SIRT2 deacetylase represses NFAT transcription factor to maintain cardiac homeostasis

Abstract

Heart failure is an aging-associated disease that is the leading cause of death worldwide. Sirtuin family members have been largely studied in the context of aging and aging-associated diseases. Sirtuin 2 (SIRT2) is a cytoplasmic protein in the family of sirtuins that are NAD⁺-dependent class III histone deacetylases. In this work, we studied the role of SIRT2 in regulating nuclear factor of activated T-cells (NFAT) transcription factor and the development of cardiac hypertrophy. Confocal microscopy analysis indicated that SIRT2 is localized in the cytoplasm of cardiomyocytes and SIRT2 levels are reduced during pathological hypertrophy of the heart. SIRT2-deficient mice develop spontaneous pathological cardiac hypertrophy, remodeling, fibrosis, and dysfunction in an age-dependent manner. Moreover, young SIRT2-deficient mice develop exacerbated agonist-induced hypertrophy. In contrast, SIRT2 overexpression attenuated agonist-induced cardiac hypertrophy in cardiomyocytes in a cell-autonomous manner. Mechanistically, SIRT2 binds to and deacetylates NFATc2 transcription factor. SIRT2 deficiency stabilizes NFATc2 and enhances nuclear localization of NFATc2, resulting in increased transcription activity. Our results suggest that inhibition of NFAT rescues the cardiac dysfunction in SIRT2-deficient mice. Thus, our study establishes SIRT2 as a novel endogenous negative regulator of NFAT transcription factor.

Keywords: NFAT transcription factor; SIRT2; cardiac hypertrophy; cardiomyocyte; heart failure; lysine acetylation; sirtuin.

Figure 1.

Pathological hypertrophy reduces SIRT2 levels in heart. A, representative confocal images of vehicle or PE (20 μm for 48 h) treated neonatal primary cardiomyocytes stained with α-actinin and SIRT2. Scale bar = 20 μm. B, Western blot analysis of vehicle- or PE- or ISO- treated (20 μm for 48 h) primary cardiomyocytes probed for SIRT2. C, quantification of SIRT2 from B. Error bars, mean ± S.D.; *, p < 0.05. D, scatter plot representing HW/TL ratio of vehicle- or ISO-treated (10 mg/kg/day for 7 days) mice. n = 9 mice; error bars, mean ± S.D.; *, p < 0.05. E, scatter plot representing fractional shortening of vehicle- or ISO-treated (10 mg/kg/day for 7 days) mice. n = 6 mice; error bars, mean ± S.D.; *, p < 0.05. F, qPCR analysis of SIRT2, ANP, BNP, and PPIA in vehicle- or ISO-treated mice hearts. Cyclophilin A (PPIA) was used as negative control. n = 4 mice; error bars, mean ± S.D.; *, p < 0.05. G, Western blot analysis of vehicle- and/or ISO-treated mice heart samples for SIRT2 and ANP. n = 4 mice. H, quantification of SIRT2 from G. I, Western blot analysis of vehicle- or ISO-treated mice for SIRT2, n = 4 mice. J, quantification of SIRT2 from I.

Figure 2.

SIRT2 deficiency induces cardiac hypertrophy. A, Western blot analysis of WT and SIRT2-KO mice hearts for SIRT2. B, scatter plot representing HW/TL ratio of WT and SIRT2-KO mice at 2 months and 9 months of age. Error bars, mean ± S.D.; n = 5 mice; *p < 0.05. C–E, scatter plots depicting left ventricular wall thickness (C), left ventricular internal diameter (D), and fractional shortening (E) of WT and SIRT2-KO mice at 2 months and 9 months of age. n = 3–5 mice; error bars, mean ± S.D.; *, p < 0.05. F, qPCR analysis of α-MHC and BNP in WT and SIRT2-KO mice hearts at 9 months of age. n = 4 mice; error bars, mean ± S.D.; *, p < 0.05. G(i), representative confocal images showing WGA staining in sections of WT and SIRT2-KO mice hearts at 9 months of age. Scale bar = 50 μm. G(ii), heart sections of 9-month-old WT and SIRT2-KO mice stained with Masson's trichrome stain showing cardiac fibrosis. n = 3–5 mice. H, graph showing relative cardiomyocyte cross-sectional area measured from G(i). I, scatter plot showing fibrosis scored in a blinded fashion from G(ii). n = 5 mice; error bars, mean ± S.D.; *, p < 0.05. J, qPCR analysis of alpha smooth muscle actin (α-SMA), fibronectin 1 (Fn1) and collagen I (Col1) in WT and SIRT2-KO mice hearts at 9 months of age. n = 3–4 mice; error bars, mean ± S.D.; *, p < 0.05.

