S. Genade

Ischemic preconditioning: infarct size is a more reliable endpoint than functional recovery.

Abstract.The search for the mechanism of preconditioning-induced cardioprotection has been hampered by controversial results obtained by workers using different animal species, experimental models, protocols and endpoints. The aim of this study was to evaluate the role of the perfusion model (retrograde vs working), the infarct size and severity of ischaemia (regional vs global) as well as the endpoint (functional recovery vs infarct size) in preconditioning. The isolated perfused rat heart was preconditioned by 3 × 5 min global ischaemia, followed by different periods of regional or global ischaemia and reperfusion. Ischaemic preconditioning of working hearts resulted in increased functional recovery after 25–35 min global ischaemia, while retrogradely perfused hearts showed no significant improvement (except after 30 min global ischaemia). In addition, the percentage reduction in functional performance during reperfusion observed in the latter group was significantly less than in working hearts. Hearts were also subjected to regional ischaemia, perfused in either retrograde or working mode and infarct size determined. Regionally ischaemic working as well as retrogradely perfused hearts when preconditioned showed a significant increase in functional recovery after 35 min ischaemia only. In contrast to global ischaemia, the percentage recovery in mechanical performance of regionally ischaemic hearts was not affected by the mode of perfusion. Preconditioning of working hearts caused a significant reduction in infarct size after both 30 and 35 min ischaemia. However, preconditioned retrogradely perfused hearts showed a significant decline in infarct size after 35 min regional ischaemia only. In conclusion, the effect of the perfusion mode on functional recovery is dependent on the size and severity of ischaemia. It also affects the ischaemic time at which infarct size reduction by prior preconditioning occurs in the retrogradely perfused heart.

At the core of survival: Autophagy delays the onset of both apoptotic and necrotic cell death in a model of ischemic cell injury

Ischemic cell injury leads to cell death. Three main morphologies have been described: apoptosis, cell death with autophagy and necrosis. Their inherent dynamic nature, a point of no return (PONR) and molecular overlap have been stressed. The relationship between a defined cell death type and the severity of injury remains unclear. The functional role of autophagy and its effects on cell death onset is largely unknown. In this study we report a differential induction of cell death, which is dependent on the severity and duration of an ischemic insult. We show that mild ischemia leads to the induction of autophagy and apoptosis, while moderate or severe ischemia induces both apoptotic and necrotic cell death without increased autophagy. The autophagic response during mild injury was associated with an ATP surge. Real-time imaging and Fluorescence Resonance Energy Transfer (FRET) revealed that increased autophagy delays the PONR of both apoptosis and necrosis significantly. Blocking autophagy shifted PONR to an earlier point in time. Our results suggest that autophagic activity directly alters intracellular metabolic parameters, responsible for maintaining mitochondrial membrane potential and cellular membrane integrity. A similar treatment also improved functional recovery in the perfused rat heart. Taken together, we demonstrate a novel finding: autophagy is implicated only in mild injury and positions the PONR in cell death.

Effect of captopril on changes in rats hearts induced by long-term irradiation

The aim of this study was to test the efficacy of captopril, an angiotensin-converting enzyme inhibitor and a known suppressor of fibrosis, in preventing late radiation-induced cardiac pathology. Myocardial functional, histochemical and ultrastructural-morphometric studies were done on perfused hearts of rats isolated 3 and 6 months after 60Co gamma irradiation with 20 Gy and age-matched controls. At each time the animals were divided into the following groups: nonirradiated controls; irradiated once with 20 Gy; irradiated as above and given daily doses of captopril; daily doses of captopril without irradiation. The results showed that captopril, while ameliorating the decrease in the indices of capillary function, increase in mast cells, fibrosis, number of atrial granules, and changes in nerve terminals, failed to prevent the progressive functional deterioration of the hearts after irradiation. These findings suggest that an intramyofiber derangement may be involved in the long-term myocardial complications of irradiation.

