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Am J Physiol Cell Physiol 295: C1302-C1315, 2008. First published September 24, 2008; doi:10.1152/ajpcell.00313.2008 Free Article
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CELLULAR AND MITOCHONDRIAL METABOLISM

Anisotropic diffusion of fluorescently labeled ATP in rat cardiomyocytes determined by raster image correlation spectroscopy

Marko Vendelin and Rikke Birkedal

Laboratory of Systems Biology, Institute of Cybernetics, Tallinn University of Technology, Tallinn, Estonia

Submitted 12 June 2008 ; accepted in final form 8 September 2008

A series of experimental data points to the existence of profound diffusion restrictions of ADP/ATP in rat cardiomyocytes. This assumption is required to explain the measurements of kinetics of respiration, sarcoplasmic reticulum loading with calcium, and kinetics of ATP-sensitive potassium channels. To be able to analyze and estimate the role of intracellular diffusion restrictions on bioenergetics, the intracellular diffusion coefficients of metabolites have to be determined. The aim of this work was to develop a practical method for determining diffusion coefficients in anisotropic medium and to estimate the overall diffusion coefficients of fluorescently labeled ATP in rat cardiomyocytes. For that, we have extended raster image correlation spectroscopy (RICS) protocols to be able to discriminate the anisotropy in the diffusion coefficient tensor. Using this extended protocol, we estimated diffusion coefficients of ATP labeled with the fluorescent conjugate Alexa Fluor 647 (Alexa-ATP). In the analysis, we assumed that the diffusion tensor can be described by two values: diffusion coefficient along the myofibril and that across it. The average diffusion coefficients found for Alexa-ATP were as follows: 83 ± 14 µm2/s in the longitudinal and 52 ± 16 µm2/s in the transverse directions (n = 8, mean ± SD). Those values are ~2 (longitudinal) and ~3.5 (transverse) times smaller than the diffusion coefficient value estimated for the surrounding solution. Such uneven reduction of average diffusion coefficient leads to anisotropic diffusion in rat cardiomyocytes. Although the source for such anisotropy is uncertain, we speculate that it may be induced by the ordered pattern of intracellular structures in rat cardiomyocytes.

anisotropy; fluorescence correlation spectroscopy


Address for reprint requests and other correspondence: M. Vendelin, Laboratory of Systems Biology, Institute of Cybernetics at Tallinn Univ. of Technology Akadeemia 21, 12618 Tallinn, Estonia (e-mail: markov{at}ioc.ee)






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