TY - JOUR A1 - Williams, Tatjana A1 - Machann, Wolfram A1 - Kühler, Leif A1 - Hamm, Henning A1 - Müller-Höcker, Josef A1 - Zimmer, Michael A1 - Ertl, Georg A1 - Ritter, Oliver A1 - Beer, Meinrad A1 - Schönberger, Jost T1 - Novel desmoplakin mutation: juvenile biventricular cardiomyopathy with left ventricular non-compaction and acantholytic palmoplantar keratoderma JF - Clinical Research in Cardiology N2 - Two sons of a consanguineous marriage developed biventricular cardiomyopathy. One boy died of severe heart failure at the age of 6 years, the other was transplanted because of severe heart failure at the age of 10 years. In addition, focal palmoplantar keratoderma and woolly hair were apparent in both boys. As similar phenotypes have been described in Naxos disease and Carvajal syndrome, respectively, the genes for plakoglobin (JUP) and desmoplakin (DSP) were screened for mutations using direct genomic sequencing. A novel homozygous 2 bp deletion was identified in an alternatively spliced region of DSP. The deletion 5208_5209delAG led to a frameshift downstream of amino acid 1,736 with a premature truncation of the predominant cardiac isoform DSP-1. This novel homozygous truncating mutation in the isoform-1 specific region of the DSP C-terminus caused Carvajal syndrome comprising severe early-onset heart failure with features of non-compaction cardiomyopathy, woolly hair and an acantholytic form of palmoplantar keratoderma in our patient. Congenital hair abnormality and manifestation of the cutaneous phenotype in toddler age can help to identify children at risk for cardiac death. KW - Desmoplakin KW - Juvenile biventricular cardiomyopathy KW - Palmoplantar keratoderma Y1 - 2011 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-141198 VL - 100 IS - 12 ER - TY - JOUR A1 - Nordbeck, Peter A1 - Bönhof, Leoni A1 - Hiller, Karl-Heinz A1 - Voll, Sabine A1 - Arias-Loza, Paula A1 - Seidlmaier, Lea A1 - Williams, Tatjana A1 - Ye, Yu-Xiang A1 - Gensler, Daniel A1 - Pelzer, Theo A1 - Ertl, Georg A1 - Jakob, Peter M. A1 - Bauer, Wolfgang R. A1 - Ritter, Oliver T1 - Impact of Thoracic Surgery on Cardiac Morphology and Function in Small Animal Models of Heart Disease: A Cardiac MRI Study in Rats JF - PLoS ONE N2 - Background Surgical procedures in small animal models of heart disease might evoke alterations in cardiac morphology and function. The aim of this study was to reveal and quantify such potential artificial early or long term effects in vivo, which might account for a significant bias in basic cardiovascular research, and, therefore, could potentially question the meaning of respective studies. Methods Female Wistar rats (n = 6 per group) were matched for weight and assorted for sham left coronary artery ligation or control. Cardiac morphology and function was then investigated in vivo by cine magnetic resonance imaging at 7 Tesla 1 and 8 weeks after the surgical procedure. The time course of metabolic and inflammatory blood parameters was determined in addition. Results Compared to healthy controls, rats after sham surgery showed a lower body weight both 1 week (267.5±10.6 vs. 317.0±11.3 g, n<0.05) and 8 weeks (317.0±21.1 vs. 358.7±22.4 g, n<0.05) after the intervention. Left and right ventricular morphology and function were not different in absolute measures in both groups 1 week after surgery. However, there was a confined difference in several cardiac parameters normalized to the body weight (bw), such as myocardial mass (2.19±0.30/0.83±0.13 vs. 1.85±0.22/0.70±0.07 mg left/right per g bw, p<0.05), or enddiastolic ventricular volume (1.31±0.36/1.21±0.31 vs. 1.14±0.20/1.07±0.17 µl left/right per g bw, p<0.05). Vice versa, after 8 weeks, cardiac masses, volumes, and output showed a trend for lower values in sham operated rats compared