E2/ER β Enhances Calcineurin Protein Degradation and PI3K/Akt/MDM2 Signal Transduction to Inhibit ISO-Induced Myocardial Cell Apoptosis

Figure 1. E2/ERβ inhibits ISO-induced cellular apoptosis in Tet-on ERβ H9c2 myocardial cells. Tet-on/ERα H9c2 cardiomyoblast cells were incubated with Dox (1 μg/mL) and E2 (10−8 M) in presence or absence of ISO (50 μM) and ICI (0.5 μM) for 24 h, then TUNEL and DAPI double-staining were performed. The images were detected by fluorescent microscope and the number of apoptotic nuclei was counted (Bar length = 100 μm). Mean ± S.D., n = 3. *** = p < 0.001 indicates significant difference with respect to the control group; ### = p < 0.001 indicates significant difference with respect to the ISO challenged group.

Figure 2. E2/ERβ inhibits ISO-induced mitochondria-dependent apoptosis in H9c2 myocardial cells. (A), Tet-on ERβ H9c2 cells were incubated with E2 (10−8 M), Dox (2 μg/mL), ICI (0.5 μM) in the presence of ISO (50 μM) for 24 h, then western blotting was performed. Cleaved caspase3 and α-tubulin were detected by Western blot. (B), H9c2 cells were incubated with E2 (10−8 M), MPP (1 μM), PHTPP (1 μM) in the presence of ISO (50 μM) for 24 h, then mitochondria isolation assay was performed. Cytochrome c and β-actin were detected by western blot. (*** = p < 0.001indicates significant difference with respect to the Control group; ### = p < 0.001 indicates significant difference with respect to the ISO challenged group).

Figure 3. E2/ERβ attenuates ISO induced calcium accumulation in H9c2 cells. Tet-on ERβ H9c2 cells were incubated with E2 (10−8 M), Dox (2 μg/mL) in the presence of ISO (50 μM) for 24 h, then fluo-4AM calcium staining was performed. The images were detected by fluorescent microscope (Bar length = 10 μm).

Figure 4. Calcineurin plays an important role in isoproterenol-induced cellular apoptosis signaling. H9c2 cells were incubated with E2 (10−8 M), CsA (1 μM) in the presence of ISO (50 μM) for 24 h, then western blotting was performed. Cleaved caspase3 and α-tubulin were detected by western blot. (*** = p < 0.001 indicates significant difference with respect to the control group; ### = p < 0.001 indicates significant difference with respect to the ISO challenged group).

Figure 5. E2 enhances calcineurin protein degradation via estrogen receptor β. (A) H9c2 cells were incubated with E2 (10−8 M), ISO (50 μM), PHTPP (1 μM) in the presence of protein synthesis inhibitor cycloheximide (1 μM) and proteasome inhibitor MG132 (1 μM) for 24 h, then western blotting was performed. Calcineurin and β-actin were detected by western blot. (B) NRVM cells were incubated with E2 (10-8 M), Nutlin-3 (1 μM) in the presence of ISO (50 μM) for 24 h, then western blotting was performed. Calcineurin and β-actin were detected by western blot. (*** = p < 0.001 indicates significant difference with respect to the Control group; (# = p < 0.05 and ### = p < 0.001 indicate significant differences with respect to the ISO challenged group).

Figure 6. E2/ERβ enhances calcineurin protein degradation via PI3K/Akt/MDM2 signaling. (A), H9c2 cells were incubated with MPP (1 μM), PHTPP (1 μM) in the presence of E2 (10−8 M) for 24 h, then western blotting was performed. p-Akt (Ser473), ERα, ERβ, and α-tubulin were detected by western blot. (B), H9c2 cells were incubated with E2 (10−8 M), MDM2 inhibitor Nutlin-3 (1 μM), PI3K inhibitor LY294002 (1 μM), PHTPP (1 μM) in the presence of ISO (50 μM) for 24 h, then western blotting was performed. p-Akt (Ser473), p-MDM2 (Ser166), calcineurin and β-actin were detected by Western blot. (C), Tet-on ERβ H9c2 cells were incubated with E2 (10−8 M), Dox (2 μg/mL), Nutlin-3 (1 μM), LY294002 (1 μM) in the presence of ISO (50 μM) for 24 h, then western blotting was performed. Calcineurin and β-actin were detected by Western blot. (*** = p < 0.001indicates significant difference with respect to the Control group; ### = p < 0.001 indicates significant difference with respect to the ISO challenged group, δδ = p < 0.01 and δδδ = p < 0.001 indicate significant differences with respect to the E2 treated group).