Effect of estrogen on global myocardial ischemia-reperfusion injury in female rats

We investigated the effects of estrogen on global myocardial ischemia-reperfusion injury in rats that were ovariectomized (Ovx), sham-operated, or ovariectomized and then given 17beta-estradiol (E(2)beta) supplementation (Ovx+E(2)beta). Hearts were excised, cannulated, perfused with and then immersed in chilled (4 degrees C) cardioplegia solution for 30 min, and then retrogradely perfused with warm (37 degrees C), oxygenated Krebs-Henseleit bicarbonate buffer for 120 min. The coronary flow rate, first derivative of left ventricular pressure, and nitrite production were all significantly lower in Ovx than in sham-operated or Ovx+E(2)beta hearts. However, coronary flow rates or nitrate production were not consistently different throughout the entire reperfusion period. Ca(2+) accumulated more in Ovx rat hearts than in sham-operated or Ovx+E(2)beta hearts, and mitochondrial respiratory function was lower in Ovx hearts than in hearts from the other two groups. Marked interstitial edema and contraction bands were seen in hematoxylin-eosin-stained sections of Ovx rat hearts but not in hearts from either of the other groups. Hematoxylin-basic fuchsin-picric acid-stained sections revealed fewer viable myocytes in hearts from the Ovx group than from the sham or Ovx+E(2)beta group. Transmission electron microscopy demonstrated more severely damaged mitochondria and ultrastructural damage to myocytes in Ovx rat hearts. Our results indicate that estrogen plays a cardioprotective role in global myocardial ischemia-reperfusion injury in female rats.     

Estrogen receptor-alpha mediates acute myocardial protection in females

Sex differences in myocardial recovery have been reported after acute ischemia and reperfusion injury. Estrogen and the estrogen receptor are critical determinants of cardiovascular sex differences. However, the mechanistic pathways responsible for these differences remain unknown. We hypothesized that estrogen receptor-alpha is an important modulator of 1) myocardial functional recovery after ischemia and 2) inflammatory signaling via MAPK. To study this, adult male and female wild-type (WT) and estrogen receptor-alpha knockout (ER1KO) mouse hearts were isolated, perfused via Langendorff model, and subjected to 20 min of ischemia and 60 min of reperfusion. Myocardial contractile function (left ventricular developed pressure and positive and negative first derivative of pressure) was continuously recorded. After ischemia-reperfusion, hearts were assessed for expression of inflammatory cytokines (ELISA) and activation of MAPK and caspase-3 (Western blot analysis). Data were analyzed with two-way ANOVA or Student's t-test, and P < 0.05 was statistically significant. ER1KO females exhibited significantly less functional recovery than WT females and were similar to WT males. Activated ERK was increased in female WT hearts compared with female ER1KO. Activated JNK was decreased in female WT hearts compared with female ER1KO. No significant differences were found between male WT, female WT, male ER1KO, and female ER1KO in activated p38 MAPK, proinflammatory cytokine expression, and proapoptotic signaling. Estrogen receptor-alpha plays a role in the protection observed in the female heart. Differential activation of MAPK may mediate this protection. Further studies are necessary to delineate these mechanistic pathways.     

Ischemic preconditioning attenuates apoptotic cell death associated with ischemia/reperfusion

