Myocardial ischemia and reperfusion: direct evidence for free radical generation by electron spin resonance spectroscopy.

Abstract

Electron spin resonance spectroscopy has recently been used by others to detect directly radical species in isolated perfused hearts. Sample processing prior to spectroscopy in this study involved pulverization of tissue, which can artifactually generate radical species. We assessed in isolated perfused hearts the influence of tissue pulverization on the identity of radical species detected by spectroscopy and then, using a processing technique less likely to induce artifacts, whether myocardial ischemia and reperfusion generate radical species. Rat and rabbit hearts (n = 8) were perfused aerobically for 10 min and freeze-clamped to -196C. Frozen tissue was processed at -196°C for spectroscopic analysis by pulverization vs. chopping. Spectra of pulverized tissue consisted of three components: a semiquinone (g = 2.004), a lipid peroxy radical (g11 = 2.04 and gL = 2.006), and a carboncentered radical that is possibly a lipid radical (gl,, = 2.002 and A' 50 G). Chopped tissue consisted of a single component, a semiquinone (g = 2.004). Rat hearts (n = 8 per group) also underwent 10-min global no-flow normothermic ischemia followed by 5-60 sec of either aerobic or anaerobic reperfusion, with frozen tissue chopped prior to spectroscopy. Spectra of ischemic tissue consisted of an iron-sulfur center and a semiquinone. Aerobic reperfusion resulted in a spectrum similar to the control but with increased amplitude that peaked after 10-15 sec of reflow. Anaerobic reperfusion yielded a spectrum identical to that of ischemic tissue. We conclude that pulverization of frozen myocardial tissue artifactually generates radical species. Using a nonpulverization technique for tissue processing, we found that myocardial ischemia and reperfusion produce radical species but that molecular oxygen is necessary for the burst of radical production during reflow.Myocardial ischemia occurs when myocardial oxygen demand exceeds oxygen supply. Unless reversed, this situation results in cell injury. The clinical event is myocardial infarction. Reperfusion of the ischemic myocardium during thrombolysis, coronary artery bypass surgery, or angioplasty can restore oxygen and energy substrates to the ischemic myocardial cell, but this process may create another form of myocardial damage, termed "reperfusion injury." This is characterized by tissue disruption, enzyme leakage, and development of contracture (1). Damage to the myocardial cell induced by a cycle of ischemia and reperfusion has been proposed to be due in part to the generation of toxic oxygen-derived free radicals (2, 3). Until recently, evidence to support the role of free radicals in myocardial cell injury has been of an indirect nature. Their production during ischemia and reperfusion has been implied through the use either of inhibitors of free radical generation or of scavengers of radical production (4-7).Nearly 2 decades ago, Beinert and coworkers (8-10) used low-temperature ESR spectroscopy to study the mechanism of electron transport in the cardiac mitochondrial respirat...

Inhibitory effect of carvedilol in the high-conductance state of the mitochondrial permeability transition pore

“…As cardiomyocytes need a constant supply of mitochondrial ATP to ensure their functions, their survival is threatened when mitochondrial dysfunction occurs. In a condition of ischemiarreperfusion, where there is increased oxidative stress, calcium and P , the mitochondrial permeability transition occurs i ŽCrompton and Costi, 1990;Halestrap et al, 1998;Baker . et al, 1988;Kristian and Siesjo, 1996 .…”

S‐Nitrosoglutathione induces formation of nitrosylmyoglobin in isolated hearts during cardioplegic ischemia — an electron spin resonance study

“…2A. The spectra reveal the presence of a ubisemiquinone radical (g : 2.0045, AHpp = 8.0 + 0.4 G) and a reduced iron-sulfur center (g = 1.94), presumably associated with the mitochondrial NADH or succinate dehydrogenase [17,18]. Perfusion with GSNO did not alter the ESR spectral pattern (Fig.…”

“…quartz tube immersed in liquid nitrogen. Samples prepared by this technique did not artifactually generate radical species [17]. Myocardial tissue homogenate was prepared in icecold phosphate-buffered saline (25 mmol/1, pH 7.4) using Brinkman homogenizer.…”

Ascorbyl free radical as a reliable indicator of free‐radical‐mediated myocardial ischemic and post‐ischemic injury

“…This signal was persistent and remained unchanged for at least 2 h at 20'C. Since it has been shown recently that pulverization of freeze-clamped ischemic heart tissues favours formation of artifactual free radicals [27], we have repeatedly compared the ESR signals generated by two samples taken from the same frozen heart, then either ground or gently chopped with a stainless-steel spatula under liquid nitrogen, and further processed by Me2S0 extraction. In all assays, the ascorbyl radical was always the only free radical species detected, with no significant differences in intensity due to processing.…”

“…Conversely, since low-temperature ESR studies of myocardial free radical content do not require the introduction of exogenous chemicals into the system, they could lead to more significant biological results. Several authors have proposed that the evolution of anisotropic ESR signals obtained at low temperatures and tentatively assigned to (a) ubisemiquinone-type radicals associated with the mitochondrial respiratory chain [21,23,24,[27][28][29][30] and (b) a paramagnetic ion complex of iron-sulfur proteins [21,23,25,27,29, 301 could reflect free-radical-induced damage throughout ischemia and reflow. These signals have been thought to include resonances from potentially hazardous oxygen-and carbon-centered free radicals.…”