Inducible cardiac-specific SRF overexpression in adult mice.

<p>Schematic representation of the genetic strategy used to obtain a cardiac-specific SRF overexpression in adult mice. Tamoxifen injections to the double transgenic mice (α-MHC-MerCreMer/CAG-flCAT-SRF) induce excision of the floxed CAT gene that allows the expression of SRF gene.</p

Impact of cardiac-specific overexpression of SRF on gene expression.

<p>qRT-PCR analysis of control (n = 7), SRF-LL (n = 6) and SRF-HL (n = 6) mouse mRNA, using cyclophilin as internal control. Data are presented as means ± s.e.m. *, ** and *** indicate significant difference at P < 0.05, P < 0.01 and P < 0.001, respectively versus the control group. (A) SRF target genes. (B) Genes involved in cardiac fibrosis. SRF-LL: low level of SRF; SRF-HL: high level of SRF.</p

A scheme illustrating the loop of regulation between SRF, CTGF and miR-133a in low and high SRF expression level in cardiomyocytes.

<p>A scheme illustrating the loop of regulation between SRF, CTGF and miR-133a in low and high SRF expression level in cardiomyocytes.</p

Impact of the level of SRF overexpression on cardiac fibrosis development.

<p>(A) Western blot analysis of vimentin protein. (B) Quantification of vimentin protein level (n = 4). Data are presented as means ± s.e.m. ** indicates significant difference at P < 0.01, respectively versus the control group. (C) Vizualisation of cardiac fibroblasts by vimentin staining of heart sections from control, SRF-LL and SRF-HL mice. Longitudinal frozen section showing the localization of cardiac fibroblasts stained with vimentin (red) and polymerized actin in myocytes labeled with Alexa-Fluor-488-phalloidin (green). These data are representative of three independent experiments. Scale bar: 80 μm. (D) Confocal microscopic view of Ki67 labeling (pink), nuclei (blue) and vinculin (green). Ki67 labeling is stronger in the SRF-HL than the SRF-LL and control mice and is exclusively located in the interstitial cells. The enlargement is of twice. These data are representative of three independent experiments. Scale bar: 80 μm. (E) Confocal microscopic view of Ki67 labeling (pink), vimentin (green) and nuclei (blue) of heart sections from SRF-HL and control mice. Top panel, scale bar: 100 μm; bottom panel corresponding to the square indicated in the top panel, scale bar: 10 μm. These data are representative of two independent experiments.</p

Cardiac CTGF expression induced by a high SRF overexpression.

<p>(A) Western blot analysis of CTGF protein. The GAPDH blot represented here is the same used for vimentin in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0139858#pone.0139858.g004" target="_blank">Fig 4</a> since CTGF and vimentin were blot on the same membrane. (B) Quantification of CTGF protein level (n = 4). Data are presented as means ± s.e.m. * and *** indicate significant difference at P < 0.05 and P < 0.001, respectively versus the control group. (C) Immunoflurescence labeling of SRF (red), vinculin (green) and nuclei (blue) (upper line), and CTGF (orange), vinculin (green) and nuclei (blue) (lower line) on serial heart sections of control, SRF-LL and SRF-HL mice. The white arrows indicate SRF-positive/CTGF-null cardiomyocytes while the orange arrows indicate SRF-positive/CTGF-positive cardiomyocytes. All these data presented in this figure are representative of three independent experiments. Scale bar: 80 μm.</p

Effects of cardiac-specific overexpression of SRF on heart dilation and cardiomyocyte architecture.

<p>(A) Western blot analysis of SRF protein (10 μg of total protein per lane). The blot was also probed for glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as a loading control. (B) Quantification of SRF protein level (n = 4). Data are presented as means ± s.e.m. *** indicates significant difference at P < 0.001, respectively versus the control group. (C) Hematoxylin-eosin staining of paraffin-embedded heart sections. The dilation of the ventricles is less marked in SRF-LL than in SRF-HL samples. LV: left ventricle; RV: right ventricle. These data are representative of three independent experiments. Scale bar: 1 mm. (D) Sections of mouse hearts were stained with Sirius red. The presence of endomyocardial fibrotic regions was observed in SRF-HL mice (white arrows). These data are representative of three independent experiments. Scale bar: 100 μm. (E) Identical fields than in D visualized with polarized light. Collagen fibers are highly birefrigent with fine fibers appearing green, and thicker fibers appearing yellow or orange (white arrows). (F) Confocal microscopy of cardiac sections labeled with anti-SRF antibody (red), anti-vinculin FITC (green) for cardiomyocyte membranes and DAPI (blue) for nuclei. SRF staining (white arrows) showing higher labeled cardiomyocyte nuclei in the SRF-HL than in the SRF-LL and in the control groups corroborating overexpression of the SRF gene. In the same way, intercalated discs (orange arrows) are substantially enlarged and irregularly shaped in the SRF-HL group compared with the SRF-LL and the control groups. These results are representative of three independent experiments. Scale bar: 80 μm. (G) Distribution of cardiomyocyte lengths and widths in the three groups of mice; n = 180 for each group (three different mice per group). SRF-LL: low level of SRF; SRF-HL: high level of SRF.</p

Involvement of SRF and of miR-133a in CTGF regulation.

