SIRT2 Acts as a Cardioprotective Deacetylase in Pathological Cardiac Hypertrophy

Abstract

Background: Pathological cardiac hypertrophy induced by stresses such as aging and neurohumoral activation is an independent risk factor for heart failure and is considered a target for the treatment of heart failure. However, the mechanisms underlying pathological cardiac hypertrophy remain largely unknown. We aimed to investigate the roles of SIRT2 in aging-related and angiotensin II (Ang II)-induced pathological cardiac hypertrophy.

Methods: Male C57BL/6J wild-type and Sirt2 knockout mice were subjected to the investigation of aging-related cardiac hypertrophy. Cardiac hypertrophy was also induced by Ang II (1.3 mg/kg/d for 4 weeks) in male C57BL/6J Sirt2 knockout mice, cardiac-specific SIRT2 transgenic (SIRT2-Tg) mice, and their respective littermates (8 to ≈12 weeks old). Metformin (200 mg/kg/d) was used to treat wild-type and Sirt2 knockout mice infused with Ang II. Cardiac hypertrophy, fibrosis, and cardiac function were examined in these mice.

Results: SIRT2 protein expression levels were downregulated in hypertrophic hearts from mice. Sirt2 knockout markedly exaggerated cardiac hypertrophy and fibrosis and decreased cardiac ejection fraction and fractional shortening in aged (24-month-old) mice and Ang II-infused mice. Conversely, cardiac-specific SIRT2 overexpression protected the hearts against Ang II-induced cardiac hypertrophy and fibrosis and rescued cardiac function. Mechanistically, SIRT2 maintained the activity of AMP-activated protein kinase (AMPK) in aged and Ang II-induced hypertrophic hearts in vivo as well as in cardiomyocytes in vitro. We identified the liver kinase B1 (LKB1), the major upstream kinase of AMPK, as the direct target of SIRT2. SIRT2 bound to LKB1 and deacetylated it at lysine 48, which promoted the phosphorylation of LKB1 and the subsequent activation of LKB1-AMPK signaling. Remarkably, the loss of SIRT2 blunted the response of AMPK to metformin treatment in mice infused with Ang II and repressed the metformin-mediated reduction of cardiac hypertrophy and protection of cardiac function.

Conclusions: SIRT2 promotes AMPK activation by deacetylating the kinase LKB1. Loss of SIRT2 reduces AMPK activation, promotes aging-related and Ang II-induced cardiac hypertrophy, and blunts metformin-mediated cardioprotective effects. These findings indicate that SIRT2 will be a potential target for therapeutic interventions in aging- and stress-induced cardiac hypertrophy.

Keywords: AMPK; LKB1; SIRT2; aging; angiotensin II; cardiac hypertrophy; deacetylation; metformin.

Figure 1. Sirt2 deficiency aggravates cardiac hypertrophy in aged mice

(A) Representative gross morphology of aged (24-month-old) WT and Sirt2-KO mice. (B) Ejection fraction and fractional shortening of WT and Sirt2-KO mice at 4 months (young) or 24 months (aged). n=11~13. ***P<0.001. (C) Heart weight-to-tibia length (HW/TL) ratios of WT and Sirt2-KO mice at 4 months (young) or 24 months (aged). n=12~13. *P<0.05, **P<0.01. (D) Left: Hematoxylin-eosin (H&E, scale bar=1 mm) staining and wheat germ agglutinin (WGA, scale bar=30 μm) staining were performed to determine the hypertrophic growth of the hearts in young and aged WT and Sirt2-KO mice. Right: Quantification of cardiomyocyte size in young and aged WT and Sirt2-KO mice (n=8~10; ***P<0.001). (E) Left: Picrosirius red (PSR, scale bar=50 μm) staining was performed to determine cardiac fibrosis in young and aged WT and Sirt2-KO mice. Right: Quantification of cardiac fibrosis in young and aged WT and Sirt2-KO mice (n=8~10; ***P<0.001). (F) Left: Western blotting showing the expression levels of phosphorylated IGF1R, AMPK and mTOR and the total protein expression levels of IGF1R, AMPK, mTOR, and SIRT1. Right: Quantification of p-IGF1R, p-AMPK, p-mTOR, and SIRT1 levels (n=4; *P<0.05). IGF1R: Insulin-like growth factor 1 receptor; AMPK: AMP-activated protein kinase; mTOR: Mammalian target of rapamycin.

