Live-cell photo-activated localization microscopy correlates nanoscale ryanodine receptor configuration to calcium...

Ca²⁺ sparks constitute the fundamental units of Ca²⁺ release in cardiomyocytes. Here we investigate how ryanodine receptors (RyRs) collectively generate these events by employing a transgenic mouse with a photo-activated label on RyR2. This allowed correlative imaging of RyR localization, by super-resolution Photo-Activated Localization Microscopy, and Ca²⁺ sparks, by high-speed imaging. Two populations of Ca²⁺ sparks were observed: stationary events and "travelling" events that spread between neighbouring RyR clusters. Travelling sparks exhibited up to 8 distinct releases, sourced from local or distal junctional sarcoplasmic reticulum. Quantitative analyses showed that sparks may be triggered by any number of RyRs within a cluster, and that acute β-adrenergic stimulation augments intra-cluster RyR recruitment to generate larger events. In contrast, RyR "dispersion" during heart failure facilitates the generation of travelling sparks. Thus, RyRs cooperatively generate Ca²⁺ sparks in a complex, malleable fashion, and channel organization regulates the propensity for local propagation of Ca²⁺ release.