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Early-life perturbations in glucocorticoid activity impacts on the structure, function and molecular composition of the adult zebrafish (Danio rerio) heart.

Wilson KS, Baily J, Tucker CS, Matrone G, Vass S, Moran C, Chapman KE, Mullins JJ, Kenyon C, Hadoke PW, Denvir MA - Mol. Cell. Endocrinol. (2015)

Bottom Line: GR Mo embryos (120 hpf) had smaller hearts with fewer cardiomyocytes, less mature striation pattern, reduced cardiac function and reduced levels of vmhc and igf mRNA compared with controls.GR Mo adult hearts were smaller with diminished trabecular network pattern, reduced expression of vmhc and altered echocardiographic Doppler flow compared to controls.Perturbations in GR activity during embryonic development results in short and long-term alterations in the heart.

View Article: PubMed Central - PubMed

Affiliation: The British Heart Foundation Centre for Cardiovascular Science, University of Edinburgh, The Queen's Medical Research Institute, Edinburgh EH16 4TJ, UK.

No MeSH data available.


Related in: MedlinePlus

Effects of glucocorticoid receptor morpholino and rescue on the embryonic heart Western blot analysis was carried out to determine whether glucocorticoid receptor protein (Gr) was reduced in isolated hearts from zebrafish embryos (5 days post fertilization (5 dpf)) or adults (120 dpf) which had been treated with either glucocorticoid receptor (GR) translational blocking morpholino (GR Mo) or with GR agonist dexamethasone (Dex) [100 μM] for 120 h (5 dpf). A) Densitometry of Western blots of Gr abundance in isolated hearts. Data are n = 3 (150 embryo heart or 10 adult hearts per n), given as a mean percentage of their respective controls (mism Mo for GR Mo and vehicle for Dex) ± SEM analysed by two-way ANOVA and Bonferroni post hoc test. *p ≤ 0.05. B) Example of Western blot of embryonic (5 dfp) and adult (120 dpf) heart tissue lysates probed with antibodies against Gr with alpha-tubulin (loading control), C) Success of GR Mo was further determined following co-injection of rescue mRNA with GR Mo and calculation of ventricle length at 120 h post fertilization (hpf) D) Calculation of cardiomyocyte number at 120 hpf. C and D) n = 3 (6 embryos per group). Data are mean ± SEM analysed by two-way ANOVA and Bonferroni post hoc test vs each individual group. *p ≤ 0.05 and **p ≤ 0.01. E) Densitometry of Western blots of Gr abundance in 120 hpf whole embryo homogenate. Data are n = 3 (10 embryo per n), data given as relative protein abundance for control (mism Mo), GR Mo, and Gr mRNA rescue (rescue) n = 3 mean ± SEM analysed by two-way ANOVA and Bonferroni post hoc test, *p ≤ 0.05, **p ≤ 0.01. F) Confocal images of isolated 120 hpf Tg (CMLC2: GFP) zebrafish hearts (green) co-stained with DAPI nuclear stain (blue), images are isolated from embryos which were treated with control morpholino (mism Mo), GR Mo and capped GR mRNA (rescue) co-injected with GR Mo. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
