A high-throughput drug screening identifies luteolin as a therapeutic candidate for pathological cardiac hypertrophy and heart failure
. 2023 Mar 14;10:1130635.
doi: 10.3389/fcvm.2023.1130635.
eCollection 2023.
Affiliations
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Front Cardiovasc Med.
.
Abstract
Background:
Pathological cardiac hypertrophy is commonly resulted from sustained pressure overload and/or metabolic disorder and eventually leads to heart failure, lacking specific drugs in clinic. Here, we aimed to identify promising anti-hypertrophic drug(s) for heart failure and related metabolic disorders by using a luciferase reporter-based high-throughput screening.
Methods:
A screen of the FDA-approved compounds based on luciferase reporter was performed, with identified luteolin as a promising anti-hypertrophic drug. We systematically examined the therapeutic efficacy of luteolin on cardiac hypertrophy and heart failure in vitro and in vivo models. Transcriptome examination was performed to probe the molecular mechanisms of luteolin.
Results:
Among 2,570 compounds in the library, luteolin emerged as the most robust candidate against cardiomyocyte hypertrophy. Luteolin dose-dependently blocked phenylephrine-induced cardiomyocyte hypertrophy and showed extensive cardioprotective roles in cardiomyocytes as evidenced by transcriptomics. More importantly, gastric administration of luteolin effectively ameliorated pathological cardiac hypertrophy, fibrosis, metabolic disorder, and heart failure in mice. Cross analysis of large-scale transcriptomics and drug-target interacting investigations indicated that peroxisome proliferator activated receptor γ (PPARγ) was the direct target of luteolin in the setting of pathological cardiac hypertrophy and metabolic disorders. Luteolin can directly interact with PPARγ to inhibit its ubiquitination and subsequent proteasomal degradation. Furthermore, PPARγ inhibitor and PPARγ knockdown both prevented the protective effect of luteolin against phenylephrine-induced cardiomyocyte hypertrophy in vitro.
Conclusion:
Our data clearly supported that luteolin is a promising therapeutic compound for pathological cardiac hypertrophy and heart failure by directly targeting ubiquitin-proteasomal degradation of PPARγ and the related metabolic homeostasis.
Keywords:
cardiac hypertrophy; fatty acid metabolism; glucose metabolism; heart failure; luteolin; peroxisome proliferator activated receptor γ.
© 2023 Wang, Shi, Wu, Peng, Wang, Liu, Yang, Wang, Li, Tian, Hong, Yang, Bai, Hu, Cheng, Li, Zhang and She.
Conflict of interest statement
The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.
Figures
Figure 1
Luteolin emerged as a therapeutic candidate for pathological cardiac hypertrophy in the FDA-approved chemical compounds screen. (A) Schematic illustration of the experimental workflow of the luciferase-based FDA-approved chemical compounds screen. (B) The scatter plot demonstrating Myh7 and Bnp luciferase activity in HEK 293 T cells treated with FDA-approved compounds. Red dots represent each FDA drug that inhibited both Myh7 and Bnp luciferase reporter activities for more than 50%. (C) The advancement criterion for screening out the most effective compound on Myh7 and Bnp luciferase activity downregulation and cardiomyocyte hypertrophy inhibition. (D) Representative immunofluorescence images (left) of α-actinin staining and quantitative results of the cell surface area (right) of NRCMs treated with PBS, PE (50 μM), PE + entinostat, PE + belinostat, or PE + luteolin for 24 h (n ≥ 50 cells per group). Scale bar, 50 μm. The data shown are representative of three independent experiments. Values are presented as mean ± SD. *P < 0.05, **P < 0.01, n.s., no significant difference. FDA, the United States Food and Drug Administration; NRCMs, Primary neonatal rat cardiomyocytes; HEK 293 T cells, human embryonic kidney 293 T cells; luc, luciferase; Bnp, b-type natriuretic peptide; Myh7, myosin heavy chain 7; PE, phenylephrine.
Figure 2
Luteolin ameliorates PE-induced cardiomyocyte hypertrophy in primary cardiomyocytes. (A) Relative cell viability of NRCMs after treatment with different concentrations of luteolin. The data shown are representative of three independent experiments. n.s., no significant difference compared to the 0 μM group. (B) Representative immunofluorescence images (left) of α-actinin staining and quantitative results of the cell surface area (right) of NRCMs treated with PBS, PE (50 μM), or PE + luteolin (5 or 10 μM) for 24 h (n ≥ 50 cells per group). Scale bar, 50 μm. The data shown are representative of three independent experiments. (C) Relative mRNA levels of cardiac hypertrophy marker genes (Anp, Bnp, and Myh7) in NRCMs treated with PBS, PE (50 μM), or PE + luteolin (5 or 10 μM) for 24 h (n = 5 independent experiments). (D) Principal component analysis showing the global sample distribution profiles between groups based on the RNA-sequencing data. (E) Volcano plot analysis showing a huge number of differentially expressed genes between the two groups. Genes with adjusted P-values less than 0.05 and a fold change larger than 1.5 was recognized as differentially expressed genes. (F) Gene set enrichment analysis of molecular events involved in cardiac hypertrophy, fibrosis, and protein synthesis in RNA-sequencing data. (G) Heatmap showing the significantly altered genes related to cardiac hypertrophy. Values are presented as mean ± SD. *P < 0.05, **P < 0.01, n.s., no significant difference. PE, phenylephrine; Anp, atrial natriuretic peptide; Bnp, b-type natriuretic peptide; Myh7, myosin heavy chain 7.
