Delayed uncoupling contributes to the protective effect of heptanol against ischaemia in the rat isolated heart

1. It is known that infusion of the gap junction uncoupler heptanol, before ischaemia or during reperfusion, limits myocardial infarct size. However, whether this cardiac effect is linked to the effect of heptanol on communication across gap junctions has not been elucidated. The aims of the present study were to examine the effect of heptanol on infarct size, arrhythmias and myocardial tissue resistance and to assess whether changes in electrical coupling correlate with cardiac protection. 2. Rat isolated, perfused hearts were subjected to a 24 min infusion of heptanol (0.05, 0.1, 0.5 or 1.0 mmol/L) followed by 20 min regional ischaemia and 60 min reperfusion, or by 70 min global no-flow ischaemia. The effective refractory period, action potential and conduction velocity were measured in papillary muscles from the right ventricle. Heptanol markedly decreased arrhythmia scores during ischaemia and reperfusion, as well as reducing infarct size to a degree similar to that induced by ischaemic preconditioning. In the prolonged ischaemia model, heptanol delayed the onset of uncoupling, increased time to plateau and decreased the maximal rate of uncoupling during ischaemia. Ischaemic preconditioning had similar effects on these parameters. In papillary muscle, heptanol reduced the conduction velocity of the action potential in a dose-dependent manner, but had no significant effect on resting potential, action potential amplitude, action potential duration, maximal upstroke of depolarization or effective refractory period. 3. These results demonstrate that treatment with the gap junction uncoupler heptanol confers cardioprotection against ischaemia and this effect is related to delayed electrical uncoupling during prolonged ischaemia.     

The influence of nifedipine and mioflazine on mitochondrial calcium overload in normoxic and ischaemic guinea-pig hearts

The influence of nifedipine (20 nM) and mioflazine (300 nM), i.e. concentrations inducing a 60-70% recovery of cardiac function during reperfusion of globally ischaemic guinea-pig working hearts, on the mitochondrial calcium content was investigated in normoxic, globally ischaemic and reperfused globally ischaemic guinea-pig working hearts. Mitochondrial calcium was determined electronmicroscopically with oxalate-pyroantimonate method. In normoxic hearts both nifedipine and mioflazine reduced the mitochondrial calcium content. Global ischaemia for 45 min and subsequent reperfusion for 25 min resulted in a pronounced mitochondrial calcium overload and damage to the cellular structure. In ischaemic and in reperfusion hearts the drugs maintained mitochondrial calcium at pre-ischaemic levels and decreased the damage to the cellular structure.     

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.     

Preserved cardiac mitochondrial function and reduced ischaemia/reperfusion injury afforded by chronic continuous hypoxia: Role of opioid receptors

Chronic continuous normobaric hypoxia (CNH) increases cardiac tolerance to acute ischaemia/reperfusion injury. The objective of this study was to find out whether the cardioprotective effect of CNH mediated by opioid receptors is associated with preservation of mitochondrial function. Rats were adapted to CNH (12% oxygen) for 3 weeks. Isolated perfused hearts were subjected to 45 min of global ischaemia and 30 min of reperfusion; subgroups were pretreated with non‐selective opioid receptor antagonist naloxone (300 nmol/L) for 10 min. Cardiac contractile function, creatine kinase activity in coronary effluent, mitochondrial respiration rate, and calcium retention capacity were assessed. Adaptation to CNH decreased myocardial creatine kinase release during reperfusion and improved the post‐ischaemic recovery of contractile function, mitochondrial state 3 and uncoupled respiration rates, and calcium retention capacity compared to the normoxic group. These protective effects were completely abolished by naloxone. The contractile recovery positively correlated with state 3 respiration and calcium retention capacity. The results suggest that the preserved mitochondrial function contributes to the protected cardiac phenotype afforded by adaptation to CNH and point to an important role of opioid receptor activation.     

