Second window of ischemic preconditioning regulates mitochondrial permeability transition pore by enhancing Bcl-2 expression

OBJECTIVE: The second window of protection (SWOP) following brief coronary artery occlusion begins at 24 h and may last up to 72 h and occurs via many unknown mechanisms. We investigated the role of the mitochondrial permeability transition pore (PTP), a non specific pore in the inner membrane of the mitochondria in this phenomenon. METHODS: Ischemic preconditioning (IP) was induced in Wistar rats by left coronary artery occlusion (four, 3-min episodes separated by 10 min of reperfusion) on day 1. On day 2, ischemia was induced with 30 min of ischemia and 120 min of reperfusion in IP and control rats. RESULTS: IP rats showed decreased myocardial infarction (MI) area vs. non-IP control rats (15.32 vs. 45.6%). Furthermore, IP rats had preserved cardiac function (heart rate, rate pressure product, coronary flow and aortic flow) and myocardial ATP (P<0.03), decreased tissue water content (73.2 vs. 90.6%), increased expression of Bcl-2, and less mitochondrial swelling, cytochrome C release and apoptosis (2.6 vs. 12.4%) when compared to sham-operated rats. Activation of the permeability transition pore with PTP activators lonidamine (10 mg/kg body weight) or atractyloside (5 mg/kg body weight) before the sustained ischemia on day 2 resulted in complete abolition of SWOP-mediated cytoprotective effects. These agents had no effect on the cytoprotective effects that took place during the first window of preconditioning. CONCLUSION: The cytoprotective effects of SWOP are dependent on PTP activation state and may involve upregulation of Bcl-2 expression.     

缺血后处理减轻大鼠肥厚心肌缺血再灌注损伤的观察

目的探讨缺血后处理对心肌肥厚大鼠离体心脏缺血再灌注损伤的影响及其信号机制。方法通过腹主动脉结扎建立大鼠心肌肥厚模型,用Landendorff装置建立心肌肥厚大鼠离体心脏缺血再灌注模型。观察缺血后处理对心肌肥厚大鼠离体缺血再灌注心脏左心室收缩压,冠状动脉流量,肌酸磷酸激酶和乳酸脱氢酶释放,心肌梗死范围,心肌组织中蛋白激酶B／Akt（Akt）、糖原合成酶激酶-3β（GSK-3β）磷酸化的影响。结果与缺血再灌注对照组相比,缺血后处理组心脏左心室收缩压、冠状动脉流量显著高,冠状动脉循环流出液中肌酸磷酸激酶、乳酸脱氢酶含量低,心肌梗死范围减小,心肌组织中磷酸化Akt（Ser473）、磷酸化GSK-3β（Set9）水平高,磷脂酰肌醇-3激酶（PI3K）抑制剂渥曼青霉素（wortmannin）能够抑制缺血后处理所致的磷酸化Akt（Ser473）、磷酸化GSK-3β（Set9）水平升高,但只能部分消除缺血后处理的心脏保护效应。结论缺血后处理能够减轻心肌肥厚大鼠离体心脏缺血再灌注损伤,PI3K／Akt／GSK-3信号途径参与介导缺血后处理对离体缺血再灌注肥厚心肌的保护作用。     

Opening of Ca-activated K channels is involved in ischemic preconditioning in canine hearts

Brief periods of ischemia that precede sustained ischemia can markedly reduce infarct size (IS), a phenomenon that is known as ischemic preconditioning (IP). Several investigators have shown that elevation of the intracellular Ca(2+) level ([Ca(2+)](i)) during the antecedent brief periods of ischemia triggers the cardioprotective mechanism of IP. Since opening of Ca(2+) activated K(+) (K(Ca)) channels is reported to be cardioprotective, we hypothesized that these channels may be involved in the cardioprotective mechanism of IP. In anesthetized open-chest dogs, myocardial ischemia/reperfusion injury was created by occlusion of the left anterior descending coronary artery (LAD) for 90 min followed by 6 h of reperfusion. First, we showed that the treatment with NS1619, a K(Ca) channel opener, reduced IS (IS in NS1619 group and control group, 19.8 +/- 5.5% vs. 45.4 +/- 3.5% of the area at risk, P < 0.05). Next, four cycles coronary occlusion for 5 min and reperfusion (IP) were performed before the 90-min occlusion with or without the infusion of potent K(Ca) channel inhibitors, iberiotoxin (IbTX) and charybdotoxin (ChTX). IP markedly reduced IS (IS in the IP group was 8.2 +/- 1.8%, P < 0.01 vs. control group). Infusion of either of K(Ca) channel blockers during IP blunted the IS-limiting effect of IP (IS in the IP + IbTX and IP + ChTX groups was 30.7 +/- 7.0% and 35.5 +/- 3.7%, respectively, P < 0.05, vs. IP group). However, the cardioprotective effect of IP was not blunted by the treatment with ChTX when treated only during reperfusion (14.0 +/- 4.1%). Thus, we conclude that the opening of K(Ca) channel is involved in early trigger phase of the molecular mechanism of IP.     

