Density of L-type calcium channels in ischaemically preconditioned porcine heart regions

Ischaemic preconditioning by brief ischaemic episodes could be explained by reduced cellular calcium ion (Ca²⁺) influx, reduced cytosolic Ca²⁺ overload and delayed cell-injury during subsequent long-lasting ischaemia. L-type calcium channels (LCC) regulate sarcolemmal Ca²⁺ influx in myocardial cells. The aim of this study was to investigate if preconditioning was associated with reduced density or altered state of LCC in the preconditioned region of the heart. To test this we compared the density and the dissociation constant of (4-)-[³H]isradipine binding to LCC in membranes from preconditioned and control regions of porcine hearts. Eight porcine hearts were regionally preconditioned by two 10-min occlusions of the mid left anterior descending artery, and each occlusion was followed by 30 min of reperfusion. Biopsies were taken from the preconditioned regions and control regions supplied by the circumflex artery at the end of the last reperfusion, and (+)-[³H]isradipine binding to membranes made from the biopsies was measured. The differences in density and dissociation constant of (4-)-[³H]isradipine binding to LCC in membranes from preconditioned and control regions were not significant. In conclusion, the proposed effect of ischaemic preconditioning to reduce Ca²⁺ influx, does not involve local changes in density or state of LCC that could be detected by (+)-[³H]isradipine binding.     

Involvement of endogenous adenosine in ischaemic preconditioning in swine

Adenosine release and the subsequent activation of adenosine receptors are involved in ischaemic preconditioning in dogs and rabbits. In the present study, we investigated whether adenosine also mediates ischaemic preconditioning in swine. Swine were used since, due to the lack of an innate collateral circulation, infarct development in this species most closely resembles that observed in humans. In 36 enflurane-anaesthetized swine the impact of increased adenosine breakdown with exogenous porcine adenosine deaminase (5 IU/ml blood/min) on global and regional myocardial function (sonomicrometry), subendocardial blood flow (ENDO, microspheres) and infarct size (IS, triphenyl tetrazolium chloride staining following 90 min ischaemia and 120 min reperfusion) were analysed. Low-flow ischaemia for 90 min at an ENDO of 0.09±0.04 (mean±SD) ml/min/g caused an IS of 13.2±9.7% (n=8) of the area at risk. Ischaemic preconditioning by a cycle of 10 min low-flow ischaemia followed by 15 min reperfusion prior to the 90-min ischaemic period (ENDO=0.06±0.03 ml/min/g) reduced IS to 2.6±3.0% (n=11, P<0.05). The interstitial adenosine concentration (microdialysis) increased from 1.60±0.87 nmol/ml to above 10 M during ischaemia; with intracoronary adenosine deaminase, the interstitial adenosine concentration fell from 1.65±0.23 to 0.12±0.07 nmol/ml and did not increase during ischaemia. Adenosine deaminase per se did not alter IS after 90 min ischaemia (n=7, ENDO=0.08±0.04 ml/min/g, IS=12.1±6.9%) but abolished the beneficial effect of ischaemic preconditioning (n=10, ENDO=0.06±0.03 ml/min/g, IS=8.8±5.8%). For any given ENDO, IS was significantly reduced in the ischaemic preconditioned group compared with the other three groups. Global and regional myocardial function were comparable among all groups of swine. We conclude that endogenous adenosine mediates ischaemic preconditioning also in swine.     

Endogenous nitric oxide attenuates hypoxic vasoconstriction of small pulmonary arteries and veins in anaesthetized cats

Ischaemic preconditioning has cardioprotective effects. Reactive oxygen species may be possible mediators. The present study investigated whether low doses of exogenous hydrogen peroxide could mimic preconditioning in isolated, Langendorff-perfused rat hearts. Hearts were subjected to two episodes of 3 min global ischaemia and 5 min reperfusion (n = 17), or were given 10 (n=15), 20 (n=10), 30 (n=20), 40 (n=18), 80 (n=17) or 160 μM (n=10) hydrogen peroxide for 10 min, followed by 10 min recovery, before 25 min global ischaemia and 60 min reperfusion, and compared with ischaemic controls of matching perfusion time (n=17 and n=23). Cardiac performance was assessed by heart rate, left ventricular systolic, end-diastolic and developed pressures, and coronary flow. Severe reperfusion arrhythmias occurred frequently in control hearts, and was attenuated by ischaemic preconditioning. All hearts pretreated with 160 μM hydrogen peroxide had severe arrhythmias throughout reperfusion, while these were not seen in any heart perfused with 20 μM hydrogen peroxide (P< 0.01 compared to controls). Ischaemia and reperfusion induced a minor decrease in heart rate, left ventricular systolic and developed pressures, and increased end-diastolic pressure. Ischaemic preconditioning attenuated the decrease of heart rate and the increase of end-diastolic pressure, and increased coronary flow, while hydrogen peroxide did not significantly attenuate these changes. In conclusion, a low dose of exogenous hydrogen peroxide before global ischaemia inhibited severe reperfusion arrhythmias, but had no other protective effects. The present work does not suggest that reactive oxygen species are important mediators of the preconditioning effects on stunning and arrhythmias in the rat heart.     

