Korean Circ J.  2025 Mar;55(3):231-247. 10.4070/kcj.2024.0093.

YAK577 Attenuates Cardiac Remodeling and Fibrosis in Isoproterenol-Infused Heart Failure Mice by Downregulating MMP12

• Zhou H
• Kee HJ
• Wan L
• Asfaha Y
• Fischer F
• Kassack MU
• Kurz T
• Kim SH
• Kee SJ
• Hong YJ
• Jeong MH

• KMID: 2565815
• DOI: http://doi.org/10.4070/kcj.2024.0093

Abstract

Background and Objectives
Heart failure is a potentially fatal event caused by diverse cardiovascular diseases, leading to high morbidity and mortality. Histone deacetylase (HDAC) inhibitors positively influence cardiac hypertrophy, fibrosis, hypertension, myocardial infarction, and heart failure, causing some side effects. We aimed to investigate the effect of the novel HDAC inhibitor YAK577 on the heart failure mouse model and its underlying mechanism.
Methods
New hydroxamic acid YAK577 was prepared via methyl-2,3-diphenylpropanoate synthesis using carboxylic acids. We used a micro-osmotic pump, including isoproterenol (ISO; 80 mg/kg/day), to induce a heart failure with reduced ejection fraction. Cardiac hypertrophy was assessed by heart weight to body weight ratio and cross-sectional area. The left ventricular (LV) function was assessed by echocardiography. Fibrosis was evaluated using picrosirius red staining. Overexpression and knockdown experiments were performed to investigate the association between HDAC8 and matrix metalloproteinase 12 (MMP12).
Results
YAK577 treatment restored ISO-induced reduction in LV fractional shortening and ejection fraction (n=9–11). YAK577 significantly downregulated cardiac hypertrophy marker genes (natriuretic peptide B, NPPB, and myosin heavy chain 7, MYH7) and cardiomyocyte size in vitro but not in vivo. YAK577 ameliorated cardiac fibrosis and fibrosis-related genes in vivo and in vitro. Additionally, YAK577 reduced elevated HDAC8 and MMP12 mRNA and protein expressions in ISO-infused mice, H9c2 cells, and rat neonatal cardiomyocytes. HDAC8 overexpression stimulated MMP12 and NPPB mRNA levels, while HDAC8 knockdown downregulated these genes.
Conclusions
YAK577 acts as a novel heart failure drug through the HDAC8/MMP12 pathway.

Keyword

YAK577; Heart failure; Fibrosis; Histone deacetylase 8; Matrix metalloproteinase 12

Figure

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  Figure 1 Novel hydroxamic acid YAK577 synthesis and its cytotoxicity effect. (A) Chemical structure of YAK577. (B) H9c2 cells treated with different concentrations of YAK577 (0.1μM, 1 μM, and 10 μM) for 24 hours. Cell viability was measured.NS = not significant.***p<0.001.

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  Figure 2 YAK577 reduced isoproterenol-induced cardiomyocyte hypertrophy in both H9c2 cells and rat neonatal cardiomyocytes. (A) Representative Alexa Fluor 488 phalloidin-stained cell images (green) of 3 different groups. Nuclei were stained with DAPI (blue). The right panel is merged images. Scale bar=50μm. (B) Cell size quantification (n=40). (C-F) NPPB and Myh7 mRNA expression levels from control and isoproterenol groups with or without YAK577 were analyzed by qRT-PCR in both H9c2 cells and rat neonatal cardiomyocytes (n=6–9). (G-J) Representative immunoblot images of Nppb and band density were quantified (n=6). Data are expressed as mean ± standard error of the mean.CMC = cardiomyocyte; DAPI = 4',6-diamidino-2-phenylindole; GAPDH = glyceraldehyde 3-phosphate dehydrogenase; ISO = isoproterenol; Myh7 = myosin heavy chain 7; Nppb = natriuretic peptide B.**p<0.01, ***p<0.001; ##p<0.01, ###p<0.001.

