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Background/aims: The embryonic stem cell-derived cardiomyocytes (ES-CMs) serve as potential sources for cardiac regenerative therapy. However, the immature sarcoplasmic reticulum (SR) function of ES-CMs prevents its application. In this report, we examined the effect of puerarin, an isoflavone compound, on SR function of murine ES-CMs., Methods: Murine ES-CMs were harvested by embryoid body-based differentiation method. Confocal calcium imaging and whole-cell patch clamps were performed to assess the function of SR. The mRNA expression levels of SR-related genes were examined by quantitative PCR. The protein expression of sarcoplasmic reticulum calcium-ATPase 2a (SERCA2a) was evaluated by immunofluorescent and western blot., Results: Long-term application of puerarin promotes basic properties of spontaneous calcium transient with increased amplitude, decay velocity, and decreased duration. Puerarin fails to alter ICa,L but increases the Ca2+ content of SR. Puerarin-treated ES-CMs have intact SR Ca2+ cycling with more SR Ca2+ reuptake. Long-term application of puerarin asynchronously upregulates the mRNA and protein expression of SERCA2a, as well as the transcripts of calsequestrin and triadin in developing ES-CMs. Application of puerarin during the stage of post-cardiac differentiation upregulates dose-dependently the transcripts of SERCA2a, phospholamban and tridin which can be reversed by the inhibitors of the PI3K/Akt and MAPK/ERK signaling pathways, but shows no effect on the protein expression of SERCA2a., Conclusion: This study demonstrates that long-term puerarin treatment enhances Ca2+ reuptake and Ca2+ content via upregulation of SERCA2a., (© 2017 The Author(s). Published by S. Karger AG, Basel.)

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., (© 2016 The Author(s) Published by S. Karger AG, Basel.)

Background/aims: Puerarin shows a wide range of biological activities, including affecting the cardiac differentiation from murine embryonic stem (mES) cells. However, little is known about its effect and mechanism of action on the self-renewal of mES cells. This study aimed to determine the effect of puerarin on the self-renewal and pluripotency of mES cells and its underlying mechanisms., Methods: RT-PCR and real-time PCR were used to detect the transcripts of core transcription factors, specific markers for multiple lineages, REST and microRNA-21 (miR-21). Colony-forming assay was performed to estimate the self-renewal capacity of mES cells. Western blotting and wortmannin were employed to explore the role of PI3K/Akt signaling pathway in the inhibitory action of puerarin on REST transcript. Transfected mES cells with antagomir21 were used to confirm the role of miR-21 in the action of puerarin on cell self-renewal., Results: Puerarin significantly decreased the percentage of the self-renewal colonies, and suppressed the transcripts of Oct4, Nanog, Sox2, c-Myc and REST. Besides, PECAM, NCAM and miR-21 were up-regulated both under the self-renewal conditions and at day 4 of differentiation. The PI3K inhibitor wortmannin successfully reversed the mRNA expression changes of REST, Nanog and Sox2. Transfection of antagomir21 efficiently reversed the effects of puerarin on mES cells self-renewal., Conclusion: Inhibition of REST-miR-21 regulatory pathway may be the key mechanism of puerarin-induced suppression of mES cells self-renewal.

Aims: The embryonic stem cell-derived cardiomyocytes (ES-CM) is one of the promising cell sources for repopulation of damaged myocardium. However, ES-CMs present immature structure, which impairs their integration with host tissue and functional regeneration. This study used murine ES-CMs as an in vitro model of cardiomyogenesis to elucidate the effect of puerarin, the main compound found in the traditional Chinese medicine the herb Radix puerariae, on t-tubule development of murine ES-CMs., Methods: Electron microscope was employed to examine the ultrastructure. The investigation of transverse-tubules (t-tubules) was performed by Di-8-ANEPPS staining. Quantitative real-time PCR was utilized to study the transcript level of genes related to t-tubule development., Results: We found that long-term application of puerarin throughout cardiac differentiation improved myofibril array and sarcomeres formation, and significantly facilitated t-tubules development of ES-CMs. The transcript levels of caveolin-3, amphiphysin-2 and junctophinlin-2, which are crucial for the formation and development of t-tubules, were significantly upregulated by puerarin treatment. Furthermore, puerarin repressed the expression of miR-22, which targets to caveolin-3., Conclusion: Our data showed that puerarin facilitates t-tubule development of murine ES-CMs. This might be related to the repression of miR-22 by puerarin and upregulation of Cav3, Bin1 and JP2 transcripts., (© 2014 S. Karger AG, Basel.)

Aims: It is important to screen and identify chemical compounds to improve the efficiency of cardiac differentiation and specialization of embryonic stem (ES) cells. The objective of this study was to investigate the effect of puerarin, a natural phytoestrogen, on the in vitro cardiac differentiation and ventricular specialization of murine ES cells., Methods: Cardiac differentiation of murine ES cells was performed by embryoid body (EB)-based differentiation method. Quantitative RT-PCR, flow cytometry and immunofluorescence were employed to identify cardiomyocytes (CMs) derived from murine ES cells (mES-CMs). Patch clamp was used to study the electrophysiological properties of CMs., Results: We found that continuous puerarin treatment significantly increased the population of ES-CMs which express typical cardiac markers and are electrophysiological intact. Puerarin treatment shifted the cardiac phenotype from pacemaker-like cells to ventricular-like cells, which were Mlc2v-positive and present typical ventricular-like AP. Puerarin up-regulated transcripts involved in cardiac differentiation and ventricular specialization of ES cells., Conclusion: Our results suggest that puerarin promotes cardiac differentiation, and significantly enhances the specialization of mES cells into ventricular-like CMs. Puerarin may be used to increase the yield of ventricular mES-CMs during in vitro differentiation., (© 2013 S. Karger AG, Basel)