Titel: Atrial Fibrillation Modelling and Targeted DNA Methylation Editing in Human Engineered Heart Tissue-Based Disease Models
Sprache: Englisch
Autor*in: Pan, Bangfen
GND-Schlagwörter: Tissue EngineeringGND
Erscheinungsdatum: 2022
Tag der mündlichen Prüfung: 2022-10-07
Atrial fibrillation (AF) and heart failure (HF) are significant contributors to morbidity and mortality worldwide. Effective treatments for these heart diseases are still limited. Furthermore, many aspects of AF pathophysiology remain incompletely understood due to a lack of suitable models. Human engineered heart tissue (EHT) derived from human induced pluripotent stem cells (hiPSCs) has proven its value as an in vitro model for disease modelling and exploration of novel therapeutic strategies. Thus, we set out to establish and refine models for AF and HF based on human EHT for both assessment and manipulation of chromatin-based signaling.
In the first part of this work, a chronic optical tachypacing model based on atrial-like EHT was established and the effects of tachypacing were evaluated, both with regard to model validation and beyond, probing chromatin signaling. To allow optical pacing, a channelrhodopsin (volvox carteri channelrhodopsin 1; VChR1) expressing hiPSC line was generated by inserting the transgene into the AAVS1 locus with CRISPR/Cas9 genome editing. VChR1-iPSCs were then differentiated into atrial-like cardiomyocytes (CMs) using an optimized embryoid body (EB)-based protocol involving retinoic acid (RA) treatment combined with reduced concentrations of activin A and BMP4. We demonstrated that RA-EHTs resembled the human atrium transcriptionally and electrophysiologically, especially compared to previous attempts of atrial tissue engineering. Next, a flexible digital optogenetic device was built for long-term optical stimulation. VChR1-RA-EHTs were then intermittently optically tachypaced to model AF. Paced EHTs displayed impaired contractile force and released more cardiac troponin I into the culture medium than unpaced EHTs. First results from next-generation sequencing (NGS) revealed altered gene expression and loss of chromatin accessibility in paced versus unpaced EHTs. In summary, our tachypaced atrial-like EHT model partially recapitulated AF pathophysiology, provided new insights into chromatin dynamics in AF and could be used to test new potential therapies.
In the second part, targeted DNA methylation editing in EHT disease models was investigated. Harnessing DNA methylation for therapy could open up new opportunities, as pathways previously considered not druggable could be addressed, targeting the root of the problem could prove favorable in terms of side effects, and the kinetics of the intervention could be especially suitable. To this end, besides the AF model mentioned above, two other disease EHT models of HF were generated or adapted in this study. Cardiac fibroblasts (CFs) were differentiated from hiPSCs and added into ventricular-like EHTs to produce fibroblast-EHTs (CF-EHTs) for fibrosis modelling. Meanwhile, a pro-hypertrophic EHT model was adapted with the treatment of ventricular-like EHTs with phenylephrine (PE) and endothelin-1 (ET-1) for 7 days. In order to manipulate DNA methylation, split-dCas9-DNMT3A and split-dCas9-TET1 were created for methylation and demethylation of specific genes, respectively. These editing tools were designed for adeno-associated virus (AAV)-mediated delivery. To this end, dCas9 was split into dCas9N and dCas9C and fused with protein inteins in order to circumvent problems arising from a limited AAV packaging capacity. In an activity test in HEK 293 cells, transfection of split-dCas9-DNMT3A plasmids led to expression and fusion of full-length dCas9 and methylation of the targeted promoter region of IL6ST. Full-length dCas9-DNMT3A protein was also detected in both HEK 293 cells and CFs after plasmids co-transfection or AAV co-transduction. However, no regulation of expression could be detected for genes targeted by AAV in CFs, likely because the absolute effect was small and masked by variability. Taken together, in the second part of this thesis, we successfully adapted and optimized two further EHT-based models and established split-dCas9 targeted DNA methylation editing tools. However, optimization of the editing tools is still needed to achieve meaningful DNA methylation editing in CFs or CMs in EHTs by AAV delivery in the future.
URL: https://ediss.sub.uni-hamburg.de/handle/ediss/9989
URN: urn:nbn:de:gbv:18-ediss-105466
Dokumenttyp: Dissertation
Betreuer*in: Oetjen, Elke
Enthalten in den Sammlungen:Elektronische Dissertationen und Habilitationen

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