Uncoupling of mitochondrial oxidative phosphorylation alters lipid peroxidation-derived free radical production but not recovery of postischemic rat hearts and post-hypoxic endothelial cells

The contribution of mitochondrial free radical production towards the initiation of lipid peroxidation (LPO) and functional injury in the post-ischemic heart is unclear. Using the isolated rat heart model, the effects of the uncoupler of mitochondrial oxidative phosphorylation dinitrophenol (DNP, 50 M final) on post-ischemic lipid peroxidation-derived free radical production and functional recovery were assessed. Hearts were subjected to 30 min total global ischemia followed by 15 min of reperfusion in the presence of DNP. As expected, DNP enhanced oxygen consumption before (11.3 ± 0.9 mol/min, p < 0.001) and during reperfusion (at 10 min: 7.9 ± 0.7 umol/min), compared to the heart with control treatment (8.2 ± 0.5 and 6.7 ± 0.3, respectively). This effect was only associated with a higher incidence of ventricular tachycardia during reperfusion (80 vs. 50% for control treatment, p < 0.05). Electron spin resonance spectroscopy (ESR) and spin trapping with u.-phenyl-tert-butylnitrone (PBN, 3 mM final) were used to monitor free radical generation during reperfusion. The vascular concentration of PBN-radical adducts (untreated: 6.4 ±1.0 nM, at 10 min) decreased in the presence of DNP (1.7 ± 0.4 nM, p < 0.01). The radical concentration inversely correlated with myocardial oxygen consumption. Total liberation of free radical adducts during the initial 10 min of reperfusion was reduced by DNP (0.59 ± 0.09 nmol, p < 0.01) compared to the respective control treatment (1.26 ± 0.16 nmol). Similar effects, prevention of PBN adduct formation and unchanged viability in the presence of DNP, were obtained with endothelial cells during post-hypoxic reoxygenation. Since inhibition of mitochondrial phosphorylation can inhibit the formation of LPO-derived free radicals after an ischemic/hypoxic interval, mitochondria may represent an important source of free radicals capable of initiating lipid peroxidative injury during reperfusion/reoxygenation. (Mol Cell Biochem 160/161: 167–177, 1996)     

Hypercholesterolemia attenuates postischemic ventricular dysfunction in the isolated rabbit heart

The effects of the chronic administration of cholesterol on the stunned myocardium have not been studied. The objective was to determine the effect of a cholesterol enriched diet on postischemic ventricular dysfunction. In group 1 (G1, n = 7 isolated rabbit hearts underwent a follow up of ventricular function during 30 min in aerobic conditions. In group 2 (G2, n = 6) G1 was repeated but the animals were subjected to a 1% cholesterol enriched diet during 4 weeks (hypercholesterolemic animals). In group 3 (G3, n = 8) hearts underwent 15 min of global ischemia followed by 30 min of reperfusion. In Group 4 (G4, n = 11) G3 was repeated, but in hypercholesterolemic animals. Since cholesterol decreased the inotropism in basal situation, and this makes the comparison between groups difficult, we performed a Group 5 (G5, n = 7), in which G4 protocol was repeated but isoproterenol (8 g/kg/min) was administered 10 min before ischemia, in order to match the preischemic inotropic state with respect to the normocholesterolemic ones. G1 and G2 maintained a stable inotropism during the 30 min of perfusion. The preischemic left ventricular developed pressure (LVDP) in G3 and G4 was 91.4± 4.3 and 70.8± 3.4 mmHg (p< 0.05), respectively, and after 30 min of reperfusion differences were not observed between G3 and G4. Nevertheless, when LVDP is expressed as a percentage, we detected an attenuation of postischemic systolic alterations in hypercholesterolemic animals (67.3± 3.6 in G4 vs. 90.8± 3.1% in G3, p< 0.05). When LVDP in G5 was increased until matching the one of G3, there were no differences after 30 min of reperfusion. Left ventricular end diastolic pressure increased 285± 46%, 61± 25% (p< 0.05 vs. G3 and G5) and 216± 25% in G3, G4 and G5 at 30 min of reperfusion. There were no differences either in the values of tau or infarct size between groups. Thus, in hypercholesterolemic animals, a decrease of the preischemic inotropism exists and there is an attenuation of the stunned myocardium. When contractility of the normo and hypercholesterolemic animals is matched, the beneficial effect disappears.     

Change in substrate metabolism in isolated mouse hearts following ischemia-reperfusion

