Restoration of myocardial contractile function during gradual reperfusion after total ischemia

Isolated guinea pig hearts subjected to 25-min total normothermic ischemia and 30-min reperfusion with the initial rate exhibited a great rise in isovolumic diastolic pressure while the contractile function recovered to 34 +/- 4% of initial value. Reperfusion with gradually increased rate from 13% of initial rate to 100% resulted in better recovery of the contractile function--to 54 +/- 3% and markedly less rise in the diastolic pressure. This coincided with 28% less inosine loss. More better recovery of the myocardial contractile function (to 80 +/- 5%) was observed in experiments in which gradual reperfusion was combined with recirculation. In both gradual reperfusion series, the recovery of the heart rate and the contractile function were much delayed during first 5 min reperfusion and were associated with a rise in coronary resistance.     

Identification and quantification of free radicals during myocardial ischemia and reperfusion using electron paramagnetic resonance spectroscopy

There is general agreement that free radicals are involved in reperfusion injury. Electron paramagnetic resonance (EPR) spectroscopy can be considered as the more suitable technique to directly measure and characterize free radical generation during myocardial ischemia and reperfusion. There are essentially two approaches used in the detection of unstable reactive species: freezing technique and spin traps. The detection of secondary free radicals or ascorbyl free radicals during reperfusion might provide an index of oxidative stress. Spin trapping can also characterize nitric oxide. EPR spectroscopy can provide important data regarding redox state and free radical metabolism but ideally, the spin traps must not interfere with cell or organism function.     

Plasma endothelin levels during myocardial ischemia and reperfusion

Endothelin, an endothelium-derived vasoconstrictive peptide, has a strong potency of coronary artery constriction. However, the role of endogeneous endothelin under pathophysiological conditions has not yet been known. In this study, we examined plasma endothelin concentration in dogs with myocardial ischemia and reperfusion. Anesthetized open-chest dogs underwent either 45 minutes occlusion of the left anterior descending coronary artery followed by 3 hours reperfusion, or 4-10 hours of continuous occlusion. Plasma concentration of endothelin from the central vein was measured by the highly sensitive enzyme-immunoassay. Plasma endothelin concentration increased 2.2-fold with the peak level at 60 minutes after release of the ligated artery, but occlusion per se caused no remarkable change. These data suggest that reperfusion of the occluded artery might be needed to increase the plasma concentration of endothelin in case of myocardial infarction.     

Ischemic preconditioning alters real-time measure of O2 radicals in intact hearts with ischemia and reperfusion

Reactive oxygen species (ROS) are believed to be involved in triggering cardiac ischemic preconditioning (IPC). Decreased formation of ROS on reperfusion after prolonged ischemia may in part underlie protection by IPC. In heart models, these contentions have been based either on the effect of ROS scavengers to abrogate IPC-induced preservation or on a measurement of oxidation products on reperfusion. Using spectrophotofluorometry at the left ventricular wall and the fluorescent probe dihydroethidium (DHE), we measured intracellular ROS superoxide (O(2)(-).) continuously in isolated guinea pig heart and tested the effect of IPC and the O(2)(-). scavenger manganese(III) tetrakis (4-benzoic acid) porphyrin chloride (MnTBAP) on O(2)(-). formation throughout the phases of preconditioning (PC), 30-min ischemia and 60-min reperfusion (I/R). IPC was evidenced by improved contractile function and reduced infarction; MnTBAP abrogated these effects. Brief PC pulses increased O(2)(-). during the ischemic but not the reperfusion phase. O(2)(-). increased by 35% within 1 min of ischemia, increased further to 95% after 20 min of ischemia, and decreased slowly on reperfusion. In the IPC group, O(2)(-). was not elevated over 35% during index ischemia and was not increased at all on reperfusion; these effects were abrogated by MnTBAP. Our results directly demonstrate how intracellular ROS increase in intact hearts during IPC and I/R and clarify the role of ROS in triggering and mediating IPC.     

