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.
Highlights
The cardiac myocyte possesses a highly organized assembly of membrane networks, contractile proteins, ion channels and buffering systems
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, mitochondria, and ryanodine receptors (RyR, ion channels that release the Ca2+ required to trigger myofibril contraction from intracellular stores)
We found that Ca2+ release patterns between the two species are similar, suggesting Ca2+ dynamics are robust to variations in cell ultrastructure
Summary
The cardiac myocyte possesses a highly organized assembly of membrane networks, contractile proteins, ion channels and buffering systems. ECC begins with the electrical activation and depolarization of the cell membrane and its transverse-tubular extensions (t-tubules) that invaginate the inner depths of the cell, causing a small flux of Ca2+ (through voltage-dependent L-type Ca2+ channels) into the dyadic cleft—the space between the t-tubules and the extensive internal Ca2+ storage network called the sarcoplasmic reticulum (SR). Structural imaging using confocal, super-resolution, and electron microscopy [4,5,6,7,8,9,10,11] are providing insights into the structural organization of the cardiac cell at different spatial scales These datasets are increasingly being used to generate accurate, spatially-extended computational models of the cell in order to investigate intracellular Ca2+ dynamics that current [Ca2+]i imaging technologies cannot resolve [12,13,14,15]. The robustness of these computational models to variations in structural organization is hard to assess, since the model geometry is strongly dependent on the datasets at hand
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