Interrelation between α-Cardiac Actin Treadmilling and Myocardin-Related Transcription Factor-A Nuclear Shuttling in Cardiomyocytes

Figures

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  Figure 1. Expression and localization of MRTFA-GFP fusion protein on neonatal rat cardiomyocytes. Cells were infected by adenovirus MRTFA-GFP at 50 MOI and starved in serum free DMEM medium for 24 h. Serum stimulation was performed by adding 20% serum (13.5% HS plus 6.5% FCS) to medium during 60 min in (A–F). (A,B) Immunofluorescence labeling of vimentin (red), and MRTFA-GFP (green). The yellow arrows indicate fibroblasts. (C,D) Immunofluorescence labeling of desmin (red), The orange arrows indicate cardiomyocytes including MRTFA-GFP localized in the nucleus (green). (E) Histogram representing percentage of cardiomyocytes (CM) and fibroblasts (FB) in cultured cells. (F) Histogram of percentage of CM and FB having MRTFA-GFP protein. (G) Percentage of three categories of MRTFA-GFP distribution in NRCs under basal conditions (serum free) at the beginning (0 min) and the end (60 min) of experiment. ns: non-significant. These data are representative of 3 independent experiments, n = X − Y (range) cells per analysis.

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  Figure 2. Impact of serum, phenylephrine, endothelin-1 and angiotensin II on MRTFA-GFP shuttling. NRCs were starved in serum-deprived DMEM medium for 24 h. Serum stress was conducted by adding serum to medium to obtain 20% final concentration (13.5% HS plus 6.5% FCS) at time 0 then followed at 60 min. NRCs were also treated with pro-hypertrophic agonists (Ang II, PE or ET-1) at 100 nM at time 0 then followed at 180 min. (A) Fluorescence image of MRTFA-GFP protein in NRCs that were starved and then stimulated by serum at time 0, 5, 20 and 60 min. Scale bar: 20 µm. Percentage of three MRTFA-GFP distribution categories in NRCs; (B) serum stimulation conditions at 0 and 60 min; (C) treatment with PE at 0 and 180 min; (D) treatment with ET-1 at 0 and 180 min; (E) treatment with Ang II at 0 and 180 min. Data are mean ± SEM. *, ** and *** indicate significant difference, respectively, at p < 0.05, p < 0.01 and p < 0.001 vs time 0, ns: non-significant. These data are representative of 3 to 4 independent experiments.

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  Figure 3. Effects of ROCK inhibitor (Y-27632) on the MRTFA-GFP shuttle. NRCs were infected by adenovirus MRTFA-GFP at 50 MOI, starved in serum-deprived DMEM medium for 24 h before stress induction. Fluorescence image of MRTFA-GFP, (A) basal conditions (serum free); (B) basal conditions and treatment with Y-27632 (10 µM); (C) treatment with Ang II (100 nM); (D) treatment with Ang II (100 nM) + Y-27632 (10 µM). Scale bar: 20 µm. These data are representative of 3 independent experiments.

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  Figure 4. Impact of ROCK inhibitor (Y-27632) on actin network in relation to MRTFA-GFP shuttle. NRCs were infected either by adenovirus MRTFA-GFP alone at 50 MOI or by both adenovirus MRTFA-GFP and Life-Act-RFP. Cells were starved in serum-deprived DMEM medium for 24 h before the treatment. The immunofluorescent staining of α-cardiac actin (red) and fluorescent image of MRTFA-GFP (green), (A) basal conditions (serum free); (B) treatment with Ang II (100 nM). (C) The fluorescent image of MRTFA-GFP (green) and Life-Ac-RFP (red) with Ang II (100 nM) treatment. The white arrows indicate F-actin at 180 min. The immunofluorescent staining of α-cardiac actin (red) and fluorescent image of MRTFA-GFP (green), (D) treatment with Y-27632 (10 µM); (E) treatment with Ang II (100 nM) + Y-27632 (10 µM). Scale bar: 5 or 15 µm.

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  Figure 5. Effects of wortmannin and LY294002, two PI3K inhibitors, on the MRTFA-GFP shuttle induced by serum. NRCs were infected by adenovirus MRTFA-GFP at 50 MOI, starved in serum-deprived DMEM medium for 24 h before stress induction of 60 min. Fluorescence image of MRTFA-GFP, (A) basal conditions (serum free); (B) treatment with serum (20%); (C) treatment with serum (20%) + wortmannin (100 nM); (D) treatment with serum (20%) + LY294002 (30 µM). Scale bar: 15 µm. These data are representative of 3 independent experiments.

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  Figure 6. Effects of wortmannin and LY294002, two PI3K inhibitors, on the MRTFA-GFP shuttle induced by PE. NRCs were infected by adenovirus MRTFA-GFP at 50 MOI, starved in serum-deprived DMEM medium for 24 h before stress induction of 180 min. Fluorescence image of MRTFA-GFP, (A) basal conditions (serum free); (B) treatment with PE (100 nM); (C) treatment with PE (100 nM) + wortmannin (100 nM); (D) treatment with PE (100 nM) + LY294002 (30 µM). Scale bar: 15 µm. These data are representative of 3 independent experiments.