Ischemia In Rabbit Hearts Research Articles

Mitochondrial depolarization and asystole in the globally ischemic rabbit heart: coordinated response to interventions affecting energy balance.

Mitochondrial membrane potential (ΔΨm) depolarization has been implicated in the loss of excitability (asystole) during global ischemia, which is relevant for the success of defibrillation and resuscitation after cardiac arrest. However, the relationship between ΔΨm depolarization and asystole during no-flow ischemia remains unknown. We applied spatial Fourier analysis to confocally recorded fluorescence emitted by ΔΨm-sensitive dye tetramethylrhodamine methyl ester. The time of ischemic ΔΨm depolarization (tmito_depol) was defined as the time of 50% decrease in the magnitude of spectral peaks reflecting ΔΨm. The time of asystole (tasys) was determined as the time when spontaneous and induced ventricular activity ceased to exist. Interventions included tachypacing (150 ms), myosin II ATPase inhibitor blebbistatin (heart immobilizer), and the combination of blebbistatin and the inhibitor of glycolysis iodoacetate. In the absence of blebbistatin, confocal images were obtained during brief perfusion with hyperkalemic solution and after the contraction failed between 7 and 15 min of ischemia. In control, tmito_depol and tasys were 24.4 ± 6.0 and 26.0 ± 5.0 min, respectively. Tachypacing did not significantly affect either parameter. Blebbistatin dramatically delayed tmito_depol and tasys (51.4 ± 8.6 and 45.7 ± 5.3 min, respectively; both P < 0.0001 vs. control). Iodoacetate combined with blebbistatin accelerated both events (tmito_depol, 12.7 ± 1.8 min; and tasys, 6.5 ± 1.1 min; both P < 0.03 vs. control). In all groups pooled together, tasys was strongly correlated with tmito_depol (R(2) = 0.845; P < 0.0001). These data may indicate a causal relationship between ΔΨm depolarization and asystole or a similar dependence of the two events on energy depletion during ischemia. Our results urge caution against the use of blebbistatin in studies addressing pathophysiology of myocardial ischemia.

Effects of tanshinone IIA on ventricular arrhythmias induced by acute myocardial ischemia

Objective To investigate the effects of Tanshinone Ⅱ A (extracted from Chinese herb medicine Salvia miltiorrhiza Bge.) on ventricular arrhythmias of rabbit hearts induced by ischemia in order to illuminate its mechanism of anti-arrhythmia.Methods Thirty rabbits were randomly (random number)divided into normal group,ischemic group and Tanshinone Ⅱ A group.Model of wedge shaped mass of rabbit left ventricular myocardium with coronary perfusion was prepared,and then by using floating glassy microelectrode,the trans-mural ECG,QT interval,the trans-mural dispersion of re-polarization (TDR) and trans-membrane action potentials from both endocardium and epicardium were simultaneously and wholly recorded.The incidence of ventricular arrhythmia in myocardium was observed after ischemia for thirty min.Results Under the condition of acute ischemia,compared with normal group,the incidence of ventricular arrhythmia and TDR were significantly increased in ischemia group (P ＜ 0.01),while incidence of ventricular arrhythmia and TDR were significantly reduced in tanshinone ⅡA group compared with ischemia group (P ＜ 0.05).The incidences of ventricular arrhythmia in normal,ischemia and Tanshinone Ⅱ A groups were 0/10,9/10 and 2/10 respectively.Conclusions Tanshinone Ⅱ A prevents ventricular arrhythmia and reduces TDR significantly in ischemic rabbit hearts. Key words: Tanshinone Ⅱ A; Ischemia; Ventricular arrhythmia; Transmural dispersion of repolarization; Action potential; Electrophysiology ; Transmural ECG; Rabbit

Abstract 18794: Temporal Relationship Between Mitochondrial Membrane Potential (ΔΨm) Depolarization and Loss of Excitability in Globally Ischemic Rabbit Heart

