Class II histone deacetylases act as signal-responsive repressors of cardiac hypertrophy

Abstract

The heart responds to stress signals by hypertrophic growth, which is accompanied by activation of the MEF2 transcription factor and reprogramming of cardiac gene expression. We show here that class II histone deacetylases (HDACs), which repress MEF2 activity, are substrates for a stress-responsive kinase specific for conserved serines that regulate MEF2-HDAC interactions. Signal-resistant HDAC mutants lacking these phosphorylation sites are refractory to hypertrophic signaling and inhibit cardiomyocyte hypertrophy. Conversely, mutant mice lacking the class II HDAC, HDAC9, are sensitized to hypertrophic signals and exhibit stress-dependent cardiomegaly. Thus, class II HDACs act as signal-responsive suppressors of the transcriptional program governing cardiac hypertrophy and heart failure.

Figure 1. Elevated HDAC Kinase Activity in Hypertrophic Hearts

(A) Schematic diagram of class II HDACs. Class II HDACs have a bipartite structure, with a C-terminal catalytic (HDAC) domain and an N-terminal extension with a MEF2 binding domain. Conserved phosphorylation sites flank the nuclear localization sequence (NLS) and a nuclear export sequence (NES) is near the C terminus. MITR is a splice variant of HDAC9 that lacks an HDAC domain. (B) Amino acid homologies surrounding the regulatory serines (arrowheads) in the class II HDACs. (C) Schematic diagram of HDAC kinase assay. Kinase assays were performed with GST-HDAC substrates. The region of each HDAC that was used as substrate is underlined in (A). A kinase from heart lysates phosphorylates the wild-type (WT) substrates, but not mutant (Mut) substrates containing alanine in place of the regulatory serine residue. (D and E) HDAC kinase assays were performed with heart extracts from thoracic aorta-banded (TAB) or sham-operated mice (D) or from wild-type (wt) or αMHC-calcineurin transgenic (Cn-Tg) mice. Values represent the average of at least three independent assays. (F) HDAC kinase assays were performed with heart extracts derived from wild-type (lane 1) and Cn-Tg (lanes 2–23) mice in the absence (lanes 1 and 2) or presence (lanes 3–23) of diverse inhibitors (upper image). Kinase activity relative to that in extracts from Cn-Tg mice (lane 2) was determined (lower image). Inhibitors were added to reactions at concentrations 20 times the IC₅₀. Combo1 contained AIP and Go6983 and combo2 contained HA1004 and Go6983. Inhibitor experiments were performed at least three times with comparable results. A representative experiment is shown. For (D–F), enzyme activity was quantified as described in Experimental Procedures.

Figure 2. Inhibition of Fetal Gene Expression and Acetylation by Signal-Resistant MITR

(A) Neonatal rat cardiomyocytes were infected with adenoviruses encoding phosphorylation site mutants of HDAC5 (Ad-HDAC5 S259/498A) or MITR (Ad-MITR S218/448A) and were then stimulated with PE for 24 hr. Transcripts for ANF, β-MHC, and GAPDH were detected by dot blot. ANF and β-MHC expression are shown relative to that in untreated, uninfected controls and normalized to the level of GAPDH. (B) Cardiomyocytes were infected with adenoviruses encoding Myc-tagged MITR or MITR S218/448A at the indicated multiplicity of infection (moi). Cells maintained in serum-free medium were stimulated with PE for 24 hr. The relative levels of β-MHC mRNA in Ad-MITR versus Ad-MITR S218/448A-infected cells were quantified by dot blot and normalized to the level of GAPDH. (C) The expression of ectopic MITR in cardiomyocyte lysates was determined by Western blotting with an anti-Myc antibody (upper image). The blot was reprobed with an anti-α-tubulin antibody to reveal total protein input (bottom image). (D) Soluble chromatin was prepared from cardiomyocytes infected with either Ad-GFP or Ad-MITR S218/448A and treated for 24 hr with FBS or PE. Chromatin was immunoprecipitated with an antibody specific for acetylated histone H3 and precipitated genomic DNA was analyzed by PCR using primers for ANF, β-MHC, GAPDH, and α-cardiac actin promoters. Positions of primers (numbers) and the GATA sites (black boxes) relative to the transcription initiation sites of each gene are shown. The lower image shows a DNA input control in which PCR amplification was performed prior to immunoprecipitation.

