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First published online 26 September 2006
doi: 10.1242/jcs.03186

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Disruption of MEF2 activity in cardiomyoblasts inhibits cardiomyogenesis

¹ Department of Biochemistry, Medical Sciences Building, University of Western Ontario, London, Ontario, N6A 5C1, Canada
² Department of Biochemistry, Microbiology, and Immunology, University of Ottawa, 451 Smyth Road, Ottawa, Ontario, K1H 8M5, Canada
³ Molecular Medicine Program, Ottawa Health Research Institute, 501 Smyth Road, Ottawa, ON K1H 8L6, Canada
⁴ Division of Molecular Cardiovascular Biology, Cincinnati Children's Medical Center ML7020, Cincinnati, OH, USA

View larger version (59K): [in a new window]   Fig. 1. (A-D) The expression of MEF2C/EnR in cardiomyoblasts inhibits the development of cardiac muscle. P19(Nkx-MEF2C/EnR) cells (C,D) and P19(control) cells (A,B) were aggregated with DMSO and fixed for immunofluorescence on day 6 of differentiation to examine the presence of cardiac muscle. Cells were stained with the anti-MyHC antibody MF20 (B,D), and nuclei were counter-stained with Hoechst dye 33258 (A,C) (Magnification 400x).

View larger version (42K): [in a new window]   Fig. 2. MEF2C/EnR expression in cardiomyoblasts initially enhances Nkx2-5 and MEF2C transcript levels and subsequently downregulates cardiac -actin, Gata4, Bmp4, Nkx2-5 and Mef2c transcripts. (A-J) P19(Nkx-MEF2C/EnR) and P19(control) cells were differentiated in the presence of DMSO. 12 µg of total RNA that was harvested on days 0, 2, 3, 4 and 6 were northern blotted and probed as indicated (A-E). Nkx2-5, MEF2C/EnR, Mef2c, and ß-actin expression were detected using RT-PCR (G-I). A 750 bp EcoRI fragment of rabbit 18S cDNA (F) or a 520 bp PCR product of mouse ß-actin (J) were used as loading standards.

View larger version (106K): [in a new window]   Fig. 3. MEF2C/EnR expressed via an Nkx2-5 enhancer abrogated heart muscle development in type-I transgenic mouse embryos. Wild-type (A,D,G,J), type-I (B,E,H,K) and type-II (C,F,I,L) embryos were harvested on E9.5 and photographed under a dissecting microscope (A-C). The heart area is indicated by a circle. Cryosections were prepared and in situ hybridization was performed with probes against Nkx2-5 (D-F), cardiac -actin (G-I), and Gata4 (J-L). The heart region (arrowhead) and head (h) are indicated.

View larger version (79K): [in a new window]   Fig. 4. MEF2C/EnR expressed via an Nkx2-5 enhancer resulted in a thin-walled myocardium in type-II founder transgenic mouse embryos. Immunofluorescence was performed with anti-MyHC antibody MF20 (D,E,F) to indicate the cardiomyocytes in sections from wild-type (A,D), type-I (B,E) and type-II (C,F) transgenic embryos. Sections were stained with Hoechst dye 33258 to indicate nuclei (A-C).

View larger version (11K): [in a new window]   Fig. 5. Mechanistic model for the effect of MEF2 disruption on the differentiation of cells into cardiac muscle. In the cardiomyoblast, MEF2C, GATA4 and Nkx2-5 are important for the maintenance of the cardiomyoblast phenotype and for subsequent differentiation into cardiomyocytes (Dodou et al., 2004; Grepin et al., 1997; Jamali et al., 2001; Reecy et al., 1999; Searcy et al., 1998; Skerjanc et al., 1998). The presence of MEF2C/EnR downregulated the expression of genes encoding Nkx2-5, MEF2C and GATA4, and inhibited the progression of cardiomyoblasts into cardiomyocytes. An initial enhancement of Nkx2-5 and MEF2C in the stem/mesoderm cell confirmed previous results showing an increase in Nkx2-5 and Gata4 expression, probably due to the relief of HDAC inhibition of cardiomyoblast formation by MEF2C/EnR (Karamboulas et al., 2006).