Maintenance of cardiac energy metabolism by histone deacetylase 3 in mice (original) (raw)
Conditional deletion of Hdac3. Knowing of the embryonic and neonatal lethality associated with loss of Hdac1 or Hdac2, we generated a conditional null allele of Hdac3 to investigate the role of Hdac3 in the adult heart. Targeting of Hdac3 was performed by introducing loxP sites upstream of exon 11 and downstream of exon 14 through homologous recombination (Figure 1A). This mutation deletes almost all of the nuclear import sequence and a carboxyterminal region that is necessary for transcriptional repression (19). Germ-line transmission was detected by Southern blot, and deletion of Hdac3 was confirmed at the genomic level (Figure 1C). Hdac3neo-loxP mice were bred to CAG-Cre (20) transgenic mice, which express Cre recombinase ubiquitously, allowing for the generation of Hdac3+/– mice. Hdac3+/– mice were intercrossed to obtain Hdac3–/– mice, which died before E9.5 due to defects in gastrulation (data not shown).
Generation of a conditional Hdac3 allele. (A) Strategy to generate a conditional Hdac3 allele. Protein, genomic structure, targeting vector, and targeted allele are shown. loxP sites were inserted upstream of exon 11 and downstream of exon 14. The neomycin cassette was removed by crossing to FLPe transgenic mice. Cre-mediated excision leaves 1 loxP site in place of exons 11 through 14. NES, nuclear export sequence; NLS, nuclear localization sequence; FRT, flipase recognition target; dta, diphtheria toxin A. (B) Representative Southern blot of genomic DNA to show germ-line transmission. WT (~13.8 kb) and targeted (~6.6 kb) bands are indicated. (C) Genotyping of Hdac3 conditional mice by genomic PCR. Primer triplex includes 1 set flanking the 5′ loxP site and a third primer downstream of the 3′ loxP site. Global deletion by CAG-Cre removes the primer within the loxP sites, resulting in 1 fragment of approximately 650 bp. Primers are shown in A.
Cardiac deletion of Hdac3 causes cardiac hypertrophy. To circumvent embryonic lethality, we deleted Hdac3 specifically in the heart by breeding homozygous Hdac3loxP/loxP mice to transgenic mice expressing Cre recombinase under the control of the α-myosin heavy chain (α_-MHC_) promoter (21). Cardiomyocyte-restricted deletion of Hdac3 (hereafter referred to as Hdac3cko for Hdac3 cardiac KO; wild-type are represented by Hdac3loxP/loxP mice) was confirmed by RT-PCR and Western blot (Figure 2, A and B). RT-PCR of Hdac3 using primers flanking the floxed region showed efficient deletion of Hdac3; however, residual expression of Hdac3 was seen using primers within the deleted region as well as by Western blot, suggesting that Hdac3 is expressed at very low levels in α_-MHC–Cre_ negative cell types such as cardiac fibroblasts (Figure 2, A and B). Expression levels of other class I and class II HDACs were not significantly altered in Hdac3cko hearts (Figure 2C).
Cardiac-specific deletion of Hdac3. (A) Semiquantitative RT-PCR showing Hdac3 transcript levels in wild-type and Hdac3cko mice using primers in exon 10, forward (F), and exon 15, reverse (R), or exon 13, forward, and exon 15, reverse. Cardiac deletion of Hdac3 results in deletion of exons 11 through 14. (B) Western blot showing reduced expression of Hdac3 in Hdac3cko hearts. (C) Real-time PCR of class I and class II Hdacs in wild-type and Hdac3cko hearts. cKO, Hdac3cko.
Hdac3cko mice were born at Mendelian ratios; however, signs of cardiac hypertrophy, assessed by heart weight (HW) to BW ratios, were apparent by 4 weeks of age and were exacerbated by 12 weeks of age, resulting in a 72% increase in HW/BW ratio compared with wild-type littermates (Figure 3A). Cardiac deletion of Hdac3 resulted in 100% lethality by 16 weeks of age, with significant lethality occurring between 12 and 14 weeks (Figure 3B). Hearts of Hdac3cko mice were hypertrophic and showed enlargement of both right and left atria (Figure 3C). Histology confirmed cardiomyocyte hypertrophy, especially in the LV free wall and septum, as well as robust interstitial fibrosis in Hdac3cko mice compared with wild-type littermates (Figure 3C). Cardiac stress markers atrial natriuretic factor (ANF, Nppa), brain natriuretic peptide (BNP, Nppb), and α-skeletal actin (Acta1) were significantly upregulated as early as 8 weeks of age in mutant mice, consistent with the hypertrophy seen by histology (Figure 3D). Expression of p21 (Cdkn1a), shown to be repressed by class I HDACs in a variety of cell types (22), was also significantly upregulated in hearts of Hdac3cko mice, supporting the role of class I HDACs as transcriptional repressors of p21.