Mitochondrial big conductance KCa channel and cardioprotection in infant rabbit heart

Chronic hypoxia increases resistance to myocardial ischemia in infants. Activation of the mitochondrial big conductance Ca(2+) -sensitive K channel (mitoBKCa) has been shown to be protective in adult hearts; however, its role in infant hearts is unknown. Hearts from normoxic or hypoxic infant rabbits were perfused with a mitoKCa opener, NS1619, or blocker Paxilline before ischemia and reperfusion. Hypoxic hearts were more resistant to ischemia than normoxic hearts as manifested by a reduction in infarct size (9 +/- 5% versus 14 +/- 5%) and an increase in recovery of left ventricular developed pressure (LVDP) (69 +/- 7% versus 51 +/- 2%). NS1619 decreased infarct size in normoxic hearts from 14 +/- 5% to 10 +/- 5% and increased recovery of LVDP from 51 +/- 2% to 65 +/- 4%, but it had no effect on hypoxic hearts. Paxilline did not affect normoxic or hypoxic hearts. Activation of mitoBKCa protects normoxic infant rabbit hearts; however, cardioprotection by chronic hypoxia in infant rabbits does not appear involve mitoBKCa.     

A role for the RISK pathway and K(ATP) channels in pre- and post-conditioning induced by levosimendan in the isolated guinea pig heart

BACKGROUND AND PURPOSE: Myocardial reperfusion injury prevents optimal salvage of the ischaemic myocardium, and adjunct therapy that would significantly reduce reperfusion injury is still lacking. We investigated whether (1) the heart could be pre- and/or post-conditioned using levosimendan (levosimendan pre-conditioning (LPC) and levosimendan post-conditioning (LPostC)) and (2) the prosurvival kinases and/or the sarcolemmal or mitochondrial K(ATP) channels are involved. EXPERIMENTAL APPROACH: Isolated guinea pig hearts were treated with two 5 min cycles of levosimendan (0.1 microM) interspersed with vehicle perfusion, or two 5 min cycles of ischaemia/reperfusion, before coronary artery ligation (CAL) for 40 min at 36.5 degrees C. Hearts were treated with mitochondrial or sarcolemmal K(ATP) channel blockers before LPC or LPostC. For post-conditioning, hearts received three 30 s cycles of ischaemia/reperfusion or levosimendan/vehicle. Hearts were pretreated with levosimendan immediately before CAL (without washout). Cardiac function, infarct size and reperfusion injury salvage kinase activity was assessed. KEY RESULTS: LPC and LPostC halved the infarct size compared with controls (P<0.05). Treatment with K(ATP) channel blockers before LPC or LPostC reversed this decrease. Pretreating hearts with levosimendan increased activity of extracellular signal-regulated kinase (ERK) 42/44 on reperfusion and had the most marked infarct-lowering effect (P<0.05). CONCLUSIONS AND IMPLICATIONS: (1) Hearts could be pharmacologically pre- and post-conditioned with levosimendan; (2) levosimendan pretreatment is the most effective way to reduce infarct size, possibly by increasing ERK 42/44 activity; (3) benefits of LPC and LPostC were abolished by both K(ATP) channel blockers and (4) LPC may be useful before elective cardiac surgery, whereas LPostC may be used after acute coronary artery events.     

ISCHAEMIC POST-CONDITIONING PROTECTS BOTH ADULT AND AGED SPRAGUE-DAWLEY RAT HEART FROM ISCHAEMIA–REPERFUSION INJURY THROUGH THE PHOSPHATIDYLINOSITOL 3-KINASE–AKT AND GLYCOGEN SYNTHASE KINASE-3β PATHWAYS

• 1 Numerous studies have demonstrated that ischaemic post‐conditioning (IPoC) protects adult rats from myocardial ischaemia‐reperfusion (I/R) injury. Recent evidence suggests compromised cardioprotection by IPoC in aged mice. The present study was designed to test the hypothesis that IPoC protects against I/R injury in aged hearts, potentially through a phosphatidylinositol 3‐kinase (PI3‐K)–Akt‐ and glycogen synthase kinase (GSK)‐3β‐dependent mechanism.
• 2 Hearts from adult (3–4 months) or aged (16–18 months) Sprague‐Dawley rats were subjected in vivo to 30 min ischaemia followed by 3 h reperfusion. Ischaemic post‐conditioning (four cycles of 10 s reperfusion–10 s ischaemia) was applied at the beginning of reperfusion, either alone or in combination with the PI3‐K inhibitor LY294002 (0.3 mg/kg). Infarct size and the phosphorylation of Akt and GSK‐3β were determined.
• 3 Ischaemic post‐conditioning reduced infarct size in both adult and aged rat hearts. This protection was accompanied by a significant increase in phosphorylation of Akt and GSK‐3β. LY294002 abolished the IPoC‐induced phosphorylation of Akt and GSK‐3β, as well as the infarct‐limiting effect of IPoC in adult and aged rats. In addition, IPoC significantly attenuated plasma concentrations of creatine kinase and lactate dehydrogenase after reperfusion in both adult and aged rats.
• 4 In conclusion, IPoC, at the onset of reperfusion, reduces myocardial infarct size in both adult and aged rat hearts, potentially through a PI3‐K‐, Akt‐ and GSK‐3β‐dependent mechanism.