Apoptosis or programmed cell death is a genetically controlled response for cells to commit suicide and is associated with DNA fragmentation or laddering. The common inducers of apoptosis include oxygen free radicals/oxidative stress and Ca2+ which are also implicated in the pathogenesis of myocardial ischemic reperfusion injury. To examine whether ischemic reperfusion injury is mediated by apoptotic cell death, isolated perfused rat hearts were subjected to 15, 30 or 60 min of ischemia as well as 15 min of ischemia followed by 30, 60, 90 or 120 min of reperfusion. At the end of each experiment, the heart was processed for the evaluation of apoptosis and DNA laddering. Apoptosis was studied by visualizing the apoptotic cardiomyocytes by direct fluorescence detection of digoxigenin-labeled genomic DNA using APOPTAG® in situ apoptosis detection kit. DNA laddering was evaluated by subjecting the DNA obtained from the hearts to 1.8% agarose gel electrophoresis and photographed under UV illumination. The results of our study revealed apoptotic cells only in the 90 and 120 min reperfused hearts as demonstrated by the intense fluorescence of the immunostained digoxigenin-labeled genomic DNA when observed under fluorescence microscopy. None of the ischemic hearts showed any evidence of apoptosis. These results were corroborated with the findings of DNA fragmentation which showed increased ladders of DNA bands in the same reperfused hearts representing integer multiples of the internucleosomal DNA length (about 180 bp). The presence of apoptotic cells and DNA fragmentation in the myocardium were completely abolished by subjecting the myocardium to repeated short-term ischemia and reperfusion which also reduced the ischemic reperfusion injury as evidenced by better recovery of left ventricular performance in the preconditioned myocardium. The results of this study indicate that reperfusion of ischemic heart, but not ischemia, induces apoptotic cell death and DNA fragmentation which can be inhibited by myocardial adaptation to ischemia.     

Bax ablation protects against myocardial ischemia-reperfusion injury in transgenic mice

The role of the proapototic Bax gene in ischemia-reperfusion (I/R) injury was studied in three groups of mice: homozygotic knockout mice lacking the Bax gene (Bax(-/-)), heterozygotic mice (Bax(+/-)), and wild-type mice (Bax(+/+)). Isolated hearts were subjected to ischemia (30 min, 37 degrees C) and then to 120 min of reperfusion. The left ventricular developed force of Bax-deficient vs. Bax(+/+) hearts at stabilization and at 120 min of reperfusion was 1,411 +/- 177 vs. 1,161 +/- 137 mg and 485 +/- 69 vs. 306 +/- 68 mg, respectively. Superior cardiac function of Bax(-/-) hearts after I/R was accompanied by a decrease in creatine kinase release, caspase 3 activity, irreversible ischemic injury, and the number of terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling-positive cardiomyocytes. Electron microscopic evaluation revealed reduced damage to mitochondria and the nuclear chromatin structure in Bax-deficient mice. In the Bax(+/-) hearts, the damage markers were moderate. The superior tolerance of Bax knockout hearts to I/R injury recommends this gene as a potential target for therapeutic intervention in patients with severe and intractable myocardial ischemia.     

Adenosine-enhanced ischemic preconditioning: adenosine receptor involvement during ischemia and reperfusion

Adenosine-enhanced ischemic preconditioning (APC) extends the cardioprotection of ischemic preconditioning (IPC) by both significantly decreasing myocardial infarct size and significantly enhancing postischemic functional recovery. In this study, the role of adenosine receptors during ischemia-reperfusion was determined. Rabbit hearts (n = 92) were used for Langendorff perfusion. Control hearts were perfused for 180 min, global ischemia hearts received 30-min ischemia and 120-min reperfusion, and IPC hearts received 5-min ischemia and 5-min reperfusion before ischemia. APC hearts received a bolus injection of adenosine coincident with IPC. Adenosine receptor (A(1), A(2), and A(3)) antagonists were used with APC before ischemia and/or during reperfusion. GR-69019X (A(1)/A(3)) and MRS-1191/MRS-1220 (A(3)) significantly increased infarct size in APC hearts when administered before ischemia and significantly decreased functional recovery when administered during both ischemia and reperfusion (P < 0.05 vs. APC). DPCPX (A(1)) administered either before ischemia and/or during reperfusion had no effect on APC cardioprotection. APC-enhanced infarct size reduction is modulated by adenosine receptors primarily during ischemia, whereas APC-enhanced postischemic functional recovery is modulated by adenosine receptors during both ischemia and reperfusion.     

