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     

Preconditioning-induced attenuation of purine metabolite accumulation during ischemia: memory and multiple cycles

Brief myocardial ischemia (ISC) is known to attenuate purine metabolite accumulation in the interstitial fluid (ISF) during subsequent ISC. We determined how this attenuated purine accululation was altered by 1) extended reperfusion (REP) and 2) multiple cycles of brief ISC. Microdialysis probes were used to assess ISF levels of the purine metabolites adenosine, inosine, and hypoxynthine in anesthetized rabbits. In one series of experiments, two 10 min periods of regional ISC were separated by 10 (n=6), 60 (n=6), or 180 (n=6) min of REP. In the 10, 60, and 180 min REP groups the increase in ISF purine metabolites during the second ISC was 47%, 55%, and 53% of that seen during the first ISC, respectively. In a second series of experiments, hearts were exposed to 120 min of ISC with (n=6) or without (n=6) five preceding cycles of transient ISC (10 min ISC; 10 min of REP). The increase in ISF purine metabolites during the multiple cycles of ISC was progressively attenuated, and there was a delay but eventual increase in ISF purine metabolites during the 120 min ISC. These data demonstrate that attenuated purine metabolite accumulation 1) is progressively greater with multiple cycles of brief ISC, 2) has a memory time of at least 180 min, and 3) is not due to high energy phosphate depletion. Received: 18 May 1999, Returned for 1. revision: 21 June 1999, 1. Revision received: 12 July 1999, Returned for 2. revision:12 August 1999, 2. Revision received: 27 September 1999, Accepted: 29 September 1999     

A role of PKC in the improvement of energy metabolism in preconditioned heart

Objectives. A possible link between activation of PKC and improvement of energy metabolism during reperfusion in ischemic preconditioning hearts was examined. Methods. Isolated perfused rat hearts were preconditioned by 5-min ischemia and 5-min reperfusion in the presence and absence of a PKC inhibitor polymyxin B (50 μM) and then subjected to 40-min sustained ischemia and subsequent 30-min reperfusion. In another set of experiments, the hearts pretreated with and without a PKC activator PMA (15 pmol/5 min) were subjected to the sustained ischemia and reperfusion. Myocardial high-energy phosphates, glycolytic intermediates and mitochondrial oxygen consumption capacity were determined at appropriate experimental sequences. Results. Preconditioning enhanced the recovery of cardiac function such as left ventricular developed pressure, heart rate and rate-pressure product of the reperfused heart, suppressed the release of creatine kinase, enhanced the reperfusion-induced restoration of myocardial high-energy phosphates, attenuated the reperfusion-induced accumulation in glucose 6-phosphate and fructose 6-phosphate contents, abolished the ischemia-induced increase in tissue lactate content and prevented the ischemia-induced decrease in mitochondrial oxygen consumption capacity. Treatment of the perfused heart with PMA mimicked the effects of preconditioning on post-ischemic contractile function, enzyme release, levels of myocardial energy store, glycolytic intermediates and lactate, and mitochondrial function. Polymyxin B-treatment abolished the preconditioning-induced recovery of post-ischemic contractile function, the suppression of the release of CK, the restoration of myocardial energy store, and the preservation of mitochondrial function, whereas it did not cancel the improvement of glycolytic intermediate levels and the reduction in tissue lactate accumulation. Post-ischemic contractile function was closely related to restoration of high-energy phosphates and mitochondrial oxygen consumption capacity in all hearts subjected to ischemia/reperfusion. Conclusion. The results suggest that activation of PKC and preservation of mitochondrial function are closely linked with each other in the preconditioned heart, which may lead to the improvement of post-ischemic contractile function. Received: 29 January 1999, Returned for 1. revision: 26 February 1999, 1. Revision received: 27 April 1999, Returned for 2. revision: 18 May 1999, 2. Revision received: 12 July 1999, Returned for 3. revision: 26 July 1999, 3. Revision received: 25 October 1999, Accepted: 3 November 1999     

NHE1-inhibitor cariporide prevents the transient reperfusion-induced shortening of the monophasic action potential after coronary ischemia in pigs

