Preconditioning of the ischaemic myocardium; involvement of the L-arginine nitric oxide pathway.

1. Short periods of coronary artery occlusion protect the heart against the effects of a subsequent prolonged period of ischaemia. This phenomenon is known as preconditioning of the ischaemic myocardium. 2. In mongrel, chloralose-urethane anaesthetized open-chest dogs, within a restricted body weight range, two 5 min periods of occlusion of the anterior descending branch of the left coronary artery markedly reduced the severity of the early ischaemic arrhythmias resulting from a prolonged (25 min) occlusion of the same coronary artery starting 20 min later. Thus, the number of ventricular premature beats (VPBs) was reduced from 528 +/- 140 in controls to 78 +/- 27 in preconditioned dogs, the incidence of ventricular fibrillation (VF) was reduced from 47% to 0% and the incidence of ventricular tachycardia (VT) from 100% to 20%. ST-segment elevation recorded from electrodes within the ischaemic area, and the degree of inhomogeneity of conduction within the ischaemic area were markedly reduced in these preconditioned dogs. 3. The incidence of VF following reperfusion of the ischaemic myocardium at the end of the 25 min occlusion period was reduced in the preconditioned dogs from 100% to 60%; there was thus a 40% survival from the combined ischaemia-reperfusion insult compared with 0% in the controls. 4. NG-nitro-L-arginine methyl ester (L-NAME) an inhibitor of the L-arginine nitric oxide pathway, given in a dose of 10 mg kg-1 intravenously on two occasions, both before the initial preconditioning occlusion and then again before the prolonged occlusion, partially attenuated the protective effects of preconditioning.(ABSTRACT TRUNCATED AT 250 WORDS)     

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.     

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.     

Effects of Bordetella pertussis toxin pretreatment on the antiarrhythmic action of ischaemic preconditioning in anaesthetized rats.

1. Bordetella pertussis toxin, which catalyses the ADP-ribosylation of certain guanine nucleotide binding proteins (G proteins), thus functionally uncoupling them from associated receptors, was examined to determine whether it modified the antiarrhythmic effect of ischaemic preconditioning in anaesthetized rats. 2. Pertussis toxin (25 micrograms kg-1, i.p., 48 h prior to heart isolation) attenuated the negative chronotropic effect of acetylcholine (ACh) in rat isolated Langendorff perfused hearts. ACh (10 microM) reduced heart rate by 4% in hearts taken from pertussis toxin-treated animals, compared to a reduction of 57% in hearts taken from animals treated only with vehicle. 3. In anaesthetized rats, ischaemic preconditioning (a single 3 min occlusion of the left main coronary artery followed by 10 min reperfusion) had a pronounced antiarrhythmic effect during a subsequent 30 min period of regional myocardial ischaemia. Compared to hearts receiving only a 30 min period of left coronary occlusion, there was a reduced mortality (67% and 0% for control and preconditioned groups, respectively; P < 0.01) and decreased incidences of ventricular tachycardia (VT) and ventricular fibrillation (VF). Pretreatment with pertussis toxin (25 micrograms kg-1, i.p., 48 h previously) did not modify the arrhythmias associated with a 30 min period of regional myocardial ischaemia, neither did it modify the reduction in mortality (from 56% to 0%; P < 0.05) associated with preconditioning. Furthermore, the decrease in total ventricular premature beat count induced by preconditioning seen in controls (from 427 +/- 130 to 95 +/- 45) was also seen in pertussis toxin-treated rats (from 252 +/- 190 to 57 +/- 25).(ABSTRACT TRUNCATED AT 250 WORDS)     

Blockade of anaesthetic-induced preconditioning in the hyperglycaemic myocardium: the regulation of different mitogen-activated protein kinases

