PARS activity can directly (in the absence of PMNs) protect cardiac myocytes against radical-mediated injury. To achieve this goal, we have investigated the effects of 3-AB (and its inactive structural analogue, 3-aminobenzoic acid, 3-ABA) [19] on the infarct size resulting from regional myocardial ischaemia and reperfusion in the isolated, buffer-perfused heart of the rat. In addition, we have investigated the role of PARS activation in the injury/necrosis caused by hydrogen peroxide in rat cardiac myoblasts. Finally, to elucidate whether the ‘cardioprotective’ effects of PARS inhibitors are due to the ability of these agents to scavenge ROS, we have compared the effects of PARS inhibitors with those of established scavengers of superoxide anions, superoxide dismutase (SOD) and 2,2,6,6-tetramethylpiperidine-1-oxyl (TEMPO) [20].
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2 Methods |
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The investigation conforms with the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH publication No 85/23, revised 1996).
2.1 Regional myocardial ischaemia and reperfusion in the isolated, buffer-perfused heart of the rat
Male Wistar rats (250–350 g) were anaesthetised with thiopentone sodium (120 mg/kg, i.p.) and received heparin (1400 U/kg, i.p.) to prevent the formation of microthrombi in the coronary circulation. A midline thoracotomy was performed and the heart was rapidly removed by cutting the great vessels and immersed in ice-cold modified Krebs–Henseleit solution which had been gassed with 95%O2/5%CO2. The heart was transferred to the perfusion apparatus and the aorta was cannulated for retrograde perfusion with modified Krebs–Henseleit solution composed of (in mM) NaCl 118.5, NaHCO3 25, KCl 4.0, MgSO4 1.2, KH2PO4 1.2, D-glucose 11.1, CaCl2 1.8 and gassed with 95%O2/5%CO2. The hearts were perfused at a constant pressure (80 mm Hg). The intracardiac temperature was measured by placement of a temperature probe in the right atrium (via the pulmonary artery) and maintained constant (at 37±0.5°C) by changing the level of a heated water-jacketed bath around it. Coronary perfusion pressure (CPP) was measured through a side arm of the perfusion cannula. An isovolumetric balloon was inserted into the left ventricle and the balloon volume was adjusted to achieve a left ventricular end diastolic pressure (LVEDP) of 5–10 mmHg. CPP was continuously recorded by a pressure transducer connected to a polygraph Grass recorder and coronary flow was measured by collection of the effluent. A suture on a round-bodied needle was placed around the left anterior descending coronary artery (LAD) and the suture ends were threaded through a small length of tubing to form a snare. The heart was allowed to stabilise for 30 min.
Following the stabilisation period, the LAD was occluded for 35 min. During this period of regional myocardial ischaemia, care was taken to maintain the intracardiac temperature at 37°C. Subsequently, the snare occluder was released to allow reperfusion of the previously ischaemic vascular bed for 2 h. The LAD was then re-occluded and Evans Blue dye (1 ml of 0.5% w/v) was injected into the heart via the aorta, to distinguish between perfused and non-perfused (area at risk) sections of the heart. The Evans Blue solution stains the perfused myocardium, while the occluded vascular bed remains uncoloured. The heart was removed from the perfusion apparatus, sectioned into slices of 3–4 mm, the right ventricular wall was removed, and the area at risk (pink) was separated from the perfused (blue) area. The area at risk was cut into small pieces and incubated with p-nitro-blue tetrazolium (0.5 mg/ml) for 20 min at 37°C. In the presence of intact dehydrogenase enzyme systems (viable perfused myocardium), NBT forms a dark blue formazan, whilst areas of necrosis lack dehydrogenase activity and therefore fail to stain [21]. The percentage infarct was determined by gravimetric analysis.
To elucidate the effects of the PARS inhibitor 3-aminobenzamide on infarct size in this model, hearts were reperfused with either buffer (control, n=9) or buffer containing 3-AB (300 µM, n=6). To ensure that any of the observed effects of 3-AB are indeed due to inhibition of PARS activity (rather than non-specific effects), we also investigated the effects of 3-ABA, an analogue of 3-AB, which has a similar structure but does not inhibit PARS activity [19], in this model. In these experiments, hearts were reperfused with either buffer containing vehicle (DMSO, final concentration 0.15%, n=6) or buffer containing 3-ABA (300 µM, n=6).