<p>(A) ChIP was performed using H9c2 cells and antibodies specific to SRF, RNApol2 and IgG. Bound SRF or CTGF promoters was amplified by qRT-PCR and normalized to input and to an additional negative control (primers spanning the first intron of Il4). Data are mean± s.e.m. ** and *** indicate significant difference at P < 0.01 and P < 0.001, respectively versus IgG. Data are representative of 4 independent experiments. (B) qRT-PCR assays of miR-133a in cardiac tissue from control (n = 7), SRF-LL (n = 6) and SRF-HL (n = 6) mouse miRNA, using miR–16 as internal control. Data are presented as means ± s.e.m. * indicates significant difference at P < 0.05, respectively versus the control group. (C) H9c2 cells were not treated (n = 7) or treated by miR-133a (50 nM) (n = 7) or antimiR-133a (50 nM) (n = 7) for 48 hours then scratched and total RNAs were extracted. Data are presented as means ± s.e.m. *, ** and *** indicate significant difference at P < 0.05, P < 0.01 and P < 0.001, respectively versus the control group. (D) Western blot analysis of SRF, CTGF and GAPDH proteins (20 μg of total protein per lane). This blot is representative of three independent experiments. (E) H9c2 cells were transduced with AdGFP (n = 5) or AdSRF-VP16 (n = 5) adenoviruses for 8 hours, the medium was changed and treated or not by miR-133a (50 nM) or antimiR-133a (50 nM). 48 hours later, cells were scratched and total RNAs extraction was done. Data are presented as means ± s.e.m. ** and *** indicate significant difference at P < 0.01 and P < 0.001, respectively versus AdGFP; §§§ indicates significant difference at P < 0.001, respectively versus AdSRF-VP16. (F) H9c2 cells were transduced with AdGFP (n = 5) or AdSRF-VP16 (n = 5) adenoviruses for 8 hours then fresh medium was added. 48 hours later, cells were scratched and total RNAs extraction was done. Data are presented as means ± s.e.m. *** indicates significant difference at P < 0.001, respectively versus AdGFP.</p

AMPK phosphorylation and AC5 expression in rat AVMs in response to AMPK activation.

Rat AVMs were incubated for 48h with 1mM AICAR. A, Western blot analysis with phosphorylated-αAMPK and pan-αAMPK antibodies evidenced an increase in AMPK phosphorylation in response to AICAR. B, Adenylyl cyclase 5 (AC5) gene expression was measured by real-time RT-PCR and normalized to TBP. Values are means±SEM of five independent cultures per experimental condition. *p<0.05 and ***p<0.001 for differences from control values.</p

AMPK phosphorylation and AC5 and AC6 gene expression in hearts from WT and AMPKα2-/- mice following TAC.

A, left ventricular mass/tibia length shows a similar hypertrophy in WT and KO mice; B, ejection fraction (%) showing the worsening of cardiac function in KO mice; C, representative western blots of αAMPK and ACC of three hearts per group performed on the same gel and mean values of phosphorylation evidencing an increase in αAMPK and ACC phosphorylation in WT-TAC mice only; D, following TAC, AC5 expression was significantly decreased in WT but not in KO mice while E, AC6 expression was decreased in both WT and KO mice. Values are means±SEM of 6–8 mice. *p$p$ $p$ $$p<0.001 versus corresponding WT.</p

Real-time measurements of cAMP in response to IBMX in control and AICAR-treated rat AVMs.

Non-treated (A) or AICAR-treated (B) rat AVMs were infected with cAMP sensor Epac2-camps adenovirus for 48h to estimate cAMP content. Following CFP excitation at 440±20nm, cAMP binding to the sensor induces a transient decrease in fluorescence resonance energy transfer (FRET) between CFP and YFP plotted as the ratio of the corrected CFP/YFP fluorescence (see Materials and Methods). Acquisitions were performed every 5 seconds. Pseudocolor images reflecting the CFP/YFP ratio were recorded at the times indicated by the letters on the graph below. The graphs below show variations of the CFP/YFP fluorescence emission ratio before, during and after IBMX application. C, comparison of the relative increase in CFP/YFP ratio in response to IBMX in control (n = 21) and AICAR-treated (n = 17) AVMs isolated from three different rats. Values are means±SEM. ***p<0.001 for differences from control values.</p