Preconditioning does not attenuate cardiac dysfunction after global ischaemia in the guinea-pig

Ischaemic preconditioning reduces infarct size, but the effects on cardiac function after global ischaemia are more controversial. Additionally, species differences may exist. The present study investigates the effects of preconditioning on cardiac performance in the globally ischaemic, Langendorff-perfused guinea-pig heart. Hearts were stabilized for 25 min, and divided into the following groups: (1) (n = 8) control perfusion for 16 min before 30-min global ischaemia and 30-min reperfusion, (2) (n = 7) two episodes of 3-min ischaemia and 5-min reperfusion before global ischaemia, (3) (n = 7) 5-min ischaemia and 10-min reperfusion before ischaemia, (4) (n = 8) control perfusion before 40-min ischaemia and 30-min reperfusion, (5) (n = 8) Preconditioning as group 2 before ischaemia as group 4, (6) (n = 9) Control perfusion before 50-min ischaemia and 30-min reperfusion, (7) (n = 10) Preconditioning as group 2 before ischaemia as group 6. A dose-dependent reduction of left ventricular systolic pressure, and increase of end-diastolic pressure was observed during reperfusion after 30-, 40- and 50-min ischaemia. Preconditioning did not influence these changes, nor did it attenuate the incidence of severe reperfusion arrhythmias or reduction of coronary flow. In conclusion, ischaemic preconditioning does not improve cardiac function during reperfusion of the globally ischaemic, isolated guinea-pig heart.     

Renal denervation abolishes the protective effects of ischaemic preconditioning on function and haemodynamics in ischaemia‐reperfused rat kidneys

Studies were conducted to investigate the role of renal sympathetic nerves in the process of acquiring ischaemic tolerance in ischaemic preconditioned ischaemia‐reperfused rat kidneys. Two periods of 3‐min occlusion of bilateral renal arteries was performed prior to 30‐min bilateral ischaemia and 90‐min reperfusion in acute renal denervated or innervated kidneys. The glomerular filtration rate (GFR), fractional excretion of sodium (FENa) and lithium (FELi), and renal blood flow (RBF) were assessed in reperfused kidneys. Ischaemic preconditioning significantly improved values for all these parameters as compared with no treated ischaemia‐reperfused kidneys. Denervation caused slight increase in GFR, diuresis and natriuresis without improving RBF after reperfusion. However, protecting effects of ischaemic preconditioning on renal function were disappeared in denervated kidneys, while in innevated kidneys the effects of ischaemic preconditioning were maintained. These results clearly showed that ischaemic preconditioning pre‐treatment protects kidneys against ischaemia–reperfusion injury, and the effects are, at least in part, mediated by sympathetic nerves, as the protective effects were abolished by denervation.     

Influence of coronary venous retroinfusion and vasodilatation on regional myocardial blood flow measurement with microspheres. An analysis of ‘microsphere loss’ from ischaemic and reperfused porcine hearts

