Heart failure after pressure overload in autosomal-dominant desminopathies: Lessons from heterozygous DES-p.R349P knock-in mice

Background Mutations in the human desmin gene (DES) cause autosomal-dominant and -recessive cardiomyopathies, leading to heart failure, arrhythmias, and AV blocks. We analyzed the effects of vascular pressure overload in a patient-mimicking p.R349P desmin knock-in mouse model that harbors the orthologue of the frequent human DES missense mutation p. R350P. Methods and results Transverse aortic constriction (TAC) was performed on heterozygous (HET) DES-p.R349P mice and wild-type (WT) littermates. Echocardiography demonstrated reduced left ventricular ejection fraction in HET-TAC (WT-sham: 69.5 ± 2.9%, HET-sham: 64.5 ± 4.7%, WTTAC: 63.5 ± 4.9%, HET-TAC: 55.7 ± 5.4%; p<0.01). Cardiac output was significantly reduced in HET-TAC (WT sham: 13088 ± 2385 μl/min, HET sham: 10391 ± 1349μl/min, WT-TAC: 8097 ± 1903μl/min, HET-TAC: 5793 ± 2517μl/min; p<0.01). Incidence and duration of AV blocks as well as the probability to induce ventricular tachycardias was highest in HET-TAC. We observed reduced mtDNA copy numbers in HET-TAC (WT-sham: 12546 ± 406, HET-sham: 13526 ± 781, WT-TAC: 11155 ± 3315, HET-TAC: 8649 ± 1582; p = 0.025), but no mtDNA deletions. The activity of respiratory chain complexes I and IV showed the greatest reductions in HET-TAC. Conclusion Pressure overload in HET mice aggravated the clinical phenotype of cardiomyopathy and resulted in mitochondrial dysfunction. Preventive avoidance of pressure overload/arterial hypertension in desminopathy patients might represent a crucial therapeutic measure

Desmin Knock-Out Cardiomyopathy: A Heart on the Verge of Metabolic Crisis

Desmin mutations cause familial and sporadic cardiomyopathies. In addition to perturbing the contractile apparatus, both desmin deficiency and mutated desmin negatively impact mitochondria. Impaired myocardial metabolism secondary to mitochondrial defects could conceivably exacerbate cardiac contractile dysfunction. We performed metabolic myocardial phenotyping in left ventricular cardiac muscle tissue in desmin knock-out mice. Our analyses revealed decreased mitochondrial number, ultrastructural mitochondrial defects, and impaired mitochondria-related metabolic pathways including fatty acid transport, activation, and catabolism. Glucose transporter 1 and hexokinase-1 expression and hexokinase activity were increased. While mitochondrial creatine kinase expression was reduced, fetal creatine kinase expression was increased. Proteomic analysis revealed reduced expression of proteins involved in electron transport mainly of complexes I and II, oxidative phosphorylation, citrate cycle, beta-oxidation including auxiliary pathways, amino acid catabolism, and redox reactions and oxidative stress. Thus, desmin deficiency elicits a secondary cardiac mitochondriopathy with severely impaired oxidative phosphorylation and fatty and amino acid metabolism. Increased glucose utilization and fetal creatine kinase upregulation likely portray attempts to maintain myocardial energy supply. It may be prudent to avoid medications worsening mitochondrial function and other metabolic stressors. Therapeutic interventions for mitochondriopathies might also improve the metabolic condition in desmin deficient hearts

Identification and characterization of new genes regulating myogenesis via a genome-wide RNAi screen in Tribolium castaneum