Opening of Ca2+-activated K+ channels is involved in ischemic preconditioning in canine hearts

Brief periods of ischemia that precede sustained ischemia can markedly reduce infarct size (IS), a phenomenon that is known as ischemic preconditioning (IP). Several investigators have shown that elevation of the intracellular Ca(2+) level ([Ca(2+)](i)) during the antecedent brief periods of ischemia triggers the cardioprotective mechanism of IP. Since opening of Ca(2+) activated K(+) (K(Ca)) channels is reported to be cardioprotective, we hypothesized that these channels may be involved in the cardioprotective mechanism of IP. In anesthetized open-chest dogs, myocardial ischemia/reperfusion injury was created by occlusion of the left anterior descending coronary artery (LAD) for 90 min followed by 6 h of reperfusion. First, we showed that the treatment with NS1619, a K(Ca) channel opener, reduced IS (IS in NS1619 group and control group, 19.8 +/- 5.5% vs. 45.4 +/- 3.5% of the area at risk, P < 0.05). Next, four cycles coronary occlusion for 5 min and reperfusion (IP) were performed before the 90-min occlusion with or without the infusion of potent K(Ca) channel inhibitors, iberiotoxin (IbTX) and charybdotoxin (ChTX). IP markedly reduced IS (IS in the IP group was 8.2 +/- 1.8%, P < 0.01 vs. control group). Infusion of either of K(Ca) channel blockers during IP blunted the IS-limiting effect of IP (IS in the IP + IbTX and IP + ChTX groups was 30.7 +/- 7.0% and 35.5 +/- 3.7%, respectively, P < 0.05, vs. IP group). However, the cardioprotective effect of IP was not blunted by the treatment with ChTX when treated only during reperfusion (14.0 +/- 4.1%). Thus, we conclude that the opening of K(Ca) channel is involved in early trigger phase of the molecular mechanism of IP.     

心肌缺血预适应引起的ATP敏感性钾电流变化

许多研究证实三磷酸腺苷敏感性钾电流 (KATP)在心肌保护的机制中起重要作用 ,但尚未有缺血预适应 (IPC)期间KATP电流变化的直接报道。本实验用全细胞膜片钳技术在豚鼠心室肌细胞上观察了多次模拟缺血 (低氧、去能量 )和再灌注期间KATP电流的变化情况。结果 :对照组 (n =9)和IPC组 (n =12 )的KATP电流分别由实验开始时的- 97± 14和 - 94± 16pA开放至第 3次短暂缺血结束时的 - 5 7± 10和 - 16± 2 0pA(P <0 .0 5 ) ,以及持续缺血 5min时的 35± 2 3和 472± 310pA(P均 <0 .0 1) ;然而在持续缺血晚期和再灌注过程中KATP通道的开放程度在两组之间无显著差异。以上这些效应可被优降糖阻断。结论 :本文首次直接观察到IPC可导致KATP通道在预适应末及随后长时间缺血早期的适度激活 ,但不影响长时间缺血晚期和再灌注过程中的开放程度 ,为进一步研究IPC的发生机制和开发KATP开放剂作为新型抗缺血性心脏病药物提供了理论基础     

Effects of preconditioning on myocardial interstitial levels of ATP and its catabolites during regional ischemia and reperfusion in the rat