2.2 Cell culture
Rat ventricular myoblasts (H9c2 cells) were obtained from the American Type Culture Collection (ATCC; Rockville, MD, USA) and grown to confluence in culture flasks containing Dulbecco's modified Eagle's medium (DMEM) supplemented with L-glutamine (3.5 mM) and 10% foetal calf serum. Cells were passaged every 2 days, removed by treatment with trypsin/EDTA and then cultured in 96-well or 6-well plates (for the measurement of PARS activity only) until they reached confluence.
2.3 Experimental design
To investigate the effects of exposure of rat cardiac myoblasts to hydrogen peroxide, cells were exposed to hydrogen peroxide (10 µM to 10 mM) for various durations (1 to 6 h) after which the cell injury was assessed (see below). To elucidate the effects of PARS inhibitors on the cell injury caused by hydrogen peroxide, cells were preincubated (10 min, 37°C) in media containing (i) the PARS inhibitors [19] 3-AB (0.1 to 30 mM), 1,5-dihydroxyisoquinoline (ISO, 0.01 to 3 mM) or nicotinamide (Nic, 0.1 to 30 mM), or (ii) their inactive (with respect to inhibition of PARS activity) structural analogues 3-ABA (0.1 to 3 mM) or nicotinic acid (Nic A, 0.1 to 30 mM) [19]. The cells were then exposed to hydrogen peroxide (1 mM) for 4 h after which time cell injury/necrosis was assessed (see below).
To elucidate which radical species mediates the cell injury caused by hydrogen peroxide, cells were preincubated with the iron chelator, deferoxamine (0.01 to 10 mM), exposed to hydrogen peroxide (1 mM) for 4 h, after which time cell injury/necrosis was assessed.
To elucidate whether hydrogen peroxide causes PARS activation, cells were exposed to hydrogen peroxide (1 to 10 mM, concentration–response study) for various durations (10 to 90 min, time-response study). Having found that a maximal increase in PARS activity occurred within 30 min after addition of hydrogen peroxide, the following study was designed to investigate whether the PARS inhibitors used do indeed inhibit PARS activity in rat cardiac myoblasts: Cells were (i) preincubated with media containing the PARS inhibitors 3-AB (3 mM), ISO (300 µM) or Nic (3 mM), or their inactive analogues 3-ABA (3 mM) or Nic A (3 mM), (ii) exposed to hydrogen peroxide (1 mM) for 30 min, and (iii) collected and PARS activity was measured as described below.
In addition, the effects of deferoxamine on the increase in PARS activity caused by hydrogen peroxide were also investigated. Cells were preincubated with media containing deferoxamine (3 mM, pre-treatment study) and then exposed to hydrogen peroxide (1 mM) for 30 min. The cells were then collected and PARS activity was measured as described below. Having found that deferoxamine inhibited PARS activity, the following study was designed to elucidate whether the observed effects was due to the prevention of the formation of hydroxyl radical (and hence prevention of DNA strand breakage) or due to a ‘direct’ inhibition of PARS activity by deferoxamine. To investigate the latter possibility, cells were exposed to hydrogen peroxide (1 mM for 30 min to cause a maximal increase in PARS activity) and then permeabilised in reaction buffer (see below) containing deferoxamine (3 mM, post-treatment study). Thus, this experiment investigated whether deferoxamine could inhibit the activity of PARS itself.
2.4 Measurement of cell injury (MTT assay)
Cell viability was determined indirectly by measuring the mitochondrial-dependent reduction of MTT (3-(4,5-dimethyliazol-2-yl)-2,5-diphenyltetrazolium bromide) to formazan (i.e. mitochondrial respiration). Cells in 96-well plates were incubated with MTT (100 µl, 0.2 mg/ml, dissolved in PBS) for 60 min at 37°C. MTT solution was removed by aspiration and cells were solubilised in 100 µl of dimethyl sulfoxide (DMSO). The amount of purple formazan formed was detected and quantified by measuring the absorbance of the solution at 550 nm using an Anthos Labtec microplate reader.
