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Control of histone H3 phosphorylation by CaMKIIδ in response to haemodynamic cardiac stress.

Awad S, Al-Haffar KM, Marashly Q, Quijada P, Kunhi M, Al-Yacoub N, Wade FS, Mohammed SF, Al-Dayel F, Sutherland G, Assiri A, Sussman M, Bers D, Al-Habeeb W, Poizat C - J. Pathol. (2014)

Bottom Line: Heart failure is associated with the reactivation of a fetal cardiac gene programme that has become a hallmark of cardiac hypertrophy and maladaptive ventricular remodelling, yet the mechanisms that regulate this transcriptional reprogramming are not fully understood.Similar changes are detected in patients with end-stage heart failure, where CaMKIIδ specifically interacts with phospho-H3.The findings reveal a novel in vivo function of CaMKIIδ in regulating H3 phosphorylation and suggest a novel epigenetic mechanism by which CaMKIIδ controls cardiac hypertrophy.

View Article: PubMed Central - PubMed

Affiliation: Cardiovascular Research Programme, King Faisal Specialist Hospital and Research Centre, Riyadh, Kingdom of Saudi Arabia.

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14–3–3 recruitment to chromatin is important for transcriptional elongation of GATA-4 and of fetal cardiac genes. (A) ChIP–qPCR performed from mouse heart tissues showing 14–3–3 binding at the ANP and β-MHC promoters in CaMKIIδ-WT or CaMKIIδ-KO mice 21 days after sham operation or TAC. qPCR reactions were also performed at GAPDH and α-actin promoters that are not responsive to pressure overload hypertrophy: results are expressed as percentage input over signals with IgG antibody and represent average ± SD (n = 3); p values from Student's t test are indicated. (B) Immunoblotting of cardiomyocytes expressing reduced 14–3–3 (si14–3–3) or normal 14–3–3 (siControl). (C) ChIP–qPCR, showing p-RNAPII S2 at the promoter and internal exon of ANP, β-MHC, GATA-4, GAPDH and actin in primary neonatal rat cardiomyocytes with siCt or si14–3–3 and stimulated with phenylephrine (PE) for 24 h. (D) ChIP–qPCR assay from mouse heart chromatin, showing p-RNAPII S2 at the ANP, β-MHC, actin and GAPDH exonic region in CaMKIIδ-WT or CaMKIIδ-KO animals, 21 days after sham operation or TAC. (E) ChIP–qPCR assay, showing p-RNAPII S2 at the ANP, β-MHC, actin and GAPDH promoter regions in CaMKIIδ-WT or CaMKIIδ-KO animals, 21 days after sham operation or TAC: results are expressed as percentage input over signals with IgG antibody, and represent average ± SD (n = 3); p values from Student's t-test are indicated
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fig06: 14–3–3 recruitment to chromatin is important for transcriptional elongation of GATA-4 and of fetal cardiac genes. (A) ChIP–qPCR performed from mouse heart tissues showing 14–3–3 binding at the ANP and β-MHC promoters in CaMKIIδ-WT or CaMKIIδ-KO mice 21 days after sham operation or TAC. qPCR reactions were also performed at GAPDH and α-actin promoters that are not responsive to pressure overload hypertrophy: results are expressed as percentage input over signals with IgG antibody and represent average ± SD (n = 3); p values from Student's t test are indicated. (B) Immunoblotting of cardiomyocytes expressing reduced 14–3–3 (si14–3–3) or normal 14–3–3 (siControl). (C) ChIP–qPCR, showing p-RNAPII S2 at the promoter and internal exon of ANP, β-MHC, GATA-4, GAPDH and actin in primary neonatal rat cardiomyocytes with siCt or si14–3–3 and stimulated with phenylephrine (PE) for 24 h. (D) ChIP–qPCR assay from mouse heart chromatin, showing p-RNAPII S2 at the ANP, β-MHC, actin and GAPDH exonic region in CaMKIIδ-WT or CaMKIIδ-KO animals, 21 days after sham operation or TAC. (E) ChIP–qPCR assay, showing p-RNAPII S2 at the ANP, β-MHC, actin and GAPDH promoter regions in CaMKIIδ-WT or CaMKIIδ-KO animals, 21 days after sham operation or TAC: results are expressed as percentage input over signals with IgG antibody, and represent average ± SD (n = 3); p values from Student's t-test are indicated

