The p38 MAPK inhibitor, SB203580, abrogates ischaemic preconditioning in rat heart but timing of administration is critical

There is debate concerning the involvement of p38 mitogen activated protein kinase (MAPK) in the mediation of ischaemic preconditioning. Pharmacological inhibition of p38 MAPK with SB203580 has been reported to block preconditioning in some studies but not in others. We hypothesised that this divergence could be due to differences in the timing of inhibitor administration. Isolated rat hearts were perfused in the Langendorff mode and subjected to 35 min regional ischaemia followed by 120 min reperfusion. Hearts were then double stained with Evans' blue and triphenyltetrazolium chloride to determine risk (R) and infarct zones (I), expressed as I/R% ratios. Preconditioned hearts were subjected to 2 times 5 min global ischaemia with 10 min intervening reperfusion. SB203580 10 μ M was perfused either during the preconditioning protocol (PC+SB-early), just prior to and during the first 15 min of the lethal ischaemia (PC+SB-late) or prior to regional ischaemia in the absence of preconditioning. Ischaemic preconditioning significantly limited infarct size (I/R 38.9 ± 3.0% in control vs 13.4 ± 2.4%, P < 0.01). In the PC+SB-early group, preconditioning was still fully protective (I/R% 14.6 ± 1.0). However, in the PC+SB-late group, SB203580 completely blocked the protection afforded by preconditioning (I/R% 33.6 ± 4.4%, P < 0.01 vs 13.4 ± 2.4% in preconditioned hearts, p < 0.05). SB203580 alone did not affect infarct size when given prior to and during regional ischaemia (I/R 36.2 ± 2.7%). These histological data are corroborated by a significant increase in p38 MAPK activation in the preconditioned hearts during sustained ischaemia in comparison with the controls. In conclusion the activation of p38 MAPK during lethal ischaemia, but not during the ischaemic preconditioning protocol, is essential for the mediation of protection and may resolve some of the earlier controversy surrounding the use of SB203580 in preconditioning studies. Received: 28 June 2000, Returned for revision: 21 July 2000, Revision received: 9 August 2000, Accepted: 13 September 2000     

Ischaemic preconditioning and a mitochondrial KATP channel opener both produce cardioprotection accompanied by F1F0-ATPase inhibition in early ischaemia

Ischaemic preconditioning gives powerful protection against prolonged ischaemia affecting several intracellular regulatory and messenger pathways, although their mutual importance is far from established. Protective, preconditioning-like effects have been reported for K_ATP channel openers, and most of the evidence points to the mitochondrial K_ATP channels. We show here that the K_ATP channel opener diazoxide, which acts selectively on the mitochondrial channel, causes potentiation of ischaemic inhibition of mitochondrial ATP synthase (F₁F₀-ATPase) along with cardioprotection. These effects are comparable with that of ischaemic preconditioning. The administration of diazoxide did not affect the cellular energy state as monitored with ³¹P NMR. The actions of both diazoxide and ischaemic preconditioning were prevented by 5-hydroxydecanoate, a specific inhibitor of the mitochondrial K_ATP channel. Thus mitochondrial K_ATP channel opening and ischaemic preconditioning must share common mechanisms of action involving mitochondrial F₁F₀-ATPase, although involvement of the energy state in protection could not be proved. Received: 2 December 2002, Returned for revision: 9 January 2003, Revision received: 26 March 2003, Accepted: 8 April 2003, Published online: 9 May 2003 RID="*" ID="*"Publishers Note In the “online-first” publication of this article figure 3 and 4 were erroneously swapped. This mistake was corrected in the printed version and in the final online version on the authors request. The publisher apologizes for this oversight. Correspondence to: I. Hassinen     

Mild Hypothermia reduces infarct size in the beating rabbit heart: A practical intervention for acute myocardial infarction?

