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

Abstract

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.

Insights on the impact of mitochondrial organisation on bioenergetics in high-resolution computational models of cardiac cell architecture

“…In a previous study, Takahashi et al [26] visualised intracellular gradients of myoglobin oxygen saturation using high spatial resolution spectrophotometry and further converted the observed oxygenation level into partial pressure of O2 (PO2) using linear regression of data and the Hill equation. Fig 3C represents [27]. Figure 3C shows that the concentration profile in the simulated confocal image of O2 distribution follows a similar profile to the radial PO2 gradient experimentally measured by Takahashi et al [26] in six different isolated cells.…”

Size Matters: Ryanodine Receptor Cluster Size Affects Arrhythmogenic Sarcoplasmic Reticulum Calcium Release

“…We might miss a small number of tiny clusters that are distant from identified clusters, but ≈90% of all Z‐line clusters are within 200 nm of other clusters 5. Although the vast majority of interior RyRs and Ca 2+ ‐spark initiation sites are near Z‐lines, some RyR clusters and sparks are observed at other cellular loci (eg, sarcomeric, near mitochondria, and perinuclear) 31, 43, 44…”

An Automated Workflow for Segmenting Single Adult Cardiac Cells from Large-Volume Serial Block-Face Scanning Electron Microscopy Data

“…Segmentations for Cell 3 were used as input in a 3D tetrahedral mesh generation program called iso2mesh 20 . Such meshes can be used for simulations of intracellular calcium dynamics 10 and bioenergetics 21 even without other details such as t-tubules or the sarcoplasmic reticulum. .…”

“…We anticipate that the segmented dataset will prove invaluable to mathematical modelers interested in studying cardiac biophysics in spatially detailed models of cardiac ultrastructure [10][11][12] . We believe that our MATLAB implemented algorithm will prove useful to many cardiac biophysicists who are interested in rapid extraction and analysis of ultrastructure content and organization without manual segmentation or prior knowledge of image processing techniques.…”