1. Figure 1 HSP70 specifically binds to S394-phosphorylated HDAC2 for the maintenance of HDAC2 S394 phosphorylation (A) ...
Figure 1 HSP70 specifically binds to S394-phosphorylated HDAC2 for the maintenance of HDAC2 S394 phosphorylation (A) peptide-precipitation. Biotin-conjugated HDAC2 phospho-mimic peptide (S394E) was mixed with cell lysate from AoB heart. Phospho-dead peptide (S394A) served as a negative control. Proteins that bound to each peptide were visualized by Coomassie brilliant blue staining. Arrowhead indicates HSP70. (B) Western blot was performed after scramble, S394A, and S394E peptide-precipitation with cell lysate from AoB heart. (C) Immunoprecipitation. Constructs with V5-tagged wild type (Hdac2 WT-V5), phospho-resistant (Hdac2 S394A-V5), or phospho-mimic (Hdac2 S394E-V5) were co-transfected with HSP70-myc in H9c2 cells. Cell lysates were immunoprecipitated (IP) with anti-V5 antibody followed by immunoblotting (IB) with anti-myc antibody. Hdac2 S394E-V5 preferentially bound to exogenously transfected HSP70-myc. (D, E) Transfection of HSP70-myc in H9c2 cells enhanced Hdac2 S394 phosphorylation (D), whereas knocking down by siRNA attenuated it in HEK293 cells (E). (F) GST-HSP70 does not directly phosphorylate Hdac2 in the cell-free condition. GST-HSP70 failed to induce phosphorylation of GST-Hdac2 (2nd lane). Both GST-HSP70 and GST-Hdac2 were mixed and Hdac2 S394 phosphorylation was measured by antibody. Note that addition of cardiomyocyte lysates induced the phosphorylation. (G) GST-HSP70 induced Hdac2 phosphorylation in H9c2 lysates. PPi, protein phosphatase inhibitor. (A–G) All results shown in the figures are representative confirmed by three independent sets of western blot analysis and the quantification results of band intensity are shown in Supplementary material online, Figures S1 and S2.
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2. Figure 2 Modulation of HDAC2 S394 phosphorylation by either CK2α1 or PP2CA and association with HSP70. (A) ...
Figure 2 Modulation of HDAC2 S394 phosphorylation by either CK2α1 or PP2CA and association with HSP70. (A) Transfection of HA-tagged CK2α1 enhanced the interaction between Hsp70 and Hdac2 in H9c2 cells. (B) Pp2ca did not bind to Hsp70. (C) Transfection of HA-PP2CA reduced the association of Hdac2 with Hsp70 in H9c2 cells. (D) Transfection of HSP70-myc prevented Hdac2 from binding to Pp2ca in H9c2 cells. (E) Knocking down of HSP70 with HSP70 siRNA enhanced the binding of Pp2ca to Hdac2 in HEK293 cells. (A–E) The physical interactions are representative of three independent set of co-immunoprecipitation.
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3. Figure 3 Phenotypes of transgenic overexpression and knockout of HSP70
Figure 3 Phenotypes of transgenic overexpression and knockout of HSP70. (A) Hsp70-transgenic mouse (TgHsp70) did not ...
Figure 3 Phenotypes of transgenic overexpression and knockout of HSP70. (A) Hsp70-transgenic mouse (TgHsp70) did not show cardiac hypertrophy as determined by HW/BW. Non-Tg: 14 mice. TgHsp70: 11 mice. The two-tailed Student’s t-test. (B) The responsiveness to ISP in wild type, Hsp70 knockout (Hsp70 KO), and TgHsp70. ISP (30 mg/kg/day) was administered for 14 days. Wild type/vehicle: 12 mice. Wild type/ISP: 11 mice. Hsp70 KO/vehicle: six mice. Hsp70 KO/ISP: seven mice. TgHsp70/vehicle: six mice. TgHsp70/ISP: eight mice. **P < 0.01. NS, not significant. One-way ANOVA followed by Dunnett T3 post hoc test. Dots represent individual mouse data. (C) ISP-induced Hdac2 S394 phosphorylation was blunted in Hsp70 KO hearts. (D) ISP failed to activate Hdac2 in Hsp70 KO hearts. HDAC activity was measured in the Hdac2-immunoprecipitates from individual mice. IgG: five mice. Wild type/vehicle: 10 mice. Wild type/ISP: nine mice. Hsp70 KO/vehicle: nine mice. Hsp70/ISP: 4 mice. **P < 0.01. NS, not significant. One-way ANOVA followed by Dunnett T3 post hoc test. (E) Increase in the phosphorylation of Hdac2 S394 in TgHsp70 hearts. (F) HDAC2 activity in TgHsp70 hearts. IgG: four mice. Non-Tg: five mice. TgHsp70: six mice. **P < 0.01. One-way ANOVA followed by Tukey’s post hoc test. (G) Changes in the interaction between Hdac2 and protein phosphatase 2A, catalytic subunit (PP2CA) in Hsp70 KO mice. HW/BW, heart weight to body weight ratio. Each dot represents one individual animal. (C, E, and G) The representative images are confirmed by three of independent experiments and quantification of band intensity for the western blot and immunoprecipitation are shown in Supplementary material online, Figure S4.