Several genetic and transgenic mouse models are currently being used for studying the regulation of myocardial contractility under normal conditions and in disease states. Little information has been provided, however, about myocardial energy metabolism in mouse hearts. We measured glycolysis, glucose oxidation and palmitate oxidation (using ³H-glucose, ¹⁴C-glucose and ³H-palmitate) in isolated working mouse hearts during normoxic conditions (control group) and following a 15 min global no-flow ischemic period (reperfusion group). Fifty min following reperfusion (10 min Langendorff perfusion + 40 min working heart perfusion) aortic flow, coronary flow, cardiac output, peak systolic pressure and heart rate were 44 ± 4, 88 ± 4, 57 ± 4, 94 ± 2 and 81 ± 4% of pre-ischemic values. Rates of glycolysis and glucose oxidation in the reperfusion group (13.6 ± 0.8 and 2.8 ± 0.2 mol/min/g dry wt) were not different from the control group (12.3 ± 0.6 and 2.5 ± 0.2 mol/min/g dry wt). Palmitate oxidation, however, was markedly elevated in the reperfusion group as compared to the control group (576 ± 37 vs. 357 ± 21 nmol/min/g dry wt, p < 0.05). This change in myocardial substrate utilization was accompanied by a marked fall in cardiac efficiency measured as cardiac output/oxidative ATP production (136 ± 10 vs. 54 ± 5 ml/mol ATP, p < 0.05, control and reperfusion group, respectively). We conclude that ischemia-reperfusion in isolated working mouse hearts is associated with a shift in myocardial substrate utilization in favour of fatty acids, in line with previous observations in rat.     

Lysophosphatidylcholine-induced myocardial damage is inhibited by pretreatment with poloxamer 188 in isolated rat heart

Lysophosphatidylcholine (LPC) accumulates in myocardial tissues and coronary sinus during ischemia, and plays important role in the development of ischemia-reperfusion injury and ischemic ventricular arrhythmia. The aim of this study was to examine whether pretreatment of poloxamer 188 (P-188), a nonionic and non-toxic surfactant, can prevent the cardiac dysfunction induced by exogenous LPC perfusion in Langendorff perfused rat heart model. LPC (6 M) significantly (p < 0.05) decreased heart rate (HR) and left ventricular developed pressure (LVDP) from 274.3 ± 23.2 to 175.0 ± 42.9/min and from 115.9 ± 11.3 to 26.7 ± 7.1 mmHg, respectively. The LPChyphen;induced reduction of HR and LVDP did not recover by washout of LPC. Pretreatment with P-188 (1 mM for 30 min) inhibited completely the LPC-induced decreases of HR and LVDP. The pretreatment with P-188 also prevented the LPC-induced increases of left ventricular end-diastolic pressure (LVEDP) and GOT release, significantly (p < 0.05). The coronary perfusion pressure (CPP) rose (p < 0.01) by the LPC perfusion from 71.9 ± 5.3 to 121.9 ± 13.0 mmHg, significantly, but pretreatment of P-188 did not affect the LPC-induced vasoconstriction. Our results suggest that exogenous LPC causes irreversible cardiac injury by the sarcolemmal membrane disruption followed by Ca overload, and this LPC-induced cardiac injury, probably, can be prevented by the pretreatment with poloxamer 188.     

A single cell model of myocardial reperfusion injury: Changes in intracellular Na+ and Ca2+ concentrations in guinea pig ventricular myocytes

To investigate the contribution of the changes in intracellular Na+ and Ca2+ concentrations ([Na+]i and [Ca2+]i) to myocardial reperfusion injury, we made an ischemia/reperfusion model in intact guinea pig myocytes. Myocardial ischemia was simulated by the perfusion of metabolic inhibitors (3.3 mM amobarbital and 5 M carbonyl cyanide m-chlorophenylhydrazone) with pH 6.6 and reperfusion was achieved by the washout of them with pH 7.4. [Na+]i increased from 7.9 ± 2.0 to 14.0 ± 3.4 mM (means ± S.E., p < 0.01) during 7.5 min of simulated ischemia (SI) and increased further to 18.8 ± 3.0 mM at 7.5 min after reperfusion. [Ca2+]i, expressed as the ratio of fluo 3 fluorescence intensity, increased to 133 ± 8% (p < 0.01) during SI and gradually returned to the control level after reperfusion. Intracellular pH decreased from 7.53 ± 0.04 to 6.31 ± 0.04 (p < 0.01) and recovered quickly after reperfusion. Reperfusion with the acidic solution or the continuous perfusion of hexamethylene amiloride (2 M) prevented the reperfusion-induced increase in [Na+]i. When the duration of SI was prolonged to 15 min, the cell response after reperfusion varied, 16 of 37 cells kept quiescent, 21 cells showed spontaneous Ca2+ waves, and 4 cells out of these 21 cells became hypercontracted. In quiescent cells, both [Na+]i and [Ca2+]i decreased immediately after reperfusion. In cells with Ca2+ waves, [Na+]i transiently increased further at the early phase of reperfusion, while [Ca+]i declined. In hypercontracted cells, [Na+]i increased as much as in Ca2+ wave cells, but [Ca2+]i increased extensively and both ion concentrations continued to increase. Reperfusion with the Ca2+-free solution prevented both the [Ca2+]i increase and morphological change. In the presence of ryanodine (10 M), the increase in [Ca2+]i after reperfusion was augmented and some cells became hypercontracted. We concluded that (1) Na+/H+ exchange is active both during SI and reperfusion, resulting in the additional [Na+]i elevation on reperfusion, (2) the [Na+]i level after reperfusion and the following Ca2+ influx via Na+/Ca2+ exchange are crucial for reperfusion cell injury, and (3) the Ca2+ buffering capacity of sarcoplasmic reticulum would also contribute to the Ca2+ regulation and cell injury after reperfusion.     