2.5 Measurement of cell necrosis (lactate dehydrogenase assay)
Loss of plasma membrane integrity (cell necrosis) was assessed by measurement of the activity of lactate dehydrogenase (LDH) in the supernatant. A 100-µl sample of cell-free supernatant was transferred into a 96-well plate. LDH activity was measured using a cytotoxicity detection kit (Boehringer Mannheim Ltd.). The kit operates on the principle that (in the first step) released LDH reduces NAD to NADH and H+ by oxidation of lactate to pyruvate. In the second reaction, 2 hydrogens are transferred from NADH and H+ to the yellow tetrazolium salt (2-[4-iodophenyl]-3-[4-nitrophenyl]-5-phenyltetrazolium chloride) by a catalyst. Thus, the increase in amount of enzyme activity in the supernatant directly correlates with the amount of formazan produced. The formazan formed is water soluble and shows a broad absorbance maximum at about 500 nm while the tetrazolium salt shows no significant absorbance at these wavelengths. So, following addition of 100 µl of kit reaction buffer to the sample of cell supernatant, the plates were incubated at room temperature in the dark for 30 min. The reaction was terminated by addition of 25 µl of 2 M HCl and the amount of formazan formed was detected and quantified by measuring the absorbance of the solution at 490 nm (reference filter 620 nm) using a Ceres microplate reader.
2.6 Measurement of PARS activity in rat cardiac myoblasts
PARS activity was measured as the ability of permeabilised cells to transfer the substrate [3H]-NAD onto nuclear proteins over a set time period as described [13]. Following the appropriate treatment and duration, the media was aspirated before addition of fresh culture medium (400 µl) and the cells were scraped and transferred to an Eppendorff tube. Following centrifugation (10 000g, 10 s) and aspiration of media, the cells were resuspended in reaction buffer (56 mM Hepes buffer containing 28 mM KCl, 28 mM NaCl, 2 mM MgCl2, 0.02% digitonin, and 125 nmoles NAD spiked with 0.25 µCi/ml [3H-NAD], pH 7.5), vortexed for 5 s and incubated at 37°C for 5 min. The reaction was terminated by the addition of 200 µl of 50% trichloroacetic acid (TCA) and the resultant precipitate was pelleted by centrifugation at 10 000g for 3 min. The protein pellet was washed twice with 50% TCA and then solubilised in 200 µl of 1 M NaOH/2% SDS overnight at 37°C in a shaking incubator. The radioactivity incorporated into protein was determined by scintillation counting. PARS activity was calculated and expressed as p moles NAD incorporated per well per minute.
2.7 Evaluation of the effects of test drugs on the reduction of cytochrome c caused by superoxide anion generation (by hypoxanthine/xanthine oxidase)
The ability of the test compounds to scavenge superoxide anions was determined in an in vitro microassay as previously described by Laight et al. [20]. The assay mixture consisted of (final concentration): 50 µl ferricytochrome c (cyt c, 100 µM), 10 µl xanthine oxidase (XO, 10 mU/ml), 20 µl hypoxanthine (Hx, 100 µM) and 10 µl catalase (200 U/ml) dissolved in phosphate buffered saline (10 mM phosphate, pH=7.4), and 10 µl of the test compounds. Changes in absorbance (A) at 550 nm were recorded at 37°C using a kinetic plate reader over 3 min. Using this technique, we have compared the effects of the PARS inhibitors 3-AB (0.1 to 3 mM), ISO (0.01 to 3 mM) and Nic (0.1 to 3 mM) with those of the established superoxide anion scavengers superoxide dismutase (SOD, 200 U/ml) or 2,2,6,6-tetramethylpiperidine-1-oxyl (TEMPO, 1 mM) [20].
2.8 Statistical analysis
All data are expressed as mean±s.e. mean of n independent experiments. Statistical comparisons between groups were made by a one way ANOVA followed by a Dunnett's test. A p value of less than 0.05 was considered to be statistically significant.
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3 Results |
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3.1 3-Aminobenzamide reduces infarct size in the isolated, perfused heart of the rat
Occlusion of the LAD resulted in an area at risk of approximately 58% of the left ventricle, which was not different between any of the groups studied (Fig. 1a). In isolated hearts which were reperfused with vehicle for 3-AB (buffer; control), occlusion of the LAD (35 min) followed by reperfusion (120 min) resulted in an infarct size of 54±5% of the area at risk (Fig. 1b). Reperfusion of the heart with buffer containing 3-AB (300 µM) caused a significant reduction in infarct size (Fig. 1b). In contrast, reperfusion of the heart with buffer containing either 3-ABA (300 µM) (Fig. 1b) or its vehicle (0.15% DMSO) did not result in a significant reduction in infarct size (0.15% DMSO, 59±7%).
