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Global gene expression profiles of cardiomyocytes differentiated from human pluripotent stem cells (hiPSC-CMs) in 3D culture exposed to ethanol

ABSTRACT: Global gene expression profiles of cardiomyocytes differentiated from human pluripotent stem cells (hiPSC-CMs) in 3D culture exposed to ethanol

PROVIDER: PRJNA517945 | ENA |

REPOSITORIES: ENA

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			Action	DRS
	SRR8508500_1.fastq.gz	Fastqsanger.gz
	SRR8508500_2.fastq.gz	Fastqsanger.gz
	SRR8508510_1.fastq.gz	Fastqsanger.gz
	SRR8508510_2.fastq.gz	Fastqsanger.gz
	SRR8508501_1.fastq.gz	Fastqsanger.gz

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Global gene expression profiles of cardiomyocytes differentiated from human pluripotent stem cells (hiPSC-CMs) in 3D culture exposed to ethanol

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.

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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.

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