Regulation of cardiomyocyte apoptosis in ischemic reperfused mouse heart by glutathione peroxidase

Apoptosis, a genetically controlled programmed cell death, has been found to play a role in ischemic reperfusion injury in several animal species including rats and rabbits. To examine whether this is also true for other animals, an isolated perfused mouse heart was subjected to 30 min of ischemia followed by 2 h of reperfusion. Experiments were terminated before ischemia (baseline), after ischemia, and at 30, 60, 90 and 120 min of reperfusion. At the end of each experiment, hearts were processed for the evaluation of apoptosis and DNA laddering. The in situ end labeling (ISEL) technique was used to detect apoptotic cardiomyocyte nuclei while DNA laddering was evaluated by subjecting the DNA obtained from the cardiomyocytes to 1.8% agarose gel electrophoresis followed by photographing under UV illumination. The results of our study revealed that apoptotic cells appear only after 60 min of reperfusion as demonstrated by the intense fluorescence of the immunostained genomic DNA when observed under fluorescence microscopy. None of the ischemic hearts showed any evidence of apoptosis. These results were corroborated with the findings of DNA fragmentation showing increased ladders of DNA bands in the same reperfused hearts representing integer multiples of the internucleosomal DNA length (about 180 bp). Since our previous studies showed a role of glutathione peroxidase (GSHPx) in apoptotic cell death, we performed identical experiments using isolated hearts from GSHPx-l knockout mice and transgenic mice overexpressing GSHPx-l. GSHPx-l knockout mice showed evidence of apoptotic cell death even after 30 min of reperfusion. Significant number of apoptotic cells were found in the cardiomyocytes as compared to non-transgenic control animals. To the contrary, very few apoptotic cells were found in the hearts of the transgenic mice overexpressing GSHPx-l. Hearts of GSHPx-l knockout mice were more susceptible to ischemia/reperfusion injury while transgenic mice overexpressing GSHPx- 1 were less susceptible to ischemia reperfusion injury compared to non-transgenic control animals. The results of this study clearly demonstrate a role of GSHPx in ischemia/reperfusion-induced apoptosis in mouse heart.     

Nitroxyl affords thiol-sensitive myocardial protective effects akin to early preconditioning

Nitric oxide (NO) donors mimic the early phase of ischemic preconditioning (IPC). The effects of nitroxyl (HNO/NO(-)), the one-electron reduction product of NO, on ischemia/reperfusion (I/R) injury are unknown. Here we investigated whether HNO/NO(-), produced by decomposition of Angeli's salt (AS; Na(2)N(2)O(3)), has a cardioprotective effect in isolated perfused rat hearts. Effects were examined after intracoronary perfusion (19 min) of either AS (1 microM), the NO donor diethylamine/NO (DEA/NO, 0.5 microM), vehicle (100 nM NaOH) or buffer, followed by global ischemia (30 min) and reperfusion (30 min or 120 min in a subset of hearts). IPC was induced by three cycles of 3 min ischemia followed by 10 min reperfusion prior to I/R. The extent of I/R injury under each intervention was assessed by changes in myocardial contractility as well as lactate dehydrogenase (LDH) release and infarct size. Postischemic contractility, as indexed by developed pressure and dP/dt(max), was similarly improved with IPC and pre-exposure to AS, as opposed to control or DEA/NO-treated hearts. Infarct size and LDH release were also significantly reduced in IPC and AS groups, whereas DEA/NO was less effective in limiting necrosis. Co-infusion in the triggering phase of AS and the nitroxyl scavenger, N-acetyl-L-cysteine (4 mM) completely reversed the beneficial effects of AS, both at 30 and 120 min reperfusion. Our data show that HNO/NO(-) affords myocardial protection to a degree similar to IPC and greater than NO, suggesting that reactive nitrogen oxide species are not only necessary but also sufficient to trigger myocardial protection against reperfusion through species-dependent, pro-oxidative, and/or nitrosative stress-related mechanisms.     

