Bilirubin Improves Gap Junction to Alleviate Doxorubicin-Induced Cardiotoxicity by Regulating AMPK-Axl-SOCS3-Cx43 Axis

Figures

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  FIGURE 1. Doxorubicin significantly induces abnormal heart rhythm in mice. Mice received a single administration of doxorubicin (20 mg/kg, i.p.) or vehicle (0.9% NaCl, i.p.). (A) Kaplan Meier survival analysis showed that the survival rate of doxorubicin-treatment mice. The Log rank (Mantel-Cox) test was used to determine significance (n = 10 of each group). (B) Body weights was measured every day for consecutive 12 days. (C) Representative images of electrocardiogram (ECG), and (D) echocardiography were examined on Day 7 after doxorubicin administration. (E) Quantitative data of the LVEF values in mice. (F) Masson staining of myocardial sections in mice. Magnification: ×100. Scale bar: 100 μm. (n = 4 of each group). Significant difference was revealed following one-way ANOVA (*p < 0.05, **p < 0.01 vs. Control group; Bonferroni post hoc tests).

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  FIGURE 2. Bilirubin protects against doxorubicin-induced cardiotoxicity. (A) Survival rate was measured every day for consecutive 12 days (n = 10 of each group). The level of myocardial enzyme including AST (B), LDH (C), CK-MB (D), α-HBDH (E) and cTnT (F) were measured by the Elisa kits. (G) Representative images of ECG and the quantitative data of heart rate (H), QT interval (I), QTc (J) and T-peak Tend Interval (K) showed the cardiac conduction function in mice. The plasma samples (n ≥ 4 of each group) were collected after the last bilirubin treatment on Day 7. Significant difference was revealed following one-way or two-way ANOVA (*p < 0.05, **p < 0.01, ***p < 0.001 vs. Control group; # p < 0.05, ## p < 0.01, ### p < 0.001 vs. Doxorubicin-treated group; Bonferroni post hoc tests).

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  FIGURE 3. Bilirubin reduces doxorubicin-induced JNK-Cx43 gap junctions’ dysfunction. (A,C) Representative Western blot images showed the levels of p-Cx43, total Cx43, p-JNK and JNK in H9C2 cells in vitro. (B,D) Representative Western blot images showed the levels of p-Cx43, total Cx43, p-JNK and JNK in heart tissues from doxorubicin-mice given different dose of bilirubin (7.5, 15, and 30 mg/kg, i.p.) for 7 days. The western blot samples were collected and analyzed (n = 4 of each group). (E) Apoptosis was assessed by flow cytometric analysis in H9C2 cells. (F) Quantitative data of the apoptosis ratio analyzed by flow cytometry. H9C2 cells were treated with bilirubin (0.1 μM) for 6 h before doxorubicin (1 μM) treatment, and the samples were collected 24 h after doxorubicin treatment (n = 4 of each group). Significant difference was revealed following one-way ANOVA (*p < 0.05, **p < 0.01, ***p < 0.001 vs. Control group; # p < 0.05, ## p < 0.01, ### p < 0.001 vs. Doxorubicin-treated group; Bonferroni post hoc tests).

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  FIGURE 4. Bilirubin increases AMPK phosphorylation and SOCS3 expression. (A,B) Representative western blot bands showed the levels of p-AMPK, AMPK, and SOCS3 in heart tissues from mice given different dosages of bilirubin (7.5, 15, and 30 mg/kg, i.p.) for 7 days, (n = 4 of each group). (C,D) Representative Western blot bands shown the levels of p-AMPK, AMPK, and SOCS3 in H9C2 cells cultured with different concentrations of bilirubin for 6 h. Significant difference was revealed following one-way ANOVA (*p < 0.05, **p < 0.01 vs. Control group; # p < 0.05, ## p < 0.01 vs. Doxorubicin-treated group; Bonferroni post hoc tests).

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  FIGURE 5. Bilirubin protects against Doxorubicin-induced cardiotoxicity in an AMPK-SOCS3 dependent manner in H9C2 cells. (A) RTCA (xCELLigence platform) was used to record the cell growth curves in H9C2 cells. (B–E) Representative western blot bands showed the levels of p-AMPK, AMPK, SOCS3, p-JNK, JNK, p-Cx43, and total Cx43 in H9C2 cells in vitro. H9C2 cells were pretreated with Compound C (2 μM) for half an hour before bilirubin treatment, and then cells were cultured with bilirubin (0.1 μM), AICAR (20 μM) and metformin (1 mM) for 6 h, followed by doxorubicin treatment (1 μM) for another 24 h. The western blot samples were collected and analyzed (n = 4 of each group). Significant difference was revealed following one-way ANOVA (*p < 0.05, ***p < 0.001 vs. Control group; # p < 0.05, ## p < 0.01, ### p < 0.001 vs. Doxorubicin-treated group; $ p < 0.05 vs. Doxorubicin and bilirubin-treated group; Bonferroni post hoc tests).

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  FIGURE 6. Bilirubin induces SOCS3 expression via activating Axl receptor in H9C2 cells. (A–E) Representative western blot bands showed the levels of p-AMPK, AMPK, Axl, SOCS3, p-JNK, JNK, p-Cx43, and total Cx43 in H9C2 cells in vitro. H9C2 cells were pretreated with R428 (1 μM) for half an hour before bilirubin treatment, and then cells were cultured with bilirubin (0.1 μM) for 6 h, followed by doxorubicin treatment (1 μM) for another 24 h. The western blot samples were collected and analyzed (n = 4 of each group). (F) Representative western blot bands showed the levels of Axl in H9C2 cells. (G) The efficiency of Axl knockdown was assessed by western blot assay. (H–J) Representative western blot bands showed the levels of SOCS3, p-JNK, JNK, p-Cx43, and total Cx43 in H9C2 cells. H9C2 cells were transfected with 100 pM Axl siRNA or control siRNA for 24 h, and then subjected to bilirubin (0.1 μM) for 6 h, followed by exposure to doxorubicin (1 μM) for another 24 h. The western blot samples were collected and analyzed (n ≥ 3 each group). Significant difference was revealed following one-way ANOVA (*p < 0.05, **p < 0.01, ***p < 0.001 vs. Control group; # p < 0.05, ## p < 0.01, ### p < 0.001 vs.…

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  FIGURE 7. Schematic indicating that moderate increase bilirubin can inhibit doxorubicin-induced gap junction’ dysfunction and conduction abnormalities via regulating AMPK-Axl-SOCS3 signaling axis. Doxorubicin significantly increases the phosphorylation of JNK and Cx43 in mouse cardiomyocytes, leading to gap junction’ dysfunction and conduction abnormalities and causing cardiotoxicity. Bilirubin induces SOCS3 expression in an AMPK-Axl-dependent manner, inhibiting JNK-Cx43 signaling pathway to improve gap junction’ function and conduction abnormalities.