The interstitial accumulation of adenine nucleotide breakdown products (ANBP) in the myocardium during ischemia has been shown to provide a useful index of the ischemic injury, whereas reperfusion ANBP washout rate has been regarded as an index of reperfusion damage. The purpose of this study was, using cardiac microdialysis, to examine in the rat model of regional ischemia/reperfusion the relationship between the duration of ischemia and these indices and to assess the profile of interstitial ATP concentrations and the beneficial effects of ischemic preconditioning (IP). The rats underwent 10, 20, 30 or 40 min of coronary artery occlusion and 50 min of reperfusion. Regional ischemia, with its duration, provoked a progressive increase in dialysate ANBP in the ischemic zone. The rate of purine washout during reperfusion exponentially declined with an increase in duration of the ischemic period. IP, induced by three 5-min episodes of ischemia, each separated by 5 min of reperfusion, significantly reduced the accumulation of ANBP during the 30-min period of sustained ischemia and resulted in a marked acceleration of reperfusion ANBP washout, indicating the improvement of postischemic microcirculation. These effects were suggested to be, at least in part, responsible for the infarct size limitation observed. Using the relationship between the duration of ischemia and ANBP washout rate, it could be demonstrated that IP produced similar facilitation of purine washout as shortening of the ischemic period in nonpreconditioned rats from 30 to approximately 7 min. Regional 20-min ischemia induced an early peak increase in interstitial fluid ATP which correlated with the maximal incidence of ventricular arrhythmias, whereas IP abolished both ATP release and arrhythmias during the sustained ischemia. These findings suggest that ATP may be an important mediator of ischemia-induced ventricular arrhythmias. Received: 20 April 1999, Returned for 1. revision: 11 May 1999, 1. Revision received: 23 June 1999, Returned for 2. revision:15 July 1999, 2. Revision received: 27 September 1999, Accepted: 29 September 1999     

Protective effects of ischemic preconditioning and application of lipoic acid prior to 90 min of hepatic ischemia in a rat model

AIM： To compare different preconditioning strategies to protect the liver from ischemia/reperfusion injury focusing on the expression of pro- and anti-apoptotic proteins. Interventions comprised different modes of ischemic preconditioning （IP） as well as pharmacologic pretreatment by α-lipoic acid （LA）.
METHODS： Several groups of rats were compared： sham operated animals, non-pretreated animals （nt）, animals receiving IP （10 rain of ischemia by clamping of the portal triad and 10 min of reperfusion） prior to sustained ischemia, animals receiving selective ischemic preconditioning （IPsel, 10 min of ischemia by selective clamping of the ischemic lobe and 10 rain of reperfusion） prior to sustained ichemia, and animals receiving 500 1μmol α-LA injected i.v. 15 min prior to the induction of 90 min of selective ischemia.
RESULTS： Cellular damage was decreased only in the LA group. TUNEL-positive hepatocytes as well as necrotic hepatocyte injury were also decreased only by LA（19 ± 2 vs 10 ± 1, P〈 0.05 and 29 ± 5 vs 12 ± 1, P 〈 0.05）. Whereas caspase 3- activities in liver tissue were unchanged, caspase 9- activity in liver tissue was decreased only by LA pretreatment （3.1 ± 0.3 vs 1.8 ± 0.2, P 〈 0.05）. Survival rate as the endpoint of liver function was increased after IP and LA pretreatment but not after IPsel. Levels of lipid peroxidation （LPO） in liver tissue were decreased in the IP as well as in the LA group compared to the nt group. Determination of pro- and anti-apoptotic proteins showed a shift towards anti-apoptotic proteins by LA. In contrast, both our IP strategies failed to influence apototic cell death.
CONCLUSION： IP, consisting of 10 min of ischemia and 10 min of reperfusion, ischemia/reperfusion injury protects only partly against of the liver prior to 90 min of selective ischemia. IPsel did not influence ischemic tolerance of the liver. LA improved tolerance to ischemia, possibly by downregulation of pro-apoptotic Bax.     

Relationship between the high-energy phosphate content and various left ventricular functional parameters of the normal and hypertrophied heart after global ischemia and reperfusion

Using an isolated rat heart preparation (Langendorff perfusion, perfusion pressure 100 cm H2O) the correlation between the high-energy phosphate content and various left ventricular (lv) functional parameters of the hypertrophied heart (spontaneous hypertensive rats lv/body weight ratio 3.6 +/- 0.5 x 10(-3) was determined after normo- (30 min) and hypothermic (25 degrees C, 120 min) cardioplegic arrest and reperfusion, and compared with normal hearts (Wistar rats lv/body weight ratio 2.0 +/- 0.3 x 10(-3). St. Thomas Hospital solution was used as the cardioplegic agent. Before ischemia hypertrophied hearts had a significantly higher developed left ventricular pressure, pressure rate product and dp/dtmax, but a significantly lower ATP and total adenine nucleotide content. Irrespective of the mode and temperature of cardiac arrest there was a strong correlation both for normal and for hypertrophied hearts between the high-energy phosphate content expressed as ATP, total adenine nucleotides or the "energy charge" and the left ventricular functional parameters pressure rate product and dp/dtmax. The correlation coefficient ranged from 0.80 to 0.89 and was highest when the ATP content was plotted against pressure rate product (r = 0.89). There was a different slope for normal and hypertrophied hearts with a steeper decline of the left ventricular function in hypertrophied hearts for any given reduction of the myocardial adenine nucleotide content. Our results indicate that a similar reduction of the ATP or total adenine nucleotide content in both the normal and hypertrophied heart reduces left ventricular function to a greater degree in the hypertrophied heart.     