     

Non-specific caspase inhibition reduces infarct size and improves post-ischaemic recovery in isolated ischaemic/reperfused rat hearts

Myocardial ischaemia and reperfusion lead to myocardial cell death due, at least in part, to apoptotic mechanisms. Although cysteinyl aspartate-specific proteinase (caspase) activation is a major event and the most-cited culprit in the development of apoptosis, its potential contribution to ischaemic myocardial cell death is largely unknown. To study the role of caspase activation, isolated rat hearts (n=6 per group) were subjected to 30 min coronary artery occlusion followed by 120 min reperfusion. A non-selective [0.1 or 0.5 microM acetyl-Tyr-Val-Ala-Asp chloromethylketone (YVAD-cmk)] or selective caspase inhibitors [0.07 or 0.2 microM acetyl-Asp-Glu-Val-Asp-cmk (Ac-DEVD-cmk, caspase-3 inhibitor); 0.07 or 0.2 microM benzoxycarbonyl-Leu-Glu-OMe-His-Asp(OMe)-fluoromethylketone (z-LEHD-fmk, caspase-9 inhibitor)] were added to the perfusate at the start of reperfusion. Non-selective caspase inhibition with 0.1 or 0.5 microM YVAD-cmk limited infarct size: (21 +/- 4%, P<0.05; 17 +/- 3%, P<0.05, respectively) compared with the ischaemic/reperfused control (32 +/- 5%). In hearts treated with 0.1 or 0.5 microM caspase II non-selective inhibitor, the fraction of terminal-deoxynucleotidyl-transferase deoxyuridine nick end labelling (TUNEL)-positive myocyte nuclei in the infarcted zone was reduced from the ischaemic/reperfused non-treated control of 11.2 +/- 2.1% to 6.2 +/- 1.6% (P<0.05) and 1.2 +/- 0.2% (P<0.05), respectively. The recovery of post-ischaemic cardiac function (coronary flow, aortic flow and left-ventricular developed pressure) improved significantly with the application of the non-selective caspase inhibitor as well. In hearts perfused with specific caspase inhibitors (caspase-3 and caspase-9) there was no significant reduction in the infarct size, no improvement in post-ischaemic cardiac function and no reduction of apoptotic cell death. We conclude that non-specific inhibition of caspases may be therapeutically beneficial in myocardial ischaemia/reperfusion-induced damage, while selective caspase inhibitors may fail to prevent such reperfusion-induced injury in our model system.     

Acute administration of cannabidiol in vivo suppresses ischaemia-induced cardiac arrhythmias and reduces infarct size when given at reperfusion

Background and purpose:

Cannabidiol (CBD) is a phytocannabinoid, with anti-apoptotic, anti-inflammatory and antioxidant effects and has recently been shown to exert a tissue sparing effect during chronic myocardial ischaemia and reperfusion (I/R). However, it is not known whether CBD is cardioprotective in the acute phase of I/R injury and the present studies tested this hypothesis.

Experimental approach:

Male Sprague-Dawley rats received either vehicle or CBD (10 or 50 µg·kg⁻¹ i.v.) 10 min before 30 min coronary artery occlusion or CBD (50 µg·kg⁻¹ i.v.) 10 min before reperfusion (2 h). The appearance of ventricular arrhythmias during the ischaemic and immediate post-reperfusion periods were recorded and the hearts excised for infarct size determination and assessment of mast cell degranulation. Arterial blood was withdrawn at the end of the reperfusion period to assess platelet aggregation in response to collagen.

Key results:

CBD reduced both the total number of ischaemia-induced arrhythmias and infarct size when administered prior to ischaemia, an effect that was dose-dependent. Infarct size was also reduced when CBD was given prior to reperfusion. CBD (50 µg·kg⁻¹ i.v.) given prior to ischaemia, but not at reperfusion, attenuated collagen-induced platelet aggregation compared with control, but had no effect on ischaemia-induced mast cell degranulation.

