Acute doxorubicin cardiotoxicity is associated with miR-146a-induced inhibition of the neuregulin-ErbB pathway

Abstract

Aims: A significant increase in congestive heart failure (CHF) was reported when the anti-ErbB2 antibody trastuzumab was used in combination with the chemotherapy drug doxorubicin (Dox). The aim of the present study was to investigate the role(s) of miRNAs in acute Dox-induced cardiotoxicity.

Methods and results: Neuregulin-1-ErbB signalling is essential for maintaining adult cardiac function. We found a significant reduction in ErbB4 expression in the hearts of mice after Dox treatment. Because the proteasome pathway was only partially involved in the reduction of ErbB4 expression, we examined the involvement of microRNAs (miRs) in the reduction of ErbB4 expression. miR-146a was shown to be up-regulated by Dox in neonatal rat cardiac myocytes. Using a luciferase reporter assay and overexpression of miR-146a, we confirmed that miR-146a targets the ErbB4 3'UTR. After Dox treatment, overexpression of miR-146a, as well as that of siRNA against ErbB4, induced cell death in cardiomyocytes. Re-expression of ErbB4 in miR-146a-overexpressing cardiomyocytes ameliorated Dox-induced cell death. To examine the loss of miR-146a function, we constructed 'decoy' genes that had tandem complementary sequences for miR-146a in the 3'UTR of a luciferase gene. When miR-146a 'decoy' genes were introduced into cardiomyocytes, ErbB4 expression was up-regulated and Dox-induced cell death was reduced.

Conclusion: These findings suggested that the up-regulation of miR-146a after Dox treatment is involved in acute Dox-induced cardiotoxicity by targeting ErbB4. Inhibition of both ErbB2 and ErbB4 signalling may be one of the reasons why those patients who receive concurrent therapy with Dox and trastuzumab suffer from CHF.

Figure 1

ErbB4 expression was decreased after Dox treatment in vivo and in vitro. (A) Immunoblotting for ErbB4 and ErbB2 in the hearts of mice after intraperitoneal injection of Dox (20 mg/g). (B) Densitometry for ErbB4 after intraperitoneal injection of Dox. Values are the means ± SE of three independent experiments (^##P < 0.01 vs. 0 h). (C) Immunoblotting for ErbB4 and ErbB2 in NRCMs after Dox treatment. (D) Immunoblotting for ErbB4 in NRCMs after Dox treatment. Because the antibody against ErbB4 recognizes the C-terminus of ErbB4, full length of ErbB4 is present at 180 kDa and cleaved-ErbB4 is present at 80 kDa. Note that cleaved-ErbB4 was not seen at 80 kDa. (E) NRCMs were pre-treated with or without proteasome inhibitor MG132 for 1 h and then treated with Dox for 24 h.

Figure 2

ErbB4 is a target for miR-146a. (A) qRT-PCR for miR-146a after Dox for 16 h at the indicated concentrations in NRCMs. Values are the means ± SE of three to four independent experiments (^#P < 0.05, ^##P < 0.01). (B) qRT-PCR for miR-146a after Dox (1 µM) for the indicated times in NRCMs. Values are the means ± SE of three to four independent experiments (^#P < 0.05). (C) Immunoblotting for ErbB4 and ErbB2 in miR-control or miR-146a-overexpressing NRCMs. (D) Densitometry for ErbB4 in miR-control or miR-146a-overexpressing NRCMs (^#P < 0.05). (E) qRT-PCR analysis for ErbB4 in miR-control or miR-146a-overexpressing NRCMs. Values are the means ± SE of four independent experiments (n.s.; not significant). (F) ErbB4 3′UTR Firefly luciferase (F-luc) activity at binding site 3 in 293T cells. Values are the means ± SE of four independent experiments (^#P < 0.05). (G) ErbB4 3′UTR F-luc activity at binding site 3 in 293T cells. Values are the means ± SE of four independent experiments (^#P < 0.05). (H) ErbB4 3′UTR F-luc activity at binding site 3 in NRCMs. Values are the means ± SE of six independent experiments (^#P < 0.05).