Superoxide anion production during reperfusion is reduced by an antineutrophil antibody after prolonged cerebral ischemia

Neutrophils may be involved in the pathophysiology of reperfusion injury following cerebral ischemia. One potential mechanism of reperfusion injury by neutrophils is through production of the superoxide anion. We hypothesized that, due to progressive endothelial damage during ischemia, neutrophil activation would be more prominent after longer periods of ischemia prior to reperfusion. Thus, neutrophils would contribute more to pathological processes such as superoxide anion formation after longer than after shorter periods of ischemia. A reversible middle cerebral artery occlusion model in rats was employed and superoxide anion concentration was measured with a cytochrome c coated electrode placed on the cortical penumbral region. Occlusion times were varied from 60 min to 2 h, and neutrophils were inhibited with an antiCD18 antibody administered prior to occlusion. Neutrophil accumulation and reduction with antibody treatment was confirmed immunohistochemically. Superoxide anion (O₂^•−) concentration was detected during the hours following 60 min of occlusion, and increased further with 2 h of occlusion. Treatment with the antiCD18 antibody had no effect on O₂^•− concentration during reperfusion in the 60–90 min occlusion groups, but O₂^•− concentration was significantly lower in the antiCD18 antibody treated group than in the control group during reperfusion after 120 min of ischemia. The antibody also reduced cortical neutrophil accumulation in the 120 min ischemia group. These results indicate for the first time that superoxide production by neutrophils becomes more important with longer periods of ischemia, and other quantitatively less important sources of superoxide predominate with shorter periods of ischemia. This phenomenon may explain some of the variation seen between different models of ischemia with different durations of ischemia when targeting reactive oxygen species, and supports an approach to combination therapy to extend the therapeutic window and reduce the deleterious effects of reperfusion.     

O2 delivery and redox state are determinants of compartment-specific reactive O2 species in myocardial reperfusion

Reperfusion of the ischemic myocardium leads to a burst of reactive O(2) species (ROS), which is a primary determinant of postischemic myocardial dysfunction. We tested the hypothesis that early O(2) delivery and the cellular redox state modulate the initial myocardial ROS production at reperfusion. Isolated buffer-perfused rat hearts were loaded with the fluorophores dihydrofluorescein or Amplex red to detect intracellular and extracellular ROS formation using surface fluorometry at the left ventricular wall. Hearts were made globally ischemic for 20 min and then reperfused with either 95% or 20% O(2)-saturated perfusate. The same protocol was repeated in hearts loaded with dihydrofluorescein and perfused with either 20 or 5 mM glucose-buffered solution to determine relative changes in NADH and FAD. Myocardial O(2) delivery during the first 5 min of reperfusion was 84.7 +/- 4.2 ml O(2)/min with 20% O(2)-saturated buffer and 354.4 +/- 22.8 ml O(2)/min with 95% O(2) (n = 8/group, P < 0.001). The fluorescein signal (intracellular ROS) was significantly increased in hearts reperfused with 95% O(2) compared with 20% O(2). However, the resorufin signal (extracellular ROS) was significantly increased with 20% O(2) compared with 95% O(2) during reperfusion. Perfusion of hearts with 20 mM glucose reduced the (.)NADH during ischemia (P < 0.001) and the (.)ROS at reperfusion (P < 0.001) compared with 5.5 mM-perfused glucose hearts. In conclusion, initial O(2) delivery to the ischemic myocardium modulates a compartment-specific ROS response at reperfusion such that high O(2) delivery promotes intracellular ROS and low O(2) delivery promotes extracellular ROS. The redox state that develops during ischemia appears to be an important precursor for reperfusion ROS production.     

Leukocytes, oxygen radicals, and myocardial injury due to ischemia and reperfusion

Ischemic myocardium generates stimuli for neutrophil chemotaxis before the final extent of irreversible ischemic injury is attained. Reperfusion accelerates the infiltration of ischemic myocardium by neutrophils. Oxygen radicals released by the activated neutrophils may exacerbate the tissue damage caused by ischemia. Neutrophil depletion by antiserum was shown to limit infarct size in dogs undergoing coronary occlusion for 90 minutes followed by reperfusion for 6 or 72 hours, but not in dogs undergoing occlusion for 4 hours. Prostacyclin, which inhibits the generation of superoxide anions by neutrophils, also limited canine myocardial injury despite no effect on collateral blood flow. Iloprost, an analogue of prostacyclin that inhibits neutrophils also reduced infarct size, while SC39902, an analogue that does not inhibit neutrophils, did not alter infarct size. The results suggest that oxygen radicals released by activated neutrophils play a role in the pathophysiology of myocardial injury due to ischemia followed by reperfusion.     

