Reperfusion duration paradox with late myocardial preconditioning in rabbits

In this study, pharmacological late preconditioning was induced in 53 rabbits with an adenosine A(1) receptor agonist (2-chloro-N(6)-cyclopentyladenosine, CCPA, 100 microg/kg), or a NO-donor (S-nitroso-N-acetyl-penicillamine, SNAP, 2.5 microg/kg/min; 75 min) vs. saline as control. Later, after 24 h, rabbits underwent a 30-min coronary occlusion and subsequent reperfusion. After 3 h of coronary artery reperfusion, infarct size was reduced with CCPA (43+/-4%) and SNAP (27+/-4%) vs. saline (56+/-4%). However, after 72 h of coronary artery reperfusion, infarct sizes were similar in all groups, demonstrating an only transient effect of late preconditioning against myocardial infarction. Combined administration of CCPA and SNAP failed to induce sustained cardioprotection.     

Role of endogenous endothelin in myocardial and coronary endothelial injury after ischaemia and reperfusion in rats: studies with bosentan, a mixed ETA-ETB antagonist.

1. Previous studies suggested that endothelin-1 (ET-1) may play a role in myocardial ischaemia and reperfusion. This study was designed to test the effect of a new nonpeptide antagonist of endothelin ETA and ETB receptors, bosentan, on myocardial infarct size, ventricular arrhythmias, and coronary endothelial dysfunction after ischaemia and reperfusion. 2. Anaesthetized male Wistar rats were subjected to 20 min ischaemia (left coronary artery occlusion) followed by 1 h (for the evaluation of coronary endothelial dysfunction) or 2 h (for the evaluation of infarct size) reperfusion, or 5 min ischaemia followed by 15 min reperfusion (for the evaluation of reperfusion arrhythmias). Vascular studies were performed on 1.5-2 mm coronary segments (internal diameter 250-300 microns) removed distal to the site of occlusion and mounted in wire myographs for isometric tension recording. Area at risk and infarct size were determined by Indian ink injection and triphenyl tetrazolium staining, using computerized analysis of enlarged sections after colour video acquisition. 3. Bosentan, administered at a dose which virtually abolished the pressor response to big ET-1 (3 mg kg-1, i.v. before ischaemia) did not affect heart rate, arterial pressure or the rate pressure product before ischaemia, during ischaemia and during reperfusion. Bosentan did not affect the incidence of reperfusion-induced ventricular fibrillation (controls: 86%, n = 14; bosentan: 93%, n = 15), and did not modify infarct size (% of area at risk: controls: 63 +/- 4, n = 10; bosentan: 60 +/- 6, n = 8).(ABSTRACT TRUNCATED AT 250 WORDS)     

The time course of cardioprotection induced by GR79236, a selective adenosine A1-receptor agonist, in myocardial ischaemia-reperfusion injury in the pig

The cardioprotective effects of the selective adenosine A1-receptor agonist, GR79236 (N-[(1S, trans)-2-hydroxycyclopentyl]adenosine), were examined in a porcine model of myocardial ischaemia-reperfusion injury. When pigs were subjected to a 50-min coronary artery occlusion followed by 3-h reperfusion, GR79236 (10 nmol/kg, i.v.) significantly reduced infarct size whether given 10 min before the onset of ischaemia or reperfusion. This effect was independent of the bradycardia induced by GR79236, as it was also observed in animals in which heart rate was maintained by electrical pacing. However, GR79236 administered 10 min after reperfusion did not reduce infarct size. GR79236 had no effect on the incidence or outcome of ventricular dysrhythmias in this pig model of infarction. Similarly, ischaemic preconditioning (IPC, 2 x 10-min ischaemia and 10-min reperfusion) significantly reduced infarct size. The selective adenosine A1-receptor antagonist, 8-cyclopentyl-1,3-dipropylxanthine (DPCPX; 3.3 micromol/kg, i.v.), abolished the haemodynamic and cardioprotective effects of GR79236 and the cardioprotective effects of IPC in anaesthetised pigs. In conclusion, GR79236 exerted a marked cardioprotective effect in a porcine model of myocardial ischaemia-reperfusion injury, provided that it was administered before reperfusion. This suggests that GR79236 may have clinical utility in the treatment of various aspects of ischaemic heart disease.     

Effects and interaction, of cariporide and preconditioning on cardiac arrhythmias and infarction in rat in vivo.