Figure 2. SIRT2 expression and activity are down-regulated in hypertrophic hearts

(A) Left: Representative western blotting showing changes in SIRT2 protein in aged (24-month-old) hearts and Ang II-induced hypertrophic hearts. Right: Quantification of SIRT2 protein levels (n=4; **P<0.01, ***P<0.001). (B) Left: Representative western blotting showing changes in acetylated Tubulin levels in aged (24-month-old) hearts and Ang II-induced hypertrophic hearts. Right: Quantification of acetylated Tubulin levels (n=4; *P<0.05, ***P<0.001). (C) SIRT2 activity in cardiac tissues of mice infused with saline or Ang II. The SIRT2 protein was purified by immunoprecipitation using an anti-SIRT2 antibody. Then an enzymatic assay was performed to determine SIRT2 protein activity (n=6; *P<0.05). (D) Left: Representative western blotting results showing the phosphorylated level of c-Src in aged and Ang II-infused mouse hearts. Right: Quantification of phosphorylated level of c-Src (n=4; ***P<0.001). (E) Left: Representative western blotting results showing the phosphorylated level of c-Src and level of SIRT2. Right: Quantification of phosphorylated c-Src and SIRT2 levels (**P<0.01, ***P<0.001, ns: not significant). Neonatal rat cardiomyocytes (NRCMs) were treated with Ang II (1 μM) for 24 hours with/without the presence of c-Src inhibitor SU6656 (5 μM).

Figure 3. Sirt2-KO aggravates Ang II-induced cardiac hypertrophy

(A) Ejection fraction and fractional shortening of WT and Sirt2-KO mice treated with saline or Ang II (1.3 mg/kg/day) for four weeks (n=17~20; *P<0.05, **P<0.01, ***P<0.001). (B) Heart weight-to-body weight (HW/BW) ratios and heart weight-to-tibia length (HW/TL) ratios of WT and Sirt2-KO mice treated with saline or Ang II (n=16~20; *P<0.05, ***P<0.001). (C) Left: Hematoxylin-eosin (H&E, scale bar=1 mm) staining and wheat germ agglutinin (WGA, scale bar=30 μm) staining were performed to determine the hypertrophic growth of the hearts in WT and Sirt2-KO mice treated with saline or Ang II. Right: Quantification of cardiomyocyte size in WT and Sirt2-KO mice treated with saline or Ang II (n=13~17; *P<0.05, ***P<0.001). (D) Left: Picrosirius red (PSR, scale bar=50 μm) staining was performed to determine cardiac fibrosis of the hearts from WT and Sirt2-KO mice treated with saline or Ang II. Right: Quantification of cardiac fibrosis in WT and Sirt2-KO mice treated with saline or Ang II (n=13~17; *P<0.05, **P<0.01). (E) Quantitative real-time PCR (qRT-PCR) was performed to analyze the mRNA levels of hypertrophic (Anp, Bnp, β-Mhc, Acta1, IL-6 and Rcan1.4) and fibrosis (Ctgf, Col1a1 and Col3a1) genes (n=6; *P<0.05, ***P<0.001). Anp: Atrial natriuretic peptide; Bnp: brain natriuretic peptide; β-Mhc: Myosin heavy chain beta; Acta1: α-sarcomeric actin; IL-6: interleukin 6; Rcan1.4: regulator of calcineurin 1.4. Ctgf: Connective tissue growth factor; Col1a1: Alpha-1 type I collagen; Collagen 3a1.

