Clustering properties of the cardiac ryanodine receptor in health and heart failure

Figures

• 

  Fig. 1.. Cellular and RyR2 regulatory changes in HF. Schematic demonstrating the physiological organisation and regulation of ryanodine receptor (RyR2) in healthy cardiomyocytes (left) and changes associated with heart failure (HF; right). RyR2 is expressed in the sarcoplasmic reticulum (SR) membrane where it localises to the dyad with the L-Type Ca2+ channel (LTCC), facilitated by junctophilin-2 (JPH2). The Na+-Ca2+ exchanger (NCX) together with SR Ca2+-ATPase (SERCA; which is regulated by phospholamban (PLB)) remove cytosolic Ca2+ from LTCC influx and RyR2 release. Failing cardiomyocytes exhibit dilation, loss and disorganisation of t-tubules, along with reduced JPH2 expression leading to dyad disruption and separation of RyR2 from LTCC. Inset shows RyR2 demonstrates enhanced phosphorylation by PKA and CaMKII (‘P’ in red circle) and oxidation (‘O’ in blue circle), with reduced FKBP (maroon hexagon) association in failing hearts. Created with BioRender.com.

• 

  Fig. 2.. Individual RyR2 nanoscale clustering parameters. Example images of RyR2 clusters in non-failing rat ventricular cardiomyocytes visualised using A) confocal, B) dSTORM, C) 3D dSTORM and D) DNA-PAINT imaging modalities, demonstrating differences in image resolution. E) Schematic demonstrating changes in individual RyR2 cluster parameters associated with i) cluster size, ii) RyR2 channel packing density within a cluster, and iii) different arrangement of RyR2 tetramers within a cluster, showing side-by-side, checkerboard and isolated configurations. Blue line represents the outline edge of a single RyR2 cluster. Panel A modified from Soeller et al., 2009 [118], image reproduced with permission; Panel B modified from Hou et al., 2015 [65], image reproduced with permission; Panel C modified from Shen et al., 2019 [68]; Panel D modified from Jayasinghe et al., 2018 [66]. Panel E created with BioRender.com.

• 

  Fig. 3.. Inter-cluster parameters. Schematic demonstrating changes in RyR2 inter-cluster parameters. A) Representation of RyR2 distribution to the z-disk, demonstrating changes in RyR2 cluster density within a cardiomyocyte and the relationship with nearest neighbour distance (NND) Blue outline represents a single RyR2 cluster. B) The functional grouping of RyR2 clusters into a Ca2+ release unit (CRU; red boundary) when separated by NND of <100–150 nm. An increase in NND reduces the number of clusters per CRU, while clusters separated by >100–150 nm are excluded from the CRU. Created with BioRender.com.

• 

  Fig. 4.. Nanoscale RyR2 cluster remodelling in heart failure. A) dSTORM images of RyR2 clusters in cardiac tissue from non-failing patients (left) and patients with end-stage heart failure (HF; right). B) dSTORM images of RyR2 clusters and CRUs in cardiac myocytes from control (left) and HF model (right) rats. C) Schematic summarising the organisation of RyR2 clusters in healthy cardiomyocytes (left) and the reported cluster remodelling in human and animal models of HF (right). Clusters in failing hearts demonstrate a reduction size and an increased distance between channels within a cluster (lower channel packing density). Inter-cluster nearest neighbour distance (NND) is reduced, with a higher cluster density and increased number of individual clusters (blue outline) per Ca2+ release unit (CRU; red boundary) in failing cardiomyocytes. Panel A images modified from Hou et al., 2021 [57]. Panel B images modified from Kolstad et al., 2018 [21]. Panel C created with BioRender.com.