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The emergence of human induced pluripotent stem cells (iPSCs) and the rise of the clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9 (CRISPR/Cas9) gene editing technology innovated the research platform for scientists based on living human pluripotent cells. The revolutionary combination of both Nobel Prize-honored techniques enables direct disease modeling especially for research focused on genetic diseases. To allow the study on mutation-associated pathomechanisms, we established robust human in vitro systems of three inherited cardiomyopathies: arrhythmogenic cardiomyopathy (ACM), dilated cardiomyopathy with juvenile cataract (DCMJC) and dilated cardiomyopathy with ataxia (DCMA).
Sendai virus vectors encoding OCT3/4, SOX2, KLF4, and c-MYC were used to reprogram human healthy control or mutation-bearing dermal fibroblasts from patients to an embryonic state thereby allowing the robust and efficient generation of in total five transgene-free iPSC lines. The nucleofection-mediated CRISPR/Cas9 plasmid delivery in healthy control iPSCs enabled precise and efficient genome editing by mutating the respective disease genes to create isogenic mutant control iPSCs. Here, a PKP2 knock-out and a DSG2 knock-out iPSC line were established to serve as a model of ACM. Moreover, a DNAJC19 C-terminal truncated variant (DNAJC19tv) was established to mimic a splice acceptor site mutation in DNAJC19 of two patients with the potential of recapitulating DCMA-associated phenotypes. In total eight self-generated iPSC lines were assessed matching internationally defined quality control criteria. The cells retained their ability to differentiate into cells of all three germ layers in vitro and maintained a stable karyotype. All iPSC lines exhibited a typical stem cell-like morphology as well as expression of characteristic pluripotency markers with high population purities, thus validating the further usage of all iPSC lines in in vitro systems of ACM, DCMA and DCMJC.
Furthermore, cardiac-specific disease mechanisms underlying DCMA were investigated using in vitro generated iPSC-derived cardiomyocytes (iPSC-CMs). DCMA is an autosomal recessive disorder characterized by life threatening early onset cardiomyopathy associated with a metabolic syndrome. Causal mutations were identified in the DNAJC19 gene encoding an inner mitochondrial membrane (IMM) protein with a presumed function in mitochondrial biogenesis and cardiolipin (CL) remodeling. In total, two DCMA patient-derived iPSC lines (DCMAP1, DCMAP2) of siblings with discordant cardiac phenotypes, a third isogenic mutant control iPSC line (DNAJC19tv) as well as two control lines (NC6M and NC47F) were directed towards the cardiovascular lineage upon response to extracellular specification cues. The monolayer cardiac differentiation approach was successfully adapted for all five iPSC lines and optimized towards ventricular subtype identity, higher population purities and enhanced maturity states to fulfill all DCMA-specific requirements prior to phenotypic investigations. To provide a solid basis for the study of DCMA, the combination of lactate-based metabolic enrichment, magnetic-activated cell sorting, mattress-based cultivation and prolonged cultivation time was performed in an approach-dependent manner. The application of the designated strategies was sufficient to ensure adult-like characteristics, which included at least 60-day-old iPSC-CMs. Therefore, the novel human DCMA platform was established to enable the study of the pathogenesis underlying DCMA with respect to structural, morphological and functional changes.
The disease-associated protein, DNAJC19, is constituent of the TIM23 import machinery and can directly interact with PHB2, a component of the membrane bound hetero-oligomeric prohibitin ring complexes that are crucial for phospholipid and protein clustering in the IMM. DNAJC19 mutations were predicted to cause a loss of the DnaJ interaction domain, which was confirmed by loss of full-length DNAJC19 protein in all mutant cell lines. The subcellular investigation of DNAJC19 demonstrated a nuclear restriction in mutant iPSC-CMs. The loss of DNAJC19 co-localization with mitochondrial structures was accompanied by enhanced fragmentation, an overall reduction of mitochondrial mass and smaller cardiomyocytes. Ultrastructural analysis yielded decreased mitochondria sizes and abnormal cristae providing a link to defects in mitochondrial biogenesis and CL remodeling. Preliminary data on CL profiles revealed longer acyl chains and a more unsaturated acyl chain composition highlighting abnormities in the phospholipid maturation in DCMA.
