Functional heterogeneity of mitochondria after cardiac cold ischemia and reperfusion revealed by confocal imaging

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Abstract
Mitochondria play a critical role in ischemia-reperfusion injury of the heart. The purpose of the present study was to analyze the intracellular region-specific functional state of mitochondria after cold ischemia-reperfusion in a rat heart transplant model. Imaging of the mitochondrial functional state in situ in nonfixed myocardial fibers was performed by confocal microscopy of mitochondrial flavoprotein autofluorescence as redox state indicator; fluorescence of Rhod-2, a specific probe for mitochondrial calcium; and of tetramethylrhodamine ethyl ester fluorescence to monitor the mitochondrial membrane potential. This imaging demonstrated that, in contrast to control fibers, 10-hr heart cold storage, heterotopic cardiac transplantation, and 24-hr reperfusion result in a highly heterogeneous mitochondrial functional state (mitochondrial calcium content, redox state, and inner membrane potential), thus suggesting local permeability transitions and heterogeneous mitochondrial damage. Imaging of in situ mitochondria allows topologic assessment of mitochondrial defects and heterogeneity, consequently providing new insights into the mechanisms of cardiac ischemia-reperfusion injury.