SIRT3-mediated inhibition of FOS through histone H3 deacetylation prevents cardiac fibrosis and inflammation

Abstract

Sirtuin 3 (SIRT3) is a deacetylase that modulates proteins that control metabolism and protects against oxidative stress. Modulation of SIRT3 activity has been proposed as a promising therapeutic target for ameliorating metabolic diseases and associated cardiac disturbances. In this study, we investigated the role of SIRT3 in inflammation and fibrosis in the heart using male mice with constitutive and systemic deletion of SIRT3 and human cardiac AC16 cells. SIRT3 knockout mice showed cardiac fibrosis and inflammation that was characterized by augmented transcriptional activity of AP-1. Consistent with this, SIRT3 overexpression in human and neonatal rat cardiomyocytes partially prevented the inflammatory and profibrotic response induced by TNF-α. Notably, these effects were associated with a decrease in the mRNA and protein levels of FOS and the DNA-binding activity of AP-1. Finally, we demonstrated that SIRT3 inhibits FOS transcription through specific histone H3 lysine K27 deacetylation at its promoter. These findings highlight an important function of SIRT3 in mediating the often intricate profibrotic and proinflammatory responses of cardiac cells through the modulation of the FOS/AP-1 pathway. Since fibrosis and inflammation are crucial in the progression of cardiac hypertrophy, heart failure, and diabetic cardiomyopathy, our results point to SIRT3 as a potential target for treating these diseases.

Fig. 1

SIRT3 deletion in knockout mice does not induce cardiac hypertrophy. Body weight a and heart weight to tibia length (HW/TL) ratio b in SIRT3 knockout (KO SIRT3) mice and their wild-type (WT) littermates. c Representative hematoxylin and eosin stained micrographs showing transverse sections from the left ventricle myocardium and quantification of cardiomyocyte cross-sectional areas. d Relative quantification of Nppb (type B natriuretic peptide) and β-myosin heavy chain (β-MHC) mRNA expression. Graphs represent the quantification of adenine phosphoribosyl transferase (Aprt)-normalized mRNA levels expressed as a percentage of the WT samples. The data are the mean ± SD (Mann–Whitney test)

Fig. 2

SIRT3 knockout mice display inflammation in the heart. a Relative quantification of Icam1, Il6, Mcp1, Socs3, and Sod2 mRNA expression in SIRT3 knockout (KO SIRT3) and wild-type (WT) mice. b Western blot analysis showing the levels of p-IκBα^Ser32/IκBα in total protein extracts and p65 in nuclear protein fractions (NE) obtained from the same samples. The data are the mean ± SD. c EMSA data showing NF-κB DNA-binding activity in the heart. Ab antibody; IC immunocomplex; NE nuclear extract. Western blot analysis showing the levels of FOS and JUN in cytosolic d and nuclear e protein fractions obtained from heart samples of KO SIRT3 and WT mice. The graphs represent the quantification of adenine phosphoribosyl transferase (Aprt)–normalized mRNA levels a or the normalized quantification of protein levels b, d, and e expressed as a percentage of the control samples ± SD. f EMSA data showing AP-1 DNA-binding activity in the heart. g Representative images of Mason’s trichrome staining and quantification of fibrosis expressed as a percentage of WT samples ± SD in the heart. h Representative M-mode transthoracic echocardiographic images and graphs representing evaluations of EF, FS, LV EDD, and LV ESD diameters, and IVRT. The Data are presented as the median ± interquartile range and were compared using the Mann–Whitney test. *P < 0.05, **P < 0.01, and ***P < 0.001 vs. WT

Fig. 3

SIRT3 overexpression attenuates inflammation in human cardiac cells. a Relative quantification of IL6, MCP1, and SOCS3 mRNA expression in human AC16 cardiac cells transfected with LacZ-carrying or SIRT3-carrying plasmids in the presence or absence of TNF-α (TNF, 10 ng/mL, 24 h). The graphs represent the quantification of the glyceraldehyde-3-phosphate dehydrogenase (GAPDH)-normalized mRNA levels, expressed as a percentage of the control samples ± SD. b Western blot analysis showing the levels of IκBα in total protein extracts, p65 in nuclear protein fractions, and FOS in cytosolic (CP) and nuclear (NE) protein fractions obtained from the same samples. The graphs represent the quantification of protein levels normalized to actin (total protein) or lamin B (nuclear protein), expressed as a percentage of the control samples ± SD. c EMSA data showing NF-κB DNA-binding activity. Ab antibody; IC immunocomplex; NE nuclear extract. d EMSA data showing AP-1 DNA-binding activity in the same samples depicted in panel a. e Relative quantification of ATF4, EDN-1, and TGFB1 mRNA expression expressed as a percentage of the control samples ± SD. The data were compared by ANOVA followed by Tukey’s post hoc test. *P < 0.05, **P < 0.01, and ***P < 0.001 vs. LacZ; ^#P < 0.05, ^##P < 0.01, and ^###P < 0.001 vs. LacZ + TNF-α. f Pearson correlation coefficient between SIRT3 and FOS gene expression in left ventricular myocardial tissue obtained from patients undergoing aortic valve replacement surgery. The relative transcript levels of the target genes, in arbitrary units, were used to calculate the Pearson correlation coefficient