Figure 4. SIRT2 regulates Ang II-induced hypertrophy in neonatal rat cardiomyocytes (NRCMs)

(A) NRCMs were treated with phosphate-buffered saline (PBS) or Ang II (1 μM) for 48 hours in the presence of AGK2 (10 μM) or DMSO. α-Actinin staining was performed to determine cell size. Representative images (Left) and quantification of cell size of total 30 NRCMs (Right) in each group are shown (scale bar=30 μm; *P<0.05, ***P<0.001). (B) NRCMs were treated as shown in (A) and qRT-PCR was performed to analyze the mRNA levels of hypertrophic genes (Anp, Bnp, and β-Mhc). *P<0.05, **P<0.01, ***P<0.001. (C) NRCMs were infected with the indicated adenovirus for 24 hours and then treated with PBS or Ang II (1 μM) for 48 hours. α-Actinin staining was performed to determine cell size. Representative images (Left) and quantification of cell size of total 30 NRCMs (Right) in each group are shown (scale bar=30 μm; *P<0.05, ***P<0.001). Ad-Ctrl: Control adenovirus; Ad-SIRT2: Adenovirus overexpressing human SIRT2. (D) NRCMs were treated as shown in (C) and qRT-PCR was performed to analyze the expression of hypertrophic genes (Anp, Bnp, and β-Mhc). **P<0.01, ***P<0.001.

Figure 5. Cardiac-specific SIRT2 overexpression represses Ang II-induced cardiac hypertrophy

(A) Ejection fraction and fractional shortening in non-transgenic (N-Tg) and cardiac-specific SIRT2 transgenic (SIRT2-Tg) mice treated with saline or Ang II (1.3 mg/kg/day) for four weeks (n=18~20; *P<0.05, **P<0.01, *** P<0.001). (B) Ratios of heart weight-to-body weight (HW/BW) or heart weight-to-tibia length (HW/TL) in N-Tg and SIRT2-Tg mice treated with saline or Ang II (n=18~20; ***P <0.001). (C) Left: Hematoxylin-eosin (H&E, scale bar=1 mm) staining and wheat germ agglutinin (WGA, scale bar=30 μm) staining were performed to determine the hypertrophic growth of the hearts in N-Tg and SIRT2-Tg mice treated with saline or Ang II. Right: Quantification of cardiomyocyte size in N-Tg and SIRT2-Tg mice treated with saline or Ang II (n=15~18; ***P <0.001). (D) Left: Picrosirius red (PSR, scale bar=50 μm) staining was performed to determine cardiac fibrosis of the hearts from N-Tg and SIRT2-Tg mice treated with saline or Ang II. Right: Quantification of cardiac fibrosis in N-Tg and SIRT2-Tg mice treated with saline or Ang II (n=15~18; *P<0.05, ***P <0.001). (E) Expression of hypertrophic (Anp, Bnp, β-Mhc, Acta1, IL-6 and Rcan1.4) and fibrotic (Ctgf, Col1a1 and Col3a1) genes in the hearts of N-Tg and SIRT2-Tg mice treated with saline or Ang II (n=6; *P<0.05, ** P<0.01, ***P<0.001).

Figure 6. SIRT2 maintains AMPK signaling in myocardial tissues

(A) Representative western blotting and quantitative results showing the phosphorylation levels of AMPK and the phosphorylation levels of its substrates ACC and Raptor in the hearts of WT, Sirt2-knockout, N-Tg and SIRT2-Tg mice infused with Ang II (n=4; *P<0.05, **P<0.01, ***P<0.001). (B) Representative western blotting and quantification results showing the phosphorylation of AMPK, ACC, and Raptor in NRCMs. NRCMs were treated with the SIRT2 inhibitor AGK2 (10 μM) or with DMSO for 24 hours or infected with adenovirus overexpressing SIRT2 (Ad-SIRT2) or control adenovirus (Ad-Ctrl) for 24 hours (**P<0.01, ***P<0.001). (C) NRCMs were infected with the indicated adenovirus for 24 hours and then treated with Ang II (1 μM) for 48 hours in the presence of the AMPK inhibitor compound C (CC, 10 μM) or DMSO. α-Actinin staining was performed to identify cells. Representative images (Left) and quantification of cell size of total 30 NRCMs in each group are shown (scale bar=30 μm; *P<0.05, ***P<0.001, ns: not significant). (D) NRCMs were treated as shown in (C) and RNA was subjected to qRT-PCR to determine the mRNA level of hypertrophic genes (Anp, Bnp, and β-Mhc). *P<0.05, **P<0.01, ***P<0.001, ns: not significant.