Insulin improves postischemic recovery of function through PI3K in isolated working rat heart

Insulin improves contractile function after ischemia, but does not increase glucose uptake in the isolated working rat heart. We tested the hypothesis that the positive inotropic effect of insulin is independent of the signaling pathway responsible for insulin-stimulated glucose uptake. We inhibited this pathway at the level of phosphatidyl inositol 3-kinase (PI3K) with wortmannin. Hearts were perfused for 70 min at physiological workload with Krebs-Henseleit buffer containing [2-³H] glucose (5 mM, 0.05 Ci/ml) and oleate (0.4 mM, 1% BSA) in the presence (WM, n = 5) or absence (control, n = 7) of wortmannin (WM, 3 mol/L). After 20 min, hearts were subjected to 15 min of total global ischemia followed by 35 min of reperfusion. Insulin (1 mU/ml) was added at the beginning of reperfusion (WM + insulin n = 8, insulin n = 8). Cardiac power before ischemia was 8.1 ± 0.7 mW. Recovery of contractile function after ischemia was significantly increased in the presence of insulin (73.5 ± 8.9% vs. 38.5 ± 6.7%, p < 0.01). The addition of wortmannin completely abolished the effect of insulin on recovery (32.6 ± 6.4%). Glucose uptake was 1.84 ± 0.32 mol/min/g dry before ischemia and was slightly elevated during reperfusion (2.68 ± 0.35 mol/min/g dry, n.s.). Insulin did not affect postischemic glucose uptake. In the presence of wortmannin, glucose uptake was lowest during reperfusion (n.s.). The results suggest that PI3K is involved in the insulin-induced improvement in postischemic recovery of contractile function. This effect of insulin is independent of its effect on glucose uptake.     

Apomorphine-induced myocardial protection is due to antioxidant and not adrenergic/dopaminergic effects

Apomorphine (Apo), a dopaminergic agonist used for treatment of Parkinson disease, is a potent antioxidant. In addition to its antioxidative effects, the dopaminergic and adrenergic effects of Apo were studied. Isolated perfused rat hearts were exposed to 25 min of no-flow global ischemia (37 degrees C) and 60 min of reperfusion (I/R, control). Drugs were introduced for the first 20 min of reperfusion. The LVDP of the control group recovered to 54.6 +/- 3.3%. Apo-treated hearts had significantly improved recovery (61.6 +/- 5%, p < 0.05). The recovery of the work index LVDP x HR was even bigger: 67.8 +/- 3.7% (Apo treatment) vs 41.7 +/- 4.6% (control, p < 0.001). Haloperidol, a dopaminergic antagonist, did not affect the recovery with Apo. Propranolol, a beta-adrenergic blocker, initially inhibited the effect of Apo. However, the recovery of the combined group (Apo + propranolol) increased and reached significance (LVDP, p < 0.05 vs control group) after cessation of propranolol perfusion. At 60 min of reperfusion this group was superior to Apo-treated hearts (LVDP, p < 0.05). Propranolol (without Apo) did not improve the hemodynamic recovery. The same pattern of recovery applies also to the recovery of the +dP/dt during the reperfusion. L-DOPA was less effective than Apo. I/R caused significant increase in carbonylation of proteins. Apomorphine inhibited the increase in carbonylation. Haloperidol did not affect this beneficial effect of Apo. L-DOPA significantly decreased the carbonylation of proteins. We conclude that the antioxidative effect of Apo is its main mechanism of cardioprotection.     

Characterization of the phosphatidylinositol-specific phospholipase C-released form of rat osseous plate alkaline phosphatase and its possible significance on endochondral ossification

S-2-(3 aminopropylamino) ethylphosphorothioic acid (WR-2721) shown to surpass radical scavenging thiols in their radioprotective efficacy in cancer-type diseases has been tested for its protective potential in the reperfused heart. We investigated the radical scavenger properties of the compound in a radical generating systemin vitro as well as in isolated rat hearts subjected to 30 min ischaemia and 30 min reperfusion with the electron-paramagnetic resonance spin trap technique. The action on high-energy phosphates is observed by means of phosphorus-31 nuclear magnetic resonance (NMR) spectroscopy while its influence on left ventricular systolic segmental length change (SSLC) during 60 min reperfusion following 60 min regional ischaemia was assessed with a fibreoptic system in anaesthetized open-chest rats. WR-2721 (0.1 mM) reduced the vascular concentration of radical adduct in isolated hearts by up to 78% (275±84% of pre-ischaemic baseline values vs 1260±413%, p<0.05) between 5 and 12.5 min reperfusion. This was accompanied by a reduction of the left ventricular end diastolic pressure to pre-ischaemic values at 30 min of reperfusion (9±6 mmHg vs 42±8 mmHg in the absence of WR-2721, p<0.02). An accelerated recovery of creatine phosphate levels (78±5% of pre-ischaemia values vs 41±5% within 60 min reperfusion; p<0.05) was observed under similar conditions with NMR-spectroscopy, although the post-ischaemic tissue content of adenosine triphosphate was not affected. The administration of WR-2721 (0.5 mmol/kg body weight) ledin situ to an accelerated restoration of contractile activity in the post-ligated left ventricular area reflected by the post-ischaemic recovery of SSLC (64±10% of pre-ischaemic values compared with 27±6% in control animals 60 min following reperfusion; p<0.02). The present data confirm an effective reduction in the exposure of the reperfused heart to endogenously released free radicals by WR-2721, a partial preservation of high-energy phosphates and an improvement in post-ischaemic contractility and encourage further investigation of such favourable action in injured myocardium.     

