Postischemic Na(+)-K(+)-ATPase reactivation is delayed in the absence of glycolytic ATP in isolated rat hearts

Normalization of intracellular sodium (Na) after postischemic reperfusion depends on reactivation of the sarcolemmal Na(+)-K(+)-ATPase. To evaluate the requirement of glycolytic ATP for Na(+)-K(+)-ATPase function during postischemic reperfusion, 5-s time-resolution 23Na NMR was performed in isolated perfused rat hearts. During 20 min of ischemia, Na increased approximately twofold. In glucose-reperfused hearts with or without prior preischemic glycogen depletion, Na decreased immediately upon postischemic reperfusion. In glycogen-depleted pyruvate-reperfused hearts, however, the decrease of Na was delayed by approximately 25 s, and application of the pyruvate dehydrogenase (PDH) activator dichloroacetate (DA) did not shorten this delay. After 30 min of reperfusion, Na had almost normalized in all groups and contractile recovery was highest in the DA-treated hearts. In conclusion, some degree of functional coupling of glycolytic ATP and Na(+)-K(+)-ATPase activity exists, but glycolysis is not essential for recovery of Na homeostasis and contractility after prolonged reperfusion. Furthermore, the delayed Na(+)-K(+)-ATPase reactivation observed in pyruvate-reperfused hearts is not due to inhibition of PDH.     

Glutathione disulfide as an index of oxidative stress during postischemic reperfusion in isolated rat hearts

The objectives of this study were to determine 1) whether reactive oxygen species generated upon postischemic reperfusion lead to oxidative stress in rat hearts, and 2) whether an exogenous prooxidant present in the early phase of reperfusion causes additional injury. Isolated buffer-perfused rat hearts were subjected to 30 min of hypothermic no-flow ischemia followed by 30 min of reperfusion. Increased myocardial content of glutathione disulfide (GSSG) and increased active transport of GSSG were used as indices of oxidative stress. To impose a prooxidant load, cumene hydroperoxide (20 M) was administered during the first 10 min of reperfusion to a separate group of postischemic hearts. Reperfusion after 30 min of hypothermic ischemia resulted in a recovery of myocardial ATP from 28% at end-ischemia to 50–60%, a release of 5% of total myocardial LDH, and an almost complete recovery of both coronary flow rate and left ventricular developed pressure. After 5 and 30 min of reperfusion, neither myocardial content of GSSG nor active transport of GSSG were increased. These indices were increased, however, if cumene hydroperoxide was administered during early reperfusion. After stopping the administration of cumene hydroperoxide, myocardial GSSG content returned to control values and GSH content increased, indicating an unimpaired glutathione reductase reaction. Despite the induction of oxidative stress, reperfusion with cumene hydroperoxide did not cause additional metabolic, structural, or functional injury when compared to reperfusion without cumene hydroperoxide. We conclude that reactive oxygen species generated upon postischemic reperfusion did not lead to oxidative stress in isolated rat hearts. Moreover, even a superimposed prooxidant load during early reperfusion did not cause additional injury.     

Postischemic injury in isolated rat hearts is not aggravated by prior depletion of myocardial glutathione

The aim of this study was to test the hypothesis that a decreased myocardial concentration of reduced glutathione (GSH) during ischemia renders the myocardium more susceptible to injury by reactive oxygen species generated during early reperfusion. To this end, rats were pretreated with L-buthionine-S,R-sulfoximine (2 mmol/kg), which depleted myocardial GSH by 55%. Isolated buffer-perfused hearts were subjected to 30 min of either hypothermic or normothermic no-flow ischemia followed by reperfusion. Prior depletion of myocardial GSH did not lead to oxidative stress during reperfusion, as myocardial concentration of glutathione disulfide (GSSG) was not increased after 5 and 30 min of reperfusion. In addition, prior depletion of GSH did not exacerbate myocardial enzyme release, nor did it impair the recoveries of tissue ATP, coronary flow rate and left ventricular developed pressure during reperfusion after either hypothermic or normothermic ischemia. Even administration of the prooxidant cumene hydroperoxide (20 M) to postischemic GSH-depleted hearts during the first 10 min of reperfusion did not aggravate postischemic injury, although this prooxidant load induced oxidative stress, as indicated by an increased myocardial concentration of GSSG. These results do not support the hypothesis that a reduced myocardial concentration of GSH during ischemia increases the susceptibility to injury mediated by reactive oxygen species generated during reperfusion. Apparently, myocardial tissue possesses a large excess of GSH compared to the quantity of reactive oxygen species generated upon reperfusion. (Mol Cell Biochem 156: 79-85, 1996)     

