Publication Date:
2022-05-25
Description:
© 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.
Description:
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.
Description:
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-
11PG).
Keywords:
Ca2+ signaling
;
Apo-aequorin expression
;
Bioluminescence
;
HES2 human embryonic stem cells
;
hESC-derived cardiospheres
;
IP3 and ryanodine receptors
Repository Name:
Woods Hole Open Access Server
Type:
Article
