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

Figures

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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.