Comparison Between Ischaemic and Anisomycin-Induced Preconditioning: Role of p38 MAPK

To further evaluate the significance of p38 MAPK as trigger or mediator in ischaemic preconditioning, anisomycin and SB 203580 were used to manipulate its activation status. Special attention was given to the concentration of the drugs and protocols used.The isolated perfused rat heart, subjected to either 25 min global ischaemia or 35 min regional ischaemia, was used as experimental model. This was preceded by anisomycin (2 or 5 μM: 3 × 5 min; 5 μM: 5 min or 10 min; 5 μM: 10 min + 10 min washout or 20 μM: 20 min) or SB 203580 (2 μM: 3 × 5 min; before and during 3 × 5 min or 1 × 5 min ischaemic preconditioning; 10 min). Endpoints were functional recovery during reperfusion and infarct size.Anisomycin, regardless of the protocol, reduced infarct size, but did not improve functional recovery. In a number of experiments activation of JNK by anisomycin was blocked by SP 600125 (10 μM). SP 600125 had no effect on the anisomycin-induced reduction in infarct size. SB 203580 when administered for 10 min before sustained ischaemia, improved functional recovery and reduced infarct size. SB 203580 could not abolish the beneficial effects of a multi-cycle preconditioning protocol, but it significantly reduced the outcome of 1 × 5 min preconditioning. In all hearts improved functional recovery and reduction in infarct size were associated with attenuation of p38 MAPK activation during sustained ischaemia-reperfusion.The results indicate that activation of p38 MAPK acts as a trigger of preconditioning, while attenuation of its activation is a prerequisite for improved recovery and a reduction in infarct size.

Myocardial membrane cholesterol: Effects of ischaemia

Evidence has recently been presented that myocardial ischaemia is associated with a significant increased mitochondrial cholesterol content, suggesting a redistribution of cholesterol within the ischaemic cell (Rouslin et al. 1980, 1982). The aim of this study was therefore to determine the effects of different periods of ischaemia and reperfusion on the cholesterol content of myocardial mitochondria, sarcoplasmic reticulum and sarcolemma. Using the isolated perfused rat heart as experimental model, it was demonstrated that increasing periods of ischaemia (15-60 min) caused a progressive loss of cholesterol from the tissue as well as from the sarcolemma and sarcoplasmic reticulum, concomitant with a significant increase in mitochondrial cholesterol content. These compositional changes were associated with a marked increase in sarcolemmal and mitochondrial microviscosity, while that of the sarcoplasmic reticulum was reduced. To gain more insight into the mechanisms controlling intracellular cholesterol distribution, control and ischaemic hearts were perfused with either exogenous cholesterol or its precursor [U-14C]acetate as an indicator of endogenous cholesterol synthesis. Perfusion with exogenous cholesterol resulted in significant increases in the membrane cholesterol content of control hearts. However, hypoxic, low flow perfusion prevented cholesterol enrichment of the sarcolemmal and sarcoplasmic reticulum membranes, while the cholesterol content of the mitochondria was increased from 99.48 +/- 12.75 to 127.61 +/- 1.84 nmols/mg protein, indicating specific incorporation into this membrane system. Incorporation of [U-14C]acetate into cholesterol in the sarcoplasmic reticulum was increased by 120% in ischaemic conditions. However, a marked redistribution of newly synthesized cholesterol was observed within the ischaemic cell: under control conditions most of the labelled cholesterol was transferred to the sarcolemma and least to the mitochondria, while this distribution pattern was reversed in ischaemia. In view of the fact that exchange of cholesterol between membranes is affected by both phospholipid polar head-group composition and acyl chain length and saturation, it is suggested that prior ischaemia-induced membrane compositional changes might lead to intracellular cholesterol redistribution. Finally, to determine whether cholesterol loss affects sarcolemmal permeability, hearts enriched in sarcolemmal cholesterol were subjected to 15 or 30 min global ischaemia followed by reperfusion and the rate of enzyme release determined. However, enzyme release was similar in treated and untreated hearts, indicating that sarcolemmal cholesterol loss probably does not affect its permeability.

A comparison between ischemic preconditioning and anti-adrenergic interventions: cAMP, energy metabolism and functional recovery