The influence of coronary venous retroinfusion and a vasoselective calcium antagonist felodipine on the microsphere loss in a porcine model of myocardial ischaemia and reperfusion was studied. Sixteen open-chest pigs underwent 45 min of myocardial ischaemia induced by occlusion of the left anterior descending coronary artery followed by 4 h of reperfusion. Either felodipine (felo-retro group, 7 nmol kg^-1, n= 6) or the corresponding amount of vehicle (vehicle-retro group, n= 5) was infused retrogradely into the coronary veins over 30 min, starting 5 min before reperfusion. In a third group, the same amount of felodipine was administered intravenously (felo-iv group, n= 5). Myocardial regional blood flow was measured with radiolabelled microspheres (ø= 15 μm) injected before ischaemia to investigate a possible loss during ischaemia. In the felo-retro group, the apparent blood flow in the ischaemic areas, expressed as a percentage of the corresponding values in the non-ischaemic areas (%-flow), were 73±15, 73±11 and 75±19 in the subendocardial, midmyocardial and subepicardial layers, respectively. The corresponding percentage flows were 64±11, 70±11 and 62±9 in the vehicle-retro group and 75±18, 77±15 and 76±11 in the felo-iv group. The differences between the groups were not satistically significant. It is concluded that in this open-chest preparation microsphere loss observed in the ischaemic and reperfused myocardium is not increased by coronary venous retroinfusion or by a concomitantly administered vasodilative agent like felodipine.     

Unchanged protein expression of sarcoplasmic reticulum Ca2+-ATPase, phospholamban, and calsequestrin in terminally failing human myocardium

The enhanced diastolic Ca²⁺ levels observed in cardiac myocytes from patients with idiopathic dilated cardiomyopathy (DCM) may be either a consequence of functional impairment of sarcoplasmic reticulum calcium- ATPase (SERCA 2) and its regulator protein phospholamban or due to a reduction in the number of SERCA 2 proteins. As different myocardial membrane preparations may lead to different accumulation of proteins, the present study evaluated two different membrane preparations, in human failing and nonfailing myocardium for comparison of SERCA 2 activity and the protein expression of SERCA 2 and phospholamban. Crude membranes and tissue homo-genates without any centrifugation steps were prepared from human nonfailing hearts (donor hearts, NF, n=18) and terminally failing hearts (heart transplant, DCM, n=18). Calsequestrin protein expression was used as an internal control for overall protein expression. In both crude membranes and homogenates maximal SERCA 2 activity (V _max) was significantly reduced in failing heart preparations (NF crude membranes, 130±8; DCM crude membranes, 102±5 nmol ATP/mg protein per minute). In contrast, the protein expression of SERCA 2 (NF crude membranes, 488±35; DCM crude membranes, 494±42; P=0.92), phospholamban (NF crude membranes, 497±51; DCM crude membranes, 496±45; P=0.98) and calsequestrin (NF crude membranes, 109±06; DCM crude membranes, 107±08; P=0.84) was unchanged in NF and DCM hearts in both preparation methods. This was also the case when the protein expression was normalized to calsequestrin protein levels. Preparation of sarcoplasmic reticulum in crude membranes led to enhanced purification and consequently higher SERCA 2, phospholamban, and calsequestrin protein levels in crude membranes than in the homogenates, which was paralleled by an increase in SERCA 2 enzyme activity. In conclusion, the altered Ca²⁺ handling in DCM may be a consequence of reduced SERCA 2 enzyme activity and not the result of differences in protein expression of the Ca²⁺ regulating proteins SERCA 2, phospholamban, and calsequestrin in human myocardium. The present study emphasizes the importance of different myocardial membrane preparations with respect to quantitative investigations of protein expression and function. Received: 2 September 1997 / Accepted: 2 December 1997     

Peroxisomal fatty acid oxidation capacity is more resistant to ischaemic and reperfusion injury than mitochondrial fatty acid oxidation capacity in feline hearts

We investigated ischaemic and postischaemic mitochondrial and peroxisomal fatty acid oxidation capacity, ATP levels and regional function in 40 anaesthetized open chest cats subjected to 10 or 40 min of regional myocardial ischaemia with or without 3 h of reperfusion (n=10 in each situation). Following 10 min of ischaemia, the mitochondrial fatty acid oxidation capacity measured in tissue extracts from ischaemic tissue (nmol min^-1 mg protein^-1) was reduced in both subepi- and subendocardium, but was normalized in reperfused tissue extracts from both wall layers (0.29±0.03 and 0.30±0.04 vs. 0.57±0.05 and 0.59±0.05, P<0.05). Peroxisomal fatty acid oxidation capacity in tissue extracts was unaffected by ischaemia and reperfusion. ATP levels and regional function measured in the LAD region was partly restored transmurally. After 40 min of LAD occlusion, mitochondrial fatty acid oxidation capacity was reduced, with higher activity in subepi- than in subendocardium (0.27±0.05 vs. 0.19±0.04, P<0.05). Reperfusion did not restore mitochondrial fatty acid oxidation capacity. Peroxisomal fatty acid oxidation capacity was increased in the ischaemic subendocardium compared with levels in non-ischaemic subendocardium (0.53±0.02 vs. 0.45±0.03, P<0.05), with normalization at the end of reperfusion. ATP levels were non-uniformly reduced during ischaemia and not repleted during reperfusion. Regional function recovered in circumferential segments but not in longitudinal segments following 40 min of ischaemia. In conclusion fatty acid oxidation enzymes seem to be more resistant to ischaemia in peroxisomes than in mitochondria. Mitochondrial fatty acid oxidation is fully reversible following shortlasting ischaemia, but remains depressed following prolonged ischaemia and reperfusion.     

