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

Figures

• 

  Figure 1. β1‐AR/PKA activation reduces HDAC5 phosphorylation. A, Time course of HDAC5 dephosphorylation 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 β1‐AR‐selective antagonist), 100 nmol/L ICI 118,551 (ICI; a β2‐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 μmol/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. β1‐AR indicates β‐adrenergic receptor; ARVM, adult rat ventricular myocytes; BNZ, N6‐benzoyl cAMP; cTnI, cardiac troponin I; HDAC5,…

• 

  Figure 2. β‐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‐μm 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 F nuc and F cyto 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…

• 

  Figure 3. ISO‐induced dephosphorylation and nuclear accumulation of HDAC5 is PKA‐dependent. A and B, Quantification of the nuclear/cytoplasmic fluorescence ratio (F nuc/F cyto) in ARVM treated with 10 nmol/L ISO or CON in the presence of 10 μmol/L H89 (PKA inhibitor) or vehicle (VEH). F nuc/F cyto 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 F cell 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 μmol/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.

• 

  Figure 4. The phosphorylation status of S259/S498, and not S279, is the key determinant of GFP‐HDAC5 localization in ARVM. A and B, F nuc/F cyto and F cell 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 μg 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. F nuc/F cyto ratios and F cell values…

• 

  Figure 5. PP2A dephosphorylates HDAC5 downstream of β‐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. β‐AR indicates β‐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.

• 

  Figure 6. B55α 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 B55α, 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 (B55α) siRNAs for 48 hours prior to treatment with 1 μmol/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 B55α: *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/B55α). 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.

• 

  Figure 7. B55α is not phospho‐regulated downstream of β‐AR activation. Lysates from ARVM treated with vehicle control or 10 nmol/L isoproterenol (ISO) for 10 minutes were subjected to standard SDS‐PAGE or PhosTag phosphate‐affinity SDS‐PAGE. Duplicate sets of samples were run on the same gels for subsequent immunoblotting with antibodies against B55α and B56δ. Unlike B56δ, B55α is not phosphorylated in response to ISO treatment (multiple bands in ISO sample on PhosTag blot probed for B56δ; absence of multiple bands in ISO sample on PhosTag blot probed for B55α). β‐AR indicates β‐adrenergic receptor; ARVM, adult rat ventricular myocytes.

• 

  Figure 8. Working model for the regulation of class IIa histone deacetylases downstream of β‐AR activation in adult cardiomyocytes. Acute stimulation of β‐adrenergic receptors (β‐ARs) does not lead to nuclear accumulation of HDAC5 through PKA‐dependent phosphorylation of HDAC5 at S279, as previously proposed.18, 19 Rather, activation of β1‐ARs in adult cardiomyocytes leads to PP2A‐mediated dephosphorylation of HDAC5 at S259/S498 and S279, likely following the recruitment of PP2A to HDAC5 by the regulatory subunit B55α. Dephosphorylation of S259/S498 is the key driver of HDAC5 nuclear accumulation downstream of β1‐AR/PKA activation in ARVM. This mechanism may act in concert with the PKA‐dependent formation of an N‐terminal HDAC4 cleavage product (HDAC4‐NT)43 to repress MEF2‐dependent gene transcription in settings of acute/transient β‐adrenergic receptor stimulation. ARVM indicates adult rat ventricular myocytes; β‐AR, β‐adrenergic receptor; HDAC5, histone deacetylase 5; MEF2, myocyte enhancing factor‐2; PKA, protein kinase A; PP2A, protein phosphatase 2A.