Isolated perfused rat hearts release secondary free radicals during ischemia reperfusion injury: Cardiovascular effects of the spin trap α-phenyl N-tert-butylnitrone

Free radicals produced during myocardial post-ischemic reperfusion are aggravating factors for functional disturbances and cellular injury. The aim of our work was to investigate the significance of the secondary free radical release during non ischemic perfusion and post-ischemic reperfusion and to evaluate the cardiovascular effects of the spin trap used. For that purpose, isolated perfused rat hearts underwent 0, 20, 30 or 60 min of a total ischemia, followed by 30 min of reperfusion. The spin trap: α-phenyl N-tert-butylnitrone (PBN) was used (3 mM). Functional parameters were recorded and samples of coronary effluents were collected and analyzed using Electron Paramagnetic Resonance (EPR) to identify and quantify the amount of spin adducts produced. During non ischemic perfusion, almost undetectable levels of free radical release were observed. Conversely, a large and long-lasting (30 min) release of spin adducts was detected from the onset of reperfusion. The free radical species were identified as alkyl and alkoxyl radicals with amounts reaching 40 times the pre-ischemic values. On the other hand, PBN showed a cardioprotective effect, allowing a significant reduction of rhythm disturbances and a better post-ischemic recovery for the hearts which were submitted to 20 min of ischemia. When the duration of ischemia increased, the protective effects of PBN disappeared and toxic effects became more important. Our results have therefore confirmed the antioxidant and protective properties of a spin trap agent such as PBN. Moreover, we demonstrated that the persistent post-ischemic dysfunction was associated with a sustained production and release of free radical species.     

Is stunning prevented by ischemic preconditioning?

In a model of global ischemia in the isolated perfused rat heart, a 20-min ischemic period followed by 30 min of reperfusion induces a decrease in isovolumic developed pressure (LVDP) and +dP/dt_max to 61 ± 6% and 61 ± 7% of baseline, respectively. Left ventricular end-diastolic pressure (LVEDP) increases to 36 ± 4 mmHg at the end of the reperfusion period. No significant necrotic area as assessed by triphenyltetrazolium chloride (TTC) was detected at the end of the reperfusion period. By an immunohistochemical method using antiactin monoclonal antibodies 10.8 ± 1.9% of unstained cells were detected in the stunned hearts and 10.3 ± 1.2% in control hearts. Preceding the ischemic episode with a cycle of 5 min of ischemia followed by 10 min of reperfusion (ischemic preconditioning) protected contractile function. LVDP and +dP/dt_max now stabilized at 89 ± 5% and 94 ± 5% of baseline respectively. LVEDP was 20 ± 2 mmHg at the end of the reperfusion period. The protection of contractile dysfunction after 20 min of ischemia was achieved also by early reperfusion of low Ca²⁺-low pH perfusate. With this intervention LVDP stabilized at 87 ± 5% of baseline. LVEDP was 12 ± 2 mmHg at the end of the reperfusion period. A positive inotropic intervention induced by a modified postextrasystolic potentiation protocol at the end of the reperfusion period increases LVDP to levels higher than baseline in the stunned hearts. However, these values were less than those obtained in control hearts. Ischemic preconditioning significantly increased the maximal inotropic response. Therefore, ischemic preconditioning diminishes the contractile dysfunction of early stunning.     

Alpha-Phenyl-Tert-Butyl-Nitrone (PBN) Attenuates Hydroxyl Radical Production During Ischemia-Reperfusion Injury of Rat Brain: An EPR Study

α-phenyl-tert-butyl-nitrone (PBN) a spin adduct forming agent is believed to have a protective action in ischemia-reperfusion injury of brain by forming adducts of oxygen free radicals including ±OH radical. Electron paramagnetic resonance (EPR) has been used to both detect and monitor the time course of oxygen free radical formation in the in vivo rat cerebral cortex. Cortical cups were placed over both cerebral hemispheres of methoxyflurane anesthetized rats prepared for four vessel occlusion-evoked cerebral ischemia. Prior to the onset of sample collection, both cups were perfused with artificial cerebrospinal fluid (aCSF) containing the spin trap agent α-(4-pyridyl-1-oxide)-N-tert butylnitrone (POBN 100 mM) for 20 min. In addition 50 mg/kg BW of POBN was administered intraperitoneally (IP) 20 min prior to ischemia in order to improve our ability to detect free radical adducts. Cup fluid was subsequently replaced every 15 min during ischemia and every 10 min during reperfusion with fresh POBN containing CSF and the collected cortical superfusates were analyzed for radical adducts by EPR spectroscopy. After a basal 10 min collection, cerebral ischemia was induced for 15 or 30 min (confirmed by EEG flattening) followed by a 90 min reperfusion. -OH radical adducts (characterized by six line EPR spectra) were detected during ischemia and 90 min reperfusion. No adduct was detected in the basal sample or after 90 min of reperfusion. Similar results were obtained when diethylenetriaminepenta-acetic acid (100 μM; DETAPAC) a chelating agent was included in the artificial CSF. Systemic administration of PBN (100 mg/kg BW) produced a significant attenuation of radical adduct during reperfusion. A combination of systemic and topical PBN (100 mM) was required to suppress -OH radical adduct formation during ischemia as well as reperfusion. PBN free radical adducts were detected in EPR spectra of the lipid extracts of PBN treated rat brains subjected to ischemia/reperfusion. Thus this study suggests that PBN's protective action in cerebral ischemia/reperfusion injury is related to its ability to prevent a cascade of free radical generation by forming spin adducts.     

