title: Expression and reconstitution of the bioluminescent Ca(2+) reporter aequorin in human embryonic stem cells, and exploration of the presence of functional IP3 and ryanodine receptors during the early stages of their differentiation into cardiomyocytes.
abstract: In order to develop a novel method of visualizing possible Ca(2+) 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 Ca(2+) reporter, apo-aequorin, in HES2 cells and then reconstituted active holo-aequorin by incubation with f-coelenterazine. The temporal nature of the Ca(2+) 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 Ca(2+) 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 Ca(2+) transients were generated from day 1 onward. That ATP was inducing Ca(2+) 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 Ca(2+) 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.
authors.affiliation: Division of Life Science & State Key Laboratory of Molecular Neuroscience, HKUST, Clear Water Bay, Hong Kong, China.
authors.forename: Harvey Y S
authors.initials: HY
authors.lastname: Chan
authors.affiliation: Stem Cell & Regenerative Medicine Consortium, Li Ka Shing Faculty of Medicine, The University of Hong Kong, Pokfulam, Hong Kong, China.
authors.forename: Man Chun
authors.initials: MC
authors.lastname: Cheung
authors.affiliation: Stem Cell & Regenerative Medicine Consortium, Li Ka Shing Faculty of Medicine, The University of Hong Kong, Pokfulam, Hong Kong, China.
authors.forename: Yi
authors.initials: Y
authors.lastname: Gao
authors.affiliation: Division of Life Science & State Key Laboratory of Molecular Neuroscience, HKUST, Clear Water Bay, Hong Kong, China.|Marine Biological Laboratory, Woods Hole, MA, 02543, USA.
authors.forename: Andrew L
authors.initials: AL
authors.lastname: Miller
authors.affiliation: Division of Life Science & State Key Laboratory of Molecular Neuroscience, HKUST, Clear Water Bay, Hong Kong, China. barnie@ust.hk.
authors.forename: Sarah E
authors.initials: SE
authors.lastname: Webb
mesh_terms: D000255:Adenosine Triphosphate / Q000494:pharmacology; D000331:Aequorin / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D015153:Blotting, Western; D002110:Caffeine / Q000494:pharmacology; D002118:Calcium / Q000378:metabolism; D002454:Cell Differentiation; D002460:Cell Line; D005456:Fluorescent Dyes / Q000737:chemistry; D057809:HEK293 Cells; D000066449:Human Embryonic Stem Cells / Q000378:metabolism*; D006801:Humans; D007093:Imidazoles / Q000737:chemistry; D053496:Inositol 1,4,5-Trisphosphate Receptors / Q000819:agonists / Q000378:metabolism*; D008163:Luminescent Measurements; D032383:Myocytes, Cardiac / Q000187:drug effects / Q000378:metabolism*; D011189:Potassium Chloride / Q000494:pharmacology; D011719:Pyrazines / Q000737:chemistry; D019837:Ryanodine Receptor Calcium Release Channel / Q000378:metabolism*; D013997:Time Factors
chemical_list: D005456:Fluorescent Dyes; D007093:Imidazoles; D053496:Inositol 1,4,5-Trisphosphate Receptors; D011719:Pyrazines; D019837:Ryanodine Receptor Calcium Release Channel; D002110:Caffeine; C017144:coelenterazine; D000331:Aequorin; D011189:Potassium Chloride; D000255:Adenosine Triphosphate; D002118:Calcium
keywords: Ca2+ signaling; HES2 human embryonic stem cells; IP3 and ryanodine receptors; apo-aequorin expression; bioluminescence; hESC-derived cardiospheres