Genomic structural variations lead to dysregulation of important coding and non-coding RNA species in dilated cardiomyopathy

The transcriptome needs to be tightly regulated by mechanisms that include transcription factors, enhancers, and repressors as well as non-coding RNAs. Besides this dynamic regulation, a large part of phenotypic variability of eukaryotes is expressed through changes in gene transcription caused by genetic variation. In this study, we evaluate genome-wide structural genomic variants (SVs) and their association with gene expression in the human heart. We detected 3,898 individual SVs affecting all classes of gene transcripts (e.g., mRNA, miRNA, lncRNA) and regulatory genomic regions (e.g., enhancer or TFBS). In a cohort of patients (n = 50) with dilated cardiomyopathy (DCM), 80,635 non-protein-coding elements of the genome are deleted or duplicated by SVs, containing 3,758 long non-coding RNAs and 1,756 protein-coding transcripts. 65.3% of the SV-eQTLs do not harbor a significant SNV-eQTL, and for the regions with both classes of association, we find similar effect sizes. In case of deleted protein-coding exons, we find downregulation of the associated transcripts, duplication events, however, do not show significant changes over all events. In summary, we are first to describe the genomic variability associated with SVs in heart failure due to DCM and dissect their impact on the transcriptome. Overall, SVs explain up to 7.5% of the variation of cardiac gene expression, underlining the importance to study human myocardial gene expression in the context of the individual genome. This has immediate implications for studies on basic mechanisms of cardiac maladaptation, biomarkers, and (gene) therapeutic studies alike

Functional characterization of the Jumonji C domain-containing protein 6 (Jmjd6) in the vascular system

