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. 2013 Sep 5;4(9):e787.
doi: 10.1038/cddis.2013.313.

Characterization of oxygen radical formation mechanism at early cardiac ischemia

X Zhu  1 , L Zuo
Affiliations

Characterization of oxygen radical formation mechanism at early cardiac ischemia

X Zhu et al. Cell Death Dis. .

Abstract

Myocardial ischemia-reperfusion (I/R) causes severe cardiac damage. Although the primary function of oxymyoglobin (Mb) has been considered to be cellular O2 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 (O2(•-)) release, as visualized in an isolated rat heart. Thus, we hypothesize that the formation of O2(•-) correlates to Mb due to a decrease in oxygen tension in the myocardium. Measurement of O2(•-) 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 O2(•-). 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 O2(•-) 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 O2(•-). This novel mechanism may shed light on the protective role of Mb in I/R injury.

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Figures

Figure 1
Figure 1
Fluorescence levels over the time course of 9 min in ischemia, ischemia with CO treatment, ischemia with SOD mimic treatment, and autofluorescence. *P<0.05, ischemia versus all other groups
Figure 2
Figure 2
Zoomed fluorescence levels over the micro time course of 60 s in ischemia, ischemia with CO treatment, ischemia with SOD mimic treatment, and autofluorescence. *P<0.05, ischemia versus all other groups
Figure 3
Figure 3
Mean fluorescence rate (fluorescence change per second; relative unit per second, RU/s) over the time course of 60 s from the start of ischemia in rat heart. (a) mean fluorescence rate in the ischemia-only group. (b) mean fluorescence rate in SOD mimic treatment group. (c) mean fluorescence rate in CO treatment group. (d) mean fluorescence rate in autofluorescence group (n=3)
Figure 4
Figure 4
Fluorescence levels over the time course of 60 s from the start of ischemia. *P<0.05, n=4; Mb-knockout (Myo−/−) mice versus wild-type (WT) mice
Figure 5
Figure 5
Mean fluorescence rate (fluorescence change per second; RU/s) over the time course of 60 s from the start of ischemia in a mouse heart. (a) mean fluorescence rate in WT mice. (b) mean fluorescence rate in Myo−/− mice (n=4)
Figure 6
Figure 6
Localization of O2•– formation during the onset of ischemia on the heart surface using confocal microscopy. (a) heart baseline fluorescence before ischemia. (b) O2•– generation in the HE-loaded heart within 3 min during ischemia. (c) O2•– generation in hearts treated with dual-fluorescence probes (HE and fluorescein lectin I-isolectin B4 (FLI-IB4)), showing that O2•– production is within the cardiomyocytes, not in the endothelial vessels
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