Proteome and phosphoproteome analysis of human cardiac organoids following nanobody therapeutics
Ontology highlight
ABSTRACT:
INSTRUMENT(S): Q Exactive HF-X
ORGANISM(S): Homo Sapiens (ncbitaxon:9606)
SUBMITTER: 
David Greening
PROVIDER: MSV000092856 | MassIVE |
SECONDARY ACCESSION(S): PXD045373
REPOSITORIES: MassIVE
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Project description:Self-organisation and coordinated morphogenesis of multiple cardiac lineages is essential for the development and function of the heart1-3. However, the absence of a human in vitro model that mimics the basic lineage architecture of the heart hinders research into developmental mechanisms and congenital defects4. Here, we describe the establishment of a reliable, lineage-controlled and high-throughput cardiac organoid platform. We show that cardiac mesoderm derived from human pluripotent stem cells robustly self-organises and differentiates into cardiomyocytes forming a cavity. Co-differentiation of cardiomyocytes and endothelial cells from cardiac mesoderm within these structures is required to form a separate endothelial layer. As in vivo, the epicardium engulfs these cardiac organoids, migrates into the cardiomyocyte layer and differentiates. We use this model to demonstrate that cardiac cavity formation is controlled by a mesodermal WNT-BMP signalling axis. Disruption of one of the key BMP targets in cardiac mesoderm, the transcription factor HAND1, interferes with cavity formation, which is consistent with its role in early heart tube and left chamber development5. Thus, the cardiac organoid platform represents a powerful resource for the quantitative and mechanistic analysis of early human cardiogenesis and defects that are otherwise inaccessible. Beyond understanding congenital heart disease, cardiac organoids provide a foundation for future translational research into human cardiac disorders.
2021-05-18 | PXD018306 | Pride
Project description:Pathogenic mutations in alpha kinase 3 (ALPK3) cause cardiomyopathy and a range of musculoskeletal defects. How ALPK3 mutations result in disease remains unclear and little is known about this atypical kinase. Using a suite of engineered human pluripotent stem cells (hPSCs) we show that ALPK3 localizes to the sarcomere, specifically at the M-Band. Both sarcomeric organization and calcium kinetics were disrupted in ALPK3 deficient hPSC derived cardiomyocytes. Further, cardiac organoids derived from ALPK3 knockout hPSCs displayed reduced force generation. Phosphoproteomic profiling of wildtype and ALPK3 null hPSC derived cardiomyocytes revealed ALPK3-dependant phospho-peptides were enriched for proteins involved in sarcomere function and protein quality control. We demonstrate that ALPK3 binds to the selective autophagy receptor SQSTM1 (Sequestome 1) and is required for the sarcomeric localization of SQSTM1. We propose that ALPK3 is a myogenic kinase with an integral role in the intracellular signaling networks underlying sarcomere maintenance required for continued cardiac contractility.
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Project description:The mammalian heart undergoes major transitions during postnatal life to acquire the physiological properties of an adult organ. Postnatal life imposes numerous adaptations including electrophysiological, structural and metabolic maturation of cardiomyocytes1, which occur coincident with loss of proliferative capacity and regenerative potential2,3. The discovery of key upstream drivers of cardiomyocyte maturation and cell cycle arrest remains one of the most important unanswered questions in cardiac biology. Discovery of these drivers would facilitate current attempts to promote cardiomyocyte maturation in vitro for drug discovery and to de-differentiate adult cardiomyocytes in vivo for regenerative medicine. A recent study has suggested that the shift from a low oxygen environment in utero towards a high oxygen environment after birth acts as a key trigger for cardiomyocyte cell cycle exit4. Moreover, it was recently demonstrated that proliferative adult cardiomyocytes reside in a hypoxic niche5 and that exposure of adult mice to gradual hypoxemia is sufficient to drive cell cycle re-entry and regeneration following infarction6. However, it is currently unclear whether postnatal changes in oxygen tension or the associated shifts in cardiomyocyte metabolism are sufficient to promote maturation and cell cycle arrest as human pluripotent stem cell (hPSC)-derived cardiomyocytes fail to mature when cultured at 21% oxygen7,8. There are considerable changes in metabolic substrate provision during early postnatal life. The mammalian heart relies on high concentrations of carbohydrates and the presence of insulin in utero but later switches to fatty acid dominated substrates present in milk and low insulin levels post-birth9. In order to adapt to these changes in substrates, cardiomyocytes upregulate the genes required for fatty acid oxidation after birth10. The importance of these metabolic adaptations for cardiomyocyte maturation has been difficult to study because genetic disruption of fatty acid oxidation components in vivo can have a broad range of negative health impacts11. Therefore, there is a need to develop alternative approaches for studying the impact of cardiomyocyte metabolism on the maturation process. hPSCs are now widely used for the generation of defined human somatic cell types, including cardiomyocytes. These cardiomyocytes have now been used extensively for developmental