Correlation Between Direct ESR Spectroscopic Measurements and Electromechanical and Biochemical Assessments of Exogenous Free Radical Injury in Isolated Rat Cardiac Myocytes

Abstract

Reactive free radical species appear to be involved in the ischemic injury of cardiac muscle, although the mechanisms by which oxygen-derived free radicals affect the heart cell function are not known. In the present study, cultured ventricular myocytes were exposed to an exogenous oxygen radical generating system. The myocyte-enriched, primary cultures were prepared from ventricles of new-born rat heart and exposed to a xanthine/xanthine oxidase (X+XO) system. The transmembrane potentials were recorded with glass microelectrodes. Cell contractions were monitored photometrically. The release of lactate dehydrogenase (LDH) in the medium was analysed. Quantitative measurement and the time course of the radical generation were performed by the electron paramagnetic resonance (EPR) spin trapping technique with the spin trap 5,5-dimethyl-1-pyroline-N-oxide (DMPO). We verified that X and XO alone had no significant functional and biochemical effects. The X+XO system produced a rapid decrease in the action potential amplitude. This effect was accompanied by a strong decrease in contractility and spontaneous rate. The time course of these functional defects were correlated with a progressive efflux of LDH from the cardiomyocytes. Prolonging the exposure to the X+XO system provoked the cessation of the spontaneous beatings and the progressive loss of the resting diastolic potential, together with a near total release of the cellular LDH. The LDH release and the functional depression were both efficiently prevented by catalase. On the contrary, superoxide dismutase (SOD) slowed down but did not protect against the functional and biochemical effects of the free radicals. In comparison, the EPR spectra obtained indicated that the X+XO system was associated with an important generation of superoxide anions but also with a small hydroxyl production. SOD scavenged the superoxide but a small ·OH production persisted. Catalase (CAT) did not modify the superoxide generation but decreased the hydroxyl adduct formation. These results suggest that, although the generation of superoxide anions by the X+XO system was higher than the hydroxyl production, the functional injury and enzyme leakage seemed mainly mediated through a hydrogen peroxide-hydroxyl radical pathway. Cultured ventricular myocytes can be thus used as a valuable model to investigate the cellular mechanism of oxidant-induced damage in the heart.

Introduction

Reactive free radical species were suggested to contribute to cellular injury during cardiac ischemia and reperfusion.1, 2 The cellular mechanisms by which oxygen-derived free radicals exert their effects on heart cell function are not fully understood. The free radical release is weak during ischemia, but increases substantially at reperfusion.3, 4, 5 At oxygen readmission, the natural scavenging systems, such as superoxide dismutase, catalase and glutathione peroxidase, decrease. If the protective processes in myocardial tissue cannot prevent the increase in free radical generation, the free radicals may initiate oxidative damage of cellular components. The highly reactive ·OH generated from $\text{O}{}_2{}^{\mathrm{<mtext>̇</mtext><mtext>-</mtext>}}$ and H₂O₂ via the iron-mediated Fenton reaction has been reported to trigger lipid peroxidation and severe cardiac tissue injury6, 7 Several groups of investigators have shown that the abnormalities which follow reperfusion of the ischemic myocardium, consist of postischemic contractile dysfunction,1, 8 ventricular arrhythmias, abnormalities in membrane permeability and modification in the fluidity of myocardial membranes.9, 10, 11 These phenomena have been correlated with the detection of free radicals in isolated rat hearts undergoing global ischemia and reperfusion.12, 13, 14, 15 Moreover, oxygen free radical-generating systems administered exogenously have been demonstrated to cause cardiac contractile dysfunction and electrophysiological abnormalities.16, 17, 18, 19 They were also reported to affect heart sarcolemmal membranes,20, 21 sarcoplasmic reticulum[22] and mitochondrial functions.[23] However, the current knowledge on the effect of oxygen free radicals is based either on studies using isolated heart preparations or ventricular tissue preparations, and the literature is poorly documented on the effect of free radical species in homogeneous preparations of isolated cardiac myocytes.24, 25, 26 Moreover, the assumption that free radical-related injury is suggested to contribute to postischemic myocardial cell dysfunction is largely based on indirect evidence. Therefore, the present study was aimed to study the dysfunction in cultured ventricular myocytes exposed to in vitro-generated oxygen radicals detected by electron spin resonance (ESR).