Infiltrating macrophages amplify doxorubicin-induced cardiac damage: role of catecholamines

Figures

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  Fig. 1. Macrophage infiltration in the heart in response to Dox. a Cardiac function was evaluated by echocardiography at different time points. The ejection fraction was reduced 5 days after treatment. Results are shown as mean ± SEM; *p < 0.05 vs “time zero” (n = 5). b Paraffin-embedded sections of heart were stained with hematoxylin and eosin (magnification × 200) or processed for immunohistochemistry (magnification × 600) and immunofluorescence (magnification × 400). Representative images of F4/80 staining reveal macrophage infiltration. c Hearts were processed for flow cytometry to evaluate F4/80 positive cells. The results are shown as mean ± SEM; *p < 0.05 vs “time zero” (n = 4). d Mice were pre-treated with Clodrosome or Encapsome and then treated with Dox for 5 days. Macrophages depletion was confirmed by both immunofluorescence (magnification × 400) and flow cytometry analysis, *p < 0.05 vs “CTRL + Encapsome”; #p < 0.05 vs “Dox + Encapsome” (n = 4). e Cardiac function was evaluated in Clodrosome and Encapsome treated mice in response to Dox. Clodrosome ameliorated cardiac function. #p < 0.05 vs “Dox + Encapsome”. f Dox-dependent increase in ANP gene expression was significantly…

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  Fig. 2. Effects of M-Dox and D-Dox treatments. a Cell viability was evaluated at 12 and 24 h. M-Dox reduced cell viability at 12 and 24 h while D-Dox induced a late reduction at 24 h. The results are shown as mean ± SEM in the bar graph; *p < 0.05 vs Basal. b Apoptosis was evaluated by western blot. Cleaved caspase 3 levels increased in both experimental conditions with a higher increase in M-Dox treated cells. Densitometric analysis is shown in the bar graph; *p < 0.05 vs Basal. c We performed co-culture experiments with RAW264.7 and H9C2 using Transwell permeable support. RAW264.7 were plated on the upper compartment of the Transwells and treatment was performed with Doxorubicin (10 μg/ml). Protein content of H9C2 was analyzed by western blot; *p < 0.05 vs Basal; #p < 0.05 vs H9C2. d Mass spectrometry analysis was performed in H9C2 whole lysates, proteins that are up- or down-regulated in response to M-Dox are reported in a heat map

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  Fig. 3. Effects of M-Dox and D-Dox on p53. a Mitochondrial levels of p53 were detected by western blot. They were significantly and early increased in response to M-Dox. Densitometric analysis is shown in the bar graph; *p < 0.05 vs “time zero”. b Cytochrome c levels were evaluated in the cytosolic extract by western blot. An early increase in Cytochrome c was found early in M-Dox treated cells. Densitometric analysis is shown in the bar graph; *p < 0.05 vs “time zero”. The levels of p53 were evaluated by western blot in whole lysates (c) and nuclear extracts (d). In both experimental conditions, whole and nuclear p53 levels were increased. Densitometric analysis is shown in the bar graph; *p < 0.05 vs “time zero”. e GRK2 levels were evaluated by western blot in cells treated with M-Dox and D-Dox for 24 h. The kinase levels were reduced in response to M-Dox but not D-Dox. Densitometric analysis is shown in the bar graph; *p < 0.05 vs “time zero”

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  Fig. 4. Alterations of mitophagy in M-Dox treated cells. a Mitochondrial levels of Parkin were evaluated by western blot. Densitometric analysis is shown in the bar graph; *p < 0.05 vs D-C; #p < 0.05 vs M-C. b Levels of LC3-II were evaluated by western blot. LC3-II levels were unchanged in response to M-Dox both in the absence and presence of E46d. Densitometric analysis is shown in the bar graph; *p < 0.05 vs M-C. c Autophagosomes were quantified at a flow cytometer. The graph shows autophagosomes in response to D-C (dotted black line), D-Dox (continuous black line), M-C (dotted red line), and M-Dox (continuous red line). d Mitochondrial biogenesis was evaluated by real-time PCR. M-Dox blocked mitochondrial biogenesis. Data are shown as mean ± SEM in the bar graph. e Live images of M-C and M-Dox treated H9C2 expressing AAV-Mito-Keima. Scale bar, 10 μm. The graph shows the ratio of red vs green-labeled cells. *p < 0.05 vs M-C

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  Fig. 5. Mitochondria alterations in response to M-Dox. a Cells treated with M-Dox were analyzed at confocal microscopy after staining with mitotracker and TMRE. Most M-Dox treated cells showed an impaired mitochondrial morphology. The graph shows the Pearson’s correlation that indicates a net reduction in TMRE staining in M-Dox samples. *p < 0.05 vs M-C. b Mitochondrial oxygen consumption of M-Dox treated cells was analyzed at 8 and 24 h by Seahorse, resulting significantly reduced; #p < 0.05 vs M-C 8 h, *p < 0.05 vs M-C 24 h

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  Fig. 6. Dox-activated macrophages release catecholamines. Norepinephrine (a) and Epinephrine (b) levels were evaluated in the culture medium of macrophages treated with Dox for 24 h (a, b) by ELISA assay. * p < 0.05 vs CTRL. c, d Norepinephrine (GC) and Epinephrine (HD) levels were evaluated by ELISA assay in plasma from Clodrosome and Encapsome treated mice after 5 days of Dox treatment. *p < 0.05 vs “CTRL + Encapsome”; #p < 0.05 vs “Dox + Encapsome”. e–h The inhibition of TH enzyme activity (e) or its silencing (f) reduced catecholamines release in the culture medium. The inhibition of TH enzyme activity is associated with reduced cleaved caspase 3 and cytochrome c levels (g). TH gene deletion confirmed the reduction in cytochrome release from mitochondria (h) supporting the suggested mechanism. Densitometric analysis is shown in the bar graph; *p < 0.05 vs M-C; #p < 0.05 vs M-Dox