Mitochondrial membrane potential (ΔΨm) depolarization has been implicated in the loss of excitability during ischemia, which could be relevant for resuscitation after cardiac arrest. The timing and degree of ΔΨm loss during no-flow ischemia in whole hearts is still poorly documented, in part due to the difficulty of estimating ΔΨm dynamics under this condition. We use a new approach based on the spatially periodic pattern present in the fluorescence emitted by potentiometric fluorophore TMRM when the dye accumulates in well-polarized mitochondria. This periodicity creates a distinct “mitochondrial” peak (MP) in the spatial Fourier spectrum corresponding to the sarcomere length (∼2 μm). Redistribution of TMRM upon ΔΨm depolarization diminishes the area under MP (MPA). We used Langendorff-perfused rabbit hearts mechanically uncoupled with blebbistatin (5.7μM) and stained with TMRM (450 nM). Confocal images were obtained from posterior left ventricular epicardium. Mitochondrial uncoupler FCCP (5 μM) and glycolytic inhibitior iodoacetate (IA, 15 mM) were used to validate MPA method. Eight hearts underwent minimum 1 hour global-ischemia; 4 hearts remained in sinus rhythm, 4 hearts were paced at 6.67 Hz to simulate ventricular fibrillation. FCCP reduced MPA to 32+/-6% of baseline; subsequent application of IA made MPA undetectable. During global ischemia ΔΨm loss started after a variable delay, was gradual and was not different between paced and non-paced hearts. MPA decreased to 50% of baseline at 44±17 (from 10 to 64) minutes and to 32% of baseline at 49±16 (from 27 to 70) minutes of ischemia. The minimum MPA value was 7±9% (range, 0-26%) of baseline and was observed at 65±9 minutes of ischemia (range, 51-72 minutes). Complete loss of excitability occurred earlier in paced than in non-paced hearts (33.2±3.1 vs. 45.0±7.2 min, p&lt;0.05) but no clear relationship between ΔΨm dynamics and loss of excitability could be found. Conclusions: In this model a significant ΔΨm loss, comparable with severe metabolic inhibition, occurs only after prolonged time of global ischemia, is not accelerated by rapid pacing and is not correlated with the loss of excitability. This suggests a limited the role of ΔΨm dynamics in the outcomes of resuscitation after sudden cardiac arrest.

A novel glutathione preparation protects the ischemic rabbit heart after oral administration

A novel glutathione preparation protects the ischemic rabbit heart after oral administration

Heart rate reduction with ivabradine improves energy metabolism and mechanical function of isolated ischaemic rabbit heart

The anti-anginal agent ivabradine slows heart rate (HR) by selectively inhibiting the I(f) current in the sinus node. We report an ex vivo study to evaluate the anti-ischaemic effect of ivabradine in terms of modulation of cardiac energy metabolism. A Langendorff-perfused rabbit heart model was subjected to low-flow ischaemia and reperfusion. Cardiac metabolism was studied by measuring cardiac high-energy phosphate contents via HPLC, mitochondrial respiration was analysed polarographically, and cardiac redox potentials by HPLC. Cardiac function was determined in terms of the recovery of developed pressure during reperfusion and release of creatine kinase (CK) (spectrophotometrically) and noradrenaline (HPLC) after reperfusion. Four concentrations of ivabradine (0.3, 1, 3, and 6 microM) were tested on aerobically perfused hearts to select the most effective without causing changes in mechanical parameters. This proved to be 3 microM, which was therefore the concentration selected for the ischaemia-reperfusion experiments. Ivabradine concentration-dependently reduced HR with a maximal effect of 41 +/- 4% at 3 microM (P < 0.001 vs. vehicle), without a negative inotropic effect. This concentration protected the heart against ischaemia-reperfusion damage by reducing the rise in diastolic pressure (from 66 +/- 3 with vehicle to 39 +/- 4 mmHg, P < 0.01) and improving developed pressure after 30 min reperfusion (39 +/- 3 vs. 18 +/- 3 mmHg with vehicle, P < 0.01). Ivabradine reduced both CK and noradrenaline release by 47% (both P < 0.05 vs. vehicle) and improved mitochondrial respiratory control index (from 6.9 +/- 0.3 to 11.9 +/- 1.3, P < 0.001). It preserved cardiac energy metabolism (ATP, from 3.7 +/- 0.3 to 11.0 +/- 0.6 microM/g dry weight, P < 0.001) and redox state (NADPH/NADP(+), from 2.5 +/- 0.5 to 4.2 +/- 0.5, P < 0.001). There was a significant correlation between HR reduction in the ivabradine-treated hearts and cardiac creatine phosphate (r = 0.574, P = 0.02) and ATP levels (ATP, r = 0.674, P = 0.0042) at the end of ischaemia. These benefits were no longer detectable during pacing. HR reduction by ivabradine confers a marked anti-ischaemic benefit. It significantly reduces cardiac energy consumption, preserves redox potentials during ischaemia, and enhances recovery at reperfusion.

