Figure 1. Normalized O₂⁻ mapping in the intact perfused heart reveals two distinct ROS peaks in response to acute OS.

Initial proof-of-concept protocol of OS entailed perfusion of hearts with 200 μm H₂O₂ for 40 min. This protocol was chosen because it results in sustained ventricular fibrillation (VF) in all hearts. A, average normalized optical O₂⁻ levels of individual hearts in response to this protocol of OS. Time of H₂O₂ perfusion for each heart is indicated by the red arrow. H₂O₂ perfusion resulted in two distinct O₂⁻ peaks (P1 and P2) in all hearts. The maximum amplitude of the delayed peak (P2) was significantly greater than P1. B, contour maps illustrating the dynamic changes in the spatial distribution of O₂⁻. Green represents regions of highest superoxide levels. Representative maps during onset of P2 perfusion demonstrate wave-like propagation of O₂⁻ increase, consistent with RIRR. C, normalized ΔΨ_m levels of 4 Tetra methyl rhodamine methylester (TMRM)-stained hearts in response to this protocol of OS, showing ΔΨ_m depolarization.