Reversibility of ultrastructural, contractile function and Ca2+ transport changes in guinea pig hearts after global ischemia

To understand the subcellular basis of contractile failure due to ischemia-reperfusion injury, effects of 20, 60, and 90 min of global ischemia followed by 30 min of reperfusion were examined in isolated guinea pig hearts. Cardiac ultrastructure and function as well as Ca2+ transport abilities of both mitochondrial and microsomal fractions were determined in control, ischemic, and reperfused hearts. Hearts were unable to generate any contractile force after 20 min of ischemia and showed a 75% recovery upon reperfusion. However, there were no significant changes in the subcellular Ca2+ transport in the 20-min ischemic or reperfused hearts. When hearts were made ischemic for 60 and 90 min, the recovery of contractile force on reperfusion was 50 and 7%, respectively. There was a progressive decrease in mitochondrial and microsomal Ca2+ binding and uptake activities after 60 and 90 min of ischemia; these changes were evident at various times of incubation period and at different concentrations of Ca2+. Mitochondrial Ca2+ transport changes were only partially reversible upon reperfusion after 60 and 90 min of ischemia, whereas the microsomal Ca2+ binding, uptake and Ca2+ ATPase activities deteriorated further upon reperfusion of the 90-min ischemic hearts. Ultrastructural changes increased with the duration of the ischemic insult and reperfusion injury was extensive in the 90-min ischemic hearts. These data show that the lack of recovery of contractile function upon reperfusion after a prolonged ischemic insult was accompanied by defects in sarcoplasmic reticulum Ca2+ transporting properties and structural damage.     

Trolox C, a lipid-soluble membrane protective agent, attenuates myocardial injury from ischemia and reperfusion

The lipophilic antioxidant Trolox C, a vitamin E analog, was administered to isolated, buffer-perfused rabbit hearts subjected to 25 min of global stop-flow ischemia and 30 min of reperfusion. In six hearts, Trolox C (200 microM) was infused for 15 min immediately prior to ischemia and for the first 15 min of reperfusion. Six control hearts received only vehicle. Gas chromatography analysis confirmed that effective myocardial levels of Trolox were attained. At 30 min reperfusion, the recovery of left ventricular developed pressure was 56 +/- 3% of baseline in control hearts versus 70 +/- 4% in Trolox-treated hearts (p < .01). There was also significant improvement in recovery of Trolox-treated hearts in diastolic pressure and both maximum and minimum values of the first derivative of left ventricular pressure (dP/dt). Creatine phosphokinase release into the coronary effluent at 30 min of reperfusion was 16.5 +/- 8.4 IU/min in untreated and 6.3 +/- 1.0 IU/min (p < .05) in Trolox-treated hearts. Thus Trolox C, a lipophilic antioxidant, attenuated myocardial injury during stop-flow ischemia and reperfusion.     

Ischemic preconditioning and superoxide dismutase protect against endothelial dysfunction and endothelium glycocalyx disruption in the postischemic guinea-pig hearts

The effect of ischemic preconditioning and superoxide dismutase (SOD) on endothelial glycocalyx and endothelium-dependent vasodilation in the postischemic isolated guinea-pig hearts was examined. Seven groups of hearts were used: group 1 underwent sham aerobic perfusion; group 2 was subjected to 40 min global ischemia without reperfusion; group 3, 40 min ischemia followed by 40 min reperfusion; group 4 was preconditioned with three cycles of 5 min global ischemia followed by 5 min of reperfusion (IPC), prior to 40 min ischemia; group 5 was subjected to IPC prior to standard ischemia/reperfusion; group 6 underwent standard ischemia/reperfusion and SOD infusion (150 U/ml) was begun 5 min before 40 min ischemia and continued during the initial 5 min of the reperfusion period; group 7 was subjected to 80 min aerobic perfusion with NO-synthase inhibitor, L-NAME, to produce a model of endothelial dysfunction independent from the ischemia/reperfusion. Coronary flow responses to acetylcholine (ACh) and sodium nitroprusside (SNP) were used as measures of endothelium-dependent and endothelium-independent vascular function, respectively. Reduction in coronary flow caused by NO-synthase inhibitor, L-NAME, served as a measure of a basal endothelium-dependent vasodilator tone. After completion of each experimental protocol, the hearts were stained with ruthenium red or lanthanum chloride for electron microscopy evaluation of the endothelial glycocalyx. While ischemia led only to a slightly flocculent appearance of the glycocalyx, in ischemia/reperfused hearts the glycocalyx was disrupted, suggesting that it is the reperfusion injury which leads to the glycocalyx injury. Moreover, the coronary flow responses to ACh and L-NAME were impaired, while the responses to SNP were unchanged in the ischemia/reperfused hearts. The disruption of the glycocalyx and the deterioration of ACh and L-NAME responses was prevented by IPC. In addition, the alterations in the glycocalyx and the impairment of ACh responses were prevented by SOD. The glycocalyx appeared to be not changed in the hearts subjected to 80 min aerobic perfusion with L-NAME. In conclusion: (1) the impairment of the endothelium-dependent coronary vasodilation is paralleled by the endothelial glycocalyx disruption in the postischemic guinea-pig hearts; (2) both these changes are prevented by SOD, suggesting the role of free radicals in the mechanism of their development; (3) both changes are prevented by IPC. We hypothesize, therefore, that alterations in the glycocalyx contribute to the mechanism of the endothelial dysfunction in the postischemic hearts.     