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4. Figure 4 Pharmacologic intervention with HSP70 blocks PE-induced enlargement of NRVCs by inhibition of HDAC2 ...
Figure 4 Pharmacologic intervention with HSP70 blocks PE-induced enlargement of NRVCs by inhibition of HDAC2 phosphorylation and activation. (A) Determination of 2-phenylethynesulfoamide (PES), an HSP70 inhibitor, doses for HSP70-inhibition in rat NRVCs. Immunoprecipitation assay of Hsp70 and Hdac2. PES reduced the binding in a dose-dependent fashion. (B) NRVCs were treated with PE (100 μmol) and/or PES (0.5 μmol or 2 μmol, respectively). Cells were visualized by immunocytochemical analysis with anti-α-actinin antibody. Scale bar: 15 μm. (C) Quantitative examination of cell size measurement of cardiomyocytes from five independent isolations administrated with vehicle (n = 55 cells), PES 0.5 (n = 72 cells), PES 2 (n = 62 cells), PE (n = 189 cells), PES 0.5 μmol/PE (n = 212 cells), and PES 2 μmol/PE (n = 65 cells). ***P < 0.001 vs. vehicle, vs. PE, ^#P < 0.05 vs. PES 0.5 μmol, one-way ANOVA followed by Dunnett T3 post hoc. Dots represent individual cell size. PE, phenylephrine. (D) PE-induced Hdac2 phosphorylation was attenuated in PES-treated NRVCs. (E) HDAC2 enzymatic activity from six experimental set. *** P < 0.001 vs. vehicle only. vs. PE, one-way ANOVA followed by Tukey’s post hoc. (A and D) Representative images of three independent set are shown. Quantifications are available in Supplementary material online, Figure S5.
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5. Figure 5 Pharmacologic intervention with HSP70 blocks cardiac hypertrophy in vivo animal model. (A) Protocol for the ...
Figure 5 Pharmacologic intervention with HSP70 blocks cardiac hypertrophy in vivo animal model. (A) Protocol for the administration of PES in the long-term AoB model (14 days). (B) PES blocks AoB-induced cardiac hypertrophy. Vehicle: seven mice. PES: five mice. AoB: five mice. AoB/PES: five mice. **P < 0.01 vs. sham/vehicle, vs. AoB, one-way ANOVA followed by Tukey’s post hoc test. (C) Representative M-mode echocardiogram. Large-scale images are available in Supplementary material online, Figure S7. (D, E) Masson Trichrome staining (D, scale bar: 300 μm) and the quantification results from five individual mice (E). **P < 0.01 vs. sham/vehicle, vs. AoB/vehicle, one-way ANOVA followed by Dunnett T3 post hoc test. (F) TGF-β-induced expression of α-SMA in mouse adult cardiac fibroblast. Scale bar: 15 μm.
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6. Figure 6 PES blocks cardiac hypertrophy and HDAC2 S394 phosphorylation in an in vivo model. (A) In vivo dosing ...
Figure 6 PES blocks cardiac hypertrophy and HDAC2 S394 phosphorylation in an in vivo model. (A) In vivo dosing schedule of PES with short-term AoB. (B) PES dose-dependent attenuation of cardiac hypertrophy induced by short-term AoB. Sham: nine mice. PES 4 mg/kg: four mice. PES 12 mg/kg: four mice. PES 40 mg/kg: five mice. AoB: 11 mice. AoB/PES 4 mg/kg: five mice. AoB/PES 12 mg/kg: four mice. AoB/PES 40 mg/kg: five mice. ***P < 0.001 vs. sham, vs. AoB, one-way ANOVA followed by Tukey’s post hoc test. (C) Upper two panels: Hsp70 induction and Hdac2 S394 phosphorylation in heart subjected to short-term AoB. Lower two panels: Immunoprecipitation analysis of Hsp70 and Hdac2 in the presence of PES in hearts subjected to AoB. (D) HDAC2 enzyme activity in three independent mouse hearts. ***P < 0.001 vs. sham, vs. AoB, one-way ANOVA followed by Tukey’s post hoc test. (E) PES did not block p53 activity in NRVCs. Western blot analysis with anti-phospho-p53, an active form of p53. (C and E) Representative images of three independent set are shown. Quantification results are shown in Supplementary material online, Figure S9.
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7. Figure 7 Diagram. Hypertrophic stress-induced phosphorylation of HDAC2 enhances the binding of HDAC2 to HSP70, which ...
Figure 7 Diagram. Hypertrophic stress-induced phosphorylation of HDAC2 enhances the binding of HDAC2 to HSP70, which is required for HDAC2 activation and the ensuing cardiac hypertrophy.
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