Xuekun Liu

Detection of hydroxyl radical in the mitochondria of ischemic-reperfused myocardium by trapping with salicylate

Although the presence of free radicals has been indicated in ischemic-reperfused heart, the exact nature and source of these free radicals are not known. The present study utilized a chemical trap, salicylic acid, to trap hydroxyl radical which could be detected as hydroxylated benzoic acid using high pressure liquid chromatography. Since the hydroxylated product is extremely stable, heart was subjected to subcellular fractionation after ischemia and reperfusion, and each fraction was separately examined for the presence of hydroxyl radical. The results indicated for the first time the presence of hydroxyl radical in the mitochondrial fraction during early reperfusion, which decreased in intensity as the reperfusion progressed.

Drug-induced heat-shock preconditioning improves postischemic ventricular recovery after cardiopulmonary bypass.

BACKGROUND Heat-stress preconditioning of mammalian heart has been found to confer protection against ischemic reperfusion injury. Heat shock is generally provided by warming the animal by mechanical means, which is often impractical in a clinical setting. Amphetamine, a sympathomimetic drug, can elevate the body temperature as a result of enhanced endogenous lipolysis. In this study, we examined the effects of heat shock induced by amphetamine on postischemic myocardial recovery in a setting of coronary revascularization for acute myocardial infarction. METHODS AND RESULTS Adult Yorkshire swine were injected with amphetamine (3 mg/kg IM) (n = 12), and body temperature was continuously monitored. For control studies, the pigs were injected with saline (n = 12). Five swine in each group were killed after 3 hours to obtain biopsies of vital organs to measure heat-shock protein (HSP) mRNAs. After 40 hours, the remaining 7 pigs in each group were placed on cardiopulmonary bypass, and the isolated, in situ heart preparations were subjected to 1 hour of occlusion of the left anterior descending coronary artery followed by 1 hour of global hypothermic cardioplegic arrest and 1 hour of reperfusion. Postischemic myocardial performance was monitored by measuring left ventricular (LV) pressure, its dP/dt, myocardial segment shortening, and coronary blood flow. Cellular injury was examined by measurement of creatine kinase release. The antioxidant enzymes superoxide dismutase and catalase were also assayed. Amphetamine treatment was associated with the induction of mRNAs for HSP 27, HSP 70, and HSP 89 in all the vital organs, including heart, lung, liver, kidney, and brain. Amphetamine also enhanced superoxide dismutase and catalase activities in the heart. Significantly greater recovery of LV contractile functions was noticed, as demonstrated by improved recovery of LV developed pressure (61% versus 52%), LV dP/dtmax (52% versus 44%), and segment shortening (46.2% versus 10%) and reduced creatine kinase release in the amphetamine group. CONCLUSIONS The results demonstrate that amphetamine can induce whole-body heat shock that can precondition the heart, enhancing cellular tolerance to ischemia-reperfusion injury. Amphetamine is a sympathomimetic drug that may be used for preconditioning.

Phospholipase D signaling in ischemic heart

Phospholipase D (PLD) activity was found to be present in the membrane fraction of rat myocardial cells by in vitro assays (36.7 +/- 4.1 nmol/mg protein per h against 1-palmitoyl-2-arachidonoyl- phosphatidylcholine) and demonstrated in intact cells by the specific transphosphatidylation reaction (in the presence of 0.02% ethanol) quantitated using n-[1-14C]butanol (201.16 +/- 7.1 pmol/min per g dry weight in the whole heart). Both methods showed a significant increase in PLD activity (by 62 and 44%, respectively) in hearts subjected to reversible (30 min) global normothermic ischemia followed by reperfusion (30 min). In hearts prelabeled with [1-14C]arachidonic acid, ischemia/reperfusion induced a significant increase in the amount of radiolabel incorporated into phosphatidic acid (PtdOH) (by 49.6%) and diacylglycerol (DG) (by 259%). DG kinase inhibition by 100 microM dioctanoylethylene glycol did not affect the ischemia/reperfusion DG and PtdOH levels while PtdOH phosphohydrolase inhibition with 40 microM propranolol produced a further increase in PtdOH (to 2.36-fold the baseline level) and a reduction in DG (to only 145% over the baseline levels). Put together, all these results suggest an activation of PLD during myocardial ischemia/reperfusion generating intracellular PtdOH, part of which is converted by PtdOH phosphohydrolase to DG. We further investigated the possible pathophysiological significance of the observed PLD activation. Stimulation of PLD with sodium oleate (20 microM) induced a significant improvement of functional recovery of ischemic hearts during reperfusion (as monitored by coronary flow and left intraventricular pressure measurements) and an attenuation of cellular injury as expressed by lactate dehydrogenase and creatine kinase release in the coronary effluent during reperfusion. These results suggest a PLD-mediated signaling in the ischemic heart which may benefit functional recovery during reperfusion.