During myocardial ischemia intracellular acid load increases as a consequence of anaerobic metabolism. Exchange of excessive protons for sodium via the sodium proton exchanger type 1 (NHE1) is supposed to cause intracellular sodium accumulation. The NHE1 inhibitor cariporide has been shown to inhibit ischemia and reperfusion-induced ventricular fibrillation (VF) but the mechanisms are not fully understood. During early reperfusion transient shortening of the action potential has been reported, which renders the heart susceptible to reentrant arrhythmias. In anesthetized pigs subjected to 10 min of left circumflex coronary artery (LCX) occlusion and reperfusion we have investigated whether NHE1 is involved in reperfusion-induced shortening of the monophasic action potential (MAP) taken with an epicardial probe over the ischemic area. In control pigs (n = 7) a moderate decrease in the duration of the MAP at 50% repolarization (MAPD₅₀) occurred during ischemia reaching 78.8 ± 5.0% of the pre-ischemic duration at 5 min (p < 0.01) and 87.3 ± 7.6% after 10 min. An additional, transient but marked shortening occurred during the first 2 min of reperfusion, which fully recovered after 4 min. At 50 sec of reperfusion MAPD₅₀ fell to 53.1 ± 8.2% of the pre-ischemic value corresponding to 90.1 ± 20.2 msec of reperfusion-induced shortening. Cariporide, 3 mg/kg i. v. 5 min before occlusion (n = 6), totally prevented reperfusion-induced MAP shortening while heaving no effect on MAPD₅₀ during ischemia. In conclusion, our data suggest that the immediate, transient, but strong action potential shortening during early reperfusion after 10 min of coronary ischemia is due to the activity of the NHE1. Received: 16 August 2000, Returned for 1. revision: 8 September 2000, 1. Revision received: 21 September 2000, Returned for 2. revision: 27 September 2000, 2. Revision received: 2 November 2000, Accepted: 7 November 2000     

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     

Diadenosine tetraphosphate (AP4A) mimics cardioprotective effect of ischemic preconditioning in the rat heart: contribution of KATP channel and PKC

Diadenosine tetraphosphate (AP4A) administration is reported to mimic the effect of ischemic preconditioning (PC) via purine 2y receptors (P2yR) and adenosine receptors. This study was designed to test the contributions of the ATP-sensitive potassium channel (K_ATP channel) and protein kinase C (PKC), two of the main regulator in PC, to the effect of AP4A. Isolated buffer-perfused rat hearts were subjected to 20 min of global ischemia (37 °C) and 20 min of reperfusion. Three cycles of 1-min ischemia and 3-min reperfusion induced PC. Chemicals were administrated for 2 min before 20 min of ischemia. AP4A (10 μM) administration was as effective as PC in improving the recovery of post-ischemic contractile function and reducing creatine kinase leakage after reperfusion, whereas adenosine (10 and 100 μM) have not effect. AP4A had not effect on reperfusion-induced arrhythmia, whereas PC significantly prevented it. These effects of AP4A and PC were reversed by co-administration of glibenclimade (K_ATP channel blocker, 100 μM) and GF109203X (PKC inhibitor, 10 μM); the effects of AP4A but not PC were reversed by co-administration of reactive blue (P2yR antagonist, 13 nM). AP4A appears to activate the K_ATP channel and PKC via P2yR mimic the effects of PC in part. The role of P2yR indicated that trigger mechanism of the effect of PC and AP4A administration might differ in rat hearts. Received: 30 September 1999, Returned for revision: 26 October 1999, Revision received: 8 December 1999, Accepted: 16 December 1999     

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     

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     

Selective modulation of endogenous nitric oxide formation in ischemia/reperfusion injury in isolated rat hearts