Pre‐conditioning activates adenosine utilization in a cost‐effective way during myocardial ischaemia

During pre‐conditioning the interstitial concentration of adenosine, in contrast to lactate, presents a die‐away curve‐pattern for every successive episode of ischaemia. This die‐away pattern might not necessarily be attributed to diminished adenosine production. The present study was undertaken to investigate whether pre‐conditioning alters the metabolic turnover of adenosine as observed by the lactate production during ischaemia. Interstitial levels of metabolites in pre‐conditioned (n=21) and non‐preconditioned (n=21) porcine hearts were monitored with microdialysis probes inserted in both ischaemic and non‐ischaemic tissue in an open chest heart model. Three subgroups perturbated with either plain microdialysis buffer (control), buffer containing adenosine (375 μM ), or buffer containing deoxyadenosine (375 μM ) were studied. All animals were subjected to 90 min of equilibrium microdialysis before 40 min of regional myocardial ischaemia and 120 min of reperfusion. Pre‐conditioning consisted of four repetitive episodes of 10 min of ischaemia and 20 min of reperfusion. Significantly higher levels of inosine and lactate were found in the ischaemic tissue of the pre‐conditioned subgroup receiving adenosine (P < 0.05) compared with the other two subgroups receiving deoxyadenosine and plain buffer, respectively. This difference was only valid for pre‐conditioned ischaemic myocardium, and hence equal amounts of inosine and lactate were produced in the non‐preconditioned ischaemic myocardium regardless of the presence of adenosine or deoxyadenosine. In the non‐ischaemic myocardium baseline levels of metabolites were measured in all subgroups. Pre‐conditioning favoured degradation of exogenous adenosine to inosine successively ending up in enhanced lactate production. This was probably because of the involvement of the hexose monophosphate pathway in the pre‐conditioned ischaemic myocardium. This route may therefore be supplementary in energy metabolism as a metabolic flow can be started by adenosine ending up in lactate without initial adenosine 5′‐triphosphate (ATP) investment. Utilization of adenosine in this way may also explain the successive die‐away pattern of adenosine seen in consecutive pre‐conditioning cycles.     

Effects of sex,gonadectomy, and oestrogen substitution on ischaemic preconditioning and ischaemia–reperfusion injury in mice

Aim: Ischaemic preconditioning (IPC) has been demonstrated to protect heart function and viability, but has been predominantly studied in male animals. Methods: We studied a possible influence of sex and oestrogen for protection in IPC. Infarct size and heart function after 40 min global ischaemia and 60 min reperfusion with or without preceding classic IPC was investigated in Langendorff‐perfused hearts. Hearts were harvested from 10‐week‐old male and female C57BL6 mice with or without gonadectomy 6 weeks earlier, or gonadectomy and substitution with 17β‐oestradiol for 4 weeks (n = 104). Results: Classic IPC reduced depression of left ventricular developed pressure (P < 0.01), attenuated the increase of end‐diastolic pressure (P < 0.01), and reduced infarct size (P < 0.01) in hearts of untreated male mice, but failed to protect untreated females which had improved functional recovery and smaller infarctions than untreated males. After gonadectomy of female mice, developed pressure was reduced (P < 0.01) and infarct size increased (P < 0.01) compared with normal females, with no protection of preconditioning. The changes were not reversed by 17β‐oestradiol substitution. In hearts of gonadectomized males, the post‐ischaemic increase of end‐diastolic pressure was attenuated (P < 0.01), and enhanced after substitution with 17β‐oestradiol (P < 0.01). The preconditioning effect disappeared after gonadectomy and gonadectomy with substitution in male mice. Conclusion: There is a sex difference in evoking preconditioning in male and female mice which is only partially dependent on sex hormones.     

