Couplons in rat atria form distinct subgroups defined by their molecular partners

Abstract

Standard local control theory, which describes Ca2+ release during excitation–contraction coupling (ECC), assumes that all ryanodine receptor 2 (RyR2) complexes are equivalent. Findings from our laboratory have called this assumption into question. Specifically, we have shown that the RyR2 complexes in ventricular myocytes are different, depending on their location within the cell. This has led us to hypothesize that similar differences occur within the rat atrial cell. To test this hypothesis, we have triple-labelled enzymatically isolated fixed myocytes to examine the distribution and colocalization of RyR2, calsequestrin (Casq), voltage-gated Ca2+ channels (Cav1.2), the sodium–calcium exchanger (Ncx) and caveolin-3 (Cav3). A number of different surface RyR2 populations were identified, and one of these groups, in which RyR2, Cav1.2 and Ncx colocalized, might provide the structural basis for ‘eager’ sites of Ca2+ release in atria. A small percentage of the dyads containing RyR2 and Cav1.2 were colocalized with Cav3, and therefore could be influenced by the signalling molecules it anchors. The majority of the RyR2 clusters were tightly linked to Cav1.2, and, whereas some were coupled to both Ca 1.2 and Ncx, none were with Ncx alone. This suggests that Cav1.2-mediated Ca2+ -induced Ca2+ release is the primary method of ECC. The two molecules studied that were found in the interior of atrial cells, RyR2 and Casq, showed significantly less colocalization and a reduced nearest-neighbour distance in the interior, compared with the surface of the cell. These differences might result in a higher excitability for RyR2 in the interior of the cells, facilitating the spread of excitation from the periphery to the centre. We also present morphometric data for all of the molecules studied, as well as for those colocalizations found to be significant.

Single molecule localization deep within thick cells; a novel super‐resolution microscope

“…The isolation of the cardiomyocytes and the labeling of the ryanodine receptor have been described in detail elsewhere ; a full description of the method is included in the supplementary information. Imaging was performed in a standard GLOX‐thiol solution (a nitrogenated TN buffer [50 mM Tris, 10 mM NaCl, pH 8.0], 0.5 mg/ml glucose oxidase, 40 µg/ml catalase, 10% (w/v) glucose and 10mM MEA).…”

Are SR Ca content fluctuations or SR refractoriness the key to atrial cardiac alternans?: insights from a human atrial model

“…Recently, the idea that the RyR2 complexes are homogeneous along the cell has been put into question. The functionality of the RyR2 has been found to depend on the location within the cell both in atrial and in ventricular myocyes (35). In particular, although we have considered the same dynamics for the RyR2 in the whole cell, the properties of the RyR2 could differ between those in junctional and nonjunctional spaces.…”

Calcium Signaling in Cardiac Myocytes

“…1 and 2) (Chen-Izu et al 2006;Schulson et al 2011). Because these nonjunctional RyRs are not located within a dyadic junction, they are not activated by I Ca (Mackenzie et al 2001).…”