Ultrastructural Quantification of Electron-Dense Strings in the Sarcoplasmic Reticulum of Rat Heart Cells

Ca2+ release from the ryanodine receptors (RyRs) in the sarcoplasmic reticulum (SR), in the form of Ca2+ sparks, is the fundamental events underlying excitation-contraction. Recent structural studies of calsequestrin has characterized the molecular size, assembly and Ca2+ binding capacity of this Ca2+ binding protein in the SR. In order to link the molecular properties of calsequestrin to the in situ behavior of RyR Ca2+ sparks, we studied the ultrastructural organization of junctional SR contents in rat heart cells using transmission electron microscopy. We found that the junctional SR lumen is usually ∼20 nm thick, with the assembled calsequestrin chains visualized as electron-dense strings (EDS) residing at the midline of the SR lumen. Between the EDS and the SR membrane of each side, there was a symmetric electron-transparent space of 5∼7 nm. Quantitative comparison between the EDS and the molecular geometry of crystallized calsequestrin suggested that cardiac SR lumen contained only a single layer of calsequestrin network. Morphometric analysis predicted that the number of calsequestrin molecules per 1000 nm2 dyadic area (roughly the luminal area beneath a single RyR) is no more than 30∼50. By comparing spark rising phase to standard Ca2+ sparklets of single L-type Ca2+ channels, we determined that a typical Ca2+ spark represents a 2∼4 pA Ca2+ release flux lasting for ∼10 ms. Based on above structural quantification, this flux would be supplied by 2000∼6000 calsequestrin molecules within an 0.04∼0.2 μm2 dyadic area, agreeing well with the size of a single dyad (or the size of 40∼200 inter-linked RyRs). Our results indicated that the calsequestrin was dyad-widely well organized for firing a single Ca2+ spark. The number and organization of calsequestrin molecules should be important factors in shaping Ca2+ sparks in healthy and disease conditions.