Figure 3
Luteolin inhibits cardiac dysfunction induced by pressure overload in mice. (A) Schematic diagram of the experimental procedure. (B) Representative echocardiography images of mice measured at 12 weeks after TAC. (C) Assessments of echocardiographic parameters of left ventricular end-diastolic diameter (LVEDd), left ventricular end-systolic diameter (LVESd), ejection fractions (EF), and fraction shortening (FS) of mice at 4 weeks after sham or TAC surgery (n = 8). (D) Heart weight (HW), HW/body weight (BW), lung weight (LW)/BW, and HW/tibia length (TL) ratios of mice at 12 weeks after sham or TAC surgery (n = 8). (E,F) Assessments of echocardiographic parameters of EF, FS, stroke volume, cardiac output, LVEDd, LVESd, left ventricular end-diastolic volume (LVEDV), and left ventricular end-systolic volume (LVESV) of mice at 12 weeks after sham or TAC surgery (n = 8). Values are presented as mean ± SD. *P < 0.05, **P < 0.01.
Figure 4
Luteolin prevents cardiac hypertrophy and fibrosis in mice. (A) Representative images of hematoxylin-eosin (H&E) staining of left ventricular cross-sections in the mice hearts at 12 weeks after sham or TAC surgery (n = 6). Scale bar, 1 mm for the top set and 25 μm for the bottom parts. (B) Quantitative results of average cross-sectional areas from the indicated groups. (C) Representative images of picrosirius red (PSR) staining of left ventricular cross-sections in the mice hearts at 12 weeks after sham or TAC surgery (n = 6). Scale bar, 50 μm. (D) Quantitative results of left ventricular interstitial collagen volume from the indicated groups. (E,F) Relative mRNA levels of hypertrophy and fibrosis marker genes in heart tissues from the indicated mice (n = 5). (G) Immunoblotting (left) and quantitation (right) of ANP, BNP, and MYH7 protein levels in the mice hearts at 12 weeks after sham or TAC surgery (n = 3). Values are presented as mean ± SD. *P < 0.05, **P < 0.01. ANP, atrial natriuretic peptide; BNP, b-type natriuretic peptide; PE, phenylephrine; MYH7, myosin heavy chain 7.
Figure 5
Luteolin enhances fatty acid metabolism and decreases glucose metabolism in the mouse failing hearts. (A) Relative mRNA levels of PPARγ coactivator-1α and PPARγ coactivator-1β in the mice hearts at 12 weeks after sham or TAC surgery (n = 5). (B,C) Relative mRNA levels of genes associated with fatty acid uptake (B) and fatty acid oxidation (C) in the mice hearts at 12 weeks after sham or TAC surgery (n = 5). (D) Relative mRNA levels of genes associated with glucose metabolism in the mice hearts at 12 weeks after sham or TAC surgery (n = 5). Values are presented as mean ± SD. *P < 0.05, **P < 0.01. Pgc-1α/β, peroxisome proliferative activated receptor-gamma coactivator-1α/β; Fabp3/4, fatty acid binding protein 3/4; Cpt1b/2, carnitine palmitoyltransferase 1b/2; Mcad, medium-chain acyl-CoA dehydrogenase; Lcad, long-chain acyl-CoA dehydrogenase; Atgl, adipose triglyceride lipase; Glut1, glucose transporter 1; Hif-1α, hypoxia-inducible factor 1α; PPARγ, peroxisome proliferator activated receptor γ; Ldha, lactate dehydrogenase A; Pkm2, pyruvate kinase M2.
Figure 6
Luteolin directly binds to and activates PPARγ during cardiac hypertrophy and HF. (A) Principal component analysis showing the global sample distribution profiles between groups based on the RNA-sequencing data. (B) Gene set enrichment analysis of molecular events involved in cardiac hypertrophy, fibrosis, and protein synthesis in RNA-sequencing data. (C) Heatmap showing the significantly altered genes related to cardiac hypertrophy. (D) Schematic diagram of the conjoint analysis. (E) Kyoto Encyclopedia of Genes and Genomes pathway enrichment analysis of the identified differentially expressed genes. (F) Individual GSEA (gene set enrichment analysis) plots of PPAR signaling pathway. (G) Schematic diagram of luteolin-protein binding from protein data bank (PDB). (H) Biotinylated protein interaction pull-down assays showing the binding of luteolin and HA-tagged PPARγ protein in HEK 293 T cells. The data shown are representative of three independent experiments. (I) PPARγ-induced PPRE luciferase activity in the treatment of luteolin at three doses (5 μM, 10 μM, and 20 μM) in HEK 293 T cells. The data shown are representative of three independent experiments. Values are presented as mean ± SD. **P < 0.01. PPARγ, peroxisome proliferator activated receptor γ; PPRE, PPAR response element.