Alpha-Phenyl-Tert-Butyl-Nitrone (PBN) Attenuates Hydroxyl Radical Production During Ischemia-Reperfusion Injury of Rat Brain: An EPR Study

-phenyl-tert-butyl-nitrone (PBN) a spin adduct forming agent is believed to have a protective action in ischemia-reperfusion injury of brain by forming adducts of oxygen free radicals including ±OH radical. Electron paramagnetic resonance (EPR) has been used to both detect and monitor the time course of oxygen free radical formation in the in vivo rat cerebral cortex. Cortical cups were placed over both cerebral hemispheres of methoxyflurane anesthetized rats prepared for four vessel occlusion-evoked cerebral ischemia. Prior to the onset of sample collection, both cups were perfused with artificial cerebrospinal fluid (aCSF) containing the spin trap agent -(4-pyridyl-1-oxide)-N-tert butylnitrone (POBN 100 mM) for 20 min. In addition 50 mg/kg BW of POBN was administered intraperitoneally (IP) 20 min prior to ischemia in order to improve our ability to detect free radical adducts. Cup fluid was subsequently replaced every 15 min during ischemia and every 10 min during reperfusion with fresh POBN containing CSF and the collected cortical superfusates were analyzed for radical adducts by EPR spectroscopy. After a basal 10 min collection, cerebral ischemia was induced for 15 or 30 min (confirmed by EEG flattening) followed by a 90 min reperfusion. -OH radical adducts (characterized by six line EPR spectra) were detected during ischemia and 90 min reperfusion. No adduct was detected in the basal sample or after 90 min of reperfusion. Similar results were obtained when diethylenetriaminepenta-acetic acid (100 μM; DETAPAC) a chelating agent was included in the artificial CSF. Systemic administration of PBN (100 mg/kg BW) produced a significant attenuation of radical adduct during reperfusion. A combination of systemic and topical PBN (100 mM) was required to suppress -OH radical adduct formation during ischemia as well as reperfusion. PBN free radical adducts were detected in EPR spectra of the lipid extracts of PBN treated rat brains subjected to ischemia/reperfusion. Thus this study suggests that PBN's protective action in cerebral ischemia/reperfusion injury is related to its ability to prevent a cascade of free radical generation by forming spin adducts.     

Isolated perfused rat hearts release secondary free radicals during ischemia reperfusion injury: cardiovascular effects of the spin trap alpha-phenyl N-tert-butylnitrone.

Free radicals produced during myocardial post-ischemic reperfusion are aggravating factors for functional disturbances and cellular injury. The aim of our work was to investigate the significance of the secondary free radical release during non ischemic perfusion and post-ischemic reperfusion and to evaluate the cardiovascular effects of the spin trap used. For that purpose, isolated perfused rat hearts underwent 0, 20, 30 or 60 min of a total ischemia, followed by 30 min of reperfusion. The spin trap: alpha-phenyl N-tert-butylnitrone (PBN) was used (3 mM). Functional parameters were recorded and samples of coronary effluents were collected and analyzed using Electron Paramagnetic Resonance (EPR) to identify and quantify the amount of spin adducts produced. During non ischemic perfusion, almost undetectable levels of free radical release were observed. Conversely, a large and long-lasting (30 min) release of spin adducts was detected from the onset of reperfusion. The free radical species were identified as alkyl and alkoxyl radicals with amounts reaching 40 times the pre-ischemic values. On the other hand, PBN showed a cardioprotective effect, allowing a significant reduction of rhythm disturbances and a better post-ischemic recovery for the hearts which were submitted to 20 min of ischemia. When the duration of ischemia increased, the protective effects of PBN disappeared and toxic effects became more important. Our results have therefore confirmed the antioxidant and protective properties of a spin trap agent such as PBN. Moreover, we demonstrated that the persistent post-ischemic dysfunction was associated with a sustained production and release of free radical species.     

