Calcium cycling protein density and functional importance to automaticity of isolated sinoatrial nodal cells are independent of cell size

Spontaneous, localized, rhythmic ryanodine receptor (RyRs) Ca2+ releases occur beneath the cell membrane during late diastolic depolarization in cardiac sinoatrial nodal cells (SANCs). These activate the Na+/Ca2+ exchanger (NCX1) to generate inward current and membrane excitation that drives normal spontaneous beating. The morphological background for the proposed functional of RyR and NCX crosstalk, however, has not been demonstrated. Here we show that the average isolated SANC whole cell labeling density of RyRs and SERCA2 is similar to atrial and ventricle myocytes, and is similar among SANCs of all sizes. Labeling of NCX1 is also similar among SANCs of all sizes and exceeds that in atrial and ventricle myocytes. Submembrane colocalization of NCX1 and cardiac RyR (cRyR) in all SANCs exceeds that in the other cell types. Further, the Cx43 negative primary pacemaker area of the intact rabbit sinoatrial node (SAN) exhibits robust positive labeling for cRyR, NCX1, and SERCA2. Functional studies in isolated SANCs show that neither the average action potential (AP) characteristics, nor those of intracellular Ca2+ releases, nor the spontaneous cycle length vary with cell size. Chelation of intracellular [Ca2+], or disabling RyRs or NCX1, markedly attenuates or abolishes spontaneous SANC beating in all SANCs. Thus, there is dense labeling of SERCA2, RyRs, and NCX1 in small-sized SANCs, thought to reside within the SAN center, the site of impulse initiation. Because normal automaticity of these cells requires intact Ca2+ cycling, interactions of SERCA, RyR2 and NCX molecules are implicated in the initiation of the SAN impulse.