The present study describes a method for rapidly cooling the whole body via its blood pool and tests whether cooling instituted after ischemia has begun can sill limit infarction. We also evaluated whether the cardiac protection seen with cooling could be added to that from ischemic preconditioning. Recently it was reported that lowering myocardial temperature by only several degrees greatly slows the extent of myocardial infarction in the beating heart experiencing regional ischemia. To further explore the potential of hypothermia for myocardial protection, rabbits underwent either a 30-, 45- or 60-min coronary artery occlusion and 3-h reperfusion. Blood from a carotid artery was allowed to circulate through a heat exchanger immersed in ice water and return to a jugular vein until the blood temperature in the left atrium reached the target temperature of 35 or 32°C. Furthermore, to elucidate the mechanism of hypothermia's protection, we also examined its effect on isolated cardiomyocytes. Rewarming began upon reperfusion in all protocols. Cooling to 32°C before a 30-min ischemia reduced infarct size from 37.3±2.5% (n=6) of the risk zone in normothermic controls to 3.6±0.3% (n=6). When cooling was begun 10 or 20 min after the onset of ischemia infarct size was still significantly smaller [8.1±1.2% and 22.8±1.8%, respectively (n=6 in each group)]. Less but significant protection was also seen with cooling to 35°:C. Cooling caused only mild bradycardia and hypotension and no apparent arrhythmias. Forty-five min of regional ischemia caused 50.7±3.3% (n=6) of risk zone to infarct in untreated hearts. Preconditioning with 5-min ischemia/10-min reperfusion reduced infarct size to 27.5±2.5% (n=6). Cooling to 32°C starting 20 min after the onset of ischemia protected the heart (28.7±2.6% infarction, n=8), and this protection could be added to the effect from ischemic preconditioning delayed the progressive increase in osmotic fragility that occurs during simulated ischemia in an additive way, but only hypothermia delayed the appearance of contracture suggesting that different mechanisms are involved. Hence blood pool cooling was easily induced and well tolerated and protected the beating heart against infarction even when hypothermia was started after the onset of coronary occlusion. We conclude that hypothermia might be a simple and useful therapy for patients presenting with acute myocardial infarction. Received: 5 January 1998, Returned for 1. revision: 3 February 1998, 1. Revision received: 24 February 1998, Returned for 2. revision: 1 April 1998, 2. Revision received: 7 April 1998, Accepted: 14 July 1998     

Limitation of infarct size and attenuation of myeloperoxidase activity by an endothelin A receptor antagonist following ischaemia and reperfusion

It has previously been shown that endothelin (ET) receptor antagonists limit myocardial ischaemia/reperfusion (I/R) injury. The mechanism behind this effect is still unclear. The aim of this study was to elucidate the possible relationship between cardioprotection by an ET_A receptor antagonist and inhibition of neutrophil accumulation or activation in the myocardium determined as myeloperoxidase (MPO) activity during I/R. Anaesthetised pigs were subjected to 45 min ischaemia by ligation of the left anterior descending coronary artery (LAD) followed by 4 h of reperfusion. Infiltration of MPO-containing cells, presumably neutrophils, into the ischaemic area was confirmed with an immunohistochemical technique using antibodies against porcine MPO. Vehicle (n = 7) or the selective ETA receptor antagonist LU 135252 (LU; n = 7) were given into the LAD during the last 10 min of ischaemia and the first 5 min of reperfusion. There were no significant differences in LAD flow, mean arterial pressure, heart rate, or rate pressure product between the groups during I/R. The area at risk was similar in the two groups. LU reduced the final infarct size to 40 ± 6 % of the area at risk compared to 80 ± 6 % in the vehicle group (P < 0.001). Endothelin-like immunoreactivity increased 2-fold in the ischaemic area in the vehicle group (P < 0.01), but not in the group given LU. MPO activity was higher (2.5x) in the ischaemic than in the non-ischaemic myocardium of the vehicle group. The MPO activity in the ischaemic myocardium was significantly lower in the group given LU (7.0 ± 1.2 units g⁻¹) than in the vehicle group (14.2 ± 1.9 units g⁻¹; P < 0.01). There was a significant correlation between the infarct size and MPO activity (P < 0.01, r = 0.68). In conclusion, local administration of the selective ET_A receptor antagonist LU during the last period of ischaemia and early reperfusion reduces the extent of myocardial necrosis and MPO activity. This suggests that LU may exert its cardioprotective effect by inhibiting neutrophil-mediated injury. Received: 20 November 2000, Returned for revision: 11 December 2000, Revision received: 9 February 2001, Accepted: 13 February 2001     

The influence of maturation and gender on the anti-arrhythnmic effect of ischaemic preconditioning in rats