Use of Spin Traps in Intact Animals Undergoing Myocardial Ischemia/Reperfusion: A New Approach to Assessing the Role of Oxygen Radicals in Myocardial “Stunning”

Numerous studies have indirectly, suggested that oxygen-derived free radicals play an important path-ogenetic role in the prolonged depression of contractile function observed in myocardium reperfused after reversible ischemia (myocardial “stunning”). In order to provide direct evidence for the oxy-radical hypothesis of stunning, we administered the spin trap, α-phenyl N-tert-butyl nitrone (PBN), to open-chest dogs undergoing a 15-min coronary artery occlusion followed by reperfusion. Plasma of local coronary venous blood was analyzed by electron paramagnetic resonance (EPR) spectroscopy. EPR signals characteristic of radical adducts of PBN appeared during ischemia and increased dramatically in the first few minutes after reperfusion. After this initial burst, the production of adducts abated but did not cease, persisting up to 3 h after reflow. The production of PBN adducts after reperfusion was inversely related to collateral flow during ischemia. PBN itself enhanced recovery of contractile function. indicating that the radicals trapped may play a pathogenetic role in myocardial stunning. Superoxide dismutase plus catalase attenuated PBN adduct production and, at the same time, improved recovery of contractile function. Antioxidant therapy given 1 min before reperfusion suppressed PBN adduct production and improved contractile recovery; however, the same therapy given 1 min after reperfusion did not suppress early radical production and did not attenuate contractile dysfunction. After i.v. administration, the elimination half-life of PBN was estimated to be approximately 4–5 h. The results demonstrate that 1) free radicals are produced in the stunned myocardium in intact animals; 2) inhibition of free radical production results in improved contractile recovery; and 3) the free radicals important in causing dysfunction are produced in the first few minutes of reperfusion. Taken together, these studies provide cogent evidence supporting the oxy-radical hypothesis of stunning in open-chest dogs. It is now critical to determine whether these results can be reproduced in conscious animal preparations.     

Progressively developed myocardial apoptotic cell death during late phase of reperfusion

Myocardial apoptosis is primarily triggered during reperfusion (R). The aim of this study was to test the hypothesis that R-induced apoptosis develops progressively during the late phase of R, and that R-induced apoptosis is associated with changes in expression of anti- and pro-apoptotic proteins and infiltrated inflammatory cells. Thirty-one dogs were subjected to 60 min of left anterior descending coronary occlusion followed by 6, 24, 48, and 72 h R, respectively. There was no group difference in collateral blood flow, measured by colored microspheres during ischemia. Necrotic cell death (TTC staining) was significantly increased during R, starting at 27 ± 2% at 6 h R and increasing to 41 ± 2% at 24 h R. There was no further change at 48 (37 ± 3%) and 72 (36 ± 6%) h R, respectively. TUNEL positive cells (% total normal nuclei) in the peri-necrotic zone progressively increased from 6 (26 ± 2^*) to 24 (38 ± 1^*), 48 (48 ± 3^*) and 72 (59 ± 4^*) h R, respectively. The number of detected TUNEL positive cells at these time points was consistent with an increased intensity of DNA ladders, identified by agarose gel electrophoresis. Compared with normal tissue, western blot analysis showed persistent reduction in expression of anti-apoptotic protein Bcl-2 from 6 (16 ± 0.8%^*) to 72 h R (78 ± 2%^*), and increase in expression of pro-apoptotic proteins including Bax from 6 (30 ± 3%^*) to 72 h R (66 ± 3%^*), and p53 from 6 (12 ± 1%^*) to 72 h R (91 ± 2%^*), respectively. Immunohistochemical staining revealed that infiltrated neutrophils (mm² myocardium) were significantly correlated with development of necrotic and apoptotic cell death from 6 to 24 h R, respectively (P < 0.05), while large macrophage infiltration seen during 48 to 72 h R were correlated with apoptotic cell death (P < 0.05). These results indicate that 1) necrosis peaked at 24 h R when apoptosis was still progressively developing during later R; 2) changes in Bcl-2 family and p53 proteins may participate in R-induced myocardial apoptosis; 3) inflammatory cells may play a role in triggering cell death during R. ^* P < 0.05 vs. normal nuclei and tissue; P < 0.01 vs. 6 h R.     

Na+ overload during ischemia and reperfusion in rat hearts: Comparison of the Na+/H+ exchange blockers EIPA,cariporide and eniporide

Intracellular myocardial Na⁺ overload during ischemia is an important cause of reperfusion injury via reversed Na⁺/Ca²⁺ exchange. Prevention of this Na⁺ overload can be accomplished by blocking the different Na⁺ influx routes. In this study the effect of ischemic inhibition of the Na⁺/H⁺ exchanger (NHE) on [Na⁺]_i, pHi and post-ischemic contractile recovery was tested, using three different NHE-blockers: EIPA, cariporide and eniporide. pHi and [Na⁺]_i were measured using simultaneous ³¹P and ²³Na NMR spectroscopy, respectively, in paced (5 Hz) isolated, Langendorff perfused rat hearts while contractility was assessed by an intraventricular balloon. NHE-blockers (3 M) were administered during 5 min prior to 30 min of global ischemia followed by 30 min drug-free reperfusion. NHE blockade markedly reduced ischemic Na⁺ overload; after 30 min of ischemia [Na⁺]_i had increased to 293 ± 26, 212 ± 6, 157 ± 5 and 146 ± 6% of baseline values in untreated and EIPA (p < 0.01 vs. untreated), cariporide (p < 0.01 vs. untreated) and eniporide (p < 0.01 vs. untreated) treated hearts, respectively. Ischemic acidosis did not differ significantly between groups. During reperfusion, however, recovery of pHi was significantly delayed in treated hearts. The rate pressure product recovered to 12.0 ± 1.9, 12.1 ± 2.1, 19.5 ± 2.8 and 20.4 ± 2.5 × 103 mmHg/min in untreated and EIPA, cariporide (p < 0.01 vs. untreated) and eniporide (p < 0.01 vs. untreated) treated hearts, respectively. In conclusion, blocking the NHE reduced ischemic Na⁺ overload and improved post-ischemic contractile recovery. EIPA, however, was less effective and exhibited more side effects than cariporide and eniporide in the concentrations used.     