Figure 3. Prevention of Hypertrophy by MITR

(A and B) Cardiomyocytes were cultured either in the absence of virus or were infected with adenoviruses encoding GFP or Myc-tagged MITR S218/448A. After stimulation with PE or FBS for 24 hr, cells were fixed and stained with antibody against sarcomeric α-actinin (A, red signal) or ANF (B, perinuclear red signal). Adenovirus-infected cells were identified by GFP fluorescence or costaining with anti-Myc antibody to reveal ectopic MITR (green signal). Scale bar equals 20 μm. (C) Cardiomyocytes were cultured and infected with adenoviruses as described above. Cells maintained in serum-free medium were stimulated with FBS, PE, ET-1, ANGII, or IGF-1. After 24 hr, RNA was isolated and the indicated transcripts were measured by dot blot. Numbers indicate the expression level of each transcript in the presence of Ad-MITR S218/448A relative to Ad-GFP and normalized to GAPDH.

Figure 4. Targeting of Mouse HDAC9

(A and B) A diagram of the HDAC9 and MITR proteins is shown above the mouse HDAC9 locus. Sizes of introns in kilobases (kb) and exons (filled boxes) are indicated. In the targeting vector, a nuclear LacZ (nLacZ) reporter was inserted in-frame with exon 4. Homologous recombination resulted in deletion of exons 4 and 5, which encode the MEF2 binding domain of HDAC9/MITR. Insertion of the nLacZ gene introduced a BamHI site that was used in conjunction with the indicated external 5′ probe to distinguish the wild-type (6.5 kb) and mutant (4.1 kb) alleles by Southern blot (B). (C) Genomic DNA from mice of the indicated genotypes was analyzed by PCR using primers specific for the 5′ and 3′ regions of the targeted mutation. (D) The positions of the primers within HDAC9 exons are shown (lower image). Targeting the HDAC9 locus results in an aberrant mRNA splicing event between exons 3 and 6, as evidenced by the 300 base pair (bp) PCR product using primers P1 and P3, which is not present in wild-type controls. Sequencing of this RT-PCR product revealed a frameshift that introduced stop codons that prevent translation of sequences downstream of exon 3. L7 transcripts were amplified as a control for cDNA integrity. (E) The consequences of HDAC9 gene disruption on the expression of mRNA transcripts for HDACs 1–8 were assessed by semi-quantitative RT-PCR using heart RNA from mice of the indicated HDAC9 genotypes.

Figure 5. Cardiac Hypertrophy in HDAC9 Mutant Mice

(A and B) HDAC9 mutant mice and wild-type littermates were sacrificed at one and eight months of age and heart weight-to-body weight ratios were determined. Values represent the mean ± standard deviation (SD). N = 5. Scale bar equals 2 mm. (C) Hypersensitivity to TAB. Six-to-eight-week-old mice were subjected to thoracic aortic banding (TAB) or to sham operation. Twenty-one days later, animals were sacrificed and the ratios of left ventricular (LV) mass-to-body weight were determined. At least five mice of each genotype were analyzed. Values represent the mean ± SD.

Figure 6. HDAC9 Mutant Mice Are Hypersensitive to Calcineurin-Mediated Hypertrophy

(A) HDAC9 mutant mice were bred with mice harboring the αMHC-calcineurin transgene (Cn-Tg). Hearts from one-month-old mice of the indicated genotype were isolated (top images), sectioned, and stained with H and E (middle and bottom images). The bottom image shows high magnification images of H and E-stained cardiomyocytes from the ventricular septum. Scale bar equals 2 mm for top and middle, 20 μm for bottom. (B) Heart weight-to-body weight ratios of mice of the indicated genotypes were determined at four weeks of age. At least ten mice of each genotype were analyzed. Values represent the mean ± standard deviation. (C) Total RNA was isolated from hearts of mice of the indicated genotype and expression of transcripts for ANF, brain natriuretic peptide (BNP), β-MHC, and GAPDH was determined by Northern blot analysis. The relative abundance of transcripts was quantified and normalized to GAPDH. (D) Wild-type or HDAC9 mutant mice were crossed to transgenic mice harboring a MEF2-dependent LacZ reporter gene. Offspring were bred with Cn-Tg mice. At least two littermates of each genotype were sacrificed at 8 weeks of age and cardiac MEF2 activity was detected by staining for β-galactosidase activity. Scale bar equals 2 mm.

Figure 7. Repression of Cardiac Hypertrophy by Class II HDACs

Class II HDACs (4, 5, 7, and 9) associate with MEF2 and inhibit hypertrophy and the fetal gene program. Stress signals stimulate an HDAC kinase that phosphorylates (P) HDACs at two conserved serine residues. HDAC kinase is also stimulated by calcineurin. When phosphorylated, HDACs bind 14-3-3, dissociate from MEF2, and are exported from the nucleus. Upon release of HDACs, MEF2 is free to associate with histone acetyltransferases (HATs) and to activate downstream target genes that drive a hypertrophic response. MEF2 also interacts with NFAT and GATA transcription factors, which have been implicated in hypertrophic gene expression.