Examination of the Effects of Heterogeneous Organization of RyR Clusters,Myofibrils and Mitochondria on Ca2+ Release Patterns in Cardiomyocytes

Spatio-temporal dynamics of intracellular calcium, Ca²⁺]_i, regulate the contractile function of cardiac muscle cells. Measuring Ca²⁺]_i flux is central to the study of mechanisms that underlie both normal cardiac function and calcium-dependent etiologies in heart disease. However, current imaging techniques are limited in the spatial resolution to which changes in Ca²⁺]_i can be detected. Using spatial point process statistics techniques we developed a novel method to simulate the spatial distribution of RyR clusters, which act as the major mediators of contractile Ca²⁺ release, upon a physiologically-realistic cellular landscape composed of tightly-packed mitochondria and myofibrils. We applied this method to computationally combine confocal-scale (~ 200 nm) data of RyR clusters with 3D electron microscopy data (~ 30 nm) of myofibrils and mitochondria, both collected from adult rat left ventricular myocytes. Using this hybrid-scale spatial model, we simulated reaction-diffusion of Ca²⁺]_i during the rising phase of the transient (first 30 ms after initiation). At 30 ms, the average peak of the simulated Ca²⁺]_i transient and of the simulated fluorescence intensity signal, F/F₀, reached values similar to that found in the literature (Ca²⁺]_i ≈1 μM; F/F₀≈5.5). However, our model predicted the variation in Ca²⁺]_i to be between 0.3 and 12.7 μM (~3 to 100 fold from resting value of 0.1 μM) and the corresponding F/F₀ signal ranging from 3 to 9.5. We demonstrate in this study that: (i) heterogeneities in the Ca²⁺]_i transient are due not only to heterogeneous distribution and clustering of mitochondria; (ii) but also to heterogeneous local densities of RyR clusters. Further, we show that: (iii) these structure-induced heterogeneities in Ca²⁺]_i can appear in line scan data. Finally, using our unique method for generating RyR cluster distributions, we demonstrate the robustness in the Ca²⁺]_i transient to differences in RyR cluster distributions measured between rat and human cardiomyocytes.