CD47 antibody protects mice from doxorubicin-induced myocardial damage by suppressing cardiomyocyte apoptosis

Abstract

Cluster of differentiation 47 (CD47) is upregulated in mouse models of doxorubicin (Dox)-induced dilated cardiomyopathy (DCM). To explore the role of CD47 in the development of DCM, in the present study, CD47 signaling was blocked by an anti-CD47 neutralizing antibody (aCD47) in mice with Dox-induced DCM. Intraperitoneal (i.p.) administration of 10 mg/kg Dox once a week significantly induced the development of DCM after 4 weeks, which was accompanied by the upregulation of CD47 expression in heart tissues. However, co-administration of Dox with 7 mg/kg aCD47 once a week significantly reduced the severity of DCM, with lower numbers of disordered and broken myofibers, reduced cardiomyocytes and infiltration of macrophages in the heart tissues of treated mice. The beneficial effects were associated with the reduced population of Annexin V⁺7-AAD^- apoptotic cells, and the attenuated formation of interstitial fibrosis and release of lactate dehydrogenase (LDH) in the aCD47-treated mice. In addition, co-administration with aCD47 effectively reduced the expression of Bax, collagen I, interleukin (IL)-6 and tumor necrosis factor (TNF)-α in murine DCM. These results were further supported by an in vitro study, in which aCD47 pre-treatment significantly reduced the Dox-induced early apoptosis of cardiomyocytes and suppressed the expression of Bax, cleaved caspase-1/3 and phosphorylation of p38 MAPK. Therefore, aCD47 attenuated DCM in mice, possibly by suppressing cardiomyocyte early apoptosis and p38 MAPK signaling. CD47 may be a useful therapeutic target in the treatment of DCM.

Keywords: CD47; apoptosis; cardiomyocytes; dilated cardiomyopathy; doxorubicin.

Figure 1

CD47 is upregulated in cardiomyocytes after Dox treatment. Adult mice were intraperitoneally (i.p.) administered with 5 and 10 mg/kg Dox once a week for 4 weeks. Mice treated with PBS were naïve controls. (A) Heart histology was analyzed by hematoxylin and eosin (H&E) staining. Representative images with x40 (upper panel) and x200 (lower panel) magnification. Red arrow, cardiomyocytes with centrally localized nuclei; blue arrow, broken myofibers. (B) Flow cytometry analysis for the expression of CD47 on CD11b^- myofibers (upper panel) and Annexin V⁺7-AAD^- apoptotic myofibers (lower panel) in the cardiac tissues of treated mice. One representative dot plot of flow cytometry analysis. (C) Representative dot plot of flow cytometry analysis for CD47 expression 24 h after treatment of primary cardiomyocytes with different concentrations of Dox. (D) Quantitative analysis of CD47 expression on CD11b^- cardiomyocytes and CD11b⁺ immune cells after treatment with different concentrations of Dox. Data are presented as the mean percentage of gate cells ± standard error. ^*P<0.05, ^**P<0.01 vs. untreated control, n=3. Two-way ANOVA with Tukey's multiple comparison's test. (E) Viability of cardiomyocytes was measured by Cell Counting kit-8 (CCK-8) after Dox treatment in vitro. Data are presented as the mean optical density over untreated controls ± standard error. ^**P<0.01 vs. untreated control, n=3. Two-way ANOVA with Tukey's multiple comparison's test. Dox, doxorubicin.

Figure 2

aCD47 significantly reduces the severity of Dox-induced dilated cardiomyopathy (DCM) in mice. Adult mice (aCD47/Dox group) were i.p. administered with both 7 mg/kg aCD47 and 10 mg/kg Dox once a week for 4 weeks. Mice treated with both IgG isotype and Dox (IgG/Dox group) or PBS and aCD47 alone (PBS and aCD47 groups) were controls. (A) Hematoxylin and eosin (H&E) staining for mouse heart tissues. Representative gross morphology and phase contrast microscope images with x200 magnification (upper two panels). Masson staining for interstitial fibrosis and immunostaining for collagen I (lower two panels). Representative images with x200 magnification. Red arrow, cells with centrally localized nuclei; blue arrow, broken and patchy myofibers; yellow arrow, interstitial fibrosis; green, collagen I. (B) Representative echocardiograms of mice 4 weeks after treatment. (C) Quantitative analysis of echocardiographic measurements. Left ventricular ejection fraction (LVEF); left ventricular fractional shortening (LVFS). (D) Quantitative analysis of cardiomyocyte area after H&E staining (upper panel; Mann-Whitney test) and fluorescence intensity of collagen I-positive fibers in heart tissues after immunostaining by ImageJ software. Data are presented as relative fluorescence intensity of positively stained cells over untreated controls. (E) Western blot analysis for the expression of collagen I, Bax and Bcl-2 in the cardiac tissues of treated mice. GAPDH was internal loading control. One representative blot. (F) Band densitometric intensity was semi-quantified by ImageJ software. (G) Flow cytometry analysis for the infiltrating CD11b⁺ macrophages in cardiac tissues. (H) The infiltrating CD11b⁺ macrophages in cardiac tissues were measured by flow cytometry and quantitatively analyzed. All quantitative data are presented as mean ± standard error. ^*P<0.05, ^**P<0.01 vs. PBS group; ^#P<0.05, ^##P<0.01 vs. IgG/Dox group (n=5-7). Two-way ANOVA with Tukey's multiple comparison's test, except where it is indicated otherwise. aCD47, anti-CD47 neutralizing antibody; Dox, doxorubicin.

