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Soon after its identification as a powerful vasoconstrictor peptide, endothelin (ET-1) was implicated as a detrimental agent involved in determining the outcome of myocardial ischaemia and reperfusion. Early experimental studies demonstrated that ETA selective and mixed ETA/ETB receptor antagonists can reduce infarct size and prevent ischaemiainduced ventricular arrhythmias in models of ischaemia/reperfusion, implying that ET-1 acts through the ETA receptor to contribute to injury and arrhythmogenesis. However, as our understanding of the physiology of ET-1 has expanded, the role of ET-1 in the ischaemic heart appears ever more complex. Recent evidence suggests that ET-1 exerts actions on the heart that are not only detrimental (vasoconstriction, inhibition of NO production, activation of inflammatory cells), but which may also contribute to tissue repair, such as inhibition of cardiomyocyte apoptosis. In addition, ET-1-induced mast cell degranulation has been linked to a homeostatic mechanism that controls endogenous ET-1 levels, which may have important implications for the ischaemic heart. Furthermore the mechanism by which ET-1 promotes arrhythmogenesis remains controversial. Some studies imply a direct electrophysiological effect of ET-1, via ETA receptors, to increase monophasic action potential duration (MAPD) and induce early after-depolarisations (EADs), while other studies support the view that coronary constriction resulting in ischaemia is the basis for the generation of arrhythmias. Moreover, ET-1 can induce cardioprotection (precondition) against infarct size and ventricular arrhythmias, through as yet incompletely understood mechanisms. To enable us to identify the most appropriate means of targeting this system in a therapeutically meaningful way we need to continue to explore the physiology of ET-1, both in the normal and the ischaemic heart.

The aims of this study were to determine if the ETBreceptor agonist, sarafotoxin 6c (S6c) reduces myocardial infarct size following myocardial ischemia and reperfusion and to investigate whether any changes in mRNA for endothelin receptors in the injured myocardium were modified by S6c pretreatment. Hypnorm/Hypnovel anesthetized rats were subjected to occlusion of the left main coronary artery for 30 minutes, followed by 120 minutes reperfusion. Animals were administered a bolus dose of S6c (0.24 nmol kg-1 i.v., n 10) or saline (n 15) 5 minutes prior to occlusion. At the end of reperfusion, hearts were stained with Evan’s Blue dye to delineate area at risk. A 1.5- to 2.0-mm thick slice was cut transmurally 1 mm below the site of ligation for assessment of infarct size by triphenyltetrazolium chloride. A further transmural slice (2.5–3-mm thick) was cut for assessment of receptor mRNA levels by RTPCR. Administration of S6c caused a transient fall in mean arterial blood pressure (MABP) prior to occlusion and attenuated the fall in MABP induced by coronary occlusion. S6c significantly reduced infarct size (13 ± 4 of area of slice at risk) compared with control hearts (35 ± 5; P< 0.05). In control hearts, there was a marked reduction in mRNA content for both ETA(50 reduction) and ETB(70 reduction) receptors in the ischemic zone, compared with non-ischemic tissue. In hearts pre-treated with S6c there was a reduction in ETA, but not ETBreceptor mRNA in the ischemic zone. This study has shown that S6c reduces myocardial infarct size and results in preservation of ETBreceptor mRNA in ischemic/reperfused tissue.

The aim of this study was to examine the changes in leukocyte adhesion and leukocyte‐induced contraction in balloon‐injured rabbit subclavian artery and to correlate these changes with vessel morphology and expression of adhesion molecules on the injured arteries.Rabbits were anaesthetized and their left subclavian arteries were injured by balloon inflation and withdrawal followed by sacrifice at 2, 24, 48 h or 8 days after injury. The left and right subclavian arteries were removed and leukocytes were isolated from autologous rabbit blood. Leukocyte‐induced contraction was measured in 5‐HT precontracted artery rings and leukocyte adhesion was measured using 51Cr‐labelled leukocytes. Immunocytochemistry using paraffin‐embedded tissue was employed to detect changes in the expression of adhesion molecules on injured arteries.Autologous leukocytes caused a contraction of rabbit subclavian artery rings, which was prevented by L‐NAME (10−3M). Balloon‐induced injury abolished the contractile response to leukocytes, which correlated with loss of carbachol‐induced relaxationBalloon injury markedly enhanced the adhesiveness of the subclavian artery for leukocytes, most notably at 24 and 48 h after injury (1.7 and 1.8 fold respectively). Increased leukocyte adhesion at these two time points correlated with an upregulation of E‐selectin, P‐selectin and VCAM‐1 expression on the remaining endothelium of the injured artery.Vessel morphology revealed that balloon inflation had induced an infiltration of inflammatory cells into the vessel wall, the greatest increase being seen at 24 h after injury.It is concluded that an increase in the expression of E‐selectin, P‐selectin and VCAM‐1 following balloon‐induced injury leads to enhanced leukocyte adhesion and migration into the injured vessel.