Preconditioning by volatile anaesthetics is blocked by hyperglycaemia. The regulation of mitogen-activated protein kinases during this effect has yet not been investigated. For infarct size measurements, anaesthetized rats were subjected to 25 min coronary artery occlusion followed by 120 min reperfusion. Control animals were not further treated. One group was preconditioned by two 5-min periods of desflurane inhalation (desflurane preconditioning, Des-preconditioning, 1MAC), each followed by 10-min washout. Four groups received glucose 50% in order to achieve blood glucose concentrations between 22.2 and 33.3 mM/l. Glucose infusion started 40 min before ischaemia (early hyperglycaemia, EH) and stopped with the onset of artery occlusion with (EH+Des-preconditioning) or without (EH) preconditioning. The other two groups received glucose during ischaemia (late hyperglycaemia, LH), again with (LH+Des-preconditioning) or without (LH) preconditioning. Additional hearts were excised for Western blot of mitogen-activated protein kinases. Infarct size was reduced from 51.7+/-9.0% in controls to 28.8+/-11.8% after Des-preconditioning (P<0.01 vs Con). Hyperglycaemia alone did not affect infarct size (EH, 51.5+/-9.0%, LH, 44.3+/-16.9%), but EH as well as LH blocked Des-preconditioning (49.1+/-12.3%, P<0.01, 48.1+/-17.6%, P<0.05 vs Des-preconditioning). All three mitogen-activated protein kinases showed a similar time course pattern of phosphorylation in the Des-preconditioning, EH and EH+Des-preconditioning group. Despite the lack of cardioprotection, mitogen-activated protein kinases are activated in hyperglycaemic myocardium. Therefore, it can be assumed that the hyperglycaemic induced blockade of Des-preconditioning is situated downstream or in parallel of these mitogen-activated protein kinases or involves different signal transduction pathways.     

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.     

Sildenafil (Viagra) reduces arrhythmia severity during ischaemia 24 h after oral administration in dogs

Sildenafil (Viagra) prolongs repolarisation in cardiac muscle, an effect that could lead to ventricular fibrillation (VF). Sildenafil (2 mg kg(-1)) was given by mouth to 12 mongrel dogs and, 24 h later, these dogs were anaesthetised, thoracotomised and subjected to a 25 min occlusion of the anterior descending coronary artery. Haemodynamic parameters were similar in this and the control group, but there were fewer and less serious ventricular arrhythmias during occlusion in the sildenafil group (VF 17 vs 60%; ventricular premature beats 140+/-52 vs 437+/-127% and episodes of ventricular tachycardia 4.0+/-3.2 vs 19.3+/-7.7%, all P<0.05). However, reperfusion VF and indices of ischaemia severity (epicardial ST-segment mapping, inhomogeneity) were not modified by the drug. Sildenafil increased the QT interval, especially during ischaemia. Our conclusion is that ischaemia-induced ventricular arrhythmias are reduced by sildenafil, but this protection is less pronounced than that following cardiac pacing or exercise.     

Involvement of protein kinase C in the delayed cytoprotection following sublethal ischaemia in rabbit myocardium.

Rabbit hearts were preconditioned with four 5 min coronary artery occlusions 24 h before 30 min coronary occlusion with 120 min reperfusion. Preconditioning significantly reduced the percentage of myocardium infarcting within the risk zone from 49.1 +/- 4.3% to 31.8 +/- 3.5% (P < 0.05). When the protein kinase C (PKC) inhibitor, chelerythrine, was administered just before preconditioning, the delayed protection against infarction 24 h later was abolished. We conclude that the delayed cytoprotective response associated with ischaemic preconditioning of myocardium is likely to involve the early activation of one or more PKC subtypes.     

