atg7-Based Autophagy Activation Reverses Doxorubicin-Induced Cardiotoxicity

Abstract

[Figure: see text].

Keywords: anthracycline; autophagy; cardiotoxicity; doxorubicin; zebrafish.

Fig 1.. Dynamic autophagy signaling in a zebrafish adult AIC (aAIC) model and modifying effects of atg7+/−.

(A) Schematics of the experimental procedure of an anthracycline-induced cardiotoxicity (AIC) in an adult (aAIC) zebrafish model (DOX, doxorubicin). (B) Dynamics of EF% in the DOX-treated zebrafish and the control group using a high-frequency echo system (n=15). (C) Representative Western blot showing temporal changes in LC3-II protein expression in the hearts of adult zebrafish with AIC. Bafilomycin A1 (30 nM) was administered 4 h before the zebrafish were sacrificed. (D to G) Quantification of LC3-II and the ratio between hearts treated with and without BafA1 in (C), n=3 hearts/group. (H) Representative images of a Western blot showing the LC3 expression in the hearts of WT and atg7+/− zebrafish in the absence or presence of 30 nM BafA1 for 4 h. (I) and (J) Quantification of the Western blot data in (H), n = 3 in each group. (K) Ventricular ejection fraction of WT and atg7−/+ zebrafish with or without DOX stress 8 weeks post injection (wpi) (n=10 fish/group). (L) Quantification of nppa and nppb gene expression by quantitative RT-PCR. n=3 per group. Student’s t test was used in (B); Mann-Whitney test in (E), (G) and (J); Kruskal-Wallis test in (D), (F), (I), and (L); one-way ANOVA followed by post hoc Tukey’s test in (K).

Fig 2.. atg7 overexpression (OE) activates autophagy and exerts deleterious effects in the early phase of aAIC.

(A) Schematics of the atg7 conditional transgenic line. (B and C) Fluorescence images of hearts in fish at 1 week after 24-h treatment with 4HT or EtOH. Signals in the cerulean channel represent cardiomyocyte-specific atg7 overexpression after conditional gene activation. (D) Relative transcript level of atg7 RNA in a wild-type sibling and zebrafish with atg7 OE and with and without 4HT treatment. (E) Representative Western blot showing increased LC3-II levels in a zebrafish with atg7 OE and control treated with BafA1 (n=3/treatment). (F) Quantification of LC3-II and the ratio of LC3-II between the hearts treated with and without BafA1 in (E). (G) Schematics of the experimental procedure for activating atg7 in the early phase of aAIC. (H and J) High-frequency echocardiography was performed at the indicated times to quantify cardiac function. One-way ANOVA followed by Tukey’s post hoc test was used. (I) Evaluation of nppa and nppb gene transcript expression by quantitative RT-PCR. (n = 3). Kruskal-Wallis test was used followed by post hoc Tukey’s test in in (D), (F) and (I). WT, wild type; DOX, doxorubicin.

Fig 3.. atg7 OE can reverse the decline in cardiac function in the late phase of aAIC.

(A) Schematics of the experimental schedule for activating atg7 in the late phase of aAIC. (B and C) High-frequency echocardiography was performed at the indicated times to quantify cardiac function. (WT, wild type; DOX, doxorubicin) (n=15). We used bright red to represent data at the early AIC phase; dark red to represent data at the late AIC phase. One-way ANOVA followed by post hoc Tukey’s test was used. (D) Evaluation of nppa and nppb gene transcript expression by quantitative RT-PCR. (n = 3). (E) Western blotting was used to assess autophagy activity in atg7;Cre fish hearts injected with DOX with or without 4HT treatment, as indicated by LC3-II expression. (F) Quantification of LC3-II and the ratio of LC3-II between the hearts treated with and without BafA1 in (E). (n = 3). Kruskal-Wallis test was used followed by post hoc Tukey’s test in (D) and (F).

Fig 4.. FDA-approved autophagy activators (FAAs) were ranked based on their therapeutic effects in a zebrafish embryonic AIC (eAIC) model.