Acetaminophen-mediated cardioprotection via inhibition of the mitochondrial permeability transition pore-induced apoptotic pathway

Our laboratory has previously reported that acetaminophen confers functional cardioprotection following cardiac insult, including ischemia/reperfusion, hypoxia/reoxygenation, and exogenous peroxynitrite administration. In the present study, we further examined the mechanism of acetaminophen-mediated cardioprotection following ischemia/reperfusion injury. Langendorff-perfused guinea pig hearts were exposed to acute treatment with acetaminophen (0.35 mM) or vehicle beginning at 15 min of a 30-min baseline stabilization period. Low-flow global myocardial ischemia was subsequently induced for 30 min followed by 60 min of reperfusion. At the completion of reperfusion, hearts were homogenized and separated into cytosolic and mitochondrial fractions. Mitochondrial swelling and mitochondrial cytochromec release were assessed and found to be significantly and completely reduced in acetaminophen- vs. vehicle-treated hearts following reperfusion. In a separate group of hearts, ventricular myocytes were isolated and subjected to fluorescence-activated cell sorting. Acetaminophen-treated hearts showed a significant decrease in late stage apoptotic myocytes compared with vehicle-treated hearts following injury (58 +/- 1 vs. 81 +/- 5%, respectively). These data, together with electron micrograph analysis, suggest that acetaminophen mediates cardioprotection, in part, via inhibition of the mitochondrial permeability transition pore and subsequent apoptotic pathway.     

乙酰胆碱对离体大鼠缺血/复灌心脏的保护作用及其机制

目的:探讨乙酰胆碱(ACh)预处理抗心肌缺血复灌(I/R)损伤作用及其与线粒体渗透性转换孔和/或线粒体ATP敏感性钾通道的关系。方法:采用离体大鼠心脏Langendorff灌流方法进行全心停灌30min,复灌120min复制I/R模型。测定心室力学指标和复灌各时间点冠脉流出液中乳酸脱氢酶(LDH)含量。实验结束测定心肌组织formazan含量的变化。结果:与单纯I/R组相比,ACh(0.1μmol/L,5min)预处理明显提高心肌细胞的formazan含量,降低复灌期间冠脉流出液中LDH含量,明显改善I/R所致的左室发展压、左心室内压最大上升和下降速率、心率与发展压乘积和左室舒张末压力的下降,缓解冠脉流量的减少。线粒体渗透性转换孔开放剂苍术苷(20μmol/L,复灌前给药20min)和线粒体ATP敏感性钾通道抑制剂5-羟基癸酸(100μmol/L,缺血前给药20min)能明显减弱ACh的保护作用。结论:在大鼠离体心脏灌流模型上,ACh预处理具有抗心脏缺血/复灌损伤的作用,这种保护作用可能与其抑制线粒体渗透性转换孔的开放和促进线粒体ATP敏感性钾通道的开放有关。     

ATP-dependent potassium channels involved in the cardiac protection induced by intermittent hypoxia against ischemia/reperfusion injury