The aim of this study was to investigate the influence of maturation and gender on the anti-arrhythmic effect of myocardial ischaemic preconditioning in rats. Coronary artery occlusion was carried out in either rats anaesthetised with sodium pentobarbitone or in rat isolated hearts. Cardiac arrhythmias occurring in the 30 min post-occlusion period were assessed. In anaesthetised 3 month (m) old male rats ischaemic preconditioning, with a 3 min temporary coronary artery occlusion, significantly reduced the total number of ventricular ectopic beats (VEBs) from 2074±206 to 490±139 and the incidence of ventricular fibrillation (VF) from 40 to 0% during a subsequent 30 min occlusion (P<0.05). In middle-aged male rats (16 m) the anti-arrhythmic effect of preconditioning was unaltered (VEBs were reduced from 1958±121 to 245±66 and VF from 70 to 0%). In 3 m old anaesthetised female rats the effect of ischaemic preconditioning was also evident (VEBs reduced from 961±170 to 154±48; P<0.05). In non-preconditioned age-matched female animals the total number of VEBs (961±170), VF (0%) and mortality (0%) were significantly (P<0.05) lower than in respective male animals. In female rats, attenuation of ischaemia-induced arrhythmic severity was most pronounced in the oestrus state. In hearts isolated from weight-matched male and female rats the incidence of ventricular tachycardia (81 vs 25%) and the total number of VEBs (351±73 vs 81±50) were significantly (P<0.05) different. It is concluded that in rats neither maturation nor gender influence the anti-arrhythmic effect of ischaemic preconditioning. However, female rats exhibit a lower level of arrhythmic activity during sustained coronary artery occlusion than male rats both in vivo and in vitro. Received: 8 April 1998, Returned for revision: 2 June 1998, Revision received: 9 July 1998, Accepted: 21 July 1998     

Ischaemic preconditioning protects against myocardial dysfunction caused by ischaemia in isolated hypertrophied rat hearts

Although ischaemic preconditioning (PC) has been shown to protect normal hearts from a subsequent ischaemic insults, its protective effect on the hypertrophied myocardium has not been widely studied. This study was designed to investigate whether ischaemic preconditioning protects hearts with hypertrophy (HYP). Cardiac HYP was produced in rats by suprarenal abdominal aortic constriction of 5 weeks' duration, and was defined as left ventricular weight: body weight [LVW: BW (mg/g)] ratio over 3.0. Isolated rat hearts were perfused with a modified Krebs-Henseleit buffer at 37°C in a Langendorff preparation. Hearts from sham-operated animals (NORM) and those with HYP underwent a PC protocol consisting of 3 min of global zero flow ischaemia, 5 min of reperfusion followed by 5 min of ischaemia and 5 min of reperfusion. This was followed by 20 min ischaemia and 45 min reperfusion. Control hearts in the HYP and NORM groups were not subjected to the PC protocol. There were, thus, four experimental groups: NORM control (n=9), NORM, PC (n=9), HYP control (n=9), HYP, PC (n=11). The recovery of function after ischaemia was evaluated by recovery of left ventricular developed pressure (LVDP) expressed as % of the initial value (LVDP%). The LVW: BW ratio for the HYP groups was 3.4 (SEM 0.08). LVDP% was higher (p<0.01) in preconditioned groups as compared with controls. In NORM control recovery was 49.3 (6.1), NORM, PC 76.5 (3.4), HYP control 39.8 (4.6) HYP, PC 70.1 (4.1). These data indicate that the ability of preconditioning to protect against ischanemic ventricular dysfunction is preserved in this model of cardiac hypertrophy.     

Opposing effects on infarction of delta and kappa opioid receptor activation in the isolated rat heart: implications for ischemic preconditioning

δ-Opioid receptors are known to participate in the protection found following ischemic preconditioning (IPC), but the role of κ-receptors in IPC is currently controversial. Langendorff-perfused rat hearts received 35 min regional ischemia and 2 h reperfusion. PC (2 cycles 5 min global ischemia) substantially reduced infarct size. Pharmacological PC with the δ-agonist DADLE (10 nmol/L) had similar protective effects. However, higher dose DADLE (1 μmol/L) had a less beneficial effect, and in conjunction with the δ-antagonist naltrindole unexpectedly increased infarct size (61.5 ± 2.0%, p < 0.05 v 45.9 ± 2.3% in controls) sugggesting a non-δ effect. The universal κ-opioid agonist bremazocine (30 nmol/L) increased infarct size (61.3 ± 1.6%, p < 0.05 v controls), an effect abrogated by the selective κ₁-antagonist nor-binaltorphimine (BNI). Since opiates are known to have anti-adrenergic effects, which hypothetically may help to mediate IPC, cyclic AMP levels were measured in DADLE and in bremazocine-treated hearts. Decreased levels of cyclic AMP at the start of the regional ischemic period were found in low dose DADLE hearts (0.485 ± 0/020, n = 8, vs controls, 0.654 ± 0.025 nmol/g wet weight, p < 0.001), but not in high dose DADLE nor in bremazocine treated hearts. Thus, in the isolated rat heart κ₁-opioid receptor activation exacerbates infarct size through an as yet unknown mechanism, suggesting that there could be an “anti-preconditioned state”. In contrast, δ-activity mediates protection which may be associated with a reduction of tissue cyclic AMP levels. Received: 16 November 1999, Accepted: 7 December 1999     

