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The IGF2BP family of RNA binding proteins consists of three paralogs that regulate intracellular RNA localization, RNA stability, and translational control. Although IGF2BP1 and 3 are oncofetal proteins, IGF2BP2 expression is maintained in many tissues, including the heart, into adulthood. Previous studies indicated that IGF2BP2 is upregulated in cardiomyocytes during cardiac stress and remodelling and returns to normal levels in recovering hearts. These results raise the possibility that IGF2BP2 might play an adaptive role during cardiac stress and recovery. Using a conditional, inducible transgenic mouse line, we found that enhanced expression of an IGF2BP2 transgene in newborn or adult hearts leads to dilated cardiomyopathy (DCM) and death within 3-4 weeks. Downregulation of the transgene after 2 weeks, however, rescues these mice, with complete recovery by 12 weeks. Hearts overexpressing IGF2BP2 downregulate sarcomeric and mitochondrial proteins and have fragmented mitochondria and elongated, thinner sarcomeres. Consistent with these results, IGF2BP2 is upregulated in patients with DCM or after myocardial infarction. These results suggest that IGF2BP2 may be an attractive target for therapeutic intervention in DCM.

Whole-genome sequencing is identifying growing numbers of non-coding variants in human disease studies, but the lack of accurate functional annotations prevents their interpretation. We describe the genome-wide landscape of distant-acting enhancers active in the developing and adult human heart, an organ whose impairment is a predominant cause of mortality and morbidity. Using integrative analysis of >35 epigenomic data sets from mouse and human pre- and postnatal hearts we created a comprehensive reference of >80,000 putative human heart enhancers. To illustrate the importance of enhancers in the regulation of genes involved in heart disease, we deleted the mouse orthologs of two human enhancers near cardiac myosin genes. In both cases, we observe in vivo expression changes and cardiac phenotypes consistent with human heart disease. Our study provides a comprehensive catalogue of human heart enhancers for use in clinical whole-genome sequencing studies and highlights the importance of enhancers for cardiac function., Identification of non-coding variants has outstripped our ability to annotate and interpret them. Dickel et al. present a compendium of over 80,000 putative human heart enhancers and demonstrate that two conserved enhancers are required for proper cardiac function in mice.

BACKGROUND Myocardial mass is a key determinant of cardiac muscle function and hypertrophy. Myocardial depolarization leading to cardiac muscle contraction is reflected by the amplitude and duration of the QRS complex on the electrocardiogram (ECG). Abnormal QRS amplitude or duration reflect changes in myocardial mass and conduction, and are associated with increased risk of heart failure and death.OBJECTIVES This meta-analysis sought to gain insights into the genetic determinants of myocardial mass.METHODS We carried out a genome-wide association meta-analysis of 4 QRS traits in up to 73,518 individuals of European ancestry, followed by extensive biological and functional assessment.RESULTS We identified 52 genomic loci, of which 32 are novel, that are reliably associated with 1 or more QRS phenotypes at p CONCLUSIONS Taken together, our findings provide new insights into genes and biological pathways controlling myocardial mass and may help identify novel therapeutic targets. (C) 2016 by the American College of Cardiology Foundation.

© 2014 . The key information processing units within gene regulatory networks are enhancers. Enhancer activity is associated with the production of tissue specific noncoding RNAs yet the existence of such transcripts during cardiac development has not been established. Using an integrated genomic approach we demonstrate that fetal cardiac enhancers generate long noncoding RNAs (lncRNAs) during cardiac differentiation and morphogenesis. Enhancer expression correlates with the emergence of active enhancer chromatin states the initiation of RNA polymerase II at enhancer loci and expression of target genes. Orthologous human sequences are also transcribed in fetal human hearts and cardiac progenitor cells. Through a systematic bioinformatic analysis we identified and characterized for the first time a catalog of lncRNAs that are expressed during embryonic stem cell differentiation into cardiomyocytes and associated with active cardiac enhancer sequences. RNA sequencing demonstrates that many of these transcripts are polyadenylated multi exonic long noncoding RNAs. Moreover knockdown of two enhancer associated lncRNAs resulted in the specific downregulation of their predicted target genes. Interestingly the reactivation of the fetal gene program a hallmark of the stress response in the adult heart is accompanied by increased expression of fetal cardiac enhancer transcripts. Altogether these findings demonstrate that the activity of cardiac enhancers and expression of their target genes are associated with the production of enhancer derived lncRNAs. •Fetal cardiac enhancers are transcribed generating enhancer derived lncRNAs.