The aim of this study was to investigate the protection afforded by intermittent hypoxia (IH) against ischemia/reperfusion injury and its effects on calcium homeostasis during ischemia/reperfusion. The roles of KATP channels in these two actions were to be explored. Isolated hearts from IH and normoxic rats were subjected to 30 min global ischemia followed by 30 min reperfusion. Cardiac function was less deteriorated during ischemia and reperfusion in the IH rat hearts compared to normoxia rat hearts. Amplitude of the maximal contracture during ischemia was lower, while time to maximal contracture was extended in IH hearts. Post-ischemic recovery of left ventricular developed pressure and +/-dP/dtmax were higher in IH hearts than in normoxic hearts. KATP antagonist glibenclamide (10 microM) completely abolished these protective effects of IH, but had no appreciable influence on normoxic hearts. In cardiomyocytes isolated from normoxic hearts, [Ca2+]i, measured as arbitrary units of fluorescence ratio (340 nm/380 nm) of fura-2, gradually increased during 20 min simulated ischemia and kept at high level during 30 min reperfusion (1.081 +/- 0.004 and 1.088 +/- 0.006 respectively, p<0.01 vs pre-ischemia perfusion). However, in cardiomyocytes isolated from IH hearts, [Ca2+]i kept at normal level during ischemia and reperfusion (1.012 +/- 0.006 and 1.021 +/- 0.002 respectively, P>0.05 vs pre-ischemia perfusion). 10 microM glibenclamide and 100 microM 5-hydroxydecanoate (a selective mitochondria KATP antagonist) respectively abolished this effect of IH; calcium overloading reappeared during ischemia (1.133 +/- 0.007 and 1.118 +/- 0.007 respectively, P<0.01) and reperfusion (1.091 +/- 0.004 and 1.095 +/- 0.012 respectivly, P<0.01). However they had no effects on simulated ischemia and reperfusion-induced calcium overloading in normoxic myocytes. 50 microM pinacidil, a KATP opener, attenuated calcium overloading during ischemia and reperfusion in normoxic myocytes, but had no effect on [Ca2+]i change in IH myocytes. These results suggested that KATP channels contributed to the cardiac protection induced by IH against ischemia/reperfusion injury; the elimination of calcium overloading during ischemia/reperfusion by IH might underlie the mechanism of protection.     

Dose-dependent protection of cardiac function by propofol during ischemia and early reperfusion in rats: effects on 15-F2t-isoprostane formation

We examined the effects of propofol (2,6-diisopropylphenol) on functional recovery and 15-F2t-isoprostane generation during ischemia-reperfusion in Langendorff-perfused rat hearts. Before the induction of 40 min of global ischemia, hearts were perfused (10 min) with propofol at 5 (lo-P) or 12 microg/mL (hi-P) in saline or with saline only (control). During ischemia, saline, lo-P, or hi-P was perfused through the aorta at 60 microL/min. During the first 15 min of reperfusion, propofol (5 or 12 microg/mL) was continued, followed by perfusion with 5 microg/mL propofol for 75 min in both propofol-treated groups. After 90 min of reperfusion (Rep-90), heart tissues were harvested for assessment of antioxidant status. In hi-P, we observed increased latency to and greater reduction of ischemic contracture relative to the lo-P or control groups. 15-F2t-Isoprostane concentrations increased during ischemia and were significantly lower in hi-P and lo-P than in control (P < 0.01). At Rep-90, myocardial functional recovery was greater in both propofol-treated groups relative to control, and it correlated positively with tissue antioxidant capacity preservation. Tissue antioxidant capacity was better preserved in hi-P than in lo-P treatment (P < 0.05). We conclude that oxidant injury occurs during ischemia and reperfusion, and propofol provides dose-dependent protection primarily by enhancing tissue antioxidant capacity and reducing lipid peroxidation.     

