E2/ER β inhibit ISO-induced cardiac cellular hypertrophy by suppressing Ca2+-calcineurin signaling

Figures

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  Fig 1. Isoproterenol induces myocardial cell hypertrophy via calcineurin related pathway. H9c2 cells were incubated with ISO (50μM) for 24 hours, and then actin staining and western blotting were performed. A: Representative blots showing levels of p-CaMKIISer286, CaMKII, p-GATA4Ser262, GATA4, p-NFATc3Ser344, NFATc3, ANP, BNP and α-tubulin detected by Western blotting. B, Western blots showing levels of ANP and BNP proteins in H9c2 cells and β-MHC and BNP levels in NRVMs and fluorescent microscope images show actin stained H9c2 showing hypertrophic effect when treated with ISO.

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  Fig 2. E2/ERβ inhibits ISO-induced cellular hypertrophy in Tet-on ERβ H9c2 myocardial cells. Tet-on/ERα H9c2 cardiacmyobalst cells were incubated with Dox (1μg/ml) and E2 (10-8M) in presence or absence of ISO (50μM) and ICI (0.5μM) for 24 hour, then Actin and DAPI double-staining were performed. A: The images were detected by fluorescent microscope. B: The cardiomyoblast cells area were measured from one independent experiment. Mean ± S.D., n = 4.

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  Fig 3. E2/ERβ inhibits ISO-induced NFAT activation in H9c2 myocardial cells. A: Tet-on ERβ H9c2 cells were incubated with E2 (10-8M), Dox (2μg/ml), ICI (0.5μM) in the presence of ISO (50μM) for 24 hours, then western blotting was performed, BNP and α-tubulin were detected. B: H9c2 cells were incubated with E2 (10-8M), ERα inhibitor MPP (1μM), ERβ inhibitor PHTPP (1μM) in the presence of ISO (50μM) for 24 hours, then cytosol/nuclear isolation assay was performed. To extract cytosolic and nuclear proteins by nuclei extraction assay, then NFATc3, HDAC1 and α-tubulin were detected by Western blot. The statistical results were shown from three independent experiments; mean ± S.D., n = 3. (***p < 0.001 significant differences from ISO group; ##p < 0.01 significant differences from ISO group).

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  Fig 4. E2/ERβ inhibit ISO-induced myocardial cell hypertrophy through calcineurin suppression. A: H9c2 cells were incubated with E2 (10-8M), CsA (1μM) in the presence of ISO (50μM) for 24 hours, then western blotting and actin staining were performed. BNP and α-tubulin were detected by Western blot. The images were detected by fluorescent microscope. The statistical results were shown from three independent experiments; mean ± S.D., n = 3. (***p < 0.001 significant differences from Control group; ##p < 0.01 significant differences from ISO group). B: H9c2 cells were incubated with E2 (10-8M), ISO (50μM), PHTPP (1μM) in the presence of CsA (1μM) for 24 hours, then western blotting was performed. p-NFATc3Ser344 and β-actin were detected by Western blot. C: NRVM cells were incubated with E2 (10-8M), PHTPP (1μM) in the presence of ISO (50μM) for 24 hours. After treatments, then western blotting was performed. p-NFATc3Ser344 and β-actin were detected by Western blot.

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  Fig 5. E2/ERβ inhibits ISO-induced calcineurin activity through SR Ca2+ cycling modification. A: H9c2 cells were incubated with E2 (10-8M), CsA (1μM) in the presence of ISO (50μM) for 24 hours, then western blotting and calcium staining were performed. PP1α, p-PLBSer16, p-I1Ser35 and α-tubulin were detected by Western blot. The images were detected by fluorescent microscope. B, C: Tet-on ERβ H9c2 cells were incubated with E2 (10-8M), Dox (2μg/ml), ICI (0.5μM), ISO (50μM) in the presence of BATPA (1μM) for 24 hours, then western blotting and fluo-4AM calcium staining were performed. PP1α, p-PLBSer16 and α-tubulin were detected by Western blot. The images were detected by fluorescent microscope. D: H9c2 cells were incubated with E2 (10-8M), MPP (1μM), PHTPP (1μM) in the presence of ISO (50μM) for 24 hours, then immunoprecipitation assay was performed. CaM, calcineurin, p-NFATc3Ser344 and α-tubulin were detected by Western blot.

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  Fig 6. E2/ER β inhibit ISO-induced myocardial cell hypertrophy through Ca2+-calcineurin signaling suppression.