Conclusions and implications:

This study demonstrates that CBD is cardioprotective in the acute phase of I/R by both reducing ventricular arrhythmias and attenuating infarct size. The anti-arrhythmic effect, but not the tissue sparing effect, may be mediated through an inhibitory effect on platelet activation.     

Paradoxically enhanced heart tolerance to ischaemia in type 1 diabetes and role of increased osmolarity

There is considerable controversy regarding the tolerance of diabetic hearts to ischaemia and the underlying mechanisms responsible for the increased heart tolerance to ischamia remain uncertain. In the present study, we observed, in vitro, type 1 diabetic heart responses to ischaemia and reperfusion at different degrees of hyperglycaemia. In addition, the possible role of increased osmolarity in cardioprotection due to hyperglycaemia was evaluated. Hearts from 3 week streptozocin-induced diabetic rats were isolated and perfused in a Langendorff apparatus and subjected to 30 min ischaemia and 30 min reperfusion. Cardiac function and the electrocardiogram were recorded. Myocardial content of osmolarity associated heat shock protein (hsp) 90, heme oxygenase (HO)-1 and anti-oxidant enzymes were determined in diabetic or hyperosmotic solution-perfused hearts using western blot. The hsp90 inhibitor 17-allylamino-17-demethoxygeldanamycin (17-AAG; 2 x 10(-7) mol/L) or the nitric oxide synthase (NOS) inhibitor Nomega-nitro-L-arginine methyl ester (1 x 10(-5) mol/L) was added to the perfusate to observe the effects of hsp90 inhibition and hsp90-associated endothelial NOS on ischaemic responses of diabetic hearts. Compared with normal control rats, diabetic hearts with severe hyperglycaemia (blood glucose > 20 mmol/L) showed markedly improved postischaemic heart function with fewer reperfusion arrhythmias. Mild hyperglycaemia (< 12 mmol/L) exhibited no significant cardioprotection. Elevated expression of hsp90 accompanied the enhanced resistance to ischaemia in diabetic hearts, which was abrogated by 17-AAG. In the presence of the NOS inhibitor, heart function was preserved, whereas reperfusion arrhythmias were increased in diabetes. Diabetic hearts also had markedly elevated HO-1 and catalase, with no significant change in superoxide dismutase. Hyperosmotic perfusion with glucose or mannitol also increased myocardial hsp90 and catalase. The present findings reveal that heart resistance to ischaemia is increased in short-term type 1 diabetes with severe hyperglycaemia. Elevated osmolarity caused by significant hyperglycaemia may contribute to the enhanced myocardial activity against oxidative injury during ischaemia and reperfusion.     

Role of protein kinase C in the reduction of infarct size by N-methyl-1-deoxynojirimycin, an alpha-1,6-glucosidase inhibitor.

Preischaemic treatment with N-methyl-1-deoxynojirimycin (MOR-14), an alpha-1,6-glucosidase inhibitor, attenuates glycogenolysis and lactate accumulation during ischaemia and markedly reduces infarct size in rabbit hearts. In the present study, we have investigated whether protein kinase C (PKC), a principal mediator of ischaemic preconditioning, is also involved in the cardioprotective effect of MOR-14. To assess the effect of PKC inhibition on infarct size in MOR-14-treated hearts, 38 rabbits were subjected to 30 min of ischaemia followed by 48 h of reperfusion. Infarct size, as a per cent of area at risk, was significantly smaller in rabbits administered 100 mg kg(-1) of MOR-14 10 min before ischaemia (17+/-2%, n=10), than in a control group (46+/-5%, n=10). This beneficial effect of MOR-14 was abolished when 5 mg kg(-1) of chelerythrine, a PKC inhibitor, was given 10 min prior to MOR-14 injection (39+/-4%, n=10), although chelerythrine alone did not alter infarct size (43+/-4%, n=8). Further, chelerythrine had no effect on MOR-14-induced attenuation of glycogen breakdown and lactate accumulation in hearts excised at 30 min of ischaemia. Immunoblot analysis of PKC in homogenates of Langendorff-perfused rabbit hearts revealed that MOR-14 significantly increased levels of PKC-epsilon in the particulate fraction at 20 and 30 min of ischaemia and in the cytosolic fraction at 30 min of ischaemia. Taken as a whole, our data suggest that PKC acts downstream of the inhibition of glycogenolysis by MOR-14 to reduce infarct size. Thus, activation of PKC is a more direct mediator of the cardioprotection afforded by MOR-14 than is inhibition of glycogenolysis.     

