A transcriptional enhancer regulates cardiac maturation [RNA-seq]
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ABSTRACT:
ORGANISM(S): Mus musculus
PROVIDER: GSE266306 | GEO | 2024/05/24
REPOSITORIES: GEO
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Project description:Cardiomyocyte maturation is crucial for generating adult cardiomyocytes and the application of human pluripotent stem cell derived-cardiomyocytes (hPSC-CMs). However, regulation at the cis-regulatory element level, and its role in heart disease remain unclear. Alpha-actinin 2 (ACTN2) levels increase during CM maturation. Here, we investigate a clinically relevant, conserved ACTN2 enhancer’s effects on CM maturation using hPSC and mouse models. Heterozygous ACTN2 enhancer deletion led to abnormal CM morphology, reduced function, and mitochondrial respiration. Transcriptomic analyses in vitro and in vivo showed disrupted CM maturation and upregulated anabolic mammalian target for rapamycin (mTOR) signaling promoting senescence and hindering maturation. As confirmation, ACTN2 enhancer deletion induced heat shock protein 90A expression, a chaperone mediating mTOR activation. Conversely, targeting the ACTN2 enhancer via enhancer CRISPR activation (enCRISPRa) promoted hPSC-CM maturation. Our studies reveal the transcriptional enhancer’s role in cardiac maturation and disease, offering insights into potentially fine-tuning gene expression to modulate cardiomyocyte physiology.
2024-05-24 | GSE266307 | GEO
Project description:Cardiovascular disease is a leading cause of death worldwide. Human pluripotent stem cell-derived cardiomyocytes (hPSC-CMs) hold immense clinical potential and recent studies have enabled generation of virtually pure hPSC-CMs with high efficiency in chemically defined and xeno-free conditions. Despite these advances, hPSC-CMs exhibit an immature phenotype and are arrhythmogenic in vivo, necessitating development of methods to mature these cells. hPSC-CMs undergo significant metabolic alterations during differentiation and maturation. A detailed analysis of the metabolic changes accompanying maturation of hPSC-CMs may prove useful in identifying new strategies to expedite the maturation process and also provide biomarkers for testing or validating hPSC-CM maturation. In this study we identified global metabolic changes which take place during long-term culture and maturation of hPSC-CMs derived from three different hPSC lines. We have identified several metabolic pathways, including phospholipid metabolism and pantothenate and Coenzyme A metabolism, which showed significant enrichment upon maturation in addition to fatty acid oxidation and metabolism. We also identified an increase in glycerophosphocholine and reduction in phosphocholine as potential metabolic biomarkers of maturation. These biomarkers were also affected in a similar manner during murine heart development in vivo. These results support that hPSC-CM maturation is associated with extensive metabolic rewiring and understanding the role of these metabolic changes in maturation process has the potential to develop novel approaches to monitor and expedite hPSC-CM maturation.
2018-04-24 | MTBLS425 | MetaboLights
Project description:Although human pluripotent stem cells-derived cardiomyocytes (hPSC-CMs) have emerged as a novel platform for heart regeneration, disease modeling, and drug screening, their immaturity significantly hinders their application. A hallmark of postnatal cardiomyocyte maturation is the metabolic substrate switch from glucose to fatty acids. We hypothesized that fatty acid supplementation would enhance hPSC-CM maturation. Fatty acid treatment induces cardiomyocyte hypertrophy and significantly increases cardiomyocyte force production. The improvement in force generation is accompanied by enhanced calcium transient peak height and kinetics, and by increased action potential upstroke velocity. Fatty acids enhance mitochondrial respiratory reserve capacity. RNA sequencing showed fatty acid treatment upregulates genes involved in fatty acid β-oxidation and downregulates genes in lipid synthesis. Signal pathway analyses reveal that fatty acid treatment results in phosphorylation of multiple intracellular kinases. Thus, fatty acids increase human cardiomyocyte hypertrophy, force generation, calcium dynamics, action potential upstroke velocity, and oxidative capacity. This enhanced maturation should facilitate hPSC-CMs usage for cell therapy, disease modeling, and drug/toxicity screens.
2018-12-19 | GSE124057 | GEO
Project description:Human pluripotent stem cell-derived cardiomyocytes (hPSC-CM) are a promising disease model, although hPSC-CMs are not fully mature cardiomyocytes. We examined exponential differentially expressed miRNA/gene to identify critical time-regulated transcripts associated with hPSC-CM differentiation. We uncovered 324 interactions among 29 differentially expressed genes and 51 miRNAs from 20.543 transcripts during the 120 days of hPSC-CM differentiation. We found 16 genes and 26 miRNAs with an inverse pattern of expression (Pearson R-values < -0.5) validated using several human and mouse databases. We further validated these findings using two hPSC-CM lines and seven sampling times over a 30-day protocol and observed 16 inverse interactions among eight genes and 12 miRNAs (Person R-values < -0.5) changing over time. Finally, we tested the top eight miRNAs by adding miRNA mimics to differentiating hPSC-CMs and found that they influenced proliferation and maturation phenotypes. These time-regulated transcripts appear to regulate single or multiple pathways affecting cardiac differentiation.