Ischemic preconditioning in rat heart: No correlation between glycogen content and return of function

We tested the hypothesis that glycogen levels at the beginning of ischemia affect lactate production during ischemia and postischemic contractile function.Isolated working rat hearts were perfused at physiological workload with bicarbonate buffer containing glucose (10 mmol/L). Hearts were subjected to four different preconditioning protocols, and cardiac function was assessed on reperfusion. Ischemic preconditioning was induced by either one cycle of 5 min ischemia followed by 5, 10, or 20 min of reperfusion (PC5/5, PC5/10, PC5/20), or three cycles of 5 min ischemia followed by 5 min of reperfusion (PC3 × 5/5). All hearts were subjected to 15 min total, global ischemia, followed by 30 min of reperfusion. We measured lactate release, timed the return of aortic flow, compared postischemic to preischemic power, and determined tissue metabolites at selected time points.Compared with preischemic function, cardiac power during reperfusion improved in groups PC5/10 and PC5/20, but was not different from control in groups PC5/5 and PC3 × 5/5. There was no correlation between preischemic glycogen levels and recovery of function during reperfusion. There was also no correlation between glycogen breakdown (or resynthesis) and recovery of function. Lactate accumulation during ischemia was lowest in group PC5/20 and highest in the group with three cycles of preconditioning (PC3 × 5/5). Lactate release during reperfusion was significantly higher in the groups with low recovery of power than in the groups with high recovery of power.In glucose-perfused rat heart recovery of function is independent from both pre- and postischemic myocardial glycogen content over a wide range of glycogen levels. The ability to utilize lactate during reperfusion is an indicator for postischemic return of contractile function.     

Pyruvate-fortified cardioplegia suppresses oxidative stress and enhances phosphorylation potential of arrested myocardium

Cardioplegic arrest for bypass surgery imposes global ischemia on the myocardium, which generates oxyradicals and depletes myocardial high-energy phosphates. The glycolytic metabolite pyruvate, but not its reduced congener lactate, increases phosphorylation potential and detoxifies oxyradicals in ischemic and postischemic myocardium. This study tested the hypothesis that pyruvate mitigates oxidative stress and preserves the energy state in cardioplegically arrested myocardium. In situ swine hearts were arrested for 60 min with a 4:1 mixture of blood and crystalloid cardioplegia solution containing 188 mM glucose alone (control) or with additional 23.8 mM lactate or 23.8 mM pyruvate and then reperfused for 3 min with cardioplegia-free blood. Glutathione (GSH), glutathione disulfide (GSSG), and energy metabolites [phosphocreatine (PCr), creatine (Cr), P(i)] were measured in myocardium, which was snap frozen at 45 min arrest and 3 min reperfusion to determine antioxidant GSH redox state (GSH/GSSG) and PCr phosphorylation potential {[PCr]/([Cr][P(i)])}. Coronary sinus 8-isoprostane indexed oxidative stress. Pyruvate cardioplegia lowered 8-isoprostane release approximately 40% during arrest versus control and lactate cardioplegia. Lactate and pyruvate cardioplegia dampened (P < 0.05 vs. control) the surge of 8-isoprostane release following reperfusion. Pyruvate doubled GSH/GSSG versus lactate cardioplegia during arrest, but GSH/GSSG fell in all three groups after reperfusion. Myocardial [PCr]/([Cr][P(i)]) was maintained in all three groups during arrest. Pyruvate cardioplegia doubled [PCr]/([Cr][P(i)]) versus control and lactate cardioplegia after reperfusion. Pyruvate cardioplegia mitigates oxidative stress during cardioplegic arrest and enhances myocardial energy state on reperfusion.     

