Journal: Journal of the American Heart Association

Article Title: β‐Adrenergic Stimulation Induces Histone Deacetylase 5 (HDAC5) Nuclear Accumulation in Cardiomyocytes by B55α‐PP2A‐Mediated Dephosphorylation

doi: 10.1161/jaha.116.004861

Figure Lengend Snippet: Figure 1. b1-AR/PKA activation reduces HDAC5 phosphorylation. A, Time course of HDAC5 dephos- phorylation by 10 nmol/L isoproterenol (ISO). Representative Western blots and grouped data from 4 independent experiments. Data obtained for each phosphorylation site were analyzed by 1-way ANOVA followed by Dunnett’s multiple comparisons tests. *P<0.05 vs 0 minutes. B, ARVM were treated with 10 nmol/L ISO or vehicle control (CON) for 10 minutes in the presence of 100 nmol/L CGP-20712A (CGP; a b1-AR-selective antagonist), 100 nmol/L ICI 118,551 (ICI; a b2-AR-selective antagonist), or vehicle (VEH). Representative Western blots and grouped data from 6 independent experiments. One-way ANOVA followed by Dunnett’s multiple comparisons tests for each phosphorylation site. *P<0.05 vs VEH. C, ARVM were treated with 500 lmol/L N6-benzoyl cAMP (BNZ) to selectively activate PKA or vehicle control (CON) for 30 minutes. Representative Western blots and grouped data from 5 independent experiments (n=4 for S498 site). Unpaired t test for each phosphorylation site. *P<0.05 vs CON. b1-AR indicates b-adrenergic receptor; ARVM, adult rat ventricular myocytes; BNZ, N6-benzoyl cAMP; cTnI, cardiac troponin I; HDAC5, histone deacetylase 5; PKA, protein kinase A.

Article Snippet: The beads were pelleted and the cleared supernatants incubated with HDAC5 antibody (Cell Signaling 20458; 1:70) or water (no antibody control) with gentle rocking at 4°C overnight.

Techniques: Activation Assay, Phospho-proteomics, Western Blot, Control, Histone Deacetylase Assay

Journal: Journal of the American Heart Association

Article Title: β‐Adrenergic Stimulation Induces Histone Deacetylase 5 (HDAC5) Nuclear Accumulation in Cardiomyocytes by B55α‐PP2A‐Mediated Dephosphorylation

doi: 10.1161/jaha.116.004861

Figure Lengend Snippet: Figure 2. b-adrenergic stimulation induces nuclear accumulation of GFP-HDAC5. A, 3-Dimensional projections of an ARVM expressing GFP-HDAC5 at baseline and 45 minutes after the addition of 10 nmol/L isoproterenol (ISO). Z-stacks (25 z-sections at 1.5-lm intervals) were acquired as described in the Methods section. The fluorescent signals from the Cell Tracker Orange and DRAQ5 dyes were used to define the cytoplasmic (cyto) and nuclear (nuc) volumes for objective quantification of the average fluorescence intensity (F) arising from the GFP-HDAC5 signal within those compartments. B and C, Quantification of Fnuc and Fcyto following treatment with 10 nmol/L ISO or vehicle control (CON) for 15 to 45 minutes. All measurements obtained at the 15- and 45-minute time points were normalized to the baseline value for each cell (denoted by the dotted line). Two-way repeated-measures ANOVA followed by Sidak’s multiple comparisons tests. *P<0.05 vs CON at the same time point. †P<0.05 vs the same treatment group at the 15-minute time point. n=17 to 22 cells from 3 hearts per group. D, ARVM were fractionated into Triton X- 100-soluble (SOL) and -insoluble (INSOL) fractions via centrifugation following treatment with 10 nmol/L ISO or CON for 45 minutes. Representative Western blots show the distribution of GFP-HDAC5, GAPDH (cytosolic protein), and histone 2B (nuclear protein). Quantitative data show GFP-HDAC5 abundance in soluble and insoluble fractions from 5 independent experiments. In each experiment, the GFP-HDAC5 signal in CON and ISO fractions was normalized to the respective input. Unpaired t tests. *P<0.05 vs CON. ARVM indicates adult rat ventricular myocytes; GFP, green fluorescent protein; HDAC5, histone deacetylase 5.