Cardiac defects resulting from cardiac deletion of Hdac3. (A) HW/BW ratios of WT and Hdac3cko showing progression of cardiac hypertrophy. (B) Kaplan-Meier survival curve showing lethality by 16 weeks in Hdac3cko mice. (C) Masson trichrome–stained sections of wild-type and Hdac3cko mice at 12 weeks. Deletion of Hdac3 results in cardiac hypertrophy, left atrial thrombus, and cardiac fibrosis, seen in blue. Original magnification, ×20 (lower panels). (D) Expression of cardiac stress markers in Hdac3cko mice at 8 weeks. mRNA transcript levels were detected by real-time RT-PCR and normalized to 18S ribosomal RNA. ANF, atrial natriuretic factor; BNF, brain natriuretic peptide; αSkActin, α-skeletal actin. (E) Electron microscopy of LV tissue from WT and Hdac3cko hearts at 8 weeks. Scale bars: 5000 nm (×4,200); 1000 nm (×16,500).
Ultrastructural analysis of the LV free wall of the adult myocardium revealed that the normal juxtaposition of sarcomeres to mitochondria (Figure 3E), which facilitates efficient myofibrillar contraction and relaxation in normal cardiomyocytes, was aberrant in Hdac3cko mutants. Instead, cardiac deletion of Hdac3 resulted in disorganized and fragmented myofibrils, associated with intracellular debris and disarrangement of mitochondria that showed reduced cristae density (Figure 3E).
To determine whether there is a correlation between Hdac3 expression levels and pathological conditions of the heart, we examined Hdac3 expression in multiple settings of hypertrophy and failure. Hdac3 levels were not significantly altered following angiotensin infusion, aortic banding, myocardial infarction, or in the Zucker diabetic fatty (ZDF) rat heart; however, Hdac3 expression was decreased following isoproterenol infusion (Supplemental Figure 1; supplemental material available online with this article; doi:10.1172/JCI35847DS1).
Functional analyses of wild-type and Hdac3cko mice were performed at 12 weeks of age by echocardiography. As shown in Figure 4, Hdac3cko mice showed diminished contractility and ventricular dysfunction, as indicated by reduced fractional shortening (39.25 ± 0.75 vs. 78.65 ± 4.35 for WT), and increased LV chamber dilatation, as assessed by systolic and diastolic internal diameters, LVIDs and LVIDd, respectively. Additionally, ECG was performed on 8- and 14-week-old mice to determine whether Hdac3cko mice have conduction system defects. Hdac3cko mice showed no overt abnormalities in their sinus rhythm compared with wild-type littermates (Supplemental Figure 2). Furthermore, continuous telemetry was performed on wild-type and Hdac3cko mice from 12 to 16 weeks of age to determine whether arrhythmias contributed to sudden death. No signs of cardiac arrhythmia were observed in Hdac3cko mice when compared with wild-type littermates (data not shown).
Echocardiographic data of Hdac3cko mice. Values show severe ventricular dysfunction in Hdac3cko hearts at 12 weeks of age. Values represent mean ± SEM. FS, fractional shortening.
Upregulation of myocardial energetic genes from cardiac deletion of Hdac3. In an effort to more precisely understand the primary cause of cardiomyopathy in Hdac3 mutant hearts, we performed microarray analysis on LVs from 5-week-old mice. At this time, mutant hearts showed moderate increases in HW/BW ratios and relatively minor changes in cardiac stress markers (Figure 3A and data not shown). Gene ontology analysis of significantly upregulated transcripts in Hdac3cko mice revealed dramatic dysregulation of cardiac metabolism in the mutant hearts (Figure 5A and Supplemental Data).
Aberrant expression of cardiac metabolism genes from cardiac deletion of Hdac3. (A) Microarray analysis was performed at 5 weeks, and gene ontology analysis was performed with PANTHER (http://www.pantherdb.org). Significantly enriched biological processes are shown and plotted as the –log(P value). (B) Expression of PPAR-regulated mitochondrial uncoupling genes is increased in Hdac3cko mice. (C and D) Expression of fatty acid uptake and oxidation genes is moderately increased in Hdac3cko hearts. FATP, fatty acid transport protein. (E) Glucose metabolism is decreased in Hdac3cko hearts. For B–E, real-time RT-PCR was performed from LV RNA of 6-week-old wild-type and Hdac3cko mice in absence or presence of Wy14,643. Transcript levels were normalized to 18S ribosomal RNA. Error bars represent SD.
