Rapamycin efficiently promotes cardiac differentiation of mouse embryonic stem cells

Abstract

To investigate the effects of rapamycin on cardiac differentiation, murine embryonic stem cells (ESCs) were induced into cardiomyocytes by 10−4 M ascorbic acid (AA), 20 nM rapamycin alone or 0.01% solvent DMSO. We found that rapamycin alone was insufficient to initiate cardiomyogenesis. Then, the ESCs were treated with AA and rapamycin (20 nM) or AA and DMSO (0.01%) as a control. Compared with control, mouse ESCs (mESCs) treated with rapamycin (20 nM) and AA yielded a significantly higher percentage of cardiomyocytes, as confirmed by the percentage of beating embryonic bodies (EBs), the immunofluorescence and FACS analysis. Rapamycin significantly increased the expression of a panel of cardiac markers including Gata4, α-Mhc, β-Mhc, and Tnnt2. Additionally, rapamycin enhanced the expression of mesodermal and cardiac transcription factors such as Mesp1, Brachyury T, Eomes, Isl1, Gata4, Nkx2.5, Tbx5, and Mef2c. Mechanistic studies showed that rapamycin inhibits Wnt/β-catenin and Notch signaling but promotes the expression of fibroblast growth factor (Fgf8), Fgf10, and Nodal at early stage, and bone morphogenetic protein 2 (Bmp 2) at later stages. Sequential treatment of rapamycin showed that rapamycin promotes cardiac differentiation at the early and later stages. Interestingly, another mammalian target of rapamycin (mTOR) inhibitor Ku0063794 (1 µM) had similar effects on cardiomyogenesis. In conclusion, our results highlight a practical approach to generate cardiomyocytes from mESCs by rapamycin.

Pharmacological inhibition of mTOR attenuates replicative cell senescence and improves cellular function via regulating the STAT3-PIM1 axis in human cardiac progenitor cells

“…The mTOR signaling pathway efficiently regulates tissue homeostasis, including the heart. Rapamycin is an mTOR inhibitor and an autophagy inducer 33 . First, several studies reported that inhibition of mTORC1 by knockdown of RAPTOR promotes cardiac differentiation 59 ; however, inhibition of mTORC2 via knockdown of its component RICTOR suppresses cardiac differentiation 60 .…”

“…Prolonged rapamycin treatment inhibits the assembly of mTORC2 31 . Rapamycin can continuously promote cardiomyogenesis throughout the process of cardiac induction 33 . Second, the present literature on the autophagy signaling pathway indicates that it plays a crucial role in cell metabolism, proliferation, differentiation, and replicative senescence of (see figure on previous page) Fig.…”

“…Several studies have reported that rapamycin efficiently promotes cardiac cell generation from the differentiation of mouse embryonic stem cells 33,34 . These observations indicate that chronic mTOR activity is important for the differentiation function of embryonic stem cells; however, the role of chronic mTOR activity in senescent hCPCs remains unclear.…”

“…The other ATPcompetitive inhibitors of mTOR, namely, PP242, have recently been demonstrated to have more potent antileukemic activity than rapamycin 32 . In addition, rapamycin can efficiently promote cardiac cell generation from the differentiation of mouse embryonic stem cells 33,34 . These observations indicate that chronic mTOR activity is important for the differentiation of embryonic stem cells into cardiac cells; however, the role of chronic mTOR activity in hCPC regulation remains unclear.…”

Inhibition of miR‑101‑3p protects against sepsis‑induced myocardial injury by inhibiting MAPK and NF‑κB pathway activation via the upregulation of DUSP1

“…The β-MHC protein content reflects the differentiation of MSCs into cardiomyocytes to a certain extent, and its gene expression level is one of the important indicators for observing the differentiation of MSCs into mature cardiomyocytes ( 36 ). A previous study found that the upregulation of α-MHC and β-MHC promoted the cardiac differentiation of mouse embryonic stem cells ( 37 ). Furthermore, in a model of LPS-induced sepsis, α-MHC and β-MHC were shown to both be significantly downregulated ( 38 ), which was also identified in the present study.…”