studies, drug screening, disease modeling, and heart repair. However, lack of maturity and inappropriate responses to pharmacological agents have been identified as limitations in 2D or embryoid body based differentiation strategies12. To improve maturity of hPSC-derived cardiomyocytes, long-term culture can be used13, although long-term cultures may not be amenable to high-throughput screening applications and adult-like maturity is still not achieved14. In an effort to better simulate heart muscle structure and function, cardiac tissue engineering to form 3D engineered heart tissue has been used15-19. However, despite these recent advances in human cardiac tissue engineering, cardiac tissues derived from hPSC still lack many features of fully mature adult heart tissue20. Moreover, engineered heart tissue fabrication, culture, mechanical loading and pacing protocols, and analysis methods using organ baths are costly, labor intensive, and the multiple handling steps induce variability. In order to facilitate higher-throughput experiments, platforms for engineered heart tissue production have been miniaturized, however, screening experiments using semi-automated force of contraction analyses have only been published in 24-well plate formats21. Therefore, we developed a novel 96-well device, the heart dynamometer (Heart-Dyno), for high-throughput functional screening of human cardiac organoids (hCOs) to facilitate screening on a larger scale. The Heart-Dyno is designed to facilitate automated formation of dense muscle bundles from minimal cells and reagents while also facilitating culture and automated force of contraction analysis without any tissue handling. Using the Heart-Dyno, we define serum-free 3D culture conditions that promote structural, electrophysiological, metabolic and proliferative maturation of hPSC-derived cardiac organoids. Furthermore, we uncover a metabolic mechanism governing cardiomyocyte cell cycle arrest through repression of a β-catenin and YAP1 dependent signalling.
2017-11-21 | PXD005736 | Pride
Project description:The mammalian heart undergoes maturation during postnatal life to meet the increased functional requirements of the adult. However, the key drivers of this process remain poorly defined. We developed as 96-well screening platform, using human pluripotent stem cell derived cardiac organoids, to determine the molecular requirements for in vitro cardiomyocyte maturation. Here, we describe gene expression changes resulting from culturing human cardiac organoids in standard cell culture conditions and under optimized maturation conditions. We assessed our maturation conditions by comparing transcriptional changes of human cardiac organoids to RNA isolated from human heart. Interesting, analysis of these data revealed that a switch to fatty acid oxidative metabolism is a key governor of cardiomyocyte maturation and mature cardiac organoids were refractory to mitogenic stimuli.
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Project description:The study of cardiac physiology and disease is hindered by physiological differences between humans and small-animal models. Here, we report the generation of multi-chambered vascularized human cardiac organoids under anisotropic stress, and their applicability to study electro-metabolic coupling in cardiac tissue. The organoids are derived from human induced pluripotent stem cells, and integrate sensors for the simultaneous measurement of oxygen uptake, extracellular field potentials and cardiac contraction at resolutions higher than 10 Hz. The microphysiological system allowed us to find that 1-Hz cardiac respiratory cycles are coupled with electrical activity rather than with mechanical activity, that calcium oscillations drive a mitochondrial respiration cycle, that the pharmaceutical or genetic inhibition of electro-mitochondrial coupling results in arrhythmogenic behaviour, and that the induction of arrythmia by the chemotherapeutic mitoxantrone can be partially reversed by the co-administration of metformin. Microphysiological cardiac systems may further facilitate the study of the mitochondrial dynamics of cardiac rhythms and advance the understanding of cardiac physiology.
2024-06-01 | GSE234907 | GEO
Project description:Stem cell organoids are powerful models for studying organ development, disease modeling, drug screening, and regenerative medicine applications. The convergence of organoid technology, tissue engineering, and artificial intelligence (AI) could potentially enhance our understanding of the design principle for organoid engineering. In this study, we utilized micropatterning techniques to create a designer library of 230 cardiac organoids with 7 geometric designs (Circle 200, Circle 600, Circle 1000, Rectangle 1:1, Rectangle 1:4, Star 1:1, and Star 1:4). We employed manifold learning techniques to analyze single organoid heterogeneity based on 10 physiological parameters. We successfully clustered and refined our cardiac organoids based on their functional similarity using unsupervised machine learning approaches, thus elucidating unique functionalities associated with geometric designs. We also highlighted the critical role of calcium rising time in distinguishing organoids based on geometric patterns and clustering results. This innovative integration of organoid engineering and machine learning enhances our understanding of structure-function relationships in cardiac organoids, paving the way for more controlled and optimized organoid design.
2024-05-17 | GSE267438 | GEO