to controls in absolute measures (782.2±57.2/260.2±33.2 vs. 805.9±84.8/310.4±48.5 mg, p<0.05 for left/right ventricular mass), but not normalized to body weight. Matching these findings, blood testing revealed only minor inflammatory but prolonged metabolic changes after surgery not related to cardiac disease. Conclusion Cardio-thoracic surgical procedures in experimental myocardial infarction cause distinct alterations upon the global integrity of the organism, which in the long term also induce circumscribed repercussions on cardiac morphology and function. This impact has to be considered when analyzing data from respective animal studies and transferring these findings to conditions in patients. KW - heart rate KW - body weight KW - surgical and invasive medical procedures KW - magnetic resonance imaging KW - blood KW - vascular surgery KW - myocardial infarction KW - cardiac ventricles Y1 - 2013 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-130064 VL - 8 IS - 8 ER - TY - THES A1 - Williams, Tatjana T1 - Untersuchungen zur Rolle des Phosphoproteins Stathmin in Listeria monocytogenes-infizierten Säugerzellen und Molekulare Charakterisierung der listeriellen Zweikomponetensysteme T1 - The role of the phosphoprotein stathmin during an infection with Listeria monocytogenes and Molecular characterization of listerial two-componentsystems N2 - Das fakultativ intrazelluläre Bakterium Listeria monocytogenes besitzt die Fähigkeit, eukaryotische Wirtszellen zu penetrieren, sich in diesen zu vermehren, fortzubewegen und zwischen den Zellen auszubreiten. Im Zuge des intrazellulären Lebenszyklus gehen Listerien Wechselwirkungen mit ver-schiedenen zellulären Proteinen ein. Als eines dieser Proteine konnte das zelluläre Phosphoprotein Stathmin identifiziert werden. Dieses Protein bindet an Untereinheiten des Tubulins und destabilisiert dadurch Mikrotubuli (MT). Es wird durch Phosphorylierung von vier spezifischen Serinresten in seiner MT-destabilisierenden Aktivität reguliert. Da Stathmin als Antwort auf externe Signale zelluläre Funktionen, z. B. Zell-Proliferation und Differenzierung reguliert, vermutet man seine Funktion in einer Art Relais welches verschiedene Signale aus dem Umfeld der Zelle integriert. In mit L. monocytogenes infizierten Wirtszellen wird Stathmin an die Oberfläche intrazellulärer Bakterien rekrutiert. Inwiefern diese Rekrutierung das Phosphorylierungsmuster von Stathmin und damit dessen Aktivität beeinflusst, konnte im Rahmen dieser Arbeit nicht geklärt werden. Stathmin knock-out Mäuse sollten sich gut eignen, um die Rolle von Stathmin während einer Infektion mit L. monocytogenes EGD in vitro und in vivo zu untersuchen. Es stellte sich heraus, dass in Stathmin(-/-)-Makrophagen der intrazelluläre Lebenszyklus der Listerien nicht signifikant beeinflusst ist. Nach intravenöser Verabreichung von 5x103 L. monocytogenes waren drei Tage nach der Infektion in Leber und Milz der knock-out Mäuse allerdings signifikant mehr Listerien nachzuweisen, als in den Organen wildtypischer Mäuse. Mittels Immunfluoreszenzmikroskopie und einem anti-Stathmin-Antiersum konnte an mit verschiedenen L. monocytogenes-Mutanten infizierten Zellen gezeigt werden, dass Stathmin mit der Oberfläche intrazellulärer Listerien kolokalisiert. Allerdings konnten dabei die Angaben in der Literatur nicht bestätigt werden, wonach für diese Kolokalisation die Expression von ActA notwendig ist. Die Ergebnisse dieser Arbeit sprechen im Gegensatz zu den publizierten Daten dafür, dass Stathmin über einen bisher noch unbekannten Mechanismus ActA-unabhängig an intrazelluläre Listerien rekrutiert wird. Zweikomponentensysteme ermöglichen Bakterien eine rasche Anpassung an sich verändernde Umweltbedingungen, da sie extra- und intrazelluläre Stimuli in zelluläre