H3K27ac acetylome signatures reveal the epigenomic reorganization in remodeled non-failing human hearts

BACKGROUND: H3K27ac histone acetylome changes contribute to the phenotypic response in heart diseases, particularly in end-stage heart failure. However, such epigenetic alterations have not been systematically investigated in remodeled non-failing human hearts. Therefore, valuable insight into cardiac dysfunction in early remodeling is lacking. This study aimed to reveal the acetylation changes of chromatin regions in response to myocardial remodeling and their correlations to transcriptional changes of neighboring genes. RESULTS: We detected chromatin regions with differential acetylation activity (DARs; Padj. < 0.05) between remodeled non-failing patient hearts and healthy donor hearts. The acetylation level of the chromatin region correlated with its RNA polymerase II occupancy level and the mRNA expression level of its adjacent gene per sample. Annotated genes from DARs were enriched in disease-related pathways, including fibrosis and cell metabolism regulation. DARs that change in the same direction have a tendency to cluster together, suggesting the well-reorganized chromatin architecture that facilitates the interactions of regulatory domains in response to myocardial remodeling. We further show the differences between the acetylation level and the mRNA expression level of cell-type-specific markers for cardiomyocytes and 11 non-myocyte cell types. Notably, we identified transcriptome factor (TF) binding motifs that were enriched in DARs and defined TFs that were predicted to bind to these motifs. We further showed 64 genes coding for these TFs that were differentially expressed in remodeled myocardium when compared with controls. CONCLUSIONS: Our study reveals extensive novel insight on myocardial remodeling at the DNA regulatory level. Differences between the acetylation level and the transcriptional level of cell-type-specific markers suggest additional mechanism(s) between acetylome and transcriptome. By integrating these two layers of epigenetic profiles, we further provide promising TF-encoding genes that could serve as master regulators of myocardial remodeling. Combined, our findings highlight the important role of chromatin regulatory signatures in understanding disease etiology

Comparative analysis of biopolymer-based scaffolds for therapeutically relevant cells

Im Rahmen dieser Arbeit wurde die Generierung somatischer, kardialer Zellen aus humanen pluripotenten Stammzellen in einen dynamischen Suspensionsbioreaktor übertragen und vergleichend mit der statischen „hängenden Tropfen“ Kultur hinsichtlich der Differenzierungseffizienz und der Reife der abgeleiteten Kardiomyozyten untersucht. Darüber hinaus wurden für die Dissoziierung der Zellen drei Enzyme vergleichend analysiert. Die Wirkung von adhäsiven, kontraktilen Biopolymeren auf die Kardiomyozytenphysiologie wurde am Beispiel von ultra-hoch viskosem Alginat untersucht. So konnte festgestellt werden, dass Kardiomyozyten auf solchen Biopolymeren eine reifere Organisation des Zytoskeletts und Geneexpression aufweisen. Des Weiteren wurde ein neuartiges Verfahren entwickelt welches die simultane Quantifizierung der Kontraktionskraft und der intrazellulären Kalziumströme erlaubt. Hierzu wurden die Kardiomyozyten unterschiedlichen Konzentrationen von Isoprenalin, ein Beta-Adrenozeptoragonist, ausgesetzt. Darüber hinaus wurde eine neuartige Methode für den 3D Druck von niedrig-konzentrierten Biopolymerlösungen entwickelt und hinsichtlich ihrer Druckauflösung charakterisiert. Kardiomyozyten wurden auf diesen Gerüststrukturen kultiviert und es konnte gezeigt werden, dass die Zytoskelette eine noch höhere Organisation aufweisen und spezifische, mit der physiologischen Reifung assoziierte Proteine, in erhöhtem Maße vorkommen.In this work, a process for the production of somatic, cardiac cells from human induced pluripotent stem cells in three-dimensional micro tissues was adapted to a dynamic, suspension-based bioreactor. The differentiation efficiency and the maturation of the generated cardiomyocytes in the bioreactor and the static hanging drop culture were analyzed. To dissociate the cardiac tissues into single cells, the performances of three enzymes were comparatively evaluated. To analyze the effect of contractile biopolymers on cardiomyocyte physiology ultra-high viscosity alginates were utilized. Cardiomyocytes cultured on these matrices for a prolonged period exhibit a matured cytoskeletal organization and gene expression. In addition, a novel approach was developed, which enables the simultaneous quantification of the generated contraction force and intracellular calcium transients. To this extent, different concentrations of the beta-adrenergic agonist isoprenaline were applied. A novel 3D printing method for the three-dimensional deposition of low concentrated biopolymer formulations was devised and evaluated concerning the printing resolution and pore formation. The culture of human cardiomyocytes on such 3D printed, biopolymer-based scaffolds led to a further maturation of the cytoskeletal organization and a higher abundance of proteins, which are associated with the maturation in vivo.Fraunhofer-Gesellschaft zur Förderung der angewandten Forschung e.V

