Rhein, a novel Histone Deacetylase (HDAC) inhibitor with antifibrotic potency in human myocardial fibrosis

Abstract

Although fibrosis depicts a reparative mechanism, maladaptation of the heart due to excessive production of extracellular matrix accelerates cardiac dysfunction. The anthraquinone Rhein was examined for its anti-fibrotic potency to mitigate cardiac fibroblast-to-myofibroblast transition (FMT). 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 during hypoxia, whereas p53 and p21 were identified as upstream regulators involved in the manifestation of cardiac fibroblast phenotypes. Mechanistically, Rhein acts inhibitory on HDAC classes I/II as enzymatic inhibitor. Rhein-mediated cellular effects were linked to the histone deacetylase (HDAC)-dependent protein stabilization of p53 under normoxic but not hypoxic conditions. Functionally, Rhein inhibited collagen contraction, indicating anti-fibrotic property in cardiac remodeling. This was accompanied by increased abundance of SMAD7, but not SMAD2/3, and consistently SMAD-specific E3 ubiquitin ligase SMURF2. In conclusion, this study identifies Rhein as a novel potent direct HDAC inhibitor that 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.

Figure 1

Rhein reduces αSMA protein abundance independently from oxygen. (A) Experimental setup of hypoxia treatment. HCF-v were adhered for 16 h, grown for 60 h under normoxic (21% O₂) or hypoxic (0.5% O₂) conditions and subsequently serum-starved for 36 h. With every change of the media (every 30 h), respective cells were treated with 35 µM Rhein throughout the duration of the experiment (96 h). (B) Rhein does not affect HIF1α stabilization. Representative blot and quantification of HIF1α abundance (n = 4). (C) mRNA expression of the direct HIF1α target Glut1. (D) Rhein-mediated αSMA protein reduction. Representative Western blot and quantification of relative protein abundance (n = 4). Antibodies in (B,D) were probed to the same membrane. All data are presented as mean ± SD. One-way-ANOVA with post-hoc Sidak’s multiple comparison, *p < 0.05, **p < 0.01, ***p < 0.001, as indicated.

Figure 2

Effect of Rhein on the secretome of cardiac fibroblast under normoxic and hypoxic environments. (A) Graph showing the global top 15 annotated canonical pathways of differential secretomes sorted by cumulative −Log₁₀ p-value. (B) Graph showing the global top 15 predicted upstream regulators of differential secretomes sorted by cumulative −Log₁₀ p-value. The graphs were generated through the use of IPA (QIAGEN Inc., https://www.qiagenbioinformatics.com/products/ingenuity-pathway-analysis).

Figure 3

Influence of Rhein administration on transcriptomic profiles in cardiac fibroblasts under normoxic and hypoxic conditions. (A) Graph showing the global top 15 canonical pathway annotations of differential transcripts sorted by cumulative −Log₁₀ p-value. (B) Graph showing the global top 15 predicted upstream regulators of differential transcripts sorted by cumulative −Log₁₀ p-value. N: Normoxia, H: Hypoxia, NR: Normoxia + Rhein, HR: Hypoxia + Rhein.

Figure 4

Effect of Rhein on HDAC activity and G2/M cell cycle phase regulation, p53 and p21 abundance and proliferation markers. (A) Representative blots and quantification showing Rhein-mediated increase of p53 and p21 abundance (n = 4). (B) Representative blots and quantification showing Rhein-driven p53 stabilization by increased HDAC inhibition-mediated acetylation at Lys382 (n = 4). 1 mM sodium butyrate (SB) was used as reference HDAC inhibitor. (C) Representative blots and quantification showing Rhein-mediated increase of p53 abundance (n = 4) under conditions described in (B). (D) Representative blots and quantification showing Rhein-mediated increase of p21 abundance (n = 4) under conditions described in (B). Antibodies in (C,D) were probed to the same membrane. (E) Enzymatic HDAC activity is inhibited in the presence of Rhein. Graph showing HDAC activity after 30 min treatment of normal cell lysates with Rhein or SB (n = 4). All data are presented as mean ± SD. One-way-ANOVA with post-hoc Sidak’s multiple comparison, *p < 0.05, **p < 0.01, ***p < 0.001, as indicated.

Figure 5

HDAC3/5 regulated downstream targets in transcriptome analyses. Rhein mediated effects were analyzed in the comparison groups NvsNR and HvsHR. Fold changes of up- and down-regulated genes are scaled in shades of red (higher expression in first condition of comparison) and green (lower in first condition), respectively. Blue color of upstream target indicates predicted inhibition. Orange and blue arrows indicate indirect activation and inhibition; yellow and gray arrows represent inconsistent effects and no prediction, respectively. The networks were generated through the use of IPA (QIAGEN Inc., https://www.qiagenbioinformatics.com/products/ingenuity-pathway-analysis).

Figure 6

Rhein functionally inhibits TGFβ1-stimulated FMT. (A) Representative Western Blot and quantification of αSMA relative protein abundance (n = 4). (B) Rhein inhibition of TGFβ1/SMAD signaling. Representative blot and quantification of phospho(Ser465/467)-SMAD2 abundance in Rhein and TGFβ1 treated cells. (C) Expression analysis of SMAD7 showing TGFβ1-mediated increase of transcription and absence of Rhein-mediated effects (n = 4). (D) Rhein increases SMAD7 abundance independently from TGFβ1. Representative blot and quantification showing increased basal SMAD7 abundance after Rhein treatment and TGFβ1-dependent SMAD7 expression (n = 4). (E) Effect of HDAC inhibition on SMAD7 stabilization. Representative blot and quantification showing increased protein abundance after Rhein and SB treatment (N = 4). (F) FPCL contraction assay. Quantitative analysis on the contraction of experimental FPCLs (n = 4). Representative overhead pictures of FPCLs before (d0) and after treatment (d0) with TGFβ1 alone or in combination with Rhein. Dashed line indicates baseline (start point at d0). All data are presented as mean ± SD. One-way-ANOVA with post-hoc Sidak’s multiple comparison, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 as indicated.