However, the assessment of mitochondrial function in iPSCs and dermal fibroblasts revealed an overall higher oxygen consumption that was even more enhanced in iPSC-CMs when comparing all three mutants to healthy controls. Excess oxygen consumption rates indicated a higher electron transport chain (ETC) activity to meet cellular ATP demands that probably result from proton leakage or the decoupling of the ETC complexes provoked by abnormal CL embedding in the IMM.
Moreover, in particular iPSC-CMs presented increased extracellular acidification rates that indicated a shift towards the utilization of other substrates than fatty acids, such as glucose, pyruvate or glutamine. The examination of metabolic features via double radioactive tracer uptakes (18F-FDG, 125I-BMIPP) displayed significantly decreased fatty acid uptake in all mutants that was accompanied by increased glucose uptake in one patient cell line only, underlining a highly dynamic preference of substrates between mutant iPSC-CMs.
To connect molecular changes directly to physiological processes, insights on calcium kinetics, contractility and arrhythmic potential were assessed and unraveled significantly increased beating frequencies, elevated diastolic calcium concentrations and a shared trend towards reduced cell shortenings in all mutant cell lines basally and upon isoproterenol stimulation. Extended speed of recovery was seen in all mutant iPSC-CMs but most striking in one patient-derived iPSC-CM model, that additionally showed significantly prolonged relaxation times. The investigations of calcium transient shapes pointed towards enhanced arrhythmic features in mutant cells comprised by both the occurrence of DADs/EADs and fibrillation-like events with discordant preferences.
Taken together, new insights into a novel in vitro model system of DCMA were gained to study a genetically determined cardiomyopathy in a patient-specific manner upon incorporation of an isogenic mutant control. Based on our results, we suggest that loss of full-length DNAJC19 impedes PHB2-complex stabilization within the IMM, thus hindering PHB-rings from building IMM-specific phospholipid clusters. These clusters are essential to enable normal CL remodeling during cristae morphogenesis. Disturbed cristae and mitochondrial fragmentation were observed and refer to an essential role of DNAJC19 in mitochondrial morphogenesis and biogenesis. Alterations in mitochondrial morphology are generally linked to reduced ATP yields and aberrant reactive oxygen species production thereby having fundamental downstream effects on the cardiomyocytes` functionality. DCMA-associated cellular dysfunctions were in particular manifested in excess oxygen consumption, altered substrate utilization and abnormal calcium kinetics. The summarized data highlight the usage of human iPSC-derived CMs as a powerful tool to recapitulate DCMA-associated phenotypes that offers an unique potential to identify therapeutic strategies in order to reverse the pathological process and to pave the way towards clinical applications for a personalized therapy of DCMA in the future.
Die Gottesknechtslieder gehören zu den Texten des Alten Testaments, die sehr häufig interpretiert worden sind. Das liegt zweifelsohne an der besonderen christlichen Rezeption, aber auch an der literaturgeschichtlichen und theologischen Bedeutung der Kapitel Jes 40–55. Eine weitere Studie dazu könnte in der Tat als Zeit- und Ressourcenverschwendung gewertet werden, wäre da nicht die theologisch herausragende Bedeutung der Gottesknechtslieder über den literarischen Horizont Deuterojesaja hinaus.
Spätestens seit den Arbeiten von Ulrich Berges steht die lange Zeit angenommene Eigenständigkeit der Textgruppe Gottesknechtslieder in Frage. Diese Sichtweise eröffnet einerseits die Möglichkeit, die Funktionalität der Lieder innerhalb Deuterojesajas neu zu beschreiben, andererseits vernachlässigt sie eine genauere Beschreibung der Funktion des Gottesknechts im historischen Kontext des zu Ende gehenden babylonischen Exils.