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3.2 Inhibitors of PARS activity attenuate the cell injury/necrosis caused by hydrogen peroxide in rat cardiac myoblasts
Exposure of rat cardiac myoblasts to hydrogen peroxide caused a concentration- and time-dependent fall in mitochondrial respiration (Fig. 2a). Hydrogen peroxide also caused a time- and concentration-dependent release of LDH into the culture medium (Fig. 2b). Inhibitors of PARS activity including 3-AB (0.1 to 30 mM), ISO (0.01 to 3 mM) and Nic (0.1 to 30 mM) caused a concentration-dependent attenuation of the impairment in mitochondrial respiration caused by hydrogen peroxide (1 mM for 4 h). The PARS inhibitors 3-AB (3 mM), ISO (300 µM) or Nic (3 mM) also attenuated the impairment in mitochondrial respiration and the release of LDH caused by exposure of rat cardiac myoblasts to hydrogen peroxide (1 mM for 4 h), while the structural analogues (negative controls) 3-ABA (3 mM) or NicA (3 mM) did not affect impairment in mitochondrial respiration or the release of LDH caused by hydrogen peroxide (Fig. 3).
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3.3 The cell injury caused by hydrogen peroxide is mediated by generation of the hydroxyl radical in rat cardiac myoblasts
The intracellular iron-chelator deferoxamine, which prevents the formation of hydroxyl radicals from hydrogen peroxide (by inhibition of the Fenton reaction), attenuated the fall in mitochondrial respiration (Fig. 4a) and the associated release of LDH (Fig. 4b) caused by exposure of rat cardiac myoblasts to hydrogen peroxide.
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3.4 Inhibitors of PARS activity attenuate the increase in PARS activity caused by hydrogen peroxide in rat cardiac myoblasts
Exposure of rat cardiac myoblasts to hydrogen peroxide (1 to 10 mM, 10 to 90 min) caused a time- and concentration-dependent increase in PARS activity (Fig. 5a,b). Exposure of the rat cardiac myoblasts to hydrogen peroxide (1 mM) for 30 min caused a peak increase in PARS activity. This increase in PARS activity was attenuated by pretreatment of the cells with the PARS inhibitors 3-AB (3 mM), ISO (300 µM) or Nic (3 mM) (Fig. 5c). In contrast, 3-ABA or NicA (negative controls) did not affect the increase in PARS activity caused by hydrogen peroxide in rat cardiac myoblasts (Fig. 5c). The increase in PARS activity was also attenuated by pretreatment of cells with deferoxamine (3 mM) (Fig. 5d) but not affected by addition of deferoxamine to permeabilised cells (post-treatment, Fig. 5d).
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3.5 PARS inhibitors do not scavenge superoxide anions in vitro
TEMPO (1 mM) abolished, and SOD (200 U/ml) significantly depressed the rate of reduction of cytochrome c by the xanthine oxidase/hypoxanthine superoxide anion generating system. In contrast, none of the PARS inhibitors, even at the highest concentrations (Fig. 6) had any significant effect on the rate of reduction of cytochrome c. Thus, none of the PARS inhibitors used were able to scavenge superoxide anions.
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4 Discussion |
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We have recently discovered that the administration, upon reperfusion, of several chemically distinct inhibitors of PARS activity (including 3-AB, ISO and Nic) reduce the myocardial infarct size caused by regional ischaemia and reperfusion in the anaesthetised rabbit [17]. We have therefore proposed [17] that the activation of PARS contributes to ischaemia-reperfusion injury of the heart. The exact mechanism of action of PARS inhibitors is still unclear, but may involve a direct protection of cardiomyocytes against reperfusion injury [17]. We demonstrate in this study that the PARS inhibitor 3-AB reduces the infarct size caused by regional myocardial ischaemia and reperfusion of the isolated, perfused heart of the rat. In contrast, 3-ABA, an analogue of 3-AB, which has a structure similar to 3-AB, but does not inhibit PARS activity in many cells [19] including rat cardiac myoblasts (this study), did not reduce infarct size in this model. These findings support the view that the cardioprotective effect of 3-AB is due to (i) inhibition of PARS activity (rather than a non-specific effect), and (ii) a direct effect on cardiomyocytes. What, then, is the mechanism by which 3-AB (or other PARS inhibitors) protect cardiac myocytes against reperfusion injury? Reperfusion of the previously ischaemic heart is associated with the generation of ROS including hydrogen peroxide [4]. We show here that exposure of rat cardiac myoblasts in culture to hydrogen peroxide causes an increase in PARS activity (within 30 min) followed by cell necrosis (within 4 h). The PARS inhibitors 3-AB, ISO or Nic attenuated the increase in PARS activity and reduced the cell injury/necrosis caused by hydrogen peroxide in these cells. In contrast, 3-ABA (the inactive analogue of 3-AB) and NicA (the inactive analogue of Nic) neither affected the increase in PARS activity nor the cell injury/necrosis caused by hydrogen peroxide. These results strongly support the conclusion that hydrogen peroxide causes strand breaks in DNA which in turn results in the activation of PARS, inhibition of mitochondrial respiration and ultimately cell death. Although we have not demonstrated that hydrogen peroxide causes strand breaks in DNA, there is evidence that hydrogen peroxide (at concentrations even lower than those used in our study) causes strand breaks in DNA in cultured cardiac myoblasts [22]. While our study was ongoing, Gilad and colleagues reported that 3-AB and Nic attenuate the impairment in mitochondrial respiration caused by hydrogen peroxide and peroxynitrite in H9c2 cells [23].