Ischemic preconditioning reduces infarct size in swine myocardium

We evaluated the hypothesis that stunning swine myocardium with brief ischemia reduces oxygen demand in the stunned region and increases tolerance of myocardium to longer periods of ischemia. Wall function was quantified with ultrasonic crystals aligned to measure wall thickening, and stunning was achieved with two cycles of left anterior descending coronary artery (LAD) occlusion (10 minutes) and reperfusion (30 minutes), after which the LAD was occluded for 60 minutes and reperfused for 90 minutes. Infarct size (as a percent of risk region) was then determined by incubating myocardium with para-nitro blue tetrazolium. Regional oxygen demand was measured as myocardial oxygen consumption before the 60-minute LAD occlusion in the stunned region; tracer microspheres were used to determine blood flow, and blood from the anterior interventricular vein and left atrium was used to calculate oxygen saturations. After the second reperfusion period, wall thickening in the stunned region was reduced to 1.4 +/- 2.4% compared with 36.7 +/- 2.5% (mean +/- SEM) before ischemia (p less than 0.001). Regional myocardial oxygen consumption after stunning (3.1 +/- 0.7 ml O2/min/100 g) was no different from regional myocardial oxygen consumption before stunning (3.7 +/- 0.6 ml O2/min/100 g). In the nine pigs "preconditioned" by stunning, infarct size was 10.4 +/- 6.3% of the risk region compared with 48.0 +/- 12.7% in the six control pigs subjected to 60 minutes of ischemia without prior stunning (p less than 0.005). The risk regions were similar (14.4 +/- 1.5% vs. 14.6 +/- 1.9% of the left ventricle, preconditioned vs. control pigs, respectively). We conclude that stunning swine myocardium with two cycles of a 10-minute LAD occlusion followed by reperfusion increases ischemic tolerance but that changes in regional demand in stunned myocardium do not predict the marked reduction in infarct size that follows a subsequent 60-minute period of ischemia.     

Reduction of infarct size in vivo with ischemic preconditioning: Mathematical evidence for protection via non-ischemic tissue

Summary We constructed a mathematical model of ischemic preconditioning based on experimental data obtained from rat hearts. In this animal model of low collateral blood flow, we found that infarct size in preconditioned hearts, expressed as a percentage of area at risk, increased as the size of the area at risk increased (r=0.76, p=0.0007). In contrast, infarct size in control hearts appeared independent of changes in area at risk. Similarly, the lateral distance between the edge of the area at risk and the edge of the area of necrosis did not vary with risk region in control hearts, but in preconditioned hearts, lateral distance decreased as the size of the area at risk increased (r=–0.67, p=0.0046). We used these findings to develop a simple model which provided mathematical relationships between lateral distance and area at risk and between infarct size and area at risk for both control and preconditioned hearts that were consistent with the experimental data. These relationships led us to propose that in preconditioned hearts 1) a protective substance may be produced or activated throughout the heart, and 2) that the protective substance may be transported by diffusion. If we assumed uniform production of protective substance in an amount proportional to the size of the ischemic and non-ischemic areas, we were able to derive, using a simple diffusion model, relationships between the above variables that were consistent with our mathematical model and with the experimental data. Although our model does not identify the protective substance, its implications provide ideas for additional crucial experiments that may enhance our understanding of ischemic preconditioning.List of abbreviations used A_N area of necrosis - A_N/A_R area of necrosis expressed as a fraction of the area at risk - A_R area at risk - C concentration of protective substance; C₁ inside the area at risk, C₂ outside the area at risk - d diffusion distance - LV left ventricle - m amount of protective substance supplied to the area at risk - s lateral distance between the edge of the infarct and the edge of the area at risk - t thickness of the area at risk - w width of the area at risk     