Sevoflurane post-conditioning protects against myocardial reperfusion injury by activation of phosphatidylinositol-3-kinase signal transduction

The mechanisms underlying myocardial protection by sevoflurane post-conditioning are unclear. In the present study, we tested two hypotheses: (i) that sevoflurane post-conditioning produces cardioprotection via a phosphatidylinositol-3-kinase (PI3-K)-dependent pathway; and (ii) combining sevoflurane and ischaemic post-conditioning offers an additional benefit against reperfusion injury. Rat isolated perfused hearts were exposed to 25 min ischaemia followed by 90 min reperfusion. Sevoflurane post-conditioning was induced by administration of sevoflurane (3.0 vol%) for 15 min from the onset of reperfusion. In some groups, 15 micromol/L LY294002, a selective PI3-K inhibitor, was coadministrated with sevoflurane. Other groups of hearts were exposed to ischaemic post-conditioning or combined sevoflurane plus ischaemic post-conditioning in the presence and absence of LY294002. After 15 min reperfusion, phosphorylation of Akt and glycogen synthase kinase 3beta (GSK3beta) was determined by Western blot analysis. Infarct size was determined by 2,3,5-triphenyltetrazolium chloride staining and subsarcolemmal mitochondrial lesions were assessed by electron microscopy after 90 min reperfusion. Sevoflurane post-conditioning significantly decreased infarct size compared with control hearts (31 +/- 2 vs 42 +/- 3%, respectively; P < 0.05), diminished mitochondrial lesions and increased phosphorylation of Akt and GSK3beta, as did ischaemic post-conditioning. However, combined sevoflurane plus ischaemic post-conditioning did not further improve the cardioprotective effects compared with either intervention alone. Sevoflurane-mediated cardioprotection was abolished or inhibited by 15 micromol/L LY294002. In conclusion, sevoflurane acts during early reperfusion after ischaemia to salvage the myocardium by activating PI3-K. The combination of sevoflurane plus ischaemic post-conditioning does not offer any additional benefit over either intervention alone.     

Role of calcitonin gene-related peptide in ischaemic preconditioning in diabetic rat hearts

1. It has been suggested that calcitonin gene-related peptide (CGRP) is involved in the protection provided by ischaemic preconditioning in rat hearts and that ischaemic preconditioning is absent in diabetic rat hearts. 2. In the present study, we tested the relationship between sensory nerve function and ischaemic preconditioning in diabetic rats. 3. In 4- and 8-week diabetic rats and age-matched non- diabetic controls, 30 min global ischaemia and 40 min reperfusion caused a significant decrease in cardiac function and a marked increase in creatine kinase (CK) release. Ischaemic preconditioning, by three cycles of 5 min ischaemia and 5 min reperfusion, improved the recovery of cardiac function and decreased CK release during reperfusion in 4-week diabetic rat hearts. However, the cardioprotection afforded by ischaemic preconditioning was lost in 8-week diabetic rat hearts. Pretreatment with CGRP for 5 min also significantly improved the recovery of cardiac function and decreased CK release in rats subjected to 4 or 8 weeks of diabetes. 4. The content of CGRP in the coronary effluent during ischaemic preconditioning was significantly increased in 4-week diabetic rat hearts (P < 0.05). However, only a slight increase in the release of CGRP was shown in 8-week diabetic rat hearts (P > 0.05). 5. In summary, the present results suggest that the protection afforded by ischaemic preconditioning is attenuated in diabetic rats and that the change may be related to the reduction in CGRP release in diabetic rat hearts.     

POSSIBLE MECHANISM OF CARDIOPROTECTIVE EFFECT OF ISCHAEMIC PRECONDITIONING IN ISOLATED RAT HEART

The present study is designed to investigate the mechanism of the cardioprotective effect of ischaemic preconditioning. Isolated perfused rat heart was subjected to global ischaemia for 30 min followed by reperfusion for 120 min. Coronary effluent was analysed for LDH and CK release to assess the degree of cardiac injury. Myocardial infarct size was estimated macroscopically using TTC staining. Four episodes of ischaemic preconditioning markedly reduced LDH and CK release in the coronary effluent and decreased myocardial infarct size. Administration of prazosin (alpha(1)adrenoceptor antagonist) before global ischaemia reduced the extent of ischaemia-reperfusion induced myocardial injury. The cardioprotective effect of ischaemic preconditioning was abolished by prazosin and colchicine (microtubule disaggregator). On the basis of these results, it may be concluded that the cardioprotective effects of ischaemic preconditioning may be mediated through stimulation of alpha(1)adrenoceptors and translocation of PKC.     

