A Wnt- and beta -catenin-dependent pathway for mammalian cardiac myogenesis.

Center for Cardiovascular Development and Department of Biochemistry, Baylor College of Medicine, Houston, TX 77030, USA.
Proceedings of the National Academy of Sciences (Impact Factor: 9.81). 06/2003; 100(10):5834-9. DOI: 10.1073/pnas.0935626100
Source: PubMed

ABSTRACT Acquisition of a cardiac fate by embryonic mesodermal cells is a fundamental step in heart formation. Heart development in frogs and avians requires positive signals from adjacent endoderm, including bone morphogenic proteins, and is antagonized by a second secreted signal, Wnt proteins, from neural tube. By contrast, mechanisms of mesodermal commitment to create heart muscle in mammals are largely unknown. In addition, Wnt-dependent signals can involve either a canonical beta-catenin pathway or other, alternative mediators. Here, we tested the involvement of Wnts and beta-catenin in mammalian cardiac myogenesis by using a pluripotent mouse cell line (P19CL6) that recapitulates early steps for cardiac specification. In this system, early and late cardiac genes are up-regulated by 1% DMSO, and spontaneous beating occurs. Notably, Wnt3A and Wnt8A were induced days before even the earliest cardiogenic transcription factors. DMSO induced biochemical mediators of Wnt signaling (decreased phosphorylation and increased levels of beta-catenin), which were suppressed by Frizzled-8Fc, a soluble Wnt antagonist. DMSO provoked T cell factor-dependent transcriptional activity; thus, induction of Wnt proteins by DMSO was functionally coupled. Frizzled-8Fc inhibited the induction of cardiogenic transcription factors, cardiogenic growth factors, and sarcomeric myosin heavy chains. Likewise, differentiation was blocked by constitutively active glycogen synthase kinase 3beta, an intracellular inhibitor of the Wntbeta-catenin pathway. Conversely, lithium chloride, which inhibits glycogen synthase kinase 3beta, and Wnt3A-conditioned medium up-regulated early cardiac markers and the proportion of differentiated cells. Thus, Wntbeta-catenin signaling is activated at the inception of mammalian cardiac myogenesis and is indispensable for cardiac differentiation, at least in this pluripotent model system.