1. Although Na+-H+ exchange (NHE) inhibitors are reported to protect the myocardium against ischaemic injury, NHE activation has also been proposed as a potential mechanism of ischaemic preconditioning-induced protection. This study was performed to test any modifiable effect of cariporide, an NHE inhibitor, on cardioprotective effects of preconditioning. 2. Anaesthetized rats were subjected to 30 min of coronary artery occlusion and 150 min of reperfusion. The preconditioning (PC) was induced by 3 min of ischaemia and 10 min of reperfusion (1PC) or three episodes of 3 min ischaemia and 5 min reperfusion (3PC). Cariporide (0.3 mg kg(-1)) an NHE inhibitor, was administered 30 min (cari(30)) or 45 min (cari(45)) before coronary ligation (n=8-11 for each group). 3. Ventricular arrhythmias during 30 min ischaemia and infarct size (measured by triphenyltetrazolium (TTC) and expressed as a per cent area at risk (%AAR)) were determined. Cari(30) reduced ventricular fibrillation (VF) incidence and infarct size (from 45 to 0% and 34+/-4 to 9+/-2%; each P<0.05), whereas cari(45) did not. Likewise, 3PC reduced these variables (to 0% and 10+/-2%; P<0.05 in each case) whereas 1PC did not. Moreover, subthreshold preconditioning (1PC) and cariporide (cari(45)), when combined, reduced VF incidence and infarct size (to 0% and 15+3%; each P<0.05 ). 4. In conclusion, changes in NHE activity do not seem to be responsible for the cardioprotective action of ischaemic preconditioning. Protective effects of NHE inhibition and subthreshold preconditioning appear to act additively.     

Effects of trimetazidine on myocardial preconditioning in anesthetized rats

Trimetazidine is a widely used anti-ischemic agent, but its effect on myocardial preconditioning in anesthetized animals has not been investigated. The aim of this study was to examine the effects of trimetazidine on ischemic preconditioning and carbachol preconditioning in anesthetized rats. Ischemic preconditioning, induced by 5-min coronary artery occlusion and 5-min reperfusion, decreased the incidence of ventricular tachycardia and abolished the occurrence of ventricular fibrillation during 30-min ischemia. Trimetazidine (10 mg/kg, i.v.) alone attenuated these parameters of arrhythmia. Carbachol infusion induced preconditioning with a marked depression of mean arterial blood pressure, heart rate and ventricular tachycardia. The marked reductions in parameters of arrhythmia induced by ischemic preconditioning and carbachol preconditioning were preserved in the presence of trimetazidine. Arrhythmia scores and myocardial infarct size were significantly reduced with ischemic preconditioning or carbachol preconditioning and were not inhibited by trimetazidine. These results show that trimetazidine protects the heart against ischemia-induced arrhythmias, reduces myocardial infarct size, preserves the effects of ischemic preconditioning and pharmacological preconditioning, and is able to mimic ischemic preconditioning in anesthetized rats.     

Delayed or second window preconditioning induced by adenosine A1 receptor activation is independent of early generation of nitric oxide or late induction of inducible nitric oxide synthase

Transient adenosine A1 receptor (A1R) activation induces a second window or delayed preconditioning against myocardial infarction 24-72 h later. Early generation of nitric oxide and delayed induction of nitric oxide synthase have been implicated in mediating delayed cardioprotection after ischemic preconditioning in rabbits. Recent evidence indicates that some of the regulatory roles of adenosine in cardiac tissue may be mediated by A1R-induced generation of nitric oxide. This study examined the role of nitric oxide in the mediation of A1R-induced delayed preconditioning against infarction. Pharmacologic preconditioning of rabbits with the selective A1R agonist 2-chloro-N6-cyclopentyladenosine 100 microg/kg (CCPA) significantly reduced myocardial infarct size compared with control animals, after 30 min regional ischemia and 2 h reperfusion in vivo 24 h later (27.3+/-4.7 vs. 46.0+/-3.7%, respectively; p = 0.001). Nonselective inhibition of nitric oxide synthase with N(G)-nitro-L-arginine methyl ester (10 mg/kg) before administration of CCPA did not affect this infarct limitation at 24 h. Selective inhibition of inducible nitric oxide synthase before the prolonged ischemic insult on day 2, with two structurally independent inducible nitric oxide synthase inhibitors, L-N(6)-(1-iminoethyl)-lysine (10 mg/kg) or aminoguanidine (300 mg/kg), did not abrogate the reduction in infarction observed by pharmacologic preconditioning with CCPA 24 h earlier. These results suggest that the second window or delayed protection against myocardial infarction observed 24 h after pharmacologic preconditioning with an adenosine A1 agonist occurs independently of either early generation of nitric oxide or subacute induction of inducible nitric oxide synthase.     