Direct activation of mitochondrial K(ATP) channels mimics preconditioning but protein kinase C activation is less effective in middle-aged rat hearts

OBJECTIVES: This study is aimed to determine whether loss of preconditioning (IP) effects in the middle-aged hearts (MA) is due to the failure of protein kinase C (PKC) activation and, if so, whether direct activation of mitochondrial ATP-sensitive potassium channels (m-K(ATP)) or PKC mimics IP. BACKGROUND: PKC is a mediator and m-K(ATP) may be its downstream effector of IP in young adult hearts (YA), but we have demonstrated that IP is not effective in MA. METHODS AND RESULTS: Isolated hearts from YA (12-week) and MA (50-week) Fischer 344 rats were preconditioned by three cycles of ischemia and reperfusion (5 min each), and the translocation of PKC isoforms and the effects on reperfusion injury were assessed. In some hearts activation of m-K(ATP) or PKC by diazoxide or 1, 2-dioctanoyl glycerol (DOG) was performed before 25 min of global ischemia/30 min of reperfusion. IP could improve the recovery of LV function and resulted in higher content of ATP after reperfusion in YA but these beneficial effects of IP was not found in MA. The effects of IP in YA were abolished by 5-hydroxydecanoate. In YA but not in MA, immunohistochemical analysis revealed that IP translocated PKC-alpha and delta from the cytosolic or membrane to the perinuclear region but immunoblotting analysis showed translocation of PKC-alpha, delta and epsilon to the membrane fraction. Pretreatment with diazoxide or DOG mimicked IP and decreased the creatine kinase release in YA. Diazoxide was also effective but effects of DOG were less in MA as compared with in YA. CONCLUSIONS: IP is not effective in MA hearts partly due to failure of translocation of PKC isoforms. Moreover, less efficacy of PKC activation by DOG as compared with activities of m-K(ATP) by diazoxide in MA may suggest that defect(s) of cell signaling downstream to PKC may also be involved in the loss of IP effects in MA.     

Postischemic recovery of mechanical performance and energy metabolism in the presence of left ventricular hypertrophy. A 31P-MRS study

The present study was undertaken to define the effects of left ventricular hypertrophy on postischemic recovery of myocardial performance and high energy phosphate metabolism. Hemodynamics and 31P-magnetic resonance spectra were monitored simultaneously in the isolated Langendorff-perfused rat heart during 30 minutes of ischemia and 30 minutes of reperfusion. Left ventricular hypertrophy was produced by either suprarenal aortic constriction or chronic thyroxine administration. In chronic pressure overload hypertrophy, minimal coronary resistance was significantly higher (p less than 0.001) and the loss of purine nucleosides in the coronary effluent during early reperfusion significantly larger (p less than 0.001) compared with both normal hearts and thyroxine-induced hypertrophied hearts. Postischemic recovery of the baseline values for left ventricular developed pressure and phosphorylation potential was 43 +/- 4% and 82 +/- 4%, respectively, in chronic pressure overload hypertrophied hearts; 86 +/- 4% and 91 +/- 3%, respectively, in normal hearts (chronic pressure overload hypertrophy versus normal hearts, p less than 0.001 and p less than 0.05); and 100 +/- 4% and 98 +/- 2%, respectively, in thyroxine-induced hypertrophied hearts (normal hearts versus thyroxine-induced hypertrophied hearts, p less than 0.05 and p less than 0.05). Recovery after reperfusion was not related to intracellular pH, ATP, phosphocreatine, or inorganic phosphate levels during ischemia. Also, recovery was not related to developed pressure or oxygen consumption before ischemia. However, recovery was inversely related to coronary resistance and directly related to coronary flow before ischemia. Thus, functional and/or anatomic alterations of the coronary vascular bed and a greater loss of purine nucleosides during reperfusion are likely responsible for the attenuated compensatory response to ischemia and reperfusion in left ventricular hypertrophy induced by chronic pressure overload. On the other hand, the excess muscle mass per se does not seem to alter recovery, since thyroxine-induced myocardial hypertrophied hearts responded at least as well as normal hearts.     

Nicorandil improves post-ischemic myocardial dysfunction in association with opening the mitochondrial K(ATP) channels and decreasing hydroxyl radicals in isolated rat hearts.

