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RhNRG-1β Protects the Myocardium against Irradiation-Induced Damage via the ErbB2-ERK-SIRT1 Signaling Pathway.

Gu A, Jie Y, Sun L, Zhao S, E M, You Q - PLoS ONE (2015)

Bottom Line: Radiation-induced heart disease (RIHD), which is a serious side effect of the radiotherapy applied for various tumors due to the inevitable irradiation of the heart, cannot be treated effectively using current clinical therapies.By activating ErbB2, rhNRG-1β maintained mitochondrial integrity, ATP production, respiratory chain function and the Krebs cycle status in irradiated cardiomyocytes.Moreover, the protection of irradiated cardiomyocytes and myocardium tissue by rhNRG-1β was at least partly mediated by the activation of the ErbB2-ERK-SIRT1 signaling pathway.

View Article: PubMed Central - PubMed

Affiliation: Department of Radiotherapy, the Affiliated Tumor Hospital of Harbin Medical University, Harbin, Heilongjiang, China.

ABSTRACT
Radiation-induced heart disease (RIHD), which is a serious side effect of the radiotherapy applied for various tumors due to the inevitable irradiation of the heart, cannot be treated effectively using current clinical therapies. Here, we demonstrated that rhNRG-1β, an epidermal growth factor (EGF)-like protein, protects myocardium tissue against irradiation-induced damage and preserves cardiac function. rhNRG-1β effectively ameliorated irradiation-induced myocardial nuclear damage in both cultured adult rat-derived cardiomyocytes and rat myocardium tissue via NRG/ErbB2 signaling. By activating ErbB2, rhNRG-1β maintained mitochondrial integrity, ATP production, respiratory chain function and the Krebs cycle status in irradiated cardiomyocytes. Moreover, the protection of irradiated cardiomyocytes and myocardium tissue by rhNRG-1β was at least partly mediated by the activation of the ErbB2-ERK-SIRT1 signaling pathway. Long-term observations further showed that rhNRG-1β administered in the peri-irradiation period exerts continuous protective effects on cardiac pump function, the myocardial energy metabolism, cardiomyocyte volume and interstitial fibrosis in the rats receiving radiation via NRG/ErbB2 signaling. Our findings indicate that rhNRG-1β can protect the myocardium against irradiation-induced damage and preserve cardiac function via the ErbB2-ERK-SIRT1 signaling pathway.

No MeSH data available.


Related in: MedlinePlus

rhNRG-1β rescues radiation-induced myocardium injury via ErbB2 signaling.(A)&(C) Immunofluorescence images showing γH2AX (red) in the nuclei (blue) of cultured adult rat cardiomyocytes (A) and rat myocardial tissue (C). (B)&(D) Western blotting analyses of γH2AX levels in the adult rat cardiomyocyte cultures (B) and rat myocardial tissue (D). β-actin expression was analyzed as an internal control. For (B) and (D), data are expressed as mean ± SEM; statistical significance is determined by one-way ANOVA and the following Bonferroni’s multiple comparisons; bdi, below detectable limit; ***, P<0.001; n = 5 per group. Scale bar: (A) = 25 μm; (C) = 100 μm. Abbreviation: ANOVA, analysis of variance; bdi, below detectable limit; DAPI, 4′,6′-diamidino-2-phenylindole; ErbB2, human epidermal growth factor receptor-2; H+NRG, Herceptin plus recombinant human neuregulin; M+NRG, Mubritinib plus recombinant human neuregulin; NRG, recombinant human neuregulin; SEM, standard error of mean.
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pone.0137337.g001: rhNRG-1β rescues radiation-induced myocardium injury via ErbB2 signaling.(A)&(C) Immunofluorescence images showing γH2AX (red) in the nuclei (blue) of cultured adult rat cardiomyocytes (A) and rat myocardial tissue (C). (B)&(D) Western blotting analyses of γH2AX levels in the adult rat cardiomyocyte cultures (B) and rat myocardial tissue (D). β-actin expression was analyzed as an internal control. For (B) and (D), data are expressed as mean ± SEM; statistical significance is determined by one-way ANOVA and the following Bonferroni’s multiple comparisons; bdi, below detectable limit; ***, P<0.001; n = 5 per group. Scale bar: (A) = 25 μm; (C) = 100 μm. Abbreviation: ANOVA, analysis of variance; bdi, below detectable limit; DAPI, 4′,6′-diamidino-2-phenylindole; ErbB2, human epidermal growth factor receptor-2; H+NRG, Herceptin plus recombinant human neuregulin; M+NRG, Mubritinib plus recombinant human neuregulin; NRG, recombinant human neuregulin; SEM, standard error of mean.

