Chronic ethanol exposure induces deleterious changes in cardiomyocytes derived from human induced pluripotent stem cells
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ABSTRACT:
INSTRUMENT(S): LTQ Orbitrap Elite
ORGANISM(S): Homo Sapiens (ncbitaxon:9606)
SUBMITTER: 
Ronghu Wu
PROVIDER: MSV000087349 | MassIVE | Sat May 01 07:10:00 BST 2021
REPOSITORIES: MassIVE
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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:Pluripotent stem cell-derived cardiomyocytes (PSC-CMs) provide an unprecedented opportunity to study human heart development and disease. A major caveat however is that they remain functionally and structurally immature in culture, limiting their potential for disease modeling and regenerative approaches. Here we address the question of how different metabolic pathways can be modulated in order to induce efficient cardiac maturation of hPSC-CMs. We show that PPAR signaling acts in an isoform-specific manner to balance the glycolysis and fatty acid oxidation (FAO) pathways. PPARd activation or inhibition results in efficient respective up- or down-regulation of the gene regulatory networks underlying FAO in hPSC-CMs. PPARd induction further increases mitochondrial and peroxisome content, enhances mitochondrial cristae formation and augments FAO flux. Lastly PPARd activation results in enhanced myofibril organization and increased numbers of bi-nucleated hPSC-CMs. Transient lactate exposure, commonly used in hPSC-CM purification protocols, induces an independent program of cardiac maturation, but when combined with PPARd activation equally results in a metabolic switch to FAO. In summary, we identify multiple axes of metabolic modifications of hPSC-CMs including a role for PPARdelta signaling in inducing the metabolic switch to FAO in hPSC-CMs. Our findings provide new opportunities to generate and use metabolically mature hPSC-CMs including for disease modeling and regenerative therapy.
2021-11-01 | GSE160987 | GEO
Project description:Human embryonic stem cell-derived cardiomyocytes (hESC-CMs) and human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) grown for 35 days on laminin-coated coverslips, a stiff matrix, were enzymatically dissociated, replated on laminin-coated coverslips and grown for further 2 days. Gene expression was compared with controls grown for 37 days on laminin-coated coverslips. Total RNA was isolated and gene expression was analyzed by RNA-sequencing (RNA-seq) on an Illumina NextSeq 550 sequencer using a High Output Flowcell for single reads (20024906; Illumina). Gene enrichment analysis based on RNA-Seq data of hESC-CMs replated for 2 days as compared with 37 days old controls was performed by using the comprehensive gene set enrichment analysis tool Enrichr for pathway analysis with KEGG Pathways 2019 Human, as well as for Gene Ontology (GO) analysis with GO Cellular Component 2018, GO Molecular Function 2018, and GO Biological Process 2018. Gene enrichment was also analyzed by Ingenuity Pathway analysis (IPA, Qiagen), especially Canonical Pathways analysis. Gene enrichment analysis revealed changes in the gene expression profile, especially of mechanosensation/-transduction-related genes and pathways in replated hESC-CMs.
2023-08-17 | GSE176154 | GEO
Project description:Methods: RNA-seq libraries were prepared using the Illumina TruSeq RNA kit and the TrueSeq method was employed for mRNA enrichment. The libraries were quantified and samples were multiplexed in each lane of the flowcell. Cluster generation was performed and then sequenced on the Illumina HiSeq2500 system. Reads were mapped on the Human Genome Reference and normalized expression table was generated. Results: RNA-seq results reveal gene expression of cardiac toxicity in hiPSC-CMs that are consistent with alcohol-induced pathophysiology observed in animal models. For example MMP9 is among the top 5 upregulated genes in ethanol-treated hiPSC-CMs, MMP9 concentrations are significantly higher in human sera of chronic alcohol abusers and MMP9 mRNA and protein levels are increased in the myocardium of rats following acute ethanol exposure. Conclusions: Data demonstrate significant alteration in gene expression, among the top 60 genes significantly altered by ethanol exposure, 8 genes are involved in ion channels, which may be in part contributing to the abnormal intracellular Ca2+ transients. Ethanol up-regulated the expression of genes associated with collagen metabolism and extracellular matrix (MMP9, EMID1, COL14A1), most of the downregulated genes are involved in cardiovascular system development (NPPB, DNAAF3), actin filament-based process (LMOD2, MYH4) and muscle contraction (MYL2). These findings are consistent with previous studies showing a correlation between alcohol exposure and defects in heart and circulatory system development.