Nifedipine limits infarct size via NO-dependent mechanisms in dogs

Objectives Amlodipine increases NO levels in coronary vessels and aorta via bradykinin-dependent mechanisms in vitro. We have previously reported that nifedipine increases cardiac NO levels in the ischemic canine hearts, suggesting that nifedipine may also have protective effects against ischemia and reperfusion injury, because the enhancement of NO production limits infarct size. We tested whether nifedipine limits infarct size via NO-dependent mechanisms. Methods In open chest dogs, the left anterior descending coronary artery was perfused with blood through a bypass tube and occluded for 90 min followed by 6 hours of reperfusion. Infarct size was assessed at 6 hours of reperfusion. Nifedipine of 3 or 6 μg/kg/min was infused into the bypass tube between 10 min prior to the onset of ischemia and 60 min of reperfusion. Results Neither systemic blood pressure nor heart rate changed during infusion of nifedipine. Infarct size was reduced by the administration of nifedipine (3 or 6 μg/kg/min) compared with the untreated condition (25.6 plusmn; 2.6 and 19.1 ± 3.5 vs. 43.4 ± 5.6 %, respectively), which was completely blunted by L-NAME (45.0 ± 3.6 and 45.4 ± 4.2 vs. 47.9 ± 3.9 % in the nifedipine (3 or 6 μg/kg/min) with L-NAME groups vs. the L-NAME group). Myeloperoxidase activity of the myocardium increased after 6 hours of reperfusion, which was attenuated by nifedipine. The limitation of infarct size and the attenuation in myeloperoxidase activity were completely blunted by L-NAME. There were no significant differences in collateral blood flow at 45 min of ischemia between each group. Conclusions We conclude that the Ca channel blocker, nifedipine, limits infarct size via NO-dependent mechanisms. Received: 21 September 2000, Returned for 1. revision: 9 October 2000, 1. Revision received: 17 January 2001, Returned for 2. revision: 5 February 2001, 2. Revision received: 13 February 2001, Accepted: 14 February 2001     

Reperfusion arrhythmias in the murine heart: their characteristics and alteration after ischemic preconditioning

In this study, we examined the features of reperfusion arrhythmias and the effect of preconditioning (PC) in the mouse for future application of genetically engineered mice to study mechanisms of this type of arrhythmia. Under pentobarbital anesthesia, reperfusion arrhythmias were induced by temporary occlusion of the left anterior descending coronary artery was occluded for periods ranging from 2 to 15 min and then reperfused. In the second protocol, hearts were preconditioned with 2- or 3-min ischemia and 5-min reperfusion prior to the 5 min of coronary occlusion. An electrocardiogram was recorded throughout the experiment, and arrhythmias were diagnosed according to the Lambeth Convention criteria. The incidences of reperfusion-induced ventricular tachycardia (VT) in hearts that received 2, 3, 5, 10 and 15-min ischemia (n = 10∼14) were 0, 9, 73, 55 and 30%, respectively. Ventricular fibrillation (VF) was not observed upon reperfusion regardless of the ischemia duration. PC with 2-min ischemia and with 3-min ischemia (n = 10 for each PC) reduced the incidences of reperfusion VT after 5-min ischemia to 40% and 10%, respectively. However, in mice that developed reperfusion VT, the VT duration was similar to that in non-preconditioned controls, ranging from 1 to 16 s. These results suggest that the relationship between ischemia duration and incidence of VT upon subsequent reperfusion is “bell shaped ” and that PC has anti-arrhythmic effects in the mouse, as it does in anesthetized rat hearts. However, there appear to be differences in the incidence of reperfusion-induced VF and the duration of reperfusion VT between these species. Thus, the present murine preparation, appears to be a useful model for studying the mechanism of reperfusion VT and PC, though it does not share all of the features of reperfusion arrhythmias with the anesthetized rat preparation. Received: 11 February 1999, Returned for revision: 9 March 1999, Revision received: 17 May 1999, Accepted: 8 June 1999     

Aminophylline inhibits adaptation to ischaemia during angioplasty Role of adenosine in ischaemic preconditioning