2022-09-15 | GSE188731 | GEO
Project description:Human pluripotent stem cell-derived cardiomyocytes (hPSC-CM) are a promising disease model, although hPSC-CMs are not fully mature cardiomyocytes. We examined exponential differentially expressed miRNA/gene to identify critical time-regulated transcripts associated with hPSC-CM differentiation. We uncovered 324 interactions among 29 differentially expressed genes and 51 miRNAs from 20.543 transcripts during the 120 days of hPSC-CM differentiation. We found 16 genes and 26 miRNAs with an inverse pattern of expression (Pearson R-values < -0.5) validated using several human and mouse databases. We further validated these findings using two hPSC-CM lines and seven sampling times over a 30-day protocol and observed 16 inverse interactions among eight genes and 12 miRNAs (Person R-values < -0.5) changing over time. Finally, we tested the top eight miRNAs by adding miRNA mimics to differentiating hPSC-CMs and found that they influenced proliferation and maturation phenotypes. These time-regulated transcripts appear to regulate single or multiple pathways affecting cardiac differentiation.
2022-09-15 | GSE188725 | GEO
Project description:Rationale: Human pluripotent stem cells-derived cardiomyocytes (hPSC-CMs) exhibit the properties of fetal CMs, which limit their applications. Various methods have been used to promote maturation of hPSC-CMs; however, there is a lack of an unbiased and comprehensive method for accurate benchmarking of hPSC-CM maturation.
Objective: We aim to develop an unbiased proteomics method integrating high-throughput top-down targeted proteomics and bottom-up global proteomics for accurate and comprehensive assessment of hPSC-CM maturation.
Methods and Results: Utilizing hPSC-CMs from early- and late-stage two-dimensional monolayer culture and three-dimensional engineered cardiac tissue, we demonstrated high reproducibility and reliability of the top-down proteomics method, which enabled simultaneous quantification of contractile protein isoform expressions and their PTMs. This method allowed for the detection of known maturation-associated contractile protein alterations, and for the first time, identified contractile protein PTMs as promising new markers of maturation. By employing a global proteomics strategy, we identified candidate maturation markers important for sarcomere organization, cardiac excitability, and Ca2+ homeostasis; and validated these markers in the developing mouse cardiac ventricles.
Conclusions: We established an unbiased proteomics method that can provide accurate and specific benchmarking of hPSC-CM maturation, and identified new markers of maturation. Furthermore, this integrated proteomics strategy laid a strong foundation for uncovering molecular basis underlying cardiac development and disease using hPSC-CMs.
| MSV000082985 | MassIVE
Project description:Perlecan (HSPG2), a multifunctional heparan sulphate proteoglycan (HSPG), is a key player in extracellular matrix maturation and stabilisation. Structurally perlecan is similar to agrin, another HSPG known to contribute to cardiomyocyte phenotype switching by reverting hypertrophy. Although perlecan is crucial for cardiac development, its role in cardiomyocyte phenotypic switching is unknown. Here we show perlecan expression increases over time during the differentiation of pluripotent stem cells to cardiomyocytes (hPSC-CMs). Haploinusuffient hPSCs (HSPG2+/-) differentiate efficiently towards cardiomyocytes with minimal transcriptomic differences seen early in differentiation, but wide-ranging changes seen in late-stage cardiomyocytes. These differences are associated with structural, contractile, metabolic, and ECM genes. In keeping, late-stageHSPG2+/-hPSC-CMs display reduced maximal metabolic capacity and increased extracellular acidification rate, characteristics of reduced maturity. Moreover, engineered heart tissues produced withHSPG2+/-hPSC-CMs exhibit increased ECM remodelling, reduced tissue thickness and force generation. Wild-type hPSC-CMs grown on a substrate coated with a perlecan peptide showed increased cardiomyocyte nucleation typical of hypertrophic growth, suggesting that perlecan plays the opposite role of agrin by promoting cellular maturation rather than hyperplasia and proliferation. These data show that perlecan promotes cardiomyocyte maturity, possibly through hypertrophic growth. Targeting perlecan-dependent signalling may, therefore, reverse the phenotypic switch common to many forms of heart failure, by improving cardiomyocyte functionality.
2024-01-23 | GSE229975 | GEO
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 goal of this study is to analyze the impact of Actn2 depletion on transcription during cardiomyocyte maturation in mouse
2020-12-14 | GSE136079 | GEO
Project description:Vascularization and maturation options for cardiac tissue engineered structures are currently intensively investigated. Therefore, the generation and characterisation of all cardiovascular cell types from human pluripotent stem cells (hPSC; either induced -iPSC- or embryonic -hESC) are of particular interest. In our group, differentiation and selection methods were described for obtaining highly pure hPSC-derived cardiomyocytes (CM; selected for αMHC), endothelial cells (EC; selected for CD31) and PDGFRβ expressing cardiac pericyte-like cells (PC). With the purpose of identifying cell type-related mechanisms in co-culture and tissue formation, gene expression profile of hPSC-derived CM, ECs, and PCs was compared to their undifferentiated progeny (hPSC) as well as to primary pericytes (hPC-PL) and fibroblasts (HFF).
2020-11-30 | GSE145957 | GEO