ObjectivesThe postulate that ischemic preconditioning caused an attenuation in ischemia induced increases in tissue cAMP, and that this may pertain to the mechanism of ischemic preconditioning, was investigated in the isolated rat heart. A significant reduction in tissue cAMP in preconditioned hearts was observed for all time periods of global ischemia studied. The significance of this observation was evaluated by comparing the effect of antiadrenergic interventions on energy metabolism and post-ischemic functional recovery of both non-preconditioned and preconditioned hearts.MethodsThe isolated perfused rat heart was used as experimental model. Six groups were studied: Non-preconditioned rat hearts: i) untreated controls (Non-PC), ii) reserpinised (Non-PC Res), iii) propranolol treated (10−7M) (Non-PC Prop); Preconditioned rat hearts: iv) preconditioned controls (PC), v) reserpinised (PC Res) and vi) propranolol (10−7M) treated (PC Prop).ResultsAfter 25 min global ischemia the concentration of cAMP was increased by 79.6% in the Non-PC group. This increase was attenuated in all of the treated groups, although in varying degrees. Energy utilization in these hearts also differed markedly between the groups. Functional recovery was however similar in all Non-PC and PC treated groups and significantly superior to that of Non-PC control hearts. Prior reserpinisation mimicked the protective effect of preconditioning on energy metabolism and functional recovery. To determine the significance of attenuation of the increase in cAMP in the protection conferred by preconditioning, hearts were pretreated with forskolin (10−6M). This caused an accumulation of tissue cAMP in preconditioned hearts to similar absolute values as seen in untreated non-preconditioned hearts during 25 min global ischemia. However, the percentage increase in forskolin-pretreated preconditioned hearts during sustained, ischemia was only 50% vs. 71% in non-preconditioned hearts treated with forskolin, confirming an attenuated β-response induced by preconditioning. Forskolin treatment of preconditioned hearts did not abolish the protective effect.ConclusionsThe findings suggest that the protection against ischemic damage conferred by preconditioning is associated with an attenuated β-adrenergic response. However, whether the changes in cAMP occurring during sustained global ischemia is the cause or consequence of the elicited protection, remains to be established.

The effect of ischaemia-reperfusion on [3H]inositol phosphates and Ins(1,4,5)P3 levels in cardiac atria and ventricles - A comparative study

In this study incorporation of [3H]inositol into inositol phosphates and phosphoinositides as well as tissue Ins(1,4,5)P3 levels of the atria and ventricles of isolated, perfused rat hearts were compared. Although the incorporation of [3H]inositol into the phosphoinositides of atria and ventricles was similar, significantly higher (2–3 fold) incorporation rates into inositol phosphates were observed in atrial tissue. Using a D-myo-[3H]Ins(1,4,5)P3 assay system, the Ins(1,4,5)P3 levels observed in atria from perfused rat hearts were also significantly higher than those obtained under the same experimental circumstances in the ventricles.Since previous studies on whole hearts showed inhibition of the phosphatidylinositol (PI) pathway during ischaemia with an immediate significant stimulation upon reperfusion [12, 20], the effects of ischaemia and 1 min postischaemic reperfusion were also examined separately in atria and ventricles. The results showed that 20 min of global ischaemia significantly depressed Ins(1,4,5)P3 levels as well as incorporation of [3H]inositol into ventricular InSP2 and InSP3. Reperfusion caused an immediate (within 1 min) increase in Ins(1,4,5)P3 levels and also [3H]inositol incorporation into all three cytosolic inositol phosphates in the ventricles. However, the effect of ischaemia and reperfusion on Ins(1,4,5)P3 levels as well as the incorporation of [3H]inositol into the inositol phosphates were less prominent in the atria. It therefore appears that the differential responses of the atria and the ventricles to an oxygen deficiency [41] are also reflected in the differences in PI metabolism during ischaemia-reperfusion.

The role of alpha1-adrenergic stimulation in inositol phosphate metabolism during post-ischaemic reperfusion

The aim of this study was to elucidate the mechanism of enhanced inositol phosphate metabolism during reperfusion. Inositol phosphate stores were prelabelled by perfusing isolated rat hearts for 1 h with [3H]inositol (1.5 microCi/ml). LiCl (10 mM) and prazosin (0.3 microM) were subsequently added 15 min before (i) 20 min control perfusion; (ii) 20 min normothermic ischaemic cardiac arrest (NICA); (iii) 20 min NICA followed by 1 min reperfusion. The ventricles were freeze-clamped before determination of isotopical incorporation of [3H]inositol into the inositol phosphates (Dowex anion exchange chromatography) and InsP3 levels (Amersham InsP3 assay system). In addition, noradrenaline release into the perfusate was also assessed (HPLC and electrochemical detection). The results showed: (i) increased noradrenaline release into the perfusate immediately after the onset of reperfusion; (ii) significant depression of [3H]inositol incorporation into inositol phosphates and InsP3 levels after 20 min NICA; (iii) reperfusion caused an immediate significant increase in isotopical incorporation of [3H]inositol into inositol phosphates as well as InsP3 levels; (iv) the alpha 1-adrenergic blocker, prazosin (0.3 microM), completely inhibited the reperfusion-induced increase in inositol phosphate metabolism. These observations suggested that increased alpha 1-adrenergic receptor stimulation by noradrenaline might be responsible for the stimulation of ventricular inositol phosphate metabolism during postischaemic reperfusion.

