3d Dstorm Imaging Reveals Disassembly Of Ryanodine Receptor Clusters In Failing Cardiomyocytes

Excitation-contraction coupling in the heart critically depends on the function of ryanodine receptors (RyRs). While advances in single-molecule localization microscopy (SMLM) have allowed 2D visualization of RyRs at the nanoscale level, the precise cellular localisation of RyRs in 3D in both health and disease has yet to be characterized. We presently utilised our recently developed 3D direct stochastic optical reconstruction microscopy (3D dSTORM) method to quantify the subcellular distribution of RyRs in normal and failing (post-infarction) rat cardiomyocytes, hypothesizing that breakup of RyR clusters may contribute to dysregulated Ca2+ homeostasis during heart failure. We specifically examined the nanoscale arrangement of two subpopulations of RyRs: sarcolemma-associated surface clusters and transverse tubule-associated interior clusters. Using 3D dSTORM, we observed a clear difference between surface and interior RyR cluster size in normal cardiomyocytes (13.5±0.6 and 16.0±0.7 RyRs/cluster, respectively, p<0.05). In failing cardiomyocytes, both surface and interior RyR cluster sizes were significantly reduced when compared to normal cells (9.7±0.7 and 10.5±0.6 RyRs/cluster, respectively, p<0.05). Interestingly, post-infarct non-failing cardiomyocytes only exhibited reduced cluster size within the cell interior (10.2±0.6 RyRs/cluster, p<0.05 vs normal), suggesting that RyR dispersion may initially occur at these sites. Ca2+ release units (CRUs), which are functional groups of RyR clusters responsible for the cooperative generation of Ca2+ sparks, contained significantly fewer RyRs in failing cells (surface: 20.6±0.8 vs. 17.0±1.1 RyRs/CRU in failing, p<0.05; interior: 24.2±0.9 vs. 18.3±1.0 RyRs/CRU in failing, p<0.05) and exhibited more dispersed organization. These alterations were paralleled by an observed slowing of Ca2+ spark kinetics and greater RyR Ca2+ leak in failing cells. Together, our results identify disassembly of RyR clusters as a novel mechanism underlying impaired cardiomyocyte Ca2+ homeostasis in heart failure.