BACKGROUND: Nicorandil has been reported to induce cardioprotection by opening the mitochondrial K(ATP) channels. However, whether nicorandil affects reactive oxygen species is unclear. METHODS AND RESULTS: The hearts of male Sprague-Dawley rats were excised and perfused on a Langendorff apparatus with Krebs-Henseleit solution with a gas mixture of 95% O(2) and 5% CO(2). 1 mmol/L of nicorandil was given 10 min before ischemia. Left ventricular developed pressure (LVDP, mmHg), +/-dP/dt (mmHg/s) and coronary flow (ml/min) were continuously monitored. All hearts were perfused for a total of 120 min consisting of a 30 min pre-ischemic period, followed by a 30 min global ischemia and 60 min reperfusion with and without 5-hydroxydecanoic acid sodium salt (5-HD), a mitochondrial K(ATP) channel blocker. The concentrations of 2,3-dihydroxybenzoic acid (2,3-DHBA), an indicator of hydroxyl radicals, in the perfusate during reperfusion period were also measured. Nicorandil significantly improved LVDP and +/-dP/dt, and increased coronary flow during reperfusion. Pretreatment with 5-HD abolished the improvement of LVDP and +/-dP/dt, and the increase in coronary flow induced by nicorandil. Nicorandil significantly attenuated the concentrations of 2,3-DHBA during reperfusion, which were restored by 5-HD. CONCLUSION: Nicorandil is protective against post-ischemic left ventricular dysfunction in association with opening the mitochondrial K(ATP) channels, decreasing hydroxyl radicals and increasing coronary flow in the isolated rat heart.     

Effects of mitochondrial K(ATP) modulators on cardioprotection induced by chronic high altitude hypoxia in rats

OBJECTIVES: Adaptation of rats to intermittent high altitude hypoxia increases the tolerance of their hearts to acute ischemia/reperfusion injury. Our aim was to examine the role of mitochondrial ATP-sensitive potassium channels (K(ATP)) in this form of protection. METHODS: Adult male Wistar rats were exposed to hypoxia of 5000 m in a barochamber for 8 h/day, 5 days a week; the total number of exposures was 24-32. A control group was kept under normoxic conditions (200 m). Infarct size (tetrazolium staining) was measured in anesthetized open-chest animals subjected to 20-min regional ischemia (coronary artery occlusion) and 4-h reperfusion. Isolated perfused hearts were used to assess the recovery of contractile function following 20-min global ischemia and 40-min reperfusion. In the open-chest study, a selective mitochondrial K(ATP) blocker, 5-hydroxydecanoate (5 mg/kg), or openers, diazoxide (10 mg/kg) or BMS-191095 (10 mg/kg), were administered into the jugular vein 5 and 10 min before occlusion, respectively. In the isolated heart study, 5-hydroxydecanoate (250 micromol/l) or diazoxide (50 micromol/l) were added to the perfusion medium 5 or 10 min before ischemia, respectively. RESULTS: In the control normoxic group, infarct size occupied 62.2+/-2.0% of the area at risk as compared with 52.7+/-2.5% in the chronically hypoxic group (P<0.05). Post-ischemic recovery of contractile function (dP/dt) reached 60.0+/-3.9% of the pre-ischemic value and it was improved to 72.4+/-1.2% by adaptation to hypoxia (P<0.05). While 5-hydroxydecanoate completely abolished these protective effects of chronic hypoxia, it had no appreciable influence in normoxic groups. In contrast, diazoxide significantly increased the recovery of contractile function and reduced infarct size in normoxic groups only. The later effect was also observed following treatment with BMS-191095. CONCLUSION: The results suggest that opening of mitochondrial K(ATP) channels is involved in the cardioprotective mechanism conferred by long-term adaptation to intermittent high altitude hypoxia.     

The reversal of the protective effects of intermittent perfusion on ischemic myocardium by hypoxic, no-substrates, zero-K+ perfusate

Intermittent perfusion during ischemia protected ischemic myocardium and improved recovery of function. These protective effects were reversed when hearts were perfused intermittently with hypoxic, no-substrates, zero-K+ buffer instead of oxygenated standard buffer containing substrates. We investigated the mechanisms of this reversal in isolated rat hearts. After 40 mins of sustained global ischemia, intracellular Na (Nai) increased by 6 times along with decrease in ATP and accumulation of lactate. During 30 mins of reperfusion, 45Ca2+ uptake reached 10.0 mumol/g dry with reduced recovery of ventricular function (LVEDP from 1 to 48 mmHg). When the 40 min period of ischemia was interrupted at 10 min intervals by 3 mins of IP, Nai didn't increase and reperfusion resulted in no increase in 45Ca2+ uptake (0.5 mumol/g dry). Recovery of function was 100% of the preischemic value without elevation of LVEDP. When hypoxic buffer without substrate and K+ was used for IP, Nai increased more rapidly with less recovery of function and more increased 45Ca2+ uptake (8 times) than sustained ischemia. These results indicate that disappearance of prevention of an increase in Nai with increased Ca2+ overload in hypoxic, no-substrates, zero-K+ IP, which resulted from accelerated ATP depletion and inhibition of Na/K pump is probably the main cause of the reversal of the protective effects.     