Protection of ischemic heart from reperfusion injury by Myo-inositol hexaphosphate, a natural antioxidant

Myo-inositol hexaphosphate (phytic acid), a highly charged antioxidant, has been found to chelate metal ions such as iron and calcium and to scavenge hydroxyl radicals, .OH. This study examined the efficacy of this antioxidant and redox agent in attenuating myocardial reperfusion injury. Sprague-Dawley rats were injected intravenously with three different doses of phytic acid (group 1, saline solution only, control; group 2, 1.5 mg/100 g; group 3, 7.5 mg/100 g; group 4, 15 mg/100 g) 30 minutes before excision of hearts. Isolated hearts were prepared by the Langendorff technique. Global ischemia was induced for 30 minutes, followed by 30 minutes of reperfusion. As expected, in group 1, reperfusion was associated with enhanced creatine kinase release, reduced coronary flow, poor recovery of ventricular function as evidenced by reduced left ventricular developed pressure and the first derivative of left ventricular pressure, and increased lipid peroxidation. Groups 3 and 4, but not group 2, demonstrated myocardial protection as evidenced by reduced creatine kinase release, improved left ventricular function and coronary flow, and decreased lipid peroxidation compared with the control group. These results suggest that potential use of this antioxidant in salvaging the heart from ischemic and reperfusion injury.

Attenuation of myocardial reperfusion injury by sulfhydryl-containing angiotensin-converting enzyme inhibitors

SummaryRecent studies have suggested the beneficial effects of angiotensin converting enzyme (ACE) inhibitors against myocardial ischemic-reperfusion injury. This study was designed to compare the cardioprotective effects of two sulfhydryl ACE inhibitors, captopril and zofenopril, with those of a nonsulfhydryl ACE inhibitor, fosinopril. The efficacy of these ACE inhibitors to scavenge oxygen radicals in vitro were also examined. Isolated rat hearts perfused by the Langendorff technique were preperfused in the presence or absence of ACE inhibitors (50 μm for 15 minutes), and the hearts were then subjected to 30 minutes of ischemia followed by 30 minutes of reperfusion. Zofenopril and captopril, but not fosinopril, improved postischemic left ventricular functions and reduced myocardial cellular injury, as evidenced by improved recovery of the first derivative of left ventricular pressure development and reduced creatine kinase release compared with control (p<.05). Coronary flow was significantly increased by captopril and zofenopril only. The same two drugs also inhibited the enhanced lipid peroxidation during reperfusion. Although significant differences were not noticed in the postischemic myocardial membrane phospholipid composition, captopril and zofenopril reduced nonesterified fatty acid contents, including palmitic, linoleic, oleic, and arachidonic acids. In vitro studies demonstrated that captopril and zofenopril were able to scavenge hydroxyl radicals. These results indicate that among three ACE inhibitors, two sulfhydryl-containing drugs, captopril and zofenopril, possess cardioprotective as well as free-radical scavenging abilites. Attenuation of phospholipid degradation and lipid peroxidation may be contributory to the protective effects observed in this study.

