Cardiomyocyte-specific TGFb1/2/3 deficiency impacts early heart maturation [snRNA-Seq].
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ABSTRACT:
ORGANISM(S): Mus musculus
PROVIDER: GSE307899 | GEO | 2026/09/10
REPOSITORIES: GEO
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Project description:Transforming growth factor β (TGFβ) is a secreted growth factor that, during development and injury, influences fibroblasts to differentiate and synthesize ECM and matricellular proteins. We observed that deletion of Tgfb1, Tgfb2, Tgfb3 genes (TGFβ ligands) from cardiomyocytes early during development results in cardiac dysfunction by 6 weeks of age with altered fibroblast activity and altered ECM content. The goal of this project was to elucidate the changes, at the transcriptional level, in cardiac fibroblasts from triple knockout (TKO) hearts lacking expression of TGFbeta1/2/3 in cardiac myocytes.
2026-09-10 | GSE298104 | GEO
Project description:Transforming growth factor β (TGFβ) is a secreted growth factor that, in early development, is produced by cardiac myocytes and impacts fibroblasts, driving differentiation and into a differentiated state to synthesize ECM and matricellular proteins. These, in turn, impact cardiomyocyte maturation through cell-cell interactions, ECM-cellular interactions, and paracrine factors. Deletion of Tgfb1, Tgfb2, Tgfb3 genes (TGFβ ligands) from cardiomyocytes during early development in a genetically modified mouse line was found to result in cardiac dysfunction by 6 weeks of age. This indicated a definitive role for TGFβ ligands produced by cardiac myocytes in cardiac development and function. The goal of this microarray project was to transcriptionally profile hearts with cardiomyocytes that were deficient in TGFβ ligand production. In order to transcriptionally profile hearts encompassing cardiomyocytes with deficient TGFb signaling, genetically-modified mouse lines were bred in which genes encoding the TGFb ligands could be knocked out specifically in cardiomyocytes in a Cre recombinase-dependent manner. This was achieved utilizing the aMHC-Cre transgenic line where Cre-recombinase is expressed exclusively in cardiomyocytes under the aMHC promoter [PMID: 9202069]. Whole hearts were harvested from 6-week old control [aMHC-Cre transgenic] and mutant [double knockouts (DKO) with TGFb 2 and 3 deleted and triple knockouts (TKO) with TGFb 1, 2 and 3 deleted] mice. Microarray analysis was performed on total RNA isolated from biological replicates of these hearts at the Gene Expression Core Facility (CCHMC) using the Affymetrix Cariom S platform. Differential gene expression was determined by bioinformatic analysis (CEL files) using Transcriptome Analysis Console 9.
2026-09-10 | GSE298103 | GEO
Project description:Transforming growth factor β (TGFβ) is a secreted growth factor that, in early development, transforms fibroblasts into a differentiated state to synthesize ECM and matricellular proteins. These, in turn, impact cardiomyocyte maturation through cell-cell interactions, ECM-cellular interactions, and paracrine factors. We observed that deletion of Tgfb1, Tgfb2, Tgfb3 genes (TGFβ ligands), together, from cardiomyocytes in the early developing heart results in cardiac dysfunction by 6 weeks of age with altered fibroblast activity and perturbed ECM content. Early postnatal hearts from Tgfb1/2/3 cardiomyocyte deleted mice are dysmorphic and cardiac fibroblasts have incorrect activity and produce inappropriate ECM with reduced stiffness, indicating effects during embryonic development itself. In order to tease out the effects of altered fibroblast programming on cardiomyocyte maturation even before birth, we generated transcriptional profiles of embryonic hearts from two models of dysregulated fibroblast programming; the first model was E17.5 embryonic hearts from our Tgfb1/2/3 cardiomyocyte deleted line and the second model was E17.5 embryonic hearts from the previously reported Tcf21-null line [PMID: 22573622], which lack cardiac fibroblasts. The rationale was that comparison of the transcriptional alterations in these two models (normalized to their respective controls) would allow us to generate a signature indicative of altered cardiomyocyte maturation resulting from dysregulated or deficient fibroblast programming during embryonic development.