Studying arrythmogenic right ventricular cardiomyopathy : dysplasia using induced pluripotent stem cells

PhD ThesisArrhythmogenic right ventricular cardiomyopathy / dysplasia (ARVC/D) is a cause of ventricular arrhythmia and heart failure in adults. Fifty percent of subjects with ARVC/D carry pathogenic variants in the genes encoding desmosomal proteins. ARVC/D is associated with changes in cardiac desmosomal ultrastructure and the cellular distribution of desmosomal proteins in cardiomyocytes. These changes may be part of a common pathway of pathogenesis for the disease. Induced pluripotent stem cell derived cardiomyocytes (iPSC-CMs) carrying ARVC/D associated variants have been reported to recapitulate features of the disease. Five iPSC-CM models of ARVC/D have been reported, all of which carry mutations in the gene encoding the desmosomal protein plakophilin 2. In this study iPSC-CMs were generated from the peripheral blood mononuclear cells of a control subject and three subjects with ARVC/D. The ARVC/D iPSC-CMs carried pathogenic variants three different desmosomal genes: plakophilin 2 (PKP2), desmoglein 2 (DSG2), and desmoplakin (DSP) that have not been studied previously using cellular or animal models. No differences were found in either the cellular distribution of desmosomal proteins or the ultrastructure of desmosomes when ARVC/D and control iPSC-CMs were compared. It was concluded that iPSC-CMs are not as robust a platform for modelling ARVC/D as had been previously reported. The expression of desmosomal genes in iPSCs were at levels similar to those seen in iPSC-CMs. The differentiation of iPSCs to iPSC-CMs was associated with a decrease in the expression of genes encoding desmosomal cadherins and an increase in those encoding arm-repeat proteins. There was also evidence of desmosomal protein expression and the presence of desmosomes in iPSC cultures. It is suggested that intercellular adhesion junctions containing desmosomal proteins have a role in the maintenance of pluripotency in iPSCs in vitro and changes in desmosomal gene and protein expression are important in defining the cardiac differentiation of iPSCs.Newcastle upon Tyne Hospitals NHS Charit