Mentions: γH2AX is considered to be the specific marker of DNA double-strand break and an important indicator of radiation-induced cellular injury, the level of which is tightly associated cardiac function in injured heart [26, 27]. To investigate whether rhNRG-1β influences the cardiomyocyte injury caused by irradiation, irradiated cardiomyocytes were subjected to immunofluorescence staining for γH2AX 6 h after irradiation (Fig 1A, S1A Fig). In contrast to the blank control (Control), cultured cardiomyocytes subjected to a single 5 Gy dose of X-ray radiation showed obvious upregulation of nuclear γH2AX levels (Vehicle, P<0.001 versus Control; n = 5 per group). The fluorescence intensity of γH2AX in the nuclei of cardiomyocytes pretreated with rhNRG-1β was markedly reduced (NRG, P<0.001 versus Vehicle; n = 5 per group), which indicated attenuation of irradiation-induced damage. However, when cardiomyocytes were exposed to Herceptin (as a ErbB2 receptor antagonist) before rhNRG-1β treatment, the protection of cells against radiation-induced injury by rhNRG-1β was nearly abolished (H+NRG, P<0.001 versus NRG, P>0.05 versus Vehicle; n = 5 per group), indicating that rhNRG-1β functions via ErbB2. The irradiation-protective effects of rhNRG-1β was also abrogated by Mubritinib (a selective small molecule inhibitor of ErbB2), which rules out the specific effects of Herceptin (M+NRG, P<0.001 versus NRG, P>0.05 versus Vehicle, P>0.05 versus H+NRG; n = 5 per group). Consistent phenomena were observed in the myocardial tissue of irradiated rats (Fig 1C, S1B Fig). Immunohistochemical staining performed 6 h after radiation also showed that rhNRG-1β pretreatment ameliorated the myocardial injury caused by irradiation, but ErbB2 blockage by Herceptin nearly eliminated the protective effects of rhNRG-1β on the myocardium (Vehicle, P<0.001 versus Control; NRG, P<0.001 versus Vehicle; H+NRG, P<0.001 versus NRG, P>0.05 versus Vehicle; n = 5 per group). The rhNRG-1β/ErbB2-mediated protection of cultured adult cardiomyocyte and myocardium tissue against irradiation was further confirmed by Western blotting analyses for γH2AX (Fig 1B and 1D). Although γH2AX-indicative DNA damage is implicated in the cell necrosis and apoptosis in myocardium [27, 28], neither necrosis-related cell lysis and inflammatory response nor typical apoptotic body and apoptosis-specific activation of caspase 3 were detected in the irradiated cardiomyocyte cultures and rat myocardium tissue (Fig 1, S2 Fig). These results suggested that rhNRG-1β rescued radiation-induced injury in cultured cardiomyocytes and rat myocardial tissue, which was mediated by ErbB2 signaling.


RhNRG-1β Protects the Myocardium against Irradiation-Induced Damage via the ErbB2-ERK-SIRT1 Signaling Pathway.

Gu A, Jie Y, Sun L, Zhao S, E M, You Q - PLoS ONE (2015)

rhNRG-1β rescues radiation-induced myocardium injury via ErbB2 signaling.(A)&(C) Immunofluorescence images showing γH2AX (red) in the nuclei (blue) of cultured adult rat cardiomyocytes (A) and rat myocardial tissue (C). (B)&(D) Western blotting analyses of γH2AX levels in the adult rat cardiomyocyte cultures (B) and rat myocardial tissue (D). β-actin expression was analyzed as an internal control. For (B) and (D), data are expressed as mean ± SEM; statistical significance is determined by one-way ANOVA and the following Bonferroni’s multiple comparisons; bdi, below detectable limit; ***, P<0.001; n = 5 per group. Scale bar: (A) = 25 μm; (C) = 100 μm. Abbreviation: ANOVA, analysis of variance; bdi, below detectable limit; DAPI, 4′,6′-diamidino-2-phenylindole; ErbB2, human epidermal growth factor receptor-2; H+NRG, Herceptin plus recombinant human neuregulin; M+NRG, Mubritinib plus recombinant human neuregulin; NRG, recombinant human neuregulin; SEM, standard error of mean.
© Copyright Policy
Related In: Results  -  Collection