Histamine release and its effects in ischaemia-reperfusion injury of the isolated rat heart

Histamine is released from the heart during ischaemia-reperfusion injury. As histamine has cardiac effects, we investigated the role of histamine in ischaemia-reperfusion injury of isolated rat hearts. A Langendorff-model with 30 min global (37 ^oC) ischaemia followed by 60 min reperfusion was employed. The effects of ischaemia alone (n= 10, group 1.1+n = 10, group 2.1, 2 different series), and ischaemia with H₁- and H₂-receptor blockade with cimetidine (10 μM, n= 10), chlorpheniramine (10 μm, n= 8), terfenadine (10 μM, n= 8), and promethazin (10 μM, n= 9), or both cimetidine and chlorpheniramine (n = 8), were studied. Histamine was measured in the coronary effluent and cardiac tissue of group 1.1. Release of histamine increased from 6.5 ± 1 pmol min^-1 before ischaemia to 19 ± 3 pmol min^-1 at the start of reperfusion. Ischaemia decreased left ventricular developed pressure to 18 ± 11 % (1.1) and 50 ± 11 % (2.1) of initial value (mean ± SEM) at the start of reperfusion. Left ventricular end-diastolic pressure increased from 0 to 79 ± 8 mmHg (1.1) and 39 ± 9 (2.1) mmHg, while left ventricular systolic pressure was unchanged (101 ± 12% in 1.1 and 101 ± 10% in 2.1). Severe arrhythmias were induced in 90 (1.1) and 30 (2.1)% of the hearts, while coronary flow decreased during reperfusion. H₂-blockade did not modify the changes in left ventricular pressures, coronary flow, or heart rate induced by ischaemia. Three different Hj-blockers increased left ventricular systolic pressure, inhibited the decrease of developed pressure, attenuated the increase of end-diastolic pressure, and totally inhibited reperfusion arrhythmias. The effect of both blockers together was similar to that of H₁-blockers alone. Coronary flow was increased during reperfusion in two of the groups with Hj-blocker compared with ischaemic controls. Increased release of histamine from ischaemic-reperfused rat hearts concurred with depression of left ventricular function and arrhythmias during early reperfusion. Cardiac dysfunction during reperfusion was attenuated by three different Hj-receptor blockers.     

Ischaemic preconditioning in the rat brain: a longitudinal magnetic resonance imaging (MRI) study

Ischaemic preconditioning in rats was studied using MRI. Ischaemic preconditioning was induced, using an intraluminal filament method, by 30 min middle cerebral artery occlusion (MCAO), and imaged 24 h later. The secondary insult of 100 min MCAO was induced 3 days following preconditioning and imaged 24 and 72 h later. Twenty-four hours following ischaemic preconditioning most rats showed small sub-cortical hyperintense regions not seen in sham-preconditioned rats. Twenty-four hours and 72 h following the secondary insult preconditioned animals showed significantly smaller lesions (24 h = 112 +/- 31 mm(3), mean +/- standard error; 72 h = 80 +/- 35 mm(3)), which were confined to the striatum, than controls (24 h = 234 +/- 32 mm(3), p = 0.026; 72 h = 275 +/- 37 mm(3), p = 0.003). In addition during lesion maturation from 24 to 72 h post-secondary MCAO, preconditioned rats displayed an average reduction in lesion size as measured by MRI whereas sham-preconditioned rats displayed increases in lesion size; this is the first report of such differential lesion volume evolution in cerebral ischaemic preconditioning. Copyright -Copyright 2001 John Wiley & Sons, Ltd.     

Hyperbaric oxygen and neutrophil accumulation/tissue damage during permanent focal cerebral ischaemia in rats

The use of hyperbaric oxygenation (HBO) for the treatment of severe brain ischaemia remains controversial. The HBO may interfere with destructive neutrophil (PMN) infiltration following ischaemia/reperfusion. The effects of HBO on PMN accumulation and the area of ischaemic tissue damage were investigated in rats having permanent focal ischaemia (4 h). The right middle cerebral arteries of a group of Wistar rats were permanently occluded. The rats were then randomly divided into those (n=7) to be treated with HBO at 2 atm for 230 min and those (n=8) to breathe air at atmospheric pressure for an equivalent period. The HBO had no effect on permanent ischaemia, as there was no significant difference in the area of ischaemic tissue damage between HBO-treated [mean (SD)] [331 (88) mm³] and non-treated animals [322 (111) mm³]. Moreover, the increase in myeloperoxidase [5.4 (4.1) compared to 2.4 (1.2) pg·g^–1 wet weight of brain] was not significantly different. The results indicate that HBO did not reduce tissue damage during 4 h of permanent focal ischaemia. Electronic Publication     