Free fatty acid metabolism during myocardial ischemia and reperfusion

Long chain free fatty acids (FFA) are the preferred metabolic substrates of myocardium under aerobic conditions. However, under ischemic conditions long chain FFA have been shown to be harmful both clinically and experimentally. Serum levels of free fatty acids frequently are elevated in patients with myocardial ischemia. The proposed mechanisms of the detrimental effects of free fatty acids include: (1) accumulation of toxic intermediates of fatty acid metabolism, such as long chain acyl-CoA thioesters and long chain acylcarnitines, (2) inhibition of glucose utilization, particularly glycolysis, during ischemia and/or reperfusion, and (3) uncoupling of oxidative metabolism from electron transfer. The relative importance of these mechanisms remains controversial. The primary site of FFA-induced injury appears to be the sarcolemmal and intracellular membranes and their associated enzymes. Inhibitors of free fatty acid metabolism have been shown experimentally to decrease the size of myocardial infarction and lessen postischemic cardiac dysfunction in animal models of regional and global ischemia. The mechanism by which FFA inhibitors improve cardiac function in the postischemic heart is controversial. Whether the effects are dependent on decreased levels of long chain intermediates and/or enhancement of glucose utilization is under investigation. Manipulation of myocardial fatty acid metabolism may prove beneficial in the treatment of myocardial ischemia, particularly during situations of controlled ischemia and reperfusion, such as percutaneous transluminal coronary angioplasty and coronary artery bypass grafting. (Mol Cell Biochem 166: 85-94, 1997)     

Biphasic changes in relaxation following reperfusion after myocardial ischemia

The present study provides evidences of left ventricular diastolic alterations following reperfusion in a model of global ischemia. Isolated perfused rabbit and rat hearts, were subjected to ischemia for 15 and 20 min respectively, followed by 30 min of reperfusion. In rabbit heart at the end of the reperfusion period, isovolumic left ventricular developed pressure (LVDP) and +dP/dt_max stabilized at 55 ± 3% and 60 ± 2% of preischemic values respectively and, in rat heart LVDP = 61 ± 8% and +dP/dt_max = 57 ± 9% of preischemic values. Stunned heart was then obtained from both species. Left ventricular end diastolic pressure (LVEDP) values stabilized at the end of reperfusion period at values higher than preischemic conditions in both species (38.9 ± 4.4 mmHg and 30.3 ± 3.1 mmHg in rabbit and rat respectively). The time constant of relaxation (T) increased early in reperfusion in both species, but then decreased and stabilized at the end of reperfusion period at values lower than preischemic values. The ratio between both maximal velocities (+P/-P), also showed a transitory impairment in relaxation, followed by normalization and stabilization at values lower than preischemic values. This biphasic pattern in relaxation was detected in both species. The changes in relaxation were dissociated from the diastolic compliance and could be the result of a transitory calcium overload and/or sarcoplasmic reticulum dysfunction. The faster myocardial relaxation at the end of reperfusion period is consistent with the decreased myofilament sensitivity, which characterizes the stunned myocardium.     

Ion transport systems as targets of free radicals during ischemia reperfusion injury

Oxidative stress is a recognized pathogenic factor in ischemia/reperfusion injury (IRI). Iron induced generation of reactive oxygen species (ROS) in vitro reduces both the Na+K+-ATPase activity and Na+-Ca2+ exchanger of synaptosomal membranes, concomitantly with alteration of physical state of membranes. Oxidative insult also leads to the loss of ability of endoplasmic reticular membranes (ER) to sequester Ca2+ as well as to the increase of Ca2+ permeability. Furthermore, ROS induces both lipid peroxidation and lipid-independent modifications of membrane proteins. Acute in vivo ischemia alters kinetic parameters of Na+K+-ATPase affecting mainly the dephosphorylation step of ATPase cycle with parallel changes of Na+-Ca2+ exchanger and alterations of physical membrane environment. Subsequent reperfusion after ischemia is associated with decrease of immuno signal for PMCA 1 isoform in hippocampus. In addition, incubation of non-ischemic membranes with cytosol from ischemic hippocampus decreases level of PMCA 1 in non-ischemic tissues. Loss of PMCA 1 protein is partially protected both by calpain- and by non-specific protease inhibitors which suggest possible activation of proteases in the reperfusion period. On the other hand, ischemia does not affect the level of Ca2+ pump (SERCA 2b) and calreticulin of intracellular Ca2+ stores. However, IRI resulted in a decrease of IP3 receptor I and altered active Ca2+ accumulation into the ER. A non-specific alteration of physical properties of total membranes such as the oxidative modifications of proteins as well as the content of lipoperoxidation products can also be detected after IRI. ROS can alter physical and functional properties of neuronal membranes. We discuss our results suggesting that ischemia-induced disturbation of ion transport systems may participate in or follow delayed death of neurons after ischemia.     

