Pacemaker Organization at the Nanoscale: Imaging of Ryanodine Receptors as Clusters in Single Sinoatrial Nodal Cells

Abstract

Ryanodine Receptors type 2 (RyR2) are the intracellular Ca2+ release channels of cardiac myocytes. They are organized into clusters of ∼ 25 RyR2 (∼10 to ∼150). When RyR2 channels are activated they produce an “invisible” Ca2+ release (single RyR2s) from the sarcoplasmic reticulum (SR) or a Ca2+ spark (RyR2 cluster). Reports suggest that SAN Ca2+ sparks contribute to pacemaker depolarization of the SAN along with other mechanisms (e.g. HCN channels, L-type Ca2+ channels, recovery from activation of repolarizing K+ currents). To better understand SAN Ca2+ signaling and pacemaker activity, we have examined the organization of RyR2 in SAN cells. We used direct stochastic optical reconstruction microscopy (dSTORM) of RyR2s in single SAN cells. SAN cells were isolated from adult rat hearts and fixed in 3% paraformaldehyde 0.01% glutaraldehyde. Cells were labeled with a mouse-anti-RyR2 primary antibody (C3-33, Thermo-Fisher) and an Alexa Fluor 647 conjugated goat-anti-mouse secondary antibody (Thermo-Fisher). Prior to image acquisition glucose oxidase imaging buffer was added to the cell dish. Images were acquired on a Nikon Ti2-E inverted microscope with a 100X NA 1.35 silicon oil-immersion objective (Nikon), and an electron multiplying CCD camera (Princeton Instruments). Cells were pre-bleached for approximately 5s to push a large proportion of fluorophores into the dark state. Image sequences were then acquired and analyzed using Nikon Elements software. RYR2 cluster size in SAN cells is variable with cluster sizes varying between ∼15-140 RyR2 channels. The shape of RyR2 clusters in SAN cells is heterogeneous ranging from circular to elongated. Edge-to-edge distances between adjacent RyR2 clusters is often < 50 nm. This is indicative of potential inter-cluster signaling. More data on the organization and localization (e. g. subsarcolemmal versus central-cellular) of RyR2 nano-signaling domains will be shown.