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fig3: Effects of glucocorticoid receptor morpholino and rescue on the embryonic heart Western blot analysis was carried out to determine whether glucocorticoid receptor protein (Gr) was reduced in isolated hearts from zebrafish embryos (5 days post fertilization (5 dpf)) or adults (120 dpf) which had been treated with either glucocorticoid receptor (GR) translational blocking morpholino (GR Mo) or with GR agonist dexamethasone (Dex) [100 μM] for 120 h (5 dpf). A) Densitometry of Western blots of Gr abundance in isolated hearts. Data are n = 3 (150 embryo heart or 10 adult hearts per n), given as a mean percentage of their respective controls (mism Mo for GR Mo and vehicle for Dex) ± SEM analysed by two-way ANOVA and Bonferroni post hoc test. *p ≤ 0.05. B) Example of Western blot of embryonic (5 dfp) and adult (120 dpf) heart tissue lysates probed with antibodies against Gr with alpha-tubulin (loading control), C) Success of GR Mo was further determined following co-injection of rescue mRNA with GR Mo and calculation of ventricle length at 120 h post fertilization (hpf) D) Calculation of cardiomyocyte number at 120 hpf. C and D) n = 3 (6 embryos per group). Data are mean ± SEM analysed by two-way ANOVA and Bonferroni post hoc test vs each individual group. *p ≤ 0.05 and **p ≤ 0.01. E) Densitometry of Western blots of Gr abundance in 120 hpf whole embryo homogenate. Data are n = 3 (10 embryo per n), data given as relative protein abundance for control (mism Mo), GR Mo, and Gr mRNA rescue (rescue) n = 3 mean ± SEM analysed by two-way ANOVA and Bonferroni post hoc test, *p ≤ 0.05, **p ≤ 0.01. F) Confocal images of isolated 120 hpf Tg (CMLC2: GFP) zebrafish hearts (green) co-stained with DAPI nuclear stain (blue), images are isolated from embryos which were treated with control morpholino (mism Mo), GR Mo and capped GR mRNA (rescue) co-injected with GR Mo. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Mentions: Isolated hearts from GR Mo embryos (120 hpf) were confirmed to have lower Gr protein abundance than controls, (18.60 ± 4.45% reduction compared to age matched controls p < 0.05. By adulthood however (120 dpf) there was no difference in heart Gr protein in GR Mo adults compared to controls confirming the transient nature of this genetically induced reduction in GR activity (Fig. 3A and B). In contrast hearts isolated from embryos treated with Dex showed no significant alteration in Gr abundance compared to controls at either 120 hpf or 120 dpf (Fig. 3A and B).The effects of GR Mo on heart size, cardiomyocyte number and total embryo protein expression were partially rescued by co-treatment with a gr-RNA (rescue), with heart size and cardiomyocyte number both greater than GR Mo alone, but still reduced compared to mism controls (Fig. 3C and D). In contrast to control isolated hearts (120 hpf), a lack of clear striation pattern was observed in GR Mo embryo hearts, however striation was observed in hearts isolated from embryos co-treated with GR Mo and gr-RNA (Fig. 3E).


