A novel anti-diabetic drug, miglitol, markedly reduces myocardial infarct size in rabbits

1. We examined whether N-hydroxyethyl-1-deoxynojirimycin (miglitol), a new human anti-diabetic drug with effects to inhibit alpha-1, 6-glucosidase glycogen debranching enzyme and reduce the glycogenolytic rate as well as to inhibit alpha-1,4-glucosidase, could reduce infarct size in the rabbit heart. Rabbits were subjected to 30-min coronary occlusion followed by 48-h reperfusion. 2. The infarct size as a percentage of area at risk was not reduced by pre-ischaemic treatment with 1 mg kg(-1) miglitol (42.7+/-4.0%, n=10) compared with the saline control group (41.7+/-2.3%, n=10). However, it was significantly and dose-dependently reduced by pre-ischaemic treatment with 5 or 10 mg kg(-1) of miglitol (25.7+/-4. 5%, n=10, and 14.6+/-2.4%, n=10, respectively) without altering the blood pressure, heart rate or blood glucose level. However, there was no evidence of an infarct-size reducing effect after pre-reperfusion treatment with 10 mg kg(-1) of miglitol (35.0+/-3.0%, n=10). 3. Another 40 rabbits given 1, 5 and 10 mg kg(-1) of miglitol or saline before ischaemia (n=10 in each) were sacrificed at 30 min of ischaemia for biochemical analysis. Miglitol preserved significantly the glycogen content, and attenuated significantly the lactate accumulation in a dose dependent manner in the ischaemic region at 30 min of ischaemia. 4. Pre-ischaemic treatment, but not pre-reperfusion treatment, with miglitol markedly reduced the myocardial infarct size, independently of blood pressure and heart rate. A dose-dependent effect of miglitol on infarct size, glycogenolysis and lactate formation suggests that the mechanism may be related to the inhibition of glycogenolysis. Thus, miglitol may be beneficial for coronary heart disease as well as diabetes mellitus.     

Benidipine reduces myocardial infarct size involving reduction of hydroxyl radicals and production of protein kinase C-dependent nitric oxide in rabbits

Japanese white rabbits underwent 30 minutes of ischemia and 48 hours of reperfusion. Benidipine (3 or 10 microg/kg, i.v.) was administered 10 minutes before ischemia with and without pretreatment with L-NAME (10 mg/kg, i.v., a NOS inhibitor), chelerythrine (5 mg/kg, i.v., a PKC blocker) or 5-HD (5 mg/kg, i.v. a mitochondrial KATP channel blocker), genistein (5 mg/kg, i.v. a protein tyrosin kinase blocker). SNAP (2.5 mg/kg/min x 70 minutes, i.v., an NO donor) was also administered 10 minutes before ischemia. Benidipine significantly reduced the infarct size in a dose-dependent manner (3 microg/kg: 29.0 +/- 2.7%, n = 8, 10 microg/kg: 23.0 +/- 2.4%, n = 10) compared with the control (41.6 +/- 3.3%, n = 10). This effect was completely blocked by L-NAME (39.9 +/- 3.6%, n = 8) and chelerythrine (35.5 +/- 2.4%, n = 8) but not by 5-HD (23.0 +/- 2.4%, n = 10) or genistein (24.6 +/- 3.1%, n = 10). SNAP also reduced the infarct size (24.6 +/- 3.1%, n = 8). Benidipine significantly increased the expression of eNOS mRNA at 30 minutes after reperfusion and significantly increased the expression of eNOS protein at 3 hours after reperfusion in the ischemic area of the left ventricle. Benidipine and SNAP significantly decreased myocardial interstitial 2,5-DHBA levels, an indicator of hydroxyl radicals, during ischemia and reperfusion. Benidipine increased myocardial interstitial NOx levels, which effect was blocked by chelerythrine, during 0 to 30 minutes and 150 to 180 minutes after reperfusion. Benidipine reduces the infarct size through PKC-dependent production of nitric oxide and decreasing hydroxyl radicals but not through involving protein tyrosine kinase or mitochondrial KATP channels in rabbits.     

