The miRNA199a/SIRT1/P300/Yy1/sST2 signaling axis regulates adverse cardiac remodeling following MI

Figures

• 

  Figure 1. miR199a-5p is dynamically regulated following MI. (a) Quantification of miR199a levels by RT-qPCR in the remote LV region after MI. (b) Quantification of cardiac miR199a levels in the infarcted LV region by RT-qPCR. (c) Serum levels of miR199a after MI. n = 10 mice per groups. All quantitative data are reported as means ± SEM. ***P < 0.001 determined by two-way ANOVA followed by Bonferroni’s post hoc test. d: day(s); w: week(s).

• 

  Figure 2. AntimiR-199a treatment protects against cardiac hypertrophy and fibrosis following MI. (a) Design of the study. (b) JunB mRNA levels in the remote non-infarcted LV region 4 weeks after MI. (c) Echocardiographic analysis of left ventricular fractional shortening in sham- and MI-operated mice 27 days after the first injection of antimiR-199a, antimiR-ctrl or PBS. (d) Representative images of hearts harvested 4 weeks post-MI and treated, or not, with antimiR-199a; Scale bar: 0.5 cm. (e) Heart weight-to-tibia length ratio. (f) Representative left ventricular sections of the indicated treatment groups stained with Masson’s trichrome; Scale bar: 25 μm. (g) Quantification of interstitial fibrosis. (h,i) WGA staining of left ventricular tissue from mice for determination of cardiac and CM hypertrophy; Scale bar: 25 μm. (j) Quantitative real-time PCR analysis of molecular markers for cardiac myocyte hypertrophy (Myh7/Myh6, Nppa) and of fibrosis-associated collagens. All quantifications derive from n = 10 mice/group, PCR performed with 2 replicates each. All quantitative data are reported as means ± SEM. ***P < 0.001 determined by two-way ANOVA followed by Bonferroni’s post hoc test. CM:…

• 

  Figure 3. Pharmacological inhibition of miR-199a prevents Sirt1 down-regulation following MI. (a) Up: the putative miR-199a-5p binding sequence within the 3′-UTR of Sirt1 mRNA; Down: the sequences and position of Sirt1 containing wild type and mutant binding site of miR-199a that cloned into the luciferase reporter vector. (b) Dual-luciferase reporter assay. (c) Determination of Sirt1 mRNA levels in the non-infarcted LV region 4 weeks after MI. (d) Sirt1 protein levels in the non-infarcted region 4 weeks after MI; Representative full western blot images are shown in supplementary material. (e) Assessment of Sirt1 protein levels by immunohistochemical analysis in the non-infarcted LV; Scale bar: 25 μm. All quantifications derive from n = 10 mice/group, PCR experiments were performed with 2 replicates each. All quantitative data are reported as means ± SEM. ***P < 0.001 determined by two-way ANOVA followed by Bonferroni’s post hoc test. GAPDH: Glyceraldehyde-3-Phosphate Dehydrogenase; TBP: TATA binding protein; other abbreviations as in Fig. 2.

• 

  Figure 4. AntimiR-199a therapy prevents Sirt1 downregulation and P300 acetylation. (a) Co-immunoprecipitation of the P300-acetyl-Lys complex. (b) Densitometric analysis of the co-immunoprecipitated P300 protein. (c) Densitometric analysis of the co-immunoprecipitated acetyl-Lys. (d) Co-immunoprecipitation of the Sirt1/P300 complex. (e) Densitometric analysis of the co-immunoprecipitated Sirt1 protein. (f) Densitometric analysis of the co-immunoprecipitated P300 protein. (g) Co-immunoprecipitation assay of the P300/Yy1 complex. (h) Densitometric analysis of the co-immunoprecipitated P300 protein. (i) Densitometric analysis of the co-immunoprecipitated Yy1protein. (j) Representative immunohistochemical images and quantifications of sST2 in the remote LV region of sham and MI mice treated with antimiR-Ctrl or animiR-199a; Scale bar: 25 μm. Representative full western blot images are shown in supplementary material. All quantifications derive from n = 10 mice/group. All quantitative data are reported as means ± SEM. ***P < 0.001 determined by two-way ANOVA followed by Bonferroni’s post hoc test. Abbreviations as in Figs. 2 and 3.

• 

  Figure 5. The miR199a-5p is dynamically regulated in CMs after cyclic stretching. (a) miR199a levels in CMs after cyclic stretching quantified by RT-qPCR. (b) miR199a levels in CFs after cyclic stretching quantified by RT-qPCR. (c) miR199a levels in ECs after cyclic stretching quantified by RT-qPCR. (d) miR199a levels in the supernatants of CMs (red), CFs (blue) and CECs (green) assessed by RT-qPCR after cyclic stretching. All quantifications derive from n = 10 independent assays/group. All quantitative data are reported as means ± SEM. ***P < 0.001 respect to control assay; determined by two-way ANOVA followed by Bonferroni’s post hoc test.

• 

  Figure 6. AntimiR-199a treatment prevents cardiac hypertrophy. (a) Design of the study using CMs under stretching. (b) Quantification of JunB mRNA levels in CMs after cyclic stretching assessed. (c) Quantification of Sirt1 mRNA levels by RT-qPCR in CMs after cyclic stretching. (d) Cell viability measured by MTT assay of CMs treated with PBS, antimiR-Ctrl or antimiR-199a. (e) LDH levels in CMs treated with PBS, antimiR-Ctrl or antimiR-199a after cyclic stretching. (f) Quantitative real-time PCR analysis of molecular markers for cardiac myocyte hypertrophy (Myh7/Myh6, Nppa and BNP). All quantifications derive from n = 10 independent assays/group. All quantitative data are reported as means ± SEM. ***P < 0.001 determined by two-way ANOVA followed by Bonferroni’s post hoc test. BNP: brain natriuretic peptide; LDH: lactate dehydrogenase; ST: stretching; other abbreviations as in Fig. 2.

• 

  Figure 7. P300 deacetylation prevents Yy1 activation leading to sST2 down-regulation. (a) Co-immunoprecipitation of the P300-acetyl-Lys complex in control and stretched CMs that were treated with PBS, antimiR-Ctrl or antimiR-199a. (b) Densitometric analysis of the co-immunoprecipitated P300 protein. (c) Densitometric analysis of the co-immunoprecipitated acetyl-Lys. (d) Co-immunoprecipitation of the Sirt1/P300 complex in control and stretched CMs that were treated with PBS, antimiR-Ctrl or antimiR-199a. (e) Densitometric analysis of the co-immunoprecipitated Sirt1 protein. (f) Densitometric analysis of the co-immunoprecipitated P300 protein. (g) Co-immunoprecipitation assay of the P300/Yy1 complex in control and stretched CMs that were treated with PBS, antimiR-Ctrl or antimiR-199a. (h) Densitometric analysis of the co-immunoprecipitated P300 protein. (i) Densitometric analysis of the co-immunoprecipitated Yy1protein. (j) sST2 protein levels in supernatant from CMs under stretching. (k) Quantitative real-time PCR analysis of molecular markers for cardiac myocyte hypertrophy (Myh7/Myh6, Nppa) and cardiac death (PTX3, LDH); sST2-Fc (100 nM) is added 30 min after antimiR-199a therapy.…