The ability of brief periods of ischaemia to protect the heartfrom subsequent ischaemia has been termed ‘ischaemic preconditioning’.In order to assess the role of adenosine receptor stimulationin this phenomenon we studied the ischaemic preconditioningeffect during angioplasty in 10 control patients and in 10 patientspre-treated with 5 mg kg^–1 aminophylline, an adenosinereceptor antagonist. The ischaemic response was assessed by analysis of the intracoronaryelectrocardiogram every 10 s during three consecutive inflationsof 90 s with a reperfusion time of 180 s. The severity of transmurallocal ischaemia was expressed as the magnitude of the ST segmentshift in relation to the time during each inflation. The controlpatients showed an improved tolerance to myocardial ischaemia:ST segment shift decreased from 1·42±0·49mV at the end of the first inflation to 1·03±0·44mV at the end of the third inflation (P<0·001). However,in patients pre-treated with aminophylline, the ischaemic responsewas not significantly different during three inflations. CONCLUSION: Aminophylline inhibits ischaemic preconditioning, as assessedby analysis of the intracoronary ST segment changes during angioplasty.This suggests that ischaemic preconditioning is mediated byadenosine receptor stimulation in humans.     

5-HD abolishes ischemic preconditioning independently of monophasic action potential duration in the heart

Objective: Blocking of the K_ATP channel with either glibenclamide or 5-hydroxydecanoate (5-HD) has been shown to abolish the infarct reducing effect of ischemic preconditioning (IPC) in hearts from several species, but the results in rat and rabbit have been equivocal. In this study we investigated if 5-HD could abolish IPC in rat and rabbit and further if IPC or IPV + 5-HD were affecting action potential duration in the rabbit heart. Methods: The rat hearts were isolated and retrogradely perfused on a Langendorff perfusion apparatus with Krebs-Henseleit buffer. The rabbit experiments were performed in an in situ model. Rat and rabbit hearts were subjected to 30 min regional ischemia by ligating a coronary artery followed by 120 min (rat) or 150 min (rabbit) of reperfusion. The preconditioning protocol was one or three cycles of 5 min ischemia plus 5 min reperfusion in the rat and one cycle of 5 min ischemia plus 10 min reperfusion in the rabbit. In the rat 5-HD was added to the reservoir before ischemic preconditioning in different concentrations, and in the rabbit 5-HD was given as a bolus 5 mg/kg intraventricularly 2 min before the preconditioning ischemia. In the rabbit epicardial monophasic action potential duration at 50% repolarization (MAPD₅₀) was measured at 1, 2 and 5 min in each of the ischemic periods using a contact pressure electrode. Infarcts were measured with tetrazolium staining and risk zone volumes with fluorescent microspheres. Results: All data are presented as infarct size in % of risk zone volume (mean ± SEM). In the rat 200 μM of 5-HD abolished the protective effect of one cycle of IPC (28.6 ± 4.7 versus 8.4 ± 0.8) and 500M of 5-HD abolished three cycles of IPC (50.7 ± 7.8 versus 8.4 ± 2.0). Control was 40.9 ± 2.8. In the rabbit 5-HD abolished IPC (41.2 ± 7.2 versus 8.1 ± 3.2). Control was 53.5 ± 12.4. MAPD₅₀ were significantly more shortened compared to control at 1 and 2 min into the 30 min ischemia for the IPC and IPC+5-HD. Conclusions: We conclude that 5-HD abolishes ischemic preconditioning when given before the preconditioning ischemia in both rat and rabbit but does not abolish into ischemia induced shortening of the action potential duration in the rabbit; thus, a role for the mitochondrial K_ATP channel and not the sarcolemmal K_ATP channel in the protective mechanism behind IPC is probable. Received: 15 July 1999, Returned for 1. revision: 17 August 1999, 1. Revision received: 13 September 1999, Returned for 2. revision: 12 October 1999, 2. Revision received: 3 November 1999, Accepted: 17 November 1999     

Solute exchange in the rabbit myocardium: Ischaemia, reflow, and myocardial necrosis

