Hypoxia-activated apoptosis of cardiac myocytes requires reoxygenation or a pH shift and is independent of p53.
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Ischemia and reperfusion activate cardiac myocyte apoptosis, which may be an important feature in the progression of ischemic heart disease. The relative contributions of ischemia and reperfusion to apoptotic signal transduction have not been established. We report here that severe chronic hypoxia alone does not cause apoptosis of cardiac myocytes in culture. When rapidly contracting cardiac myocytes were exposed to chronic hypoxia, apoptosis occurred only when there was a decrease in extracellular pH ([pH](o)). Apoptosis did not occur when [pH](o) was neutralized. Addition of acidic medium from hypoxic cultures or exogenous lactic acid stimulated apoptosis in aerobic myocytes. Hypoxia-acidosis-mediated cell death was independent of p53: equivalent apoptosis occurred in cardiac myocytes isolated from wild-type and p53 knockout mice, and hypoxia caused no detectable change in p53 abundance or p53-dependent transcription. Reoxygenation of hypoxic cardiac myocytes induced apoptosis in 25-30% of the cells and was also independent of p53 by the same criteria. Finally, equivalent levels of apoptosis, as demonstrated by DNA fragmentation, were induced by ischemia-reperfusion, but not by ischemia alone, of Langendorff-perfused hearts from wild-type and p53 knockout mice. We conclude that acidosis, reoxygenation, and reperfusion, but not hypoxia (or ischemia) alone, are strong stimuli for programmed cell death that is substantially independent of p53. (+info)
Protective effects of preconditioning in cultured rat endothelial cells: effects on neutrophil adhesion and expression of ICAM-1 after anoxia and reoxygenation.
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BACKGROUND: Preconditioning with brief periods of ischemia protects the coronary endothelium against acute and chronic reperfusion injury, but the mechanisms of this endothelial protection remain unknown. We hypothesized that preconditioning protects endothelial cells through a decreased production of endothelial adhesion molecules such as intercellular adhesion molecule-1 (ICAM-1), leading to a lesser adhesion of neutrophils to the endothelium. METHODS AND RESULTS: Cultured rat aortic endothelial cells were subjected to 6-hour anoxia followed by various durations of reoxygenation. Preconditioning was induced by 1-hour anoxia and 1-hour reoxygenation. ICAM-1 gene expression was measured by polymerase chain reaction, and the percentage of cells expressing ICAM-1 was assessed by confocal laser fluorescence microscopy. Anoxia/reoxygenation increased expression of ICAM-1, with a peak occurring after 6 hours of reoxygenation for mRNA and 9 hours for protein. Preconditioning prevented the increase in ICAM-1. Similar reductions were observed with the free radical scavenger N-2 mercaptopropionyl glycine (MPG). The inhibitory effect of preconditioning on ICAM-1 expression was abolished by an inhibitor of protein kinase C, an inhibitor of nitric oxide synthesis, and by MPG but was not affected by an adenosine receptor antagonist. Finally, both preconditioning and MPG partially prevented the increased adhesion of human neutrophils to reoxygenated endothelial cells. CONCLUSIONS: Preconditioning prevented reoxygenation-induced, free radical-mediated expression of ICAM-1 by a mechanism involving activation of protein kinase C and production of nitric oxide and free radicals, and this is associated with a lesser adhesion of neutrophils to endothelial cells. Such prevention of neutrophil adhesion may contribute to the protective effect of preconditioning against reperfusion-induced endothelial injury. (+info)
Effects and interaction, of cariporide and preconditioning on cardiac arrhythmias and infarction in rat in vivo.
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1. Although Na+-H+ exchange (NHE) inhibitors are reported to protect the myocardium against ischaemic injury, NHE activation has also been proposed as a potential mechanism of ischaemic preconditioning-induced protection. This study was performed to test any modifiable effect of cariporide, an NHE inhibitor, on cardioprotective effects of preconditioning. 2. Anaesthetized rats were subjected to 30 min of coronary artery occlusion and 150 min of reperfusion. The preconditioning (PC) was induced by 3 min of ischaemia and 10 min of reperfusion (1PC) or three episodes of 3 min ischaemia and 5 min reperfusion (3PC). Cariporide (0.3 mg kg(-1)) an NHE inhibitor, was administered 30 min (cari(30)) or 45 min (cari(45)) before coronary ligation (n=8-11 for each group). 3. Ventricular arrhythmias during 30 min ischaemia and infarct size (measured by triphenyltetrazolium (TTC) and expressed as a per cent area at risk (%AAR)) were determined. Cari(30) reduced ventricular fibrillation (VF) incidence and infarct size (from 45 to 0% and 34+/-4 to 9+/-2%; each P<0.05), whereas cari(45) did not. Likewise, 3PC reduced these variables (to 0% and 10+/-2%; P<0.05 in each case) whereas 1PC did not. Moreover, subthreshold preconditioning (1PC) and cariporide (cari(45)), when combined, reduced VF incidence and infarct size (to 0% and 15+3%; each P<0.05 ). 4. In conclusion, changes in NHE activity do not seem to be responsible for the cardioprotective action of ischaemic preconditioning. Protective effects of NHE inhibition and subthreshold preconditioning appear to act additively. (+info)
Coronary vasoconstriction to endothelin-1 increases with age before and after ischaemia and reperfusion.