SummaryThe mammalian telencephalon plays critical roles in cognition, motor function, and emotion. Though many of the genes required for its development have been identified, the distant-acting regulatory sequences orchestrating their in vivo expression are mostly unknown. Here, we describe a digital atlas of in vivo enhancers active in subregions of the developing telencephalon. We identified more than 4,600 candidate embryonic forebrain enhancers and studied the in vivo activity of 329 of these sequences in transgenic mouse embryos. We generated serial sets of histological brain sections for 145 reproducible forebrain enhancers, resulting in a publicly accessible web-based data collection comprising more than 32,000 sections. We also used epigenomic analysis of human and mouse cortex tissue to directly compare the genome-wide enhancer architecture in these species. These data provide a primary resource for investigating gene regulatory mechanisms of telencephalon development and enable studies of the role of distant-acting enhancers in neurodevelopmental disorders.

We present a literature review of collaborative enterprises between psychiatrists and primary care physicians in Israel and other countries. Also described are local psychiatric liaison initiatives in Israel, as well as landmark studies of collaborative psychiatric care. These studies demonstrate the superiority of community psychiatric liaison models in the treatment of patients suffering from depressive anxiety disorders and somatization disorder. In light of the mental health reform process currently underway in Israel, it is important to develop, implement and assess such liaison models.

The functional consequences of genetic variation in mammalian regulatory elements are poorly understood. We report the in vivo dissection of three mammalian liver enhancers at single nucleotide resolution via a massively parallelized reporter assay. For each enhancer, we synthesized a library of >100,000 mutant haplotypes with 2–3% divergence from wild-type. Each haplotype was linked to a unique sequence tag embedded within a transcriptional cassette. We introduced each enhancer library into mouse liver and measured the relative activities of individual haplotypes en masse by sequencing of the transcribed tags. Linear regression yielded highly reproducible estimates of the impact of every possible single nucleotide change on enhancer activity. The functional impact of most mutations was modest, with ~22% impacting activity by >1.2-fold, and only ~3% by >2-fold. These results suggest that mammalian enhancers are relatively robust to single nucleotide changes. Several, but not all positions with higher impact showed evidence for purifying selection, or co-localized with known liver-associated transcription factor binding sites, demonstrating the value of empirical high-resolution functional analysis.

A transgenic mouse model for conditional induction of long-term hibernation via myocardium-specific expression of a VEGF-sequestering soluble receptor allowed the dissection of the hibernation process into an initiation and a maintenance phase. The hypoxic initiation phase was characterized by peak levels of K(ATP) channel and glucose transporter 1 (GLUT1) expression. Glibenclamide, an inhibitor of K(ATP) channels, blocked GLUT1 induction. In the maintenance phase, tissue hypoxia and GLUT1 expression were reduced. Thus, we employed a combined “-omics” approach to resolve this cardioprotective adaptation process. Unguided bioinformatics analysis on the transcriptomic, proteomic and metabolomic datasets confirmed that anaerobic glycolysis was affected and that the observed enzymatic changes in cardiac metabolism were directly linked to hypoxia-inducible factor (HIF)-1 activation. Although metabolite concentrations were kept relatively constant, the combination of the proteomic and transcriptomic dataset improved the statistical confidence of the pathway analysis by 2 orders of magnitude. Importantly, proteomics revealed a reduced phosphorylation state of myosin light chain 2 and cardiac troponin I within the contractile apparatus of hibernating hearts in the absence of changes in protein abundance. Our study demonstrates how combining different “-omics” datasets aids in the identification of key biological pathways: chronic hypoxia resulted in a pronounced adaptive response at the transcript and the protein level to keep metabolite levels steady. This preservation of metabolic homeostasis is likely to contribute to the long-term survival of the hibernating myocardium., Graphical Abstract Research Highlights ► The hibernation process was dissected into an initiation and a maintenance phase. ► Glibenclamide, an inhibitor of K(ATP) channels, blocked GLUT1 induction. ► The maintenance phase was characterized by attenuated tissue hypoxia. ► Phosphorylation of myosin light chain 2 and cardiac troponin I was reduced. ► Combining of proteomics and transcriptomics improved the bioinformatic pathway analysis.

