The expression of calcium-sensing receptor in mouse embryonic stem cells (mESCs) and its influence on differentiation of mESC into cardiomyocytes

Abstract

The calcium-sensing receptor (CaSR), a G protein coupled receptor, is involved in a number of physiological and pathological processes. Embryonic stem cells (ESCs) have a potential role to differentiate into all types of cells. Whether CaSR is functionally expressed in ESCs is unclear. In this study, the expression and distribution of CaSR in 129 mouse ES-D3 cell lines were detected by Western blotting and immunofluorescence; and the intracellular calcium concentration ([Ca2+]i) was measured using Laser Confocal Scanning Microscopy. Mouse embryonic stem cells (mESCs) were cultured to embryoid bodies (EBs) and the differentiation of EBs into cardiomyocytes was induced by icariin (ICA). The cardiac specific proteins, a-Actinin and cardiac troponin-I (cTnI), were analyzed by immunofluorescence, and the differentiation rate was analyzed by flow cytometry. The expression of cardiac-specific transcription factors, Nkx2.5 and GATA-4, was detected by Western blotting.We found that the CaSR protein exists in both mESCs and mESC-derived cardiomyocytes (mESC-CMs). Increasing extracellular calcium or neomycin (an agonist of CaSR) increased [Ca2+]i and the differentiation rate. These effects were abolished by inhibition of CaSR, phospholipase C, IP3 receptor and Ca2+ ATPase, or by depletion of the sarcoplasmic reticulum Ca2+ store, respectively. Activation of CaSR up-regulated protein expression of Nkx2.5 and GATA4 in EBs at an early stage of ICA-induced differentiation.In conclusion, CaSR is functionally expressed in mESCs, and activation of CaSR is involved in the differentiation of mESCs into cardiomyocytes by facilitating the expression of NKx2.5 and GATA-4.