Remote Postconditioning is More Potent than Classic Postconditioning in Reducing the Infarct Size in Anesthetized Rabbits

Postconditioning confers protection to the heart after a potentially lethal episode of prolonged ischemia. There is evidence that it may also be protective when applied at a distal artery. In the present study, we sought to determine whether remote postconditioning within the heart (local) or outside the heart (distal) is effective in salvaging the ischemic heart in vivo and to compare its effect with that of the classic postconditioning. Twenty seven open chest New Zealand white anesthetized male rabbits were divided into four groups and were exposed to 30 min regional myocardial ischemia (isc), after ligation of a prominent coronary artery, followed by 3 h reperfusion (rep) after releasing the snare. Control group (n = 7) was subjected to no additional interventions, postC group (n = 6) was subjected to four cycles of 1 min isc/1 min rep of the same coronary artery at the beginning of reperfusion, remote local postC group (n = 7) to four cycles of 1 min isc/1 min rep of another coronary artery 30 s before the end of index isc and remote distal postC group (n = 7) to four cycles of 1 min isc/1 min rep of another (carotid) artery again 30 s before the end of index isc. Infarct size (I) and area at risk (R) were delineated with the aid of TTC staining and green fluorescent microspheres respectively and their ratio was expressed in percent (%I/R). Remote local and remote distal postC reduced the % I/R ratio (17.7 +/- 1.7% and 18.4 +/- 1.6%, respectively vs 47.0 +/- 2.5% in the control group, P < 0.01). Classic PostC had an intermediate protective effect (33.1 +/- 1.7%, P < 0.05 vs all the other groups). Remote postconditioning consisted of 1 min isc/1 min rep protects the ischemic rabbit heart in vivo, independently of the site of the remote artery. This intervention seems to confer a stronger protection than the classic postconditioning.

Effects of potassium aspartate and magnesium on ventricular arrhythmia in ischemia-reperfusion rabbit heart

The aim of this study was to determine if the potassium aspartate and magnesium (PAM) prevent reperfusion-induced ventricular arrhythmias (RIVA) in ischemia-reperfusion (IR) rabbit heart. Thirty rabbits were randomly divided into control, ischemia and PAM groups. Arterially-perfused rabbit left ventricular preparations were made, and transmural ECG as well as action potentials from both endocardium and epicardium were simultaneously recorded in the whole process of all experiments. In control group rabbit ventricular wedge preparations were continuously perfused with Tyrode's solution, and in ischemia group and PAM groups the perfusion of Tyrode's solution was stopped for 30 min. Then the ischemia group was reperfused with Tyrode's solution and the PAM group with Tyrode's solution containing 2.42 mg/L PAM, respectively. ECG, QT interval, transmural repolarization dispersion (TDR) and action potentials from epicardium and endocardium were simultaneously recorded, and the RIVA of the wedge preparation was observed. Compared with control group, TDR and incidence of RIVA were significantly increased in ischemia group (P<0.05). The incidence of RIVA in control, ischemia and PAM group was 0/10, 9/10 and 1/10, respectively. Compared with ischemia group, TDR and incidence of RIVA were significantly reduced in PAM group (P<0.05). Potassium aspartate and magnesium significantly reduce TDR and prevent ventricular arrhythmia in ischemic rabbit heart.

A107. Intrinsic signals of myocardial apoptosis

A protocol consisted of normaxia, ischemia, and reperfusion was employed to obtain the myocardial apoptosis signals by identifying different mitochondrial redox states. Ischemia in rabbit heart was induced by LAD occlusion and fluorescent signals are collected by applying two different fluorescence techniques: fluorometry, and cryoimaging. The first one is employed to detect FP (Flavoprotein) and NADH (Nicotinamide Adenine Dinucletide) signals in vivo during the experiments and the latter one used for low temperature fluorescence imaging.

