Transcriptome analysis of Tbx18-induced pacemaker cells (TBX18-iPMs) in comparison to ventricular cardiomyocytes
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ABSTRACT:
PROVIDER: PRJNA531698 | ENA |
REPOSITORIES: ENA
Dataset's files
| Action | DRS | |||
|---|---|---|---|---|
| SRR8871520_1.fastq.gz | Fastqsanger.gz | |||
| SRR8871520_2.fastq.gz | Fastqsanger.gz | |||
| SRR8871521_1.fastq.gz | Fastqsanger.gz | |||
| SRR8871521_2.fastq.gz | Fastqsanger.gz | |||
| SRR8871522_1.fastq.gz | Fastqsanger.gz |
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Project description:To understand the transcriptional landscape of Tbx18-induced cardiac pacemaker cells. The control cells were ventricular myocytes expressing GFP.
2022-08-25 | GSE129545 | GEO
Project description:Background: Embryonic trancription factor TBX18 re-expression has been demonstrated to be capable of converting ventricular myocytes to pacemakers cells. The goals of this study are to investigate the effects of TBX18 on myocytes and non-myocytes at day 3, day 6 and day 14, as well as tracking the key node of reprogramming process and mechanism. Methods: Neonatal rat ventricular myocytes (NRVC) were treated by adeno-TBX18 or GFP for 3 days to investigate the early effects of TBX18 on myocytes and non-myocytes. Except for these 2 treatments, Adeno-TBX18 with one specific Tgfβ inhibitor, A83-01 (0.2uM) was treated for NRVC for 14 days for explore the effects of Tgfβ inhibition on fibroblast activation and myocyte reprogramming process. Attached NRVC monolayers were digested with 0.25% trypsin (Cat# LS003707, Corning, NY) for 5 min at 37 °C, and filtered through a 100-μm cell strainer (Fischer Scientific, Waltham, MA) to generate a single-cell suspension. Single cells were resuspended to a concentration of 700-2,000 cells per microliter. Gel beads-in-emulsion (GEMs) were generated with Chromium X (10X Genomics, Pleasanton, CA) and RNA libraries were prepared using the Chromium Next GEM Single Cell 3' GEM kit, v3.1 (10X Genomics Inc., Pleasanton, CA) with a targeted cell recovery of 4,000-10,000 cells per reaction. Quality control of cDNA and scRNA-seq libraries were performed using the Bioanalyzer High Sensitivity kit (Agilent Technologies Inc., CA). Libraries were sequenced using Novaseq 6000 (Illumina Inc., CA) with a minimum depth of 50,000–65,000 read pairs per cell. Result: Upon QA/QC filtering, 13,437 cells met the quality thresholds for subsequent analysis. We focused on three cell types: cardiomyocytes (CMs, Tnni3-positive), total FBs (Pdgfrb and Tcf21-positive) with 2 subpopulations, including myoFBs (Pdgfrb, Tcf21, Acta2-high) and quiescent FBs (Pdgfrb, Tcf21, Acta2-low). Pathway analysis further revealed that upregulated DEGs by TBX18 are involved in fibroblast activation and ECM remodeling. Among those, Tgfβ signaling, which is one of the major molecular mediators of fibrosis in the heart, figured prominently in TBX18-transduced CMs and FBs. This was evidenced by upregulated expression of Tgfβ ligands and profibrotic genes such as Tgfb1, Tgfb2, and Acta2 in TBX18-NRVCs. Accordingly, higher proportion of activated myofibroblasts (39.3% vs. 25.4%) and lower proportion of quiescent FBs were observed in TBX18-NRVCs compared to control (Fig. 2F). To test the causative role of Tgfβ signaling in triggering fibrosis upon TBX18 re-expression, monolayers of NRVCs were treated with a small molecule inhibitor of type I Tgfβ receptors, A83-01. Treatment with A83-01 significantly decreased the population of myoFBs from 68.0% in TBX18-NRVC to 49.5% of total cell population at d14. Heat map analysis of all genes indicated that TBX18-CMs show upregulation of fibroblast-related genes, which is mitigated with A83-01 treatment. The upregulated DEGs in TBX18-FBs pointed to ECM remodeling as well as inflammatory response involving Tgfβ and other cytokines. Again, treatment with A83-01 abrogated the rise of these pathways. Profibrotic genes such as Tgfb2, Tgfb3, Acta2, and inflammatory genes were upregulated in FBs transduced with Ad-TBX18, which was mitigated by A83-01 treatment. The upregulated gene clusters in TBX18-FBs were also upregulated in TBX18-CMs, particularly, interferon signaling, interleukin-4 regulation of apoptosis, which may have led to the loss of TBX18-CMs over time. The inflammatory and profibrotic pathways were suppressed by treatment with A83-01 and may explain the higher population of TBX18-CMs with A83-01 at d14.We also quantified the number of de novo iPMs based on: Tnni3high, Hcn4+, Nkx2-5low, Gja1low through our scRNA-seq analysis and demonstrated the proportions of Hcn4+ iPMs was similar in TBX18-CMs regardless of Tgfβ inhibition but was higher compared to control. Conclusion: Tgfβ signaling inhibition with a83-01 prevents against myofibroblast activation, cardiomyocytes apoptosis and inflammatory response induced by TBX18, and short term treatment of A8301 does not strongly interfere the reprogramming process and efficiency.