The isolated blood-perfused rat heart: an inappropriate model for the study of ischaemia- and infarction-related ventricular fibrillation

1. Well-characterized in vivo and in vitro models exist for the study of ischaemia- and infarction-related ventricular fibrillation (VF). In rats in vivo, VF appears to occur in distinct acute ischaemia- (early) and infarction-related (late) phases. Interestingly, isolated buffer-perfused rat hearts do not develop late VF. This raises the possibility that unidentified components of the blood may be responsible for late VF. We thus sought to characterize an isolated blood-perfused rat heart in order to investigate the possible influence of blood components on arrhythmias arising from ischaemia and infarction. 2. Hearts, excised from male Wistar rats, were perfused in the Langendorff mode with blood from support rats (male Wistar, 350-430 g) via an extracorporeal circuit. Perfused hearts underwent left coronary artery occlusion for 240 min or a sham procedure (n=10 group(-1)). 3. Only 10% of ischaemic hearts developed late VF (90-240 min). Tissue myeloperoxidase activity (an index of neutrophil accumulation) increased during ischaemia from 0.017+/-0.004 (six fresh hearts) to 0.056+/-0.005 units mg protein(-1) (P<0.05) at 240 min, but values were similar in sham hearts (0.083+/-0.013). Likewise, the decline (-1 vs 240 min of ischaemia shown) in circulating total white blood cells from 6.8+/-0.5 to 1.9+/-0.2 x 10(3) micro l(-1) and in platelets from 441+/-32 to 274+/-16 x 10(3) micro l(-1) (both P<0.05) was similar in time-matched sham hearts (data not shown). 4 Surprisingly, only 10% of ischaemic hearts developed early VF (0-90 min), although the incidence of early ventricular tachycardia was 100% in these hearts (P<0.05 vs sham hearts). Blood K+ values were normal (hyperkalaemia suppresses VF). 5 Although late VF was absent in blood-perfused hearts, it would be premature to conclude from this that late VF is not mediated by blood components. This is because the similar neutrophil accumulation in ischaemic and sham hearts, the decline in numbers of circulating blood components, and the unexpected paucity of early VF all question the validity of the model.     

The effects of Z13752A, a combined ACE/NEP inhibitor, on responses to coronary artery occlusion; a primary protective role for bradykinin

The effects on the responses to coronary artery occlusion of a combined ACE/NEP inhibitor (Z13752A) were examined in anaesthetized dogs. A 1 h infusion of Z13752A (128 microgram kg(-1) min(-1) intravenously) decreased arterial blood pressure (by 11+/-3%; P<0. 05) and increased coronary blood flow (by 12+/-4%, P<0.05). There were no other significant haemodynamic changes. Z13752A inhibited both NEP and ACE enzymes both in dog plasma and in tissue (lung ACE; kidney NEP). Pressor responses to angiotensin I in vivo were inhibited and systemic vasodilator responses to bradykinin were potentiated. When the left anterior descending coronary artery was occluded for 25 min, Z13752A markedly reduced the severity of the resultant ventricular arrhythmias. No ventricular fibrillation (VF) occurred (compared to 7/16 in the controls; P<0.05), and ventricular tachycardia (VT) was reduced (VT in 2/9 dogs treated with Z13752A cp. 16/16 of controls; episodes of VT 0.2+/-0.1 c.p. 10.7+/-3.3; P<0. 05). Reperfusion of the ischaemic myocardium led to VF in all control dogs but occurred less frequently in dogs given Z13752A (survival from the combined ischaemia-reperfusion insult 67% c.p. 0% in controls; P<0.05). Z13752A reduced two other indices of ischaemia severity; epicardial ST-segment elevation and inhomogeneity of electrical activation. These protective effects of Z13752A during ischaemia and reperfusion were abolished by the administration of icatibant (0.3 mg kg(-1), i.v.) a selective antagonist of bradykinin at B(2) receptors; the ischaemic changes in dogs given both icatibant and Z13752A were similar to those in the controls. We conclude that this ACE/NEP inhibitor is effective at reducing the consequences of coronary artery occlusion in this canine model and that this protection is primarily due to potentiation of released bradykinin.British Journal of Pharmacology (2000) 129, 671 - 680     

Pacing-induced delayed protection against arrhythmias is attenuated by aminoguanidine, an inhibitor of nitric oxide synthase.