The role of nitric oxide (NO) in ischemia/reperfusion injury is controversial. We tested the role of inducible NOS (iNOS) in the ischemia/reperfusion injury in isolated rat hearts using the selective iNOS inhibitor S-methylisothiourea sulfate (SMT) and the non-selective NOS inhibitor N^G-nitro-L-arginine methyl ester (L-NAME). After 15 min of stabilization in Langendorff mode, hearts were perfused either with normal Krebs-Henseleit buffer, buffer containing 100 μM L-NAME, 0.5 μM SMT or 50 μM SMT for 5 min and were subjected to 25 min of ischemia followed by 30 min of reperfusion. Left ventricular developed pressure (LVDP) and total coronary flow (CF) were recorded continuously. After ischemia/reperfusion, a marked expression of iNOS protein was demonstrated by Western blotting, while virtually no iNOS protein was present in hearts without ischemia/reperfusion. Regional myocardial blood flow (RMBF) was measured with colored microspheres. Coronary vasoactive concentration of L-NAME and SMT depressed myocardial function as shown by decreased LVDP, dP/dt_max and coronary .ow before ischemia. After ischemia the recovery of the total CF was impaired in L-NAME and 50 μM SMT pretreated hearts which was related to homogenous RMBF decrease in the right and left ventricle compared to that in control group. Low concentration SMT (0.5 μM) showed no coronary vasoactive effects before ischemia and attenuated ischemia/reperfusion injury indicated by lower ischemic contracture at 25 min of ischemia and reduced CK and LDH release during reperfusion. Thus, NOS inhibition did not affect blood flow distribution in rat hearts either in the pre-ischemic or reperfusion period. Selective iNOS inhibition reduced ischemic injury by reducing ischemic contracture and CK as well as LDH release during reperfusion. Received: 20 March 2002, Returned for 1. revision: 8 April 2002, 1. Revision received: 14 August 2002, Returned for 2. revision: 5 September 2002, 2. Revision received: 3 February 2003, Accepted: 18 February 2003, Published online: 16 April 2003     

Effects of BIIB513 on ischemia-induced arrhythmias and myocardial infarction in anesthetized rats

Na⁺/H⁺ exchange (NHE) plays an important role in the regulation of the intracellular pH (pH_i) and in cardiac cell injury induced by ischemia and reperfusion. In the present study, we investigated the effects of BIIB513, a selective NHE-1 inhibitor on myocardial ischemia induced arrhythmias and myocardial infarction, provoked by 30 minutes of left main coronary artery occlusion followed by 2 hours of reperfusion in an anesthetized rat model. Intravenous administration of BIIB513 (0.01–3.0 mg/kg) did not induce changes in blood pressure or heart rate. BIIB513 (0.01, 0.1, 0.3, 1.0, 3.0 mg/kg) given prior to the coronary artery occlusion dose-dependently reduced ventricular premature beats, ventricular tachycardia, and a complete suppression of ventricular fibrillation down to the dose of 0.1 mg/kg. BIIB513 (0.01, 0.1, 0.3, 1.0, 3.0 mg/kg) given prior to the coronary artery occlusion dose-dependently reduced the infarct size with an ED50 value of 0.16 mg/kg. BIIB513 (1.0 mg/kg) given prior to reperfusion also reduced infarct size by 47.3 ± 13.1%. The reduction in infarct size was accompanied by a decrease in circulating levels of creatine phosphokinase (CPK). In conclusion, the present study demonstrates the cardioprotective ability of NHE-1 inhibition during myocardial ischemia and reperfusion by reducing serious ventricular arrhythmias and myocardial infarct size in anesthetized rats. Received: 18 November 1999, Returned for 1.revision: 9 December 1999, 1.Revision received: 2 May 2000, Returned for 2.revision: 24 May 2000, 2.Revision received: 5 June 2000, Accepted: 7 June 2000     

Antioxidant properties of acetaminophen and cardioprotection

The acute administration of acetaminophen to isolated, perfused guinea pig hearts appears to have cardioprotective effects against the injury/mechanical dysfunction caused by global, low-flow, myocardial ischemia and reperfusion. In the current study we selected ischemia/reperfusion and administration of sodium pentobarbital as perturbations of the electrical stability of the myocardium. We investigated their ability to induce ventricular arrhythmias and changes in the characteristics of monophasic action potentials in the absence and presence of acetaminophen (0.35 mmol/l). The numbers of ventricular premature beats and ventricular salvos encountered in the presence of pentobarbital were significantly (P < 0.05) reduced by acetaminophen. The combined frequency of these arrhythmias was 0.14 ± 0.06/min vs 0.03 ± 0.01/min (P < 0.05) in the absence and presence of acetaminophen, respectively. The incidence of ventricular salvos increased steadily in vehicle-treated hearts after administration of pentobarbital. No such trend was seen with acetaminophen. After 10 min of global, low-flow myocardial ischemia, MAP50 and MAP90 (monophasic action potentials at 50 and 90 % repolarization, respectively) decreased without acetaminophen (e.g. MAP50, 31 ± 4 ms) but did not change during the same time interval with acetaminophen (e.g. MAP50, 57 ± 6 ms)(P < 0.05). During ischemia and reperfusion, acetaminophen attenuated the release of hydroxyl radicals and peroxynitrite. Collectively these data reveal cardioprotective, antioxidant behavior of acetaminophen. Under selected conditions (e.g. those causing release of free radicals and other oxidants) such behavior might also prevent ventricular arrhythmias. Received: 30 October 2000, Returned for 1. revision: 20 November 2000, 1. Revision received: 12 December 2000, Returned for 2. revision: 2 January 2001, 2. Revision received: 14 January 2001, Accepted: 31 January 2001     