Article Snippet: The beads were pelleted and the cleared supernatants incubated with HDAC5 antibody (Cell Signaling 20458; 1:70) or water (no antibody control) with gentle rocking at 4°C overnight.

Techniques: Expressing, Control, Centrifugation, Western Blot, Histone Deacetylase Assay

Journal: Journal of the American Heart Association

Article Title: β‐Adrenergic Stimulation Induces Histone Deacetylase 5 (HDAC5) Nuclear Accumulation in Cardiomyocytes by B55α‐PP2A‐Mediated Dephosphorylation

doi: 10.1161/jaha.116.004861

Figure Lengend Snippet: Figure 3. ISO-induced dephosphorylation and nuclear accumulation of HDAC5 is PKA-dependent. A and B, Quantification of the nuclear/ cytoplasmic fluorescence ratio (Fnuc/Fcyto) in ARVM treated with 10 nmol/L ISO or CON in the presence of 10 lmol/L H89 (PKA inhibitor) or vehicle (VEH). Fnuc/Fcyto ratios were normalized to the baseline value obtained for each cell to account for differences in the basal distribution of GFP-HDAC5 between cells. Two-way ANOVA followed by Tukey’s multiple comparisons test. *P<0.05 vs CON within the same pretreatment group (VEH/H89). †P<0.05 vs VEH within the same treatment group (CON/ISO). n=17 to 30 cells from 3 hearts per group. C, Quantification of Fcell at the 45-minute time point, normalized to baseline. No significant differences were detected by 2-way ANOVA. D, ARVM were treated with 10 nmol/L ISO or CON in the presence of 10 lmol/L H89 or vehicle (VEH). Representative Western blots and grouped data from 5 independent experiments. Unpaired t test. *P<0.05 vs VEH. ARVM indicates adult rat ventricular myocytes; CON, control; cTnI, cardiac troponin I; GFP, green fluorescent protein; HDAC5, histone deacetylase 5; ISO, isoproterenol; PKA, protein kinase A.

Article Snippet: The beads were pelleted and the cleared supernatants incubated with HDAC5 antibody (Cell Signaling 20458; 1:70) or water (no antibody control) with gentle rocking at 4°C overnight.

Techniques: De-Phosphorylation Assay, Western Blot, Control, Histone Deacetylase Assay

Journal: Journal of the American Heart Association

Article Title: β‐Adrenergic Stimulation Induces Histone Deacetylase 5 (HDAC5) Nuclear Accumulation in Cardiomyocytes by B55α‐PP2A‐Mediated Dephosphorylation

doi: 10.1161/jaha.116.004861

Figure Lengend Snippet: Figure 4. The phosphorylation status of S259/S498, and not S279, is the key determinant of GFP-HDAC5 localization in ARVM. A and B, Fnuc/Fcyto and Fcell for ARVM expressing wildtype (WT) or nonphosphorylatable variants (S259/498A, S279A) of GFP-HDAC5. All individual data points, as well as the median and interquartile range, are shown. Kruskal-Wallis 1-way analysis of ranks followed by Dunn’s multiple comparisons test. *P<0.05, n=69 to 98 cells from 3 to 6 hearts per group. C, Western blot showing comparable expression of GFP-HDAC5 variants in ARVM. D, ARVM expressing GFP-HDAC5 variants or GFP alone were cotransduced with adenoviruses encoding a MEF2-luciferase reporter. ARVM were lysed and the luminescence readings from 25 lg protein were normalized to an internal control (ARVM expressing the luciferase reporter but not GFP/GFP-HDAC5). One-way ANOVA followed by Tukey’s multiple comparisons test. *P<0.05 vs GFP, †P<0.05 vs WT. n=6 to 7 independently transduced samples from 2 hearts per group. E and F, ARVM expressing WT or nonphosphorylatable GFP-HDAC5 variants were treated with 10 nmol/L isoproterenol (ISO) or vehicle control (CON) for 45 minutes. Fnuc/Fcyto ratios and Fcell values were normalized to the baseline value obtained for each cell to account for differences in the basal distribution of GFP-HDAC5 between cells. Two-way ANOVA followed by Tukey’s multiple comparisons test. *P<0.05 vs CON for the same GFP-HDAC5 variant. †P<0.05 vs WT ISO, ‡P<0.05 vs S259/498A ISO. n=17 to 40 cells from 3 hearts per group. ARVM indicates adult rat ventricular myocytes; GFP, green fluorescent protein; HDAC5, histone deacetylase 5; MEF, myocyte enhancing factor.

