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

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 CaCl 2 , indicating that holo-f-aequorin was functional in these cells. Furthermore, following the addition of exogenous ATP, an inositol trisphosphate receptor (IP 3 R) agonist, small Ca 2+ transients were generated from day 1 onward. That ATP was inducing Ca 2+ release from functional IP 3 Rs was demonstrated by treatment with 2-APB, a known IP 3 R 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, IP 3 Rs are present and continually functional at these early stages of cardiomyocyte differentiation.

L‐type voltage‐gated calcium channels in stem cells and tissue engineering

“…Calcium channels, and particularly the L-VGCCs, regulate the early differentiation of ESCs into cardiomyocytes. Chan et al cultured mouse embryos with nifedipine or verapamil, which caused their hearts to develop an enlarged left ventricle and a long, thin outflow tract 91. In fully differentiated adult cardiomyocytes, Ca v 1.2 is believed to be the predominant L-VGCCs expressed in ventricular myocytes, whereas both Ca v 1.2 and Ca v 1.3 are expressed in atrial cells as well as sinoatrial and atrioventricular node cells 92.…”