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OBJECTIVE: Ageing is known to be associated with changes within the heart. We investigated whether the coronary response to endothelin-1 (ET) and sarafotoxin S6c (S6c) is altered with increasing age, before and after cardioplegic arrest. METHODS: Using an isolated rat heart model, increasing concentrations of ET and S6c were administered to rats of different ages (group I = one month; group II = five months; group III = 21 months). An identical series of experiments was performed following the addition of indomethacin and NG-nitro-L-arginine methyl ester (L-NAME) to the Krebs perfusion fluid. In a third series of experiments, increasing doses of ET-1 were added to hearts following 4 h of cardioplegic arrest at 4 degrees C. RESULTS: Coronary flows are expressed as a percentage of initial coronary flow +/- SEM. There was a greater decrease in coronary flow in the older rats for all doses of ET-1. ET-1 (10(-9) M) reduced coronary flows to 72.8 +/- 3.7, 53.2 +/- 6.7 and 56.5 +/- 10.7% for groups I-III respectively (P = 0.01 I vs. II; P = 0.1 I vs. III). A similar response to ET-1 was seen in hearts perfused with indomethacin and L-NAME when compared to those perfused without (P = NS). Perfusion with ET-1 (10(-9) M) following 4 h of cardioplegic arrest reduced coronary flows to 40.5 +/- 4.9, 26.8 +/- 4.8 and 24.1 +/- 3.9%, respectively (P = 0.08 I vs. II; P = 0.03 I vs. III). Perfusion with S6c (10(-10) M) produced coronary flows of 93.3 +/- 5.5, 77.0 +/- 3.5 and 73.9 +/- 3.9% for groups I-III, respectively (P = 0.03 I vs. II; P = 0.01 I vs. III). Perfusion with S6c (10(-9) M) in the presence of L-NAME and indomethacin reduced coronary flows to 85.7 +/- 3.0, 81.6 +/- 2.2 and 74.6 +/- 3.6% (P = NS I vs. II; P = 0.03 I vs. III). CONCLUSIONS: The coronary vasoconstrictor response to ET-1 and S6c increases with age. The increased vasoconstriction in response to ET-1 is independent of the decrease in NO release seen with ageing. (+info)
Acute cardiac inflammatory responses to postischemic reperfusion during cardiopulmonary bypass.
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OBJECTIVES: The investigation centers on whether there is a reperfusion-induced specific cardiac inflammatory reaction after bypass surgery. BACKGROUND: Cardiopulmonary bypass (CPB) leads to systemic inflammation. Additionally, cardiac inflammation due to reperfusion could occur. Knowledge about nature and time course of this reaction might help to develop cardioprotective interventions. METHODS: In 12 patients receiving coronary bypass grafts, arterial and coronary venous blood was obtained before onset of CPB, and 1, 5, 10, 25, 35 and 75 min after cardiac reperfusion. Plasma levels of IL6 and IL8 were measured by immunoassay. CD11b, CD41, and CD62 on blood cells were quantified by flow cytometry. Measurement of CD41, a platelet marker, on neutrophils and monocytes allowed detection of leukocyte-platelet microaggregates. RESULTS: Transcardiac veno-arterial difference of IL6 rose in the 10th and 25th min of reperfusion (from 0 to 7 pg/ml; p < 0.05), and after 75 min (15 pg/ml). IL8 did not change. CD11b on neutrophils (PMN) decreased transcardially to 95, 88 and 82% of the initial level in the 5th, 10th, and 75th min, respectively, suggesting sequestration of activated neutrophils. CD62 on platelets rose about 30% in the 75th min. Initially, leukocyte-platelet microaggregates were formed during coronary passage (+31% of the arterial level for PMN, +23% for monocytes). During reperfusion, coaggregates were retained (PMN: -1% and -7% in the 5th and 10th min, monocytes: -22%, -13% and -12% in the 1st, 5th and 10th min. CONCLUSIONS: During early reperfusion after aortic declamping, the coronary bed is already a source of proinflammatory stimuli and target for activated leukocytes, partly in conjunction with platelets. Mitigation of these phenomena might help to improve cardiac function after CPB especially in patients at risk. (+info)
Cardiomyocytes from hearts with left ventricular dysfunction after ischemia-reperfusion do not manifest contractile abnormalities.