The anti-diabetic drug miglitol is protective against anginal ischaemia through a mechanism independent of regional myocardial blood flow in the dog

1. In the present study, we attempted to clarify whether the antidiabetic drug miglitol, an alpha-glucosidase inhibitor, has a protective effect against anginal ischaemia. We had reported previously that miglitol reduces myocardial infarct size through inhibition of glycogenolysis during ischaemia in rabbits. However, the effect of miglitol on anginal ischaemia remains unknown. 2. In open-chest beagle dogs with a severely stenosed left anterior descending coronary artery, an epicardial electrode was attached to the surface of the risk area of the left ventricle and a microdialysis probe was implanted into the myocardium to measure ST segment changes and interstitial lactate accumulation. The first episode of anginal ischaemia was induced by atrial pacing and phenylephrine infusion (50-100 microg/min) for 10 min. The second episode of anginal ischaemia was induced 210 min after the first episode. Miglitol (10 mg/kg, i.v.) was administered to the miglitol group (n = 10) 30 min before the second episode of anginal ischaemia, whereas saline was administered to the control group (n = 10). Regional myocardial blood flow was measured using coloured microspheres. 3. There was no significant difference in regional myocardial blood flow in the risk and non-risk areas between the first and second episodes of anginal ischaemia and between the miglitol and control groups. During the first and second episodes of anginal ischaemia, the ST segment was decreased to a similar extent in the control group. Although ST segment depression during the first episode of anginal ischaemia was similar in both groups, ST segment depression during the second episode of anginal ischaemia was significantly attenuated in the miglitol-treated group compared with the control group (1.3 +/- 0.4 vs 2.2 +/- 0.4 mV, respectively). Miglitol significantly attenuated myocardial interstitial lactate accumulation in the risk area. 4. In conclusion, in the present study miglitol improved ST segment depression and attenuated the accumulation of myocardial interstitial lactate during anginal ischaemia without altering regional myocardial blood flow. Miglitol has an anti-anginal ischaemia effect via a mechanism that is independent of regional myocardial blood flow.     

Angiotensin II reduces infarct size and has no effect on post-ischaemic contractile dysfunction in isolated rat hearts

1. In order to test the hypothesis that angiotensin II exacerbates myocardial ischaemia-reperfusion (IR) injury, we examined the effects of graded angiotension II concentrations of angiotensin II on IR injury in both working and non-working (Langendorff) isolated rat hearts. 2. Non-working hearts were subjected to 30 min aerobic perfusion (baseline) then 25 min of global, no-flow ischaemia followed by 30 min of reperfusion either in the absence (control, n=7) or presence of 1 (n=6) or 10 nM (n=5) angiotensin II). Recoveries of LV developed pressure and coronary flow after 30 min reperfusion in control hearts (58+/-9 and 40+/-8% of baseline levels, respectively) were no different from hearts treated with 1 or 10 nM angiotensin II. Infarct size (determined at the end of reperfusion by triphenyltetrazolium chloride staining) was reduced by angiotensin II in a concentration-dependent manner (from a control value of 27+/-3 to 18+/-4% and 9+/-3% of the LV, respectively). 3. Working hearts were subjected to 50 min pre-ischaemic (pre-I) aerobic perfusion then 30 min of global, no-flow ischaemia followed by 30 min of reperfusion either in the absence (control, n=14) or presence of 1 (n=8), 10 (n=7) or 100 nM (n=7) angiotensin II). In controls, post-ischaemic (post-I) left ventricular (LV) work and efficiency of oxygen consumption were depressed (43+/-9 and 42+/-10% of pre-I levels, respectively). The presence of angiotensin II throughout IR had no effect on LV work compared with control. 4. Thus, angiotensin II reduces infarct size in a concentration-dependent manner but has no effect on contractile stunning associated with IR in isolated rat hearts.     

Role of protein kinase C, K(ATP) channels and DNA fragmentation in the infarct size-reducing effects of the free radical scavenger T-0970

1. In the present study, we investigated the effect of 1-(3-tert-butyl-2-hydroxy-5-methoxyphenyl)-3-(3-pyridylmethyl) urea hydrocloride (T-0970), a novel water-soluble low-molecular weight free radical scavenger, on the generation of hydroxyl radicals in vivo and on myocardial infarct size in an in vivo model of myocardial infarction in rabbits. 2. T-0970 scavenged hydroxyl radicals generated in the myocardium during reperfusion, as assessed by using a microdialysis technique and HPLC in an in vivo model with 30 min coronary occlusion and 30 min reperfusion in rabbits. 3. Another group of rabbits was subjected to 30 min coronary occlusion and 48 h reperfusion. The control group (n = 10) was infused with saline for 190 min from 10 min before occlusion to 180 min after reperfusion. The treatment group (T-0970 group; n = 10) was injected with a bolus 2.5 mg/kg T-0970 and then infused with T-0970 for 190 min from 10 min before reperfusion to 180 min after reperfusion at a rate of 100 microg/kg per min. The T-0970 + CHE group (n = 5) was given chelerythrine (CHE; a selective inhibitor of protein kinase C (PKC); 5 mg/kg, i.v.) 10 min before the administration of T-0970. The T-0970 + 5-HD group (n = 5) was given 5-hydroxydecanoate (5-HD; an inhibitor of mitochondrial K(ATP) channels; 5 mg/kg, i.v.) 10 min before the administration of T-0970. The CHE and 5-HD groups were given CHE (5 mg/kg, i.v.) and 5-HD (5 mg/kg, i.v.) 20 min before reperfusion, respectively. After 48 h reperfusion, infarct size was measured histologically and expressed as a percentage of the area at risk (AAR). In another series of experiments, the control (n = 5) and T-0970 (n = 5) groups were killed 4 h after reperfusion following 30 min coronary occlusion and DNA fragmentation in myocytes was assessed using in situ dUTP nick end-labelling (TUNEL) at the light microscopic level. 4. Infarct size, as a percentage of AAR, in the T-0970 group was significantly reduced compared with the control group (21+/-4 vs 41+/-4%, respectively; P<0.05). This reduction of infarct size by T-0970 was abolished by pretreatment with CHE and 5-HD. Neither CHE nor 5-HD alone had any effect on infarct size. The percentage of infarcted myocytes with DNA fragmentation by TUNEL in the T-0970 group was significantly reduced compared with the number in the control group (4.0+/-1.5 vs 10.7+/-1.9%, respectively; P<0.05). 5. T-0970, a free radical scavenger, improved reperfusion injury. This effect seemed to be mediated by activation of PKC, the opening of mitochondrial K(ATP) channels and inhibition of DNA fragmentation.     