2024-09-27 | GSE189815 | GEO
Project description:In this study, we performed two-dimensional liquid chromatography coupled to tandem mass spectrometry (2D-LC-MS/MS) to characterize the global transcriptome and proteome changes in Tbx18-iPMs compared to control, GFP-overexpressing neonatal rat ventricular myocytes (NRVMs). 67% of protein abundances changed significantly (4,475 of 6,661 proteins; p<0.05) at a false discovery rate of 1.6%. Downregulation was broad among metabolic pathways, such as TCA cycle, oxidative phosphorylation, glycolysis and fatty acid oxidation. Ion channels associated with ventricular excitation-contraction coupling (sodium, calcium, and potassium channels, Connexin 43, among others) were likewise downregulated. Pacemaker phenotype was characterized by upregulation of the pacemaker current channel (HCN4) but also mechnosensitive Piezo1, Trp channels Trpp2 (PKD2) and TrpM7, and Connexin45. Consistent with emergent pacemaker morphology, there was broad upregulation of cytoskeletal proteins and their small GTPase regulators. Extracellular matrix and growth factor/cytokine/interferon genes were also dynamically upregulated, indicating that Tbx18-iPMs are primed for sinoatrial node integration.
2022-08-26 | PXD013351 | Pride
Project description:We addressed the question of which protein kinases are expressed in cardiomyocytes. We assessed the changes during postnatal development, comparing profiles in rat neonatal ventricular cardiomyocytes (NVMs) with adult ventricular cardiomyocytes (AVMs). Neonatal and adult rat ventricular cardiomyocytes prepared according to established procedures (Marshall et al. PLoS ONE 2010 5(4):e10027; Fuller and Sugden, FEBs Lett. 1989 247:209-12; Rodrigues and Severson In Biochemical Techniques in the Heart (McNeill, J. H., Ed.) pp 101-115, CRC Press, New York.). mRNA expression profiles compared using Affymetrix rat genome 230 2.0 arrays.
2015-01-01 | E-MTAB-2832 | biostudies-arrayexpress
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Project description:Single cell RNA-seq analysis of TBX18-driven reprogramming of ventricular myocytes to cardiac pacemaker cells
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Project description:Study on changes in gene expression in primary cultures of neonatal rat ventricular cardiomyocytes to electric stimulation. Through comparing non-stimulated, stimulated and blebbistatin supplemented and stimulated cultures we set out to identify the genes that are specifically activated by electric pulsing separate from cardiomyocyte contractions. After initial recovery phase, primary cultures of neonatal rat ventricular cardiomyocytes were cultured for 3 days without pulsing, with electric pulsing or with electric pulsing combined with blebbistatin. Per treatment: 3 arrays, with samples obtained from 3 separate series of cardiomyocyte isolation and culturing. Per array: sample prepared from pooled (1:1) RNA from duplicate experiments.
2009-12-10 | E-GEOD-15856 | biostudies-arrayexpress
Project description:ChIP assay in adult ventricular cardiomyocytes
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2021-04-13 | GSE169580 | GEO
Project description:ChIP assay was performed to study the genomic location of SRF myocytes cistrome in adult ventricular cardiomyocytes. The myocytes were stimulated by phenylephrine (PE) combined with adenoviral overexpression of SRF serine-103 mutants.
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