Conclusion: Proteins containing a Jumonji C (JmjC) domain appear in almost all living organisms and catalyze a variety of oxidation reactions. Therefore, they are important regulators in many biological processes such as proliferation and differentiation. They act either as protein hydroxylases, histone demethylases or by regulate mRNA splicing. Given the fact that some of the JmjC domain-containing proteins are shown to be upregulated in response to hypoxia as well as the dependency of JmjC domain catalytic activity on oxygen led to the assumption of an involvement in angiogenesis. For Jmjd6, a member of the JmjC domain-containing protein family, a regulatory involvement in mRNA splicing has been shown. The Jmjd6-/- mouse dies perinatally due to several severe organ malformations, especially in the heart. Despite the pale appearance, the growth retardation and the cardiac defects, it is unclear whether these mice exhibit defects of cells comprising the vasculature. Therefore, the involvement of Jmjd6 in angiogenesis was examined in vitro using angiogenesis assays as well as in vivo using the Jmjd6+/- mouse. An siRNA-mediated knockdown of Jmjd6 in ECs significantly impaired the formation of capillary-like networks in the tube formation assay as well as sprouting in the spheroid assay. Moreover, after siRNA-mediated knockdown of Jmjd6 in ECs cell migration was significantly reduced. These findings were confirmed in the matrigel plug assay in vivo. Implanted matrigel plugs of Jmjd6+/- mice exhibited significantly less perfused vessels compared to wildtype littermates. Furthermore, cultured lung ECs from Jmjd6+/- mice exhibited impaired network forming activity ex vivo compared to cells isolated from wildtype littermates. To elucidate the mechanisms underlying the requirement of Jmjd6 in angiogenesis, an Affymetrix exon-array was performed, which allows detection of changes in gene expression as well as splicing. The siRNA-mediated knockdown of Jmjd6 altered the expression of genes known to play a role in vascular biology. The bioinformatic assessment of alternative splice variants revealed that Jmjd6 silencing affects the splicing of the VEGF receptor 1 (Flt1). Differential splicing of Flt1 was shown to generate a short and soluble form of Flt1 (sFlt1), which sequestrates VEGF and PlGF, and thereby inhibits angiogenesis. In particular, a significant increase in sFlt1 expression was observed. Jmjd6 was recently reported to hydroxylate the splicing factor U2AF65. Therefore, we investigated whether U2AF65 might mediate Flt1 splicing and binds to Flt1 mRNA. Indeed, U2AF65 co-immunoprecipitated with Jmjd6 in ECs, while an interaction of U2AF65 with sFlt1 was demonstrated. Moreover, inhibition of Jmjd6 catalytic function by reduced oxygen concentration altered splicing of Flt1 resulted in an increase of the sFlt1 splice variant. Finally, saturating concentrations of VEGF or PlGF or neutralizing antibodies against sFlt1 significantly reduced the inhibition of sprouting caused by Jmjd6 knockdown in vitro. Collectively, our results indicate that Jmjd6 has an essential role in the oxygen-dependent regulation of angiogenesis by controlling the splicing of Flt1 mRNA, thereby adjusting the generation of the anti-angiogenic short splice variant sFlt1. Several publications demonstrated a major importance for sFlt1 as a biomarker for many severe human diseases such as preeclampsia, sepsis, cancer, myocardial infarction as well as chronic heart failure. Therefore, the identification of the molecular mechanism behind the generation of sFlt1 might enable the development of new or more precise clinical markers for the diagnosis of the corresponding diseases. Furthermore, the discovery of the enzymes involved in the generation of sFlt1 provides further possibilities to modulate sFlt1 levels and thereby may potentially gives rise to the development of new therapies.Proteine, die eine JmjC-Domäne enthalten, gehören zu einer evolutionär hochkonservierten Familie von Oxygenasen. Aufgrund ihres evolutionär frühen Entstehens konnten Mitglieder dieser Proteinfamilie in einer Vielzahl von Lebewesen nachgewiesen werden. Es wurde gezeigt, dass Proteine mit einer JmjC-Domäne als Regulatoren von wichtigen biologischen Prozessen agieren, zum Beispiel in der Zellproliferation und in der Zelldifferenzierung während der embryonalen Entwicklung. Die enzymatische Reaktion, die von der JmjC Domäne katalysiert wird, benötigt zweiwertiges Eisen (Fe(II)), Oxoglutarat und Sauerstoff als Kofaktoren. Die Mitglieder dieser Proteinfamilie katalysieren eine Vielzahl von enzymatischen Reaktionen, wie Protein-Hydroxylierung, Protein-Demethylierung und DNA-Reparaturprozesse. Eine Beteiligung von Jmjd6 konnte bis jetzt bei zwei enzymatischen Reaktionen nachgewiesen werden. Zum einen hydroxyliert Jmjd6 posttranslational Proteine, die direkt am mRNA Spleißen beteiligt sind und agiert dadurch als Regulator des alternativen Spleißen spezifischer mRNAs, zum anderen wurde eine Demethylierung von zwei spezifischen Histon-Argininen durch Jmjd6 nachgewiesen. Die Jmjd6-/- Mäuse haben ein anämisches Erscheinungsbild, sind wachstumsretardiert und weisen sowohl schwerwiegende subkutane Ödeme als auch eine Vielzahl von Differenzierungsdefekten und Organmissbildungen auf. Letztere sind vor allem im Gehirn, den Nieren, der Leber, den Augen und dem Herzen zu beobachten. Aufgrund der kardialen Fehlbildungen sterben die Jmjd6-/- Mäuse innerhalb von Minuten nach der Geburt. Vor dem Beginn dieser Studie war nichts über das Vorhandensein von angiogenen Defekten in Jmjd6-/- Mäusen bekannt, allerdings ließen das anämische Erscheinungsbild, das verzögerte Wachstum und die kardialen Fehlbildungen auf eine Beeinflussung des Blutgefäßsystems dieser Mäuse schließen. Die Hif-Hydroxylase FIH1 ist bisher das einzige Mitglied der JmjC-Proteinfamilie für die eine Bedeutung in Angiogenese und Ischämie-vermittelter Neovaskularisation gezeigt wurde. Aufgrund dieser Vorbefunde, wurde in der vorliegenden Studie der Einfluss von Jmjd6 auf die Angiogenese sowohl in vitro in humanen Endothelzellen als auch in vivo in Jmjd6+/- Mäusen untersucht. Weiterhin wurde der von Jmjd6 in humanen Endothelzellen vermittelte molekulare Mechanismus unter Anwendung eines Affymetrix Exon Arrays untersucht um mRNAs zu identifizieren, die nach einer siRNA-vermittelten Suppression von Jmjd6 möglicherweise ein alternatives Spleißmuster aufweisen.... Zusammenfassend zeigen die in dieser Arbeit enthaltenen Daten, dass Jmjd6 eine essentielle Rolle in der sauerstoffabhängigen Regulation von Angiogenese spielt. Mechanistisch reguliert Jmjd6 das Spleißen von sFlt1 und moduliert damit die extrazelluläre Konzentration dieses Angiogeneseinhibitors

Samtykkekravet i pasient- og brukerrettighetsloven §§ 4-1 og 4-2. Med særlig fokus på lovmessigheten av å benytte hypotetisk samtykke som selvstendig kompetansegrunnlag for inngripen

Avhandlingen søker å redegjøre for hva som forstås med de lovregulerte vilkårene for å kunne avgi et gyldig samtykke etter pasient- og brukerrettighetsloven §§ 4-1 og 4-2. Avhandlingens hovedproblemstilling er lovmessigheten av hypotetisk samtykke som kompetansegrunnlag for inngripen. Denne problemstillingen ses spesielt under det forhold at et hypotetisk samtykke krenker den lovfestede retten til pasientautonomi. Spesielt i denne sammenheng er at hypotetisk samtykke som kompetansegrunnlag for inngripen stammer fra Rt. 1998 s. 1538 Caudia equina- dommen. Pasient- og brukerrettighetsloven som har lovfestet samtykkekravet er nyere enn denne dommen. I teorien eksisterer det allikevel en viss usikkerhet knyttet til hvorvidt hypotetisk samtykke er gyldig etter lovens ordlyd

Analyzing the release of copeptin from the heart in acute myocardial infarction using a transcoronary gradient model

Copeptin is the C-terminal end of pre-provasopressin released equimolar to vasopressin into circulation and recently discussed as promising cardiovascular biomarker amendatory to established markers such as troponins. Vasopressin is a cytokine synthesized in the hypothalamus. A direct release of copeptin from the heart into the circulation is implied by data from a rat model showing a cardiac origin in hearts put under cardiovascular wall stress. Therefore, evaluation of a potential release of copeptin from the human heart in acute myocardial infarction (AMI) has been done

Analysis of hypoxia induced alternative splicing.