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pone.0137337.g001: rhNRG-1β rescues radiation-induced myocardium injury via ErbB2 signaling.(A)&(C) Immunofluorescence images showing γH2AX (red) in the nuclei (blue) of cultured adult rat cardiomyocytes (A) and rat myocardial tissue (C). (B)&(D) Western blotting analyses of γH2AX levels in the adult rat cardiomyocyte cultures (B) and rat myocardial tissue (D). β-actin expression was analyzed as an internal control. For (B) and (D), data are expressed as mean ± SEM; statistical significance is determined by one-way ANOVA and the following Bonferroni’s multiple comparisons; bdi, below detectable limit; ***, P<0.001; n = 5 per group. Scale bar: (A) = 25 μm; (C) = 100 μm. Abbreviation: ANOVA, analysis of variance; bdi, below detectable limit; DAPI, 4′,6′-diamidino-2-phenylindole; ErbB2, human epidermal growth factor receptor-2; H+NRG, Herceptin plus recombinant human neuregulin; M+NRG, Mubritinib plus recombinant human neuregulin; NRG, recombinant human neuregulin; SEM, standard error of mean.
Mentions: γH2AX is considered to be the specific marker of DNA double-strand break and an important indicator of radiation-induced cellular injury, the level of which is tightly associated cardiac function in injured heart [26, 27]. To investigate whether rhNRG-1β influences the cardiomyocyte injury caused by irradiation, irradiated cardiomyocytes were subjected to immunofluorescence staining for γH2AX 6 h after irradiation (Fig 1A, S1A Fig). In contrast to the blank control (Control), cultured cardiomyocytes subjected to a single 5 Gy dose of X-ray radiation showed obvious upregulation of nuclear γH2AX levels (Vehicle, P<0.001 versus Control; n = 5 per group). The fluorescence intensity of γH2AX in the nuclei of cardiomyocytes pretreated with rhNRG-1β was markedly reduced (NRG, P<0.001 versus Vehicle; n = 5 per group), which indicated attenuation of irradiation-induced damage. However, when cardiomyocytes were exposed to Herceptin (as a ErbB2 receptor antagonist) before rhNRG-1β treatment, the protection of cells against radiation-induced injury by rhNRG-1β was nearly abolished (H+NRG, P<0.001 versus NRG, P>0.05 versus Vehicle; n = 5 per group), indicating that rhNRG-1β functions via ErbB2. The irradiation-protective effects of rhNRG-1β was also abrogated by Mubritinib (a selective small molecule inhibitor of ErbB2), which rules out the specific effects of Herceptin (M+NRG, P<0.001 versus NRG, P>0.05 versus Vehicle, P>0.05 versus H+NRG; n = 5 per group). Consistent phenomena were observed in the myocardial tissue of irradiated rats (Fig 1C, S1B Fig). Immunohistochemical staining performed 6 h after radiation also showed that rhNRG-1β pretreatment ameliorated the myocardial injury caused by irradiation, but ErbB2 blockage by Herceptin nearly eliminated the protective effects of rhNRG-1β on the myocardium (Vehicle, P<0.001 versus Control; NRG, P<0.001 versus Vehicle; H+NRG, P<0.001 versus NRG, P>0.05 versus Vehicle; n = 5 per group). The rhNRG-1β/ErbB2-mediated protection of cultured adult cardiomyocyte and myocardium tissue against irradiation was further confirmed by Western blotting analyses for γH2AX (Fig 1B and 1D). Although γH2AX-indicative DNA damage is implicated in the cell necrosis and apoptosis in myocardium [27, 28], neither necrosis-related cell lysis and inflammatory response nor typical apoptotic body and apoptosis-specific activation of caspase 3 were detected in the irradiated cardiomyocyte cultures and rat myocardium tissue (Fig 1, S2 Fig). These results suggested that rhNRG-1β rescued radiation-induced injury in cultured cardiomyocytes and rat myocardial tissue, which was mediated by ErbB2 signaling.

Bottom Line: Radiation-induced heart disease (RIHD), which is a serious side effect of the radiotherapy applied for various tumors due to the inevitable irradiation of the heart, cannot be treated effectively using current clinical therapies.By activating ErbB2, rhNRG-1β maintained mitochondrial integrity, ATP production, respiratory chain function and the Krebs cycle status in irradiated cardiomyocytes.Moreover, the protection of irradiated cardiomyocytes and myocardium tissue by rhNRG-1β was at least partly mediated by the activation of the ErbB2-ERK-SIRT1 signaling pathway.

View Article: PubMed Central - PubMed

Affiliation: Department of Radiotherapy, the Affiliated Tumor Hospital of Harbin Medical University, Harbin, Heilongjiang, China.

ABSTRACT
Radiation-induced heart disease (RIHD), which is a serious side effect of the radiotherapy applied for various tumors due to the inevitable irradiation of the heart, cannot be treated effectively using current clinical therapies. Here, we demonstrated that rhNRG-1β, an epidermal growth factor (EGF)-like protein, protects myocardium tissue against irradiation-induced damage and preserves cardiac function. rhNRG-1β effectively ameliorated irradiation-induced myocardial nuclear damage in both cultured adult rat-derived cardiomyocytes and rat myocardium tissue via NRG/ErbB2 signaling. By activating ErbB2, rhNRG-1β maintained mitochondrial integrity, ATP production, respiratory chain function and the Krebs cycle status in irradiated cardiomyocytes. Moreover, the protection of irradiated cardiomyocytes and myocardium tissue by rhNRG-1β was at least partly mediated by the activation of the ErbB2-ERK-SIRT1 signaling pathway. Long-term observations further showed that rhNRG-1β administered in the peri-irradiation period exerts continuous protective effects on cardiac pump function, the myocardial energy metabolism, cardiomyocyte volume and interstitial fibrosis in the rats receiving radiation via NRG/ErbB2 signaling. Our findings indicate that rhNRG-1β can protect the myocardium against irradiation-induced damage and preserve cardiac function via the ErbB2-ERK-SIRT1 signaling pathway.

No MeSH data available.


Related in: MedlinePlus