Diese Arbeit versucht mit den Methoden der historisch-kritischen Exegese zu rekonstruieren, wie eine prophetische Figur vom exilierten Teil Israels erwartet, als Gottesknecht zu wirken, aber zur Kenntnis nehmen muss, dass ihre Zeitgenossen dazu nicht bereit sind. Nach ihrem Scheitern, das in den Gottesknechtslieder verarbeitet ist, wird das prophetische Tun dieser Figur allerdings als Verwirklichung des Knechtsauftrags gedeutet. Die Besonderheit dieser Deutung des Knechtsauftrags im Kontext des Werkes Jes 40–55 liegt in seiner Erklärung des Leidens des Knechts: Der Knecht leidet wegen der Vergehen seiner Zeitgenossen. Innerhalb des alttestamentlichen Diskurses zum Problem der Theodizee liegt somit das Modell einer synchronen Schuldverkettung vor. Diese Bedeutung, die dem Tod des Knechts zugeschrieben wird, führt sogar im klassischen Tun-Ergehen-Zusammenhang zur Vorstellung eines guten Ergehens des Knechts im Jenseits. Die erste Vorstellung von Auferstehung kann somit im 4. Gottesknechtslied verortet werden.
Die Besonderheit des Knechtsauftrags zeigt eine weitere bedeutende theologische Weichenstellung im Alten Testament: Der Knechtsauftrag beinhaltet die Etablierung der göttlichen Rechts- und Heilsordnung für alle Nationen, dessen Licht der Knecht ist. Die Gottesknechtslieder sind also auch unverzichtbarer Bestandteil in der Wende zur Vorstellung eines Heilsuniversalismus. Diese Arbeit bietet demzufolge nicht nur eine Synthese der wichtigsten Forschungsfragen in Bezug auf die Gottesknechtslieder, sondern beschreibt ausführlich die Relevanz dieser Textzeugnisse innerhalb alttestamentlicher Theologie.
From the history of the Church, we gather that one of the most major tests that confronted the early Christian community was whether everyone who wanted to become a Christian also of necessity had to become a Jew as a pre-requisite for entrance into the new community of believers. The issue at stake is whether one qualifies to be a Christian through adherence to the Jewish identity, which centres on circumcision and the observance of the Mosaic legal code. The crisis resulted to the convocation of the Jerusalem Council (cf. Acts 15), which tasked itself with the definition of the Christian identity. The Council bases its definition of Christian identity, separable from adherence to the Jewish cultural practice (a form of cultural imperialism), solely on election by God in Jesus Christ. Moreover, the event of the Pentecost in Jerusalem demonstrated what the nature of the spreading of the message of this new community of believers in Jesus Christ should be: that people from other cultures, “Parthians, Medes and Elamites; people from Mesopotamia, Judaea and Capadocia, Ponthus and Asia, Phrygia and Pamphylis, Egypt and the parts of Libya around Cyrene; as well as visitors from Rome, Jews and Proselytes alike, Cretans and Arabs” (Acts 2: 9-11), could understand the message that Peter communicated to them through the force of the breath of the risen Jesus in their own mother tongue, without first becoming Jews. Against the background of this crucial point in the history of the early Church and in consideration of the Second Vatican Council, this dissertation seeks to address the problem of identity, unity and diversity in the Christian religion with special reference to Africa. It proposes that the traditional African Rites of Initiation that mark the transition from one stage of life to the other and therefore the existential and essential transformation of the individual and group offer with their rich symbolisms a very fertile ground for dialogue with the Christian religion. It views the various Rites of Initiation (from birth and ritual circumcision over puberty and adult to marriage and funeral rites) as vital and immutable seminal points in the life of the individual African and his/her society at large. These Rites that express in various ways the African holistic view and conception of life and reality are, in terms of their religious symbolism, meaning and function, analogous to their Christian counterparts (such as baptism, confirmation, Eucharist, ordination, marriage) and can as a result be conveniently accepted or at least incorporated even if in modified forms as authentic African initiation rites for African Christians. Without being syncretistic, such an incorporation and modification at one and the same time recognizes and respects the cultural identity of the African and marks his/her transformation and acceptance of his/her new identity, modelled on Christ. In this way, the African Christian will be enabled to live, articulate and express his/her faith within his/her own historical-cultural milieu. On the whole, the presentation is predictive and prescriptive with regard to what the relationship and dialogue between Christianity and the African Traditional Religion should be or should not be. It is an honest effort to make the Christian message relevant to the African in his/her own perceptual and conceptual world-view. This task remains a steady challenge to African Christians who want to maintain at one and the same time and at the same level their African identity and their Christian calling. The balancing and reconciling of these two identities in a correlating rather than confrontational manner remains a task for the Church of today and tomorrow. The dissertation is a foundational contribution to building up and sharpening consciousness for this problem.