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

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 is 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/μm2. (mean±SEM, n = 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/μm2 (8 cells, p ≤ 0.05) and created more superclusters. Our numerical SANC model showed that superclustering substantially decreased the AP cycle length (APCL) by creating Ca release hotspots that initiated larger LCRs under any condition. Increasing RyR density prolonged APCL in the basal state but shortened APCL during βAR stimulation. With no change in RyR network, βAR stimulation of only SR Ca pump and ion currents shortened APCL on average from 414.9 to 284 ms. When realistic higher RyR density and superclustering were added to the model, APCL further shortened to 231.9 ms. Thus, dynamic nanoscale changes in RyR network provide a new powerful pacemaker mechanism.