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.
Project description:Histone deacetylases (HDAC) are epigenetic regulators of trascription. Preclinical studies have determined that HDAC inhibition can prevent kidney interstitial fibrosis in acute kidney injury. The purpose of this study was to determine if overexpression of HDAC1 in kidney fibroblasts significantly affects the fibroblast transcriptome. Transforming growth factor beta-1 (TGFB1) causes fibroblasts to differentiate into myofibroblasts leading to extracelluar matrix secretion. A subset of our cells were treated with TGFB1 for 24 hours to determine if this in combination with HDAC1 overexpression significantly alters the transcriptomes.
Project description:Deletion of Tmem43 encoding nuclear membrane protein TMEM43, specifically in mouse cardiac myocytes leads to cardiac fibrosis and dysfunction. Temporal RNA-seq identified early and late trascriptomic changes and implicates TGFb1 in the pathogenesis of ACM caused by Tmem43 haploinsufficiency.
Project description:To examine whether commonly used human cultured fibroblast systems model human cardiac fibroblasts, we tested three different fibroblast cell preparations: primary human cardiac fibroblasts (NHCF), human dermal fibroblasts (NHDF), and immortalized human cardiac fibroblasts (iHCF).
Project description:Hematopoietic mutations in epigenetic regulators like DNA methyltransferase 3 alpha (DNMT3A) drive clonal hematopoiesis of indeterminate potential (CHIP) and are associated with adverse prognosis in patients with heart failure (HF). The interactions between CHIP-mutated cells and other cardiac cell types remain unknown. Here, we identify fibroblasts as potential interaction partners of CHIP-mutated monocytes using combined transcriptomic data from peripheral blood mononuclear cells of HF patients with and without CHIP and cardiac tissue. We demonstrate that DNMT3A inactivation augments macrophage-to-cardiac fibroblasts interactions and induces cardiac fibrosis in mice and humans. Mechanistically, DNMT3A inactivation increases the release of heparin-binding epidermal growth factor (EGF)-like growth factor (HB-EGF) to activate cardiac fibroblasts. These findings not only identify a novel pathway of DNMT3A CHIP-driver mutation-induced instigation and progression of HF, but may also provide a rationale for the development of new anti-fibrotic strategies.
Project description:Study of the gene expression patterns of specific cardiac cell populations, including monocytes (CD45+, CD11b+, F4/80-, LyC6+), endothelial cells (CD45-, CD31+), macrophages (CD45+, CD11b+, F4/80+, LyC6-), and fibroblasts (CD45-, GP38+), in response to at-RA and 4-oxo-RA treatments. Female C57BL/6J mice aged 12 weeks were intraperitoneally injected with 30 mg/kg body weight at-RA (Sigma-Aldrich), 30 mg/kg body weight 4-oxo-RA (Sigma-Aldrich), or an equivalent volume of DMSO in phosphate-buffered saline (PBS) as the vehicle control. After 24 hours of injection, the mice were euthanized for analysis.
