|Authors||C. Soeller, I. Jayasinghe, P. Li, A. Holden and M. Cannell|
|Title||Three-Dimensional High-Resolution Imaging of Cardiac Proteins to Construct Models of Intracellular Ca2+ Signalling|
|Afilliation||Center for Biomedical Computing (SFF), Scientific Computing|
|Project(s)||Center for Biomedical Computing (SFF)|
|Publication Type||Journal Article|
|Year of Publication||2009|
Quantitative understanding of the Ca2+ handling in cardiac ventricular myocytes requires accurate knowledge of cardiac ultrastructure and protein distribution. We have therefore developed high-resolution imaging and analysis approaches to measure the threedimensional distribution of immuno-labelled proteins with confocal microscopy. Labelling of single rat cardiac myocytes with an antibody for the z-line marker \alpha-actinin revealed a complex architecture of sarcomere misalignment across single cells. Double immuno-labelling was used to relate the z-line structure to the distribution of ryanodine receptors (RyRs, the intracellular Ca2+ release channels), and the transverse tubular system. Both RyR and transverse tubular system distributions exhibited frequent dislocations from the simple planar geometry generally assumed in existing mathematical models. To investigate potential effects of these irregularities on Ca2+ dynamics we determined the three-dimensional distribution of RyR clusters within an extended section of a single rat ventricular myocyte to construct a model of stochastic Ca2+ dynamics with a measured Ca2+ release unit (CRU) distribution. Calculations with this model were compared to a second model in which all CRUs where placed on flat planes at a sarcomere spacing of 1.8 μm. The model with a realistic CRU distribution supported Ca2+ waves that spread axially along the cell at velocities of \~50 μm/s. By contrast, in the model with planar CRU distribution axial wave spread was slowed \~two-fold and wave propagation often nearly faltered. These results demonstrate that spatial features of the CRU distribution on multiple length scales between may significantly affect intracellular Ca2+ dynamics and must be captured in detailed mechanistic models to achieve quantitative as well as qualitative insight.