Die Entwicklung der Muskulatur ist seit Jahrzehnten Gegenstand intensiver Forschungen in der Fruchtfliege Drosophila melanogaster. Dennoch gibt es noch viele offene Fragen zu beantworten. Um ein vollständigeres Bild dieses Entwicklungsprozesses zu erhalten, wählte ich in dieser Arbeit einen artenübergreifenden Ansatz. Zur Identifikation neuer Gene und Signalwege, die an der Myogenese beteiligt sind, nahm ich an dem genomweiten RNAi-Screen iBeetle im rotbraunen Reismehlkäfer Tribolium castaneum teil. Für die tiefergehende Analyse der gefundenen Kandidatengene wurde aufgrund der Vielzahl an vorhandenen genetischen Methoden Drosophila verwendet. Das erste Kapitel dieser Arbeit handelt vom iBeetle-Screen mit dem Hauptaugenmerk auf Muskelphänotypen und die Eingrenzung möglicher Kandidatengene. Ausgehend von 3500 gescreenten Genen und 126 Genen mit dokumentiertem Muskelphänotyp konnte ich in einem Rescreen die Muskeldefekte bei 29 von 52 getesteten Genen reproduzieren. Um Stamm-spezifische Defekte und off-target Effekte auszuschließen wurde mit den 15 interessantesten der 29 positiv getesteten Gene ein zweiter Rescreen durchgeführt. Von diesen Genen wurden sieben für weitere Analysen in Drosophila ausgewählt. Im zweiten Kapitel dieser Arbeit wird beleuchtet, ob Tribolium als zu Drosophila komplementäre Screening-Plattform genutzt werden kann. Diese wurde zur Identifizierung möglicher neuer Komponenten des Integrin-Signalwegs genutzt, der bei der Zell-Zell-Adhäsion eine wichtige Rolle spielt. Ist der Integrin-Signalweg gestört führt dies unter anderem zu einem Verlust der Zell-Zell-Adhäsion zwischen dem dorsalen und ventralen Flügelepithelium, wodurch sich im Flügel mit Flüssigkeit gefüllte Blasen bilden, sogenannte wing blister. Ich konnte erfolgreich demonstrieren, dass mehrere Gene, die in Tribolium nach ihrem knockdown einen wing blister Phänotyp zur Folge hatten, auch in Drosophila nach RNAi wing blister Phänotypen verursachen. Das letzte Kapitel widmet sich der Analyse des Hauptkandidatengens. In Tribolium resultierte der knockdown von nostrin in dünneren Muskeln, ein Phänotyp, der in Tribolium ebenfalls nach dem knockdown von bereits bekannten Genen, die an der Myoblastenfusion beteiligt sind, beobachtet werden kann. nostrin kodiert für ein Mietglied der F-BAR-Proteinfamilie, von der bekannt ist, dass sie die Biegung von Membranen fördern und mit Faktoren interagieren, welche die Umstrukturierung von Aktin regulieren. Beides sind Prozesse, die auch wichtige Aspekte bei der Myoblastenfusion darstellen. Meine Ergebnisse sprechen dafür, dass Nostrin in Drosophila während der Myoblastenfusion zusammen mit dem F-BAR-Protein Cip4 redundant wirkt, da der Verlust beider Proteine einen schwachen Fusionsdefekt in Embryonen zur Folge hatte, der in den jeweiligen Einzelmutanten nicht beobachtet werden konnte. Außerdem konnte ich zeigen, dass der Verlust von nostrin und cip4 in adulten Fliegen eine starke Störung in der Morphologie der longitudinalen viszeralen Muskulatur zur Folge hat, die nicht auf eine beeinträchtigte Myoblastenfusion zurückzuführen ist. In diesem Zusammenhang konnte ich außerdem zeigen, dass die Normalentwicklung der adulten longitudinalen Darmmuskeln offenbar über einen Zwischenschritt der Dedifferenzierunung und Fragmentierung in mononukleäre Myoblasten erfolgt, wobei die normale Redifferenzierung und Morphogenese nach erneuter Myoblastenfusion offenbar die Aktivitäten von nostrin und cip4 benötigt.Although muscle development in the fruitfly Drosophila melanogaster has been studied quite extensively in the last decades there still remain many open questions. In this work I chose an inter-species approach to gain a full understanding of this developmental process. I participated in the genome-wide RNAi screen iBeetle in the red flour beetle Tribolium castaneum to identify novel genes and signaling pathways involved in myogenesis. Because of the huge number of genetic tools available I used Drosophila for in-depth analysis of the newly identified genes. The first chapter of this thesis describes the iBeetle screen with the main focus of attention on muscle phenotypes and the selection of possible candidate genes. Starting from 3500 screened genes and 126 documented muscle phenotypes, I was able to reproduce the muscle phenotypes of 29 out of 52 genes I tested in a rescreen. A second rescreen was performed with the 15 most promising genes of the previously positive tested 29 genes to exclude strain-specific defects and off-target effects. Seven of these genes were selected for deeper analysis in Drosophila. The second chapter deals with the question of whether Tribolium can be used as a complementary screening platform to Drosophila. To address this issue the identification of candidates for new components of the integrin signaling pathway was used, which plays a major role during cell-cell-adhesion. Disruptions during integrin signaling lead to a loss of the cell-cell-adhesion between the dorsal and the ventral wing epithelium, which leads to liquid filled blisters on the wing surface, so called wing blisters. I was able to successfully demonstrate that several genes resulting in a wing blister phenotype after knockdown in Tribolium also lead to wing blisters in Drosophila after RNAi. The last chapter focuses on the analysis of my main candidate gene nostrin. In Tribolium the knockdown of nostrin results in thinner muscles, a phenotype commonly observed in Tribolium after knockdown of genes already known to be involved in myoblast fusion. nostrin encodes a protein of the F-BAR domain protein family, which are known to promote membrane curvature and to interact with actin remodeling molecules. Both of these processes are important aspects during myoblast fusion. My results indicate that in Drosophila Nostrin acts redundantly with the F-BAR protein Cip4 during myoblast fusion. The loss of both proteins results in a weak fusion defect during embryonic myogenesis, which can not be observed in the respective single mutants. Furthermore, I was able to show that the loss of nostrin and cip4 leads to a strong misalignment of the longitudinal visceral musculature in adult flies which can not be explained by a disrupted myoblast fusion. In this context I also was able to show that the development of the adult longitudinal visceral musculature in the wildtype seems to occur via an intermediate step of dedifferentiation and fragmentation into mononucleated myoblasts. The regular redifferentiation and morphogenesis after renewed myoblast fusion seems to be dependent on the activity of nostrin and cip4