<p>Differentially expressed probe sets are given in bold. The number of the corresponding genes is given in brackets.</p

GO analysis of upregulated genes.

1<p>Underlined genes match to the GO term: 0006096∼glycolysis (p-value = 2.1×10⁻³).</p>2<p>Underlined genes match to the GO term: 0043066∼negative regulation of apoptosis (p-value = 0.012).</p

Hypoxia-dependent alternative exon inclusion.

<p><b>A</b>) Scheme of alternative splicing of cassette exons. Introns are represented as black lines; constitutive exons as grey boxes and the cassette exon as white box. Primers are indicated as arrows and span the alternatively spliced exon. Cassette exons in <i>cask</i> (<b>B</b>), <i>sptan1</i> (<b>C</b>) and <i>pign</i> (<b>D</b>) are more often skipped under hypoxic then under normoxic conditions. Radioactive RT-PCR: N = normoxia, H = hypoxia, M = DNA size ladder. The precentage of exon inclusion is given below. SD = standard deviation. qRT-PCR: closed bars = normoxia, open bars = hypoxia. Two isoform specific forward primer (i1 and i2) spanning the exon/exon borders and one common reverse primer (i1/2) were used. Expression is normalized to <i>rplp0</i>. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0042697#s2" target="_blank">Results</a> from n = 3 experiments are shown. * = p-value<0.05, ** = p-value<0.01 (Student's t-test).</p

Hypoxia-dependent alternative N-termini.

<p>RT- and qRT-PCR analyses show alternative promoter usage for <i>cugbp1</i> (<b>A</b>), <i>tpm1</i> (<b>B</b>) and <i>robo1</i> (<b>C</b>) leading to protein isoforms with different N-termini. Schemes of alternative splicing events: Introns are represented as black lines; exons as boxes; primer pairs (arrows) used to amplify different isoforms (i1, i2) or total amounts of the mRNAs (t) are indicated. White exons are exclusive for isoform 1, which expression is changed under hypoxic conditions. Grey exons are common to both isoforms and primers spanning such regions were used to visualize changes in the total amount of the corresponding mRNA. Black exons are exclusive for isoform 2, which is transcribed by an alternative promoter. RT-PCR: N = normoxia, H = hypoxia, M = DNA size ladder. qRT-PCR: closed bars = normoxia, open bars = hypoxia. Expression is normalized to <i>rplp0</i>. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0042697#s2" target="_blank">Results</a> from n = 2 (RT-PCR) and n = 3 (qRT-PCR) experiments are shown. * = p-value<0.05, ** = p-value<0.01 (Student's t-test).</p

Identification and Characterization of Hypoxia-Regulated Endothelial Circular RNA

Rationale: Circular RNAs (circRNAs) are noncoding RNAs generated by back splicing. Back splicing has been considered a rare event, but recent studies suggest that circRNAs are widely expressed. However, the expression, regulation, and function of circRNAs in vascular cells is still unknown. Objective: Here, we characterize the expression, regulation, and function of circRNAs in endothelial cells. Methods and Results: Endothelial circRNAs were identified by computational analysis of ribo-minus RNA generated from human umbilical venous endothelial cells cultured under normoxic or hypoxic conditions. Selected circRNAs were biochemically characterized, and we found that the majority of them lacks polyadenylation, is resistant to RNase R digestion and localized to the cytoplasm. We further validated the hypoxia-induced circRNAs cZNF292, cAFF1, and cDENND4C, as well as the downregulated cTHSD1 by reverse transcription polymerase chain reaction in cultured endothelial cells. Cloning of cZNF292 validated the predicted back splicing of exon 4 to a new alternative exon 1A. Silencing of cZNF292 inhibited cZNF292 expression and reduced tube formation and spheroid sprouting of endothelial cells in vitro. The expression of pre-mRNA or mRNA of the host gene was not affected by silencing of cZNF292. No validated microRNA-binding sites for cZNF292 were detected in Argonaute high-throughput sequencing of RNA isolated by cross-linking and immunoprecipitation data sets, suggesting that cZNF292 does not act as a microRNA sponge. Conclusions: We show that the majority of the selected endothelial circRNAs fulfill all criteria of bona fide circRNAs. The circRNA cZNF292 exhibits proangiogenic activities in vitro. These data suggest that endothelial circRNAs are regulated by hypoxia and have biological functions