Intravenous atenolol and esmolol maintain the protective effect of ischemic preconditioning in vivo

Catecholamines bind to alpha- and beta-adrenoreceptors and are capable of preconditioning ischemic myocardium. Our purpose was to investigate the effect of acute either short or prolonged i.v. administration of beta-adrenoreceptor antagonists on ischemic preconditioning in vivo. Fifty-five anesthetized rabbits were divided into 10 groups (n=5-7 per group) and were subjected to 30-min regional ischemia of the heart after ligation of a prominent left coronary artery and 3-h reperfusion after releasing the snare. Ischemic preconditioning was obtained by three cycles of 5-min ischemia separated by 10-min reperfusion. beta-Adrenoreceptor blockade was obtained by the long acting beta-adrenoreceptor antagonist atenolol or by the short acting esmolol, which were given as a short 5-min infusion or as a prolonged 45-min infusion, starting respectively 20 min before and ending 15 min before the beginning of sustained ischemia, or starting 45 min before and ending immediately before the beginning of sustained ischemia. Atenolol was given at a rate of 0.2 mg min(-1) during 5 min or at a rate of 0.088 mg min(-1) as a 45-min infusion. Esmolol was given as an initial dose of 500 microg kg(-1) within 1 min, followed by a 4-min infusion at a rate of 50 microg kg(-1) min(-1) or as an initial dose of 3.4 mg within 1 min, followed by a 44-min infusion at a rate of 0.15 mg min(-1). Blood pressure and heart rate were continuously monitored. The infarcted and risk areas were delineated with the aid of tetrazolium chloride staining and fluorescent Zn-Cd particles. Infarct size was expressed in percent of the area at risk. All the animals without preconditioning developed an infarct size ranging between 36.3+/-2.4% and 49.6+/-7.6% (P=NS) and all the preconditioning groups developed an infarct size ranging between 14.9+/-1.2% and 21.0+/-2.2% (P=NS). All the preconditioning groups, independently of the use of beta-adrenoreceptor antagonists, had a smaller infarct size than the control group, which developed an infarct size of 47.3+/-2.5% (P<0.01). Intravenous atenolol and esmolol, independent of timing and mode of administration, does not seem to interfere with protection afforded by ischemic preconditioning in vivo.     

Arrhythmias induced by myocardial ischaemia-reperfusion are sensitive to ionotropic excitatory amino acid receptor antagonists

We have investigated the effects of (+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-imine hydrogen maleate (MK801), a non-competitive N-methyl-D-aspartate (NMDA) ionotropic excitatory amino acid receptor antagonist, and 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX), a non-NMDA ionotropic excitatory amino acid receptor antagonist, ketamine and memantine, NMDA receptor channel blockers, on ventricular arrhythmias induced by myocardial ischaemia and myocardial ischaemia-reperfusion. Coronary artery occlusion caused 100 +/- 2% ventricular tachycardia, in saline treated group, and 60 +/- 3% ventricular fibrillation. 66 +/- 6% of the animals recovered from ventricular fibrillation, while in 34 +/- 4% of animals the ventricular fibrillation caused mortality. The incidence of ventricular tachycardia, ventricular fibrillation and mortality was not modified by treatment of rats with MK801 (0.3 mg/kg i.v.), CNQX (1 mg/kg i.v.), ketamine (10 mg/kg) and memantine (1.5 mg/kg), injected 5 min prior to occlusion. Reperfusion caused severe arrhythmias which started within 5 +/- 2 s. For instance, in the saline treated group, the incidence of ventricular tachycardia was 100 +/- 5%, while ventricular fibrillation occurred in 87 +/- 3% of the animals and lasted 90 +/- 12 s. The mortality was 62 +/- 6%. The incidence of ventricular tachycardia, ventricular fibrillation and mortality induced by reperfusion was greatly (P < 0.01) reduced in animals treated with MK801 (0.3 mg/kg i.v.), CNQX (1 mg/kg i.v.), ketamine (10 mg/kg) and memantine (1.5 mg/kg), injected 5 min prior to occlusion. Therefore, reperfusion-induced arrhythmias, but not ischaemia-induced arrhythmias, are sensitive to NMDA/non-NMDA ionotropic excitatory amino acid receptor antagonists.     

Pharmacological evidence that inducible nitric oxide synthase is a mediator of delayed preconditioning

Brief periods of myocardial ischaemia preceding a subsequent more prolonged ischaemic period 24-72 h later confer protection against myocardial infarction ('delayed preconditioning' or the 'second window' of preconditioning). In the present study, we examined the effects of pharmacological modifiers of inducible nitric oxide synthase (iNOS) induction and activity on delayed protection conferred by ischaemic preconditioning 48 h later in an anaesthetized rabbit model of myocardial infarction. Rabbits underwent a myocardial preconditioning protocol (four 5 min coronary artery occlusions) or were sham-operated. Forty-eight hours later they were subjected to a sustained 30 min coronary occlusion and 120 min reperfusion. Infarct size was determined with triphenyltetrazolium staining. In rabbits receiving no pharmacological intervention, the percentage of myocardium infarcted within the risk zone was 43.9+5.0% in sham-operated animals and this was significantly reduced 48 h after ischaemic preconditioning with four 5 min coronary occlusions to 18.5+5.6% (P<0.01). Administration of the iNOS expression inhibitor dexamethasone (4 mg kg(-1) i.v) 60 min before ischaemic preconditioning completely blocked the infarct-limiting effect of ischaemic preconditioning (infarct size 48.6+/-6.1%). Furthermore, administration of aminoguanidine (300 mg kg(-1), s.c.), a relatively selective inhibitor of iNOS activity, 60 min before sustained ischaemia also abolished the delayed protection afforded by ischaemic preconditioning (infarct size 40.0+/-6.0%). Neither aminoguanidine nor dexamethasone per se had significant effect on myocardial infarct size. Myocardial risk zone volume during coronary ligation, a primary determinant of infarct size in this non-collateralized species, was not significantly different between intervention groups. There were no differences in systolic blood pressure, heart rate, arterial blood pH or rectal temperature between groups throughout the experimental period. These data provide pharmacological evidence that the induction of iNOS, following brief periods of coronary occlusion, is associated with increased myocardial tolerance to infarction 48 h later.     

