54Detection, quantification and visualization of ryanodine receptor phosphorylation in human atrial myocytes using a novel ratiometric immunofluorescent analysis

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Purpose: Calcium release through the ryanodine receptor (RyR2) plays a central role in the regulation of cardiac contraction and rhythm, and is modulated by RyR2 phosphorylation. However, little is known about the distribution of phosphorylated RyR2s in isolated cardiac myocytes and the purpose of the present study was to develop an immunofluorescent ratio-metric approach to quantify and visualize the spatial distribution of phosphorylated RyR2s.

Methods: Forty-nine human atrial myocytes from nine patients were labeled with anti-phospho Ser-2808 (Red) and anti-RyR2 (Green) antibodies and detected automatically using a custom made algorithm based on: 1) Enhancing the contrast of both green and red-labeled images using a histogram stretching intensity transformation. 2) Removing background noise by using an adaptive median filter that estimates the noise level. 3) Enhancing the location of all green-labeled RyRs with a 2D Gaussian filter with a standard deviation of 0.5 microns followed by segmentation using a multilevel watershed algorithm. 4) Eliminating non-specific staining by setting the maximal RyR diameter to 1.2 microns. 5) Detection of red-labeled ser2808 phosphorylated RyR2s by checking if a cluster of red pixels was present at the location of each green labeled RyR2. Red clusters that overlapped at least 20% of the area of a green-labeled RyR2 were accepted if the normalized intensity of the overlapping area was above 15%.

Results: Superimposing detected RyR2s on the original confocal image revealed a detection efficiency near 95%, and visual inspection of superimposed original confocal images confirmed that all red ser2808 clusters coincided with a green RyR2. Moreover, stimulation of RyR2 phosphorylation with the beta-adrenergic agonist was used as a positive control, and it significantly increased the ser2808:total RyR2 ratio from 0.32±0.03 to 0.52±0.06 (p<0.01).

Conclusion: We have developed a novel immunofluorescent ratiometric approach that allows quantifying and visualizing RyR2 phosporylation in isolated human atrial myocytes. This approach may also apply to other proteins with a punctate distribution that are modulated by phosphorylation, and should be useful to study how disease affects the distribution of such proteins under basal or phosphorylating conditions.

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