Massive atrial natriuretic peptide (ANP) release in ischemia reperfusion

Recent studies reported the stimulatory effects of Ca 2+ channel and al receptor agonists [1,2] on ANP release by atrial tissue. Therefore the increased cytosolic Ca z÷ [3] and release of endogenous catecholamines [4] occurring during ischemia-reperfusion may be associated with ANP release. Hypoxia has previously been shown to be a potent stimulus for ANP release by the isolated perfused heart [5,6], as well as in conscious animals [7] and humans [8]. In view of the marked differences in coronary flow rates in ischemia and hypoxia, and the role of coronary hemodynamics in ANP secretion [9], ANP release during reperfusion was monitored in this study after exposure of the isolated rat heart to either 15 or 25 minutes normothermic ischemic arrest (representing reversible and irreversible ischemic injury, respectively [10]). ANP released during ischemia alone was evaluated by determining the amounts released into the recirculating buffer of hearts subjected to 35 minutes normothermic cardioplegic arrest (a condition causing overt ischemic damage). To eliminate the possible contribution of a high K+-cardioplegic solution to ANP release, myocardial ANP release was also monitored during exposure to 90 minutes of hypothermic arrest, a procedure that has been shown to offer complete protection against ischemic damage [11]. Male Wistar rats (weight +200 g), fed ad libitum until experimentation were used in all experiments. The hearts were perfused retrogradely for 20 minutes, during which time the pulmonary vein was cannulated. Atrial stretch was then induced by perfusion in the working mode for 10 minutes (preload 15 cm H20; afterload 100 cm H20). The perfusion conditions and buffer, as well as induction of total global ischemia, have been described previously [10]. The hearts were reperfused in the retrograde mode for 10 minutes, followed by 5 minutes perfusion in the working mode. Normothermic (37°C) as well as hypothermic (20°C) cardioplegic arrests were induced by using 20 ml Plasmaiyte B (Sabax, South Africa) to which CaC12 (1.25 mM) and KC1 (25 mM) were added (final composition in mM: Na ÷ 130; K ÷ , 29; C1109; bicarbonate 28; Ca 2+ 1.25). The cardioplegic solution was gassed with 95% 02/5% CO2 and administered for 1 minute at 10-minute intervals at a pressure of 40 mmHg during the arrest period. Reperfusion of these hearts was done as described above. At least six hearts were studied in each group. Samples for measurement of perfusate ANP were collected over a period of 1 minute at the following time intervals during the control perfusion period: 10 and 20 minutes retrograde perfusion, 5 and 10 minutes working heart perfusion. During reperfusion after normothermic or hypothermic arrest, samples were collected at 1, 2, and 5 minutes of retrograde perfusion and at 1, 2, and 5 minutes of working heart perfusion. In the case of the cardioplegic hearts, the recirculating cardioplegic fluid was collected at the end of the cardioplegic arrest. Samples were kept frozen at -70°C until assayed. At the end of the experimental period, the hearts were blotted dry, dried in an oven for 24 hours, and weighed. ANP release was expressed as fentomol/g dry weight/min. Perfusate ANP was analyzed using Amershams ~ANP [12~I] radioreceptor assay system. This system utilizes a high specific activity (3 [125I] iodotyrasy128) aANP tracer, together with a specific and sensitive ANP binding receptor preparation from bovine adrenal gland. Crossreactivity with human and rat a-ANP is 100% and 73%, respectively. The assay involves a 90-minute room temperature incubation and measures aANP in the range 1-128 fentomol/tube. The ED50 and the intraassay coefficient of variation averaged 25-29 fmol and 6%, respectively. Nonspecific binding was 4%. The standard curve and unknowns were evaluated by a RIA-calc computer program. In view of the fact that similar values were obtained in the amounts of ANP released during control perfusion, the values of the four series of experiments were pooled (Table 1). Atrial stretch, induced by switching from retrograde to working heart perfusion, caused an 88-100% increase (p < 0.001) in ANP release. The severity of ischemic damage markedly affected the pattern and amounts of ANP released: (a) Reperfusion in the retrograde mode after induction of 15 minutes ischemia (reversible damage) caused a massive ANP release within 2 minutes (62059 -+ 14037 and 49466 + 8604 fmoles/g/min). Although the release tended to decline after 5 minutes of retrograde reperfusion, the amounts of ANP released were still significantly higher (25384 fmoles/g/min) than that observed during atrial stretch in the control period. ANP release during reperfusion in the working mode after 15 minutes of ischemia was similar to that occurring during the corresponding control period, indicating that the washout of the ischemia-induced ANP release was probably completed. (b) Irreversible damage (25 minutes ischemia) was associated with an initial lesser degree of ANP release, which might be due to lower coronary flow rates during retrograde perfusion (mean coronary flow rates during first 2 minutes of retrograde perfusion after 15 and 25 minutes of ischemia were 16 -+ 1 and 11 -+ 1 ml/min, respectively). However, ANP release remained high throughout the reperfusion period and was not further affected by atrial stretch. (c) The magnitude of ANP released during ischemia is reflected by the massive amounts of ANP discharged into the cardioplegic fluid. Although high concentrations of KCI have been shown to induce ANP release in rat hypothalamus [12], it was unable to do so in Langendorff-

