The sirtuin SIRT6 blocks IGF-Akt signaling and development of cardiac hypertrophy by targeting c-Jun

Abstract

Abnormal activation of insulin-like growth factor (IGF)-Akt signaling is implicated in the development of various diseases, including heart failure. However, the molecular mechanisms that regulate activation of this signaling pathway are not completely understood. Here we show that sirtuin 6 (SIRT6), a nuclear histone deacetylase, functions at the level of chromatin to directly attenuate IGF-Akt signaling. SIRT6-deficient mice developed cardiac hypertrophy and heart failure, whereas SIRT6 transgenic mice were protected from hypertrophic stimuli, indicating that SIRT6 acts as a negative regulator of cardiac hypertrophy. SIRT6-deficient mouse hearts showed hyperactivation of IGF signaling-related genes and their downstream targets. Mechanistically, SIRT6 binds to and suppresses the promoter of IGF signaling-related genes by interacting with c-Jun and deacetylating histone 3 at Lys9 (H3K9). We also found reduced SIRT6 expression in human failing hearts. These findings disclose a new link between SIRT6 and IGF-Akt signaling and implicate SIRT6 in the development of cardiac hypertrophy and failure.

Figure 1

SIRT6 deficiency causes cardiac hypertrophy and degenerative changes in the heart. (a) Western blot showing SIRT6 expression in human heart samples from representative normal control hearts (NF1, NF3, NF6; n = 8) and failing hearts (HF4, HF6, HF12, HF10; n = 24) (Supplementary Table 1). Tubulin was used as a loading control. (b) Representative western blots showing SIRT6 expression in heart samples from mice subjected to TAC or chronic infusion of ISO or angiotensin II (Ang II). n = 8 mice per group. (c) HW/BW ratio of wild-type (WT) and SIRT6 knockout (SIRT6-KO) mice at 8 weeks of age. n = 13 mice per group. Data are presented as means ± s.d. (d,e) Left ventricular internal diameter (LVID; n = 7 mice per group) and fractional shortening (FS; n = 10 mice per group) of WT and SIRT6 knockout mice at 8 weeks of age as determined by echocardiography. Data are presented as means ± s.d. (f) mRNA levels of the indicated genes in heart samples of WT and SIRT6 knockout mice. n = 6 mice per group. Data are presented as the mean ± s.d. For c–f, Student’s t test was used to calculate the P values. (g) Whole-heart sections stained with H&E showing concentric hypertrophy in SIRT6 knockout hearts (top row; scale bar, 2 mm); left ventricular muscle sections stained with wheat germ agglutinin (WGA) to demarcate cell boundaries (second row; scale bars, 10 µm) or Masson’s trichrome to detect fibrosis (third row; scale bars, 40 µm); and electron micrographs showing vacuolization (red arrows) and degeneration of mitochondria in SIRT6 knockout hearts (bottom row; scale bars, 1 µm). (h,i) Representative western blots showing the expression of the indicated fibrosis markers, cytoskeletal proteins and apoptotic markers in WT and SIRT6 knockout hearts. n = 6 per group. The line between the blots indicates two different gels. RNA Pol, RNA polymerase; SMA, α-smooth muscle actin; SMM, smooth muscle myosin.

Figure 2

Cardiac-specific deletion of SIRT6 causes cardiac hypertrophy and fibrosis. (a) HW/TL ratio of control mice (Cre, α-MHC–Cre; Flox, SIRT6^flox/flox) and SIRT6^flox/flox–α-MHC–Cre mice injected with tamoxifen to generate a cardiac-specific SIRT6 deletion (cSIRT6-KO). Data are presented as the mean ± s.d. n = 9–15 mice per group. Student’s t test was used to calculate the P value. (b) Heart sections of cardiac-specific SIRT6 knockout and control mice stained with WGA to demarcate cell boundaries (top; scale bars, 10 µm) or with Masson’s trichrome to detect fibrosis (bottom; scale bars, 40 µm). (c,d) Left ventricular wall thickness and fractional shortening of control and cardiac-specific SIRT6 knockout mice before and after tamoxifen (TO) injection. n = 5 mice per group. (e) mRNA levels of the indicated genes in heart samples of control and cardiac-specific SIRT6 knockout mice. Data are presented as the mean ± s.d. n = 4 per group. (f) Western blots showing the expression of the indicated cell death markers in the same hearts as in e. For c–e, Student’s t test was used to calculate the P value.

