Abstract 17484: Simultaneous Monitoring of Reactive Oxygen Species, Calcium and Mitochondrial Potential During Myocardial Ischemia by Real-Time Two-Photon Imaging of Perfused Rat Hearts
Background: We established a real-time two-photon imaging to monitor mitochondrial membrane potential (ΔΨm) in the perfused heart. Loss of ΔΨm and subsequent mitochondrial collapse is a crucial step in the cell death process during myocardial ischemia/reperfusion; reactive oxygen species (ROS) and Ca2+ are considered as the two major regulators of this step. We aimed to visualize the spatio-temporal relationship between ROS, Ca2+ and ΔΨm loss at cellular level in the perfused heart subjected to ischemia/reperfusion using this imaging system.
Methods and Results: Langendorff-perfused rat hearts were loaded with 5-(and-6)-chloromethyl-2′,7′-dichlorodihydrofluorescein diacetate acetyl-ester, rhod-2, and tetramethylrhodamine ethyl-ester, fluorescent indicators of ROS, Ca2+, and ΔΨm, respectively. Under the two-photon excitation, spatio-temporal changes were simultaneously monitored. During 30 min ischemia/30 min reperfusion, ROS were mainly generated and progressively accumulated during early phase of ischemia (∼10 min), and the rate of ROS accumulation was variable from cell to cell. Irreversible ΔΨm loss occurred in an all-or-none manner depending on cellular ROS level, with a clear cut-off value. Cellular Ca2+ level concomitantly increased during ischemia with variable cell-to-cell specific rates. While cells were undergoing modest ROS increase, the level of ROS and Ca2+ did not correlate; but once ROS level reached a threshold level, Ca2+ level increased robustly. Ischemic preconditioning attenuated both the increase of ROS and Ca2+ during ischemia and protected against ΔΨm loss. A free radical scavenger N-(2-mercaptopropionyl)-glycine attenuated the ROS accumulation but not the Ca2+ increase; it did not protect against ΔΨm loss. Thus, reducing only ROS accumulation does not suffice to prevent mitochondrial collapse and cardiomyocyte death; suppression of both ROS and Ca2+ may be required.
Conclusions: We successfully established a real-time imaging to monitor ROS, Ca2+ and ΔΨm in perfused heart. This novel two-photon imaging provides deeper insights into the mechanisms of cardiomyocyte death during ischemia/reperfusion, and is useful in establishing anti-ischemia/reperfusion therapy that targets mitochondria.
- © 2010 by American Heart Association, Inc.