Our finding that the iron-chelator, deferoxamine, attenuates the increase in PARS activity and the cell injury/necrosis caused by hydrogen peroxide in rat cardiac myoblasts strongly suggests that the injury caused by hydrogen peroxide is mediated by hydroxyl radical formation. The hydroxyl radical also mediates the DNA strand breakage, the activation of PARS, and the cell injury/death caused by hydrogen peroxide in many cell types [24, 27, 28]. Deferoxamine did not have any effect on the increase in PARS activity when added after exposure to hydrogen peroxide (i.e. as a post-treatment to permeabilised cells), indicating that the protection afforded by deferoxamine was not due to a ‘direct’ inhibition of the activity of PARS.
Reperfusion of the previously ischaemic myocardium leads to the generation of ROS including superoxide anions, hydrogen peroxide, hydroxyl radicals as well as peroxynitrite [1–3]. All of these radicals cause DNA strand breaks in cultured cells [13, 15, 22, 24, 28]. There is good evidence that inhibitors of PARS activity including 3-AB and Nic do not reduce the degree of DNA damage suggesting that these agents do not function as radical scavengers [13, 28]. Indeed, we demonstrate here that the PARS inhibitors 3-AB, ISO or Nic do not scavenge superoxide anions (generated in vitro). Thus, it appears unlikely that the beneficial effects of the PARS inhibitors in conditions of oxidant stress (e.g. ischaemia–reperfusion injury) are due to the scavenging of ROS.
Interestingly, the reduction in myocardial infarct size caused by 3-AB in the anaesthetised rat is associated with a reduction in myeloperoxidase activity (an indicator of the number of PMN's within a tissue) in the ischaemic-reperfused myocardium [18]. As infiltration and activation of PMN's contributes to the pathophysiology of reperfusion injury [25], it is possible that prevention by 3-AB of the infiltration of neutrophils into the ischaemic-reperfused myocardium contributes to the mechanism of the cardioprotective effects of 3-AB. Indeed, 3-AB also attenuates the zymosan-stimulated extravasation of neutrophils in the perfused mesentery of the mouse by increasing the ‘rate of detachment’ of neutrophils from the endothelium [26]. However, our findings that 3-AB reduces (by 40–50%) the infarct size caused by either global myocardial ischaemia and reperfusion in the isolated, perfused heart of the rabbit [17] or by regional myocardial ischaemia and reperfusion in the isolated perfused heart of the rat [this study] do not support this. Thus, a substantial reduction in myocardial infarct size by 3-AB can be demonstrated in models of myocardial infarction in which the tissue injury is independent of neutrophils.
In conclusion, this study demonstrates that the PARS inhibitor 3-AB (but not its inactive analogue 3-ABA) reduces the infarct size caused by regional myocardial ischaemia and reperfusion in the isolated, buffer-perfused heart of the rat. In addition, inhibition of PARS activity reduces the cell injury/necrosis caused by hydrogen peroxide (mediated by hydroxyl radical) in rat cardiac myoblasts. These findings demonstrate that inhibitors of PARS activity can directly protect cardiac myocytes in vitro against oxidant stress. The ‘PARS Suicide Hypothesis’ states that the excessive activation of PARS under conditions of oxidant stress causes cell death by depletion of NAD and ATP [16]. Thus, further studies are necessary to elucidate whether the direct protection by PARS inhibitors of cardiac myocytes/blasts against oxidant stress is secondary to the prevention of the fall in high energy phosphates.
Time for primary review 22 days.
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Acknowledgements |
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Joanne Bowes is the recipient of a British Heart Foundation (BHF) PhD studentship (FS 96/015). Michelle McDonald is supported by a grant from the Joint Research Board of St Bartholomews Hospital Medical College (G724). Christoph Thiemermann is a Senior Research Fellow of the BHF (FS 96/018). We would like to thank Liz Wood for her help with the cell culture.
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