Generation of free radicals and the damage done to the sarcoplasmic reticulum during reperfusion injury following brief ischemia in the canine heart

Free radical generation was studied by the electron spin resonance (ESR) technique using alpha-phenyl N tert butyl nitrone (PBN) in a brief ischemia-reperfusion model of the canine heart, and correlated with biochemical changes of the sarcoplasmic reticulum (SR). ESR spect a (aH = 0.3-0.4 mT, aN = 1.43-1.58 mT) were observed as PEN spin adducts, which peaked at levels 5-fold above the control levels at 5 min after reperfusion. The simulated coupling constants of PEN spin adducts suggested that the sample should contain at least 2 carbon-centered radicals at 5 min after reperfusion (radical A: aH = 0.350 mT, aN = 1.485 mT; radical B: aH = 0.370 mT, aN = 1.615 mT). At this time point, a significant reduction in Ca-ATPase activity of the SR was found without degradation of the major ATPase protein. Superoxide dismutase (SOD) significantly reduced the intensity of the PEN spin adduct signals and preserved the Ca-ATPase activity of the SR to 80% of the control level. Reperfusion injury after brief ischemia may be the result of inactivation of intracellular Ca-ATPase by free radicals generated during reperfusion, and SOD contributes to the protective effect by scavenging the radicals.