1. Cardiac pacing, in anaesthetized dogs, protects against ischaemia and reperfusion-induced ventricular arrhythmias when this is initiated 24 h after the pacing stimulus. Now we have examined whether this delayed cardioprotection afforded by cardiac pacing is mediated through nitric oxide. 2. Twenty-two dogs were paced (4 x 5 min periods at 220 beats min(-1)) by way of the right ventricle, 24 h prior to a 25 min period of coronary artery occlusion. Nine of these dogs were given the inhibitor of induced nitric oxide synthase, aminoguanidine (50 mg kg(-1) i.v.), 0.5 h prior to coronary artery occlusion. Sham-operated non-paced dogs with and without aminoguanidine treatment served as controls. 3. Pacing markedly (P<0. 05) reduced arrhythmia severity (ventricular fibrillation, VF, during occlusion 15%; survival from the combined ischaemia-reperfusion insult 62%) compared to control, sham-operated, unpaced dogs (VF during occlusion 58%; survival 17%). This protection was attenuated by the administration of aminoguanidine prior to coronary artery occlusion (survival from the combined ischaemia-reperfusion insult 11%, which was significantly (P<0.05) less than in the paced dogs not given aminoguanidine and similar to the controls). Aminoguanidine had no significant effects on coronary artery occlusion when given to dogs that had not been paced. In the dose used aminoguanadine transiently elevated systemic arterial pressure by a mean of 20 mmHg and reduced heart rate by a mean of 22 beats min(-1). 4. These results suggest that nitric oxide, probably derived from induced nitric oxide synthase, contributes significantly to the delayed cardioprotection afforded by cardiac pacing.     

Efficacy of ischaemic preconditioning in the eNOS overexpressed working mouse heart model

We recently demonstrated that exogenous nitric oxide (NO) acts as a trigger for preconditioning in the isolated rat heart model. There is however little data concerning the effects of elevated cardiac endothelial nitric oxide synthase (eNOS) expression on myocardial tolerance to ischaemia. Similarly, the effects of gender and eNOS overexpression on ischaemic preconditioning is unknown. We hypothesized that: 1) eNOS overexpression increases myocardial tolerance to ischaemia, and, 2) eNOS overexpressed hearts cannot be preconditioned, since the hearts are already maximally protected. Male and female wild-type and transgenic mice that overexpress eNOS exclusively in cardiac myocytes were perfused in the working heart mode with a modified Krebs-Henseleit buffer at a pre-load of 12.5 mm Hg and afterload of 50 mm Hg. Cardiac output, coronary flow, peak aortic systolic pressure and total work were determined before hearts were preconditioned by 4x5 min cycles of ischaemia/reperfusion, and then subjected to 20 min total global ischaemia, followed by reperfusion. Reperfusion function and myocardial infarct size were used as endpoints. Pre-ischaemic mechanical function (rate pressure product and cardiac output) was similar for wild-type and transgenic mice of both sexes. The eNOS overexpressed hearts had smaller infarcts than the hearts from their wild-type littermates (26.9+/-1.4% vs. 37.0+/-2.1% for controls, P<0.05). Preconditioning the eNOS overexpressed hearts resulted in infarct sizes comparable with control non-preconditioned hearts (27.5+/-2.0% vs. 26.9+/-1.4% for controls). Myocardial cGMP levels were elevated during sustained ischaemia in the transgenic hearts when compared with wild-type hearts (22.43+/-1.63 pmol/g ww vs 16.54+/-1.48 pmol/g ww, P<0.05). Preconditioning also elevated myocardial cGMP levels during sustained ischaemia in the wild-type hearts (26.77+/-2.81 pmol/g ww, P<0.05). We conclude that: 1) basal mechanical function is similar for both wild-type and transgenic mice of both sexes, 2) reperfusion function and infarct size was also similar for both sexes under both control conditions and after preconditioning, 3) the transgenic mice are more tolerant of ischaemia as reflected by their smaller myocardial infarcts, and, 4) the eNOS overexpressed mouse heart cannot be preconditioned regardless of whether mechanical function or infarct size is used as an end-point. These hearts may be maximally protected against ischaemia/reperfusion injury by their elevated endogenous NO levels.     