Figure 3

SIRT6 overexpression blocks the cardiac hypertrophic response. (a) [³H]-leucine incorporation into total cellular protein normalized to the DNA content of neonatal rat cardiomyocytes infected with WT SIRT6 adenovirus (Ad.SIRT6) or control adenovirus (Ad.GFP) and then treated with phenylephrine (PE) (20 µm) for 48 h. Data are presented as the mean ± s.d. n = 5 independent experiments. C.p.m. counts per minute. (b) Sarcomere organization, as determined by immunostaining cells for sarcomeric α-actinin (green), and ANF release (red), as determined by staining cells with an ANF-specific antibody, in cardiomyocytes infected with the indicated adenoviruses (Ad.mock indicates empty vector) and stimulated with phenylephrine. DAPI stain marks the position of nuclei. Scale bars, 10 µm. (c) HW/TL ratios in nontransgenic (NTg) and SIRT6 transgenic (SIRT6-Tg) mice subjected to sham or to TAC treatment for 4 weeks. Data are presented as the mean ± s.d. n = 8 mice per group. (d,e) Left ventricular wall thickness and fractional shortening, as measured by echocardiography, of the same hearts as in c. n = 6 mice per group. For c–e, one-way analysis of variance (ANOVA) was applied to calculate the P value. (f) Heart sections of nontransgenic and SIRT6 transgenic mice subjected to sham or TAC treatment and stained with WGA to demarcate cell boundaries (top; scale bars, 10 µm) or Masson’s trichrome to detect fibrosis (bottom; scale bars, 40 µm). (g) Quantification of myocyte cross-sectional area in nontransgenic and SIRT6 transgenic mice subjected to sham or to TAC treatment. Data are presented as the mean ± s.d. n = 5 mice per group. Student’s t test was used to calculate the P value. NS, not significant.

Figure 4

SIRT6 is a negative regulator of IGF signaling. (a) Representative western blots showing increased expression and phosphorylation of IGF signaling–related proteins in SIRT6 knockout hearts compared to WT control hearts. The expression of p38 and tropomyosin (tropo.) were not changed in these hearts and were therefore used as loading controls. n = 6 mice per group. The antibodies used for detecting ERK and GSK3 recognized the ERK1/2 and GSK3α/β isoforms, respectively. The ‘p-’ prefix indicates the phosphorylated form. (b) Representative western blots showing increased expression of transcription and translation factors in SIRT6 knockout hearts. n = 6 mice per group. (c) Representative western blots showing expression of IGF1R, p-IGF1R and SIRT1 in heart lysates of WT, SIRT1 knockout and SIRT6 knockout mice. n = 3 mice per group. (d) Real-time PCR analysis of IGF signaling–related genes in heart samples of WT and SIRT6 knockout mice. n = 6 mice per group. (e) ChIP analysis to detect SIRT6 binding to the promoters of the indicated genes performed with a SIRT6-specific antibody or IgG control antibody in WT or SIRT6 knockdown (Sirt6KD) cardiomyocytes. The occupancy of SIRT6 to promoters is shown relative to background signals with IgG control antibody. n = 4 independent experiments. Data are presented as the mean ± s.d. Student’s t test was applied to calculate the P value. *P < 0.001.

