Epigenomes in Cardiovascular Disease.

If unifying principles could be revealed for how the same genome encodes different eukaryotic cells and for how genetic variability and environmental input are integrated to impact cardiovascular health, grand challenges in basic cell biology and translational medicine may succumb to experimental dissection. A rich body of work in model systems has implicated chromatin-modifying enzymes, DNA methylation, noncoding RNAs, and other transcriptome-shaping factors in adult health and in the development, progression, and mitigation of cardiovascular disease. Meanwhile, deployment of epigenomic tools, powered by next-generation sequencing technologies in cardiovascular models and human populations, has enabled description of epigenomic landscapes underpinning cellular function in the cardiovascular system. This essay aims to unpack the conceptual framework in which epigenomes are studied and to stimulate discussion on how principles of chromatin function may inform investigations of cardiovascular disease and the development of new therapies

Loss of the Putative Catalytic Domain of HDAC4 Leads to Reduced Thermal Nociception and Seizures while Allowing Normal Bone Development

Histone deacetylase 4 (HDAC4) has been associated with muscle & bone development [1]–[6]. N-terminal MEF2 and RUNX2 binding domains of HDAC4 have been shown to mediate these effects in vitro. A complete gene knockout has been reported to result in premature ossification and associated defects resulting in postnatal lethality [6]. We report a viral insertion mutation that deletes the putative deacetylase domain, while preserving the N-terminal portion of the protein. Western blot and immuno-precipitation analysis confirm expression of truncated HDAC4 containing N-terminal amino acids 1-747. These mutant mice are viable, living to at least one year of age with no gross defects in muscle or bone. At 2–4 months of age no behavioral or physiological abnormalities were detected except for an increased latency to respond to a thermal nociceptive stimulus. As the mutant mice aged past 5 months, convulsions appeared, often elicited by handling. Our findings confirm the sufficiency of the N-terminal domain for muscle and bone development, while revealing other roles of HDAC4

Histone deacetylase adaptation in single ventricle heart disease and a young animal model of right ventricular hypertrophy.

BackgroundHistone deacetylase (HDAC) inhibitors are promising therapeutics for various forms of cardiac diseases. The purpose of this study was to assess cardiac HDAC catalytic activity and expression in children with single ventricle (SV) heart disease of right ventricular morphology, as well as in a rodent model of right ventricular hypertrophy (RVH).MethodsHomogenates of right ventricle (RV) explants from non-failing controls and children born with a SV were assayed for HDAC catalytic activity and HDAC isoform expression. Postnatal 1-day-old rat pups were placed in hypoxic conditions, and echocardiographic analysis, gene expression, HDAC catalytic activity, and isoform expression studies of the RV were performed.ResultsClass I, IIa, and IIb HDAC catalytic activity and protein expression were elevated in the hearts of children born with a SV. Hypoxic neonatal rats demonstrated RVH, abnormal gene expression, elevated class I and class IIb HDAC catalytic activity, and protein expression in the RV compared with those in the control.ConclusionsThese data suggest that myocardial HDAC adaptations occur in the SV heart and could represent a novel therapeutic target. Although further characterization of the hypoxic neonatal rat is needed, this animal model may be suitable for preclinical investigations of pediatric RV disease and could serve as a useful model for future mechanistic studies

Physiological Biomimetic Culture System for Pig and Human Heart Slices

RATIONALE: Preclinical testing of cardiotoxicity and efficacy of novel heart failure therapies faces a major limitation: the lack of an in situ culture system that emulates the complexity of human heart tissue and maintains viability and functionality for a prolonged time. OBJECTIVE: To develop a reliable, easily reproducible, medium-throughput method to culture pig and human heart slices under physiological conditions for a prolonged period of time. METHODS AND RESULTS: Here, we describe a novel, medium-throughput biomimetic culture system that maintains viability and functionality of human and pig heart slices (300 µm thickness) for 6 days in culture. We optimized the medium and culture conditions with continuous electrical stimulation at 1.2 Hz and oxygenation of the medium. Functional viability of these slices over 6 days was confirmed by assessing their calcium homeostasis, twitch force generation, and response to β-adrenergic stimulation. Temporal transcriptome analysis using RNAseq at day 2, 6, and 10 in culture confirmed overall maintenance of normal gene expression for up to 6 days, while over 500 transcripts were differentially regulated after 10 days. Electron microscopy demonstrated intact mitochondria and Z-disc ultra-structures after 6 days in culture under our optimized conditions. This biomimetic culture system was successful in keeping human heart slices completely viable and functionally and structurally intact for 6 days in culture. We also used this system to demonstrate the effects of a novel gene therapy approach in human heart slices. Furthermore, this culture system enabled the assessment of contraction and relaxation kinetics on isolated single myofibrils from heart slices after culture. CONCLUSIONS: We have developed and optimized a reliable medium-throughput culture system for pig and human heart slices as a platform for testing the efficacy of novel heart failure therapeutics and reliable testing of cardiotoxicity in a 3D heart model