Figure 7. SIRT2 deacetylates and activates LKB1

(A) Representative western blotting and quantitative results showing LKB1 phosphorylation at Ser428 in the hearts of aged WT and Sirt2-KO mice and the hearts of Ang II-induced WT, Sirt2-KO, N-Tg and SIRT2-Tg mice (n=4; **P<0.01, ***P<0.001). (B) Subcellular location of LKB1 in NRCMs. NRCMs were infected with Ad-SIRT2 for 24 hours, followed by immunofluorescence to detect the SIRT2 and LKB1 protein. Top: Representative immunofluorescence showing the location of LKB1 (red) in NRCMs, SIRT2-overexpressing cells were indicated by green fluorescence protein (GFP, green), scale bar=30 μm. Bottom: The quantification of the cytosol-to-nuclear ratio of LKB1 protein in 20 NRCMs without (-) and with (+) SIRT2-overexpression (***P<0.001). (C) Representative immunoprecipitation, western blotting and quantitative results showing LKB1 acetylation in hypertrophic hearts. Endogenous LKB1 was purified by immunoprecipitation with anti-LKB1 antibody from the hearts from aged WT and Sirt2-KO mice or WT, Sirt2-KO, N-Tg, and SIRT2-Tg mice infused with Ang II. Western blotting was performed with the indicated antibodies (n=4; ***P<0.001). (D) NRCMs were infected with the indicated adenovirus for 24 hours and then treated with Ang II (1 μM) for 48 hours. α-Actinin staining was performed to determine cell size. Representative images (Left) and quantification of cell size of total 30 NRCMs (Right) in each group are shown (scale bar=30 μm; ***P<0.001, ns: not significant). (E) NRCMs were treated as shown in (D) and qRT-PCR was performed to analyze the expression of hypertrophic genes (Anp, Bnp, and β-Mhc). *P<0.05, **P<0.01, ***P<0.001, ns: not significant.

Figure 8. Sirt2 deficiency blunts the cardioprotective function of metformin

(A) Left: Representative western blotting showing the phosphorylation of AMPK at Thr172 in the hearts of WT and Sirt2-KO mice treated with Ang II (1.3 mg/kg/day) and metformin (200 mg/kg/day). Veh: Vehicle; Met: Metformin. Right: Quantification of phosphorylated AMPK levels (n=6; **P<0.01, ***P<0.001, ns: not significant). (B) Ejection fraction and fractional shortening of WT and Sirt2-KO mice treated with Ang II and metformin (n=15~17; *P<0.05, **P<0.01, ***P<0.001, ns: not significant). (C) Ratios of heart weight-to-body weight (HW/BW) or heart weight-to-tibia length (HW/TL) in WT and Sirt2-KO mice treated with Ang II and metformin (n=15~24; ***P<0.001, ns: not significant). (D) Left: Hematoxylin-eosin (H&E, scale bar=1 mm) staining and wheat germ agglutinin (WGA, scale bar=30 μm) staining were performed to determine the hypertrophic growth of the hearts in WT and Sirt2-KO mice treated with Ang II and metformin. Right: Quantification of cardiomyocyte size of the hearts in WT and Sirt2-KO mice treated with Ang II and metformin (n=14~24; ***P<0.001, ns: not significant). (E) Left: Picrosirius red (PSR, scale bar=50 μm) staining was performed to determine cardiac fibrosis of the hearts from WT and Sirt2-KO mice treated with Ang II and metformin. Right: Quantification of cardiac fibrosis of the hearts in WT and Sirt2-KO mice treated with Ang II and metformin (n=14~24; ***P<0.001, ns: not significant).