Improved postischemic ventricular functional recovery by amphetamine is linked with its ability to induce heat shock

Heat shock has been shown to increase the cellular tolerances to ischemic injury. In this study, we examined the effects of heat shock induced by amphetamine on postischemic myocardial functional recovery in a setting of coronary revascularization for acute myocardial infarction. Intramuscular injection of amphetamine (3 mg/kg, i.m.) to pigs increased the body temperature to 42.5°C within 1 h, and maintained this temperature for an additional 2 h. Fourty h after the amphetamine injection, the pigs were placed on by cardiopulmonary bypass and then isolated,in situ heart preparations were subjected to 1 h of global hypothermic cardioplegic arrest and 1 h of normothermic reperfusion. Postischemic myocardial performance was monitored by measuring left ventricular (LV) pressure, its dp/dt, myocardial segmental shortening (%SS), and coronary blood flow. Cellular injury was examined by measuring creatine kinase (CK) release. Biochemical measurements included quantification of plasma catecholamines and study of the induction of heat shock gene expression and antioxidative enzymes in the heart tissue. The results of this study indicated significantly greater recovery of LV contractile functions by amphetamine as demonstrated by improved recovery of LVDP (61% vs 52%), dp/dtmax (52% vs 44%), and segmental shortening (46.2% vs 10%). Myocardial CK release was significantly reduced in the amphetamine group. Furthermore, amphetamine pretreatment was associated with the induction of heat shock protein (HSP) 27 mRNA and stimulated Cu/Zn-superoxide dismutase and catalase levels, suggesting that amphetamine mediated improved postischemic ventricular recovery might be linked with its ability to induce heat shock and stimulate antioxidant enzymes.

Oxygen-derived free radicals and hemolysis during open heart surgery

Reperfusion injury occurs during open-heart surgery after prolonged cardioplegic arrest. Cardiopulmonary bypass also is known to cause hemolysis. Since reperfusion of ischemic myocardium is associated with the generation of oxygen free radicals, and since free radicals can attack a protein molecule, it seems reasonable to assume that hemolysis might be the consequence of free radical attack on hemoglobin protein. The results of this study demonstrated that reperfusion following ischemic arrest caused an increase in free hemoglobin and free heme concentrations, simultaneously releasing free iron and generating hydroxyl radicals. In vitro studies using pure hemoglobin indicated that superoxide anion generated by the action of xanthine oxidase on xanthine could release iron from the heme ring and cause deoxygenation of oxyhemoglobin into ferrihemoglobin. This study further demonstrated that before the release of iron from the heme nucleus, oxyhemoglobin underwent deoxygenation to ferrihemoglobin. The released iron can catalyze the Fenton reaction, leading to the formation of cytotoxic hydroxyl radical (OH·). In fact, the formation of OH. in conjunction with hemolysis occurs during cardiac surgery, and when viewed in the light of the in vitro results, it seems likely that oxygen-derived free radicals may cause hemolysis during cardiopulmonary bypass and simultaneously release iron from the heme ring, which can catalyze the formation of OH·.

Preservation of membrane phospholipids by propranolol, pindolol, and metoprolol: a novel mechanism of action of beta-blockers.

In this study, we examined the effects of three different beta-blockers, propranolol, pindolol, and metoprolol, on membrane phospholipid preservation in the ischemic and reperfused rat heart. Isolated rat hearts were perfused with Krebs-Henseleit bicarbonate buffer by the Langdendorff technique in the presence or absence of propranolol, pindolol, or metroprolol (20 microM each) for 15 mins at 37 degrees C. Hearts where then either made ischemic alone at 37 degrees C for 30 mins, or followed by 30 mins of reperfusion. Coronary flow and perfusate creatine kinase content were monitored during both pre- and post-ischemic periods. At the end of the experiment, hearts were frozen by freeze-clamping at liquid nitrogen temperature. Membrane phospholipids, fatty acid composition of these phospholipids, non-esterified free fatty acids, and myocardial thiobabituric acid (TBA) reactive product were examined in these hearts. The beta-blocker-treated hearts exhibited significantly less lipid peroxidation than the control hearts (P less than 0.05), as indicated by decreased formation of TBA reactive product and the higher percentage of unsaturated fatty acids in the phosphatidylcholine (PC) in heart. In addition, compared to the control group, less accumulation of free fatty acids was observed in the propranolol and pindolol treated groups. Finally, reduced myocardial creatine kinase release and enhanced recovery of coronary flow indicated significant myocardial preservation by these beta-blockers. The efficacy of these beta-blockers were in the following order: propranolol, pindolol, metoprolol. These results suggest that beta-blockers could also protect an ischemic heart from reperfusion injury by preserving the membrane phospholipids.