Similarities between ischemic preconditioning and 17beta-estradiol mediated cardiomyocyte KATP channel activation leading to cardioprotective and antiarrhythmic effects during ischemia/reperfusion in the intact rabbit heart

The aims of our present work were to assess whether treatment with either ischemic preconditioning (IPC) or 17beta-estradiol or both combined produce proarrhythmic or antiarrhythmic effects, and whether opening of the sarcolemmal or mitochondrial KATP channels is relatable to this effect; to assess biochemically the effects of IPC and/or 17beta-estradiol on oxidant stress and antioxidant defenses in the myocardium; to examine the effects of nitric oxide (NO) synthase inhibitor, Nomega-nitro-L-arginine methyl ester (L-NAME) pretreatment in rabbits treated with either IPC or 17beta-estradiol (because 17beta-estradiol evoked NO release has been implicated in KATP activation and IPC); and examine the effects of ischemic preconditioning and 17beta-estradiol on myocardial energy metabolism during ischemia and reperfusion in a well-standardized model of reperfusion arrhythmias in anesthetized adult male New Zealand White rabbits (n = 124) subjected to 30 minutes occlusion of the left coronary artery followed by 120 minutes of reperfusion. Pretreatment with either 17beta-estradiol (10 microg/kg, i.v.) or one cycle of ischemic preconditioning prior to the period of coronary occlusion offers significant infarct size reduction (18.6 +/- 2.2% and 19.4 +/- 1.9%, respectively versus 40.1 +/- 3.9% in saline control and 39.2 +/- 3.2% in vehicle control groups; P < 0.01) and antiarrhythmic effects. Both 17beta-estradiol and ischemic preconditioning treatment significantly attenuated the incidence of life-threatening arrhythmias like sustained VT (13% and 13%, respectively versus 100% in saline control and 100% in vehicle control groups; P < 0.001) and other arrhythmias (25% and 25%, respectively versus 100% in saline control and 100% in vehicle control groups; P < 0.001), and were quite effective in increasing the number of animals that survived without developing any arrhythmia during ischemia and reperfusion. 5-hydroxydecanoate(5-HD; 5 mg/kg, i.v.) alone offered no cardioprotective and antiarrhythmic activities. Pretreatment with 5-HD but not HMR 1883 (3 mg/kg, i.v.) abolished the beneficial effects of 17beta-estradiol and ischemia preconditioning on reperfusion-induced arrhythmias and cardioprotection suggesting that such effects have been achieved via the selective activation of cardiomyocyte mitochondrial KATP channels rather than sarcolemmal KATP channels. The reduced reperfusion arrhythmic incidence and durations induced by estrogen was not significantly altered by ICI 182 720 (2.5 mg/kg, i.v.). The lack of effect of ICI 182 720 on antiarrhythmic and infarct-limiting effects of 17beta-estradiol and ischemic preconditioning suggest that these favorable effects are rapid, direct, and non-genomic effects. This study demonstrates similarities between 17beta-estradiol and ischemic preconditioning of the rabbit myocardium in terms of cardioprotection, antiarrhythmic, and metabolic activities. Ischemic preconditioning and 17beta-estradiol appear to share a final common effector; the mitochondrial KATP channel.     

The role of mitochondrial K(ATP) channels in antiarrhythmic effects of ischaemic preconditioning in dogs