3D bioprinted heart patches for cardiac regeneration

BACKGROUND: epicardial patch transplantation is a promising approach to restore some of the cardiac function lost after myocardial infarction (MI). Advances in 3D bioprinting, 3D cell culture and transplantation methods at surgery have provided hope that this approach could soon benefit heart failure patients. The optimal content of 3D bioprinted patches (the “bioink” extruded by a 3D bioprinter) is not known. Patches containing a suspension of 3D vascularised cardiac spheroids (VCS; 3D aggregates of cells / microtissues) in hydrogel may confer an advantage compared to freely suspended cells or hydrogel without cells. The mechanisms underlying the benefit of epicardial patch transplantation approaches have not been fully elucidated and this is needed for widespread clinical translation. To be fully compatible with cardiothoracic surgical approaches in future, patches should be transplantable by minimally invasive robotic approaches. METHOD: Alginate-gelatin (AlgGel) patches were optimised ex vivo for cardiac applications, followed by in vivo transplantation of patches in mice modelling MI. For the ex vivo optimisation phase, three different bioprinters were used to bioprint patches with different bioink contents which were incubated up to 28 days and analysed. For the in vivo phase, new patches were 3D bioprinted using the optimal methods determined in the previous (ex vivo) experiments and surgically transplanted to the epicardium in infarcted mice. For these in vivo experiments, we cultured mixed cardiac cells: induced pluripotent stem cell derived cardiomyocytes (iCMs), human coronary artery endothelial cells (HCAECs) and cardiac fibroblasts (CFs). Cells were cultured using hanging drops to generate VCS which were suspended in AlgGel to create bioink for 3D bioprinting of patches. Study control groups (in vivo) were: the same cells freely suspended in AlgGel, AlgGel without cells, MI without treatment and sham surgery (no MI and no treatment). The primary outcome was cardiac function (left ventricular ejection fraction, LVEF%) measured up to day 28 post surgery. Additional analyses included: electrical mapping, histology, cell quantification by flow cytometry and mRNA (gene expression) profiling. Alongside these experiments, we developed novel surgical robotic minimally invasive instruments designed to transplant similar patches at human scale. We prototyped a heart patch transplanter device and demonstrated its potential utility in a world-first operation on a pig cadaver. RESULTS: Ex vivo patches incubated for 28 days allowed for self-organisation of endothelial cells into networks and contractile activity within patches. In vivo transplantation of patches in mice modelling MI resulted in a “return to baseline” improvement in median LVEF%. Our results measured median baseline (pre-surgery) LVEF% for all mice at 66%. Post-surgery, LVEF% was 58% for Sham (non-infarcted) and 41% for MI (no treatment) mice. Patch transplantation increased LVEF%: 55% (acellular; p=0.012), 59% (cells; p=0.106), 64% (spheroids; p=0.010). The VCS group was associated with improved electrical mapping profiles, lower infarct sizes, changes in host immune cell numbers and a gene expression (mRNA) profile which was closest to sham mice (with no MI). As proof-of-concept, similar scaled-up AlgGel patches were successfully transplanted in a porcine cadaver using a prototyped robotic minimally invasive surgical instrument. CONCLUSION: Epicardial transplantation of patches improves cardiac function in mice modelling MI. The use of VCS in alginate-gelatin bioink seems to offer advantages compared to freely suspended cells or hydrogel alone. The fact that hydrogel alone without cells confers some restoration of myocardial function suggests that the mechanism is not fully accounted for by the cellular portion of the bioink. Further studies are needed with a focus on whether host immune cell modulation is a key mechanism underlying the benefit of this approach. Since our most successful treatment group (VCS) had a similar transcriptome compared to non-infarcted (sham) mice, further studies should also include transcriptomic analyses to confirm reproducibility of this finding. If it is confirmed that immuno-genetic mechanisms underly patch-based approaches to myocardial protection after MI, this may change the focus of treatment strategies and avoid wasted resources and potentially patient harm (from treatments which are not aligned with the underlying mechanism). Our robotic minimally invasive patch transplantation operation represents a first step on a potential pathway towards transplantation at human surgery (without the need for traditional open surgery). For translatability, patch development should work towards being compatible with robotic and/or minimally invasive transplantation

Comparative Gene Expression Analysis of Mouse and Human Cardiac Maturation

Understanding how human cardiomyocytes mature is crucial to realizing stem cell-based heart regeneration, modeling adult heart diseases, and facilitating drug discovery. However, it is not feasible to analyze human samples for maturation due to inaccessibility to samples while cardiomyocytes mature during fetal development and childhood, as well as difficulty in avoiding variations among individuals. Using model animals such as mice can be a useful strategy; nonetheless, it is not well-understood whether and to what degree gene expression profiles during maturation are shared between humans and mice. Therefore, we performed a comparative gene expression analysis of mice and human samples. First, we examined two distinct mice microarray platforms for shared gene expression profiles, aiming to increase reliability of the analysis. We identified a set of genes displaying progressive changes during maturation based on principal component analysis. Second, we demonstrated that the genes identified had a differential expression pattern between adult and earlier stages (e.g., fetus) common in mice and humans. Our findings provide a foundation for further genetic studies of cardiomyocyte maturation