Effects of the prostanoid EP3-receptor agonists M&B 28767 and GR 63799X on infarct size caused by regional myocardial ischaemia in the anaesthetized rat

1. This study investigates the effects of two agonists of the prostanoid EP3-receptor (M&B 28767 and GR 63799X) on the infarct size caused by regional myocardial ischaemia and reperfusion in the anaesthetized rat. 2. One hundred and sixty-seven, male Wistar rats were anaesthetized (thiopentone, 120 mg kg(-1) i.p.), ventilated (8-10 ml kg(-1), 70 strokes min(-1), inspiratory oxygen concentration: 30%; PEEP: 1-2 mmHg) and subjected to occlusion of the left anterior descending coronary artery (LAD, for 7.5, 15, 25, 35, 45 or 60 min) followed by reperfusion (2 h). Infarct size was determined by staining of viable myocardium with a tetrazolium stain (NBT), histological evaluation by light and electron microscopy and determination of the plasma levels of cardiac troponin T. 3. M&B 28767 (0.5 microg kg(-1) min(-1), i.v., n=7) or GR 63799X (3 microg kg(-1) min(-1), i.v., n=7) caused significant reductions in infarct size from 60+/-3% (25 min ischaemia and 2 h reperfusion; saline-control, n=8) to 39+/-6 and 38+/-4% of the area at risk, without causing a significant fall in blood pressure. Pretreatment of rats with 5-hydroxydecanoate (5-HD), an inhibitor of ATP-sensitive potassium channels, attenuated the cardioprotective effects of both EP3-receptor agonists. The reduction in infarct size afforded by M&B 28767 was also abolished by glibenclamide and the protein kinase C (PKC) inhibitors staurosporine and chelerythrine. 4. Thus, M&B 28767 and GR 63799X reduce myocardial infarct size in the rat by a mechanism(s) which involves the activation of PKC and the opening of ATP-sensitive potassium channels.     

Cardioprotective properties of humoral factors released from rat hearts subject to ischemic preconditioning

Myocardial protection can be achieved by transfer of coronary effluent from ischemically preconditioned to non-preconditioned hearts. This study was designed to test the hypothesis that preconditioned effluent from rat hearts purified by Sep-Pak C-18 cartridges could induce remote cardioprotection against ischemia/reperfusion (I/R) injury through the activation of protein kinase C signaling pathway. Buffer-perfused rat hearts were subject to 30 min ischemia and 60 min reperfusion. The myocardial I/R injury was assessed by postischemic contractile function recovery and infarct size. The protective effect of coronary effluent collected during ischemic preconditioning (IPC) was tested in non-preconditioned hearts in presence or absence of a PKC inhibitor, chelerythrine. Infarct size was 17 +/- 2% in preconditioned versus 37 +/- 1% in control hearts (P < 0.001). Hearts perfused with fresh preconditioned effluent had infarct sizes of 16 +/- 3% versus 36 +/- 1% in hearts treated with non-preconditioned effluent. The cardioprotective effect was lost when the effluent was left at room temperature during 24 h (infarct size, 40 +/- 3%) or heated to 70 degrees C (26 +/- 4%, P < 0.05) or 100 degrees C (39 +/- 1%, P < 0.001). The lyophilized effluent was stable for 30 days, and its purification in a Sep-Pak C-18 column resulted in a hydrophobic fraction that reduced the infarct size to 17 +/- 2% versus 38 +/- 2% for the hydrophilic fraction. Chelerythrine (100 microM) inhibited the reduction of infarct size induced by IPC (35 +/- 4%) or hydrophobic fraction (37 +/- 3%). Recovery of the contractile function at reperfusion was higher in preconditioned group (74 +/- 6% versus 17 +/- 7% in control, P < 0.001) and hydrophobic fraction (66 +/- 7% versus 8 +/- 4% in hydrophilic fraction, P < 0.001). Similarly, chelerythrine was able to abrogate the contractile function recovery (12 +/- 6%, P < 0.001 versus preconditioned group and 19 +/- 7%, P < 0.001 versus hydrophobic fraction). In conclusion, the cardioprotective factors released in the coronary effluent by IPC are thermolabile hydrophobic substances with molecular weights higher than 3.5 kDa and acting through PKC activation.     

