© The Author(s), 2016. This article is distributed under the terms of the Creative Commons Attribution License. The definitive version was published in Science China Life Sciences 59 (2016): 811-824, doi:10.1007/s11427-016-5094-6.
In order to develop a novel method of visualizing possible Ca2+ signaling during the early differentiation of hESCs into cardiomyocytes and avoid some of the inherent problems associated with using fluorescent reporters, we expressed the bioluminescent Ca2+ reporter, apo-aequorin, in HES2 cells and then reconstituted active holo-aequorin by incubation with f-coelenterazine. The temporal nature of the Ca2+ signals generated by the holo-f-aequorin-expressing HES2 cells during the earliest stages of differentiation into cardiomyocytes was then investigated. Our data show that no endogenous Ca2+ transients (generated by release from intracellular stores) were detected in 1–12-day-old cardiospheres but transients were generated in cardiospheres following stimulation with KCl or CaCl2, indicating that holo-f-aequorin was functional in these cells. Furthermore, following the addition of exogenous ATP, an inositol trisphosphate receptor (IP3R) agonist, small Ca2+ transients were generated from day 1 onward. That ATP was inducing Ca2+ release from functional IP3Rs was demonstrated by treatment with 2-APB, a known IP3R antagonist. In contrast, following treatment with caffeine, a ryanodine receptor (RyR) agonist, a minimal Ca2+ response was observed at day 8 of differentiation only. Thus, our data indicate that unlike RyRs, IP3Rs are present and continually functional at these early stages of cardiomyocyte differentiation.
This work was
supported by the Hong Kong Theme-based Research Scheme award
(T13-706/11-1), the Hong Kong Research Grants Council (RGC) General
Research Fund awards (662113, 16101714, 16100115), the ANR/RGC
joint research scheme award (A-HKUST601/13), and the Innovation and
Technology Commission (ITCPD/17-9). HYSC was supported by a Hong
Kong University Grants Council post-graduate studentship (T13-706/11-
HES2 human embryonic stem cells
IP3 and ryanodine receptors
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