The effect of a nitric oxide (NO) donor and the influence of endogenous NO in modulating ischaemia-induced arrhythmias was assessed in anaesthetised rats. The nitric oxide donor C87-3754 (1 mg/kg) caused a significant reduction in arterial blood pressure before coronary artery ligation but did not influence the incidence or severity of ventricular arrhythmias during a 30-min period of myocardial ischaemia [60 and 58% incidence of ventricular fibrillation (VF) in control and treated rats, respectively]. When the hearts were preconditioned by a short (3 min) coronary artery occlusion before the 30-min period of ischaemia, there was a marked reduction in both the number of ventricular ectopic beats (260 ± 65 vs. 812 ± 256 beats/min in controls; p < 0.05) and the incidence of ventricular fibrillation (9 vs. 67% in controls; p < 0.05). Neither NG-nitro-L-arginine methyl ester (L-NAME; 10-100 mg/kg) nor methylene blue (1-50 mg/kg) attenuated this marked antiarrhythmic effect of preconditioning. L-NAME caused a significant increase in blood pressure with all doses used, whereas methylene blue did not increase blood pressure. Both L-NAME and methylene blue attenuated ventricular arrhythmias in nonpreconditioned hearts. L-NAME reduced the number of ventricular ectopic beats (from 812 ± 256 to 318 ± 81 beats/min at 10 mg/kg; p < 0.05), whereas methylene blue decreased the incidence of VF from 67 to 20% at a dose of 50 mg/kg (p < 0.05). These findings suggest that neither endogenous nor exogenously administered NO reduces ischaemic arrhythmias in anaesthetised rats. Furthermore, the antiarrhythmic effect of preconditioning in this species appears to be independent of NO. The antiarrhythmic effects seen with both methylene blue and L-NAME may be the result of actions other than inhibition of the production or actions of NO.

The purpose of this investigation was to provide a detailed analysis of the effects of the thromboxane antagonist L655,240 (0.3 mg/kg i.v.) on early ischemia-and reperfusion-induced arrhythmias in a canine model of coronary artery occlusion. In a dose that abolished the pulmonary response to U46619, L655,240 attenuated markedly the severity of those arrhythmias that resulted from reperfusion of the myocardium; survival from the combined occlusion-reperfusion insult was increased from 10 in control animals to 70 in dogs administered L655,240. Drug intervention did not significantly alter the total number of arrhythmias during the period of ischemia, but a detailed analysis of the different types of arrhythmia that occurred during this period showed that L655,240 significantly reduced those arrhythmias in phase 1a (0–10 min of occlusion) without affecting the later phase 1b arrhythmias. This was particularly shown in the marked reduction in the number of salvos (couplets and triplets) during this period. Neither those arrhythmias occurring later in the ischaemia period (phase 1b) nor the total number of single ectopics and salvos or the incidence and duration of ventricular tachycardia was modified by L655,240. These results reveal that thromboxane antagonism protects especially against reperfusion-induced ventricular fibrillation and against early (phase 1a) ischemia-induced arrhythmias, possibly implicating a role for thromboxane in the genesis of these cardiac rhythm disturbances.

The hemodynamic profile and antiarrhythmic properties of pirsidomine, a nitric oxide donor, were examined in pigs. Intravenous administration of pirsidomine (1 mg/kg) to chloralose-anesthetized open-chest pigs resulted in a decreased afterload, and a reduced myocardial contractility and myocardial oxygen consumption (assessed by rate-pressure product), with no alterations in heart rate. After induction of regional myocardial ischemia by occlusion of the left anterior descending coronary artery, pigs given pirsidomine experienced fewer ventricular ectopic beats (119 ± 29) than control animals did (217 ± 53; p < 0.05), seen primarily as a reduction in the number of couplets and triplets. Although the incidence of ventricular fibrillation was unaffected by pirsidomine, the time to onset of this arrhythmia was significantly prolonged by this intervention (21.3 ± 0.9 min versus 16.1 ± 2.5 min in controls; p < 0.05). Further- more, the ST-segment depression seen throughout the 30-min occlusion period in controls was not sustained beyond 5 min postocclusion in pirsidomine-treated pigs. Taken together, and in the absence of an ex vivo antiplatelet effect with this dose of pirsidomine, these results suggest that the antiarrhythmic effect of pirsidomine lies in its hemodynamic effects, resulting in a reduction of ischemia. The ex vivo effect of pirsidomine on free radical generation from isolated leukocytes was also investigated. Luminol-enhanced chemilu-minescence produced by leukocytes in response to phorbol myristate acetate was markedly depressed in cells isolated from blood withdrawn after administration of pirsidomine, compared with cells tested before drug administration. The results of this study suggest that pirsidomine exerts both an antiarrhythmic and anti-ischemic effect and may also reduce myocardial injury associated with free-radical generation from activated white blood cells.