Early-life perturbations in glucocorticoid activity impacts on the structure, function and molecular composition of the adult zebrafish (Danio rerio) heart.

Wilson KS, Baily J, Tucker CS, Matrone G, Vass S, Moran C, Chapman KE, Mullins JJ, Kenyon C, Hadoke PW, Denvir MA - Mol. Cell. Endocrinol. (2015)

Effects of glucocorticoid receptor morpholino and rescue on the embryonic heart Western blot analysis was carried out to determine whether glucocorticoid receptor protein (Gr) was reduced in isolated hearts from zebrafish embryos (5 days post fertilization (5 dpf)) or adults (120 dpf) which had been treated with either glucocorticoid receptor (GR) translational blocking morpholino (GR Mo) or with GR agonist dexamethasone (Dex) [100 μM] for 120 h (5 dpf). A) Densitometry of Western blots of Gr abundance in isolated hearts. Data are n = 3 (150 embryo heart or 10 adult hearts per n), given as a mean percentage of their respective controls (mism Mo for GR Mo and vehicle for Dex) ± SEM analysed by two-way ANOVA and Bonferroni post hoc test. *p ≤ 0.05. B) Example of Western blot of embryonic (5 dfp) and adult (120 dpf) heart tissue lysates probed with antibodies against Gr with alpha-tubulin (loading control), C) Success of GR Mo was further determined following co-injection of rescue mRNA with GR Mo and calculation of ventricle length at 120 h post fertilization (hpf) D) Calculation of cardiomyocyte number at 120 hpf. C and D) n = 3 (6 embryos per group). Data are mean ± SEM analysed by two-way ANOVA and Bonferroni post hoc test vs each individual group. *p ≤ 0.05 and **p ≤ 0.01. E) Densitometry of Western blots of Gr abundance in 120 hpf whole embryo homogenate. Data are n = 3 (10 embryo per n), data given as relative protein abundance for control (mism Mo), GR Mo, and Gr mRNA rescue (rescue) n = 3 mean ± SEM analysed by two-way ANOVA and Bonferroni post hoc test, *p ≤ 0.05, **p ≤ 0.01. F) Confocal images of isolated 120 hpf Tg (CMLC2: GFP) zebrafish hearts (green) co-stained with DAPI nuclear stain (blue), images are isolated from embryos which were treated with control morpholino (mism Mo), GR Mo and capped GR mRNA (rescue) co-injected with GR Mo. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
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fig3: Effects of glucocorticoid receptor morpholino and rescue on the embryonic heart Western blot analysis was carried out to determine whether glucocorticoid receptor protein (Gr) was reduced in isolated hearts from zebrafish embryos (5 days post fertilization (5 dpf)) or adults (120 dpf) which had been treated with either glucocorticoid receptor (GR) translational blocking morpholino (GR Mo) or with GR agonist dexamethasone (Dex) [100 μM] for 120 h (5 dpf). A) Densitometry of Western blots of Gr abundance in isolated hearts. Data are n = 3 (150 embryo heart or 10 adult hearts per n), given as a mean percentage of their respective controls (mism Mo for GR Mo and vehicle for Dex) ± SEM analysed by two-way ANOVA and Bonferroni post hoc test. *p ≤ 0.05. B) Example of Western blot of embryonic (5 dfp) and adult (120 dpf) heart tissue lysates probed with antibodies against Gr with alpha-tubulin (loading control), C) Success of GR Mo was further determined following co-injection of rescue mRNA with GR Mo and calculation of ventricle length at 120 h post fertilization (hpf) D) Calculation of cardiomyocyte number at 120 hpf. C and D) n = 3 (6 embryos per group). Data are mean ± SEM analysed by two-way ANOVA and Bonferroni post hoc test vs each individual group. *p ≤ 0.05 and **p ≤ 0.01. E) Densitometry of Western blots of Gr abundance in 120 hpf whole embryo homogenate. Data are n = 3 (10 embryo per n), data given as relative protein abundance for control (mism Mo), GR Mo, and Gr mRNA rescue (rescue) n = 3 mean ± SEM analysed by two-way ANOVA and Bonferroni post hoc test, *p ≤ 0.05, **p ≤ 0.01. F) Confocal images of isolated 120 hpf Tg (CMLC2: GFP) zebrafish hearts (green) co-stained with DAPI nuclear stain (blue), images are isolated from embryos which were treated with control morpholino (mism Mo), GR Mo and capped GR mRNA (rescue) co-injected with GR Mo. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
Mentions: Isolated hearts from GR Mo embryos (120 hpf) were confirmed to have lower Gr protein abundance than controls, (18.60 ± 4.45% reduction compared to age matched controls p < 0.05. By adulthood however (120 dpf) there was no difference in heart Gr protein in GR Mo adults compared to controls confirming the transient nature of this genetically induced reduction in GR activity (Fig. 3A and B). In contrast hearts isolated from embryos treated with Dex showed no significant alteration in Gr abundance compared to controls at either 120 hpf or 120 dpf (Fig. 3A and B).The effects of GR Mo on heart size, cardiomyocyte number and total embryo protein expression were partially rescued by co-treatment with a gr-RNA (rescue), with heart size and cardiomyocyte number both greater than GR Mo alone, but still reduced compared to mism controls (Fig. 3C and D). In contrast to control isolated hearts (120 hpf), a lack of clear striation pattern was observed in GR Mo embryo hearts, however striation was observed in hearts isolated from embryos co-treated with GR Mo and gr-RNA (Fig. 3E).

Bottom Line: GR Mo embryos (120 hpf) had smaller hearts with fewer cardiomyocytes, less mature striation pattern, reduced cardiac function and reduced levels of vmhc and igf mRNA compared with controls.GR Mo adult hearts were smaller with diminished trabecular network pattern, reduced expression of vmhc and altered echocardiographic Doppler flow compared to controls.Perturbations in GR activity during embryonic development results in short and long-term alterations in the heart.

View Article: PubMed Central - PubMed

Affiliation: The British Heart Foundation Centre for Cardiovascular Science, University of Edinburgh, The Queen's Medical Research Institute, Edinburgh EH16 4TJ, UK.

No MeSH data available.


Related in: MedlinePlus