We demonstrated in a previous study [Takahashi, E., K. Sato, H. Endoh, Z.-L. Xu, and K. Doi. Am. J. Physiol. 275 (Heart Circ. Physiol. 44): H225-H233, 1998] that significant radial gradients of intracellular PO₂ may be produced in an uncoupled actively respiring, single isolated cardiomyocyte of the rat. The present study was designed to further determine whether such intracellular PO₂ gradients can be a limiting factor of oxidative metabolism in uncoupled cardiomyocytes. The NAD(P)H fluorescence of a single cardiomyocyte was captured by a digital charge-coupled device camera and quantitated with a subcellular spatial resolution by a ratio-imaging technique. In the conditions that we demonstrated significant radial PO₂ gradients (cells treated with 1 µM carbonyl cyanide m-chlorophenylhydrazone and superfused with 2.09% or 3.14% O₂ gas at 27°C), we demonstrated significant augmentation of NAD(P)H fluorescence near the core of an individual cell. The heterogeneous fluorescence pattern was not found in the control cell, whereas fluorescence intensity averaged over the cell was increased by hypoxia. These results suggest the possibility that oxidative phosphorylation near the core of actively respiring, uncoupled cardiomyocytes may be severely suppressed due to insufficient diffusional oxygen supply (hypoxic core) even if regions near the sarcolemma are adequately oxygenated.

reduced nicotinamide adenine dinucleotide fluorescence; mitochondrial respiration; intracellular PO₂ gradients; oxidative phosphorylation; oxygen diffusion

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