Cardiac energetics is tightly regulated by the PPAR and the estrogen-related receptor (ERR) families of nuclear hormone receptors (23), and PPARα cardiac overexpression results in diabetic cardiomyopathy (24). Expression levels for PPARα, PPARγ, ERRα, and PGC-1α were unchanged in Hdac3cko hearts compared with wild-type littermates, suggesting the phenotype is independent of changes in receptor or coactivator expression (Supplemental Figure 3). To determine whether the cardiac hypertrophy and ventricular dysfunction in Hdac3 mutant mice resulted from rampant nuclear receptor–dependent gene activation, we assayed known PPARα target genes in ventricles of Hdac3cko mice. PPARα has been shown to regulate expression of the mitochondrial uncoupling proteins UCP2 and UCP3 (25). Accordingly, transcript levels for both UCP2 and UCP3 were significantly upregulated at baseline (6.5-fold and 2.9-fold, respectively), and this induction was increased upon administration of Wy14,643, a synthetic PPARα-agonist (Figure 5B).
Real-time PCR analysis of genes encoding proteins involved in fatty acid import, transport, and esterification (fatty acid transport protein [FATP, _Slc27a1_], CD36, and fatty acyl-CoA synthetase [FACS, _Acsl1_]) showed modest to insignificant changes at baseline. Following administration of Wy14,643, these levels were upregulated (Figure 5C), consistent with previous studies showing PPAR ligands to be rate limiting under physiological conditions (24).
The expression of PPARα-responsive genes involved in fatty acid oxidation was also analyzed by real-time PCR. Similar to the expression of PPARα-dependent genes involved in fatty acid import, induction of PPARα-responsive genes involved in fatty acid oxidation was modest at baseline. Surprisingly, expression levels of muscle carnitine palmitoyl transferase-1 (M-CPT1) were unchanged in Hdac3cko mice compared with wild-type mice, and Wy14,643 treatment had no effect on M-CPT1 transcript levels in wild-type or Hdac3cko hearts (Figure 5D); however, additional enzymes involved in mitochondrial fatty acid oxidation were significantly upregulated at baseline, including long- and very long–chain acyl-CoA dehydrogenase (LCAD, Acadl, and VLCAD, Acadvl, respectively) (data not shown). Conversely, acyl-CoA oxidase 1 (ACOX) was significantly increased in Hdac3cko hearts and was further upregulated in response to Wy14,643 (Figure 5D). ACOX is the first enzyme in peroxisomal fatty acid β-oxidation, suggesting Hdac3cko hearts possess greater fatty acid oxidation potential than wild-type littermates.
Decreased expression of genes involved in glucose utilization from cardiac deletion of Hdac3. In diabetic cardiomyopathies, increased expression of genes involved in fatty acid import and fatty acid oxidation is coupled to a decreased utilization of the glucose oxidation pathway (26). To determine whether Hdac3cko mice show defects in glucose uptake and utilization, we examined expression levels of the glucose transporters GLUT1 and GLUT4. GLUT1 levels were relatively unchanged in Hdac3cko mice compared with wild-type (data not shown), whereas GLUT4 expression was significantly downregulated in Hdac3cko mice (Figure 5E) and was further decreased in response to Wy14,643 (Figure 5E). GLUT1 controls basal glucose uptake while GLUT4 regulates glucose transport in an insulin-sensitive–dependent manner. Downregulation of GLUT4 in Hdac3cko mice is consistent with the phenotype resulting from excessive PPARα activity.
Pyruvate dehydrogenase kinase 4 (PDK4) regulates the pyruvate dehydrogenase complex through phosphorylation and subsequent inactivation. PDK4 levels and activity are increased in diabetic hearts (27). Similarly, Hdac3cko mice showed an increase in expression of PDK4 that was further elevated upon administration of Wy14,643 (Figure 5E).
Localization of Hdac3 to the promoters of PPARα-responsive genes. To further investigate the regulation of PPARα-responsive genes by Hdac3, we performed ChIP on multiple genes upregulated in Hdac3cko hearts from neonatal rat myocytes. Immunoprecipitation of Hdac3 or PPARα was able to robustly pull down the PPAR response element within the promoters of the UCP2, UCP3, FACS, FATP, and PDK4 genes compared with the negative anti-HA control or the negative exon 5 of UCP2 control (Figure 6A). These findings indicate that Hdac3 resides in a repressive complex with PPARα under basal physiological conditions.