Effects of cold cardioplegia on pH, Na, and Ca in newborn rabbit hearts

Many studies suggest myocardial ischemia-reperfusion (I/R) injury results largely from cytosolic proton (H(i))-stimulated increases in cytosolic Na (Na(i)), which cause Na/Ca exchange-mediated increases in cytosolic Ca concentration ([Ca]i). Because cold, crystalloid cardioplegia (CCC) limits [H]i, we tested the hypothesis that in newborn hearts, CCC diminishes H(i), Na(i), and Ca(i) accumulation during I/R to limit injury. NMR measured intracellular pH (pH(i)), Na(i), [Ca]i, and ATP in isolated Langendorff-perfused newborn rabbit hearts. The control ischemia protocol was 30 min for baseline perfusion, 40 min for global ischemia, and 40 min for reperfusion, all at 37 degrees C. CCC protocols were the same, except that ice-cold CCC was infused for 5 min before ischemia and heart temperature was lowered to 12 degrees C during ischemia. Normal potassium CCC solution (NKCCC) was identical to the control perfusate, except for temperature; the high potassium (HKCCC) was identical to NKCCC, except that an additional 11 mmol/l KCl was substituted isosmotically for NaCl. NKCCC and HKCCC were not significantly different for any measurement. The following were different (P < 0.05). End-ischemia pH(i) was higher in the CCC than in the control group. Similarly, CCC limited increases in Na(i) during I/R. End-ischemia Na(i) values (in meq/kg dry wt) were 115 +/- 16 in the control group, 49 +/- 13 in the NKCCC group, and 37 +/- 12 in the HKCCC group. CCC also improved [Ca]i recovery during reperfusion. After 40 min of reperfusion, [Ca](i) values (in nmol/l) were 302 +/- 50 in the control group, 145 +/- 13 in the NKCCC group, and 182 +/- 19 in the HKCCC group. CCC limited ATP depletion during ischemia and improved recovery of ATP and left ventricular developed pressure and decreased creatine kinase release during reperfusion. Surprisingly, CCC did not significantly limit [Ca]i during ischemia. The latter is explained as the result of Ca release from intracellular buffers on cooling.     

Proteasome mediates removal of proteins oxidized during myocardial ischemia

Numerous proteins are known to be lost following myocardial ischemia/reperfusion yet little is known about the mediating proteinases. This study examines the hypothesis that proteasome plays a significant role in the removal of proteins oxidized during myocardial ischemia. Proteasome was inhibited by perfusing isolated rat hearts with buffer containing lactacystin, 2 micromol/L, for 10 min, which resulted in 51 and 42% decreases in 20S and 26S proteasome activities that persisted for a minimum of 90 min. Lactacystin pretreatment had minor effects on postischemic recovery of isolated hearts exposed to 30 min global ischemia and 60 min reperfusion. Protein carbonyl content of lactacystin-pretreated ischemic hearts was significantly (P < 0.05) increased. One band with approximate molecular mass of 50 kDa is known to contain oxidized actin. Actin degradation was quantitated by analysis of 3-methylhistidine which was significantly (P < 0.05) decreased by 15% following 30 min ischemia and 60 min reperfusion. Pretreatment of ischemic hearts with lactacystin prevented much of the loss (-6.5%) of 3-methylhistidine. Probing immunoprecipitated actin with an antibody specific for ubiquitin revealed no bands containing ubiquitinated homologues of this protein. These observations support the conclusion that proteasome mediates removal of some of the proteins oxidized during myocardial ischemia/reperfusion, and that at least oxidized actin is removed by the 20S proteasome.     

Myocardial protection from ischemic preconditioning is not blocked by sub-chronic inhibition of carnitine palmitoyltransferase I