Cytosolic calcium in the ischemic rabbit heart: assessment by pH- and temperature-adjusted rhod-2 spectrofluorometry

Cytosolic calcium ([Ca(2+)](i)) mediates ischemia-reperfusion (I/R) injury, but magnitude and time course of I/R-induced [Ca(2+)](i) overload remain unclear. Fluorescent indicators are commonly used to measure [Ca(2+)](i), and have a dissociation constant (K(d)) that depends on pH and temperature. We hypothesized that changes of K(d) during I/R lead to misleading interpretations of [Ca(2+)](i) recordings. (1) In isolated rabbit hearts (n=4 each), intracellular pH was measured during I/R at 37 degrees C, 20 degrees C, and 4 degrees C with and without cardioplegic arrest by (31)P-NMR-spectroscopy. (2) K(d) for rhod-2 and calcium was determined at varying pH and temperature in in vitro experiments. (3) Isolated rabbit hearts were subjected to I/R, and [Ca(2+)](i) was recorded by surface rhod-2 spectrofluorometry. Finally, [Ca(2+)](i) was computed using either the conventional K(d), or the pH- and temperature-adjusted K(d). K(d)(Ca(2+)Rhod-2) remained stable between pH 7.1 and 6.8, but increased exponentially with lower pH and/or temperature. Calculations using a static K(d) indicated that [Ca(2+)](i) rose only mildly during warm ischemia and did not rise during cardioplegic arrest, while a large Ca(2+) influx appeared to occur during early reperfusion. When the pH and temperature-adjusted K(d) was used for calculation, [Ca(2+)](i) rose significantly during ischemia (431+/-37% during 20 min 37 degrees C ischemia, and 78+/-19% during 20 min cardioplegic arrest at 20 degrees C). During early reperfusion, [Ca(2+)](i) decreased rapidly, without significant further [Ca(2+)](i) elevation. In contrast to previous reports, [Ca(2+)](i) accumulation occurs during unprotected ischemia as well as hypothermic ischemia with cardioplegic arrest, without further net Ca(2+) influx on reperfusion. This finding has important implications for timing of protective strategies during myocardial ischemia.

P1075 opens mitochondrial K ATP channels and generates reactive oxygen species resulting in cardioprotection of rabbit hearts

We have recently proposed that opening of mitochondrial K ATP channels (mitoK ATP) acts as a trigger for preconditioning (PC) by causing mitochondria to produce reactive oxygen species (ROS). Controversy exists as to whether the putative sarcolemma-selective K ATP channel opener P1075 also opens mitoK ATP channels and may be cardioprotective. We purified mitoK ATP channels from either rabbit heart, rat heart or rat brain and reconstituted the proteins into liposomes. mitoK ATP channels from each of these tissues were opened by P1075 with EC 50 values of 60–90 nM. We next tested whether P1075 causes rabbit cardiomyocytes to produce ROS in a K ATP-dependent fashion. Mitochondrial ROS production was monitored by the appearance of fluorescence as reduced MitoTracker™ Red was oxidized. P1075 (100 μM) led to a 44 ± 9% increase in ROS generation ( P < 0.001 vs. untreated cells), which was similar to the increase seen with 50 μM diazoxide, a selective mitoK ATP channel opener (49 ± 9%, P < 0.001 vs. untreated cells). The effect of P1075 was equally potent at a concentration of 150 nM. The P1075-induced increase in ROS production was blocked by 50 μM glibenclamide (GLI), a non-selective K ATP blocker, and by 5-hydroxydecanoate (1 mM), a highly selective mitoK ATP blocker (–6 ± 14% and +4 ± 12%, respectively; P = n.s). In isolated rabbit hearts, P1075 (150 nM) markedly reduced infarct size compared to control animals (10.6 ± 8.1% of the area at risk vs. 31.5 ± 5.6%, P < 0.05). GLI (5 μM) as well as 5-hydroxydecanoate (200 μM) completely blocked P1075’s anti-infarct effect (31.7 ± 9.5% and 27.7 ± 4.6% infarction, respectively; P = n.s. vs. untreated hearts). These data provide strong evidence that P1075 does open mitoK ATP channels and protects the ischemic rabbit heart in a mitoK ATP-dependent manner.

Cardioprotection by metformin is abolished by a nitric oxide synthase inhibitor in ischemic rabbit hearts.