Sarcoplasmic reticulum function in the "stunned" myocardium

Transient ischemia does not induce myocardial necrosis but may be associated with prolonged contractile dysfunction ("stunned" myocardium). It has been suggested that alteration of the excitation-contraction coupling system (sarcoplasmic reticulum) could be responsible for this phenomenon. We tested this hypothesis by characterizing sarcoplasmic reticulum (SR) function in an isolated rat heart model of "stunned" myocardium (hearts reperfused after 10 min of normothermic global ischemia). At the end of the ischemic period oxalate-supported Ca-uptake was depressed either in the whole homogenate or in isolated SR (to 47% and 22% of control values, respectively). During reperfusion Ca-uptake of the whole heart homogenate recovered almost completely whereas slight but significant depression persisted in isolated SR (48 +/- 2 vs 67 +/- 4 nmol/min x mg, P less than 0.01). In the presence of ruthenium red or ryanodine, two inhibitors of SR Ca-release channels, Ca-uptake was stimulated. Both in the whole heart homogenate and in isolated SR, such stimulation was remarkably smaller after reperfusion than in control conditions (P less than 0.001) suggesting reduced conductivity state of the SR Ca-release channels. Ca-stimulated, magnesium-dependent ATPase activity was remarkably reduced during ischemia and postischemic reperfusion induced only incomplete recovery (93 +/- 18 vs 169 +/- 14 nmol ATP/min x mg protein, P less than 0.05). We conclude that complex modifications of SR function occur in the "stunned" myocardium and could contribute to the contractile impairment found in this condition.     

Contribution of Na+/H+ exchange to Na+ overload in the ischemic hypertrophied hyperthyroid rat heart

OBJECTIVE: The mechanisms responsible for intracellular ion homeostasis in ischemic hypertrophied myocardium are not fully known. Moderately hypertrophied hyperthyroid hearts (T3) are characterized by the bioenergetic changes and increased Na+/H+ exchange (NHE) activity comparable with those observed in humans and experimental models of hypertrophy. Here we test the hypothesis whether NHE inhibition in T3 heart improves ion homeostasis during ischemia and contractile function during recovery. METHODS: We compared intracellular H+ (H+i) and Na+ (Na+i) accumulations during 28 min global ischemia in isolated perfused T3 and euthyroid (EUT) rat hearts with and without NHE inhibition by using 31P and 23Na NMR. Heart function was measured during control perfusion and 30 min following ischemic insult. RESULTS: In T3 hearts ischemia caused: (1) faster and greater Na+i accumulation (534+/-25% of preischemic level versus 316+/-22% in EUT, P<0.001); (2) lower acidification (pH(i) 6.66+/-0.66 versus 6.12+/-0.12 in EUT, P<0.001); and (3) faster hydrolysis of ATP. NHE inhibition (amiloride 1 mM) in T3 hearts lead to: (1) delayed and lower Na+i accumulation by 35+/-5%; (2) faster and greater acidification (pH(i) 6.45+/-0.15, P<0.05); (3) delayed ATP degradation; and (4) improved heart function during recovery. When NHE was inhibited, all T3 hearts (n=11) recovered 68+/-10% of their preischemic rate pressure product (RPP), while only two untreated T3 hearts (from 11) recovered approximately 40% of preischemic RPP. CONCLUSIONS: These data suggest that NHE inhibition could be useful intervention for the prevention of ischemic/reperfusion cell injury and could improve the function of the hypertrophied heart after acute ischemia.     