The K(Ca) channel as a trigger for the cardioprotection induced by kappa-opioid receptor stimulation -- its relationship with protein kinase C

We first determined whether the cardioprotection resulting from kappa opioid receptor (kappa-OR) stimulation was blocked by the K(Ca) channel inhibitor, paxilline (Pax), administered before or during ischaemic insults in vitro. In isolated rat hearts, 30 min of ischaemia and 120 min of reperfusion induced infarction and increased lactate dehydrogenase (LDH) release. In isolated ventricular myocytes subjected to 5 min of metabolic inhibition and anoxia followed by 10 min of reperfusion, the percentage of live cells and the amplitude of the electrically induced intracellular Ca(2+) ([Ca(2+)](i)) transient decreased, while diastolic [Ca(2+)](i) increased. Pretreatment with 10 microM U50,488H, a kappa-OR agonist, attenuated the undesirable effects of ischaemic insults in both preparations. The beneficial effects of kappa-OR stimulation, that were abolished by 5 microM nor-BNI, a kappa-OR antagonist, were also abolished by 1 microM Pax administered before ischaemic insults or 20 microM atractyloside, an opener of the mitochondrial permeability transition pore. Activation of protein kinase C (PKC) with 0.1 microM phorbol 12-myristate 13-acetate decreased the infarct size and LDH release in isolated rat hearts subjected to ischaemia/reperfusion, and these effects were abolished by blockade of PKC with its inhibitors, 10 microM GF109203X or 5 microM chelerythrine, and more importantly by 1 microM Pax. On the other hand, the cardioprotective effects of opening the K(Ca) channel with 10 microM NS1619 were not altered by either PKC inhibitor. In conclusion, the high-conductance K(Ca) channel triggers cardioprotection induced by kappa-OR stimulation that involves inhibition of MPTP opening. The K(Ca) channel is located downstream of PKC.     

Effect of duration of ischaemia on reduction of myocardial infarct size by inhibition of neutrophil accumulation using an anti-CD18 monoclonal antibody.

1. Neutrophil accumulation is a characteristic feature of the inflammatory response in myocardial tissue which has undergone a period of ischaemia. The aim of this study was to examine whether inhibition of myocardial neutrophil infiltration, using an antibody to the CD18 leukocyte adhesion molecule, was effective in reducing infarct size in anaesthetized rabbits. 2. Anaesthetized rabbits underwent coronary artery occlusion (CAO) for periods of 30 or 45 min followed by reperfusion for 3 h. Animals were treated intravenously 10 min prior to reperfusion with IB4, a monoclonal antibody to CD18 (1 mg kg-1) or saline (1 ml kg-1). In one group undergoing 45 min CAO, a control antibody, OKMI (1 mg kg-1) was given. 3. Following either 30 or 45 min of CAO, administration of IB4 resulted in a < 75% inhibition in neutrophil accumulation in the area at risk myocardium (AR) compared with control animals. 4. With the 30 min occlusion period, IB4 significantly reduced myocardial infarct size, 27.2 +/- 3.2% vs 67.4 +/- 5.6% in the saline control group (n = 5 P < 0.01). In contrast, IB4 did not reduce infarct size following a 45 min period of ischaemia. 5. In the same animals administration of IB4 significantly inhibited oedema formation in skin elicited by intradermal administration of the neutrophil chemoattractant f-Met-Leu-Phe, but had no effect on coronary microvascular plasma protein leakage in the AR. 6. Our results indicate that infiltrating neutrophils exacerbate tissue injury following a relatively short, 30 min period of myocardial ischaemia in the rabbit.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effects of acute and chronic treatment with the sodium hydrogen exchanger 1 (NHE-1) inhibitor cariporide on myocardial infarct mass in rabbits with hypercholesterolaemia