Mechanisms whereby glucose deprivation triggers metabolic preconditioning in the isolated rat heart

Transient glucose deprivation of the heart [GLU (-)] confers a preconditioning-like protection against subsequent ischemic/reperfusion (I/R). The mechanisms involved remain unclear. We hypothesized that GLU (-) would induce the classic ischemic preconditioning activated signaling cascade. Potential metabolic consequences and putative cell signaling events induced by transient glucose deprivation were evaluated as candidate mediators of this cardioprotection.Isolated glucose-perfused rat hearts were subjected to 30 min global ischemia followed by 30 min reperfusion (index I/R). Cardiac contractile recovery following I/R was used as the functional end-point in these studies. Metabolic preconditioning was stimulated by 15 min GLU (-) followed by 10 min glucose repletion prior to the index I/R. The potential metabolic consequences of GLU (-) were evaluated by using excess octanoate (11 mM OCT Hi) or 11 mM 2-deoxy-D-glucose (2-DG) in place of GLU (-) and by combining GLU (-) with fuels known to inhibit glycolysis supply (20 mM pyruvate or 1 mM octanoate, OCT Lo). The roles of -adrenoceptors, -adrenoceptors, adenosine receptors, protein kinase C (PKC) and mitochondrial K_ATP channels were investigated using inhibitors prazosin (10 M), propranolol (10 M), 8-(p-sulfophenyl) theophylline, (SPT 100 M), chelerythrine (CHEL 10 M) and 5-hydroxydecanoate (5 HD 100 M) respectively.GLU (-) increased mechanical recovery (59.8 ± 4.0 vs. 32.3 ± 4.7%; p < 0.01). Protection was abolished by pyruvate 26.6 ± 3.1; SPT 36.6 ± 3.0; CHEL 35 ± 4.8 or 5 HD 23.8 ± 3.3%. In a separate set of experiments, the specificity of SPT in this model was tested by preconditioning with adenosine (100 M) (34.7 ± 4 vs. control 16.8 ± 1.3%, p = 0.01) and blocking this protection with the same dose of SPT (16.3 ± 1.5%) used in the GLU (-) studies. Protection was unaltered by prazosin (50.2 ± 3.3%), propranolol (55.5 ± 4.0%), or OCT Lo (50.2 ± 2.5%). Protection was not mimicked by OCT Hi (35.6 ± 3.8%) or 2-DG (34 ± 4.3%).Transient glucose deprivation does not seem to achieve preconditioning-like cardioprotection by decreased glycolysis. Rather, the signal system may involve enhanced adenosine release, PKC, and activation of the mitochondrial K_ATP channel.     

Release of 6-KETO-PGF1α and thromboxane B2 in late appearing cardioprotection induced by the stable PGI analogue: 7-OXO-PGI

We have shown earlier that prostacylin (PGI₂) and its stable analogue: 7-oxo-prostacyclin(7-OXO) may induce a prolonged, late appearing (24–48 h after drug administration), dose dependent protection of the heart from harmful consequences of a subsequent severe ischaemic stress, such as myocardial ischaemia, life-threatening ventricular arrhythmias and early ischaemic morphological changes. In an other study we observed that a similar but shortlived (less than 1 h) cardioprotection, induced by preconditioning brief coronary artery occlusions, is greatly reduced by blockade of the cyclooxygenase pathway, suggesting that prostanoids might play a role in this shortlasting protection.Objective of our present study was to elucidate the importance of some arachidonic acid (AA) metabolites, such as PGI₂ and thromboxane A₂ (TXA₂) in the mechanism of the late appearing, prolonged cardioprotection. Estimation of the metabolites: 6-keto-PGF₁ (6-KETO) and thromboxane B₂ (TXB₂) was made from the perfusate of isolated Langendorff hearts of guinea-pigs pretreated with 50 g/kg 7-OXO, 24 and 48 h before preparation. Pretreatment alone produced a slight, but significant elevation of 6-KETO (from 206±11 to 284±19 pg/ml/min after 24 h, and to 261±18 pg/ml/min after 48 h). No change was seen in TXB₂ production. Global ischaemia for 25 min (followed by 25 min reperfusion) markedly increased the release of both AA metabolites; maximal values were observed in the third min of reperfusion (6-KETO from 206±11 to 1275±55 pg/ml/min and TXB₂ from 29±4 to 172±12 pg/ml/min). All values returned to the preischaemic level by the 25th min of reperfusion. Ischaemic increase in 6-KETO level was significantly higher in the perfusate of hearts from pretreated animals (1507±73 pg/ml/min after 24 h, and 1398±54 pg/ml/min after 48 h) that in those of untreated controls. There was no difference in TXB₂ values. Thus both basal and ischaemic release of PGI₂ increased 24 and 48 h after pretreatment with 7-OXO but not TXA₂ production. Results suggest that endogenous prostanoids might play a role in late appearing cardioprotection.     

