Givinostat reduces adverse cardiac remodeling through regulating fibroblasts activation

Figures

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  Fig. 1. Givinostat effect on infarcted heart and cardiac fibrosis. a Echocardiographic measurements indicate an amelioration in fractional shortening (FS), left ventricular end diastolic volume (LVEDV), left ventricular end systolic volume (LVESV), left ventricular end diastolic diameter (LVEDD) and wall thickness (WT). Representative M-mode images of control mice (left panel) and after injection with saline (middle panel) or Givinostat (right panel) in AMI mice. b Western blot analysis shows an increased acetylated H3 protein levels in the whole heart tissue after Givinostat administration compared to saline group. c After Givinostat treatment, four sections per mouse were analyzed by Masson’s trichrome staining. Scale bar represent 1 mm. Percentage of fibrotic area: fibrotic area/total area × 100. Quantitative RT-PCR illustrates the Givinostat effect on the expression of fibrosis-related genes (Col1a1, Col1a2 and Col3a1) at different time points. N = 6 mice for time point. Error bars represent ± SEM. Student’s t test, *p < 0.05, **p < 0.01, #p < 0.001

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  Fig. 2. Inflammatory process assays. a The graphs display the mRNA levels of interleukins il-1α and il-β after Givinostat administration in infarcted hearts. b Expression levels of tnf-α and f4/80 genes in AMI mice treated with Givinostat after 1 day. c Sections of AMI mouse heart treated with saline (left panels) and Givinostat (right panels) at day 7 composed of cells positive for F4/80 (magenta, upper images) and MMP9 (magenta, lower images). Scale bars represent 100 and 10 µm respectively. The graphs highlight F4/80 and MMP9 infiltrating areas (mm2) normalized to the total area of cardiac tissue, in the animals treated with Givinostat and in the control group. d Quantitative RT-PCR demonstrates a reduction of mmp9 expression in M1 macrophages and an overexpression of bcl2, plgf and bmp2 genes in M2 macrophages after Givinostat exposure. e Effect of Givinostat on body weight at different time points and on heart weight normalized to the tibia length, at the end of the treatment in AMI mice. N = 6 mice for time point. Error bars represent ± SEM. Student’s t test, *p < 0.05, **p < 0.01, #p < 0.001

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  Fig. 3. Histological analysis. a Apoptotic cardiomyocytes (α-SARC, magenta) were detected by TUNEL labeling (yellow) in SHAM and AMI mice at day 7. Scale bar represent 65 μm. The graph illustrates the ratio between the number of double positive cells (α-SARC+ and TUNEL+ cells) and the nuclei at different time points. b Cross sectional area (CSA) in the border zone at day 30 stained with wheat germ agglutinin (WGA, yellow). Scale bar represent 50 μm. Plot indicates myocytes CSA (μm2) in both the border and the contralateral zone at different time points. c Apoptotic cells were detected by TUNEL labeling (yellow) at day 7. Scale bar represent 100 μm. The graph indicates the ratio between the number of whole TUNEL positive cells and the nuclei at different time points. d α-SMA positive vessels labeled in magenta after 30 days. Scale bar represent 100 μm. The corresponding diagram shows the ratio number of α-SMA positive vessels on total area (mm2) at different time points. e Isolectin B4 positive capillaries (yellow) in the border zone at day 30. Scale bar represent 100 μm. Graphs explain the proportion number of ISO/B4 positive capillaries/cell number in the border and the contralateral…

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  Fig. 4. Pro-fibrotic and EndMT gene expression. a Quantitative RT-PCR analysis for mmp-9, tgf-β and other genes related to EndMT (twist1, twist2, snail1, snail2, bmp-7) in the whole cardiac tissue after treatment with Givinostat compared to saline injected group. b Western blot analysis for TGF-β. The graph indicates quantification of protein bands normalized to GAPDH in AMI Givinostat vs. AMI Saline mice at different time points. N = 6 mice for time point. Error bars represent ± SEM. Student’s t test, *p < 0.05, **p < 0.01, #p < 0.001

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  Fig. 5. Givinostat effect on cardiac fibroblasts (FBs). a FBs morphology cultured in FBS supplemented media (left panel) and after starvation and hypoxia without and with Givinostat treatment (middle and right panels). Scale bar represent 200 μm. b Immunofluorescence for Ki67 (magenta) on FBs FBS (left panel) and after starvation and hypoxia (middle and right panels). Scale bar represent 10 μm. Graph is related to percentage of Ki67 positivity normalized to total number of nuclei (right panel). c ChIP assay performed in FBs treated with Givinostat or with PBS using an antibody against acetylated H3. Mock IP is included as a negative control. Graphs represent a percentage of input precipitated chromatin for nos3, bmp7, and e-cad promoters. d Relative gene expression related to EndMT mechanism in regards to FBs. e Immunofluorescence identifying double positive cells labeled for von Willenbrant Factor (vWF, magenta) and Vimentin (yellow). Scale bar represent 50 μm. The chart defines the percentage of double positive cells normalized to total cell number in FBs treated with Givinostat and the relative control. Error bars represent ± SEM. Student’s t test, *p < 0.05, **p < 0.01, #p < 0.001

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  Fig. 6. Givinostat effect on HUVECs and perturbation studies. a Representative images of capillary-like structures formed by HUVECs pre-treated with Noggin or BMP7 and exposed to Givinostat/FBs-conditioned media or PBS after hypoxia. Diagrams quantifying nodes, junctions, total length segments, branches, total length and total segment length formed by HUVECs using ImageJ angiogenesis software. Scale bars represent 200 μm. b Relative gene expression regarding EndMT mechanism (tgf-β, snail1, twist1/2, n-cad, e-cad, smad1/2/3, sox9) angiogenesis (hif-1α and vegf) and apoptosis (bcl2) in the different culture media. Error bars represent ± SEM. Student’s t test, *p < 0.05, **p < 0.01, #p < 0.001