Glutathione protection against dive-associated ischemia/reperfusion in ringed seal tissues

Ischemia/reperfusion is a potentially hazardous condition that increases reactive oxygen species (ROS) production and oxidative damage. Seals of the phocid family experience repetitive episodes of ischemia/reperfusion during and after a dive as a consequence of preferential distribution of blood flow to the central nervous system and reduction or elimination of perfusion in most vascular beds. Previous studies showed that ROS production is higher in ringed seal than in domestic pig tissues as a direct consequence of the ischemia/reperfusion associated with the diving response; however, oxidative damage is not related to this high ROS production. Apparently, antioxidant enzyme activities participate in the antioxidant protection in ringed seal tissues. In the present study we addressed the potential antioxidant protection of the glutathione system against dive-induced ischemia/reperfusion in ringed seal tissues. Total glutathione (GSH-Eq = GSH + 2GSSG), glutathione (GSH) and glutathione disulfide (GSSG), the ratio GSSG:GSH-Eq, the activities of the enzymes glutathione disulfide reductase (GR) and glucose-6-phosphate dehydrogenase (G6PDH), as well as lipid peroxidation (TBARS) and carbonyl proteins, were measured in ringed seal and domestic pig heart, kidney, liver, lung and muscle samples. In heart, kidney, lung and muscle GSH-Eq and GSH content was higher in seal than in pig (p < 0.05). GSSG content was higher in seal than in pig heart kidney, liver and muscle (p < 0.05). GR and G6PDH activities were higher in all seal than in pig tissues (p < 0.05). GSSG:GSH-Eq ratio was higher in pig than in seal heart, and lung (p < 0.05). TBARS content was higher in pig than in seal lung (p < 0.05). Higher content of carbonyl proteins was present in pig than in seal heart, kidney, liver and muscle (p < 0.05). These results suggest that the glutathione levels and the activity of enzymes involved in its recycling are efficient mechanisms that ameliorate protein and lipid oxidative damage and protect ringed seal tissues against dive-induced ischemia/reperfusion.     

Effect of Dichloroacetate on Regional Energy Metabolites and Pyruvate Dehydrogenase Activity During Ischemia and Reperfusion in Gerbil Brain

The objective of this study was to determine whether administration of dichloroacetate (DCA), an activator of pyruvate dehydrogenase (PDH), improves recovery of energy metabolites following transient cerebral ischemia. Gerbils were pretreated with DCA, and cerebral ischemia was produced using bilateral carotid artery occlusion for 20 min, followed by reperfusion up to 4 h. DCA had no effect on the accumulation of lactic acid and the decrease in ATP and phosphocreatine (PCr) during the 20-min insult, nor on the recovery of these metabolites measured at 20 and 60 min reperfusion. However, at 4 h reperfusion, levels of ATP and PCr were significantly higher in DCA-treated animals than in controls, as PCr exhibited a secondary decrease in caudate nucleus of control animals. PDH was markedly inhibited at 20 min reperfusion in both groups, but was reactivated to a greater extent in DCA-treated animals at 60 min and 4 h reperfusion. These results demonstrate that DCA had no effect on the initial recovery of metabolites following transient ischemia. However, later in reperfusion, DCA enhanced the postischemic reactivation of PDH and prevented the secondary failure of energy metabolism in caudate nucleus. Thus, inhibition of PDH may limit the recovery of energy metabolism following cerebral ischemia.     

Allopurinol-Enhanced Postischemic Recovery in the Isolated Pat Heart Involves Repletion of High-Energy Phosphates

