Calcium signaling in the cardiac differentiation of mouse embryonic stem cells

Abstract

 Intracellular Ca2+ mobilization via secondary messengers modulates multiple cell functions. Cyclic Adenosine 5’-Diphosphate-Ribose (cADPR) is one of the most well recognized endogenous Ca2+ mobilizing messengers. In mammalian, cADPR is mainly formed by CD38, a multi-functional enzyme, from nicotinamide adenine dinucleotide (NAD). It has previously been shown that the cADPR/CD38/Ca2+pathway mediates many cardiac functions, such as regulating the excitation-contraction coupling in cardiac myocytes and modulating the Ca2+ homeostasis during the ischemia injury of the heart. Thus it is reasonable to propose that the cADPR/CD38/Ca2+ pathway plays a role in cardiogenesis. The pluripotent mouse embryonic stem (mES) cells which can be induced to differentiate into all cell types provide an ideal model for studying cardiogenesis. The first part of this dissertation is to determine the role of CD38/cADPR/Ca2+pathwayin the cardiomyogenesis of mES cells. The data showed that CD38 expression was markedly up-regulated during the in vitro embryoid body (EB) differentiation of mouse ES cells, which indicated a regulatory role of CD38 in the differentiation process. Lentivirus mediated shRNA provides a convenient method to knockdown the expression of CD38 in mES cells. Surprisingly, beating clusters appeared earlier and more in CD38 knockdown EBs than that in control EBs. Likewise, the expressions of several cardiac markers were up regulated in CD38 knockdown EBs. In addition, more cardiomyocytes (CMs) existed in CD38 knockdown or 8-Br-cADPR, a cADPR antagonist, treated EBs than those in control EBs. On the other hand, over-expression of CD38 in mouse ES cells significantly inhibited CM differentiation. Moreover, we showed that CMs derived from the CD38 knock down mES cells possessed the functional properties characteristic of CMs derived fromnormal ES cells. Last, we showed that the CD38-cADPR pathway negatively modulated the FGF4-Erks1/2cascade during CM differentiation of mES cells, and transiently inhibition of Erk1/2 blocked the enhancive effects of CD38 knockdown on the differentiation of CM from mES cells. Taken together, our data indicate that the CD38/cADPR/Ca2+ signaling pathway suppresses the cardiac differentiation of mES cells. One of the main goals of the researches on cardiac differentiation of ES cells is to enhance the production of CMs from ES cells, thereby providing sufficient amount of functional intact CMs for the treatment of severe heart disease. Nitric oxide (NO) has been found to be a powerful cardiogenesis inducer of mES cells, in that it can significantly increase the yield of ES-derived CM. The second objective of this dissertation is to explore the mechanism underlying the NO facilitated cardiomyogenesis of mES cells. We found that the NO did induce intracellular Ca2+ increases in mES cells, and this Ca2+ increase was due to internal Ca2+ release from ER through theIP3 pathway. Therefore, the expression of IP3 receptors (IP3Rs) in mES cells were knocked down by lentivirus-mediated shRNAs. Interestingly, only type 3 IP3R (IP3R3) knockdown significantly inhibited the NO induced Ca2+ release in mES cells. Moreover, NO facilitated cardiogensis of mES cells was abolished in IP3R3 knockdown EBs. In summary, our results indicate that the IP3R3-Ca2+ pathway is required for NO facilitated cardiomyogenesis of mES cells.