Intestinal ischemia preconditions myocardium: role of protein kinase C and mitochondrial K(ATP) channel

OBJECTIVE: The present study was designed to test the hypothesis that intestinal ischemia results in an early preconditioning against myocardial infarction and that the mechanism of the early preconditioning involves the activation of protein kinase C-mitochondrial K(ATP) channel signaling pathway in anesthetized rats. METHODS: Rats were either preconditioned with a 25-min occlusion of the superior mesenteric artery followed by 15 min of reperfusion or underwent a 40-min sham period. Subsequently, all rats were subjected to a sustained 30 min of coronary occlusion and 180 min of reperfusion. Infarct size was determined by triphenyltetrazolium chloride staining. RESULTS: In sham-operated rats receiving no pharmacological intervention, the percentage of myocardial infarct within the area at risk and left ventricle was 73+/-4% and 31+/-2%, respectively, and these were significantly reduced to 44+/-4% and 23+/-1% (P<0.01) after intestinal ischemia preconditioning. Intravenous injection of protein kinase C inhibitors chelerythrine (5 mg/kg) and staurosporine (50 microg/kg) or a specific mitochondrial K(ATP) channel inhibitor 5-hydroxydecanoate (5 mg/kg) 5 min before sustained myocardial ischemia abolished the preconditioning afforded by intestinal ischemia. However, hexamethonium, a ganglion blocker, did not attenuate the preconditioning. CONCLUSIONS: These data provide pharmacological evidence that protein kinase C and mitochondrial K(ATP) channel are involved in the mechanism of the early preconditioning induced by intestinal ischemia.     

Nitrite confers protection against myocardial infarction: role of xanthine oxidoreductase, NADPH oxidase and K(ATP) channels

Reduction of nitrite to nitric oxide during ischemia protects the heart against injury from ischemia/reperfusion. However the optimal dose of nitrite and the mechanisms underlying nitrite-induced cardioprotection are not known. We determined the ability of nitrite and nitrate to confer protection against myocardial infarction in two rat models of ischemia/reperfusion injury and the role of xanthine oxidoreductase, NADPH oxidase, nitric oxide synthase and K(ATP) channels in mediating nitrite-induced cardioprotection. In vivo and in vitro rat models of myocardial ischemia/reperfusion injury were used to cause infarction. Hearts (n=6/group) were treated with nitrite or nitrate for 15 min prior to 30 min regional ischemia and 180 min reperfusion. Xanthine oxidoreductase activity was measured after 15 min aerobic perfusion and 30 min ischemia. Nitrite reduced myocardial necrosis and decline in ventricular function following ischemia/reperfusion in the intact and isolated rat heart in a dose- or concentration-dependent manner with an optimal dose of 4 mg/kg in vivo and concentration of 10 microM in vitro. Nitrate had no effect on protection. Reduction in infarction by nitrite was abolished by the inhibition of flavoprotein reductases and the molybdenum site of xanthine oxidoreductase and was associated with an increase in activity of xanthine dehydrogenase and xanthine oxidase during ischemia. Inhibition of nitric oxide synthase had no effect on nitrite-induced cardioprotection. Inhibition of NADPH oxidase and K(ATP) channels abolished nitrite-induced cardioprotection. Nitrite but not nitrate protects against infarction by a mechanism involving xanthine oxidoreductase, NADPH oxidase and K(ATP) channels.     

Effects of regression of left ventricular hypertrophy following atenolol or bunazosin therapy on ischemic cardiac function and myocardial metabolism in spontaneously hypertensive rats.

The effects of regression of left ventricular hypertrophy following atenolol and bunazosin therapy on ischemic cardiac function and myocardial metabolism in spontaneously hypertensive rats (SHR) were studied. Atenolol (50 mg/kg/day) and bunazosin (5 mg/kg/day) were administered to SHR from 19 to 26 weeks of age, whereas tap water was given to control SHR and normotensive Wistar-Kyoto rats (WKY). Both atenolol and bunazosin significantly decreased arterial blood pressure and significantly decelerated the increase in left ventricular weight in SHR. At the end of the long-term treatment, hearts were removed and perfused by the working heart technique for 15 min, and then global ischemia was induced for either 10 or 30 min. The ischemic heart was reperfused for 30 min. The pressure-rate product and the extent of recovery of the coronary flow after reperfusion following 30 min of ischemia in the bunazosin-treated SHR were significantly higher than those in the control SHR and the atenolol-treated SHR. The levels of adenosine triphosphate (ATP), creatine phosphate (CrP), and energy charge potential in the SHR heart reperfused after 30 min of ischemia were significantly lower than those in the reperfused WKY. Both atenolol and bunazosin improved the restoration of ATP and CrP in SHR after reperfusion following 30 min of ischemia. In conclusion, antihypertensive therapy with either atenolol or bunazosin was effective in preventing cardiac hypertrophy and ischemic damage caused by different mechanisms. Factors resulting from stimulation of the cardiac alpha 1 adrenoceptor may play an important role in the development of hypertensive cardiac hypertrophy, just as factors resulting from stimulation of the beta 1-adrenoceptor do.     