Signale umwandeln. Um die 16 in der Genomsequenz von L. mono-cytogenes EGDe identifizierten Zweikomponentensysteme charakterisieren zu können, wurden individuelle Mutanten konstruiert, in denen individuelle Response Regulatorgene deletiert sind. Die erhaltenen Mutanten wurden in vitro und in vivo auf ihr Wachstumsverhalten hin untersucht. Es zeigte sich, dass unter den angewandten Kultur- und Versuchsbedingungen keines der Zweikomponentensysteme eine signifikante Rolle bei der Anpassung an Temperatur, sowie an oxidativen oder osmotischen Stress spielt. Die Zugabe von 5 % Ethanol hatte einen stark hemmenden Effekt auf das Wachstum von 4 Mutanten, wohingegen zwei andere Mutanten in Gegenwart des Alkohols deutlich besser wuchsen. Unter anaeroben Bedingungen konnte kein Unterschied im Wachstum beobachtet werden. Die Expression wichtiger Virulenzgene war in keiner der untersuchten Mutanten im Vergleich zum Ausgangsstamm verändert. Die intrazelluläre Replikation sowie intrazelluläre Bewegung und Ausbreitung im Zellrasen waren durch die Deletion der Response Regulatorgene nicht beeinträchtigt. Abgesehen von geringen Unterschieden in der Invasivität einiger Deletionsmutanten für Cos-1 und Caco-2 Zellen zeigte sich keiner der Response Regulatoren für den intrazellulären Lebenszyklus von L. monocytogenes erforderlich. Es zeigte sich, dass der in der vorliegenden Arbeit verwendete L. monocyto-genes-Wildstamm auch bei der für die Flagellenexpression normalerweise nicht-permissiven Temperatur von 37° C noch beweglich ist. Die L. monocytogenes ΔdegU-Mutante war dagegen auf Weichagar temperaturunabhängig unbeweglich. Die elektronenmikroskopische Analyse ergab, dass dieser Stamm im Gegensatz zum Wildtyp auch bei 24° C keine Flagellen ausbildet. Durch vergleichende Proteomanalysen konnte gezeigt werden, dass L. monocytogenes ΔdegU bei 24° C wesentliche Proteine des Flagellenapparates nicht synthetisiert. Mittels Transkriptomanalysen konnten die Ergebnisse der Proteomanalysen bestätigt werden. Es wurden neben Genen, die für Proteine der Flagellenbiosynthese und Chemotaxis codieren, noch weitere Gene identifiziert, die offensichtlich unter der transkriptionellen Kontrolle des Response Regulators DegU stehen. Die Ergebnisse der in vivo Studien zeigten, dass L. monocytogenes ΔdegU deutlich virulenzattenuiert ist. Für die restlichen L. monocytogenes ΔTCS-Mutanten waren im Vergleich zum Wildtyp die Unterschiede in Leber und Milz nur leicht verändert und statistisch nicht signifikant. N2 - The facultative intracellular pathogen Listeria monocytogenes is able to penetrate eukaryotic host cells, to multiply and move intracellularly and to spread between the cells. In the course of the intracellular lifecycle the listeriae interact with different cellular proteins. The cellular phosphoprotein stathmin was identified as one of these proteins. This protein binds to the tubulin subunits thereby destabilizing microtubules. It is regulated in its microtubule-destabilizing activity via phosphorylation of four specific serine residues. Since stathmin regulates cellular functions, i. e. cell proliferation and differentiation, its function is presumed in acting as a type of relay integrating different signals of the cell´s environment. In L. monocytogenes infected host cells stathmin is recruited to the bacterial cell surface. Whether and to which extent this recruitment influences the phosphorylation pattern of stathmin and thereby its activity, could not be identified during this work. Stathmin knock-out mice should be valuable for experimentally testing the influence of stathmin in an infection with L. monocytogenes in vitro and in vivo. It appeared that the listerial intracellular lifecycle is not significantly influenced in stathmin(-/-) macrophages. However, three