Figure 3

aCD47 suppresses the expression of cardiac myofiber early apoptosis and production of pro-inflammatory cytokines in mice with Dox-induced DCM. (A) Cardiac myofiber apoptosis in the treated mice was analyzed by flow cytometry analysis. Annexin V⁺7-AAD^- cells were identified as early apoptotic cells. Annexin V⁺7-AAD⁺ cells were identified as late apoptotic cells. Representative dot plot. (B) Quantitative analysis of apoptotic cells after flow cytometry analysis. ^**P<0.01 vs. untreated mice; ^##P<0.05 vs. mice treated with Dox alone, n=5-7. Two-way ANOVA with Tukey's multiple comparison's test. (C) Different doses of aCD47 (3.5, 7 and 14 mg/kg) on apoptosis of cardiac myofiber in mice with DCM. Representative dot plot (left panel). Quantitative analysis of early apoptotic cells (right panel). ^*P<0.05 and ^**P<0.01 vs. untreated mice; ^#P<0.05 vs. mice treated with Dox alone (Dox/0 group), n=3/group. Two-way ANOVA with Tukey's multiple comparison's test. (D) Measurement of LDH release in serum. Data are presented as mean ± standard error (Mann-Whitney test). (E) Quantitative analysis of Bax and Bcl-2 expression in cardiac tissues of treated mice in Fig. 2E. Data are presented as the ratio of band densitometric intensity over untreated control. (F) Ratio of Bcl-2/Bax expression in lung tissues of treated mice analyzed by western blot analysis in Fig. 2E. (G) Immunostaining for Bax (red) in the cardiac tissues of treated mice. Representative images with x200 magnification. (H) Quantitative analysis for Bax expression in the stained cells by ImageJ software. Data are presented as the fluorescence intensity over controls. (I and J) ELISA analysis for the expression of TNF-a in serum and IL-6 in heart protein extracts. Data are presented as mean ± standard error. ^**P<0.01 vs. PBS group; ^#P<0.05, ^##P<0.01 vs. IgG/Dox group (n=5-7). Two-way ANOVA with Tukey's multiple comparison's test, except where it is indicated otherwise. aCD47, anti-CD47 neutralizing antibody; Dox, doxorubicin; LDH, lactate dehydrogenase.

Figure 4

aCD47 reduces Dox-induced cardiomyocyte early apoptosis. The cultured primary cardiomyocytes were pre-treated with 1 µg/ml aCD47 for 1 h, which was followed by treatment with 10 µM Dox (aCD47/Dox group) for 24 h. The cells treated with both Dox and isotype IgG (IgG/Dox), untreated or treated with aCD47 alone (aCD47 group) were controls. (A) Flow cytometry analysis for Annexin V⁺7-AAD^- apoptotic cardiomyocytes 24 h after treatment. Representative dot plots. (B) Quantitative analysis for apoptotic cardiomyocytes analyzed by flow cytometry. (C) Cell viability was determined by CCK-8 assay. Data are presented as the ratio of optical density at 450 nm (OD450 nm) over untreated control. (D) LDH release into the supernatant of the treated cells. Data are presented as the fold increase over untreated controls. Bar plot data in all panels represent the mean ± standard error. n=3. ^**P<0.01 vs. 0 group; ^#P<0.05 vs. IgG/Dox group. Two-way ANOVA followed by a Tukey's multiple comparison's test. aCD47, anti-CD47 neutralizing antibody; Dox, doxorubicin; LDH, lactate dehydrogenase.

Figure 5

aCD47 reduces the expression of Bax and p-p38 MAPK in Dox-treated cardiomyocytes. (A) Immunostaining for the expression of cTnT, cleaved caspase-1/3, Bax and p-p38 MAPK in the treated cardiomyocytes. Cardiomyocytes were identified as cTnT-positive cells (green). Red, cells positively stained for cleaved caspase-1/3, Bax and p-p38 MAPK. Representative images with x200 magnification. (B) Semi-quantitative analysis of positively stained cells by ImageJ software. Data are presented as the relative intensity of positively stained cells over untreated controls. (C) Western blot analysis for the expression of pro-caspase-1, cleaved caspase-1, cleaved caspase-3, Bax and Bcl-2 in the treated cardiomyocytes. GAPDH was internal loading control. One representative blot. (D) p-p38 MAPK⁺ cells after Dox and aCD47 treatment were analyzed by flow cytometry. Data are presented as the percentage of p-p38 MAPK⁺ cells. (E) Association between the percentage of p-p38 MAPK⁺ cells and apoptotic cardiomyocytes after treatment. Bar plot data in all panels are represented as mean ± standard error. n=3. ^*P<0.05, ^**P<0.01 vs. 0 group; ^#P<0.05, ^##P<0.01 vs. IgG/Dox group. Two-way ANOVA followed by a Tukey's multiple comparison's test. aCD47, anti-CD47 neutralizing antibody; Dox, doxorubicin; p-, phosphorylated; cTnT, cardiac troponin T.