In order to develop a novel method of visualizing possible Ca2＋ signaling during the early differentiation of hESCs into cardi- omyocytes and avoid some of the inherent problems associated with using fluorescent repo...In order to develop a novel method of visualizing possible Ca2＋ signaling during the early differentiation of hESCs into cardi- omyocytes and avoid some of the inherent problems associated with using fluorescent reporters, we expressed the biolumines- cent Ca2＋ reporter, apo-aequorin, in HES2 cells and then reconstituted active holo-aequorin by incubation withf-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 re- lease from intracellular stores） were detected in 1-12-day-old cardiospheres but transients were generated in cardiospheres following stimulation with KC1 or CaC12, 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 minima/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.展开更多
基金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)+2 种基金the ANR/RGC joint research scheme award(A-HKUST601/13)the Innovation and Technology Commission(ITCPD/17-9)supported by a Hong Kong University Grants Council post-graduate studentship(T13-706/11-11PG)
文摘In order to develop a novel method of visualizing possible Ca2＋ signaling during the early differentiation of hESCs into cardi- omyocytes and avoid some of the inherent problems associated with using fluorescent reporters, we expressed the biolumines- cent Ca2＋ reporter, apo-aequorin, in HES2 cells and then reconstituted active holo-aequorin by incubation withf-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 re- lease from intracellular stores） were detected in 1-12-day-old cardiospheres but transients were generated in cardiospheres following stimulation with KC1 or CaC12, 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 minima/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.