We investigated the cardioprotective effect of acute and chronic sodium hydrogen exchanger 1 (NHE-1) inhibition with cariporide under pathological conditions in rabbits fed an atherogenic diet (0.25% cholesterol, 3% coconut oil), an experimental model of atherosclerosis. New Zealand White rabbits were fed over 4 weeks with normal diet or with atherogenic diet and randomized in 3 subgroups (n=7 in each group); placebo, acute cariporide (0.3 mg/kg, 10 min. before occlusion of left anterior descending coronary artery and chronic cariporide (4 weeks 0.1% in chow). In the final infarction experiments the animals were subjected to 30 min. of myocardial ischaemia by occlusion of a branch of the left anterior descending coronary artery followed by 2 hr of reperfusion. Infarct mass was evaluated by triphenyl-tetrazolium chloride staining and the infarct size expressed as a percentage of area at risk. Besides the assessment of aortic endothelium-dependent function aortic and cardiac vessels were inspected for atherosclerotic lesions. In cholesterol-fed rabbits, the infarct size was significantly increased when compared with normal diet animals (63+/-3% versus 41+/-3%). Acute cariporide treatment reduced the infarct size in normal diet rabbits to 14%+/-3% (66% decrease, P<0.05) as well as in atherogenic diet rabbits to 22+/-3% (65% decrease, P<0.05). Chronic treatment with cariporide also reduced the infarct size significantly: normal diet 19+/-2% (53% decrease, P<0.05), atherogenic diet 32+/-3% (49% decrease, P<0.05). Total cholesterol serum levels in rabbits with atherogenic diet were significantly higher (15.3+/-2.7 mmol/l) than those on a standard diet (0.65+/-0.08 mmol/l). Chronic cariporide treatment significantly attenuated the increase of serum cholesterol (7.9+/-1.9 mmol/l) and improved the lipoprotein pattern. Although the aortas and heart vessels of hypercholesterolaemic animals were without any histological evidence of atherosclerosis they developed endothelial dysfunction (reduced endothelium-dependent relaxation by ACh), which was prevented by chronic cariporide treatment. Acute and chronic treatment with the NHE-1 inhibitor cariporide significantly reduced infarct mass. This effect was associated with improved endothelial function.     

Anandamide reduces infarct size in rat isolated hearts subjected to ischaemia-reperfusion by a novel cannabinoid mechanism

Although the endocannabinoids 2-arachidonoylglycerol (2-AG) and anandamide share a similar pharmacology, 2-AG reportedly limits myocardial ischaemia-reperfusion injury whereas anandamide does not. We therefore investigated whether or not anandamide reduces infarct size and which, if any, of the known cannabinoid-signalling pathways are involved. Rat isolated perfused hearts were subjected to global, no-flow ischaemia (30 min) and reperfusion (1 h). Agonists were present from 5 min before ischaemia until the end of reperfusion. Antagonists, where used, were present throughout the protocol. Recovery of left ventricular developed pressure and coronary flow was incomplete in control hearts and not significantly affected by any drug treatment. In vehicle-treated hearts, 26+/-3% (n=13) of the left ventricle was infarcted at the end of reperfusion. Infarction of the left ventricle was significantly reduced after 1 microM anandamide (10+/-1%, n=7) or 1 microM methanandamide (12+/-4%, n=6) but not 1 microM HU210. Neither ACPA (1 microM; CB1 receptor agonist) nor JWH133 (1 microM; CB2 receptor agonist), individually or combined significantly affected infarct size. Anandamide (1 microM) did not reduce infarct size in the presence of the CB1 receptor antagonist rimonabant (SR141716A, 1 microM) or the CB2 receptor antagonist, SR144528 (1 microM). Despite sensitivity to CB1 and CB2 receptor antagonists, the infarct-limiting action of anandamide was not mimicked by agonists selective for CB1 or CB2 receptors suggesting the involvement of a novel cannabinoid site of action.     

Role of mitochondrial KATP channels and protein kinase C in ischaemic preconditioning

1. Activation of mitochondrial KATP (mitoKATP) channels and protein kinase C (PKC) has been implicated in cardioprotective mechanisms of ischaemic preconditioning (IPC). However, the exact role of these events in early IPC remains unclear. 2. Isolated and perfused rat hearts underwent IPC with three cycles of 5 min ischaemia and 5 min reperfusion. The heart was subjected to 30 min global ischaemia followed by 120 min reperfusion. Flavoprotein oxidation was monitored to assess mitoKATP channel activity. Cardioprotection was evaluated by recovery of isovolumic left ventricular (LV) function and infarct size. 3. Diazoxide (50 mgr;mol/L) increased flavoprotein oxidation and conferred cardioprotection in a manner sensitive to the selective mitoKATP channel blocker 5-hydroxydecanoate (5-HD; 0.5 mmol/L). 4. Pretreatment with 0.5 mmol/L 5-HD abrogated IPC-induced flavoprotein oxidation and cardioprotection, whereas late treatment with 5-HD after IPC required a higher dose (2 mmol/L) to abolish flavoprotein oxidation and cardioprotection afforded by IPC. 5. Pretreatment with the PKC inhibitors Ro318425 (1 micro mol/L) and chelerythrine (5 micro mol/L) abolished IPC-induced flavoprotein oxidation and cardioprotection, whereas late treatment with Ro318425 required a higher dose (4 micro mol/L) to abolish flavoprotein oxidation and cardioprotection. 6. In conclusion, these results suggest that activation of mitoKATP channels is the trigger and the mediator of IPC and that PKC plays a crucial role in both phases of mitoKATP channel activation, although mitoKATP channels and PKC may be more activated during the mediator phase.     