2019-01-31 | GSE125917 | 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:Mitochondria play a crucial role in the differentiation and maturation of human cardiomyocytes (CMs). To identify mitochondrial pathways and regulators that are involved in cardiac differentiation and maturation, we examined human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs). Proteomic analysis was performed on enriched mitochondrial protein extracts isolated from hiPSC-CMs differentiated from dermal fibroblasts (dFCM) and cardiac fibroblasts (cFCM), at different days of differentiation (between 12 and 115 days), and also from adult and neonatal mouse hearts for comparison. Mitochondrial proteins with a ≥2-fold change between differentiation time points in dFCMs and cFCMs, and between adult versus neonatal mouse hearts, were subjected to Ingenuity Pathway Analysis (IPA), and some upregulated proteins were validated by immunoblotting. The highest significant upregulation was in metabolic pathways for fatty acid oxidation (FAO), the tricarboxylic acid (TCA) cycle, oxidative phosphorylation (OXPHOS) and branched chain amino acid (BCAA) catabolism. The top upstream regulators predicted by IPA were- peroxisome proliferator-activated receptor gamma coactivator 1 alpha (PGC1-a), the insulin receptor and the retinoblastoma protein (Rb) transcriptional repressor. In addition, IPA and immunoblotting showed substantial upregulation of the mitochondrial LonP1 protease, which regulates mitochondrial proteostasis, energetics and metabolism. Using this proteomics approach, we have identified key metabolic and intracellular signaling pathways that are up- and down- regulated during the biogenesis of mitochondria in differentiating and maturing cardiac myocytes.
2022-05-19 | PXD014317 | Pride
Project description:Proteomic investigation of hiPSC-CMs post-Cfz treatment
2021-05-01 | MSV000087350 | MassIVE
Project description:Human pluripotent stem cell-derived cardiomyocytes (hPSC-CMs) show immense promise for patient-specific disease modeling, cardiotoxicity screening, and regenerative therapy development. However, hPSC-CMs in culture have not recapitulated the structural or functional properties of adult CMs in vivo thus far. To gain global insight into hPSC-CM biology, we established a multi-omics method for analyzing the hPSC-CM metabolome and proteome from the same cell culture, creating multi-dimensional profiles of hPSC-CMs. Specifically, we developed a sequential extraction to capture metabolites and proteins from the same hPSC-CM monolayer cultures, and analyzed these extracts using high-resolution mass spectrometry (MS).
2021-08-22 | MSV000088010 | MassIVE
Project description:Gene expression in the human foetal heart for a comparison with cardiomyocytes derived from human pluripotent stem cells. Human foetal heart samples were collected from the individual chambers of the heart at various stages of development. This provided the opportunity to perform gene expression analysis and identify genes involved in the formation of the heart in each of the four chambers and at different stages of development. The dataset can be used to benchmark human pluripotent stem cell derived cardiomyocytes (hPSC-CMs) in terms of their maturation state by comparing it to the foetal heart samples. Two commercially available reference RNA sets are included in the analysis in order to characterize future sets of hPSC-CMs. Microarray experiments were performed on human foetal heart samples from first and second trimester hearts, separated in atria and ventricles.
2015-07-22 | E-GEOD-71148 | biostudies-arrayexpress
Project description:Analysis of the microRNA profile exression in hiPSC-CMs. Results provide important information of the miRNAs expressed in hiPSC-CMs under control conditions.
2018-05-01 | GSE111984 | GEO