Mentions: To establish the functional link between H3 phosphorylation, 14–3–3 recruitment and the transcriptional reprogramming of fetal cardiac genes, we assessed 14–3–3 binding at fetal cardiac genes in CaMKIIδ wild-type and KO mice subjected to sham operation or TAC. 14–3–3 binding increased significantly at the ANP and β-MHC promoters and exonic region after stress in wild-type mice, but this effect was significantly reduced in CaMKIIδ-KO mice (Figure 6A). Phosphorylation of RNAPII at serine 2 (p-RNAPII S2) is associated with transcription elongation, which requires 14–3–3 [30]. Therefore, we assessed p-RNAPII S2 at the promoter and exonic region of fetal cardiac genes and of GATA-4 in neonatal rat cardiomyocytes transfected with control siRNA (siCT) or siRNA against 14–3–3 (si14–3–3) after stimulation with the hypertrophic agonist phenylephrine (Figure 6B). As previously reported [30], 14–3–3 knockdown reduced p-RNAPII S2 (Figure 6B). p-RNAPII S2 was relatively low at the promoters of ANP, β-MHC and GATA-4 in both siCtr- and si14–3–3-transfected cells (Figure 6C). p-RNAPII S2 increased at ANP, β-MHC and GATA-4 exonic regions in cells with normal expression of 14–3–3, but not in cells with reduced 14–3–3 levels (Figure 6C). We also observed increased p-RNAPII S2 at ANP and β-MHC exonic regions, while it decreased at the promoter region of both genes in wild-type mice subjected to TAC, but not in CaMKIIδ-KO mice after TAC (Figure 6D, E). Taken together, these results suggest that 14–3–3 binding to phosphorylated H3 is important for the recruitment of RNAPII and elongation of fetal cardiac genes and GATA-4 during cardiac hypertrophy.


Control of histone H3 phosphorylation by CaMKIIδ in response to haemodynamic cardiac stress.

Awad S, Al-Haffar KM, Marashly Q, Quijada P, Kunhi M, Al-Yacoub N, Wade FS, Mohammed SF, Al-Dayel F, Sutherland G, Assiri A, Sussman M, Bers D, Al-Habeeb W, Poizat C - J. Pathol. (2014)