Inability of desferrioxamine to limit tissue injury in the ischaemic and reperfused rabbit heart

The role of iron-catalysed hydroxyl radical production in development of myocardial injury was examined in an in vivo rabbit heart with regional ischaemia (45 min) and reperfusion (3 h). Open-chest anaesthetised rabbits were assigned to control (saline) or desferrioxamine-treated [30 mg/kg subcutaneously (s.c.) plus 1, 10, 20, or 100 mg/kg/h intravenously (i.v.) starting 15 min before ischaemia] groups. Haemodynamics and ECG were monitored throughout. Following reperfusion, hearts were excised and perfused in vitro with buffer. The artery was religated, and fluorescent particles were injected to delineate the ischaemic zone. Tetrazolium staining was used to define necrosis. Planimetry was performed on photographed heart slices for calculation of the size of the hearts and of the ischaemic and infarcted zones. Infarct size (as a percentage of ischaemic zone size) in control hearts was 51.2 +/- 5.8% (n = 10) and in the groups treated with desferrioxamine 1, 10, 20, and 100 mg/kg/h it was 53.1 +/- 11.7% (n = 8), 45.3 +/- 6.3% (n = 9), 65.1 +/- 10.1% (n = 6), and 52.5 +/- 9.4% (n = 7), respectively (p = NS vs. control in each instance). In addition, no antiarrhythmic effects were observed at any dose. Thus, iron-catalysed hydroxyl radical damage may not play a role in the pathogenesis of tissue injury under these conditions.     

Activation of ATP-dependent potassium channels is a trigger but not a mediator of ischaemic preconditioning in pigs

1. Activation of ATP-dependent potassium channels (K(ATP)) is involved in ischaemic preconditioning (IP). In isolated buffer-perfused rabbit hearts, activation of mitochondrial K(ATP)--through a generation of free radicals--acted as a trigger rather than a mediator of IP; the isolated buffer-perfused heart preparation, however, favours free radical generation. In contrast, in vivo studies in rats and dogs suggested that activation of K(ATP) acts as a mediator of IP's protection. A detailed analysis on the role of K(ATP) in IP's protection in vivo by varying the time and dose of K(ATP) blocker administration is, however, lacking. 2. In 54 enflurane-anaesthetized pigs, the left anterior descending coronary artery was perfused by an extracorporeal circuit. Infarct size (IS, %, TTC) following 90 min sustained low-flow ischaemia and 120 min reperfusion was 26.6+/-3.5 (s.e.m.) (n=8). IP with one cycle of 10 min ischaemia and 15 min reperfusion reduced IS to 6.5+/-2.1 (n=7, P<0.05). Blockade of K(ATP) with glibenclamide (0.5 mg kg(-1) i.v., 50 microg min(-1) continuous infusion) starting 10 min before or immediately following the preconditioning ischaemia abolished IS reduction by IP (20.7+/-2.7, n=7 and 21.9+/-6.6, n=6, respectively) while having no effect on IS per se (22.2+/-5.2, n=7), supporting a trigger role of K(ATP) in IP. In contrast, starting glibenclamide following the preconditioning ischaemia 10 min prior to the sustained ischaemia did not prevent IS reduction by IP (3.7+/-2.3, n=6), even when its bolus dose was increased to 1.5 mg kg(-1) (26.6+/-3.8 with IP vs 37.5+/-2.9 without IP; n=7 and 6 respectively, P<0.05), thereby refuting a mediator role of K(ATP) in IP. 3. In conclusion, activation of K(ATP) in the immediate reperfusion following the preconditioning ischaemia is pivotal for triggering IP.     

