Characterization of oxygen radical formation mechanism at early cardiac ischemia

Myocardial ischemia–reperfusion (I/R) causes severe cardiac damage. Although the primary function of oxymyoglobin (Mb) has been considered to be cellular O(2) storage and supply, previous research has suggested that Mb is a potentially protective element against I/R injury. However, the mechanism of its protective action is still largely unknown. With a real-time fluorescent technique, we observed that at the onset of ischemia, there was a small burst of superoxide (O(2)(•–)) release, as visualized in an isolated rat heart. Thus, we hypothesize that the formation of O(2)(•–) correlates to Mb due to a decrease in oxygen tension in the myocardium. Measurement of O(2)(•–) production in a Langendorff apparatus was performed using surface fluorometry. An increase in fluorescence was observed during the onset of ischemia in hearts perfused with a solution of hydroethidine, a fluorescent dye sensitive to intracellular O(2)(•–). The increase of fluorescence in the ischemic heart was abolished by a superoxide dismutase mimic, carbon monoxide, or by Mb-knockout gene technology. Furthermore, we identified that O(2)(•–) was not generated from the intracellular endothelium but from the myocytes, which are a rich source of Mb. These results suggest that during the onset of ischemia, Mb is responsible for generating O(2)(•–). This novel mechanism may shed light on the protective role of Mb in I/R injury.