A key energy-saving adaptation to chronic hypoxia that enables cardiomyocytes to withstand severe ischemic insults is hibernation, i.e., a reversible arrest of contractile function. Whereas hibernating cardiomyocytes represent the critical reserve of dysfunctional cells that can be potentially rescued, a lack of a suitable animal model has hampered insights on this medically important condition. We developed a transgenic mouse system for conditional induction of long-term hibernation and a system to rescue hibernating cardiomyocytes at will. Via myocardium-specific induction (and, in turn, deinduction) of a VEGF-sequestering soluble receptor, we show that VEGF is indispensable for adjusting the coronary vasculature to match increased oxygen consumption and exploit this finding to generate a hypoperfused heart. Importantly, ensuing ischemia is tunable to a level at which large cohorts of cardiomyocytes are driven to enter a hibernation mode, without cardiac cell death. Relieving the VEGF blockade even months later resulted in rapid revascularization and full recovery of contractile function. Furthermore, we show that left ventricular remodeling associated with hibernation is also fully reversible. The unique opportunity to uncouple hibernation from other ischemic heart phenotypes (e.g., infarction) was used to determine the genetic program of hibernation; uncovering hypoxia-inducible factor target genes associated with metabolic adjustments and induced expression of several cardioprotective genes. Autophagy, specifically self-digestion of mitochondria, was identified as a key prosurvival mechanism in hibernating cardiomyocytes. This system may lend itself for examining the potential utility of treatments to rescue dysfunctional cardiomyocytes and reverse maladaptive remodeling.

Oxygen is the ultimate source of oxidizing power for disulfide bond formation, suggesting that under limiting oxygen proper protein folding might be compromised. We show that secretion of vascular endothelial growth factor (VEGF), a protein with multiple disulfide bonds, was indeed impeded under hypoxia and was partially restored by artificial increase of oxidizing equivalents with diamide. Physiologically, the oxireductase endoplasmic reticulum oxidoreductin-1 (Ero1)-L alpha, but not other proteins in the relay of disulfide formation, was strongly upregulated by hypoxia and independently by hypoglycemia, two known accompaniments of tumors. Further, we provide genetic evidence that induction of Ero1-L alpha by hypoxia and hypoglycemia is mediated by the transcription factor hypoxia-inducible factor 1 (HIF-1) but is independent of p53. In natural human tumors, Ero1-L alpha mRNA was specifically induced in hypoxic microenvironments coinciding with that of upregulated VEGF expression. To establish a physiological relevance to modulations in Ero1-L alpha levels, we showed that even a modest, two- to three-fold reduction in Ero1-L alpha production via siRNA leads to significant inhibition of VEGF secretion, a compromised proliferation capacity and enhanced apoptosis. Together, these findings demonstrate that hypoxic induction of Ero1-L alpha is the key adaptive response in a previously unrecognized HIF-1-mediated pathway that operates to improve protein secretion under hypoxia and might be harnessed for inhibiting tumor growth via inhibiting VEGF-driven angiogenesis.

Objective— Proangiogenic therapy is a promising avenue for the treatment for chronic heart failure and a potentially powerful modality for reversing adverse cardiac remodeling. There is a concern, however, that adverse remodeling might enter an irreversible stage, and become refractory to treatments. The present study aims to determine whether neovascularization therapy is feasible at end stage heart failure and its capacity to reverse adverse cardiac remodeling during progressive disease stages. Methods and Results— Using a conditional transgenic mouse system for generating escalating levels of myocardium-specific vascular deficit and resultant stepwise development of heart remodeling, we show that left ventricular dilatation and fibrosis precede ventricular hypertrophy, but that interstitial fibrosis is progressive and eventually results in heart failure. Vascular endothelial growth factor–mediated neovascularization was efficient even at the end stage of disease, and rescued compromised contractile function. Remarkably, remodeling was also fully reversed by neovascularization during early and late stages. Adverse remodeling could not be rescued, however, at the end stage of the disease, thus defining a point of no return and indentifying a critical level of fibrosis as the key determinant to be considered in intended reversal. Conclusion— The study supports the notion of a restricted golden time for remodeling reversal but not for vascular endothelial growth factor–induced neovascularization, which is feasible even during advanced disease stages.