The effects of allopurinol (AP) on functional and metabolic recovery of the isolated rat heart after global ischemia were studied. Hearts were subjected to aerobic perfusion (30 min), cardioplegic infusion (5 min), normothermic ischemia (37 min), and reperfusion (50 min) which was started with secondary cardioplegic infusion (10 min). AP was injected into rats (44 mg/kg body wt ip 2 h before heart excision) and added to cardioplegic solution (2 mM) prior and after ischemia. AP treatment significantly improved postischemic recovery of the function and reduced the leakage of lactate dehydrogenase from reperfused hearts. These beneficial effects were accompanied by a better preservation of tissue content of ATP, the total adenine nucleotides, phosphocreatine, and the total creatine at the end of reperfusion. Inhibition of xanthine oxidase by AP substantially decreased pre- and postischemic release of xanthine and uric acid and increased postischemic release of hypoxanthine into the coronary effluent. Despite this, AP treated hearts did not exhibit a reduction in hydroxyl radical adduct formation in the effluents at reperfusion assessed by the spin-trap measurements. The results suggest that AP may protect the heart from ischemia/reperfusion injury due to enhanced energy provision rather than by prevention of oxygen-derived free radical formation.     

Nitrate tolerance aggravates postischemic myocardial apoptosis and impairs cardiac functional recovery after ischemia

Objectives: This study examined the effects of nitrate tolerance (NT) on myocardial ischemia reperfusion (MI/R) injury and elucidated the potential mechanisms involved. Furthermore, the effects of GSH on postischemic myocardial apoptosis in NT rats were investigated. Methods and results: Male Sprague–Dawley rats were randomized to receive nitroglycerin (60 μg/kg/h) or saline for 12 h followed by 40 min of MI and 4 h of reperfusion. Myocardial apoptosis, infarct size, nitrotyrosine formation, plasma CK and LDH activity, and cardiac function were determined. MI/R resulted in significant apoptotic cell death, which was further increased in animals with NT. In addition, NT further increased plasma CK and LDH activity, enlarged infarct size, and impaired cardiac functional recovery after ischemia. Myocardial nitrotyrosine, a footprint for cytotoxic reactive nitrogen species formation, was further enhanced in the NT heart after MI/R. Treatment of NT animals with exogenous GSH inhibited nitrotyrosine formation, reduced apoptosis, decreased infarct size, and improved cardiac functional recovery. Conclusion: Our results demonstrate that nitrate tolerance markedly enhances MI/R injury and that increased peroxynitrite formation likely plays a role in this pathologic process. In addition, our results suggest that GSH could decrease peroxynitrite formation and reduce MI/R injury in nitrate tolerant hearts.     

Machine perfusion at 20°C reduces preservation damage to livers from non-heart beating donors

We previously reported that machine perfusion (MP) performed at 20 °C enhanced the preservation of steatotic rat livers. Here, we tested whether rat livers retrieved 30 min after cardiac arrest (NHBDs) were better protected by MP at 20 °C than with cold storage. We compared the recovery of livers from NHBDs with organs obtained from heart beating donors (HBDs) preserved by cold storage. MP technique: livers were perfused for 6 h with UW-G modified at 20 °C. Cold storage: livers were perfused in situ and preserved with UW solution at 4 °C for 6 h. Both MP and cold storage preserved livers were reperfused with Krebs-Heinselet buffer (2 h at 37 °C). AST and LDH release and mitochondrial glutamate dehydrogenase (GDH) levels were evaluated. Parameters assessed included: bile production and biliary enzymes; tissue ATP; reduced and oxidized glutathione (GSH/GSSG); protein–SH group concentration. Livers preserved by MP at 20 °C showed significantly lower hepatic damage at the end of reperfusion compared with cold storage. GDH release was significantly reduced and bile production, ATP levels, GSH/GSSG and protein–SH groups were higher in livers preserved by MP at 20 °C than with cold storage. The best preserved morphology and high glycogen content was obtained with livers submitted to MP at 20 °C. Liver recovery using MP at 20 °C was comparable to recovery with HBDs. MP at 20 °C improves cell survival and gives a better-quality of preservation for livers obtained from NHBDs and may provide a new method for the successful utilization of marginal livers.     