Roles of mitochondrial ATP-sensitive K channels and PKC in anti-infarct tolerance afforded by adenosine A1 receptor activation

OBJECTIVES: This study intended to assess the role of mitochondrial ATP-sensitive potassium (mitoK ATP) channels and the sequence of signal transduction with protein kinase C (PKC) and adenosine A1 receptors in rabbits. BACKGROUND: To our knowledge, the link between trigger receptors of preconditioning, PKC and mitoK ATP channels has not been examined in a whole heart model of infarction. METHODS: In the first series of experiments, myocardial infarction was induced in isolated buffer-perfused rabbit hearts by 30-min global ischemia and 2-h reperfusion. Infarct size in the left ventricle was determined by tetrazolium staining and expressed as a percentage of area at risk (i.e., the whole left ventricle) (%IS/AR). In the second series of experiments, mitochondria were isolated from the heart, and their respiratory function was examined using glutamate as a substrate. RESULTS: Pretreatment with R-phenylisopropyladenosine (R-PIA, 1 micromol/liter), an A1-receptor agonist, reduced %IS/AR from 49.8 +/- 6.5% to 13.4 +/- 2.9%. This protection was abolished by calphostin C, a PKC inhibitor, and by 5-hydroxydecanoate (5-HD), a selective inhibitor of mitoK ATP channels. A selective mitoK ATP channel opener, diazoxide (100 micromol/liter), mimicked the effect of R-PIA on infarct size (%IS/AR = 11.6 +/- 4.0%), and this protective effect was also abolished by 5-HD. However, calphostin C failed to block the infarct size-limiting effect of diazoxide. Neither calphostin C nor 5-HD alone modified %IS/AR. State III respiration (QO2) and respiratory control index (RCI) were reduced after 30 min of ischemia (QO2 = 147.3 +/- 5.3 vs. 108.5 +/- 12.3, RCI = 22.3 +/- 1.1 vs. 12.1 +/- 1.8, p < 0.05). This mitochondrial dysfunction was persistent after 10 min of reperfusion (QO2 = 96.1 +/- 15.5, RCI = 9.5 +/- 1.9). Diazoxide significantly attenuated the respiratory dysfunction after 30 min of ischemia (QO2 = 142.8 +/- 9.7, RCI = 16.2 +/- 0.8) and subsequent 10-min reperfusion (QO2 = 135.3 +/- 7.2, RCI = 19.1 +/- 0.8). CONCLUSIONS: These results suggest that mitoK ATP channels are downstream of PKC in the mechanism of infarct-size limitation by A1-receptor activation and that the anti-infarct tolerance afforded by opening of mitoK ATP channels is associated with preservation of mitochondrial function during ischemia/reperfusion.     

Activation of kappa-opioid receptors at reperfusion affords cardioprotection in both rat and mouse hearts

The temporal properties of kappa-opioid receptor (kappa-OR) mediated cardioprotection are less well characterised than delta-opioid receptor (delta-OR) responses. This study was aimed at delineating the time course of kappa-OR-mediated protection in two experimental models: an in vivo rat model of regional myocardial infarction (30 min of left coronary artery occlusion with 120 min of reperfusion), and an in vitro perfused murine heart model (undergoing 25 min of global ischemia and 45 min of reperfusion). In the rat model, the selective kappa-OR agonist U50, 488 (0.1 mg/kg, IV bolus), administered either 10 min prior to ischemia or 5 min prior to reperfusion, significantly reduced infarct size (38 +/- 3% and 43 +/- 2% infarct size/area-at-risk (IS/AAR), respectively; P < 0.05) compared to untreated rats (56 +/- 1% IS/AAR). Administration of U50, 488 10 s after onset of reperfusion failed to elicit protection. Cardioprotection with U50,448 administered immediately prior to reperfusion was abolished by a kappa-OR antagonist, (0.1 mg/kg nor-BNI), given 10 min prior to reperfusion. In the in vitro murine model, untreated hearts exhibited 28 +/- 2% (IS/AAR) infarct size. Infusion of U50, 488 (at a final 100 nM concentration) significantly limited infarct size in mouse hearts when applied at the onset of reperfusion (15 +/- 2% IS/AAR; P < 0.05), yet failed to afford protection when infused prior to ischemia. Additionally, in both models studied, treatment with either wortmannin or 5-hydroxydecanoate (5-HD) abrogated the protective effects of U50,488 applied just prior to reperfusion. In summary, kappa-ORs afford cardioprotection primarily when activated prior to and not after reperfusion. This protection may involve activation of the PI3 kinase (PI3K) pathway and mitochondrial (mito) K (ATP) channels.