Determination of Regional Diffusion Coefficients of Fluorescent ATP in Rat Cardiomyocytes

Abstract

Results from several experiments indicate the existence of restrictions to diffusion of ADP/ATP in permeabilized rat cardiomyocytes. Decreased rate of diffusion is necessary to explain measurements of kinetics of respiration, sarcoplasmic reticulum loading with calcium, and kinetics of ATP-sensitive potassium channels. Determining intracellular diffusion coefficients allows for estimation of the effect of these restrictions on bioenergetics. Previously we extended raster image correlation spectroscopy (RICS) methods and demonstrated the existence of anisotropy in the diffusion of fluorescently labeled ATP in rat cardiomyocytes. Specifically, longitudinal and transverse diffusion coefficient was, respectively, ∼2 and ∼3.5 times smaller than in solution. We attempted to extend our method in order to construct regional maps of diffusion coefficients. Our efforts using a commercial confocal microscope failed due to shortcomings in protocol automation, photon detection and noise properties at high scanning speeds. In order to solve these issues, we custom built a confocal microscope and wrote software to automate the protocol for performing RICS measurements with varying scan speeds and rotation angles. During calibration, we demonstrated that the direction of the scan can influence autocorrelation function calculated according to RICS protocols through non-symmetric point spread function. With the new system, we have been able to measure regional diffusion coefficients in permeabilized rat cardiomyocytes. As expected, there is a sharp change of the diffusion coefficient on the border of the permeabilized cell. Interestingly, the values of diffusion coefficients estimated for the regions surrounding the cell are similar to the ones determined in plain solution. While there has been some variation in diffusion coefficients within the cell, the present precision of the method does not allow us to distinguish the diffusion coefficients in regions smaller than few micrometers. For that, further development and experiments are needed.