Three-dimensional high-resolution imaging of cardiac proteins to construct models of intracellular Ca2+ signalling in rat ventricular myocytes

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 three-dimensional distribution of immunolabelled proteins with confocal microscopy. Labelling of single rat cardiac myocytes with an antibody to the Z-line marker alpha-actinin revealed a complex architecture of sarcomere misalignment across single cells. Double immunolabelling 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 with a second model in which all CRUs were placed on flat planes. The model with a realistic CRU distribution supported Ca2+ waves that spread axially along the cell at velocities of similar to 50 mu m s(-1). By contrast, in the model with planar CRU distribution the axial wave spread was slowed roughly twofold and wave propagation often nearly faltered. These results demonstrate that spatial features of the CRU distribution on multiple length scales may significantly affect intracellular Ca2+ dynamics and must be captured in detailed mechanistic models to achieve quantitative as well as qualitative insight.