Glucose requirement for postischemic recovery of perfused working heart

The quantitative importance of glycolysis in cardiomyocyte reenergization and contractile recovery was examined in postischemic, preload-controlled, isolated working guinea pig hearts. A 25-min global but low-flow ischemia with concurrent norepinephrine infusion to exhaust cellular glycogen stores was followed by a 15-min reperfusion. With 5 mM pyruvate as sole reperfusion substrate, severe contractile failure developed despite normal sarcolemmal pyruvate transport rate and high intracellular pyruvate concentrations near 2 mM. Reperfusion dysfunction was characterized by a low cytosolic phosphorylation potential [( ATP]/[( ADP][Pi]) due to accumulations of inorganic phosphate (Pi) and lactate. In contrast, with 5 mM glucose plus pyruvate as substrates, but not with glucose as sole substrate, reperfusion phosphorylation potential and function recovered to near normal. During the critical ischemia-reperfusion transition at 30 s reperfusion the cytosolic creatine kinase appeared displaced from equilibrium, regardless of the substrate supply. When under these conditions glucose and pyruvate were coinfused, glycolytic flux was near maximum, the glyceraldehyde-3-phosphate dehydrogenase/3-phosphoglycerate kinase reaction was enhanced, accumulation of Pi was attenuated, ATP content was slightly increased, and adenosine release was low. Thus, glucose prevented deterioration of the phosphorylation potential to levels incompatible with reperfusion recovery. Immediate energetic support due to maximum glycolytic ATP production and enhancement of the glyceraldehyde-3-phosphate dehydrogenase/3-phosphoglycerate kinase reaction appeared to act in concert to prevent detrimental collapse of [ATP]/[( ADP][Pi]) during creatine kinase dysfunction in the ischemia-reperfusion transition. Dichloroacetate (2 mM) plus glucose stimulated glycolysis but failed fully to reenergize the reperfused heart; conversely, 10 mM 2-deoxyglucose plus pyruvate inhibited glycolysis and produced virtually instantaneous de-energization during reperfusion. The following conclusions were reached. (1) A functional glycolysis is required to prevent energetic and contractile collapse of the low-flow ischemic or reperfused heart (2). Glucose stabilization of energetics in pyruvate-perfused hearts is due in part to intensification of glyceraldehyde-3-phosphate dehydrogenase/3-phosphoglycerate kinase activity. (3) 2-Deoxyglucose depletes the glyceraldehyde-3-phosphate pool and effects intracellular phosphate fixation in the form of 2-deoxyglucose 6-phosphate, but the cytosolic phosphorylation potential is not increased and reperfusion failure occurs instantly. (4) Consistent correlations exist between cytosolic ATP phosphorylation potential and reperfusion contractile function. The findings depict glycolysis as a highly adaptive emergency mechanism which can prevent deleterious myocyte deenergization during forced ischemia-reperfusion transitions in presence of excess oxidative substrate.     

Parameters related to oxygen free radicals in erythrocytes,plasma and epidermis of the hairless rat

The following parameters related to oxygen free radicals (OFR) were determined in erythrocytes and the epidermis of hairless rats: catalase (CAT), glutathione peroxidase (GPx), glutathione reductase (GR), reduced (GSH) and oxidized (GSSG) glutathione, glutathione S-transferase (GST), superoxide dismutase (SOD) and thiobarbituric acid reactive substances (TBARS). GSH, GSSG and TBARS were also analyzed in plasma. In erythrocytes, the Pearson correlation coefficients (r) were significant (p < 0.001) between glutathione and other parameters as follows: GSH correlated negatively with GSSG (r = -0.665) and TBARS (r = -0.669); GSSG correlated positively with SOD (r = 0.709) and TBARS (r = 0.752). Plasma GSSG correlated negatively with erythrocytic thermostable GST activity (r = -0.608; p=0.001) and with erythrocytic total GST activity (r = -0.677; p < 0.001). In epidermis (p < 0.001 in all cases), GSH content correlated with GSSG (r = 0.682) and with GPx (r = 0.663); GSSG correlated with GPx (r = 0.731) and with GR (r = 0.794). By multiple linear regression analysis some predictor variables (R(2)) were found: in erythrocytes, thermostable GST was predicted by total GST activity and GSSG, GSSG content was predicted by GSH and by the GSH/GSSG ratio and GPx activity was predicted by GST, CAT and SOD activities; in epidermis, GSSG was predicted by GR and SOD activities and GR was predicted by GSSG, TBARS and GPx. It is concluded that the hairless rat is a good model for studying OFR-related parameters simultaneously in blood and skin, and that it may provide valuable information about other animals under oxidative stress.     