Objective: Coronary occlusion in the rabbit reduces the delivery of particulate tracers to close to zero, but exchange of diffusible solutes, derived from non-arterial sources, continues at a significant level. We investigated the relationships between the exchange of diffusible solutes during coronary occlusion and the extent of myocardial necrosis and between duration of ischaemia and the extent of recovery of solute exchange during reflow. Methods: In an anaesthetised rabbit model of regional ischaemia and reflow, solute exchange is measured using the voltammetric hydrogen clearance technique. The area at risk and infarct size are determined ex vivo with monastral blue and nitroblue tetrazolium staining, respectively. Three groups are studied: control perfusion for 130 minutes (group A); 30 minutes coronary ligation followed by 90 minutes reflow (group B) and 40 minutes coronary ligation followed by 90 minutes reflow (group C). Results: There was no significant difference in area at risk between the groups B and C (50±2% and 45±5%; p=ns) or in infarct size when expressed relative to the area at risk (42±7% and 55±5%; p=ns). During coronary ligation hydrogen clearance remained constant at 22±4% of the control region in group B and 32±4% in group C, at the same time period in group A it was 87±2% (ANOVA=p<0.05, with a significant non-linear trend). Although the duration of ischaemia and the level of solute exchange during ischaemia did not correlate individually with the extent of myocardial necrosis, together they showed a significant correlation (ANOVA; p<0.05). Following coronary occlusion, hydrogen clearance recovered to 72±9% after 30 minutes ischaemia but only to 57±5% following 40 minutes ischaemia and was 95±2% in the control group (ANOVA between the three groups p<0.05 with a significant linear trend). Myocardial hydration fell in the apical region following coronary ligation by 27±5% in group B and by 25±5% in group C, and rose on reperfusion but only to 80±3% in group B and 83±3% in group C of their preligation values. Conclusion: In collateral deficient myocardium, the extent of myocardial necrosis is dependent on the level of solute exchange occurring during ischaemia. The level of solute exchange during reflow is dependent on the duration of ischaemia. Received: 4 December 1997, Returned for 1. revision: 12 January 1998, 1. Revision received: 16 April 1998, Accepted: 13 May 1998     

Brief preconditioning ischemia alters diacylglycerol content and composition in rabbit heart

In order to give further insight into the potential role of PKC in beneficial effects of ischemic preconditioning, we have characterized the production of diacylglycerol, the endogenous activator of PKC, and its molecular species composition in ischemic control and preconditioned hearts. Preconditioning was induced by 1 cycle of 5 min of ischemia followed by 5 min of reperfusion. In control and preconditioned groups, hearts were harvested under deep anesthesia at baseline (preischemia) and at 2,5 and 10 min into the sustained coronary artery occlusion, i.e., preceding myocyte death. Diacylglycerol content and fatty acid composition were analysed by thin-layer chromatography (TLC) and high-performance liquid chromatography (HPLC), respectively. Myocardial diacylglycerol content was increased at 2 min into the sustained ischemia in the control group (481 ± 34 vs 292 ± 64 ng.mg⁻¹ at baseline; p < 0.05), but was comparable to the baseline value at 5 and 10 min. In the preconditioned group, diacylglycerol production remained unchanged throughout the 10-min test ischemia (317 ± 17 at 2 min vs 312 ± 38 ng.mg⁻¹ at baseline; p = NS). A detailed analysis of the molecular species composition at the time of 2 min revealed a reduced contribution of phosphatidy-linositol to diacylglycerol production in preconditioned myocardium (global correlation coefficient 0.57 vs 0.66 in control myocardium) with a trend toward an enrichment of diacylglycerol composition with some species originating from phosphatidylcholine. Thus, our study revealed that brief preconditioning ischemia: (1) prevents the increase of diacylglycerol content in the early minutes of the sustained ischemia, and (2) emphasizes the contribution of phosphatidylcholine in diacylglycerol formation to the detriment of that of phosphatidylinositol. Received: 12 October 1999, Returned for 1.revision: 3 November 1999, 1.Revision received: 6 January 2000, Returned for 2.revision: 14 February 2000, 2.Revision received: 21 April 2000, Accepted: 9 May 2000     

Cardioprotective effects of chronic hypoxia and ischaemic preconditioning are not additive

The objective of the work was to examine whether adaptation to intermittent high altitude hypoxia and ischaemic preconditioning provide additive protection of the heart against subsequent acute ischaemic injury. Adult male rats were exposed to hypoxia (7000 m, 8 h/day, 24–30 exposures) in a hypobaric chamber. Susceptibility of their hearts to ischaemia-induced ventricular arrhythmias and infarction was evaluated in open-chest animals subjected to 30-min coronary artery occlusion and 4-h reperfusion. Preconditioning was induced by either two (PC1) or five (PC2) occlusions of the same artery for 5 min, each followed by 5-min reperfusion. Adaptation to hypoxia decreased the arrhythmia score from 2.75 ± 0.13 in normoxic controls to 2.17 ± 0.18. Both PC1 and PC2 reduced the arrhythmia score in the controls (0.15 ± 0.10 and 0.71 ± 0.24, respectively), as well as in the hypoxic groups (0.40 ± 0.15 and 0.27 ± 0.15, respectively). The infarct size was reduced from 66.6 ± 2.3% of the area at risk in the controls to 50.2 ± 1.9% in the adapted rats. PC1 conferred further protection in adapted animals (38.4 ± 2.8%) but this combined effect was of the same magnitude as that of preconditioning in the controls (37.5 ± 1.6%). Similar results were obtained using PC2. It is concluded that adaptation to hypoxia decreases the efficiency of ischaemic preconditioning; cardioprotective effects of these two phenomena are not additive. The results are consistent with the view that the mechanisms of protection conferred by chronic hypoxia and preconditioning may share the same signalling pathway. Received: 23 April 2001, Returned for revision: 22 May 2001, Revision received: 25 July 2001, Accepted: 20 August 2001     