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)     

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.     

A comparison of the cardioprotective effects of calcium antagonists from different classes upon ischaemic damage in the guinea-pig working heart

Summary The cardioprotective effects of nifedipine, verapamil, diltiazem, bepridil, CERM 11956, lidoflazine, mioflazine and the coronary vasodilator dipyridamole were evaluated in the guinea-pig working heart with respect to cardiac function and high energy phosphate content after 45 min of global ischaemia and 25 min of reperfusion.All drugs, with the exception of dipyridamole, induced a negative inotropic effect, which resulted in a decrease of the aortic pressure (AoP), of its first derivative dAoP/dt and the cardiac output. To compare the anti-ischaemic effect of the calcium antagonists, concentrations were selected that reduced the dAoP/dt by 10% (EC₁₀) and 30% (EC₃₀), respectively. With the exception of nifedipine at the EC₁₀ and bepridil and CERM 11956 at the EC₃₀, perfusion with the calcium antagonists and dipyridamole (3 mol/l) improved the recovery of contractile function after global ischaemia and reperfusion to a value between 60 and 80% of the controls in normoxic hearts.Pretreatment with nifedipine, verapamil, diltiazem, lidoflazine and mioflazine, but not with bepridil, CERM 11956 and dipyridamole led to slightly increased ATP levels in ischaemic hearts as compared to the control value in ischaemic hearts. After subsequent reperfusion for 25 min, for all drugs, ATP levels were further enhanced to 50% of the level in normoxic hearts; phosphocreatine levels reached normoxic values. In particular at the EC₃₀, the effects of calcium antagonists on cardiac function varied in accordance with their known pharmacological and physiological profile. However, there appeared to exist no direct relationship between their beneficial effects on contractile activity and those on the levels of high energy phosphates after ischaemia and reperfusion.Send offprint requests to J. G. Hugtenburg at the above address     

Cytochrome P450 2J3/epoxyeicosatrienoic acids mediate the cardioprotection induced by ischaemic post-conditioning, but not preconditioning, in the rat

1. Cytochrome P450 (CYP) epoxygenases and their arachidonic acid metabolites play a protective role against ischaemia-reperfusion injury. In the present study, we investigated whether endogenous CYP2J3/epoxyeicosatrienoic acid (EET) mediates the cardioprotective effects of ischaemic preconditioning (IPC) and ischaemic post-conditioning (IPost). 2. Male Wistar rats were subjected to two cycles of IPC, consisting of 5 min ischaemia and 5 min reperfusion, followed by 45 min occlusion and 2 h reperfusion; IPost consisted of three cycles of 30 s reperfusion and 30 s re-occlusion at the onset of reperfusion. The selective CYP epoxygenase inhibitor N-methylsulphonyl-6-(2-propargyloxyphenyl)hexanamide (MS-PPOH; 3 mg/kg) was administered 10 min before ischaemia or during ischaemia 10 min before reperfusion started. Cardiac function was measured continuously with a angiocatheter connected to a fluid-filled pressure transducer and myocardial infarct size was assessed by triphenyl tetrazolium chloride staining at the end of the experiment. 3. Subjecting rats to IPC and IPost similarly improved cardiac function and reduced myocardial infarct size. Interestingly, IPost, but not IPC, significantly increased CYP2J3 mRNA (1.75 ± 0.22 vs 1.0; P < 0.05) and protein (1.62 ± 0.22 vs 1.0; P < 0.05), as well as 11,12-EET synthesis compared to I/R (6.2 ± 0.2 vs 2.9 ± 0.2 ng/mg wet weight, respectively; P < 0.01). Administration of MS-PPOH before ischaemia significantly decreased 11,12-EET synthesis in both IPC and IPost compared with I/R rats (2.1 ± 0.2, 3.2 ± 0.3 and 2.9 ± 0.2 ng/mg wet weight, respectively; P < 0.01), but decreased the cardioprotective effects, as evidenced by cardiac function and myocardial infarct size, of IPost only. 4. These data indicate that endogenous activation of CYP2J3/EET may be an essential trigger leading to the protective effects of IPost, but not IPC, in the rat heart.     

