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Purification of H3 and H4 Histone Proteins and the Quantification of Acetylated Histone Marks in Cells and Brain Tissue
Published on: November 30, 2018
Deacetylation of Histone H4 Accompanying Cardiomyogenesis is Weakened in HDAC1-Depleted ES Cells
Orazio Angelo Arcidiacono1,2, Jana Krejčí3, Jana Suchánková4
1Institute of Biophysics of the Czech Academy of Sciences, Královopolská 135, 612 65, Brno, Czech Republic. o.arcidiacono@gmail.com.
Abstract:
Cell differentiation into cardiomyocytes requires activation of differentiation-specific genes and epigenetic factors that contribute to these physiological processes. This study is focused on the in vitro differentiation of mouse embryonic stem cells (mESCs) induced into cardiomyocytes. The effects of clinically promising inhibitors of histone deacetylases (HDACi) on mESC cardiomyogenesis and on explanted embryonic hearts were also analyzed. HDAC1 depletion caused early beating of cardiomyocytes compared with those of the wild-type (wt) counterpart. Moreover, the adherence of embryonic bodies (EBs) was reduced in HDAC1 double knockout (dn) mESCs. The most important finding was differentiation-specific H4 deacetylation observed during cardiomyocyte differentiation of wt mESCs, while H4 deacetylation was weakened in HDAC1-depleted cells induced to the cardiac pathway. Analysis of the effect of HDACi showed that Trichostatin A (TSA) is a strong hyperacetylating agent, especially in wt mESCs, but only SAHA reduced the size of the beating areas in EBs that originated from HDAC1 dn mESCs. Additionally, explanted embryonic hearts (e15) responded to treatment with HDACi: all of the tested HDACi (TSA, SAHA, VPA) increased the levels of H3K9ac, H4ac, H4K20ac, and pan-acetylated lysines in embryonic hearts. This observation shows that explanted tissue can be maintained in a hyperacetylation state several hours after excision, which appears to be useful information from the view of transplantation strategy and the maintenance of gene upregulation via acetylation in tissue intended for transplantation.
Insights
Histone deacetylase 1 (HDAC1) depletion accelerates cardiomyocyte differentiation in mouse embryonic stem cells (mESCs). Histone deacetylase inhibitors (HDACi) also impact cardiomyocyte development and maintain hyperacetylation in explanted embryonic hearts.
Area of Science:
- Epigenetics and Developmental Biology
- Stem Cell Biology
- Cardiovascular Research
Background:
- Cell differentiation into cardiomyocytes involves specific genes and epigenetic factors.
- Histone deacetylases (HDACs) play a crucial role in regulating gene expression during differentiation.
- Understanding HDAC function is key to controlling cardiomyogenesis for therapeutic applications.
Purpose of the Study:
- To investigate the role of HDAC1 in mouse embryonic stem cell (mESC) differentiation into cardiomyocytes.
- To analyze the effects of histone deacetylase inhibitors (HDACi) on mESC cardiomyogenesis and explanted embryonic hearts.
- To explore the potential of HDACi in maintaining epigenetic states for tissue transplantation.
Main Methods:
- In vitro differentiation of mESCs into cardiomyocytes.
- Analysis of HDAC1 depletion using knockout mESC lines.
- Treatment of mESCs and explanted embryonic hearts with HDAC inhibitors (TSA, SAHA, VPA).
- Assessment of histone acetylation levels (H4, H3K9ac, H4K20ac, pan-acetylated lysines) via Western blotting and immunofluorescence.
Main Results:
- HDAC1 depletion led to earlier cardiomyocyte beating and reduced embryonic body adherence.
- Differentiation-specific H4 deacetylation was observed in wild-type mESCs, weakened in HDAC1-depleted cells.
- HDAC inhibitors TSA and SAHA modulated acetylation and beating areas in mESC-derived cardiomyocytes.
- Explanted embryonic hearts treated with HDACi exhibited increased global histone acetylation, persisting for hours post-excision.
Conclusions:
- HDAC1 is a critical regulator of mESC cardiomyogenesis, influencing differentiation timing and efficiency.
- HDAC inhibitors can modulate epigenetic states during cardiomyocyte development.
- Explanted embryonic tissues can be maintained in a hyperacetylated state using HDACi, suggesting potential applications in transplantation strategies for preserving gene upregulation.
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