Reduced free radical generation during reperfusion of hypothermically arrested hearts

Several studies indicate the presence of hydroxyl radical (OH·) as well as its involvement in the myocardial reperfusion injury. A transition metal-like iron is necessary for the conversion of superoxide anion (O2−) to a highly reactive and cytotoxic hydroxyl radical (OH·). In the present study, we have examined the generation of OH· and free iron in reperfused hearts following either normothermic (37°C) or hypothermic ischemia (5°C). Employing the Langendorff technique, isolated rat hearts were subjected to global ischemia for 30 min at 37°C or 5°C and were then reperfused for 15 min at 37°C. The results of the study suggest that both the OH· generation in myocardium and free iron release into perfusate were significantly lower in hearts made ischemic at 5°C as compared to 37°C. Release of myoglobin and lactic acid dehydrogenase into perfusate also followed a similar pattern. Furthermore, in in vitro studies, chemically generated O2− at 5°C caused a significantly lower rate of oxidation of oxymyoglobin as well as generation of OH° and free iron as compared to 37°C. These results suggest that (1) reperfusion of hypothermic ischemic heart is associated with a reduction in the generation of OH· and cellular damage compared to that of normothermic ischemic heart, and (2) myoglobin, an intracellular protein, is a source of free iron and plays a role in the reperfusion injury mediated by free radicals.

Attenuation of myocardial reperfusion injury by reducing intracellular calcium overloading with dihydropyridines

The effects of three different dihydropyridine (DHP) calcium channel antagonists, nisoldipine, nimodipine, and nifedipine, on myocardial ischemic and reperfusion injury were studied using isolated rat hearts subjected to ischemia and reperfusion. Hearts were perfused with Krebs-Henseleit bicarbonate buffer containing 0, 4, 16, 64 and 100 nM concentrations of the above dihydropyridines for 15 min. Global ischemia was then induced by terminating the aortic flow for 30 min at 37 degrees, followed by 30 min of reperfusion. Left ventricular (LV) functional (LV developed pressure, its first derivative and coronary flow) and biochemical parameters (creatine kinase release) were monitored prior to ischemia and during reperfusion. In separate group of hearts, intracellular free Ca2+ ([Ca2+]i) was monitored with an intracellular calcium analyzer using a fluorescent Ca2+ indicator (Fura-2 AM). Tissue Ca2+ was also measured by atomic absorption spectroscopy after perfusing the hearts with ion-free cold buffer to wash out extracellular Ca2+. Significant recovery of the coronary flow was observed in all hearts treated with a high concentration (100 nM) of DHPs compared with the control group (P < 0.05), while a lower dose of nisoldipine (16 nM) and nifedipine (64 nM) also improved the coronary flow effectively. Reduction of myocardial creatine kinase release and improvement of the recovery of LV developed pressure, dp/dtmax, were achieved by DHPs in a concentration-dependent manner. A higher concentration of DHPs also decreased the formation of myocardial thiobarbituric acid reactive substances, although these compounds did not possess direct free radical scavenging effects in vitro. Tissue Ca2+ content was reduced significantly in treated groups. The rise of [Ca2+]i during ischemia and reperfusion appeared to be attenuated by these DHPs. The concentration-response study of the three DHPs showed the effective concentrations for reducing [Ca2+]i to be 16, 64 and 100 nM nisoldipine, nifedipine and nimodipine, respectively, in this experimental setting. The above results indicate that pretreatment with DHPs can attenuate the myocardial reperfusion injury by modulating Ca2+ overloading and decreasing the susceptibility of the membrane to free radical attack.