An endogenous protectant effect of cardiac cyclic GMP against reperfusion-induced ventricular fibrillation in the rat heart.

1. After a period of myocardial ischaemia, reperfusion of the myocardium can elicit cardiac arrhythmias. Susceptibility to these arrhythmias declines with time, such that a preceding period of more than approximately 40 min ischaemia is associated with few reperfusion-induced arrhythmias. We have tested the hypothesis that this decline in susceptibility occurs, in part, because of protection by endogenous guanosine 3':5'-cyclic monophosphate (cyclic GMP). 2. Rat isolated hearts were subjected to 60 min left regional ischaemia followed by reperfusion (n = 10 per group). Methylene blue (20 microM), a soluble guanylate cyclase inhibitor, raised the incidence of reperfusion-induced ventricular fibrillation (VF) from 10% in control hearts to 80% (P < 0.05). This effect of methylene blue was abolished by co-perfusion with zaprinast (100 microM), a phosphodiesterase inhibitor which, in the rat heart, is cyclic GMP-specific (specific for the type-V phosphodiesterase isozyme). 3. Methylene blue reduced cyclic GMP levels in the ischaemic, non-ischaemic and reperfused myocardium (P < 0.05) to 50 +/- 10, 52 +/- 12 and 70 +/- 7 fmol mg-1 tissue wet weight, respectively from control values of 143 +/- 38, 147 +/- 43 and 156 +/- 15 fmol mg-1. Co-perfusion with zaprinast prevented this effect, and cyclic GMP levels were actually elevated (P < 0.05) to 366 +/- 102, 396 +/- 130 and 293 +/- 22 fmol mg-1 in ischaemic, non-ischaemic and reperfused myocardium, respectively. Zaprinast by itself also elevated cyclic GMP content. Cyclic AMP levels were not affected by zaprinast or methylene blue. 4. In conclusion, when endogenous cardiac cyclic GMP synthesis is reduced, susceptibility to reperfusion-induced VF after sustained ischaemia is substantially increased. The effect is prevented by inhibiting cyclic GMP degradation. Therefore cyclic GMP appears to be an endogenous intracellular cardioprotectant, and its actions may account for the low susceptibility to VF normally encountered in hearts reperfused after sustained ischaemia.     

Hyperoxia confers myocardial protection in mechanically ventilated rats through the generation of free radicals and opening of mitochondrial ATP-sensitive potassium channels

1. One hour exposure to hyperoxia has been shown previously to limit a subsequent ischaemia-reperfusion injury in spontaneously breathing rats. We tested the cardioprotective effect of a shorter period of hyperoxia during mechanical ventilation and the possible contribution of reactive oxygen species (ROS) and mitochondrial ATP-sensitive potassium (mitoK(ATP)) channels. 2. Mechanically ventilated rats were exposed to normoxia (Fi O2 = 0.3) or hyperoxia (Fi O2 = 1.0) for 30 min and pH, P CO2, PO2, heart rate, airway and blood pressure were measured at baseline and after 30 min mechanical ventilation. Isolated hearts were subsequently subjected to 30 min ischaemia and 120 min reperfusion. Infarct size and left ventricular end-diastolic pressure (LVEDP), developed pressure (LVDP) and coronary flow (CF) were measured. In order to investigate the role of ROS and KATP channels within the mechanism leading to cardioprotection, the free radical scavenger N-acetylcysteine (NAC; 150 mg/kg) was infused in mechanically ventilated rats and the KATP channel blockers glibenclamide (200 mmol/L) or 5-hydroxydecanoate (10 mmol/L) were infused in isolated hearts immediately before ischaemia. 3. No differences were detected in P CO2, pH, heart rate, airway and blood pressure between the groups. However, the PO2 in hyperoxic groups was significantly higher compared with that in normoxic groups (P < 0.01). After 30 min ischaemia, we found that hyperoxic preconditioning significantly improved CF (P < 0.01), LVDP (P < 0.01) and LVEDP (P < 0.01) and reduced the extent of infarct size in the reperfused heart compared with the normoxic group (P < 0.01). When rats were pretreated either with NAC before hyperoxic ventilation or with K(ATP) channel blockers before ischaemia, myocardial protection was abolished. 4. Hyperoxic mechanical ventilation, prior to ischaemia, reduces myocardial reperfusion injury. This is likely to occur through the induction of oxidative stress, which leads to myocyte mitoKATP channel opening.