Abstract
Excitation-contraction coupling in the heart crucially depends on the function of ryanodine receptors (RyRs). Recent advances in single-molecule localization microscopy have enabled nanoscale visualization of RyR organization, yet the precise geometric arrangement of RyRs in both health and disease has yet to be characterized. Here, we employed our recently developed 3D dSTORM method to quantify the subcellular, nanoscale organization of RyRs in normal and failing (post-infarction) rat cardiomyocytes, proposing that disassembly of RyR clusters promotes impaired Ca2+ homeostasis in this condition. We specifically investigated the nanoscale organization of interior RyRs which are associated with the transverse tubule network; a subpopulation of RyRs that can only be quantitatively characterized in 3D. We observed a significant reduction in RyR cluster size in failing cardiomyocytes when compared to sham-operated controls (5.0±0.3 vs. 7.8±0.4 RyRs/cluster, p<0.05). RyR cluster groupings localized within 100 nm of their neighbours comprise Ca2+ release units (CRUs), which are thought to cooperatively generate Ca2+ sparks. 3D imaging revealed an average of 21±0.9 RyRs/CRU in sham cells, but only 15.1±1.3 RyRs/CRU in failing cells (p<0.05). Failing cardiomyocytes also contained more RyR clusters / CRU than sham cells (2.7±0.1 vs 2.9±0.1, p<0.05), associated with slower Ca2+ spark kinetics (time to peak = 7.7±0.5 vs 10.5±0.4, p<0.05). When failing cardiomyocytes were treated with a CaMKII blocker (Autocamtide-1-related Inhibitory Peptide, AIP), Ca2+ spark characteristics were restored to sham levels. AIP administration also reversed the RyR dispersal observed at both the cluster and CRU level. Importantly, stimulation of CaMKII by the EPAC activator 8-CPT reproduced RyR dispersion in healthy cardiomyocytes. Together, our results suggest that RyR cluster disassembly in failing cardiomyocytes is driven by CaMKII phosphorylation, and promotes impaired cardiomyocyte Ca2+ homeostasis in this disease.
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