We investigated the effects of metformin on myocardial metabolism during ischemia by 31P-nuclear magnetic resonance (NMR) in isolated rabbit hearts. Metformin was administered 60 min prior to induction of global ischemia, or in combination with a nitric oxide synthase inhibitor, N(G)-nitro-L-arginine methyl ester (L-NAME), at 5 min or 60 min prior to the ischemia. Normothermic global ischemia was then carried out for 45 min. Twenty-eight hearts were divided into 4 experimental groups consisting of 7 hearts each: a control (C) group; an M group receiving metformin treatment alone; an M+L (5) group receiving metformin treatment with L-NAME at 5 min before ischemia; and an M+L (60) group receiving metformin treatment with L-NAME at 60 min before ischemia. During ischemia, the decrease in adenosine triphosphate (ATP) was significantly inhibited in the M group in comparison with the C group (p < 0.01). However, this preservation of ATP in the M group was inhibited in the M+L (5) group during ischemia. In contrast, in the M+L (60) group, this preservation of ATP in the M group was not inhibited during, but not at the end of, ischemia. These results suggest that metformin has a significant beneficial effect for improving the myocardial energy metabolism during myocardial ischemia. This cardioprotection may be more dependent on nitric oxide synthase during ischemia than during pre-ischemia.

Anti-arrhythmic peptide N-3-(4-hydroxyphenyl)propionyl Pro-Hyp-Gly-Ala-Gly-OH reduces dispersion of action potential duration during ischemia/reperfusion in rabbit hearts.

During ischemia, cardiac gap junctions close and neighboring cells uncouple. This leads to slow conduction, increased dispersion of APD90 (duration from action potential beginning to 90% of repolarization), nonuniform anisotropy, and unidirectional conduction block, all of which favor the induction of reentry arrhythmias. It has been suggested that anti-arrhythmic peptides increase gap junction conductance during states of reduced coupling. The aim of this study was to test the effect of the anti-arrhythmic peptide N-3-(4-hydroxyphenyl)propionyl Pro-Hyp-Gly-Ala-Gly-OH (HP-5) (10(-10) ) on dispersion of epicardial APD90 during both normokalemic and hypokalemic ischemia/reperfusion in isolated perfused rabbit hearts. HP-5 did not affect average APD90, heart rate, left ventricular contractility (LVP dP/dtmax), or mean coronary flow. HP-5 significantly reduced the epicardial APD dispersion during hypokalemic ischemia (HP-5 treated: 24.1 +/- 3.4 ms, untreated: 33.9 +/- 3.1 ms, p < 0.05 versus untreated) and during normokalemic reperfusion but not during normokalemic ischemia or control conditions. In addition, among untreated hearts subjected to hypokalemic ischemia/reperfusion, seven of 10 developed ventricular fibrillation, whereas only three of nine hearts perfused with HP-5 developed ventricular fibrillation. These results show that HP-5 is able to reduce APD90 dispersion during hypokalemic ischemia in rabbit hearts.

Protection from AMP 579 can be added to that from either cariporide or ischemic preconditioning in ischemic rabbit heart.

AMP 579, an adenosine A /A receptor agonist, is cardioprotective when administered at reperfusion. Pretreatment with the Na /H exchanger inhibitor cariporide or ischemic preconditioning (PC) also limits infarct size. To gain insight into the mechanism of AMP 579 we investigated whether its protection could be added to that from either cariporide or PC. rabbit hearts were subjected to 45 min of regional ischemia followed by 3 h of reperfusion. Infarct size in the control group was 55.8 +/- 3.9% of the risk zone. PC significantly reduced infarct size to 26.0 +/- 6.7% (p<0.05). AMP 579 (30 micro g/kg) given just before reperfusion followed by 3 micro g/kg/min infusion for 70 min also limited infarct size (32.1 +/- 1.8%,) but the combination of AMP 579 and PC showed a significantly greater limitation of infarct size (5.5 +/- 2.7%, p < 0.05). Because cariporide pretreatment was so protective (8.5 +/- 3.7% infarction), we had to increase the ischemic insult to 60 min to test for any additive effect of the combination of AMP 579 + cariporide. Infarct size in the untreated group was 66.0 +/- 4.9% of the risk zone. Cariporide (0.5 mg/kg) 5 min prior to ischemia significantly reduced infarct size to 41.5 +/- 7.7%. When cariporide pre-treatment was combined with AMP 579 at reperfusion, infarction was further limited (14.2 +/- 4.5%). Because AMP 579's protection can be added to that of either cariporide or PC, AMP 579's mechanism of protection probably differs from either of them. The combination of AMP 579 + cariporide was particularly efficacious and could be useful in the surgical setting.

Manipulations in glycogen metabolism and the failure to influence infarct size in the ischaemic rabbit heart.