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.     

Upstream signaling of protein kinase C-epsilon in xenon-induced pharmacological preconditioning. Implication of mitochondrial adenosine triphosphate dependent potassium channels and phosphatidylinositol-dependent kinase-1

Xenon elicits preconditioning of the myocardium via protein kinase C-epsilon. We determined the implication of (1) the mitochondrial adenosinetriphosphate dependent potassium (K(ATP)) channels and (2) the 3'phosphatidylinositol-dependent kinase-1 (PDK-1) in activating protein kinase C-epsilon. For infarct size measurements, anaesthetized rats were subjected to 25 min of coronary artery occlusion followed by 120 min of reperfusion. Rats received xenon 70% during three 5-min periods before ischaemia with or without the K(ATP) channel blocker 5-hydroxydecanoate or Wortmannin as PI3K/PDK-1 inhibitor. For Western blot, hearts were excised at five time points after xenon preconditioning (Control, 15, 25, 35, 45 min). Infarct size was reduced from 42+/-6% (mean+/-S.D.) to 27+/-8% after xenon preconditioning (P<0.05). Western blot revealed an increased activation of PKC-epsilon after 45 min and of PDK-1 after 25 min during xenon preconditioning. 5-hydroxydecanoate and Wortmannin blocked both effects. PKC-epsilon is activated downstream of mitochondrial K(ATP) channels and PDK-1. Both pathways are functionally involved in xenon preconditioning.     

Cardioprotective effects of N-hydroxyguanidine PR5 in myocardial ischaemia and reperfusion in rats

1. The potential for the N-hydroxyguanidine compound PR5 (N-(3, 4-dimethoxy-2-chlorobenzylideneamino)-N'-hydroxyguanidine) as a cardioprotective agent in heart ischaemia and reperfusion injury was investigated using rat models. 2. Administration of 1-10 mg kg-1 of PR5 5 min before 10 min of left coronary artery occlusion, followed by 20 min reperfusion, strongly inhibited reperfusion burst of arrhythmias and markedly improved the survival of the animals (e.g. ventricular fibrillation incidence 93 vs 43% (P<0.05); mortality 47 vs 0% (P<0.05), for controls and for 3 mg kg-1 of PR5, respectively). 3. Administration of 3 mg kg-1 of PR5 1 min before reperfusion to rats subjected to 10 min occlusion, 20 min reperfusion was most effective in reducing arrhythmias and decreasing mortality (43 vs 0%, P<0.05), but effects were also seen when PR5 was administered 0, 1 and 5 min after start of reperfusion. 4. Coronary occlusion/reperfusion (10 - 20 min) increased malondialdehyde (MDA) of rat hearts (0.88+/-0.13 for sham vs 1.45+/-0.12 nmol mg-1 protein for ischaemic; P<0.05). In rats where 3 mg kg-1 PR5 were administered 1 min before reperfusion the increase was attenuated (MDA being 1.04+/-0.12; P<0.05 vs ischaemic). 5. PR5 caused a substantial reduction of the infarction size in rats subjected to 180 min left coronary artery occlusion, followed by 120 min of reperfusion; the necrotic zone being 326+/-32 mg for controls vs 137+/-21 mg for animals treated with 3x3 mg kg-1 of PR5 (P<0.01). 6. PR5 reduced the elevation of the ST-segment of the ECGs, as well as caused pronounced attenuation of the rapid blood pressure drop seen at the start of reperfusion following coronary artery occlusion. 7 We conclude that the N-hydroxyguanidine PR5 provides remarkable protection against ischaemia and reperfusion induced myocardial necrosis and life-threatening arrhythmias. These effects of PR5 are discussed in relation to a recently discovered ability of N-hydroxyguanidines to function as electron acceptors at the xanthine oxidase enzyme.     