Effects of sustained low-flow ischemia and reperfusion on Ca2+ transients and contractility in perfused rat hearts

We investigated changes in cytoplasmic Ca²⁺ concentration ([Ca²⁺]_i) and in left ventricular contractility during sustained ischemia and reperfusion in isolated beating rat hearts. Hearts from male Sprague-Dawley rats were perfused retrogradely and were loaded with 4 M fura-2. Low-flow global ischemia was induced by reducing perfusion flow to 10% and by electric pacing. The hearts were exposed to ischemia for 10 min or 30 min and then reperfused. [Ca²⁺]_i was measured by monitoring the ratio of 500 nm fluorescence excited at 340 and 380 nm while simultaneously measuring left ventricular pressure (LVP). To determine diastolic [Ca²⁺]_i, background autofluorescence was subtracted. LVP rapidly decreased from 82.3 ± 8.2 to 17.1 ± 2.9 mmHg , whereas the amplitude of the Ca²⁺ transient did not change significantly during the first 1 min of ischemia. After 10 min of ischemia, the amplitude decreased to 60.8 ± 10.6% (p < 0.05) and diastolic [Ca²⁺]_i increased by 26.3 ± 2.9% (p < 0.001) compared with the pre-ischemic value (n = 8). When the hearts were reperfused after 10 min of ischemia, the amplitude of the Ca²⁺ transient and LVP recovered to 79.0 ± 7.2% and 73.2 ± 7.5 mmHg, respectively. Whereas diastolic [Ca²⁺]_i decreased to the pre-ischemic value. In the hearts exposed to 30 min of ischemia (n = 10), diastolic [Ca²⁺]_i increased even further by 32.7 ± 5.3% at the end of ischemia and continued increasing during the 10 min of reperfusion by 42.6 ± 15.6%. Six of 10 hearts developed ventricular fibrillation (VF) and intracellular Ca²⁺ overload after reperfusion. Recovery of LVP after reperfusion was significantly smaller in the hearts exposed to 30 min of ischemia than in the hearts exposed to 10 min of ischemia (58.9 ± 11.7 vs. 97.2 ± 3.0% of pre-ischemic value, p < 0.05). Diastolic [Ca²⁺]_i also increased under hypoxic conditions (N₂ bubbling) in this model. These results suggest that increases in diastolic [Ca²⁺]_i might play an important role in myocardial contractile dysfunction and viability in ischemia-reperfusion injury.     

Magnesium-deficiency potentiates free radical production associated with postischemic injury to rat hearts: Vitamin E affords protection

Preexisting magnesium deficiency may alter the susceptibility of rat hearts to postischemic oxidative injury (free radicals). This was examined in rats maintained for 3 weeks on a magnesium-deficient (Mg-D) diet with or without concurrent vitamin E treatment (1.2 mg/day, SC). Magnesium-sufficient (Mg-S) rats received the same diet supplemented with 100 mmol Mg/kg feed. Following sacrifice, isolated working hearts were subjected to 30-, 40-, or 60-min global ischemia and 30-min reperfusion. Postischemic production of free radicals was monitored using electron spin resonance (ESR) spectroscopy and spin trapping with -phenyl-N-tert butylnitrone (PBN, 3 mM final); preischemic and postischemic effluent samples were collected and then extracted with toluene. PBN/alkoxyl adduct(s) (PBN/RO·; _H = 1.93 G,_N = 13.63 G) were the dominant signals detected in untreated Mg-S and Mg-D postischemic hearts, with comparably higher signal intensities observed for the Mg-D group following any ischemic duration. Time courses of postischemic PBN/RO· detection were biphasic for both groups (maxima: 2–4 and 8.5–12.5 min), and linear relationships between the extent of PBN/RO· production and the severity of both mechanical dysfunction and tissue injury were determined. Following each duration of ischemia, Mg-D hearts displayed greater levels of total PBN adduct production (1.7 –2.0 times higher) and lower recovery of cardiac function (42–48% less) than Mg-S hearts. Pretreating Mg-D rats with vitamin E prior to imposing 40-min ischemia/reperfusion, led to a 49% reduction in total PBN/RO· production, a 55% lower LDH release and a 2.2-fold improvement in functional recovery, compared to untreated Mg-D hearts. These data suggest that magnesium deficiency predisposes postischemic hearts to enhanced oxidative injury and functional loss, and that antioxidants may offer significant protection against pro-oxidant influence(s) of magnesium deficiency.