In the adult heart, a variety of stresses induce re-expression of a fetal gene program in association with myocyte hypertrophy and heart failure. Here we show that histone deacetylase-2 (Hdac2) regulates expression of many fetal cardiac isoforms. Hdac2 deficiency or chemical histone deacetylase (HDAC) inhibition prevented the re-expression of fetal genes and attenuated cardiac hypertrophy in hearts exposed to hypertrophic stimuli. Resistance to hypertrophy was associated with increased expression of the gene encoding inositol polyphosphate-5-phosphatase f (Inpp5f) resulting in constitutive activation of glycogen synthase kinase 3beta (Gsk3beta) via inactivation of thymoma viral proto-oncogene (Akt) and 3-phosphoinositide-dependent protein kinase-1 (Pdk1). In contrast, Hdac2 transgenic mice had augmented hypertrophy associated with inactivated Gsk3beta. Chemical inhibition of activated Gsk3beta allowed Hdac2-deficient adults to become sensitive to hypertrophic stimulation. These results suggest that Hdac2 is an important molecular target of HDAC inhibitors in the heart and that Hdac2 and Gsk3beta are components of a regulatory pathway providing an attractive therapeutic target for the treatment of cardiac hypertrophy and heart failure.

Background-A number of distinct stress signaling pathways in myocardium cause cardiac hypertrophy and heart failure.Class II histone deacetylases (HDACs) antagonize several stress-induced pathways and hypertrophy. However, cardiac hypertrophy induced by transgenic overexpression of the homeodomain only protein, HOP, can be prevented by the nonspecific HDAC inhibitors trichostatin A and valproic acid, suggesting that alternate targets that oppose class II HDAC function might exist in myocardium. We tested the effects of several HDAC inhibitors, including a class I HDAC-selective inhibitor, SK-7041, on cardiac hypertrophy induced by angiotensin II (Ang II) treatment or aortic banding (AB). Methods and Results-Cardiac hypertrophy was induced by chronic infusion of Ang II or by AB in mice or rats and evaluated by determining the ratio of heart weight to body weight or to tibia length, cross-sectional area, or echocardiogram. Cardiac hypertrophy induced by Ang II or AB for 2 weeks was significantly reduced by simultaneous administration of trichostatin A, valproic acid, or SK-7041. Echocardiogram revealed that exaggerated left ventricular systolic dimensions were relieved by HDAC inhibitors. HDAC inhibitors partially reversed preestablished cardiac hypertrophy and improved survival of AB mice. The expressions of atrial natriuretic factor, ␣-tubulin, ␤-myosin heavy chain, and interstitial fibrosis were reduced by HDAC inhibition. Conclusions-These results suggest that the predominant effect of HDAC inhibition, mainly mediated by class I HDACs, is to prevent cardiac hypertrophy in response to a broad range of agonist and stretch stimuli.

A novel method for dynamic ventilation imaging of the full respiratory cycle from four-dimensional computed tomography (4D CT) acquired without added contrast is presented. Three cases with 4D CT images obtained with respiratory gated acquisition for radiotherapy treatment planning were selected. Each of the 4D CT data sets was acquired during resting tidal breathing. A deformable image registration algorithm mapped each (voxel) corresponding tissue element across the 4D CT data set. From local average CT values, the change in fraction of air per voxel (i.e. local ventilation) was calculated. A 4D ventilation image set was calculated using pairs formed with the maximum expiration image volume, first the exhalation then the inhalation phases representing a complete breath cycle. A preliminary validation using manually determined lung volumes was performed. The calculated total ventilation was compared to the change in contoured lung volumes between the CT pairs (measured volume). A linear regression resulted in a slope of 1.01 and a correlation coefficient of 0.984 for the ventilation images. The spatial distribution of ventilation was found to be case specific and a 30% difference in mass-specific ventilation between the lower and upper lung halves was found. These images may be useful in radiotherapy planning.

Cited2 is a cAMP-responsive element-binding protein (CBP)͞p300 interacting transcriptional modulator and a proposed negative regulator for hypoxia-inducible factor (HIF)-1␣ through its competitive binding with HIF-1␣ to CBP͞p300. Disruption of the gene encoding Cited2 is embryonic lethal because of defects in the development of heart and neural tube. Morphological and Doppler echocardiographic analyses of Cited2 ؊/؊ embryos reveal severe cardiovascular abnormalities, including pulmonic arterial stenosis and ventricular septal defects accompanied by high peak outflow velocities, features of the human congenital cardiac defect termed tetralogy of Fallot. The mRNA levels of several HIF-1␣-responsive genes, such as vascular endothelial growth factor (VEGF), Glut1, and phosphoglycerate kinase 1, increased in the Cited2 ؊/؊ hearts. The increase of VEGF levels is significant, because defects in the Cited2 ؊/؊ embryos closely resemble the major defects observed in the VEGF transgenic embryos. Finally, compared with wild-type, cultured fibroblasts from Cited2 ؊/؊ embryos demonstrate an enhanced expression of HIF-1␣-responsive genes under hypoxic conditions. These observations suggest that functional loss of Cited2 is responsible for defects in heart and neural tube development, in part because of the modulation of HIF-1 transcriptional activities in the absence of Cited2. These findings demonstrate that Cited2 is an indispensable regulatory gene during prenatal development.