2026-09-10 | GSE297874 | GEO
Project description:<p>Cardiac fibrosis, characterized by excessive extracellular matrix (ECM) deposition in the myocardium, is a critical target for heart disease treatments. Pw1 (paternally expressed gene 3) is an imprinted gene expressed from the paternally allele, and de novo purine biosynthesis (DNPB) is a crucial pathway for nucleotide synthesis. However, the roles of PW1 and DNPB in ECM production by cardiac fibroblasts during myocardial ischemia are not yet understood. To induce myocardial damage, we performed left anterior descending coronary artery ligation. We generated Pw1CreER-2A-eGFP and Pw12A-CreER knock-in mouse lines to evaluate the expression of the two Pw1 alleles in normal and injured hearts. Bisulfite sequencing was employed to analyze the DNA methylation of the Pw1 imprinting control region. We identified the phosphoribosylformylglycinamidine synthase (Pfas) gene, encoding the DNPB enzyme PFAS, as a direct target of PW1 using chromatin immunoprecipitation sequencing and real-time quantitative polymerase chain reaction. The role of DNPB in ECM production and cardiac fibrosis post-injury was examined in vitro using cultured cardiac fibroblasts and in vivo with Pfas-deficient mice. Our study demonstrates that myocardial infarction reduces DNA methylation at the imprinting control region of the maternally imprinted gene Pw1, triggering a switch from monoallelic imprinting to biallelic expression of Pw1 in cardiac fibroblasts. In activated cardiac fibroblasts, increased Pw1 expression promotes purine biosynthesis and induces ECM production by transcriptionally activating the DNPB factor Pfas. Notably, we identified that DNPB is essential for ECM production in activated fibroblasts and that loss of Pfas in fibroblasts limits cardiac fibrosis and improves heart function after heart injury. This study reveals that post-injury, the imprinting of Pw1 is disrupted, highlighting a novel role for the downstream DNPB pathway in cardiac fibrogenesis. Targeting DNPB presents a promising therapeutic strategy for improving cardiac repair following injury.</p>
2024-12-03 | MTBLS11364 | MetaboLights
Project description:miRNA pattern of cardic myocytes, cardiac fibroblasts and cardiac endothelial cells isolated from adult mice hearts at different time points after transaortic constriction (TAC) or sham surgery.
2020-07-01 | GSE66974 | GEO
Project description:Growth differentiating factor (GDF)15 is a TGFβ superfamily cytokine and a reported biomarker of heart failure. Myocardial expression of GDF15 is increased in heart failure. Yet, the mechanisms that control synthesis and release of GDF15 as well as the autocrine/paracrine functions of GDF15 on fibroblast function is lacking. Thus, the aim of this study was to investigate signaling pathways and functions of GDF15 in cardiac fibroblasts. Cardiac fibroblasts and cardiac myocytes were isolated from adult C57/BL6 mice, maintained in primary culture and stimulated with recombinant (r)GDF15 or recombinant (r)CCN2. Short-term stimulation (30 minutes) of cardiac fibroblasts demonstrated a GDF15-induced activation of several intracellular signaling pathways including a concentration-dependent increase of phospho-Smad3(Ser423/425) (p<0.05, n=3), phospho-AKT(Ser473) (p<0.05, n=3) and phospho-IκBα(Ser32/36) (p<0.05, n=3) levels. However, rGDF15 did not phosphorylate Smad3(Ser423/425) or IκBα(Ser32/36) in cardiac myocyte. Cardiac fibroblasts exposed to rGDF15 for 48 hours displayed differentiation towards myofibroblasts reflected by increased levels of the differentiation marker α-smooth muscle actin (SMA) similar to cardiac fibroblasts stimulated with TGFβ. The effect of GDF15 on α-SMA was dose-dependent ranging from 500 nM - 20 nM rGDF15 (p<0.05, n=3). Differentiation towards a myofibroblast phenotype in the presence of GDF15 was also supported by higher matrix metalloproteinase (MMP) enzyme activity in the cell culture medium (6±1 fold increase, n=3, p<0.05) and increased expression and release of MMP-3, 9 and 13. Immunoreactive GDF15 was predominantly found in cardiac myocytes. Recombinant CCN2 substantially induced GDF15 expression in cardiac myocytes, but not in cardiac fibroblasts. In conclusion, our data demonstrate that GDF15 is a paracrine factor in myocardial tissue and specifically regulated by CCN2 in cardiac myocytes. GDF15 has similar effects as TGFβ on fibroblasts by activation of intracellular signaling pathways and differentiation to a myofibroblasts phenotype.