(A) Schematics of the experimental procedure for an embryonic AIC (eAIC) model. (B and C) Representative Western blot of LC3-II in eAIC and LC3-II quantification (n=4/group). (D and E) Tg(GFP-LC3) zebrafish were used to quantify LC3-II induction. Arrows indicate LC3 aggregates. (n=3/group). (F and G) Autophagy activators exert therapeutic effects, and autophagy inhibitors exert detrimental effects on eAIC, as indicated by changes in both mortality and cardiac function. (H) The rank of the top 18 FAA drugs based on a composite score of their therapeutic effects based on survival rate, heart rate, heart function and phenotypes. Mann-Whitney test in (C); Kruskal-Wallis test followed by post hoc Tukey’s test in (D), (F) and (G); Kruskal-Wallis test followed by Bonferroni post hoc test in (G).

Fig 5.. Top-ranking FAAs used with the eAIC model exerted therapeutic effects in the zebrafish aAIC model in a time-dependent fashion.

(A) Schematics of the experimental procedure for drug administration in the early phase of aAIC. (B and C) High-frequency echocardiography was performed to evaluate cardiac function. (D) Kaplan–Meier survival curves showing the survival of DOX-stressed adult fish after drug administration in the early phase. n=15~25. (E) Schematics of the experimental procedure for drug administration in the late phase of aAIC. (F and G) High-frequency echocardiography was performed 8 wpi to evaluate cardiac functions. (H) Kaplan–Meier survival curves showing the survival of DOX-stressed adult zebrafish after drug administration in the late phase. n=15~25. Log-rank test was used in (D) and (H) for comparisons; one-way ANOVA followed by post hoc Tukey’s test in (B), (C), (F) and (G). wpi, weeks after DOX injection. WT, wild type; DOX, doxorubicin.

Fig 6.. Top-ranking FAAs, including spironolactone (Spi), pravastatin (Pra), minoxidil (Mino) and rapamycin (Rapa), exerted therapeutic effects in a mouse AIC model in a time-dependent fashion.

(A) Schematics of the experimental procedure for drug administration in the early phase of the mouse AIC model. (B to D) Echocardiography was performed to evaluate cardiac functions. (n=5) (E) Schematics of the experimental procedure for drug administration to the AIC model mice in the late phase. (F to H) Echocardiography was performed to evaluate cardiac functions. (n=5) (I and J) All four drugs attenuated the increase in serum LDH and CKMB. (n=8). (K) All four drugs reversed body weight loss in the AIC model mice. (n=10). One-way ANOVA followed by post hoc Tukey’s test was used. Log-rank test was used in (K) for comparisons with the model group. We applied a 0.5% aqueous solution of sodium carboxymethylcellulose (CMC) as a vehicle.

Fig 7.. Spironolactone (Spi) and rapamycin (Rapa) activated autophagosome formation in an Atg7-dependent fashion.

(A) Representative Western blot and quantification of the relative amounts of LC3-II in the hearts from mice injected with a single bolus of DOX. Activated LC3-II and an increased response to BafA1 (n=5/treatment) were observed. (B) Representative Western blots and quantification of Atg7 from the hearts of mice administered the four drugs daily in the later phase. (C) Representative Western blot and quantification of Atg7 from the H9C2 cardiac cell line. Spi and Rapa induced an increase in Atg7 protein levels. (D) H9C2 cells were transiently transfected with mRFP-GFP-LC3 adenovirus and then treated with Spi and Rapa. Representative images of GFP-LC3 and mRFP-LC3 puncta are shown. (E) Quantification of the yellow puncta (autophagosomes) and red puncta (autolysosomes) is shown in (D). (F) qRT-PCR was used to confirm that Atg7 transcripts were reduced by Atg7 siRNA. (G) Quantitative analysis of the yellow puncta (autophagosomes) and red puncta (autolysosomes) showing that Atg7 siRNA ablates the induction of autophagosomes induced by Spi or Rapa. Kruskal-Wallis test was used followed by post hoc Tukey’s test in (A) and (C); one-way ANOVA followed by post hoc Tukey’s test in (B), (E) and (G); student’s test in (F).

Fig 8.. Top2b is required for atg7-mediated autophagy in the early but not the late phase of AIC.

(A) Temporal changes of Atg7 protein level post the first dose of DOX in the mouse AIC model. N=5 mice per group. 4 wpi would be considered as the early phase, because it is equal to 1 week post the last dose of DOX. (B) H9C2 cells were transfected with the indicated siRNA. Cell lysates were analyzed by Western blots to check the knock down effects. H9C2 cells transfected with NC RNAi or Top2b RNAi were treated by DOX for 3 h (C) or 18 h (D), then levels of Atg7 and LC3II were examined by Western blots. Kruskal-Wallis test was used followed by post hoc Tukey’s test.