14–3–3 recruitment to chromatin is important for transcriptional elongation of GATA-4 and of fetal cardiac genes. (A) ChIP–qPCR performed from mouse heart tissues showing 14–3–3 binding at the ANP and β-MHC promoters in CaMKIIδ-WT or CaMKIIδ-KO mice 21 days after sham operation or TAC. qPCR reactions were also performed at GAPDH and α-actin promoters that are not responsive to pressure overload hypertrophy: results are expressed as percentage input over signals with IgG antibody and represent average ± SD (n = 3); p values from Student's t test are indicated. (B) Immunoblotting of cardiomyocytes expressing reduced 14–3–3 (si14–3–3) or normal 14–3–3 (siControl). (C) ChIP–qPCR, showing p-RNAPII S2 at the promoter and internal exon of ANP, β-MHC, GATA-4, GAPDH and actin in primary neonatal rat cardiomyocytes with siCt or si14–3–3 and stimulated with phenylephrine (PE) for 24 h. (D) ChIP–qPCR assay from mouse heart chromatin, showing p-RNAPII S2 at the ANP, β-MHC, actin and GAPDH exonic region in CaMKIIδ-WT or CaMKIIδ-KO animals, 21 days after sham operation or TAC. (E) ChIP–qPCR assay, showing p-RNAPII S2 at the ANP, β-MHC, actin and GAPDH promoter regions in CaMKIIδ-WT or CaMKIIδ-KO animals, 21 days after sham operation or TAC: results are expressed as percentage input over signals with IgG antibody, and represent average ± SD (n = 3); p values from Student's t-test are indicated
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
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fig06: 14–3–3 recruitment to chromatin is important for transcriptional elongation of GATA-4 and of fetal cardiac genes. (A) ChIP–qPCR performed from mouse heart tissues showing 14–3–3 binding at the ANP and β-MHC promoters in CaMKIIδ-WT or CaMKIIδ-KO mice 21 days after sham operation or TAC. qPCR reactions were also performed at GAPDH and α-actin promoters that are not responsive to pressure overload hypertrophy: results are expressed as percentage input over signals with IgG antibody and represent average ± SD (n = 3); p values from Student's t test are indicated. (B) Immunoblotting of cardiomyocytes expressing reduced 14–3–3 (si14–3–3) or normal 14–3–3 (siControl). (C) ChIP–qPCR, showing p-RNAPII S2 at the promoter and internal exon of ANP, β-MHC, GATA-4, GAPDH and actin in primary neonatal rat cardiomyocytes with siCt or si14–3–3 and stimulated with phenylephrine (PE) for 24 h. (D) ChIP–qPCR assay from mouse heart chromatin, showing p-RNAPII S2 at the ANP, β-MHC, actin and GAPDH exonic region in CaMKIIδ-WT or CaMKIIδ-KO animals, 21 days after sham operation or TAC. (E) ChIP–qPCR assay, showing p-RNAPII S2 at the ANP, β-MHC, actin and GAPDH promoter regions in CaMKIIδ-WT or CaMKIIδ-KO animals, 21 days after sham operation or TAC: results are expressed as percentage input over signals with IgG antibody, and represent average ± SD (n = 3); p values from Student's t-test are indicated
Mentions: To establish the functional link between H3 phosphorylation, 14–3–3 recruitment and the transcriptional reprogramming of fetal cardiac genes, we assessed 14–3–3 binding at fetal cardiac genes in CaMKIIδ wild-type and KO mice subjected to sham operation or TAC. 14–3–3 binding increased significantly at the ANP and β-MHC promoters and exonic region after stress in wild-type mice, but this effect was significantly reduced in CaMKIIδ-KO mice (Figure 6A). Phosphorylation of RNAPII at serine 2 (p-RNAPII S2) is associated with transcription elongation, which requires 14–3–3 [30]. Therefore, we assessed p-RNAPII S2 at the promoter and exonic region of fetal cardiac genes and of GATA-4 in neonatal rat cardiomyocytes transfected with control siRNA (siCT) or siRNA against 14–3–3 (si14–3–3) after stimulation with the hypertrophic agonist phenylephrine (Figure 6B). As previously reported [30], 14–3–3 knockdown reduced p-RNAPII S2 (Figure 6B). p-RNAPII S2 was relatively low at the promoters of ANP, β-MHC and GATA-4 in both siCtr- and si14–3–3-transfected cells (Figure 6C). p-RNAPII S2 increased at ANP, β-MHC and GATA-4 exonic regions in cells with normal expression of 14–3–3, but not in cells with reduced 14–3–3 levels (Figure 6C). We also observed increased p-RNAPII S2 at ANP and β-MHC exonic regions, while it decreased at the promoter region of both genes in wild-type mice subjected to TAC, but not in CaMKIIδ-KO mice after TAC (Figure 6D, E). Taken together, these results suggest that 14–3–3 binding to phosphorylated H3 is important for the recruitment of RNAPII and elongation of fetal cardiac genes and GATA-4 during cardiac hypertrophy.

Bottom Line: Heart failure is associated with the reactivation of a fetal cardiac gene programme that has become a hallmark of cardiac hypertrophy and maladaptive ventricular remodelling, yet the mechanisms that regulate this transcriptional reprogramming are not fully understood.Similar changes are detected in patients with end-stage heart failure, where CaMKIIδ specifically interacts with phospho-H3.The findings reveal a novel in vivo function of CaMKIIδ in regulating H3 phosphorylation and suggest a novel epigenetic mechanism by which CaMKIIδ controls cardiac hypertrophy.

View Article: PubMed Central - PubMed

Affiliation: Cardiovascular Research Programme, King Faisal Specialist Hospital and Research Centre, Riyadh, Kingdom of Saudi Arabia.

Show MeSH
Related in: MedlinePlus