Objective: The aim of the current study was to examine neointima formation in balloon injured left subclavian artery of rabbits subjected to two different methods of leukocyte depletion at the time of injury. Methods: Angioplasty of the left subclavian artery was performed in leukopenic male New Zealand White rabbits. Depletion of circulating leukocytes was induced by either mustine hydrochloride or an antibody against leukocyte common antigen (anti-LCA) before angioplasty. Left and right subclavian arteries were removed 28 days after injury for morphological analysis and measurement of neointimal size. At the same time, leukocytes were isolated from autologous rabbit blood for 51Cr-labelling for assessment of leukocyte adhesion to injured and non-injured artery segments. Results: Leukopenia decreased neointima formation in injured arteries (neointimal area was 0.09±0.03 mm2 in mustine-treated arteries, n = 8, vs. 0.56±0.07 mm2 in control arteries, n = 7; P<0.001 and 0.07±0.01 mm2 in anti-LCA treated arteries, n = 9, vs. 0.22±0.04 mm2 in non immune serum-treated arteries, n = 9; P<0.001). Adventitial fibrosis was also significantly (P<0.05) decreased by both leukopenic interventions. Neither medial nor adventitial area was modified in any of the groups. No differences in leukocyte adhesion were observed between injured and non-injured arteries in any of the experimental groups at the 28 day time point. Conclusion: These results suggest that leukocytes play a major role in the development of two of the major characteristics of the response to balloon injury, namely formation of neointima and adventitial fibrosis, that currently limit the success of clinical angioplasty. Elucidation of the fine mechanisms involved in leukocyte-mediated injury may lead to the discovery of novel therapeutic targets for the prevention of restenosis.

The aim of this study was to investigate whether the endothelinB ETBreceptor agonist sarafotoxin 6c S6c can protect against ischaemiainduced cardiac arrhythmias. Arrhythmias were induced by a 30 min period of coronary artery occlusion in pentobarbitoneanaesthetized male rats, or in Langendorffperfused rat hearts. Rats or rat hearts were administered a bolus dose of vehicle or S6c 0.8 nmolkg i.v. or 108M into the coronary circulation, respectively 5 min before the onset of ischaemia. In vivo administration of S6c significantly reduced the incidence of ventricular fibrillation VF from 59 to 13 and the number of premature ventricular beats. This effect was associated with a transient fall in mean arterial blood pressure. In isolated hearts, S6c reduced significantly both the incidences of ventricular tachycardia VT and VF while having no statistically significant effect on coronary perfusion pressure. This is the first report to show that stimulation of ETBreceptors, with a bolus dose of S6c, has an antiarrhythmic effect on rat hearts both in vivo and in vitro, suggestive of a direct effect on the myocardium.

The aim of this study was to investigate whether the endothelinB ETBreceptor agonist sarafotoxin 6c S6c can protect against ischaemiainduced cardiac arrhythmias. Arrhythmias were induced by a 30 min period of coronary artery occlusion in pentobarbitoneanaesthetized male rats, or in Langendorffperfused rat hearts. Rats or rat hearts were administered a bolus dose of vehicle or S6c 0.8 nmolkg i.v. or 10-8M into the coronary circulation, respectively 5 min before the onset of ischaemia. In vivo administration of S6c significantly reduced the incidence of ventricular fibrillation VF from 59 to 13 and the number of premature ventricular beats. This effect was associated with a transient fall in mean arterial blood pressure. In isolated hearts, S6c reduced significantly both the incidences of ventricular tachycardia VT and VF while having no statistically significant effect on coronary perfusion pressure. This is the first report to show that stimulation of ETBreceptors, with a bolus dose of S6c, has an antiarrhythmic effect on rat hearts both in vivo and in vitro, suggestive of a direct effect on the myocardium.

