Structural and functional heterogeneity in cardiac RyR signalling nanodomains revealed with quantitative single molecule mapping toolkit

Technical Summary: Structural and Functional Heterogeneity in Cardiac RyR Signalling Nanodomains Revealed with Quantitative Single Molecule Mapping Toolkit

Problem Statement
The spatial organization of Type-2 ryanodine receptor (RyR2) channels and their tethering protein, junctophilin-2 (JPH2), is a critical determinant of cardiac calcium (Ca²⁺) release fidelity. While it is established that heart failure disrupts the transverse-tubule system, dyadic architecture, and RyR clustering, the specific nature of nanoscale remodelling in right ventricular (RV) failure induced by pulmonary artery hypertension (PAH) remains poorly understood. Previous studies suggest that disease alters protein abundance and phosphorylation, but it is unclear how the quantitative stoichiometry, spatial co-clustering, and cell-wide heterogeneity of RyR2 and JPH2 are altered within individual subsarcolemmal Ca²⁺-release nanodomains. Furthermore, the functional consequence of these specific structural changes on the propagation of spontaneous Ca²⁺ waves has not been directly linked to experimentally derived molecular maps.

Methodology
The authors developed an integrated analysis pipeline combining multiplexed DNA-PAINT (Points Accumulation for Imaging in Nanoscale Topography) with quantitative single-molecule counting (qPAINT) and image-based stochastic simulation.

1. Sample Preparation: Ventricular cardiomyocytes were enzymatically isolated from control rats and rats with PAH-induced RV failure (induced by monocrotaline, MCT). Cells were fixed and subjected to Exchange-PAINT imaging.
2. Imaging Protocol: RyR2 and JPH2 were immunolabelled with antibodies conjugated to unique DNA docking strands. Sequential imaging rounds were performed using complementary fluorescent imager strands (Atto655) to visualize each target. Data was acquired via Total Internal Reflection Fluorescence (TIRF) microscopy.
3. Data Processing Pipeline (PyME): Using the open-source Python Microscopy Environment (PyME), the authors implemented a workflow including:
   • Event Filtering & Drift Correction: Removal of low-quality localizations and correction of lateral drift.
   • Coalescence: Merging temporally adjacent events to prevent oversampling of single docking strands.
   • Segmentation: Identification of RyR clusters based on density maps.
   • qPAINT Analysis: Calculation of the "QIndex" (inverse of the characteristic dark time) to estimate the absolute number of accessible RyR and JPH2 targets within each segmented cluster.
4. Spatial Analysis:
   • Stoichiometry: Calculation of RyR:JPH2 ratios per cluster.
   • Co-localisation: Computation of Pearson's correlation and Manders coefficients within moving windows, and Euclidean Distance Transform (EDT) to measure the distance required to encompass 50% of the co-label.
   • Heterogeneity: Application of Gaussian Random Field (GRF) analysis and empirical semi-variograms to quantify spatial heterogeneity across length scales (100–1000 nm).
5. Simulation: Experimentally derived 2D maps of RyR and JPH2 positions were used as structural templates for a stochastic reaction-diffusion model of Ca²⁺ release. The model incorporated JPH2-dependent modulation of RyR sensitivity to simulate spontaneous Ca²⁺ wave propagation.

Key Results

• Quantitative Stoichiometry: In MCT-induced failing cells, the median RyR:JPH2 stoichiometric ratio decreased by 14% compared to controls. Crucially, the variance of this ratio increased significantly in failing cells, indicating that the reduction in co-clustering is not uniform but highly heterogeneous across the cell surface.
• Spatial Decoupling: While global co-localisation coefficients showed negligible changes, local variance in Manders coefficients increased 2.3-fold in failing cells. EDT analysis revealed a >2-fold increase in the distance required to encompass 50% of JPH2 signal relative to RyR, suggesting JPH2 recedes from or becomes less concentrated around RyR domains in disease.
• Scale-Specific Heterogeneity: Semi-variogram analysis showed divergent remodelling patterns. RyR maps in failing cells exhibited lower semivariance (reduced density fluctuations) compared to controls, whereas JPH2 maps showed increased semivariance, particularly at length scales approaching 1000 nm. This indicates that disease alters the magnitude of spatial density fluctuations differently for the two proteins.
• Simulation Outcomes: When used as templates for Ca²⁺ wave simulations, maps from failing cells supported faster transverse and longitudinal wave propagation (23% and 16% increases, respectively) compared to control maps. The simulations demonstrated that the fragmented RyR architecture and heterogeneous JPH2 distribution in failing cells are sufficient to support faster, saltatory recruitment of release sites, despite the loss of local synchrony.

Significance and Claims
The paper claims to establish a transferable toolkit that links molecular nanostructure to cellular function by integrating quantitative molecular counting with spatial statistics and image-based simulation. The primary findings suggest that RV failure is characterized not merely by a uniform loss of protein or structural disarray, but by spatially heterogeneous remodelling of the RyR2-JPH2 interface.

The authors posit that this heterogeneity—specifically the increased variability in local stoichiometry and the divergent spatial fluctuations of JPH2—creates a substrate for dysregulated Ca²⁺ signalling. The simulation results support the hypothesis that while synchronous Ca²⁺ release may be impaired, the resulting heterogeneous architecture can facilitate the emergence of spontaneous Ca²⁺ waves and arrhythmogenic propagation. The study concludes that this integrated approach provides a framework for understanding how nanoscale structural defects contribute to macroscopic cellular dysfunction in heart failure, moving beyond population averages to resolve local molecular mismatches.