We determined the role of apelin-13 (apelin) on cardiac differentiation of mESCs and hESCs and assessed its synergistic effects at both cellular and molecular levels. The optimal dosage of apelin on cardiac differentiation of mESCs and hESCs was examined initially. Then a cardiac differentiation protocol including apelin and mesodermal differentiation factors (BMP-4, activin-A, and bFGF) was established by administering them in a developmentally specific temporal sequence. This study found that apelin promoted cardiac differentiation of both mouse and human ESCs. Furthermore, this novel peptide exerted synergistic effects on the differentiation efficiency when treated with mesodermal differentiation factors and generated a rich homogeneous pool of cardiac cells from the ESCs.
The effects of apelin on the ESCs were initially evaluated through the ability to produce more contractile EBs. The contractile mEBs appeared following 6 days of exposure to apelin, which was significantly earlier than the control group containing 20% FBS. Similarly, an increased percentage of contractile hEBs by apelin was also found. These findings could be attributed to the apelin-induced migration and aggregation of ESCs committed to cardiac lineage, allowing homotypic cellular interactions of mesodermally fated EBs to enhance cardiac differentiation. The 2 populations of cardiac progenitor cells in the development of primary and secondary heart fields are known to migrate and interact to develop into the heart and great vessels 
. Myocardial regulatory genes that are activated in both lineages include Gata4-6 (zinc-finger-containing transcription factors of the GATA family), Nkx2.5 (development of first and secondary heart fields), and Isl1 (key transcriptional regulators in the secondary heart field) 
. This study demonstrated up-regulation of GATA4, Nkx2.5, and Isl1, indicating enhancement of early cardiac differentiation transcriptional activity. Apelin enhanced the expression of Isl1 on day 3 and both GATA4 and NKX2.5 after day 5 of culture in the mESCs. These findings support the role of apelin in the migration of cardiac progenitor cells into the development of both heart fields. This effect of apelin was confirmed by the higher percentage of cTNT+
, connexin-43, and α-sarcomeric actin in the apelin-treated EBs, indicating increased homogeneity of the cardiac lineage committed cells.
The study then sought to determine the synergistic effects of apelin in enhancing the cardiac differentiation efficiency of 2 protocols employing mesodermal differentiation factors 
. Compared to apelin or the two protocols alone, the administration of apelin, BMP-4, activin-A and bFGF significantly enhanced the cardiac differentiation efficiency. Higher percentage of contractile EBs, up-regulation of early and mature cardiac markers, and increased percentage of cTNT+
cells confirmed the synergistic effect of apelin in promoting cardiac differentiation. The presence of apelin appears to have promoted the recruitment and aggregation of mesodermally committed progenitor cells 
Although induced contractility was not found, a previously published study reported another mechanism involving the up-regulation of myosin light chain 2 L gene expression by apelin 
. The report suggested that the apelin/APJ signaling was possibly through mitogen-activated protein kinase/p70S6 pathway downstream of Cripto 
. In other studies, it was demonstrated that apelin did not modulate L-type Ca2+
or voltage-activated K+
currents in isolated adult rat ventricular myocytes and did not change the intracellular baseline calcium level 
. These data are consistent with this study's findings regarding the Ca2+
transient data in which the exposure of apelin on hESCs significantly increased the peak contractile force while the Ca2+
transient remained unchanged. Other studies found that apelin-13 was the predominant isoform in cardiac tissue and the most potent endogenous inotropic agents tested 
. Addition of apelin was shown to increase the sarcomere shortening by 1.4 fold in single mature cardiomyocytes resulting in increased contractility of a whole rat heart and the atrial strips 
. These results support our finding that apelin not only enhanced the differentiation and maturation of ESC-derived cardiac cells, but also enhanced their contractile function. Although the mechanism of inotropic actions of apelin remains unclear, it has been found to be independent of angiotensin II, endothelin-1, catecholamines and nitric oxide release 
. Some studies suggested that it might be related to the increased availability of activator Ca2+
and/or increased Ca2+
. Although some studies reported a marginal increase in calcium transient in mature cardiomyocytes with the addition of apelin in vitro, many studies have found no effect of apelin on Ca2+
. Finally, It was reported that the effect of apelin on rat myocyte contractility was modulated through the sacrolemmal sodium-hydrogen ion exchanger which led to sensitization of myofilaments to Ca2+
. These findings are consistent with our data that the Ca2+
transient did not change between the apelin-treated and non-treated groups.
In conclusion, this study demonstrated that apelin enhanced cardiac differentiation when combined with the mesodermal differentiation factors, BMP-4, activin-A and bFGF. The underlying mechanism could be attributed to the recruitment and enhanced aggregation of the mesodermal progenitor cells. The Combined Protocols A and B systematically generated a more homogeneous population of cardiac lineage committed cells. Isolation of the cells at an earlier time point may possibly yield a pool of cardiac lineage committed progenitor cells. Future studies will establish an effective cell sorting strategy to select the cardiac progenitor cells.