STED Nanoscopy of Cardiac RyR2 Clusters and Sub-Structure Analysis After Myocardial Infarction

Abstract

Cardiac ryanodine receptor (RyR2) Ca2+ release channels occur as multi-channel clusters at subcellular Ca2+ release sites in cardiomyocytes. RyR2 clusters are thought to underlie important signal amplification mechanisms including coupled gating of physically associated Ca2+ release channels. The nature of RyR2 channel organization in intact intracellular channel clusters is not known.We have investigated the nanostructure of RyR2 clusters using Stimulated Emission Depletion (STED) nanoscopy with ∼60 nm lateral resolution and indirect immunofluorescence in cardiomyocytes. We compared RyR2 clusters in adult cardiomyocytes from sham hearts with hearts 8 weeks after myocardial infarction (post-MI).Peripheral RyR2 clusters were detected at a density of 1.3 ± 0.2 clusters per μm2 in control cells (2237 clusters) and 1.6 ± 0.1 in diseased cells (1752 clusters). Individual clusters were analyzed by incrementally detecting signal edges which defined intra-cluster substructures and intensity peaks. Within individual clusters, nearest-neighbour distances between intensity peaks showed an asymmetric distribution with a maximum at 100 ± 1 nm in healthy cardiomyocytes. In post-MI cells this distribution showed a left shift with a maximum at 85 ± 3 nm (p < 0.05 Mann-Whitney test; mean distance sham 150.6 ±3.2 nm vs post-MI 129.5 ± 2.7 nm).In conclusion, immuno-labeled cardiac RyR2 clusters showed distinct non-randomly organized sub-structures as evidenced by characteristic intra-cluster peak-to-peak distances. Our data suggest that continuous RyR2 clusters contain substructures which occur repeatedly as densely organized groups of RyR2 channels. In addition, we showed altered RyR2 clusters after myocardial infarction with smaller intra-cluster peak-to-peak distances which implies tighter channel packing. We are preparing a working mathematical model of normal RyR2 cluster function and the role of potential substructures and their changes in heart disease.