1. In the canine a single brief (5 min) coronary artery occlusion protects the myocardium against the severe ventricular arrhythmias and reduces the ischaemic changes that result from a subsequent, more prolonged (25 min) occlusion. The main purpose of the present study was to examine whether mitochondrial K(ATP) channels are involved in this protection. 2. In chloralose-urethane anaesthetized dogs, preconditioning (PC) was induced by a single 5 min period occlusion of the left anterior descending (LAD) coronary artery, 20 min prior to a 25 min occlusion of the same artery. In some of these PC dogs 5-hydroxydecanoate (5-HD; 150 micro g kg(-1) min(-1) by intracoronary infusion) was given over a period of 30 min either before, or after PC. In other dogs the mitochondrial K(ATP) channel opener diazoxide (1 mg kg(-1); i.c.) was given, either alone or in the presence of 5-HD. Control dogs (infused with saline) were simply subjected to a 25 min occlusion and reperfusion. 3. Compared to controls, both PC and diazoxide significantly reduced the number of ventricular premature beats (VPBs; 295+/-67 to 89+/-28 and 19+/-11, respectively; P<0.05), the number of episodes of ventricular tachycardia (VT; 8.3+/-4.2 to 1.6+/-0.9 and 0.2+/-0.1; P<0.05) and the incidences of VT (100 to 43 and 33%; P<0.05) and ventricular fibrilation (VF; 60 to 0 and 17%; P<0.05) during the 25 min occlusion of the LAD. Further, 43% of the PC dogs and 58% of the diazoxide treated dogs survived the combined ischaemia-reperfusion insult (cp. 0% in the controls; P<0.05). The protection afforded by PC and diazoxide was abolished by 5-HD, especially when it was given prior to the PC occlusion. In the presence of 5-HD, three out of 10 dogs fibrillated during the PC occlusion and another three dogs died following reperfusion. Furthermore, there were no survivors in this group from the prolonged ischaemia/reperfusion insult. 5-HD given after PC only attenuated the antiarrhythmic protection. 4. Opening of mitoK(ATP) channels prior to ischaemia by preconditioning and diazoxide protects the myocardium against ischaemia and reperfusion-induced arrhythmias. This protection is abolished if the opening of these channels is prevented by the prior administration of 5-HD but only attenuated if 5-HD is given after preconditioning. The results indicate that opening of mitoK(ATP) channels prior to ischaemia is mandatory for protection against ischaemia and reperfusion-induced arrhythmias.     

Selective 5-lipoxygenase inhibitor BW A4C does not influence progression of tissue injury in a canine model of regional myocardial ischaemia and reperfusion.

The effects of BW A4C, a selective arachidonate 5-lipoxygenase (5-LO) inhibitor, on the progression of myocardial tissue injury were examined in anaesthetised, open-chest beagle dogs subjected to 90-min occlusion of the left anterior descending coronary artery (LAD) followed by 120-min reperfusion. Regional myocardial blood flow (RMBF, microspheres), segment shortening (sonomicrometry), and infarct size (tetrazolium stain) as an index of tissue injury were measured. Control animals (group 1, n = 11) received an infusion of vehicle [50% vol/vol glycofurol and distilled water, 47 ml at 12 ml h-1, intravenously (i.v.)] beginning 15 min before ischaemia and continuing until the end of reperfusion. Treated animals received either 10 (group 2, n = 11) or 50 micrograms kg-1 min-1 (group 3, n = 5) BW A4C i.v. in the same period. The infarct/risk zone ratio (I/R) in group 1 (24.1 +/- 6.0%) was not significantly different from that of group 2 (28.0 +/- 8.4%) or group 3 (46.1 +/- 6.7%). The close inverse relationship observed in controls between I/R ratio and collateral flow was not altered by either dose of BW A4C. Segment shortening during ischaemia (-0.2 +/- 2.7, -2.4 +/- 1.7, and -1.5 +/- 1.7%) and reperfusion (4.9 +/- 2.8, 1.0 +/- 1.8, and -1.0 +/- 1.9%) and during an isoprenaline infusion to unmask stunned myocardium (14.7 +/- 3.0, 14.7 +/- 2.6, and 7.4 +/- 1.7%) were not significantly different between groups 1, 2, and 3.(ABSTRACT TRUNCATED AT 250 WORDS)     

Antidiabetic drug miglitol inhibits myocardial apoptosis involving decreased hydroxyl radical production and Bax expression in an ischaemia/reperfusion rabbit heart

1 We examined whether antidiabetic drug miglitol could reduce ischaemia/reperfusion-induced myocardial apoptosis by attenuating production. 2 Japanese white rabbits were subjected to 30-min coronary occlusion followed by 4-h reperfusion with miglitol (10 mg kg(-1), i.v., n=20) or saline (n=20). The infarct area was determined by myoglobin staining, and the infarct size (IS) was expressed as a percentage of the area at risk. DNA fragmentation was assessed by TUNEL method and DNA ladder formation. The expression of Bcl-XL and Bax was detected by immunohistochemical analysis and Western blot analysis. Myocardial interstitial 2,5-DHBA levels, an indicator of hydroxyl radicals, were measured during 30-min ischaemia and 30-min reperfusion in the absence (n=10) or presence of miglitol (10 mg kg(-1), i.v., n=10) using a microdialysis technique. 3 The IS was significantly reduced in the miglitol group (22.4+/-3.4%, n=10) compared to the control group (52.8+/-3.5%, n=10). Miglitol significantly decreased the 2,5-DHBA level during ischaemia and reperfusion and suppressed the incidence of TUNEL-positive myocytes in the ischaemic region (from 10.7+/-3.4 to 4.1+/-3.0%) and the intensity of DNA ladder formation. Miglitol significantly decreased the incidence of Bax-positive myocytes in the ischaemic region (7.4+/-1.7 vs 13.7+/-1.9% of the control) and significantly attenuated the upregulation of Bax protein in the ischaemic regions (from 179+/-17 to 90+/-12% of sham). There was no difference in the expression of Bcl-XL between the two groups. 4 These data suggest that miglitol reduces myocardial apoptosis by attenuating production of hydroxyl radicals and suppressing the upregulation of the expression of Bax protein.     