Ischemic Preconditioning of the Isolated Perfused Working Guinea Pig Heart

Dear Sir, Ischemic preconditioning–induced protection against regional and global ischemia has repeatedly been demonstrated in all animal species routinely used for experimental purposes (e.g., dogs, pigs, rabbits, rats). The guinea pig appears to be the exception: apart from a few studies on isolated guinea pig papillary muscles [1,2] and retrogradely perfused guinea pig hearts [3], relatively little is known about the response of such hearts to preconditioning. Hypoxic preconditioning of papillary muscles [2] has been shown to enhance posthypoxic recovery of developed tension and to attenuate rhythmic activity, whereas in the perfused heart model it prevented the impairment of hypoxic coronary vasodilation associated with ischemia-reperfusion [3]. As far as we know, the classical endpoint of preconditioning, namely, infarct size reduction, has not yet been evaluated in these animals. In view of the controversy regarding a role for adenosine in the protection conferred by prior preconditioning, the guinea pig may be a useful model to further elucidate the role of this compound [4]. According to Van Belle and coworkers [5], these animals do not produce signi~cant amounts of adenosine during hypoxia or ischemia, and inosine represents the major metabolite released upon reperfusion. It has been suggested by Schaper [4] that should the guinea pig heart be preconditioned, it could be the “kiss of death” for the adenosine hypothesis. In our laboratory the isolated perfused working rat heart has been used extensively as an experimental model in the study of the mechanism of preconditioning, using functional recovery during reperfusion as the endpoint [6]. We therefore decided to use this technique to evaluate whether prior ischemic preconditioning improves the functional performance of the working guinea pig heart during reperfusion after sustained ischemia. Male and female guinea pigs (300–400 g) were anesthetized by intraperitoneal injection of sodium pentobarbital (50 mg/kg). Animals were fed ad libitum until experimentation. Institutional and international ethics guidelines were observed in the handling of experimental animals. The isolated perfused working heart, as described previously [6], was used as the experimental model. Hearts were perfused in the Langendorff mode (retrogradely) for a period of 30 minutes for stabilization, followed by 15 minutes of perfusion in the working mode (preload 15 cm H2O; afterload 80 mmHg). Total global ischemia was used for induction of preconditioning as well as for sustained ischemia. At the end of sustained ischemia, hearts were reperfused for 30 minutes (20 minutes retorgrade, 10 minutes working heart). Measurements of functional performance were made after 15 minutes of working heart perfusion during the stabilization period and after 30 minutes of reperfusion. Three different preconditioning protocols (1 3 10 minutes, 1 3 5 minutes, 3 3 5 minutes) were evaluated, followed by 25 minutes of global ischemia and 30 minutes of reperfusion (Fig. 1). Data are expressed as the mean 6 SEM. Differences between groups were evaluated using ANOVA with Bonferroni’s correction for multiple comparisons. P values , 0.05 were considered signi~cant. The functional performance of hearts at the end of the stabilization period was similar in all groups and data were therefore pooled (Table 1). Exposure of hearts to 25 minutes of global ischemia caused a signi~cant reduction in all parameters measured during reperfusion in all four groups. Preconditioning protocols b (1 3 10 minutes) and c (1 3 5 minutes) had no bene~cial effect on functional recovery. However, preconditioning with 3 3 5 minutes global ischemia improved functional recovery (PSP and total work were signi~cantly higher than in groups a, b, and c). Two observations were made in this study: (1) the work performance of the isolated perfused guinea pig heart under basal conditions is signi~cantly lower