Development and function of the human heart depend on the dynamic control of tissue-specific gene expression by distant-acting transcriptional enhancers. To generate an accurate genome-wide map of human heart enhancers, we used an epigenomic enhancer discovery approach and identified ∼6,200 candidate enhancer sequences directly from fetal and adult human heart tissue. Consistent with their predicted function, these elements were markedly enriched near genes implicated in heart development, function and disease. To further validate their in vivo enhancer activity, we tested 65 of these human sequences in a transgenic mouse enhancer assay and observed that 43 (66%) drove reproducible reporter gene expression in the heart. These results support the discovery of a genome-wide set of non-coding sequences highly enriched in human heart enhancers which is likely to facilitate down-stream studies of the role of enhancers in development and pathological conditions of the heart.

Sequence polymorphisms in a 58-kilobase (kb) interval on chromosome 9p21 confer a markedly increased risk of coronary artery disease (CAD), the leading cause of death worldwide. The variants have a substantial effect on the epidemiology of CAD and other life-threatening vascular conditions because nearly one-quarter of Caucasians are homozygous for risk alleles. However, the risk interval is devoid of protein-coding genes and the mechanism linking the region to CAD risk has remained enigmatic. Here we show that deletion of the orthologous 70-kb non-coding interval on mouse chromosome 4 affects cardiac expression of neighbouring genes, as well as proliferation properties of vascular cells. Chr4(Delta70kb/Delta70kb) mice are viable, but show increased mortality both during development and as adults. Cardiac expression of two genes near the non-coding interval, Cdkn2a and Cdkn2b, is severely reduced in chr4(Delta70kb/Delta70kb) mice, indicating that distant-acting gene regulatory functions are located in the non-coding CAD risk interval. Allele-specific expression of Cdkn2b transcripts in heterozygous mice showed that the deletion affects expression through a cis-acting mechanism. Primary cultures of chr4(Delta70kb/Delta70kb) aortic smooth muscle cells exhibited excessive proliferation and diminished senescence, a cellular phenotype consistent with accelerated CAD pathogenesis. Taken together, our results provide direct evidence that the CAD risk interval has a pivotal role in regulation of cardiac Cdkn2a/b expression, and suggest that this region affects CAD progression by altering the dynamics of vascular cell proliferation. ispartof: NATURE vol:464 issue:7287 pages:409-U103 ispartof: location:England status: published

Rationale We have recently established a transgenic mouse model for conditional induction of long-term hibernation via myocardium-specific induction of a VEGF-sequestering soluble receptor. Objective Using a combined ‘-omics’ approach, we aim to resolve the cardioprotective response that preserves myocardial viability under chronic hypoxia by integrating mRNA, protein and metabolite changes in unsupervised network analysis. Methods and results A genome array, difference in gel electrophoresis and proton nuclear magnetic resonance spectroscopy were employed to dissect the hibernation process into an initiation and a maintenance phase. The initiation phase was characterised by peak levels of K(ATP) channel and glucose transporter 1 (GLUT1) expression. Glibenclamide, an inhibitor of K(ATP) channels, blocked GLUT1 induction. In the maintenance phase, tissue hypoxia and GLUT1 expression were reduced and metabolite concentrations were kept relatively constant. Unguided bioinformatics analysis on the combined datasets confirmed that anaerobic glycolysis was affected and that the observed enzymatic changes in cardiac metabolism were directly linked to hypoxia-inducible factor (HIF)-1 activation. Notably, the combination of the proteomic and transcriptomic datasets improved the statistical confidence of the pathway analysis by two orders of magnitude, with HIF–hypoxia–Akt signalling and glycolysis being the most significant. Conclusions We demonstrate how combining different ‘-omics’ datasets aids in the identification of key biological pathways: chronic hypoxia resulted in a pronounced adaptive response at the transcript and the protein level to keep metabolite levels steady. This preservation of metabolic homoeostasis is likely to contribute to the long-term survival of the hibernating myocardium.