Evidence against a role of inducible nitric oxide synthase in the endothelial protective effects of delayed preconditioning

Preconditioning the heart with brief periods of ischaemia induces delayed endothelial protection against reperfusion injury, but the precise mechanisms involved in this endogenous protein are still unclear. Induction of the type II-nitric oxide synthase (iNOS) acts as a mediator of the preconditioning against myocardial infarction and stunning. The present study was designed to assess whether iNOS also contributes to the delayed endothelial protective effects of preconditioning. Rats were subjected to 20 min ischaemia followed by 60 min reperfusion 24 h after sham surgery or preconditioning (one cycle or 2 min ischaemia/5 min reperfusion and two cycles of 5 min ischaemia/5 min reperfusion). At the end of the reperfusion, coronary segments were removed distal to the site of occlusion and mounted in wire myographs. Ischaemia-reperfusion (I/R) decreased the endothelium-dependent relaxations to acetylcholine (maximal relaxations: sham, 66+/-5%; I/R, 40+/-1%; P<0.05) and this impairment was prevented by preconditioning (maximal relaxation: 61+/-6%). Administration of N-(3-aminomethyl)benzyl)acetaminide (1400W), a highly selective inhibitor for iNOS, 10 min before prolonged ischaemia did not modify the beneficial effect of preconditioning (maximal relaxation: 66+/-5%). These data show that preconditioning induces delayed protection against reperfusion-injury. However, in contrast to the myocytes, these endothelial protective effects of delayed preconditioning do not involve iNOS.     

Contribution of peroxynitrite to the beneficial effects of preconditioning on ischaemia-induced arrhythmias in rat isolated hearts

We studied the effects of urate, a peroxynitrite scavenger, on ischaemia- and peroxynitrite-induced preconditioning in rat isolated hearts. Isolated hearts perfused with Krebs-Henseleit solution were preconditioned either by 3 min of coronary artery occlusion or by peroxynitrite administration (1 microM) for 3 min, followed by 10 min of reperfusion and 30 min of coronary artery occlusion. Both ischaemia and peroxynitrite produced a marked reduction in arrhythmias. Urate (1 mM) added to the perfusate 10 min prior to ischaemic preconditioning or peroxynitrite infusion and maintained until coronary artery occlusion, markedly reversed the beneficial effects in the ischaemic and peroxynitrite-treated groups. Urate administration in the peroxynitrite-treated group increased the incidence of ventricular tachycardia from 57% (n = 11) to 100% (n = 6) and total ventricular fibrillation from 0% (n=0) to 44% (n=4). Similarly, urate augmented the incidence of ventricular tachycardia from 47% (n=8) to 85% (n = 6) in the ischaemic preconditioning group. On its own, urate did not affect the severity of cardiac arrhythmias. Peroxynitrite infusion caused a marked increase in the effluent nitrate levels, from 0.05 +/- 0.1 microM (n = 5) to 0.4 +/- 0.2 microM (n = 6), and urate significantly decreased these levels to 0.08 +/- 0.03 microM (n = 9). These results suggest that peroxynitrite at low concentrations contributes to the beneficial effects of preconditioning on ischaemia-induced arrhythmias in rat isolated hearts.     

Cardioprotective effects of nicorandil in rabbits anaesthetized with halothane: potentiation of ischaemic preconditioning via KATP channels

1. The roles of ATP-sensitive K+ channels (KATP channels) in ischaemic or pharmacological preconditioning in the rabbit heart remain unclear. Infarct limitation by ischaemic preconditioning was abolished by the KATP channel blocker glibenclamide under ketamine/xylazine anaesthesia, but not under anaesthesia induced by pentobarbital. Infarct limitation by the KATP channel opener pinacidil was detected under ketamine/xylazine anaesthesia, but not under pentobarbital anaesthesia. Thus, these effects appear to be anaesthetic dependent. 2. In the present study, we examined whether nicorandil (a KATP channel opener nitrate) exhibits cardioprotective actions under halothane anaesthesia, another commonly used volatile anaesthetic. Control animals were subjected to 40 min coronary occlusion and 120 min reperfusion. Before 40 min ischaemia, the nicorandil group received nicorandil (100 microg/kg per min, i.v., for 10 min), the 5' preconditioning (PC) group received 5 min ischaemia/20 min reperfusion, the 2.5'PC group received 2.5 min preconditioning ischaemia/20 min reperfusion, the nicorandil +2.5'PC group received both nicorandil and 2.5 min ischaemia/20 min reperfusion, the nicorandil +2.5'PC + 5-hydroxydecanoate (5HD) group received both nicorandil and 2.5 min ischaemia/20 min reperfusion in the presence of 5-hydroxydecanoate (5HD; a KATP blocker) and the 5HD group received 5 mg/kg, i.v., 5HD alone. Myocardial infarct size in control (n = 7), nicorandil (n = 5), 5'PC (n = 8), 2.5'PC (n = 5), nicorandil + 2.5'PC (n = 5), nicorandil + 2.5'PC + 5HD (n = 5) and 5HD (n = 4) groups averaged 44.4 +/- 3.6, 41.7 +/- 5.7, 17.8 +/- 3.2,* 34.1 +/- 4.8, 21.3 +/- 4.2,* 39.1 +/- 5.6 and 38.9 +/- 5.0% of the area at risk, respectively (*P <0.05 vs control). 3. Thus, nicorandil alone did not have an infarct size-limiting effect in halothane-anaesthetized rabbits. However, the results suggest that even when nicorandil alone does not demonstrate a direct cardioprotective effect, it may enhance ischaemic preconditioning via KATP channels. Key words: ATP-sensitive K+ (KATP) channel, ischaemic preconditioning, myocardial infarction, nicorandil, rabbit.     

