Towards Computational Modeling of Excitation-Contraction Coupling in Cardiac Myocytes: Reconstruction of Structures and Proteins From Confocal Imaging

Abstract

Computational models of excitation-contraction (EC) coupling in myocytes are valuable tools for studying the signaling cascade that transduces transmembrane voltage into mechanical responses. A key component of these models is the appropriate description of structures involved in EC coupling, such as the sarcolemma and ion channels. This study aims at developing an approach for spatial reconstruction of these structures. We exemplified our approach by reconstructing clusters of ryanodine receptors (RyRs) together with the sarcolemma of rabbit ventricular myocytes. The reconstructions were based on dual labeling and three-dimensional (3D) confocal imaging of segments of fixed and permeabilized myocytes lying flat or on end. The imaging led to 3D stacks of cross-sections through myocytes. Methods of digital image processing were applied to deconvolve, filter and segment these stacks. Finally, we created point meshes representing RyR distributions together with volume and surface meshes of the sarcolemma. We suggest that these meshes are suitable for computational studies of structure-function relationships in EC coupling. We propose that this approach can be extended to reconstruct other structures and proteins involved in EC coupling.

Subcellular Structures and Function of Myocytes Impaired During Heart Failure Are Restored by Cardiac Resynchronization Therapy

“…The sarcolemma was detected by thresholding of the WGA image stacks followed by median filtering. 24 The threshold was calculated from image statistics and set to mode+stddev. Image stacks were segmented in outer sarcolemmal, t-system, intra- and extracellular regions by morphological operators.…”

The Current and Future Landscape of SERCA Gene Therapy for Heart Failure: A Clinical Perspective

“…For example, there are interspecies differences in t-tubule structure, [43][44][45] the density of calcium-transporting proteins, 44,46,47 and the relative importance of NCX and SERCA2a for the removal of Ca 2 + in diastole.…”

Modeling Effects of L-Type Ca2+ Current and Na+-Ca2+ Exchanger on Ca2+ Trigger Flux in Rabbit Myocytes with Realistic T-Tubule Geometries

“…The model geometry was derived from the published structural data in rabbit ventricular myocytes (Sachse et al, 2008, 2009; Savio-Galimberti et al, 2008). The model contains one repeating unit inside the cell that includes: realistic surface sarcolemma; realistic t-tubule and its surrounding half-sarcomeres (Figures 1A,B).…”

“…Measurements of Ca 2+ sparks in rabbits and other species imply that a cluster of LCCs is likely involved in gating a cluster of RyRs (Lipp and Niggli, 1998; Bridge et al, 1999; Scriven et al, 2000, 2010; Takagishi et al, 2000; Inoue and Bridge, 2003; Altamirano and Bers, 2007; Dan et al, 2007; Poláková et al, 2008; Sobie and Ramay, 2009; Louch et al, 2010). Furthermore, 3D visualizations of fragments from rabbit ventricular myocytes have revealed that the majority of RyR clusters are adjacent to the t-system and that these clusters are irregularly distributed along t-tubules (Dan et al, 2007; Sachse et al, 2009). Thus here three patches (∼200 nm in diameter with the same LCC current density) were placed randomly along the t-tubule (Figure 1A).…”

“…Scanning confocal microscopy has yielded sub-micrometer resolution details of 3D structure of the rabbit t-system (Sachse et al, 2008, 2009) including wider t-tubule cross-sections compared to rodents, and structural variations such as constrictions and flattening (Savio-Galimberti et al, 2008). These large structural differences of the t-system, as well as variations in the density and expression of proteins, suggest that the local and global Ca 2+ dynamics during myocyte contraction and relaxation may be species-dependent (Bers, 2001).…”