RNAi Screen in Tribolium Reveals Involvement of F-BAR Proteins in Myoblast Fusion and Visceral Muscle Morphogenesis in Insects

In a large-scale RNAi screen in Tribolium castaneum for genes with knock-down phenotypes in the larval somatic musculature, one recurring phenotype was the appearance of larval muscle fibers that were significantly thinner than those in control animals. Several of the genes producing this knock-down phenotype corresponded to orthologs of Drosophila genes that are known to participate in myoblast fusion, particularly via their effects on actin polymerization. A new gene previously not implicated in myoblast fusion but displaying a similar thin-muscle knock-down phenotype was the Tribolium ortholog of Nostrin, which encodes an F-BAR and SH3 domain protein. Our genetic studies of Nostrin and Cip4, a gene encoding a structurally related protein, in Drosophila show that the encoded F-BAR proteins jointly contribute to efficient myoblast fusion during larval muscle development. Together with the F-Bar protein Syndapin they are also required for normal embryonic midgut morphogenesis. In addition, Cip4 is required together with Nostrin during the profound remodeling of the midgut visceral musculature during metamorphosis. We propose that these F-Bar proteins help govern proper morphogenesis particularly of the longitudinal midgut muscles during metamorphosis

TC003132 is essential for the follicle stem cell lineage in telotrophic Tribolium oogenesis