Preischemic and postischemic treatment with a new Na+/H+-exchange inhibitor, FR183998, shows cardioprotective effects in rats with cardiac ischemia and reperfusion

This study describes the pharmacologic profile of a new Na+/H(+)-exchange inhibitor, FR183998, in anesthetized rats. FR183998 had a potent inhibitory effect on Na+/H+ exchange of rat lymphocytes with median inhibitory (IC50) value of 0.3 nM. Treatment with FR183998 (0.01-0.32 mg/kg, i.v.) reduced or completely abolished ventricular fibrillation and mortality induced by 5-min ischemia followed by reperfusion, when it was administered not only 5 min before ischemia but also 1 min before reperfusion. Myocardial infarct size induced by 30-min ischemia and 60-min reperfusion was reduced significantly in a dose-dependent manner by FR183998 (0.1-1.0 mg/kg, i.v.) when the drug was administered preischemically or at an early phase of ischemia. The ventricular tachycardia and the ventricular fibrillation observed during the ischemic period also were suppressed significantly. These results indicate that FR183998 has a strong inhibitory effect on Na+/H+ exchange and suggest that treatment with FR183998 either before or immediately after the onset of ischemia can prevent the occurrence of arrhythmias and myocardial cell necrosis in situations of ischemia and reperfusion.     

The effect of body temperature on myocardial protection conferred by ischaemic preconditioning or the selective adenosine A1 receptor agonist GR79236, in an anaesthetized rabbit model of myocardial ischaemia and reperfusion

1 The cardioprotective effect of N-[(1S, trans)-2-hydroxycyclopentyl]adenosine (GR79236), an adenosine A1 receptor agonist, was compared with that produced by ischaemic preconditioning in an anaesthetized rabbit model of myocardial ischaemia and reperfusion. In addition, we examined the effect of different body core temperatures on GR79236- or ischaemic preconditioning-induced cardioprotection when administered prior to ischaemia, and on cardioprotection induced by GR79236 administered 10 min prior to the onset of reperfusion. 2 When rabbits were subjected to 30 min occlusion of the left coronary artery, followed by 2 h reperfusion, GR79236 (3 x 10(-8) mol kg-1 i.v. (10.5 microg kg-1 i.v.)) or ischaemic preconditioning (5 min ischaemia followed by 5 min reperfusion), administered or applied 10 min prior to the occlusion, significantly limited the development of infarction. The cardioprotective effect of ischaemic preconditioning was significantly greater than that seen after administration of GR79236. Pre-treatment with the selective adenosine A1 receptor antagonist 8-cyclopentyl-1,3-dipropylxanthine (DPCPX, 3.3 x 10(-6) mol kg-1 (1 mg kg-1 i.v.)), prevented the cardioprotective effect of GR79236, but not that of ischaemic preconditioning. 3 Maintaining body core temperature at 38.5 degrees C rather than at 37.0 degrees C did not influence infarct size in control groups of rabbits, but reduced the cardioprotective effect of GR79236 when administered 10 min prior to occlusion or 10 min prior to the onset of reperfusion. The cardioprotective effect of ischaemic preconditioning was not temperature-dependent. 4 In conclusion, myocardial protection conferred by GR79236 in anaesthetized rabbits is mediated via adenosine A1 receptors. Myocardial protection can be conferred when GR79236 is administered before the onset of ischaemia or reperfusion, and is reduced when body core temperature is maintained at 38.5 degrees C rather than at 37.0 degrees C. In contrast, myocardial protection conferred by ischaemic preconditioning is not reduced by adenosine A1 receptor blockade, or by maintaining body core temperature at 38.5 degrees C rather than at 37.0 degrees C. These findings point to distinct differences in the mechanisms of induction of myocardial protection by adenosine A1 receptor agonist and ischaemic preconditioning. They also highlight the need for careful control of body core temperature when investigating the phenomenon of cardioprotection.     

