Anti-Fas gene therapy prevents doxorubicin-induced acute cardiotoxicity through mechanisms independent of apoptosis

Abstract

Activation of Fas signaling is a key mediator of doxorubicin cardiotoxicity, which involves both cardiomyocyte apoptosis and myocardial inflammation. In this study, acute cardiotoxicity was induced in mice by doxorubicin, and some mice simultaneously received an intramuscular injection of adenoviral vector encoding mouse soluble Fas (sFas) gene (Ad.CAG-sFas), an inhibitor of Fas/Fas ligand interaction. Two weeks later, left ventricular dilatation and dysfunction were apparent in the LacZ-treated control group, but both were significantly mitigated in the sFas-treated group. The in situ nick-end labeling-positive rate were similar in the two groups, and although electron microscopy revealed cardiomyocyte degeneration, no apoptotic structural features and no activation of caspases were detected, suggesting an insignificant role of apoptosis in this model. Instead, sFas treatment reversed doxorubicin-induced down-regulation of GATA-4 and attenuated ubiquitination of myosin heavy chain and troponin I to preserve these sarcomeric proteins. In addition, doxorubicin-induced significant leukocyte infiltration, fibrosis, and oxidative damage to the myocardium, all of which were largely reversed by sFas treatment. sFas treatment also suppressed doxorubicin-induced p53 overexpression, phosphorylation of c-Jun N-terminal kinase, c-Jun, and inhibitor of nuclear factor-kappaB, as well as production of cyclooxygenase-2 and monocyte chemoattractant protein-1, and it restored extracellular signal-regulated kinase activation. Therefore, sFas gene therapy prevents the progression of doxorubicin-induced acute cardiotoxicity, with accompanying attenuation of the cardiomyocyte degeneration, inflammation, fibrosis, and oxidative damage caused by Fas signaling.

Figure 1

Effects of sFas gene delivery on LV geometry and function evaluated with echocardiography and cardiac catheterization 2 weeks after doxorubicin injection. A: Representative M-mode echocardiograms at the level of the ventricles in each group. B: Comparison of hemodynamic parameters among the groups. LVDd, left ventricular end-diastolic dimension; %FS, %fractional shortening; LVSP, left ventricular peak systolic pressure. Sal, saline treatment; sFas, sFas gene transfer; LacZ, LacZ gene transfer; DOX, doxorubicin treatment. *P < 0.05 versus the saline-treated sham group; ^#P < 0.05 versus the doxorubicin-treated group with LacZ gene transfer.

Figure 2

Effects of sFas gene delivery on cardiac histology and protein expression evaluated 2 weeks after doxorubicin injection. A: Representative photomicrographs are shown for each group. HE, hematoxylin-eosin staining; CD45, immunohistochemical staining for CD45; Sirius Red, Sirius red staining; 8-OHdG, immunohistochemical staining for 8-OHdG; 4-HNE, immunohistochemical staining for 4-HNE. The arrow in CD45 points to the immunopositive cell. Scale bars = 20 μm. B: Western blot for 4-HNE with its densitometric analysis. *P < 0.05 versus the sham group; ^#P < 0.05 versus the doxorubicin-treated group with LacZ gene transfer.

Figure 3

Effects of sFas gene delivery on myocardial expression of inflammatory mediators and oxidative damage in doxorubicin cardiotoxicity. A: Western blots for cyclooxygenase-2 (COX-2), MCP-1, and TGF-β1. B: Enzyme-linked immunosorbent assay for myocardial TNF-α. *P < 0.05 versus the sham group; ^#P < 0.05 versus the doxorubicin-treated group with LacZ gene transfer.

Figure 4

Effects of sFas gene delivery on expression of Fas, Fas ligand and apoptosis-related mediators in acute doxorubicin cardiotoxicity. A: Western blots for Fas and Fas ligand. B: Double immunofluorescence for TUNEL (green) and myoglobin (red) to separately evaluate TUNEL positivity for cardiomyocytes and nonmyocytes. Arrows point to TUNEL-positive cells. The left panel shows a TUNEL-positive cardiomyocyte while the right panel does a TUNEL-positive nonmyocyte. Scale bars = 10 μm. C: Western blots for caspase-8 and -3 and their uncleaved (pro-) forms. Densitometries of their cleaved (activated) forms are not shown because they were undetected. D: Western blots for Bcl-2 and Bax and their densitometric analyses. *P < 0.05 versus the sham group; ^#P < 0.05 versus the doxorubicin-treated group with LacZ gene transfer.

Figure 5

Effects of sFas gene delivery on cardiomyocyte ultrastructure and expression of GATA-4 and sarcomeric proteins in doxorubicin cardiotoxicity. A: Electron microphotographs. Doxorubicin treatment induced marked degenerative changes, and the damage was significantly attenuated by sFas gene delivery. Scale bars = 1 μm. Graph showing the percent volume comprised of myofibrils assessed by ultrastructural morphometry. *P < 0.05 versus the sham group; ^#P < 0.05 versus the doxorubicin-treated group with LacZ gene transfer. B: Western blots for GATA-4, MHC, and troponin I. *P < 0.05 versus the sham group; ^#P < 0.05 versus the doxorubicin-treated group with LacZ gene transfer.

Figure 6

Effects of sFas gene delivery on expression of p53 and ubiquitination of sarcomeric proteins in acute doxorubicin cardiotoxicity. A: Western blots for p53. *P < 0.05 versus the sham group; ^#P < 0.05 versus the doxorubicin-treated group with LacZ gene transfer. B: Immunoprecipitation and Western blots for polyubiquitinated myosin heavy chain (MHC) and troponin I (TnI).

Figure 7

Effects of sFas gene delivery on myocardial expression of JNK, c-Jun, ERK, p-38 MAPK, and IκB and of their respective phosphorylated forms in doxorubicin cardiotoxicity. Western blots for JNK, p-JNK, c-Jun, and p-Jun (A); ERK and p-ERK (B); p38 and p-p38 (C); and IκB and p-IκB (D) with the densitometric analyses. *P < 0.05 versus the sham group; ^#P < 0.05 versus the doxorubicin-treated group with LacZ gene transfer.