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<0.05) and created more superclusters. Our new 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. This mechanism may help explain athletic bradycardia at rest and high exercise heart rates, with its deterioration contributing to the age-related loss of heart rate reserve. Author summary The heartbeat begins in the sinoatrial node, the heart’s natural pacemaker. During stress or exercise, pacemaker cells accelerate their firing rate via β-adrenergic (“fight-or-flight”) stimulation. Although Ca signals are known to drive this acceleration via electrogenic Na/Ca exchanger, how the underlying ryanodine receptor (RyR) Ca release channels are organized at the nanoscale and how this organization changes during stimulation remain unclear. Here, we used super-resolution microscopy (dSTORM) to visualize individual RyRs in rabbit pacemaker cells. We found that RyRs form heterogeneous clusters that include rare, very large “superclusters.” β-adrenergic stimulation increased overall RyR density, enlarged clusters, and markedly increased the number of superclusters. To understand how these structural changes affect pacemaker function, we developed a computational sinoatrial node cell model in which each RyR was represented explicitly and arranged according to our imaging data. Numerical model simulations revealed that superclusters act as Ca-release hotspots that fire early during diastolic depolarization and recruit neighboring RyR clusters to fire via Ca-induce-Ca-release mechanism. When combined with β-adrenergic enhancement of Ca cycling and membrane currents, the superclustering and RyR abundance synergistically accelerated action potential firing. Together, our results identified dynamic nanoscale RyR network reorganization as a key structural mechanism in fight-or-flight response.