Cardiac Pacemaker Cell Function at a Super-Resolution Scale of SIM: Distribution of RyRs, Calcium Dynamics, and Numerical Modeling

Abstract

Previous confocal microscopy studies of SA-node cells revealed (in tangential sections) hierarchical clustering of calcium release channels (RyRs). Further numerical model simulations demonstrated that such hierarchy can enable propagating Local-Calcium-Releases (LCRs), crucial events of pacemaker cell operation. RyR distributions in 3D remain, however, unknown, leaving substantial uncertainty in constructing realistic models of SA-node cells. Here we approached the problem by super-resolution structured illumination microscopy (SIM) achieving approximately double the resolution of conventional microscopy to measure both 3D-distribution of RyRs and 2D-calcium dynamics in isolated rabbit SA-node cells. Each 3D-distribution of RyR clusters was reconstructed with 150 nm z-sectioning to capture the entire cell (bottom-to-top). We developed a novel computer algorithm to detect and characterize fluorescence signals of RyR clusters, including their sizes (volumes) and genuine distances to their nearest neighbors in 3D. We analyzed 8 cells with RyR localization mainly at the cell surface and found from 1107 to 2420 (mean=2070) RyR clusters in each cell. Distributions for nearest neighbor (center-to-center) distance were essentially (>95%) within the range 200-800 nm (mean=405 nm) that is substantially smaller than we previously reported (mean∼800 nm), indicating that many small clusters (lacking in confocal scans) now become resolved by SIM and also many neighboring clusters previously missed in 2D-projections now become revealed by the new 3D-analysis. Our cell-mid-section SIM measurements of calcium dynamics (using fluo-4) revealed that LCRs occur mainly within ∼1µm under the cell surface, in agreement with immunofluorescence data on RyR cluster localization. The new SIM results on Ca dynamics and anatomical information are included in new numerical modeling of SA-node cell. The larger number of clusters and shorter distances facilitate calcium-induced-calcium-release and LCR propagation observed experimentally.