A new FGF1 variant protects against adriamycin-induced cardiotoxicity via modulating p53 activity

Fig. 1. Decreased endogenous FGF1 levels in ADR-treated cardiomyocytes and FGF1ΔHBSalleviated ADR-induced cardiac dysfunction. A) The mRNA expression of Fgf1 in heart tissues (n = 6). B) The mRNA expression of Fgf1 in primary cardiomyocytes. C) The mRNA expression of Fgf1 in H9c2 cells. D) The protein expression of FGF1 in heart tissues was analyzed by Western blot and quantification of the relative protein levels (n = 6). E) The protein expression of FGF1 in primary cardiomyocytes was analyzed by Western blot and quantification of the relative protein levels. F) The protein expression of FGF1 in H9c2 cells was analyzed by Western blot and quantification of the relative protein levels. G-I) Cardiac function was examined by echocardiography. G) Representative M-mode echocardiograms. H) Ejection fraction (n = 4–5). I) fractional shortening (n = 5–6). GAPDH as an internal control. Three independent experiments were performed in primary cardiomyocytes and H9c2 cells. Data are presented as means ± SD. *P < 0.05; N.S., not significant. Ctrl: Control.

Fig. 2. FGF1ΔHBSameliorated ADR-induced cardiac pathological abnormalities. A) Cardiac histology, tested by H&E staining. B–C) Cardiac tissue FITC-conjugated WGA staining and quantification of myocytes cross-sectional areas (n = 6). D-F) The mRNA levels of hypertrophic markers β-myosin heavy chain (Myh7) (n = 4–6); atrial natriuretic peptide (Anp) (n = 6); brain natriuretic peptide (Bnp) in cardiac tissues (n = 5–6). G-J) Cardiac fibrotic response. G) determined by Masson's trichrome staining of collagen deposition (collagen is blue) and I) related quantitative analysis (n = 6). H) assessed by Sirius Red staining of collagen accumulation (collagen is red) and J) related quantitative analysis (n = 6). K) The mRNA expression of myocardial fibrosis markers connective tissue growth factor (Ctgf) (n = 5–6). L) transforming growth factor β (Tgfb) (n = 5–6) M − P) Cardiac inflammation damage. M) detected by IHC staining with tumor necrosis factor-α (TNF-α) (brown considered positive staining) and N) related quantitative analysis (n = 6). O) Relative mRNA levels of inflammatory markers Tnfa (n = 6) and P) interleukin 6 (Il6) (n = 4–6). Data are presented as means ± SD. *P < 0.05; N.S., not…

Fig. 3. FGF1ΔHBSameliorated ADR-induced oxidative stress A) Representative images of DHE staining in cardiac tissues followed by quantification of fluorescence intensity (n = 6). B) IHC staining using 4-Hydroxynonenal (4-HNE) antibody in cardiac tissues (brown considered positive staining) and related quantitative analysis (n = 6). C) Nuclear factor erythroid 2-related factor 2 (Nrf2) nucleus accumulation (indicated by black arrows) was detected by IHC staining with Nrf2 antibody (brown) and related quantitative analysis (n = 6). D-G) The mRNA levels of catalase (Cat) (n = 5–6); superoxide dismutase (Sod) (n = 6); heme oxygenase-1 (Ho-1), (n = 5–6); NAD(P)H quinone oxidoreductase 1 (Nqo1) in the cardiac tissues (n = 6). H–K) H9c2 cells were transfected with NC-shRNA or Nrf2-shRNA for 24h, then H9c2 cells were treated with PBS or 100 ng/ml FGF1ΔHBS for 2 h, and subsequently treated with ADR (1 μM) for 24h. H) The Nrf2 and I) 4-HNE protein expressions were detected by Western blot and J-K) quantification of the relative protein levels. GAPDH as an internal control. Three independent experiments were performed in H9c2 cells. Data are presented as means ± SD. *P < 0.05, **P < 0.001. A + F:…