High afterload during 10 min of regional ischaemia affects diastolic creep but not systolic function in reperfused (stunned) myocardium

The effect of afterload during regional ischaemia on myocardial stunning was studied in 15 pentobarbital anaesthetized cats. 10 min occlusion of the left anterior descending artery (LAD) was followed by 60 min of reperfusion. Afterload was decreased by intravenous infusion of nitroglycerine 3–8 μg kg^-1 min^-1 in group I (n=8); left ventricular peak systolic pressure (LVSP) 84±4 mmHg (mean±SEM) during coronary artery occlusion. In group II (n=7) LVSP was increased to 188±10 mmHg by inflating an intraaortic balloon during coronary artery occlusion. Regional function in the LAD perfused region was evaluated by cross-oriented sonomicrometry. Myocardial tissue blood flow was evaluated by radio-labelled microspheres. Afterload alterations did not affect regional systolic shortening (10.8±2.0% vs. 11.0±1.5% in group I and II, respectively, after 60 min of reperfusion). However, increased end-diastolic dimensions (diastolic creep) in both the circumferential and longitudinal segments were markedly more pronounced in the high afterload group (group II). Also important, the markedly increased myocardial tissue blood flow during reperfusion in group II as compared with group I (2.30±0.18 vs.  1.34±0.08 mL min^-1 g^-1 and 2.58±0.23 vs. 1.49±0.07 mL min^-1 g^-1 in subepicardial and subendocardial layers in the LAD perfused region) suggests that increased diastolic creep increased metabolic demands. This study indicates that passive stretching of the ischaemic area during coronary artery occlusion is an important mechanism behind diastolic creep.     

Effects of intermittent lower limb ischaemia on coronary blood flow and coronary resistance in pigs

Aim: Intermittent limb ischaemia prior to cardiac ischaemia is a cardioprotective stimulus. This study was to investigate whether this peripheral stimulus had any effects on basal coronary blood flow and resistance, and to explore its potential mechanisms by studying the effect of femoral nerve transection and Katp blockade by glibenclamide. Methods: Remote ischaemic preconditioning (rIPC) was induced by four 5‐min cycles of lower limb ischaemia. Coronary resistance was measured using standard formulae and coronary blood flow in the left anterior descending artery (LAD) by a flow probe. In experiment 1, coronary ischaemia was induced by inflation of a cuff placed around the mid‐LAD, and inflated until cessation of flow. Left ventricular (LV) function was assessed using dp/dt and Tau at 1 and 30 min of ischaemia. Experiment 1: 20 pigs were randomized to control (n = 6), rIPC (n = 7) or femoral nerve transection + rIPC (n = 7) groups. The femoral nerve was transected before the rIPC protocol. All data were collected at fixed heart rates of 120 bpm. Coronary resistance was decreased and flow was increased significantly by the rIPC stimulus (P = 0.003, P = 0.016, paired t‐test), and these changes were preserved after femoral nerve transection. Experiment 2: 19 pigs were randomized to control (n = 5), rIPC (n = 8) or glibenclamide‐treated rIPC (n = 6) groups. Data were collected at baseline, and during incremental pacing between 120 and 180 bpm. Results: Experiment 1: Coronary resistance was decreased and flow was increased significantly by rIPC stimulus (P = 0.003, P = 0.016, paired t‐test), and these changes were preserved after femoral nerve transaction. rIPC was associated with superior LV function (dp/dt_max) at 30 min, compared with controls and the rIPC + femoral nerve transaction group. Experiment 2: Coronary resistance was significantly lower, and LAD flow was significantly higher in rIPC group (P < 0.0001, P = 0.0008, two‐way anova ). These effects were reversed in the glibenclamide group. Conclusion: The rIPC stimulus leads to reduced coronary resistance and increased flow. This effect, while modified by glibenclamide appears to be a generic effect of remote ischaemia rather than a direct preconditioning effect.     