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  Figure 3 YAK577 ameliorated left ventricular dysfunction but not cardiac hypertrophy in isoproterenol-induced heart failure in mice. (A) The schematic diagram of the experimental protocol. The pump indicates the infusion of a micro-osmotic pump. (B) HW/BW in 3 different groups (n=9–11). (C) Wet LW/BW in 3 different groups (n=9–11). (D, E) Representative images of H&E staining and wheat germ agglutinin staining, as well as its cross-sectional area of interventricular septum muscles (n=6). Top panel, wheat germ agglutinin staining; bottom panel, H&E staining. Scale bar=50μm. (F) Representative M-mode echocardiograms in 3 different groups. (G-K) Quantification of heart rate, LVIDs, LVIDd, LVEF, and LVFS (n=9–11). Data are expressed as mean ± standard error of the mean.H&E = hematoxylin and eosin; HW/BW = heart weight to body weight ratio; ISO = isoproterenol; LVEF = left ventricular ejection fraction; LVFS = left ventricular fraction shortening; LVIDd = left ventricular end-diastolic internal diameter; LVIDs = left ventricular end-systolic internal diameter; LW/BW = lung weight to body weight ratio; NS = not significant.*p<0.05, ***p<0.001; ###p<0.001.

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  Figure 4 YAK577 attenuated cardiac fibrosis in isoproterenol-induced heart failure mice. (A) Representative images of picrosirius red staining in 3 different groups of the isoproterenol-induced heart failure model. Scale bar=100μm. (B) Quantification of fibrosis (n=5–9). (C-K) mRNA levels of COL1A1, FN1, MMP3, MMP12, MMP14, TIMP1, TIMP4, LGALS3, and HDAC8 (n=8–10) were determined by qRT-PCR. (L) Representative Western blot images of Col1A1 and MMP12 protein levels (n=6). β-actin was used as a loading control. MMP12 was detected as 54 kDa and 22 kDa (active form) proteins (n=6–8). (M-O) Quantification of Col1A1 and MMP12 proteins. (P-R) Representative Western blot images of galectin-3 and HDAC8 proteins. Protein expression was quantified by ImageJ (n=4–6). Data are reported as the mean±standard error of the mean.GAPDH = glyceraldehyde 3-phosphate dehydrogenase; HDAC8 = histone deacetylase 8; ISO = isoproterenol; MMP12 = matrix metalloproteinase 12.*p<0.05, **p<0.01, and ***p<0.001; #p<0.05, ##p<0.01, and ###p<0.001.

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  Figure 5 YAK577 mitigated the isoproterenol or Hdac8 overexpression–induced Mmp12 expression in H9c2 cells and rat neonatal cardiomyocytes. (A-D) mRNA expression of HDAC8 and MMP12 in H9c2 cells and rat neonatal cardiomyocytes treated with isoproterenol (10 μM) and YAK577 (0.1 μM) (n=6–11). (E-J) Representative western blotting images and quantification of HDAC8 and MMP12 (n=4–10). β-actin was used as a loading control. (K-M) mRNA levels of HDAC8, NPPB, and MMP12 in Hdac8-overexpressed H9c2 cells with or without YAK577 (n=8–10).Data are reported as the mean±standard error of the mean.CMC = cardiomyocyte; GAPDH = glyceraldehyde 3-phosphate dehydrogenase; HDAC8 = histone deacetylase 8; ISO = isoproterenol; MMP12 = matrix metalloproteinase 12.*p<0.05, **p<0.01, and ***p<0.001; #p<0.05, ##p<0.01, and ###p<0.001.

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  Figure 6 YAK577 attenuated TGF-β1-induced fibrosis and MMP12 levels in primary rat cardiac fibroblasts. (A-D) mRNA levels of TGF-β1, FN1, MMP12, and HDAC8 (n=7–12) in TGF-β1-treated rat cardiac fibroblasts with or without YAK577. (E-G) Representative Western blot images and quantification of Col1A1 and Fn (n=8–10). β-actin was used as a loading control. (H-J) Representative images of western blotting and quantification of HDAC8 and MMP12 (n=11–14). Data are reported as the mean±standard error of the mean.GAPDH = glyceraldehyde 3-phosphate dehydrogenase; HDAC8 = histone deacetylase 8; MMP12 = matrix metalloproteinase 12; TGF-β1 = transforming growth factor β1.*p<0.05, **p<0.01, and ***p<0.001; ##p<0.01 and ###p<0.001.

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