Involvement of superoxide and nitric oxide in the genesis of reperfusion arrhythmias in rats.

To assess the role of reactive oxygen species and nitric oxide (NO) in the genesis of reperfusion-induced arrhythmias, the effects of reactive oxygen species scavengers and NO synthase inhibitors on the incidence of ventricular fibrillation and irreversible ventricular fibrillation (mortality) were examined. Hearts of anesthetized rats were subjected to 4 min regional ischemia followed by 4 min reperfusion. The animals were treated i.v. with superoxide dismutase, a O2- scavenger, catalase, a H2O2 scavenger, dimethylthiourea, a .OH scavenger, or NG-nitro-L-arginine methyl ester (L-NAME) and NG-nitro-L-arginine (L-NNA), NO synthase inhibitors. Superoxide dismutase (430 and 4300 U/kg/min) reduced the mortality from 93% to 43% and 57%, respectively, whereas treatment with catalase or dimethylthiourea did not affect these arrhythmias. L-NAME (0.1 and 0.3 mg/kg/min) reduced the mortality from 93% to 50% and 43%, respectively. L-NNA (0.3 mg/kg/min) reduced the mortality from 93% to 50%. This reduction by the NO synthase inhibitors was abolished by administration of L-Arg. However, L-Arg blocked neither a small increase in systolic blood pressure nor a decrease in heart rate elicited by the NO synthase inhibitors. The combinated treatment of superoxide dismutase (4300 U/kg/min) with L-NAME (0.3 mg/kg/min) reduced the mortality from 93% to 7%. These results suggest that the genesis of reperfusion-induced arrhythmias observed in this model may be in part due to O2- and NO.

Measurement of oxygen-derived free radical generation in the regionally-ischemic rat heart by the chemiluminescence method.

Generation of oxygen-derived free radicals (oxy-radicals) in the stored rat heart was measured by chemiluminescence. Hearts subjected (ischemia) or not subjected (non-ischemia) to a 4-min regional ischemia were frozen in liquid nitrogen and stored until assayed. The frozen myocardium was ground and oxygenated by mixing it with phosphate-buffered saline (Po2: 194 mmHg) containing lucigenin. The chemiluminescence intensity of ischemic myocardium was larger than that of non-ischemic myocardium. Recombinant human superoxide dismutase significantly decreased these intensities. These results indicate that O2- is one of the major oxy-radical species in the rat heart and that the generation of oxyradicals is enhanced by regional ischemia for 4 min.

A simple chemiluminescence method for measuring oxygen-derived free radicals generated in oxygenated rat myocardium.

We have developed a simple and reproducible method employing chemiluminescence to measure oxygen-derived free radicals generated in oxygenated myocardium. Isolated perfused rat hearts were frozen in liquid nitrogen during the control perfusion (non-ischemia), after 30 min of global ischemia (ischemia), or after 20 min of reperfusion following 30-min global ischemia (reperfusion). The frozen hearts were ground to a fine powder and then oxygenated by mixing with phosphate-buffered saline (PBS) (pH 7.4 and PO2 183-194 mmHg) containing lucigenin. The mixed solution was injected into a chemiluminescence detection flow cell. The chemiluminescence intensity increased in relation to the lucigenin and myocardium contents of the test solution. However, it was not affected by the temperature of PBS in the range of 25 degrees C to 50 degrees C. The chemiluminescence intensity of ischemic myocardium was 2.5 times larger than that of non-ischemic or reperfused myocardium (P < 0.01). Recombinant human superoxide dismutase (r-h-SOD) reduced dose-dependently the chemiluminescence intensity induced by oxygenation of ischemic myocardium, while chemically inactivated r-h-SOD did not.

[Effect of recombinant human superoxide dismutase (r-h-SOD) on reperfusion-induced irreversible arrhythmia in anesthetized rats].

Using anesthetized rats, we performed a dose-response study of the ability of r-h-SOD to reduce the mortality caused by reperfusion-induced irreversible arrhythmia. The hearts were subjected to regional ischemia by the occlusion of the left coronary artery for 4 min followed by reperfusion for 7 min. First, the relation between the anti-arrhythmic effect of r-h-SOD and the size of the ischemic zone was examined. The protective effect of r-h-SOD was easily detected in rats with an ischemic zone size of 40-55% of the total heart weight, but not in rats with other zone sizes. Intravenous infusion of r-h-SOD at rates of 430 to 130,000 U/kg/min beginning 1 min after regional ischemia significantly reduced the mortality after reperfusion. R-h-SOD at the doses used did not affect the blood pressure, heart rate or arrhythmia during the ischemic period. R-h-SOD at rates of 130 to 130,000 U/kg/min was intravenously infused into anesthetized rats, and its plasma level was measured at 3 min after the beginning of the infusion. The plasma level of r-h-SOD increased dose-dependently (8.5 to 6,600 U/ml). In conclusion, r-h-SOD reduced mortality after reperfusion over a wide range of doses and its effective plasma level was estimated to be 28 to 6,600 U/ml in this model.

[Analysis of factors influencing reperfusion-induced arrhythmia in the anesthetized rat heart: the influence of ischemic zone size and hemodynamics].

The influence of ischemic zone size and hemodynamics on the reperfusion-induced arrhythmia in the anesthetized rat heart was examined. The heart was subjected to regional ischemia for 4 min by the occlusion of LAD followed by reperfusion for 7 min. After the reperfusion, 64% of the rats died due to irreversible VF. The size of the ischemic zone, the increase in heart rate during ischemia and the incidence of ischemic arrhythmia (VPBs and/or VT) of the animals that died after the reperfusion were significantly greater or higher than those of the surviving animals. There were positive correlations between the size of the ischemic zone and the increase of heart rate during ischemia and the incidence of ischemic arrhythmia. These results indicate that the size of the ischemic zone is a main factor that determines the degree of reperfusion-induced arrhythmia and also contributes to the increase in heart rate and the occurrence of arrhythmia during ischemia. Therefore, in addition to monitoring the blood pressure, heart rate and electrocardiogram, measurement of the size of the ischemic zone is essential for the evaluation of drug effects on reperfusion-induced arrhythmia using this animal model.