Diversity of Ca2+ signaling in developing cardiac cells

During embryonic and postnatal development, the mammalian heart undergoes rapid morphological changes with cellular differentiation that at the ultrastructural level encompasses altered expression and organization of the proteins and organelles associated with Ca2+ signaling. Here the development and roles of the releasable Ca2+ stores located within the sarco/endoplasmic reticulum and possibly within the nuclear envelopes are addressed. Confocal Ca2+ imaging experiments were carried out on (i) neonatal rat cardiomyocytes, (ii) pluripotent P19 stem cells, differentiated to a cardiac phenotype by culturing with 1% dimethylsulfoxide (DMSO) in hanging droplets, and (iii) mouse embryonic cardiomyocytes isolated for short-time culture at embryonic day 9-18. The Ca2+ release channels in neonatal and "cardiac" P19 cell were activated versus inhibited by targeting ryanodine (Ry) receptors with caffeine versus Ry and IP3 receptors with adenosine 5'-triphosphate (ATP) or histamine versus U-73122, a phospholipase c (PLC) inhibitor. The neonatal cells displayed four recognizable phenotypes, of which two had specialized Ca2+ stores releasable via either Ry or IP3 receptors, and two had both types of receptors, either controlling functionally separate stores or with some degree of overlap, so that caffeine could deplete the stores releasable by ATP. The P19 cells showed variable presence of IP3-mediated Ca2+ stores, and caffeine releasable stores that gained prominence in the "cardiac" phenotype, but were absent in a "neuronal" phenotype. The different roles of Ca2+ stores were seen clearly in the mouse embryonic cells. Some cells from early stages of development (E 9-10) had Ca2+ waves that increased in intensity during the diastolic interval and could trigger synchronous electrical excitation (via Na-Ca exchanger [NCX] and excitatory Ca2+ and Na+ channels). At later stages of development (E 18) we observed diastolic Ca2+ sparks that appeared to originate from the nuclear envelope, while the Ca2+ signals during excitation were faster and stronger in the nuclear region than in the surrounding cytoplasmic regions. However, we also found cells where the nuclear Ca2+ signals were weaker and showed afterglow compared to the cytosolic Ca2+ transients. We conclude that the Ca2+ stores in cardiac cells during embryogenesis and postnatal development, that is, before the maturation of the t-tubular system and in stem cells with cardiac phenotype, show considerable diversity with respect to the pharmacology of the release channels and that regional differences in Ca2+ signaling are observed centered in, at, and around the nucleus. We suggest that the causal relationship excitation and subcellular Ca2+ signals in developing cardiac cells is different from that of adult cells and that the developing cardiomyocytes show a diversity that in later stages of development may be reflected in the different properties of atrial, ventricular, and pacemaker cells.