Expression and reconstitution of the bioluminescent Ca2+ 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
Chan, Harvey Y. S.
Cheung, Man Chun
Miller, Andrew L.
Webb, Sarah E.
MetadataShow full item record
KeywordCa2+ signaling; Apo-aequorin expression; Bioluminescence; HES2 human embryonic stem cells; hESC-derived cardiospheres; IP3 and ryanodine receptors
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.
© 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.
Suggested CitationScience China Life Sciences 59 (2016): 811-824
The following license files are associated with this item:
Showing items related by title, author, creator and subject.
Expression and function of ryanodine receptor related pathways in PCB tolerant Atlantic killifish (Fundulus heteroclitus) from New Bedford Harbor, MA, USA Fritsch, Erika B.; Stegeman, John J.; Goldstone, Jared V.; Nacci, Diane E.; Champlin, Denise; Jayaraman, Saro; Connon, Richard E.; Pessah, Isaac N. (2014-12)Atlantic killifish (Fundulus heteroclitus) thrive in New Bedford Harbor (NBH), MA, highly contaminated with polychlorinated biphenyls (PCBs). Resident killifish have evolved tolerance to dioxin-like (DL) PCBs, whose toxic ...
Ryanodine receptor and FK506 binding protein 1 in the Atlantic killifish (Fundulus heteroclitus) : a phylogenetic and population-based comparison Holland, Erika B.; Goldstone, Jared V.; Pessah, Isaac N.; Whitehead, Andrew; Reid, Noah M.; Karchner, Sibel I.; Hahn, Mark E.; Nacci, Diane E.; Clark, Bryan W.; Stegeman, John J. (2017-09)Non-dioxin-like polychlorinated biphenyls (NDL PCBs) activate ryanodine receptors (RyR), microsomal Ca2+ channels of broad significance. Teleost fish may be important models for NDL PCB neurotoxicity, and we used sequencing ...
Morphometric analysis of human embryonic stem cell-derived ventricular cardiomyocytes : determining the maturation state of a population by quantifying parameters in individual cells Chan, Harvey Y. S.; Keung, Wendy; Li, Ronald A.; Miller, Andrew L.; Webb, Sarah E. (Hindawi Publishing Corp, 2015-07)Quantitative methods were established to determine the level of maturation of human embryonic stem cell-derived ventricular cardiomyocytes (hESC-vCMs) that were treated with different metabolic stimulants (i.e., isoproterenol ...