Age-related changes of antioxidant enzyme activities, glutathione status and lipid peroxidation in rat erythrocytes after heat stress

The aim of this study was to determine whether exposure to heat stress would lead to oxidative stress and whether this effect varied with different exposure periods. We kept 1-, 6- and 12-month-old male Wistar rats at an ambient temperature of either 22 degrees C or 40 degrees C for 3 and 7 days and measured glucose-6-phosphate dehydrogenase (G-6-PD), Cu,Zn-superoxide dismutase (Cu,Zn-SOD), catalase (CAT), selenium-dependent glutathione peroxidase (Se-GSH-Px) and glutathione-S-transferase (GST) activities and levels of thiobarbituric acid-reactive substances (TBARS), reduced glutathione (GSH) and oxidized glutathione (GSSG) in erythrocytes and determined GSH/GSSG ratio, total glutathione and the redox index. G-6-PD and CAT activities were found to be significantly increased in 1- and 6-month-old rats after 3 and 7 days of heat stress, but G-6-PD activities decreased in 12-month-old rats. Cu, Zn-SOD activity decreased in 1-month-old rats after heat stress, whereas it increased in 6- and 12-month-old rats. GST activity increased in all groups. GSH and total GSH levels and GSH/GSSG ratios decreased in 1- and 6-month-old rats but they increased in 12-month-old rats after heat stress. GSSG levels increased in 1- and 6-month-old rats but decreased in 12-month-old rats after heat stress. TBARS levels increased in all groups. Seven days of stress is more effective in altering enzyme activities and levels of GSH, GSSG and TBARS. When the effects of both heat stress and aging were examined together, it was interesting to note that they mostly influenced G-6-PD activity.     

Cyclic strain modulates resistance to oxidant stress by increasing G6PDH expression in smooth muscle cells

Vascular smooth muscle cells (VSMC) may be subjected to mechanical forces, such as cyclic strain, that promote the formation of reactive oxygen species (ROS). We hypothesized that VSMC modulate this adverse milieu by increasing the expression of glucose-6-phosphate dehydrogenase (G6PDH) to maintain or restore intracellular glutathione (GSH) levels. Cyclic strain increased superoxide formation, which resulted in diminished GSH because of an increase in oxidized glutathione formation; there was also an increase in glutathione peroxidase and glutathione reductase activities. G6PDH activity and protein expression were enhanced concomitant with decreases in GSH levels and remained elevated until intracellular GSH levels were restored. To confirm the role of G6PDH in repleting GSH stores, we inhibited G6PDH activity with DHEA or inhibited enzyme expression with an antisense oligodeoxynucleotide. Diminished G6PDH activity or expression was associated with persistently depleted GSH levels and inhibition of the cyclic strain-mediated increase in glutathione reductase activity. These observations demonstrate that cyclic strain promotes oxidant stress in VSMC, which, in turn, induces G6PDH expression. When G6PDH is inhibited, GSH levels are not restored because of impaired glutathione reductase activity. These data suggest that G6PDH is a critical determinant of the response to oxidant stress in VSMC.     

Cardioprotective effects of stretch are mediated by activation of sarcolemmal, not mitochondrial, ATP-sensitive potassium channels