Figure 3

Both miR-146a overexpression and ErbB4 knockdown reduced NRCMs survival after Dox treatment. (A) MTT assay of NRCMs transduced with miR-control or miR-146a with or without Dox treatment for 24 h. Values are the means ± SE of six independent experiments (^#P < 0.05, ^##P < 0.01). (B) Immunoblotting for ErbB4, bcl-2, and cleaved caspase 3 after Dox in NRCMs. (C) Immunoblotting for ErbB4 in NRCMs transduced with control-siRNA (scrambled) or ErbB4-siRNAs. (D) Densitometry for ErbB4 in NRCMs transduced with ErbB4-siRNA. Values are the means ± SE of three independent experiments (^#P < 0.05, ^##P < 0.01). (E) MTT assay of NRCMs transduced with control-siRNA or ErbB4-siRNA with or without Dox treatment for 24 h. Values are the means ± SE of six independent experiments (^##P < 0.01). (F) Immunoblotting for ErbB4, pAkt, tAkt, and cleaved caspase 3 in NRCMs, infected with control-siRNA or ErbB4-siRNAs with or without Dox treatment for 24 h. (G) Flow cytometric analysis of TMRE in NRCMs transduced with control-siRNA or ErbB4-siRNA with or without Dox treatment for 24 h. The X-axis represents TMRE intensity (PE intensity) and the Y-axis represents cell number. Red populations indicate the TMRE high group and blue populations indicate the TMRE low group. (H) The ratio of TMRE intensity with Dox compared with without Dox for each group. Values are the means ± SE of three to four independent experiments (^#P < 0.05).

Figure 4

miR-146a enhanced Dox-induced apoptosis in NRCMs. (A) Immunoblotting for ErbB4, pAkt, tAkt, bcl-2, and cleaved caspase 3 in NRCMs transduced with miR-control, miR-146a, or ErbB4-siRNA2 in the presence or absence of Dox for the indicated time periods. (B) Flow cytometric analysis of TMRE in NRCMs transduced with miR-control of miR-146a with or without Dox for 24 h. (C) The ratio of TMRE intensity with Dox compared with without Dox for each group. Values are the means ± SE of three to four independent experiments (^##P < 0.01). (D) Immunoblotting for cleaved caspase 3 in miR-control or miR-146a-overexpressing NRCMs infected with either lacZ or ErbB4 with or without Dox for 24 h. (E) Densitometry for cleaved caspase 3 in miR-control or miR-146a-overexpressing NRCMs infected with either lacZ or ErbB4 with or without Dox for 24 h. Values are the means ± SE of four independent experiments (^#P < 0.05, ^##P < 0.01).

Figure 5

Reduction in endogenous miR-146a ameliorated Dox-induced apoptosis in NRCMs. (A) qRT-PCR analysis for miR-146a in NRCMs, infected with control-decoy (no binding site) or decoy-miR-146a (anti-miR-146a × 6) using lentivirus vector. Values are the means ± SE of three independent experiments (^##P < 0.05). (B) Immunoblotting for ErbB4 in NRCMs infected with control-decoy or decoy-miR-146a. (C) Densitometry for ErbB4 in NRCMs infected with control-decoy or decoy-miR-146a. Values are the means ± SE of four independent experiments (^#P < 0.05). (D) Immunoblotting for ErbB4, bcl-2, and cleaved caspase 3 in NRCMs infected with control or decoy gene in the presence or absence of Dox for the indicated time periods. (E) Densitometry for ErbB4 in NRCMs infected with control or decoy gene in the presence or absence of Dox. Values are the means ± SE of four independent experiments (^#P < 0.05). (F) Densitometry for cleaved caspase 3 in NRCMs infected with control or decoy gene in the presence or absence of Dox. Values are the means ± SE of four independent experiments (^#P < 0.05).

Figure 6

AnnexinV and PI staining of NRCMs. (A) Dot-plot analysis of AnnexinV/PI staining showed that apoptotic cells (Q4) and dead cells (Q2 + Q4) were increased after Dox treatment for 24 h. (B) The percentages of apoptotic cells after Dox treatment. (C) The percentages of dead cells after Dox treatment. (D) The percentages of live cells after Dox treatment. Values are the means ± SE of six independent experiments (^#P < 0.05, ^##P < 0.01).