Objective: The aims were to examine the effect of pretreatment with Bordetella pertussis toxin on the antiarrhythmic effect of ischaemic preconditioning in order to determine the possible involvement of inhibitory G proteins in this phenomenon; and (2) to characterise the model used by varying the duration of a single preconditioning occlusion of the left coronary artery, the reperfusion time, and the duration of the subsequent prolonged coronary artery occlusion. Methods: Isolated rat hearts perfused with Krebs Henseleit solution at constant flow (8-10 ml·min–1) were subjected to a single preconditioning occlusion of the left coronary artery (either 1 or 3 min) followed, up to 60 min later, by a prolonged occlusion of 30 or 60 min (n=56). The ventricular arrhythmias during occlusion were compared to those from control rats in which the artery was occluded for 30 or 60 min but without preconditioning (n=29 and 14 respectively). Results: Protection against ventricular arrhythmias was most pronounced when a 3 min preconditioning occlusion was used followed by a 10 min reperfusion period: reduction in ventricular premature beats (VPB) during the 30 min occlusion from 514(SEM 119) in control hearts to 79(29) in preconditioned hearts (p<0.01). This protection was still apparent when the reperfusion time was extended to 30 min [VPB 52(16); p<0.01] but lost when reperfusion was extended to 1 h. Rendering Gi proteins non-functional (ascertained by responses to acetylcholine) resulted in loss of this antiarrhythmic effect of preconditioning [VPB 241(93) v 226(120) for non-preconditioned hearts]. Conclusions: The antiarrhythmic effects of preconditioning can be demonstrated in isolated rat hearts perfused at constant flow with an artificial medium and this protection is lost following treatment with Bordetella pertussis toxin 48 h previously. Cardiovascular Research 1993;27:674-680

Objective: The aim of this study was to investigate the influence of endogenously released and exogenously applied endothelin-1 (ET-1) on ischaemia-induced arrhythmias. Methods: Ischaemia was induced in pentobarbitone-anaesthetised rats by ligation of a coronary artery for 30 min. To determine the role of endogenous ET-1 in ischaemic arrhythmias, either the ETA receptor antagonist BQ123 (50 μg/kg/min, i.v.; n=10) or the ETB receptor antagonist PD161721 (0.1 or 1 mg/kg i.v.; n=10 per group) was administered before the onset of ischaemia. To assess the influence of exogenous ET-1 on arrhythmias, ET-1 (1.6 nmol/kg i.v.) was administered 5 min before ischaemia in the absence (n=12) or presence of BQ123 (n=10) or PD161721 (n=10). The total number of ventricular ectopic beats (VEBs) were counted and expressed as median (Q1−Q3) and the incidence of ventricular fibrillation (VF) and ventricular tachycardia (VT) in each group was determined. Mean arterial blood pressure (MABP) and heart rate (HR) were measured. Results: In control animals (n=20), the incidence of VF was 65% and the total VEB count was 2775 (1870–4041). Both BQ123 and the higher dose of PD161721 reduced the VEB count to 654 (348–1489; P<0.05) and 782 (432–1153; P<0.05) respectively. Only PD161721 reduced the incidence of VF (to 10%; P<0.05). Administration of ET-1 reduced VEBs to 1530 (1204–2017); P<0.05) and the incidence of VF to 17% (P<0.05). Neither PD161721 nor BQ123 modified this antiarrhythmic effect of ET-1, but rather enhanced the reduction in arrhythmias. Before occlusion, ET-1 caused a transient fall in MABP (from 107±3 to 63±3 mmHg; P<0.05). PD161721, but not BQ123, partially blocked this effect. Upon occlusion, MABP fell in control animals (from 106±4 to 67±4 mmHg at 1 min post-occlusion; P<0.05). This was significantly attenuated by ET-1, although neither of the antagonists were able to block this effect of ET-1. Conclusions: ET-1 released endogenously during ischaemia is arrhythmogenic whereas exogenous application of ET-1 may, under certain conditions, be antiarrhythmic.

