Diabetes Metab J.  2024 Jul;48(4):716-729. 10.4093/dmj.2023.0031.

Diabetes Promotes Myocardial Fibrosis via AMPK/EZH2/PPAR-γ Signaling Pathway

• Li SS
• Pan L
• Zhang ZY
• Zhou MD
• Chen XF
• Qian LL
• Dai M
• Lu J
• Yu ZM
• Dang S
• Wang RX

• KMID: 2558039
• DOI: http://doi.org/10.4093/dmj.2023.0031

Abstract

Background
Diabetes-induced cardiac fibrosis is one of the main mechanisms of diabetic cardiomyopathy. As a common histone methyltransferase, enhancer of zeste homolog 2 (EZH2) has been implicated in fibrosis progression in multiple organs. However, the mechanism of EZH2 in diabetic myocardial fibrosis has not been clarified.
Methods
In the current study, rat and mouse diabetic model were established, the left ventricular function of rat and mouse were evaluated by echocardiography and the fibrosis of rat ventricle was evaluated by Masson staining. Primary rat ventricular fibroblasts were cultured and stimulated with high glucose (HG) in vitro. The expression of histone H3 lysine 27 (H3K27) trimethylation, EZH2, and myocardial fibrosis proteins were assayed.
Results
In STZ-induced diabetic ventricular tissues and HG-induced primary ventricular fibroblasts in vitro, H3K27 trimethylation was increased and the phosphorylation of EZH2 was reduced. Inhibition of EZH2 with GSK126 suppressed the activation, differentiation, and migration of cardiac fibroblasts as well as the overexpression of the fibrotic proteins induced by HG. Mechanical study demonstrated that HG reduced phosphorylation of EZH2 on Thr311 by inactivating AMP-activated protein kinase (AMPK), which transcriptionally inhibited peroxisome proliferator-activated receptor γ (PPAR-γ) expression to promote the fibroblasts activation and differentiation.
Conclusion
Our data revealed an AMPK/EZH2/PPAR-γ signal pathway is involved in HG-induced cardiac fibrosis.

Keyword

AMP-activated protein kinases; Diabetic cardiomyopathies; Enhancer of zeste homolog 2 protein; PPAR gamma

Figure

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  Fig. 1. Histone H3 lysine 27 trimethylation (H3K27me3) was up-regulated in left ventricle of streptozotocin (STZ)-induced diabetic rats and mice. (A) Left ventricular ejection fraction (LVEF) and fractional shortening (FS) were measured by echocardiography in the diabetic and non-diabetic group rats (n=4 in each group). (B) Mason staining of ventricular tissues obtained from nondiabetic and diabetic rats (scale bar 300 μm). (C) Protein levels of extracellular matrix-related genes and α-smooth muscle actin (α-SMA) were evaluated by Western blot in rat ventricular tissues (n=3 in each group). (D) Western blot analysis of the expression of phospho-enhancer of zeste homolog 2 (p-EZH2), EZH2, and H3K27me3 in ventricular tissues from non-diabetic and diabetic rats (n=3 in each group). (E) EZH2 mRNA levels were measured in non-diabetic and diabetic rat ventricular tissues (n=4 in each group). (F) LVEF and FS were measured by echocardiography in the diabetic and non-diabetic group mice (n=6 in each group). (G) Western blot analysis of the expression of EZH2 and H3K27me3 in ventricular tissues from non-diabetic and diabetic mice (n=6 in each group). Data are presented as mean±standard error of the mean. TGF-β, transforming growth factor-β; GAPDH, glyceraldehyde 3-phosphate dehydrogenase. aP<0.05, bP<0.01.

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  Fig. 2. High glucose (HG) promoted the transformation of cardiac fibroblasts (CFs) into myofibroblasts and reduced the phosphorylation of enhancer of zeste homolog 2 (EZH2) at T311. (A) Protein levels of extracellular matrix-related genes and α-smooth muscle actin (α-SMA) were analyzed in primary CFs after HG treatment (30 mM) for 72 hours (n=3 in each group). (B) Representative pictures of immunofluorescence of α-SMA expression in CFs (red, α-SMA; blue, 4´,6-diamidino-2-phenylindole [DAPI]; scale bar 50 μm). (C) Transwell assays were performed on CFs under HG treatment for 72 hours (scale bar 200 μm, n=3 in each group). (D) Scratching tests were performed on CFs under HG treatment for 72 hours (scale bar 400 μm, n=4 in each group). (E) Western blot analysis of the expression of phospho-EZH2 (p-EZH2), EZH2, and histone H3 lysine 27 trimethylation (H3K27me3) in CFs under HG treatment for 72 hours (n=3 in each group). Data are presented as mean±standard error of the mean. TGF-β, transforming growth factor-β; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; NG, normal glucose. aP<0.05, bP<0.01.