Postconditioning inhibits myocardial apoptosis during prolonged reperfusion via a JAK2-STAT3-Bcl-2 pathway

Background  

Postconditioning (PostC) inhibits myocardial apoptosis after ischemia-reperfusion (I/R) injury. The JAK2-STAT3 pathway has anti-apoptotic effects and plays an essential role in the late protection of preconditioning. Our aim was to investigate the anti-apoptotic effect of PostC after prolonged reperfusion and the role of the JAK2-STAT3 pathway in the anti-apoptotic effect of PostC.     

Early anti-apoptosis treatment reduces myocardial infarct size after a prolonged reperfusion

OBJECTIVE : Significant myocardial apoptosis occurs in ischemia/reperfused hearts. However, the contribution of apoptosis to the development of myocardial injury remains controversial. The present study attempted to obtain evidence that inhibition of apoptosis at early reperfusion can reduce myocardial infarction after prolonged reperfusion. METHODS : Adult male rats were subjected to 30 min ischemia and 4 (apoptosis assay) or 24 h (myocardial infarction determination) of reperfusion and treated with vehicle, SB 239063, insulin or insulin plus wortmannin. RESULTS : Treatment with SB 239063 or insulin markedly decreased myocardial apoptosis (10.6 +/- 1.5% and 7.9 +/- 0.9% respectively, P < 0.01 vs. vehicle) and significantly reduced infarct size (43 +/- 3.6% and 35 +/- 2.9%, respectively, P < 0.01 vs. vehicle). Most interestingly, inhibition of insulin signaling with wortmannin to block insulin signaling not only blocked insulin's anti-apoptotic effect, but also abolished its infarct reduction property. CONCLUSION : These data indicate that apoptosis contributes to the development of myocardial infarction, and inhibition of apoptosis at early reperfusion reduces the myocardial infarction.     

Selective antioxidant enzymes during ischemia/reperfusion in myocardial infarction

The study of ischemia/reperfusion injury included 25 patients in the acute phase of myocardial infarction (19 perfused, 6 remained non-reperfused as evaluated according to the time course of creatine kinase and CK-MB isoenzyme activity) and a control group (21 blood donors). Plasma level of malondialdehyde was followed as a marker of oxidative stress. Shortly after reperfusion (within 90 min), a transient increase of malondialdehyde concentration was detected. The return to the baseline level was achieved 6 h after the onset of therapy. The activity of a free radical scavenger enzyme, plasma glutathione peroxidase (GPx), reached its maximum 90 min after the onset of treatment and returned to the initial value after 18 h. The specificity of the GPx response was confirmed by comparing with both non-reperfused patients and the control group, where no significant increase was detected. The erythrocyte Cu,Zn-superoxide dismutase (SOD) did not exhibit significant changes during the interval studied in perfused patients, probably due to the stability of erythrocyte metabolism. In non-reperfused patients, a decrease of SOD was found during prolonged hypoxia. These results help to elucidate the mechanisms of fast activation of plasma antioxidant system during the reperfusion after myocardial infarction.     

Myeloperoxidase enhances nitric oxide catabolism during myocardial ischemia and reperfusion

