A new Fight-or-Flight Pacemaker Mechanism via Ryanodine Receptor abundance and superclustering

Abstract The sinoatrial node is the primary cardiac pacemaker. Individual sinoatrial node cells (SANCs) generate spontaneous rhythmic action potentials (APs) that initiate each heartbeat. The mechanism of SANC automaticity and its modulation by autonomic nervous system are based on the coupled function of molecules of both the cell membrane (ion channels, exchangers, and pumps) and the sarcoplasmic reticulum (SR), which generates rhythmic local Ca releases (LCRs). While LCRs are generated by ryanodine receptors (RyRs), the molecular-scale RyR network structure remains unknown. Here we performed single-molecule localization of RyRs via direct Stochastic Optical Reconstruction Microscopy (dSTORM) in rabbit SANCs in basal conditions and 5 minutes after β-adrenergic receptor (βAR) stimulation by isoproterenol. RyRs form clusters of various sizes, with a mean density of 67.7±13.2 RyR/μm 2 . (Mean±SEM, 6 cells). While the majority of cluster sizes ranged from 3 to 32 RyRs, each cell had a few substantially larger clusters (>76 RyRs), dubbed superclusters. βAR stimulation significantly increased the RyR density to 119.1±22.6 RyR/μm 2 (8 cells, p