2017-02-19 | GSE55252 | GEO
Project description:Activation of fibroblasts and formation of myofibroblasts are essential for granulation tissue formation following injury. In fibrotic reactions, excessive deposition of ECM by the activated fibroblasts determines scar formation and functional failure. Although these events critically depend on the activity of a plethora of growth factors and cytokines, TGFβ1 is a unique player controlling the immune response and proliferation of many cell types. Different cell types contribute to its release and activation, which is also regulated by the interaction with the ECM and by mechanical forces. The aim of this study was to elaborate whether fibroblast-derived TGFβ1 plays a critical role during these processes. The data demonstrate a dynamic expression of TGFβ1 during tissue repair. Cell-specific ablation of Tgfb1 in fibroblasts revealed that deletion of TGFβ1 attenuates bleomycin-induced skin fibrosis and delays maturation of granulation tissue in skin wounds. Absence of fibroblast-derived TGFβ1 induced vascular alterations (less vascular density and branching, hemorrhage) in early wound healing, potentially influenced by coincident alterations in the formation of stable ECM structure. This can be explained by paracrine regulation of endothelial cells or pericytes by fibroblast-released TGFβ1 and by impaired expression of pro-angiogenic factors in TGFβ1-deficient fibroblasts. Our findings provide novel mechanistic insights into the central role of fibroblast-derived TGFβ1 for early stages of tissue repair and fibrosis in the skin.
2025-07-14 | GSE298182 | GEO
Project description:Background: Fibrosis, defined by the excessive accumulation of extracellular matrix (ECM) proteins, including type I collagen, results in a stiff scar tissue that impairs organ function and can cause death. The effector cells of fibrosis are myofibroblasts, an activated form of fibroblast. In gingiva (gums), fibrotic responses are predominantly hyperproliferative (hyperplasia), where transforming growth factor (TGF)-β is overexpressed, and via canonical signaling pathway acts on gingival fibroblasts to induce fibroproliferative genes such as cellular communications network factor 2 (CCN2). Targeting of TGFβ for therapeutic gain however is challenging due its pleotropic nature, and therefore identifying new and targetable alternatives is warranted. We have shown that mechanical tension as a result of stiff ECM increases TGFβ-driven expression of CCN2 in gingival fibroblasts. We further identified yes-activated protein (YAP1), a mechanosensitive transcription (co)factor that translocate to nucleus in response to mechanical tension in gingival fibroblasts. To test if YAP is the perpetuator of TGFβ-mediated fibroproliferative gene induction, I used celastrol, a recently identified YAP/TAZ (transcriptional co-activator PDZ-binding motif) inhibitor, whose anti-fibrotic potential is not yet elucidated -- projecting it as a potential treatment option for gingival hyperplasia. Methods: Human gingival fibroblasts (HGFs) in culture were serum starved overnight and pretreated with 500nM Celastrol or DMSO for 45 minutes followed by stimulation with or without TGFβ1 for 6 hours (RT-PCR) and 24 hours (Western blot). mRNA samples collected at 6 hours were also analysed using BulkRNAsequencing and functional cluster analysis. Celastrol’s capacity to prevent the TGFβ induced proliferation of HGFs was investigated with BrdU cell proliferation assay. Celastrol’s ability to block TGFβ-mediated YAP nuclear localization in HGFs was assessed using immunofluorescent staining of YAP protein. Results: In response to added TGFβ1, the gene and protein expression of fibrotic marker and mediator CCN2 (a known YAP1 target) was significantly downregulated in the presence of Celastrol. Similarly, of the 1364 genes induced