Specification of arteries and veins is a key process for establishing functional vasculature during embryogenesis and involves distinctly different signaling mechanisms. Vascular endothelial growth factor-A (VEGFA) is required for differentiation of arteries; however, the upstream angiogenic factor for vein specification is unknown. Klippel-Trenaunay syndrome (KTS) is a congenital vascular disease associated with capillary and venous malformations (VMs), but not with arterial defects. We have previously reported that upregulation of angiogenic factor AGGF1 is associated with KTS, but the molecular mechanism is not clear. Here, we show that AGGF1 is involved in establishing venous identity in zebrafish embryos. Overexpression of AGGF1 led to increased angiogenesis and increased lumen diameter of veins, whereas knockdown of AGGF1 expression resulted in defective vasculogenesis and angiogenesis. Overexpression of AGGF1 increased expression of venous markers (e.g. flt4), but had little effect on arterial markers (e.g. notch5). Knockdown of AGGF1 expression resulted in a loss of venous identity (loss of expression of flt4, ephb4 and dab2), but had no effect on the expression of arterial development. We further show that AGGF1 activates AKT, and that decreased AGGF1 expression inhibits AKT activation. Overexpression of constitutively active AKT rescues the loss of venous identity caused by AGGF1 downregulation. Our study establishes AGGF1 as an angiogenic factor with an important role in the specification of vein identity and suggests that AGGF1-mediated AKT signaling is responsible for establishing venous cell fate. We propose that increased AGGF1 expression leads to increased vein differentiation by inducing activation of AKT signaling, resulting in VMs s in KTS patients.

Cytochrome P450 (Cyp)17A1 has both 17α-hydroxylase and 17,20-lyase activities, which are involved in the steroidogenic pathway that produces androgens and estrogens. Previously, a phenotype of all-male cyp17a1-deficient zebrafish generated by transcription activatorlike effector nuclease has been reported. In the current study, the mechanisms relating to Cyp17a1 that are involved in the development of sexual traits, especially gonadal differentiation and testicular development, were characterized. We found that the cyp17a1-deficient fish at 3 months postfertilization (mpf) were all fertile males with normal testis and spermatogenesis but compromised male-typical mating behaviors and secondary sex characters (SSCs), including breeding tubercles, body pigmentation, and anal fin coloration. These results demonstrate that spermatogenesis and testicular development are not as susceptible to androgen deficiency compared with the formation of male-typical SSCs and mating behaviors in zebrafish. The differentiation of the juvenile ovary into the mature ovary failed during the critical sexual differentiation stage. This all-male phenotype of the cyp17a1-deficient fish could be restored with testosterone or estradiol treatment. For testicular development in cyp17a1-deficient fish, a gradually increasing number of spermatozoa and testis hypertrophy from 3 to 6 mpf were observed, accompanied by constitutively upregulated pituitary gonadotropin FSH subunit β (fshβ). The hypertrophic testis and enhanced spermatogenesis in the cyp17a1-deficient fish at 6 mpf could be effectively rescued by fshβ depletion. These results confirm that adequate estrogen is essential for maintaining ovarian differentiation, and they provide new insight into the role of FSHβ in male testicular development and spermatogenesis.

Hop is an unusual homeodomain protein that was first identified in the developing heart where it functions downstream of Nkx2.5 to modulate cardiac gene expression. Hop functions through interactions with histone deacetylase (HDAC) 2 to mediate repression of cardiac-specific genes, and recent studies show that HDAC activity and HDAC2 expression are decreased in people with chronic obstructive pulmonary disease. Here, we show that Hop is expressed in airway epithelium coincident with HDAC2, and expression is induced by the combination of dexamethasone and cAMP in parallel with induction of surfactant protein gene expression. Hop functions in the developing pulmonary airway, acting downstream of Nkx2.1 and GATA6, to negatively regulate surfactant protein expression. Loss of Hop expression in vivo results in defective type 2 pneumocyte development with increased surfactant production and disrupted alveolar formation. Thus Hop represents a novel regulator of pulmonary maturation that is induced by glucocorticoids to mediate functionally important HDAC-dependent negative feedback regulation.

1α,25(OH)D (vitamin D) is crucial for mineral homeostasis in mammals, but the precise effects of 1α,25(OH)D in adipose tissue remain to be clarified in vivo. The initial 25-hydroxylation is catalyzed by liver microsomal cytochrome P450 2R1 (CYP2R1), which is conserved in vertebrates. To probe the physiological function(s) of 1α,25(OH)D in teleosts, we generated two independent cyp2r1-deficient zebrafish lines. These mutants exhibit retarded growth and increased obesity, especially in the visceral adipose tissue (VAT). These defects could be rescued with 25(OH)D treatments. ChIP-PCR analyses demonstrated that pgc1a is the target of the vitamin D receptor in the liver and VAT of zebrafish. Significantly decreased protein levels of Pgc1a, impaired mitochondrial biogenesis, and free fatty acid oxidation are also observed in the cyp2r1 mutant VAT. Our results demonstrate that regulation of 1α,25(OH)D during lipid metabolism occurs through the regulation of Pgc1a for mitochondrial biogenesis and oxidative metabolism within zebrafish VAT.