Fibroblast growth factor-2-mediated protection of cardiomyocytes from the toxic effects of doxorubicin requires the mTOR/Nrf-2/HO-1 pathway

Figures

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  Figure 1. FGF-2 isoforms prevent Doxorubicin-induced toxicity in cardiomyocytes Panels A-H show the effects of Doxorubicin (Dox) exposure for 24 hours in the presence and absence of Lo- or Hi-FGF-2 pre-incubation, as indicated. (A) and (B) show, respectively, representative images of myocytes stained with the Live-Dead assay: Calcein-AM (green, live cells) / Ethidium homodimer (red, dead cells), and the corresponding graph with the percentage of cell death in attached cells. (C) LDH released in culture medium, assessed by Absorbance at 490 nm (n=6). (D), (E), (F) show, respectively, relative protein levels of cleaved (active) caspase-3 (19 kDa), p53 (53 kDa), and Bnip-3 (~30 kDa). Data for the graphs were obtained from the corresponding western blots shown in panel (G); images of the same membranes stained for Ponceau S are also included, and served to adjust for minor variations in protein loading. (H) ATP and ADP levels relative to controls, as indicated (n=6). Data is plotted as mean ± SEM and statistically significant differences are shown by brackets between groups; a P<0.05 was considered significant.

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  Figure 2. FGF-2 isoforms attenuate the effects of Doxorubicin on reactive oxygen species (ROS), Nrf-2 and its downstream target heme oxygenase 1 (HO-1) Panel (A) Relative ROS as measured by the fluorescence intensity of the 2′,7′ –dichlorofluorescin diacetate (DCFDA), n=8, in the absence or presence of Dox and FGF-2 isoform pre-treatment, as indicated. (B) Western blots for Nrf-2, and HO-1, as indicated. Nrf-2 migrates as a 100 kDa band, while HO-1 is at 32 kDa. Quantitative assessments of immunoreactive bands are included in panel C. Panel (C) relative Nrf-2 and HO-1 protein (n=3) as well as corresponding mRNA levels, assessed by q-PCR, (n=4), as indicated. For the mRNA or protein determinations, cardiomyocytes were exposed to Dox for, respectively, 8 or 24 hours, in the presence and absence of Lo- or Hi-FGF-2 pre-treatment. For western blot analysis, the densitometry values of the probed proteins were corrected using the densitometry values of the whole lane Ponceau S stain of the same membrane. For qPCR analysis, all target RNA levels were normalized to rat RNA polymerase II (RP2) levels. Data is plotted as mean ± SEM. Bracket indicate groups whose values are show statistically…

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  Figure 3. In the presence of Doxorubicin, FGF-2 isoforms promote p62/SQSTM1 upregulation which is further increased by Chloroquine (CQ) Panel (A) Western blot showing p62/SQSTM1 immunoreactivity, in the absence and presence of the lysosomal/autophagy flux inhibitor CQ, in cardiomyocytes exposed or not to Dox and FGF-2 pre-treatment, as indicated. Corresponding densitometric data are shown in panel B. Panel (B), relative protein levels of p62/SQSTM1, in response to CQ. Densitometry of the Ponceau S (scan of the whole lane) was used to adjust for minor loading variations. Panel (C), relative levels of p62/SQSTM1 mRNA in cardiomyocytes exposed, or not, to Dox and FGF-2 isoforms, as indicated; n=4. Data is plotted as mean ± SEM. For the mRNA or protein determinations, cardiomyocytes were exposed to Dox for, respectively, 8 or 24 hours, in the presence and absence of Lo- or Hi-FGF-2 pre-treatment. Rat RNA polymerase II (RP2) levels were used to normalize the target mRNA. Statistically significant differences (P<0.5) between groups are indicated by brackets.

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  Figure 4. FGF-2 isoforms prevent the Dox-induced downregulation of transcription factor EB (TFEB) and lysosomal associated membrane protein-1 (LAMP-1) Panel (A) Relative mRNA levels of TFEB in cardiomyocytes exposed to Dox for 8 hours in the presence and absence of pre-incubation with FGF-2 isoforms. Rat RNA polymerase II (RP2) levels were used to normalize the target mRNA. (B) Western blot for LAMP-1, and corresponding graph after 24-hour exposure to Dox in the presence and absence of Lo- or Hi-FGF-2 pre-incubation. Ponceau S stain of the same membrane, used to correct for variations in loading. Brackets indicate groups displaying statistically significant differences.

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  Figure 5. The mTOR pathway mediates the FGF-2 induced effects on Nrf-2 and HO-1 Panel (A) Relative levels of active (phospho-Ser2448 mTOR)/total mTOR (n=4). The corresponding western blot is shown in Supplementary Figure 4A. (B) Cardiomyocyte viability as estimated by the Calcein-AM (fluorescence intensity) assay, in the absence (empty columns) or presence (shaded columns) of Rapamycin, Dox, and FGF-2 isoforms (n=8), as indicated. (C) and (D). Relative protein levels for, respectively, Nrf-2 and HO-1 in the absence (empty columns) or presence (shaded columns) of Rapamycin (n=3). Data is plotted as mean ± SEM and statistical differences are shown by brackets where significant P<0.05. The corresponding western blot is shown in Panel (E).

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  Figure 6. The mTOR and Heme Oxygenase-1 (HO-1) activities mediate the pro-cell survival effects of FGF-2 isoforms Panels (A) and (B). Relative caspase 3/7 activity, and LDH release, respectively, in cardiomyocytes exposed to Dox, and FGF-2 isoform pre-treatment, in the absence or presence of Rapamycin (mTOR inhibitor) and Tin-Protorphyrin (Tin-PP), as indicated;n=4. Panels (C) and (D). p53, or cleaved 19 kDa caspase-3 levels, respectively, in cardiomyocytes exposed to Dox, and FGF-2 isoform pre-treatment, in the absence or presence of Rapamycin and Tin-PP, as indicated, n=3. Corresponding western blots are included in Supplementary Figure 4C. Ponceau S stain of the same membrane, used to adjust for minor variations in loading. Data is plotted as mean ± SEM and brackets denote groups presenting statistically significant (P<0.05) differences.

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  Figure 7. The proposed mechanism of cardioprotection against Doxorubicin by FGF-2 isoforms FGF-2 mediated protection against Dox is shown to be via restoration of Nrf-2 and robust upregulation of its target HO-1 in cardiomyocytes. mTOR re-activation is essential for FGF-2 mediated protection and restoration/upregulation of Nrf-2/HO-1.