Abstract Background Stem cells are undifferentiated cells with a potential for self-renewal, which are essential to support normal development and homeostasis. To gain insight into the molecular mechanisms underlying adult stem cell biology and organ evolution, we use the telotrophic ovary of the beetle Tribolium. To this end, we participated in a large-scale RNAi screen in the red flour beetle Tribolium, which identified functions in embryonic and postembryonic development for more than half of the Tribolium genes. Results We identified TC003132 as candidate gene for the follicle stem cell linage in telotrophic Tribolium oogenesis. TC003132 belongs to the Casein Kinase 2 substrate family (CK2S), which in humans is associated with the proliferative activity of different cancers. Upon TC003132 RNAi, central pre-follicular cells are lost, which results in termination of oogenesis. Given that also Notch-signalling is required to promote the mitotic activity of central pre-follicular cells, we performed epistasis experiments with Notch and cut. In addition, we identified a putative follicle stem cell population by monitoring the mitotic pattern of wild type and TC003132 depleted follicle cells by EdU incorporations. In TC003132 RNAi these putative FSCs cease the expression of differentiation makers and are eventually lost. Conclusions TC003132 depleted pre-follicular cells neither react to mitosis or endocycle stimulating signals, suggesting that TC003132 provides competence for differentiation cues. This may resemble the situation in C. elegans were CK2 is required to maintain the balance between proliferation and differentiation in the germ line. Since the earliest effect of TC003132 RNAi is characterized by the loss of putative FSCs, we posit that TC003132 crucially contributes to the proliferation or maintenance of follicle stem cells in the telotrophic Tribolium ovary

Additional file 1: of TC003132 is essential for the follicle stem cell lineage in telotrophic Tribolium oogenesis

Table S1. EdU positive cells in wildtype. Table S2. PH3 positive cells. Table S3. EdU positive cells in TC003132 RNAi. Table S4. EdU positive cells in Cut RNAi. Methods. (DOCX 26 kb

Interdependence of PRC1 and PRC2 for recruitment to Polycomb Response Elements

Polycomb Group (PcG) proteins are epigenetic repressors essential for control of development and cell differentiation. They form multiple complexes of which PRC1 and PRC2 are evolutionary conserved and obligatory for repression. The targeting of PRC1 and PRC2 is poorly understood and was proposed to be hierarchical and involve tri-methylation of histone H3 (H3K27me3) and/or monoubiquitylation of histone H2A (H2AK118ub). Here, we present a strict test of this hypothesis using the Drosophila model. We discover that neither H3K27me3 nor H2AK118ub is required for targeting PRC complexes to Polycomb Response Elements (PREs). We find that PRC1 can bind PREs in the absence of PRC2 but at many PREs PRC2 requires PRC1 to be targeted. We show that one role of H3K27me3 is to allow PcG complexes anchored at PREs to interact with surrounding chromatin. In contrast, the bulk of H2AK118ub is unrelated to PcG repression. These findings radically change our view of how PcG repression is targeted and suggest that PRC1 and PRC2 can communicate independently of histone modifications

Screens in fly and beetle reveal vastly divergent gene sets required for developmental processes

Background: Most of the known genes required for developmental processes have been identified by genetic screens in a few well-studied model organisms, which have been considered representative of related species, and informative-to some degree-for human biology. The fruit fly Drosophila melanogaster is a prime model for insect genetics, and while conservation of many gene functions has been observed among bilaterian animals, a plethora of data show evolutionary divergence of gene function among more closely-related groups, such as within the insects. A quantification of conservation versus divergence of gene functions has been missing, without which it is unclear how representative data from model systems actually are. Results: Here, we systematically compare the gene sets required for a number of homologous but divergent developmental processes between fly and beetle in order to quantify the difference of the gene sets. To that end, we expanded our RNAi screen in the red flour beetle Tribolium castaneum to cover more than half of the protein-coding genes. Then we compared the gene sets required for four different developmental processes between beetle and fly. We found that around 50% of the gene functions were identified in the screens of both species while for the rest, phenotypes were revealed only in fly (similar to 10%) or beetle (similar to 40%) reflecting both technical and biological differences. Accordingly, we were able to annotate novel developmental GO terms for 96 genes studied in this work. With this work, we publish the final dataset for the pupal injection screen of the iBeetle screen reaching a coverage of 87% (13,020 genes). Conclusions: We conclude that the gene sets required for a homologous process diverge more than widely believed. Hence, the insights gained in flies may be less representative for insects or protostomes than previously thought, and work in complementary model systems is required to gain a comprehensive picture. The RNAi screening resources developed in this project, the expanding transgenic toolkit, and our large-scale functional data make T. castaneum an excellent model system in that endeavor