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 structureinduced 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

[Ca²⁺]_i is robust to alterations in RyR cluster distribution.

<p>[Ca²⁺]_i (top panel) and [F4Ca]_i (bottom panel) are shown at t = 30 ms, at the z-disc transverse plane. Results on the left column were generated from a simulation using RyR cluster distribution properties of the rat. Results on the right column were generated from a simulation using RyR cluster distribution properties from human myocyte measurements.</p

RyR cluster simulation algorithm workflow.

<p>(1) segment RyR clusters from original data; (2) calculate the nearest-neighborhood distances for the RyR clusters; (3) Identify Z-disc planes from RyR cluster data; (4) Convert phalloidin stack into binary image stack; (5) Identify Z-plane positions on the binary myofibrils stack from (4) and set rest of the planes to background colour; (6) Calculate background distance transform for z-disc stack and decompose to radial and axial components; (7) Determine W, window of voxels available for RyR cluster simulation; (8) Use W, nearest-neighborhood distances and axial and radial z-disc distance statistics as input for RyR cluster simulation algorithm.</p

A hybrid-scale spatial model of myofbirils, mitochondria and RyR clusters.

<p>(A): Confocal image of a tissue section from the left ventricule of an adult male Wistar rat; numbered cells were processed for RyR cluster distribution analysis and development of a novel computational fusion algorithm (see <a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1004417#pcbi.1004417.s017" target="_blank">S1 Text</a>). (B): 3D rendering of cell number 1 in (A) showing green immuno-labeling of RyR clusters and red, phalloidin staining of myofibrillar actin. (C): Electron micrographs of a 240 nm tissue section from another left ventricular sample of a similar male Wistar rat were acquired at different tilts (left) to construct a 3D electron tomogram; the stack was manually segmented (right) for myofibrils and mitochondria. More details on the data acquisition are in the Materials and Methods. (D): One image slice from the electron tomogram with an overlay of the FE computational mesh (mitochondrial regions in green) and the simulated RyR clusters (red spheres) from the computational fusion algorithm; the mesh is partly removed for visualization. (E): A 3D view of the RyR clusters and the mitochondrial regions. (F): 3D view of the predicted Fluo-4-bound Ca²⁺ at the end of 30 ms; an isosurface of the solution field is partially in view at the mid-plane of the half-sarcomere model.</p

4–6 orphaned RyR clusters contribute to a missing “spark” in line scans.

<p>View of [F4Ca]_i in the default, “healthy” model and “diseased” models (rows 2 to 4) with increasing number of un-activated RyR clusters; regions of interest are encircled in black and subsequent images show the un-activated RyR clusters in these regions, highlighted in red. The line plot for the default model is shown in all the plots for easy comparison with the line plots from the increasingly “diseased” RyR cluster distributions. All images were acquired at t = 30 ms.</p

Spatial heterogeneity in [Ca²⁺]_i during the rising phase of the Ca²⁺ transient.

<p>(A): The average cytosolic freely diffusing [Ca²⁺]_i (left) and Fluo-4 bound Ca²⁺, [F4Ca]_i (right). (B) and (C) show time-lapse snapshots of freely diffusing Ca²⁺ and F4Ca at the z-disc, transverse plane; black arrows mark regions of high [Ca²⁺]_i due to mitochondrial clustering; red arrows mark regions of high [Ca²⁺]_i due to several ryr clusters in close proximity. (C): simulated line scans of [Ca²⁺]_i (middle) and [F4Ca]_i (right). The line position is shown on the cell cross-section on the left.</p

Biochemical rate constants, diffusion constants and initial concentrations used for the Ca²⁺ simulations.

<p>Biochemical rate constants, diffusion constants and initial concentrations used for the Ca²⁺ simulations.</p