days after intravenous application of 5x103 L. monocytogenes EGD the bacterial load in liver and spleen of stathmin knock-out mice was significantly higher than in organs of wildtype mice. By means of immunfluorescence microscopy with an anti-stathmin antiserum it could be revealed that within infected cells stathmin colocalizes with the listerial cell surface. Data from the literature, however, suggesting that this recruitment of stathmin is dependent upon the expression of ActA, could not be confirmed implicating stathmin to be recruited in an up to now unknown ActA-independent manner. Two-component systems enable bacteria to efficiently adapt to changes in the environment by converting extra- and intracellular signals into cellular responses. In order to characterize the 16 identified two-component systems of L. monocytogenes EGDe in detail individual mutants with deletions in each one of the response regulator genes were constructed. The growth characteristics of the mutants were examined in in vitro and in vivo experiments. It appeared that under the culture and test conditions applied, neither one of the TCS plays a role for adapting to temperature, as well as oxidative or osmotic stress. The addition of 5 % ethanol severely impaired growth of 4 of the mutants, whereas growth of two other mutatns was enhanced. No difference in growth between wildtype listeriae and the individual mutants was observed when the cells were grown anaerobically. Expression of important virulencefactor genes was not altered in the mutants tested compared to the wildtype strain. Intracellular replication as well as intracellular movement and spreading was not affected by either one of the response regulator gene deletions. Despite a slightly reduced invasiveness of a few deletion mutants for Cos-1 and Caco-2 cells none of the response regulators appeared to be necessary for the intracellular lifecycle of L. monocytogenes. The wildtype strain used throughout this work proved to be motile even at 37° C, a temperature usually non-permissive for flagellin expression. In contrast, the L. monocytogenes ΔdegU mutant displayed a non-motile phenotype on semisolid agar irrespective of the temperature applied. Electron microscopical analysis demonstrated that, unlike the wildtype strain EGD, this deletion mutant did not produce flagella, not even at 24° C. Comparative proteomics revealed that at 24° C L. monocytogenes ΔdegU did not produce the major components of the flagella apparatus. The results of transcriptome analysis were in line with the results of the proteome analysis and, aside of genes for flagella biosynthesis and chemotaxis, identified other genes obviously under transcriptional regulation by DegU. In vivo studies revealed that only L. monocytogenes ΔdegU showed a conspicuous virulence attenuation. There were significantly less bacteria in liver and spleen compared to an infection with the wildtype strain L. monocytogenes EGD. For the other L. monocytogenes ΔTCS-mutants the difference of bacterial counts in liver and spleen compared to the wildtype was slightly altered but not statistically significant. KW - Phosphoproteine KW - Säugetiere KW - Listeria monocytogenes KW - Infektion KW - Listeria monocytogenes KW - Stathmin KW - Zweikomponentensystem KW - Listeria monocytogenes KW - stathmin KW - two-componentsystems Y1 - 2005 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-15388 ER - TY - JOUR A1 - Bernt, Alexander A1 - Rangrez, Ashraf Y. A1 - Eden, Matthias A1 - Jungmann, Andreas A1 - Katz, Sylvia A1 - Rohr, Claudia A1 - Müller, Oliver J. A1 - Katus, Hugo A. A1 - Sossalla, Samuel T. A1 - Williams, Tatjana A1 - Ritter, Oliver A1 - Frank, Derk A1 - Frey, Norbert T1 - Sumoylation-independent activation of Calcineurin-NFAT-signaling via SUMO2 mediates