Abstract 1535: Calsequestrin Contributes to the Emergence of Ryanodine Receptor Mediated Calcium Signaling in Developing Cardiac Myocytes
Intracellular Ca stores play an essential role in contractility of cardiac cells from very early stages of development. Whereas early cardiac contractility is governed by inositol 1,4,5-trisphosphate receptor (IP3R)-operated Ca signaling, contraction of adult myocytes relies almost entirely on Ca release from ryanodine receptor (RyR2) channels. The developmental transition between the two signaling systems in cardiac myocytes is not sufficiently defined. In the present study, we investigated the properties and role of IPR3 and RyR2 Ca stores in cardiomyocytes at early stages of differentiation and their dependency on the major intra-SR Ca binding protein calsequestrin (CASQ2). Myocytes were differentiated in vitro from embryonic stem cells (ESC, murine R1 cell line) and changes of cytosolic [Ca] studied by confocal imaging at 24 hours after beginning of spontaneous contractions. Myocytes exhibited spontaneous cell-wide Ca spikes and focal Ca release signals, i.e. puffs that were inhibited by PLC inhibitors, consistent with the role of IP3R stores in generation of these signals. Application of caffeine induced small transient increases in cytosolic Ca; however, caffeine had no effect on the properties of Ca spikes and puffs. Overexpression of CASQ2 resulted in a marked increase of caffeine-induced Ca transients. Moreover, CASQ2-expressing cells exhibited Ca sparks with spatiotemporal properties similar to those in adult myocytes. CASQ2 overexpression had no effect on the amplitude of Ca spikes but led to a significant acceleration of their rising face. The properties of Ca puffs were not altered by CASQ2. We conclude that components of RyR2 signaling appear at very early stages of differentiation, and, disconnected from IP3R-controlled Ca stores, remain functionally silent due to lack of a sufficient Ca reserve. CASQ2 plays a key role in forming functional RyR2 Ca stores in early cardiac development.