Expression and activity of the glutamate transporter EAAT2 in cardiac hypertrophy: implications for ischaemia reperfusion injury

The expression and activity of the glutamate transporter, excitatory amino acid transporter 2 (EAAT2), in cardiac hypertrophy were investigated with respect to glutamate’s potential as a cardioprotective agent. Sarcolemmal vesicles (SV) isolated from hypertrophic hearts of male spontaneously hypertensive rats (SHR) or normotrophic hearts from age-matched male Wistar Kyoto rats (WKY) were used to measure the relative level of EAAT2 expression by Western blotting and the initial rate of 0–0.3 mM l-[¹⁴C]glutamate uptake. The effects of 20-min global normothermic ischaemia ±0.5 mM glutamate on cardiac function were measured in isolated working SHR/WKY hearts. In a separate series of hearts, glutamate, lactate and ATP levels were measured. Both the level of EAAT2 expression and the V _max for sodium-dependent l-[¹⁴C]glutamate uptake were significantly greater in SHR SV compared to WKY SV. The reperfusion cardiac output (CO) of SHR hearts was significantly worse than that of the WKY hearts (24.3±2.2 ml/min vs 39.8±3.3 ml/min, n=7/9±SE, p<0.01). The addition of 0.5 mM l-glutamate improved the SHR reperfusion CO to 45.2±5 ml/min, (n=6±SE, p<0.01) but had no effect on WKYs (46.2±3.8 ml/min, n=6±SE). SHR with 0.5 mM l-glutamate had higher glutamate levels at the start of ischaemia, plus higher glutamate and ATP levels at the end of ischaemia compared to any other group. These results suggest that increased glutamate transporter expression and activity in the SHR hearts helped facilitate glutamate entry into the SHR cardiomyocytes leading to improved myocardial metabolism during ischaemia and better functional recovery on reperfusion.     

Various inotropic effects of angiotensin II in post‐ischaemic rat hearts depending on ischaemic time with possible involvement of protein kinase C

Aims: The present study investigated if the inotropic effect of angiotensin II (AngII) is altered during post‐ischaemic reperfusion in hearts subjected to mild and severe ischaemia. The possible involvement of protein kinase C (PKC) in the change in the inotropic effect was also investigated. Methods: Isolated Langendorff‐perfused rat hearts were perfused under constant flow with oxygenated Krebs–Henseleit buffer and paced at 360 beats min^?1. A saline‐filled balloon catheter inserted into the left ventricle was used for measurement of contractile force. In the first series of experiments, hearts were subjected to continuous perfusion, 15‐ or 25‐min global ischaemia followed by 45‐min reperfusion. At the end of reperfusion, 0.1 μmol L^?1 AngII was infused for 5 min. In a second series of experiments, AngII was infused in hearts subjected to 25‐min ischaemia followed by 45‐min reperfusion in the absence or presence of the PKC inhibitor chelerythrine chloride (5 μmol L^?1). Results: The current study demonstrates that AngII exerts a positive inotropic effect in normoxic hearts with an increase of left ventricular developed pressure (LVDP) by 11% (P < 0.05 vs. prior to AngII infusion). In post‐ischaemic hearts subjected to 15‐min ischaemia no effect of AngII was observed. In hearts subjected to 25 min of ischaemia, however, AngII evoked a negative inotropic response with a decrease of LVDP by 18% (P < 0.05 vs. prior to AngII infusion). The negative inotropic effect of AngII was inhibited by the PKC inhibitor chelerythrine chloride. Conclusions: AngII exerts negative inotropic effect in severely injured post‐ischaemic heart, possibly through the PKC pathway.     

Pretreatment with insulin before ischaemia reduces infarct size in Langendorff‐perfused rat hearts