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)     

Quinaprilat reduces myocardial infarct size involving nitric oxide production and mitochondrial KATP channel in rabbits

This study examined whether quinaprilat, an angiotensin-converting enzyme inhibitor, reduces the infarct size, and investigated the mechanisms for its infarct size-reducing effect, in rabbits. Japanese white rabbits underwent 30 min of ischemia and 48 h of reperfusion. Quinaprilat (100 microg/kg/h or 300 microg/kg/h for 70 min, IV) was administered 20 min before ischemia with or without pretreatment with Nomega-nitro-l-arginine methyl ester (l-NAME) (10 mg/kg, IV, a nitric oxide synthase inhibitor), 5-hydroxydecanoic acid sodium salt (5-HD) or posttreatment with 5-HD (5 mg/kg, IV, a mitochondrial KATP channel blocker). The area at risk as a percentage of the left ventricle was determined by Evans blue dye and the infarct size was determined as a percent of the area at risk by triphenyl tetrazolium chloride staining. Using a microdialysis technique, myocardial interstitial levels of 2,5-dihydroxybenzoic acid (2,5-DHBA), an indicator of hydroxyl radicals, and NOx, an indicator of nitric oxide, were measured before, during, and after 30 min of ischemia. Quinaprilat significantly reduced the infarct size in a dose-dependent manner (30.1 +/- 3%, n = 10, and 27.6 +/- 2%, n = 7, respectively) compared with the control (46.5 +/- 4%, n = 10). The infarct size-reducing effect of quinaprilat was completely blocked by pretreatment with l-NAME (43.8 +/- 2%, n = 8) and 5-HD (50.1 +/- 3%, n = 8) and posttreatment with 5-HD (50.3 +/- 2%, n = 8), respectively. Quinaprilat did not affect the myocardial interstitial 2,5-DHBA level but significantly increased the NOx level during ischemia and reperfusion. Quinaprilat reduces myocardial infarct size involving NO production and mitochondrial KATP channels in rabbits without collateral circulation.     

ATENOLOL DEPRESSES POST-ISCHAEMIC RECOVERY IN THE ISOLATED RAT HEART

Metabolic events during ischaemia are probably important in determining post-ischaemic myocardial recovery. The aim of this study was to assess the effects of the beta-blocker atenolol and the high energy demand in an ischaemia-reperfusion model free of neurohormonal and vascular factors. We exposed Langendorff-perfused isolated rat hearts to low-flow ischaemia (30 min) and reflow (20 min). Three groups of hearts were used: control hearts (n =11), hearts that were perfused with 2.5 micrograms l-1atenolol (n =9), and hearts electrically paced during ischaemia to distinguish the effect of heart rate from that of the drug (n =9). The hearts were freeze-clamped at the end of reflow to determine high-energy phosphates and their metabolites. During ischaemia, the pressure-rate product was 2.3+/-0.2, 5.2+/-1.1, and 3.3+/-0.3 mmHg 10(3)min in the control, atenolol and paced hearts, respectively. In addition, the ATP turnover rate, calculated from venous (lactate), oxygen uptake and flow, was higher in atenolol (11.2+/-1.7 micromol min-1) and paced (8.1+/-0.8 micromol min-1) hearts than in control (6.2+/-0.8 micromol min-1). At the end of reflow, the pressurexrate product recovered 75.1+/-6.4% of baseline in control vs 54.1+/-9.1 and 48.8+/-4.4% in atenolol and paced hearts (P<0.05). In addition, the tissue content of ATP was higher in the control hearts (15.8+/-1. 0 micromol g(dw)(-1)) than in atenolol (10.5+/-2.6 micromol g(dw)(-1)) and paced (10.9+/-1.3 micromol g(dw)(-1)) hearts. Thus, by suppressing the protective effects of down-regulation, both atenolol and pacing apparently depress myocardial recovery in this model.     

Edaravone reduces myocardial infarct size and improves cardiac function and remodelling in rabbits

1. In the present study, we investigated the effect of 3-methyl-1-phenyl-2-pyrazolin-5-one (edaravone), a free radical scavenger, on myocardial infarct (MI) size and cardiac function in an in vivo model of MI in rabbits. We further investigated the contribution of hydroxyl radicals, superoxide and nitric oxide (NO) to its effects. 2. Anaesthetized open-chest Japanese white male rabbits were subjected to 30 min coronary occlusion and 48 h reperfusion. The control group (n = 10) was injected with saline 10 min before reperfusion. The edaravone group (n = 10) was injected with a bolus of 3 mg/kg edaravone 10 min before reperfusion. The edaravone + N(G)-nitro-L-arginine methyl ester (L-NAME) group (n = 5) was given 10 mg/kg, i.v., L-NAME 10 min before the administration of 3 mg/kg edaravone. The L-NAME group (n = 5) was given 10 mg/kg, i.v., L-NAME 20 min before reperfusion. Infarct size was measured using the triphenyl tetrazolium chloride method and is expressed as a percentage of area at risk. Cardiac function was assessed by echocardiography 14 days after infarction. 3. In another series of experiments, rabbits were subjected to 30 min coronary occlusion and 30 min reperfusion and myocardial interstitial 2,3-dihydroxybenzoic acid (DHBA) and 2,5-DHBA levels, indicators of hydroxyl radical, were measured using a microdialysis technique. 4. Infarct size in the edaravone group was significantly reduced compared with that in the control group (27.4 +/- 6.8 vs 43.4 +/- 6.8%, respectively; P < 0.05). The edaravone-induced reduction of infarct size was abolished by pretreatment with L-NAME. Myocardial interstitial levels of 2,3-DHBA and 2,5-DHBA increased 20 and 30 min after ischaemia and peaked at 10 min reperfusion in the control group. Edaravone significantly inhibited the increase in 2,3-DHBA and 2,5-DHBA levels seen during reperfusion. Dihydroethidium staining showing in situ detection of superoxide was less intense in ischaemic myocardium in the edaravone-treated group compared with the control group. Edaravone improved cardiac function and left ventricular remodelling 14 days after infarction. 5. In conclusion, edaravone significantly reduces MI size and improves cardiac function and LV remodelling by decreasing hydroxyl radicals and superoxide in the myocardium and increasing the production of NO during reperfusion in rabbits.     