Myocardial ischemia and reperfusion reduce the levels of cyclic ADP-ribose in rat myocardium

Cyclic ADP-ribose (cADPR) is a novel Ca²⁺-mobilizing second messenger in mammalian cells including cardiomyocytes. It is unknown whether myocardial ischemia and reperfusion affect the metabolism of cADPR in the myocardium. The present study therefore examined the effects of myocardial ischemia and reperfusion on the concentrations of myocardial cADPR using high-performance liquid chromatography. Basal levels of cADPR in rat myocardium were 5.3 ± 1.8 nmol· mg⁻¹ protein. Myocardial ischemia for 30 min significantly decreased cADPR concentrations to 2.1 ± 0.4 nmol·mg⁻¹ protein. During reperfusion, cADPR was maintained at ischemic levels. The activity of ADP-ribosyl cyclase was expressed as the conversion rate of nicotinamide guanine dinucleotide (NGD⁺) to cyclic GDP-ribose. Myocardial ischemia and reperfusion did not alter the activity of ADP-ribosyl cyclase. However, cADPR hydrolase activity, as measured by the conversion rate of cADPR to ADP-ribose, was significantly elevated by ischemia and reperfusion. To determine the mechanism resulting in the enhancement of cADPR hydrolase activity, we examined the effects of changes in ADP, ATP, pH, and PO₂ on the conversion rate of cADPR to ADPR. Alterations of ADP, ATP, or pH in myocardial tissue had no effect on the degradation of cADPR, whereas a decrease in tissue PO₂ markedly increased the hydrolysis of cADPR. These results suggest that myocardial ischemia and reperfusion decrease cADPR in the myocardium by increasing its hydrolysis. Tissue hypoxia may be one of the important mechanisms to activate cADPR hydrolase. Received: 24 July 2001/Returned for 1. revision: 20 August 2001/1. Revision received: 25 October 2001/Returned for 2. revision: 20 November 2001/2. Revision received: 12 December 2001/Accepted: 2 January 2002     

Cardiac and coronary vascular effects of chronically administered estrogen in the dog

The acute administration of cunjugated equine estrogen (CEE) to dogs significantly attenuated the severity and incidence of ventricular arrhythmias during ischemia and reperfusion. We hypothesized that one of the cardioprotective mechanisms of estrogen might be the ability to maintain electrical stability of the heart during ischemia. The current study was conducted to determine the effect of chronic administration of estrogen, simulating hormone replacement therapy, on the ventricular arrhythmias of ischemia and reperfusion, Chronically-treated (100 μg/kg/week CEE, or vehicle) male beagles were anesthetized and subjected to regional ischemia (20 min) and reperfusion. Although there was a trend towards a lower incidence of arrhythmias during ischemia in estrogen-treated dogs, values did not achieve significance at P<0.05. Baseline coronary vascular resistance was significantly higher in estrogen-treated dogs (2.3 vs 1.5 mmHg/ml/min/100 g, P<0.05) indicating an increase in vasomotor tone. There was also an increase in the time it took hyperemic coronary blood flow to reach a peak value upon reperfusion (71 sec in estrogen-treated dogs vs 12 sec in vehicle-treated dogs, P<0.05). This slower reflow is consistent with increased coronary vascular resistance upon reflow in estrogen-treated dogs. We conclude that the chronic administration of CEE to male dogs increased coronary vascular tone, and impaired the rate of reperfusion, but did not decrease the incidence of ventricular arrhythmias caused by ischemia. Received: 31 July 1997, Returned for revision: 18 September 1997, Revision received: 19 October 1997, Accepted: 4 November 1997     

Reduction of oxygen delivery during post-ischemic reperfusion protects the isolated guinea pig heart