Local promoter architecture of dysregulated transcripts. (A) ChIP assays were performed from neonatal rat myocytes. Chromatin was immunoprecipitated with antibodies against HA, HDAC3, or PPARα. Primers flank the PPAR-responsive elements of each gene, and precipitated DNA was analyzed by PCR. Nonimmunoprecipitated sample served as an input control. (B) Global histone acetylation is unchanged in Hdac3cko hearts. Histones were isolated from wild-type and Hdac3cko hearts and subjected to Western blot analysis using antibodies against acetyl-H3 (ac-K-H3), acetyl-H4 (ac-K-H4), pan–acetyl-lysine (ac-K), and H3. (C) Representative quantitative ChIP on dysregulated transcripts performed in triplicate from myocytes isolated from wild-type and Hdac3cko hearts using anti–acetyl-H3 for immunoprecipitation.
Histone acetylation is locally increased in Hdac3cko hearts. To determine whether loss of Hdac3 results in global changes in histone acetylation, we isolated histones from wild-type and Hdac3cko hearts and examined the acetylation states of histones H3 and H4. Western blot analyses for acetyl-H3, acetyl-H4, and pan–acetyl-lysine revealed no global changes in histone acetylation in Hdac3cko hearts (Figure 6B). The localization of Hdac3 to the promoters of multiple PPAR-responsive genes suggested that changes in histone acetylation might occur locally at the promoters of upregulated transcripts. To investigate this, we isolated cardiomyocytes from wild-type and Hdac3cko mice and performed quantitative ChIP assays. Immunoprecipitation on the PPAR-responsive elements with acetyl-H3 revealed increased local acetylation levels at the promoters of multiple PPAR-responsive genes in Hdac3cko myocytes compared with wild-type controls but not on the promoters of genes unaltered in Hdac3cko, such as Gapdh (Figure 6C).
Cardiac deletion of Hdac3 causes ligand-induced lipid accumulation. The ligand-inducible activation of multiple PPAR-responsive genes in cardiomyocytes of Hdac3cko mice suggests these mice are sensitive to increased fatty acids in the circulation. A hallmark of the diabetic heart is lipid accumulation in myocytes due to augmented fatty acid import (28). To determine whether Hdac3 mutant mice show myocardial lipid accumulation following increases in circulating fatty acids, we subjected Hdac3cko and wild-type mice to a 24-hour fast, which induces circulating fatty acids that can subsequently serve as ligands for PPARs. After fasting, hearts were excised and triglycerides were quantified. Mice fed ad libitum showed no significant difference in triglyceride content between Hdac3cko and wild-type mice (Figure 7A). However, Hdac3cko fasted mice showed a dramatic increase in myocardial triglycerides compared with wild-type controls (Figure 7A). Quantification of serum fatty acid and triglyceride levels showed there to be no significant difference between wild-type and Hdac3cko mice (data not shown). Consistent with these findings, oil red O staining of histological sections from fed and fasted wild-type and Hdac3cko hearts showed no significant staining in fed mice of either genotype (data not shown), whereas fasted myocytes from Hdac3cko mice showed a pronounced increase in neutral lipid accumulation (Figure 7B). Neutral lipids could be readily visualized throughout both ventricular free walls as well as the ventricular septum; however, the atria remained free of lipids. These results further point to Hdac3 as an important regulator of PPAR and other transcription factors that govern myocardial energy metabolism.
Myocardial lipid accumulation and mitochondrial dysfunction in Hdac3cko mice. (A) Increased triglycerides in Hdac3cko hearts after fasting. Triglycerides were extracted from wild-type and Hdac3cko hearts at 8 weeks and quantified. (B) Oil red O staining of 8-week-old wild-type mice and cardiac deletion of Hdac3 following a 24-hour fast. Red droplets indicate neutral lipids. Hdac3cko mice show substantial lipid accumulation. (C) Hdac3cko hearts show impaired mitochondrial function. Complex I activity, NADH oxidase activity, and free radicals were determined from 8-week-old wild-type and Hdac3cko mice.
Mitochondrial dysfunction resulting from cardiac deletion of Hdac3. To further define the metabolic abnormalities resulting from cardiac-specific deletion of Hdac3, we compared mitochondrial function in wild-type and Hdac3cko mice. Mitochondrial dysfunction is intimately linked to diabetic cardiomyopathy through defects in glucose utilization and increased fatty acid oxidation (29). Increased fatty acid oxidation results in increased reducing equivalents to the electron transport chain, which in turn generates free radicals and leads to mitochondrial uncoupling (30). Free fatty acids are able to directly promote free radical production through the inhibition of complex I of the electron transport chain (31–33). Consistently, Hdac3cko mice showed a 25% reduction in complex I activity accompanied by a 39% reduction in NADH oxidase activity (Figure 7C). Additionally, free radical production was increased 2-fold in Hdac3cko mice compared with wild-type littermates (Figure 7C).