Sirtuin 2 Exerts Regulatory Functions on Radiation-Induced Myocardial Fibrosis in Mice by Mediating H3K27 Acetylation of Galectin-3 Promoter

Abstract

Background: Sirtuin 2 (SIRT2) and galectin-3 have been shown to protect the heart against fibrosis. However, their impacts on radiation-induced myocardial fibrosis (RIMF) remain to be elucidated. To deepen this understanding, the current study sought to explore the effects of SIRT2 and galectin-3 on RIMF and the underlying mechanisms.

Methods: Galectin-3 knockout mice were obtained, and a radiation-induced heart damage (RIHD) mouse model was induced by local radiation exposure to the heart. Lentivirus transfection was then performed, and heart function, fibrosis of heart tissues, and levels of SIRT2, galectin-3, and fibrosis-related markers collagen type-I/-III and matrix metalloproteinase (MMP)2/MMP9 were respectively assessed by echocardiography, hematoxylin-eosin and Masson staining, reverse transcription-quantitative polymerase chain reaction, Western blot, and immunofluorescence staining. Additionally, Western blot and chromatin immunoprecipitation were used to test H3K27 acetylation levels and the binding of H3K27ac to galectin-3, respectively.

Results: After radiation exposure, heart tissues from the galectin-3 knockout mice had a smaller fibrotic area compared to normal mice, with reduced expression levels of collagen type-I/-III and MMP2/MMP9. SIRT2 was down-regulated and galectin-3 was up-regulated after RIHD treatment. The histone deacetylase inhibitor sirtinol promoted galectin-3 expression and H3K27 acetylation in a time-dependent manner, and increased H3K27ac enrichment in the galectin-3 promoter. Overexpression of SIRT2 down-regulated H3K27ac, collagen type-I/-III, and MMP2/MMP9 expression levels, and reduced the fibrotic area in mouse heart tissues. However, these effects were reversed by the additional overexpression of galectin-3.

Conclusions: SIRT2 facilitates deacetylation of H3K27 to inhibit galectin-3 transcription, thus ameliorating RIMF in mice.

Keywords: Galectin-3; H3K27 acetylation; Myocardial fibrosis; Radiotherapy; Sirtuin 2.

Figure 1

Galectin-3 knockout mitigates radiation-induced myocardial fibrosis in mice. (A-B) Reverse transcription-quantitative polymerase chain reaction (RT-qPCR) and western blot to measure galectin-3 expression in heart tissues from the knockout (KO) group of mice; (C) Echocardiography and left ventricular ejection fraction (LVEF) values after 1, 3, 5 months-radiation exposure in the mouse heart; (D) Hematoxylin-eosin (HE) and Masson staining of mouse heart tissues; (E) Western blot to detect Coll-I and Coll-III expression in mouse heart tissues; (F) Immunofluorescence staining of heart tissue sections from the wild type (WT) and KO groups of mice; (G) Western blot to detect matrix metalloproteinase 2 (MMP2) and matrix metalloproteinase 9 (MMP9) expression in mouse heart tissues. n = 9. * p < 0.05 versus the WT group. coll, collagen type; GAPDH, glyceraldehyde-3-phosphate dehydrogenase.

Figure 2

H3K27 acetylation motivates galectin-3 transcription. (A) UCSC database prediction of H3K27 acetylation peak value in the galectin-3 promoter; (B-C) Reverse transcription-quantitative polymerase chain reaction (RT-qPCR) and western blot to measure mRNA and protein expression of galectin-3 and protein expression of H3K27ac in cardiac fibroblasts (CFs) after 12- and 24-h sirtinol treatment; (D) Chromatin immunoprecipitation (ChIP) assay to test the binding relationship between galectin-3 and H3K27ac. The cellular experiment was independently repeated thrice. * p < 0.05 versus the control group. GAPDH, glyceraldehyde-3-phosphate dehydrogenase; IgG, Immunoglobulin G.

Figure 3

Sirtuin 2 (SIRT2) regulates H3K27 deacetylation and inhibits the expression of galectin-3. (A-B) Reverse transcription-quantitative polymerase chain reaction (RT-qPCR) and western blot testing of galectin-3 expression in heart tissues from radiation-induced heart damage (RIHD) mouse models; (C) Echocardiography assessment of heart functions of RIHD model mice; (D) Hematoxylin-eosin (HE) and Masson staining observations of fibrosis condition in heart tissues of RIHD mouse models; (E-F) Western blot and immunofluorescence staining monitoring results of collagen type (Coll)-I and Coll-III expression in tissues from RIHD mouse models; (G) Western blot detection of matrix metalloproteinase 2 (MMP2) and matrix metalloproteinase 9 (MMP9) expression in heart tissues from RIHD mouse models; (H-I) RT-qPCR and western blot evaluation of SIRT2 expression in heart tissues of RIHD mouse models; (J-K) RT-qPCR and western blot to testify the transfection efficiency of overexpression (oe)-SIRT2; (L) Chromatin immunoprecipitation (ChIP) to measure H3K27ac levels in the galectin-3 promoter region; (M) Immunofluorescence staining of heart tissues; (N) Western blot detection of galectin-3 expression in heart tissues. n = 9. * p < 0.05 versus the control or oe-negative control (NC) group. GAPDH, glyceraldehyde-3-phosphate dehydrogenase; IgG, immunoglobulin G.

Figure 4

Overexpression of galectin-3 can counteract the ameliorating effects of Sirtuin 2 (SIRT2) overexpression on radiation-induced myocardial fibrosis in mice. (A-B) Reverse transcription-quantitative polymerase chain reaction (RT-qPCR) and western blot testing of transfection efficiency; (C) Western blot measurements of collagen type (Coll)-I and Coll-III expression in heart tissues; (D) Immunofluorescence staining of heart tissues; (E) Hematoxylin-eosin (HE) and Masson staining observations of fibrosis condition in mouse heart tissues; (F) Western blot analysis of matrix metalloproteinase 2 (MMP2) and matrix metalloproteinase 9 (MMP9) expression in heart tissues. n = 9. * p < 0.05 versus the overexpression (oe)-negative control (NC) or oe-SIRT2 + oe-NC group. GAPDH, glyceraldehyde-3-phosphate dehydrogenase.