Myocardial ischaemic preconditioning is characterized by a reduction in the rate of glycolysis. Brief myocardial ischaemia also reduces the glycogen content of the heart. The first objective was to determine whether augmenting glucose oxidation by activation of the pyruvate dehydrogenase complex would prevent the infarct limitation of ischaemic preconditioning. The second part of the study evaluates whether glycogen depletion before ischaemia mimics the infarct-limiting effect of ischaemic preconditioning. Dichloroacetate (300 + 150 mg kg(-1)), an activator of the pyruvate dehydrogenase complex, was administered intravenously in the anaesthetized open-chest rabbit. All animals underwent 45 min of regional ischaemia and 3 h of reperfusion. Ischaemic preconditiong was elicited by 5 min of coronary occlusion. Control rabbits, those with ischaemic preconditioning with no dichloroacetate, received a saline vehicle. An isolated perfused rabbit heart model was employed to test the second hypothesis. Hearts were depleted of glycogen by perfusing them with a substrate-free buffer. Infarction was assessed by triphenyl tetrazolium chloride and area at risk determined with fluorescent particles. (a) Pyruvate dehydrogenase complex activation experiments. Treatment with dichloroacetate alone did not alter infarct size (58 +/- 7% control vs. 60 +/- 5% dichloroacetate). Addition of dichloroacetate did not attenuate the infarct-limiting effect of ischaemic preconditioning as evidenced by a similar reduction in infarct size in the ischaemic preconditioning group (22 +/- 5%) and in the ischaemic preconditioning + dichloroacetate group (27 +/- 7%). (b) Glycogen depletion experiments. Compared with control hearts with a normal glycogen content (4.84 +/- 0.15 mg g(-1) wet weight), glycogen depleted and ischaemic preconditioning hearts had reduced glycogen content before ischaemia (2.15 +/- 0.26, 1.62 +/- 0.17 mg g(-1) wet weight, respectively; P < 0.01). Glycogen depletion did not reduce infarct size: 25.0 +/- 4.5% cf. 27.9 +/- 3.4% in the control group. However, ischaemic preconditioning resulted in a significant reduction of infarct size (11.5 +/- 2.3% vs. 27.9 +/- 3.4% control; P < 0.01). Augmentation of oxidative glycolysis by dichloroacetate in in situ rabbit hearts does not alter the effect of ischaemic preconditioning, and glycogen depletion in the isolated rabbit heart does not influence infarct size after subsequent coronary occlusion.

Manipulations in glycogen metabolism and the failure to influence infarct size in the ischaemic rabbit heart

Background and objective: Myocardial ischaemic preconditioning is characterized by a reduction in the rate of glycolysis. Brief myocardial ischaemia also reduces the glycogen content of the heart. The first objective was to determine whether augmenting glucose oxidation by activation of the pyruvate dehydrogenase complex would prevent the infarct limitation of ischaemic preconditioning. The second part of the study evaluates whether glycogen depletion before ischaemia mimics the infarct-limiting effect of ischaemic preconditioning. Methods: Dichloroacetate (300 + 150 mg kg−1), an activator of the pyruvate dehydrogenase complex, was administered intravenously in the anaesthetized open-chest rabbit. All animals underwent 45 min of regional ischaemia and 3 h of reperfusion. Ischaemic preconditiong was elicited by 5 min of coronary occlusion. Control rabbits, those with ischaemic preconditioning with no dichloroacetate, received a saline vehicle. An isolated perfused rabbit heart model was employed to test the second hypothesis. Hearts were depleted of glycogen by perfusing them with a substrate-free buffer. Infarction was assessed by triphenyl tetrazolium chloride and area at risk determined with fluorescent particles. Results: (a) Pyruvate dehydrogenase complex activation experiments. Treatment with dichloroacetate alone did not alter infarct size (58 ± 7% control vs. 60 ± 5% dichloroacetate). Addition of dichloroacetate did not attenuate the infarct-limiting effect of ischaemic preconditioning as evidenced by a similar reduction in infarct size in the ischaemic preconditioning group (22 ± 5%) and in the ischaemic preconditioning + dichloroacetate group (27 ± 7%). (b) Glycogen depletion experiments. Compared with control hearts with a normal glycogen content (4.84 ± 0.15 mg g−1 wet weight), glycogen depleted and ischaemic preconditioning hearts had reduced glycogen content before ischaemia (2.15 ± 0.26, 1.62 ± 0.17 mg g−1 wet weight, respectively; P < 0.01). Glycogen depletion did not reduce infarct size: 25.0 ± 4.5% cf. 27.9 ± 3.4% in the control group. However, ischaemic preconditioning resulted in a significant reduction of infarct size (11.5 ± 2.3% vs. 27.9 ± 3.4% control; P < 0.01). Conclusions: Augmentation of oxidative glycolysis by dichloroacetate in in situ rabbit hearts does not alter the effect of ischaemic preconditioning, and glycogen depletion in the isolated rabbit heart does not influence infarct size after subsequent coronary occlusion.