Fig. 4

SIRT3 modulates inflammation and FOS levels in neonatal rat cardiomyocytes. Relative quantification of Icam1, Il6, Tnf-α a, Fos b, Atf4, Edn-1, and Tgfb1 d mRNA expression in neonatal rat cardiomyocytes overexpressing Sirt3 (AdSIRT3) or GFP control vector (AdGFP; 30 IFU per cell, for 48 h) in the presence or absence of TNF-α (TNF, 10 ng/mL, 24 h). The graphs represent the quantification of the Gapdh–normalized mRNA levels expressed as a percentage of the control samples ± SD. c Western blot analysis showing FOS protein levels in total protein extracts obtained from the same samples depicted in panel a. The graph represents the quantification of protein levels normalized to GAPDH expressed as a percentage of the control samples ± SD. The data were compared by ANOVA followed by Tukey’s post hoc test. *P < 0.05, **P < 0.01, and ***P < 0.001 vs. AdGFP; ^#P < 0.05, ^##P < 0.01, and ^###P < 0.001 vs. AdGFP + TNF-α

Fig. 5

FOS is not transcriptionally regulated by STAT3 or MAPK. a Relative quantification of IL6 and MCP1 mRNA expression in human AC16 cardiac cells transfected with LacZ-carrying, SIRT3-carrying, or SIRT3H248Y-carrying plasmids in the presence or absence of TNF-α. b Relative quantification of FOS mRNA expression in human AC16 cardiac cells transfected with LacZ-carrying or SIRT3-carrying plasmids in the presence or absence of TNF-α. Western blot analysis showing the protein levels of phosphorylated ERK1/2^{Thr202/Tyr204}/ERK1/2 c, phosphorylated STAT3^Ser727/STAT3 d, and phosphorylated p38^{Thr180/Tyr182}/p38 MAPK e in total protein extracts obtained from human AC16 cardiac cells transfected with LacZ-carrying or SIRT3-carrying plasmids in the presence or absence of TNF-α (TNF, 10 ng/mL, 24 h). f Relative quantification of FOS mRNA levels in human AC16 cardiac cells incubated in the presence or absence of the p38 MAPK inhibitor SB202190 (10 μmol/mL, 24 h). The graphs represent the quantification of protein levels normalized to actin and are expressed as a percentage of the control samples ± SD. g Relative quantification of FOS, ATF4, and EDN-1 mRNA levels in human AC16 cardiac cells incubated in the presence or absence of the AP-1 inhibitor SR-11302 (10 μmol/L, 24 h). The graphs represent the quantification of the GAPDH-normalized mRNA levels a, b, f, and g or the quantification of protein levels normalized to GAPDH c–e, expressed as a percentage of the control samples ± SD. Comparisons were performed by ANOVA followed by Tukey’s posttest (panels a–e) or unpaired two-tailed Student’s t-test (panels f and g). a–e *P < 0.05, **P < 0.01, and ***P < 0.001 vs. LacZ; ^#P < 0.05, ^##P < 0.01, and ^###P < 0.001 vs. LacZ + TNF-α; ^┼P < 0.05, ^┼┼P < 0.01, and ^┼┼┼P < 0.001 vs. SIRT3; ^&P < 0.05, ^&&P < 0.01, and ^&&&P < 0.001 vs. SIRT3 + TNF-α. f and g *P < 0.05, **P < 0.01, and ***P < 0.001 vs. Control

Fig. 6

SIRT3 attenuates FOS/AP-1 signaling via H3K27 deacetylation at the chromatin. Western blot analysis showing the protein levels of acetylated histone H3 in nuclear protein extracts obtained from human AC16 cardiac cells transfected with a LacZ-carrying or SIRT3-carrying plasmids or b scrambled siRNA (siRNA control) or SIRT3 siRNA. The graphs represent the quantification of protein levels normalized to lamin B expressed as a percentage of the control samples ± SD. ChIP assay for the determination of H3K27 acetylation levels at the FOS promoter in human AC16 cardiac cells transfected with LacZ or SIRT3-carrying plasmid c, scrambled siRNA (siRNA control) or SIRT3 siRNA d. The data are the mean ± SD, and comparisons were performed by unpaired two-tailed Student’s t-test. *P < 0.05, **P < 0.01, and ***P < 0.001 vs. LacZ a and c or siRNA control b and d