  • [Show abstract] [Hide abstract]
    ABSTRACT: A complex regulatory network of morphogens and transcription factors is essential for normal cardiac development. Nkx2-5 is among the earliest known markers of cardiac mesoderm that is central to the regulatory pathways mediating second heart field (SHF) development. Here, we have examined the specific requirements for Nkx2-5 in the SHF progenitors. We show that Nkx2-5 potentiates Wnt signaling by regulating the expression of the R-spondin3 (Rspo3) gene during cardiogenesis. R-spondins are secreted factors and potent Wnt agonists that in part regulate stem cell proliferation. Our data show that Rspo3 is markedly downregulated in Nkx2-5 mutants and that Rspo3 expression is regulated by Nkx2-5. Conditional inactivation of Rspo3 in the Isl1 lineage resulted in embryonic lethality secondary to impaired development of SHF. More importantly, we find that Wnt signaling is significantly attenuated in Nkx2-5 mutants and that enhancing Wnt/β-catenin signaling by pharmacological treatment or by transgenic expression of Rspo3 rescues the SHF defects in the conditional Nkx2-5(+/-) mutants. We have identified a previously unrecognized genetic link between Nkx2-5 and Wnt signaling that supports continued cardiac growth and proliferation during development. Identification of Rspo3 in cardiac development provides a new paradigm in temporal regulation of Wnt signaling by cardiac-specific transcription factors.
    Development 08/2014; 141(15):2959-71. · 6.27 Impact Factor
  • [Show abstract] [Hide abstract]
    ABSTRACT: Matrine, one of the main components extracted from Sophora flavescens, has exhibited pharmacological effects on the differentiation in rat liver oval cells. However, its function and mechanism have not yet been fully elucidated. To further investigate them, an in vitro model was established using a rat liver oval cell line called WB‑F344 and treated with matrine. Initially, a significant increase in the number of monodansylcadaverine‑positive cells and in the levels of microtubule‑associated protein 1A/1B‑light chain 3 (LC3)‑II, which is a specific marker for detecting autophagy, was observed in matrine‑treated cells. This indicated that autophagy was stimulated by matrine, which was further confirmed by transmission electron microscopy. Additionally, the apoptotic oval cells were easily detected under matrine treatment using an Annexin‑V‑fluorescein isothiocyanate/propidium iodide assay, indicating that autophagy and apoptosis were synchronously induced by matrine. A decrease in B‑cell lymphoma (Bcl‑2) mRNA expression, but an increase in Bcl2‑associated X protein (Bax) mRNA expression were observed in matrine‑treated cells, which led to an upregulation of the Bax/Bcl‑2 ratio, a molecular marker for determining the extent of apoptosis. Next, the molecular mechanism of matrine‑induced autophagy and apoptosis was analyzed in WB‑F344 cells. β‑catenin degradation was downregulated by matrine and rapamycin, a foregone chemical agonist of autophagy, whereas it was upregulated by 3‑methyladenine, a specific inhibitor of autophagy. Additionally, β‑catenin activation induced an increase in LC3‑II levels and reversed the Bax/Bcl‑2 mRNA ratio under matrine treatment, whereas inhibition of β‑catenin by RNA interference induced a decrease of the LC3‑II amount and of the Bax/Bcl‑2 mRNA ratio. Finally, matrine treatment attenuated p53; however, with little or no change in LC3‑II levels, but a decrease in β‑catenin levels occurred in WB‑F344 cells upon treatment with pifithrin‑α, a chemical inhibitor of p53, revealing that p53, interfering with β‑catenin, may not be involved in matrine‑induced autophagy in WB‑F344 cells. These results demonstrate that β‑catenin is involved in matrine‑induced autophagy and apoptosis in WB‑F344 cells, while β‑catenin is negatively regulated by autophagy and positively by p53, indicating that β‑catenin may be involved in the crosstalk between autophagy and apoptosis in WB‑F344 cells.
    Molecular Medicine Reports 04/2014; · 1.48 Impact Factor
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: Cardiomyocytes (CMs) differentiated from human embryonic stem cells (hESCs) are a promising and potentially unlimited cell source for myocardial repair and regeneration. Recently, multiple methodologies-primarily based on the optimization of growth factors-have been described for efficient cardiac differentiation of hESCs. However, the role of extracellular matrix (ECM) signaling in CM differentiation has not yet been explored fully. This study examined the role of ECM signaling in the efficient generation of CMs from both H7 and H9 ESCs. The hESCs were differentiated on ECM substrates composed of a range of fibronectin (FN) and laminin (LN) ratios and gelatin and evaluated by the fluorescence activated cell scanning (FACS) analysis on day 14. Of the ECM substrates examined, the 70:30 FN:LN reproducibly generated the greatest numbers of CMs from both hESC lines. Moreover, the LN receptor integrin β4 (ITGB4) and FN receptor integrin β5 (ITGB5) genes, jointly with increased phosphorylated focal adhension kinase and phosphorylated extracellular signal-regulated kinases (p-ERKs), were up-regulated over 13-fold in H7 and H9 cultured on 70:30 FN:LN compared with gelatin. Blocking studies confirmed the role of all these molecules in CM specification, suggesting that the 70:30 FN:LN ECM promotes highly efficient differentiation of CMs through the integrin-mediated MEK/ERK signaling pathway. Lastly, the data suggest that FN:LN-induced signaling utilizes direct cell-to-cell signaling from distinct ITGB4(+) and ITGB5(+) cells.
    BioResearch open access. 08/2014; 3(4):150-61.


Available from