Article Snippet: The beads were pelleted and the cleared supernatants incubated with HDAC5 antibody (Cell Signaling 20458; 1:70) or water (no antibody control) with gentle rocking at 4°C overnight.

Techniques: Phospho-proteomics, Expressing, Western Blot, Luciferase, Control, Variant Assay, Histone Deacetylase Assay

Journal: Journal of the American Heart Association

Article Title: β‐Adrenergic Stimulation Induces Histone Deacetylase 5 (HDAC5) Nuclear Accumulation in Cardiomyocytes by B55α‐PP2A‐Mediated Dephosphorylation

doi: 10.1161/jaha.116.004861

Figure Lengend Snippet: Figure 5. PP2A dephosphorylates HDAC5 downstream of b-AR activation. ARVM were treated with 10 nmol/L isoproterenol (ISO) or vehicle control for 10 minutes in the presence of vehicle (VEH), 30 or 100 nmol/L okadaic acid (OKA). Representative Western blots and grouped data from 5 independent experiments. One-way ANOVA followed by Dunnett’s multiple comparisons tests for each phosphorylation site. *P<0.05 vs VEH. b-AR indicates b-adrenergic receptor; ARVM, adult rat ventricular myocytes; cTnI, cardiac troponin I; GFP, green fluorescent protein; HDAC5, histone deacetylase 5; PLN, phospholamban; PP2A, protein phosphatase 2A.

Article Snippet: The beads were pelleted and the cleared supernatants incubated with HDAC5 antibody (Cell Signaling 20458; 1:70) or water (no antibody control) with gentle rocking at 4°C overnight.

Techniques: Activation Assay, Control, Western Blot, Phospho-proteomics, Histone Deacetylase Assay

Journal: Journal of the American Heart Association

Article Title: β‐Adrenergic Stimulation Induces Histone Deacetylase 5 (HDAC5) Nuclear Accumulation in Cardiomyocytes by B55α‐PP2A‐Mediated Dephosphorylation

doi: 10.1161/jaha.116.004861

Figure Lengend Snippet: Figure 6. B55a targets PP2A to HDAC5 for dephosphorylation. A, ARVM expressing GFP or GFP-HDAC5 were treated with 10 nmol/L isoproterenol (ISO) or vehicle control (CON) for 10 minutes. GFP/GFP-HDAC5-containing complexes were immunoprecipitated from cell lysates, separated by SDS-PAGE and the resulting Western blots probed for B55a, PP2AC, and PP2AA. Representative Western blots and grouped data from 4 independent experiments. Unpaired t tests. *P<0.05 vs CON. B, NRVM expressing GFP-HDAC5 were transfected with scrambled (Scr) or PPP2R2A (B55a) siRNAs for 48 hours prior to treatment with 1 lmol/L ISO or CON for 60 minutes. Representative Western blots and grouped data from 4 independent experiments. Two-way ANOVA following by Sidak’s multiple comparisons tests. For B55a: *P<0.05 vs Scr within the same treatment group (CON/ISO). For S259: *P<0.05 vs CON within the same siRNA group (Scr/B55a). AdV indicates adenovirus; ARVM, adult rat ventricular myocytes; GFP, green fluorescent protein; HDAC5, histone deacetylase 5; NRVM, neonatal rat ventricular myocytes; PP2A, protein phosphatase 2A.

