During spontaneous cardiac differentiation of mouse embryonic stem cells (mESCs, cell line E14), the effect of progesterone on calcium regulation was investigated. Calcium (Ca2+) release from sarcoplasmic reticulum (SR) regulates various cellular functions including the smooth or skeletal muscle contraction. The cardiac L-type Ca2+ channel plays a key role in excitation-contraction coupling of cardiomyocytes and contraction-related gene expression. The mESCs formed mouse embryonic bodies (mEBs) by hanging-drop for 4 days, and mEBs were suspended for 2 days in differentiation medium; DMEM/F:12, 15% charcoal-dextran-treated FBS, β-mercaptoethanol, minimal essential medium NEAA, and penicillin/streptomycin. Then, mEBs were attached onto 6-well culture plates and differentiated into cardiomyocytes. We analysed mRNA expressions for the cardiac lineage markers and calcium-regulating genes. Percentage of beating mEBs was time-dependently increased during differentiation. Differentiated mEBs showed the highest beating ratio (92.11 ± 2.98%) after attachment for 12 days. Beating ratio was decreased to 64.86 ± 4.25% in progesterone-treated mEBs. The mRNA levels of cardiac markers such as Tbx20, Isl1, Foxh1, cTn1, and RyR2 were increased, and troponin protein was observed in beating mEBs via immunocytochemistry. Expression of calcium/contraction regulating genes including Trpv2, Ryr2, CaM2, and Mlck3 was down-regulated by progesterone treatment. These results indicate that progesterone has influences on cardiac differentiation and contraction of cardiomyocytes through regulating intercellular calcium ion.
This research was supported by a grant (15182MFDS460) from the Ministry of Food and Drug Safety in 2015.
Ouabain facilitates cardiac differentiation of mouse embryonic stem cells through ERK1/2 pathway