Aim: To compare the possible role of Akt and mammalian target of rapamycin (mTOR) in mediating cardioprotection against ischaemia under three different conditions: (1) During ischaemic preconditioning (IPC), (2) when insulin was given as a pretreatment agent (InsPC) and (3) when insulin was given as a reperfusion cell survival agent (Ins_R). Methods: Isolated perfused rat hearts were subjected to IPC (3 × 5 min) or InsPC (50 mU mL^?1; 3 × 5 min), before 30 min of regional ischaemia followed by 120 min of reperfusion ± 1L‐6‐hydroxymethyl‐chiro‐inositol‐2‐[(R)‐2‐O‐methyl‐3‐O‐octadecylcarbonate] (HIMO) (20 μm ; Akt inhibitor) or rapamycin (1 nm ; mTOR inhibitor). In addition, insulin (3 mU mL^?1) was given at the onset of reperfusion, ±HIMO or rapamycin. Risk zone (R) and infarct size (I) were determined with Evans blue and tetrazolium staining respectively. Western blot analysis was performed on tissue from Langendorff‐perfused rat hearts and cell lysates from cultured HL1 cells. Results: IPC, InsPC and Ins_R treatment resulted in a significant reduction in infarct size compared to controls (all P < 0.05). This protective effect of IPC and insulin was abolished by the inhibitors. However, the putative Akt inhibitor, although capable of abolishing cardioprotection induced by insulin, was not able to inhibit insulin‐induced phosphorylation of Akt in Langendorff‐perfused rat hearts and cultured HL1 cells. The target for this compound therefore remains to be determined. Conclusion: IPC and insulin (either as InsPC or Ins_R) appear to activate mTOR, and this kinase seems to play an essential role in cardioprotection against ischaemia and reperfusion injury as rapamycin blocked the protection.     

The novel non-peptide selective endothelin A receptor antagonist LU 135 252 protects against myocardial ischaemic and reperfusion injury in the pig

The aim of the study was to investigate the efficacy of the novel non-peptide selective endothelin A (ET_A) receptor antagonist LU 135 252 to limit the extent of myocardial ischaemic and reperfusion injury. Administration of LU 135 252 (1 and 5 mg kg^–1 i.v.) to anaesthetised pigs reduced mean arterial pressure (MAP) from 91 ± 4 to 79 ± 3 mmHg (P < 0.05) and 96 ± 3–82 ± 3 mmHg (P < 0.01), respectively. Heart rate, coronary blood flow and coronary vascular resistance were not affected by LU 135 252. The infarct size induced by 45-min ligation of the left anterior descending coronary artery (LAD) followed by 4-h reperfusion in pigs was 81 ± 5% of the area at risk in control animals given vehicle (n = 8). In pigs receiving 1 mg kg^–1 (n = 6) or 5 mg kg^–1 (n = 8) of LU 135 252 i.v. 20 min before ischaemia the infarct size was reduced to 64 ± 3% (P < 0.05) and 35 ± 4% (P < 0.001), respectively, of the area at risk. During the reperfusion period there was a non-significant trend towards a higher coronary blood flow and a lower coronary vascular resistance in the groups given LU 135 252 compared to controls. Myocardial overflow of ET-like immunoreactivity was increased during the reperfusion period but it was not affected by administration of LU 135 252. It is concluded that administration of the selective ET_A receptor antagonist LU 135 252 effectively protects the myocardium from ischaemia/reperfusion injury, indicating that the ET_A receptor subtype is involved in the development of ischaemia/reperfusion injury.     

Gi-mediated muscarinic adenylyl cyclase inhibition in timolol-treated stunned porcine myocardium

The Gi-mediated muscarinic receptor-adenylyl cyclase system was examined in stunned myocardium induced by either three or five brief ischaemic periods after β-adrenoceptor blockade by timolol (0.1 mg kg^-1). The mid-left anterior descending coronary artery was occluded for 2, 10 and 2 min in four pigs, and for 2, 2, 5, 10 and 2 min in four other pigs. All the ischaemic periods were separated by 30 min of reperfusion and the biopsies were obtained 60 min after the last ischaemic period. Segment length function was measured in the ischaemic region and in the control region supplied by the left circumflex artery. In the two groups, the percentage systolic shortening was reduced equally, to 59±9 and 58±10% of control in the region subjected to ischaemia and only minimally in the control region. The biopsies from the stunned region from both groups showed: (1) no change in either the affinity for carbachol or the number of binding sites of the muscarinic receptors; (2) no alterations in messenger RNA encoding for the α subunit-2 of the inhibitory guanine nucleotide binding protein, as demonstrated by northern blot and solution hybridization; (3) no change in membrane-bound inhibitory guanine nucleotide binding protein, as shown by enzyme immunoassay utilizing a specific anti-peptide antibody, and (4) unchanged inhibition of stimulated adenylyl cyclase activity. These results suggest that there is an intact inhibitory guanine nucleotide binding protein-mediated muscarinic receptor adenylyl cyclase system in the stunned porcine myocardium.