Immediate response of myocardium to pressure overload includes transient regulation of genes associated with mitochondrial bioenergetics and calcium availability

Ventricular hypertrophy is one of the major myocardial responses to pressure overload (PO). Most studies on early myocardial response focus on the days or even weeks after induction of hypertrophic stimuli. Since mechanotransduction pathways are immediately activated in hearts undergoing increased work load, it is reasonable to infer that the myocardial gene program may be regulated in the first few hours. In the present study, we monitored the expression of some genes previously described in the context of myocardial hypertrophic growth by using the Northern blot technique, to estimate the mRNA content of selected genes in rat myocardium for the periods 1, 3, 6, 12 and 48 h after PO stimuli. Results revealed an immediate switch in the expression of genes encoding alpha and beta isoforms of myosin heavy chain, and up-regulation of the cardiac isoform of alpha actin. We also detected transitory gene regulation as the increase in mitochondrial cytochrome c oxidase 1 gene expression, parallel to down-regulation of genes encoding sarco(endo)plasmic reticulum Ca+2 ATPase and sodium-calcium exchanger. Taken together, these results indicate that initial myocardial responses to increased work load include alterations in the contractile properties of sarcomeres and transitory adjustment of mitochondrial bioenergetics and calcium availability

Histone Deacetylases Control Neurogenesis in Embryonic Brain by Inhibition of BMP2/4 Signaling

Background Histone-modifying enzymes are essential for a wide variety of cellular processes dependent upon changes in gene expression. Histone deacetylases (HDACs) lead to the compaction of chromatin and subsequent silencing of gene transcription, and they have recently been implicated in a diversity of functions and dysfunctions in the postnatal and adult brain including ocular dominance plasticity, memory consolidation, drug addiction, and depression. Here we investigate the role of HDACs in the generation of neurons and astrocytes in the embryonic brain. Principal Findings As a variety of HDACs are expressed in differentiating neural progenitor cells, we have taken a pharmacological approach to inhibit multiple family members. Inhibition of class I and II HDACs in developing mouse embryos with trichostatin A resulted in a dramatic reduction in neurogenesis in the ganglionic eminences and a modest increase in neurogenesis in the cortex. An identical effect was observed upon pharmacological inhibition of HDACs in in vitro-differentiating neural precursors derived from the same brain regions. A reduction in neurogenesis in ganglionic eminence-derived neural precursors was accompanied by an increase in the production of immature astrocytes. We show that HDACs control neurogenesis by inhibition of the bone morphogenetic protein BMP2/4 signaling pathway in radial glial cells. HDACs function at the transcriptional level by inhibiting and promoting, respectively, the expression of Bmp2 and Smad7, an intracellular inhibitor of BMP signaling. Inhibition of the BMP2/4 signaling pathway restored normal levels of neurogenesis and astrogliogenesis to both ganglionic eminence- and cortex-derived cultures in which HDACs were inhibited. Conclusions Our results demonstrate a transcriptionally-based regulation of BMP2/4 signaling by HDACs both in vivo and in vitro that is critical for neurogenesis in the ganglionic eminences and that modulates cortical neurogenesis. The results also suggest that HDACs may regulate the developmental switch from neurogenesis to astrogliogenesis that occurs in late gestation

Chromatin Remodeling Pathways in Smooth Muscle Cell Differentiation, and Evidence for an Integral Role for p300

Phenotypic alteration of vascular smooth muscle cells (SMC) in response to injury or inflammation is an essential component of vascular disease. Evidence suggests that this process is dependent on epigenetic regulatory processes. P300, a histone acetyltransferase (HAT), activates crucial muscle-specific promoters in terminal (non-SMC) myocyte differentiation, and may be essential to SMC modulation as well.We performed a subanalysis examining transcriptional time-course microarray data obtained using the A404 model of SMC differentiation. Numerous chromatin remodeling genes (up to 62% of such genes on our array platform) showed significant regulation during differentiation. Members of several chromatin-remodeling families demonstrated involvement, including factors instrumental in histone modification, chromatin assembly-disassembly and DNA silencing, suggesting complex, multi-level systemic epigenetic regulation. Further, trichostatin A, a histone deacetylase inhibitor, accelerated expression of SMC differentiation markers in this model. Ontology analysis indicated a high degree of p300 involvement in SMC differentiation, with 60.7% of the known p300 interactome showing significant expression changes. Knockdown of p300 expression accelerated SMC differentiation in A404 cells and human SMCs, while inhibition of p300 HAT activity blunted SMC differentiation. The results suggest a central but complex role for p300 in SMC phenotypic modulation.Our results support the hypothesis that chromatin remodeling is important for SMC phenotypic switching, and detail wide-ranging involvement of several epigenetic modification families. Additionally, the transcriptional coactivator p300 may be partially degraded during SMC differentiation, leaving an activated subpopulation with increased HAT activity and SMC differentiation-gene specificity

