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Rhein, a novel Histone Deacetylase (HDAC) inhibitor with antifibrotic potency

ABSTRACT: Rationale: Myocardial fibrosis manifests progressively in several forms of cardiomyopathies. Although fibrosis depicts a reparative mechanism, maladaptation of the heart due to excessive production of extracellular matrix accelerates cardiac dysfunction. Objective: The anthraquinone Rhein, a compound from rhubarb, was examined for its anti-fibrotic potency to mitigate cardiac fibroblast-to-myofibroblast transition (FMT). Methods and Results: Primary human ventricular cardiac fibroblasts were subjected to hypoxia and characterized with proteomics, transcriptomics and cell functional techniques. Knowledge based analyses of the omics data revealed a modulation of fibrosis-associated pathways and cell cycle due to Rhein administration, whereas p53 and p21 were identified as upstream regulators involved in the manifestation of cardiac fibroblast phenotypes. Mechanistically, Rhein-mediated cellular effects were linked to the histone deacetylase (HDAC)-dependent acetylation status of p53 a posttranslational modification that acts protein stabilizing. Direct enzymatic testing revealed an inhibitory potency of Rhein for HDAC classes I/II. Functionally, Rhein inhibited collagen contraction in response to protein abundance of SMAD7, endogenous inhibitor of TGFβ1 action, thus demonstrating its anti-fibrotic property in cardiac remodeling. Conclusion: In conclusion, this study identifies Rhein as a novel potent HDAC inhibitor and provides evidence that Rhein may contribute to the treatment of cardiac fibrosis as anti-fibrotic agent. As readily available drug with approved safety, Rhein constitutes a promising potential therapeutic approach in the supplemental and protective intervention of cardiac fibrosis.

OTHER RELATED OMICS DATASETS IN: PXD016731

ORGANISM(S): Homo sapiens

PROVIDER: GSE136039 | GEO | 2020/04/17

REPOSITORIES: GEO

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Project description:Cardiomyopathy is characterized by the deposition of extracellular matrix by activated resident cardiac fibroblasts, called myofibroblasts. A current lack of therapeutic approaches to blunt the development of pathological fibrosis and ventricle chamber stiffening that ultimately leads to heart failure necessitates the investigation of the mechanisms that underlie myofibroblast activation. We previously observed that increased degradation of p53 occurs during the cardiac fibroblast expansion phase that acutely follows pressure overload. We created tissue-specific mouse models to delete p53 in quiescent resident cardiac fibroblasts and in activated myofibroblasts, then evaluated consequences to cardiac physiology, cellular biology, and fibrotic remodeling. We then performed RNA sequencing to further elucidate the effects of p53 deletion in cardiac fibroblasts on gene expression at the single cell level. We confirmed these results with a series of in vitro experiments in primary mouse cardiac fibroblasts. Results - Here, we demonstrate that p53 knockout in resident cardiac fibroblasts significantly accelerates proliferation and temporarily insulates the heart from fibrotic remodeling and functional decline in response to pressure overload, but ultimately results in a more deleterious phenotype than in wildtype animals. Conversely, p53 knockout in activated myofibroblasts does not result in a delay in functional loss, and is uniformly deleterious to the heart. Single-cell RNA sequencing revealed that p53 knockout is associated with the development of constitutively mitotic fibroblasts, and increased recruitment of fibroblasts into a main activation trajectory that involves four states: quiescent, early transitional, late transitional, and fully activated. A quiescent, functionally specialized vascular support fibroblast population that is ubiquitous in the wildtype animal is additionally recruited into activation in the p53 knockout condition, resulting in the eventual extreme functional decline seen in these animals. In vitro experimentation using viral deletion of p53 supports these findings - quiescent fibroblasts lacking p53 are resistant to activation, but activated myofibroblasts lacking p53 do not reverse their activation. This was found to be due to parallel hyperactivation of Cdkn2a/Rb1-mediated G1 cell cycle arrest in the p53 knockout condition, which was supported by in vivo studies. Conclusions - These data establish p53 as a major regulator of the myofibroblast activation response to pressure overload. Moreover, the single-cell studies establish an activation trajectory for resident cardiac fibroblasts, and elucidate the fundamental importance of clinical intervention prior to irreversible myofibroblast activation seen in chronic stages of pressure overload.

Project description:Aims and introduction: Cardiac fibrosis is the hallmark of cardiac disease, particularly myocardial infarction (MI), and is one of the most prevalent causes of heart failure. MI is intricately linked to inflammation, extracellular matrix (ECM) remodelling, and cardiac fibrosis, however the mechanisms underpinning these events remain poorly understood. We recently identified that ECM1 has potential to play a key role in cardiac wound healing but the cellular origins in the heart and specific mechanisms of ECM1 in fibrosis remain elusive. Therefore, we investigated the spatiotemporal, cell specific, expression profile of ECM1 in the healthy and diseased mouse and human heart and investigate ECM1 dependent human cardiac fibroblast (HuCFb) cell signalling mechanisms. Methods and results: Western blotting, immunohistochemistry (IHC) and mRNA in-situ hybridisation revealed that ECM1 protein expression was significantly upregulated in human ischaemic and dilated heart failure patients, and predominantly localised interstitially to fibrotic, inflammatory, and peri-vascular areas. ECM1 specific analysis of the Litviňuková et al. (2020) single-cell and -nuclei RNA sequencing (sc/snRNAseq) dataset of healthy human hearts, and the Farbehi et al. (2019) scRNAseq dataset of mouse hearts post-MI demonstrated that ECM1 expression originates from fibroblasts, macrophages, and pericytes/vascular mural cells in the heart. Further, we identify that post-MI ECM1 is expressed in a temporal dynamic flux from unactivated fibroblasts in sham-operated mice, to M1 macrophages (M1MΦ) at day-3, and myofibroblasts and M2MΦ at day-7. Phosphoproteomics of ECM1 treated human cardiac fibroblast cells (HuCFb) alongside ECM1 to HuCFb cell surface protein binding pull-down and mass spectrometry, revealed that ECM1 signalling goes via proteins associated with Rho protein signal transduction, cell-cell adhesion, microtubule dynamics, and cell chemotaxis pathways, potentially initiated by ECM1 to CTNND1 binding on the cell surface. Further mechanistic analyses identified that ECM1 inhibits HuCFb cell migration and stimulates inflammatory (IL-6 and IL-1β mRNA expression), fibrotic (TGF-β1, TGF-β2 and Col1a2 mRNA expression), and non-canonical Wnt signalling pathways (Wnt5a mRNA expression and JNK1/2/3 and MYPT1 phosphorylation). Conclusions: This study demonstrates that ECM1 expression originates from macrophages and fibroblasts in the mouse and human heart. ECM1 has significant effects on HuCFb migration, inflammatory, fibrotic, Rho protein, and Wnt5a/non-canonical Wnt signalling pathways. Together, ECM1 plays an important role in cardiac wound healing and may represent a novel mechanism of inflammation-fibrosis crosstalk and progression post-MI.

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Rhein, a novel Histone Deacetylase (HDAC) inhibitor with antifibrotic potency

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