Objective: To further characterise the influence of oxygen delivery during early reperfusion (first 5 min) in the isolated guinea pig heart, three modes of coronary reperfusion were chosen, differing with respect to reperfusion flow and arterial PO₂. Methods: Isolated working guinea pig hearts underwent ischemia and reperfusion (15 min each). Reperfusion was at constant pressure (Group 1, 60 mmHg, n = 7) or at constant flow (Group 2, 5 ml/min, n = 7) with a PO₂ of 600 mmHg. Group 3 (n = 8) was reperfused at 5 ml/min with a PO₂ of 300 mmHg for 5 min and a PO₂ of 600 mmHg thereafter. Lactate release and oxygen consumption were determined during reperfusion. Glutathione release served to assess myocardial oxidative stress. Results: After ischemia, hearts in Group 1 (mean coronary flow 14.4±1.1 ml/min during the first 5 min of reperfusion) performed external heart work at 31 ± 2 % of the pre-ischemic level. Performance in Group 2 recovered to 50 ± 3 % and in Group 3 to 68 ± 3 %. Myocardial oxygen consumption during early reperfusion (2nd min) was lowest in Group 3 (1.9 μmol/min) and highest in Group 1 (8.3 μmol/min). No difference in lactate release was observed. Release of glutathione during the first 5 min of reperfusion was 43.8 ± 7.9 nmol in Group 1, but only 3.6 ± 0.7 in Group 2 (p < 0.05). Conclusions: In isolated guinea pig hearts, controlled oxygen delivery during post-ischemic reperfusion by both, reduction of coronary flow and PO₂, improves recovery of pump function. The effect is accompanied by less oxidative stress, as indicated by lowered rates of glutathione release. Received: 1 December 1998, Returned for 1. revision: 4 January 1999, 1. Revision received: 28 January 1999, Returned for 2. revision: 8 February 1999, 2. Revision received: 18 February 1999, Accepted: 2 March 1999     

Xanthine oxidase contributes to preconditioning's preservation of left ventricular developed pressure in isolated rat heart: developed pressure may not be an appropriate end-point for studies of preconditioning

Studies of preconditioning frequently use the isolated rat heart model in which recovery of post-ischemic function is the end-point. However, function following an episode of ischemia/reperfusion represents a composite of both stunning, which is related to free radical production and is not attenuated by preconditioning, and tissue salvage, the primary effect of preconditioning. Brief ischemia/reperfusion is also known to diminish adenosine release during subsequent ischemia by a mechanism independent of preconditioning's anti-infarct effect. Reduced purine release would diminish generation of free radicals by xanthine oxidase in rat heart and thus produce less stunning. In this paradigm preserved post-ischemic function in rat heart might look similar to salvage by preconditioning, but its mechanism would be quite different and not be relevant to the xanthine oxidase-deficient human heart. This hypothesis was tested in isolated rat hearts. Control or ischemically preconditioned hearts were subjected to 30 min of global ischemia and 60 min of reperfusion, either in the presence or absence of 25 μmol/l allopurinol, an inhibitor of xanthine oxidase. In non-preconditioned hearts allopurinol increased left ventricular developed pressure after 60 min of reperfusion from 26 ± 5 mmHg in control hearts to 47 ± 7 mmHg, whereas developed pressure in preconditioned hearts following reperfusion was 59 ± 5 mmHg and was unaffected by allopurinol. Developed pressure in non-preconditioned hearts treated with allopurinol was midway between that for untreated control and preconditioned hearts suggesting that at least 50 % of the recovery of developed pressure in preconditioned hearts may be related to free radical-induced stunning. In xanthine oxidase-deficient rabbit hearts, return of function was not different between non-preconditioned and preconditioned hearts. Therefore, post-ischemic developed pressure in the rat is significantly affected by purine-dependent stunning, and, hence, may be an unreliable marker of tissue salvage and also a poor index of what might be cardioprotective in man. Received: 2 May 2001, Returned for 1. revision: 25 May 2001, 1. Revision received: 13 June 2001, Returned for 2. revision: 18 June 2001, 2. Revision received: 11 July 2001, Accepted: 18 July 2001     

Acute coronary vascular and myocardial perfusion effects of conjugated equine estrogen in the anesthetized dog