NF-kappaB Mediated Transcriptional Repression of Acid Modifying Hormone Gastrin

Helicobacter pylori is a major pathogen associated with the development of gastroduodenal diseases. It has been reported that H. pylori induced pro-inflammatory cytokine IL1B is one of the various modulators of acid secretion in the gut. Earlier we reported that IL1B-activated NFkB down-regulates gastrin, the major hormonal regulator of acid secretion. In this study, the probable pathway by which IL1B induces NFkB and affects gastrin expression has been elucidated. IL1B-treated AGS cells showed nine-fold activation of MyD88 followed by phosphorylation of TAK1 within 15 min of IL1B treatment. Furthermore, it was observed that activated TAK1 significantly up-regulates the NFkB subunits p50 and p65. Ectopic expression of NFkB p65 in AGS cells resulted in about nine-fold transcriptional repression of gastrin both in the presence and absence of IL1B. The S536A mutant of NFkB p65 is significantly less effective in repressing gastrin. These observations show that a functional NFkB p65 is important for IL1B-mediated repression of gastrin. ChIP assays revealed the presence of HDAC1 and NFkB p65 along with NCoR on the gastrin promoter. Thus, the study provides mechanistic insight into the IL1B-mediated gastrin repression via NFk

Fasting and High-Fat Diet Alter Histone Deacetylase Expression in the Medial Hypothalamus

Increasing attention is now being given to the epigenetic regulation of animal and human behaviors including the stress response and drug addiction. Epigenetic factors also influence feeding behavior and metabolic phenotypes, such as obesity and insulin sensitivity. In response to fasting and high-fat diets, the medial hypothalamus changes the expression of neuropeptides regulating feeding, metabolism, and reproductive behaviors. Histone deacetylases (HDACs) are involved in the epigenetic control of gene expression and alter behavior in response to a variety of environmental factors. Here, we examined the expression of HDAC family members in the medial hypothalamus of mice in response to either fasting or a high-fat diet. In response to fasting, HDAC3 and −4 expression levels increased while HDAC10 and −11 levels decreased. Four weeks on a high-fat diet resulted in the increased expression of HDAC5 and −8. Moreover, fasting decreased the number of acetylated histone H3- and acetylated histone H4-positive cells in the ventrolateral subdivision of the ventromedial hypothalamus. Therefore, HDACs may be implicated in altered gene expression profiles in the medial hypothalamus under different metabolic states

β-Hydroxy-β-Methylbutyrate (HMB) Promotes Neurite Outgrowth in Neuro2a Cells

β-Hydroxy-β-methylbutyrate (HMB) has been shown to enhance cell survival, differentiation and protein turnover in muscle, mainly activating phosphoinositide-3-kinase/protein kinase B (PI3K/Akt) and mitogen-activated protein kinases/ extracellular-signal-regulated kinases (MAPK/ERK) signaling pathways. Since these two pathways are related to neuronal survival and differentiation, in this study, we have investigated the neurotrophic effects of HMB in mouse neuroblastoma Neuro2a cells. In Neuro2a cells, HMB promotes differentiation to neurites independent from any effects on proliferation. These effects are mediated by activation of both the PI3K/Akt and the extracellular-signal-regulated kinases (ERK1/2) signaling as demonstrated by the use of specific inhibitors of these two pathways. As myocyte-enhancer factor 2 (MEF2) family of transcription factors are involved in neuronal survival and plasticity, the transcriptional activity and protein levels of MEF2 were also evaluated. HMB promoted MEF2-dependent transcriptional activity mediated by the activation of Akt and ERK1/2 pathways. Furthermore, HMB increases the expression of brain glucose transporters 1 (GLUT1) and 3 (GLUT3), and mTOR phosphorylation, which translates in a higher protein synthesis in Neuro2a cells. Furthermore, Torin1 and rapamycin effects on MEF2 transcriptional activity and HMB-dependent neurite outgrowth support that HMB acts through mTORC2. Together, these findings provide clear evidence to support an important role of HMB in neurite outgrowth.This project has been funded by Abbott Nutrition R&D