Ghrelin induces cardiac lineage differentiation of human embryonic stem cells through ERK1/2 pathway

Background: Ghrelin, an endogenous ligand for growth hormone secretagogue receptor (GHS-R), shows cardioprotective activity and regulates the differentiation of several mesoderm-derived cells, including myocytes, adipocytes and osteoblasts. The effect of ghrelin on cardiogenesis and its underlying mechanism, however, have not been studied in detail. Methods: The effects of ghrelin on cardiomyocyte differentiation were tested both in human embryonic stem cells (hESCs) cultured in embryoid body (EB)-based differentiation protocol, and in hESCs transplanted into rat hearts. The signaling mechanisms of ghrelin were further investigated under the EB-based culture condition. Results: The generation of beating EBs and the expression of cardiac-specific markers including cardiac troponin I (cTnI) and alpha-myosin heavy chain (alpha-MHC) were 2 to 3-fold upregulated by ghrelin. Although GHS-R1 alpha protein was expressed in differentiated EBs, the effects of exogenous ghrelin were unchanged by D-[lys(3)]-GHRP-6, a specific GHS-R1 alpha antagonist. Moreover, des-acyl ghrelin, which does not bind to GHS-R1 alpha, displayed similar effects with ghrelin. Importantly, activation of ERK1/2, but not Akt, was induced by ghrelin in the newly-formed EBs, and the ghrelin-induced effects of cardiomyocyte differentiation were abolished by adding specific ERK1/2 inhibitor PD98059, but not specific PI3K inhibitor Wortmannin. In addition, ghrelin promoted the differentiation of grafted hESCs into Sox9- and Flk1-positive mesodermal/cardiac progenitor cells in rat hearts. Conclusions: These results suggest that ghrelin induces cardiomyocyte differentiation from hESCs via the activation of the ERK1/2 signaling pathway. Our study, therefore, indicates that using ghrelin may be an effective strategy to promote the differentiation of hESCs into cardiomyocytes. (C) 2012 Elsevier Ireland Ltd. All rights reserved.