Article Snippet: The beads were pelleted and the cleared supernatants incubated with HDAC5 antibody (Cell Signaling 20458; 1:70) or water (no antibody control) with gentle rocking at 4°C overnight.

Techniques: De-Phosphorylation Assay, Expressing, Control, Immunoprecipitation, SDS Page, Western Blot, Transfection, Histone Deacetylase Assay

Journal: International Journal of Biological Sciences

Article Title: FGF2 Alleviates Microvascular Ischemia-Reperfusion Injury by KLF2-mediated Ferroptosis Inhibition and Antioxidant Responses

doi: 10.7150/ijbs.85692

Figure Lengend Snippet: FGF2 activates KLF2 through the AMPK-HDAC5 pathway. ( A ) Skeletal muscle tissues' WB assay for AMPK, p-AMPK, p-HDAC5, and HDAC5. AMPK and p-AMPK protein levels from (A) are quantified in (B) , normalised to GAPDH band density. HDAC5 protein level from (A) is evaluated in (C) , normalised in respect to Histone 3 band density, coupled with p-HDAC5 level from (A), normalised in relation to GAPDH band density. (D) Using DAPI labelling, skeletal muscle slices labelled with HDAC5 and the EC marker CD31 reveal distinct nuclei. Scale bars indicate 100 μm. Nuclear HDAC5 and CD31 double-positive cells computation (E) . ( F ) Images from a dual luciferase reporter gene test. ( G ) Relative luciferase activity was plotted as a box plots. ( H ) Skeletal muscle tissues' WB assay for AMPK, p-AMPK, p-HDAC5, KLF2, HDAC5, and NFE2L2. ( I ) Quantification of protein level of AMPK, p-AMPK, p-HDAC5, KLF2 from (H) with normalized to GAPDH band density, HDAC5 and NFE2L2 protein levels from (H) with normalized to Histone 3 band density. ( J-L ) Images of skeletal muscle slices with DAPI-stained nuclei that have had HDAC5, KLF2, NFE2L2, and EC marker CD31 stained. Scale bars: 100 μm. ( M-O ) Quantification of nuclear HDAC5, KLF2, NFE2L2 and CD31 double-positive cells, the percentages of double positive cells versus total CD31 positive cells are indicated. ( P ) Skeletal muscle tissues' WB assay for SOD1, SLC7A11, and GPX4. SOD1, SLC7A11, and GPX4 protein levels are evaluated in (Q) and normalised based on GAPDH band density. ( R ) Photographs of skeletal muscle slices with distinct nuclei shown by DAPI staining and DHE and 4-HNE staining. Scale bars: 100 μm. Immunofluorescence data from (R) are measured in (S) , which reveals the average optical density of 4-HNE and the degree of ROS. ( T ) Content of Fe 2+ was plotted as a histogram. ( U ) A histogram showing the content of MDA. ( V ) A histogram showing the GSH content. ( W ) Schematic representation of proposed molecular mechanisms highlighting the roles of AMPK-HDAC5 signaling, KLF2, NFE2L2, oxidative stress, ferroptosis in the pathophysiology of I/R-induced microvascular injury in extremities. Data are presented as the mean ± SD (n = 3-5 per group). Significance: ns, not significant; * P < 0.05.

Article Snippet: The following companies provided the specific primary antibodies that were needed: HO-1 (10701-1-AP), NQO1 (11451-1-AP), SOD1 (24127-1-AP), AMPK (66536-1-lg), NFE2L2 (16396-1-AP), GAPDH (60004-1-lg) and Histone-H3 (17168-1-AP) from Proteintech Group; SLC7A11 (DF12509) and KLF2 (DF13602) from Affinity; GPX4 (381958) from ZEN Bio; p-AMPK (2535) from abcam; HDAC5 (A0632) and p-HDAC5 (AP0202) from Abclonal; 4-HNE (MAB3249-SP) from R&D Systems.

Techniques: Marker, Luciferase, Activity Assay, Staining, Immunofluorescence