Sub-cellular network analysis of ryanodine receptor positioning in control and phosphorylated states

Abstract

Type 2 ryanodine receptors (RyR2) are large (mass=2.2 MegaDalton) proteins expressed in heart cells that mediate the release of calcium ions (Ca2+) that, in turn, modulate contraction of the heart. In this work, we analyze the sub-cellular spatial distribution of RyR2 using data from superresolution microscopy, an imaging technique that allows highly accurate positioning (<10 nm in x and y; <40 nm in z) of the RyR2 within the heart muscle cell. In particular, we present the first work to examine network measures of extracted RyR2 locations to examine the clustering behaviour of these channels. We collected images from two groups of healthy cardiac cells; the control consisted of 8 cells, while the second group of 8 cells were treated with a chemical cocktail to phosphorylate the RyR2 and to inhibit dephosphorylation. We examined the classification accuracy (using random forest classifier) and the group differences (using Mann-Whitney statistical test and Bonferroni multiple comparison correction) based on several network measures, at multiple proximity thresholds. Several network measures we examined revealed features (e.g. network clustering) that enabled us to differentiate between these two populations (p<0.00045) with high classification accuracy (>95% at proximity thresholds 200 and 250 nm). Our findings may help in better understanding Ca2+ signaling during contraction and give insight into the changes that underlie its regulation.