Figure 3.

SIRT2 deficiency augments agonist-induced cardiac hypertrophy. A, scatter plot showing HW/TL ratio of 2-month-old WT and SIRT2-KO mice treated with vehicle or isoproterenol (5 mg/kg/day) for 7 days. n = 5 mice; error bars, mean ± S.D.; *, p < 0.05. B, scatter plot depicting left ventricular wall thickness of 2-month-old WT and SIRT2-KO mice treated with vehicle or ISO. n = 5 mice; error bars, mean ± S.D.; *, p < 0.05. C, scatter plot depicting fractional shortening of 2-month-old WT and SIRT2-KO mice treated with vehicle or ISO. n = 5 mice; error bars, mean ± S.D.; *, p < 0.05. D, heart sections of 2-month-old WT and SIRT2-KO mice treated with vehicle or ISO stained with Masson's trichrome stain to detect fibrosis. n = 5 mice. E, graph showing relative fibrosis scored in a blinded fashion from D. n = 5 mice; error bars, mean ± S.D.; *, p < 0.05.

Figure 4.

SIRT2 depletion induces hypertrophy in cardiomyocytes. A, Western blot analysis confirming depletion of SIRT2 in cardiomyocytes transfected with SIRT2-specific siRNA. B, [³H]leucine incorporation into cellular proteins of neonatal rat cardiomyocytes transfected with control or SIRT2 siRNA. n = 5 mice; error bars, mean ± S.D.; *, p < 0.05. c.p.m., counts per minute. C, Western blot analysis of control and SIRT2-knockdown (SIRT2-KD) cardiomyocytes treated with vehicle or PE (20 μm) or ISO (20 μm) for 48 h and probed for ANP and SIRT2. D, representative confocal images of control and SIRT2-KD cardiomyocytes treated with vehicle or PE (20 μm) or ISO (20 μm) for 48 h and further stained with ANP and α-actinin or myomesin. Scale bar = 20 μm. E, Western blot analysis showing overexpression of SIRT2 in cardiomyocytes. F, [³H]leucine incorporation into total cellular protein of neonatal rat cardiomyocytes infected with SIRT2 (Ad-SIRT2) or control adenovirus (Ad-GFP) and then treated with vehicle or PE (20 μm) or ISO (20 μm) for 48 h. n = 5; error bars, mean ± S.D.; *, p < 0.05. c.p.m., counts per minute. G, Western blot analysis of Ad-null or Ad-SIRT2 expressing cardiomyocytes treated with vehicle or PE (20 μm) or ISO (20 μm) for 48 h and harvested after puromycin treatment (1 μm), and probed for puromycin, ANP, and SIRT2. H, quantification of puromycin incorporation from G. I, representative confocal images of control and SIRT2 overexpressing cardiomyocytes treated with vehicle or PE (20 μm) or ISO (20 μm) for 48 h and stained with ANP and α-actinin or myomesin. Scale bar = 20 μm. J, graph showing quantification of relative cardiomyocyte size measured from I.

Figure 5.