Women generally exhibit angina rather than myocardial infarction as the first manifestation of heart disease. Postmenopausal use of hormone replacement therapy, specifically estrogens, is associated with reduced incidence of major cardiac events suggesting estrogen may protect the heart during ischemia. We recently showed that acute administration of conjugated equine estrogens prior to ischemia attenuated the ventricular arrhythmias of ischemia as well as those of reperfusion. This study looks at basal effects of estrogen on coronary blood flow and the effects of estrogen on regional blood flow during ischemia to determine if estrogen exerts its antiarrhythmic effects during ischemia by altering blood flow. Under conditions of natural blood flow, estrogen caused cyclic changes in blood flow. When coronary blood flow was controlled and limited, estrogen increased coronary perfusion pressure (118±8 mmHg vs. 85±10 mmHg in non-treated dogs, P<0.05) demonstrating an overall vasoconstrictor effect. Coronary blood flow and regional myocardial perfusion were determined before and during ischemia in anesthetized dogs with and without acutely-administered estrogen. Colored microspheres were injected at steady state prior to ischemia, and during steady state myocardial ischemia. Conjugated equine estrogen (10 μg/kg), administered about 6 min before ischemia, had no effect on regional perfusion under steady state conditions, nor in the non-ischemic zone during ischemia. Perfusion in the subepicardial and subendocardial ischemic zones in estrogen-treated dogs was significantly lower than in non-treated dogs [0.14±0.01 ml/min/g vs. 0.23±0.02 ml/min/g (P<0.05) in the epicardial ischemic zone; and, 0.15±0.02 ml/min/g vs. 0.22±0.03 ml/min/g (P<0.05) in the endocardial ischemic zone]. We conclude that the acute, systemic administration of estrogen in the anesthetized dog decreases regional perfusion in the ischemic myocardium and causes significant coronary vasoconstriction when flow is controlled and limited. Received: 3 March 1998, Returned for 1. revision: 28 April 1998, 1. Revision received: 29 May 1998, Accepted: 18 June 1998     

The role of p38 mitogen-activated protein kinase in myocardial ischemia/reperfusion injury; relationship to ischemic preconditioning

Myocardial mitogen-activated protein kinases can be activated by ischemia and reperfusion, and they may play important roles in the evolution of ischemic injury. Considerable work has been performed to evaluate the role of different MAPK signaling pathways in ischemia/reperfusion injury. The focus of this review is the p38 MAPK pathway, specifically whether activation of the p38 MAPK signaling pathway is beneficial or detrimental. Different studies have come to conflicting conclusions. This review will examine the literature on the role of p38 MAPK in myocardial ischemia/reperfusion injury, highlight areas of controversy and areas of general agreement, examine possible downstream targets of p38 during acute ischemia, and attempt to draw some conclusions. Received: 25 February 2002/Returned for revision: 14 March 2002/Revision received: 4 April 2002/Accepted: 8 April 2002     

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     

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     

SB 203580, an inhibitor of p38 MAPK, abolishes infarct-limiting effect of ischemic preconditioning in isolated rabbit hearts

There is debate concerning the involvement of p38 mitogen-activated protein kinase (MAPK) in ischemic preconditioning (PC). At the center of the controversy are data obtained after administration of SB 203580, a specific inhibitor of p38 MAPK. Whereas several studies have reported that SB 203580 abolishes the cardioprotective effect of PC, others claim that this compound is actually cardioprotective against ischemia. Many of these latter observations have been made in isolated myocardial cells. Accordingly the present study was designed to test the effect of SB 203580 in a model of preconditioning in intact rabbit hearts in which infarct size was the end-point. Isolated hearts experienced 30 min of regional ischemia followed by 120 min of reperfusion. Infarct size was measured with triphenyltetrazolium chloride. In control hearts infarction was 30.2 ± 3.3% of the risk zone. PC with 5 min of global ischemia and 10 min of reperfusion before the 30-min period of ischemia significantly reduced infarct size to 10.2 ± 2.4% (P < 0.05 vs. control). SB 203580 (2 μ M) added to the perfusate for 20 min starting 5 min before the index ischemia totally blocked the protection from PC (27.4 ± 3.3% infarction). SB 203580 alone had no effect on infarct size (28.6 ± 4.6% infarction). These results reveal that SB 203580 does not affect infarct size on its own, but selectively blocks preconditioning's anti-infarct effect in the intact rabbit heart. Received: 21 August 2000, Returned for revision: 30 August 2000, Revision received: 2000, Accepted: 6 September 2000