by TGFβ1 (>2-fold), 657 genes showed a >50% decrease in the presence of Celastrol including genes involved in fibrotic responses such as type 4 collagen, interleukin 11, etc., Functional cluster analysis revealed ECM, Wnt/cytokine signaling, focal adhesion clusters induced by added TGFβ1, were downregulated in a Celastrol sensitive fashion. Also, basal level proliferation of HGFs was repressed in the presence of Celastrol. Additionally, Celastrol also successfully blocked TGFβ-mediated nuclear translocation of YAP as seen with immunofluorescence microscopy. Conclusion: These results are consistent with our hypothesis that targeting YAP/TAZ may be useful in blocking the profibrotic effects of TGFβ and that celastrol or other YAP inhibitors are eligible candidates as a treatment option for gingival hyperplasia.
2024-12-04 | GSE249428 | GEO
Project description:Cells from three adult, wild-type, FVB hearts were separated into cardiomyocyte and nonmyocyte fractions using Langendorff perfusion, collagenase digestion and gravity filtration; total RNA was prepared immediately from myocytes, while nonmyocytes were passaged twice to yield a culture of largely fibroblasts from which total RNA was prepared. 6 cardiac polyadenylated RNA (mRNA and lncRNA) and small RNA (microRNA) profiles of isolated cardiomyocytes and fibroblasts from 12-wk FVB/NJ mouse hearts were generated on Illumina HiSeq 2000 instruments.
2014-06-14 | E-GEOD-58453 | biostudies-arrayexpress
Project description:Background: Fibrosis is a common pathology in many cardiac disorders and is driven by the activation of resident fibroblasts. The global post-transcriptional mechanisms underlying fibroblast-to-myofibroblast conversion in the heart have not been explored. Methods: Genome-wide changes of RNA transcription and translation during human cardiac fibroblast activation were monitored with RNA sequencing and ribosome profiling. We then used a RNA-binding protein-based analyses to identify translational regulators of fibrogenic genes. The integration with cardiac ribosome occupancy levels of 30 dilated cardiomyopathy patients demonstrates that these post-transcriptional mechanisms are also active in the diseased fibrotic human heart. Results: We generated nucleotide-resolution translatome data during the TGFβ1-driven cellular transition of human cardiac fibroblasts to myofibroblasts. This identified dynamic changes of RNA transcription and translation at several time points during the fibrotic response, revealing transient and early-responder genes. Remarkably, about one-third of all changes in gene expression in activated fibroblasts are subject to translational regulation and dynamic variation in ribosome occupancy affects protein abundance independent of RNA levels. Targets of RNA-binding proteins were strongly enriched in post-transcriptionally regulated genes, suggesting genes such as MBNL2 can act as translational activators or repressors. Ribosome occupancy in the hearts of patients with dilated cardiomyopathy suggested the same post-transcriptional regulatory network was underlying cardiac fibrosis. Key network hubs include RNA-binding proteins such as PUM2 and QKI that work in concert to regulate the translation of target transcripts in human diseased hearts. Furthermore, silencing of both PUM2 and QKI inhibits the transition of fibroblasts toward pro-fibrotic myofibroblasts in response to TGFβ1. Conclusions: We reveal widespread translational effects of TGFβ1 and define novel post-transcriptional regulatory networks that control the fibroblast-to-myofibroblast transition. These networks are active in human heart disease and silencing of hub genes limits fibroblast activation. Our findings show the central importance of translational control in fibrosis and highlight novel pathogenic mechanisms in heart failure.
2019-10-03 | GSE131111 | GEO