SIRT2 deficiency increases NFATc2 levels in mice hearts. A, Western blot analysis of 2- and 9-month-old WT and SIRT2-KO mice hearts samples for NFATc2, calcineurin, and SIRT2. B, qPCR analysis of IFN-γ and ADSSL1 in 9-month-old WT and SIRT2-KO mice hearts. n = 3–4 mice; error bars, mean ± S.D.; *, p < 0.05. C, representative confocal images showing control and SIRT2-KD cardiomyocytes stained for calcineurin and SIRT2. Scale bar = 20 μm for upper panel, 10 μm for lower panel. D, graph showing quantification of calcineurin from C. Error bars, mean ± S.D.; *, p < 0.05. E, scatter plot showing calcineurin activity assay in 9-month-old WT and SIRT2-KO mice hearts. n = 5 mice; error bars, mean ± S.D.; *, p < 0.05. F, graph showing the calcium transients in control and SIRT2-KD cardiomyocytes treated with either vehicle or PE. G, graph showing the calcium transients in Ad-null and Ad-SIRT2 overexpressing cardiomyocytes treated with either vehicle or PE.

Figure 6.

SIRT2 deacetylates NFATc2 and inhibits its activity. A, Western blot analysis of SIRT2 interaction with NFATc2. SIRT2 was immunoprecipitated from heart lysates and probed for its interaction with NFATc2. B, in vitro deacetylation assay where recombinant acetylated-NFATc2 was incubated with wildtype or SIRT2-H187Y in the presence of NAD⁺. NFATc2 acetylation was analyzed by Western blotting. C, representative confocal images showing control and SIRT2-KD cardiomyocytes stained for NFATc2. Scale bar = 5 μm. D, graph showing quantification of NFATc2 from Fig. 4C. Error bars, mean ± S.D.; *, p < 0.05. E, luciferase activity in vehicle- or AGK2-treated cells transfected with NFAT-Luc plasmid. n = 3–5; error bars, mean ± S.D.; *, p < 0.05. F, luciferase activity in control and SIRT2-KD cells treated with vehicle or PE (20 μm) for 48 h. n = 3; error bars, mean ± S.D.; *, p < 0.05. G, luciferase activity in control and SIRT2 overexpressing cardiomyocytes transfected with NFAT-Luc plasmid. Error bars, mean ± S.D.; n = 4; *, p < 0.05. H, luciferase activity in wildtype SIRT2 and SIRT2-H187Y overexpressing cells treated with vehicle or PE (20 μm) for 48 h. n = 3; error bars, mean ± S.D.; *, p < 0.05.

Figure 7.

NFAT inhibition rescues hypertrophy in SIRT2-depleted cardiomyocytes. A, luciferase reporter assay for cells treated with vehicle or NFAT inhibitor (VIVIT, 1 μm). B, [³H]leucine incorporation into total cellular protein of control and SIRT2-KD cardiomyocytes treated with vehicle or NFAT inhibitor (VIVIT, 1 μm). n = 5; error bars, mean ± S.D.; *, p < 0.05. c.p.m., counts per minute. C, representative confocal images showing ANP expression in vehicle or NFAT inhibitor (VIVIT, 1 μm) treated control and SIRT2-KD cardiomyocytes. D, scatter plot representing HW/TL ratio of 9-month-old WT and SIRT2-KO mice treated with either vehicle or NFAT inhibitor VIVIT for 14 days. n = 5 mice; error bars, mean ± S.D.; *, p < 0.05. E, scatter plot depicting left ventricular wall thickness of 9-month-old WT and SIRT2-KO mice treated with either vehicle or NFAT inhibitor VIVIT for 14 days. n = 5 mice; error bars, mean ± S.D.; *, p < 0.05. F, scatter plot depicting fractional shortening of 9-month-old WT and SIRT2-KO mice treated with either vehicle or NFAT inhibitor VIVIT (10 mg/kg) for 14 days. n = 5 mice; error bars, mean ± S.D.; *, p < 0.05. G, schematic representation of the proposed mechanism. Under physiological conditions, SIRT2 deacetylases NFATc2 and excludes it from the nucleus, leading to its degradation, and represses its transcriptional activity. During cardiac stress SIRT2 levels decrease in heart. resulting in enhanced acetylation of NFATc2 and transcriptional hyperactivation. Therefore, NFAT remains active inside the nucleus and induces cardiac hypertrophy program.