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  Fig. 3. Enhancer of zeste homolog 2 (EZH2) inhibition attenuated high glucose (HG)-induced fibroblast differentiation and extracellular matrix (ECM) protein synthesis. (A) Western blot analysis of the expression of phospho-EZH2 (p-EZH2), EZH2, and histone H3 lysine 27 trimethylation (H3K27me3) in cardiac fibroblasts (CFs) after HG and GSK126 (500 nM) treatment (n=3 in each group). (B) Inhibition of EZH2 with GSK126 in CFs and Western blot analysis of protein levels of ECM-related genes and α-smooth muscle actin (α-SMA; n=3 in each group). (C) Representative images of immunofluorescence images of α-SMA expression in CFs with different treatment (red, α-SMA; blue, 4´,6-diamidino-2-phenylindole [DAPI]; scale bar 50 μm). (D) Inhibition of EZH2 with GSK126 under HG treatment for 72 hours, transwell assay was performed (scale bar 200 μm, n=3 in each group). (E) Inhibiting of EZH2 with GSK126 under HG treatment for 72 hours, scratching tests was performed (scale bar 400 μm, n=8 in each group). (F) CFs proliferation were measured with cell counting kit 8 (CCK-8) under HG treatment of for 72 hours with/without GSK126 (n=3 in each group). Data are presented as mean±standard error of the mean. GAPDH, glyceraldehyde 3-phosphate dehydrogenase; TGF-β, transforming growth factor-β; NG, normal glucose; DMSO, dimethyl sulfoxide; OD, optical density. aP< 0.05, bP<0.01.

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  Fig. 4. Peroxisome proliferator-activated receptor γ (PPAR-γ) activation attenuated high glucose (HG)-induced fibroblasts differentiation and extracellular matrix (ECM) proteins synthesis. (A) PPAR-γ mRNA levels were measured in cardiac fibroblasts (CFs) after HG and GSK126 (500 nM, n=3 in each group). (B) Activation of PPAR-γ with rosiglitazone (50 μM) in CFs and Western blot analysis of protein levels of ECM-related genes and α-smooth muscle actin (α-SMA; n=3 in each group). (C) Immunofluorescence images of α-SMA expression in CFs with different treatment (red, α-SMA; blue, 4´,6-diamidino-2-phenylindole [DAPI]; scale bar 50 μm). (D) Activation of PPAR-γ with rosiglitone under HG treatment for 72 hours, transwell assay was performed (scale bar 200 μm, n=3 in each group). (E) Activation of PPAR-γ with rosiglitone under HG treatment for 72 hours (scale bar 400 μm, n=6 in each group). Data are presented as mean±standard error of the mean. TGF-β, transforming growth factor-β; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; NG, normal glucose; DMSO, dimethyl sulfoxide. aP<0.05, bP<0.01.

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  Fig. 5. Activation of AMP-activated protein kinase (AMPK) promoted enhancer of zeste homolog 2 (EZH2) T311 phosphorylation. (A) Western blot analysis the expression of phospho-AMPK (p-AMPK) and AMPK in cardiac fibroblasts (CFs) under high glucose (HG) treatment for 72 hours (n=3 in each group). (B) Western blot analysis of the expression of p-AMPK and AMPK in tat myocardial tissue with diabetic (n=3 in each group). (C) Western blot analysis of the expression of p-AMPK, AMPK, p-EZH2, and EZH2 in CFs after HG and activator of AMPK (A769662, 10 μM) treatment (n=3 in each group). (D) Western blot analysis of the expression of p-AMPK, AMPK, p-EZH2, EZH2, and histone H3 lysine 27 trimethylation (H3K27me3) in myocardial tissues of diabetic mice treated with AMPK agonist A769662 (n=3 in each group). Data are presented as mean±standard error of the mean. NG, normal glucose; t-AMPK, total AMPK; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; DMSO, dimethyl sulfoxide. aP<0.05, bP<0.01.

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  Fig. 6. The mechanism of diabetes promoting myocardial fibrosis via AMP-activated protein kinase (AMPK)/enhancer of zeste homolog 2 (EZH2)/peroxisome proliferator-activated receptor γ (PPAR-γ) signaling pathway. Under normal glucose, phosphorylation of AMPK phosphorylates EZH2, which inhibits the trimethylation activity EZH2, leading to expression of PPAR-γ and inhibition of myocardial fibroblasts activation. Under the condition of diabetics, high glucose inactivates AMPK, increases trimethylation activity EZH2 by reducing the phosphorylation of EZH2 at T311, which represses transcription of PPAR-γ and suppressed diabetic fibrosis. P, phosphorylation; EED, embryonic ectoderm development; SUZ12, suppressor of zest 12; H3K27me3, histone H3 lysine 27 trimethylation; TGF-β, transforming growth factor-β; α-SMA, α-smooth muscle actin; GSK126, competitive inhibitor of PRC2.

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