Reduction by prostaglandin E1 or prostaglandin E0 of myocardial infarct size in the rabbit by activation of ATP-sensitive potassium channels.

1. This study examined whether pretreatment of rabbits with infusions of prostaglandin E1 (PGE1) or prostaglandin E0 (PGE0) (which were terminated prior to the onset of ischaemia) reduce myocardial infarct size arising from coronary artery occlusion (60 min) and reperfusion (120 min). In addition, we investigated whether the observed cardioprotective effects of these two prostaglandins were due to the activation of ATP-sensitive potassium (KATP) channels. 2. In the anaesthetized rabbit, infarct size (expressed as a percentage of the area at risk) after 60 min of coronary artery occlusion followed by 2 h of reperfusion was 59 +/- 4% (n = 10). PGE1 or PGE0 treatment (1.0 micrograms kg-1 min-1), administered as 1 h pretreatments (0.05 ml min-1, i.v.), significantly reduced infarct size to 44 +/- 6% (n = 6) or 42 +/- 1% (n = 6), respectively. PGE1 or PGE0 pretreatment resulted in a significant reduction in mean arterial blood pressure, which returned to baseline within 15 min of discontinuation of the infusion (i.e. prior to LAL ligation). 3. The reduction in infarct size afforded by PGE1 was abolished by pretreatment of rabbits with the KATP channel blockers, glibenclamide (60 +/- 4%; n = 8) or 5-hydroxydecanoate (58 +/- 6%; n = 6). Similarly, glibenclamide also largely attenuated the reduction in infarct size afforded by PGE0 (52 +/- 3%; n = 8). 4. We propose that a 1 h pretreatment of PGE1 or PGE0 reduces infarct size by activating protein kinase C resulting in the opening of KATP channels.     

Na+/H+ exchange inhibitors reverse lactate-induced depression in postischaemic ventricular recovery.

1. By use of pharmacological approaches, the present study examined the hypothesis that the deleterious effect of lactate on postischaemic ventricular recovery may be mediated, at least in part, by enhanced activation of the Na+/H+ exchanger at the time of reperfusion. 2. Spontaneously beating isolated hearts of the rat were subjected to 15 min zero-flow global ischaemia followed by 30 min reperfusion. The effects of lactate (10, 20 or 40 mM) were studied by adding it 20 min before ischaemia whereas reperfusion was carried out with lactate-free buffer. 3. Pretreatment with 20 or 40 mM lactate significantly reduced postischaemic recovery of developed force to 17 +/- 3% and 16 +/- 4% of preischaemic values (P < 0.05) compared to a 78 +/- 4% recovery in control hearts. Similarly, recovery in ventricular rate was significantly reduced to 34 +/- 7.6% and 38 +/- 12% with 20 and 40 mM lactate, respectively compared to 97.5 +/- 6.4% recovery in control hearts. At a concentration of 10 mM, lactate was without effect on either force or ventricular rate recovery. 4. Coadministration of either of two Na+/H+ exchange inhibitors, amiloride (174 microM) or 5-N,N-hexamethylene amiloride (HMA, 1 microM) with lactate and inclusion of the two drugs during the first 5 min of reperfusion resulted in reversal of lactate-induced inhibition of force recovery with observed recoveries of 69 +/- 6.7% and 64 +/- 5% with amiloride and HMA, respectively. Similarly, recovery in ventricular rate was significantly enhanced to 92 +/- 10% and 89 +/- 6% with amiloride and HMA, respectively compared to 38 +/- 12% recovery in control hearts.(ABSTRACT TRUNCATED AT 250 WORDS)