To determine whether sarcolemmal and/or mitochondrial ATP-sensitive potassium (K(ATP)) channels (sarcK(ATP), mitoK(ATP)) are involved in stretch-induced protection, isolated isovolumic rat hearts were assigned to the following protocols: nonstretched hearts were subjected to 20 min of global ischemia (Is) and 30 min of reperfusion, and before Is stretched hearts received 5 min of stretch + 10 min of no intervention. Stretch was induced by a transient increase in left ventricular end-diastolic pressure (LVEDP) from 10 to 40 mmHg. Other hearts received 5-hydroxydecanoate (5-HD; 100 microM), a selective inhibitor of mitoK(ATP), or HMR-1098 (20 microM), a selective inhibitor of sarcK(ATP), before the stretch protocol. Systolic function was assessed through left ventricular developed pressure (LVDP) and maximal rise in velocity of left ventricular pressure (+dP/dt(max)) and diastolic function through maximal decrease in velocity of left ventricular pressure (-dP/dt(max)) and LVEDP. Lactate dehydrogenase (LDH) release and ATP content were also measured. Stretch resulted in a significant increase of postischemic recovery and attenuation of diastolic stiffness. At 30 min of reperfusion LVDP and +dP/dt(max) were 87 +/- 4% and 92 +/- 6% and -dP/dt(max) and LVEDP were 95 +/- 9% and 10 +/- 4 mmHg vs. 57 +/- 6%, 53 +/- 6%, 57 +/- 10%, and 28 +/- 5 mmHg, respectively, in nonstretched hearts. Stretch increased ATP content and did not produce LDH release. 5-HD did not modify and HMR-1098 prevented the protection achieved by stretch. Our results show that the beneficial effects of stretch on postischemic myocardial dysfunction, cellular damage, and energetic state involve the participation of sarcK(ATP) but not mitoK(ATP).     

Substrate-dependent proton load and recovery of stunned hearts during pyruvate dehydrogenase stimulation

Stimulation of pyruvate dehydrogenase (PDH) improves functional recovery of postischemic hearts. This study examined the potential for a mechanism mediated by substrate-dependent proton production and intracellular pH. After 20 min of ischemia, isolated rabbit hearts were reperfused with or without 5 mM dichloroacetate (DCA) in the presence of either 5 mM glucose, 5 mM glucose + 2.5 mM lactate, or 5 mM glucose + 2.5 mM pyruvate. DCA inhibits PDH kinase, increasing the proportion of dephosphorylated, active PDH. Unlike pyruvate or glucose alone, lactate + glucose did not support the effects of DCA on the recovery of rate-pressure product (RPP) (without DCA, RPP = 14,000 +/- 1,200, n = 6; with DCA, RPP = 13,700 +/- 1,800, n = 9). Intracellular pH, from (31)P nuclear magnetic resonance spectra, returned to normal within 2.1 min of reperfusion with all substrates except for lactate + glucose + DCA or lactate + DCA, which delayed pH recovery for up to 12 min (at 2.1 min pH = 6. 00 +/- 0.08, lactate + glucose + DCA; pH = 6.27 +/- 0.34, for lactate + DCA). Hearts were also reperfused after 10 min of ischemia with 0.5 mM palmitate + 5 mM DCA and either 2.5 mM pyruvate or 2.5 mM lactate. Again, intracellular pH recovery was delayed in the presence of lactate. PDH activation in the presence of lactate also decreased coupling of oxidative metabolism to mechanical work. These findings have implications for therapeutic use of stimulated carbohydrate oxidation in stunned hearts.     

Oxidation and release of glutathione from myocardium during early reperfusion

Glutathione (GSH) is an important intracellular defense against reactive oxygen metabolites. Reaction of GSH with peroxides generates oxidized glutathione (GSSG). We hypothesized that reperfusion would cause oxidation of GSH and release of GSSG as a potential marker of intracellular oxidative reactions. Ten dogs underwent 90 min left anterior descending (LAD) occlusion and 30 min reperfusion. Coronary sinus (CS) plasma was sampled from the great cardiac vein, which drains the LAD region, and from the aorta at pre-ischemia (I), 90 min ischemia, and during reperfusion (R). We found that both GSSG and GSH increased in coronary sinus plasma during early reperfusion. (Formula: see text) Measured GSSG did not arise from autoxidation of plasma GSH. GSH and GSSG release from myocardium not only may be evidence of intracellular oxidative injury, but loss of GSH also could impair metabolism of peroxides during early reperfusion and predispose to further injury.     

