Puerarin Exerts a Delayed Inhibitory Effect on the Proliferation of Cardiomyocytes Derived from Murine ES Cells via Slowing Progression through G2/M Phase

Abstract

Objective: Puerarin, which shows beneficial and protective effects on cardiovascular diseases, is the main isoflavone extracted from Pueraria lobata (kudzu) root. The aim of this study was to investigate the effects of puerarin on in vitro myocardial proliferation and its underlying mechanism. Methods: Myocardial differentiation of transgenic embryonic stem (ES) cells was performed by embryoid body-based differentiation method. The proliferation assay of cardiomyocytes (CMs) derived from ES cells (ES-CMs) was performed by EdU (5-Ethynyl-2'-deoxyuridine) staining. Flow cytometry was employed to determine the cell cycle distribution and apoptosis of purified ES-CMs. Quantitative real-time PCR was utilized to study the transcription of genes related to cell cycle progression. Signaling pathways relating to proliferation were studied by western blot analysis and application of specific inhibitors. Results: Puerarin exerted a delayed inhibitory effect on the proliferation of ES-CMs at the early-stage differentiation. Meanwhile, puerarin slowed progression through G2/M phase without inducing apoptosis of ES-CMs. Further assays showed that puerarin up-regulated the transcription of Cyclin A2, Cyclin B1 and Cdk1 in ES-CMs. The ERK1/2 specific inhibitor PD0325901 and the PI3K specific inhibitor Wortmannin successfully reversed puerarin-induced up-regulation of Cdk1 but not Cyclin A2 and B1. Conclusion: These findings suggest that puerarin inhibits CM proliferation via slowing progression through G2/M phase during early-stage differentiation.

Puerarin Enhances Ca2+ Reuptake and Ca2+ Content of Sarcoplasmic Reticulum in Murine Embryonic Stem Cell-Derived Cardiomyocytes via Upregulation of SERCA2a

“…Researches demonstrated that signal pathways such as PI3K/Akt and MAPK/ERK were involved in biological activities of puerarin [2, 17, 20, 23]. Consistently, we found that the upregulation of SERCA2a, PLB and Trd induced by puerarin are abolished by inhibitors of both PI3K/Akt and MAPK/ERK signaling, suggesting their roles in puerarin-mediated functional maturation of mES-CMs through SERCA2a.…”

“…ES cell-derived CMs (ES-CMs) are widely accepted as a promising resource for cell replacement therapy of cardiac disease and a powerful model for investigating the development, maturation as well as pathological remodeling of heart [1-2]. However, accumulating data suggest that current culture system produces primitive ES-CMs with undeveloped structure and contractile function [1, 3-4].…”

“…Puerarin also shows acute inhibitory effects on the functional ion channels, such as Na + channels [12], inward rectified K + channels [21] and LTCC [22], in isolated CMs. Accumulated studies have shown that the PI3K/Akt, JAK/STAT, JNK, ERK1/2 and MAPK signaling pathways mediate the bio-effects of puerarin on various types of cells, i.e CMs [2, 17, 20], astrocytes [23], neurons [24], and retinal ganglion cells [10]. Our previous research found that puerarin could enhance the specialization of mES cells into ventricular-like CMs [25], inhibits the proliferation of mES-CMs [2], while improves the t-tubule development of mES-CMs [4], suggesting an ameliorated excitation-contraction coupling of these cells in the presence of puerarin.…”

Dynasore suppresses cell proliferation, migration, and invasion and enhances the antitumor capacity of cisplatin via STAT3 pathway in osteosarcoma

“…Quantitative real-time PCR was performed in the CFX Connect™ Real-Time PCR Detection System (Bio-Rad, USA) by using TB Green TM Premix Ex Taq TM (TAKARA, Japan). The CT values were normalized to GAPDH expression, and mRNA expressions were calculated via 2 −ΔΔCt method as described in previous research 20 . The primers used in present study are listed in Table 1.…”

Skeletal Extracellular Matrix Supports Cardiac Differentiation of Embryonic Stem Cells: a Potential Scaffold for Engineered Cardiac Tissue

“…The transgenic mESCs (clone αPIG44) was maintained as previously described [24]. Briefly, undifferentiated mESCs were cultured in IMDM containing 15% FCS, 1% nonessential amino acids, 2mM L-glutamine, 0.1 mM β-mercaptoethanol, 100 U/mL penicillin, 100 µg/mL streptomycin and 1000 U/mL leukemia inhibitory factor (LIF, Chemicon).…”