17β-Estradiol and/or estrogen receptor alpha signaling blocks protein phosphatase 1 mediated ISO induced cardiac hypertrophy

Figures

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  Fig 1. ISO treatment leads to hypertrophic cellular morphology in H9c2 cardiomyocytes in a time-dependent manner. A, Fluorescence images of Tet-on/ERα H9c2 cells treated with ISO for 2 h, 12 h, 24 h and 36 h, respectively. As evident from actin staining, the cell surface area was enhanced after ISO stimulation in a time dependent manner. B, Quantification of cell surface area from above experiment. The bar graph represents data from three independent experiments and shows mean ± S.D. Student’s t–test was performed to arrive at significance. “**”, p < 0.01 for control versus ISO. C, Tet-on/ERα H9c2 cells were incubated with ISO, ISO plus E2 or Dox or ICI or combined treatments for 24 hours and actin staining was performed. E2 or Dox or combined treatment reduced the cell surface area, which was negated by ICI treatment. D, Tet-on/ERα H9c2 cell surface area from above experiments was quantified. The bar graph (mean ± S.D. of response) represents data from three independent experiments. 30 cells were counted per condition in each experiment. “*”, p < 0.05 for control versus ISO; “#”, or “+”, p < 0.05 for ISO versus combined treatments. E, Cells were incubated with ISO, ISO plus E2 or Dox…

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  Fig 2. ISO-induced calcium accumulation in H9c2 cardiomyoblast cells was reduced by E2 and/or ERα. Tet-on/ERα H9c2 cardiomyoblast cells were incubated with E2 and/or Dox in presence or absence of ISO for 24 h, and followed by calcium staining. The intracytoplasmic calcium accumulation upon ISO induction was reduced by E2 and/ or Dox treatment as visualized by fluorescent microscope.

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  Fig 3. PP1cα expression was enhanced by ISO treatment, which was reversed by E2 or Dox and/ or melatonin treatment. A, Tet-on/ERα H9c2 cells were incubated with different concentrations of ISO for 24 h. Western blot analysis showed that protein expression level of PP1cα was enhanced while Ser16 phospho-PLB protein was decreased in a dose dependent manner. However, protein expression of unphosphorylated PLB did not change significantly in presence of ISO alone. “**”, p < 0.05 for ISO versus control; “***”, p < 0.01 for ISO versus control. B & C, Tet-on/ERα H9c2 cells were pre-treated with either E2 or Dox or melatonin or combination treatments with or without ISO for 24 h. PP1cα expression was inhibited by E2 or Dox or melatonin treatment at both transcriptional level as determined by RT-PCR (“***”, p < 0.05; “###”, p< 0.05 versus untreated) and translational level (“*”, p < 0.05; “**”, p < 0.01) by western blot analysis, respectively.

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  Fig 4. PP1cα overexpression mediated cardiomyoblast hypertrophy was attenuated by E2 and/ or Dox treatment, which was reversed by treatment with specific ERα inhibitor. A & B, Tet-on/ERα H9c2 cells were transfected with overexpressing vectors carrying PP1cα. At 12 h, actin staining was performed which showed that cell surface area was increased by 2 folds in PP1cα transfected cells as compared to cells transfected with empty vectors alone. At 24 h, further increase in cell surface area was observed by approximately 1.5 folds as compared to 12 h and cell surface area quantification was performed. Student’s t-test, “**”, p < 0.01 for control versus PP1cα 12 h; “##”, p < 0.01 for control versus PP1cα 24 h. C & D, Tet-on/ERα H9c2 cells were pre-treated with either E2 or Dox, or melatonin or combination treatments, followed by transfection with PP1cα overexpressing vectors for 24 h. As evident by actin staining, E2 or Dox or combination treatments reduced hyper growth effect induced by PP1cα overexpression. However, treatment with melatonin reversed the anti-hypertrophic effect of E2, Dox and/ or combination treatments in PP1cα transfected cells. The bar graph (mean ± S.D.) represents data…

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  Fig 5. Graphic summary. In ISO-induced cardiomyoblast hypertrophy model, we demonstrated that ISO treatment enhanced intracytoplasmic calcium accumulation which was correlated with increased PP1cα expression and E2 treatment suppressed the hypertrophic effect caused by ISO treatment. However, inhibiting E2 signaling by ICI or melatonin treatment resulted in significant loss of protective anti-hypertrophic effect. Furthermore, we showed that ISO induced cardiomyocyte hypertrophy was mediated by PP1cα over-expression and E2 treatment was sufficient to block PP1cα expression and exert protective effect against hypertrophy induced by ISO treatment.