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.Author Summary: Calcium (Ca2+) acts as a signal for many functions in the heart cell, from its primary role in triggering contractions during the heartbeat to acting as a signal for cell growth. Cellular function is tightly coupled to its ultra-structural organization. Spatially-realistic and biophysics-based computational models can provide quantitative insights into structure-function relationships in Ca2+ signaling. We developed a novel computational model of a rat ventricular myocyte that integrates structural information from confocal and electron microscopy datasets that were independently acquired and includes: myofibrils (protein complexes that contract during the heartbeat), mitochondria (organelles that provide energy for contraction), and ryanodine receptors (RyR, ion channels that release the Ca2+ required to trigger myofibril contraction from intracellular stores). Using this model, we examined [Ca2+]i dynamics throughout the cell cross-section at a much higher resolution than previously possible. We estimated the size of structural maladaptation that would cause disease-related alterations in [Ca2+]i dynamics. Using our methods for data integration, we also tested whether reducing the density of RyRs in human cardiomyocytes (~40% relative to rat) would have a significant effect on [Ca2+]i. We found that Ca2+ release patterns between the two species are similar, suggesting Ca2+ dynamics are robust to variations in cell ultrastructure.