Fig. 4. FGF1ΔHBSreduced ADR-induced cardiac apoptosis. A) Apoptotic cells (indicated by black arrows) was measured by TUNEL assay in mice heart tissues followed by quantification of apoptosis rate (n = 6). B) The apoptosis of H9c2 cells was detected by flow cytometry analysis using Annexin V-FITC/PI staining followed by related quantitative analysis. C) The protein expression of p53 in heart tissues was analyzed by Western blot and quantification of the relative protein levels (n = 6). D) H9c2 cells were treated with PBS or 100, 250, 500 ng/ml FGF1ΔHBS for 2 h followed with or without addition of 1 μM ADR for 24 h and harvested for Western blot and quantification of the relative protein levels. GAPDH as an internal control. Three independent experiments were performed in H9c2 cells. Data are presented as means ± SD. *P < 0.05. A + FΔHBS: ADR + FGF1ΔHBS.

Fig. 5. FGF1ΔHBSreduced ADR-induced cardiacinjuryby degrading p53. A-D) H9c2 cells were pretreated with PBS or 100 ng/ml FGF1ΔHBS in the presence or absence of nutlin-3a (10 μM) for 2 h, and then treated with or without ADR (1 μM) for 24h. The ROS accumulation of H9c2 cell was analyzed by A) DCFH-DA staining and B) flow cytometry of DCF followed by quantification of fluorescence intensity. C) The p53 and cleaved caspase-3 protein expressions were detected by Western blot and quantification of the relative protein levels. D) H9c2 cells were stained using Annexin V-FITC/PI followed by related quantitative analysis. E) H9c2 cells were treated with PBS or 100 ng/ml FGF1ΔHBS for 24h followed by 10 μM CHX treatment for indicated time and harvested for Western blot and quantification of the relative protein levels. GAPDH as an internal control. Three independent experiments were performed in H9c2 cells. Data are presented as means ± SD. *P < 0.05. C: Control; F: FGF1ΔHBS; N: Nutlin-3a; A: ADR; A + F: ADR + FGF1ΔHBS; A + F + N: ADR + FGF1ΔHBS + Nutlin-3a.

Fig. 6. FGF1ΔHBSreversed ADR-disturbed the protein expression of MDM2,Sirt1and Ac-p53 in cardiomyocytes. A) The related protein expressions were detected by Western blot in heart tissues and quantification of the relative protein levels (n = 6). B) Primary cardiomyocytes were treated with PBS or 100 ng/ml FGF1ΔHBS for 2 h followed with or without 1 μM ADR for 24 h. The related protein expression were detected by Western blot and quantification of the relative protein levels, GAPDH as an internal control. Three independent experiments were performed in primary cardiomyocytes. Data are presented as means ± SD. *P < 0.05; N.S., not significant.

Fig. 7. The effect of FGF1ΔHBSon MDM2-induced p53 ubiquitination is mediated by Sirt1. A) The immunoblotting of immunoprecipitated p53 with antibody recognized ubiquitin in primary cardiomyocytes. B) H9c2 cells were transfected with NC-shRNA or Sirt1-shRNA for 24h, then H9c2 cells were treated with PBS or 100 ng/ml FGF1ΔHBS for 2 h, and subsequently treated with or without ADR (1 μM) for 24h. B–F) The protein expression of Sirt1, MDM2, Ac-p53, p53 were analyzed by Western blot followed by quantification of the relative protein levels. G) Schematic illustration for FGF1ΔHBS protection against ADR-induced cardiotoxicity. Three independent experiments were performed in primary cardiomyocytes and H9c2 cells. GAPDH as an internal control. Data are presented as means ± SD. *P < 0.05; N.S., not significant. A + FΔHBS: ADR + FGF1ΔHBS.

Fig. 8. Sirt1-CKO mice reduced FGF1ΔHBSmediated cardiac protection and reversed the effect of FGF1ΔHBSon the expression of MDM2 and Ac-p53. A) Representative images of TUNEL staining. B) Cardiac tissues were stained by DHE (Red) followed by D) quantification of fluorescence intensity (n = 6). C) Cardiac tissue FITC-conjugated WGA staining and E) quantification of myocytes cross-sectional areas (n = 6). F-J) The expression of Sirt1, MDM2, Ac-p53, p53 in cardiac tissues were analyzed by Western blot followed by quantification of the relative protein levels (n = 6). GAPDH as an internal control. Data are presented as means ± SD. *P < 0.05; N.S., not significant. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)