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OBJECTIVES: This study evaluated contractile function in cardiomyocytes isolated from hearts with global left ventricular dysfunction following ischemia-reperfusion. BACKGROUND: Ischemia followed by reperfusion is associated with transient contractile dysfunction, termed "stunning." It is not clear whether this phenomenon is primarily due to intrinsic cardiomyocyte contractile dysfunction. METHODS: Global contractile dysfunction was induced in isolated perfused rat hearts (n = 8) using a model of transient global ischemia (20 min) followed by reperfusion (20 min). Hearts perfused uninterrupted for 40 min were used as controls (n = 8). Cardiomyocytes were isolated using enzymatic digestion and were studied under varying degrees of inotropy (using increasing extracellular calcium [Ca2+]o) and loading conditions (varying extracellular perfusate viscosity). Mechanical function was studied with video edge detection and intracellular calcium ([Ca2+]i) kinetics using fura-2 AM. RESULTS: Global ischemia-reperfusion increased left ventricle (LV) end diastolic pressure (450% vs. 33%, p < 0.01) and reduced LV developed pressure (9% vs. 33%, p < 0.01), LV positive (3% vs. 26%, p < 0.01) and negative (5% vs. 33%, p < 0.01) dP/dt. However, cells isolated from these hearts did not manifest contractile dysfunction. In fact, cell shortening (p < 0.0001) and peak rate of cell shortening (p < 0.05) and increase in [Ca2+]i with each contraction (p < 0.024) were higher in these cells during stimulation with [Ca2+]o of 1 to 10 mmol/liter. The EC50 values for calcium dose response and the slope of the relation between change in [Ca2+]i and change in cell length were no different between the groups. Cell loading (with increasing superfusate viscosity from 1 cp to 300 cp) also did not reveal any abnormalities in cells from the hearts subjected to ischemia-reperfusion. CONCLUSIONS: Cardiomyocytes isolated from hearts with ischemia-reperfusion-induced LV dysfunction or "stunning" have normal contractile function and normal [Ca2+]i transients, when studied both in the unloaded and loaded state. Our data suggest that nonmyocyte factors such as abnormalities in extracellular matrix or abnormal myocyte-interstitial tissue coupling may be important for the genesis of cardiac contractile failure in the stunned heart. (+info)
Sevoflurane and isoflurane protect the reperfused guinea pig heart by reducing postischemic adhesion of polymorphonuclear neutrophils.
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BACKGROUND: Polymorphonuclear neutrophils (PMNs) contribute to reperfusion injury. Because volatile anesthetics can reduce PMN adhesion in the reperfused, nonworking heart, the authors analyzed whether this action of volatile anesthetics affects cardiac performance after ischemia and reperfusion and further clarified the underlying mechanism. METHODS: Isolated guinea pig hearts perfused with crystalloid buffer and performing pressure-volume work were used. Hearts were subjected to 15 min global ischemia and 20 min reperfusion. In the intervention groups an intracoronary bolus of 3 x 10(6) PMNs was applied in the second min of reperfusion, either in the absence or presence of 0.5 or 1 minimum alveolar concentration sevoflurane or isoflurane. The number of sequestered PMNs was calculated from the difference between coronary input and output (coronary effluent) of PMNs. Performance of external heart work, determined pre- and postischemically, served as criterion for recovery of myocardial function. Additionally, the expression of the integrin CD11b on the cell surface of PMN was measured before and after coronary passage. RESULTS: Injection of PMN in the reperfusion phase, but not under nonischemic conditions, reduced recovery of external heart work significantly (from 55+/-7% to 19+/-11%). Addition of sevoflurane or isoflurane in concentrations of 0.5 and 1 minimum alveolar concentration to the perfusate reduced postischemic PMN adhesion from 36+/-8% to basal values (20+/-7%) and prevented decline of cardiac function. CD11b expression on PMNs increased significantly during postischemic coronary passage under control conditions. Again, both anesthetics in both concentrations inhibited that activation. CONCLUSIONS: Volatile anesthetics reduce PMN adhesion in the reperfused coronary system and thereby preserve cardiac function. Reduced expression of the adhesion molecule CD11b on PMNs in the presence of sevoflurane or isoflurane is, at least in part, responsible for the cardioprotective effect. (+info)
Alterations in sarcoplasmic reticulum function and gene expression in ischemic-reperfused rat heart.
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In view of the critical role of sarcoplasmic reticular (SR) Ca(2+) release and the Ca(2+) pump in cardiac contraction-relaxation, this study was undertaken to assess the status of SR function, protein content, and gene expression in isolated rat hearts subjected to global ischemia for 30 min followed by 60 min of reperfusion (I/R). Attenuated recovery of contractile function in the I/R hearts was associated with reduced SR Ca(2+) uptake, Ca(2+) release, and ryanodine-binding activities. mRNA levels and protein contents for SR Ca(2+) pump ATPase and Ca(2+) release channels were markedly depressed in the I/R hearts. Perfusion of hearts with superoxide dismutase plus catalase, well-known scavengers of oxyradicals, prevented the I/R-induced alterations in cardiac function and partially prevented SR Ca(2+) transport activities and mRNA abundance. In hearts perfused with xanthine plus xanthine oxidase or H(2)O(2), changes similar to those in the I/R hearts were observed. These results indicate that oxyradicals may participate in depressing the SR Ca(2+) handling and gene expression in the I/R heart. It is suggested that treatment of hearts with antioxidants may improve the recovery of cardiac function by preserving the SR function and partially protecting the SR gene expression. (+info)