Figure 4. CRU dispersion provides the structural basis for silent RyR leak in HF.

A mathematical model of the dyad was employed to examine the effects of CRU dispersion on Ca²⁺ sparks and non-spark mediated RyR leak. (A). As dSTORM imaging indicated an increased fraction of small CRUs in HF (Figure 2C), small idealized CRUs were initially modelled with as few as 4 RyRs. Simulated Ca²⁺ sparks (300 consecutive simulations) were never detected for the smallest CRUs, based on an experimentally determined spark detection threshold of ΔF/F₀ = 0.4. Higher probability of visible spark generation (fidelity) was observed for larger CRUs. (B). Real CRU geometries obtained by dSTORM imaging were employed to simulate sparks from larger dyads. Four configurations were modelled with varying numbers of constituent RyR clusters, but similar total RyR number (≈55). While the single-cluster CRU exhibited high Ca²⁺ spark fidelity, lower probability of spark generation was observed in dispersed, multi-cluster CRUs (fidelity indicated by colour scale). These data support that CRU rearrangement during HF promotes silent RyR leak, due to an increased fraction of both small CRUs as well as larger CRUs with dispersed, irregular configurations.

Figure 4—figure supplement 1. Development and characterization of the mathematical dyadic model.

(A) Graphical representation of the computational domain of the model. The junctional SR (jSR) was shaped to encompass idealized RyR geometries or dSTORM-based CRU contours. (B) To understand the role of the jSR geometry in determining the maximum inter-cluster distance permissive of functional coupling, and thus CRU definition, we spread two square 16-RyR clusters at variable distances up to 144 nm edge-to-edge (i.e. 4 RyR lengths). Coupling could occur at distances up to ~ 150 nm but only when the jSR was contiguous between clusters. (C) Similar effects could be observed in the realistic 7-cluster geometry, where a fully contiguous jSR was sufficient to functionally couple clusters at distances between 100 and 150 nm.