Evidence of the role of angiotensin AT(1) receptors in remote renal preconditioning of myocardium

The underlying mechanisms of myocardial preconditioning after a brief period of renal ischemia-reperfusion are unknown. The phenomenon itself and the involvement of AT(1) angiotensin receptors was therefore investigated using an in vivo rat model of acute myocardial infarction. Anesthetized rats underwent a left thoracotomy and pericardiotomy. A laparotomy was performed to expose the left renal artery. Animals were then preconditioned with four episodes, each consisting of 5 min of renal artery occlusion followed by 10 min of reperfusion, or underwent a 60 min sham period of anesthesia. Subsequently, the left coronary artery was occluded for 30 min and reperfused for 2 h. Area at risk and infarct size was estimated along with hemodynamic and temperature data recording. The infarct-to-risk ratio was limited from 56.36 +/- 2.36 and 50.65 +/- 4.71 in control to 12.0 +/- 0.48 and 12.57 +/- 0.44 in preconditioned hearts, by volume and weight methods. The protective effect of renal preconditioning on the myocardium was not abolished by losartan 1 mg/kg (10.11 +/- 0.12 and 11.2 +/- 1.4) pretreatment whereas it was completely attenuated by losartan 5 mg/kg (46.5 +/- 1.0 and 47.8 +/- 1.4) administration. Thus we can infer that angiotensin AT(1) receptors participate in renal preconditioning of the myocardium in in vivo rat hearts.     

d,l-sotalol enhances baroreflex sensitivity in conscious rats surviving acute myocardial infarction.

The aim of the present study was to characterize the influence of D,L-sotalol on arterial baroreflex sensitivity (BRS) which is generally considered as an estimate of vagal activity and has prognostic value in patients after myocardial infarction (MI). Conscious rats were studied 3 days after left anterior descending coronary artery ligation (n= 5) or sham-operation (SH, n= 6). BRS was determined by linear regression analysis of the RR-interval (interval between heart beats) and mean arterial pressure changes evoked by i.v. bolus injections of methoxamine (inducing reflex bradycardia, RB) and nitroprusside (inducing reflex tachycardia, RT). In MI-rats heart rate and mean arterial pressure were not significantly different from values in SH-rats, left ventricular end-diastolic pressure was increased and contractility was depressed. The BRS (RB: MI: 0.48 +/- 0.04(*), SH: 0.79 +/- 0.08; RT: MI: 0.41 +/- 0.05(*), SH: 0.86 +/- 0.08 ms mmHg(-1)) ((*)P< 0.05 vs SH) was markedly reduced. d, l -Sotalol (1.5 mg kg(-1)i.v.) reduced heart rate (MI: -11 +/- 3 %(*), SH: -11 +/- 3 %(*)) and mean arterial pressure only moderately [MI: -6 +/- 4 %(n.s.), SH: -7 +/- 2 %(*)], while BRS depression in MI-rats was completely neutralized [RB: MI: 1.08 +/- 0.14(*), SH: 1.19 +/- 0.11(*); RT: MI: 0.84 +/- 0.08(*), SH: 0.88 +/- 0.12 (n.s.) ms mmHg(-1)] ((*)P< 0.05 vs pretreatment). The BRS is reduced in rats early after MI, indicating a depressed reflex vagal activity. Treatment with D,L-sotalol at a dose with little effect on heart rate and mean arterial pressure markedly enhances and, thus, restores BRS in MI-rats. These data suggest that D,L-sotalol has both peripheral and central effects leading to an increase of reflex vagal control of heart rate in rats.     

Delayed protection against ventricular arrhythmias by monophosphoryl lipid-A in a canine model of ischaemia and reperfusion.