Figure 5

SIRT6 is a co-repressor of c-Jun transcriptional activity. (a) ChIP analysis to detect H3K9 acetylation at the promoters of the indicated IGF signaling–related genes in WT and SIRT6 knockout heart samples. Antibodies to acetylated H3K9 (AcH3K9) and to H3 were used, and acetylated H3K9 levels are shown relative to total H3 levels. n = 4 independent experiments. Data are presented as the mean ± s.d. (b) SIRT6 binding to c-Jun, as determined by western blots of SIRT6 or control IgG antibody immunoprecipitates from rat cardiomyocytes. WCL, whole-cell lysate. (c) Luciferase activity in cell extracts from control or SIRT6 knockdown (SIRT6-KD) 293T cells transfected with a c-Jun–dependent luciferase reporter plasmid. n = 5 independent experiments. Data are presented as the mean ± s.d. RLU, relative light units. Student’s t test was applied to calculate the P value. *P < 0.001. (d) Luciferase activity in rat cardiomyocytes cotransfected with the c-Jun–dependent luciferase reporter plasmid and plasmids expressing WT SIRT6 (SIRT6-WT) or mutant SIRT6 (SIRT6-H133Y); the cells were serum starved overnight and induced with FBS or with the c-Jun activator phorbol 12-myristate 13-acetate (PMA). n = 8 independent experiments. Data are presented as the mean ± s.d. ANOVA was applied to calculate the P value. *P < 0.001. (e) ChIP analysis using a c-Jun–specific antibody to detect c-Jun occupancy at promoters of IGF signaling–related genes in WT or SIRT6 knockout mouse heart homogenates. n = 4 independent experiments. Data are presented as the mean ± s.d. *P < 0.001 compared to WT (Student’s t test). (f) ChIP analysis using a SIRT6-specific antibody to detect SIRT6 occupancy at the promoters of the indicated genes in control (WT) and c-Jun knockdown (c-Jun-KD) rat cardiomyocytes. SIRT6 occupancy at the promoters is shown relative to the background signal with IgG control antibody. *P < 0.001 (Student’s t test). n = 4 independent experiments. Data are presented as the mean ± s.d. (g) Western blots showing c-Jun knockdown in rat cardiomyocytes.

Figure 6

Inhibition of c-Jun or IGF signaling blocks hypertrophy of SIRT6-deficient hearts. (a) Western blots showing effects of the indicated concentrations of the AP-1 inhibitor Tan-IIa on IGF1R and p-IGF1R abundance in WT and SIRT6 knockout MEFs. (b) [³H]-leucine incorporation into total cellular protein in control and SIRT6 knockdown neonatal rat cardiomyocytes infected with control (Ad.mock) or c-Jun dominant-negative (Ad.c-Jun-DN) adenovirus vectors. Data are presented as the mean ± s.d. n = 6–8 independent experiments. *P < 0.001 (Student’s t test). (c) Confocal imaging of ANF in the same group of cardiomyocytes as in b. Scale bars, 10 µm. (d,e) HW/TL ratio (d) and fractional shortening (FS) (e) of WT and SIRT6 knockout mice injected with vehicle, the AP-1 inhibitor Tan-IIa or the IGF1R inhibitor PQ401 for 2 weeks. n = 6–9 mice per group. Data are presented as the mean ± s.d. *P < 0.001 (ANOVA). (f) HW/TL ratio of control (Sirt6^flox/flox) and cardiac-specific SIRT6 knockout mice injected with vehicle or the IGF1R inhibitor PQ401 for 2 weeks. n = 6 mice per group. Data are presented as the mean ± s.d. *P < 0.001 (Student’s t test). (g) Under normal conditions, SIRT6 inhibits the expression of IGF signaling–related genes by deacetylating histones and repressing c-Jun activity, thereby restraining IGF signaling. Under pathological stress, cardiac SIRT6 expression is reduced, leading to increased acetylation of H3K9 (K9Ac) at the promoters of IGF signaling genes and c-Jun–mediated transcriptional activation. Increased expression of multiple IGF-Akt signaling–related genes leads to the development of cardiac hypertrophy, fibrosis and heart failure.