Effects of an HMG-CoA reductase inhibitor in combination with an ACE inhibitor or angiotensin II type 1 receptor antagonist on myocardial metabolism in ischemic rabbit hearts.

We investigated the effects of a 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitor, pravastatin, an angiotensin converting enzyme (ACE) inhibitor, temocaprilat, and an angiotensin II type 1 (AT1) receptor antagonist, CV-11974, on myocardial metabolism during ischemia in isolated rabbit hearts using phosphorus 31-nuclear magnetic resonance (31P-NMR) imaging. Forty-five minutes of continuous normothermic global ischemia was carried out. Pravastatin, temocaprilat, CV-11974 or a nitric oxide synthase inhibitor, L-NAME was administered from 60 min prior to the global ischemia. Japanese white rabbits were divided into the following experimental groups, a control group (n=7), a group treated with pravastatin (P group; n=7), a group treated with pravastatin and temocaprilat (P+T group; n=7), a group treated with pravastatin and CV-11974 (P+CV group; n=7), and a group treated with pravastatin and L-NAME (P+L-NAME group; n=7). During ischemia, P group, as well as either P+T group or P+CV group, showed a significant inhibition of the decreases in adenosine triphosphate (ATP) and intracellular pH (pHi) (p<0.01, respectively, at the end of ischemia compared to the control group as well as P+L-NAME group), and a significant inhibition of the increase in inorganic phosphate (Pi) (p<0.01, respectively, compared with the control group as well as P+L-NAME group). These results suggest that pravastatin significantly improved myocardial energy metabolism during myocardial ischemia. This beneficial effect was dependent on NO synthase. However, this beneficial effect was not enhanced by either temocaprilat or CV-11974.

K(ATP) channel opening during ischemia: effects on myocardial noradrenaline release and ventricular arrhythmias.

Cardioprotection by K(ATP) channel openers during ischemia is well documented although ill understood. Proarrhythmic effects may be an important drawback. K(ATP) channel modulation influences neurotransmitter release during ischemia in brain synaptosomes. Therefore, we studied the effects of K(ATP) channel modulation on myocardial noradrenaline release and arrhythmias in ischemic rabbit hearts. Isolated rabbit hearts were perfused according to Langendorff and stimulated. Local electrograms were recorded and K+-selective electrodes were inserted in the left ventricular free wall. Cromakalim (3 microM) or glibenclamide (3 microM) was added 20 min prior to induction of global ischemia. After 15, 20, or 30 min of ischemia, hearts were reperfused and noradrenaline content of the first 100 ml of reperfusate was measured. Cromakalim (n = 16) prevented the second rise of extracellular [K(+)] in accordance with its cardioprotective effect. Cromakalim significantly reduced noradrenaline release after 15 min (mean, 169 +/- SEM 97 pmol/gr dry weight vs. control 941 +/- 278; p < 0.05) and 20 min of ischemia (230 +/- 125 pmol/gr dry wt vs. control 1,460 +/- 433; p < 0.05), but after 30 min of ischemia, the difference in noradrenaline release was no longer significant (cromakalim 2,703 +/- 1,195 pmol/gr dry wt vs. control 5,413 +/- 1,310; p = 0.08). Ventricular fibrillation or ventricular tachycardia occurred in 10 of 13 control hearts (77%) (n = 19), in six of 10 glibenclamide-treated hearts (60%) (n = 15), and in six of 14 cromakalim-treated hearts (43%) (p = NS). Cromakalim significantly accelerated onset of ventricular tachycardia or fibrillation (mean +/- SEM onset after 12.5 +/- 1.6 min ischemia vs. control 16.2 +/- 0.7 min; p < 0.05). Noradrenaline release occurred only in cromakalim-treated hearts with early-onset arrhythmias whereas no noradrenaline release was observed in cromakalim-treated hearts without ventricular tachycardia or fibrillation. Our results show that activation of the K(ATP) channel by cromakalim during ischemia reduces myocardial noradrenaline release and postpones the onset of irreversible damage, contributing to the cardioprotective potential of K(ATP) openers during myocardial ischemia.