Bacterial endotoxin reduces the severity of ventricular arrhythmias which occur when a coronary artery is occluded several hours later. We have now examined in anaesthetised dogs the effects on ischaemia and reperfusion-induced arrhythmias, of a non-toxic derivative component of the endotoxin molecule of the lipid-A (monophosphoryl lipid-A). This was given intravenously, in doses of 10 and 100 microg kg(-1), 24 h prior to coronary artery occlusion. Arrhythmia severity was markedly reduced by monophosphoryl lipid-A. During ischaemia, ventricular premature beats were reduced from 315+/-84 in the vehicle controls to 89+/-60 (with the lower dose of monophosphoryl lipid-A) and 53+/-23 (P<0.05) with the higher dose. The incidence of ventricular tachycardia was reduced from 75% to 25% (P<0.05) and 31% (P<0.05), and the number of episodes of ventricular tachycardia from 13.4+/-4.9 per dog to 1.1+/-1.1 (P<0.05) and 1. 2+/-0.9 (P<0.05) after doses of 10 and 100 microg kg(-1), respectively. The incidence of ventricular fibrillation during occlusion and reperfusion in the control group was 96% (15/16), i.e., only 6% (1/16) dogs survived the combined ischaemia-reperfusion insult. Monophosphoryl lipid-A (100 microg kg(-1)) significantly reduced the incidence of occlusion-induced ventricular fibrillation (from 50% to 7%; P<0.05), and increased survival following reperfusion to 54% (P<0.05). Monophosphoryl lipid-A also significantly reduced ischaemia severity as assessed from ST-segment elevation recorded from epicardial electrodes as well as the degree of inhomogeneity of electrical activation within the ischaemia area. There were no haemodynamic differences prior to coronary occlusion between vehicle controls and monophosphoryl lipid-A-treated dogs. These results demonstrate that monophosphoryl lipid-A reduces arrhythmia severity 24 h after administration. Although the precise mechanisms are still unclear, there is some evidence that nitric oxide and prostanoids (most likely prostacyclin) may be involved because the dual inhibition of nitric oxide synthase and cyclooxygenase enzymes by administration of aminoguanidine and meclofenamate abolished the marked antiarrhythmic protection resulted from monophosphoryl lipid-A treatment 24 h previously.     

Evidence for the involvement of peroxynitrite in ischaemic preconditioning in rat isolated hearts

1. The aim of this study was to investigate the involvement of peroxynitrite, reactive metabolite originating from nitric oxide and superoxide, in preconditioning of the ischaemic myocardium in rat isolated hearts. 2. Isolated hearts perfused with Krebs-Henseleit solution were preconditioned either by 3 min of coronary artery occlusion (CAO) or by peroxynitrite administration at three different concentrations (0.1, 1, 10 microM) for 3 min, followed by 10 min reperfusion and 30 min of CAO. Peroxynitrite, at 1 microM concentration, decreased the incidence of VT from 100% (n=14) to 62% (n=13) and abolished the occurrence of VF (50% in the control group). 3. N-2-mercaptopropionylglycine (MPG, 1 microM - 10 mM) produced a concentration-dependent inhibition of peroxynitrite signals in luminol chemiluminescence and 67+/-1% inhibition was observed at 100 microM (n=7). MPG (at 300 microM, n=7) added to the perfusate 10 min prior to ischaemic preconditioning or peroxynitrite infusion and maintained until CAO, significantly reversed the beneficial effects of the ischaemic and peroxynitrite-treated groups. MPG administration in the peroxynitrite-treated group increased the incidence of VT from 62% (n=13) to 100% (n=10) and total VF from 0% (n=0) to 67% (n=10). Similarly, MPG elevated the incidence of VT from 50% (n=10) to 100% (n=8) in the ischaemic preconditioned group. On its own, MPG did not affect the severity of cardiac arrhythmias. 4. These results suggest that endogenously produced peroxynitrite plays a significant role in the antiarrhythmic effect of ischaemic preconditioning in the rat isolated hearts.     

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.