Isorhapontigenin protects against doxorubicin-induced cardiotoxicity via increasing YAP1 expression

Figures

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  Figure 1. YAP1 expression was decreased in human failing hearts and in Dox-induced rat cardiotoxicity model. The expression changes of genes related to Hippo pathway in the human hearts with dilated cardiomyopathy (A) and in Dox-stimulated NRCMs were exhibited by heatmap (B). The change of YAP1 mRNA expression in the human failing heart tissue was confirmed by qRT-PCR (C), n = 4 in control group and n = 7 in heart failure group. The change of Yap1 mRNA expression in Dox-induced rat cardiotoxicity model was confirmed by qRT-PCR (D). NRCMs were treated with Dox (1 μmol/L) at different time point (0, 6 and 12 h). The mRNA expression of Yap1 in NRCMs was measured by qRT-PCR (E). The protein expression of YAP1 in NRCMs was measured by Western blot (F) and IF assay (G, scale bar was 20 μm). The subcellular protein changes of YAP1 were measured by Western blot (H). The target genes changes of YAP1 were measured by qRT-PCR (I). Data were expressed as mean ± SEM, n = 4; ∗∗P < 0.01, ∗∗∗P < 0.001 vs. control group. Dox, doxorubicin; NRCMs, neonatal rat cardiomyocytes; qRT-PCR, quantitative real-time polymerase chain reaction; YAP1, Yes-associated protein 1; IF, immunofluorescence.

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  Figure 2. YAP1 protected against Dox-induced cardiomyocytes apoptosis. Yap1 was overexpressed by using recombinant adenovirus following Dox (1 μmol/L, 12 h) stimulation in NRCMs. The morphology of NRCMs was observed by EVOS FL Auto and scale bar was 200 μm (A). The change of cell viability was measured by CCK-8 (B). The TUNEL staining positive NRCMs and nuclear condensation were assayed and scale bar was 400 μm (C). TUNEL-positive cells were quantified and analyzed from four different views (D). The protein change of cleaved caspase 3 was measured by Western blot (E). The changes of mitochondrial membrane potential were detected by TMRE staining (F) and quantified (G). Data were expressed as mean ± SEM, n = 4. ∗P < 0.05, ∗∗P < 0.01 vs. control group; #P < 0.05, ##P < 0.01 vs. Dox group. YAP1, Yes-associated protein 1; Dox, doxorubicin; TUNEL, TdT-mediated dUTP nick end labeling; TMRE, tetramethylrhodamine ethyl ester perchlorate; CCK-8, cell counting kit-8.

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  Figure 3. YAP1 protected against Dox-induced cardiomyocytes apoptosis through TEAD1. The silent efficiency of siRNA to Tead1 was confirmed by qRT-PCR (A). The cell viability change of NRCMs was assayed by CCK-8 (B). The protein changes of cleaved caspase 3, TEAD1 and YAP1 were measured by Western blot (C). The morphology of NRCMs was observed by EVOS FL Auto and scale bar was 200 μm (D). NRCMs were stained by TUNEL and DAPI. TUNEL positive cells and nuclear condensation were observed by EVOS FL Auto, and scale bar was 400 μm (E). The change of mitochondrial membrane potential was measured by TMRE staining (F). Data were expressed as mean ± SEM, n = 4; ∗P < 0.05, ∗∗P < 0.01 vs. control group; ##P < 0.01 vs. Dox group; $$P < 0.01 vs. Dox+YAP1 group. CCK8, cell counting kit-8; TEAD1, TEA domain transcription factor proteins 1; TUNEL, TdT-mediated dUTP nick end labeling; TMRE, tetramethylrhodamine ethyl ester perchlorate; DAPI, 4′,6-diamidino-2-phenylindole; YAP1, Yes-associated protein 1.

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  Figure 4. The effects of isorhapontigenin (Isor, 1–10 μmol/L) on Dox-induced cardiomyocytes injury. Cardiomyocytes were pre-incubated different concentrations (10, 5, 2.5 and 1 μmol/L) of Isor for 1 h and co-treated with Dox (1 μmol/L) for 12 h. The cell morphology was observed by EVOS FL Auto and scale bar was 200 μm (A). Cell viability was assayed by CCK-8 (B). Representative graphs of nuclear condensation and TUNEL-staining positive cell were measured by IF and analyzed (C). The protein changes of cleaved caspase 3 were assayed by Western blot (D). Changes of mitochondrial membrane potential were assayed by TMRE staining (E). Data were expressed as mean ± SEM, n = 6; ∗∗∗P < 0.001 vs. control group; #P < 0.05, ##P < 0.01 vs. Dox group; ns means no significant differences vs. Dox group. CCK-8, cell counting kit-8; IF, immunofluorescence; TMRE, tetramethylrhodamine ethyl ester perchlorate.