Objective: The aims of this study were to determine the morphological and functional consequences of balloon angioplasty of the left subclavian artery of Froxfield heritable hyperlipidaemic (FHHL) rabbits and the influence of oral <$O_SCPLOW>L<$C_SCPLOW>-arginine therapy on these changes. Methods: Sixteen-week-old FHHL rabbits were subjected to balloon injury of the left subclavian artery under halothane anaesthesia. Control rabbits (n = 7) were given free access to food and normal tap water. <$O_SCPLOW>L<$C_SCPLOW>-Arginine-treated rabbits were given <$O_SCPLOW>L<$C_SCPLOW>-arginine (5 g·l–1) in the drinking water for 2 days prior to angioplasty and then for either 2 weeks (n = 7) or 4 weeks (n = 7) after surgery. All rabbits were euthanised 28–30 days after surgery and blood and tissue removed for quantification of neointimal size and determination of endothelial function using isolated vessel tension studies. The ability of the endothelium to prevent platelet aggregation was determined by challenging a vessel ring with carbachol when incorporated into a whole blood sample in which platelet aggregation was induced with collagen. Results: Balloon injury in non-treated rabbits resulted in the development of marked intimal hyperplasia (18.8[3.6]% of the area within the internal elastic lamina) while endothelial function remained intact. Maximum responses to carbachol and calcimycin were, respectively, a 66.6[14.7]% and 46.9[12.9]% relaxation of 5HT-induced tone, compared to 58.0[3.2]% and 39.8[9.4]% in non-injured vessels. Maximum contractile responses to 5HT and KCl were unaffected by injury. <$O_SCPLOW>L<$C_SCPLOW>-Arginine therapy alone had no effect on the vasodilator function of the endothelium, but reduced the endothelium-dependent inhibition of platelet aggregation (68.4[7.8] vs 109[10]% of the maximum extent of platelet aggregation in non-treated and 2-week <$O_SCPLOW>L<$C_SCPLOW>-arginine-treated non-injured vessels, respectively). <$O_SCPLOW>L<$C_SCPLOW>-Arginine significantly reduced the extent of neointimal formation (7.2[3.9]% of the area within the IEL; P<0.05 vs. non-treated group). However, <$O_SCPLOW>L<$C_SCPLOW>-arginine significantly attenuated the relaxant responses to both carbachol (26.5[10.4]% and 31.4[9.4]% for 2- and 4-week <$O_SCPLOW>L<$C_SCPLOW>-arginine groups) and calcimycin (38.7[15.4]% and 16.4[10.7]%) in the injured artery (P<0.05 compared to non-treated controls). Conclusions: <$O_SCPLOW>L<$C_SCPLOW>-Arginine reduces neointimal formation following balloon catheter injury in heritable hypercholesterolaemic rabbits, which is consistent with previous findings in normocholesterolaemic models. However, in the presence of hypercholesterolaemia, <$O_SCPLOW>L<$C_SCPLOW>-arginine has a detrimental effect on endothelial function following injury. This may be a consequence of the presence of lipids in the vascular wall on nitric oxide synthase activity.

Study objective – It has been suggested that the balance between the release from the heart of prostacyclin and thromboxane A2 is a major determinant of the severity of arrhythmias during ischaemia and reperfusion. This has been examined in a dog model. Design – Three different cyclo-oxygenase inhibitors in doses adequate to prevent formation of both prostacyclin and thromboxane - aspirin (7 mg·kg−1), flurbiprofen (3 mg·kg−1), and sodium meclofanate (2 mg·kg−1) with or without nafazatrom or dazmegrel -were given prior to a combined occlusion-reperfusion insult, and the severity of resulting arrhythmias examined. Material – Adult greyhound dogs were used: controls n = 29; aspirin n = 10; flurbiprofen n = 10; sodium meclofenamate ± nafazatrom or dazmegrel n = 22. Measurements and main results – None of the interventions modified the severity of ischaemic arrhythmias induced by coronary artery occlusion and release. The numbers of ventricular extrasy-stoles during the 40 min occlusion period were similar in all groups: controls 653(SEM 109); aspirin 690(187); flurbiprofen 454(132); meclofenamate 833(218). Ventricular fibrillation from the combined occlusion-reperfusion insult was also similar; controls 83%; aspirin 80%; flurbiprofen 80%; meclofenamate 100%. In the doses used all three inhibitors prevented the increase in plasma concentrations of thromboxane A2 [from 104(23) to 166(34) pg·ml−1] and prostacyclin [from 450(80) to 720(110) pg-ml−1] seen in control untreated dogs subjected to coronary artery occlusion. The addition of sodium meclofenamate to either nafazatrom (10 mg·kg−1 orally) or dazmegrel (3 mg·kg−1 intravenously), which when given alone are markedly protective, abolished this protection. Conclusion – The results show the importance of maintaining prostacyclin release in modifying the severity of ischaemic and reperfusion arrhythmias, and again suggest that prostacyclin is an “endogenous antiarrhythmic substance".