Amiloride和耗竭细胞内糖原对心肌缺血再灌注损伤的影响

本工作在离体大鼠等容收缩心脏模型上,观察在缺血前给予ａｍｉｌｏｒｉｄｅ和耗竭心肌细胞内糖原以减少Ｎａ＋－Ｈ＋交换的底物对缺血后再灌注损伤的影响,以探讨Ｎａ＋－Ｈ＋交换和Ｎａ＋－Ｃａ２＋交换机制在心肌缺血后再灌注损伤中的发病学意义。结果表明,Ａｍｉｌｏｒｉｄｅ及耗竭心肌细胞内糖原均能提高心脏血液动力学的恢复,心肌组织乳酸脱氢酶（ＬＤＨ）漏出及丙二醛（ＭＤＡ）生成减少,线粒体中谷胱甘肽过氧化物酶有较高的活性;心肌细胞内Ｎａ＋,Ｃａ２＋超负荷减轻。Ａｍｉｌｏｒｉｄｅ的心肌保护作用可能与其抑制再灌注初期的细胞膜Ｎａ＋－Ｈ＋交换机制有关。耗竭细胞内糖原因减少缺血末细胞内Ｈ＋的堆积,使Ｎａ＋－Ｈ＋交换底物减少而抑制Ｎａ＋－Ｈ＋交换机制。     

Fasudil prevents KATP channel-induced improvement in postischemic functional recovery

Whereas activation of ATP-dependent potassium (K(ATP)) channels greatly improves postischemic myocardial recovery, the final effector mechanism for K(ATP) channel-induced cardioprotection remains elusive. RhoA is a GTPase that regulates a variety of cellular processes known to be involved with K(ATP) channel cardioprotection. Our goal was to determine whether the activity of a key rhoA effector, rho kinase (ROCK), is required for K(ATP) channel-induced cardioprotection. Four groups of perfused rat hearts were subjected to 36 min of zero-flow ischemia and 44 min of reperfusion with continuous measurements of mechanical function and (31)P NMR high-energy phosphate data: 1) untreated, 2) pinacidil (10 microM) to activate K(ATP) channels, 3) fasudil (15 microM) to inhibit ROCK, and 4) both fasudil and pinacidil. Pinacidil significantly improved postischemic mechanical recovery [39 +/- 16 vs. 108 +/- 4 mmHg left ventricular diastolic pressure (LVDP), untreated and pinacidil, respectively]. Fasudil did not affect reperfusion LVDP (41 +/- 13 mmHg) but completely blocked the marked improvement in mechanical recovery that occurred with pinacidil treatment (54 +/- 15 mmHg). Substantial attenuation of the postischemic energetic recovery was also observed. These data support the hypothesis that ROCK activity plays a role in K(ATP) channel-induced cardioprotection.     

Relation between energy metabolism,glycolysis, noradrenaline release and duration of ischemia

We studied the effect of 12–36 min of global ischemia followed by 36 min of reperfusion in Langendorff perfused rabbit hearts (n = 26). Metabolism was determined in terms of peak and total release of purines (adenosine, inosine, hypoxanthine), lactate and noradrenaline during reperfusion; and myocardial content of nucleotides (ATP, ADP, AMP), glycogen and noradrenaline at the end of reperfusion. An inverse relationship (r = –0.79) existed between duration of ischemia and developed pressure post-ischemia. Early during reperfusion, after 12 min of ischemia, the purine concentration (peak release) increased 100x (p < 0.01), that of lactate and noradrenaline lOx (p < 0.05) . Total purine release rose with progression of the ischemic period (30x after 36 min of ischemia; p < 0.01), concomitant with a reduction in nucleotide content. Lactate release was independent from the duration of ischemia, although glycogen had declined by 30% (p < 0.01) after 36 min of ischemia. The acid insoluble glycogen fraction, which presumably contains proglycogen, increased substantially during short-term ischemia. Peak noradrenaline increased 100x and 200x (p < 0.05) after 24 and 36 min of ischemia, respectively. Total noradrenaline release due to various periods of ischemia mirrored its peak release. Function recovery was inversely related to total purine and noradrenaline efflux (both r =–0.81); it correlated with tissue nucleotide content (r = 0.84). In conclusion, larger amounts of noradrenaline are released only after a substantial drop in myocardial ATP. During severe ischemia ATP consumption more than limited ATP production by anaerobic glycolysis, is a key factor affecting recovery on subsequent reperfusion. In contrast to lactate efflux, purine and noradrenaline release are useful markers of ischemic and reperfusion damage.