cardiomyocyte hypertrophy JF - Scientific Reports N2 - The objective of this study was to identify unknown modulators of Calcineurin (Cn)-NFAT signaling. Measurement of NFAT reporter driven luciferase activity was therefore utilized to screen a human cardiac cDNA-library (~10\(^{7}\) primary clones) in C2C12 cells through serial dilutions until single clones could be identified. This extensive screening strategy culminated in the identification of SUMO2 as a most efficient Cn-NFAT activator. SUMO2-mediated activation of Cn-NFAT signaling in cardiomyocytes translated into a hypertrophic phenotype. Prohypertrophic effects were also observed in mice expressing SUMO2 in the heart using AAV9 (Adeno-associated virus), complementing the in vitro findings. In addition, increased SUMO2-mediated sumoylation in human cardiomyopathy patients and in mouse models of cardiomyopathy were observed. To decipher the underlying mechanism, we generated a sumoylation-deficient SUMO2 mutant (ΔGG). Surprisingly, ΔGG replicated Cn-NFAT-activation and the prohypertrophic effects of native SUMO2, both in vitro and in vivo, suggesting a sumoylation-independent mechanism. Finally, we discerned a direct interaction between SUMO2 and CnA, which promotes CnA nuclear localization. In conclusion, we identified SUMO2 as a novel activator of Cn-NFAT signaling in cardiomyocytes. In broader terms, these findings reveal an unexpected role for SUMO2 in cardiac hypertrophy and cardiomyopathy, which may open the possibility for therapeutic manipulation of this pathway. KW - Calcineurin-NFATsignaling KW - activation KW - SUMO2 KW - cardiac hypertrophy Y1 - 2016 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-167525 VL - 6 IS - 35758 ER - TY - JOUR A1 - Zink, Miriam A1 - Seewald, Anne A1 - Rohrbach, Mareike A1 - Brodehl, Andreas A1 - Liedtke, Daniel A1 - Williams, Tatjana A1 - Childs, Sarah J. A1 - Gerull, Brenda T1 - Altered expression of TMEM43 causes abnormal cardiac structure and function in zebrafish JF - International Journal of Molecular Sciences N2 - Arrhythmogenic cardiomyopathy (ACM) is an inherited heart muscle disease caused by heterozygous missense mutations within the gene encoding for the nuclear envelope protein transmembrane protein 43 (TMEM43). The disease is characterized by myocyte loss and fibro-fatty replacement, leading to life-threatening ventricular arrhythmias and sudden cardiac death. However, the role of TMEM43 in the pathogenesis of ACM remains poorly understood. In this study, we generated cardiomyocyte-restricted transgenic zebrafish lines that overexpress eGFP-linked full-length human wild-type (WT) TMEM43 and two genetic variants (c.1073C>T, p.S358L; c.332C>T, p.P111L) using the Tol2-system. Overexpression of WT and p.P111L-mutant TMEM43 was associated with transcriptional activation of the mTOR pathway and ribosome biogenesis, and resulted in enlarged hearts with cardiomyocyte hypertrophy. Intriguingly, mutant p.S358L TMEM43 was found to be unstable and partially redistributed into the cytoplasm in embryonic and adult hearts. Moreover, both TMEM43 variants displayed cardiac morphological defects at juvenile stages and ultrastructural changes within the myocardium, accompanied by dysregulated gene expression profiles in adulthood. Finally, CRISPR/Cas9 mutants demonstrated an age-dependent cardiac phenotype characterized by heart enlargement in adulthood. In conclusion, our findings suggest ultrastructural remodeling and transcriptomic alterations underlying the development of structural and functional cardiac defects in TMEM43-associated cardiomyopathy. KW - TMEM43 KW - arrhythmogenic cardiomyopathy KW - zebrafish KW - CRISPR/Cas9 Y1 - 2022 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-286025 SN - 1422-0067 VL - 23 IS - 17 ER -