Critical timing of mitochondrial K(ATP) channel opening for enhancement of myocardial tolerance against infarction

Objective The present study was designed to assess the relationship between the timing of a mitoK_ATP channel opener, diazoxide, and its infarct size-limiting effect. Methods In isolated rabbit hearts, infarction was induced by 30 min of global ischemia and 2 h of reperfusion, and infarct size was determined by tetrazolium staining and expressed as a percentage of the left ventricle (%IS/LV). Diazoxide, a mitoK_ATP channel selective opener, and/or 5-hydroxydecanoate (5-HD), a mitoKATP channel blocker, were infused before or after the onset of ischemia. When these agents were infused during the ischemic period, they were dissolved in a hypoxic buffer at concentrations 10-fold higher than those in the pre-ischemic period, and the infusion rate was set at 2 % of the pre-ischemic coronary flow. Results In untreated controls, %IS/LV was 53.2 ± 4.1 (SE). Pretreatment with diazoxide (100 μM) with a 10-min washout period reduced %IS/LV to 7.8 ± 2.4 and this protection was abolished by co-infusion of 5-HD (50 μM). Pre-ischemic infusion of diazoxide without a washout period reduced %IS/LV to 7.3 ± 1.4, and infusion of diazoxide from 10 min after the onset of ischemia also limited %IS/LV to 14.9 ± 4.6. However, diazoxide infusion from 25 min after the onset of ischemia failed to reduce infarct size (%IS/LV = 54.5 ± 7.2). Furthermore, pretreatment with 5-HD (50 μM) also completely abolished the protection afforded by early post-ischemic diazoxide infusion (%IS/LV = 48.3 ± 6.5). Neither infusion of 5-HD nor the anoxic vehicle alone during ischemia modified %IS/LV. Conclusion These findings suggest that opening of mitoK_ATP channels before ischemia and during early ischemia, but not that upon reperfusion, is important for enhancement of myocardial tolerance against infarction. Received: 6 November 2000, Returned for revision: 21 November 2000, Revision received: 24 January 2001, Accepted: 25 January 2001     

Ischaemic preconditioning limits infarct size in the rat heart.

OBJECTIVE: One of the mechanisms by which ischaemic preconditioning is thought to protect against later prolonged ischaemia is via a reduction in ATP utilisation during ischaemia. The ATP "wastage" that occurs during ischaemia is thought to be due to mitochondrial ATPase activity, which may be prevented in ischaemic preconditioning by the binding of a specific inhibitor protein. As the rat is known to have less inhibitor protein than other species, this study was designed to determine whether the rat heart could be ischaemically preconditioned. METHODS: Rats were anaesthetised with pentobarbitone, the chest opened and the hearts ischaemically preconditioned with a 5 min occlusion of the left main coronary artery followed by 10 min reperfusion. The hearts were then subjected to a 45 min occlusion followed by 3 h reperfusion. Control hearts were treated identically but without ischaemic preconditioning. Infarct size was measured using triphenyl tetrazolium and expressed as a percentage of the region at risk, measured with fluorescent particles. RESULTS: Infarct size as a percent of the risk area in the ischaemically preconditioned group (n = 8) was 31.4(SEM 6.1)%, versus 61.0(4.8)% in control hearts (n = 8) (p < 0.005). CONCLUSIONS: These results show that rat hearts can be ischaemically preconditioned and suggest that the protective mechanism involved in this phenomenon is not mediated through the endogenous inhibition of mitochondrial ATPase. An overall reduction in mitochondrial ATP "wastage" may not be the sole mechanism in the protection seen in ischaemic preconditioning.