The Molecular Architecture of Calcium Microdomains in Rat Cardiomyocytes

We have used standard indirect immunofluorescence techniques in combination with wide-field microscopy and image deconvolution to assess the distribution of proteins implicated in excitation-contraction coupling and Ca(2+) homeostasis in adult rat cardiomyocytes. We begin by discussing our earlier results and summarizing what is known about the molecular architecture of this species to provide a rationale for the work presented here. The previous results showed that the dyads contain Ca(2+) channels and ryanodine receptors, but few Na(+) channels or Na(+)/Ca(2+) exchangers. The latter proteins were not colocalized elsewhere on the membrane, and we have now found that they appear to be minimally associated with caveolin-3. None of the molecules examined are distributed uniformly in the membranes in which they are located but are organized into discrete clusters attached to the underlying cytoskeleton, an arrangement that, at the level of light microscopy, does not appear to be affected by the enzymatic dissociation used to study single cells. Analysis of how the clusters are organized and distributed throughout the volume of the cell suggests that there may be differences in excitation-contraction coupling between the cell surface and the interior.