Minimal contribution of IP3R2 in cardiac differentiation and derived ventricular-like myocytes from human embryonic stem cells.

Type 2 inositol 1,4,5-trisphosphate receptor (IP3R2) regulates the intracellular Ca2+ release from endoplasmic reticulum in human embryonic stem cells (hESCs), cardiovascular progenitor cells (CVPCs), and mammalian cardiomyocytes. However, the role of IP3R2 in human cardiac development is unknown and its function in mammalian cardiomyocytes is controversial. hESC-derived cardiomyocytes have unique merits in disease modeling, cell therapy, and drug screening. Therefore, understanding the role of IP3R2 in the generation and function of human cardiomyocytes would be valuable for the application of hESC-derived cardiomyocytes. In the current study, we investigated the role of IP3R2 in the differentiation of hESCs to cardiomyocytes and in the hESC-derived cardiomyocytes. By using IP3R2 knockout (IP3R2KO) hESCs, we showed that IP3R2KO did not affect the self-renewal of hESCs as well as the differentiation ability of hESCs into CVPCs and cardiomyocytes. Furthermore, we demonstrated the ventricular-like myocyte characteristics of hESC-derived cardiomyocytes. Under the α1-adrenergic stimulation by phenylephrine (10 µmol/L), the amplitude and maximum rate of depolarization of action potential (AP) were slightly affected in the IP3R2KO hESC-derived cardiomyocytes at differentiation day 90, whereas the other parameters of APs and the Ca2+ transients did not show significant changes compared with these in the wide-type ones. These results demonstrate that IP3R2 has minimal contribution to the differentiation and function of human cardiomyocytes derived from hESCs, thus provide the new knowledge to the function of IP3R2 in the generation of human cardiac lineage cells and in the early cardiomyocytes.

Functional expression of the Ca2+ signaling machinery in human embryonic stem cells.

Emerging evidence suggests that Ca2+ signals are important for the self-renewal and differentiation of human embryonic stem cells (hESCs). However, little is known about the physiological and pharmacological properties of the Ca2+-handling machinery in hESCs. In this study we used RT-PCR and Western blotting to analyze the expression profiles of genes encoding Ca2+-handling proteins; we also used confocal Ca2+ imaging and pharmacological approaches to determine the contribution of the Ca2+-handling machinery to the regulation of Ca2+ signaling in hESCs. We revealed that hESCs expressed pluripotent markers and various Ca2+-handling-related genes. ATP-induced Ca2+ transients in almost all hESCs were inhibited by the inositol-1,4,5-triphosphate receptor (IP3R) blocker 2-APB or xestospongin C. In addition, Ca2+ transients were induced by a ryanodine receptor (RyR) activator, caffeine, in 10%-15% of hESCs and were blocked by ryanodine, whereas caffeine and ATP did not have additive effects. Moreover, store-operated Ca2+ entry (SOCE) but not voltage-operated Ca2+ channel-mediated Ca2+ entry was observed. Inhibition of sarco/endoplasmic reticulum (ER) Ca2+-ATPase (SERCA) by thapsigargin induced a significant increase in the cytosolic free Ca2+ concentration ([Ca2+]i). For the Ca2+ extrusion pathway, inhibition of plasma membrane Ca2+ pumps (PMCAs) by carboxyeosin induced a slow increase in [Ca2+]i, whereas the Na+/Ca2+ exchanger (NCX) inhibitor KBR7943 induced a rapid increase in [Ca2+]i. Taken together, increased [Ca2+]i is mainly mediated by Ca2+ release from intracellular stores via IP3Rs. In addition, RyRs function in a portion of hESCs, thus indicating heterogeneity of the Ca2+-signaling machinery in hESCs; maintenance of low [Ca2+]i is mediated by uptake of cytosolic Ca2+ into the ER via SERCA and extrusion of Ca2+ out of cells via NCX and PMCA in hESCs.