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

Abstract

BackgroundClass IIa histone deacetylase (HDAC) isoforms such as HDAC5 are critical signal‐responsive repressors of maladaptive cardiomyocyte hypertrophy, through nuclear interactions with transcription factors including myocyte enhancer factor‐2. β‐Adrenoceptor (β‐AR) stimulation, a signal of fundamental importance in regulating cardiac function, has been proposed to induce both phosphorylation‐independent nuclear export and phosphorylation‐dependent nuclear accumulation of cardiomyocyte HDAC5. The relative importance of phosphorylation at Ser259/Ser498 versus Ser279 in HDAC5 regulation is also controversial. We aimed to determine the impact of β‐AR stimulation on the phosphorylation, localization, and function of cardiomyocyte HDAC5 and delineate underlying molecular mechanisms.Methods and ResultsA novel 3‐dimensional confocal microscopy method that objectively quantifies the whole‐cell nuclear/cytoplasmic distribution of green fluorescent protein tagged HDAC5 revealed the β‐AR agonist isoproterenol to induce β1‐AR‐mediated and protein kinase A‐dependent HDAC5 nuclear accumulation in adult rat cardiomyocytes, which was accompanied by dephosphorylation at Ser259/279/498. Mutation of Ser259/Ser498 to Ala promoted HDAC5 nuclear accumulation and myocyte enhancer factor‐2 inhibition, whereas Ser279 ablation had no such effect and did not block isoproterenol‐induced nuclear accumulation. Inhibition of the Ser/Thr phosphatase PP2A blocked isoproterenol‐induced HDAC5 dephosphorylation. Co‐immunoprecipitation revealed a specific interaction of HDAC5 with the PP2A targeting subunit B55α, as well as catalytic and scaffolding subunits, which increased >3‐fold with isoproterenol. Knockdown of B55α in neonatal cardiomyocytes attenuated isoproterenol‐induced HDAC5 dephosphorylation.Conclusionsβ‐AR stimulation induces HDAC5 nuclear accumulation in cardiomyocytes by a mechanism that is protein kinase A‐dependent but requires B55α‐PP2A‐mediated dephosphorylation of Ser259/Ser498 rather than protein kinase A‐mediated phosphorylation of Ser279.

Nucleocytoplasmic shuttling: The ins and outs of quantitative imaging

“…Robust IF detection of HDAC5 in adult cardiomyocytes is hampered by the low abundance of this protein in this cell type. Therefore, most studies investigating the impact of hypertrophic stimuli on the nuclear/cytoplasmic distribution of HDAC5 in adult cardiomyocytes have transiently expressed GFP‐tagged HDAC5 fusion proteins using adenoviruses, and then imaged the same cells or populations of cells before and after treatment with pharmacological agents . In our studies in adult rat ventricular myocytes (ARVM), significant variation in GFP‐HDAC5 expression is apparent between cells in the same dish and from independent experiments (Figure A,B).…”

“…GFP‐HDAC5 fluorescence intensities in nuclear and cytoplasmic compartments from the entire cell volume were measured to determine the subcellular distribution of GFP‐HDAC5. This method was less subjective and more accurate than previous methods that had been used to investigate HDAC5 nucleocytoplasmic shuttling in adult cardiomyocytes, and demonstrated unequivocally that acute β‐adrenergic receptor activation induces the nuclear accumulation of GFP‐HDAC5 in this cell type …”

“…Additional considerations for both image acquisition and analysis come in to play, as reviewed in De Mey et al For example, avoiding excessive photobleaching (and with it, phototoxicity), while maintaining an acceptable signal‐to‐noise ratio, is critical in 4D microscopy. In Weeks et al, 4D imaging using SDCM was performed to investigate the impact of β‐adrenergic signalling on the subcellular distribution of GFP‐HDAC5, as there were mixed reports in the literature regarding this phenomenon in adult cardiomyocytes. Z‐stacks (spanning 36 μm in 1.5 μm steps) of ARVM expressing GFP‐HDAC5 were acquired at baseline and at multiple timepoints after the addition of agonists to activate the signalling pathway of interest.…”

“…Accurate identification of nuclear and cytoplasmic regions can be achieved by labelling cells with fluorescent dyes and thresholding the signals from such dyes to generate binary layers corresponding to each subcellular compartment (see Section and Figure ). This allows quantification of F arising from the fluorescent fusion protein within those compartments in an automated and unbiased manner . If the same cells have been imaged at multiple timepoints, F values can be divided by the baseline value for each cell.…”

“…In our studies, cells are labelled with Cell Tracker Orange (Molecular Probes) the day before imaging, and nuclei are labelled with DRAQ5 (Biostatus) 10 minutes prior to image acquisition. This allows objective definition of nuclear and cytoplasmic compartments for quantification of the fluorescent signal arising from GFP‐tagged fusion proteins localised to those compartments . Numerous Cell Tracker reagents with different excitation/emission spectra are available, making them compatible for imaging alongside other fluorescent compounds and AFP proteins.…”