INTRODUCTION: Histone acetylation/deacetylation represents a central mechanism for the control of gene expression. Histone deacetylases (HDACs) deacetylate histones and transcription factors, causing transcriptional repression critical to the maintenance of the normal adult cardiac myocyte phenotype. Export of HDAC5 from the nucleus is associated with derepression of the fetal gene program and induction of hypertrophy in the cardiac myocyte. Prior studies of HDAC5 localization in adult cardiomyocytes have employed rabbit cells. METHODS: Because many laboratories are restricted from working with USDA-regulated species such as rabbits, we sought to develop a quantitative assay to monitor signal-dependent nuclear export of HDAC5 in adult rat ventricular myocytes (ARVMs). RESULTS: We demonstrate that GFP-tagged HDAC5 expressed from an adenoviral vector appropriately localizes in nuclei of cultured ARVMs within 24 h post infection. Nuclear localized GFP-HDAC5 undergoes quantitative nuclear export (approximately 20-30% within 2 h) in response to hypertrophic agonists such as endothelin-1 (ET-1) and prostaglandin-F2alpha (PGF2alpha), as well as the direct protein kinase C (PKC) activator and phorbol myristate acetate (PMA). Nuclear export of HDAC5 in ARVMs is blocked by Gö6976, a small molecule inhibitor of PKC and protein kinase D (PKD). DISCUSSION: These data suggest that GFP-HDAC5 is appropriately functional in ARVMs 24 h post infection, and that translocation events can be quantitated for the study of hypertrophy or the identification of antihypertrophic agents in adult cardiac myocytes.
INTRODUCTION: Histone acetylation/deacetylation represents a central mechanism for the control of gene expression. Histone deacetylases (HDACs) deacetylate histones and transcription factors, causing transcriptional repression critical to the maintenance of the normal adult cardiac myocyte phenotype. Export of HDAC5 from the nucleus is associated with derepression of the fetal gene program and induction of hypertrophy in the cardiac myocyte. Prior studies of HDAC5 localization in adult cardiomyocytes have employed rabbit cells. METHODS: Because many laboratories are restricted from working with USDA-regulated species such as rabbits, we sought to develop a quantitative assay to monitor signal-dependent nuclear export of HDAC5 in adult rat ventricular myocytes (ARVMs). RESULTS: We demonstrate that GFP-tagged HDAC5 expressed from an adenoviral vector appropriately localizes in nuclei of cultured ARVMs within 24 h post infection. Nuclear localized GFP-HDAC5 undergoes quantitative nuclear export (approximately 20-30% within 2 h) in response to hypertrophic agonists such as endothelin-1 (ET-1) and prostaglandin-F2alpha (PGF2alpha), as well as the direct protein kinase C (PKC) activator and phorbol myristate acetate (PMA). Nuclear export of HDAC5 in ARVMs is blocked by Gö6976, a small molecule inhibitor of PKC and protein kinase D (PKD). DISCUSSION: These data suggest that GFP-HDAC5 is appropriately functional in ARVMs 24 h post infection, and that translocation events can be quantitated for the study of hypertrophy or the identification of antihypertrophic agents in adult cardiac myocytes.
Authors: Kate L Weeks; Antonella Ranieri; Agnieszka Karaś; Bianca C Bernardo; Alexandra S Ashcroft; Chris Molenaar; Julie R McMullen; Metin Avkiran Journal: J Am Heart Assoc Date: 2017-03-25 Impact factor: 5.501