Fluorescence imaging-based analysis of the mitochondrial permeability transition pore opening in cardiomyocyte-derived H9c2 cells

Abstract

　Mitochondrial function is the key factor in the regulation of energy metabolism and is closely related to the generation and prevention of lifestyle-related diseases. We established a method to observe the opening of the mitochondrial permeability transition pore using fluorescence microscopy and permeabilized cardiomyocyte-derived H9c2 cells. After calcein loading and permeabilization of the cell membrane with digitonin, the cellular calcein fluorescence coincided with that of the mitochondrial fluorescent probe tetramethylrhodamine methylester. Elevation of extracellular Ca²⁺ concentration, which was accompanied by an increase in intramitochondrial Ca²⁺ concentration, induced a decrease in calcein fluorescence intensity. This decrease was inhibited by cyclosporine A, an inhibitor of mitochondrial permeability transition pore. Further, the decrease was inhibited by ruthenium red, an inhibitor of the mitochondrial uniporter, and enhanced by diltiazem, which inhibits the mitochondrial Na⁺-Ca²⁺ exchanger. The present imaging method would be useful for further studies on the regulation of mitochondrial function under physiological and various pathological conditions related to lifestyle-related diseases.