Figure 7
Luteolin inhibits cardiac hypertrophy in a PPARγ-dependent manner. (A) Representative immunofluorescence images of α-actinin staining of NRCMs treated with PBS, PE (50 μM), PE + luteolin (10 μM), or PE + luteolin + GW9662 (20 μM) for 24 h (n ≥ 50 cells per group). Scale bar, 50 μm. (B) Quantitative results of the cell surface area of NRCMs from the indicated groups. The data shown are representative of three independent experiments. (C,D) Immunoblotting analysis (C) and quantitative results (D) of ANP and BNP in cultured NRCMs treated with vehicle (PBS), PE (50 μM), PE + luteolin (10 μM), or PE + luteolin + GW9662 (20 μM) for 24 h. The data shown are representative of three independent experiments. (E) Immunoblotting analysis (top) and quantitative results (bottom) of PPARγ in cultured WT and PPARγ knockdown H9C2. (F,G) Immunoblotting analysis (F) and quantitative results (G) of ANP and BNP in cultured cultured WT and PPARγ knockdown H9C2 treated with vehicle (PBS), PE (50 μM), and PE + luteolin (10 μM) for 24 h. The data shown are representative of three independent experiments. Values are presented as mean ± SD. *P < 0.05, **P < 0.01, n.s., no significant difference. PPARγ, peroxisome proliferator activated receptor γ; NRCM, primary neonatal rat cardiomyocyte; ANP, atrial natriuretic peptide; BNP, b-type natriuretic peptide; PE, phenylephrine; WT, wild-type.
Figure 8
Luteolin elevates the stability of PPARγ via inhibiting PPARγ ubiquitination. (A) Relative mRNA levels of PPARγ in the mice hearts at 12 weeks after sham or TAC surgery (n = 5). Values are presented as mean ± SD. n.s., no significant difference. (B) Immunoblotting analysis (left) and quantitative results (right) of PPARγ in the mice hearts at 12 weeks after sham or TAC surgery (n = 5). Values are presented as mean ± SD. **P < 0.01, n.s., no significant difference. (C) Immunoblotting analysis (left) and quantitative results (right) of PPARγ protein in NRCMs exposed to CHX (100 μM) for the indicated time with or without the luteolin treatment. The data shown are representative of three independent experiments. Values are presented as mean ± SD. *P < 0.05 compared to the control group. #P < 0.05 compared to the PE group. (D) PPARγ protein levels in NRCMs exposed to MG132 (10 μM) or Chlq (25 μM) in the presence of CHX (100 μM) for 4 h. The data shown are representative of three independent experiments. (E) The ubiquitination levels of PPARγ in HEK 293 T cells transfected with HA-tagged PPARγ and Myc-tagged Ub exposed to DMSO or luteolin (10 μM) in the presence of MG132 (10 μM). The data shown are representative of three independent experiments. (F) IP analyses of the interaction between TRIM55 and PPARγ in HEK 293 T cells transfected indicated plasmids exposed to DMSO or luteolin (10 μM). The data shown are representative of three independent experiments. (G) PPARγ protein levels in HEK 293 T cells transfected with HA-tagged PPARγ and Flag-tagged TRIM55 exposed to DMSO or luteolin (10 μM) in the presence of CHX (100 μM) for 12 h. The data shown are representative of three independent experiments. (H) The ubiquitination levels of PPARγ in HEK 293 T cells transfected with HA-tagged PPARγ, Flag-tagged TRIM55, and Myc-tagged Ub exposed to DMSO or luteolin (10 μM) in the presence of MG132 (10 μM). The data shown are representative of three independent experiments. (I) Schematic illustrating the model that luteolin is a promising therapeutic compound for pathological cardiac hypertrophy and heart failure by directly targeting PPARγ ubiquitin-proteasomal degradation and metabolic homeostasis. PPARγ, peroxisome proliferator activated receptor γ; DMSO, dimethyl sulfoxide; CHX, cycloheximide; Ub, ubiquitin; PE, phenylephrine; TRIM55, tripartite motif containing 55; NRCM, primary neonatal rat cardiomyocyte; PPRE, PPAR response element.
References
Grant support
This work was supported by grants from the National Science Foundation of China (81970364, 82270390, 82170595 and 81970070), the Hubei Province Innovation Platform Construction Project (20204201117303072238), and the Hubei Provincial Engineering Research Center of Comprehensive Care for Heart-Brain Diseases.