Ischaemic preconditioning and mast cell histamine release: microdialysis of isolated rat hearts.

The aim of this study was to investigate the feasibility of intramyocardium kinetics of histamine release by microdialysis in the isolated rat heart and ascertain if the inhibition of histamine release is implicated in the antiarrhythmic effect of preconditioning. A 30 min normothermic global ischaemia model followed by 30 min reperfusion was carried out in the control group (n= 9). In the preconditioning group (n= 8) there was a 5 min global ischaemia followed by 10 min of reperfusion. A mast cell stabilizing group received the disodium cromoglycate ( 10 micro M, n= 10). The last group received a mast cell degranulator, compound 48/80 (1micro g ml (-1), n= 10). In the control group, the histamine release during reperfusion was significantly different from the basal concentration ( 18.4 +/- 6.5 vs 1.9 +/- 0.5 nM, P< 0.05) and was associated with a maximal period of severe arrhythmias. The ischaemic preconditioning modified the histamine release kinetics with an early mast cell degranulation ( 9.7 +/- 1.5 nM) and a significant decrease in the total period of severe arrhythmias in comparison with the control group ( P< 0.05). In the disodium cromoglycate group, the histamine release during reperfusion decreased ( 3.1 +/- 0.7 nM) and was associated with a maximal period of severe arrhythmias. In the C48/80 group, the increase in the histamine released during reperfusion ( 21.2 +/- 5.0 nM) was associated with a maximal period of severe arrhythmias. These results showed firstly the feasibility of kinetic histamine release in myocardium interstitial fluid on the isolated rat heart and secondly that the inhibition of histamine release did not play a direct role in the antiarrhythmic effect of preconditioning.     

The KATP channel blocker HMR 1883 does not abolish the benefit of ischemic preconditioning on myocardial infarct mass in anesthetized rabbits

Previous experimental studies showed that the benefit of ischemic preconditioning (IPC) is abolished by K(ATP) channel blockade with glibenclamide. However, the newly discovered K(ATP) channel blocker HMR 1883 (1-[[5-[2-(5-chloro-o-anisamido)ethyl]-methoxyphenyl]sulfonyl]-3-m ethylthiourea) shows marked antifibrillatory activity in the dose range of 3 mg/kg to 10 mg/kg i.v. in various experimental models without affecting blood glucose levels. In order to investigate in a head to head comparison glibenclamide and HMR 1883 with respect to their influence on IPC, experiments were performed in rabbits with ischemia-reperfusion using myocardial infarct mass as final read out. Male New Zealand White rabbits (2.6-3.0 kg) were subjected to 30-min occlusion of a branch of the left descending coronary artery (LAD) followed by 2-h reperfusion. For IPC experiments the LAD was additionally occluded for two periods of 5 min, each followed by 10-min reperfusion, before the long-term ischemia. Infarct mass was evaluated by TTC staining and expressed as a percentage of area at risk. Rabbits (n=7/group) were randomly selected to receive (i.v.) saline vehicle 5 min prior to the 30-min occlusion period in infarct studies without IPC or to receive glibenclamide (0.3 mg/kg) or HMR 1883 (3 mg/kg) in IPC experiments, these substances being given 5 min prior to the first preconditioning or 5 min prior to the long-term ischemia of 30 min. Myocardial risk mass as a percentage of left ventricular mass did not differ between groups. The same was true for the ratio of left ventricular mass to 100 g body weight. Myocardial infarct mass as a percentage of the area at risk in the saline vehicle group without IPC was 41+/-3%. Whereas glibenclamide significantly increased infarct mass (from 41+/-3% to 55+/-4%), HMR 1883 did not affect it. IPC reduced infarct mass from 41+/-3% to 21+/-4% (P<0.05 vs. control without IPC). Glibenclamide given prior to IPC or prior to the long-term ischemia totally abolished the IPC effect (42+/-2% and 55+/-4%, respectively; P<0.05 vs. control). In contrast, HMR 1883 under the same conditions did not affect infarct size when given prior to IPC or prior to the long-term ischemia (21+/-3% and 26+/-2%, respectively). The monophasic action potential duration (MAP50) was reduced from 103+/-3 ms under normoxic conditions to 82+/-2 ms, 5 min after ischemia in the absence of drugs. This ischemia-induced shortening of the MAP was prevented by both HMR 1883 (MAP50 103+/-3 ms) and glibenclamide (MAP50 106+/-3 ms). In conclusion, although both K(ATP) channel blockers prevented ischemia-induced shortening of MAP, HMR 1883 did not abolish the beneficial effects of IPC on myocardial infarct mass in rabbits, whereas glibenclamide totally reversed this cardioprotective effect of IPC. This suggests that the sarcolemmal ATP-sensitive potassium channels are not involved in the mechanism of IPC.