Impaired microvascular function during myocardial ischemia and reperfusion is associated with recruitment of polymorphonuclear neutrophils (PMN) and has been attributed to decreased bioavailability of nitric oxide (NO). Whereas myeloperoxidase (MPO), a highly abundant, PMN-derived heme protein facilitates oxidative NO consumption and impairs vascular function in animal models of acute inflammation, its capacity to function in this regard during human myocardial ischemia and reperfusion remains unknown. Plasma samples from 30 consecutive patients (61 +/- 14 years, 80% male) presenting with acute myocardial infarction were collected 9 +/- 4 h after vessel recanalization and compared to plasma from healthy control subjects (n = 12). Plasma levels of MPO were higher in patients than in control subjects (1.4 +/- 0.9 vs 0.3 +/- 0.2 ng/mg protein, respectively, p < 0.0001). The addition of hydrogen peroxide to patient plasma resulted in accelerated rates of NO consumption compared to control subjects (0.53 +/- 0.25 vs 0.068 +/- 0.039 nM/s/mg protein, respectively, p < 0.0001). Myocardial tissue from patients with the same pathology revealed intense recruitment of MPO-positive PMN localized along infarct-related vessels as well as diffuse endothelial distribution of non-PMN-associated MPO immunoreactivity. Endothelium-dependent microvascular function, as assessed by an acetylcholine-dependent increase in forearm blood flow in 75 patients with symptomatic coronary artery disease, inversely correlated with MPO plasma levels (r = -0.75, p < 0.005). Plasma from patients undergoing myocardial reperfusion contained increased levels of MPO, which catalytically consumed NO in the presence of H(2)O(2). Given the correlation between intravascular MPO levels and forearm vasomotor function in patients with coronary artery disease, MPO appears to be an important modulator of vasomotor function in inflammatory vascular disease and a potential therapeutic target for treatment.     

Microdialysis separately monitors myocardial interstitial myoglobin during ischemia and reperfusion

Direct monitoring of myoglobin efflux during ischemia and reperfusion has been limited because of inherent sample collection problems in the ischemic region. Recently, the cardiac dialysis technique has offered a powerful method for monitoring myocardial interstitial levels of low-molecular-weight compounds in the cardiac ischemic region. In the present study, we extended the molecular target to high-molecular-weight compounds by use of microdialysis probes with a high-molecular-mass cutoff and monitored myocardial interstitial myoglobin levels. A dialysis probe was implanted in the left ventricular free wall in anesthetized rabbits. The main coronary artery was occluded for 60 or 120 min. We examined the effects of myocardial ischemia and reperfusion on myocardial interstitial myoglobin levels. Interstitial myoglobin increased within 15 min of ischemia and continued to increase during 120 min of ischemia, whereas blood myoglobin increased at 45 min of ischemia. Lactate and myoglobin in the interstitial space increased during the same period. At 60 min of ischemia, reperfusion markedly accelerated interstitial myoglobin release. The interstitial myoglobin level was fivefold higher at 0-15 min of reperfusion than at 60-75 min of coronary occlusion. The dialysis technique permits earlier detection of myoglobin release and separately monitors myoglobin release during ischemia and reperfusion. Myocardial interstitial myoglobin levels can serve as an index of myocardial injury evoked by ischemia or reperfusion.     

Identification of free radicals in myocardial ischemia/reperfusion by spin trapping with nitrone DMPO

The spin trapping ESR technique was applied to investigate oxygen-derived radicals in ischemic and post-ischemic rat hearts. Using 5,5'-dimethyl-l-pyrroline-N-oxide, carbon-centered radicals were identified during ischemia and oxy-radical adducts (superoxide anion radical, O.-2 and hydroxyl radicals, .OH) in post-ischemic rat heart. The formation of these spin adducts was inhibited by superoxide dismutase, suggesting that superoxide plays a role in the adducts' formation. The results demonstrate that oxygen derived free radicals are important byproducts of abnormal oxidative metabolism during myocardial ischemic and reperfusion injuries.     

Ultrastructure of coronary microvessels during heart reperfusion after prolonged ischemia under conditions of various forms of artificial hypothermia

It has been found that, in children with Roger’s disease corrected under conditions of two fundamentally different procedures of anesthetic management, myocardial reperfusion after cardiac arrest under artificial hypothermic circulation is accompanied with obstruction of more than 70% of coronary bed microvessels, one-third of them being blocked with hydropic endothelial cells or their cystiform fragments. It hampers or even totally prevents their functioning in the postoperative period. The content of necrotic cells increases, while the three “working” cell types demonstrate a decrease in myocropinocytotic transport. Circulatory arrest during perfusionless hypothermia and immersion reperfusion do not result in a dramatic change of general morphology of microvessels as compared to the control group maintaining the endothelial cell population unaltered. Ultrastructural organization of endothelial microvessels displayed no features of intracellular regeneration. However, a heterogenic response of the structures responsible for transendothelial transfer of macromolecules provides the basis for the recovery of endothelium structure and function, as a patient’s temperature reaches the standard value.