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

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. HCF-v culture and chronic hypoxia treatment Primary HCF-v of 5 different donors were purchased (Lonza, Basel, Switzerland; Promocell, Heidelberg, Germany) and cultured in FGMTM-3 medium (Lonza, Basel, Switzerland) (37°C, 5% CO2). Prior treatment HCF-v were seeded in 145mm dishes. After 16h, cells were subjected to the hypoxic workstation (Xvivo System (Biospherix, Parish, NY, USA) set to 0.5% O2, 5% CO2, 94.5% N2 and 37°C) with N2-pre-gassed DMEM (10% FCS, 1% glutamine, 1% antibiotic/antimycotic). After 60h, sub-confluent cells were washed (1x PBS) and serum-starved nitrogenated serum-free media (DMEM, 1% glutamine, 1% antibiotic/antimycotic) for 36h. Normoxic control cells were treated in parallel in a second incubator of the hypoxic workstation (21% O2, 74% N2, 5% CO2 and 37°C). For Rhein treatment, 35µM Rhein was added during normoxic or hypoxic treatment for a total incubation time of 96h.