Ischemic preconditioning (IP) triggers cardioprotection via a signaling pathway that converges on mitochondria. The effects of the inhibition of carnitine palmitoyltransferase I (CPT-I), a key enzyme for transport of long chain fatty acids (LCFA) into the mitochondria, on ischemia/reperfusion (I/R) injury are unknown. Here we investigated, in isolated perfused rat hearts, whether sub-chronic CPT-I inhibition (5 days i.p. injection of 25 mg/kg/day of Etomoxir) affects I/R-induced damages and whether cardioprotection by IP can be induced after this inhibition. Effects of global ischemia (30 min) and reperfusion (120 min) were examined in hearts harvested from Control (untreated), Vehicle- or Etomoxir-treated animals. In subsets of hearts from the three treated groups, IP was induced by three cycles of 3 min ischemia followed by 10 min reperfusion prior to I/R. The extent of I/R injury under each condition was assessed by changes in infarct size as well as in myocardial contractility. Postischemic contractility, as indexed by developed pressure and dP/dt(max), was similarly affected by I/R, and was similarly improved with IP in Control, Vehicle or Etomoxir treated animals. Infarct size was also similar in the three subsets without IP, and was significantly reduced by IP regardless of CPT-I inhibition. We conclude that CPT-I inhibition does not affect I/R damages. Our data also show that IP affords myocardial protection in CPT-I inhibited hearts to a degree similar to untreated animals, suggesting that a long-term treatment with the metabolic anti-ischemic agent Etomoxir does not impede the possibility to afford cardioprotection by ischemic preconditioning.     

Protection of the ischemic diabetic heart by L-propionylcarnitine therapy

Diabetics suffer from an increased incidence of myocardial infarction and are less likely to survive an ischemic insult. Since L-propionylcarnitine (LPC) has been shown to protect against ischemic/reperfusion injury, we hypothesized that LPC may be of even greater benefit to the diabetic heart. Diabetes was induced by i.v. streptozotocin, 60 mg/kg; duration: 12 wks. The chronic effect of LPC was determined by daily i.p. injections (100 mg/kg) for 8 wks. The acute effects of LPC were determined by adding it to the perfusion medium (5 mM) of control and diabetic hearts. Initial cardiac contractile performance of isolated perfused working hearts was assessed by varying left atrial filling pressure. Hearts were then subjected to 90 min of low flow global ischemia followed by 30 min reperfusion. Chronic LPC treatment had no effect on initial cardiac performance in either control or diabetic hearts. Acute addition of LPC to the perfusion medium enhanced pump performance of control hearts, but had no effect in diabetic hearts. Both acute and chronic LPC significantly improved the ability of control and diabetic hearts to recover cardiac contractile performance after ischemia and reperfusion, however, chronic treatment was more effective in diabetic hearts.     

Transduction of anti-cell death protein FNK protects isolated rat hearts from myocardial infarction induced by ischemia/reperfusion

Artificial anti-cell death protein FNK, a Bcl-x(L) derivative with three amino acid-substitutions (Y22F, Q26N, and R165K) has enhanced anti-apoptotic and anti-necrotic activity and facilitates cell survival in many species and cell types. The objectives of this study were (i) to investigate whether the protein conjugated with a protein transduction domain (PTD-FNK) reduces myocardial infarct size and improves post-ischemic cardiac function in ischemic/reperfused rat hearts, and (ii) to understand the mechanism(s) by which PTD-FNK exerts a protective effect. Isolated rat hearts were subjected to 35-min global ischemia, followed by 120-min reperfusion using the Langendorff methods. PTD-FNK (a total of 30 microl) was injected intramuscularly into the anterior wall of the left ventricle either at 1 min after induction of global ischemia (group A) or at 30 min after induction of global ischemia (at 5 min before reperfusion) (group B). In group A, infarct size was significantly reduced from 47.8+/-6.8% in the control to 30.4+/-5.2, 28.7+/-3.8, and 30.4+/-6.8% with PTD-FNK at 5, 50, and 500 nmol/l, respectively (p<0.05). Temporal recovery of left ventricular developed pressure at 60 min and 120 min after reperfusion was significantly better in PTD-FNK (50 and 500 nmol/l)-treated groups than in the control (p<0.05). In contrast, PTD-FNK treatment had no effect on group B. Western blot analysis showed that PTD-FNK markedly inhibited procaspase-3 cleavage (activation of caspase-3) and reduced the number of nuclei stained by a terminal deoxynucleotidyl transferase-mediated deoxyuridine 5-triphoshate nick-end labeling (TUNEL) assay. These findings suggest that PTD-FNK reduces the volume of myocardial infarction with corresponding functional recovery, at least in part, through the suppression of myocardial apoptosis following ischemia/reperfusion.