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  Figure 5. Isorhapontigenin (Isor) relieved Dox-caused inhibition of YAP1 expression in NRCMs. NRCMs were pre-incubated with different concentrations of Isor (10, 5, 2.5 and 1 μmol/L) for 1 h followed by Dox stimulation for 12 h. The mRNA expression of Yap1 was measured by qRT-PCR (A). The protein expression of YAP1 was measured by Western blot (B). IF assay was used to detect the protein changes of YAP1 in NRCMs following Isor (10 μmol/L) and Dox (1 μmol/L) co-treatment for 12 h (C). Scale bar = 20 μm. Western blot results show the change of YAP1 protein in nucleic and cytoplasmic fraction of NRCMs following Isor (10 μmol/L) and Dox (1 μmol/L) co-treatment for 12 h (D). The mRNA changes of Ctgf (E) and Areg (F) were measured by qRT-PCR. Data were expressed as mean ± SEM, n = 6; ∗∗P < 0.01, ∗∗∗P < 0.001 vs. control group; #P < 0.05, ##P < 0.01 vs. Dox group; ns means no significant differences vs. Dox group. Dox, doxorubicin; NRCMs, neonatal rat cardiomyocytes; qRT-PCR, quantitative real-time polymerase chain reaction; CTGF, connective tissue growth factor; AREG, amphiregulin; YAP1, Yes-associated protein 1.

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  Figure 6. YAP1 was involved in the protection of isorhapontigenin (Isor) on Dox-induced cardiotoxic effects. NRCMs were transfected with sgRNA targeting Yap1 or treated with YAP1 inhibitor Dob (10 μmol/L) before Dox (1 μmol/L) and Isor (10 μmol/L) co-treatment. Cell viability was measured by CCK-8 (A). The cell morphology was observed by light microscopy, and scale bar was 200 μm (B) and (F). The nuclear condensation and TUNEL positive staining cells were observed and scale bar was 400 μm (C) and (G). The protein change of cleaved caspase 3 was measured by Western blot (D) and (H). The mitochondrial membrane potential changes of NRCMs were detected by TMRE staining, and scale bar was 400 μm (E) and (I). Data were expressed as mean ± SEM, n = 6; ∗∗P < 0.01 vs. control group; #P < 0.05, ##P < 0.01 vs. Dox group; $P < 0.05, $$P < 0.01 vs. Dox+Isor group. Isor, isorhapontigenin; Dox, doxorubicin; NRCMs, neonatal rat cardiomyocytes; Dob, dobutamine; TMRE, tetramethylrhodamine ethyl ester perchlorate; YAP1, Yes-associated protein 1.

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  Figure 7. Isor improved Dox-induced cardiac dysfunction in vivo. C57BL/6 mice were pre-treated with Isor (30 mg/kg/day) for 1 week followed with Dox stimulation for 2 weeks. The gross hearts from mice were shown (A). The hematoxylin–eosin (HE) staining of heart tissues was shown, and scale bar was 100 μm (B) and (C). Wheat germ agglutinin (WGA) staining of heart tissues was shown, and scale bar was 100 μm (D). Heart weight to body weight ratio (HW/BW) was shown (E), n = 7 in control group, n = 6 in Dox group and Dox+Isor group. Representative echocardiographic graphs are shown (F). The echocardiographic parameters were measured, including cardiac output (CO), ejection fraction (EF), fractional shortening (FS) (G), left ventricular anterior wall thickness (LVAW) at systolic end-stage and diastolic end-stage (H) and (I) and left ventricular posterior wall thickness (LVPW) at systolic end-stage and diastolic end-stage (J) and (K), n = 7 in control group, n = 6 in Dox group and n = 8 in Dox+Isor group. Data were expressed as mean ± SEM; ∗P < 0.05, ∗∗P < 0.01 vs. control group; #P < 0.05, ##P < 0.01 vs. Dox group. Isor, isorhapontigenin; Dox, doxorubicin.

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  Figure 8. Isor alleviated Dox-induced cardiomyocytes apoptosis and increased YAP1 expression in vivo. The heart tissue of mice was stained with TUNEL and observed by EVOS FL Auto. Representative graphs are shown and scale bar is 100 μm (A). The change of cleaved caspase 3 levels in the heart tissue of mice was measured by Western blot (B). The protein level of YAP1 in the heart of mice was assayed by IHC and scale bar is 100 μm (C). The protein and mRNA levels of Yap1 in the heart of mice were measured by Western blot (D) and qRT-PCR (E). The mRNA expressions of YAP1′ target genes (Ctgf and Areg) were assayed by qRT-PCR (F) and (G). Data were expressed as mean ± SEM, n = 6; ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001 vs. control group; #P < 0.05, ##P < 0.01 vs. Dox group. YAP1, Yes-associated protein 1; Isor, isorhapontigenin; Dox, doxorubicin; IHC, immunohistochemistry; qRT-PCR, quantitative real-time polymerase chain reaction; CTGF, connective tissue growth factor; AREG, amphiregulin.