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First published online 19 December 2006
doi: 10.1242/jcs.03340

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Expression and function of -smooth muscle actin during embryonic-stem-cell-derived cardiomyocyte differentiation

Sophie Clément^1,*,, Michael Stouffs^1,, Esther Bettiol^1,, Sandy Kampf^1,, Karl-Heinz Krause^1,, Christine Chaponnier² and Marisa Jaconi^1,

¹ Department of Geriatrics, Laboratory of Ageing, Geneva Hospital, Chêne-Bourg, Geneva, Switzerland
² Department of Pathology and Immunology, Faculty of Medicine, CMU, Geneva, Switzerland

View larger version (82K): [in this window] [in a new window]   Fig. 1. Distribution and quantitative evaluation of actin isoforms during heart development. (A) 4-µm sections of embryos at E9.5 (a-c), E13 (d-f) and E17 (g-i), and hearts from 2-week-old mice (j-l) were used for immunochemistry with anti -SMA (a,d,g,j), anti SKA (b,e,h,k) and anti -CAA (c,f,i,l) antibodies. Bar, 100 µm. (B) The percentage of -SMA-positive, -SKA-positive and -CAA-positive cardiomyocytes was calculated using the software KS400.

View larger version (53K): [in this window] [in a new window]   Fig. 2. Temporal and spatial distribution of actin isoforms during ES cell differentiation. (A) EBs fixed at different time points (6, 8, 12 and 15 days) were triple-stained with antibodies against all three anti-actin isoforms (-SMA, green; -SKA, blue; -CAA, red). In overlay images (d, h, l and p), beating areas are highlighted by a dashed white line. Bar, 200 µm. (B) Overlay image of a magnified beating area at day 8 co-stained with -SMA (green), -SKA (blue) and -CAA (red) antibodies. Inset, magnified part of the beating area. Bar, 20 µm. Images were acquired with a confocal microscope using either 10x (A) or 63x oil immersion objectives (B). (C) Whole-cell extracts of undifferentiated ES cell (D0) and EBs at days 6, 7, 8, 12 and 15 were analyzed by SDS-PAGE and immunoblotted with anti-SMA, anti-SKA and anti-CAA antibodies. (D) Schematic representation of expression of actin isoform during EB differentiation. Width of each bar correlates with the qualitative changes of the three actins over time.

View larger version (53K): [in this window] [in a new window]   Fig. 3. Differential effect of SMA-FP and SKA-FP on beating and myofibril organization. (A) Schematic representation of the protocol used to treat EBs with FPs twice a day with 5 or 10 µg/ml of SMA-FP, or SKA-FP from days 6 to 8. At day 8, the capacity of the treated ES cells to differentiate into fully differentiated cardiomyocytes was assessed by determining the appearence of spontaneous beating. (B) Percentage of beating EBs at day 8 in untreated EBs (white bars), SMA-FP-treated (striped bars) and SKA-FP (black bars) treated EBs. Error bars represent s.e.m. of a total of five independent experiments (**P0.001). (C) Western blot analysis of untreated EBs (lanes 1) and EBs treated with 10 µg/ml SMA-FP (lanes 2) or 10 µg/ml SKA-FP (lanes 3). Proteins were immunoblotted with anti-total actin (1C4), anti-SMA, anti-CAA and anti-SKA antibodies. (D) 3D reconstruction of confocal microscopy images of untreated EBs (a) and SMA-FP-treated EBs (b) fixed at day 8 and double-stained with anti- actinin (red) and anti-SMA (green) antibodies. (E) 3D reconstruction of confocal microscopy images of untreated (a) and SKA-FP treated (b) EBs fixed at day 8 and double-stained with anti- actinin (red) and anti-SKA (green) antibodies. Bars, 10 µm. Insets in b, magnification of the myofibrils.

View larger version (63K): [in this window] [in a new window]   Fig. 4. Design of shRNAs and their effect on -SMA expression in lung fibroblasts (A) Schematic representation of shRNA constructs targeting -SMA gene (shSMA1-2-3). A shRNA sequence inactive against all actin isoforms was used as control (shCont). The resulting entry vectors were then recombined into pLenti6/BLOCK-iTTM RNAi Vector. The loop sequence CGAA is indicated in red. (B) Untransduced mouse lung fibroblasts (a), and mouse lung fibroblasts transduced with shCont (b), shSMA1 (c), shSMA2 (d) and sh-SMA3 (e) were stained for -SMA (green). DAPI (blue) was used to stain nuclei. Bar, 20 µm.

View larger version (32K): [in this window] [in a new window]   Fig. 5. Effect of shRNA targeting -SMA in ES cells. (A) Proteins of undifferentiated cells (lanes 1 and 4, untransduced ES cells; lanes 2 and 5, ES cells transduced with lentivectors expressing shCont; lanes 3 and 6, ES cells transduced with lentivectors expressing shSMA3) were submitted to SDS-PAGE (Coomassie Blue, lanes 1-3), transferred onto nitrocellulose and blotted with anti-Oct-4, a marker of pluripotenciality (lanes 4-6). (B) Cell cycle analysis by FACS cytometry of propidium iodure labeled untransduced (top), shCont (middle) and shSMA3 (bottom) ESC. (C) Proteins of EBs at day 6 (lanes 1 and 4, untransduced ESC; lanes 2 and 5, transduced with lentivectors expressing shCont; lanes 3 and 6, ES cells transduced with lentivectors expressing shSMA3) were submitted to SDS-PAGE (lanes 1-3; Coomassie Blue staining) and immunoblotted with anti--SMA, anti--SKA and anti--cytoplasmic actin antibodies (lanes 4-6). (D) The percentage of beating EBs at day 8 was assessed under all three conditions. Bars represent s.e.m. of five independent experiments (**P 0.001). (E) Impact of -SMA downregulation on the expression of cardiac transcription factors Nkx2.5 (black bars) and MEF2C (white bars) was evaluated by real-time RT-PCR. Bars represent s.e.m. of three independent experiments.*P0.05, **P0.001.

View larger version (16K): [in this window] [in a new window]   Fig. 6. Effect of FPs on cardiomyocyte beating activity. (A) SMA-FP and SKA-FP (50 µg/ml) were applied on differentiated EBs at day 8 for 2 hours. (B) The frequency of beating was estimated by counting under the microscope the number of beats per 30 seconds of about 50 EBs during a 2-hours period without any treatment () or following a treatment with SMA-FP () or SKA-FP (). (C) 20-second movies of beating EBs were recorded and the variation of grey level in regions at the periphery of EB was calculated over time. Plots are representative of the EBs beating activity before (a and c) and after 2 hours treatment with 50 µg/ml of either SMA-FP (b) or SKA-FP (d). (D) To obtain beating rate spectrums of the frequency plots, Fourier transformation was performed on plots shown in C (see Materials and Methods for details). Numbers on the y-axis represent an `index of arrhythmia'.