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First published online 13 February 2007
doi: 10.1242/jcs.03386

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Stimulation of ES-cell-derived cardiomyogenesis and neonatal cardiac cell proliferation by reactive oxygen species and NADPH oxidase

¹ Department of Physiology, Justus-Liebig-University Giessen, Aulweg 129, 35392 Giessen, Germany
² Department of Cell Biology, GKSS Research Center, Teltow, Germany

View larger version (29K): [in this window] [in a new window]   Fig. 1. Decay of the proliferation capacity of ES cell-derived- and neonatal cardiac cells after cell isolation from embryoid bodies and neonatal mouse hearts. (A,B) Decay of the numbers of BrdU-positive cell nuclei of cardiac cells. (C,D) Decay in Ki-67 immunoreactivity in cardiac cells. (E,F) Nuclear/cytoplasmic (n/c) cyclin D1 distribution in cardiac cells. (G,H) Decay in retinoblastoma (Rb) phosphorylation in cardiac cells during different times of cell culture. (I,J) Increase in p27kip1 expression in the cell nuclei of cardiac cells during different times of cell culture. *P<0.05, significantly different to day 1 of cell culture.

View larger version (18K): [in this window] [in a new window]   Fig. 2. Stimulation of cardiomyogenesis of ES cells by H2O2. (A) ES-cell-derived embryoid bodies were treated on day 4 of differentiation with 500 nM H2O2 either in the presence and absence of the antioxidants vitamin E (100 µM) and NMPG (100 µM). H2O2 significantly increased the number of spontaneously contracting embryoid bodies on day 8 of cell culture, which was totally abolished in the presence of antioxidants. (B) Treatment of embryoid bodies with H2O2 significantly upregulated the cardiac-specific genes -actin, ANP, -MHC, MLC2a and MLC2v. (C) Upregulation of -actinin, MLC2a and cMLC1 protein by H2O2. The bar chart shows densitometric analyses of three independent experiments. *P<0.05, significantly different to the untreated control.

View larger version (42K): [in this window] [in a new window]   Fig. 3. Role of the NADPH oxidase in cardiomyogenesis of ES cells. (A) mRNA expression of Nox isoforms during ES-cell differentiation within embryoid bodies. Effects of H2O2 on protein expression of NADPH oxidase subunits in ES cell-derived cardiac cells (B) and neonatal cardiac cells isolated from mouse hearts (C). (D) Effects of H2O2 on Nox-1 and Nox-4 mRNA expression in embryoid bodies. (E) Inhibition of H2O2-induced cardiomyogenesis by the NADPH oxidase antagonists DPI (100 nM) and apocynin (10 µM). *P<0.05, significantly different to the untreated control.

View larger version (28K): [in this window] [in a new window]   Fig. 4. Semiquantitative analysis of MEF2C, Nkx-2.5 and GATA-4 protein expression 24 hours after treatment with 500 nM H2O2. Untreated and treated embryoid bodies were labelled with antibodies directed against the respective protein and immunofluorescence corrected for background fluorescence was quantified by confocal laser scanning microscopy and computer-assisted image analysis. The images show representative immunostainings. Bar, 200 µm. The bar chart represents pooled data from three experiments. *P<0.05, significantly different to the untreated control.

View larger version (37K): [in this window] [in a new window]   Fig. 5. Redox-sensitive gene activation of the cardiac-specific transcription factors Mef 2c (A), Dtef (B), Gata4 (C), Nkx2.5 (D) as well as the cardiac cell differentiation associated growth factor Bmp10 (E) in embryoid bodies upon treatment with 500 nM H2O2. Note that preincubation with the antioxidants vitamin E (100 µM) and NMPG (100 µM) totally abolished the activation of the Mef2c and Dtef genes, whereas gene activation already in the absence of H2O2 occurred when Gata4, Nkx2.5 and Bmp10 mRNA expression was investigated. *P<0.05, significantly different to the untreated control.

View larger version (32K): [in this window] [in a new window]   Fig. 6. Increase of ES-cell-derived- and neonatal cardiac cell numbers following treatment with 100 nM H2O2. Cardiac cells were treated with H2O2 for 24 hours. Subsequently, cell numbers were analyzed following immunohistochemical staining for sarcomeric -actinin and Sytox Green counterstaining cell nuclei by microscopical inspection. The images in panel A show representative ES-cell-derived cardiomyocytes (a,b) and neonatal cardiomyocytes (c,d), which remained either untreated (a,c) or were treated with H2O2 (b,d). Bar, 20 µm. (B) Mean ± s.e.m. of 11 experiments. *P<0.05, significantly different to the untreated control.

View larger version (52K): [in this window] [in a new window]   Fig. 7. Analysis of proliferation markers in ES-cell-derived- and neonatal cardiac cells following treatment for 24 hours with 100 nM H2O2. Analysis of BrdU-positive cell nuclei in ES-cell-derived (A) and neonatal (B) cardiac cells. Nuclear/cytoplasmic (n/c) distribution of cyclin D1 in ES cell-derived (C) and neonatal (D) cardiac cells. Nuclear expression of Ki67 in ES cell-derived-(E) and neonatal (F) cardiac cells. Phosphorylation of Rb protein in ES-cell-derived (G) and neonatal (H) cardiac cells. Nuclear expression of p27kip1 in ES-cell-derived (I) and neonatal (J) cardiac cells. *P<0.05, significantly different to the untreated control.