Sirt3 blocks the cardiac hypertrophic response by augmenting Foxo3a-dependent antioxidant defense mechanisms in mice
Nagalingam R. Sundaresan, … , Ayman Isbatan, Mahesh P. Gupta
Nagalingam R. Sundaresan, … , Ayman Isbatan, Mahesh P. Gupta
Published August 3, 2009
Citation Information: J Clin Invest. 2009;119(9):2758-2771. https://doi.org/10.1172/JCI39162.
View: Text | PDF
Research Article Aging

Sirt3 blocks the cardiac hypertrophic response by augmenting Foxo3a-dependent antioxidant defense mechanisms in mice

Abstract

Sirtuin 3 (SIRT3) is a member of the sirtuin family of proteins that promote longevity in many organisms. Increased expression of SIRT3 has been linked to an extended life span in humans. Here, we have shown that Sirt3 protects the mouse heart by blocking the cardiac hypertrophic response. Although Sirt3-deficient mice appeared to have normal activity, they showed signs of cardiac hypertrophy and interstitial fibrosis at 8 weeks of age. Application of hypertrophic stimuli to these mice produced a severe cardiac hypertrophic response, whereas Sirt3-expressing Tg mice were protected from similar stimuli. In primary cultures of cardiomyocytes, Sirt3 blocked cardiac hypertrophy by activating the forkhead box O3a–dependent (Foxo3a-dependent), antioxidant–encoding genes manganese superoxide dismutase (MnSOD) and catalase (Cat), thereby decreasing cellular levels of ROS. Reduced ROS levels suppressed Ras activation and downstream signaling through the MAPK/ERK and PI3K/Akt pathways. This resulted in repressed activity of transcription factors, specifically GATA4 and NFAT, and translation factors, specifically eukaryotic initiation factor 4E (elf4E) and S6 ribosomal protein (S6P), which are involved in the development of cardiac hypertrophy. These results demonstrate that SIRT3 is an endogenous negative regulator of cardiac hypertrophy, which protects hearts by suppressing cellular levels of ROS.

Authors

Nagalingam R. Sundaresan, Madhu Gupta, Gene Kim, Senthilkumar B. Rajamohan, Ayman Isbatan, Mahesh P. Gupta

×

Figure 9

Sirt3 binds, deacetylates, and activates Foxo3a.

Options: View larger image (or click on image) Download as PowerPoint
Sirt3 binds, deacetylates, and activates Foxo3a. (A) Lysate of cells ove...
(A) Lysate of cells overexpressed with Flag or Flag.Sirt3 was subjected to immunoprecipitation with Flag-M2 agarose beads. The resulting complex was analyzed by Western blotting with anti-Foxo3a antibody. (B) Lysate of cells infected with Ad.Sirt3 virus was subjected to immunoprecipitation with anti-Foxo3a antibody, and the resulting complex was analyzed by Western blotting with anti-Sirt3 antibody. (C) Sirt3 deacetylates Foxo3a. Cells were overexpressed with Flag.Foxo3a and then treated with H2O2 to induce protein acetylation. Flag.Foxo3a was immunoprecipitated and subjected to deacetylation with Sirt3 or Sirt1 in the presence or absence of NAD in the buffer. Protein acetylation was determined by Western blotting with anti–Ac-K and anti-Flag antibodies. (D) Cytoplasmic and nuclear fractions of cardiomyocytes infected with Ad.Sirt3 or Ad.Smut viruses were prepared and analyzed by Western blotting with Foxo3a antibody. Histone 3 and GAPDH were utilized as nuclear and cytoplasmic markers, respectively. (E) Confocal analysis of Foxo3a localization (red) in cardiomyocytes infected with Ad.Sirt3 or Ad.Smut and treated with PE. Positions of nuclei were determined by DAPI stain (blue). Original magnification, ×1,000. (F) Overexpression of Sirt3 activates Foxo3a-dependent promoters. Neonatal rat cardiomyocytes were infected with viruses synthesizing Sirt1, Sirt3, or a mutant protein. The next morning cells were transfected with a Foxo3a expression plasmid and a Foxo3a responsive/luciferase reporter plasmid in different combinations as indicated. Luciferase activity was determined 48 hours after transfection. Sirt3 overexpression significantly activated Foxo3a-dependent promoter. Mean ± SEM, n = 4.