STZ-induced diabetes alters the oxidation state of sulfhydryl (SH) groups on RyR2

The objective of present study was to investigate the role of antioxidant enzymes (superoxide dismutase and glutathione peroxidase), intracellular calcium and protein kinase C at various intervals of in-situ ischaemic preconditioned rabbit heart using isolated cardiac myocytes preparation. The heart of male Newzealand White rabbits were exposed and left anterior descending branch (LAD) of left coronary artery was ligated for 5 min., repetfused for 10 min., again ligated for 30 min. and then repetfused for 30 min. This protocol was divided into six groups (n=8/group). The antioxidant enzymes, lipid peroxidation (malondialdehyde), intracellular caldum and protein kinase C were estimated in the cardiac mywytes from three anatomical regions of the heart. There was a significant increase in antioxidant enzymes, intracellular calcium and protein kinase C obtained after ischaemic preconditioning. Hence antioxidant enzymes, intracellular calcium and protein kinase C play a significant role in protection provided by ischaemic preconditioning.

Role of cardiac ATP-sensitive K+ channels induced by HMG CoA reductase inhibitor in ischemic rabbit hearts.

Although 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitors can protect the myocardium against ischemic injury, the mechanisms of their effect have not yet been characterized at the cellular level. Therefore, we investigated the role of cardiac ATP-sensitive K+ (K(ATP)) channels induced by the HMG-CoA reductase inhibitor known as pravastatin on the myocardial metabolism during ischemia by phosphorus 31-nuclear magnetic resonance (31P-NMR) in isolated rabbit hearts. Forty-five min of continuous normothermic global ischemia was carried out. Pravastatin with or without the K(ATP) channel blocker glibenclamide or the nitric oxide synthase inhibitor L-NAME was administered beginning 60 min prior to the global ischemia. Twenty-eight hearts were divided into 4 experimental groups consisting of 7 hearts each: the control group, the P group consisting of pravastatin treatment, the P+G group consisting of pravastatin treatment with glibenclamide, and the P+L group consisting of pravastatin treatment with L-NAME. During ischemia, the decreases in adenosine triphosphate (ATP) and intracellular pH (pHi) were significantly inhibited in the P group in comparison with Control group (at end of ischemia, respectively; both p<0.01), as was the increase in inorganic phosphate (Pi) (at end of ischemia, p<0.01). However, the decreases in ATP and pHi and the increase in Pi were not inhibited in the P+G group during ischemia. The P+L group also showed no inhibition of the aforementioned parameters during the same period. These results suggest that pravastatin has a significant beneficial effect for improving the myocardial energy metabolism, which is provided by K(ATP) channels and nitric oxide (NO), during myocardial ischemia. The cardioprotection of HMG-CoA reductase inhibitor may be caused by the K(ATP) channels that are mediated by the NO.

Inhibition of adenosine uptake and augmentation of ischemia-induced increase of interstitial adenosine by cilostazol, an agent to treat intermittent claudication.

Cilostazol (Pletal), a quinolinone derivative with a cyclic nucleotide phosphodiesterase type 3 (PDE3) inhibitory activity, was recently approved by the Food and Drug Administration for treatment of symptoms of intermittent claudication (IC). However, the underlying mechanisms of action are not entirely clear. In this study, we showed that cilostazol inhibited adenosine uptake into cardiac ventricular myocytes, coronary artery smooth muscle, and endothelial cells with a median effective concentration (EC50) approximately 10 microM. In vivo, cilostazol increased cardiac interstitial adenosine levels after a 2-min ischemia in rabbit hearts (329 +/- 92% increase vs. 102 +/- 29% ischemia alone). The combination of cilostazol and 2-min ischemia reduced infarction from subsequent 30-min regional ischemia and 3 h of reperfusion (infarct size was 18 +/- 4% vs. 53 +/- 3% in the hearts with 2-min ischemia alone or 48 +/- 2% in the hearts treated with cilostazol alone). In contrast, milrinone had no effect on either adenosine uptake or interstitial adenosine levels. These data show that cilostazol, unlike milrinone, inhibits adenosine uptake, and thus potentiates adenosine accumulation from a 2-min ischemia. Future studies are needed to investigate the role of adenosine in the treatment of IC by cilostazol.